Biomarkers for Head-and-Neck Cancers and Precancers

Abstract

The invention provides markers and methods for detecting head-and-neck precancers, (including OPLs), cancers and related disease conditions in a subject. The invention also provides localization and imaging methods for head-and-neck precancers (including OPLs) and cancers, along with kits for carrying out methods of the invention. The invention further provides therapeutic applications for head-and-neck precancers (including OPLs) and cancers which employ head-and-neck precancer and cancer markers, polynucleotides encoding the markers, and binding agents for the markers.

Description

RELATED APPLICATIONS

[0001] This application is a Divisional of U.S. application Ser. No. 12/866,567, filed Nov. 17, 2010, which is a national stage application of International Patent Application No. PCT/CA2009/000154, filed Feb. 6, 2009, which claims priority to Canadian Patent Application No. 2,643,611, filed Nov. 10, 2008 and Canadian Patent Application No. 2,618,163, filed on Feb. 7, 2008 all of which are incorporated herein by reference, as if set forth in their entirety

FIELD OF THE INVENTION

[0002] The invention relates to markers for head-and-neck cancers, including oral cancers. The invention also relates to markers for head-and-neck precancers--including, but not limited to, oral leukoplakia with hyperplasia or dysplasia. The invention further relates to methods for assessing the status of head-and-neck tissue and oral tissue, and methods for the detection, diagnosis, prediction, and therapy of head-and-neck disease. In one aspect, the invention relates to biomarkers of head-and-neck squamous cell carcinoma and biomarkers of head-and-neck precancers (including, but not limited to, oral leukoplakia with hyperplasia or dysplasia), and methods for detecting, diagnosing, predicting, and treating these and related conditions. In a further aspect, the invention relates to biomarkers of oral leukoplakia with hyperplasia or dysplasia, and methods for detecting, diagnosing, predicting, and treating same.

BACKGROUND OF THE INVENTION

[0003] Annually, about 500,000 cancer-related deaths are estimated in the United States alone. Of these, approximately 13,000 are attributed to head-and-neck including oral squamous-cell carcinoma (HNOSCC), making it the sixth most common cause of cancer deaths and the fourth most prevalent cancer in men worldwide (1).

[0004] A lack of biomarkers for early detection and risk assessment is clearly reflected by the fact that more than 50% of all patients with head-and-neck squamous-cell carcinoma (HNSCC) have advanced disease at the time of diagnosis (2). The five-year survival rate of HNSCC patients is less than 50%, and the prognosis of advanced HNSCC cases has not changed much over the past three decades, except in a few advanced centers (2). Conceivably, improvement in understanding of the steps leading to tumorigenesis will provide the ability to identify and predict malignant progression at an earlier stage of HNSCC lesions, in turn leading to more effective treatment and reduction of morbidity and mortality.

[0005] The precancerous lesions, potentially malignant lesions, premalignant lesions, and squamous intraepithelial lesions (SILs) of the head and neck (oral cavity, oropharynx, and larynx)--which are clinically usually defined as "leukoplakia"--remain the main controversial topic in head and neck pathology as regards classification, histological diagnosis, and treatment (3-5). The transition from a normal epithelium to squamous cell carcinoma (SCC) of the head and neck is a lengthy, comprehensive and multistage process, causally related to progressive accumulation of genetic changes leading to the selection of a clonal population of transformed epithelial cells (6). The whole spectrum of histological changes occurring in this process has been recently cumulatively designated potentially malignant lesions or SILs, ranging from squamous hyperplasia to carcinoma in situ (CIS) (3). In their evolution, some cases of potentially malignant lesions and SILs are self-limiting and reversible, some persist, and some progress to SCC in spite of careful follow-up and treatment.

[0006] Oral squamous-cell carcinoma (OSCC), the most common form of HNOSCC, is often preceded by clinically-well-defined lesions, such as leukoplakia, causally linked with chronic exposure of the oral mucosa to carcinogens or growth promoters in tobacco and alcohol; leukoplakias with dysplasia are termed "oral premalignant lesions" (OPLs) (3, 6). The presence of dysplastic areas in the oral epithelium is associated with a likely progression to cancer; however, it is not an accurate predictor of cancer risk (6, 7). The major challenge in oral tumorigenesis is the identification of proteins that may serve as markers to differentiate the high-risk leukoplakic lesions from more benign lesions for early intervention to reduce the morbidity associated with this devastating disease. Rapid advances in treatment modalities and improvements in the early detection of head-and-neck cancers have not significantly impacted the overall survival rates of cancer patients.

[0007] Currently, there are no clinically-established biomarkers to facilitate the diagnosis or prognosis of head-and-neck cancer and oral leukoplakia. It is expected that identification of novel protein markers or therapeutic targets will ultimately improve patient care and survival. Thus, much effort has been focused on genomics- and proteomics-based identification of biomarkers that can detect the disease in early stages, predict the risk of malignant transformation in patients with oral leukoplakia, and/or predict the clinical outcome in HNOSCC patients after treatment of primary tumors. It is hoped that these biomarkers will transform clinical practice by including cancer screening and diagnosis based on molecular markers as a complement to histopathology.

[0008] In the post-genomics era, proteomics combined with mass spectrometry (MS) has become a powerful paradigm for the examination of proteins in a global manner, and the consequent discovery of cancer risk markers and drug targets. While transcriptomics provides a tool for unraveling gene-expression networks, proteomics links these networks to protein products and provides further insight into post-translational modifications that regulate cellular functions, thereby complementing genomic analyses (reviewed in Ralhan (8)). Identification of differentially expressed proteins in HNSCCs using proteomics revealed that expression patterns of proteins may have some predictive power for clinical outcome and personalized risk assessment (8-16)

[0009] Differential tagging with isotopic reagents, such as isotope-coded affinity tags (ICAT) (17) or the more recent variation that uses isobaric tagging reagents, iTRAQ (Applied Biosystems, Foster City, Calif.), followed by multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis, is emerging as one of the more powerful methodologies in the search for disease biomarkers. Recent studies using iTRAQ reagents resulted in identification and relative quantification of proteins leading to a discovery of potential cancer markers (PCMs) for human cancers (17-23).

SUMMARY OF THE INVENTION

[0010] As discussed herein, the inventors used iTRAQ labeling in combination with multidimensional LC-MS/MS analysis of head-and-neck cancer in order to compare protein profiles of HNSCC and non-cancerous head-and-neck tissues. The inventors also used iTRAQ labeling in combination with multidimensional LC-MS/MS analysis of oral leukoplakia with dysplasia (oral premalignant lesions, or OPLs) in order to compare protein profiles of OPLs and normal head-and-neck tissues. These studies were designed to identify potential biomarkers, and to identify, in a global fashion, molecular pathways that are deregulated in head-and-neck and oral cancer, thereby aiding in drug-target discovery.

[0011] The iTRAQ experiments were performed on resected HNSCC, excised OPLs, and non-cancerous tissue homogenates. The rationale for using whole tissue homogenates as opposed to laser-capture-microdissection (LCM)-procured tumor cells has been discussed (21, 23). There are at least two major advantages in the analysis of tissue homogenates: the relevant proteins are much more abundant in the tissues of interest than in bodily fluids, and there is an automatic link between a protein that is differentially expressed and the tumor itself. Such a link would need to be demonstrated if the differentially expressed proteins were to be discovered in a bodily fluid (e.g., blood), as every tissue or organ can potentially discharge into blood. Furthermore, the tumor microenvironment plays an important role in cancer progression (24); examination of protein expression in tissues from a homogenate of different cell types takes into account the contributions of the tumor microenvironment.

[0012] The protein expression profiles of HNSCCs and OPLs were compared with non-cancerous head-and-neck tissues (controls) using the iTRAQ-labelling technique in combination with multidimensional LC-MS/MS analysis. In the iTRAQ technology, primary amines are tagged, thereby potentially allowing the tagging of most tryptic peptides. The multiplexing ability afforded by the iTRAQ reagents, which are available in four different tags, was ideally suited for the present study, as it provided a means to perform a proteomic analysis of both paired and non-paired non-cancerous (histologically normal) head-and-neck tissues, while simultaneously comparing them against the cancer samples. This strategy helps to identify proteins that might be differentially expressed due to manifestation of field cancerization (25-27) in clinically normal mucosa, and may be useful in designing strategies for risk prediction of disease recurrence or second primary tumor development.

[0013] Some of the overexpressed proteins that were identified in the tissues by the iTRAQ technology and LC MS/MS analysis were confirmed by immunohistochemistry and Western blotting. These approaches ensured that the selected proteins demonstrated a consistent pattern of overexpression in HNSCCs and OPLs, and greatly increased confidence in the observations stemming from iTRAQ analysis. Apart from their potential utility as biomarkers for HNSCC and OPLs, these proteins also provide valuable insight into the previously unknown molecular networks and mechanisms that govern the normal-to-malignant conversion of epithelium.

[0014] Using the above techniques, the inventors identified markers associated with head-and-neck tissues including oral tissues. Thus, the invention relates to novel markers for head-and-neck including oral tissues, including markers of head-and-neck including oral disease, and compositions comprising same.

[0015] In one aspect, the invention provides marker sets that distinguish head-and-neck cells or tissue, diseases, or phases thereof. Also provided are uses of these marker sets. A marker set may include a plurality of polypeptides and/or a plurality of polynucleotides encoding such polypeptides, including at least one marker listed in Table 5 and optionally including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the markers listed therein. In specific aspects, the markers include at least 2, 3, 4, or 5 polypeptides listed in Table 5. In another aspect, the protein marker set includes protein clusters or proteins in pathways including markers listed in Table 5 and Table 2. In yet another aspect, the invention provides markers in Table 1 that are up-regulated or down-regulated or expressed in cancer samples as compared to the non-cancer samples.

[0016] In another aspect, the invention provides marker sets that distinguish oral cells or tissue, diseases, or phases thereof. Also provided are uses of these marker sets. A marker set may include a plurality of polypeptides and/or a plurality of polynucleotides encoding such polypeptides, including at least one marker listed in Table 5 and optionally including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the markers listed therein. In specific aspects, the markers include at least 2, 3, 4, or 5 polypeptides listed in Table 5. In another aspect, the protein marker set includes protein clusters or proteins in pathways including markers listed in Table 5. In yet another aspect, the invention provides markers in 5 and optionally Table 8 including 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of the markers listed therein that are up-regulated or down-regulated or expressed in OPL samples as compared to the normal samples. In specific aspects, the OPL markers include at least 2, 3, 4, or 5 polypeptides listed in Table 6 and 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 listed in Table 5. In another aspect, the protein marker set includes protein clusters or proteins in pathways including markers listed in Table 5 (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32).

[0017] Up-regulated markers identified in Table 1 (up-regulated in cancer samples versus non-cancer samples), and Table 6 (up-regulated in OPL samples versus non-cancer samples), including but not limited to native-sequence polypeptides, isoforms, chimeric polypeptides, all homologs, fragments, and precursors of the markers, including modified forms of the polypeptides and derivatives are referred to and defined herein as "head-and-neck cancer marker(s) and OPL marker(s)". Polynucleotides encoding head-and-neck cancer markers are referred to and defined herein as "head-and-neck cancer polynucleotide marker(s)", "polynucleotides encoding head-and-neck cancer markers", or "polynucleotides encoding the cancer marker(s)". Polynucleotides encoding OPL markers are referred to and defined herein as "OPL polynucleotide marker(s)", "polynucleotides encoding OPL markers", or "polynucleotides encoding the precancer marker(s)". The head-and-neck cancer markers and head-and-neck cancer polynucleotide markers are sometimes collectively referred to herein as "cancer marker(s)", while the OPL markers and OPL polynucleotide markers are sometimes collectively referred to herein as "OPL marker(s)".

[0018] Up-regulated head-and-neck cancer markers listed in Table 1 (in cancer sample versus non-cancer sample), those listed in Table 1 or 2, and polynucleotides encoding the markers, have application in the determination of the status of the head-and-neck cell or tissue and in the detection of a head-and-neck disease such as head-and-neck cancer. Thus, the markers can be used for diagnosis, monitoring (i.e., monitoring progression or therapeutic treatment), prognosis, treatment, or classification of a head-and-neck disease (e.g., head-and-neck cancer), HNSCC or related conditions or as markers before surgery or after relapse. The invention also contemplates methods for assessing the status of a head-and-neck tissue, and methods for the diagnosis and therapy of a head-and-neck disease.

[0019] Up-regulated OPL markers listed in Table 6 (in OPL sample versus normal sample), those listed in Table 6 or Table 7, and polynucleotides encoding the markers, have application in the determination of the status or phase of the head-and-neck/oral cell or tissue and in the detection of a head-and-neck disease such as oral leukoplakia with hyperplasia or dysplasia or head-and-neck cancer. Thus, the markers can be used for diagnosis, monitoring (i.e., monitoring progression or therapeutic treatment), prognosis, treatment, or classification of a head-and-neck disease (e.g., oral leukoplakia with hyperplasia or dysplasia or OPLs), HNSCC or related conditions or as markers before surgery or after relapse. The invention also contemplates methods for assessing the status of a head-and-neck tissue, and methods for the diagnosis and therapy of a head-and-neck disease.

[0020] In accordance with methods of the invention, OPL and head-and-neck cancer can be assessed or characterized, for example, by detecting the presence in the sample of (a) an OPL or head-and-neck cancer marker or fragment thereof; (b) a metabolite which is produced directly or indirectly by an OPL or head-and-neck cancer marker; (c) a transcribed nucleic acid or fragment thereof having at least a portion with which an OPL polynucleotide marker or a head-and-neck cancer polynucleotide marker is substantially identical; and/or (c) a transcribed nucleic acid or fragment thereof, wherein the nucleic acid hybridizes with an OPL polynucleotide marker or a head-and-neck cancer polynucleotide marker.

[0021] The levels of OPL markers or head-and-neck cancer markers or OPL polynucleotide markers or head-and-neck cancer polynucleotide markers in a sample may be determined by methods as described herein and generally known in the art. The expression levels may be determined by isolating and determining the level of nucleic acid transcribed from each OPL polynucleotide markers or head-and-neck cancer polynucleotide marker. Alternatively or additionally, the levels of OPL markers or head-and-neck cancer markers translated from mRNA transcribed from an OPL polynucleotide markers or a head-and-neck cancer polynucleotide marker respectively may be determined.

[0022] In an aspect, the invention provides a method for characterizing or classifying a head-and-neck sample including detecting a difference in the expression of a first plurality of head-and-neck cancer markers or head-and-neck cancer polynucleotide markers relative to a control, the first plurality of markers including or consisting of at least 2, 3, 4, or 5 of the markers corresponding to the markers listed in Table 5, and optionally 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or up to all of the markers listed therein or those listed in Table 2 or those that are up-regulated in cancer versus control tissue in Table 1. In specific aspects, the plurality of markers consists of at least 3, 4, or 5 of the markers listed in Table 1.

[0023] In an aspect, the invention provides a method for characterizing or classifying an OPL or a head-and-neck including detecting a difference in the expression of a first plurality of OPL markers or head-and-neck markers or OPL or head-and-neck polynucleotide markers relative to a control, the first plurality of markers including or consisting of at least 2, 3, 4, or 5 of the markers corresponding to the markers listed in Table 5, and optionally 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 of the markers listed therein or those listed in Table 7 or those that are up-regulated in OPL versus control tissue in Table 6. In specific aspects, the plurality of markers consists of at least 3, 4 or 5 of the markers listed in Table 6.

[0024] In an aspect, a method is provided for characterizing a head-and-neck tissue by detecting OPL markers/head-and-neck cancer markers or OPL polynucleotide markers/head-and-neck cancer polynucleotide markers associated with a head-and-neck tissue stage or phase, or head-and-neck disease in a subject including: [0025] (a) obtaining a sample from a subject; [0026] (b) detecting or identifying in the sample OPL markers/head-and-neck cancer markers or OPL polynucleotide markers/head-and-neck cancer polynucleotide markers; and [0027] (c) comparing the detected amount with an amount detected for a standard.

[0028] In an embodiment of the invention, a method is provided for detecting OPL markers or head-and-neck cancer markers or OPL polynucleotide markers or head-and-neck cancer polynucleotide markers associated with OPL or head-and-neck cancer in a patient including: [0029] (a) obtaining a sample from a patient; [0030] (b) detecting in the sample OPL markers or head-and-neck cancer markers or OPL polynucleotide markers or head-and-neck cancer polynucleotide markers; and [0031] (c) comparing the detected amount with an amount detected for a standard.

[0032] The term "detect" or "detecting" includes assaying, imaging or otherwise establishing the presence or absence of the target OPL markers or head-and-neck cancer markers or polynucleotides encoding the markers, subunits thereof, or combinations of reagent bound targets, and the like, or assaying for, imaging, ascertaining, establishing, or otherwise determining one or more factual characteristics of a head-and-neck tissue phase or head-and-neck disease including OPL, cancer, metastasis, stage, or similar conditions. The term encompasses diagnostic, prognostic, and monitoring applications for the OPL markers or head-and-neck cancer markers and polynucleotides encoding these markers.

[0033] The invention also provides a method of assessing whether a patient is afflicted with or has a pre-disposition for head-and-neck disease, in particular OPL or head-and-neck cancer, the method including comparing: [0034] (a) levels of OPL or head-and-neck cancer markers or polynucleotides encoding OPL or head-and-neck cancer markers associated with the head-and-neck disease in a sample from the patient; and [0035] (b) normal levels of OPL or head-and-neck cancer markers or polynucleotides encoding OPL or head-and-neck cancer markers associated with the head-and-neck disease in samples of the same type obtained from control patients not afflicted with the disease, wherein altered levels of the OPL or head-and-neck cancer markers or the polynucleotides relative to the corresponding normal levels of OPL or head-and-neck cancer markers or polynucleotides is an indication that the patient is afflicted with head-and-neck disease.

[0036] In an aspect of a method of the invention for assessing whether a patient is afflicted with or has a pre-disposition for head-and-neck cancer, higher levels of head-and-neck cancer markers (e.g., YWHAZ, stratifin, S100A7) in a sample relative to the corresponding normal levels is an indication that the patient is afflicted with head-and-neck cancer.

[0037] In an aspect of a method of the invention for assessing whether a patient is afflicted with or has a pre-disposition for OPL/head-and-neck cancer, higher levels of OPL markers (e.g., YWHAZ, stratifin, hnRNPK) in a sample relative to the corresponding normal levels is an indication that the patient is afflicted with OPL.

[0038] In another aspect of a method of the invention for assessing whether a patient is afflicted with or has a pre-disposition for head-and-neck cancer, lower levels of head-and-neck cancer markers (e.g., alpha-1-antitrypsin, KPSG lumican) in a sample relative to the corresponding normal levels is an indication that the patient is afflicted with head-and-neck cancer.

[0039] In another aspect of a method of the invention for assessing whether a patient is afflicted with or has a pre-disposition for OPL/head-and-neck cancer, lower levels of OPL/head-and-neck cancer markers (e.g., alpha-1-antitrypsin, peroxiredoxin 2) in a sample relative to the corresponding normal levels is an indication that the patient is afflicted with OPL (oral leukoplakia with dysplasia).

[0040] In a further aspect, a method for screening a subject for head-and-neck disease is provided including (a) obtaining a biological sample from a subject; (b) detecting the amount of OPL or head-and-neck cancer markers associated with the disease in said sample; and (c) comparing said amount of OPL or head-and-neck cancer markers detected to a predetermined standard, where detection of a level of OPL or head-and-neck cancer markers that differs significantly from the standard indicates head-and-neck disease.

[0041] In an embodiment, a significant difference between the levels of OPL or head-and-neck cancer marker levels in a patient and normal levels is an indication that the patient is afflicted with or has a predisposition to head-and-neck disease.

[0042] In a particular embodiment the amount of head-and-neck cancer marker(s) (e.g., prothymosin alpha, APC-binding protein EB1) detected is greater than that of a standard and is indicative of head-and-neck disease, in particular head-and-neck cancer. In another particular embodiment the amount of head-and-neck cancer marker(s) (e.g., alpha-1-antitrypsin, KPSG lumican) detected is lower than that of a standard and is indicative of head-and-neck disease, in particular head-and-neck cancer.

[0043] In a particular embodiment the amount of OPL/head-and-neck marker(s) (e.g., YWHAZ, stratifin, hnRNPK) detected is greater than that of a standard and is indicative of head-and-neck disease, in particular OPL (oral leukoplakia with hyperplasia or dysplasia)/head-and-neck cancer. In another particular embodiment the amount of OPL/head-and-neck marker(s) (e.g., alpha-1-antitrypsin, peroxiredoxin 2) detected is lower than that of a standard and is indicative of head-and-neck disease, in particular OPL/head-and-neck cancer.

[0044] In aspects of the methods of the invention, the methods are non-invasive for detecting head-and-neck disease which in turn allow for diagnosis of a variety of conditions or diseases associated with the head-and-neck.

[0045] In particular, the invention provides a non-invasive non-surgical method for detection, diagnosis or prediction of head-and-neck disease (e.g., OPL or oral leukoplakia with hyperplasia or dysplasia and head-and-neck cancer or HNSCC) in a subject including: obtaining a sample of blood, plasma, serum, urine or saliva or a tissue sample from the subject; subjecting the sample to a procedure to detect OPL markers and head-and-neck cancer markers or OPL polynucleotide markers and head-and-neck cancer polynucleotide markers in the blood, plasma, serum, urine, saliva or tissue; detecting, diagnosing, and predicting head-and-neck disease by comparing the levels of OPL markers and head-and-neck cancer markers or OPL polynucleotide markers and head-and-neck cancer polynucleotide markers to the levels of marker(s) or polynucleotide(s) obtained from a control subject with no head-and-neck disease.

[0046] In an embodiment, head-and-neck disease is detected, diagnosed, or predicted by determination of increased levels of markers (e.g., one or more Table 1 up-regulated markers, preferable Table 5 up-regulated markers and more preferably one or more Table 2 up-regulated markers) when compared to such levels obtained from the control.

[0047] In an embodiment, head-and-neck disease is detected, diagnosed, or predicted by determination of increased levels of markers (e.g., one or more Table 6 up-regulated markers, preferable Table 5 up-regulated OPL markers and more preferably one or more Table 7 up-regulated markers) when compared to such levels obtained from the control.

[0048] In another embodiment, head-and-neck disease is detected, diagnosed, or predicted by determination of decreased levels of markers (e.g., one or more Table 1 down-regulated markers) when compared to such levels obtained from the control.

[0049] In another embodiment, head-and-neck disease is detected, diagnosed, or predicted by determination of decreased levels of OPL markers (e.g., one or more markers in Table 6 such as, Cystatin B or DLC1) when compared to such levels obtained from the control.

[0050] The invention also provides a method for assessing the aggressiveness or indolence of a head-and-neck disease in particular OPL (e.g., staging hyperplasia or dysplasia or degree of differentiation-mild dysplasia or severe dysplasia) or cancer (e.g., staging), the method including comparing: [0051] (a) levels of OPL or head-and-neck cancer markers or polynucleotides encoding OPL or head-and-neck cancer markers associated with the head-and-neck disease in a patient sample; and [0052] (b) normal levels of the OPL or head-and-neck cancer markers or the polynucleotides in a control sample.

[0053] In an embodiment, a significant difference between the levels in the sample and the normal levels is an indication that the head-and-neck disease, in particular OPL or cancer, is aggressive or indolent. In a particular embodiment, the levels of OPL or head-and-neck cancer markers are higher than normal levels. In another particular embodiment, the levels of OPL or head-and-neck cancer markers are lower than normal levels.

[0054] In an embodiment, a method is provided for diagnosing and/or monitoring OPL and HNSCC including comparing: [0055] (a) levels of YWHAZ or polynucleotides encoding YWHAZ in a sample from the patient; and [0056] (b) normal levels of YWHAZ or polynucleotides encoding same in samples of the same type obtained from control patients not afflicted with OPL or head-and-neck cancer or having a different stage of OPL or head-and-neck cancer, wherein altered levels of YWHAZ or polynucleotides encoding same compared with the corresponding normal levels is an indication that the patient is afflicted with OPL or HNSCC.

[0057] In an embodiment, a method is provided for diagnosing and/or monitoring OPL or HNSCC including comparing: [0058] (a) levels of S100 A7 or polynucleotides encoding S100 A7 in a sample from the patient; and [0059] (b) normal levels of S100 A7 or polynucleotides encoding same in samples of the same type obtained from control patients not afflicted with head-and-neck cancer or having a different stage of head-and-neck cancer, wherein altered levels of S100 A7 or polynucleotides encoding same compared with the corresponding normal levels is an indication that the patient is afflicted with HNSCC.

[0060] In an embodiment, a method is provided for diagnosing and/or monitoring oral leukoplakia with hyperplasia or dysplasia (OPL)/HNSCC including comparing: [0061] (a) levels of hnRNPK or polynucleotides encoding hnRNPK in a sample from the patient; and [0062] normal levels of hnRNPK or polynucleotides encoding same in samples of the same type obtained from control patients not afflicted with leukoplakia with hyperplasia or dysplasia (OPL)/head-and-neck cancer or having a different stage of leukoplakia with hyperplasia or dysplasia (OPL) or head-and-neck cancer, wherein altered levels of hnRNPK or polynucleotides encoding same compared with the corresponding normal levels is an indication that the patient is afflicted with leukoplakia with hyperplasia or dysplasia (OPL)/HNSCC.

[0063] In an embodiment, a method is provided for diagnosing and/or monitoring HNSCC and leukoplakia with hyperplasia or dysplasia (OPL) including comparing [0064] (a) levels of stratifin or polynucleotides encoding stratifin in a sample from the patient; and [0065] (b) normal levels of stratifin or polynucleotides encoding same in samples of the same type obtained from control patients not afflicted with head-and-neck cancer or leukoplakia with hyperplasia or dysplasia (OPL) or having a different stage of leukoplakia with hyperplasia or dysplasia (OPL) or head-and-neck cancer, wherein altered levels of stratifin or polynucleotides encoding same compared with the corresponding normal levels is an indication that the patient is afflicted with HNSCC.

[0066] In an aspect, the invention provides a method for determining whether a cancer has metastasized or is likely to metastasize in the future, the method including comparing: [0067] (a) levels of OPL or head-and-neck cancer markers or polynucleotides encoding OPL or head-and-neck cancer markers in a patient sample; and [0068] (b) normal levels (or non-metastatic levels) of the OPL or head-and-neck cancer markers or polynucleotides in a control sample.

[0069] In an embodiment, a significant difference between the levels in the patient sample and the normal levels is an indication that the cancer has metastasized or is likely to metastasize in the future.

[0070] In another aspect, the invention provides a method for monitoring the progression of head-and-neck disease, in particular OPL or head-and-neck cancer in a patient the method including: [0071] (a) detecting OPL or head-and-neck cancer markers or polynucleotides encoding the markers associated with the disease in a sample from the patient at a first time point; [0072] (b) repeating step (a) at a subsequent point in time; and [0073] (c) comparing the levels detected in (a) and (b), thereby monitoring the progression of the head-and-neck disease.

[0074] The invention contemplates a method for determining the effect of an environmental factor on the head-and-neck tissue, or head-and-neck disease including comparing OPL or head-and-neck cancer polynucleotide markers or OPL or head-and-neck cancer markers in the presence and absence of the environmental factor.

[0075] The invention also provides a method for assessing the potential efficacy of a test agent for inhibiting head-and-neck disease, and a method of selecting an agent for inhibiting head-and-neck disease.

[0076] The invention contemplates a method of assessing the potential of a test compound to contribute to a head-and-neck disease including: [0077] (a) maintaining separate aliquots of head-and-neck diseased cells in the presence and absence of the test compound; and [0078] (b) comparing the levels of OPL or head-and-neck cancer markers or polynucleotides encoding the markers associated with the disease in each of the aliquots.

[0079] A significant difference between the levels of OPL or head-and-neck cancer markers or polynucleotides encoding the markers in an aliquot maintained in the presence of (or exposed to) the test compound relative to the aliquot maintained in the absence of the test compound, indicates that the test compound potentially contributes to head-and-neck disease.

[0080] The invention further relates to a method of assessing the efficacy of a therapy for inhibiting head-and-neck disease in a patient. A method of the invention includes comparing: (a) levels of OPL or head-and-neck cancer markers or polynucleotides encoding the markers associated with disease in a first sample from the patient obtained from the patient prior to providing at least a portion of the therapy to the patient; and (b) levels of OPL or head-and-neck cancer markers or polynucleotides encoding the markers associated with disease in a second sample obtained from the patient following therapy.

[0081] In an embodiment, a significant difference between the levels of OPL or head-and-neck cancer markers or polynucleotides encoding the markers in the second sample relative to the first sample is an indication that the therapy is efficacious for inhibiting head-and-neck disease.

[0082] In a particular embodiment, the method is used to assess the efficacy of a therapy for inhibiting head-and-neck disease (e.g., OPL or head-and-neck cancer), where lower levels of OPL or head-and-neck cancer markers or polynucleotides encoding same in the second sample relative to the first sample, is an indication that the therapy is efficacious for inhibiting the disease.

[0083] The "therapy" may be any therapy for treating head-and-neck disease, in particular OPL or head-and-neck cancer, including but not limited to therapeutics, radiation, immunotherapy, gene therapy, and surgical removal of tissue. Therefore, the method can be used to evaluate a patient before, during, and after therapy.

[0084] Certain methods of the invention employ binding agents (e.g., antibodies) that specifically recognize OPL or head-and-neck cancer markers.

[0085] In an embodiment, the invention provides methods for determining the presence or absence of head-and-neck disease, in particular OPL or head-and-neck cancer, in a patient, including the steps of (a) contacting a biological sample obtained from a patient with one or more binding agent that specifically binds to one or more OPL or head-and-neck cancer markers associated with the disease; and (b) detecting in the sample an amount of marker that binds to the binding agent, relative to a predetermined standard or cut-off value, thereby determining the presence or absence of head-and-neck disease in the patient.

[0086] In another embodiment, the invention relates to a method for diagnosing and monitoring a head-and-neck disease, in particular OPL or head-and-neck cancer, in a subject by quantifying one or more OPL or head-and-neck cancer markers associated with the disease in a biological sample from the subject including (a) reacting the biological sample with one or more binding agent specific for the OPL or head-and-neck cancer markers (e.g., an antibody) that are directly or indirectly labelled with a detectable substance; and (b) detecting the detectable substance.

[0087] In another aspect the invention provides a method of using an antibody to detect expression of one or more head-and-neck marker in a sample, the method including: (a) combining antibodies specific for one or more head-and-neck marker with a sample under conditions which allow the formation of antibody:marker complexes; and (b) detecting complex formation, wherein complex formation indicates expression of the marker in the sample. Expression may be compared with standards and is diagnostic of a head-and-neck disease, in particular OPL or HNSCC.

[0088] Embodiments of the methods of the invention involve (a) reacting a biological sample from a subject with antibodies specific for one or more OPL or head-and-neck cancer markers which are directly or indirectly labelled with an enzyme; (b) adding a substrate for the enzyme wherein the substrate is selected so that the substrate, or a reaction product of the enzyme and substrate forms fluorescent complexes; (c) quantifying one or more OPL or head-and-neck cancer markers in the sample by measuring fluorescence of the fluorescent complexes; and (d) comparing the quantified levels to levels obtained for other samples from the subject patient, or control subjects.

[0089] In another embodiment the quantified levels are compared to levels quantified for control subjects (e.g., normal or benign) without a head-and-neck disease (e.g., OPL or cancer) wherein an increase in head-and-neck marker levels compared with the control subjects is indicative of head-and-neck disease.

[0090] In a further embodiment the quantified levels are compared to levels quantified for control subjects (e.g., normal or benign) without a head-and-neck disease (e.g., OPL or cancer) wherein a decrease in head-and-neck marker levels compared with the control subjects is indicative of head-and-neck disease.

[0091] A particular embodiment of the invention includes the following steps [0092] (a) incubating a biological sample with first antibodies specific for one or more OPL or head-and-neck cancer markers which are directly or indirectly labelled with a detectable substance, and second antibodies specific for one or more head-and-neck cancer markers which are immobilized; [0093] (b) detecting the detectable substance thereby quantifying OPL or head-and-neck cancer markers in the biological sample; and [0094] (c) comparing the quantified OPL or head-and-neck cancer markers with levels for a predetermined standard.

[0095] The standard may correspond to levels quantified for samples from control subjects without OPL or head-and-neck cancer (normal or benign), with a different disease stage, or from other samples of the subject. In an embodiment, increased levels of OPL or head-and-neck cancer markers as compared to the standard may be indicative of head-and-neck precancer or cancer. In another embodiment, lower levels of OPL or head-and-neck cancer markers as compared to a standard may be indicative of head-and-neck precancer or cancer.

[0096] OPL or HNSCC marker levels can be determined by constructing an antibody microarray in which binding sites include immobilized, preferably monoclonal, antibodies specific to a substantial fraction of marker-derived OPL or HNSCC marker proteins of interest.

[0097] Other methods of the invention employ one or more polynucleotides capable of hybridizing to one or more polynucleotides encoding OPL or HNSCC markers. Thus, methods can be used to monitor a head-and-neck disease (e.g., OPL or cancer) by detecting OPL or head-and-neck cancer polynucleotide markers associated with the disease.

[0098] Thus, the present invention relates to a method for diagnosing and monitoring a head-and-neck disease (e.g., OPL or head-and-neck cancer, HNSCC or related condition) in a sample from a subject including isolating nucleic acids, preferably mRNA, from the sample; and detecting OPL or HNSCC marker polynucleotides associated with the disease in the sample. The presence of different levels of OPL or HNSCC marker polynucleotides in the sample compared to a standard or control may be indicative of head-and-neck disease, disease stage, and/or a negative or positive prognosis (e.g., longer progression-free and overall survival).

[0099] In embodiments of the invention, OPL or head-and-neck cancer marker polynucleotide positive tumors (e.g., higher levels of the polynucleotides compared to a control normal or benign sample) are a negative diagnostic indicator. Positive OPLs or tumors can be indicative of premalignant lesions with variable risk of disease progression/head-and-neck cancer, advanced stage disease, lower progression-free survival, and/or overall survival.

[0100] In other embodiments of the invention, OPL or head-and-neck cancer marker polynucleotide negative tumors (e.g., lower levels of the polynucleotides compared to a control normal or benign tissue) are a negative diagnostic indicator. Negative OPL or tumors can be indicative of premalignant lesions with variable risk of disease progression/head-and-neck cancer, advanced stage disease, lower progression-free survival, and/or overall survival.

[0101] The invention provides methods for determining the presence or absence of a head-and-neck disease in a subject including detecting in the sample levels of nucleic acids that hybridize to one or more polynucleotides encoding OPL or head-and-neck cancer markers associated with the disease, comparing the levels with a predetermined standard or cut-off value, thereby determining the presence or absence of head-and-neck disease in the subject. In an embodiment, the invention provides methods for determining the presence or absence of OPL or head-and-neck cancer, such as HNSCC in a subject including (a) contacting a sample obtained from the subject with oligonucleotides that hybridize to one or more polynucleotides encoding OPL or head-and-neck cancer markers; and (b) detecting in the sample a level of nucleic acids that hybridize to the polynucleotides relative to a predetermined cut-off value, thereby determining the presence or absence of OPL or head-and-neck cancer in the subject.

[0102] Within certain embodiments, the amount of polynucleotides that are mRNA are detected via polymerase chain reaction using, for example, oligonucleotide primers that hybridize to one or more polynucleotides encoding OPL or HNSCC markers, or complements of such polynucleotides. Within other embodiments, the amount of mRNA is detected using a hybridization technique, employing oligonucleotide probes that hybridize to one or more polynucleotides encoding OPL or HNSCC markers, or complements thereof.

[0103] When using mRNA detection, the method may be carried out by combining isolated mRNA with reagents to convert to cDNA according to standard methods; treating the converted cDNA with amplification reaction reagents (such as cDNA PCR reaction reagents) in a container along with an appropriate mixture of nucleic acid primers; reacting the contents of the container to produce amplification products; and analyzing the amplification products to detect the presence of one or more OPL or HNSCC polynucleotide markers in the sample. For mRNA the analyzing step may be accomplished using Northern Blot analysis to detect the presence of OPL or HNSCC polynucleotide markers. The analysis step may be further accomplished by quantitatively detecting the presence of OPL or HNSCC polynucleotide markers in the amplification product, and comparing the quantity of marker detected against a panel of expected values for the known presence or absence of the markers in normal and malignant tissue derived using similar primers.

[0104] Therefore, the invention provides a method wherein mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to one or more OPL or HNSCC polynucleotide markers to produce amplification products; (d) analyzing the amplification products to detect an amount of mRNA encoding the OPL or HNSCC markers; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal and diseased tissue (e.g., premalignant or malignant tissue) derived using similar nucleic acid primers.

[0105] In particular embodiments of the invention, the methods described herein utilize the OPL or HNSCC polynucleotide markers placed on a microarray so that the expression status of each of the markers is assessed simultaneously.

[0106] In a particular aspect, the invention provides a head-and-neck microarray including a defined set of genes (i.e., at least 2, 3 4, or 5 genes listed in Table 5 or Table 2) whose expression is significantly altered by head-and-neck phase or head-and-neck disease. The invention further relates to the use of the microarray as a prognostic tool to predict head-and-neck phase or head-and-neck disease. In an embodiment, the head-and-neck microarray discriminates between head-and-neck disease resulting from different etiologies.

[0107] In a particular aspect, the invention provides an OPL microarray including a defined set of genes (i.e., at least 2, 3, 4, or 5 genes listed in Table 5 or Table 7 whose expression is significantly altered by head-and-neck phase or head-and-neck disease. The invention further relates to the use of the microarray as a prognostic tool to predict head-and-neck phase or head-and-neck disease. In an embodiment, the OPL microarray discriminates between head-and-neck disease resulting from different etiologies.

[0108] In an embodiment, the invention provides for oligonucleotide arrays including marker sets described herein. The microarrays provided by the present invention may include probes to markers able to distinguish head-and-neck phase or disease. In particular, the invention provides oligonucleotide arrays including probes to a subset or subsets of at least 5 to 10 gene markers up to a full set of markers which distinguish head-and-neck disease.

[0109] The invention also contemplates a method including administering to cells or tissues imaging agents that carry labels for imaging and bind to OPL or HNSCC markers and optionally other markers of a head-and-neck disease, and then imaging the cells or tissues.

[0110] In an aspect the invention provides an in vivo method including administering to a subject an agent that has been constructed to target one or more OPL or HNSCC markers.

[0111] In a particular embodiment, the invention contemplates an in vivo method including administering to a mammal one or more agent that carries a label for imaging and binds to one or more OPL or HNSCC marker, and then imaging the mammal.

[0112] According to a particular aspect of the invention, an in vivo method for imaging OPL or head-and-neck cancer is provided including: [0113] (a) injecting a patient with an agent that binds to one or more OPL or HNSCC cancer marker, the agent carrying a label for imaging the head-and-neck cancer; [0114] (b) allowing the agent to incubate in vivo and bind to one or more OPL or HNSCC cancer marker associated with the head-and-neck cancer; and [0115] (c) detecting the presence of the label localized to the OPL or HNSCC cancer.

[0116] In an embodiment of the invention the agent is an antibody which recognizes an OPL or head-and-neck cancer marker. In another embodiment of the invention the agent is a chemical entity which recognizes an OPL or head-and-neck cancer marker.

[0117] An agent carries a label to image an OPL or head-and-neck marker and optionally other markers. Examples of labels useful for imaging are radiolabels, fluorescent labels (e.g., fluorescein and rhodamine), nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography ("PET") scanner, chemiluminescers such as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-range radiation emitters, such as isotopes detectable by short-range detector probes can also be employed.

[0118] The invention also contemplates the localization or imaging methods described herein using multiple markers for a head-and-neck disease (e.g., leukoplakia with hyperplasia or dysplasia (OPL) or head-and-neck cancer, HNSCC or related conditions).

[0119] The invention also relates to kits for carrying out the methods of the invention. In an embodiment, a kit is for assessing whether a patient is afflicted with a head-and-neck disease (e.g., or leukoplakia with hyperplasia or dysplasia (OPL) or head-and-neck cancer or HNSCC or related conditions) and it includes reagents for assessing one or more head-and-neck cancer markers or polynucleotides encoding the markers.

[0120] The invention further provides kits including marker sets described herein. In an aspect the kit contains a microarray ready for hybridization to target OPL or HNSCC oligonucleotide markers, plus software for the data analyses.

[0121] The invention also provides a diagnostic composition including an OPL or HNSCC marker or a polynucleotide encoding the marker. A composition is also provided including a probe that specifically hybridizes to OPL or HNSCC polynucleotide markers, or a fragment thereof, or an antibody specific for OPL or HNSCC markers or a fragment thereof. In another aspect, a composition is provided including one or more OPL or head-and-neck cancer polynucleotide marker specific primer pairs capable of amplifying the polynucleotides using polymerase chain reaction methodologies. The probes, primers or antibodies can be labeled with a detectable substance.

[0122] Still further the invention relates to therapeutic applications for head-and-neck diseases, in particular OPL or head-and-neck cancer, employing OPL or head-and-neck cancer markers and polynucleotides encoding the markers, and/or binding agents for the markers.

[0123] In an aspect, the invention relates to compositions including markers or parts thereof associated with a head-and-neck disease, or antibodies specific for OPL or HNSCC markers associated with a head-and-neck disease, and a pharmaceutically acceptable carrier, excipient, or diluent. A method for treating or preventing a head-and-neck disease, in particular OPL or head-and-neck cancer (e.g., HNSCC), in a patient is also provided including administering to a patient in need thereof, markers or parts thereof associated with a head-and-neck disease, antibodies specific for OPL or HNSCC markers associated with a head-and-neck disease, or a composition of the invention. In an aspect the invention provides a method of treating a patient afflicted with or at risk of developing a head-and-neck disease (e.g., OPL or head-and-neck cancer) including inhibiting expression of OPL or head-and-neck cancer markers.

[0124] In an aspect, the invention provides antibodies specific for OPL or HNSCC markers associated with a disease (e.g., leukoplakia with hyperplasia or dysplasia or HNSCC) that can be used therapeutically to destroy or inhibit the disease (e.g., the growth of OPL or HNSCC marker expressing cancer cells), or to block OPL or HNSCC marker activity associated with a disease. In an aspect, OPL or HNSCC markers may be used in various immunotherapeutic methods to promote immune-mediated destruction or growth inhibition of tumors expressing OPL or HNSCC markers.

[0125] The invention also contemplates a method of using OPL or head-and-neck cancer markers or parts thereof, or antibodies specific for OPL or HNSCC markers in the preparation or manufacture of a medicament for the prevention or treatment of a head-and-neck disease (e.g., leukoplakia with hyperplasia or dysplasia (OPL) or head-and-neck cancer, HNSCC or related conditions).

[0126] Another aspect of the invention is the use of OPL or HNSCC markers, peptides derived therefrom, or chemically produced (synthetic) peptides, or any combination of these molecules, for use in the preparation of vaccines to prevent a head-and-neck disease and/or to treat a head-and-neck disease.

[0127] The invention contemplates vaccines for stimulating or enhancing in a subject to whom the vaccine is administered production of antibodies directed against one or more HNSCC markers.

[0128] The invention also provides a method for stimulating or enhancing in a subject production of antibodies directed against one or more OPL or HNSCC marker. The method includes administering to the subject a vaccine of the invention in a dose effective for stimulating or enhancing production of the antibodies.

[0129] The invention further provides a method for treating, preventing, or delaying recurrence of a head-and-neck disease (e.g., OPL or head-and-neck cancer, HNSCC or related conditions). The method includes administering to the subject a vaccine of the invention in a dose effective for treating, preventing, or delaying recurrence of a head-and-neck disease (e.g., OPL or head-and-neck cancer, HNSCC or related conditions).

[0130] The invention contemplates the methods, compositions, and kits described herein using additional markers associated with a head-and-neck disease (e.g., OPL or head-and-neck cancer, HNSCCm or related conditions). The methods described herein may be modified by including reagents to detect the additional markers, or polynucleotides for the markers.

[0131] In particular, the invention contemplates the methods described herein using multiple markers for OPL or HNSCC cancer. Therefore, the invention contemplates a method for analyzing a biological sample for the presence of OPL or HNSCC markers and polynucleotides encoding the markers, and other markers that are specific indicators of cancer, in particular head-and-neck cancer. The methods described herein may be modified by including reagents to detect the additional markers, or nucleic acids for the additional markers.

[0132] In embodiments of the invention the methods, compositions and kits use one or more of the markers listed in Table 5, in particular those listed in Table 2 and Table 7. In another embodiment, the method uses a panel of markers selected from the markers listed in Table 5, and in one embodiment of those listed in Table 2 and Table 7 in particular a panel including two, three or four or more of the markers in Table 5.

[0133] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE TABLES AND DRAWINGS

Tables

[0134] Table 1. iTRAQ ratios for HNSCC and non-cancerous head-and-neck tissue samples. HNSCC samples (C1-05, cancer of the buccal mucosa; C6-C10, cancer of the tongue), non-paired non-cancerous samples (N1, N4, N5), and paired non-cancerous samples (N2, N3) versus the pooled non-paired non-cancerous control. Grey boxes, not detected; NQ, not quantified; 9999, no expression observed in the pooled sample

[0135] Table 2. Receiver-operating characteristics from the iTRAQ ratios of a panel of three best-performing biomarkers-YWHAZ, stratifin, and S100A7-individually and as a panel.

[0136] Table 3. Receiver-operating characteristics from the IHC scores of a panel of three best-performing biomarkers-YWHAZ, stratifin, and S100A7-individually and as a panel.

[0137] Table 4. Comparison of receiver-operating characteristics from the iTRAQ ratios of the panel of three best-performing biomarkers. Non-paired non-cancerous tissues give better sensitivity and specificity as comparators than do paired non-cancerous tissues.

[0138] Table 5. Differentially-expressed proteins not previously described in OPLs and head-and-neck malignancies and cancer.

[0139] Table 6. Average iTRAQ ratios for OPLs and histologically-normal control oral tissue samples. Ratios are from the comparison between OPLs (D1-D6) and the pooled normal sample, and the comparison between histologically-normal oral tissues (N1-N3) and the pooled normal sample. N4-N6 are histologically-normal oral tissues analyzed in an earlier iTRAQ analysis using the same pooled normal control to demonstrate consistent iTRAQ ratios in different experiments analyzed over different time periods. ND, not detected; NQ, not quantified

[0140] Table 7. Receiver-operating characteristics from (A) the iTRAQ ratios and (B) IHC scores of a panel of three best-performing biomarkers-YWHAZ, stratifin, and hnRNPK-individually and as a panel.

[0141] Table 8. Analysis of Stratifin and YWHAZ in HNOSCCs: correlation with clinicopathological parameters.

[0142] Table 9a. Analysis of overexpression of hnRNPK protein in oral lesions and correlation with clinicopathological parameters.

[0143] Table 9b. Biomarker analysis of hnRNPK (nuclear/cytoplasmic) in oral lesions.

[0144] Table 10. Alternate accession numbers for OPL proteins.

[0145] Table 11. Peptide sequences and coverage for HNSCC and OPL.

[0146] Table 12. Clinicopathological parameters of patients with oral premalignant lesions (OPLs).

[0147] Table 13. Antibodies used for immunohistochemistry and Western Blotting: sources and dilutions.

[0148] Table 14. RT-PCR analysis primers and PCR conditions.

[0149] Table 15. Molecules identified in the Networks and their cellular functions.

Figures

[0150] FIG. 1 provides a flow diagram for online 2D LC-MS/MS analysis. In position 1, ports 1-2, 3-4, 5-6, 7-8, and 9-10 are connected; in position 2, ports 2-3, 4-5, 6-7, 8-9, and 10-1 are connected. In the diagram, the valves are shown at the initial (Time=0 min) positions.

[0151] FIG. 2 shows a receiver-operating-characteristic curves of a panel of three best-performing biomarkers, YWHAZ, stratifin, and S100 A7: (a) iTRAQ ratios, and (b) IHC scores.

[0152] FIG. 3 presents an immunohistochemical verification of iTRAQ-discovered potential cancer markers, YWHAZ, stratifin, and S100 A7 in HNSCCs and non-cancerous head-and-neck tissues. Positive staining is brown and is intense in HNSCCs. The left panel shows the noncancerous (histologically normal) tissues and the right panel depicts the HNSCC tissue sections. Panel A: the HNSCC sample shows intense cytoplasmic and nuclear staining for YWHAZ, while the normal mucosa shows no detectable immunostaining Panel B: the HNSCC tissue section shows cytoplasmic staining for stratifin in tumor cells, while the normal mucosa shows no detectable immunostaining Panel C: the HNSCC tissue section shows intense cytoplasmic staining for S100A7 in tumor cells, while the normal mucosa shows no detectable immunoreactivity. All panels show .times.200 magnifications.

[0153] FIG. 4 depicts Western blot analyses of YWHAZ, Stratifin and S100 A7 in HNSCCs and paired noncancerous head-and-neck tissues. Equal amounts of protein lysates from HNSCCs and paired non-cancerous head-and-neck tissues were employed. See the text for details. The Panels show increased expression of (i) YWHAZ, (ii) stratifin, and (iii) S100A7 in HNSCCs (C1-C3) as compared to paired non-cancerous head-and-neck tissues (N1-N3). Alpha-tubulin (Panel 4) was used as the loading control.

[0154] FIG. 5 shows RT-PCR analyses of YWHAZ, stratifin, and S100 A7 in HNSCCs and non-cancerous head-and-neck tissues: Panel (i) shows increased levels of YWHAZ transcripts in HNSCCs (C1-C3) as compared to the non-cancerous head-and-neck tissues that show basal levels (N2 and N3) and no detectable level (N1) of YWHAZ transcripts. Panel (ii) shows increased levels of stratifin transcripts in HNSCCs (C1-C3) as compared to the non-cancerous head-and-neck tissues that show basal level (N3) and no detectable level (N1 and N2) of stratifin transcripts. Panel (iii) shows increased levels of S100A7 transcripts in HNSCCs (C1-C3) as compared to the non-cancerous head-and-neck tissues that show basal level (N3) and no detectable level (N1 and N2) of S100A7 transcripts. .beta.-Actin (Panel iv) was used as a control for normalizing the quantity of RNA used.

[0155] FIG. 6 provides receiver-operating-characteristic curves of a panel of three best-performing biomarkers, YWHAZ, stratifin, and hnRNPK: (A) iTRAQ ratios, and (B) IHC scores.

[0156] FIG. 7 presents an immunohistochemical verification of iTRAQ-discovered potential biomarkers, YWHAZ, stratifin, hnRNPK, S100A7, and PTHA in OPLs and histologically normal oral tissues. Verification of the panel of these 5 potential biomarkers was carried out using an independent set of 30 OPLs and 21 histological normal oral tissues. Representative photomicrographs are shown here. Positive staining is brown and is intense in OPLs. The upper panel shows the normal tissues and the lower panel the OPL tissue sections. Panel A: the OPL sample shows intense cytoplasmic and nuclear staining for YWHAZ, while the normal mucosa shows no detectable immunostaining Panel B: the OPL tissue section exhibits cytoplasmic staining for stratifin in epithelial cells, while the normal mucosa shows no detectable immunostaining Panel C: the OPL tissue section shows nuclear staining for hnRNPK in epithelial cells, while no detectable immunostaining is evident in the normal mucosa. Panel D: the OPL sample shows intense cytoplasmic staining for S100A7 in epithelial cells, while the normal mucosa displays no detectable immunoreactivity. Panel E: the OPL sample exhibits intense nuclear staining for PTHA in epithelial cells, while no detectable immunostaining is evident in the normal sample. All panels show .times.100 magnifications.

[0157] FIG. 8A illustrates Western blot analyses of YWHAZ, stratifin, hnRNPK, S100A7 and PTHA in representative OPLs and histologically normal oral tissues. The OPLs and histologically normal oral tissues (n=3) were selected randomly from the same cohort of tissues as used for IHC analysis and the results shown are representative of 3 independent experiments. Equal amounts of protein lysates from OPLs (D1-D3) and histologically normal oral tissues (N1-N3) were used. The panels show increased expression of (i) YWHAZ, (ii) stratifin, (iii) hnRNPK, (iv) S100A7, and (v) PTHA in OPLs (D1-D3) as compared to the histologically normal oral tissues (N1-N3). .alpha.-tubulin (Panel (vi)) was used as the loading control.

[0158] FIG. 8B depicts RT-PCR analyses of YWHAZ, stratifin, hnRNPK, S100A7, and PTHA in representative OPLs and histologically normal oral tissues selected randomly, as used for IHC and Western blot analysis and the results shown are representative of 3 independent experiments: Panel (i) shows increased levels of YWHAZ transcripts in OPLs (D1-D3) as compared to the histologically normal oral tissues (N1-N3) that did not show detectable levels of YWHAZ transcripts. Panel (ii) demonstrates increased levels of stratifin transcripts in OPLs (D1-D3) as compared to the histologically normal oral tissues (N1-N3) that show basal level (N1) and no detectable level (N2 and N3) of stratifin transcripts. Panel (iii) shows increased levels of hnRNPK transcripts in OPLs (D1-D3) as compared to the histologically normal oral tissues (N1-N3), in which no detectable levels of hnRNPK transcripts are evident. Panel (iv) exhibits increased levels of S100A7 transcripts in OPLs (D1-D3) as compared to no detectable levels in the histologically normal oral tissues (N1-N3). Panel (v) shows increased levels of PTHA transcripts in OPLs (D1-D3) as compared to the histologically normal oral tissues (N1-N3) in which no detectable levels of PTHA transcripts are evident. .beta.-Actin (Panel iv) was used as a control for normalizing the quantity of RNA used.

[0159] FIG. 8C shows network analysis using ingenuity pathways analysis (IPA) software. Differentially expressed proteins identified in OPLs in comparison with normal oral tissues were analyzed using the IPA software. Network analysis classified proteins into 2 networks on the basis of function cited previously in literature. Above panel shows merged network of the pathways identified using IPA software. Bold lines (-) show direct interactions/regulation while dashed lines (---) show indirect interactions/regulation of proteins at the ends of line. Proteins shown in red color are upregulated and in green color are down-regulated in OPLs in comparison with normal tissues.

[0160] FIGS. 9A and 9B illustrate identification of stratifin and YWHAZ, respectively, in HNOSCCs by mass spectrometry. The peptides for which MS/MS spectra are shown are colored red and in a larger font. Those that are common between stratifin and YWHAZ are shown in purple. Other peptides observed are in blue. The matched b ions are shown in green, and the matched y ions in red.

[0161] FIG. 10 provides an immunohistochemical analysis of stratifin in head-and-neck cancer tissues. Paraffin-embedded HNOSCC tissue sections and non-malignant mucosa were stained using anti-stratifin antibody (all .times.100 magnifications): Panel a shows normal oral mucosa with no detectable stratifin immunostaining; Panel b shows HNOSCC with strong cytoplasmic and nuclear stratifin immunostaining in the tumor cells; Panel c shows HNOSCC negative control with lack of staining in the tumor cells.

[0162] FIG. 11 shows a box-plot analysis. The box plot shows distribution of total scores of stratifin in HNOSCCs and non-malignant head-and-neck tissues.

[0163] FIG. 12 sets forth a co-immunoprecipitation assay and Western blot analysis. Immunoprecipitation assays of stratifin, YWHAZ, NF.kappa.B, Bcl-2, and .beta.-catenin proteins were carried out using specific antibodies in head-and-neck cancer cells, HSC2. FIG. 12a shows immunoblot analysis for stratifin, demonstrating the binding of stratifin with YWHAZ, NF.kappa.B, Bcl-2, and .beta.-catenin, and the lack of binding in the negative control. Similarly, reverse immunoprecipitation assays were carried out using specific antibodies for YWHAZ, NF.kappa.B, Bcl-2, and .beta.-catenin. FIG. 12b shows immunoblot analysis for: (i) YWHAZ, (ii) NF.kappa.B, (iii) Bcl-2, and (iv) .beta.-catenin confirming the binding of these proteins with stratifin.

[0164] FIG. 13 illustrates a Kaplan-Meier estimation of cumulative proportion of disease-free survival: 13a, stratifin protein expression; the median time for disease-free survival (no recurrence/metastasis) in patients with stratifin-positive tumors was 19 months, whereas in those with stratifin-negative tumors it was 38 months (p=0.06). 13b, YWHAZ protein expression; the median time for disease-free survival (no recurrence/metastasis) in patients with YWHAZ-positive tumors was 23 months, whereas in those with YWHAZ-negative tumors it was 35 months (p=0.08). 13c, concomitant stratifin and YWHAZ expressions; the median time for disease-free survival of patients with HNOSCCs showing concomitant expressions of stratifin and YWHAZ (Stratifin+/YWHAZ+) was 13 months, as compared to patients with tumors that did not show increased expression of either of these proteins with the median time for disease-free survival being 38 months (p=0.019).

[0165] FIG. 14 shows an immunohistochemical analysis of hnRNPK in head-and-neck cancer tissues. Paraffin-embedded sections of histologically normal mucosa, leukoplakia with no evidence of dysplasia or with dysplasia and HNOSCCs were stained using anti-hnRNPK monoclonal antibody as described herein. (a) Normal oral mucosa with no detectable hnRNPK immunostaining (b) Leukoplakic lesion with no dysplasia showing nuclear hnRNPK immunostaining (c) Leukoplakic lesion with no dysplasia showing nuclear and cytoplasmic hnRNPK immunostaining (d) Dysplasia depicting nuclear hnRNPK immunostaining in epithelial cells. (e) Dysplasia depicting nuclear and cytoplasmic hnRNPK immunostaining in epithelial cells. (f) HNOSCC section illustrating only nuclear hnRNPK immunostaining in the tumor cells. (g) HNOSCC section showing both cytoplasmic and nuclear staining in tumor cells. (h) HNOSCC section showing no immunostaining in tumor cells for hnRNPK protein serving as a negative control. Arrow shows nuclear staining of hnRNPK in panels b, d, and f, and nuclear and cytoplasmic staining in panel c, e, and g. a-h, original magnification.times.200

[0166] FIG. 15 presents receiver-operating characteristic curves of hnRNPK (nuclear/cytoplasmic) in (a) normal vs. leukoplakia with no evidence of dysplasia; (b) normal vs. dysplasia; and (c) normal vs. HNOSCCs. Bold line shows ROC analysis for nuclear hnRNPK. Dashed line shows ROC analysis for cytoplasmic hnRNPK.

[0167] FIG. 16 depicts an evaluation of hnRNPK expression (nuclear/cytoplasmic) as a biomarker for risk prediction of oral leukoplakia and prognosis of HNOSCCs. The figure shows estimated (a) positive predictive value (PPV) and (b) negative predictive value (NPV) for nuclear/cytoplasmic hnRNPK expression as prognostic biomarkers for disease progression of leukoplakia. Panels c and d show PPV and NPV for recurrence in HNOSCC patients, respectively.

[0168] FIG. 17 illustrates a Kaplan-Meier estimation of cumulative proportion of disease-free survival showing: (a) significantly reduced time for disease progression (p<0.001; median time=17 months) in leukoplakia patients showing increased cytoplasmic expression of hnRNPK as compared to median time of 35 months in the patients showing no/faint immunostaining of hnRNPK in cytoplasm; (b) median time for disease progression (34 months) was observed in leukoplakia patients showing intense nuclear expression of hnRNPK (n=78) as compared to patients who did not show increased nuclear hnRNPK; and (c) Median time for disease-free survival (no recurrence/metastasis) in HNOSCC patients showing cytoplasmic immunostaining of hnRNPK was 11 months, whereas in those patients showing no/faint hnRNPK-immunostaining in cytoplasm it was 41 months (p=0.004). In patients showing increased nuclear expression disease (d), free survival was 14 months as compared to HNOSCCs that showed mild or moderate nuclear immunostaining (median disease-free survival=57 months, p=0.07).

[0169] FIG. 18 depicts validation of hnRNPK expression in oral lesions. (a) Western blot analysis of hnRNPK in normal mucosa, leukoplakia and HNOSCC tissues. Equal amount of protein lysates from these tissues were electrophoresced on 12% SDS-PAGE and transferred to PVDF membrane. The membrane was incubated with respective primary antibodies and secondary antibodies as described herein, and the signal detected by enhanced chemiluminescence method. Panel (a) shows increased expression of hnRNPK in leukoplakia (L) and HNOSCCs (T) as compared to paired non-malignant head-and-neck tissues (N). Actin was used as control for equal loading of protein in SDS-PAGE (lower panel). (b) RT-PCR analysis of hnRNPK in normal mucosa, leukoplakia and HNOSCC tissues. Panel shows increased levels of hnRNPK transcripts in leukoplakia (L) and HNOSCCs (T) as compared to the non-malignant head-and-neck tissues that showed basal levels (N) of hnRNPK transcripts. .beta.-actin, used as a control to normalize the quantity of RNA used for each RT-PCR reaction, is shown in the lower panel.

[0170] FIG. 19A-E presents CID spectra of the single-peptide identifications in the HNSCC and OPL.

[0171] FIG. 20 illustrates negative and positive controls for IHC in OPLs.

[0172] FIGS. 21A and 21B depict CID spectra of the peptide identifications for hnRNPK in OPLs.

[0173] FIG. 22 sets forth a Kaplan-Meier analysis of OPLs with no evidence of dysplasia showing overexpression of hnRNPK.

DETAILED DESCRIPTION OF THE INVENTION

[0174] Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been used for the analysis of biological samples labeled with isobaric mass tags (iTRAQ) to identify proteins that are differentially expressed in human head-and-neck squamous-cell carcinomas (HNSCCs) in relation to non-cancerous head-and-neck tissues (control) for cancer biomarker discovery. Fifteen individual samples (cancer and non-cancerous tissues) were compared against a pooled non-cancerous control (prepared by pooling equal amounts of proteins from six noncancerous tissues) in five sets by online and offline separation. Eight hundred and eleven (811) non-redundant proteins in HNSCCs were identified, including structural proteins, signaling components, enzymes, receptors, transcription factors and chaperones.

[0175] A panel of proteins showing consistent differential expression in HNSCC relative to the non-cancerous controls was discovered. Some of the proteins include stratifin (14-3-3 sigma), YWHAZ (14-3-3 zeta), three calcium-binding proteins of the S100 family, S100A 2, 5100A 7 (psoriasin) and S100A 11 (calgizarrin), prothymosin alpha (PTHA), L-lactate dehydrogenase A chain (LDH-A), glutathione S transferase-Pi, APCbinding protein EB1, and fascin. Peroxiredoxin2, carbonic anhydrase I, flavin reductase, histone H3, and polybromo-1D (BAF180) were underexpressed in HNSCCs.

[0176] A panel of the three best performing biomarkers--YWHAZ, stratifin, and S100A7--achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating cancerous from non-cancerous head-and-neck tissues (Table 7A). Verification of differential expression of YWHAZ, stratifin and S100A7 proteins in clinical samples of HNSCCs and paired and non-paired non-cancerous tissues by immunohistochemistry (Table 7B), immunoblotting, and RT-PCR confirmed their overexpression in head-and-neck cancer. Verification of YWHAZ, stratifin and S100A7 in an independent set of HNSCCs achieved a sensitivity of 0.92 and a specificity of 0.87 in discriminating cancerous from non-cancerous head-and-neck tissues, thereby confirming their overexpressions and utility as credible cancer biomarkers.

[0177] The inventors also used iTRAQ labeling in combination with multidimensional LC-MS/MS analysis of oral leukoplakia with dysplasia (oral premalignant lesions, or OPLs) in order to compare protein profiles of OPLs and normal head-and-neck tissues. Nine individual samples (6 OPLs and 3 normal tissues) were compared against a pooled normal control (prepared by pooling equal amounts of proteins from six noncancerous tissues) in five sets by online and offline separation.

[0178] The LC-MS/MS analyses collectively resulted in identification of 459 non-redundant proteins; 216 were identified as single hits with more than 95% confidence. Of all the proteins identified, only 17 were differentially expressed in OPLs relative to normal control (observed in .gtoreq.3 out of the 6 samples and with >50% showing differential expression). Of these, 15 proteins were confidently identified with a minimum of two peptide matches in each case. Two proteins, parathymosin and DLC1 were identified by single peptides. All these 17 proteins are given in Table 6, along with two structural proteins: .beta.-actin and gelsolin precursor as controls. The heat map in Table 6 also depicts the variations in the levels of overexpressed and underexpressed proteins in individual OPL and histological normal tissues versus the pooled normal control. These differential expression levels were averages of the replicate analyses: 56.4% of the ratios varied by less than 10% from their respective averages shown, and 82.0% varied by less than 20%.

[0179] Thirteen proteins that did not meet the aforementioned initial criteria--IGL2, P37AUF1 (HNRPD), SOD2, PKM2, ROA1HNRNPA1, HSP27, cofilin, glyceraldehyde-3-phosphate dehydrogenase, NDP kinase B, elongation factor 2, CALM3, PEBP, and S100A7--were also included in Table 5 for further analysis, as these proteins are of biological relevance in cancer development. Of these, 11 proteins were confidently identified with a minimum of two peptide matches in each case. p37AUF1 (hnRNP D) was identified by a single peptide with a confidence of 99%. SOD2 was identified by more than one unique peptides, however, the best matching peptide was identified with a confidence of 93%. Although, individually this peptide did not meet the inventors' stipulated criteria for acceptance, manual verification of the spectrum showed good sequence coverage for this peptide. Furthermore, the cumulative score which included the lower confidence peptide matches was >2.0 and corresponded with a confidence of 99%.

[0180] The best-performing proteins that can differentiate between OPLs and normal tissues were identified by determining the individual receiver-operating characteristic (ROC) curves of the proteins in Table 7. The three proteins with the highest AUC values-YWHAZ, stratifin and hnRNPK--are listed in Table 7A together with their individual and collective figures-of-merit, including sensitivity and specificity. As a panel, these three biomarkers achieved a sensitivity of 0.83 and a specificity of 0.74 in discriminating OPLs from histological normal oral tissues (Table 7A and FIG. 6A).

[0181] The panel of three potential biomarkers, YWHAZ, stratifin and hnRNPK, and two other proteins with high AUC values, S100A7 (0.56) and PTHA (0.56), were chosen for verification in an independent set of OPLs (30 cases) and normal tissues (21 cases) by IHC. Representative levels of expression and subcellular localizations of all the five proteins in oral dysplastic tissues in comparison with normal tissues are shown in FIG. 7A-E. These data were further verified by Western blot analysis (FIG. 8A) at the protein level, as well as RT-PCR analysis at the mRNA level (FIG. 8B). The differential expression suggested by iTRAQ ratios tended to be moderate, while the results of Western and RT-PCR analyses tended to show more extreme differential expression. Thus, Western and RT-PCR analyses, verified the differential expression reported by the iTRAQ analysis in trend but not in scale. This discrepancy of scale has also been noted in other studies ascribed to compression of the dynamic range of iTRAQ ratios (21). Importantly, in IHC analysis, the biomarker panel of YWHAZ, stratifin, and hnRNPK achieved a sensitivity of 0.91, specificity of 0.95, and predictive value of 0.96 (Table 7B and FIG. 7B) in discriminating OPLs from histological normal oral tissues.

[0182] To gain insight into the plausible biological processes in which these proteins might be involved, the inventors used the Ingenuity pathway analysis tools (Ingenuity Systems, Inc. software) and discovered two major networks in OPLs (FIG. 8C). The network comprised of 23 proteins identified in this study that are primarily involved in inflammation, molecular transport, cellular movement, cellular signaling, proliferation, gene expression and cancer. To the best of the inventors' knowledge, this is the first study reporting differential expressions of p37AUF1 (HNRPD) and histone H2B.1 in OPLs.

[0183] Accordingly, the inventors describe herein methods for detecting the presence of a head-and-neck disease (e.g., OPL or head-and-neck cancer) in a sample, the absence of a disease (e.g., OPL or head-and-neck cancer) in a sample, the stage or grade of the disease, and other characteristics of head-and-neck diseases that are relevant to prevention, diagnosis, characterization, and therapy of head-and-neck diseases such as OPL or cancer in a patient, for example, the benign, premalignant or malignant nature of a head-and-neck cancer, the metastatic potential of a head-and-neck cancer, assessing the histological type of neoplasm associated with a head-and-neck cancer, the indolence or aggressiveness of a leukoplakia with hyperplasia or dysplasia or head-and-neck cancer, and other characteristics of head-and-neck diseases that are relevant to prevention, diagnosis, characterization, and therapy of head-and-neck diseases such as OPL or cancer in a patient. Methods are also provided for assessing the efficacy of one or more test agents for inhibiting a head-and-neck disease, assessing the efficacy of a therapy for a head-and-neck disease, monitoring the progression of a head-and-neck disease, selecting an agent or therapy for inhibiting a head-and-neck disease, treating a patient afflicted with a head-and-neck disease, inhibiting a head-and-neck disease in a patient, and assessing the disease (e.g., carcinogenic) potential of a test compound.

[0184] Abbreviation Index.

[0185] For convenience, certain abbreviations used in the description, tables, figures, and appended claims are defined here: iTRAQ, isobaric tags for relative and absolute quantification; LC, liquid chromatography; MS/MS, tandem mass spectrometry; PCM, potential cancer marker; HNSCC, head-and-neck squamous cell carcinoma; LCM, laser capture microdissection; PBS, phosphate-buffered saline; SCX, strong cation exchange; ID, internal diameter; RP, reverse phase; IDA, information-dependent acquisition; TBS, tris-buffered saline; SFN, stratifin or 14-3-3 sigma; YWHAZ, 14-3-3 zeta; LDH-A, L-lactate dehydrogenase A; SD, standard deviation; ROC, receiver-operating characteristics; PPIA, peptidyl prolyl isomerase A; PV, predictive values; PPV, positive predictive values; PTHA, prothymosin alpha; PKM2, pyruvate kinase isozyme M2; AUC, area under the curve; RSD, relative standard deviation; TMA, tissue microarray.

[0186] Glossary.

[0187] For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

[0188] The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Furthermore, it is to be understood that "a", "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition or method comprising "a head-and-neck marker" includes two or more OPL or head-and-neck cancer markers. The term "about" means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, of the number to which reference is being made.

[0189] "Head-and-neck disease" refers to any disorder, disease, condition, syndrome or combination of manifestations or symptoms recognized or diagnosed as a disorder of the head and neck, including but not limited to hyperplasia, dysplasia and cancer precursors, head-and-neck cancer or carcinoma.

[0190] "Head-and-neck cancer" or "head-and-neck carcinoma" includes malignant head-and-neck disease including but not limited to squamous cell and adenocarcinomas.

[0191] Biomarkers of head-and-neck precancers includes OPL markers including but not limited to oral leukoplakia with hyperplasia or dysplasia.

[0192] The terms "sample", "biological sample", and the like mean a material known or suspected of expressing or containing one or more OPL or head-and-neck cancer polynucleotide markers or one or more OPL or head-and-neck cancer markers. A test sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample. The sample can be derived from any biological source, such as tissues, extracts, or cell cultures, including cells (e.g., tumor cells), cell lysates, and physiological fluids, such as, for example, whole blood, plasma, serum, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, synovial fluid, peritoneal fluid, lavage fluid, and the like. The sample can be obtained from animals, preferably mammals, most preferably humans. The sample can be treated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, distillation, extraction, concentration, inactivation of interfering components, the addition of reagents, and the like.

[0193] In embodiments of the invention the sample is a mammalian tissue sample. In a particular embodiment, the tissue is head-and-neck tissue.

[0194] In another embodiment the sample is a human physiological fluid. In a particular embodiment, the sample is human serum.

[0195] The samples that may be analyzed in accordance with the invention include polynucleotides from clinically relevant sources, preferably expressed RNA or a nucleic acid derived therefrom (cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter). The target polynucleotides can comprise RNA, including, without limitation total cellular RNA, poly(A).sup.+ messenger RNA (mRNA) or fraction thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (i.e., cRNA; see, for example, Linsley & Schelter, U.S. patent application Ser. No. 09/411,074, or U.S. Pat. Nos. 5,545,522; 5,891,636; or 5,716,785). Methods for preparing total and poly(A).sup.+ RNA are well known in the art, and are described generally, for example, in Sambrook et al., (1989, Molecular Cloning--A Laboratory Manual (2.sup.nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and Ausubel et al., eds. (1994, Current Protocols in Molecular Biology, Vol. 2, Current Protocols Publishing, New York). RNA may be isolated from eukaryotic cells by procedures involving lysis of the cells and denaturation of the proteins contained in the cells. Additional steps may be utilized to remove DNA. Cell lysis may be achieved with a non-ionic detergent, followed by microcentrifugation to remove the nuclei and hence the bulk of the cellular DNA. (See Chirgwin et al., 1979, Biochemistry 18:5294-5299). Poly(A)+RNA can be selected using oligo-dT cellulose (see Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). In the alternative, RNA can be separated from DNA by organic extraction, for example, with hot phenol or phenol/chloroform/isoamyl alcohol.

[0196] It may be desirable to enrich mRNA with respect to other cellular RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA). Most mRNAs contain a poly(A) tail at their 3' end allowing them to be enriched by affinity chromatography, for example, using oligo(dT) or poly(U) coupled to a solid support, such as cellulose or Sephadex.TM. (see Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 2, Current Protocols Publishing, New York). Bound poly(A)+mRNA is eluted from the affinity column using 2 mM EDTA/0.1% SDS.

[0197] A sample of RNA can comprise a plurality of different mRNA molecules each with a different nucleotide sequence. In an aspect of the invention, the mRNA molecules in the RNA sample comprise at least 100 different nucleotide sequences.

[0198] Target polynucleotides can be detectably labeled at one or more nucleotides using methods known in the art. The label is preferably uniformly incorporated along the length of the RNA, and more preferably, is carried out at a high degree of efficiency. The detectable label can be a luminescent label, fluorescent label, bio-luminescent label, chemiluminescent label, radiolabel, and colorimetric label. In a particular embodiment, the label is a fluorescent label, such as a fluorescein, a phosphor, a rhodamine, or a polymethine dye derivative. Commercially available fluorescent labels include, for example, fluorescent phosphoramidites, such as FluorePrime (Amersham Pharmacia, Piscataway, N.J.), Fluoredite (Millipore, Bedford, Mass.), FAM (ABI, Foster City, Calif.), and Cy3 or Cy5 (Amersham Pharmacia, Piscataway, N.J.).

[0199] Target polynucleotides from a patient sample can be labeled differentially from polynucleotides of a standard. The standard can comprise target polynucleotides from normal individuals (i.e., those not afflicted with or pre-disposed to head-and-neck disease), in particular pooled from samples from normal individuals. The target polynucleotides can be derived from the same individual, but taken at different time points, and thus indicate the efficacy of a treatment by a change in expression of the markers, or lack thereof, during and after the course of treatment.

[0200] The terms "subject", "individual", and "patient" refer to a warm-blooded animal such as a mammal. In particular, the terms refer to a human. A subject, individual or patient may be afflicted with or suspected of having or being pre-disposed to head-and-neck disease or a condition as described herein. The terms also includes domestic animals bred for food or as pets, including horses, cows, sheep, poultry, fish, pigs, cats, dogs, and zoo animals.

[0201] Methods herein for administering an agent or composition to subjects/individuals/patients contemplate treatment as well as prophylactic use. Typical subjects for treatment include persons susceptible to, suffering from or that have suffered a condition or disease described herein. In particular, suitable subjects for treatment in accordance with the invention are persons that are susceptible to, suffering from or that have suffered head-and-neck cancer.

[0202] The term "head-and-neck marker" refers to a marker associated with normal or diseased head-and-neck tissue and includes or consists of one or more of the polypeptides that are up-regulated in cancer samples as compared to normal samples in Table 1 and Table 7, those listed in Table 5, in particular The term includes native-sequence polypeptides, isoforms, chimeric polypeptides, complexes, all homologs, fragments, precursors, and modified forms and derivatives of the markers.

[0203] A head-and-neck marker may be associated with a head-and-neck disease, in particular it may be an OPL or head-and-neck cancer marker. The term "OPL or head-and-neck cancer marker" includes a marker associated with OPL or head-and-neck cancer, in particular a marker listed in Table 5.

[0204] The terms "YWHAZ", "YWHAZ polypeptide", and "YWHAZ protein" include human YWHAZ, in particular the native-sequence polypeptide, isoforms, chimeric polypeptides, all homologs, fragments, precursors, complexes, and modified forms and derivatives of human YWHAZ. The amino acid sequence for native human YWHAZ includes the amino acid sequences referenced in NCBI Gene ID: Q86V33, including GenBank Accession Nos. P29213, P29312, Q32P43, Q5XJ08, Q6GPI2, Q6IN74, Q6NUR9, Q6P3U9, and the exemplary sequences shown in SEQ ID NO: 24 (GenBank Accession No. P63104; sequence provided in Appendix 1). Corresponding terms for "S100A7", "stratifin", and "hnRNPK" have similar meanings. The amino acid sequence for native human S100A7 includes the amino acid sequences referenced in NCBI Gene ID: P31151, including GenBank Accession Nos. Q6FGE3, Q9H1E2, and the exemplary sequences shown in SEQ ID NO: 13 (GenBank Accession No. P31151; sequence provided in Appendix 1). The amino acid sequence for native human stratifin includes the amino acid sequences referenced in NCBI Gene ID: P31947, including GenBank Accession Nos. Q6FH30, Q6FH51, Q96DH0, and the exemplary sequences shown in SEQ ID NO: 23 (GenBank Accession No. P31947; sequence provided in Appendix 1). The amino acid sequence for native human hnRNPK includes the amino acid sequences referenced in NCBI Gene ID: gi|48429103, NP.sub.--002131.2, P61978.1 including GenBank Accession Nos. 574678.1, NP.sub.--112552.1, AAB20770.1, NP.sub.--112553.1, X72727.1, 1J5K_A, CAA51267.1, 1KHM_A, AB209562.1, 1ZZI_A, BAD92799.1, 1ZZI_B, BC000355.2, 1ZZJ_A, AAH00355.1, 1ZZJ_B, BC014980.1, 1ZZJ_C, AAH14980.1, 1ZZK_A, and the exemplary sequences shown in SEQ ID NO: 25 (GenBank Accession No. P61978.1; sequence provided in Appendix 1).

[0205] A "native-sequence polypeptide" includes a polypeptide having the same amino acid sequence of a polypeptide derived from nature. Such native-sequence polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term specifically encompasses naturally occurring truncated or secreted forms of a polypeptide, polypeptide variants including naturally occurring variant forms (e.g., alternatively spliced forms or splice variants), and naturally occurring allelic variants.

[0206] The term "polypeptide variant" means a polypeptide having at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity, particularly at least about 70-80%, more particularly at least about 85%, still more particularly at least about 90%, most particularly at least about 95% amino acid sequence identity with a native-sequence polypeptide. Particular polypeptide variants have at least 70-80%, 85%, 90%, 95% amino acid sequence identity to the sequences identified in Table 5 or 2 or 7. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added to, or deleted from, the N- or C-terminus of the full-length or mature sequences of the polypeptide, including variants from other species, but excludes a native-sequence polypeptide. In aspects of the invention variants retain the immunogenic activity of the corresponding native-sequence polypeptide.

[0207] Percent identity of two amino acid sequences, or of two nucleic acid sequences is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues in a polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid or nucleic acid sequence identity can be achieved in various conventional ways, for instance, using publicly available computer software including the GIG program package (Devereux J. et al., Nucleic Acids Research 12(1): 387, 1984); BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410, 1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., J. Mol. Biol. 215: 403-410, 1990). Skilled artisans can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Methods to determine identity and similarity are codified in publicly-available computer programs.

[0208] An allelic variant may also be created by introducing substitutions, additions, or deletions into a polynucleotide encoding a native polypeptide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations may be introduced by standard methods, such as site-directed mutagenesis and PCR-mediated mutagenesis. In an embodiment, conservative substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue with a similar side chain. Amino acids with similar side chains are known in the art and include amino acids with basic side chains (e.g., Lys, Arg, His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains (e.g., Gly, Asp, Glu, Ser, Thr, Tyr, and Cys), non-polar side chains (e.g., Ala, Val, Leu, Iso, Pro, Trp), beta-branched side chains (e.g., Thr, Val, Iso), and aromatic side chains (e.g., Tyr, Phe, Trp, His). Mutations can also be introduced randomly along part or all of the native sequence, for example, by saturation mutagenesis. Following mutagenesis the variant polypeptide can be recombinantly expressed and the activity of the polypeptide may be determined.

[0209] Polypeptide variants include polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of a native polypeptide which include fewer amino acids than the full length polypeptides. A portion of a polypeptide can be a polypeptide which is for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids in length. Portions in which regions of a polypeptide are deleted can be prepared by recombinant techniques and can be evaluated for one or more functional activities such as the ability to form antibodies specific for a polypeptide.

[0210] A naturally occurring allelic variant may contain conservative amino acid substitutions from the native polypeptide sequence or it may contain a substitution of an amino acid from a corresponding position in a polypeptide homolog, for example, a murine polypeptide.

[0211] An OPL or head-and-neck marker may be part of a chimeric or fusion protein. A "chimeric protein" or "fusion protein" includes all or part (preferably biologically active) of an OPL or head-and-neck marker operably linked to a heterologous polypeptide (i.e., a polypeptide other than a head-and-neck marker). Within the fusion protein, the term "operably linked" is intended to indicate that an OPL or head-and-neck marker and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the N-terminus or C-terminus of an OPL or head-and-neck marker. A useful fusion protein is a GST fusion protein in which an OPL or head-and-neck marker is fused to the C-terminus of GST sequences. Another example of a fusion protein is an immunoglobulin fusion protein in which all or part of an OPL or head-and-neck marker is fused to sequences derived from a member of the immunoglobulin protein family. Chimeric and fusion proteins can be produced by standard recombinant DNA techniques.

[0212] A modified form of a polypeptide referenced herein includes modified forms of the polypeptides and derivatives of the polypeptides, including post-translationally modified forms such as glycosylated, phosphorylated, acetylated, methylated or lapidated forms of the polypeptides. For example, an N-terminal methionine may be cleaved from a polypeptide, and a new N-terminal residue may or may not be acetylated. In particular, for chaperonin 10 the first residue, methionine, can be cleaved and the second first residue, alanine can be N-acetylated.

[0213] Oral premalignant lesions (OPL) or head-and-neck cancer markers may be prepared by recombinant or synthetic methods, or isolated from a variety of sources, or by any combination of these and similar techniques.

[0214] "Oral premalignant lesions (OPL) or head-and-neck cancer polynucleotide marker(s)", "polynucleotides encoding the marker(s)", and "polynucleotides encoding oral premalignant lesions (OPL) or head-and-neck cancer markers" refer to polynucleotides that encode OPL or head-and-neck cancer markers including native-sequence polypeptides, polypeptide variants including a portion of a polypeptide, an isoform, precursor, complex, a chimeric polypeptide, or modified forms and derivatives of the polypeptides. An OPL or head-and-neck cancer polynucleotide marker includes or consists of one or more of the polynucleotides encoding the polypeptides listed in Table 5.

[0215] In an aspect, a polynucleotide of the invention encodes YWHAZ (SEQ ID NO:24), for example a polynucleotide sequence referenced in NCBI Gene ID. NM.sub.--003406, NP.sub.--003397.1, NM.sub.--145690.1, or NP.sub.--663723.1, more particularly GenBank Accession Nos. gi|21735625; gi|119612231; gi|85567424; gi|4507953; gi|83754473; gi|68085578; gi|119612234; gi|30354619; gi|14278218; gi|119612230; gi|68085278; gi|14278221; gi|6137540; gi|14278219; gi|68085909; gi|6137543; gi|83754467; gi|68085317; gi|83754468; gi|80477445; gi|49119653; gi|119612233; gi|14278220; gi|189953; gi|6137539; gi|68084347; gi|899459; gi|6137544; gi|75517642; gi|71533983; gi|119612232; gi|52000887; gi|119612235; gi|83754474 (and see, for example, SEQ ID NOs. NM.sub.--003406 in Appendix 1), or a fragment thereof.

[0216] In an aspect, a polynucleotide of the invention encodes stratifin (SEQ ID NO: 23), for example a polynucleotide sequence referenced in NCBI Gene ID. NM.sub.--006142.3 or NP.sub.--006133.1, more particularly GenBank Accession Nos. gi|398953; gi|2702355; gi|7981260; gi|49456765; gi|12653125; gi|187302; gi|12654345; gi|62738853; gi|16306737; gi|23940; gi|62738854; gi|5454052; gi|23270783; gi|61680850; gi|61680851; gi|2702353; gi|49456807; gi|119628191; gi|12804273 (and see, for example, SEQ ID NOs. NM.sub.--006142.3 in Appendix 1), or fragment thereof.

[0217] In an aspect, a polynucleotide of the invention encodes S100 A7 (SEQ ID NO: 13), for example a polynucleotide sequence referenced in NCBI Gene ID. NM.sub.--002963.3 or NP.sub.--002954.2, more particularly GenBank Accession Nos. gi|4389153; gi|400892; gi|4389154; gi|49457324; gi|190668; gi|11228051; gi|12053626; gi|5542542; gi|5542543; gi|21961601; gi|119573712; gi|157835758, (and see, for example, SEQ ID NOs. NM.sub.--002963.3 in Appendix 1) or a fragment thereof.

[0218] In an aspect, a polynucleotide of the invention encodes hnRNPK (SEQ ID NO: 25), for example a polynucleotide sequence referenced in NCBI Gene ID: gi|48429103, NP.sub.--002131.2, P61978.1, more particularly GenBank Accession Nos. 574678.1, NP.sub.--112552.1, AAB20770.1, NP.sub.--112553.1, X72727.1, IJ5K_A, CAA51267.1, 1KHM_A, AB209562.1, 1ZZI_A, BAD92799.1, 1ZZI_B, BC000355.2, 1ZZJ_A, AAH00355.1, 1ZZJ_B, BC014980.1, 1ZZJ_C, AAH14980.1, 1ZZK_A, and see, for example, SEQ ID NOs. P61978.1 in Appendix 1.

[0219] Oral premalignant lesion (OPL) or head-and-neck cancer polynucleotide markers include complementary nucleic acid sequences, and nucleic acids that are substantially identical to these sequences (e.g., having at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity).

[0220] Oral premalignant lesion (OPL) or head-and-neck cancer polynucleotide markers also include sequences that differ from a native sequence due to degeneracy in the genetic code. As one example, DNA sequence polymorphisms within the nucleotide sequence of an OPL or head-and-neck marker may result in silent mutations that do not affect the amino acid sequence. Variations in one or more nucleotides may exist among individuals within a population due to natural allelic variation. DNA sequence polymorphisms may also occur which lead to changes in the amino acid sequence of a polypeptide.

[0221] Oral premalignant lesion (OPL) or head-and-neck cancer polynucleotide markers also include nucleic acids that hybridize under stringent conditions, preferably high stringency conditions to an OPL or head-and-neck cancer polynucleotide marker, in particular an OPL or head-and-neck cancer polynucleotide marker. Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, 6.0.times.sodium chloride/sodium citrate (SSC) at about 45.degree. C., followed by a wash of 2.0.times.SSC at 50.degree. C. may be employed. The stringency may be selected based on the conditions used in the wash step. By way of example, the salt concentration in the wash step can be selected from a high stringency of about 0.2.times.SSC at 50.degree. C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65.degree. C.

[0222] Oral premalignant lesion (OPL) or head-and-neck cancer polynucleotide markers also include truncated nucleic acids or nucleic acid fragments and variant forms of the nucleic acids that arise by alternative splicing of an mRNA corresponding to a DNA.

[0223] The OPL or head-and-neck cancer polynucleotide markers are intended to include DNA and RNA (e.g., mRNA) and can be either double stranded or single stranded. A polynucleotide may, but need not, include additional coding or non-coding sequences, or it may, but need not, be linked to other molecules and/or carrier or support materials. The polynucleotides for use in the methods of the invention may be of any length suitable for a particular method. In certain applications the term refers to antisense polynucleotides (e.g., mRNA or DNA strand in the reverse orientation to sense cancer polynucleotide markers).

[0224] "Statistically different levels", "significantly altered levels", or "significant difference" in levels of markers in a patient sample compared to a control or standard (e.g., normal levels or levels in other samples from a patient) may represent levels that are higher or lower than the standard error of the detection assay. In particular embodiments, the levels may be 1.5, 2, 3, 4, 5, or 6 times higher or lower than the control or standard.

[0225] "Microarray" and "array" refer to nucleic acid or nucleotide arrays or protein or peptide arrays that can be used to detect biomolecules associated with head and neck cell or tissue phase and head-and-neck disease, for instance to measure gene expression. A variety of arrays are made in research and manufacturing facilities worldwide, some of which are available commercially. By way of example, spotted arrays and in situ synthesized arrays are two kinds of nucleic acid arrays that differ in the manner in which the nucleic acid materials are placed onto the array substrate. A widely used in situ synthesized oligonucleotide array is GeneChip.TM. made by Affymetrix, Inc. Oligonucleotide probes that are 20- or 25-base long can be synthesized in silico on the array substrate. These arrays can achieve high densities (e.g., more than 40,000 genes per cm.sup.2). Generally spotted arrays have lower densities, but the probes, typically partial cDNA molecules, are much longer than 20- or 25-mers. Examples of spotted cDNA arrays include LifeArray made by Incyte Genomics and DermArray made by IntegriDerm (or Invitrogen). Pre-synthesized and amplified cDNA sequences are attached to the substrate of spotted arrays. Protein and peptide arrays also are known (see for example, Zhu et al., Science 293:2101 (2001).

[0226] "Binding agent" refers to a substance such as a polypeptide or antibody that specifically binds to one or more OPL or head-and-neck cancer markers. A substance "specifically binds" to one or more OPL or head-and-neck cancer markers if is reacts at a detectable level with one or more OPL or head-and-neck cancer markers, and does not react detectably with peptides containing an unrelated or different sequence. Binding properties may be assessed using an ELISA, which may be readily performed by those skilled in the art (see, for example, Newton et al., Develop. Dynamics 197: 1-13, 1993).

[0227] A binding agent may be a ribosome, with or without a peptide component, an aptamer, an RNA molecule, or a polypeptide. A binding agent may be a polypeptide that includes one or more OPL or head-and-neck marker sequence, a peptide variant thereof, or a non-peptide mimetic of such a sequence. By way of example, a YWHAZ sequence may be a peptide portion of a YWHAZ that is capable of modulating a function mediated by YWHAZ.

[0228] An aptamer includes a DNA or RNA molecule that binds to nucleic acids and proteins. An aptamer that binds to a protein (or binding domain) of an OPL or head-and-neck marker or an OPL polynucleotide marker or head-and-neck cancer polynucleotide marker can be produced using conventional techniques, without undue experimentation. (For example, see the following publications describing in vitro selection of aptamers: Klug et al., Mol. Biol. Reports 20:97-107 (1994); Wallis et al., Chem. Biol. 2:543-552 (1995); Ellington, Curr. Biol. 4:427-429 (1994); Lato et al., Chem. Biol. 2:291-303 (1995); Conrad et al., Mol. Div. 1:69-78 (1995); and Uphoff et al., Curr. Opin. Struct. Biol. 6:281-287 (1996)).

[0229] Antibodies for use in the present invention include but are not limited to monoclonal or polyclonal antibodies, immunologically active fragments (e.g., a Fab or (Fab).sub.2 fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain F.sub.v molecules (Ladner et al., U.S. Pat. No. 4,946,778), chimeric antibodies, for example, antibodies which contain the binding specificity of murine antibodies, but in which the remaining portions are of human origin, or derivatives, such as enzyme conjugates or labeled derivatives.

[0230] Antibodies, including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art. Isolated native or recombinant OPL or head-and-neck cancer markers may be utilized to prepare antibodies. (See, for example, Kohler et al. (1975), Nature 256:495-497; Kozbor et al. (1985), J. Immunol Methods 81:31-42; Cote et al. (1983), Proc Natl Acad Sci 80:2026-2030; and Cole et al. (1984), Mol Cell Biol 62:109-120 for the preparation of monoclonal antibodies; Huse et al. (1989), Science 246:1275-1281 for the preparation of monoclonal Fab fragments; and, Pound (1998), Immunochemical Protocols, Humana Press, Totowa, N.J. for the preparation of phagemid or B-lymphocyte immunoglobulin libraries to identify antibodies). Antibodies specific for an OPL or head-and-neck marker may also be obtained from scientific or commercial sources.

[0231] In an embodiment of the invention, antibodies are reactive against an OPL or head-and-neck marker if they bind with a K.sub.a of greater than or equal to 10.sup.-7 M.

[0232] Markers.

[0233] The invention provides a set of markers correlated with head-and-neck disease. In an aspect, the invention provides a set of markers identified as useful for detection, diagnosis, prevention and therapy of head-and-neck disease including or consisting of one or more of the markers listed in Table 5. In another aspect, the invention provides the head-and-neck or OPL cancer markers in Table 2 and Table 7 for detection, diagnosis and prognosis of a head-and-neck disease. The invention also provides a method of using OPL or head-and-neck cancer markers listed in Table 5 or Table 2 or Table 7, to distinguish head-and-neck disease.

[0234] In an embodiment, the markers include or consist of 1, 2, 3, 4 or more other markers listed in Table 5, Table 2, and Table 7.

[0235] In embodiments of the invention, a marker is provided which is selected from the group consisting of the polypeptides set forth in Table 5 which polypeptides are up-regulated biomarkers in OPL or head-and-neck cancer and optionally at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides set forth in Table 5 and/or 2 or polypeptides in Table 1 and Table 5 and/or 6 or polypeptides in Table 7 that are up-regulated biomarkers in OPL or head-and-neck cancer.

[0236] In embodiments of the invention, a marker is provided which is selected from the group consisting of at least one marker of Table 2 and at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides set forth in Table 5.

[0237] In embodiments of the invention, a marker is provided which is selected from the group consisting of at least one marker of Table 7 and at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides set forth in Table 5.

[0238] The invention provides marker sets that distinguish head-and-neck disease and uses therefor. In an aspect, the invention provides a method for classifying a head-and-neck disease including detecting a difference in the expression of a first plurality of OPL or head-and-neck cancer markers or OPL or head-and-neck cancer polynucleotide markers relative to a control, the first plurality of OPL or head-and-neck cancer markers or OPL or head-and-neck cancer polynucleotide markers including or consisting of at least 2, 3, 4, or 5 of the markers listed in Table 5. In specific aspects, the plurality of markers consists of the markers listed in Table 2 and optionally at least 5 to 10 of the markers listed in Table 5. In specific aspects, the plurality of markers consists of the markers listed in Table 7 and optionally at least 5 to 10 of the markers listed in Table 5. In specific aspects, a control includes markers derived from a pool of samples from individual patients with no head-and-neck disease.

[0239] Any of the markers provided herein may be used alone or with other markers of head-and-neck disease, or with markers for other phenotypes or conditions. Additionally, all of the sequences provided herein are representative only; there may be other sequences for particular protein or coding sequences or related sequences. The invention is not intended to be limited to the sequences herein provided.

[0240] Detection Methods.

[0241] A variety of methods can be employed for the diagnostic and prognostic evaluation of head-and-neck disease or head-and-neck status involving one or more OPL or head-and-neck cancer markers and polynucleotides encoding the markers, and the identification of subjects with a predisposition to head-and-neck diseases or that are receptive to in vitro fertilization and embryo transfer procedures. Such methods may, for example, utilize head-and-neck cancer polynucleotide markers, and fragments thereof, and binding agents (e.g., antibodies) against one or more OPL or head-and-neck cancer markers, including peptide fragments. In particular, the polynucleotides and antibodies may be used, for example, for (1) the detection of the presence of OPL or head-and-neck cancer polynucleotide marker mutations, or the detection of either over- or under-expression of OPL or head-and-neck marker mRNA relative to a non-disorder state or different head and neck cell or tissue phase, or the qualitative or quantitative detection of alternatively spliced forms of OPL or head-and-neck cancer polynucleotide marker transcripts which may correlate with certain conditions or susceptibility toward such conditions; and (2) the detection of either an over- or an under-abundance of one or more OPL or head-and-neck cancer markers relative to a non-disorder state or a different head and neck cell or tissue phase or the presence of a modified (e.g., less than full length) OPL or head-and-neck marker which correlates with a disorder state or a progression toward a disorder state.

[0242] The invention contemplates a method for detecting the phase of a head-and-neck tissue, in particular a secretory head-and-neck tissue, including producing a profile of levels of one or more OPL or head-and-neck marker associated with a known head and/or neck cell or tissue phase and/or polynucleotides encoding the markers, and optionally other markers associated with the phase in cells from a patient, and comparing the profile with a reference to identify a profile for the test cells indicative of the phase. In an aspect, the head-and-neck cancer markers include those of Table 5, preferably Table 2 or fragments thereof. In an aspect, the OPL markers include those of Table 5, preferably Table 7 or fragments thereof.

[0243] The invention also contemplates a method for detecting a head-and-neck disease, in particular an OPL or head-and-neck cancer, including producing a profile of levels of one or more head-and-neck marker associated with a head-and-neck disease and/or polynucleotides encoding the markers, and other markers associated with head-and-neck disease in cells from a patient, and comparing the profile with a reference to identify a profile for the test cells indicative of disease. In an aspect, the head-and-neck cancer markers are one or more of those listed in Table 5 or Table 2. In an aspect, the OPL markers are one or more of those listed in Table 5 or Table 7.

[0244] The methods described herein may be used to evaluate the probability of the presence of malignant or pre-malignant cells, for example, in a group of cells freshly removed from a host. Such methods can be used to detect tumors, quantify their growth, and help in the diagnosis and prognosis of head-and-neck disease. The methods can be used to detect the presence of cancer metastasis, as well as confirm the absence or removal of all tumor tissue following surgery, cancer chemotherapy, and/or radiation therapy. They can further be used to monitor cancer chemotherapy and tumor reappearance.

[0245] The methods described herein can be adapted for diagnosing and monitoring head-and-neck tissue status or a head-and-neck disease by detecting one or more OPL or head-and-neck cancer markers or polynucleotides encoding the markers in biological samples from a subject. These applications require that the amount of markers or polynucleotides quantified in a sample from a subject being tested be compared to a predetermined standard or cut-off value. The standard may correspond to levels quantified for another sample or an earlier sample from the subject, or levels quantified for a control sample. Levels for control samples from healthy subjects, different head-and-neck tissue phases, or subjects with a head-and-neck disease may be established by prospective and/or retrospective statistical studies. Healthy subjects who have no clinically evident disease or abnormalities may be selected for statistical studies. Diagnosis may be made by a finding of statistically different levels of detected head-and-neck cancer markers associated with disease or polynucleotides encoding same, compared to a control sample or previous levels quantified for the same subject.

[0246] The methods described herein may also use multiple markers for a head-and-neck disease, in particular OPL and head-and-neck cancer. Therefore, the invention contemplates a method for analyzing a biological sample for the presence of one or more OPL or head-and-neck cancer markers and polynucleotides encoding the markers, and other markers that are specific indicators of a head-and-neck disease. The methods described herein may be modified by including reagents to detect the additional markers, or polynucleotides for the markers.

[0247] Nucleic Acid Methods/Assays.

[0248] As noted herein a head-and-neck disease or phase may be detected based on the level of OPL or head-and-neck cancer polynucleotide markers in a sample. Techniques for detecting polynucleotides such as polymerase chain reaction (PCR) and hybridization assays are well known in the art.

[0249] Probes may be used in hybridization techniques to detect OPL or head-and-neck cancer polynucleotide markers. The technique generally involves contacting and incubating nucleic acids (e.g., recombinant DNA molecules, cloned genes) obtained from a sample from a patient or other cellular source with a probe under conditions favorable for the specific annealing of the probes to complementary sequences in the nucleic acids. After incubation, the non-annealed nucleic acids are removed, and the presence of nucleic acids that have hybridized to the probe if any are detected.

[0250] Nucleotide probes for use in the detection of nucleic acid sequences in samples may be constructed using conventional methods known in the art. Suitable probes may be based on nucleic acid sequences encoding at least 5 sequential amino acids from regions of an OPL or head-and-neck cancer polynucleotide marker, preferably they include 10-200, more particularly 10-30, 10-40, 20-50, 40-80, 50-150, 80-120 nucleotides in length.

[0251] The probes may include DNA or DNA mimics (e.g., derivatives and analogues) corresponding to a portion of an organism's genome, or complementary RNA or RNA mimics. Mimics are polymers including subunits capable of specific, Watson-Crick-like hybridization with DNA, or of specific hybridization with RNA. The nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone.

[0252] DNA can be obtained using standard methods such as polymerase chain reaction (PCR) amplification of genomic DNA or cloned sequences. (See, for example, in Innis et al., eds., 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif.). Computer programs known in the art can be used to design primers with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences). Controlled robotic systems may be useful for isolating and amplifying nucleic acids.

[0253] A nucleotide probe may be labeled with a detectable substance such as a radioactive label that provides for an adequate signal and has sufficient half-life such as .sup.32P, .sup.3H, .sup.14C or the like. Other detectable substances that may be used include antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and luminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. Labeled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (2nd ed.). The nucleic acid probes may be used to detect head-and-neck cancer polynucleotide markers, preferably in human cells. The nucleotide probes may also be useful in the diagnosis of a head-and-neck disease involving one or more OPL or head-and-neck cancer polynucleotide markers, in monitoring the progression of such disorder, or monitoring a therapeutic treatment.

[0254] The detection of OPL or head-and-neck cancer polynucleotide markers may involve the amplification of specific gene sequences using an amplification method such as polymerase chain reaction (PCR), followed by the analysis of the amplified molecules using techniques known to those skilled in the art. Suitable primers can be routinely designed by one of skill in the art.

[0255] By way of example, at least two oligonucleotide primers may be employed in a PCR based assay to amplify a portion of a polynucleotide encoding one or more OPL or head-and-neck marker derived from a sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the OPL or head-and-neck marker. The amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis.

[0256] In order to maximize hybridization under assay conditions, primers and probes employed in the methods of the invention generally have at least about 60%, preferably at least about 75%, and more preferably at least about 90% identity to a portion of a polynucleotide encoding an OPL or head-and-neck marker; that is, they are at least 10 nucleotides, and preferably at least 20 nucleotides in length. In an embodiment the primers and probes are at least about 10-40 nucleotides in length.

[0257] Hybridization and amplification techniques described herein may be used to assay qualitative and quantitative aspects of OPL or head-and-neck cancer polynucleotide marker expression. For example, RNA may be isolated from a cell type or tissue known to express a head-and-neck cancer polynucleotide marker and tested utilizing the hybridization (e.g., standard Northern analyses) or PCR techniques referred to herein.

[0258] The primers and probes may be used in the above-described methods in situ (i.e., directly on tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections).

[0259] In an aspect of the invention, a method is provided employing reverse transcriptase-polymerase chain reaction (RT-PCR), in which PCR is applied in combination with reverse transcription. Generally, RNA is extracted from a sample tissue using standard techniques (e.g., guanidine isothiocyanate extraction as described by Chomcynski and Sacchi, Anal. Biochem. 162:156-159, 1987) and is reverse transcribed to produce cDNA. The cDNA is used as a template for a polymerase chain reaction. The cDNA is hybridized to a set of primers, at least one of which is specifically designed against a head-and-neck marker sequence. Once the primer and template have annealed a DNA polymerase is employed to extend from the primer, to synthesize a copy of the template. The DNA strands are denatured, and the procedure is repeated many times until sufficient DNA is generated to allow visualization by ethidium bromide staining and agarose gel electrophoresis.

[0260] Amplification may be performed on samples obtained from a subject with a suspected head-and-neck disease and an individual who is not afflicted with a head-and-neck disease. The reaction may be performed on several dilutions of cDNA spanning at least two orders of magnitude. A statistically significant difference in expression in several dilutions of the subject sample as compared to the same dilutions of the non-disease sample may be considered positive for the presence of a head-and-neck disease.

[0261] In an embodiment, the invention provides methods for determining the presence or absence of a head-and-neck disease in a subject including (a) contacting a sample obtained from the subject with oligonucleotides that hybridize to OPL or head-and-neck cancer polynucleotide markers; and (b) detecting in the sample a level of nucleic acids that hybridize to the polynucleotides relative to a predetermined cut-off value, and therefrom determining the presence or absence of a head-and-neck disease in the subject. In an aspect, the head-and-neck disease is OPL or cancer and the OPL or head-and-neck cancer markers are one or more of those listed in Table 5, or in another embodiment Table 2 or in another embodiment Table 7.

[0262] The invention provides a method wherein an OPL or head-and-neck marker mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to one or more OPL or head-and-neck marker polynucleotides, to produce amplification products; (d) analyzing the amplification products to detect amounts of mRNA encoding OPL or head-and-neck polynucleotide markers; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal and malignant tissue derived using similar nucleic acid primers.

[0263] Oral premalignant lesion (OPL) or head-and-neck cancer marker-positive samples or alternatively higher levels in patients compared to a control (e.g., non-cancerous tissue) may be indicative of late stage disease, and/or that the patient is not responsive to chemotherapy. Alternatively, negative samples or lower levels compared to a control (e.g., non-cancerous tissue or negative samples) may be indicative of progressive disease and shorter overall survival.

[0264] In another embodiment, the invention provides methods for determining the presence or absence of OPL or head-and-neck cancer in a subject including (a) contacting a sample obtained from the subject with oligonucleotides that hybridize to one or more OPL or head-and-neck cancer polynucleotide markers; and (b) detecting in the sample levels of nucleic acids that hybridize to the polynucleotides relative to a predetermined cut-off value, and therefrom determining the presence or absence of OPL or head-and-neck cancer in the subject. In an embodiment, the OPL or head-and-neck cancer polynucleotide markers encode one or more polypeptides listed in Table 5.

[0265] In particular, the invention provides a method wherein YWHAZ, S100 A7, hnRNPK and/or stratifin mRNA is detected by (a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to a polynucleotide encoding YWHAZ, S100 A7, hnRNPK, and/or stratifin, to produce amplification products; (d) analyzing the amplification products to detect an amount of mRNA encoding YWHAZ, S100 A7, hnRNPK, and/or stratifin; and (e) comparing the amount of mRNA to an amount detected against a panel of expected values for normal and malignant tissue derived using similar nucleic acid primers.

[0266] Oral premalignant lesion or head-and-neck cancer marker-positive samples or alternatively higher levels, in particular significantly higher levels of YWHAZ, S100 A7, hnRNPK and/or stratifin polynucleotides in patients compared to a control (e.g., normal or benign) are indicative of head-and-neck cancer. Negative samples or lower levels (e.g., of alpha-1-antitrypsin or KPSG lumican polynucleotides) compared to a control (e.g., normal or benign) may also be indicative of progressive disease and poor overall survival.

[0267] Oligonucleotides or longer fragments derived from an OPL or head-and-neck cancer polynucleotide marker may be used as targets in a microarray. The microarray can be used to simultaneously monitor the expression levels of large numbers of genes and to identify genetic variants, mutations, and polymorphisms. The information from the microarray may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.

[0268] The preparation, use, and analysis of microarrays are well known to a person skilled in the art. (See, for example, Brennan, T. M. et al. (1995), U.S. Pat. No. 5,474,796; Schena, et al. (1996), Proc. Natl. Acad. Sci. 93:10614-10619; Baldeschweiler et al. (1995), PCT Application WO 95/251116; Shalon, D. et al. (1995), PCT application WO 95/35505; Heller, R. A. et al. (1997), Proc. Natl. Acad. Sci., 94:2150-2155; and Heller, M. J. et al. (1997), U.S. Pat. No. 5,605,662).

[0269] Thus, the invention also includes an array including one or more OPL or head-and-neck cancer polynucleotide markers (in particular the markers listed in Table 5) and/or markers listed in Table 2 or Table 7 or other markers. The array can be used to assay expression of OPL or head-and-neck cancer polynucleotide markers in the array. The invention allows the quantification of expression of one or more OPL or head-and-neck cancer polynucleotide markers.

[0270] Microarrays typically contain at separate sites nanomolar quantities of individual genes, cDNAs, or ESTs on a substrate (e.g., nitrocellulose or silicon plate), or photolithographically prepared glass substrate. The arrays are hybridized to cDNA probes using conventional techniques with gene-specific primer mixes. The target polynucleotides to be analyzed are isolated, amplified and labeled, typically with fluorescent labels, radiolabels or phosphorous label probes. After hybridization is completed, the array is inserted into the scanner, where patterns of hybridization are detected. Data are collected as light emitted from the labels incorporated into the target, which becomes bound to the probe array. Probes that completely match the target generally produce stronger signals than those that have mismatches. The sequence and position of each probe on the array are known, and thus by complementarity, the identity of the target nucleic acid applied to the probe array can be determined.

[0271] Microarrays are prepared by selecting polynucleotide probes and immobilizing them to a solid support or surface. The probes may include DNA sequences, RNA sequences, copolymer sequences of DNA and RNA, DNA and/or RNA analogues, or combinations thereof. The probe sequences may be full or partial fragments of genomic DNA, or they may be synthetic oligonucleotide sequences synthesized either enzymatically in vivo, enzymatically in vitro (e.g., by PCR), or non-enzymatically in vitro.

[0272] The probe or probes used in the methods of the invention can be immobilized to a solid support or surface which may be either porous or non-porous. For example, the probes can be attached to a nitrocellulose or nylon membrane or filter covalently at either the 3' or the 5' end of the polynucleotide probe. The solid support may be a glass or plastic surface. In an aspect of the invention, hybridization levels are measured to microarrays of probes consisting of a solid support on the surface of which are immobilized a population of polynucleotides, such as a population of DNA or DNA mimics, or, alternatively, a population of RNA or RNA mimics. A solid support may be a nonporous or, optionally, a porous material such as a gel.

[0273] In accordance with embodiments of the invention, a microarray is provided including a support or surface with an ordered array of hybridization sites or "probes" each representing one of the markers described herein. The microarrays can be addressable arrays, and in particular positionally addressable arrays. Each probe of the array is typically located at a known, predetermined position on the solid support such that the identity of each probe can be determined from its position in the array. In preferred embodiments, each probe is covalently attached to the solid support at a single site.

[0274] Microarrays used in the present invention are preferably (a) reproducible, allowing multiple copies of a given array to be produced and easily compared with each other; (b) made from materials that are stable under hybridization conditions; (c) small (e.g., between 1 cm.sup.2 and 25 cm.sup.2, between 12 cm.sup.2 and 13 cm.sup.2, or 3 cm.sup.2); and (d) include a unique set of binding sites that will specifically hybridize to the product of a single gene in a cell (e.g., to a specific mRNA, or to a specific cDNA derived therefrom). However, it will be appreciated that larger arrays may be used particularly in screening arrays, and other related or similar sequences will cross hybridize to a given binding site.

[0275] In accordance with an aspect of the invention, the microarray is an array in which each position represents one of the markers described herein (e.g., the markers listed in Table 1 and optionally Table 2 and Table 7). Each position of the array can include a DNA or DNA analogue based on genomic DNA to which a particular RNA or cDNA transcribed from a genetic marker can specifically hybridize. A DNA or DNA analogue can be a synthetic oligomer or a gene fragment. In an embodiment, probes representing each of the OPL or head-and-neck cancer markers and OPL or head-and-neck cancer polynucleotide markers is present on the array. In a preferred embodiment, the array includes at least 5 of the OPL or head-and-neck cancer polynucleotide markers.

[0276] Probes for the microarray can be synthesized using N-phosphonate or phosphoramidite chemistries (Froehler et al., 1986, Nucleic Acid Res. 14:5399-5407; McBride et al., 1983, Tetrahedron Lett. 24:246-248). Synthetic sequences are typically between about 10 and about 500 bases, 20-100 bases, or 40-70 bases in length. Synthetic nucleic acid probes can include non-natural bases, such as, without limitation, inosine. Nucleic acid analogues such as peptide nucleic acid may be used as binding sites for hybridization (see, e.g., Egholm et al., 1993, Nature 363:566-568; U.S. Pat. No. 5,539,083).

[0277] Probes can be selected using an algorithm that takes into account binding energies, base composition, sequence complexity, cross-hybridization binding energies, and secondary structure (see Friend et al., International Patent Publication WO 01/05935, published Jan. 25, 2001).

[0278] Positive control probes (e.g., probes known to be complementary and hybridize to sequences in the target polynucleotides) and negative control probes (e.g., probes known to not be complementary and hybridize to sequences in the target polynucleotides) are typically included on the array. Positive controls can be synthesized along the perimeter of the array or synthesized in diagonal stripes across the array. A reverse complement for each probe can be next to the position of the probe to serve as a negative control.

[0279] The probes can be attached to a solid support or surface, which may be made from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, gel, or other porous or nonporous material. The probes can be printed on surfaces such as glass plates (see Schena et al., 1995, Science 270:467-470). This method may be particularly useful for preparing microarrays of cDNA. (See, also, DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; and Schena et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 93:10539-11286).

[0280] High-density oligonucleotide arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface can be produced using photolithographic techniques for synthesis in situ (see Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270) or other methods for rapid synthesis and deposition of defined oligonucleotides (Blanchard et al., Biosensors & Bioelectronics 11:687-690). Using these methods oligonucleotides (e.g., 60-mers) of known sequence are synthesized directly on a surface such as a derivatized glass slide. The array produced may be redundant, with several oligonucleotide molecules per RNA.

[0281] Microarrays can be made by other methods including masking (Maskos and Southern, 1992, Nuc. Acids. Res. 20:1679-1684). In an embodiment, microarrays of the present invention are produced by synthesizing polynucleotide probes on a support wherein the nucleotide probes are attached to the support covalently at either the 3' or the 5' end of the polynucleotide.

[0282] The invention provides microarrays including a disclosed marker set. In one embodiment, the invention provides a microarray for distinguishing head-and-neck disease samples including a positionally-addressable array of polynucleotide probes bound to a support, the polynucleotide probes including a plurality of polynucleotide probes of different nucleotide sequences, each of the different nucleotide sequences including a sequence complementary and hybridizable to a plurality of genes, the plurality consisting of at least 2, 3, 4, 5, or 6 of the genes corresponding to the markers listed in Table 5 and optionally at least 2 to 18, 5 to 16, 10 to 15, 13-21, 2-21, 2-32, 22-32 or 13-32 of the genes corresponding to the markers listed in Table 2. An aspect of the invention provides microarrays including at least 4, 5, or 6 of the polynucleotides encoding the markers listed in Table 5.

[0283] The invention provides gene marker sets that distinguish head-and-neck disease and uses therefor. In an aspect, the invention provides a method for classifying a head-and-neck disease including detecting a difference in the expression of a first plurality of genes relative to a control, the first plurality of genes consisting of at least 3, 4, 5, or 6 of the genes encoding the markers listed in Table 5. In specific aspects, the plurality of genes consists of at least 4 or 5 of the genes encoding the markers listed in Table 5 and optionally at least 2 to 18, 5 to 16, 10 to 15-21, 2-21, 2-32, 22-32, or 13-32 of the genes corresponding to the markers listed in Table 5. In another specific aspect, the control includes nucleic acids derived from a pool of samples from individual control patients.

[0284] The invention provides a method for classifying a head-and-neck disease by calculating the similarity between the expression of at least 3, 4, 5, or 6 polynucleotides encoding markers listed in Table 5 in a sample to the expression of the same markers in a control pool, including the steps of: [0285] (a) labeling nucleic acids derived from a sample, with a first fluorophore to obtain a first pool of fluorophore-labeled nucleic acids; [0286] (b) labeling with a second fluorophore a first pool of nucleic acids derived from two or more head-and-neck disease samples, and a second pool of nucleic acids derived from two or more control samples; [0287] (c) contacting the first fluorophore-labeled nucleic acid and the first pool of second fluorophore-labeled nucleic acid with a first microarray under conditions such that hybridization can occur, and contacting the first fluorophore-labeled nucleic acid and the second pool of second fluorophore-labeled nucleic acid with a second microarray under conditions such that hybridization can occur, detecting at each of a plurality of discrete loci on the first microarray a first fluorescent emission signal from the first fluorophore-labeled nucleic acid and a second fluorescent emission signal from the first pool of second fluorophore-labeled genetic matter that is bound to the first microarray and detecting at each of the marker loci on the second microarray the first fluorescent emission signal from the first fluorophore-labeled nucleic acid and a third fluorescent emission signal from the second pool of second fluorophore-labeled nucleic acid; [0288] (d) determining the similarity of the sample to patient and control pools by comparing the first fluorescence emission signals and the second fluorescence emission signals, and the first emission signals and the third fluorescence emission signals; and [0289] (e) classifying the sample as head-and-neck disease where the first fluorescence emission signals are more similar to the second fluorescence emission signals than to the third fluorescent emission signals, and classifying the sample as non-head-and-neck disease where the first fluorescence emission signals are more similar to the third fluorescence emission signals than to the second fluorescent emission signals, wherein the first microarray and the second microarray are similar to each other, exact replicas of each other, or are identical, and wherein the similarity is defined by a statistical method such that the cell sample and control are similar where the p value of the similarity is less than 0.01.

[0290] In aspects of the invention, the array can be used to monitor the time course of expression of one or more OPL or head-and-neck cancer polynucleotide markers in the array. This can occur in various biological contexts such as tumor progression.

[0291] The array is also useful for ascertaining differential expression patterns of OPL or head-and-neck cancer polynucleotide markers, and optionally other markers, in normal and abnormal cells. This may provide a battery of nucleic acids that could serve as molecular targets for diagnosis or therapeutic intervention.

[0292] Protein Methods.

[0293] Binding agents may be used for a variety of diagnostic and assay applications. There are a variety of assay formats known to the skilled artisan for using a binding agent to detect a target molecule in a sample. (For example, see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). In general, the presence or absence of a head-and-neck disease (e.g., cancer) in a subject may be determined by (a) contacting a sample from the subject with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined standard or cut-off value.

[0294] In particular embodiments of the invention, the binding agent is an antibody. Antibodies specifically reactive with one or more head-and-neck marker, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect one or more head-and-neck marker in various samples (e.g., biological materials). They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of expression of one or more head-and-neck marker, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of one or more head-and-neck marker. Antibodies may also be used to screen potentially therapeutic compounds in vitro to determine their effects on disorders (e.g., OPL or head-and-neck cancer) involving one or more OPL or head-and-neck cancer markers, and other conditions. In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies.

[0295] In an aspect, the invention provides a method for monitoring or diagnosing a head-and-neck disease (e.g., OPL or cancer) in a subject by quantifying one or more OPL or head-and-neck cancer markers in a biological sample from the subject including reacting the sample with antibodies specific for one or more OPL or head-and-neck cancer markers, which are directly or indirectly labeled with detectable substances and detecting the detectable substances. In a particular embodiment of the invention, OPL or head-and-neck cancer markers are quantified or measured.

[0296] In an aspect of the invention, a method for detecting a head-and-neck disease (e.g., OPL or cancer) is provided including: [0297] (a) obtaining a sample suspected of containing one or more OPL or head-and-neck cancer markers associated with a head-and-neck disease; [0298] (b) contacting said sample with antibodies that specifically bind to the OPL or head-and-neck cancer markers under conditions effective to bind the antibodies and form complexes; [0299] (c) measuring the amount of OPL or head-and-neck cancer markers present in the sample by quantifying the amount of the complexes; and [0300] (d) comparing the amount of OPL or head-and-neck cancer markers present in the samples with the amount of OPL or head-and-neck cancer markers in a control, wherein a change or significant difference in the amount of OPL or head-and-neck cancer markers in the sample compared with the amount in the control is indicative of a head-and-neck disease.

[0301] In an embodiment, the invention contemplates a method for monitoring the progression of a head-and-neck disease (e.g., OPL or cancer) in an individual, including: [0302] (a) contacting antibodies which bind to one or more OPL or head-and-neck cancer markers with a sample from the individual so as to form complexes including the antibodies and one or more OPL or head-and-neck cancer markers in the sample; [0303] (b) determining or detecting the presence or amount of complex formation in the sample; [0304] (c) repeating steps (a) and (b) at a point later in time; and [0305] (d) comparing the result of step (b) with the result of step (c), wherein a difference in the amount of complex formation is indicative of disease, disease stage, and/or progression of the disease in said individual.

[0306] The amount of complexes may also be compared to a value representative of the amount of the complexes from an individual not at risk of, or afflicted with, a head-and-neck disease at different stages. A significant difference in complex formation may be indicative of advanced disease (e.g., advanced head-and-neck cancer, or an unfavourable prognosis).

[0307] In aspects of the invention for diagnosis and monitoring of OPL or head-and-neck cancer, the OPL or head-and-neck cancer markers are one or more of those upregulated in cancer samples as compared to normal samples in Table 1, those listed in Table 5, and/or YWHAZ, S100 A7, and/or stratifin, and/or hnRNPK.

[0308] In embodiments of the methods of the invention, YWHAZ, S100 A7, hnRNPK and/or stratifin is detected in samples and higher levels, in particular significantly higher levels compared to a control (normal or benign) is indicative of the prognosis of OPL or head-and-neck cancer patient outcome.

[0309] In aspects of the invention for characterizing head-and-neck disease the OPL or head-and-neck cancer markers include YWHAZ, S100 A7, hnRNPK and/or stratifin and fragments thereof.

[0310] Antibodies may be used in any known immunoassays that rely on the binding interaction between antigenic determinants of one or more head-and-neck marker and the antibodies. Immunoassay procedures for in vitro detection of antigens in fluid samples are also well known in the art. (See, for example, Paterson et al., Int. J. Can. 37:659 (1986) and Burchell et al., Int. J. Can. 34:763 (1984) for a general description of immunoassay procedures.) Qualitative and/or quantitative determinations of one or more head-and-neck marker in a sample may be accomplished by competitive or non-competitive immunoassay procedures in either a direct or indirect format. Detection of one or more head-and-neck marker using antibodies can be done utilizing immunoassays which are run in either the forward, reverse or simultaneous modes. Examples of immunoassays are radioimmunoassays (RIA), enzyme immunoassays (e.g., ELISA), munofluorescence, immunoprecipitation, latex agglutination, hemagglutination, histochemical tests, and sandwich (immunometric) assays. These terms are well understood by those skilled in the art. A person skilled in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

[0311] According to an embodiment of the invention, an immunoassay for detecting one or more OPL or head-and-neck cancer markers in a biological sample includes contacting binding agents that specifically bind to OPL or head-and-neck cancer markers in the sample under conditions that allow the formation of first complexes including a binding agent and OPL or head-and-neck cancer markers and determining the presence or amount of the complexes as a measure of the amount of OPL or head-and-neck cancer markers contained in the sample. In a particular embodiment, the binding agents are labeled differently or are capable of binding to different labels.

[0312] Antibodies may be used to detect and quantify one or more OPL or head-and-neck cancer markers in a sample in order to diagnose and treat pathological states. In particular, the antibodies may be used in immunohistochemical analyses, for example, at the cellular and sub-subcellular level, to detect one or more OPL or head-and-neck cancer markers, to localize them to particular head-and-neck cells and tissues (e.g., tumor cells and tissues), and to specific subcellular locations, and to quantify the level of expression.

[0313] Immunohistochemical methods for the detection of antigens in tissue samples are well known in the art. For example, immunohistochemical methods are described in Taylor, Arch. Pathol. Lab. Med. 102:112 (1978). Briefly, in the context of the present invention, a tissue sample obtained from a subject suspected of having a head-and-neck-related problem is contacted with antibodies, preferably monoclonal antibodies recognizing one or more head-and-neck cancer markers. The site at which the antibodies are bound is determined by selective staining of the sample by standard immunohistochemical procedures. The same procedure may be repeated on the same sample using other antibodies that recognize one or more OPL or head-and-neck cancer markers. Alternatively, a sample may be contacted with antibodies against one or more OPL or head-and-neck cancer markers simultaneously, provided that the antibodies are labeled differently or are able to bind to a different label. The tissue sample may be normal head-and-neck tissue, an OPL, or a cancer tissue or a benign tissue.

[0314] An antibody microarray in which binding sites include immobilized, preferably monoclonal, antibodies specific to a substantial fraction of marker-derived OPL or head-and-neck cancer markers of interest can be utilized in the present invention. Antibody arrays can be prepared using methods known in the art (see, for example, Zhu et al., Science 293:2101 (2001) and reference 20).

[0315] Antibodies specific for one or more OPL or head-and-neck marker may be labelled with a detectable substance and localised in biological samples based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g., .sup.3H, .sup.14C, .sup.35s, .sup.125I, .sup.131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods)), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualised by electron microscopy.

[0316] One of the ways an antibody can be detectably labeled is to link it directly to an enzyme. The enzyme when later exposed to its substrate will produce a product that can be detected. Examples of detectable substances that are enzymes are horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase, malate dehydrogenase, ribonuclease, urease, catalase, glucose-6-phosphate, staphylococcal nuclease, delta-5-steriod isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, triose phosphate isomerase, asparaginase, glucose oxidase, and acetylcholine esterase.

[0317] For increased sensitivity in an immunoassay system a fluorescence-emitting metal atom such as Eu (europium) and other lanthanides can be used. These can be attached to the desired molecule by means of metal-chelating groups such as DTPA or EDTA.

[0318] A bioluminescent compound may also be used as a detectable substance. Bioluminescence is a type of chemiluminescence found in biological systems where a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent molecule is determined by detecting the presence of luminescence. Examples of bioluminescent detectable substances are luciferin, luciferase and aequorin.

[0319] Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against one or more head-and-neck cancer markers. By way of example, if the antibody having specificity against one or more OPL or head-and-neck marker is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.

[0320] Methods for conjugating or labelling the antibodies discussed above may be readily accomplished by one of ordinary skill in the art. (See, for example, Inman, Methods In Enzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B, Jakoby and Wichek (eds.), Academic Press, New York, p. 30, 1974; and Wilchek and Bayer, "The Avidin-Biotin Complex in Bioanalytical Applications", Anal. Biochem. 171:1-32, 1988 re methods for conjugating or labelling the antibodies with enzyme or ligand binding partner.)

[0321] Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect one or more head-and-neck cancer markers. Generally, antibodies may be labeled with detectable substances and one or more head-and-neck cancer markers may be localised in tissues and cells based upon the presence of the detectable substances.

[0322] In the context of the methods of the invention, the sample, binding agents (e.g., antibodies specific for one or more OPL or head-and-neck cancer markers), or one or more OPL or head-and-neck cancer markers may be immobilized on a carrier or support. Examples of suitable carriers or supports are agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene, gabbros, filter paper, magnetite, ion-exchange resin, plastic film, plastic tube, glass, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The support material may have any possible configuration including spherical (e.g., bead), cylindrical (e.g., inside surface of a test tube or well, or the external surface of a rod), or flat (e.g., sheet, test strip). Thus, the carrier may be in the shape of, for example, a tube, test plate, well, beads, disc, sphere, etc. The immobilized antibody may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling. An antibody may be indirectly immobilized using a second antibody specific for the antibody. For example, mouse antibody specific for a head-and-neck marker may be immobilized using sheep anti-mouse IgG Fc fragment specific antibody coated on the carrier or support.

[0323] Where a radioactive label is used as a detectable substance, one or more OPL or head-and-neck marker may be localized by radioautography. The results of radioautography may be quantified by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.

[0324] Time-resolved fluorometry may be used to detect a signal. For example, the method described by Christopoulos T K and Diamandis E P in Anal Chem 1992:64:342-346 may be used with a conventional time-resolved fluorometer.

[0325] In accordance with an embodiment of the invention, a method is provided wherein one or more OPL or head-and-neck marker antibodies are directly or indirectly labelled with enzymes, substrates for the enzymes are added wherein the substrates are selected so that the substrates, or a reaction product of an enzyme and substrate, form fluorescent complexes with a lanthanide metal (e.g., europium, terbium, samarium, and dysprosium, preferably europium and terbium). A lanthanide metal is added and one or more OPL or head-and-neck cancer markers are quantified in the sample by measuring fluorescence of the fluorescent complexes. Enzymes are selected based on the ability of a substrate of the enzyme, or a reaction product of the enzyme and substrate, to complex with lanthanide metals such as europium and terbium. Suitable enzymes and substrates that provide fluorescent complexes are described in U.S. Pat. No. 5,3112,922 to Diamandis. Examples of suitable enzymes include alkaline phosphatase and .beta.-galactosidase. Preferably, the enzyme is alkaline phosphatase.

[0326] Examples of enzymes and substrates for enzymes that provide such fluorescent complexes are described in U.S. Pat. No. 5,312,922 to Diamandis. By way of example, when the antibody is directly or indirectly labelled with alkaline phosphatase the substrate employed in the method may be 4-methylumbelliferyl phosphate, 5-fluorosalicyl phosphate, or diflunisal phosphate. The fluorescence intensity of the complexes is typically measured using a time-resolved fluorometer (e.g., a CyberFluor 615 Immunoanalyzer (Nordion International, Kanata, Ontario)).

[0327] One or more OPL or head-and-neck marker antibodies may also be indirectly labelled with an enzyme. For example, the antibodies may be conjugated to one partner of a ligand binding pair, and the enzyme may be coupled to the other partner of the ligand binding pair. Representative examples include avidin-biotin, and riboflavin-riboflavin binding protein. In an embodiment, the antibodies are biotinylated, and the enzyme is coupled to streptavidin. In another embodiment, an antibody specific for OPL or head-and-neck marker antibody is labeled with an enzyme.

[0328] In accordance with an embodiment, the present invention provides means for determining one or more OPL or head-and-neck cancer markers in a sample by measuring one or more head-and-neck cancer markers by immunoassay. It will be evident to a skilled artisan that a variety of immunoassay methods can be used to measure one or more head-and-neck cancer markers. In general, an immunoassay method may be competitive or non-competitive. Competitive methods typically employ an immobilized or immobilizable antibody to one or more OPL or head-and-neck marker and a labeled form of one or more OPL or head-and-neck marker. Sample OPL or head-and-neck cancer markers and labeled OPL or head-and-neck cancer markers compete for binding to antibodies to OPL or head-and-neck cancer markers. After separation of the resulting labeled OPL or head-and-neck cancer markers that have become bound to antibodies (bound fraction) from that which has remained unbound (unbound fraction), the amount of the label in either bound or unbound fraction is measured and may be correlated with the amount of OPL or head-and-neck cancer markers in the test sample in any conventional manner (e.g., by comparison to a standard curve).

[0329] In an aspect, a non-competitive method is used for the determination of one or more OPL or head-and-neck cancer markers, with the most common method being the "sandwich" method. In this assay, two antibodies to OPL or head-and-neck cancer markers are employed. One of the antibodies to OPL or head-and-neck cancer markers is directly or indirectly labeled (sometimes referred to as the "detection antibody") and the other is immobilized or immobilizable (sometimes referred to as the "capture antibody"). The capture and detection antibodies can be contacted simultaneously or sequentially with the test sample. Sequential methods can be accomplished by incubating the capture antibody with the sample, and adding the detection antibody at a predetermined time thereafter (sometimes referred to as the "forward" method); or the detection antibody can be incubated with the sample first and then the capture antibody added (sometimes referred to as the "reverse" method). After the necessary incubation(s) have occurred, to complete the assay, the capture antibody is separated from the liquid test mixture, and the label is measured in at least a portion of the separated capture antibody phase or the remainder of the liquid test mixture. Generally, it is measured in the capture antibody phase since it includes OPL or head-and-neck cancer markers bound by ("sandwiched" between) the capture and detection antibodies. In an embodiment, the label may be measured without separating the capture antibodies and liquid test mixture.

[0330] In a typical two-site immunometric assay for OPL or head-and-neck cancer markers, one or both of the capture and detection antibodies are polyclonal antibodies or one or both of the capture and detection antibodies are monoclonal antibodies (i.e. polyclonal/polyclonal, monoclonal/monoclonal, or monoclonal/polyclonal). The label used in the detection antibody can be selected from any of those known conventionally in the art. The label may be an enzyme or a chemiluminescent moiety, but it can also be a radioactive isotope, a fluorophor, a detectable ligand (e.g., detectable by a secondary binding by a labeled binding partner for the ligand), and the like. In a particular aspect, the antibody is labelled with an enzyme which is detected by adding a substrate that is selected so that a reaction product of the enzyme and substrate forms fluorescent complexes. The capture antibody may be selected so that it provides a means for being separated from the remainder of the test mixture. Accordingly, the capture antibody can be introduced to the assay in an already immobilized or insoluble form, or can be in an immobilizable form, that is, a form which enables immobilization to be accomplished subsequent to introduction of the capture antibody to the assay. An immobilized capture antibody may include an antibody covalently or non-covalently attached to a solid phase such as a magnetic particle, a latex particle, a microtiter plate well, a bead, a cuvette, or other reaction vessel. An example of an immobilizable capture antibody is antibody which has been chemically modified with a ligand moiety, e.g., a hapten, biotin, or the like, and which can be subsequently immobilized by contact with an immobilized form of a binding partner for the ligand, e.g., an antibody, avidin, or the like. In an embodiment, the capture antibody may be immobilized using a species specific antibody for the capture antibody that is bound to the solid phase.

[0331] The above-described immunoassay methods and formats are intended to be exemplary and are not limiting.

[0332] Computer Systems.

[0333] Analytic methods contemplated herein can be implemented by use of computer systems and methods described below and known in the art. Thus, the invention provides computer readable media including one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers (e.g., markers of OPL or head-and-neck cancer). "Computer readable media" refers to any medium that can be read and accessed directly by a computer, including but not limited to magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. Thus, the invention contemplates computer readable medium having recorded thereon markers identified for patients and controls.

[0334] "Recorded" refers to a process for storing information on computer readable medium. The skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures including information on one or more OPL or head-and-neck cancer markers, and optionally other markers.

[0335] A variety of data processor programs and formats can be used to store information on one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and other markers on computer readable medium. For example, the information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and MicroSoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. Any number of dataprocessor structuring formats (e.g., text file or database) may be adapted in order to obtain computer readable medium having recorded thereon the marker information.

[0336] By providing the marker information in computer readable form, one can routinely access the information for a variety of purposes. For example, one skilled in the art can use the information in computer readable form to compare marker information obtained during or following therapy with the information stored within the data storage means.

[0337] The invention provides a medium for holding instructions for performing a method for determining or whether a patient has a head-and-neck disease (e.g., head-and-neck cancer) or a pre-disposition to a head-and-neck disease (e.g., cancer), including determining the presence or absence of one or more head-and-neck cancer markers, and/or polynucleotides encoding one or more head-and-neck cancer markers, and optionally other markers, and based on the presence or absence of the one or more head-and-neck cancer markers, and/or polynucleotides encoding one or more head-and-neck cancer markers, and optionally other markers, determining uterine head-and-neck receptivity, head-and-neck disease (e.g., cancer) or a pre-disposition to a head-and-neck disease (e.g., cancer), and optionally recommending a procedure or treatment.

[0338] The invention also provides in an electronic system and/or in a network, a method for determining whether a subject has a head-and-neck disease (e.g., OPL or cancer) or a pre-disposition to a head-and-neck disease (e.g., OPL or cancer), including determining the presence or absence of one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers (e.g., OPL or cancer markers), and based on the presence or absence of the one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers, determining whether the subject has a head-and-neck disease (e.g., OPL or cancer) or a pre-disposition to a head-and-neck disease (e.g., OPL or cancer), and optionally recommending a procedure or treatment.

[0339] The invention further provides in a network, a method for determining whether a subject is receptive to in vitro fertilization, has a head-and-neck disease (e.g., OPL or cancer) or a pre-disposition to a head-and-neck disease (e.g., OPL or cancer) including: (a) receiving phenotypic information on the subject and information on one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers associated with samples from the subject; (b) acquiring information from the network corresponding to the one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers; and (c) based on the phenotypic information and information on the one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers, determining whether the subject is receptive to in vitro fertilization, has a head-and-neck disease (e.g., OPL or cancer) or a pre-disposition to a head-and-neck disease (e.g., OPL or cancer); and (d) optionally recommending a procedure or treatment.

[0340] The invention still further provides a system for identifying selected records that identify a diseased head-and-neck cell or tissue (e.g., premalignant cell or tissue and/or cancer cell or tissue) or an head and neck cell or tissue phase. A system of the invention generally includes a digital computer; a database server coupled to the computer; a database coupled to the database server having data stored therein, the data including records of data including one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other OPL or head-and-neck cancer markers, and a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records which match the desired selection criteria.

[0341] In an aspect of the invention a method is provided for detecting OPL or head-and-neck cancer tissue or cells using a computer having a processor, memory, display, and input/output devices, the method including the steps of: [0342] (a) creating records of one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers of OPL or cancer identified in a sample suspected of containing head-and-neck precancer or cancer cells or tissue; [0343] (b) providing a database including records of data including one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally other markers of Premalignant lesions or cancer; and [0344] (c) using a code mechanism for applying queries based upon a desired selection criteria to the data file in the database to produce reports of records of step (a) which provide a match of the desired selection criteria of the database of step (b) the presence of a match being a positive indication that the markers of step (a) have been isolated from cells or tissue that are head-and-neck precancer or cancer cells or oral leukoplakia or head-and-neck cancer tissue.

[0345] The invention contemplates a business method for determining whether a subject is receptive to in vitro fertilization, has a head-and-neck disease (e.g., OPL or cancer) or a pre-disposition to OPL or head-and-neck cancer including: (a) receiving phenotypic information on the subject and information on one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding the markers, and optionally other markers, associated with samples from the subject; (b) acquiring information from a network corresponding to one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding the markers, and optionally other markers; and (c) based on the phenotypic information, information on one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding the markers, and optionally other markers, and acquired information, determining whether the subject is receptive to in vitro fertilization, has a head-and-neck disease (e.g., OPL or cancer) or a pre-disposition to a head-and-neck disease (e.g., OPL or cancer); and (d) optionally recommending a procedure or treatment.

[0346] In an aspect of the invention, the computer systems, components, and methods described herein are used to monitor disease or determine the stage of disease.

[0347] Imaging Methods.

[0348] Binding agents, particularly antibodies, specific for one or more OPL or head-and-neck cancer markers may also be used in imaging methodologies in the management of a head-and-neck disease.

[0349] In an aspect, the invention provides a method for imaging oral leukoplakia with one or more OPL markers and tumors associated with one or more head-and-neck cancer markers.

[0350] The invention also contemplates imaging methods described herein using multiple markers for a head-and-neck disease. Preferably, each agent is labeled so that it can be distinguished during the imaging.

[0351] In an embodiment the method is an in vivo method and a subject or patient is administered one or more agents that carry an imaging label and that are capable of targeting or binding to one or more OPL or head-and-neck cancer markers. The agent is allowed to incubate in vivo and bind to the OPL or head-and-neck cancer markers associated with head-and-neck cells or tissues of a particular phase or associated with diseased cells or tissues, (e.g., an OPL or a head-and-neck tumor). The presence of the label is localized to the head-and-neck cells or tissues, and the localized label is detected using imaging devices known to those skilled in the art.

[0352] The agent may be an antibody or chemical entity that recognizes the OPL or head-and-neck cancer markers. In an aspect of the invention the agent is a polyclonal antibody or monoclonal antibody, or fragments thereof, or constructs thereof including but not limited to, single chain antibodies, bifunctional antibodies, molecular recognition units, and peptides or entities that mimic peptides. The antibodies specific for the head-and-neck cancer markers used in the methods of the invention may be obtained from scientific or commercial sources, or isolated native OPL or head-and-neck cancer markers or recombinant OPL or head-and-neck cancer markers may be utilized to prepare antibodies etc. as described herein.

[0353] An agent may be a peptide that mimics the epitope for an antibody specific for an OPL or head-and-neck marker and binds to the marker. The peptide may be produced on a commercial synthesizer using conventional solid phase chemistry. By way of example, a peptide may be prepared that includes either tyrosine, lysine, or phenylalanine to which N.sub.2S.sub.2 chelate is complexed (see U.S. Pat. No. 4,897,255). An anti-endocrine marker peptide conjugate is then combined with a radiolabel (e.g., sodium .sup.99mTc pertechnetate or sodium .sup.188Re perrhenate) and it may be used to locate a head-and-neck marker producing cell or tissue (e.g., tumor).

[0354] The agent carries a label to image the OPL or head-and-neck cancer markers. The agent may be labelled for use in radionuclide imaging. In particular, the agent may be directly or indirectly labelled with a radioisotope. Examples of radioisotopes that may be used in the resent invention are the following: .sup.277Ac, .sup.211At, .sup.128Ba, .sup.131Ba, .sup.7Be, .sup.204Bi, .sup.205Bi, .sup.206Bi, .sup.76Br, .sup.77Br, .sup.82Br, .sup.109Cd, .sup.47Ca, .sup.11C, .sup.14C, .sup.36Cl, .sup.48Cr, .sup.51Cr, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.165Dy, .sup.155Eu, .sup.18F, .sup.153Gd, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.72Ga, .sup.198Au, .sup.3H .sup.166Ho, .sup.111In, .sup.113mIn, .sup.115mIn, .sup.123I, .sup.125I, .sup.131I, .sup.189Ir, .sup.191mIr, .sup.192Ir, .sup.194Ir, .sup.52Fe, .sup.55Fe, .sup.59Fe, .sup.177Lu, .sup.15O, .sup.191m-191Os, .sup.109Pd, .sup.32P, .sup.33P, .sup.42K, .sup.226Ra, .sup.186Re, .sup.188Re, .sup.82mRb, .sup.153Sm, .sup.46Sc, .sup.47Sc, .sup.72Se, .sup.75Se, .sup.105Ag, .sup.22Na, .sup.24Na, .sup.89Sr, .sup.35s, .sup.38s, .sup.177Ta, .sup.96Tc, .sup.99mTc, .sup.201Tl, .sup.202Tl, .sup.113Sn, .sup.117mSn, .sup.121Sn, .sup.166Yb, .sup.169Yb, .sup.175Yb, .sup.88Y, .sup.90Y, .sup.62Zn and .sup.65Zn. Preferably the radioisotope is .sup.131I, .sup.125I, .sup.123I, .sup.111I, .sup.99mTc, .sup.90Y, .sup.186Re, .sup.188Re, .sup.32P, .sup.153Sm, .sup.67Ga, .sup.201Tl, .sup.77Br, or .sup.18F, and is imaged with a photoscanning device.

[0355] Procedures for labeling biological agents with the radioactive isotopes are generally known in the art. U.S. Pat. No. 4,302,438 describes tritium labeling procedures. Procedures for iodinating, tritium labeling, and .sup.35S labeling especially adapted for murine monoclonal antibodies are described by Goding, J. W. (supra, pp. 124-126) and the references cited therein. Other procedures for iodinating biological agents, such as antibodies, binding portions thereof, probes, or ligands, are described in the scientific literature (see Hunter and Greenwood, Nature 144:945 (1962); David et al., Biochemistry 13:1014-1021 (1974); and U.S. Pat. Nos. 3,867,517 and 4,376,110). Iodinating procedures for agents are described by Greenwood, F. et al., Biochem. J. 89:114-123 (1963); Marchalonis, J., Biochem. J. 113:299-305 (1969); and Morrison, M. et al., Immunochemistry, 289-297 (1971). .sup.99m Tc-labeling procedures are described by Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: The Radioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982) and the references cited therein. Labelling of antibodies or fragments with technetium-.sup.99m are also described for example in U.S. Pat. Nos. 5,317,091; 4,478,815; 4,478,818; 4,472,371; Re 32,417; and 4,311,688. Procedures suitable for .sup.111In-labeling biological agents are described by Hnatowich, D. J. et al., J. Immun. Methods, 65:147-157 (1983), Hnatowich, D. et al., J. Applied Radiation, 35:554-557 (1984), and Buckley, R. G. et al., F.E.B.S. 166:202-204 (1984).

[0356] An agent may also be labeled with a paramagnetic isotope for purposes of an in vivo method of the invention. Examples of elements that are useful in magnetic resonance imaging include gadolinium, terbium, tin, iron, or isotopes thereof (See, for example, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986) Magn. Reson. Med. 3, 336-340; Wolf, G L., (1984) Physiol. Chem. Phys. Med. NMR 16, 93-95; Wesbey et al. (1984), Physiol. Chem. Phys. Med. NMR 16, 145-155; Runge et al. (1984), Invest. Radiol. 19, 408-415 for discussions on in vivo nuclear magnetic resonance imaging.)

[0357] In the case of a radiolabeled agent, the agent may be administered to the patient, it is localized to the cell or tissue (e.g., tumor) having a head-and-neck marker with which the agent binds, and is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography. (See, for example, A. R. Bradwell et al., "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer Detection and Therapy; R. W. Baldwin et al. (eds.), pp. 65-85 (Academic Press 1985).) A positron emission transaxial tomography scanner, such as designated Pet VI located at Brookhaven National Laboratory, can also be used where the radiolabel emits positrons (e.g., .sup.11C, .sup.18F, .sup.15O, and .sup.13N).

[0358] Whole body imaging techniques using radioisotope labeled agents can be used for locating diseased cells and tissues (e.g., primary tumors and tumors which have metastasized). Antibodies specific for OPL or head-and-neck cancer markers, or fragments thereof having the same epitope specificity, are bound to a suitable radioisotope, or a combination thereof, and administered parenterally. For OPL or head-and-neck cancer, administration preferably is intravenous. The bio-distribution of the label can be monitored by scintigraphy, and accumulations of the label are related to the presence of OPL or head-and-neck cancer cells. Whole body imaging techniques are described in U.S. Pat. Nos. 4,036,945 and 4,311,688. Other examples of agents useful for diagnosis and therapeutic use that can be coupled to antibodies and antibody fragments include metallothionein and fragments (see U.S. Pat. No. 4,732,864). These agents are useful in diagnosis staging and visualization of cancer, in particular OPL or head-and-neck cancer, so that surgical and/or radiation treatment protocols can be used more efficiently.

[0359] An imaging agent may carry a bioluminescent or chemiluminescent label. Such labels include polypeptides known to be fluorescent, bioluminescent or chemiluminescent, or, that act as enzymes on a specific substrate (reagent), or can generate a fluorescent, bioluminescent or chemiluminescent molecule. Examples of bioluminescent or chemiluminescent labels include luciferases, aequorin, obelin, mnemiopsin, berovin, a phenanthridinium ester, and variations thereof and combinations thereof. A substrate for the bioluminescent or chemiluminescent polypeptide may also be utilized in a method of the invention. For example, the chemiluminescent polypeptide can be luciferase and the reagent luciferin. A substrate for a bioluminescent or chemiluminescent label can be administered before, at the same time (e.g., in the same formulation), or after administration of the agent.

[0360] An imaging agent may include a paramagnetic compound, such as a polypeptide chelated to a metal (e.g., a metalloporphyrin). The paramagnetic compound may also include a monocrystalline nanoparticle, e.g., a nanoparticle including a lanthanide (e.g., Gd) or iron oxide; or, a metal ion such as a lanthanide. As used herein, "lanthanide" refers to elements of atomic numbers 58 to 70, a transition metal of atomic numbers 21 to 29, 42 or 44, a Gd(III), a Mn(II), or an element including a Fe element. Paramagnetic compounds can also include a neodymium iron oxide (NdFeO.sub.3) or a dysprosium iron oxide (DyFeO.sub.3). Examples of elements that are useful in magnetic resonance imaging include gadolinium, terbium, tin, iron, or isotopes thereof (See, for example, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al. (1986), Magn. Reson. Med. 3, 336-340; Wolf, G. L. (1984), Physiol. Chem. Phys. Med. NMR 16, 93-95; Wesbey et al. (1984), Physiol. Chem. Phys. Med. NMR 16, 145-155; Runge et al. (1984), Invest. Radiol. 19, 408-415 for discussions on in vivo nuclear magnetic resonance imaging.)

[0361] An image can be generated in a method of the invention by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS) image, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI) or equivalent.

[0362] Computer assisted tomography (CAT) and computerized axial tomography (CAT) systems and devices well known in the art can be utilized in the practice of the present invention. (See, for example, U.S. Pat. Nos. 6,151,377; 5,946,371; 5,446,799; 5,406,479; 5,208,581; and 5,109, 97.) The invention may also utilize animal imaging modalities, such as MicroCAT.TM. (ImTek, Inc.).

[0363] Magnetic resonance imaging (MRI) systems and devices well known in the art can be utilized in the practice of the present invention. For a description of MRI methods and devices, see, for example, U.S. Pat. Nos. 6,151,377; 6,144,202; 6,128,522; 6,127,825; 6,121,775; 6,119,032; 6,115,446; 6,111,410; 602,891; 5,555,251; 5,455,512; 5,450,010; 5,378,987; 5,214,382; 5,031,624; 5,207,222; 4,985,678; 4,906,931; and 4,558,279. MRI and supporting devices are commercially available, for example, from Bruker Medical GMBH; Caprius; Esaote Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; Intermagnetics General Corporation; Lunar Corp.; MagneVu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems; including imaging systems, by, e.g., Silicon Graphics. The invention may also utilize animal imaging modalities such as micro-MRIs.

[0364] Positron emission tomography imaging (PET) systems and devices well known in the art can be utilized in the practice of the present invention. For example, a method of the invention may use the system designated Pet VI located at Brookhaven National Laboratory. For descriptions of PET systems and devices, see, for example, U.S. Pat. Nos. 6,151,377; 6,072,177; 5,900,636; 5,608,221; 5,532,489; 5,272,343; and 5,103,098. Animal imaging modalities such as micro-PETs (Concorde Microsystems, Inc.) can also be used in the invention.

[0365] Single-photon emission computed tomography (SPECT) systems and devices well known in the art can be utilized in the practice of the present invention. (See, for example, U.S. Pat. Nos. 6,115,446; 6,072,177; 5,608,221; 5,600,145; 5,210,421; 5,103,098.) The methods of the invention may also utilize animal imaging modalities, such as micro-SPECTs.

[0366] Bioluminescence imaging includes bioluminescence, fluorescence, and chemiluminescence and other photon detection systems and devices that are capable of detecting bioluminescence, fluorescence, or chemiluminescence. Sensitive photon detection systems can be used to detect bioluminescent and fluorescent proteins externally; see, for example, Contag (2000), Neoplasia 2:41-52; adn Zhang (1994), Clin. Exp. Metastasis, 12:87-92. The methods of the invention can be practiced using any such photon detection device, or variation or equivalent thereof, or in conjunction with any known photon detection methodology, including visual imaging. By way of example, an intensified charge-coupled device (ICCD) camera coupled to an image processor may be used in the present invention. (See, e.g., U.S. Pat. No. 5,650,135.) Photon detection devices are also commercially available from Xenogen, Hamamatsue.

[0367] Screening Methods.

[0368] The invention also contemplates methods for evaluating test agents or compounds for their ability to inhibit a head-and-neck disease (e.g., OPL or cancer), potentially contribute to a head-and-neck disease (e.g., OPL or cancer). Test agents and compounds include but are not limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments (e.g., Fab, F(ab).sub.2, and Fab expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules. The agents or compounds may be endogenous physiological compounds or natural or synthetic compounds.

[0369] The invention provides a method for assessing the potential efficacy of a test agent for inhibiting a head-and-neck disease (e.g., OPL or cancer) in a patient, the method including comparing: [0370] (a) levels of one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers in a first sample obtained from a patient and exposed to the test agent; and [0371] (b) levels of one or more OPL or head-and-neck cancer markers and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers, in a second sample obtained from the patient, wherein the sample is not exposed to the test agent, wherein a significant difference in the levels of expression of one or more head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers, and optionally the other markers, in the first sample, relative to the second sample, is an indication that the test agent is potentially efficacious for inhibiting a head-and-neck disease (e.g., OPL or cancer) in the patient.

[0372] The first and second samples may be portions of a single sample obtained from a patient or portions of pooled samples obtained from a patient.

[0373] In an aspect, the invention provides a method of selecting an agent for inhibiting a head-and-neck disease (e.g., OPL or cancer) in a patient including: [0374] (a) obtaining a sample from the patient; [0375] (b) separately maintaining aliquots of the sample in the presence of a plurality of test agents; [0376] (c) comparing one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers, in each of the aliquots; and [0377] (d) selecting one of the test agents which alters the levels of one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers in the aliquot containing that test agent, relative to other test agents.

[0378] In a further aspect, the invention provides a method of selecting an agent for inhibiting or enhancing a OPL or head and neck cell or tissue phase in a patient including: [0379] (a) obtaining a sample of OPL or head and neck cell or tissue in a selected phase; [0380] (b) separately maintaining aliquots of the sample in the presence of a plurality of test agents; [0381] (c) comparing one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers, in each of the aliquots; and [0382] (d) selecting one of the test agents which alters the levels of one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers in the aliquot containing that test agent, relative to other test agents.

[0383] Still another aspect of the present invention provides a method of conducting a drug discovery business including: [0384] (a) providing one or more methods or assay systems for identifying agents that inhibit a head-and-neck disease (e.g., OPL or head-and-neck cancer) or affect a OPL or head and neck cell or tissues phase in a patient; [0385] (b) conducting therapeutic profiling of agents identified in step (a), or further analogs thereof, for efficacy and toxicity in animals; and [0386] (c) formulating a pharmaceutical preparation including one or more agents identified in step (b) as having an acceptable therapeutic profile.

[0387] In certain embodiments, the subject method can also include a step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.

[0388] The invention also contemplates a method of assessing the potential of a test compound to contribute to a head-and-neck disease (e.g., OPL or head-and-neck cancer) including: [0389] (a) maintaining separate aliquots of cells or tissues from a patient with a head-and-neck disease (e.g., OPL or cancer) in the presence and absence of the test compound; and [0390] (b) comparing one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding OPL or head-and-neck cancer markers, and optionally other markers in each of the aliquots.

[0391] A significant difference between the levels of the markers in the aliquot maintained in the presence of (or exposed to) the test compound relative to the aliquot maintained in the absence of the test compound, indicates that the test compound possesses the potential to contribute to a head-and-neck disease (e.g., OPL or head-and-neck cancer).

[0392] Kits.

[0393] The invention also contemplates kits for carrying out the methods of the invention. Kits may typically include two or more components required for performing a diagnostic assay. Components include but are not limited to compounds, reagents, containers, and/or equipment.

[0394] The methods described herein may be performed by utilizing pre-packaged diagnostic kits including one or more specific OPL or head-and-neck marker polynucleotide or antibody described herein, which may be conveniently used, e.g., in clinical settings to screen and diagnose patients and to screen and identify those individuals exhibiting a predisposition to developing a head-and-neck disease.

[0395] In an embodiment, a container with a kit includes a binding agent as described herein. By way of example, the kit may contain antibodies or antibody fragments which bind specifically to epitopes of one or more OPL or head-and-neck cancer markers and optionally other markers, antibodies against the antibodies labelled with an enzyme; and a substrate for the enzyme. The kit may also contain microtiter plate wells, standards, assay diluent, wash buffer, adhesive plate covers, and/or instructions for carrying out a method of the invention using the kit.

[0396] In an aspect of the invention, the kit includes antibodies or fragments of antibodies which bind specifically to an epitope of one or more polypeptide listed in Table 1 that is upregulated in cancer samples as compared to normal samples, or those listed in Table 5 or Table 2 and means for detecting binding of the antibodies to their epitope associated with tumor cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to use or at the concentration of use, where the vials may include one or more dosages. Where the kits are intended for in vivo use, single dosages may be provided in sterilized containers, having the desired amount and concentration of agents. Containers that provide a formulation for direct use, usually do not require other reagents, as for example, where the kit contains a radiolabelled antibody preparation for in vivo imaging.

[0397] In an aspect of the invention, the kit includes antibodies or fragments of antibodies which bind specifically to an epitope of one or more polypeptide listed in Table 6 that is upregulated in OPL samples as compared to normal samples, or those listed in Table 5 or Table 7 and means for detecting binding of the antibodies to their epitope associated with OPL cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to use or at the concentration of use, where the vials may include one or more dosages. Where the kits are intended for in vivo use, single dosages may be provided in sterilized containers, having the desired amount and concentration of agents. Containers that provide a formulation for direct use, usually do not require other reagents, as for example, where the kit contains a radiolabelled antibody preparation for in vivo imaging.

[0398] A kit may be designed to detect the level of polynucleotides encoding one or more OPL or head-and-neck cancer polynucleotide markers in a sample. In an embodiment, the polynucleotides encode one or more polynucleotides encoding a polypeptide listed in Table 6 that is upregulated in OPL samples as compared to normal samples, or those listed in Table 5 or Table 7 or the polynucleotides encode one or more polynucleotides encoding a polypeptide listed in Table 1 that is upregulated in cancer samples as compared to normal samples, or those listed in Table 5 or Table 2. Such kits generally include at least one oligonucleotide probe or primer, as described herein, that hybridizes to a polynucleotide encoding one or more OPL or head-and-neck cancer markers. Such an oligonucleotide may be used, for example, within a PCR or hybridization procedure. Additional components that may be present within the kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate detection of a polynucleotide encoding one or more OPL or head-and-neck cancer markers.

[0399] The invention provides a kit containing a microarray described herein ready for hybridization to target OPL or head-and-neck cancer polynucleotide markers, plus software for the data analysis of the results. The software to be included with the kit includes data analysis methods, in particular mathematical routines for marker discovery, including the calculation of correlation coefficients between clinical categories and marker expression. The software may also include mathematical routines for calculating the correlation between sample marker expression and control marker expression, using array-generated fluorescence data, to determine the clinical classification of the sample.

[0400] The reagents suitable for applying the screening methods of the invention to evaluate compounds may be packaged into convenient kits described herein providing the necessary materials packaged into suitable containers.

[0401] The invention contemplates a kit for assessing the presence of head-and-neck cells, wherein the kit includes antibodies specific for one or more OPL or head-and-neck cancer markers, or primers or probes for polynucleotides encoding same, and optionally probes, primers or antibodies specific for other markers associated with a head-and-neck disease (e.g., OPL or cancer).

[0402] The invention relates to a kit for assessing the suitability of each of a plurality of test compounds for inhibiting a head-and-neck disease (e.g., OPL or head-and-neck cancer) in a patient. The kit includes reagents for assessing one or more OPL or head-and-neck cancer markers or polynucleotides encoding same, and optionally a plurality of test agents or compounds.

[0403] Additionally the invention provides a kit for assessing the potential of a test compound to contribute to a head-and-neck disease (e.g., OPL or cancer). The kit includes head-and-neck diseased cells (e.g., OPL or cancer cells) and reagents for assessing one or more OPL or head-and-neck cancer markers, polynucleotides encoding same, and optionally other markers associated with a head-and-neck disease.

[0404] Therapeutic Applications.

[0405] One or more OPL or head-and-neck cancer markers may be targets for immunotherapy. Immunotherapeutic methods include the use of antibody therapy, in vivo vaccines, and ex vivo immunotherapy approaches.

[0406] In one aspect, the invention provides one or more OPL or head-and-neck marker antibodies that may be used systemically to treat a head-and-neck disease associated with the marker. In particular, the head-and-neck disease is OPL or head-and-neck cancer and one or more head-and-neck marker antibodies may be used systemically to treat OPL or head-and-neck cancer. Preferably antibodies are used that target the tumor cells but not the surrounding non-tumor cells and tissue.

[0407] Thus, the invention provides a method of treating a patient susceptible to, or having a disease (e.g., OPL or cancer) that expresses one or more OPL or head-and-neck marker, in particular, a marker up-regulated in OPL or head-and-neck cancer (for example, an up-regulated marker in Table 6, or that of Table 5 and/or 7 or an up-regulated marker in Table 1, or that of Table 5 and/or 2), including administering to the patient an effective amount of an antibody that binds specifically to one or more OPL or head-and-neck marker.

[0408] In another aspect, the invention provides a method of inhibiting the growth of OPL or tumor cells expressing one or more OPL or head-and-neck cancer markers, including administering to a patient an antibody which binds specifically to one or more OPL or head-and-neck cancer markers in an amount effective to inhibit growth of the tumor cells.

[0409] One or more OPL or head-and-neck marker antibodies may also be used in a method for selectively inhibiting the growth of, or killing a cell expressing one or more OPL marker (e.g., OPL cell expressing one or more OPL marker) or head-and-neck marker (e.g., tumor cell expressing one or more head-and-neck cancer marker) including reacting one or more head-and-neck marker antibody immunoconjugate or immunotoxin with the cell in an amount sufficient to inhibit the growth of, or kill the cell.

[0410] By way of example, unconjugated antibodies to OPL or head-and-neck cancer markers may be introduced into a patient such that the antibodies bind to OPL or head-and-neck cancer marker expressing cancer cells and mediate growth inhibition of such cells (including the destruction thereof), and the tumor, by mechanisms which may include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, altering the physiologic function of one or more OPL or head-and-neck cancer markers, and/or the inhibition of ligand binding or signal transduction pathways. In addition to unconjugated antibodies to OPL or head-and-neck cancer markers, one or more OPL or head-and-neck cancer marker antibodies conjugated to therapeutic agents (e.g., immunoconjugates) may also be used therapeutically to deliver the agent directly to one or more OPL or head-and-neck cancer marker expressing tumor cells and thereby destroy the tumor. Examples of such agents include abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; and biological response modifiers such as lymphokines, interleukin-1, interleukin-2, interleukin-6, granulocyte macrophage colony stimulating factor, granulocyte colony stimulating factor, or other growth factors.

[0411] Cancer immunotherapy using one or more OPL or head-and-neck cancer marker antibodies may utilize the various approaches that have been successfully employed for cancers, including but not limited to colon cancer (Arlen et al., 1998, Crit Rev Immunol 18: 133-138), multiple myeloma (Ozaki et al., 1997, Blood 90: 3179-3186; Tsunenati et al., 1997, Blood 90: 2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res 52: 2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunther Emphasis Tumor Immunol 19: 93-101), leukemia (Zhong et al., 1996, Leuk Res 20: 581-589), colorectal cancer (Moun et al., 1994, Cancer Res 54: 6160-6166); Velders et al., 1995, Cancer Res 55: 4398-4403), and breast cancer (Shepard et al., 1991, J. Clin Immunol 11: 117-127).

[0412] In the practice of a method of the invention, OPL or head-and-neck cancer marker antibodies capable of inhibiting the growth of precancer or cancer cells expressing OPL or head-and-neck cancer markers are administered in a therapeutically effective amount to OPL or cancer patients whose lesions or tumors express or overexpress one or more OPL or head-and-neck cancer markers. The invention may provide a specific, effective and long-needed treatment for OPL or head-and-neck cancer. The antibody therapy methods of the invention may be combined with other therapies including chemotherapy and radiation.

[0413] Patients may be evaluated for the presence and level of expression or overexpression of one or more OPL or head-and-neck cancer markers in diseased cells and tissues (e.g., OPLs or tumors), in particular using immunohistochemical assessments of tissue, quantitative imaging as described herein, or other techniques capable of reliably indicating the presence and degree of expression of one or more OPL or head-and-neck cancer markers. Immunohistochemical analysis of OPL or tumor biopsies or surgical specimens may be employed for this purpose.

[0414] Head-and-neck marker antibodies useful in treating disease (e.g., OPL or cancer) include those that are capable of initiating a potent immune response against the disease (e.g., OPL or tumor) and those that are capable of direct cytotoxicity. In this regard, OPL or head-and-neck marker antibodies may elicit cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites or complement proteins.

[0415] Oral premalignant lesions or head-and-neck marker antibodies that exert a direct biological effect on tumor growth may also be useful in the practice of the invention. Such antibodies may not require the complete immunoglobulin to exert the effect. Potential mechanisms by which such directly cytotoxic antibodies may act include inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism by which a particular antibody exerts an anti-tumor effect may be evaluated using any number of in vitro assays designed to determine ADCC, antibody-dependent macrophage-mediated cytotoxicity (ADMMC), complement-mediated cell lysis, and others known in the art.

[0416] The anti-tumor activity of a particular head-and-neck cancer marker antibody, or combination of head-and-neck cancer marker antibodies, may be evaluated in vivo using a suitable animal model. Xenogenic cancer models, where human cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice, may be employed.

[0417] The methods of the invention contemplate the administration of single head-and-neck marker antibodies as well as combinations, or "cocktails", of different individual antibodies such as those recognizing different epitopes of other markers. Such cocktails may have certain advantages inasmuch as they contain antibodies that bind to different epitopes of head-and-neck cancer markers and/or exploit different effector mechanisms or combine directly cytotoxic antibodies with antibodies that rely on immune effector functionality. Such antibodies in combination may exhibit synergistic therapeutic effects. In addition, the administration of one or more head-and-neck marker specific antibodies may be combined with other therapeutic agents, including but not limited to chemotherapeutic agents, androgen-blockers, and immune modulators (e.g., IL2, GM-CSF). The head-and-neck marker specific antibodies may be administered in their "naked" or unconjugated form, or may have therapeutic agents conjugated to them.

[0418] The head-and-neck marker specific antibodies used in the methods of the invention may be formulated into pharmaceutical compositions including a carrier suitable for the desired delivery method. Suitable carriers include any material which when combined with the antibodies retains the function of the antibody and is non-reactive with the subject's immune systems. Examples include any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16.sup.th Edition, A. Osal., ed., 1980).

[0419] One or more head-and-neck marker specific antibody formulations may be administered via any route capable of delivering the antibodies to the a disease (e.g., tumor) site. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Preferably, the route of administration is by intravenous injection. Antibody preparations may be lyophilized and stored as a sterile powder, preferably under vacuum, and then reconstituted in bacteriostatic water containing, for example, benzyl alcohol preservative, or in sterile water prior to injection.

[0420] Treatment will generally involve the repeated administration of the antibody preparation via an acceptable route of administration such as intravenous injection (IV), at an effective dose. Dosages will depend upon various factors generally appreciated by those of skill in the art, including the type of disease and the severity, grade, or stage of the disease, the binding affinity and half life of the antibodies used, the degree of head-and-neck marker expression in the patient, the extent of circulating head-and-neck markers, the desired steady-state antibody concentration level, frequency of treatment, and the influence of any chemotherapeutic agents used in combination with the treatment method of the invention. Daily doses may range from about 0.1 to 100 mg/kg. Doses in the range of 10-500 mg antibodies per week may be effective and well tolerated, although even higher weekly doses may be appropriate and/or well tolerated. A determining factor in defining the appropriate dose is the amount of a particular antibody necessary to be therapeutically effective in a particular context. Repeated administrations may be required to achieve disease inhibition or regression. Direct administration of one or more head-and-neck marker antibodies is also possible and may have advantages in certain situations.

[0421] Patients may be evaluated for serum cancer markers in order to assist in the determination of the most effective dosing regimen and related factors. The head-and-neck cancer assay methods described herein, or similar assays, may be used for quantifying circulating head-and-neck marker levels in patients prior to treatment. Such assays may also be used for monitoring throughout therapy, and may be useful to gauge therapeutic success in combination with evaluating other parameters such as serum levels of head-and-neck cancer markers.

[0422] The invention further provides vaccines formulated to contain one or more head-and-neck marker or fragment thereof.

[0423] In an embodiment, the invention provides a method of vaccinating an individual against one or more head-and-neck marker listed in Table 1 and optionally one or more maker listed in Table 2, including the step of inoculating the individual with the marker or fragment thereof that lacks activity, wherein the inoculation elicits an immune response in the individual thereby vaccinating the individual against the marker.

[0424] The use in anti-cancer therapy of a tumor antigen in a vaccine for generating humoral and cell-mediated immunity is well known and, for example, has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63: 231-237; and Fong et al., 1997, J. Immunol. 159: 3113-3117). These and similar methods can be practiced by employing one or more head-and-neck cancer markers, or fragment thereof, or head-and-neck cancer polynucleotide markers and recombinant vectors capable of expressing and appropriately presenting head-and-neck marker immunogens.

[0425] By way of example, viral gene delivery systems may be used to deliver one or more head-and-neck cancer polynucleotide markers. Various viral gene delivery systems which can be used in the practice of this aspect of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbus virus (Restifo, 1996, Curr. Opin. Immunol. 8: 658-663). Non-viral delivery systems may also be employed by using naked DNA encoding one or more head-and-neck cancer marker or fragment thereof introduced into the patient (e.g., intramuscularly) to induce an anti-tumor response.

[0426] Various ex vivo strategies may also be employed. One approach involves the use of cells to present one or more head-and-neck marker to a patient's immune system. For example, autologous dendritic cells which express MHC class I and II, may be pulsed with one or more head-and-neck marker or peptides thereof that are capable of binding to MHC molecules, to thereby stimulate the patients' immune systems (see, for example, Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate 29: 371-380).

[0427] Anti-idiotypic head-and-neck marker specific antibodies can also be used in therapy as a vaccine for inducing an immune response to cells expressing one or more head-and-neck marker. The generation of anti-idiotypic antibodies is well known in the art and can readily be adapted to generate anti-idiotypic head-and-neck cancer marker specific antibodies that mimic an epitope on one or more head-and-neck cancer markers (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin Invest 96: 334-342; and Herlyn et al., 1996, Cancer Immunol Immunother 43: 65-76). Such an antibody can be used in anti-idiotypic therapy as presently practiced with other anti-idiotypic antibodies directed against antigens associated with disease (e.g., tumor antigens).

[0428] Genetic immunization methods may be utilized to generate prophylactic or therapeutic humoral and cellular immune responses directed against cells expressing one or more head-and-neck cancer marker. One or more DNA molecules encoding head-and-neck cancer markers, constructs including DNA encoding one or more head-and-neck cancer markers/immunogens and appropriate regulatory sequences may be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded head-and-neck cancer markers/immunogens. The head-and-neck cancer markers/immunogens may be expressed as cell surface proteins or be secreted. Expression of one or more head-and-neck cancer markers results in the generation of prophylactic or therapeutic humoral and cellular immunity against the disease (e.g., cancer). Various prophylactic and therapeutic genetic immunization techniques known in the art may be used.

[0429] The invention further provides methods for inhibiting cellular activity (e.g., cell proliferation, activation, or propagation) of a cell expressing one or more OPL or head-and-neck marker. This method includes reacting immunoconjugates of the invention (e.g., a heterogeneous or homogenous mixture) with the cell so that OPL or head-and-neck cancer markers form complexes with the immunoconjugates. A subject with a neoplastic or preneoplastic condition can be treated when the inhibition of cellular activity results in cell death.

[0430] In another aspect, the invention provides methods for selectively inhibiting a cell expressing one or more OPL or head-and-neck marker by reacting any one or a combination of the immunoconjugates of the invention with the cell in an amount sufficient to inhibit the cell. Amounts include those that are sufficient to kill the cell or sufficient to inhibit cell growth or proliferation.

[0431] Vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids, may be used to deliver polynucleotides encoding OPL or head-and-neck cancer markers to a targeted organ, tissue, or cell population. Methods well known to those skilled in the art may be used to construct recombinant vectors that will express antisense polynucleotides for OPL or head-and-neck cancer markers. (See, for example, the techniques described in Sambrook et al. (supra) and Ausubel et al. (supra).)

[0432] Methods for introducing vectors into cells or tissues include those methods discussed herein and which are suitable for in vivo, in vitro and ex vivo therapy. For ex vivo therapy, vectors may be introduced into stem cells obtained from a patient and clonally propagated for autologous transplant into the same patient (See U.S. Pat. Nos. 5,399,493 and 5,437,994). Delivery by transfection and by liposome are well known in the art.

[0433] Genes encoding OPL or head-and-neck cancer markers can be turned off by transfecting a cell or tissue with vectors that express high levels of a desired OPL or head-and-neck marker-encoding fragment. Such constructs can inundate cells with untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases.

[0434] Modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA or PNA, to the regulatory regions of a gene encoding a head-and-neck marker including but not limited to OPL marker, i.e., the promoters, enhancers, and introns. Preferably, oligonucleotides are derived from the transcription initiation site, e.g., between -10 and +10 regions of the leader sequence. The antisense molecules may also be designed so that they block translation of mRNA by preventing the transcript from binding to ribosomes. Inhibition may also be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Therapeutic advances using triplex DNA were reviewed by Gee J E et al., in: Huber and Carr (1994), Molecular and Immunologic Approaches, Futura Publishing Co, Mt Kisco, N.Y.

[0435] Ribozymes are enzymatic RNA molecules that catalyze the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. The invention therefore contemplates engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a head-and-neck marker.

[0436] Specific ribozyme cleavage sites within any potential RNA target may initially be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU, and GUC. Once the sites are identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be determined by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0437] One or more OPL or head-and-neck cancer markers and polynucleotides encoding the markers, and fragments thereof, may be used in the treatment of a head-and-neck disease (e.g., OPL or head-and-neck cancer) in a subject. In an aspect the OPL or head-and-neck cancer markers and polynucleotides encoding the markers are OPL or head-and-neck cancer markers that are down-regulated in OPL or head-and-neck cancer, for example, mucin 5B, alpha 1 anti-trypsin, and one or more of the down-regulated markers listed in Table 2. The markers or polynucleotides may be formulated into compositions for administration to subjects suffering from a head-and-neck disease. Therefore, the present invention also relates to a composition including one or more OPL or head-and-neck cancer markers or polynucleotides encoding the markers, or a fragment thereof, and a pharmaceutically acceptable carrier, excipient or diluent. A method for treating or preventing a head-and-neck disease in a subject is also provided including administering to a patient in need thereof, one or more OPL or head-and-neck cancer markers or polynucleotides encoding the markers, or a composition of the invention.

[0438] The invention further provides a method of inhibiting a head-and-neck disease (e.g., OPL or head-and-neck cancer) in a patient including: [0439] (a) obtaining a sample including diseased cells from the patient; [0440] (b) separately maintaining aliquots of the sample in the presence of a plurality of test agents; [0441] (c) comparing levels of one or more OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers in each aliquot; [0442] (d) administering to the patient at least one of the test agents which alters the levels of the OPL or head-and-neck cancer markers, and/or polynucleotides encoding one or more OPL or head-and-neck cancer markers in the aliquot containing that test agent, relative to the other test agents.

[0443] An active therapeutic substance described herein may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the substance from the action of enzymes, acids and other natural conditions that may inactivate the substance. Solutions of an active compound as a free base or pharmaceutically acceptable salt can be prepared in an appropriate solvent with a suitable surfactant. Dispersions may be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils.

[0444] The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA, 1985). On this basis, the compositions include, albeit not exclusively, the active substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[0445] The compositions are indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment. The compositions of the invention may be administered concurrently, separately, or sequentially with other therapeutic agents or therapies. The therapeutic activity of compositions, agents, and compounds may be identified using a method of the invention and may be evaluated in vivo using a suitable animal model.

[0446] The inventors' study is a significant advancement in this direction as it lays major thrust on determining the clinical impact of a proteomics based biomarker in predicting the high risk leukoplakia, as early as hyperplasia, and clinical outcome in HNOSCC patients after treatment of primary tumors. The unique features of the inventors' study are its prospective nature, the large number of patients in this type of disease setting, and the length of follow-up of leukoplakia and HNOSCC patients. Furthermore, in support of the proposed role of hnRNPK as a transformation-related protein, its overexpression in early oral lesions is a very important unique finding of this study and herein the inventors present clinical evidence to establish its link with progression potential of leukoplakia. To the inventors' knowledge, this is the first investigation to demonstrate the clinical application of a candidate biomarker identified using MS-based tissue proteomics in identifying early oral premalignant lesions that may be at high risk of disease progression.

[0447] Most studies on leukoplakia focus on dysplastic lesions, while knowledge of molecular alterations in oral hyperplasias is meager. As per the existing literature, the malignant transformation potential is often linked to the severity of dysplasia; in comparison the hyperplastic lesions have received less attention, primarily because lesions undergo spontaneous regression. However, the lesions that do not regress need identification and biomarkers to predict the risk of malignant transformation. In this context the inventors' study assumes importance, because not only does it show aberrant hnRNPK expression as early as in hyperplasia, but the follow-up study also points to the relevance of cytoplasmic hnRNPK in predicting the risk of disease progression in leukoplakia patients with hyperplasia and HNOSCCs.

[0448] It is noteworthy that studies on molecular analysis of leukoplakia with hyperplasia are very limited, because these patients often do not come to the clinics since their lesions are small and do not pose any overt clinical problem. However, it is extremely important to target this patient population for risk assessment and early intervention for cancer prevention in high risk cases. Hence, the inventors' findings are important and warrant further validation in larger independent studies on oral hyperplastic lesions. Furthermore, the cytoplasmic expression of hnRNPK protein observed in epithelial cells of a subset of hyperplastic and dysplastic lesions points to a potential role in development and progression during early stages of oral tumorigenesis, while the overexpression in HNOSCCs and association with poor prognosis suggests a sustained involvement in frank malignancy as well.

[0449] The inventors' promising findings will stimulate other groups to undertake large scale studies to evaluate hnRNPK's potential as an indicator of risk of progression of leukoplakia and role in development and progression during early stages of head and neck/oral tumorigenesis. Furthermore, targeting hnRNPK might be a new chemopreventive/therapeutic strategy in head-and-neck and oral cancer.

[0450] The present invention is described in the following non-limiting Examples, which are set forth to illustrate and to aid in an understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXAMPLES

[0451] In Examples 1-13, the inventors demonstrate the identification of a consistently increased expression of a panel of proteins, including stratifin (14-3-3.sigma.) and YWHAZ (14-3-3.zeta.), that may serve as cancer biomarkers. In Examples 14-20, the prognostic utility of these two candidate biomarkers for head-and-neck/oral squamous cell carcinoma (HNOSCC) is described. In Examples 21-27, the clinical significance and utility of one of the OPL markers, hnRNPK, in early premalignant stages and in development, progression, and prognosis of premalignant lesions and confirmed/frank head-and-neck malignancies, is described.

Example 1

Samples and Reagents

[0452] Head-and-neck cancer and oral leukoplakia tissues were retrieved from an in-house, dedicated, research head-and-neck tissue bank, with approval from the Human Ethics Committee of All India Institute of Medical Sciences, New Delhi, India. With patient consent, biopsies/excised tissue specimens of oral leukoplakia and surgically resected specimens of HNSCCs, and paired non-cancerous tissues (each taken from a distant site) were collected and banked from patients undergoing treatment at the Department of Otorhinolaryngology, All India Institute of Medical Sciences. Normal tissues with no evidence of cancer (non-paired noncancerous controls) were collected from patients attending the Dental Outpatient Department of All India Institute of Medical Sciences for tooth extraction, after consent of the patients. After excision, tissues were flash-frozen in liquid nitrogen within 20 min of devitalization and stored at -80.degree. C. until further use; one tissue piece was collected in 10% formalin and embedded in paraffin for histopathological analysis. The clinical and pathological data were recorded in a pre-designed proforma. These included clinical TNM staging (tumor, node, metastasis based on International Union Against Cancer's classification of malignant tumors, 1988), site of the lesion, histopathological differentiation, age, and gender of the patients.

[0453] The histologic diagnosis (dysplasia for OPLs and histological normal oral epithelium for controls) was rendered using microscopic examination of hematoxylin-and-eosin-stained frozen section of each research tissue block. The histologic diagnosis for each HNSCC sample was reconfirmed using microscopic examination of a hematoxylin-and-eosin-stained frozen section of each research tissue block. The tissue from the mirror face of the histologic section was then washed three times in approximately 1 ml of phosphate-buffered saline (PBS) with a cocktail of protease inhibitors as described previously (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 .mu.M leupeptin, 1 .mu.g/ml aprotinin, and 1 .mu.M pepstatin) (21). The washed tissue was then homogenized in 0.5 ml PBS with protease inhibitors, using a handheld homogenizer. These homogenates were then flash frozen in liquid nitrogen and stored at -80.degree. C. until use. Samples were thawed and clarified by centrifugation and the protein concentration determined by a Bradford-type assay using Bio-Rad protein quantification reagent (Bio-Rad, Mississauga, ON, Canada).

[0454] The iTRAQ experiments were performed in five sets of four samples each for the HNSCC samples. A pool of non-paired non-cancerous head-and-neck tissue homogenates was used as a control in each set of experiments: equal amounts of total protein from the lysates of six non-cancerous samples (non-paired controls) were pooled to generate a common reference "control sample" against which all the HNSCC samples were compared. Each sample contained 200 .mu.g of proteins. Trypsin digestion and labeling were performed according to the manufacturer's (Applied Biosystems') protocol; however, as double the manufacturer's recommended amounts were used, two individual vials of each reagent were used for labeling each sample. iTRAQ labeling was performed as follows: control (non-paired non-cancerous pool), iTRAQ reagent 114; two cancer samples, iTRAQ 115 and 117; individual non-cancerous tissue sample (paired or non-paired sample), iTRAQ 116. A total of five iTRAQ sets were analyzed resulting in ten cancer (five buccal mucosa and five tongue) samples and two paired non-cancerous plus three non-paired noncancerous samples being compared to the control sample. The paired non-cancerous samples originated from patients with cancer that were resected from sites a minimum of 2 cm away from the advancing edge of the cancer. Each iTRAQ set was analyzed with one run each of online 2D LC-MS/MS and offline 2D LC-MS/MS analyses.

[0455] The experiments with OPLs were performed in three sets of four samples; the same pool of non-cancerous oral-tissue homogenates was used as a control in each set of experiments. Each analytical set comprised 4-100 .mu.g of each sample labeled as follows: control (normal pool) was labeled with one iTRAQ tag; two OPL samples were labeled with two other iTRAQ tags; and an individual histological normal tissue sample was labeled with the fourth iTRAQ tag. Thus a total of six OPLs and three histological normal samples were compared to the control sample in three iTRAQ sets. The order in which the samples were labeled within each of these three sets was randomized to minimize any systematic error and bias. The iTRAQ analysis of these samples was performed with one run of online reverse phase LC-MS/MS for preliminary examinations, and three replicate runs per set of online two-dimensional LC-MS/MS analyses.

Example 2

Strong Cation Exchange (SCX) Separation Conditions

[0456] For the offline 2D LC-MS/MS analysis, each set of labeled samples was first separated by SCX fractionation using an HP1050 high-performance liquid chromatograph (Agilent, Palo Alto, Calif., U.S.) with a 2.1-mm internal diameter (ID).times.100-mm length polysulfoethyl A column packed with 5-.mu.m beads with 300 .ANG. pores (The Nest Group, Southborough, Mass.) as described previously (21). A 2.1-mm ID.times.10-mm length guard column of the same material was fitted immediately upstream of the analytical column. Separation was performed as previously described (21). Briefly, each pooled sample set was diluted with the loading buffer (15 mM KH2PO4 in 25% acetonitrile, pH 3.0) to a total volume of 2 ml and the pH adjusted to 3.0 with phosphoric acid. Samples were then filtered using a 0.45-.mu.m syringe filter (Millipore, Cambridge, ON, Canada) before loading onto the column. Separation was performed using a linear binary gradient over one hour. Buffer A was identical in composition to the loading buffer, while Buffer B was Buffer A containing 350 mM KCl. Fractions were collected every two minutes using an SF-2120 Super Fraction Collector (Advantec MFs, Dublin, Calif.), after an initial wait of 2 minutes to accommodate the void volume. This resulted in a total of 30 SCX fractions per sample set. These fractions were dried by speed vacuuming (Thermo Savant SC110 A, Holbrook, N.Y.) and resuspended in 30 .mu.l of 0.1% formic acid each.

[0457] For the online 2D LC-MS/MS analysis, an SCX cartridge (BioX-SCX, LC Packings, The Netherlands) was plumbed upstream of the reverse phase (RP) desalting cartridge and analytical column. This SCX cartridge was connected through a second valve on the Switchos unit as shown in FIG. 1. Samples were separated on this SCX cartridge using 10 .mu.l step elutions with increasing concentration of ammonium acetate (10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 500 mM and 1M). Each step elution was loaded onto the RP desalting column using the switching program as shown in FIG. 1, where the eluting peptides were desalted before loading onto the analytical column that was subsequently brought inline with the desalting column. The flow path used for these steps was designed to ensure that there was never any flow reversal through either of the cartridges (SCX or RP). Separation on the RP analytical column was effected as described for the second stage of the offline LC-MS/MS analysis described below.

Example 3

LC-MS/MS Run Conditions

[0458] The SCX fractions from 6 to 30 were analyzed by nanoLC-MS/MS using the LC Packings Ultimate instrument (Amsterdam, The Netherlands) fitted with a 10-.mu.l sample loop. Samples were loaded, using a .mu.l pick-up mode, onto a 5-mm RP C18 pre-column (LC Packings) at 50 .mu.l/min and washed for 4 min before switching the precolumn inline with the separation column. The separation column used was either a 75-.mu.m ID.times.150-mm length PepMap RP column from LC Packings packed with 3 .mu.m C18 beads with 100 .ANG. pores, or an in-house equivalent packed with similar beads (Kromasil; The Nest Group, Southborough, Mass.). The flow rate used for separation on the RP column was 200 nl/min with the following gradient:

TABLE-US-00001 Time (min) 0 10 15 125 145 150 160 162 188 % B 5 5 15 35 60 80 80 5 Stop

[0459] Samples were analyzed on a QSTAR Pulsar i mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, Calif.) in information-dependent acquisition (IDA) mode with the scan cycles set up to perform a 1-s MS scan followed by five MS/MS scans of the five most abundant peaks for 2 s each. Every fourth scan the peak that was closest in intensity to the threshold of 10 counts was selected for MS/MS. Data acquisition was performed without any repetitions and with a dynamic exclusion of 30 s. Relative protein abundances were determined using the MS/MS scans of iTRAQ-labeled peptides (17). The iTRAQ-labeled peptides fragmented under collision-induced dissociation (CID) conditions to give reporter ions at 114.1, 115.1, 116.1, and 117.1 Th.

[0460] The ratios of peak areas of the iTRAQ reporter ions reflect the relative abundances of the peptides and, consequently, the proteins in the samples. Larger, sequence-information-rich fragment ions were also produced under these conditions and gave the identity of the protein from which the peptide was derived.

[0461] The OPL samples were analyzed on a Q-STAR Pulsar-i hybrid quadrupole/time-of-flight tandem mass spectrometer (Applied Biosystems/MDS SCIEX, Foster City, Calif.) in information-dependent acquisition (IDA) mode with the scan cycles set up to perform a 1-s MS scan followed by five MS/MS scans of the five most abundant ions for 2 s each. The method was also set up to select the least abundant ions in the MS scan that are nearest to a threshold of 10 counts on every fourth scan. Data acquisition was performed without any repetitions and with a dynamic exclusion of 30 s. Relative protein abundances were determined using the 114.1, 115.1, 116.1 and 117.1 Th reporter ions in the MS/MS scans of the iTRAQ-labeled peptides (23). The ratios of the peak areas of the iTRAQ reporter ions reflect the relative abundances of the peptides and the relative concentrations of the proteins in the samples. Larger, sequence-information-rich fragment ions were also produced under these MS/MS conditions and gave the identity of the protein from which the peptide originated.

Example 4

Data Analysis

[0462] The software used for data acquisition was Analyst QS 1.1 (Applied Biosystems/MDS SCIEX). Data were analyzed using ProteinPilot (21, 28) and the database searched was the Celera human database (human KBMS 20041109) with a total of 178, 243 entries, both provided by Applied Biosystems Inc. Identified proteins were grouped by the software to minimize redundancy. All peptides used for the calculation of protein ratios were unique to the given protein or proteins within the group; peptides that were common to other isoforms or proteins of the same family that were reported separately were ignored. The ProteinPilot cutoff score used was 1.3, which corresponds to a confidence limit of 95%.

Example 5

Statistical Analysis

[0463] The average iTRAQ ratios from different runs were calculated for each protein in the offline and online analyses. Thereafter, the iTRAQ ratios for each protein in the two analyses were averaged. Proteins that were selected for further analysis met the following criteria: (1) detection in .gtoreq.6 out of the 10 cancer samples, .gtoreq.50% of which showed differential expression.gtoreq.1.5-fold relative to the control sample, and/or (2) known to be of interest from other studies. These proteins are listed in Table 1 along with two housekeeping proteins (to contrast the performance of the potential biomarkers).

[0464] For the OPL samples, the average iTRAQ ratios from the replicates were calculated for each protein. Proteins selected for further statistical analysis met the following criteria: (1) detection in .gtoreq.3 of 6 OPLs, and .gtoreq.50% of which showed differential expression.gtoreq.50% higher than the control sample, and/or (2) known to be of interest based on their biological functions or associations with tumorigenesis. These proteins are listed in Table 6 along with two housekeeping proteins (to contrast the performance of the potential biomarkers).

Example 6

Biomarker Panel Analysis

[0465] To identify a panel of best-performing proteins that can distinguish between HNSCC and non-cancerous tissues, each protein in Table 1 was individually assessed for its ability to discriminate between normal and cancer samples by evaluating its receiver operator characteristic (ROC) curve based on the iTRAQ ratios. Plotting ROC curves and calculating the area-under-the-curve (AUC) and other attributes were performed using the ROCR package within the R statistical computing environment (29). Proteins giving the highest AUC values were selected for biomarker panel analysis and used as input variables into a Naive Bayes model, implemented in JAVA (30) using the WEKA package (31). Given a sample i that has iTRAQ ratios (or IHC scores, see later) in the vector x(i), the Naive Bayes model has the form:

P(i=cancer|x(i))=P(cancer).times.P(x(i))|i=cancer)/P(x(i))

[0466] where P(i=cancer|x(i)) is the probability that i is a cancer sample given its x(i) values. This is the posterior probability and is calculated using Bayes theorem. A value.gtoreq.0.5 is considered a positive hit. P(x(i)|i=cancer) is the probability that within the cancer samples, x(i) exists within them. P(cancer) is the probability of i being a cancer sample; this is the prior probability. P(x(i)) is the probability of i occurring and is a normalization factor. Nine trials of three-fold cross validation were used for each biomarker panel input into the Naive Bayes model. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each trial.

[0467] To identify a panel of best-performing proteins that can distinguish between OPL and normal tissues, each protein in Table 6 was individually assessed for its ability to discriminate between histological normal and OPL samples by evaluating its receiver-operating characteristic (ROC) performance based on the iTRAQ ratio values in terms of sensitivity and specificity using the ROCR package within the R statistical computing environment (29, 30). Proteins giving the highest AUC values were selected for biomarker panel analysis and used as input variables into a Naive Bayes model, implemented in JAVA (30) using the WEKA package (31). Nine trials of three-fold cross-validation were used for each biomarker panel input into the Naive Bayes model. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each trial and the averages are shown in Table 7A. The ROC curve for the panel of the three-best biomarkers-stratifin, YWHAZ (14-3-3 zeta), and heterogeneous nuclear ribonucleoprotein K (hnRNPK)--is depicted in FIG. 6A.

Example 7

Verification of Candidate Potential Cancer Markers (PCMS) by Immunohistochemistry

[0468] The three best-performing proteins from the above biomarker panel analysis were selected for immunohistochemical verification using an independent, larger sample set (Table 12). Antibodies against these three biomarkers were available commercially (Santa Cruz Biotechnology Inc., Santa Cruz, Calif., Table 13). Each antibody was first optimized with respect to dilution and the use of microwave heating in citrate buffer (0.01 M, pH 6.0) to expose the antigen ("antigen retrieval"). Paraffin-embedded sections (5 .mu.m) of human HNSCCs (25 cases) and paired head-and-neck non-cancerous tissues from these patients (25 samples), as well as non-paired non-cancerous head-and-neck tissues (10 samples) were collected on gelatin-coated slides. For histopathological analysis, representative sections were stained with hematoxylin and eosin; immunostaining was done on serial sections as previously described (32). Following the application of a protein blocker for 10 min, deparaffinized tissue sections were first incubated with the primary antibodies for 1 h at room temperature or for 16 h at 4.degree. C., followed by the respective secondary antibody conjugated with biotin. The primary antibody was detected using the streptavidin-biotin complex (DAKO LSAB plus kit, DAKO Cytomation, Denmark) and diaminobenzidine as chromogen. Slides were washed with 3.times. Tris-buffered saline (TBS, 0.1 M, pH=7.4) after every step. Finally, the sections were counterstained with Mayer's hematoxylin and mounted with DPX mountant. In the negative controls, the primary antibody was replaced by non-immune mouse IgG of the same isotype to ensure specificity. HNSCC tissue sections with known immunopositivity for specific proteins were used as positive controls in each batch of sections analyzed (32).

Example 8

Evaluation of Immunohistochemical Staining

[0469] The immunopositive staining was evaluated in five areas. Sections were scored as positive if epithelial cells showed immunopositivity in the cytoplasm, plasma membrane and/or nucleus when judged independently by two scorers who were blinded to the clinical outcome, i.e., the slides were coded and the pathologists did not have prior knowledge of the local tumor burden, lymphonodular spread, and grading of the tissue samples, while scoring the immunoreactivity. First, a quantitative score was performed by estimating the percentage of immunopositive-stained cells: 0<10% cells, 1=10-30% cells, 2=30-50% cells, 3=50-70% cells, and 4=>70% cells. Second, the intensity of staining was scored by evaluating the average staining intensity of the positive cells (0, none; 1, weak; 2, intermediate; and 3, strong). Finally, a total score (ranging from 0 to 7) was obtained by adding the quantitative score and the intensity score for each of the 60 sections. The immunohistochemical data were subjected to statistical analysis as described above for the MS results.

Example 9

Western Blot Analysis of Proteins in HNSCCS and Normal Tissues

[0470] Whole-cell lysates were prepared from five HNSCCs and five non-cancerous head-and-neck tissues. Frozen tissue samples were homogenized and lysed in a buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM ethylenediamine tetraacetate (pH 8.0), 1% Nonidet P-40, 100 mM sodium fluoride, 1 mM phenylmethylenesulfonyl fluoride, and 2 .mu.l/ml protease inhibitor cocktail (Sigma). Protein concentrations were determined using the Bradford reagent (Sigma), and equal amounts of proteins (80 .mu.g/lane) from the HNSCCs and non-cancerous tissues were resolved on 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel. The proteins were then electro-transferred onto polyvinylidene-difluoride (PVDF) membranes. After blocking with 5% non-fat powdered milk in TBS (0.1 M, pH=7.4), blots were incubated with the respective primary antibodies (1:200 dilution) at 4.degree. C. overnight. The protein abundance of alpha-tubulin was used as a control for protein loading, and was determined with mouse monoclonal anti-alpha-tubulin antibody (Clone B7, Santa Cruz Biotechnology Inc.). Membranes were incubated with the respective secondary antibody, HRP-conjugated rabbit/goat/mouse anti-IgG (goat anti-rabbit IgG, 1:5000; rabbit anti-goat IgG, 1:4000; or rabbit anti-mouse IgG, 1:2000, DAKO Cytomation, Denmark), and diluted with 1% bovine serum albumin for 2 h at room temperature. After each step, blots were washed three times with Tween (0.2%)-TBS. Protein bands were detected by the enhanced chemiluminescence method (Santa Cruz Biotechnology Inc.) on XO-MAT film.

[0471] The panel of the three-best biomarkers for OPLs-stratifin, YWHAZ and hnRNPK-together with two additionally promising proteins-S100A7 and prothymosin alpha (PTHA)-were evaluated for their performances using IHC on an independent set of 30 OPLs and 21 histological normal oral tissues. The inventors included S100A7 because it had high individual AUC value and was identified as one of the best-performing PCMs in the inventors' earlier iTRAQ analysis of HNOSCCs (23); it is important to determine whether overexpression of S100A7 occurs in early stages in the development of HNOSCC. PTHA was included because it also had high individual AUC value and had been reported to be important in other cancers (33-36). The sources of the antibodies and dilutions used for IHC are given in Table 13. After histological confirmations of dysplasia in OPLs and normal oral mucosa in the control tissues, paraffin-embedded tissue sections were processed for immunohistochemistry (23).

[0472] Briefly, after antigen retrieval, tissue sections were incubated with the primary antibodies (See Table 13 for details) for 16 h at 4.degree. C., followed by the respective biotin conjugated secondary antibodies and detected using streptavidin-biotin complex (DAKO LSAB plus kit, DAKO Cytomation, Glostrup, Denmark) and diaminobenzidine as the chromogen. In the negative controls, the primary antibody was replaced by isotype specific non-immune mouse IgG to ensure specificity. HNOSCC sections with known immunopositivity for respective proteins as reported earlier (23) were used as positive control in each batch of sections analyzed (FIG. 20).

Example 10

Evaluation of Immunohistochemical Staining

[0473] Immunopositive staining was evaluated in five areas of the tissue sections as described (32). Sections were scored as positive if epithelial cells showed immunopositivity in the cytoplasm, plasma membrane, and/or nucleus when observed by two evaluators who were blinded to the clinical outcome. These sections were scored as follows: 0, <10% cells; 1, 10-30% cells; 2, 30-50% cells; 3, 50-70% cells; and 4, >70% cells showed immunoreactivity. Sections were also scored semi-quantitatively on the basis of intensity as follows: 0, none; 1, mild; 2, moderate; and 3, intense. Finally, a total score (ranging from 0 to 7) was obtained by adding the scores of percentage positivity and intensity for each of the 51 sections (30 OPLs and 21 histologically normal tissues). The immunohistochemical data were subjected to statistical analysis as described above for the iTRAQ ratios.

Example 11

Western Blot Analysis of 14-3-3 Proteins in OPLS and Normal Tissues

[0474] Whole-cell lysates were prepared from 3 OPLs and 3 normal oral tissues using lysis buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM sodium chloride, 10 mM magnesium chloride, 1 mM EDTA (pH 8.0), 1% Nonidet P-40, 100 mM sodium fluoride, 1 mM phenylmethylene sulphonylfluoride, and 2 .mu.l/ml protease inhibitor cocktail (23). Equal amounts of proteins (80 .mu.g/lane) from OPLs and normal tissues were resolved on 12% SDS-polyacrylamide gels. The proteins were then electro-transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% non-fat milk in TBS (0.1 M, pH 7.4), blots were incubated at 4.degree. C. overnight with the respective antibodies (details given in Table 13). The sources of the antibodies and dilutions used for IHC for OPL markers are given in Table 13. Protein abundance of .alpha.-tubulin served as a control for protein loading. Membranes were incubated with the respective secondary antibodies, horseradish peroxidase-conjugated rabbit/goat/mouse anti-IgG diluted at the appropriate dilution in 1% BSA for 2 h at room temperature. After each step, blots were washed three times with Tween (0.2%)-Tris-buffer saline (TTBS). Protein bands were detected by the enhanced chemiluminescence method (ECL, Santa Cruz Biotechnology Inc.) on XO-MAT film.

Example 12

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis

[0475] To determine if overexpression of the five proteins in OPLs were due to increase in the transcript levels, RT-PCR analysis was performed using total RNA isolated from OPLs and normal oral mucosa (3 each), using gene-specific primers for YWHAZ, stratifin, hnRNPK, S100A7 and PTHA, and beta actin as a control (See Table 14 for gene-specific primer sequences (synthesized by Microsynth, Switzerland) and PCR conditions used). Synthesis of complementary DNAs (cDNAs) were carried out by reverse transcription of 2.0 .mu.g of total RNA using MMLV reverse transcriptase. PCR amplification was carried out in a total volume of 20 .mu.l containing 3 .mu.l reverse transcribed cDNA, 10.times.PCR buffer, 10 mM dNTPs, 20 .mu.M of each primer and 1 U of Taq polymerase. After 5 min of initial denaturation, 32 amplification cycles of 1 min at 94.degree. C.-1 min at specific annealing temperature and 1 min at 72.degree. C.-were carried out, followed by a 10-min elongation at 72.degree. C. .beta.-actin was used as a control to optimize the amounts of cDNAs generated. PCR products were separated on 1.5% agarose gel, stained with ethidium bromide, and visualized with Chemilmager IS-4400 (Alpha Innotech Corp., CA) (23).

Example 13

Network Analysis

[0476] The 30 proteins listed in Table 6 were used for network analysis. HUGO or SwissProt accession numbers were imported into the Ingenuity Pathway Analysis (IPA) Software (Ingenuity Systems, Mountain View, Calif.). The IPA database consists of proprietary ontology representing 300 000 biologic objects ranging from genes, proteins, and molecular and cellular processes. More than 11 200 human genes are currently represented in the database. The proteins were categorized based on location, cellular components, and reported or suggested biochemical, biologic, and molecular functions using the software. The identified proteins were mapped to networks that were generated based on evidence from existing literature available in the Ingenuity database and then ranked by score. A score of 3 or higher has a 99.9% confidence level of not being generated by random chance alone and was used as the cutoff for identifying protein networks. The molecules identified in the networks and their cellular functions are given in Table 15.

[0477] Provided below is a summary of the results obtained by the inventors in connection with the experiments of Examples 1-13:

[0478] The offline and online 2D LC-MS/MS analyses collectively resulted in the identification of a total of 811 non-redundant proteins. Only a few of these proteins displayed consistent differential expression in the HNSCC samples (measured in .gtoreq.6 out of the 10 samples and with .gtoreq.50% showing .gtoreq.1.5-fold differential expression relative to the control sample) that warranted further analysis. These proteins, all confidently identified with .gtoreq.two peptide matches (except APC-binding protein EB1 and superoxide dismutase [Mn]) are given in Table 1 along with two structural proteins, actin and .beta.-2-tubulin, as controls. (See Table 11 for peptide sequences and coverage, and FIG. 19A for the CID spectra of the single-peptide identifications.) As the nanoLC analyses were performed on 25 SCX fractions, the acquired data files were searched in two groups out of necessity (the version of ProteinPilot software available at that time was incapable of handling a large number of data files each with a large amount of data). Fractions 6-15 were, therefore, searched in one group, while fractions 16-30 were searched in a second group.

[0479] The ProteinPilot result files from these two halves were then exported into an Excel spreadsheet where the proteins of interest from the two searches were combined by averaging the ratios for the protein in each sample. It is noteworthy that each of the ratios reported by searching either half of the fractions is itself comprised of the ratios from multiple peptides identified in the given protein. ProteinPilot automatically only includes unique and high-confidence matches of peptides for any particular protein in the ratios reported (i.e., it excludes those that are shared between different isoforms of any protein or low-confidence matches to peptides). These averaged ratios from the offline and online analyses were then again averaged and reported in Table 1. Of all the individual expression ratios (two offline and two online), 56.4% varied by less than 10% from their respective average shown, and 82.0% varied by less than 20%. It is reassuring that the expression ratios from different analyses and separate handling were comparable.

[0480] Nine proteins that did not meet the cutoff criteria stated above-cytokeratin 14, polybromo 1D, PKM2, annexin A1, nucleophosmin 1, Hsp27, cystatin B, GRP 94, and MARCKS-were also included in Table 1 for further analysis, as these had been reported in head-and-neck cancer or are of biological relevance in cancer. The HNSCCs analyzed included five squamous-cell carcinomas (SCCs) of buccal mucosa and five SCCs of the tongue. The rationale for the choice of these two SCC types was to determine if there are site-specific protein expressions or not. The best-performing proteins that can differentiate between HNSCC and non-cancerous tissues were identified by determining the individual receiver-operator characteristic (ROC) curves of the proteins in Table 1 (as described in the Experimental section). The three proteins with the highest AUC values--YWHAZ, Stratifin and S100 A7--are listed in Table 2 together with their individual and collective figures-of-merit, including sensitivity (cancer samples correctly identified as cancer samples) and specificity (normal samples correctly identified as normal samples). As a panel, the three best-performing biomarkers achieved a sensitivity of 0.92 and a specificity of 0.91 in discriminating HNSCC from non-cancerous head-and-neck tissues (Table 2 and FIG. 2a).

[0481] A number of proteins, e.g., prothymosin alpha and APC-binding protein EB1, were predominantly overexpressed in SCCs of buccal mucosa (Table 1) and showed some promise in differentiating between SCCs of buccal mucosa and the tongue; however, as the number of samples are small, this possibility will need to be fully investigated in a future study involving more samples of both types.

[0482] Verification of candidate protein biomarkers is a necessary step in moving from the initial discovery to possible application. The panel of three best-performing biomarkers identified by MS analysis-YWHAZ, Stratifin and S100 A7-were chosen for verification in a different and larger set of HNSCCs and non-cancerous head-and-neck tissues. Verification exercises included immunohistochemical (FIG. 3) and Western blot analyses (FIG. 4) at the protein level, as well as RT-PCR analysis (FIG. 5) at the mRNA level. All verification results support the above MS findings. In the immunohistochemical analysis, the biomarker panel of YWHAZ, Stratifin and S100A7 achieved a sensitivity of 0.92 and a specificity of 0.87 (Table 3 and FIG. 2b) in discriminating HNSCCs from non-cancerous head-and-neck tissues. The paired non-cancerous head-and-neck tissues obtained from HNSCC patients might have altered protein expressions prior to histological changes.

[0483] To investigate this possibility, the noncancerous tissues were segregated into paired and non-paired groups and evaluated separately with the HNSCCs. Significantly, the panel of the three biomarkers-YWHAZ, Stratifin and S100A7-appears to perform better in discriminating HNSCC tissues against the non-paired non-cancerous head-and-neck tissues (sensitivity, 0.96; specificity, 0.96) than against the paired non-cancerous tissues (sensitivity, 0.92; specificity, 0.83) (see Table 4). These results appear to support the notion of protein-expression alterations prior to histological changes and caution the use of only paired samples.

[0484] The LC-MS/MS analyses of OPLs collectively resulted in identification of 439 non-redundant proteins; 216 were identified as single hits with more than 95% confidence. Of all the proteins identified, only 17 passed the inventors' criteria for further statistical analysis (vide supra). Of this subset, 15 proteins were confidently identified with a minimum of two peptide matches in each case (See Table 11 for peptide sequences and coverage). Two proteins, parathymosin and DLC 1 were identified by single peptides (See FIG. 19B-E for the CID spectra of the single-peptide identifications). These 17 proteins are given in Table 6, along with two structural proteins, .beta.-actin and gelsolin precursor, as controls. Table 6 also depicts the variations in the levels of overexpressed and underexpressed proteins in individual OPL and histological normal tissues versus the pooled normal control. These differential expression levels were averages of the replicate injections: 56.4% of the ratios varied by less than 10% from their respective averages shown, and 82.0% varied by less than 20%.

[0485] Thirteen proteins that did not meet the aforementioned initial criteria-IGL2, p37AUF1 (hnRNPD), SOD2, PKM2, hnRNPA1, HSP27, cofilin, glyceraldehyde-3-phosphate dehydrogenase, NDP kinase B, elongation factor 2, CALM3, PEBP and S100A7-were also included in Table 6 for further analysis, as they are of biological relevance in cancer development. Of these, 11 proteins were confidently identified with a minimum of two peptide matches in each case (see Table 11B-E for peptide sequences and coverage). p37AUF1 (hnRNPD) was identified by a single peptide with a confidence of 99% (see FIG. 19D for the CID spectra of the single-peptide identification). SOD2 was identified by more than one unique peptide; however, the best-matching peptide was identified with a confidence of only 93%. Although this peptide did not meet the inventors' stipulated criteria for acceptance, manual verification of the spectrum showed good sequence coverage for this peptide (FIG. 19E). Furthermore, the cumulative score, which included the lower confidence peptide matches, was >2.0 and corresponded to a confidence of 99%.

[0486] The best-performing proteins that can differentiate between OPLs and normal tissues were identified by determining the individual ROC curves of the proteins in Table 7. The three proteins with the highest AUC values-YWHAZ, stratifin and hnRNPK--are listed in Table 7A together with their individual and collective figures-of-merit, including sensitivity and specificity. As a panel, these three biomarkers achieved a sensitivity of 0.83 and a specificity of 0.74 in discriminating OPLs from histological normal oral tissues (Table 7A and FIG. 6A).

[0487] Verification of Candidate Biomarkers by Immunohistochemistry, Western Blot, and RT-PCR Analyses.

[0488] The panel of three potential biomarkers, YWHAZ, stratifin and hnRNPK, and two other proteins with high AUC values, S100A7 (0.56) and PTHA (0.56), were chosen for verification in an independent set of OPLs (30 cases) and normal tissues (21 cases) by IHC. Representative levels of expression and subcellular localizations of all five proteins in oral dysplastic tissues in comparison with normal tissues are shown in FIG. 7A-E. (FIG. 20 depicts the positive and negative controls used for each protein analyzed by IHC.) These data were further verified by Western blot analysis (FIG. 8A) at the protein level, as well as RT-PCR analysis at the mRNA level (FIG. 8B).

[0489] The differential expression suggested by iTRAQ ratios tended to be moderate, whereas the results of Western and RT-PCR analyses tended to show more extreme differential expression. Thus, Western and RT-PCR analyses, verified the differential expression reported by the iTRAQ analysis in trend, but not in scale. This discrepancy of scale has also been noted in other studies and has been ascribed to compression of the dynamic range of iTRAQ ratios (21). Specifically, in that study the inventors determined that a two-fold differential expression as determined by iTRAQ analysis was in reality closer to a four-fold differential expression in an absolute quantification study that was performed on the same samples. Importantly, in IHC analysis, the biomarker panel of YWHAZ, stratifin, and hnRNPK achieved a sensitivity of 0.91 and a specificity of 0.95 (Table 8B and FIG. 6B) in discriminating OPLs from histological normal oral tissues.

[0490] Network Analysis.

[0491] To gain insight into the plausible biological processes in which these proteins might be involved, the inventors used the Ingenuity Pathway Analysis tools (Ingenuity Systems, Inc. software) and discovered two major networks (Table 15) in OPLs (the merged network is shown in FIG. 8C). To the best of the inventors' knowledge, ours is the first study reporting differential expressions of p37AUF1 and histoneH2B.1 in OPLs. These proteins and their cellular functions are listed in Table 11.

[0492] Without being bound by theory, the results obtained in the experiments of Examples 1-13 are discussed below:

[0493] Multidimensional LC-MS/MS has been used for the analysis of clinical samples of HNSCCs labeled with isobaric mass tags (iTRAQ) to identify proteins that are differentially expressed in head-and-neck cancer in relation to non-cancerous head-and-neck tissues. The expression ratios were consistent between the online and offline 2D LC-MS/MS methods used, demonstrating that the methodologies were rugged and reproducible, even though the conditions and details used in peptide elution from the SCX columns in the two methods were different. Expectedly, the numbers of proteins identified by the online analysis (431) was lower than those identified by the offline analysis (580), due to the lower capacity of the SCX cartridge used in the former (50 ng of total peptides versus up to 1 mg in the latter). However, the online analysis was advantageous in terms of shorter data acquisition time and lower amounts of total sample required.

[0494] Development of HNSCC is a multistep process that often involves field cancerization, a phenomenon in which not only the site of the primary tumor, but the entire mucosa of the upper aerodigestive tract, is prone to undergoing malignant transformation or progression at multiple sites (37). It is now evident that molecular changes underlying field cancerization are not localized to areas with altered histology, but may persist beyond the histological border of precancerous lesions; a large fraction of the carcinogen-exposed field may harbor molecular aberrations without presenting clinical or morphological symptoms (6 and references therein). Identification of proteins with altered expression as a manifestation of field cancerization is important in identification of biomarkers for prediction of risk of recurrence, as well as for development of second primary tumors in patients treated for HNSCC. Thus, the selection of normal controls for HNSCCs in a differential expression analysis, including the current study, is non-straightforward and requires careful planning.

[0495] To address this issue, the inventors have included two types of non-cancerous histologically confirmed normal tissues in the inventors' analysis: (1) noncancerous tissues obtained from HNSCC patients from a site distant to the tumor, and (2) normal tissues obtained from individuals with no evidence of cancer or pre-cancerous lesions. In a recent proteomic study, Roesch-Ely et al. (12) investigated changes in protein expressions occurring in different stages of tumorigenesis and field cancerization in HNSCCs. A number of reported differentially expressed proteins, including calgizarrin, stratifin, histone H4, and cystatin A, are also identified in this current study. This study appears to be the first reporting differential expression of calmodulin-like protein 5, polybromo-1D, APC-binding protein EB1, .alpha.-1-antitrypsin precursor, carbonic anhydrase I, mast cell tryptase .beta. III, histone H2B.1, L-plastin, and peptidylprolyl isomerase A (PPIA) in HNSCC.

[0496] Among the differentially expressed proteins identified, no single protein emerged as a unique marker for HNSCC. However, a panel of three best-performing biomarkers YWHAZ, Stratifin and S100 A7 performed satisfactorily, as determined by both MS and immunohistochemistry on independent sets of samples. Significantly, YWHAZ has previously been identified by the inventors to be overexpressed in oral cancer at the mRNA level and has subsequently been verified using immunohistochemical analysis (38, 39). This serves as an independent validation of and complements the current results. Furthermore, YWHAZ has also been reported to be overexpressed in stomach cancer (40), and in breast and prostate tumor model systems (41, 42). More importantly, YWHAZ is not overexpressed in lung-cancer tissue samples (43), thus illustrating the fact that this protein can provide some selectivity in discriminating among different cancer types.

[0497] Stratifin has been reported to be overexpressed in HNSCC. A recent proteomic study reported a 3.6-fold straffin overexpression (44), thus corroborating the results obtained in this study. A second independent study also showed stratifin overexpression in the range of 2.8-9.1-fold in cancer samples (45). In addition, a study of 300 patients with pancreatic ductal adenocarcinoma showed stratifin overexpression in 82% of primary infiltrating adenocarcinomas, while another 15% showed weak immunopositivity. Overexpression of stratifin correlated with poor prognosis (46). Interestingly and significantly, stratifin was reported to be down-regulated in HNSCCs by Roesch-Ely et al. (12), whereas the inventors observed consistent overexpression of stratifin in iTRAQ and in IHC verification analysis. The HNSCCs in the study of Roesch-Ely et al. (12) are from the German population with tobacco smoking and alcohol consumption being the major risk factors, while the clinical samples in this study, and in Lo et al. (15, 44) and Chen et al. (45) are from Asian populations, where in addition, chewing tobacco and/or betel quid, and bidi smoking are important risk factors. These differences in the risk factors may account for the observed variations in stratifin expression and warrant in-depth investigation in a larger study.

[0498] 14-3-3 proteins recognize phosphoserine/threonine (pS/T)-containing motifs used by a variety of signal transduction pathways to bind over 200 target proteins that play important roles in the regulation of various cellular processes, including mitogenic and cell-survival signaling, cell-cycle control and apoptotic cell death, epithelial-mesenchymal transition, and cell adhesion, invasion and metastasis (46). The involvement of 14-3-3 proteins in the regulation of oncogenes and tumor suppressor genes points to a potential role in turmorigenesis (47); multiple pathways can be targeted by modulation of these proteins, underscoring their potential as candidate drug targets. Although it might be argued that 14-3-3 proteins are, therefore, too pleiotropic to be targets for therapeutic inhibition, it has been shown that simultaneous inactivation of all 14-3-3 proteins sensitizes cancer cells to DNA-damaging agents. Selective inactivation of stratifin leads to an increased sensitivity towards cancer chemotherapeutic agents. Recent studies have shown that stratifin forms homodimers, while YWHAZ forms homodimers and also heterodimers with other isoforms (48-50). Stratifin has been extensively investigated; by contrast, YWHAZ remains largely unexplored. It is noteworthy that the potential success of strategies aimed at modulating 14-3-3 availability in the cell for cancer therapy is provided in studies showing that reducing cellular 14-3-3 increases chemosensitivity (51, 52).

[0499] Cytokeratin 14 has also been demonstrated to be overexpressed in many squamous cell carcinomas. A study using immunohistochemical analysis demonstrated that 67 of 74 cases of squamous cell carcinomas showed immunoreactivity regardless of origin, suggesting cytokeratin to be a useful marker for squamous cell carcinoma (53). Another study, comparing mRNA levels of cytokeratin 14 between oral squamous cell carcinoma and leukoplakia samples reported that the former showed a higher amount of cytokeratin 14 (54).

[0500] Prothymosin alpha, found to be overexpressed here in HNSCC, has recently been proposed to be a potential marker of proliferation in patients with thyroid cancer (55). This protein was implicated in various other cancers, including gastric, lung, liver, colon, and breast cancers (33-36, 56, 57). Prothymosin alpha was proposed to be a surrogate marker for the diagnosis of estrogen-negative breast-cancer cases (56), and a urinary marker for the detection and monitoring of bladder cancer (58). Prothymosin alpha expression has been observed in lymph nodes and tonsils (59). This expression in lymph nodes of HNSCC patients would correlate with locoregional spread of the disease and may be a determinant of disease prognosis. Prothymosin alpha is a small, highly acidic, nuclear protein that has been proposed to play a role in cell proliferation and immune regulation (60).

[0501] Protein changes related to cytoskeletal reorganization, cellular metabolism, and protein-protein interactions have been observed, based on which a model for its immunological mode of action has been proposed (60). Interestingly, some of the proteins identified in that study, such as L-plastin, HSP90, vinculin, aldolaseA, meosin, and galectin 3 are found to be overexpressed in the present study as well, although not all of them are included in Table 1.

[0502] L-plastin is expressed by hematopoetic cells and by most human cancer cell lines, including human submandibular gland cell lines (61, 62); yet, its functional importance in tumor tissues is controversial: its expression correlates with tumor progression in colon cancer, but not in breast cancer; in melanoma, L-plastin phosphorylation promotes tumor cell invasion (reviewed in 61). Intriguingly, L-plastin has also been proposed to represent a novel target for cancer therapy, and the constitutive activity of its promoter in non-hematopoetic tumors presents novel perspectives for cancer gene therapy using L-plastin promoter-driven viral vectors (61).

[0503] S100A7, a small calcium-binding protein of the S100 protein family, originally identified in psoriatic keratinocytes, is up-regulated in abnormally differentiating keratinocytes, squamous carcinomas of different organs, and in a subset of breast tumors (62-66). Incidentally, S100A7 was also identified in oral premalignant epithelia by microarray analysis and proposed to be a marker for invasion (63). It has been hypothesized to play a role in breast-tumor progression by promoting angiogenesis and enhancing the selection of cells that overcome their anti-invasive function (64). This hypothesis has also been suggested to explain why S100A7 expression is high in high-grade or estrogen-receptor negative tumors, as these are associated with increased hypoxia and reactive oxygen species (ROS), a scenario in which the angiogenic effects of S100A7 are most important.

[0504] It is noteworthy that increased hypoxia and ROS also occur in head-and-neck tumors and might explain the observed changes in S100A7 expression here. Another study in breast cancer showed that BRCA1 is a transcriptional repressor of S100A7. BRCA1 and c-Myc form a complex on S100A7 promoter, and BRCA1-mediated repression is dependent on a functional c-Myc (68). Furthermore, BRCA1 mutations in tumors abrogate the repression of S100A7. In the absence of BRCA1, S100A7 is induced by topoisomerase II poison and etoposide, as well as increases the cellular sensitivity to etoposide, suggesting a mechanism for BRCA1-mediated resistance to etoposide (68). Incidentally, BRCA1 alterations have been reported in head-and-neck cancer (69, 70). However, a correlation, if any, between BRCA1 alterations and S100A7 expression in head-and-neck cancer remains to be demonstrated.

[0505] Calgizzarin (S100 A11) has also been previously linked with cancer and was reported as a potential marker for head-and-neck cancer (18). Likewise, S100 A2, which shows overexpression in HNSCC is also known to be overexpressed in other forms of cancer, such as non-small cell lung cancer and uterine leiomyoma (71, 72). It has been demonstrated that calgizzarin plays an anti-apoptotic role in uterine leiomyosarcoma cell line (72). Fascin has been discovered to be an early marker for esophageal squamous cell carcinoma (73). The inventors have previously reported pyruvate kinase M2 to be overexpressed in head-and-neck cancers (17, 21). Several studies suggest that PKM2 is present primarily in a dimeric form in tumors, and is useful as a biomarker in their early detection (74-78). PKM2 overexpression in tumor cells is explained on the basis of its key role in the generation of ATP in the glycolytic pathway. Under hypoxic conditions that are typical for tumors, this pathway is a critical route by which tumors satisfy the higher energy requirements needed for proliferation (reviewed in 79, 80).

[0506] Two of the more interesting proteins discovered in this current HNSCC study are the APC-binding protein EB1 and polybromo-1D. End-binding protein 1 (EB1) was initially discovered as a protein that binds adenomatous polyposis coli protein (APC) at its C-terminal region (81). More recently, however, it has also been shown to bind tubulin and has been detected to associate with the microtubules that form the mitotic spindle during mitosis (82).

[0507] The EB1 interaction with APC is of particular interest as APC is a tumor suppressor whose inactivation leads to a significantly enhanced level of susceptibility for malignant transformation in colorectal cancer (82). Among others, APC binds to .beta.-catenin and possibly controls .beta.-catenin's availability in the cytoplasm (82). By virtue of APC's binding to tubulin, EB1 participates in microtubule-dependent processes, including intracellular vesicle trafficking, organization of organelles within the cell, and even cell migration (82).

[0508] One possible proposed explanation for the mechanism of action of EB1, is that overexpression of EB1, at least in esophageal squamous cell carcinoma, affects the interaction between APC and .beta.-catenin, and that this overexpression correlates with the nuclear accumulation of .beta.-catenin (82). Normally, APC in combination with glycogen synthase kinase 3P (GSK 3.beta.) and axin forms a destruction complex that phosphorylates free .beta.-catenin in the cytoplasm, which in turn targets it for ubiquitination and degradation (82). Disruption of APC interaction with .beta.-catenin by EB 1 overexpression leads to increased levels of .beta.-catenin in the nucleus, which in turn binds to T-cell factor/lymphoid-enhancing factor (TCF/LEF) and activates transcription of target genes such as c-myc and cyclin D1. Thus, the overexpression of EB1 is thought to play a role in the development of esophageal squamous cell carcinoma by indirectly causing the activation of the .beta.-catenin/TCF pathway (83). It is, therefore, possible that overexpression of EB1 in this study could be the first evidence for the same process occurring in HNSCC.

[0509] Polybromo 1D (PB1), also known as BRG1-associated factor 180 (BAF180), is a relatively new member of the SWI/SNF-B (PBAF) chromatin remodeling complex that is a homolog of the yeast rsc protein complex, which is required for progression through mitosis (84). In fact, antibodies against BAF180 localize to the kinetochores during mitosis (84). The fact that both PB1 and EB1 are known to be involved with mitosis is also noteworthy, but requires further investigation to ascertain if a direct relationship between the two exists. Other studies have shown that in yeast, rsc can act as an activator as well as a suppressor of transcription, and that it can be functionally linked with the PKC pathway (85, 86).

[0510] Additionally, it was shown that temperature-sensitive mutants of one of the proteins (nps1) in the rsc complex, when placed at the restrictive temperature, can be rescued by the overexpression of not only the yeast homolog of PKC, PKC1 (as well as other proteins downstream of the PKC1 signal pathway), but also by Bim1p, which is the yeast homolog of EB1 (85). It was further demonstrated that there is no direct interaction between Pkc1p and Bim1p, or any activation of BIM1 transcription or post-transcriptional regulation by Pkc1p, but that suppression of the activity of overexpressed Pkc1p requires a functional Bim1p (85).

[0511] In addition to the possibility of this potentially significant link between PB1 and EB1, there is also independent evidence suggesting that PB1 is a tumor suppressor and that this activity is found in lung cancer but not in breast cancer (86). This was verified when transfection of BAF180 gene into breast tumor cell lines, possessing a truncated version of the same gene, resulted in growth inhibition (86). Other members of this complex that have been associated with cancer include hSNF5/INI1 and BRG1 itself. HSNF5/INI1 mutations have been found in malignant rhabdoid tumors, while mutations in BRG1 have been noted in various cell lines including carcinomas of the breast, lung, pancreas and prostate. Implication of other members of the PBAF complex in suppression of various cancers, in addition to the above evidence which suggests that PB1 may be a tumor suppressor itself, makes PB1 an exciting discovery in the inventors' study. In light of PB1's suggested tumor suppressor role, the presently observed lower expression of this protein in the HNSCC samples is consistent with expectations and warrants in-depth investigation of its role in head-and-neck tumorigenesis.

[0512] Thus, the use of iTRAQ-labeling of head-and-neck cancers combined with LCMS/MS has led to the discovery of several novel, differentially expressed proteins in these tumors. A panel of the three best-performing biomarkers achieved a sensitivity of 0.92 and a specificity of 0.91. This performance was verified using immunohistochemistry on a larger, independent set of clinical samples of HNSCCs.

[0513] The unique features of the OPL study are its prospective nature, the large number of patients in this type of disease setting, and the length of follow-up of leukoplakia and HNOSCC patients. Table 7 also shows the analysis of three histological normal samples used in a previous exercise to demonstrate consistency and validity over a period of six months (23). The number of OPLs examined in this study-six for the LC-MS/MS and 30 for subsequent verifications--was relatively modest, but was necessitated by the small number and size of OPL samples available. Nevertheless, the inventors successfully demonstrated the utility of iTRAQ-labeling of small OPL biopsy specimens and detection of a large number of expressed proteins that led to the discovery of a panel of candidate OPL biomarkers.

[0514] Replicate analyses demonstrated that the expression ratios were reproducible-82% were within 20% of the averages shown in Table 6. Differential expressions analyses of the proteomes between OPLs and histological normal oral tissues revealed 30 proteins that merit further examination and verification. A panel of three potential biomarkers selected by ROC analyses and two other biologically relevant proteins that had high AUC values were successfully verified to be overexpressed using an independent and larger set of OPLs and histological normal tissues by IHC and Western analyses, thus confirming findings of the iTRAQ analyses. In addition, RT-PCR analyses showed increased levels of transcripts for all five proteins, suggesting that the increased protein expressions were due to upregulation at the transcriptional level.

[0515] The inventors' approach enabled identifying in oral premalignant lesions a large number of proteins, including mediators of inflammatory response, redox system, proteases, chaperones, transcriptional regulators, calcium binding proteins, metabolic enzymes, and proteins involved in cell proliferation and growth, intermediary metabolism, signal transduction, cell cycle regulation, cell death, cell motility and cell morphology. Pathway analyses unraveled important novel links between inflammation and cancer. Importantly, it showed direct interaction between all the three proteins-YWHAZ, stratifin and hnRNPK--that constitute the panel of OPL biomarkers (87). The mechanism involved in upregulation of YWHAZ and stratifin in OPLs remains unknown.

[0516] hnRNPK is an RNA-binding protein that regulates gene expression at both transcriptional and translational levels (88, 89). Therefore, the inventors speculate that hnRNPK may be involved in regulation of YWHAZ and stratifin in OPLs. The data analysis also suggests that hnRNPK directly regulates the expression of COX2, enzyme implicated in the synthesis of prostaglandins, which are mediators of the inflammatory response. The inventors' earlier in vitro and in vivo studies demonstrated that COX-2 activation and NF-kB overexpression are parallel events occurring in early precancerous stages of tobacco-associated oral carcinogenesis and these events remain elevated down the tumorigenic pathway (90), while others demonstrated a role for 14-3-3 proteins in the nuclear export of p65-I.kappa..beta.-alpha complexes (91) and the role of 14-3-3 in phosphorylation of beta-catenin by AKT that promotes beta-catenin transcriptional activity (92) as well as in apoptosis and cell adhesion emphasizing the oncogenic character of 14-3-3 zeta (YWHAZ) (93). Tobacco carcinogens, including tobacco specific nitrosamines (TSNA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), appear to activate these proteins involved in the inflammatory response of epithelial cells and initiation of the carcinogenic cascade.

[0517] The other important links identified are regulation of NF-kB by SERPINA1 and phosphatidyl ethanolamine binding protein 1 (PEBP1). SERPINA1 has been shown to reduce the activation of NF-kB; decrease in SERPINA1 levels in OPLs may partly account for activated NF-kB in OPLs. Pathway analyses also showed, for the first time, that deregulation of calcium-associated proteins (cystatin B, FABP5, and S100A7) and mitochondrial dysfunction (SOD2) may play an important role in the development of OPLs. Importantly, YWHAZ was found to directly interact with nucleophosmin (NPM1), an important protein involved in increased cellular proliferation, and with HSP90B1, which has been reported to increase the activity of ERK. HSP90B1, YWHAZ, nucleophosmin1, parathymosin (PTMS), SOD2, and PEBP1 are all involved in the inhibition of apoptosis.

[0518] Furthermore, HSP90B1, SOD2, and stratifin are associated with increased cell viability. Increased expressions of hnRNPK, PTHA, stratifin, SOD2 and nucleophosmin1, and reduced expression of DLC1, play a role in hyperproliferation of cells. Increased cell proliferation and inhibition of apoptosis are two hallmarks of cancer, and the inventors' data suggest that both events are occurring in early, premalignant stages. Alterations in cytoskeleton are important events in tumorigenesis; deregulation of YWHAZ and its interaction with MARCKs, beta actin and tubulin identified herein suggest the implication of cytoskeletal reorganization in development of oral dysplasias.

[0519] It is noteworthy that invasion is an important event in the progression of dysplasia as well as of cancer, and five proteins identified in the inventors' study-underexpressions of DLC1, IGHG1 and FABP5, and overexpressions of S100A7 and PEBP1--are all involved in cell invasion. Significantly, this is the first report of p37AUF1 (hnRNPD) expression in OPLs, and pathway analyses suggest its potential interaction with stratifin and scaffold protein b (SAFB). PEBP1 is oncogenic, while FABP5 and SERPINA1 are metastatic. Taken together, the discovery of these alterations in OPLs suggests that these proteins may be associated with the potential of malignant transformation. The findings herein certainly warrant additional validation; elucidation of functional significance in future studies will provide further insight into the biology of development and progression of OPLs.

[0520] None of the aforementioned proteins can be classified individually as specific biomarkers for OPLs. However, a panel of the three best-performing biomarkers: YWHAZ, stratifin, and hnRNPK confer satisfactory performance. The current study clearly demonstrates overexpression of YWHAZ in OPLs, suggesting its involvement in early stages of oral carcinogenesis. Mechanistically, YWHAZ overexpression increases p53 protein degradation via hyperactivation of the phosphoinositide 3-kinase (PI3K)--Akt signaling pathway that phosphorylates MDM2 (92-95). Replacement of p53 leads to luminal cell apoptosis. YWHAZ is known to be involved in diverse cellular processes, many of which are deregulated in HNOSCCs (39). The inventors' ongoing studies on functional analysis of YWHAZ in oral cancer cells have also shown its involvement in the activation of PI3K--Akt signaling pathway and cytoskeletal reorganization (data not shown).

[0521] Stratifin, another member of the 14-3-3 protein family, was overexpressed in OPLs and emerged as one of the best-performing potential biomarkers. The inventors' present data suggest that the increase in stratifin expression is an early event in the development of cancer. Importantly, their recent proteomic analysis showed overexpression of stratifin in HNOSCCs (23); underscoring its importance in head-and-neck tumorigenesis (87). Interestingly and significantly, stratifin was reported to be down-regulated in HNSCCs by Roesch-Ely et al., (12), whereas the inventors observed consistent overexpression of stratifin in iTRAQ and in IHC verification analyses. The HNSCCs in the study of Roesch-Ely et al. (12) were from the German population with tobacco smoking and alcohol consumption being the major risk factors; while the clinical samples in this study, and in Lo et al. (44), were from Asian populations, where in addition, chewing tobacco and/or betel quid, bidi smoking and HPV infection are important risk factors. In support of these observations, Bhawal et al., (96) reported that hypermethylation of stratifin promoter is not a frequent event in HNSCC. Moreover, IKK alpha, a catalytic subunit of the IKK complex, has been shown to protect the stratifin locus from hypermethylation; this function serves to maintain genomic stability in keratinocytes.

[0522] Heterogeneous nuclear ribonucleoprotein K protein (hnRNPK), identified by iTRAQ analysis and verified by IHC in OPLs, is an interesting protein that has been strongly implicated as a key player of tumorigenesis (87) hnRNPK is overexpressed, aberrantly localized, and associated with poor prognosis in colorectal cancer (88), while its transcriptional upregulation was reported in OSCC (89). In view of a role of hnRNPK as a transformation-related protein, its overexpression in OPLs is an important finding; in-depth studies are warranted to establish its link, if any, with transformation potential of OPLs.

[0523] Prothymosin alpha (PTHA), overexpressed in a subset of OPLs, has been proposed to be a proliferation marker in patients with thyroid cancer (55). This protein was implicated in various other cancers, including gastric, lung, liver, colon, and breast cancers (33-36, 55-60). S100A7, a small calcium-binding protein, is upregulated in abnormally differentiating keratinocytes, squamous carcinomas of different organs, and in a subset of breast tumors (65, 67). S100A7 has been identified in oral premalignant epithelia by microarray analysis and proposed to be a marker for invasion (66). It is postulated to play a role in breast-tumor progression in association with increased hypoxia and reactive oxygen species (ROS) by promoting angiogenesis (67). Increased hypoxia and ROS also occur in OPLs and HNOSCCs, and might explain the observed changes in S100A7 expression here. Reciprocal negative regulation between S100A7 and .beta.-catenin signaling has been shown to play an important role in tumor progression of OSCC.

[0524] Thus, proteomic analyses of OPLs revealed the integrated importance of alterations in multiple cellular processes and suggested novel links between inflammation and premalignancy, some of which may serve as potential chemopreventive/therapeutic targets. Validation of the panel of OPL biomarkers in larger studies will ascertain its clinical utility and long-term patient follow up will evaluate the potential of these biomarkers for predicting the risk of malignant transformation in OPLs.

Example 14

Tissues

[0525] Following institutional human ethics committee approval, 51 anonymized HNOSCCs and 39 non-malignant head-and-neck tissues dating from 2002 and 2006 were retrieved from the Research Tissue Bank at All India Institute of Medical Sciences, New Delhi, India. The tissue specimens, surgically resected human HNOSCCs and non-malignant tissues (taken from a distant site) had been collected from patients undergoing curative surgery (with prior written patient consents). After excision, tissues were immediately snap-frozen in liquid nitrogen and stored at -80.degree. C. in the Research Tissue Bank. One piece from each patient was collected in 10% formalin and embedded in paraffin for histopathological analysis; the rest was banked. Clinical and pathological data were recorded in a pre-designed performa; these included clinical TNM staging (tumor, node, and metastasis classification of malignant tumors of the International Union Against Cancer (UICC)) (97), site of the lesion, histopathological differentiation, age, and gender.

Example 15

Follow-Up Study

[0526] Fifty-one HNOSCC patients who underwent treatment of primary HNOSCC between 2002 and 2006 were investigated and evaluated in the head-and-neck cancer follow-up clinic. Survival status of the patients was verified and regularly updated from the records of the Tumor Registry, Institute Rotary Cancer Hospital, as of May, 2007. Patients were monitored for a maximum period of 42 months. As per the hospital protocol, HNOSCC patients with T.sub.1 and T.sub.2 tumors were treated with radical radiotherapy or surgery alone, whereas the majority of patients with T.sub.3 and T.sub.4 diseases were treated using a combination of radical surgery followed by postoperative radical radiotherapy. The patients were revisited clinically on a regular basis and the time to recurrence was recorded. If a patient died, the survival time was censored at the time of death; the medical history, clinical examination, and radiological evaluation were used to determine whether the death had resulted from recurrent cancer (relapsing patients) or from any other cause.

[0527] Disease-free survivors were defined as patients free from clinical and radiological evidence of local, regional, or distant relapse at the time of the last follow-up. Loco-regional relapse/death was observed in 17 of 51 (30%) patients monitored in this study. Thirty-four patients who did not show recurrence were alive until the end of the follow-up period. Only disease-free survival was evaluated in the present study, as the number of deaths due to disease progression did not allow a reliable statistical analysis. Disease-free survival was expressed as the number of months from the date of surgery to the loco-regional relapse.

Example 16

Immunohistochemistry

[0528] Paraffin-embedded sections (5 .mu.m thick) of human oral normal tissues (n=39) and HNOSCCs (n=51) were collected on gelatin-coated slides. For histopathological analysis, representative sections were stained with hematoxylin and eosin, whereas immunostaining was performed on serial sections as described previously (23, 98). Briefly, the sections were deparaffinized in xylene, hydrated, and pretreated in a microwave oven in citrate buffer (0.01 M (pH=6.0)) for antigen retrieval. The sections were incubated with hydrogen peroxide (0.3% v/v) in methanol for 20 min to quench the endogenous peroxidase activity. Non-specific binding was blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS, 0.01 M, pH=7.2) for 1 h. Thereafter, slides were incubated with the primary antibody, (1 .mu.g/ml) (anti-14-3-3.sigma., goat polyclonal antibody, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) for 16 h at 4.degree. C. and washed with PBS. The primary antibody was detected using the streptavidin-biotin complex (Dako LSAB plus kit, Dako, Copenhagen, Denmark) and diaminobenzidine as the chromogen. All incubations were performed at room temperature in a moist chamber. Slides were washed with 3.times. Tris-buffered saline (TBS, 0.1 M, pH=7.4) after every step. Finally, the sections were counterstained with Mayer's hematoxylin and mounted with DPX mountant. In negative controls, the primary antibody was replaced by non-immune mouse IgG of the same isotype to ensure specificity.

Example 17

Positive Criteria for Immunohistochemical Staining

[0529] Immunopositive staining was evaluated in five areas of the tissue section. For stratifin expression, sections were scored as positive if epithelial cells showed immunopositivity in the cytoplasm, plasma membrane, and/or nucleus as evaluated by two independent scorers blinded to the clinical outcome (the slides were coded and the scorers did not have prior knowledge of the local tumor burden, lymphonodular spread, and grading of the tissue samples). These sections were rated based on the percentage of cells showing immunopositivity as follows: 0, <10%; 1, 10-30%; 2, 30-50%; 3, 50-70%; and 4, >70%. Sections were also rated on the basis of stain intensity as follows: 0, none; 1, mild; 2, moderate; 3, intense, as described by Perathoner et al. (99). Finally, a total score (ranging from 0 to 7) was obtained by adding the scores of percentage positivity and intensity. The sections were considered positive if the total score was .gtoreq.5 (23).

Example 18

Cell Culture

[0530] Human oral squamous carcinoma cell line, HSC2, was used in this study. Cells were grown in monolayer cultures in Dulbecco's modified eagle medium (DMEM-F12) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, Mo.), 100 g/ml streptomycin and 100 U/ml Penicillin in a humidified incubator (5% carbon-dioxide, 95% air) at 37.degree. C. as described (23).

Example 19

Co-Immunoprecipitation

[0531] Co-immunoprecipitation (Co-IP) assays were carried out as described earlier (100). Briefly, oral cancer cells, HSC2, were rinsed in ice-cold PBS and lysed in lysis buffer. Lysates were incubated on ice for 30 min and cell debris was removed by centrifugation. Lysates were pre-cleared by adding 20:1 of Protein A-Sepharose (GE Healthcare Biosciences, Sweden), followed by overnight incubation with polyclonal stratifin, YWHAZ, and NF.kappa.B antibodies, or monoclonal .beta.-catenin and Bcl-2 antibodies (1:200 dilution) (Santa Cruz Biotechnology, CA) on a rocker at 4.degree. C. Immunocomplexes were pulled down by incubating with Protein A-Sepharose for 2 h at 4.degree. C., followed by washing with 4.times. ice-cold lysis buffer to eliminate non-specific interactions. In negative controls, the primary antibody was replaced by non-immune mouse IgG of the same isotype to ensure specificity. Protein A-Sepharose-bound immunocomplexes were then resuspended in Laemelli sample buffer (10 mM Tris, 10% v/v glycerol, 2% w/v SDS, 5 mM EDTA, 0.02% bromophenol blue, and 6% .beta.-mercaptoethanol, pH=7.4), boiled for 5 min, and analyzed by Western blotting using specific antibodies.

[0532] The proteins were electro-transferred onto polyvinylidenedifluoride (PVDF) membrane. After blocking with 5% non-fat powdered milk in TBS (0.1 M, pH=7.4), blots were incubated with anti-14-3-3a antibody (1:200 dilution) at 4.degree. C. overnight. alpha-Tubulin served as a control for protein loading and was determined with mouse monoclonal anti-alpha-tubulin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.). Membranes were incubated with secondary antibody, HRP-conjugated goat/mouse anti-IgG (Dako CYTOMATION, Denmark), diluted at an appropriate dilution in 1% BSA, for 2 h at room temperature. After each step, blots were washed with 3.times. Tween (0.2%)-TBS (TTBS). Protein bands were detected by the enhanced chemiluminescence method (Santa Cruz Biotechnology, CA) on XO-MAT film.

Example 20

Statistical Analysis

[0533] The immunohistochemical data were subjected to statistical analysis using SPSS 10.0 software. The relationship between the protein expression and clinicopathological parameters were tested by Chi-Square and Fischer's exact test. Two sided p-values were calculated and p.ltoreq.0.05 was considered to be significant. Box plots were prepared to determine the distribution of total score of stratifin expression in HNOSCCs and non-malignant tissues. The correlation of stratifin and or YWHAZ staining with patient survival was evaluated using life tables constructed from survival data with Kaplan-Meier plots.

[0534] Provided below is a summary of the results obtained by the inventors in connection with the experiments of Examples 14-20:

[0535] To determine the clinical significance of stratifin and YWHAZ in head-and-neck tumorigenesis, their expressions were analyzed in HNOSCCs (51 cases) and non-malignant tissues (39 cases) using immunohistochemistry. Significant increase in stratifin expression was observed in the HNOSCCs as compared to the non-malignant mucosa (p=0.03, Odd's Ratio (OR)=3.8, 95% CI=1.6-9.2). Kaplan-Meier survival analysis reveals correlation of stratifin overexpression with reduced disease-free survival of HNOSCC patients (p=0.06). The most intriguing finding is the significant decrease in median disease-free survival (13 months) in HNOSCC patients showing overexpression of both stratifin and YWHAZ proteins, as compared to patients that did not show overexpression of these proteins (median disease-free survival=38 months, p=0.019), underscoring their utility as adverse prognosticators for HNOSCCs. Co-immunoprecipitation assays show the formation of stratifin-YWHAZ heterodimers in HNOSCC and interactions with NF.kappa.B, .beta.-catenin, and Bcl-2 proteins. These results indicate the involvement of these proteins in the development of head-and-neck cancer and their association with adverse disease outcome. The amino acid sequences of stratifin and YWHAZ with peptides identified by MS and MS/MS are given in FIGS. 9A and 9B, respectively.

[0536] Immunohistochemical Analysis of Stratifin in HNOSCCs and Non-Malignant Tissues.

[0537] Results of the immunohistochemical analysis of stratifin expression in HNOSCCs and non-malignant mucosa, and the relationship with clinicopathological parameters are summarized in Table 8. Chi-Square analysis shows significant increase in stratifin expression in HNOSCCs as compared to non-malignant mucosa (p=0.03, OR=3.8, 95% CI=1.6-9.2). In histologically normal oral tissues, 31% of the cases show weak immunostaining of stratifin (FIG. 10a). Increased stratifin expression was observed in 63% of HNOSCCs. Intense nuclear/membranous staining, in addition to cytoplasmic staining, was observed in the epithelial cells of HNOSCCs (FIG. 10b). No immunostaining was observed in tissue sections used as negative controls where the primary antibody was replaced by isotype specific IgG (FIG. 10c). No significant correlation was observed between stratifin overexpression and clinicopathological parameters including age, gender, histological differentiation, tumor stage, and nodal status of HNOSCCs (Table 8). Increased expressions of stratifin were observed in HNOSCCs with a median score of 6 (range 4-7) as compared to non-malignant (histologically normal) oral tissues median score of 5 (range 4-6) shown in the box plot analysis in FIG. 11.

[0538] Co-Immunoprecipitation.

[0539] To determine the functional significance of stratifin in head-and-neck carcinogenesis, the inventors identified its binding partners in oral cancer cells, HSC2, using co-IP assays followed by Western blotting. IP of stratifin reveals its binding to YWHAZ NF.kappa.B, .beta.-catenin, and Bcl-2 proteins as shown in FIG. 12a. Reverse IP assay using specific antibodies for these proteins followed by Western blotting confirmed their binding to stratifin (FIG. 12b). No band was observed in the immunoblot analysis of the negative controls. It is noteworthy that all these proteins--stratifin, YWHAZ, NF.kappa.B, .beta.-catenin and Bcl-2--have 14-3-3 binding motif, Mode 1, as previously reported by the inventors (39).

[0540] Association of Stratifin and YWHAZ Expression with Disease Outcome.

[0541] Kaplan-Meier Survival analysis reveals reduced disease-free survival for HNOSCC patients overexpressing stratifin (p=0.06, FIG. 13a). The median disease-free survival was 19 months in HNOSCC patients showing stratifin overexpression as compared to 38 months in HNOSCC patients who did not. Patients with YWHAZ-positive tumors had a shorter disease-free survival (median=23 months) than YWHAZ-negative tumors (median=35 months; p=0.08, FIG. 13b). Most remarkably, HNOSCC patients showing overexpressions of both stratifin and YWHAZ have a significantly decreased median disease-free survival of 13 months (p=0.019, FIG. 13c) in HNOSCCs, as compared to patients showing no overexpression of these two proteins (median=38 months), underscoring the utility of these proteins as adverse prognosticators for HNOSCCs.

[0542] Without being bound by theory, the results obtained in the experiments of Examples 14-20 are discussed below:

[0543] This investigation is one of the very few studies that demonstrate the prognostic utility of candidate biomarkers identified using MS-based proteomics. Recently, a comparison of protein profiles in tumor-distant head-and-neck tissues with clinical outcomes was reported to reveal a significant association between aberrant profiles and tumor-relapse events, suggesting that proteomic profiling in conjunction with protein identification may have significant predictive power for clinical outcome (12). The concept of using a panel of biomarkers for the purpose of improved diagnostics has taken hold in recent years. For example, DeSouza et al. (21) and Dube et al. (101) demonstrated that the use of a panel of three biomarkers-chaperonin 10, pyruvate kinase M2, and .alpha.-1-antitrypsin-markedly increases the sensitivity and specificity for differentiating between endometrial carcinoma and non-malignant endometrial tissues. For head-and-neck cancers, Example 1 determined that a panel of biomarkers-stratifin, YWHAZ, and S100 A7-performs better than any of the individual biomarkers for the detection of HNOSCC (23). In this current study, it is found that a panel of two biomarkers-stratifin and YWHAZ-shows promise as prognostic markers for HNOSCCs. Although not wishing to be bound by any particular theory, the enhanced performance of the combination of stratifin and YWHAZ versus either protein individually, in prognosing the clinical outcome of HNOSCCs, can be understood on the basis of their biological functions detailed below.

[0544] Results of in vitro studies demonstrate the formation of stratifin-YWHAZ heterodimers and binding of stratifin to NF.kappa.B, .beta.-catenin, and Bcl-2, implicating stratifin's involvement in many cellular processes associated with tumorigenesis. Similar to the present invention, Bhawal et al. (96) have very recently reported increased expression of the stratifin transcript and protein in OSCCs. These results are in accordance with findings of Chen et al. (45) and Lo et al. (15, 44) who reported overexpression of stratifin in OSCCs. In addition, Lo et al. (15) demonstrated ten-fold increases in stratifin expression in HPV18-positive OSCCs in comparison with HPV18-negative OSCCs. It is of note that all these studies are on Asian populations. By contrast, studies on European populations reported decreased expression of stratifin in HNSCCs (12). If this geographical difference stands up to further scrutiny, it points to the importance of genetic and/or risk factors in developing HNOSCCs. Furthermore, epigenetic inactivation of stratifin has been shown to be associated with p16 gene silencing and HPV negativity in OSCCs (102). Thus these results suggest different mechanisms at work in HNOSCC tumorigenesis exhibiting overexpression of stratifin and tumorigenesis that does not. Whether these different mechanisms can be attributed to the presence or absence of HPV infection and/or differences in risk factors, such as smoking and drinking in the European and Western populations, and chewing of betel quid and/or tobacco in the Asian population, remains to be determined.

[0545] Overexpression of stratifin has been observed in other human cancers. Perathoner et al. (99) suggested that stratifin overexpression promotes tumor proliferation and/or prevents apoptotic signal transduction in colorectal carcinoma. Samuel et al. (103) demonstrated the role of stratifin in prevention of apoptosis by influencing the sub-cellular distribution of the pro-apoptotic protein, Bax, in colorectal cancer cells. Deletion of stratifin has been correlated with increased sensitivity of colorectal cells to doxorubicin. Similarly, Guweidhi et al. (104) proposed an anti-apoptotic role for stratifin in pancreatic cancer cells by inhibiting bad-mediated apoptosis. Liu et al. (105) showed that elevated stratifin expression contributes considerably to the observed drug resistance in MCF7/AdVp3000 cells. Stratifin has been shown to be a pivotal MDM2 regulator, involved in blocking a variety of MDM2 activities, including MDM2-mediated cytoplasmic localization of p53. Stratifin overexpression leads to destabilization of MDM2 by enhancing its self-ubiquitination and, thereby, stabilizing the cellular p53 (106). In previous studies, the inventors reported that p53 mutations are infrequent in OSCCs in the Indian population, despite stratifin overexpression; a currently unknown mechanism must be involved in stabilizing p53 in these OSCC patients (107, 108). The present inventors have also reported overexpression of MDM2 and cyclinD1 in OSCCs (109). Investigation of the relationship between stratifin overexpression, p53 stabilization, MDM2 and cyclinD1 expressions in HNOSCCs is currently underway.

[0546] The inventors, in a recent study aimed at delineation of early changes in expression of proteins in hyperplasia, demonstrated increased expressions of NF.kappa.B and COX-2 in early pre-malignant stages of the development of oral cancer and sustained elevation along the tumorigenic pathway (90). Furthermore, increased expression of YWHAZ in different stages of the development of OSCC and YWHAZ's involvement in cell signaling pathways involved in inflammation, cell proliferation and abrogation of apoptosis during oral carcinogenesis (39) was shown. Herein the inventors extend these findings by demonstrating the binding of stratifin with YWHAZ, thus suggesting the formation of stratifin-YWHAZ heterodimers and binding to NF-kB in oral cancer. These findings are supported by the study of Aguilera et al. (91) which showed the requirement of 14-3-3 proteins for efficient export of the p65 sub-unit of NF-kB. Taken together with the inventors' earlier findings of YWHAZ it is hypothesized that 14-3-3 proteins may be an important link between chronic inflammation and cancer that warrants further investigation.

[0547] The Co-IP results show stratifin binding also with .beta.-catenin and Bcl-2 proteins. Earlier, the inventors showed that these proteins interact with YWHAZ thereby supporting the hypothesis that these complexes may be responsible for altered functions of stratifin. Fang et al. (92) recently showed that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates .beta.-catenin at Ser-552 in vitro and in vivo, causing its dissociation from cell-cell contacts and accumulation in both the cytosol and nucleus, and enhancing its interaction with YWHAZ via a binding motif containing Ser-552. This phosphorylation of .beta.-catenin by AKT increases .beta.-catenin's transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of .beta.-catenin plays a critical role in tumor invasion and development. The oncogenic role of YWHAZ has been proposed in a recent study using siRNA for knocking down its expression in cancer cells (93). Down-regulation of YWHAZ sensitizes cells to stress--induced apoptosis and JNK/p38 signaling; in addition, it enforces cell-cell contacts and expression of adhesion proteins. YWHAZ's oncogenic properties is also supported by a Web-based meta-analysis (Onco-mine) that reveals its overexpression in various types of carcinomas (39, 93). To unravel the functional significance of 14-3-3 proteins in tumor development, the present inventors have investigated the functional significance of the interactions between stratifin and YWHAZ, and their respective roles in the development and progression of HNOSCC. All indications are that targeting specific 14-3-3 isoforms may serve as a plausible strategy for cancer therapy.

[0548] Hence, stratifin is overexpressed in HNOSCCs relative to normal tissues. Increased concomitant expression of stratifin and YWHAZ serves as adverse prognosticator in HNOSCCs and underscores the importance of these proteins in head-and-neck tumorigenesis. Increased expression of stratifin forming stratifin-YWHAZ heterodimers and binding to NF.kappa.B, .beta.-catenin, and Bcl-2 proteins suggest the implication of these complexes in diverse cellular processes in head-and-neck carcinogenesis. It is submitted that targeting the stratifin-YWHAZ heterodimer, using small molecule modulator/peptide inhibitor that intervenes with 14-3-3 client protein interactions, would serve as a plausible therapeutic strategy for head-and-neck cancer.

Example 21

Patients and Clinicopathological Data Collection and Tumor and Biopsy Specimens

[0549] The Institutional Human Ethics Committee of the All India Institute of Medical Sciences (AIIMS), New Delhi, India, approved this study prior to its commencement. Tissue specimens were obtained from diagnostic or therapeutic procedures from 199 patients with oral leukoplakia (with no dysplasia (n=115) or with dysplasia (n=84) (Table 9A)) attending the Outpatient Clinic of the Departments of Surgical Disciplines and Otolaryngology, AIIMs, and from 100 HNOSCC patients undergoing curative cancer surgery during the period 2002-2007, after obtaining patient consents. Wherever possible non-malignant tissues (n=30) were taken from a site distant from the surgically resected HNOSCC patients. Non-malignant normal oral tissues (n=25) were also collected from the patients attending the Outpatient Department of Dental Surgery for tooth extraction. Taken together, these 55 non-malignant oral tissues with histological evidence of normal epithelium constituted the normal group. After excision, tissues were immediately snap-frozen in liquid nitrogen and stored at -80.degree. C. in the Research Tissue Bank till further use; one part of the tissue was collected in 10% formalin and embedded in paraffin for histopathological and immunohistochemical analyses.

[0550] The histopathological assessment scoring was based on the architectural and cytological changes of grading epithelial dysplasia described in the WHO classification and recently reviewed (3). For each case, the pathologist recorded the grade and details of the criteria on which the decision was based. Leukoplakic lesions were classified into two groups: (a) lesions with no dysplasia, (b) lesions with dysplasia.

[0551] Histologically confirmed oral normal epithelia, leukoplakia with evidence of no dysplasia or with dysplasia, and HNOSCCs as revealed by H&E staining were used for immunohistochemistry (32). Patient demographic, clinical, and pathological data were recorded in a pre-designed form as described previously (32). The information documented included clinical TNM staging (tumor, node, metastasis based on the Union International Center le Cancer TNM classification of malignant tumors 1998), site of the lesion, histopathological differentiation, age, gender, and tobacco consumption habits.

Example 22

Follow-Up Study

[0552] One hundred patients with oral leukoplakia who underwent treatment between 2002-2005 were followed up in the head-and-neck follow-up clinic at regular time intervals with the maximum follow-up period included in this study being 36 months. All small leukoplakic lesions (size < or =4.times.4 cm) were completely excised, while incisional biopsy was done for the large diffuse lesions (size >4.times.4 cm). The patients were followed every six months, and the status of each lesion was defined and recorded using following the criteria: (a) static: if it was within .+-.2 mm size on the largest diameter; (B) progressed: if it had grown more than 2 mm from the original (size of the residuam, if partially excised); and (c) regressed: if it was reduced in size by more than 2 mm from the original residuam. For the lesions that were excised completely-disease progression was defined as development of a new lesion after excision of the primary lesion at the same site, or at another site in the oral cavity. The lesions that had progressed as per the above mentioned criteria were re-biopsied and leukoplakic lesions with dysplasia were excised.

[0553] Seventy-seven HNOSCC patients who underwent treatment from 2002-2007 were also investigated and evaluated in the head-and-neck cancer follow-up clinic at regular time intervals. Survival status of the HNOSCC patients was verified and updated from the records of the Tumor Registry, Institute Rotary Cancer Hospital, AIIMs, as of May 2008. HNOSCC patients were monitored for a maximum period of 76 months. As per the hospital protocol, HNOSCC patients with T.sub.1 and T.sub.2 tumors were treated with radical radiotherapy or surgery alone, whereas the majority of patients with T.sub.3 and T.sub.4 diseases were treated by radical surgery followed by postoperative radical radiotherapy. The patients were revisited clinically on a regular basis and the time to recurrence was recorded.

[0554] If a patient died, the survival time was censored at the time of death; the medical history, clinical examination, and radiological evaluation were used to determine whether the death had resulted from recurrent cancer (relapsing patients) or from any other causes. Disease-free survivors were defined as patients free from clinical and radiological evidence of local, regional, or distant relapse at the time of the last follow-up. Loco-regional relapse/death was observed in 61 of 77 (79%) patients monitored during the follow-up. Sixteen patients who did not show recurrence were alive until the end of the follow-up period. Only disease-free survival was evaluated in the present study, as the number of deaths due to disease progression did not allow a reliable statistical analysis. Disease-free survival was expressed as the number of months from the date of surgery to loco-regional relapse.

Example 23

Immunohistochemistry

[0555] Paraffin-embedded sections (5 .mu.m) of human oral non-malignant tissues (n=55), leukoplakic lesions (with no dysplasia (n=115) or with dysplasia (n=84)) and HNOSCCs (n=100) were collected on gelatin-coated slides. Immunohistochemistry conditions were optimized and evaluated by three of the inventors. In brief, the sections were deparaffinized in xylene, hydrated in gradient alcohol, and pretreated in a microwave oven for 10 min in Tris-EDTA buffer (0.01 M, pH=9) for antigen retrieval. The sections were incubated with hydrogen peroxide (0.3% v/v) in methanol for 30 min to quench the endogenous peroxidase activity, followed by blocking with 1% bovine serum albumin (BSA) to preclude nonspecific binding. Thereafter, the slides were incubated with mouse monoclonal anti-hnRNPK antibody (1 .mu.g/ml, ab23644, Abcam Inc., Cambridge, Mass.) for 16 h at 4.degree. C. The primary antibody was detected using the streptavidin-biotin complex with the Dako LSAB plus kit (Dako CYTOMATION, Glostrup, Denmark) and diaminobenzidine as the chromogen (25). All procedures were carried out at room temperature unless otherwise specified. Slides were washed with Tris-buffered saline (TBS, 0.1 M, pH=7.4), 3-5 times after every step. Finally, the sections were counterstained with Mayer's hematoxylin and mounted with D.P.X. mountant. In the negative control tissue sections, the primary antibody was replaced by isotype-specific non-immune mouse IgG. A section from colorectal cancer tissue was used as a positive control in each batch of immunohistochemistry. The sections were evaluated by light microscopic examination.

Example 24

Evaluation of Immunohistochemical Staining

[0556] Each slide was evaluated for hnRNPK immunoreactivity using a semiquantitative scoring system for both staining intensity and the percentage of positive epithelial cells. Immunopositive staining was evaluated in randomly selected five areas of the tissue section. For hnRNPK protein expression, sections were scored as positive if epithelial cells showed immunopositivity in the nucleus/cytoplasm when observed independently by three of the inventors, who were blinded to the clinical outcome. (The slides were coded and the scorers did not have prior knowledge of the local tumor burden, lymphonodular spread, and grading of the tissue samples.) The tissue sections were scored based on the % of immunostained cells as: 0-10%=0; 10-30%=1; 30-50%=2; 50-70%=3 and 70-100%=4. Sections were also scored semi-quantitatively on the basis of staining intensity as negative=0; mild=1; moderate=2; intense=3 (17). Finally, a total score was obtained by adding the score of percentage positivity and intensity.

Example 25

Statistical Analyses

[0557] The immunohistochemical data were subjected to statistical analyses using the SPSS 10.0 software (Chicago). Sensitivity and specificity were calculated and quantified using receiver-operating characteristic (ROC) analyses. The predictive value (PV) describes the proportion of correctly classified cases. Based on sensitivity and specificity values for hnRNPK, a cutoff.gtoreq.5 was defined as positive criterion for hnRNPK immunopositivity for statistical analyses. The relationships between hnRNPK protein expression and clinicopathologic parameters were tested using Chi-Square and Fischer's exact test. Two-sided p values were calculated and p<0.05 was considered to be significant.

[0558] For the follow-up study of 100 leukoplakia cases, let T denote the failure time, i.e., the first time the progression is diagnosed after excision of the leukoplakic lesions. For these data, the positive and negative predictive values as functions of time are defined as follows:

PV.sub.nuclear(t)=Prob(T.ltoreq.t AND Progression|hnRNPK(nuclear).gtoreq.5);

PV.sub.nuclear(t)=Prob(T>t OR No Progression|hnRNPK(nuclear)<5);

0.ltoreq.t.ltoreq.36,

and, analogously, for cytoplasmic hnRNPK. Similarly, PPV and NPV were calculated for recurrence in HNOSCCs (where t runs from 0 to 76 months). These probabilities are estimated from the observed accumulated incidences over the respective time periods. The correlation of hnRNPK staining with patient survival was evaluated using life tables constructed from survival data with Kaplan-Meier plots.

Example 26

Immunoblot Analysis of hnRNPK in Oral Tissues

[0559] Whole-cell lysates were prepared from oral non-malignant, leukoplakia and HNOSCC tissues by homogenization in lysis buffer containing 50 mM Tris-Cl (pH=7.5), 150 mM NaCl, 10 mM MgCl.sub.2, 1 mM ethylenediamine tetraacetate (EDTA, pH=8.0), 1% Nonidet P-40, 100 mM sodium fluoride, 1 mM phenylmethylene sulfonylfluoride (PMSF) and 2 .mu.l/ml protease inhibitor cocktail (Sigma) as previously described (32). Protein concentration was determined using the Bradford reagent (Sigma) and equal amounts of proteins (80 .mu.g/lane) were resolved on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The proteins were then electro-transferred onto polyvinylidenedifluoride (PVDF) membrane. After blocking with 5% non-fat powdered milk in Tris-buffered saline (TBS, 0.1 M, pH=7.4), blots were incubated with anti-hnRNPK monoclonal antibody (1 .mu.l/ml, Abcam Inc., Cambridge, Mass.) at 4.degree. C. overnight. Protein abundance of actin (goat polyclonal antibody, Santa Cruz Biotechnology, CA) served as a control for protein loading in each lane. Membranes were incubated with HRP-conjugated anti-mouse/goat secondary antibody, G (DAKO Cytomation, Glostrup, Denmark), diluted at an appropriate dilution in 1% BSA, for 2 h at room temperature. After each step, blots were washed three times with Tween (0.1%)-Tris-buffer saline (TTBS). Protein bands were detected by the enhanced chemiluminescence method (ECL, Santa Cruz Biotechnology, CA) on XO-MAT film.

Example 27

Reverse Transcription--PCT

[0560] Representative frozen tissue specimens of histologically confirmed oral normal tissues, leukoplakia and HNOSCCs were used for extraction of total RNA using the TRI reagent (Sigma, Mo.) as previously described (29). First-strand cDNA was synthesized using 2 .mu.g RNA with oligo dT as the primer with MMLV reverse transcriptase. PCR was carried out using hnRNPK specific primers forward-(5'AGCAGAGCTCGGAATCTTCCTCTT3' SEQ ID NO: 749) and reverse-(5'ATCAGCACTGAAACC AAC CA TGCC3' SEQ ID NO: 750) (Accession No. NM 002140). 20 .mu.l of each PCR product was used for electrophoresis on a 1.2% agarose gel stained with ethidium bromide. The gel was visualized with UV light and photographed.

[0561] Provided below is a summary of the results obtained by the inventors in connection with the experiments of Examples 21-27:

[0562] Identification of hnRNP K in Oral Premalignant Lesions by Mass Spectrometry.

[0563] The tandem MS spectra for the two peptides, A: DLAGSIIGK (SEQ ID NO: 368) and B: IDEPLEGSEDR (SEQ ID NO: 369), identified from hnRNPK in iTRAQ-labeled oral leukoplakia with dysplasia by LC-MS are given in FIGS. 21A and 21B, respectively. In each case, the topmost panel shows the confidence and score for each peptide, the sequence, theoretical mass, theoretical mass/charge ratio, charge state and difference between experimental and theoretical mass of the peptide. The expected product ion masses highlighted in the table in the middle are matched with the detected peaks in each spectrum. The lowermost panel in each case shows the location of the peptide within the context of the intact protein sequence.

[0564] Immunohistochemical Analysis of hnRNP K Expression in Oral Lesions.

[0565] To determine the clinical significance of hnRNPK protein in head-and-neck tumorigenesis, its expression was analyzed in different stages of HNOSCC development by means of immunohistochemistry using a specific monoclonal antibody. Of the 55 normal tissues analyzed, 51 cases (93%) showed faint or no detectable hnRNPK immunostaining in the nucleus/cytoplasm of the epithelial cells (Table 9a, FIG. 14a). However, four normal tissues showed nuclear expression of hnRNPK, as per positivity criteria defined in the Methods section; all these four tissues were obtained from a site adjacent to the tumor from HNOSCC patients, and thus the increased hnRNPK expression in these histological normal tissues may be a manifestation of field cancerization.

[0566] Chi square trend analysis showed significant increase in nuclear staining of hnRNPK in different stages of head-and-neck/oral tumorigenesis (normal, leukoplakia and HNOSCCs; Table 9a, p.sub.trend<0.001). Of the 199 leukoplakias analyzed, 141 cases (71%) showed significant increase in nuclear hnRNPK immunostaining in comparison with the normal tissues (p<0.001, Odds ratio (OR)=30.9, 95% CI=10.7-89.7, Table 9a). Oral leukoplakia is a clinical terminology and histologically these lesions are classified into leukoplakia with no dysplasia or with dysplasia for disease management. Of the 199 leukoplakias, 115 cases showed no histological evidence of dysplasia; 78/115 (68%) cases showed significant increase in nuclear hnRNPK immunoreactivity in comparison with the normal tissues (p<0.001, OR=26.8, 95% CI=9.1-79.9, Table 1a and FIG. 14b). Importantly, progressive increase in nuclear expression of hnRNPK was observed in 75% dysplasias (63 of 84 cases, FIG. 14d) in comparison with normal tissues (p<0.001, OR=38.2, 95% CI=11.7-113.1).

[0567] It is noteworthy that 26 of 199 leukoplakia cases showed cytoplasmic localization of hnRNPK, in addition to its nuclear expression, as shown in FIGS. 14c and 14e, respectively. Chi square analysis revealed that leukoplakias showing cytoplasmic hnRNPK staining are at 4.3-fold higher risk for cancer development (p<0.001, 95% CI=2.2-7.2, Table 9a). The majority of HNOSCCs (78%) showed nuclear localization of hnRNPK in tumor cells (FIG. 140. In addition to nuclear staining, intense hnRNPK staining was also observed in the cytoplasm of tumors cells in 38 of 100 HNOSCCs analyzed (FIG. 14g).

[0568] The clinicopathological parameters of HNOSCCs patients and their correlation with nuclear/cytoplasmic expression of hnRNPK are shown in Table 9a. Increased cytoplasmic staining of hnRNPK showed a significant association with de-differentiation of HNOSCCs (p=0.001). Furthermore, no significant correlation between nuclear/cytoplasmic hnRNPK and other clinicopathological parameter, including gender, tumor size, nodal status or tobacco consumption of HNOSCC patients was found. No immunostaining was observed in tissue sections used as negative controls where the primary antibody was replaced by isotype specific IgG (FIG. 14h), while the positive control (colorectal cancer) showed nuclear expression of hnRNPK protein (data not shown).

[0569] Evaluation of hnRNP K as Potential Biomarker for Diagnosis and/or Prognosis.

[0570] Receiver-operating characteristic (ROC) curve analysis was used to determine the potential of hnRNPK as a biomarker to distinguish leukoplakia and HNOSCCs from normal oral epithelium. The values for area-under-the-curve (AUC) were 0.822, 0.872 and 0.869 for leukoplakia without dysplasia (FIG. 15a), with dysplasia (FIG. 15b), and cancer (FIG. 15c), respectively, with respect to normal oral tissues based on the total score for nuclear immunostaining (Table 9b). The positive predictive values (PPV) were 92.7, 92.3, and 92.3, respectively, for nuclear immunostaining in the three groups. Similarly, ROC analysis was used for determination of AUC and PPV for cytoplasmic hnRNPK staining in all these three groups as shown in Table 9b and FIG. 16a-c.

[0571] hnRNP K Expression as a Predictor of Disease Progression and Prognosis.

[0572] Significantly, the follow-up data sets of 100 leukoplakia patients for three years and 77 HNOSCC cases for seven years were used to assess the prognostic value of hnRNPK for predicting disease progression in patients with leukoplakia and cancer recurrence in HNOSCC patients after completion of primary treatment. Both positive predictive and negative predictive values of the prognostic test are of paramount importance in this context, with the former to correctly identify cases that need early intervention, and with the latter to gauge, in the most accurate way, where such intervention with its monumental personal impacts can and should be avoided.

[0573] FIG. 16 shows the estimated PPVs and NPVs for nuclear and cytoplasmic hnRNPK expression as prognostic biomarkers. FIGS. 16a and 16b for disease progression of leukoplakia; and for cancer recurrence in HNOSCC patients (FIGS. 16c and 16d). Even though the absolute numbers that give rise to these estimates are only moderate (23 cases), cytoplasmic hnRNPK expression in leukoplakias (12 cases) appears to be a promising biomarker for disease progression, with PPV.sub.cyto(18 months)=44.4 and PPV.sub.cyto (36 months)=66.7 (12 disease progressions). These values are compared with the PPVs of overall hnRNPK expression (both nuclear and cytoplasmic immunopositivity--PPV.sub.overall), PPV.sub.overall (18 months)=15.0 and PPV.sub.overall (36 months)=23.0. These result in estimated ratios or relative positive predictive values of cytoplasmic hnRNPK expression for disease progression of 2.96 and 2.90 for 18 and 36 months, respectively.

[0574] On the other hand, high nuclear hnRNPK expression (22 cases) appears not, by itself, to define a biomarker of high prognostic value (PPV.sub.nuc (36 months)=28.2, compared with PPV.sub.overall (36 months)=23). Of note, the negative predictive value of cytoplasmic hnRNPK expression in leukoplakias is very high (NPV.sub.cyto (36 months)=86.6). Based on the inventors' data, the additional prognostic value which hnRNPK, in either its nuclear or cytoplasmic expression, provides for predicting (PPV) or excluding (NPV) cancer recurrence in HNOSCC patients is: PPV.sub.nuc(76 months)/PPV.sub.overall(76 months)=68.9/61.0; PPV.sub.cyto(76 months)/PPV.sub.overall(76 months)=81.3/61.0; NPV. (76 months)/NPV.sub.overall (76 months)=68.9/39.0; and NPV.sub.cyto (76 months)/NPV.sub.overall (76 months)=53.3/39.0. Based on these analyses, the most significant improvement over clinicopathological criteria that cytoplasmic hnRNPK appears to offer as a marker is in predicting disease progression in leukoplakia patients and prognosis of HNOSCCs.

[0575] While PP Vs. and NP Vs. quantify the estimated predictive power of the marker, the strength of the statistical association of hnRNPK expression with poor prognosis was assessed by Kaplan-Meier survival analysis. Log-rank test showed significantly reduced time for disease progression (p<0.001; median time=17 months) in leukoplakia patients showing increased cytoplasmic expression of hnRNPK (18 cases), as compared to the median time of 35 months in the patients showing no/faint immunostaining of hnRNPK in the cytoplasm (FIG. 17a). Leukoplakia patients showing intense nuclear hnRNPK expression (78 cases) had poor prognosis, as compared to patients who did not show increased nuclear hnRNPK (p=0.004, FIG. 17b), though there was no significant difference in median time for disease progression. The inventors' findings clearly underscore the potential of cytoplasmic hnRNPK as a marker for predicting disease progression in leukoplakia patients. Of the 100 leukoplakia patients, 83 cases showed no histological evidence of dysplasia and similar correlations of cytoplasmic and nuclear expression were observed with disease progression for these leukoplakias (FIGS. 4a and 4b, respectively). However, similar statistical analysis could not be carried out for dysplasias due to the small number of cases in this group (17 cases).

[0576] In addition, Kaplan-Meier survival analysis showed significantly reduced disease free survival (p=0.004; median survival 11 months) in HNOSCC patients harboring increased cytoplasmic expression of hnRNPK, as compared to median disease-free survival of 41 months in the patients showing no/faint cytoplasmic hnRNPK immunostaining (FIG. 17c). Similarly, reduced disease-free survival of 14 months was observed in HNOSCC patients showing intense nuclear expression of hnRNPK, as compared to patients who did not show increased nuclear hnRNPK (median survival of 57 months); although this could not reach a statistically significant value of p<0.05 (FIG. 17d). These findings clearly demonstrate the potential of nuclear hnRNPK as a biomarker for diagnosis, and cytoplasmic hnRNPK as a potential marker for predicting poor prognosis of HNOSCCs.

[0577] Immunoblotting and RT-PCR.

[0578] The overexpression of hnRNPK in oral lesions was further validated by immunoblotting and RT-PCR analyses in the same tissue samples as used for immunohistochemical analysis. Immunoblot analysis showed a single intense band of 64 kDa, confirming the increased expression of hnRNPK in oral leukoplakias and HNOSCCs, as compared to the normal tissues (FIG. 18A). RT-PCR analysis demonstrated increased levels of hnRNPK transcripts in the same tissue specimens of leukoplakias and HNOSCCs in comparison with normal tissues (FIG. 18B), thus supporting the immunohistochemical findings and suggesting transcriptional upregulation of hnRNPK in these tissues.

[0579] Without being bound by theory, the results obtained in the experiments of Examples 21-27 are discussed below:

[0580] hnRNPK overexpression in early oral lesions is a very important unique finding of this study, providing clinical evidence to establish its link with progression potential of leukoplakia in support of its proposed role as a transformation-related protein. To the inventors' knowledge, this is the first investigation to demonstrate the clinical application of a candidate biomarker identified using MS-based tissue proteomics in identifying early oral premalignant lesions that may be at high risk of disease progression. The salient findings of the inventors' study are: i) nuclear hnRNPK expression increases progressively from oral normal tissues to hyperplasia, dysplasia and frank malignancy and may serve as a plausible diagnostic marker for HNOSCCs; ii) cytoplasmic accumulation of hnRNPK is significantly increased from leukoplakia to cancer; (iii) aberrant subcellular localization (cytoplasmic accumulation) of hnRNPK is a predictor of disease progression in leukoplakia patients and disease recurrence in HNOSCC patients; iv) cytoplasmic hnRNPK is associated with poor prognosis of HNOSCCs; and v) hnRNPK is transcriptionally upregulated in head and neck tumorigenesis.

[0581] Expression profiling of different cancer types and mechanistic studies have strongly implicated hnRNPK as a key player in human cancers. To the inventors' knowledge, this study is the first report demonstrating increased expression of hnRNPK in oral leukoplakia by immunohistochemistry. The significantly increased nuclear expression of hnRNPK in oral hyperplastic lesions points to this alteration being an early event in the development of premalignant lesions and is in accord with its role as a transcriptional regulator of growth promoting genes such as myc and src and promoter of cell proliferation (111-117).

[0582] The major challenge in oral tumorigenesis is the identification of proteins that may serve as markers to predict high risk leukoplakias for early intervention. Most studies on leukoplakia focus on dysplastic lesions, while knowledge of molecular alterations in oral hyperplasia is meager. As per the existing literature, the malignant transformation potential is often linked to the severity of dysplasia; in comparison the hyperplastic lesions have received less attention, primarily because these lesions often undergo spontaneous regression. However, the lesions that do not regress need identification and biomarkers to predict the risk of malignant transformation.

[0583] In this context, the study assumes importance, because not only does it show aberrant hnRNPK expression as early as in hyperplasia, but the follow-up study also points to the relevance of cytoplasmic hnRNPK in predicting the risk of disease progression in leukoplakia patients with hyperplasia and HNOSCCs. It is noteworthy that studies on molecular analysis of leukoplakia with hyperplasia are very limited, because these patients often do not come to the clinics since their lesions are small and do not pose any overt clinical problem. However, it is extremely important to target this patient population for risk assessment and early intervention for cancer prevention in high risk cases. Hence, the inventors' findings are important and warrant further validation in larger independent studies on oral hyperplastic lesions. Furthermore, the cytoplasmic expression of hnRNPK protein observed in epithelial cells of a subset of hyperplastic and dysplastic lesions points to a potential role in development and progression during early stages of oral tumorigenesis, while the overexpression in HNOSCCs and association with poor prognosis suggests a sustained involvement in frank malignancy as well.

[0584] In this context, the aberrant cytoplasmic accumulation of hnRNPK protein in a small subset of leukoplakias (26/199, 13% cases) and larger proportion of HNOSCCs (38%) and its potential of risk prediction is noteworthy. The cumulative risk of leukoplakia to transform into OSCC range from 3.6 to 19.8%, 0.4 to 38%, 3 to 33%, and 0 to 20% in different studies; the calculated average amounted to 3% to 8.1% (35). Based on meta-analysis of several follow-up studies of leukoplakia patients an overall rate of 5% transformations in 5 years, resulting in an average annual transformation rate of 1% has been reported by Hunter et al. (2). This 3 year follow-up study showed disease progression in 23/100 leukoplakia patients, 12/23 showed cytoplasmic accumulation of hnRNPK. Long-term follow-up of these leukoplakia patients is needed to establish the link between cytoplasmic hnRNPK and risk of cancer development.

[0585] Kaplan-Meier survival analysis revealed association of cytoplasmic hnRNPK with disease progression of leukoplakia and poor prognosis of HNOSCC. Furthermore, analysis of the predictive potential of hnRNPK revealed its utility as a marker to identify high risk leukoplakia and aggressive HNOSCCs, supporting the association observed by Kaplan Meier analysis. These findings also suggest that leukoplakic lesions with cytoplasmic hnRNPK protein expression are at high risk of disease progression and warrant early intervention as well. The potential mechanistic link between cytoplasmic hnRNPK expression and potential of malignant transformation remains to be established. Efforts are currently underway to demonstrate the role of hnRNPK in malignant transformation of cell cultures established from oral hyperplastic lesions in vitro.

[0586] The poor prognosis of HNOSCC patients showing aberrant cytoplasmic hnRNPK protein expression also supports a role for this protein in progression of HNOSCCs. Interestingly, aberrant cytoplasmic hnRNPK protein expression has also been observed in colorectal cancers (88). Importantly, nasopharyngeal carcinoma patients showing cytoplasmic hnRNPK were reported to have significantly reduced distant metastasis free survival (118). The cytoplasmic hnRNPK expression may be attributed to the presence of a N-terminal bipartite nuclear localization signal and a hnRNPK-specific nuclear shuttling signal that confer the capacity for bidirectional transport across the nuclear envelope (114). Recently, the K nuclear shuttling (KNS) domain, a well-known signal for nuclear import and export, has also been shown to be responsible for the transactivation activity of hnRNPK protein (119). The cytoplasmic accumulation of hnRNPK is controlled by extracellular signal-regulated kinase (ERK)-dependent serine phosphorylation (Ser284 and Ser353) (120). In the cytoplasm, hnRNPK functions as a translational regulator of specific mRNAs, such as c-myc mRNA, renin mRNA, human papillomavirus type 16 L2 capsid protein mRNA, and reticulocyte-15-lipoxygenase (r15-LOX) mRNA (121-124). HPV 16 and 18 have been associated with a large proportion of HNOSCCs, especially among non-consumers of tobacco, though the molecular mechanisms underlying the development of HPV associated HNOSCCS are under intense investigation. Recent proteomic analysis of HPV positive and HPV negative OSCCs have revealed differences in protein expression patterns; whether hnRNPK plays different roles in these tumor subtypes remains to be investigated (15). In the cytoplasm, hnRNPK functions as a specific activator of c-Src and is a substrate of this tyrosine kinase. c-Src-dependent phosphorylation modulates the r15-LOX mRNA-binding activity of hnRNPK and its function in the control of mRNA translation during erythroid cell maturation (113, 114, 124). Taken together with the diverse influence of hnRNPK on gene expression and mechanisms regulating hnRNPK subcellular localization, it is speculated that gene dysregulation resulting from cytoplasmic accumulation of hnRNPK may play an important role in tumorigenesis.

[0587] Hence, hnRNPK has herein been shown to be over-expressed in oral lesions--early premalignant stages of tumorigenesis and in frank tumors in comparison with normal oral tissues both at protein and transcript levels. Furthermore, its subcellular localization--predominantly nuclear in hyperplasias, but present in both cytoplasm and nucleus in a subset of hyperplasias and dysplasias and increasing cytoplasmic expression in tumor cells, suggests that nuclear-cytoplasmic translocation may have an important role in malignant transformation of oral cancer cells. The most important finding is that cytoplasmic hnRNPK is a predictor of disease progression in leukoplakia patients and poor prognostic marker for HNOSCCs, hence targeting hnRNPK might be a new chemopreventive/therapeutic strategy in head and neck/oral cancer. Large scale studies are warranted to further evaluate hnRNPK's potential as an indicator of risk of progression of leukoplakia and role in development and progression during early stages of head and neck/oral tumorigenesis.

[0588] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims The present invention is not to be limited in scope by the specific embodiments described herein, since such embodiments are intended as but single illustrations of one aspect of the invention, and any functionally equivalent embodiments are within the scope of thereof. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0589] All publications, patents, and patent applications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate the cited references by virtue of prior invention.

[0590] Set out below are full citations for the references cited herein. [0591] 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J et al. (2008) Cancer statistics, 2008. CA Cancer J Clin 58: 71-96. [0592] 2. Hunter K D, Parkinson E K, Harrison P R (2005) Profiling early head-and-neck cancer. Nat Rev Cancer 5:127-35. [0593] 3. Warnakulasuriya s, Reibel J, Bouquot J, Dabelsteen E (2008) Oral epithelial dysplasia classification systems: predictive value, utility, weaknesses and scope for improvement. J Oral Pathol Med 37: 127-33. [0594] 4. Gale N, Michaels L, Luzar B, Poljak M, Zidar N, Fischinger J, Cardesa A. (2008) Current review on squamous intraepithelial lesions of the larynx. Histopathology. 2008 Aug. 25. [Epub ahead of print] [0595] 5. Mahajan M, Hazarey V K. An assessment of oral epithelial dysplasia using criteria of Smith and Pindborg's grading system and Ljubljiana Grading system in oral precancer lesions. J Oral Maxillofac Pathol 2004; 8: 73-81. [0596] 6. Warnakulasuriya K A, Ralhan R (2007) Clinical, pathological, cellular and molecular lesions caused by oral smokeless tobacco. J Oral Pathol Med 36: 63-77. [0597] 7. Brennan M, Migliorati C A, Lockhart P B, Wray D, Al-Hashimi I et al. (2007) Management of oral epithelial dysplasia: a review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 103:1-12. [0598] 8. Ralhan R. (2007) Diagnostic potential of Genomic and Proteomic signatures in Oral cancer. Review. Int J of Human Genetics. 7, 57-66. [0599] 9. Linkov, F., Lisovich, A., Yurkovetsky, Z., Marrangoni, A., Velikokhatnaya, L., Nolen, B., Winans, M., Bigbee, W., Siegfried, J., Lokshin, A., Ferris, R L. (2007) Early detection of head-and-neck cancer: development of a novel screening tool using multiplexed immunobead-based biomarker profiling. Cancer Epidemiol Biomarkers Prev. 16, 102-7. [0600] 10. Koike, H., Uzawa, K., Nakashima, D., Shimada, K., Kato, Y., Higo, M., Kouzu, Y., Endo, Y., Kasamatsu, A., Tanzawa, H. (2005) Identification of differentially expressed proteins in oral squamous cell carcinoma using a global proteomic approach. Int J Oncol. 27, 59-67. [0601] 11. Melle, C., Ernst, G., Schimmel, B., Bleul, A., Koscielny, S., Wiesner, A., Bogumil, R., Moller, U., Osterloh, D., Halbhuber, K. J., von Eggeling, F. (2003) Biomarker discovery and identification in laser microdissected head-and-neck squamous cell carcinoma with ProteinChip technology, two-dimensional gel electrophoresis, tandem mass spectrometry, and immunohistochemistry. Mol Cell Proteomics. 2, 443-52. [0602] 12. Roesch-Ely, M., Nees, M., Karsai, S., Ruess, A., Bogumil, R., Warnken, U., Schnolzer, M., Dietz, A., Plinkert, P. K., Hofele, C., Bosch, F. X. (2007) Proteomic analysis reveals successive aberrations in protein expression from healthy mucosa to invasive head-and-neck cancer. Oncogene 26, 54-64. [0603] 13. Drake, R. R., Cazare, L. H., Semmes, O. J., Wadsworth, J. T. (2005) Serum, salivary and tissue proteomics for discovery of biomarkers for head-and-neck cancers. Expert Rev. Mol Diagn. 5, 93-100. [0604] 14. Balys, R., Alaoui-Jamali, M., Hier, M., Black, M., Domanowski, G., Rochon, L., Jie, S. (2006) Clinically relevant oral cancer model for serum proteomic eavesdropping on the tumour microenvironment. J Otolaryngol. 35, 157-66. [0605] 15. Lo, W. Y., Lai, C. C., Hua, C. H., Tsai, M. H., Huang, S. Y., Tsai, C. H., Tsai, F. J. (2007) S100A8 is identified as a biomarker of HPV18-infected oral squamous cell carcinomas by suppression subtraction hybridization, clinical proteomics analysis, and immunohistochemistry staining J Proteome Res. 6(6), 2143-51. [0606] 16. Gygi, S. P., Rist, B., Gerber, S. A., Turecek, F., Gelb, M. H., Aebersold, R. (1999) Quantitative analysis of complex protein mixtures using isotope--coded affinity tags. Nat Biotechnol. 17, 994-99. [0607] 17. DeSouza, L., Diehl, G., Rodrigues, M. J., Guo, J., Romaschin, A. D., Colgan, T. J., Siu, K. W. M. (2005) Search for cancer markers from endometrial tissues using differentially labeled tags iTRAQ and cICAT with multidimensional liquid chromatography and tandem mass spectrometry. J Proteome Res. 4, 377-86. [0608] 18. DeSouza, L., Diehl, G., Yang, E. C. C., Guo, J., Rodrigues, M. J., Romaschin, A. D., Colgan, T. J., Siu, K. W. M. (2005) Proteomic Analysis of the Proliferative and Secretory Phases of the Human Endometrium: Protein Identification and Differential Protein Expression. Proteomics. 5, 270-81. [0609] 19. Li, H., Desouza, L. V., Ghanny, S., Li, W., Romaschin, A. D., Colgan, T. J., Siu, K. W. M. (2007) Identification of Candidate Biomarker Proteins Released by Human Cervical Cancer Cells Using Two-Dimensional Liquid Chromatography/Tandem Mass Spectrometry. J Proteome Res. 6, 2648-55. [0610] 20. Guo, J., Colgan, T. J., DeSouza, L., Rodrigues, M. J., Romaschin, A D., Siu, K. W. M. (2005) Direct analysis of laser capture microdissected endometrial carcinoma and epithelium by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom. 19, 2762-66. [0611] 21. DeSouza, L. V., Grigull, J., Ghanny, S., Dube, V., Romaschin, A. D., Colgan, T. J., Siu, K. W. M. (2007) Endometrial carcinoma biomarker discovery and verification using differentially tagged clinical samples with multidimensional liquid chromatography and tandem mass spectrometry. Mol Cell Proteomics (2007) 6:1170-82. [0612] 22. DeSouza L V, et al. Multiple reaction monitoring of mTRAQ labeled peptides enables absolute quantification of endogenous levels of a potential cancer marker in cancerous and normal endometrial tissues. J Proteome Res (2008). [0613] 23. Ralhan R, et al. Discovery and verification of head-and-neck cancer biomarkers by differential protein expression analysis using iTRAQ-labeling and multidimensional liquid chromatography and tandem mass spectrometry. Mol Cell Proteomics (2008); 7:1162-73 [0614] 24. Ariztia, E. V., Lee, C. J., Gogoi, R., Fishman, D. A. (2006) The tumor microenvironment: key to early detection. Crit Rev Clin Lab Sci. 43, 393-425. [0615] 25. Slaughter D P, Southwick H. W., Smejkal, W. `Field cancerisation` in oral stratified squamous epithelium. Cancer (1953) 6:963-8. [0616] 26. Braakhuis B J, et al. Expanding fields of genetically altered cells in head-and-neck squamous carcinogenesis. Semin Cancer Biol (2005) 15:113-20. [0617] 27. Garcia S B, et al. Field cancerization, clonality, and epithelial stem cells: the spread of mutated clones in epithelial sheets. J Pathol (1999) 187:61-81. [0618] 28. Shilov, I. V., Seymour, S. L., Patel, A. A., Loboda, A., Tang, W. H., Keating, S. P., Hunter, C. L., Nuwaysir, L. M., Schaeffer, D. A. (2007) The Paragon Algorithm: A next generation search engine that uses sequence temperature values and feature probabilities to identify peptides from tandem mass spectra. Mol Cell Proteomics. May 27, [Epub ahead of print]. [0619] 29. The R Development Core Team. The R Project for Statistical Computing. http://www.rproject.org. [0620] 30. Sun Developer Network (SDN). Java.sun.com: The Source for Java Developers. http://java.sun.com. [0621] 31. Witten, I. H. and Frank, E. (2005) Data Mining: Practical machine learning tools and techniques. 2nd Edition. Morgan Kaufmann. San Francisco. [0622] 32. Arora, S., Kaur, J., Sharma, C., Mathur, M., Bahadur, S., Shukla, N. K., Deo, S. V., Ralhan, R. (2005) Stromelysin 3, Ets-1, and vascular endothelial growth factor expression in oral precancerous and cancerous lesions: correlation with microvessel density, progression, and prognosis. Clin Cancer Res. 11, 2272-84. [0623] 33. Leys, C. M., Nomura, S., LaFleur, B. J., Ferrone, S., Kaminishi, M., Montgomery, E., Goldenring. J. R. (2007) Expression and prognostic significance of prothymosin-alpha and ERp57 in human gastric cancer. Surgery. 141, 41-50. [0624] 34. Sasaki, H., Nonaka, M., Fujii, Y., Yamakawa, Y., Fukai, I., Kiriyama, M. and Sasaki, M. (2001) Expression of the prothymosin-a gene as a prognostic factor in lung cancer. Surg Today. 31, 936-938. [0625] 35. Wu, C. G., Habib, N. A., Mitry, R. R., Reitsma, P. H., van Deventer, S. J. and Chamuleau, R. A. (1997) Over-expression of hepatic prothymosin alpha, a novel marker for human hepatocellular carcinoma. Br J Cancer. 76, 1199-1204. [0626] 36. Mori, M., Barnard, G. F., Staniunas. R. J., Jessup. J. M., Steele. G. D. Jr and Chen, L. B. (1993) Prothymosin-alpha mRNA expression correlates with that of c-myc in human colon cancer. Oncogene. 8, 2821-2826. [0627] 37. Slaughter, D. P., Southwick H. W., Smejkal, W. (1953) Cancer 6, 963-968. [0628] 38. Arora, S., Matta, A., Shukla, N. K., Deo, S. V. and Ralhan R. (2005) Identification of differentially expressed genes in oral squamous cell carcinoma. Mol Carcinog. 42, 97-108. [0629] 39. Matta, A.; Bahadur, S.; Duggal, R.; Gupta, S. D.; Ralhan, R. Over-expression of 14-3-3zeta is an early event in oral cancer. BMC Cancer 2007, 7, 169. [0630] 40. Jang, J. S., Cho, H. Y., Lee, Y. J., Ha, W. S. and Kim, H. W. (2004) The differential proteome profile of stomach cancer: identification of the biomarker candidates. Oncol Res. 14, 491-499. [0631] 41. Meehan, K. L., Sadar, M. D. (2004) Quantitative profiling of LNCaP prostate cancer cells using isotope-coded affinity tags and mass spectrometry. Proteomics. 4, 1116-34. [0632] 42. Li, D. Q, Wang, L., Fei, F., Hou, Y. F., Luo, J. M., Wei-Chen, Zeng, R., Wu, J., Lu, J. S., Di, G. H., Ou, Z. L., Xia, Q. C, Shen, Z. Z., Shao, Z. M. (2006) Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry. Proteomics. 6(11): 3352-68. [0633] 43. Qi, W., Liu, X., Qiao, D. and Martinez, J. D. (2005) Isoform-specific expression of 14-3-3 proteins in human lung cancer tissues. Int J Cancer. 113, 359-363. [0634] 44. Lo, W. Y., Tsai, M. H., Tsai, Y., Hua, C. H., Tsai, F. J., Huang, S. Y., Tsai, C. H. and Lai, C. C. (2007) Identification of over-expressed proteins in oral squamous cell carcinoma(OSCC) patients by clinical proteomic analysis. Clin Chim Acta. 376, 101-107. [0635] 45. Chen, J., He, Q. Y., Yuen, A. P. and Chiu J. F. (2004) Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis. Proteomics. 4: 2465-2475. [0636] 46. Hustinx, S. R., Fukushima, N., Zahurak, M. L., Riall, T. S., Maitra, A., Brosens, L., Cameron, J. L., Yeo, C. J., Offerhaus, G. J., Hruban, R. H. and Goggins, M. (2005) Expression and prognostic significance of 14-3-3sigma and ERM family protein expression in periampullary neoplasms. Cancer Biol Ther. 4, 596-601. [0637] 47. Hermeking, H., Benzinger, A. (2006) 14-3-3 proteins in cell cycle regulation. Semin Cancer Biol. 16, 183-92. [0638] 48. Tzivion, G., Gupta, V. S., Kaplunm L., Balan, V. (2006) 14-3-3 proteins as potential oncogenes. Semin Cancer Biol. 16, 203-13. [0639] 49. Verdoodt, B., Benzinger, A., Popowicz, G. M., Holak, T. A., Hermeking, H. (2006) Characterization of 14-3-3 sigma dimerization determinants: requirement of homodimerization for inhibition of cell proliferation. Cell Cycle. 5, 2920-6. [0640] 50. Gardino, A. K., Smerdon, S. J., Yaffe, M. B. (2006) Structural determinants of 14-3-3 binding specificities and regulation of subcellular localization of 14-3-3-ligand complexes: a comparison of the X-ray crystal structures of all human 14-3-3 isoforms. Semin Cancer Biol. 16, 173-82. [0641] 51. Rosell, R., Cecere, F., Santarpia, M., Reguart, N., Taron, M. (2006) Predicting the outcome of chemotherapy for lung cancer. Curr Opin Pharmacol. 6, 323-31. [0642] 52. Qi, W., Martinez, J. D. (2003) Reduction of 14-3-3 proteins correlates with increased sensitivity to killing of human lung cancer cells by ionizing radiation. Radiat. Res. 160, 217-23. [0643] 53. Chu, P. G., Lyda, M. H. and Weiss, L. M. (2001) Cytokeratin 14 expression in epithelial neoplasms: a survey of 435 cases with emphasis on its value in differentiating squamous cell carcinomas from other epithelial tumours. Histopathology. 39, 9-16. [0644] 54. Ohkura, S., Kondoh, N., Hada, A., Arai, M., Yamazaki, Y., Sindoh, M., Takahashi, M., Matsumoto, I. and Yamamoto, M. (2005) Differential expression of the keratin-4, -13, -14, -17 and transglutaminase 3 genes during the development of oral squamous cell carcinoma from leukoplakia. Oral Oncol. 41, 607-613. [0645] 55. Letsas, K. P., Vartholomatos, G., Tsepi, C., Tsatsoulis, A., Frangou-Lazaridis, M. (2007) Fine-needle aspiration biopsy-RT-PCR expression analysis of prothymosin alpha and parathymosin in thyroid: novel proliferation markers? Neoplasma. 54, 57-62. [0646] 56. Traub, F., Jost, M., Hess, R., Schorr, K., Menzel, C., Budde, P., Schulz-Knappe, P., Lamping, N., Pich, A., Kreipe, H. and Tammen, H. (2006) Peptidomic analysis of breast cancer reveals a putative surrogate marker for estrogen receptor-negative carcinomas. Lab Invest. 86, 246-53. [0647] 57. Tsitsiloni, O. E., Stiakakis, J., Koutselinis, A., Gogas, J., Markopoulos, C., Yialouris, P., Bekris, S., Panoussopoulos, D., Kiortsis, V., Voelter, W. and Haritos, A. A. (1993) Expression of alpha-thymosins in human tissues in normal and abnormal growth. Proc Natl Acad Sci. 90, 9504-9507. [0648] 58. Tzai, T S., Tsai, Y. S., Shiau, A L, Wu, C. L., Shieh, G. S., Tsai, H. T. (2006) Urine prothymosin-alpha as novel tumor marker for detection and follow-up of bladder cancer. Urology. 67, 294-9. [0649] 59. Gallego, R., Roson, E., Garcia-Caballero, T., Fraga, M., Forteza, J., Dominguez, F., Beiras, A. (1992) Prothymosin alpha expression in lymph nodes and tonsils: an optical and ultrastructural study. Acta Anat (Basel). 43, 219-22 [0650] 60. Skopeliti, M., Kratzer, U., Altenberend, F., Panayotou, G., Kalbacher, H., Stevanovic, S., Voelter, W., Tsitsilonis, O. E. (2007) Proteomic exploitation on prothymosin alphainduced mononuclear cell activation. Proteomics. 7, 1814-1824. [0651] 61. Samstag, Y., Klemke, M. (2007) Ectopic expression of L-plastin in human tumor cells: Diagnostic and therapeutic implications. Adv Enzyme Regul. February 28, [Epub ahead of print]. [0652] 62. Kasamatsu, A., Uzawa, K., Nakashima, D., Kouzu, Y., Endo, Y., Koike, H., Yokoe, H., Harada, K., Sato, M., Tanzawa, H. (2006) A proteomics approach to characterizing human submandibular gland cell lines by fluorescent two-dimensional differential in-gel electrophoresis. Int. J. Mol. Med. 17, 253-60. [0653] 63. Banerjee, A. G., Bhattacharyya, I., Vishwanatha, J. K. (2005) Identification of genes and molecular pathways involved in the progression of premalignant oral epithelia. Mol. Cancer. Ther. 4, 865-75. [0654] 64. Moubayed, N., Weichenthal, M., Harder, J., Wandel, E., Sticherling, M., Glaser, R. (2007) Psoriasin (S100A7) is significantly up-regulated in human epithelial skin tumours. J Cancer Res Clin Oncol. 133, 253-61. [0655] 65. Skliris, G. P., Lewis, A., Emberley, E., Peng, B., Weebadda, W. K., Kemp, A., Davie, J. R., Shiu, R. P., Watson, P. H., Murphy, L. C. (2007) Estrogen receptor-beta regulates psoriasin (S100A7) in human breast cancer.

Breast Cancer Res Treat. 104, 75-85. [0656] 66. Fukuzawa, H., Kiyoshima, T., Kobayashi, I., Ozeki, S., Sakai, H. (2006) Transcription promoter activity of the human S100A7 gene in oral squamous cell carcinoma cell lines. Biochem. Biophys. Acta. 1759, 171-6. [0657] 67. Krop, I., Marz, A., Carlsson, H., Li, X., Bloushtain-Qimron, N., Hu, M., Gelman, R., Sabel, M. S., Schnitt, S., Ramaswamy, S., Kleer, C. G., Enerback, C., Polyak, K. (2005) A putative role for psoriasin in breast tumor progression. Cancer Res. 65, 11326-34. [0658] 68. Kennedy, R. D., Gorski, J. J., Quinn, J. E., Stewart, G. E., James, C. R., Moore, S., Mulligan, K., Emberley, E. D., Lioe, T. F., Morrison, P. J., Mullan, P. B., Reid, G., Johnston, P. G., Watson, P. H., Harkin, D. P. (2005) BRCA1 and c-Myc associate to transcriptionally repress psoriasin, a DNA damage-inducible gene. Cancer Res. 65, 10265-72. [0659] 69. Sparano, A., Quesnelle, K. M., Kumar, M. S., Wang, Y., Sylvester, A. J., Feldman, M., Sewell, D. A., Weinstein, G. S., Brose, M. S. (2006) Genome-wide profiling of oral squamous cell carcinoma by array-based comparative genomic hybridization. Laryngoscope. 116, 735-41. [0660] 70. Vora, H. H., Shah, N. G., Patel, D. D., Trivedi, T. I., Choksi, T. J. (2003) BRCA1 expression in leukoplakia and carcinoma of the tongue. J Surg Oncol. 83, 232-40. [0661] 71. Zech, V. F., Dlaska, M., Tzankov, A. and Hilbe, W. (2006) Prognostic and diagnostic relevance of hnRNP A2/B1, hnRNP B1 and S100 A2 in non-small cell lung cancer. Cancer Detect Prev. 30, 395-402. [0662] 72. Kanamori, T., Takakura, K., Mandai, M., Kariya, M., Fukuhara, K., Sakaguchi, M., Huh, N. H., Saito, K., Sakurai, T., Fujita, J. and Fujii, S. (2004) Increased expression of calcium-binding protein S100 in human uterine smooth muscle tumors. Mol Hum Reprod. 10, 735-42. [0663] 73. Zhang, H., Xu, L., Xiao, D., Xie, J., Zeng, H., Cai, W., Niu, Y., Yang, Z., Shen, Z. and Li, E. (2006) Fascin is a potential biomarker for early-stage oesophageal squamous cell carcinoma. J Clin Pathol. 59, 958-964. [0664] 74. Zhang, B., Chen, J. Y., Chen, D. D., Wang, G. B., Shen, P. (2004) Tumor type M2 pyruvate kinase expression in gastric cancer, colorectal cancer and controls. World J Gastroenterol. 10, 1643-6. [0665] 75. Ahmed, A. s, Dew, T., Lawton, F. G., Papadopoulos, A. J., Devaja, O., Raju, K. S., Sherwood, R. A. (2007) M2-PK as a novel marker in ovarian cancer. A prospective cohort study. Eur J Gynaecol Oncol. 28, 83-8. [0666] 76. Goonetilleke, K. S., Mason, J. M., Siriwardana, P., King, N. K., France, M. W., Siriwardena, A. K. (2007) Diagnostic and prognostic value of plasma tumor M2 pyruvate kinase in periampullary cancer: evidence for a novel biological marker of adverse prognosis. Pancreas. 34, 318-24. [0667] 77. Kumar, Y., Tapuria, N., Kirmani, N., Davidson, B. R. (2007) Tumor, M2-pyruvate kinase: a gastrointestinal cancer marker. Eur J Gastroenterol Hepatol. 19, 265-76. [0668] 78. Stetak, A., Veress, R., Ovadi, J., Csermely, P., Keri, G., Ullrich, A. (2007) Nuclear translocation of the tumor marker pyruvate kinase M2 induces programmed cell death. Cancer Res. 67, 1602-8. [0669] 79. Kim, J. W., Dang, C. V. (2006) Cancer's molecular sweet tooth and the Warburg effect. Cancer Res. 66, 8927-30. [0670] 80. Shaw, R. J. (2006) Glucose metabolism and cancer. Curr Opin Cell Biol. 18, 598-608. [0671] 81. Juwana, J. P., Henderikx, P., Mischo, A., Wadle, A., Fadle, N., Gerlach, K., Arends, J. W., Hoogenboom, H., Pfreundschuh, M. and Renner, C. (1999) EB/RP gene family encodes tubulin binding proteins. Int J Cancer. 81, 275-284. [0672] 82. Wang, Y., Zhou, X., Zhu, H., Liu, S., Zhou, C., Zhang, G., Xue, L., Lu, N., Quan, L., Bai, J., Zhan, Q. and Xu, N. (2005) Overexpression of EB1 in human esophageal squamous cell carcinoma (ESCC) may promote cellular growth by activating beta-catenin/TCF pathway. Oncogene. 24, 6637-6645. [0673] 83. Xue, Y., Canman, J. C., Lee, C. S., Nie, Z., Yang, D, Moreno, G. T., Young, M. K., Salmon, E. D. and Wang, W. (2000) The human SWI/SNF-B chromatin-remodeling complex is related to yeast Rsc and localizes at kinetochores of mitotic chromosomes. Proc Natl Acad Sci. 97, 13015-13020. [0674] 84. Martens, J. A. and Winston, F. (2003) Recent advances in understanding chromatin remodeling by Swi/Snf complexes. Curr Opin Genet Dev. 13, 136-142. [0675] 85. Hosotani, T., Koyama, H., Uchino, M., Iyakawa/T. and Tsuchiya/E. (2001) PKC1, a protein kinase C homologue of Saccharomyces cerevisiae, participates in microtubule function through the yeast EB1 homologue, BIM1. Genes Cells. 6, 775-788. [0676] 86. Sekine, I., Sato, M., Sunaga, N., Toyooka, S., Peyton, M., Parsons, R., Wang, W., Gazdar, A. F. and Minna., J. D. (2005) The 3p21 candidate tumor suppressor gene BAF180 is normally expressed in human lung cancer. Oncogene. 24, 2735-2738. [0677] 87. Ralhan R, Leroi V. DeSouza, Ajay Matta, Satyendra Chandra Tripathi, Shaun Ghanny, Siddartha DattaGupta, Alok Thakar and Siu K W M. iTRAQ-Multidimensional Liquid Chromatography and Tandem Mass Spectrometry based Identification of Potential Biomarkers of Oral Epithelial Dysplasia and Novel Networks between Inflammation and Premalignancy. Journal of Proteome Research. [0678] 88. Carpenter B, et al. Heterogeneous nuclear ribonucleoprotein K is over expressed aberrantly localized and is associated with poor prognosis in colorectal cancer. Br J Cancer (2006) 95:921-7. [0679] 89. Roychoudhury P, et al. Evidence for heterogeneous nuclear ribonucleoprotein K overexpression in oral squamous cell carcinoma. Br J Cancer (2007) 97:574-5. [0680] 90. Sawhney, M.; Rohatgi, N.; Kaur, J.; Shishodia, S.; Sethi, G.; Gupta, S. D.; Deo, S. V.; Shukla, N. K.; Aggarwal, B. B.; Ralhan, R. Expression of NF-kappaB parallels COX-2 expression in oral precancer and cancer: association with smokeless tobacco. Int. J. Cancer 2007, 120, 2545-56. [0681] 91. Aguilera, C.; Fernandez-Majada, V.; Ingles-Esteve, J.; Rodilla, V.; Bigas, A.; Espinosa, L. Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins. J. Cell. Sci. 2006, 119, 3695-704. [0682] 92. Fang, D.; Hawke, D.; Zheng, Y.; Xia, Y.;, Meisenhelder, J.; Nika, H.; Mills, G. B.; Kobayashi, R.; Hunter, T.; Lu, Z.; Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J. Biol. Chem. 2007, 82, 11221-9. [0683] 93. Niemantsverdriet, M.; Wagner, K.; Visser, M.; Backendorf, C. Cellular functions of 14-3-3zeta in apoptosis and cell adhesion emphasize its oncogenic character. Oncogene 2007, (Epub ahead of print) [0684] 94. Danes, C. G., Wyszomierski, S. L.; Lu, J.; Neal, C. L.; Yang, W.; Yu, D. 14-3-3 zeta down-regulates p53 in mammary epithelial cells and confers luminal filling. Cancer. Res. 2008, 68, 1760-7. [0685] 95. Wilker, E.; Yaffe, M. B. 14-3-3 Proteins-a focus on cancer and human disease. J. Mol. Cell. Cardiol. 2004, 37, 633-42. [0686] 96. Bhawal, U. K.; Tsukinoki, K.; Sasahira, T.; Sato, F.; Mori, Y.; Muto, N.; Sugiyama, M.; Kuniyasu, H. Methylation and intratumoural heterogeneity of 14-3-3 sigma in oral cancer. Oncol. Rep. 2007, 18, 817-24. [0687] 97. http://www.uicc.org. [0688] 98. Cregger, M.; Berger, A. J.; Rimm, D. L. Immunohistochemistry and quantitative analysis of protein expression. Arch. Pathol. Lab. Med. 2006, 130, 1026-30. [0689] 99. Perathoner, A.; Pirkebner, D.; Brandacher, G.; Spizzo, G.; Stadlmann, S.; Obrist, P.; Margreiter, R.; Amberger, A. 14-3-3sigma expression is an independent prognostic parameter for poor survival in colorectal carcinoma patients. Clin. Cancer Res. 2005, 11, 3274-9. [0690] 100. Matta, A.; DeSouza, L. V.; Bahadur, S.; Gupta, S. D.; Ralhan, R.; Siu, K. W. Prognostic Significance of Head-and-neck cancer Biomarkers Previously Discovered Using iTRAQ-Multidimensional Liquid Chromatography and Tandem Mass Spectrometry. J. Proteome. Research. 2008, 7, 2078-87. [0691] 101. Dube V, Grigull J, DeSouza L, Ghanny s, Colgan T J, Romaschin A D, Siu K W M. Verification of head-and-neck tissue biomarkers previously discovered using mass-tagging and multidimensional liquid chromatography/tandem mass spectrometry by means of immunohistochemistry in a tissue microarray format. J. Proteome Res. 2007; July; 6(7):2648-55. [0692] 102. Gasco, M.; Bell, A. K.; Heath, V.; Sullivan, A.; Smith, P.; Hiller, L.; Yulug, I.; Numico, G.; Merlano, M.; Farrell, P. J.; Tavassoli, M.; Gusterson, B.; Crook, T. Epigenetic inactivation of 14-3-3 sigma in oral carcinoma: association with p16(INK4a) silencing and human papilloma virus negativity. Cancer Res. 2002, 62, 2072-6. [0693] 103. Samuel, T.; Weber, H. O.; Rauch, P.; Verdoodt, B.; Eppel, J. T.; McShea, A.; Hermeking, H.; Funk, O. The G2/M regulator 14-3-3sigma prevents apoptosis through sequestration of Bax. J. Biol. Chem. 2001, 276, 45201-6. [0694] 104. Guweidhi, A.; Kleeff, J.; Giese, N.; El, Fitori. J; Ketterer, K.; Giese, T.; Buchler, M. W.; Korc, M.; Friess, H. Enhanced expression of 14-3-3sigma in pancreatic cancer and its role in cell cycle regulation and apoptosis. Carcinogenesis 2004, 25, 1575-85. [0695] 105. Liu, Y.; Liu, H.; Han, B.; Zhang, J. T. Identification of 14-3-3sigma as a contributor to drug resistance in human breast cancer cells using functional proteomic analysis. Cancer Res. 2006, 66, 3248-55. [0696] 106. Yang, H. Y.; Wen, Y. Y.; Lin, Y. L.; Pham, L.; Su, C. H.; Yang, H.; Chen, J.; Lee, M. H. Roles for negative cell regulator 14-3-3sigma in control of MDM2 activities. Oncogene 2007. [0697] 107. Ralhan, R.; Agarwal, S.; Nath, N.; Mathur, M.; Wasylyk, B.; Srivastava, A. Correlation between p53 gene mutations and circulating antibodies in betel- and tobacco-consuming North Indian population. Oral Oncol. 2001, 37, 243-50. [0698] 108. Ralhan, R.; Sandhya, A.; Meera, M.; Bohdan, W.; Nootan, S. K. Induction of MDM2-P2 transcripts correlates with stabilized wild-type p53 in betel- and tobacco-related human oral cancer. Am. J. Pathol. 2000, 157, 587-96. [0699] 109. Soni, S.; Kaur, J.; Kumar, A.; Chakravarti, N.; Mathur, M.; Bahadur, S.; Shukla, N. K.; Deo, S. V.; Ralhan, R. Alterations of Rb pathway components are frequent events in patients with oral epithelial dysplasia and predict clinical outcome in patients with squamous cell carcinoma. Oncology 2005, 68, 314-25. [0700] 110. Matta A, Tripathi S C, DeSouza L V, Bahadur s, DattaGupta S, Ralhan R* and Siu K W M. Clinical Significance of Heterogenous ribonucleoprotein K in Head and Neck Tumorigenesis: A Potential Biomarker Previously Discovered Using iTRAQ-Multidimensional Liquid Chromatography and Tandem Mass Spectrometry., Clinical Cancer Research, submitted. [0701] 111. Evans J R, Mitchell S A, Spriggs K A et al. Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo. Oncogene 2003; 22:8012-20. [0702] 112. Adolph D, Flach N, Mueller K, Ostareck D H, Ostareck-Lederer A. Deciphering the cross talk between hnRNPK and c-Src: the c-Src activation domain in hnRNPK is distinct from a second interaction site. Mol Cell Biol 2007; 27:1758-70. [0703] 113. Naarmann Is, Harnisch C, Flach N et al. mRNA silencing in human erythroid cell maturation: hnRNPK controls the expression of its regulator c-Src. J Biol Chem 2008; 283:18461-72 [0704] 114. Ostrowski J, Bomsztyk K. Nuclear shift of hnRNPK protein in neoplasms and other states of enhanced cell proliferation. Br J Cancer 2003; 89:1493-501. [0705] 115. Bomsztyk K, Denisenko O, Ostrowski J. hnRNPK: one protein multiple processes. Bioessays 2004; 26:629-38. [0706] 116. He Y, Brown M A, Rothnagel J A, Saunders N A, Smith R. Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation. J Cell Sci 2005; 118:3173-83. [0707] 117. Michael W M, Eder Ps, Dreyfuss G. The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNPK protein. EMBO J 1997; 16: 3587-98. [0708] 118. Sun Y, Yi H, Zhang P F et al. Identification of differential proteins in nasopharyngeal carcinoma cells with p53 silence by proteome analysis. FEBS Lett 2007; 581:131-9. [0709] 119. Chan J Y, Huang S M, Liu S T, Huang C H. The transactivation domain of heterogeneous nuclear ribonucleoprotein K overlaps its nuclear shuttling domain. Int J Biochem Cell Biol 2008; 40:2078-89. [0710] 120. Habelhah H, Shah K, Huang L et al. ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation. Nat Cell Biol 2001; 3:325-30. [0711] 121. Evans J R, Mitchell S A, Spriggs K A et al. Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo. Oncogene 2003; 22:8012-20. [0712] 122. Skalweit A, Doller A, Huth A et al. Posttranscriptional control of renin synthesis: identification of proteins interacting with renin mRNA 3'-untranslated region. Circ Res 2003; 92:419-27. [0713] 123. Collier B, Goobar-Larsson L, Sokolowski M, Schwartz S. Translational inhibition in vitro of human papillomavirus type 16 L2 Mrna mediated through interaction with heterogenous ribonucleoprotein K and poly(rC)-binding proteins 1 and 2. J Biol Chem 1998; 273:22648-56. [0714] 124. Reimann I, Huth A, Thiele H, Thiele B J. Suppression of 15-lipoxygenase synthesis by hnRNP El is dependent on repetitive nature of LOX mRNA 3'-UTR control element DICE. J Mol Biol 2002; 315: 965-74.

TABLE-US-00002 [0714] TABLE 1 iTRAQ ratios for HNSCC and non-cancerous head-and-neck tissue samples. ##STR00001## ##STR00002##

TABLE-US-00003 TABLE 2 Receiver-operating characteristics from the iTRAQ ratios of a panel of three best-performing biomarkers - YWHAZ, stratifin, and S100A7 - individually and as a panel. BIOMARKER PANEL SENS SPEC PPV NPV AUC YWHAZ 0.30 1.00 1.00 0.42 1.00 SFN 0.80 1.00 1.00 0.71 0.98 S100 CBP A7 0.70 1.00 1.00 0.63 0.90 YWHAZ, STRATIFIN, 0.92 0.91 0.95 0.85 0.96 S100A7

TABLE-US-00004 TABLE 3 Receiver-operating characteristics from the IHC scores of a panel of three best-performing biomarkers - YWHAZ, stratifin, and S100A7 - individually and as a panel. BIOMARKER PANEL SENS SPEC PPV NPV AUC YWHAZ 1.00 0.71 0.71 1.00 0.90 SFN 0.92 0.60 0.62 0.91 0.85 S100 CBP A7 0.96 0.71 0.71 0.96 0.90 YWHAZ, STRATIFIN, 0.92 0.87 0.83 0.94 0.91 S100A7

TABLE-US-00005 TABLE 4 Comparison of receiver-operating characteristics from the iTRAQ ratios of the panel of three best-performing biomarkers. COMPARISON SENS SPEC PPV NPV AUC Cancer vs. 0.92 0.83 0.85 0.92 0.89 Paired Normal Cancer vs. 0.96 0.96 0.98 0.90 0.97 Non-Paired Normal

TABLE-US-00006 TABLE 5 Differentially-expressed proteins not previously described in OPLs and head-and-neck malignancies and cancer. Expression SEQ in Head-and- Expression ID. No. Protein Name Gene Name Accession No. Neck Cancer in OPLs 1. Calmodulin-like CALML5 Peptide: spt|Q9NZT1 Up-regulated protein 5 mRNA/DNA coding: NM_017422 2. Polybromo-1D PBRM1 Peptide: trm|Q86U86 Down-regulated mRNA/DNA coding: NM_181042 3. APC-binding protein MAPRE1 Peptide: spt|Q15691 Up-regulated EB1 mRNA/DNA coding: NM_012325 4. Carbonic anhydrase I CA1 Peptide: spt|P00915 Down-regulated mRNA/DNA coding: NM_001738 5. Mast cell tryptase tryptaseB Peptide: trm|Q96RZ7 Down-regulated beta III mRNA/DNA coding: NM_024164 6. Histone H3 HIST2H3A Peptide: trm|Q71DI3 Down-regulated mRNA/DNA coding: NM_021059 7. Plastin 3 PLS3 Peptide: spt|P13797 Up-regulated mRNA/DNA coding: NM_005032 8. Histone H4 HIST1H4A Peptide: spt|P62805 Down-regulated mRNA/DNA coding: NM_003495 9. Cyclophilin A PPIA Peptide: trm|Q6NTE9 Up-regulated mRNA/DNA coding: NM_021130 10. PACAP MGC29506 Peptide: trm|Q8WU39 Down-regulated (proapoptotic caspase mRNA/DNA coding: NM_016459 adaptor protein) 11. LDH A LDHA Peptide: spt|P00338 Up-regulated mRNA/DNA coding: NM_005566 12. KSPG Luminican LUM Peptide: spt|P51884 Down-regulated mRNA/DNA coding: NM_002345 13. S100 CBP A7 S100A7 Peptide: spt|P31151 Up-regulated *Up-regulated mRNA/DNA coding: NM_002963.3 14. Peroxiredoxin 2 PRDX2 Peptide: spt|P32119 Down-regulated Down-regulated mRNA/DNA coding: NM_181738 15. Superoxide dismutase SOD2 Peptide: trm|AAH16934 Up-regulated *Up-regulated 2 (SOD2 protein) mRNA/DNA coding: BC016934 16. Alpha 1 Anti-Trypsin SERPINA1 or Peptide: spt|P01009 Down-regulated Down-regulated Precursor AAT mRNA/DNA coding: NM_001002236 17. MARCKS MACS Peptide: gb|AAA59555.1 Up-regulated Down-regulated mRNA/DNA coding: M68956 18. GRP-94 HSP90B1 Peptide: spt|P14625 Down-regulated Up-regulated mRNA/DNA coding: NM_003299 19. Prothymosin Alpha PTMA Peptide: spt|P06454 Up-regulated Up-regulated mRNA/DNA coding: NM_001099285 20. Histone H2B.1 HIST2H2BE Peptide: spt|Q16778 Down-regulated Up-regulated mRNA/DNA coding: NM_003528 21. Nucleophosmin 1 NPM1 Peptide: gb|AAH16768.1 Up-regulated Up-regulated mRNA/DNA coding: BC016768 22. PK M2 PKM2 Peptide: spt|P14618 Up-regulated mRNA/DNA coding: NM_182471 23. Stratifin SFN spt|P31947 Up-regulated mRNA/DNA coding: 24. YWHAZ trm|Q86V33 Up-regulated mRNA/DNA coding: 25. hnRNPK spt|P61978 Up-regulated mRNA/DNA coding: 18. HSP90B1 spt|P14625 Up-regulated mRNA/DNA coding: 26. Parathymosin PTHM trm|O15256 Up-regulated mRNA/DNA coding: 27. Cystatin B spt|P04080 Down-regulated mRNA/DNA coding: P04080 28. DLC1 trm|Q6NSB4 Down-regulated mRNA/DNA coding: 29. FABP5 spt|Q01469 Down-regulated mRNA/DNA coding: 30. IGHG1 protein gb|AAH25314.1 Down-regulated mRNA/DNA coding: 31. Calgizzarin spt|P31949 Down-regulated mRNA/DNA coding: 32. IGL 2* trm|Q8N5F4 Up-regulated mRNA/DNA coding: 33. P37AUF1* trm|Q12771 Up-regulated mRNA/DNA coding: 22. PKM2* spt|P14618 Up-regulated mRNA/DNA coding: 34. ROA1HNRNPA1* sptIP09651 Up-regulated mRNA/DNA coding: 35. Hsp27* sptIP04792 Up-regulated mRNA/DNA coding: P04792 36. Cofilin* spt|P23528 Up-regulated mRNA/DNA coding: 37. Glyceraldehyde-3- emb|CAA25833.1 Up-regulated phosphate Dehydrogenase* mRNA/DNA coding: 38. NDP Kinase B* spt|P22392 Up-regulated mRNA/DNA coding: 39. Elongation Factor 2* spt|P13639 Up-regulated mRNA/DNA coding: 40. PE Binding protein* spt|P30086 Up-regulated mRNA/DNA coding: 41. CALM 3* spt|P27482 Up-regulated mRNA/DNA coding:

TABLE-US-00007 TABLE 6 Average iTRAQ ratios for OPLs and histologically-normal control oral tissue samples. ##STR00003## ##STR00004##

TABLE-US-00008 TABLE 7 Receiver-operating characteristics from (A) the iTRAQ ratios and (B) IHC scores of a panel of three best-performing biomarkers - YWHAZ, stratifin, and hnRNPK - individually and as a panel. Biomarker Sensitivity Specificity PPV NPV AUC A. iTRAQ analysis YWHAZ 0.33 1.0 1.0 0.43 0.78 Stratifin 0.81 1.0 1.0 0.75 0.82 hnRNPK 0.17 1.0 1.0 0.38 0.78 Panel of the three 0.83 0.74 0.87 0.69 0.85 B. IHC analysis YWHAZ 0.90 0.95 0.96 0.87 0.93 Stratifin 0.77 0.95 0.96 0.74 0.93 hnRNPK 0.80 0.91 0.92 0.76 0.89 Panel of the three 0.91 0.95 0.96 0.88 0.97

TABLE-US-00009 TABLE 8 Analysis of Stratifin and YWHAZ in HNOSCCs: correlation with clinicopathological parameters. Total Clinicopathological Cases STRATIFIN.sup.+ STRATIFIN.sup.+-YWHAZ.sup.+ STRATIFIN.sup.+/YWHAZ.sup.+ Features N n (%) n (%) n (%) Non-malignant tissue 39 12 (31) 8 (20) 25 (64) HNOSCC# 51 32 (63) 28 (55) 43 (84) Differentiation* WDSCC 29 18 (62) 16 (55) 26 (90) MDSCC 19 12 (63) 10 (53) 15 (79) PDSCC 3 2 (67) 2 (67) 2 (67) Tumor Stage T.sub.1 6 5 (83) 5 (83) 6 (100) T.sub.2 15 6 (40) 4 (27) 11 (73) T.sub.3 13 9 (69) 8 (61) 12 (92) T.sub.4 17 12 (71) 11 (65) 14 (82) Nodal Status N.sup.- 28 17 (61) 15 (54) 23 (82) N.sup.+ 23 15 (65) 13 (56) 20 (87) *WDSCC, well differentiated squamous cell carcinoma; MDSCC, moderately differentiated squamous cell carcinoma; PDSCC, poorly differentiated squamous cell carcinoma #For HNOSCCs vs. Non-malignant tissues: a) Stratifin.sup.+ (p = 0.03, OR = 3.8, 95% CI = 1.6-9.2); b) YWHAZ.sup.+ (p = 0.024, OR = 2.8, 95% CI = 1.2-6.8); c) Stratifin.sup.+-YWHAZ.sup.+ (p = 0.001, OR = 4.7, 95% CI = 1.8-12.2); d) SFN.sup.+/YWHAZ.sup.+ (p = 0.027, OR = 3.1, 95% CI = 1.1-8.2).

TABLE-US-00010 TABLE 9a Analysis of overexpression of hnRNP K protein in oral lesions and correlation with clinicopathological parameters. Clinicopathological Total Nuclear Positivity Cytoplasmic Positivity Features Cases N (%) p-value OR (95% CI) N (%) p-value OR (95% CI) NORMAL 55 4 (7) -- -- LEUKOPLAKIA 199 141 (71) <0.001.sup.a 30.9 (10.7-89.7) 26 (13) <0.001.sup.b 4.3 (2.2-7.2) NO DYSPLASIA 115 78 (68) <0.001.sup.c 26.8 (9.1-79.9) 18 (16) <0.001.sup.d 4.3 (1.8-6.3) DYSPLASIA 84 63 (75) <0.001.sup.e 38.2 (11.7-113.1) 8 (10) <0.001.sup.f 5.8 (2.5-13.4) HNOSCC 100 78 (78) <0.001.sup.g 45.2 (14.7-138.8) 38 (38) Age (Median, 53 yrs) <53 49 34 (69) 0.05 2.7 (1.1-7.5) 16 (33) 0.28 -- .gtoreq.53 51 44 (86) 22 (43) Gender Male 75 59 (78) 0.78 -- 28 (37) 0.81 -- Female 25 19 (76) 10 (40) Differentiation 0.001 WDSCC 45 33 (73) 0.31 -- 9 (20) -- MDSCC 49 39 (79) 24 (49) PDSCC 6 6 (100) 5 (83) Tumor Stage 0.19 T.sub.1 4 4 (100) 0.42 -- 3 (75) -- T.sub.2 35 28 (80) 15 (43) T.sub.3 25 17 (68) 6 (24) T.sub.4 36 29 (81) 14 (39) Nodal Status 0.52 N.sub.0 33 24 (73) 0.37 -- 14 (42) -- N.sub.1-4 67 54 (81) 24 (36) Habits 0.41 Non consumer 22 19 (86) 0.28 -- 10 (45) -- Tobacco consumer 78 59 (75) 28 (36) # Nuclear staining: .sup.aNormal vs. Leukoplakia; .sup.cNormal vs. Leukoplakia with no evidence of dysplasia; .sup.eNormal vs. Dysplasia; .sup.gNormal vs. HNOSCCs; N/L/HNOSCCs: p < 0.001; ## Cytoplasmic staining: .sup.bLeukoplakia vs. HNOSCCs; .sup.dLeukoplakia with no evidence of dysplasia vs. HNOSCCs; .sup.fDysplasia vs. HNOSCCs; N/L/HNOSCCs: p < 0.001

TABLE-US-00011 TABLE 9b Biomarker analysis of hnRNP K (nuclear/cytoplasmic) in oral lesions. hnRNP K Sensitivity Specificity PPV AUC I) Nuclear staining Normal vs. Leukoplakia 67.0 92.7 94.8 0.822 (No Dysplasia) Normal vs. Leukoplakia 74.1 92.3 93.7 0.872 (Dysplasia) Normal vs. OSCCs 78.0 92.3 95.1 0.869 II) Cytoplasmic staining Normal vs. Leukoplakia 15.3 100 100 0.577 (No Dysplasia) Normal vs. Leukoplakia 8.3 100 100 0.543 (Dysplasia) Normal vs. OSCCs 38.1 100 100 0.709

TABLE-US-00012 TABLE 10 Alternate accession numbers for OPL proteins. Accession # Protein Name Alternate Accession #s spt|P61978 hnRNPK gi|48429103, NP_002131.2, S74678.1, NP_112552.1, AAB20770.1, NP_112553.1, X72727.1, 1J5K_A, CAA51267.1, 1KHM_A, AB209562.1, 1ZZI_A, BAD92799.1, 1ZZI_B, BC000355.2, 1ZZJ_A, AAH00355.1, 1ZZJ_B, BC014980.1, 1ZZJ_C, AAH14980.1, 1ZZK_A, S43363 spt|P14625 HSP90B1 gi|119360, AAH66656.1, X15187.1, M26596.1, CAA33261.1, AAA58621.1, M33716.1, AY040226.1, AAA68201.1, AAK74072.1, BC066656.1, NP_003290.1 trm|O15256 Parathymosin gi|74705500, Y13586.1, CAA73913.1 trm|Q6NSB4 DLC1 gi|74758095, BC070299.1, AAH70299.1 spt|Q01469 FABP5 gi|232081, AAH70303.1, M94856.1, I56326, AAA58467.1, NP_001435.1, BT007449.1, XP_001127657.1, AAP36117.1, XP_001128089.1, BC019385.2, XP_001718427.1, AAH19385.1, 1B56_A, BC070303.1, 1JJJ_A gb|AAH25314.1 IGHG1 protein gi|19263707 trm|Q8N5F4 IGL 2* gi|74728989, BC032452.1, AAH32452.1 trm|Q12771 P37AUF1* gi|74754454, U02019.1, AAC50056.1, A54601 prf|0904262A SOD2* gi|223632 spt|P14618 PKM2* gi|20178296, BC007952.2, M23725.1, AAH07952.3, AAA36449.1, BC012811.2, M26252.1, AAH12811.3, AAA36672.1, BC035198.1, X56494.1, AAH35198.1, CAA39849.1, AF025439.1, AY352517.1, AAC39559.1, AAQ15274.1, S30038, AK222927.1, S64635, BAD96647.1, NP_002645.3, AC020779.10, NP_872270.1, CH471082.1, 1T5A_A, EAW77884.1, 1T5A_B, BC000481.2, 1T5A_C, AAH00481.3, 1T5A_D, BC007640.1, 1ZJH_A, AAH07640.1 sptIP09651 ROA1HNRNPA1* gi|133254, AAH74502.1, X12671.1, BC103707.1, CAA31191.1, AAI03708.1, X06747.1, NZ_SHDD041214211, CAA29922.1, NP_002127.1, X04347.1, 1HA1_A, CAA27874.1, 1L3K_A, X79536.1, 1PGZ_A, CAA56072.1, 1PO6_A, BC002355.2, 1U1K_A, AAH02355.1, 1U1L_A, BC009600.1, 1U1M_A, AAH09600.1, 1U1N_A, BC012158.1, 1U1O_A, AAH12158.1, 1U1P_A, BC033714.1, 1U1Q_A, AAH33714.1, 1U1R_A, BC052296.1, 1UP1_A, AAH52296.1, 2H4M_C, BC070315.1, 2H4M_D, AAH70315.1, 2UP1_A, BC074502.1 spt|P23528 Cofilin* gi|116848, AAH11005.1, D00682.1, BC012265.1, BAA00589.1, AAH12265.1, U21909.1, BC012318.1, AAA64501.1, AAH12318.1, X95404.1, BC018256.2, CAA64685.1, AAH18256.1, BT006846.1, NP_005498.1, AAP35492.1, 1Q8G_A, BC011005.2, 1Q8X_A emb|CAA25833.1 Glyceraldehyde-3- gi|31645, 1U8F, Phosphate Dehydrogenase* 1ZNQ spt|P22392 NDP Kinase B* gi|127983, NP_002503.1, X58965.1, 1NSK_L, CAB37870.1, 1NSK_N, M36981.1, 1NSK_O, AAA36369.1, 1NSK_R, L16785.1, 1NSK_T, AAA60228.1, 1NSK_U, BC002476.2, 1NUE_A, AAH02476.1, 1NUE_B, A49798, 1NUE_C, NP_001018146.1, 1NUE_D, NP_001018147.1, 1NUE_E, NP_001018148.1, 1NUE_F, NP_001018149.1 spt|P13639 Elongation Factor 2* gi|119172, BC126259.1, X51466.1, AAI26260.1, CAA35829.1, M19997.1, Z11692.1, AAA50388.1, CAA77750.1, EFHU2, AY942181.1, NP_001952.1, AAX34409.1 spt|P30086 PE Binding protein gi|1352726, BC031102.1, D16111.1, AAH31102.1, BAA03684.1, S76773.1, X75252.1, AAD14234.1, CAA53031.1, I53745, X85033.1, NP_002558.1, CAA59404.1, 1BD9_A, BC008714.2, 1BD9_B, AAH08714.1, 1BEH_A, BC017396.1, 1BEH_B, AAH17396.1 spt|P27482 CALM 3* gi|115502, CAI11029.1, M58026.1, BC031889.1, AAA36356.1, AAH31889.1, X13461.1, AAA21893.1, CAA31809.1, NP_005176.1, AL732437.12, 1GGZ_A

TABLE-US-00013 TABLE 11 # # SEQUENCE PEP- MAX Seq Times ACCESSION PROTEIN COVERAGE TIDES PEPTIDE Confidence ID Seen gb|AAC13868.1 Glutathione 38.27761279 6 AFLASPEYVNLPINGNGKQ 98.99999499 42 17 S Trans- ferase-P DQQEAALVDMVNDGVEDLR 98.99999499 43 17 FQDGDLTLYQSNTILR 98.99999499 44 17 MLLADQGQSWK 98.99999499 45 2 PPYTVVYFPVR 98.99999499 46 1 TLGLYGKDQQEAALVDMVNDGVEDLR 98.99999499 47 2 spt|P00338 LDH A 20.64380626 7 DQLIYNLLK 98.99999499 48 2 FIIPNVVK 98.99999499 49 3 GEMMDLQHGSLFLR 98.99999499 50 2 LVIITAGAR 98.99999499 51 11 QVVESAYEVIK 98.99999499 52 5 VIGSGCNLDSAR 98.99999499 53 7 VTLTSEEEAR 98.99999499 54 12 spt|P01002 Alpha 1 29.90430593 9 AVLTIDEK 98.99999499 55 2 Anti- Trypsin Precursor DTEEEDFHVDQVTTVK 98.99999499 56 2 FLENEDRR 97.99999595 57 1 GTEAAGAMFLEAIPM 98.99999499 58 3 GTEAAGAMFLEAIPMSIPPEVK 98.99999499 59 1 LQHLENELTHDIITK 98.99999499 60 1 SVLGQLGITK 98.99999499 61 2 TDTSHHDQDHPTPNK 98.99999499 62 1 VFSNGADLSGVTEEAPLK 98.99999499 63 5 spt|P04060 Cystatin B 54.08163071 6 CGAPSATQPATAETQMIADQVR 98.99999499 64 2 GAPSATOPATAETQMIADQVR 98.99999499 65 2 MMCGAPSATQPATAETQHIADQVR 98.99999499 66 21 PSATQPATAETQHIADQVR 98.99999499 67 4 SQVVAGTNYFIK 98.99999499 68 14 VHVGDEDFVHLR 98.99999499 69 10 spt|P04083 Annexin A1 34.49275404 9 ALYEAGER 98.99999499 70 7 DITSDTSGDFR 98.99999499 71 6 GLGTDEDTLIEILASR 98.99999499 72 50 GTDVNVFNTILTTR 98.99999499 73 9 GVDEATIIDILTK 98.99999499 74 4 KGTDVNVFNTILTTR 98.99999499 75 8 NALLSLAK 98.99999499 76 6 QAWFIENEEQEYVQTVK 98.99999499 77 1 TPAQFDADELR 98.99999499 78 6 spt|P063S4 Prothy- 35.45464443 5 AAEDDEDDDVDTK 98.99999499 79 1 mosin Alpha AAEDDEDDDVDTKK 98.99999499 80 2 EVVEEAENGR 98.99999499 81 3 KEVVEEAENGR 98.99999499 82 5 SDAAVDTSSEITTK 98.99999499 83 6 spt|P07565 Detoxin 5.013927445 4 DFEPSLGPVCPFR 94.99999881 84 1 precursor DLPPDTTLLDLQNNK 98.99999499 85 1 ELHLDNNK 97.99999595 86 1 VSPGAFTPLVK 98.99999499 87 1 spt|P68670 Vimertin 58.81057382 28 ADLSEAANR 98.99999499 88 2 DGQVINETSQHHDDLE 98.99999499 89 5 DNLAEDIMR 98.99999499 90 10 EEAENTLQSFR 98.99999499 91 4 EKLQEEMLQR 98.99999499 92 6 EMEENFAVEAANYQDTIGR 98.99999499 93 17 ETNLDSLPLVDTHSK 98.99999499 94 8 EYQDLLNVK 98.99999499 95 2 FADLSEAANR 98.99999499 96 10 ILLAELEQLK 98.99999499 97 3 ISLPLPNFSSLNLR 98.99999499 98 5 KLLEGEESR 98.99999499 99 7 LGDLYEEEMR 98.99999499 100 13 LLEGEESR 94.99999861 101 1 LLQDSVDFSLADAINTEFK 98.99999499 102 2 LQDEIQNMK 98.99999499 103 3 LQDEIQNMKEEMAR 98.99999499 104 9 LQEEMLQR 98.99999499 105 10 NLDSLPLVDTHSK 98.99999499 106 1 NLQEAEEWYK 98.99999499 107 3 QDVDNASLAR 98.99999499 108 1 QQYESVAAK 98.99999499 109 5 QVDQLTNDK 98.99999499 110 2 QVQSLTCEVDALK 98.99999499 111 7 RQVDQLTNDK 98.9999969 112 1 TNEKVELQELNDR 98.99999499 113 5 VELQELNDR 98.99999499 114 7 VEVERDNLAEDIMR 98.99999499 115 2 spt|P13928 Annexin A8 17.4311921 6 AYEEDYGSSLEEDIQADTSGYLER 98.99999499 116 1 EGVIIEILASR 97.99999595 117 1 GIGTNEQAIIDVLTK 97.99999595 118 1 NALLSLVGSDP 96.9999969 119 1 SSSHFNPDPDAETLYK 98.99999499 120 1 TLSSMHMEDTSGDYK 98.99999499 121 2 spt|P14618 PK M2 39.81132209 18 AEGSDVANAVLDGADCIMLSGETAK 98.99999499 122 14 APIIAVTR 98.99999499 123 17 EAEAAIYHLQLFEELR 98.99999499 124 3 EAEAAIYHLQLFEELRR 98.99999499 125 1 FDEILEASDGIMVAR 98.99999499 126 3 GADFLVTEVENGGSLGSK 98.99999499 127 17 GDLGIEIPAEK 98.99999499 128 3 GDYPLEAVR 98.99999499 129 10 GSGTAEVELK 98.99999499 130 1 GVNLPGAAVDLPAVSEK 98.99999499 131 5 HVDDGLISLQVK 98.99999499 132 7 KASDVHEVR 98.99999499 133 1 LAPITSDPTEATAVGAVEASFK 98.99999499 134 3 LDIDSPFITAR 98.99999499 135 21 NTGIICTIGPASR 98.99999499 136 10 RFDEILEASDGIMVAR 98.99999499 137 17 TATESFASDPILYRPVAVALDTK 98.99999499 138 6 VNFAMNVGK 98.99999499 139 2 spt|14625 GRP 94 18.31382257 6 FAFQAEVNR 98.99999499 140 1 GVVDSDDLPLNVSR 98.99999499 141 2 IYFMAGSSR 98.99999499 142 1 LGVIEDHSNR 98.99999499 143 4 LISLTDENALSGNEELTVK 98.99999499 144 1 NLLHVTDTGVGMTR 98.99999499 145 11 spt|P26038 Meosin 31.25 17 ALELEQER 98.99999499 146 5 ALTSELANAR 98.99999499 147 8 APDFVFYAPR 98.99999499 148 5 AQQELEEQTR 98.99999499 149 3 EALLQASR 96.9999969 150 4 EKEELMER 98.99999499 151 4 FYPEDVSEELIQDITQR 98.99999499 152 2 IGFPWSEIR 96.9999969 153 1 IQVWHEEHR 98.99999499 154 3 ISQLEMAR 98.99999499 155 6 KAQQELEEQTR 98.99999499 156 10 KTQEQLALEMAELTAR 98.99999499 157 1

QLFDQVVK 96.9999969 158 1 TAMSTPHVAEPAENEQDEQDENGAEAS 96.9999969 159 1 TANDMIHAENMR 98.99999499 160 3 TQEQLALEMAELTAR 98.99999499 161 1 VTTMDAELEFAIQPNTTGK 98.99999499 162 2 spt|P29034 S100A 2 17.52577275 2 ELPSFVGEK 98.99999499 163 13 ELPSFVGEKVDEEGLK 98.99999499 164 1 spt|P31151 S100A 7 33.00000131 3 GTNYLADVFEK 98.99999499 165 4 GTNYLADVFEKK 98.99999499 166 2 SIIGMIDMFHK 98.99999499 167 32 spt|P31947 SFN 75.4629612 16 ADLHTLSEDSYK 96.9999969 168 1 DNLTLWTADNAGEEGGEAPQEPQS 98.99999499 169 4 DSTLIMQLLR 98.99999499 170 180 EEKGPEVR 97.99999595 171 1 EMPPTNPIR 98.99999499 172 9 GAVEKGEELSCEER 98.99999499 173 20 GEELSCEER 98.99999499 174 10 LAEQAER 98.99999499 175 3 NLLSVAYK 98.99999499 176 56 SAYQEAMDISK 98.99999499 177 4 SAYQEAMDISKK 98.99999499 178 10 SNEEGSEEKGPEVR 98.99999499 179 56 TTFDEAMADLHTLSEDSYK 98.99999499 180 50 VLSSIEQK 98.99999499 181 47 YEDMAAFMK 98.99999499 182 8 YLAEVATGDDK 98.99999499 183 9 spt|P31949 Calgizzarin 36.19047701 2 ISSPTETER 98.99999499 184 6 TEFLSFMNTELAAFTK 98.99999499 185 6 spt|P36952 Maspin 11.46666631 2 GDTANEIGQVLHFENVK 98.99999499 186 1 Precursor VCLEITEDGGDSIEVPGAR 94.99999681 187 1 spt|P51884 KSPG 23.66863936 6 FNALQYLR 97.99999595 188 1 Lumican ISNPDEYFK 96.9999969 189 1 NIPTVNENLENYYLEVNQLEK 98.99999499 190 3 NNQIDHDEK 98.99999499 191 13 SLEDLQLTHNK 98.99999499 192 11 SLEYLDLSFNQIAR 98.99999499 193 10 spt|P60709 Beta Actin 72.5333333 34 AALVVDNGSGMCK 98.99999499 194 5 AGFAGDDAPR 98.99999499 195 66 ALDFEQEMATAASSSSLEK 98.99999499 196 6 AVFPSIVGR 98.99999499 197 36 AVFPSIVGRPR 98.99999499 198 68 DDDIAALVVDNGSGMCK 98.99999499 199 88 DESGPSIVHR 98.99999499 200 2 DLTDYLMK 98.99999499 201 24 DLYANTVLSGGTTMYPGIADR 98.99999499 202 15 DSYVGDEAQSK 98.99999499 203 32 DSYVGDEAQSKR 98.99999499 204 8 EITALAPSTMK 98.99999499 205 38 GFAGDDAPR 98.99999499 206 1 GYSFTTTAER 98.99999499 207 10 HQGVMVGMGQK 98.99999499 208 1 IWHHTFYNELR 98.99999499 209 4 KDLYANTVLSGGTTMYPGIADR 98.99999499 210 7 LCYVALDFEQEMATAASSSSLEK 98.99999499 211 1 LLTEAPLNPK 98.99999499 212 2 QEYDESGPSIVHR 98.99999499 213 6 SGGTTMYPGIADR 98.99999499 214 2 SYELPDGQVITIGNER 98.99999499 215 101 TALAPSTMK 98.99999499 216 3 TEAPLNPK 98.99999499 217 18 TTGIVMDSGDGVTHTVPIYEGY 98.99999499 218 6 TTGIVMDSGDGVTHTVPIYEGYALPH 98.99999499 219 1 TTGIVMDSGDGVTHTVPIYEGYALPHAlt 98.99999499 220 15 VALDFEQEMATAASSSSLEK 98.99999499 221 2 VAPEEHPV 97.99999595 222 2 VAPEEHPVL 95.99999785 223 2 VAPEEHPVLL 98.99999499 224 5 VAPEEHPVLLTEAPLNPK 96.9999969 225 1 VLSGGTTMYPGIADR 98.99999499 226 3 YPIEHGIVTNWDDMEK 98.99999449 227 33 spt|P62805 Histone H4 48.0392158 5 DAVTYTEHAK 98.99999499 228 15 DNIQGITKPAIR 98.99999499 229 2 ISGLIYEETR 98.99999499 230 10 TVTAMDVVYALK 98.99999499 231 1 VFLENVIR 97.99999595 232 7 spt|Q9NZT1 Calmodule- 58.21917653 6 AFDQDGDGHITVDELR 98.99999499 233 3 like protein 5 AFSAVDTDGNGTINAQELGAALK 98.99999499 234 1 AGLEDLQVAFR 98.99999499 235 4 AMAGLGQPLPQEELDAMIR 98.99999499 236 2 NLSEAQLR 98.99999499 237 1 VNYEEFAR 98.99999499 238 1 trm|O60744 Thio- 39.28571343 3 EKLEATINELV 98.99999499 239 1 redoxin Delta 3 TAFQEALDAAGDK 98.99999499 240 4 VGEFSGANK 98.99999499 241 10 trm|Q6NTE9 PPIA 52.38095522 8 EGMNVEAMER 98.99999499 242 14 FEDENFILK 98.99999499 243 3 IIPGFMCQGGDFTR 98.99999499 244 21 ITIADCGQLE 98.9999969 245 1 KITIADCGQLE 98.99999499 246 2 SIYGEKFEDENFILK 98.99999499 247 8 VNPTVFFDIAVDGEPLGR 98.99999499 248 54 VSFELFADK 98.99999499 249 1 trm|Q71DI3 Histone H3 30.14706777 2 EIAQDFKTDLR 94.99999881 250 1 SAPATGGVK 98.99999499 251 4 trm|Q36V33 YWHAZ 39.37500119 13 DNLTLWTSDTQGDEAEAGEGGEN 98.99999499 252 3 DSTLIMQLLR 98.99999499 253 180 EMQPTHPIR 98.99999499 254 8 GIVDQSQQAYQEAFEISK 98.99999499 255 6 GIVDQSQQAYQEAFEISKK 98.99999499 256 2 LAEQAER 98.99999499 257 3 MDKNELVQK 98.99999499 258 4 NLLSVAYK 98.99999499 259 56 SVTEQGAELSNEER 98.99999499 260 8 TAFDEAIAELDTLSEESYK 98.99999499 261 45 VVSSIEQK 98.99999499 262 9 YDDMAACMK 98.99999499 263 7 YLAEVAAGDDKK 98.99999499 264 9 trm|QEWU39 PACAP 26.93412836 3 ELSELVYTDVLDR 98.99999499 265 11 NWQDYGVR 98.99999499 266 5 TCLHYLGEFGEDQIYEAHQQGR 98.99999499 267 1 trm|Q96IH1 FSCN1 6.200000107 5 ASAETVDPASLWEY 98.99999499 268 1 DVPWGVDSLITLAFQDQR 98.99999499 269 1 FLIVAHDDGR 98.99999499 270 7 KVTGTLDANR 98.99999499 271 3 YLAADKDGNVTCER 98.99999499 272 2 trm|Q96R27 Mast cell 18.02575141 5 DDMLCAGNTR 98.9999969 273 1 lyplase beta III EQHLYYQDQLLPVSR 98.99999499 274 2 IVGGQEAPR 98.99999499 275 9 VPIMENHICDAK 98.99999499 276 2 YHLGAYTGDDVR 98.99999499 277 6 trm|Q9UE88 Histone 19.80198026 3 AMGIMNSFVNDIFER 98.99999499 278 4

H2B.1 LLLPGELAK 98.99999499 279 2 QVHPDTGISSK 98.99999499 280 11 OPLs spt|P29956 MARCKS 9.64 2 AAEEPSKVEEK 99.00 281 2 EAPAEGEAAEPGSPTAAEGEAASAASS 99.00 282 1 spt|P06748 Nucleo 6.18 2 GPSSVEDIK 99.00 283 5 phosmin 1 VTLATLK 96.00 284 1 gb|AAH25314.1 IGHG1 21.49 12 ALPAPIEK 99.00 285 12 protein DYFPEPVTV 98.00 286 1 FNWYVDGVEVHNAK 99.00 287 5 GFYPSDIAVEWESNGQPENNYK 99.00 288 4 GPSVFPLAPSSK 99.00 289 2 NQVSLTCLVK 99.00 290 1 QVQLVQSGAEVK 99.00 291 3 STSGGTAALGCLVK 99.00 292 2 THTCPPCPAPELLGGPSVFLFPPKPK 99.00 293 2 TPEVTCVVVDVSHEDPEVK 99.00 294 3 TTPPVLDSDGSFFLYSK 99.00 295 24 VVSVLTVLHQDWLNGK 99.00 296 2 spt|P01009 Alpha 1 8.13 3 AVLTIDEK 99.00 297 4 Anti- Trypsin Precursor DTEEEDFHVDQVTTVK 99.00 298 2 LSITGTYDLK 99.00 299 1 spt|P04080 Cystatin B 36.73 4 GAPSATQPATAETQHIADQVR 99.00 300 1 MMCGAPSATQPATAETQHIADQVR 99.00 301 12 SQVVAGTNYFIK 99.00 302 3 VFQSLPHENKPLTLSNYQTNK 99.00 303 2 spt|P06454 PTHA 25.45 5 AAEDDEDDDVDTK 99.00 304 9 AAEDDEDDDVDTKK 99.00 305 14 EVVEEAENGR 99.00 306 6 RAAEDDEDDDVDTK 99.00 307 1 SDAAVDTSSEITTK 99.00 308 17 spt|P14625 GRP 94 3.74 10 DDEVDVDGTVEEDLGK 99.00 309 5 EFEPLLNWMK 99.00 310 4 EVEEDEYK 99.00 311 3 FAFQAEVNR 99.00 312 1 KEAESSPFVER 99.00 313 4 LGVIEDHSNR 99.00 314 3 LISLTDENALSGNEELTVK 99.00 315 4 TDDEVVQREEEAIQLDGLNASQIR 99.00 316 1 TVLDLAVVLFETATLR 99.00 317 1 VFITDDFHDMIMPK 99.00 318 2 spt|P27482 Calmodulin 20.27 5 AADTDGDGQVNYEEFVR 99.00 319 4 related protein-NB1 ADQLTEEQVTEFK 99.00 320 4 LSDEEVDEMIR 99.00 321 1 SLGQNPTEAELR 99.00 322 2 VFDKDGNGFVSAAELR 99.00 323 5 spt|P30086 PE Binding 15.05 3 GNDISSGTVLSDYVGSGPPK 99.00 324 1 protein PEBP LYTLVLTDPDAPSR 99.00 325 1 VLTPTQVK 99.00 326 1 spt|P31151 S100A7 24.00 4 GTNYLADVFEK 99.00 327 1 KIDFSEFLSLLGDIATDYHK 99.00 328 3 QSHGAAPCSGGSQ 99.00 329 1 SIIGMIIDMFHK 99.00 330 2 spt|P31947 Stretifin 26.24 16 DSTLIMQLLR 99.00 331 52 EKVETELQGVCDTVLGLLDSHLIK 99.00 332 13 EKVETELQGVCDTVLGLLDSHLIKEAGD 99.00 333 2 EMPFTNPIR 99.00 334 4 GAVEKGEELSCEER 99.00 335 38 GEELSCEER 99.00 336 7 GSEEKGPEVR 97.00 337 1 LAEQAER 99.00 338 2 NLLSVAYK 99.00 339 62 SAYQEAMDISKK 99.00 340 1 SNEEGSEEKGPEVR 99.00 341 42 TTFDEAMADLHTLSEDSYK 99.00 342 4 VETELQGVCDTVLGLLDSHLIK 99.00 343 2 VLSSIEQK 99.00 344 26 YEDMAAFMK 99.00 345 2 YLAEVATGDDK 99.00 346 9 spt|P60709 Beta Actin 27.47 21 AGFAGDDAPR 99.00 347 83 AVFPSIVGR 99.00 348 14 AVFPSIVGRPR 99.00 349 73 DDDIAALVVDNGSGMCK 99.00 350 20 DLYANTVLSGGTTMYPGIADR 99.00 351 8 DSYVGDEAQSK 99.00 352 29 DSYVGDEAQSKR 99.00 353 36 EITALAPSTMK 99.00 354 34 GYSFTTTAER 99.00 355 5 HQGVMVGMGQK 99.00 356 4 IIAPPER 97.00 357 7 tWHHTFYNELR 98.00 358 4 LCYVALDFEQEMATAASSSSLEK 99.00 359 12 LDLAGRDLTDYLMKILTERGYSFTTTAEF 97.00 360 2 QEYDESGPSIVHR 99.00 361 1 SYELPDGQVITIGNER 99.00 362 199 TEAPLNPK 99.00 363 37 TTGIVMDSGDGVTHTVPIYEGYALPHAIL 99.00 364 3 VAPEEHPVLL 99.00 365 2 VAPEEHPVLLTEAPLNPK 99.00 366 48 YPIEHGIVTNWDDMEK 99.00 367 7 spt|P61978 hetero- 2.38 3 DLAGSIIGK 99.00 368 2 geneous nuclear protein K IDEPLEGSEDR 99.00 369 1 ILSISADIETIGEILK 96.00 370 2 spt|Q01469 FABP5 28.15 5 ATVQQLEGR 99.00 371 7 ELGVGIALR 99.00 372 17 FEETTADGR 99.00 373 3 GFDEYMK 96.00 374 4 TTQFSCTLGEKFEETTADGR 99.00 375 1 trm|O15256 Parathy- 19.05 1 AAEEEDEADPKR 99.00 376 8 mosin trm|Q12771 P37AUF1* 2.80 1 GFGFVLFK 99.00 377 5 trm|Q6NSB4 Hp 3.20 1 VGYVSGWGR 99.00 378 2 Protein, DLC1 spt|P63104 YWHAZ 13.75 8 DSTLIMQLLR 99.00 379 50 GIVDQSQQAYQEAFEISK 99.00 380 1 LAEQAER 99.00 381 2 NLLSVAYK 99.00 382 59 SVTEQGAELSNEER 99.00 383 7 TAFDEAIAELDTLSEESYK 99.00 384 4 VVSSIEQK 99.00 385 10 YLAEVAAGDDKK 99.00 386 2 trm|Q8N5F4 NGL2* 23.81 4 AGVETTTPSK 99.00 387 21 DTERPSGIPER 98.00 388 1 SYELTQPPSVSVSPGQTAR 99.00 389 2 SYSCQVTHEGSTVEK 99.00 390 17 spt|P06396 Gelsolin 2.30 2 AQPVQVAEGSEPDGFWEALGGK 99.00 391 1 Precursor TPSAAYLWVGTGASEAEK 99.00 392 6 amb|CAA25833.1 eraide- 18.72 8 AGAHLQGGAK 99.00 393 11 hyde3- phosphate Dehydrogen

GALQNIIPASTGAAK 99.00 394 19 LTGMAFR 98.00 395 3 QASEGPLK 99.00 396 9 TVDGPSGK 99.00 397 4 VIPELDGK 99.00 398 16 VPTANVSVVDLTCR 99.00 399 10 VVDLMAHMASKE 99.00 400 2 spt|P14618 PKM2* 9.04 8 APIIAVTR 99.00 401 10 GADFLVTEVENGGSLGSK 99.00 402 1 GDLGIEIPAEK 99.00 403 6 GDYPLEAVR 99.00 404 2 IENHEGVR 99.00 405 5 KASDVHEVR 99.00 406 1 LDIDSPPITAR 99.00 407 9 NTGIICTIGPASR 99.00 408 4 spt|P04792 Hsp27* 18.85 5 AQLGGPEAAK 99.00 409 21 LATQSNEITIPVTFESR 99.00 410 9 QDEHGYISR 99.00 411 6 QLSSGVSEIR 99.00 412 1 VSLDVNHFAPDELTVK 99.00 413 8 spt|P09651 ROA1- 7.50 2 EDSQRPGAHLTVK 99.00 414 3 HNRNPA1* SESPKEPEQLR 99.00 415 4 spt|P13639 Elongation 1.17 2 GEGQLGPAER 99.00 416 1 Factor 2* SDPVVSYR 99.00 417 1 spt|P14625 HSP9081 3.74 10 DDEVDVDGTVEEDLGK 99.00 418 5 EFEPLLNWMK 99.00 419 4 EVEEDEYK 99.00 420 3 FAFQAEVNR 99.00 421 1 KEAESSPFVER 99.00 422 4 VGVIEDHSNR 99.00 423 3 LISLTDENALSGNEELTVK 99.00 424 4 TDDEVVQREEEAIQLDGLNASQIR 99.00 425 1 TVLDLAVVLFETATLR 99.00 426 1 VFITDDFHDMMPK 99.00 427 2 spt|P22392 NDP 11.54 2 GLVGEIK 99.00 428 4 Kinase B* NIHGSDSVK 99.00 429 1 spt|P23528 Coflin* 4.82 4 ASGVAVSDGVIK 99.00 430 24 spt|P31940 Calgizzarin 8.57 2 ISSPTETER 98.00 431 1 NQKDPGVLDR 99.00 432 2 spt|P32119 Peroxi- 4.04 6 ATAVVDGAFK 99.00 433 18 redoxin 2 GLFIIDGK 99.00 434 2 KEGGLGPLNIPLLADVTR 99.00 435 2 LSEDYGVLK 99.00 436 1 QITVNDLPVGR 99.00 437 10 TDEGIAYR 99.00 438 6 prt|0904262A SOD2* 13.73 1 TLVVHEK 93.00 439 1 spt|Q16778 Histone 15.87 3 AMGIMNSFVNDIFER 99.00 440 4 H28.1 LLLPGELAK 99.00 441 7 QVHPDTGISSK 99.00 442 68 OFFLINE gb|AAA59555.1 MARCKS 59.33734775 11 AAEEPSKVEEK 98.99999499 443 2 AEDGATPSPSNETPK 98.00000095 444 3 AEDGATPSPSNETPKK 94.99999881 445 1 AVAPEKPPASDETK 99.99999995 446 1 EAGEGGEAEAPAAEGGK 99.00000095 447 1 EAPAEGEAAEPGSPTAAEGEAASAASS 98.99999499 448 3 EELQANGSAPAADKEEPAAAGSGAASP 99.00000095 449 3 GEAAAERPGEAAVASSPSK 99.00000095 450 6 GEPAAAAAPEAGASPVEK 99.00000095 451 9 GSAPAADKEEPAAAGSGAASPSAAEK 98.99999499 452 1 VNGDASPAAAESGAK 99.00000095 453 7 gb|AAC13869.1 Gluta- 61.90476418 4 AFLASPEYVNLPINGNGKQ 99.00000095 454 2 thione S Trans- ferase-P ALPGQLKPFETLLSQNQGGK 99.00000095 455 27 ASCLYGQLPK 99.00000095 456 1 FQDGDLTLYQSNTILR 98.00000191 457 1 gb|AAH16768.1 Nucleo- 12.24489808 3 GPSSVEDIK 99.00000095 458 1 phosmin 1 MSVQPTVSLGGFEITPPVVLR 98.99999499 459 1 MTDQEAIQDLWQWR 98.99999499 460 2 pir|KRHUE Cyto- 69.4915235 5 ASLENSLEETK 99.00000095 461 2 keratin 14 DAEEWFFTK 99.00000095 462 2 ILTATVDNANVLLQIDNAR 98.99999499 463 2 KVVSTHEQVLR 99.00000095 464 2 VVSTHEQVLR 99.00000095 465 6 spt|P00338 LDH A 52.58767671 14 ATLKDQLIYNLLKEEQTPQNK 98.99999499 466 8 DQLIYNLLK 98.99999499 467 2 DQLIYNLLKEEQTPQNK 99.00000095 468 2 FIIPNVVK 94.99999881 469 2 GEMMDLQHGSLFLR 98.99999499 470 2 KSADTLWGIQK 97.99999695 471 2 LVIITAGAR 99.00000095 472 20 OVVESAYEVIK 99.00000095 473 32 RVHPVSTMIK 99.00000095 474 8 SADTLWGIQK 99.00000095 475 3 TLHPDLGTDKDKEQWK 98.99999499 476 2 VHPVSTMIK 99.00000095 477 2 VIGSGCNLDSAR 99.00000095 478 31 VTLTSEEEAR 99.00000095 479 55 spt|P00915 Carbonic 66.15384817 13 ASPDWGYDDK 99.00000095 480 2 anhydrasel ASPDWGYDDKNGPEQIVSK 99.00000095 481 3 EIINVGHSFHVNFEDNDNR 99.00000095 482 71 GGPFSDSYR 99.00000095 483 2 HDTSLKPISV 99.00000095 484 1 HDTSLKPISVSYNPATAK 99.00000095 485 49 LYPIANGNNQSPVDIK 99.00000095 486 31 SAELHVAHWNSAK 99.00000095 487 3 SLLSNVEGDNAVPMQHNNRPTQPLK 99.00000095 488 73 SSSEQLAQFR 99.00000095 489 2 VLDALQAIK 99.00000095 490 6 YSAELHVAHWNSAK 99.00000095 491 22 YSSLAEAASK 99.00000095 492 3 spt|P01009 Alpha 1 55.74162602 9 DTEEEDFHVDQVTTVK 99.00000095 493 7 Anti- Trypsin Precursor EDPQGDAAQK 98.00000191 494 1 GTEAAGAMFLEAIPM 95.99999785 495 1 KLSSWVLLMK 98.9999969 496 1 LGMFNIQHCK 99.00000095 497 1 LQHLENELTHDIITK 98.99999499 498 3 LSITGTYDLK 99.00000095 499 2 LVDKFLEDVK 99.99999499 500 1 VFSNGADLSGVTEEAPLK 99.00000095 501 15 spt|P04063 Annexin A1 75.36231875 14 AAYLQETGKPLDETLK 99.00000095 502 10 AAYLQETGKPLDETLKK 99.00000095 503 35 ALYEAGER 99.00000095 504 8 CATSKPAFFAEK 99.00000095 505 4 DITSDTSGDFR 99.00000095 506 40 GGPGSAVSPYPTFNPSSDVAALHK 99.00000095 507 14 GLGTDEDTLIEILASR 99.00000095 508 427

GTDVNVFNTILTTR 99.00000095 509 25 GVDEATIIDILTK 99.00000095 510 75 KGTDVNVFNTILTTR 99.00000095 511 14 NALLSLAK 98.00000191 512 2 SEDFGVNEDLADSDAR 99.00000095 513 25 SEIDMNDIK 99.00000095 514 1 TPAQFDADELR 99.00000095 515 12 spt|P04792 Hsp27 34.55497324 7 DGVVEITGK 99.00000095 516 4 KYTLPPGVDPTQVSSSLSPEGTLTVEAP 99.00000095 517 5 LATQSNEITIPVTFESR 98.99999499 518 14 QDEHGYISR 95.99999785 519 2 QLSSGVSEIR 98.99999499 520 2 TKDGVVEITGK 99.00000095 521 6 VSLDVNHFAPDELTVK 99.00000095 522 15 spt|P06454 Prothy- 23.63636345 6 AAEDDEDDDVDTK 98.00000191 523 1 mosin alpha AAEDDEDDDVDTKK 98.99999499 524 1 EVVEEAENGR 99.00000095 525 13 EVVEEAENGRDAPAN 98.99999499 526 1 KEVVEEAENGR 99.00000095 527 1 SDAAVDTSSEITTK 99.00000095 528 54 spt|P07585 Decorin 38.16158089 5 DLPPDTTLLDLQNNK 99.00000095 529 7 Precursor NLHALILVNNK 99.00000095 530 3 SSGIENGAFQGMK 99.00000095 531 3 VSPGAFTPLVK 99.00000095 532 3 VVQCSDLGLDKVPK 99.00000095 533 3 spt|P06670 Vismentin 63.21585774 29 ADLSEAANR 98.00000191 534 1 DGQVINETSQHHDDLE 98.99999499 535 6 DNLAEDIMR 99.00000095 536 9 EEAENTLQSFR 99.00000095 537 6 EKLQEEMLQR 98.99999499 538 1 EMEENFAVEAANYQDTIGR 98.99999499 539 16 ETNLDSLPLVDTHSK 98.99999499 540 4 EYQDLLNVK 98.99999499 541 2 FADLSEAANR 99.00000095 542 7 ILLAELEQLK 98.99999499 543 18 ILLAELEQLKGQGK 98.99999499 544 14 ISLPLPNFSSLNLR 98.99999499 545 55 KLLEGEESR 98.99999499 546 1 KVESLQEEIAFLK 98.99999499 547 15 KVESLQEEIAFLKK 98.99999499 548 1 LGDLYEEEMR 99.00000095 549 7 LLQDSVDFSLADAINTEFK 98.99999499 550 18 LQDEIQNMKEEMAR 98.99999499 551 4 LQEEMLQR 99.00000095 552 4 NLDSLPLVDTHSK 98.99999499 553 2 NLQEAEEWYK 98.99999499 554 4 QDVDNASLAR 99.00000095 555 78 QQYESVAAK 97.99999595 556 2 QVDQLTNDK 98.00000191 557 2 QVQSLTCEVDALK 98.99999499 558 8 RQVDQLTNDK 97.99999595 559 1 TVETRDGQVINETSQHHDDLE 98.99999499 560 2 VELQELNDR 98.99999499 561 5 VEVERDNLAEDIMR 98.99999499 562 2 spt|P14625 GRP 94 26.40099525 14 DDEVDVDGTVEEDLGK 98.99999499 563 2 EFEPLLNWMK 96.9999969 564 2 EGVKFDESEK 98.99999499 565 2 ELISNASDALDK 99.00000095 566 1 FQSSHRPTDITSLDQYVER 99.00000095 567 2 GLFDEYGSK 99.00000095 568 1 GVVDSDDLPLNVSR 99.00000095 569 21 KIADDKYNDTFWK 98.99999499 570 2 LGVIEDHSNR 99.00000095 571 1 LISLTDENALSGNEELTVK 98.99999499 572 6 NLLHVTDTGVGVTR 98.99999499 573 6 RVFITDDFHDMMPK 99.00000095 574 3 SILFVPTSAPR 98.99999499 575 9 VFITDDFHDMMPK 98.99999499 576 2 spt|P29034 S100A 2 27.83505023 6 ELPSFVGEK 99.00000095 577 6 ELPSFVGEKVDEEGLK 99.00000095 578 14 ELPSFVGEKVDEEGLKK 99.00000095 579 13 GEKVDEEGLK 94.99999881 580 1 VDEEGLKK 98.99999499 581 6 YSCQEGDKFK 99.00000095 582 7 spt|P30043 Flavin 39.51219616 7 CLTTDEYDGHSTYPSHQYQ 99.00000095 583 1 reductase HDLGHFMLR 99.00000095 584 2 LPSEGPRPAHVVVGDVLQAADVDK 99.00000095 585 9 LQAVTDDHIR 99.00000095 586 1 NDLSPTTVMSEGAR 99.00000095 587 14 PAHVVVGDVLQAADVDK 98.99999499 588 4 TVAGQDAVIVLLGTR 98.99999499 589 4 spt|P31151 S100A 7 98.00000191 7 ENFPNFLSACDKK 99.00000095 590 9 GTNYLADVFEK 99.00000095 591 22 GTNYLADVFEKK 99.00000095 592 7 IDKPSLLTMMK 99.00000095 593 2 KGTNYLADVFEKK 95.99999785 594 2 QSHGAAPCSGGSQ 99.00000095 595 3 SIIGMIIDMFHK 99.00000095 596 300 spt|P31947 SFN 89.35185075 22 ADNAGEEGGEAPQEPQS 99.00000095 597 13 DNLTLWTADNAGEEGGEAPQEPQ 96.9999969 598 2 DNLTLWTADNAGEEGGEAPQEPQS 99.00000095 599 53 DSTLIMQLLR 99.00000095 600 515 EMPFTNPIR 99.00000095 601 14 GAVEKGEELSCEER 99.00000095 602 26 GEELSCEER 99.00000095 603 16 LAEQAER 99.00000095 604 9 NLLSVAYK 99.00000095 605 35 RYLAEVATGDDK 99.00000191 606 1 SAYQEARDISK 99.00000095 607 9 SAYQEAMDISKK 99.00000095 608 29 SNEGSEEKGPEVR 99.00000095 609 9 STLIMQLLR 98.99999499 610 1 SVFHYEIANSPEEAISLAK 98.99999499 611 1 TADNAGEEGGEAPQEPQS 98.99999499 612 6 TIFDEAMADLHTLSEDS 98.99999499 613 1 TTFDEAMADLHTLSEDSYK 99.00000096 614 396 VLSSIEQK 99.00000096 615 6 YEDMAAFMK 99.00000095 616 12 YLAEVATGDDK 99.00000095 617 19 YLAEVATGDDKK 99.99999499 618 2 spt|P31949 Calgizzarin 23.80952386 8 DGYNYTLSK 99.00000095 619 1 ISSPTETER 99.00000096 620 13 TEFLSFMNTELAAFTK 99.00000096 621 15 spt|P32119 Peroxi- 64.64645459 11 ATAVVDGAFK 99.00000095 622 3 redoxin 2 EGGLGPLNIPLLADVTR 99.00000096 623 4 GLFIIDGK 99.00000096 624 14 IGKPAPDFK 98.00000095 625 22 KEGGLGPLNIPLLADVTR 99.00000095 626 21 LSEDYGVLK 99.00000095 627 7 LVQAFQYTDEHGEVCPAGWKPGSDTIK 99.00000095 628 62 QITVNDLPVGR 99.00000095 629 41 QYTDEHGEVCPAGWKPGSDTIKPNVDC 97.00000286 630 1 RLSEDYGVLK 99.00000095 631 5

TDEGIAYR 99.00000095 632 13 spt|P51684 KSPG 56.80473447 13 FNALQYLR 99.00000095 633 7 Lumican ILGPLSYSK 99.00000095 634 3 ISETSLPPDMYECLR 99.00000095 635 4 ISNIPDEYFK 99.00000095 636 4 LKEDAVSAAFK 99.00000095 637 8 LPSGLPVSLLTLYLDNNK 99.00000095 638 17 NIPTVNENLENYYLEVNQLEK 99.00000095 639 5 NNQIDHIDEK 99.00000095 640 2 RFNALQYLR 99.00000095 641 2 SLEDLQLTHNK 99.00000095 642 10 SLEYLDLSFNQIAR 99.00000095 643 8 SVPMVPPGIK 94.99999981 644 1 VANEVTLN 95.99999785 645 1 spt|P60709 Beta Actin 83.46666694 49 AGFAGDDAPR 99.00000095 646 77 ALDFEQEMATAASSSSLEK 99.00000095 647 13 AVFPSIVGR 99.00000095 648 61 AVFPSIVGRPR 99.00000095 649 303 CPEALFQPSFLGMESCGIHETTFNSIMK 98.00000499 650 30 DDDIAALVVDNGSGMCK 99.00000095 651 172 DDIAALVVDNGSGMCK 99.00000095 652 3 DLTDYLMK 99.00000095 653 29 DLYANTVLSGGTTMYPGIADR 99.00000095 654 54 DSYVGDEAQSK 99.00000095 655 39 DSYVGDEAQSKR 99.00000095 656 3 EITALAPSTMK 99.00000095 657 46 GFAGDDAPR 99.00000095 658 1 GIHETTFNSIMK 99.00000095 659 3 GIVTNWDDMEK 99.00000095 660 1 GQKDSYVGDEAQSK 99.00000095 661 8 GYSFTTTAER 99.00000095 662 33 HQGVMVGMGQK 98.99999499 663 14 HQGVMVGMGQKDSYVGDEAQSK 98.00000191 664 1 IIAPPER 97.00000286 665 8 IIAPPERK 99.00000095 666 3 IWHHTFYNELR 99.00000095 667 512 KQLYANTVLSGGTTMYPGIADR 99.00000095 668 99 KQEYDESGPSIVHR 99.00000095 669 2 KYPIEHGIVTNWDDMEK 99.00000095 670 1 LCYVALDFEQEMATAASSSSLEK 98.99999499 671 12 LLTEAPLNPK 99.00000095 672 2 PIEHGIVTNWDDMEK 98.99999499 673 2 QEYDESGPSIVHR 99.00000095 674 36 RVAPEEHPVLLTEAPLNPK 98.99999499 675 2 SGGTTMYPGIADR 99.00000095 676 6 SKQEYDESGPSIVHR 99.00000095 677 2 SYELPDGQVI 99.00000095 678 5 SYELPDGQVITI 98.99999499 679 6 SYELPDGQVITIGNER 99.00000095 680 702 TTGIVMDSGDGVTH 98.99999499 681 4 TTGIVMDSGDGVTHTVPIYEGY 99.00000095 682 5 TTGIVMDSGDGVTHTVPIYEGYALPH 99.00000095 683 1 TTGIVMDSGDGVTHTVPIYEGYALPHAIL 99.00000095 684 69 TVLSGGTTMYPGIADR 99.00000095 685 3 VAPEEHPV 99.00000095 686 9 VAPEEHPVL 97.00000286 687 4 VAPEEHPVLL 99.00000095 688 8 VAPEEHPVLLTEA 98.99999499 689 4 VAPEEHPVLLTEAPLN 98.99999499 690 8 VAPEEHPVLLTEAPLNPK 99.00000095 691 509 VAPEEHPVLLTEAPLNPKANR 99.00000095 692 3 YPIEHGIVTNWDDMEK 99.00000095 693 120 YVALDFEQEMATAASSSSLEK 94.99999881 694 2 spt|Q15891 APC- 8.655223858 1 KPLTSSSAAPQRPISTQR 99.00000095 695 12 binding protein EB1 trm|Q86U86 Polybromo- 29.01124954 2 AAQQQQPSASPR 98.00000191 696 2 1D RPNETFHLATRK 99.00000095 697 1 trm|Q86V33 YVMAZ 77.49999762 16 DNLTLWTSDTQGDEAEAGEGGEN 99.00000095 698 10 DSTLIMQLLR 99.00000095 699 753 EKIETELR 96.9999969 700 1 GIVDQSQQAYQEAFEISK 99.00000095 701 21 GIVDQSQQAYQEAFEISKK 99.00000095 702 32 LAEQAER 99.00000095 703 10 MDKNELVQK 98.00000191 704 1 NLLSVAYK 99.00000095 705 38 SDTQGDEAEAGEGGEN 99.99999995 706 3 STLIMQLLR 98.99999499 707 2 SVTEQGAELSNEER 99.00000095 708 113 TAFDEAIAELDTLSEESYK 99.00000095 709 899 VVSSIEQK 99.00000095 710 7 YDDMAACMK 99.00000095 711 3 YLAEVAAGDDKK 99.00000095 712 11 trm|Q86Y16 L Ptastin 52.53968239 9 AESMLQQADK 99.00000095 713 1 HVIPMNPNTDDLPK 98.99999499 714 2 IDINMSGFNETDDLKR 98.99999499 715 2 IKVPVDWSK 98.99999499 716 2 MINLSVPDTIDER 94.99999881 717 1 SGNLTEDDKHNNAK 99.00000095 718 4 TISSSLAVVDLIDAIQPGCINYDLVK 98.99999499 719 2 VNKPPYPK 99.00000095 720 1 VYALPEDLVEVKPK 95.00000785 721 2 trm|Q81Z29 Tubulin, 40.67415585 10 ALTVPELTQQMFDAK 98.99999499 722 1 beta, 2 AVLVDLEPGTMDSVR 98.99999499 723 6 EIVHLQAGQCGNQIGAK 98.99999499 724 2 GHYTEGAELVDSVLDVVR 98.99999499 725 4 IMNTFSVVPSPK 98.99999499 726 1 IREEYPDR 97.99999595 727 1 LHFFMPGFAPLTSR 98.99999499 728 7 LTTPTYGDLNHLVSATMSGVTTCLR 98.99999489 729 4 SGPFGQIFRPDNFVFGQSGAGNNWAK 98.99999499 730 34 TAVCDIPPR 98.99999499 731 2 trm|Q96AM7 Superoxide 52.85714269 1 HHAAYVNNLNVTEEK 99.00000095 732 1 dismutase [Mn] trm|Q96IH1 FSCN1 66.60000086 8 ASAETVDPASLWEY 99.00000095 733 3 DVPWGVDSLITLAFQDQR 98.99999499 734 4 EVPGPDCR 99.00000095 735 1 FLIVAHDDGR 99.00000095 736 3 KVTGTLDANR 98.99999499 737 1 LVARPEPATGYTLEFR 98.99999499 738 2 WSLQSEAHR 94.99999681 739 1 YLAADKDGNVTCER 99.00000095 740 5

TABLE-US-00014 TABLE 12 Clinicopathological parameters of patients with Oral Premalignant Lesions (OPLs) Age/Gender Site Tissue Histopathology Tobacco habits A) ITRAQ Analysis: Discovery of Biomarkers 1. 35/F GBS Leukoplakia Dysplasia Areca nut chewing (.times.5-6 yrs) 2. 42/M BM Leukoplakia Dysplasia Bidi smoking (.times.10 yrs) 3. 50/M BM Leukoplakia Dysplasia Pan masala and bidi smoking (.times.15-18 yrs) 4. 42/M GBS Leukoplakia Dysplasia Gutkha chewing (.times.20 yrs) and Bidi smoking (.times.6 yrs) 5. 42/M T Leukoplakia Dysplasia Gutkha chewing and bidi smoking (.times.5 yrs) 6. 50/M SP Leukoplakia Dysplasia Gutkha chewing and bidi smoking (.times.35 yrs) B) Immunohistochemical Analysis: Verification of Biomarkers 1. 23/M BM Leukoplakia Dysplasia Khaini chewing (.times.4 yrs) 2. 36/M LIP Leukoplakia Dysplasia Khaini chewing (.times.10-12 yrs) 3. 40/F GBS Leukoplakia Dysplasia Khaini chewing (.times.14 yrs) 4. 22/M GBS Leukoplakia Dysplasia Khaini chewing (.times.5 yrs) 5. 45/F BM Leukoplakia Dysplasia Gutkha chewing (.times.8 yrs) 6. 22/M BM Leukoplakia Dysplasia Gutkha chewing (.times.3-4 yrs) 7. 50/F GBS Leukoplakia Dysplasia Gutkha chewing (.times.5 yrs) 8. 18/M GBS Leukoplakia Dysplasia Gutkha and khaini chewing (.times.5 yrs) 9. 35/M BM Leukoplakia Dysplasia Gutkha and khaini chewing (.times.10 yrs) 10. 40/F T Leukoplakia Dysplasia Gutkha and khaini chewing (.times.6 yrs) 11. 31/M BM Leukoplakia Dysplasia Betel quid with khaini chewing (.times.8 yrs) 12. 42/M BM Leukoplakia Dysplasia Bidi smoking (.times.18 yrs) 13. 50/M BM Leukoplakia Dysplasia Bidi smoking (.times.30 yrs) 14. 46/M BM Leukoplakia Dysplasia Bidi smoking (.times.18 yrs) 15. 54/M BM Leukoplakia Dysplasia Bidi smoking (.times.27 yrs) 16. 45/M BM Leukoplakia Dysplasia Bidi smoking (.times.12 yrs) 17. 50/M BM Leukoplakia Dysplasia Bidi smoking (.times.20 yrs) 18. 35/M T Leukoplakia Dysplasia Bidi smoking (.times.8 yrs) 19. 55/M T Leukoplakia Dysplasia Bidi smoking (.times.45 yrs) 20. 43/M BM Leukoplakia Dysplasia Cigarette smoking (.times.21 yrs) 21. 50/M BM Leukoplakia Dysplasia Cigarette smoking (.times.11 yrs) 22. 27/M BM Leukoplakia Dysplasia Gutkha chewing (.times.2 yrs) and cigarette smoking (.times.5 yrs) 23. 35/M BM Leukoplakia Dysplasia Gutkha and cigarette smoking (.times.10 yrs) 24. 24/M BM Leukoplakia Dysplasia Gutkha chewing (.times.3 yrs) and cigarette smoking (.times.1 yrs) 25. 54/M BM Leukoplakia Dysplasia Ghutka, khaini chewing (.times.15-16 yrs) and hookah smoking (.times.15-16 yrs) 26. 36/M BM Leukoplakia Dysplasia Gutkha and pan masala chewing (.times.8 yrs); bidi smoking (.times.10-12 yrs) 27. 66/M BM Leukoplakia Dysplasia Gutkha and khaini (.times.8 yrs) and bidi smoking (5 yrs) 28. 39/M BM Leukoplakia Dysplasia Betel quid with khaini (.times.2 yrs) and cigarette smoking (.times.5 yrs) 29. 45/M A Leukoplakia Dysplasia Betel quid with khaini (.times.20 yrs) and cigarette smoking (.times.25 yrs) 30. 70/M BM Leukoplakia Dysplasia No habit of addiction (NHA) All the patients were from India. Abbreviations: M: Male; F: Female; Site: A; Alvcolus, BM: Buccal Mucosa, GBS: Gigivo-buccal sulcus, T: Tongue; SP: Soft palate; `Khaini` is a mixture of tobacco, lime and menthol or aromatic spices; `Gutkha` usually contains powdered tobacco, betel nut, catechu, lime and flavors; Pan masala is a mixture of spices such as cardamon, lime, menthol, catechu and betel nuts; `Bidi` is tobacco hand rolled in Temburini leaf.

TABLE-US-00015 TABLE 13 Antibodies used for immunohistochemistry and Western Blotting: sources and dilutions. Dilution Dilution used for used for Western Company Clone ID IHC Blot Antibody Anti-14-3-3.zeta. Santa Cruz C-16 1:200 1:500 (YWHAZ) Rabbit polyclonal antibody Anti-14-3-3.sigma. (Stratifin) Santa Cruz N-14 1:200 1:200 Goat polyclonal antibody Anti-Psoriasin (S100A7) Santa Cruz 47C1068 1:100 1:200 mouse monoclonal antibody Anti-Pro-thymosin.alpha. Santa Cruz N-18 1:75 1:100 (PTHA) Goat polyclonal antibody Anti-hnRNPK mouse Abcam ab23644 1:400 1:400 monoclonal antibody Anti-Tubulin .alpha. mouse Santa Cruz B7 -- 1:200 monoclonal antibody Secondary Antibody: Goat Anti-rabbit IgG DAKO -- 1:5000 1:5000 Rabbit Anti-Goat IgG DAKO -- 1:4000 1:4000 Rabbit Anti-Mouse IgG DAKO -- 1:2000 1:2000

TABLE-US-00016 TABLE 14 RT-PCR Analysis Primers and PCR conditions: Annealing No. of Gene Tempera- amplifi- Product (Accession Primer Sequence ture cation size #) (SEQ ID NO:) (.degree. C.) cycles (bp) 14-3-3.zeta. 5'-ATGTACTTGGAAAAAGGCCG-3' 54 32 400 (NM_001135 (SEQ ID NO: 741) 699) 5'-CCCTGCTCTTGAGGAGCTTA-3' (SEQ ID NO: 742) Stratifin 5'- 57 32 396 (NM_006142) AGAGACACAGAGTCCGGCATTGG- 3' (SEQ ID NO: 743) 5'- TCCACCTTCTCCCGGTACTCACGC- 3' (SEQ ID NO: 744) S100A7 5'- 57 32 121 (Psoriasin) CTTCCTTAGTGCCTGTGACAAAAA- (NM_002963) 3' (SEQ ID NO: 745) 5'- AAGGACAGAAACTCAGAAAAATC AATCT-3' (SEQ ID NO: 746) Prothymosin 5'- 65 32 326 Alpha ATGTCAGACGCAGCCGTAGACACC (NP_0010927 A-3' (SEQ ID NO: 747) 55.1) 5'CTAGTCATCCTCGTCGGTCTTCTG- 3' (SEQ ID NO: 748) HnRNPK 5'AGCAGAGCTCGGAATCTTCCTCT 54 32 123 (NM_002140) T-3' (SEQ ID NO: 749) 5'ATCAGCACTGAAACCAACCATGC C-3' (SEQ ID NO: 750) Beta actin 5'- 62 32 421 (NM_001101. CAGCCATGTACGTTGCTATCCAG-3' 3) (SEQ ID NO: 751) 5'-GTTTCGTGGATGCCACAGGAC-3' (SEQ ID NO: 752)

TABLE-US-00017 TABLE 15 Molecules identified in the Networks and their cellular functions ID Molecules in Network Score Focus genes Top Functions 1 .uparw.ACTB, Actin, ADRA2C, ALOX15, ANG, COX2, .dwnarw.CSTB, 39 15 Molecular DDX1, ERK, .dwnarw. FABP5, .uparw.HNRPD, .uparw. HNRPK, .uparw. HSP90B1, Transport, KIF1C, LIMA1, LOX, .dwnarw. MARCKS, MLXIP, NFkB, .uparw. NPM1 Cancer, Cellular (includes EG: 4869), PCBP2, PCBP1 (includes Movement EG: 29371), .uparw.PEBP1, PHACTR1, REM1, .uparw.S100A7, SAFB, .dwnarw.SERPINA1, .uparw. SFN, .uparw. SOD2, SYNPO2, .uparw.TUBB, WTAP, YWHAD, .uparw.YWHAZ 2 AKAP13, AKT1, AKT2, .uparw. CALML3, COX2, .dwnarw. DLC1, 15 7 Cancer, Cell-To- ERRFI1, HMG1L1, .uparw.HNRPD, IGH-1A, .dwnarw.IGHG1, IGHG3, Cell Signalling IGKV1-117, IL33, KRT19, KRT72, LCK, LOX, .uparw.NPM1 and Interaction, (includes EG: 4869), NR3C1, PCAF, PRKDC, .uparw.PTMS, Hematological RELA, retinoic acid, RGS3, SLC25A4, SLC2A1, System Development SUMO1, TAT, TCL1B, .uparw.TLR1, TPD52, VAV1, YBX1 and Function

TABLE-US-00018 Appendix 1 SEQ ID NO: 23: Stratifin: Accn #P31947 (Up-regulated in OPL and Up-regulated in HNCa) gi|398953|sp|P31947.1|1433S_HUMAN 14-3-3 protein sigma (Stratifin) (Epithelial cell marker protein 1) MERASLIQKAKLAEQAERYEDMAAFMKGAVEKGEELSCEERNLLSVAYKNVVGGQRA AWRVLSSIEQKSNEEGSEEKGPEVREYREKVETELQGVCDTVLGLLDSHLIKEAGDAES RVFYLKMKGDYYRYLAEVATGDDKKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLAL NFSVFHYEIANSPEEAISLAKTTFDEAMADLHTLSEDSYKDSTLIMQLLRDNLTLWTAD NAGEEGGEAPQEPQS SEQ ID NO: 24: YWHAZ: Accn # P63104 (Up-regulated in OPL and Up-regulated in HNCa) gi|52000887|sp|P63104.1|1433Z_HUMAN 14-3-3 protein zeta/delta (Protein kinase C inhibitor protein 1) (KCIP-1) MDKNELVQKAKLAEQAERYDDMAACMKSVTEQGAELSNEERNLLSVAYKNVVGARR SSWRVVSSIEQKTEGAEKKQQMAREYREKIETELRDICNDVLSLLEKFLIPNASQAESKV FYLKMKGDYYRYLAEVAAGDDKKGIVDQSQQAYQEAFEISKKEMQPTHPIRLGLALNF SVFYYEILNSPEKACSLAKTAFDEAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDTQG DEAEAGEGGEN SEQ ID NO: 753: S100 A2: Accn # P29034 (Up-regulated in HNCa) gi|114152869|sp|P29034.3|S10A2_HUMAN Protein S100-A2 (S100 calcium-binding protein A2) (Protein S-100L) (CAN19) MMCSSLEQALAVLVTTFHKYSCQEGDKFKLSKGEMKELLHKELPSFVGEKVDEEGLKK LMGSLDENSDQQVDFQEYAVFLALITVMCNDFFQGCPDRP SEQ ID NO: 13: S100 A7: Accn # P31151 (Protein of interest Up-regulated in OPL and Up-regulated in HNCa) gi|400892|sp|P31151.3|S10A7_HUMAN Protein S100-A7 (S100 calcium-binding protein A7) (Psoriasin) MSNTQAERSIIGMIDMFHKYTRRDDKIDKPSLLTMMKENFPNFLSACDKKGTNYLADV FEKKDKNEDKKIDFSEFLSLLGDIATDYHKQSHGAAPCSGGSQ SEQ ID NO: 19: Prothymosin alpha: Accn # P06454 (Up-regulated in OPL and Up-regulated in HNCa) gi|135834|sp|P06454.2|PTMA_HUMAN Prothymosin alpha [Contains: Thymosin alpha-1] MSDAAVDTSSEITTKDLKEKKEVVEEAENGRDAPANGNAENEENGEQEADNEVDEEEE EGGEEEEEEEEGDGEEEDGDEDEEAESATGKRAAEDDEDDDVDTKKQKTDEDD SEQ ID NO: 754: Fascin: Accn # Q96IH1 (Up- regulated in HNCa) gi|74732058|sp|Q96IH1|Q96IH1_HUMAN FSCN1 protein SASSTATMTANGTAEAVQIQFGLINCGNKYLTAEAFGFKVNASASSLKKKQIWTLEQPP DEAGSAAVCLRSHLGRYLAADKDGNVTCEREVPGPDCRFLIVAHDDGRWSLQSEAHR RYFGGTEDRLSCFAQTVSPAEKWSVHIAMHPQVNIYSVTRKRYAHLSARPADEIAVDR DVPWGVDSLITLAFQDQRYSVQTADHRFLRHDGRLVARPEPATGYTLEFRSGKVAFRD CEGRYLAPSGPSGTLKAGKATKVGKDELFALEQSCAQVVLQAANERNVSTRQGMDLS ANQDEETDQETFQLEIDRDTKKCAFRTHTGKYWTLTATGGVQSTASSKNASCYFDIEW RDRRITLRASNGKFVTSKKNGQLAASVETAGDSELFLMKLINRPIIVFRGEHGFIGCRKV TGTLDANRSSYDVFQLEFNDGAYNIKDSTGKYWTVGSDSAVTSSGDTPVDFFFEFCDY NKVAIKVGGRYLKGDHAGVLKASAETVDPASLWEY SEQ ID NO: 31: Calgizzarin: Accn # P31949 ((Down- regulated in OPL and Up-regulated in HNCa) gi|1710818|sp|P31949.2|S10AB_HUMAN Protein S100-A11 (S100 calcium-binding protein A11) (Protein S100C) (Calgizzarin) (MLN 70) MAKISSPTETERCIESLIAVFQKYAGKDGYNYTLSKTEFLSFMNTELAAFTKNQKDPGVL DRMMKKLDTNSDGQLDFSEFLNLIGGLAMACHDSFLKAVPSQKRT SEQ ID NO: 755: Maspin: Accn # P36952 (Up- regulated in HNCa) gi|547892|sp|P36952.1|SPB5_HUMAN Serpin B5 precursor (Maspin) (Protease inhibitor 5) MDALQLANSAFAVDLFKQLCEKEPLGNVLFSPICLSTSLSLAQVGAKGDTANEIGQVLH FENVKDIPFGFQTVTSDVNKLSSFYSLKLIKRLYVDKSLNLSTEFISSTKRPYAKELETVD FKDKLEETKGQINNSIKDLTDGHFENILADNSVNDQTKILVVNAAYFVGKWMKKFPESE TKECPFRLNKTDTKPVQMMNMEATFCMGNIDSINCKIIELPFQNKHLSMFILLPKDVEDE STGLEKIEKQLNSESLSQWTNPSTMANAKVKLSIPKFKVEKMIDPKACLENLGLKHIFSE DTSDFSGMSETKGVALSNVIHKVCLEITEDGGDSIEVPGARILQHKDELNADHPFIYIIRH NKTRNIIFFGKFCSP SEQ ID NO: 756: Annexin A8: Accn # P13928 (Up- regulated in HNCa) gi|126302519|sp|P13928.2|ANXA8_HUMAN Annexin A8 (Annexin-8) (Annexin VIII) (Vascular anticoagulant-beta) (VAC-beta) MAWWKAWIEQEGVTVKSSSHFNPDPDAETLYKAMKGIGTNEQAIIDVLTKRSNTQRQQ IAKSFKAQFGKDLTETLKSELSGKFERLIVALMYPPYRYEAKELHDAMKGLGTKEGVIIE ILASRTKNQLREIMKAYEEDYGSSLEEDIQADTSGYLERILVCLLQGSRDDVSSFVDPGL ALQDAQDLYAAGEKIRGTDEMKFITILCTRSATHLLRVFEEYEKIANKSIEDSIKSETHGS LEEAMLTVVKCTQNLHSYFAERLYYAMKGAGTRDGTLIRNIVSRSEIDLNLIKCHFKKM YGKTLSSMIMEDTSGDYKNALLSLVGSDP SEQ ID NO: 1: Calmodulin like protein 5: Accn # Q9NZT1 (Up-regulated in HNCa) gi|14285407|sp|Q9NZT1.1|CALL5_HUMAN Calmodulin-like protein 5 (Calmodulin- like skin protein) MAGELTPEEEAQYKKAFSAVDTDGNGTINAQELGAALKATGKNLSEAQLRKLISEVDS DGDGEISFQEFLTAARKARAGLEDLQVAFRAFDQDGDGHITVDELRRAMAGLGQPLPQ EELDAMIREADVDQDGRVNYEEFARMLAQE SEQ ID NO: 757: GST P: Accn # AAC13869.1 (Up-regulated in HNCa) gi|726098|gb|AAC13869.1| glutathione S-transferase-Plc [Homo sapiens] MPPYTVVYFPVRGRCAALRMLLADQGQSWKEEVVTVETWQEGSLKASCLYGQLPKFQ DGDLTLYQSNTILRHLGRTLGLYGKDQQEAALVDMVNDGVEDLRCKYVSLIYTNYEV GKDDYVKALPGQLKPFETLLSQNQGGKTFIVGDQISFADYNLLDLLLIHEVLAPGCLDA FPLLSAYVGRLSARPKLKAFLASPEYVNLPINGNGKQ SEQ ID NO: 11: LDH A: Accn # P00338 (Up-regulated in HNCa) gi|126047|sp|P00338.2|LDHA_HUMAN L-lactate dehydrogenase A chain (LDH-A) (LDH muscle subunit) (LDH-M) (Renal carcinoma antigen NY-REN-59) (Cell proliferation-inducing gene 19 protein) MATLKDQLIYNLLKEEQTPQNKITVVGVGAVGMACAISILMKDLADELALVDVIEDKL KGEMMDLQHGSLFLRTPKIVSGKDYNVTANSKLVIITAGARQQEGESRLNLVQRNVNIF KFIIPNVVKYSPNCKLLIVSNPVDILTYVAWKISGFPKNRVIGSGCNLDSARFRYLMGER LGVHPLSCHGWVLGEHGDSSVPVWSGMNVAGVSLKTLHPDLGTDKDKEQWKEVHKQ VVESAYEVIKLKGYTSWAIGLSVADLAESIMKNLRRVHPVSTMIKGLYGIKDDVFLSVP CILGQNGISDLVKVTLTSEEEARLKKSADTLWGIQKELQF SEQ ID NO: 9: PPIA: Accn # P62937 (Up-regulated in HNCa) gi|51702775|sp|P62937.2|PPIA_HUMAN Peptidyl- prolyl cis-trans isomerase A (PPIase A) (Rotamase A) (Cyclophilin A) (Cyclosporin A-binding protein) MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGF MCQGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKT EWLDGKHVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE SEQ ID NO: 3: APC binding protein EB1: Accn # Q15691 (Up-regulated in HNCa) gi|20138589|sp|Q15691.3|MARE1_HUMAN Microtubule-associated protein RP/EB family member 1 (APC-binding protein EB1) (End-binding protein 1) (EB1) MAVNVYSTSVTSDNLSRHDMLAWINESLQLNLTKIEQLCSGAAYCQFMDMLFPGSIAL KKVKFQAKLEHEYIQNFKILQAGFKRMGVDKIIPVDKLVKGKFQDNFEFVQWFKKFFD ANYDGKDYDPVAARQGQETAVAPSLVAPALNKPKKPLTSSSAAPQRPISTQRTAAAPK AGPGVVRKNPGVGNGDDEAAELMQQVNVLKLTVEDLEKERDFYFGKLRNIELICQENE GENDPVLQRIVDILYATDEGFVIPDEGGPQEEQEEY SEQ ID NO: 15: SOD (Mn): Accn # AAH16934 (Up-regulated in HNCa) gi|16877367|gb|AAH16934.1| SOD2 protein [Homo sapiens] MLSRAVCGTSRQLAPALGYLGSRQKHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAY VNNLNVTEEKYQEALAKGRFQAERREAVPGRGDPREPGPIRTGLSVEENSLRICTGSEFS RHDSLSFKHMVYLIVEGVPRWV SEQ ID NO: 7: L-Plastin: Accn # P13797 (Up-regulated in HNCa) gi|2506254|sp|P13797.3|PLST_HUMAN Plastin-3 (T-plastin) MATTQISKDELDELKEAFAKVDLNSNGFICDYELHELFKEANMPLPGYKVREIIQKLML DGDRNKDGKISFDEFVYIFQEVKSSDIAKTFRKAINRKEGICALGGTSELSSEGTQHSYSE EEKYAFVNWINKALENDPDCRHVIPMNPNTDDLFKAVGDGIVLCKMINLSVPDTIDERA INKKKLTPFIIQENLNLALNSASAIGCHVVNIGAEDLRAGKPHLVLGLLWQIIKIGLFADIE LSRNEALAALLRDGETLEELMKLSPEELLLRWANFHLENSGWQKINNFSADIKDSKAYF HLLNQIAPKGQKEGEPRIDINMSGFNETDDLKRAESMLQQADKLGCRQFVTPADVVSG NPKLNLAFVANLFNKYPALTKPENQDIDWTLLEGETREERTFRNWMNSLGVNPHVNHL YADLQDALVILQLYERIKVPVDWSKVNKPPYPKLGANMKKLENCNYAVELGKHPAKF SLVGIGGQDLNDGNQTL TLALVWQLMRRYTLNVLEDLGDGQKANDDIIVNWVNRTLSEAGKSTSIQSFKDKTISSS LAVVDLIDAIQPGCINYDLVKSGNLTEDDKHNNAKYAVSMARRIGARVYALPEDLVEV KPKMVMTVFACLMGRGMKRV SEQ ID NO: 10: PACAP: Accn # Q8WU39 (Down-regulated in HNCa) gi|74730663|sp|Q8WU39|Q8WU39_HUMAN PACAP protein MRLSLPLLLLLLGAWAIPGGLGDRAPLTATAPQLDDEEMYSAHMPAHLRCDACRAVA YQMWQNLAKAETKLHTSNSGGRRELSELVYTDVLDRSCSRNWQDYGVREVDQVKRL TGPGLSEGPEPSISVMVTGGPWPTRLSRTCLHYLGEFGEDQIYEAHQQGRGALEALLCG GPQGACSEKVSATREEL SEQ ID NO: 6: Histone H3: Accn # Q71DI3 (Down-regulated in HNCa) gi|74758899|sp|Q71DI3.3|H32_HUMAN Histone H3.2 (H3/m) (H3/o) MARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPGTVALREIRRYQK STELLIRKLPFQRLVREIAQDFKTDLRFQSSAVMALQEASEAYLVGLFEDTNLCAIHAKR VTIMPKDIQLARRIRGERA SEQ ID NO: 8: Histone H4: Accn # P62805

(Down-regulated in HNCa) gi|51317339|sp|P62805.2|H4_HUMAN Histone H4 MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVL KVFLENVIRDAVTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG SEQ ID NO: 16: Alpha-1-antitrypsin: Accn # P01009 (Down-regulated in OPL and Down- regulated in HNCa) gi|1703025|sp|P01009.3|A1AT_HUMAN Alpha-1- antitrypsin precursor (Alpha-1 protease inhibitor) (Alpha-1-antiproteinase) MPSSVSWGILLLAGLCCLVPVSLAEDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAF SLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGF QELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKK QINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQV TTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDI ITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKA VHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPT QK SEQ ID NO: 12: KPSG lumican: Accn # P51884 (Down-regulated in HNCa) gi|20141464|sp|P51884.2|LUM_HUMAN Lumican precursor (Keratan sulfate proteoglycan lumican) (KSPG lumican) MSLSAFTLFLALIGGTSGQYYDYDFPLSIYGQSSPNCAPECNCPESYPSAMYCDELKLKS VPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGRVFSKLKQLK KLHINHNNLTESVGPLPKSLEDLQLTHNKITKLGSFEGLVNLTFIHLQHNRLKEDAVSAA FKGLKSLEYLDLSFNQIARLPSGLPVSLLTLYLDNNKISNIPDEYFKRFNALQYLRLSHNE LADSGIPGNSFNVSSLVELDLSYNKLKNIPTVNENLENYYLEVNQLEKFDIKSFCKILGPL SYSKIKHLRLDGNRISETSLPPDMYECLRVANEVTLN SEQ ID NO: 5: Mast cell tryptase beta III: Accn # Q96RZ7 (Down-regulated in HNCa) gi|74761085|sp|Q96RZ7|Q96RZ7_HUMAN Mast cell tryptase beta III MLNLLLLALPVLASRAYAAPAPGQALQRVGIVGGQEAPRSKWPWQVSLRVRDRYWM HFCGGSLIHPQWVLTAAHCVGPDVKDLAALRVQLREQHLYYQDQLLPVSRIIVHPQFYT AQIGADIALLELEEPVNVSSHVHTVTLPPASETFPPGMPCWVTGWGDVDNDERLPPPFP LKQVKVPIMENHICDAKYHLGAYTGDDVRIVRDDMLCAGNTRRDSCQVATAPHTFPAP S SEQ ID NO: 20: Histone H2B.1: Accn # Q16778 (Up-regulated in OPL and Down- regulated in HNCa) gi|7387736|sp|Q16778.3|H2B2E_HUMAN Histone H2B type 2-E (H2B.q) (H2B/q) (H2B-GL105) MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKVLKQVHPDTGISSK AMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKA VTKYTSSK SEQ ID NO: 758: Vimentin: Accn # P08670 (Down-regulated in HNCa) gi|55977767|sp|P08670.4|VIME_HUMAN Vimentin MSTRSVSSSSYRRMFGGPGTASRPSSSRSYVTTSTRTYSLGSALRPSTSRSLYASSPGGVY ATRSSAVRLRSSVPGVRLLQDSVDFSLADAINTEFKNTRTNEKVELQELNDRFANYIDK VRFLEQQNKILLAELEQLKGQGKSRLGDLYEEEMRELRRQVDQLTNDKARVEVERDNL AEDIMRLREKLQEEMLQREEAENTLQSFRQDVDNASLARLDLERKVESLQEEIAFLKKL HEEEIQELQAQIQEQHVQIDVDVSKPDLTAALRDVRQQYESVAAKNLQEAEEWYKSKF ADLSEAANRNNDALRQAKQESTEYRRQVQSLTCEVDALKGTNESLERQMREMEENFA VEAANYQDTIGRLQDEIQNMKEEMARHLREYQDLLNVKMALDIEIATYRKLLEGEESRI SLPLPNFSSLNLRETNLDSLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDLE SEQ ID NO: 14: Peroxiredoxin 2: Accn # P32119 (Down-regulated in OPL and Down- regulated in HNCa) gi|2507169|sp|P32119.5|PRDX2_HUMAN Peroxiredoxin-2 (Thioredoxin peroxidase 1) (Thioredoxin-dependent peroxide reductase 1) (Thiol-specific antioxidant protein) (TSA) (PRP) (Natural killer cell-enhancing factor B) (NKEF-B) MASGNARIGKPAPDFKATAVVDGAFKEVKLSDYKGKYVVLFFYPLDFTFVCPTEIIAFS NRAEDFRKLGCEVLGVSVDSQFTHLAWINTPRKEGGLGPLNIPLLADVTRRLSEDYGVL KTDEGIAYRGLFIIDGKGVLRQITVNDLPVGRSVDEALRLVQAFQYTDEHGEVCPAGWK PGSDTIKPNVDDSKEYFSKHN SEQ ID NO: 4: Carbonic anhydrase I: Accn # P00915 (Down-regulated in HNCa) gi|115449|sp|P00915.2|CAH1_HUMAN Carbonic anhydrase 1 (Carbonic anhydrase I) (Carbonate dehydratase I) (CA-I) MASPDWGYDDKNGPEQWSKLYPIANGNNQSPVDIKTSETKHDTSLKPISVSYNPATAK EIINVGHSFHVNFEDNDNRSVLKGGPFSDSYRLFQFHFHWGSTNEHGSEHTVDGVKYSA ELHVAHWNSAKYSSLAEAASKADGLAVIGVLMKVGEANPKLQKVLDALQAIKTKGKR APFTNFDPSTLLPSSLDFWTYPGSLTHPPLYESVTWIICKESISVSSEQLAQFRSLLSNVEG DNAVPMQHNNRPTQPLKGRTVRASF SEQ ID NO: 759: Flavin reductase: Accn # P30043 (Down-regulated in HNCa) gi|1706870|sp|P30043.3|BLVRB_HUMAN Flavin reductase (FR) (NADPH-dependent diaphorase) (NADPH-flavin reductase) (FLR) (Biliverdin reductase B) (BVR-B) (Biliverdin-IX beta- reductase) (Green heme-binding protein) (GHBP) MAVKKIAIFGATGQTGLTTLAQAVQAGYEVTVLVRDSSRLPSEGPRPAHVVVGDVLQA ADVDKTVAGQDAVIVLLGTRNDLSPTTVMSEGARNIVAAMKAHGVDKVVACTSAFLL WDPTKVPPRLQAVTDDHIRMHKVLRESGLKYVAVMPPHIGDQPLTGAYTVTLDGRGPS RVISKHDLGHFMLRCLTTDEYDGHSTYPSHQYQ SEQ ID NO: 760: Cytokeratin 14: Accn # NP_000517 (Up-regulated in HNCa) gi|15431310|ref|NP_000517.2|keratin 14 [Homo sapiens] MTTCSRQFTSSSSMKGSCGIGGGIGGGSSRISSVLAGGSCRAPSTYGGGLSVSSSRFSSGG AYGLGGGYGGGFSSSSSSFGSGFGGGYGGGLGAGLGGGFGGGFAGGDGLLVGSEKVT MQNLNDRLASYLDKVRALEEANADLEVKIRDWYQRQRPAEIKDYSPYFKTIEDLRNKIL TATVDNANVLLQIDNARLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLARADL EMQIESLKEELAYLKKNHEEEMNALRGQVGGDVNVEMDAAPGVDLSRILNEMRDQYE KMAEKNRKDAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSM KASLENSLEETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLE QEIATYRRLLEGEDAHLSSSQFSSGSQSSRDVTSSSRQIRTKVMDVHDGKVVSTHEQVL RTKN SEQ ID NO: 2: Polybromo-ID: Accn # Q86U86 (Down-regulated in HNCa) gi|73921624|sp|Q86U86.1|PB1_HUMAN Protein polybromo-1 (hPB1) (Polybromo-1D) (BRG1- associated factor 180) (BAF180) MGSKRRRATSPSSSVSGDFDDGHHSVSTPGPSRKRRRLSNLPTVDPIAVCHELYNTIRDY KDEQGRLLCELFIRAPKRRNQPDYYEVVSQPIDLMKIQQKLKMEEYDDVNLLTADFQLL FNNAKSYYKPDSPEYKAACKLWDLYLRTRNEFVQKGEADDEDDDEDGQDNQGTVTE GSSPAYLKEILEQLLEAIVVATNPSGRLISELFQKLPSKVQYPDYYAIIKEPIDLKTIAQRIQ NGSYKSIHAMAKDIDLLAKNAKTYNEPGSQVFKDANSIKKIFYMKKAEIEHHEMAKSSL RMRTPSNLAAARLTGPSHSKGSLGEERNPTSKYYRNKRAVQGGRLSAITMALQYGSES EEDAALAAARYEEGESEAESITSFMDVSNPFYQLYDTVRSCRNNQGQLIAEPFYHLPSK KKYPDYYQQIKMPISLQQIRTKLKNQEYETLDHLECDLNLMFENAKRYNVPNSAIYKRV LKLQQVMQAKKKELARRDDIEDGDSMISSATSDTGSAKRKSKKNIRKQRMKILFNVVL EAREPGSGRRLCDLFMVKPSKKDYPDYYKIILEPMDLKIIEHNIRNDKYAGEEGMIEDM KLMFRNARHYNEEGSQVYNDAHILEKLLKEKRKELGPLPDDDDMASPKLKLSRKSGISP KKSKYMTPMQQKLNEVYEAVKNYTDKRGRRLSAIFLRLPSRSELPDYYLTIKKPMDME KIRSHMMANKYQDIDSMVEDFVMMFNNACTYNEPESLIYKDALVLHKVLLETRRDLE GDEDSHVPNVTLLIQELIHNLFVSVMSHQDDEGRCYSDSLAEIPAVDPNFPNKPPLTFDII RKNVENNRYRRLDLFQEHMFEVLERARRMNRTDSEIYEDAVELQQFFIKIRDELCKNGE ILLSPALSYTTKHLHNDVEKERKEKLPKEIEEDKLKREEEKREAEKSEDSSGAAGLSGLH RTYSQDCSFKNSMYHVGDYVYVEPAEANLQPHIVCIERLWEDSAGEKWLYGCWFYRP NETFHLATRKFLEKEVFKSDYYNKVPVSKILGKCVVMFVKEYFKLCPENFRDEDVFVCE SRYSAKTKSFKKIKLWTMPISSVRFVPRDVPLPVVRVASVFANADKGDDEKNTDNSEDS RAEDNFNLEKEKEDVPVEMSNGEPGCHYFEQLHYNDMWLKVGDCVFIKSHGLVRPRV GRIEKVWVRDGAAYFYGPIFIHPEETEHEPTKMFYKKEVFLSNLEETCPMTCILGKCAVL SFKDFLSCRPTEIPENDILLCESRYNESDKQMKKFKGLKRFSLSAKVVDDEIYYFRKPIVP QKEPSPLLEKKIQLLEAKFAELEGGDDDIEEMGEEDSEVIEPPSLPQLQTPLASELDLMPY TPPQSTPKSAKGSAKKEGSKRKINMSGYILFSSEMRAVIKAQHPDYSFGELSRLVGTEW RNLETAKKAEYEERAAKVAEQQERERAAQQQQPSASPRAGTPVGALMGVVPPPTPMG MLNQQLTPVAGMMGGYPPGLPPLQGPVDGLVSMGSMQPLHPGGPPPHHLPPGVPGLP GIPPPGVMNQGVAPMVGTPAPGGSPYGQQVGVLGPPGQQAPPPYPGPHPAGPPVIQQPT TPMFVAPPPKTQRLLHSEAYLKYIEGLSAESNSISKWDQTLAARRRDVHLSKEQESRLPS HWLKSKGAHTTMADALWRLRDLMLRDTLNIRQAYNLENV SEQ ID NO: 22: PK M2: Accn # P14618 (Up-regulated in OPL and Up-regulated in many cancers and possibly HNCa) gi|20178296|sp|P14618.4|KPYM_HUMAN Pyruvate kinase isozymes M1/M2 (Pyruvate kinase muscle isozyme) (Pyruvate kinase 2/3) (Cytosolic thyroid hormone-binding protein) (CTHBP) (THBP1) MSKPHSEAGTAFIQTQQLHAAMADTFLEHMCRLDIDSPPITARNTGIICTIGPASRSVETL KEMIKSGMNVARLNFSHGTHEYHAETIKNVRTATESFASDPILYRPVAVALDTKGPEIRT GLIKGSGTAEVELKKGATLKITLDNAYMEKCDENILWLDYKNICKVVEVGSKIYVDDG LISLQVKQKGADFLVTEVENGGSLGSKKGVNLPGAAVDLPAVSEKDIQDLKFGVEQDV DMVFASFIRKASDVHEVRKVLGEKGKNIKIISKIENHEGVRRFDEILEASDGIMVARGDL GIEIPAEKVFLAQKMMIGRCNRAGKPVICATQMLESMIKKPRPTRAEGSDVANAVLDGA DCIMLSGETAKGDYPLEAVRMQHLIAREAEAAIYHLQLFEELRRLAPITSDPTEATAVGA VEASFKCCSGAIIVLTKSGRSAHQVARYRPRAPIIAVTRNPQTARQAHLYRGIFPVLCKD PVQEAWAEDVDLRVNFAMNVGKARGFFKKGDVVIVLTGWRPGSGFTNTMRVVPVP SEQ ID NO: 761: Annexin A1: Accn # P04083 (Up-regulated in HNCa) gi|113944|sp|P04083.2|ANXA1_HUMAN Annexin A1 (Annexin-1) (Annexin I) (Lipocortin I) (Calpactin II) (Chromobindin-9) (p35) (Phospholipase A2 inhibitory protein)

MAMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPTFNPSSDVAALHKAIMVK GVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLEEVVLALLKTPAQ FDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYREELKRDLAKDITSDTSGDFR NALLSLAKGDRSEDFGVNEDLADSDARALYEAGERRKGTDVNVFNTILTTRSYPQLRR VFQKYTKYSKHDMNKVLDLELKGDIEKCLTAIVKCATSKPAFFAEKLHQAMKGVGTR HKALIRIMVSRSEIDMNDIKAFYQKMYGISLCQAILDETKGDYEKILVALCGGN SEQ ID NO: 21: Nucleophosmin I: Accn # AAH16768.1 (Up-regulated in HNCa) gi|16876992|gb|AAH16768.1|Nucleophosmin (nucleolar phosphoprotein B23, numatrin) [Homo sapiens] MEDSMDMDMSPLRPQNYLFGCELKADKDYHFKVDNDENEHQLSLRTVSLGAGAKDE LHIVEAEAMNYEGSPIKVTLATLKMSVQPTVSLGGFEITPPVVLRLKCGSGPVHISGQHL VAVEEDAESEDEEEEDVKLLSISGKRSAPGGGSKVPQKKVKLAADEDDDDDDEEDDDE DDDGDDFDDEEAEEKAPVKKSIRDTPAKNAQKSNQNGKDSKPSSTPRSKGQESFKKQE KTPKTPKGPSSVEDIKAKMQASIEKGGSLPKVEAKFINCVKNCFRMTDQEAIQDLWQW RKSL SEQ ID NO: 35: Hsp 27: Accn # P04792 (Up- regulated in OPL and Up-regulated in HNCa) gi|19855073|sp|P04792.2|HSPB1_HUMAN Heat shock protein beta-1 (HspB1) (Heat shock 27 kDa protein) (HSP 27) (Stress-responsive protein 27) (SRP27) (Estrogen-regulated 24 kDa protein) (28 kDa heat shock protein) MTERRVPFSLLRGPSWDPFRDWYPHSRLFDQAFGLPRLPEEWSQWLGGSSWPGYVRPL PPAAIESPAVAAPAYSRALSRQLSSGVSEIRHTADRWRVSLDVNHFAPDELTVKTKDGV VEITGKHEERQDEHGYISRCFTRKYTLPPGVDPTQVSSSLSPEGTLTVEAPMPKLATQSN EITIPVTFESRAQLGGPEAAKSDETAAK SEQ ID NO: 27: Cystatin B: Accn # P04080 (Down-regulated in OPL and Down-regulated in HNCa) gi|1706278|sp|P04080.2|CYTB_HUMAN Cystatin-B (Stefin-B) (Liver thiol proteinase inhibitor) (CPI-B) MMCGAPSATQPATAETQHIADQVRSQLEEKENKKFPVFKAVSFKSQVVAGTNYFIKVH VGDEDFVHLRVFQSLPHENKPLTLSNYQTNKAKHDELTYF SEQ ID NO: 18: GRP 94: Accn # P14625 (Up- regulated in OPL and Down-regulated in HNCa) gi|119360|sp|P14625.1|ENPL_HUMAN Endoplasmin precursor (Heat shock protein 90 kDa beta member 1) (94 kDa glucose-regulated protein) (GRP94) (gp96 homolog) (Tumor rejection antigen 1) MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQL DGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLIS LTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNK MTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADP RGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEE EAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDE YKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVR RVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMI KKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERM KEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNV AKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVA SQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDD KTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETA EDTTEDTEQDEDEEMDVGTDEEEETAKESTAEKDEL SEQ ID NO: 17: MARCKS: Accn # gb|AAA59555.1 (Down-regulated in OPL and protein of interest; possibly up-regulated in HNCa) gi|187387|gb|AAA59555.1| myristoylated alanine-rich C-kinase substrate MGAQFSKTAAKGEAAAERPGEAAVASSPSKANGQENGHVKVNGDASPAAAESGAKEE LQANGSAPAADKEEPAAAGSGAASPSAAEKGEPAAAAAPEAGASPVEKEAPAEGEAAE PGSPTAAEGEAASAASSTSSPKAEDGATPSPSNETPKKKKKRFSFKKSFKLSGFSFKKNK KEAGEGGEAEAPAAEGGKDEAAGGAAAAAAEAGAASGEQAAAPGEEAAAGEEGAAG GDSQEAKPQEAAVAPEKPPASDETKAAEEPSKVEEKKAEEAGASAAACEAPSAAGLVC PRRGGSPRGGARGRRSLNQACAAPSQEAQPECSPEAPPAEAAE SEQ ID NO: 25: gi|48429103|sp|P61978.1|HNRPK_HUMAN Heterogeneous nuclear ribonucleoprotein K (hnRNP K) (Transformation up-regulated nuclear protein) (TUNP). (Up-regulated in OPL and HNCa) METEQPEETFPNTETNGEFGKRPAEDMEEEQAFKRSRNTDEMVELRILLQSKNAGAVIG KGGKNIKALRTDYNASVSVPDSSGPERILSISADIETIGEILKKIIPTLEEGLQLPSPTATSQ LPLESDAVECLNYQHYKGSDFDCELRLLIHQSLAGGIIGVKGAKIKELRENTQTTIKLFQ ECCPHSTDRVVLIGGKPDRVVECIKIILDLISESPIKGRAQPYDPNFYDETYDYGGFTMMF DDRRGRPVGFPMRGRGGFDRMPPGRGGRPMPPSRRDYDDMSPRRGPPPPPPGRGGRGG SRARNLPLPPPPPPRGGDLMAYDRRGRPGDRYDGMVGFSADETWDSAIDTWSPSEWQ MAYEPQGGSGYDYSYAGGRGSYGDLGGPIITTQVTIPKDLAGSIIGKGGQRIKQIRHESG ASIKIDEPLEGSEDRIITITGTQDQIQNAQYLLQNSVKQYSGKFF SEQ ID NO: 1: >gi|119360|sp|P14625.1|ENPL_HUMAN Endoplasmin precursor (Heat shock protein 90 kDa beta member 1) (94 kDa glucose-regulated protein) (GRP94) (gp96 homolog) (Tumor rejection antigen 1) (Up-regulated in OPL) MRALWVLGLCCVLLTFGSVRADDEVDVDGTVEEDLGKSREGSRTDDEVVQREEEAIQL DGLNASQIRELREKSEKFAFQAEVNRMMKLIINSLYKNKEIFLRELISNASDALDKIRLIS LTDENALSGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGTSEFLNK MTEAQEDGQSTSELIGQFGVGFYSAFLVADKVIVTSKHNNDTQHIWESDSNEFSVIADP RGNTLGRGTTITLVLKEEASDYLELDTIKNLVKKYSQFINFPIYVWSSKTETVEEPMEEE EAAKEEKEESDDEAAVEEEEEEKKPKTKKVEKTVWDWELMNDIKPIWQRPSKEVEEDE YKAFYKSFSKESDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDYIKLYVR RVFITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLKVIRKKLVRKTLDMI KKIADDKYNDTFWKEFGTNIKLGVIEDHSNRTRLAKLLRFQSSHHPTDITSLDQYVERM KEKQDKIYFMAGSSRKEAESSPFVERLLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNV AKEGVKFDESEKTKESREAVEKEFEPLLNWMKDKALKDKIEKAVVSQRLTESPCALVA SQYGWSGNMERIMKAQAYQTGKDISTNYYASQKKTFEINPRHPLIRDMLRRIKEDEDD KTVLDLAVVLFETATLRSGYLLPDTKAYGDRIERMLRLSLNIDPDAKVEEEPEEEPEETA EDTTEDTEQDEDEEMDVGTDEEEETAKESTAEKDEL SEQ ID NO: 26: >gi|74705500|sp|O15256|O15256_HUMAN Parathymosin. (Up-regulated in OPL). RRRTGLRRKKKKLPRMERRKMKGKKKDEEEEEEDDEGPALKRAAEEEDEADPKRQKT ENGASA SEQ ID NO: 28: >gi|74758095|sp|Q6NSB4|Q6NSB4_HUMAN HP protein. (Down-regulated in OPL). MSRISQMTAARSPPRLHMAMWSTRFATSVRTNAVQRILGGHLDAKGSFPWQAKMVSH HNLTTGATLINEQWLLTTAKNLFLNHSENATAKDIAPTLTLYVGKKQLVEIEKVVLHPN YSQVDIGLIKLKQKVSVNERVMPICLPSKDYAEVGRVGYVSGWGRNANFKFTDHLKYV MLPVADQDQCIRHYEGSTVPEKKTPKSPVGVQPILNEHTFCAGMSKYQEDTCYGDAGS AFAVHDLEEDTWYATGILSFDKSCAVAEYGVYVKVTSIQDWVQKTIAEN SEQ ID NO: 29: >gi|232081|sp|Q01469.3|FABP5_HUMAN Fatty acid-binding protein, epidermal (E-FABP) (Fatty acid-binding protein 5) (Psoriasis- associated fatty acid-binding protein homolog) (PA-FABP). (Down-regulated in OPL). MATVQQLEGRWRLVDSKGFDEYMKELGVGIALRKMGAMAKPDCIITCDGKNLTIKTES TLKTTQFSCTLGEKFEETTADGRKTQTVCNFTDGALVQHQEWDGKESTITRKLKDGKL VVECVMNNVTCTRIYEKVE SEQ ID NO: 30: >gi|19263707|gb|AAH25314.1|IGHG1 protein [Homo sapiens] (Down-regulated in OPL). MDWTWRFLFVVAAATSVQSQVQLVQSGAEVKKPGSSVKVSCKASGDSFNSLAINWVR QAPGQGLEWMGGIIPIFGTTNYAQRFQGRVTFTADESTGRAYMELTSLRSEDTAVYYCA SRFISETNFCFKFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K SEQ ID NO: 32: >gi|74728989|sp|Q8N5F4|Q8N5F4_HUMAN IGLC1 protein (Up-regulated in OPL). MAWTPLLLPLLTFCTVSEASYELTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSG QTPVLVIYDDTERPSGIPERFSGSSSGTVATLTLSGAQVEDEADYYCYSSDSSGNHWVFG GGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS SEQ ID NO: 33: >gi|74754454|sp|Q12771|Q12771_HUMAN P37 AUF1 (Up-regulated in OPL). MSEEQFGGTGRRHANGGGRRSAGDEEGAMVAATQGAAAAREADAGPGAEPRLEAPK GSAESEGAKIDASKNEEDEGKMFIGGLSWDTTKKDLKDYFSKFGEVVDCTLKLDPITGR SRGFGFVLFKESESVDKVMDQKEHKLNGKVIDPKRAKAMKTKEPVKKIFVGGLSPDTP EEKIREYFGGFGEVESIELPMDNKTNKRRGFCFITFKEEEPVKKIMEKKYHNVGLSKCEI KVAMSKEQYQQQQQWGSRGGFAGRARGEFRNSSEAGEGLELPPNSIHCWQLSV SEQ ID NO: 762: >gi|223632|prf||0904262A dismutase,Cu/Zn superoxide (Up-regulated in OPL). ATKAVCVLKGBGPVZGIIBFZZKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTS AGPHFNPLSRKHGGPKDEERHVGBLGBVTABKBGVABVSIZBSVISLSGBHCIIGRTLVV HEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ SEQ ID NO: 34: >gi|133254|sp|P09651.4|ROA1_HUMAN Heterogeneous nuclear ribonucleoprotein A1 (hnRNP core protein A1) (Helix-destabilizing protein) (Single-strand RNA-binding protein) (Up-regulated in OPL). MSKSESPKEPEQLRKLFIGGLSFETTDESLRSHFEQWGTLTDCVVMRDPNTKRSRGFGF VTYATVEEVDAAMNARPHKVDGRVVEPKRAVSREDSQRPGAHLTVKKIFVGGIKEDTE EHHLRDYFEQYGKIEVIEIMTDRGSGKKRGFAFVTFDDHDSVDKIVIQKYHTVNGHNCE VRKALSKQEMASASSSQRGRSGSGNFGGGRGGGFGGNDNFGRGGNFSGRGGFGGSRG

GGGYGGSGDGYNGFGNDGGYGGGGPGYSGGSRGYGSGGQGYGNQGSGYGGSGSYDS YNNGGGRGFGGGSGSNFGGGGSYNDFGNYNNQSSNFGPMKGGNFGGRSSGPYGGGG QYFAKPRNQGGYGGSSSSSSYGSGRRF SEQ ID NO: 36: >gi|116848|sp|P23528.3|COF1_HUMAN Cofilin-1 (Cofilin, non-muscle isoform) (18 kDa phosphoprotein) (p18). (Up-regulated in OPL). MASGVAVSDGVIKVFNDMKVRKSSTPEEVKKRKKAVLFCLSEDKKNIILEEGKEILVGD VGQTVDDPYATFVKMLPDKDCRYALYDATYETKESKKEDLVFIFWAPESAPLKSKMIY ASSKDAIKKKLTGIKHELQANCYEEVKDRCTLAEKLGGSAVISLEGKPL SEQ ID NO: 37: >gi|31645|emb|CAA25833.1| glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens]. (Up-regulated in OPL). MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKFH GTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHLQGG AKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGIVEGLM TTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELDGKLTGMAF RVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVVSSDFNSDTHS STFDAGAGIALNDHFVKLISWYDNEFGYSNRVVDLMAHMASKE SEQ ID NO: 38: >gi|127983|sp|P22392.1|NDKB_HUMAN Nucleoside diphosphate kinase B (NDP kinase B) (NDK B) (nm23-H2) (C-myc purine-binding transcription factor PUF). (Up-regulated in OPL). MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPF FPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIH GSDSVKSAEKEISLWFKPEELVDYKSCAHDWVYE SEQ ID NO: 39: >gi|119172|sp|P13639.4|EF2_HUMAN Elongation factor 2 (EF-2). (Up-regulated in OPL). MVNFTVDQIRAIMDKKANIRNMSVIAHVDHGKSTLTDSLVCKAGIIASARAGETRFTDT RKDEQERCITIKSTAISLFYELSENDLNFIKQSKDGAGFLINLIDSPGHVDFSSEVTAALRV TDGALVVVDCVSGVCVQTETVLRQAIAERIKPVLMMNKMDRALLELQLEPEELYQTFQ RIVENVNVIISTYGEGESGPMGNIMIDPVLGTVGFGSGLHGWAFTLKQFAEMYVAKFAA KGEGQLGPAERAKKVEDMMKKLWGDRYFDPANGKFSKSATSPEGKKLPRTFCQLILDP IFKVFDAIMNFKKEETAKLIEKLDIKLDSEDKDKEGKPLLKAVMRRWLPAGDALLQMIT IHLPSPVTAQKYRCELLYEGPPDDEAAMGIKSCDPKGPLMMYISKMVPTSDKGRFYAFG RVFSGLVSTGLKVRIMGPNYTPGKKEDLYLKPIQRTILMMGRYVEPIEDVPCGNIVGLV GVDQFLVKTGTITTFEHAHNMRVMKFSVSPVVRVAVEAKNPADLPKLVEGLKRLAKSD PMVQCIIEESGEHIIAGAGELHLEICLKDLEEDHACIPIKKSDPVVSYRETVSEESNVLCLS KSPNKHNRLYMKARPFPDGLAEDIDKGEVSARQELKQRARYLAEKYEWDVAEARKIW CFGPDGTGPNILTDITKGVQYLNEIKDSVVAGFQWATKEGALCEENMRGVRFDVHDVT LHADAIHRGGGQIIPTARRCLYASVLTAQPRLMEPIYLVEIQCPEQVVGGIYGVLNRKRG HVFEESQVAGTPMFVVKAYLPVNESFGFTADLRSNTGGQAFPQCVFDHWQILPGDPFD NSSRPSQVVAETRKRKGLKEGIPALDNFLDKL SEQ ID NO: 40: >gi|1352726|sp|P30086.3|PEBP1_HUMAN Phosphatidylethanolamine-binding protein 1 (PEBP-1) (Prostatic-binding protein) (HCNPpp) (Neuropolypeptide h3) (Raf kinase inhibitor protein) (RKIP) [Contains: Hippocampal cholinergic neurostimulating peptide (HCNP)] (Up-regulated in OPL). MPVDLSKWSGPLSLQEVDEQPQHPLHVTYAGAAVDELGKVLTPTQVKNRPTSISWDGL DSGKLYTLVLTDPDAPSRKDPKYREWHHFLVVNMKGNDISSGTVLSDYVGSGPPKGTG LHRYVWLVYEQDRPLKCDEPILSNRSGDHRGKFKVASFRKKYELRAPVAGTCYQAEW DDYVPKLYEQLSGK SEQ ID NO: 41: >gi|115502|sp|P27482.2|CALL3_HUMAN Calmodulin-like protein 3 (Calmodulin-related protein NB-1) (CaM-like protein) (CLP) (Up-regulated in OPL). MADQLTEEQVTEFKEAFSLFDKDGDGCITTRELGTVMRSLGQNPTEAELRDMMSEIDR DGNGTVDFPEFLGMMARKMKDTDNEEEIREAFRVFDKDGNGFVSAAELRHVMTRLGE KLSDEEVDEMIRAADTDGDGQVNYEEFVRVLVSK

Sequence CWU 1

1

7631146PRTHUMAN 1Met Ala Gly Glu Leu Thr Pro Glu Glu Glu Ala Gln Tyr Lys Lys Ala 1 5 10 15 Phe Ser Ala Val Asp Thr Asp Gly Asn Gly Thr Ile Asn Ala Gln Glu 20 25 30 Leu Gly Ala Ala Leu Lys Ala Thr Gly Lys Asn Leu Ser Glu Ala Gln 35 40 45 Leu Arg Lys Leu Ile Ser Glu Val Asp Ser Asp Gly Asp Gly Glu Ile 50 55 60 Ser Phe Gln Glu Phe Leu Thr Ala Ala Arg Lys Ala Arg Ala Gly Leu 65 70 75 80 Glu Asp Leu Gln Val Ala Phe Arg Ala Phe Asp Gln Asp Gly Asp Gly 85 90 95 His Ile Thr Val Asp Glu Leu Arg Arg Ala Met Ala Gly Leu Gly Gln 100 105 110 Pro Leu Pro Gln Glu Glu Leu Asp Ala Met Ile Arg Glu Ala Asp Val 115 120 125 Asp Gln Asp Gly Arg Val Asn Tyr Glu Glu Phe Ala Arg Met Leu Ala 130 135 140 Gln Glu 145 21689PRTHUMAN 2Met Gly Ser Lys Arg Arg Arg Ala Thr Ser Pro Ser Ser Ser Val Ser 1 5 10 15 Gly Asp Phe Asp Asp Gly His His Ser Val Ser Thr Pro Gly Pro Ser 20 25 30 Arg Lys Arg Arg Arg Leu Ser Asn Leu Pro Thr Val Asp Pro Ile Ala 35 40 45 Val Cys His Glu Leu Tyr Asn Thr Ile Arg Asp Tyr Lys Asp Glu Gln 50 55 60 Gly Arg Leu Leu Cys Glu Leu Phe Ile Arg Ala Pro Lys Arg Arg Asn 65 70 75 80 Gln Pro Asp Tyr Tyr Glu Val Val Ser Gln Pro Ile Asp Leu Met Lys 85 90 95 Ile Gln Gln Lys Leu Lys Met Glu Glu Tyr Asp Asp Val Asn Leu Leu 100 105 110 Thr Ala Asp Phe Gln Leu Leu Phe Asn Asn Ala Lys Ser Tyr Tyr Lys 115 120 125 Pro Asp Ser Pro Glu Tyr Lys Ala Ala Cys Lys Leu Trp Asp Leu Tyr 130 135 140 Leu Arg Thr Arg Asn Glu Phe Val Gln Lys Gly Glu Ala Asp Asp Glu 145 150 155 160 Asp Asp Asp Glu Asp Gly Gln Asp Asn Gln Gly Thr Val Thr Glu Gly 165 170 175 Ser Ser Pro Ala Tyr Leu Lys Glu Ile Leu Glu Gln Leu Leu Glu Ala 180 185 190 Ile Val Val Ala Thr Asn Pro Ser Gly Arg Leu Ile Ser Glu Leu Phe 195 200 205 Gln Lys Leu Pro Ser Lys Val Gln Tyr Pro Asp Tyr Tyr Ala Ile Ile 210 215 220 Lys Glu Pro Ile Asp Leu Lys Thr Ile Ala Gln Arg Ile Gln Asn Gly 225 230 235 240 Ser Tyr Lys Ser Ile His Ala Met Ala Lys Asp Ile Asp Leu Leu Ala 245 250 255 Lys Asn Ala Lys Thr Tyr Asn Glu Pro Gly Ser Gln Val Phe Lys Asp 260 265 270 Ala Asn Ser Ile Lys Lys Ile Phe Tyr Met Lys Lys Ala Glu Ile Glu 275 280 285 His His Glu Met Ala Lys Ser Ser Leu Arg Met Arg Thr Pro Ser Asn 290 295 300 Leu Ala Ala Ala Arg Leu Thr Gly Pro Ser His Ser Lys Gly Ser Leu 305 310 315 320 Gly Glu Glu Arg Asn Pro Thr Ser Lys Tyr Tyr Arg Asn Lys Arg Ala 325 330 335 Val Gln Gly Gly Arg Leu Ser Ala Ile Thr Met Ala Leu Gln Tyr Gly 340 345 350 Ser Glu Ser Glu Glu Asp Ala Ala Leu Ala Ala Ala Arg Tyr Glu Glu 355 360 365 Gly Glu Ser Glu Ala Glu Ser Ile Thr Ser Phe Met Asp Val Ser Asn 370 375 380 Pro Phe Tyr Gln Leu Tyr Asp Thr Val Arg Ser Cys Arg Asn Asn Gln 385 390 395 400 Gly Gln Leu Ile Ala Glu Pro Phe Tyr His Leu Pro Ser Lys Lys Lys 405 410 415 Tyr Pro Asp Tyr Tyr Gln Gln Ile Lys Met Pro Ile Ser Leu Gln Gln 420 425 430 Ile Arg Thr Lys Leu Lys Asn Gln Glu Tyr Glu Thr Leu Asp His Leu 435 440 445 Glu Cys Asp Leu Asn Leu Met Phe Glu Asn Ala Lys Arg Tyr Asn Val 450 455 460 Pro Asn Ser Ala Ile Tyr Lys Arg Val Leu Lys Leu Gln Gln Val Met 465 470 475 480 Gln Ala Lys Lys Lys Glu Leu Ala Arg Arg Asp Asp Ile Glu Asp Gly 485 490 495 Asp Ser Met Ile Ser Ser Ala Thr Ser Asp Thr Gly Ser Ala Lys Arg 500 505 510 Lys Ser Lys Lys Asn Ile Arg Lys Gln Arg Met Lys Ile Leu Phe Asn 515 520 525 Val Val Leu Glu Ala Arg Glu Pro Gly Ser Gly Arg Arg Leu Cys Asp 530 535 540 Leu Phe Met Val Lys Pro Ser Lys Lys Asp Tyr Pro Asp Tyr Tyr Lys 545 550 555 560 Ile Ile Leu Glu Pro Met Asp Leu Lys Ile Ile Glu His Asn Ile Arg 565 570 575 Asn Asp Lys Tyr Ala Gly Glu Glu Gly Met Ile Glu Asp Met Lys Leu 580 585 590 Met Phe Arg Asn Ala Arg His Tyr Asn Glu Glu Gly Ser Gln Val Tyr 595 600 605 Asn Asp Ala His Ile Leu Glu Lys Leu Leu Lys Glu Lys Arg Lys Glu 610 615 620 Leu Gly Pro Leu Pro Asp Asp Asp Asp Met Ala Ser Pro Lys Leu Lys 625 630 635 640 Leu Ser Arg Lys Ser Gly Ile Ser Pro Lys Lys Ser Lys Tyr Met Thr 645 650 655 Pro Met Gln Gln Lys Leu Asn Glu Val Tyr Glu Ala Val Lys Asn Tyr 660 665 670 Thr Asp Lys Arg Gly Arg Arg Leu Ser Ala Ile Phe Leu Arg Leu Pro 675 680 685 Ser Arg Ser Glu Leu Pro Asp Tyr Tyr Leu Thr Ile Lys Lys Pro Met 690 695 700 Asp Met Glu Lys Ile Arg Ser His Met Met Ala Asn Lys Tyr Gln Asp 705 710 715 720 Ile Asp Ser Met Val Glu Asp Phe Val Met Met Phe Asn Asn Ala Cys 725 730 735 Thr Tyr Asn Glu Pro Glu Ser Leu Ile Tyr Lys Asp Ala Leu Val Leu 740 745 750 His Lys Val Leu Leu Glu Thr Arg Arg Asp Leu Glu Gly Asp Glu Asp 755 760 765 Ser His Val Pro Asn Val Thr Leu Leu Ile Gln Glu Leu Ile His Asn 770 775 780 Leu Phe Val Ser Val Met Ser His Gln Asp Asp Glu Gly Arg Cys Tyr 785 790 795 800 Ser Asp Ser Leu Ala Glu Ile Pro Ala Val Asp Pro Asn Phe Pro Asn 805 810 815 Lys Pro Pro Leu Thr Phe Asp Ile Ile Arg Lys Asn Val Glu Asn Asn 820 825 830 Arg Tyr Arg Arg Leu Asp Leu Phe Gln Glu His Met Phe Glu Val Leu 835 840 845 Glu Arg Ala Arg Arg Met Asn Arg Thr Asp Ser Glu Ile Tyr Glu Asp 850 855 860 Ala Val Glu Leu Gln Gln Phe Phe Ile Lys Ile Arg Asp Glu Leu Cys 865 870 875 880 Lys Asn Gly Glu Ile Leu Leu Ser Pro Ala Leu Ser Tyr Thr Thr Lys 885 890 895 His Leu His Asn Asp Val Glu Lys Glu Arg Lys Glu Lys Leu Pro Lys 900 905 910 Glu Ile Glu Glu Asp Lys Leu Lys Arg Glu Glu Glu Lys Arg Glu Ala 915 920 925 Glu Lys Ser Glu Asp Ser Ser Gly Ala Ala Gly Leu Ser Gly Leu His 930 935 940 Arg Thr Tyr Ser Gln Asp Cys Ser Phe Lys Asn Ser Met Tyr His Val 945 950 955 960 Gly Asp Tyr Val Tyr Val Glu Pro Ala Glu Ala Asn Leu Gln Pro His 965 970 975 Ile Val Cys Ile Glu Arg Leu Trp Glu Asp Ser Ala Gly Glu Lys Trp 980 985 990 Leu Tyr Gly Cys Trp Phe Tyr Arg Pro Asn Glu Thr Phe His Leu Ala 995 1000 1005 Thr Arg Lys Phe Leu Glu Lys Glu Val Phe Lys Ser Asp Tyr Tyr 1010 1015 1020 Asn Lys Val Pro Val Ser Lys Ile Leu Gly Lys Cys Val Val Met 1025 1030 1035 Phe Val Lys Glu Tyr Phe Lys Leu Cys Pro Glu Asn Phe Arg Asp 1040 1045 1050 Glu Asp Val Phe Val Cys Glu Ser Arg Tyr Ser Ala Lys Thr Lys 1055 1060 1065 Ser Phe Lys Lys Ile Lys Leu Trp Thr Met Pro Ile Ser Ser Val 1070 1075 1080 Arg Phe Val Pro Arg Asp Val Pro Leu Pro Val Val Arg Val Ala 1085 1090 1095 Ser Val Phe Ala Asn Ala Asp Lys Gly Asp Asp Glu Lys Asn Thr 1100 1105 1110 Asp Asn Ser Glu Asp Ser Arg Ala Glu Asp Asn Phe Asn Leu Glu 1115 1120 1125 Lys Glu Lys Glu Asp Val Pro Val Glu Met Ser Asn Gly Glu Pro 1130 1135 1140 Gly Cys His Tyr Phe Glu Gln Leu His Tyr Asn Asp Met Trp Leu 1145 1150 1155 Lys Val Gly Asp Cys Val Phe Ile Lys Ser His Gly Leu Val Arg 1160 1165 1170 Pro Arg Val Gly Arg Ile Glu Lys Val Trp Val Arg Asp Gly Ala 1175 1180 1185 Ala Tyr Phe Tyr Gly Pro Ile Phe Ile His Pro Glu Glu Thr Glu 1190 1195 1200 His Glu Pro Thr Lys Met Phe Tyr Lys Lys Glu Val Phe Leu Ser 1205 1210 1215 Asn Leu Glu Glu Thr Cys Pro Met Thr Cys Ile Leu Gly Lys Cys 1220 1225 1230 Ala Val Leu Ser Phe Lys Asp Phe Leu Ser Cys Arg Pro Thr Glu 1235 1240 1245 Ile Pro Glu Asn Asp Ile Leu Leu Cys Glu Ser Arg Tyr Asn Glu 1250 1255 1260 Ser Asp Lys Gln Met Lys Lys Phe Lys Gly Leu Lys Arg Phe Ser 1265 1270 1275 Leu Ser Ala Lys Val Val Asp Asp Glu Ile Tyr Tyr Phe Arg Lys 1280 1285 1290 Pro Ile Val Pro Gln Lys Glu Pro Ser Pro Leu Leu Glu Lys Lys 1295 1300 1305 Ile Gln Leu Leu Glu Ala Lys Phe Ala Glu Leu Glu Gly Gly Asp 1310 1315 1320 Asp Asp Ile Glu Glu Met Gly Glu Glu Asp Ser Glu Val Ile Glu 1325 1330 1335 Pro Pro Ser Leu Pro Gln Leu Gln Thr Pro Leu Ala Ser Glu Leu 1340 1345 1350 Asp Leu Met Pro Tyr Thr Pro Pro Gln Ser Thr Pro Lys Ser Ala 1355 1360 1365 Lys Gly Ser Ala Lys Lys Glu Gly Ser Lys Arg Lys Ile Asn Met 1370 1375 1380 Ser Gly Tyr Ile Leu Phe Ser Ser Glu Met Arg Ala Val Ile Lys 1385 1390 1395 Ala Gln His Pro Asp Tyr Ser Phe Gly Glu Leu Ser Arg Leu Val 1400 1405 1410 Gly Thr Glu Trp Arg Asn Leu Glu Thr Ala Lys Lys Ala Glu Tyr 1415 1420 1425 Glu Glu Arg Ala Ala Lys Val Ala Glu Gln Gln Glu Arg Glu Arg 1430 1435 1440 Ala Ala Gln Gln Gln Gln Pro Ser Ala Ser Pro Arg Ala Gly Thr 1445 1450 1455 Pro Val Gly Ala Leu Met Gly Val Val Pro Pro Pro Thr Pro Met 1460 1465 1470 Gly Met Leu Asn Gln Gln Leu Thr Pro Val Ala Gly Met Met Gly 1475 1480 1485 Gly Tyr Pro Pro Gly Leu Pro Pro Leu Gln Gly Pro Val Asp Gly 1490 1495 1500 Leu Val Ser Met Gly Ser Met Gln Pro Leu His Pro Gly Gly Pro 1505 1510 1515 Pro Pro His His Leu Pro Pro Gly Val Pro Gly Leu Pro Gly Ile 1520 1525 1530 Pro Pro Pro Gly Val Met Asn Gln Gly Val Ala Pro Met Val Gly 1535 1540 1545 Thr Pro Ala Pro Gly Gly Ser Pro Tyr Gly Gln Gln Val Gly Val 1550 1555 1560 Leu Gly Pro Pro Gly Gln Gln Ala Pro Pro Pro Tyr Pro Gly Pro 1565 1570 1575 His Pro Ala Gly Pro Pro Val Ile Gln Gln Pro Thr Thr Pro Met 1580 1585 1590 Phe Val Ala Pro Pro Pro Lys Thr Gln Arg Leu Leu His Ser Glu 1595 1600 1605 Ala Tyr Leu Lys Tyr Ile Glu Gly Leu Ser Ala Glu Ser Asn Ser 1610 1615 1620 Ile Ser Lys Trp Asp Gln Thr Leu Ala Ala Arg Arg Arg Asp Val 1625 1630 1635 His Leu Ser Lys Glu Gln Glu Ser Arg Leu Pro Ser His Trp Leu 1640 1645 1650 Lys Ser Lys Gly Ala His Thr Thr Met Ala Asp Ala Leu Trp Arg 1655 1660 1665 Leu Arg Asp Leu Met Leu Arg Asp Thr Leu Asn Ile Arg Gln Ala 1670 1675 1680 Tyr Asn Leu Glu Asn Val 1685 3268PRTHUMAN 3Met Ala Val Asn Val Tyr Ser Thr Ser Val Thr Ser Asp Asn Leu Ser 1 5 10 15 Arg His Asp Met Leu Ala Trp Ile Asn Glu Ser Leu Gln Leu Asn Leu 20 25 30 Thr Lys Ile Glu Gln Leu Cys Ser Gly Ala Ala Tyr Cys Gln Phe Met 35 40 45 Asp Met Leu Phe Pro Gly Ser Ile Ala Leu Lys Lys Val Lys Phe Gln 50 55 60 Ala Lys Leu Glu His Glu Tyr Ile Gln Asn Phe Lys Ile Leu Gln Ala 65 70 75 80 Gly Phe Lys Arg Met Gly Val Asp Lys Ile Ile Pro Val Asp Lys Leu 85 90 95 Val Lys Gly Lys Phe Gln Asp Asn Phe Glu Phe Val Gln Trp Phe Lys 100 105 110 Lys Phe Phe Asp Ala Asn Tyr Asp Gly Lys Asp Tyr Asp Pro Val Ala 115 120 125 Ala Arg Gln Gly Gln Glu Thr Ala Val Ala Pro Ser Leu Val Ala Pro 130 135 140 Ala Leu Asn Lys Pro Lys Lys Pro Leu Thr Ser Ser Ser Ala Ala Pro 145 150 155 160 Gln Arg Pro Ile Ser Thr Gln Arg Thr Ala Ala Ala Pro Lys Ala Gly 165 170 175 Pro Gly Val Val Arg Lys Asn Pro Gly Val Gly Asn Gly Asp Asp Glu 180 185 190 Ala Ala Glu Leu Met Gln Gln Val Asn Val Leu Lys Leu Thr Val Glu 195 200 205 Asp Leu Glu Lys Glu Arg Asp Phe Tyr Phe Gly Lys Leu Arg Asn Ile 210 215 220 Glu Leu Ile Cys Gln Glu Asn Glu Gly Glu Asn Asp Pro Val Leu Gln 225 230 235 240 Arg Ile Val Asp Ile Leu Tyr Ala Thr Asp Glu Gly Phe Val Ile Pro 245 250 255 Asp Glu Gly Gly Pro Gln Glu Glu Gln Glu Glu Tyr 260 265 4261PRTHUMAN 4Met Ala Ser Pro Asp Trp Gly Tyr Asp Asp Lys Asn Gly Pro Glu Gln 1 5 10 15 Trp Ser Lys Leu Tyr Pro Ile Ala Asn Gly Asn Asn Gln Ser Pro Val 20 25 30 Asp Ile Lys Thr Ser Glu Thr Lys His Asp Thr Ser Leu Lys Pro Ile 35 40 45 Ser Val Ser Tyr Asn Pro Ala Thr Ala Lys Glu Ile Ile Asn Val Gly 50 55 60 His Ser Phe His Val Asn Phe Glu Asp Asn Asp Asn Arg Ser Val Leu 65 70 75 80 Lys Gly Gly Pro Phe Ser Asp Ser Tyr Arg Leu Phe Gln Phe His Phe 85 90 95 His Trp Gly Ser Thr Asn Glu His Gly Ser Glu His Thr Val Asp Gly 100 105 110 Val Lys Tyr Ser Ala Glu Leu His Val Ala His Trp Asn Ser Ala Lys 115 120 125 Tyr Ser Ser Leu Ala Glu Ala Ala Ser Lys Ala Asp Gly Leu Ala Val 130 135 140 Ile Gly Val Leu Met Lys Val Gly Glu Ala Asn Pro Lys Leu Gln Lys 145 150 155

160 Val Leu Asp Ala Leu Gln Ala Ile Lys Thr Lys Gly Lys Arg Ala Pro 165 170 175 Phe Thr Asn Phe Asp Pro Ser Thr Leu Leu Pro Ser Ser Leu Asp Phe 180 185 190 Trp Thr Tyr Pro Gly Ser Leu Thr His Pro Pro Leu Tyr Glu Ser Val 195 200 205 Thr Trp Ile Ile Cys Lys Glu Ser Ile Ser Val Ser Ser Glu Gln Leu 210 215 220 Ala Gln Phe Arg Ser Leu Leu Ser Asn Val Glu Gly Asp Asn Ala Val 225 230 235 240 Pro Met Gln His Asn Asn Arg Pro Thr Gln Pro Leu Lys Gly Arg Thr 245 250 255 Val Arg Ala Ser Phe 260 5233PRTHUMAN 5Met Leu Asn Leu Leu Leu Leu Ala Leu Pro Val Leu Ala Ser Arg Ala 1 5 10 15 Tyr Ala Ala Pro Ala Pro Gly Gln Ala Leu Gln Arg Val Gly Ile Val 20 25 30 Gly Gly Gln Glu Ala Pro Arg Ser Lys Trp Pro Trp Gln Val Ser Leu 35 40 45 Arg Val Arg Asp Arg Tyr Trp Met His Phe Cys Gly Gly Ser Leu Ile 50 55 60 His Pro Gln Trp Val Leu Thr Ala Ala His Cys Val Gly Pro Asp Val 65 70 75 80 Lys Asp Leu Ala Ala Leu Arg Val Gln Leu Arg Glu Gln His Leu Tyr 85 90 95 Tyr Gln Asp Gln Leu Leu Pro Val Ser Arg Ile Ile Val His Pro Gln 100 105 110 Phe Tyr Thr Ala Gln Ile Gly Ala Asp Ile Ala Leu Leu Glu Leu Glu 115 120 125 Glu Pro Val Asn Val Ser Ser His Val His Thr Val Thr Leu Pro Pro 130 135 140 Ala Ser Glu Thr Phe Pro Pro Gly Met Pro Cys Trp Val Thr Gly Trp 145 150 155 160 Gly Asp Val Asp Asn Asp Glu Arg Leu Pro Pro Pro Phe Pro Leu Lys 165 170 175 Gln Val Lys Val Pro Ile Met Glu Asn His Ile Cys Asp Ala Lys Tyr 180 185 190 His Leu Gly Ala Tyr Thr Gly Asp Asp Val Arg Ile Val Arg Asp Asp 195 200 205 Met Leu Cys Ala Gly Asn Thr Arg Arg Asp Ser Cys Gln Val Ala Thr 210 215 220 Ala Pro His Thr Phe Pro Ala Pro Ser 225 230 6136PRTHUMAN 6Met Ala Arg Thr Lys Gln Thr Ala Arg Lys Ser Thr Gly Gly Lys Ala 1 5 10 15 Pro Arg Lys Gln Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala 20 25 30 Thr Gly Gly Val Lys Lys Pro His Arg Tyr Arg Pro Gly Thr Val Ala 35 40 45 Leu Arg Glu Ile Arg Arg Tyr Gln Lys Ser Thr Glu Leu Leu Ile Arg 50 55 60 Lys Leu Pro Phe Gln Arg Leu Val Arg Glu Ile Ala Gln Asp Phe Lys 65 70 75 80 Thr Asp Leu Arg Phe Gln Ser Ser Ala Val Met Ala Leu Gln Glu Ala 85 90 95 Ser Glu Ala Tyr Leu Val Gly Leu Phe Glu Asp Thr Asn Leu Cys Ala 100 105 110 Ile His Ala Lys Arg Val Thr Ile Met Pro Lys Asp Ile Gln Leu Ala 115 120 125 Arg Arg Ile Arg Gly Glu Arg Ala 130 135 7627PRTHUMAN 7Met Ala Thr Thr Gln Ile Ser Lys Asp Glu Leu Asp Glu Leu Lys Glu 1 5 10 15 Ala Phe Ala Lys Val Asp Leu Asn Ser Asn Gly Phe Ile Cys Asp Tyr 20 25 30 Glu Leu His Glu Leu Phe Lys Glu Ala Asn Met Pro Leu Pro Gly Tyr 35 40 45 Lys Val Arg Glu Ile Ile Gln Lys Leu Met Leu Asp Gly Asp Arg Asn 50 55 60 Lys Asp Gly Lys Ile Ser Phe Asp Glu Phe Val Tyr Ile Phe Gln Glu 65 70 75 80 Val Lys Ser Ser Asp Ile Ala Lys Thr Phe Arg Lys Ala Ile Asn Arg 85 90 95 Lys Glu Gly Ile Cys Ala Leu Gly Gly Thr Ser Glu Leu Ser Ser Glu 100 105 110 Gly Thr Gln His Ser Tyr Ser Glu Glu Glu Lys Tyr Ala Phe Val Asn 115 120 125 Trp Ile Asn Lys Ala Leu Glu Asn Asp Pro Asp Cys Arg His Val Ile 130 135 140 Pro Met Asn Pro Asn Thr Asp Asp Leu Phe Lys Ala Val Gly Asp Gly 145 150 155 160 Ile Val Leu Cys Lys Met Ile Asn Leu Ser Val Pro Asp Thr Ile Asp 165 170 175 Glu Arg Ala Ile Asn Lys Lys Lys Leu Thr Pro Phe Ile Ile Gln Glu 180 185 190 Asn Leu Asn Leu Ala Leu Asn Ser Ala Ser Ala Ile Gly Cys His Val 195 200 205 Val Asn Ile Gly Ala Glu Asp Leu Arg Ala Gly Lys Pro His Leu Val 210 215 220 Leu Gly Leu Leu Trp Gln Ile Ile Lys Ile Gly Leu Phe Ala Asp Ile 225 230 235 240 Glu Leu Ser Arg Asn Glu Ala Leu Ala Ala Leu Leu Arg Asp Gly Glu 245 250 255 Thr Leu Glu Glu Leu Met Lys Leu Ser Pro Glu Glu Leu Leu Leu Arg 260 265 270 Trp Ala Asn Phe His Leu Glu Asn Ser Gly Trp Gln Lys Ile Asn Asn 275 280 285 Phe Ser Ala Asp Ile Lys Asp Ser Lys Ala Tyr Phe His Leu Leu Asn 290 295 300 Gln Ile Ala Pro Lys Gly Gln Lys Glu Gly Glu Pro Arg Ile Asp Ile 305 310 315 320 Asn Met Ser Gly Phe Asn Glu Thr Asp Asp Leu Lys Arg Ala Glu Ser 325 330 335 Met Leu Gln Gln Ala Asp Lys Leu Gly Cys Arg Gln Phe Val Thr Pro 340 345 350 Ala Asp Val Val Ser Gly Asn Pro Lys Leu Asn Leu Ala Phe Val Ala 355 360 365 Asn Leu Phe Asn Lys Tyr Pro Ala Leu Thr Lys Pro Glu Asn Gln Asp 370 375 380 Ile Asp Trp Thr Leu Leu Glu Gly Glu Thr Arg Glu Glu Arg Thr Phe 385 390 395 400 Arg Asn Trp Met Asn Ser Leu Gly Val Asn Pro His Val Asn His Leu 405 410 415 Tyr Ala Asp Leu Gln Asp Ala Leu Val Ile Leu Gln Leu Tyr Glu Arg 420 425 430 Ile Lys Val Pro Val Asp Trp Ser Lys Val Asn Lys Pro Pro Tyr Pro 435 440 445 Lys Leu Gly Ala Asn Met Lys Lys Leu Glu Asn Cys Asn Tyr Ala Val 450 455 460 Glu Leu Gly Lys His Pro Ala Lys Phe Ser Leu Val Gly Ile Gly Gly 465 470 475 480 Gln Asp Leu Asn Asp Gly Asn Gln Thr Leu Thr Leu Ala Leu Val Trp 485 490 495 Gln Leu Met Arg Arg Tyr Thr Leu Asn Val Leu Glu Asp Leu Gly Asp 500 505 510 Gly Gln Lys Ala Asn Asp Asp Ile Ile Val Asn Trp Val Asn Arg Thr 515 520 525 Leu Ser Glu Ala Gly Lys Ser Thr Ser Ile Gln Ser Phe Lys Asp Lys 530 535 540 Thr Ile Ser Ser Ser Leu Ala Val Val Asp Leu Ile Asp Ala Ile Gln 545 550 555 560 Pro Gly Cys Ile Asn Tyr Asp Leu Val Lys Ser Gly Asn Leu Thr Glu 565 570 575 Asp Asp Lys His Asn Asn Ala Lys Tyr Ala Val Ser Met Ala Arg Arg 580 585 590 Ile Gly Ala Arg Val Tyr Ala Leu Pro Glu Asp Leu Val Glu Val Lys 595 600 605 Pro Lys Met Val Met Thr Val Phe Ala Cys Leu Met Gly Arg Gly Met 610 615 620 Lys Arg Val 625 8103PRTHUMAN 8Met Ser Gly Arg Gly Lys Gly Gly Lys Gly Leu Gly Lys Gly Gly Ala 1 5 10 15 Lys Arg His Arg Lys Val Leu Arg Asp Asn Ile Gln Gly Ile Thr Lys 20 25 30 Pro Ala Ile Arg Arg Leu Ala Arg Arg Gly Gly Val Lys Arg Ile Ser 35 40 45 Gly Leu Ile Tyr Glu Glu Thr Arg Gly Val Leu Lys Val Phe Leu Glu 50 55 60 Asn Val Ile Arg Asp Ala Val Thr Tyr Thr Glu His Ala Lys Arg Lys 65 70 75 80 Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys Arg Gln Gly Arg 85 90 95 Thr Leu Tyr Gly Phe Gly Gly 100 9165PRTHUMAN 9Met Val Asn Pro Thr Val Phe Phe Asp Ile Ala Val Asp Gly Glu Pro 1 5 10 15 Leu Gly Arg Val Ser Phe Glu Leu Phe Ala Asp Lys Val Pro Lys Thr 20 25 30 Ala Glu Asn Phe Arg Ala Leu Ser Thr Gly Glu Lys Gly Phe Gly Tyr 35 40 45 Lys Gly Ser Cys Phe His Arg Ile Ile Pro Gly Phe Met Cys Gln Gly 50 55 60 Gly Asp Phe Thr Arg His Asn Gly Thr Gly Gly Lys Ser Ile Tyr Gly 65 70 75 80 Glu Lys Phe Glu Asp Glu Asn Phe Ile Leu Lys His Thr Gly Pro Gly 85 90 95 Ile Leu Ser Met Ala Asn Ala Gly Pro Asn Thr Asn Gly Ser Gln Phe 100 105 110 Phe Ile Cys Thr Ala Lys Thr Glu Trp Leu Asp Gly Lys His Val Val 115 120 125 Phe Gly Lys Val Lys Glu Gly Met Asn Ile Val Glu Ala Met Glu Arg 130 135 140 Phe Gly Ser Arg Asn Gly Lys Thr Ser Lys Lys Ile Thr Ile Ala Asp 145 150 155 160 Cys Gly Gln Leu Glu 165 10189PRTHUMAN 10Met Arg Leu Ser Leu Pro Leu Leu Leu Leu Leu Leu Gly Ala Trp Ala 1 5 10 15 Ile Pro Gly Gly Leu Gly Asp Arg Ala Pro Leu Thr Ala Thr Ala Pro 20 25 30 Gln Leu Asp Asp Glu Glu Met Tyr Ser Ala His Met Pro Ala His Leu 35 40 45 Arg Cys Asp Ala Cys Arg Ala Val Ala Tyr Gln Met Trp Gln Asn Leu 50 55 60 Ala Lys Ala Glu Thr Lys Leu His Thr Ser Asn Ser Gly Gly Arg Arg 65 70 75 80 Glu Leu Ser Glu Leu Val Tyr Thr Asp Val Leu Asp Arg Ser Cys Ser 85 90 95 Arg Asn Trp Gln Asp Tyr Gly Val Arg Glu Val Asp Gln Val Lys Arg 100 105 110 Leu Thr Gly Pro Gly Leu Ser Glu Gly Pro Glu Pro Ser Ile Ser Val 115 120 125 Met Val Thr Gly Gly Pro Trp Pro Thr Arg Leu Ser Arg Thr Cys Leu 130 135 140 His Tyr Leu Gly Glu Phe Gly Glu Asp Gln Ile Tyr Glu Ala His Gln 145 150 155 160 Gln Gly Arg Gly Ala Leu Glu Ala Leu Leu Cys Gly Gly Pro Gln Gly 165 170 175 Ala Cys Ser Glu Lys Val Ser Ala Thr Arg Glu Glu Leu 180 185 11332PRTHUMAN 11Met Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu Glu 1 5 10 15 Gln Thr Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly 20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu 35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met Asp 50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly 65 70 75 80 Lys Asp Tyr Asn Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr Ala 85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg 100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr Ser 115 120 125 Pro Asn Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu Thr 130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Val Leu Gly Glu 180 185 190 His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Met Asn Val Ala Gly 195 200 205 Val Ser Leu Lys Thr Leu His Pro Asp Leu Gly Thr Asp Lys Asp Lys 210 215 220 Glu Gln Trp Lys Glu Val His Lys Gln Val Val Glu Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro 260 265 270 Val Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Asp Asp Val Phe 275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Leu Val 290 295 300 Lys Val Thr Leu Thr Ser Glu Glu Glu Ala Arg Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe 325 330 12338PRTHUMAN 12Met Ser Leu Ser Ala Phe Thr Leu Phe Leu Ala Leu Ile Gly Gly Thr 1 5 10 15 Ser Gly Gln Tyr Tyr Asp Tyr Asp Phe Pro Leu Ser Ile Tyr Gly Gln 20 25 30 Ser Ser Pro Asn Cys Ala Pro Glu Cys Asn Cys Pro Glu Ser Tyr Pro 35 40 45 Ser Ala Met Tyr Cys Asp Glu Leu Lys Leu Lys Ser Val Pro Met Val 50 55 60 Pro Pro Gly Ile Lys Tyr Leu Tyr Leu Arg Asn Asn Gln Ile Asp His 65 70 75 80 Ile Asp Glu Lys Ala Phe Glu Asn Val Thr Asp Leu Gln Trp Leu Ile 85 90 95 Leu Asp His Asn Leu Leu Glu Asn Ser Lys Ile Lys Gly Arg Val Phe 100 105 110 Ser Lys Leu Lys Gln Leu Lys Lys Leu His Ile Asn His Asn Asn Leu 115 120 125 Thr Glu Ser Val Gly Pro Leu Pro Lys Ser Leu Glu Asp Leu Gln Leu 130 135 140 Thr His Asn Lys Ile Thr Lys Leu Gly Ser Phe Glu Gly Leu Val Asn 145 150 155 160 Leu Thr Phe Ile His Leu Gln His Asn Arg Leu Lys Glu Asp Ala Val 165 170 175 Ser Ala Ala Phe Lys Gly Leu Lys Ser Leu Glu Tyr Leu Asp Leu Ser 180 185 190 Phe Asn Gln Ile Ala Arg Leu Pro Ser Gly Leu Pro Val Ser Leu Leu 195 200 205 Thr Leu Tyr Leu Asp Asn Asn Lys Ile Ser Asn Ile Pro Asp Glu Tyr 210 215 220 Phe Lys Arg Phe Asn Ala Leu Gln Tyr Leu Arg Leu Ser His Asn Glu 225 230 235 240 Leu Ala Asp Ser Gly Ile Pro Gly Asn Ser Phe Asn Val Ser Ser Leu 245 250 255 Val Glu Leu Asp Leu Ser Tyr Asn Lys Leu Lys Asn Ile Pro Thr Val 260 265 270 Asn Glu Asn Leu Glu Asn Tyr Tyr Leu Glu Val Asn Gln Leu Glu Lys 275 280 285 Phe Asp Ile Lys Ser Phe Cys Lys Ile Leu Gly Pro Leu Ser Tyr Ser 290 295 300 Lys Ile Lys His Leu Arg Leu Asp Gly Asn Arg Ile Ser Glu Thr Ser 305 310 315 320 Leu Pro Pro Asp Met Tyr Glu Cys Leu Arg Val Ala Asn Glu Val Thr 325 330 335 Leu Asn 13101PRTHUMAN 13Met Ser Asn Thr Gln Ala Glu Arg Ser Ile Ile Gly Met Ile Asp Met 1 5 10 15 Phe His Lys Tyr Thr Arg Arg Asp Asp Lys Ile Asp Lys Pro Ser Leu 20 25 30 Leu Thr Met Met Lys Glu Asn Phe Pro Asn Phe Leu Ser Ala Cys Asp 35 40 45 Lys Lys Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys Lys Asp Lys 50 55 60 Asn Glu Asp

Lys Lys Ile Asp Phe Ser Glu Phe Leu Ser Leu Leu Gly 65 70 75 80 Asp Ile Ala Thr Asp Tyr His Lys Gln Ser His Gly Ala Ala Pro Cys 85 90 95 Ser Gly Gly Ser Gln 100 14198PRTHUMAN 14Met Ala Ser Gly Asn Ala Arg Ile Gly Lys Pro Ala Pro Asp Phe Lys 1 5 10 15 Ala Thr Ala Val Val Asp Gly Ala Phe Lys Glu Val Lys Leu Ser Asp 20 25 30 Tyr Lys Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr 35 40 45 Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asn Arg Ala Glu Asp 50 55 60 Phe Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln 65 70 75 80 Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly Leu 85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr Arg Arg Leu Ser 100 105 110 Glu Asp Tyr Gly Val Leu Lys Thr Asp Glu Gly Ile Ala Tyr Arg Gly 115 120 125 Leu Phe Ile Ile Asp Gly Lys Gly Val Leu Arg Gln Ile Thr Val Asn 130 135 140 Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp 165 170 175 Lys Pro Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu 180 185 190 Tyr Phe Ser Lys His Asn 195 15140PRTHUMAN 15Met Leu Ser Arg Ala Val Cys Gly Thr Ser Arg Gln Leu Ala Pro Ala 1 5 10 15 Leu Gly Tyr Leu Gly Ser Arg Gln Lys His Ser Leu Pro Asp Leu Pro 20 25 30 Tyr Asp Tyr Gly Ala Leu Glu Pro His Ile Asn Ala Gln Ile Met Gln 35 40 45 Leu His His Ser Lys His His Ala Ala Tyr Val Asn Asn Leu Asn Val 50 55 60 Thr Glu Glu Lys Tyr Gln Glu Ala Leu Ala Lys Gly Arg Phe Gln Ala 65 70 75 80 Glu Arg Arg Glu Ala Val Pro Gly Arg Gly Asp Pro Arg Glu Pro Gly 85 90 95 Pro Ile Arg Thr Gly Leu Ser Val Glu Glu Asn Ser Leu Arg Ile Cys 100 105 110 Thr Gly Ser Glu Phe Ser Arg His Asp Ser Leu Ser Phe Lys His Met 115 120 125 Val Tyr Leu Ile Val Glu Gly Val Pro Arg Trp Val 130 135 140 16418PRTHUMAN 16Met Pro Ser Ser Val Ser Trp Gly Ile Leu Leu Leu Ala Gly Leu Cys 1 5 10 15 Cys Leu Val Pro Val Ser Leu Ala Glu Asp Pro Gln Gly Asp Ala Ala 20 25 30 Gln Lys Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn 35 40 45 Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu Tyr Arg Gln 50 55 60 Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser Pro Val Ser 65 70 75 80 Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys Ala Asp Thr 85 90 95 His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr Glu Ile Pro 100 105 110 Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg Thr Leu Asn 115 120 125 Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly Leu Phe Leu 130 135 140 Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp Val Lys Lys 145 150 155 160 Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp Thr Glu Glu 165 170 175 Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr Gln Gly Lys 180 185 190 Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val Phe Ala Leu 195 200 205 Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro Phe Glu Val 210 215 220 Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val 225 230 235 240 Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile Gln His Cys 245 250 255 Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu Gly Asn Ala 260 265 270 Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln His Leu Glu 275 280 285 Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu Asn Glu Asp 290 295 300 Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile Thr Gly Thr 305 310 315 320 Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr Lys Val Phe 325 330 335 Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro Leu Lys 340 345 350 Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp Glu Lys Gly 355 360 365 Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser Ile 370 375 380 Pro Pro Glu Val Lys Phe Asn Lys Pro Phe Val Phe Leu Met Ile Glu 385 390 395 400 Gln Asn Thr Lys Ser Pro Leu Phe Met Gly Lys Val Val Asn Pro Thr 405 410 415 Gln Lys 17332PRTHUMAN 17Met Gly Ala Gln Phe Ser Lys Thr Ala Ala Lys Gly Glu Ala Ala Ala 1 5 10 15 Glu Arg Pro Gly Glu Ala Ala Val Ala Ser Ser Pro Ser Lys Ala Asn 20 25 30 Gly Gln Glu Asn Gly His Val Lys Val Asn Gly Asp Ala Ser Pro Ala 35 40 45 Ala Ala Glu Ser Gly Ala Lys Glu Glu Leu Gln Ala Asn Gly Ser Ala 50 55 60 Pro Ala Ala Asp Lys Glu Glu Pro Ala Ala Ala Gly Ser Gly Ala Ala 65 70 75 80 Ser Pro Ser Ala Ala Glu Lys Gly Glu Pro Ala Ala Ala Ala Ala Pro 85 90 95 Glu Ala Gly Ala Ser Pro Val Glu Lys Glu Ala Pro Ala Glu Gly Glu 100 105 110 Ala Ala Glu Pro Gly Ser Pro Thr Ala Ala Glu Gly Glu Ala Ala Ser 115 120 125 Ala Ala Ser Ser Thr Ser Ser Pro Lys Ala Glu Asp Gly Ala Thr Pro 130 135 140 Ser Pro Ser Asn Glu Thr Pro Lys Lys Lys Lys Lys Arg Phe Ser Phe 145 150 155 160 Lys Lys Ser Phe Lys Leu Ser Gly Phe Ser Phe Lys Lys Asn Lys Lys 165 170 175 Glu Ala Gly Glu Gly Gly Glu Ala Glu Ala Pro Ala Ala Glu Gly Gly 180 185 190 Lys Asp Glu Ala Ala Gly Gly Ala Ala Ala Ala Ala Ala Glu Ala Gly 195 200 205 Ala Ala Ser Gly Glu Gln Ala Ala Ala Pro Gly Glu Glu Ala Ala Ala 210 215 220 Gly Glu Glu Gly Ala Ala Gly Gly Asp Ser Gln Glu Ala Lys Pro Gln 225 230 235 240 Glu Ala Ala Val Ala Pro Glu Lys Pro Pro Ala Ser Asp Glu Thr Lys 245 250 255 Ala Ala Glu Glu Pro Ser Lys Val Glu Glu Lys Lys Ala Glu Glu Ala 260 265 270 Gly Ala Ser Ala Ala Ala Cys Glu Ala Pro Ser Ala Ala Gly Leu Val 275 280 285 Cys Pro Arg Arg Gly Gly Ser Pro Arg Gly Gly Ala Arg Gly Arg Arg 290 295 300 Ser Leu Asn Gln Ala Cys Ala Ala Pro Ser Gln Glu Ala Gln Pro Glu 305 310 315 320 Cys Ser Pro Glu Ala Pro Pro Ala Glu Ala Ala Glu 325 330 18803PRTHUMAN 18Met Arg Ala Leu Trp Val Leu Gly Leu Cys Cys Val Leu Leu Thr Phe 1 5 10 15 Gly Ser Val Arg Ala Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu 20 25 30 Glu Asp Leu Gly Lys Ser Arg Glu Gly Ser Arg Thr Asp Asp Glu Val 35 40 45 Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp Gly Leu Asn Ala Ser 50 55 60 Gln Ile Arg Glu Leu Arg Glu Lys Ser Glu Lys Phe Ala Phe Gln Ala 65 70 75 80 Glu Val Asn Arg Met Met Lys Leu Ile Ile Asn Ser Leu Tyr Lys Asn 85 90 95 Lys Glu Ile Phe Leu Arg Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu 100 105 110 Asp Lys Ile Arg Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly 115 120 125 Asn Glu Glu Leu Thr Val Lys Ile Lys Cys Asp Lys Glu Lys Asn Leu 130 135 140 Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg Glu Glu Leu Val 145 150 155 160 Lys Asn Leu Gly Thr Ile Ala Lys Ser Gly Thr Ser Glu Phe Leu Asn 165 170 175 Lys Met Thr Glu Ala Gln Glu Asp Gly Gln Ser Thr Ser Glu Leu Ile 180 185 190 Gly Gln Phe Gly Val Gly Phe Tyr Ser Ala Phe Leu Val Ala Asp Lys 195 200 205 Val Ile Val Thr Ser Lys His Asn Asn Asp Thr Gln His Ile Trp Glu 210 215 220 Ser Asp Ser Asn Glu Phe Ser Val Ile Ala Asp Pro Arg Gly Asn Thr 225 230 235 240 Leu Gly Arg Gly Thr Thr Ile Thr Leu Val Leu Lys Glu Glu Ala Ser 245 250 255 Asp Tyr Leu Glu Leu Asp Thr Ile Lys Asn Leu Val Lys Lys Tyr Ser 260 265 270 Gln Phe Ile Asn Phe Pro Ile Tyr Val Trp Ser Ser Lys Thr Glu Thr 275 280 285 Val Glu Glu Pro Met Glu Glu Glu Glu Ala Ala Lys Glu Glu Lys Glu 290 295 300 Glu Ser Asp Asp Glu Ala Ala Val Glu Glu Glu Glu Glu Glu Lys Lys 305 310 315 320 Pro Lys Thr Lys Lys Val Glu Lys Thr Val Trp Asp Trp Glu Leu Met 325 330 335 Asn Asp Ile Lys Pro Ile Trp Gln Arg Pro Ser Lys Glu Val Glu Glu 340 345 350 Asp Glu Tyr Lys Ala Phe Tyr Lys Ser Phe Ser Lys Glu Ser Asp Asp 355 360 365 Pro Met Ala Tyr Ile His Phe Thr Ala Glu Gly Glu Val Thr Phe Lys 370 375 380 Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg Gly Leu Phe Asp Glu 385 390 395 400 Tyr Gly Ser Lys Lys Ser Asp Tyr Ile Lys Leu Tyr Val Arg Arg Val 405 410 415 Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys Tyr Leu Asn Phe 420 425 430 Val Lys Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 435 440 445 Glu Thr Leu Gln Gln His Lys Leu Leu Lys Val Ile Arg Lys Lys Leu 450 455 460 Val Arg Lys Thr Leu Asp Met Ile Lys Lys Ile Ala Asp Asp Lys Tyr 465 470 475 480 Asn Asp Thr Phe Trp Lys Glu Phe Gly Thr Asn Ile Lys Leu Gly Val 485 490 495 Ile Glu Asp His Ser Asn Arg Thr Arg Leu Ala Lys Leu Leu Arg Phe 500 505 510 Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr Val 515 520 525 Glu Arg Met Lys Glu Lys Gln Asp Lys Ile Tyr Phe Met Ala Gly Ser 530 535 540 Ser Arg Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg Leu Leu Lys 545 550 555 560 Lys Gly Tyr Glu Val Ile Tyr Leu Thr Glu Pro Val Asp Glu Tyr Cys 565 570 575 Ile Gln Ala Leu Pro Glu Phe Asp Gly Lys Arg Phe Gln Asn Val Ala 580 585 590 Lys Glu Gly Val Lys Phe Asp Glu Ser Glu Lys Thr Lys Glu Ser Arg 595 600 605 Glu Ala Val Glu Lys Glu Phe Glu Pro Leu Leu Asn Trp Met Lys Asp 610 615 620 Lys Ala Leu Lys Asp Lys Ile Glu Lys Ala Val Val Ser Gln Arg Leu 625 630 635 640 Thr Glu Ser Pro Cys Ala Leu Val Ala Ser Gln Tyr Gly Trp Ser Gly 645 650 655 Asn Met Glu Arg Ile Met Lys Ala Gln Ala Tyr Gln Thr Gly Lys Asp 660 665 670 Ile Ser Thr Asn Tyr Tyr Ala Ser Gln Lys Lys Thr Phe Glu Ile Asn 675 680 685 Pro Arg His Pro Leu Ile Arg Asp Met Leu Arg Arg Ile Lys Glu Asp 690 695 700 Glu Asp Asp Lys Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr 705 710 715 720 Ala Thr Leu Arg Ser Gly Tyr Leu Leu Pro Asp Thr Lys Ala Tyr Gly 725 730 735 Asp Arg Ile Glu Arg Met Leu Arg Leu Ser Leu Asn Ile Asp Pro Asp 740 745 750 Ala Lys Val Glu Glu Glu Pro Glu Glu Glu Pro Glu Glu Thr Ala Glu 755 760 765 Asp Thr Thr Glu Asp Thr Glu Gln Asp Glu Asp Glu Glu Met Asp Val 770 775 780 Gly Thr Asp Glu Glu Glu Glu Thr Ala Lys Glu Ser Thr Ala Glu Lys 785 790 795 800 Asp Glu Leu 19111PRTHUMAN 19Met Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp 1 5 10 15 Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn Gly Arg Asp 20 25 30 Ala Pro Ala Asn Gly Asn Ala Glu Asn Glu Glu Asn Gly Glu Gln Glu 35 40 45 Ala Asp Asn Glu Val Asp Glu Glu Glu Glu Glu Gly Gly Glu Glu Glu 50 55 60 Glu Glu Glu Glu Glu Gly Asp Gly Glu Glu Glu Asp Gly Asp Glu Asp 65 70 75 80 Glu Glu Ala Glu Ser Ala Thr Gly Lys Arg Ala Ala Glu Asp Asp Glu 85 90 95 Asp Asp Asp Val Asp Thr Lys Lys Gln Lys Thr Asp Glu Asp Asp 100 105 110 20126PRTHUMAN 20Met Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys 1 5 10 15 Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg 20 25 30 Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr Lys Val Leu Lys Gln 35 40 45 Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn 50 55 60 Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg 65 70 75 80 Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln 85 90 95 Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val 100 105 110 Ser Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ser Lys 115 120 125 21294PRTHUMAN 21Met Glu Asp Ser Met Asp Met Asp Met Ser Pro Leu Arg Pro Gln Asn 1 5 10 15 Tyr Leu Phe Gly Cys Glu Leu Lys Ala Asp Lys Asp Tyr His Phe Lys 20 25 30 Val Asp Asn Asp Glu Asn Glu His Gln Leu Ser Leu Arg Thr Val Ser 35 40 45 Leu Gly Ala Gly Ala Lys Asp Glu Leu His Ile Val Glu Ala Glu Ala 50 55 60 Met Asn Tyr Glu Gly Ser Pro Ile Lys Val Thr Leu Ala Thr Leu Lys 65 70 75 80 Met Ser Val Gln Pro Thr Val Ser Leu Gly Gly Phe Glu Ile Thr Pro 85 90 95 Pro Val Val Leu Arg Leu Lys Cys Gly Ser Gly Pro Val His Ile Ser 100 105 110 Gly Gln His Leu Val Ala Val Glu Glu Asp Ala Glu Ser Glu Asp Glu 115

120 125 Glu Glu Glu Asp Val Lys Leu Leu Ser Ile Ser Gly Lys Arg Ser Ala 130 135 140 Pro Gly Gly Gly Ser Lys Val Pro Gln Lys Lys Val Lys Leu Ala Ala 145 150 155 160 Asp Glu Asp Asp Asp Asp Asp Asp Glu Glu Asp Asp Asp Glu Asp Asp 165 170 175 Asp Gly Asp Asp Phe Asp Asp Glu Glu Ala Glu Glu Lys Ala Pro Val 180 185 190 Lys Lys Ser Ile Arg Asp Thr Pro Ala Lys Asn Ala Gln Lys Ser Asn 195 200 205 Gln Asn Gly Lys Asp Ser Lys Pro Ser Ser Thr Pro Arg Ser Lys Gly 210 215 220 Gln Glu Ser Phe Lys Lys Gln Glu Lys Thr Pro Lys Thr Pro Lys Gly 225 230 235 240 Pro Ser Ser Val Glu Asp Ile Lys Ala Lys Met Gln Ala Ser Ile Glu 245 250 255 Lys Gly Gly Ser Leu Pro Lys Val Glu Ala Lys Phe Ile Asn Cys Val 260 265 270 Lys Asn Cys Phe Arg Met Thr Asp Gln Glu Ala Ile Gln Asp Leu Trp 275 280 285 Gln Trp Arg Lys Ser Leu 290 22531PRTHUMAN 22Met Ser Lys Pro His Ser Glu Ala Gly Thr Ala Phe Ile Gln Thr Gln 1 5 10 15 Gln Leu His Ala Ala Met Ala Asp Thr Phe Leu Glu His Met Cys Arg 20 25 30 Leu Asp Ile Asp Ser Pro Pro Ile Thr Ala Arg Asn Thr Gly Ile Ile 35 40 45 Cys Thr Ile Gly Pro Ala Ser Arg Ser Val Glu Thr Leu Lys Glu Met 50 55 60 Ile Lys Ser Gly Met Asn Val Ala Arg Leu Asn Phe Ser His Gly Thr 65 70 75 80 His Glu Tyr His Ala Glu Thr Ile Lys Asn Val Arg Thr Ala Thr Glu 85 90 95 Ser Phe Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val Ala Leu 100 105 110 Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly Leu Ile Lys Gly Ser Gly 115 120 125 Thr Ala Glu Val Glu Leu Lys Lys Gly Ala Thr Leu Lys Ile Thr Leu 130 135 140 Asp Asn Ala Tyr Met Glu Lys Cys Asp Glu Asn Ile Leu Trp Leu Asp 145 150 155 160 Tyr Lys Asn Ile Cys Lys Val Val Glu Val Gly Ser Lys Ile Tyr Val 165 170 175 Asp Asp Gly Leu Ile Ser Leu Gln Val Lys Gln Lys Gly Ala Asp Phe 180 185 190 Leu Val Thr Glu Val Glu Asn Gly Gly Ser Leu Gly Ser Lys Lys Gly 195 200 205 Val Asn Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu Lys 210 215 220 Asp Ile Gln Asp Leu Lys Phe Gly Val Glu Gln Asp Val Asp Met Val 225 230 235 240 Phe Ala Ser Phe Ile Arg Lys Ala Ser Asp Val His Glu Val Arg Lys 245 250 255 Val Leu Gly Glu Lys Gly Lys Asn Ile Lys Ile Ile Ser Lys Ile Glu 260 265 270 Asn His Glu Gly Val Arg Arg Phe Asp Glu Ile Leu Glu Ala Ser Asp 275 280 285 Gly Ile Met Val Ala Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu 290 295 300 Lys Val Phe Leu Ala Gln Lys Met Met Ile Gly Arg Cys Asn Arg Ala 305 310 315 320 Gly Lys Pro Val Ile Cys Ala Thr Gln Met Leu Glu Ser Met Ile Lys 325 330 335 Lys Pro Arg Pro Thr Arg Ala Glu Gly Ser Asp Val Ala Asn Ala Val 340 345 350 Leu Asp Gly Ala Asp Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly 355 360 365 Asp Tyr Pro Leu Glu Ala Val Arg Met Gln His Leu Ile Ala Arg Glu 370 375 380 Ala Glu Ala Ala Ile Tyr His Leu Gln Leu Phe Glu Glu Leu Arg Arg 385 390 395 400 Leu Ala Pro Ile Thr Ser Asp Pro Thr Glu Ala Thr Ala Val Gly Ala 405 410 415 Val Glu Ala Ser Phe Lys Cys Cys Ser Gly Ala Ile Ile Val Leu Thr 420 425 430 Lys Ser Gly Arg Ser Ala His Gln Val Ala Arg Tyr Arg Pro Arg Ala 435 440 445 Pro Ile Ile Ala Val Thr Arg Asn Pro Gln Thr Ala Arg Gln Ala His 450 455 460 Leu Tyr Arg Gly Ile Phe Pro Val Leu Cys Lys Asp Pro Val Gln Glu 465 470 475 480 Ala Trp Ala Glu Asp Val Asp Leu Arg Val Asn Phe Ala Met Asn Val 485 490 495 Gly Lys Ala Arg Gly Phe Phe Lys Lys Gly Asp Val Val Ile Val Leu 500 505 510 Thr Gly Trp Arg Pro Gly Ser Gly Phe Thr Asn Thr Met Arg Val Val 515 520 525 Pro Val Pro 530 23248PRTHUMAN 23Met Glu Arg Ala Ser Leu Ile Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Glu Asp Met Ala Ala Phe Met Lys Gly Ala Val Glu Lys 20 25 30 Gly Glu Glu Leu Ser Cys Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Gly Gln Arg Ala Ala Trp Arg Val Leu Ser Ser 50 55 60 Ile Glu Gln Lys Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu 65 70 75 80 Val Arg Glu Tyr Arg Glu Lys Val Glu Thr Glu Leu Gln Gly Val Cys 85 90 95 Asp Thr Val Leu Gly Leu Leu Asp Ser His Leu Ile Lys Glu Ala Gly 100 105 110 Asp Ala Glu Ser Arg Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr 115 120 125 Arg Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys Lys Arg Ile Ile 130 135 140 Asp Ser Ala Arg Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys Lys 145 150 155 160 Glu Met Pro Pro Thr Asn Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe 165 170 175 Ser Val Phe His Tyr Glu Ile Ala Asn Ser Pro Glu Glu Ala Ile Ser 180 185 190 Leu Ala Lys Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu 195 200 205 Ser Glu Asp Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 210 215 220 Asp Asn Leu Thr Leu Trp Thr Ala Asp Asn Ala Gly Glu Glu Gly Gly 225 230 235 240 Glu Ala Pro Gln Glu Pro Gln Ser 245 24245PRTHUMAN 24Met Asp Lys Asn Glu Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala 1 5 10 15 Glu Arg Tyr Asp Asp Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln 20 25 30 Gly Ala Glu Leu Ser Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr 35 40 45 Lys Asn Val Val Gly Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser 50 55 60 Ile Glu Gln Lys Thr Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg 65 70 75 80 Glu Tyr Arg Glu Lys Ile Glu Thr Glu Leu Arg Asp Ile Cys Asn Asp 85 90 95 Val Leu Ser Leu Leu Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Ala 100 105 110 Glu Ser Lys Val Phe Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr 115 120 125 Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln 130 135 140 Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met 145 150 155 160 Gln Pro Thr His Pro Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val 165 170 175 Phe Tyr Tyr Glu Ile Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala 180 185 190 Lys Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu 195 200 205 Glu Ser Tyr Lys Asp Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn 210 215 220 Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly 225 230 235 240 Glu Gly Gly Glu Asn 245 25463PRTHUMAN 25Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn 1 5 10 15 Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala 20 25 30 Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu 35 40 45 Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn 50 55 60 Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp 65 70 75 80 Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr 85 90 95 Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu 100 105 110 Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp 115 120 125 Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp 130 135 140 Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile 145 150 155 160 Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr 165 170 175 Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val 180 185 190 Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile 195 200 205 Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro 210 215 220 Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr 225 230 235 240 Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg 245 250 255 Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro 260 265 270 Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly 275 280 285 Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala 290 295 300 Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu 305 310 315 320 Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met 325 330 335 Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp 340 345 350 Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly 355 360 365 Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly 370 375 380 Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly 385 390 395 400 Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu 405 410 415 Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp 420 425 430 Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln 435 440 445 Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ser Gly Lys Phe Phe 450 455 460 2663PRTHUMAN 26Arg Arg Arg Thr Gly Leu Arg Arg Lys Lys Lys Lys Leu Pro Arg Met 1 5 10 15 Glu Arg Arg Lys Met Lys Gly Lys Lys Lys Asp Glu Glu Glu Glu Glu 20 25 30 Glu Asp Asp Glu Gly Pro Ala Leu Lys Arg Ala Ala Glu Glu Glu Asp 35 40 45 Glu Ala Asp Pro Lys Arg Gln Lys Thr Glu Asn Gly Ala Ser Ala 50 55 60 2798PRTHUMAN 27Met Met Cys Gly Ala Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr 1 5 10 15 Gln His Ile Ala Asp Gln Val Arg Ser Gln Leu Glu Glu Lys Glu Asn 20 25 30 Lys Lys Phe Pro Val Phe Lys Ala Val Ser Phe Lys Ser Gln Val Val 35 40 45 Ala Gly Thr Asn Tyr Phe Ile Lys Val His Val Gly Asp Glu Asp Phe 50 55 60 Val His Leu Arg Val Phe Gln Ser Leu Pro His Glu Asn Lys Pro Leu 65 70 75 80 Thr Leu Ser Asn Tyr Gln Thr Asn Lys Ala Lys His Asp Glu Leu Thr 85 90 95 Tyr Phe 28281PRTHUMAN 28Met Ser Arg Ile Ser Gln Met Thr Ala Ala Arg Ser Pro Pro Arg Leu 1 5 10 15 His Met Ala Met Trp Ser Thr Arg Phe Ala Thr Ser Val Arg Thr Asn 20 25 30 Ala Val Gln Arg Ile Leu Gly Gly His Leu Asp Ala Lys Gly Ser Phe 35 40 45 Pro Trp Gln Ala Lys Met Val Ser His His Asn Leu Thr Thr Gly Ala 50 55 60 Thr Leu Ile Asn Glu Gln Trp Leu Leu Thr Thr Ala Lys Asn Leu Phe 65 70 75 80 Leu Asn His Ser Glu Asn Ala Thr Ala Lys Asp Ile Ala Pro Thr Leu 85 90 95 Thr Leu Tyr Val Gly Lys Lys Gln Leu Val Glu Ile Glu Lys Val Val 100 105 110 Leu His Pro Asn Tyr Ser Gln Val Asp Ile Gly Leu Ile Lys Leu Lys 115 120 125 Gln Lys Val Ser Val Asn Glu Arg Val Met Pro Ile Cys Leu Pro Ser 130 135 140 Lys Asp Tyr Ala Glu Val Gly Arg Val Gly Tyr Val Ser Gly Trp Gly 145 150 155 160 Arg Asn Ala Asn Phe Lys Phe Thr Asp His Leu Lys Tyr Val Met Leu 165 170 175 Pro Val Ala Asp Gln Asp Gln Cys Ile Arg His Tyr Glu Gly Ser Thr 180 185 190 Val Pro Glu Lys Lys Thr Pro Lys Ser Pro Val Gly Val Gln Pro Ile 195 200 205 Leu Asn Glu His Thr Phe Cys Ala Gly Met Ser Lys Tyr Gln Glu Asp 210 215 220 Thr Cys Tyr Gly Asp Ala Gly Ser Ala Phe Ala Val His Asp Leu Glu 225 230 235 240 Glu Asp Thr Trp Tyr Ala Thr Gly Ile Leu Ser Phe Asp Lys Ser Cys 245 250 255 Ala Val Ala Glu Tyr Gly Val Tyr Val Lys Val Thr Ser Ile Gln Asp 260 265 270 Trp Val Gln Lys Thr Ile Ala Glu Asn 275 280 29135PRTHUMAN 29Met Ala Thr Val Gln Gln Leu Glu Gly Arg Trp Arg Leu Val Asp Ser 1 5 10 15 Lys Gly Phe Asp Glu Tyr Met Lys Glu Leu Gly Val Gly Ile Ala Leu 20 25 30 Arg Lys Met Gly Ala Met Ala Lys Pro Asp Cys Ile Ile Thr Cys Asp 35 40 45 Gly Lys Asn Leu Thr Ile Lys Thr Glu Ser Thr Leu Lys Thr Thr Gln 50 55 60 Phe Ser Cys Thr Leu Gly Glu Lys Phe Glu Glu Thr Thr Ala Asp Gly 65 70 75 80 Arg Lys Thr Gln Thr Val Cys Asn Phe Thr Asp Gly Ala Leu Val Gln 85 90 95 His Gln Glu Trp Asp Gly Lys Glu Ser Thr Ile Thr Arg Lys Leu Lys 100 105 110 Asp Gly Lys Leu Val Val Glu Cys Val Met Asn Asn Val Thr Cys Thr 115 120 125 Arg Ile Tyr Glu Lys Val Glu 130 135 30470PRTHUMAN 30Met Asp Trp Thr Trp Arg Phe Leu Phe Val Val Ala Ala Ala Thr Ser 1 5 10 15 Val Gln Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Ser Phe 35 40 45 Asn Ser Leu Ala Ile Asn Trp Val

Arg Gln Ala Pro Gly Gln Gly Leu 50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Ile Phe Gly Thr Thr Asn Tyr Ala 65 70 75 80 Gln Arg Phe Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr Gly 85 90 95 Arg Ala Tyr Met Glu Leu Thr Ser Leu Arg Ser Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Ser Arg Phe Ile Ser Glu Thr Asn Phe Cys Phe Lys 115 120 125 Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 130 135 140 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 145 150 155 160 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 165 170 175 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 180 185 190 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 195 200 205 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 210 215 220 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 225 230 235 240 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 245 250 255 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 260 265 270 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 275 280 285 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 290 295 300 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 305 310 315 320 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 325 330 335 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 340 345 350 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 355 360 365 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 370 375 380 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 385 390 395 400 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 405 410 415 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 420 425 430 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 435 440 445 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 450 455 460 Ser Leu Ser Pro Gly Lys 465 470 31105PRTHUMAN 31Met Ala Lys Ile Ser Ser Pro Thr Glu Thr Glu Arg Cys Ile Glu Ser 1 5 10 15 Leu Ile Ala Val Phe Gln Lys Tyr Ala Gly Lys Asp Gly Tyr Asn Tyr 20 25 30 Thr Leu Ser Lys Thr Glu Phe Leu Ser Phe Met Asn Thr Glu Leu Ala 35 40 45 Ala Phe Thr Lys Asn Gln Lys Asp Pro Gly Val Leu Asp Arg Met Met 50 55 60 Lys Lys Leu Asp Thr Asn Ser Asp Gly Gln Leu Asp Phe Ser Glu Phe 65 70 75 80 Leu Asn Leu Ile Gly Gly Leu Ala Met Ala Cys His Asp Ser Phe Leu 85 90 95 Lys Ala Val Pro Ser Gln Lys Arg Thr 100 105 32233PRTHUMAN 32Met Ala Trp Thr Pro Leu Leu Leu Pro Leu Leu Thr Phe Cys Thr Val 1 5 10 15 Ser Glu Ala Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser 20 25 30 Pro Gly Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys 35 40 45 Lys Tyr Ala Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Thr Pro Val Leu 50 55 60 Val Ile Tyr Asp Asp Thr Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Ser Ser Gly Thr Val Ala Thr Leu Thr Leu Ser Gly Ala 85 90 95 Gln Val Glu Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Ser Asp Ser Ser 100 105 110 Gly Asn His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 33286PRTHUMAN 33Met Ser Glu Glu Gln Phe Gly Gly Thr Gly Arg Arg His Ala Asn Gly 1 5 10 15 Gly Gly Arg Arg Ser Ala Gly Asp Glu Glu Gly Ala Met Val Ala Ala 20 25 30 Thr Gln Gly Ala Ala Ala Ala Arg Glu Ala Asp Ala Gly Pro Gly Ala 35 40 45 Glu Pro Arg Leu Glu Ala Pro Lys Gly Ser Ala Glu Ser Glu Gly Ala 50 55 60 Lys Ile Asp Ala Ser Lys Asn Glu Glu Asp Glu Gly Lys Met Phe Ile 65 70 75 80 Gly Gly Leu Ser Trp Asp Thr Thr Lys Lys Asp Leu Lys Asp Tyr Phe 85 90 95 Ser Lys Phe Gly Glu Val Val Asp Cys Thr Leu Lys Leu Asp Pro Ile 100 105 110 Thr Gly Arg Ser Arg Gly Phe Gly Phe Val Leu Phe Lys Glu Ser Glu 115 120 125 Ser Val Asp Lys Val Met Asp Gln Lys Glu His Lys Leu Asn Gly Lys 130 135 140 Val Ile Asp Pro Lys Arg Ala Lys Ala Met Lys Thr Lys Glu Pro Val 145 150 155 160 Lys Lys Ile Phe Val Gly Gly Leu Ser Pro Asp Thr Pro Glu Glu Lys 165 170 175 Ile Arg Glu Tyr Phe Gly Gly Phe Gly Glu Val Glu Ser Ile Glu Leu 180 185 190 Pro Met Asp Asn Lys Thr Asn Lys Arg Arg Gly Phe Cys Phe Ile Thr 195 200 205 Phe Lys Glu Glu Glu Pro Val Lys Lys Ile Met Glu Lys Lys Tyr His 210 215 220 Asn Val Gly Leu Ser Lys Cys Glu Ile Lys Val Ala Met Ser Lys Glu 225 230 235 240 Gln Tyr Gln Gln Gln Gln Gln Trp Gly Ser Arg Gly Gly Phe Ala Gly 245 250 255 Arg Ala Arg Gly Glu Phe Arg Asn Ser Ser Glu Ala Gly Glu Gly Leu 260 265 270 Glu Leu Pro Pro Asn Ser Ile His Cys Trp Gln Leu Ser Val 275 280 285 34372PRTHUMAN 34Met Ser Lys Ser Glu Ser Pro Lys Glu Pro Glu Gln Leu Arg Lys Leu 1 5 10 15 Phe Ile Gly Gly Leu Ser Phe Glu Thr Thr Asp Glu Ser Leu Arg Ser 20 25 30 His Phe Glu Gln Trp Gly Thr Leu Thr Asp Cys Val Val Met Arg Asp 35 40 45 Pro Asn Thr Lys Arg Ser Arg Gly Phe Gly Phe Val Thr Tyr Ala Thr 50 55 60 Val Glu Glu Val Asp Ala Ala Met Asn Ala Arg Pro His Lys Val Asp 65 70 75 80 Gly Arg Val Val Glu Pro Lys Arg Ala Val Ser Arg Glu Asp Ser Gln 85 90 95 Arg Pro Gly Ala His Leu Thr Val Lys Lys Ile Phe Val Gly Gly Ile 100 105 110 Lys Glu Asp Thr Glu Glu His His Leu Arg Asp Tyr Phe Glu Gln Tyr 115 120 125 Gly Lys Ile Glu Val Ile Glu Ile Met Thr Asp Arg Gly Ser Gly Lys 130 135 140 Lys Arg Gly Phe Ala Phe Val Thr Phe Asp Asp His Asp Ser Val Asp 145 150 155 160 Lys Ile Val Ile Gln Lys Tyr His Thr Val Asn Gly His Asn Cys Glu 165 170 175 Val Arg Lys Ala Leu Ser Lys Gln Glu Met Ala Ser Ala Ser Ser Ser 180 185 190 Gln Arg Gly Arg Ser Gly Ser Gly Asn Phe Gly Gly Gly Arg Gly Gly 195 200 205 Gly Phe Gly Gly Asn Asp Asn Phe Gly Arg Gly Gly Asn Phe Ser Gly 210 215 220 Arg Gly Gly Phe Gly Gly Ser Arg Gly Gly Gly Gly Tyr Gly Gly Ser 225 230 235 240 Gly Asp Gly Tyr Asn Gly Phe Gly Asn Asp Gly Gly Tyr Gly Gly Gly 245 250 255 Gly Pro Gly Tyr Ser Gly Gly Ser Arg Gly Tyr Gly Ser Gly Gly Gln 260 265 270 Gly Tyr Gly Asn Gln Gly Ser Gly Tyr Gly Gly Ser Gly Ser Tyr Asp 275 280 285 Ser Tyr Asn Asn Gly Gly Gly Arg Gly Phe Gly Gly Gly Ser Gly Ser 290 295 300 Asn Phe Gly Gly Gly Gly Ser Tyr Asn Asp Phe Gly Asn Tyr Asn Asn 305 310 315 320 Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly Arg 325 330 335 Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro Arg 340 345 350 Asn Gln Gly Gly Tyr Gly Gly Ser Ser Ser Ser Ser Ser Tyr Gly Ser 355 360 365 Gly Arg Arg Phe 370 35205PRTHUMAN 35Met Thr Glu Arg Arg Val Pro Phe Ser Leu Leu Arg Gly Pro Ser Trp 1 5 10 15 Asp Pro Phe Arg Asp Trp Tyr Pro His Ser Arg Leu Phe Asp Gln Ala 20 25 30 Phe Gly Leu Pro Arg Leu Pro Glu Glu Trp Ser Gln Trp Leu Gly Gly 35 40 45 Ser Ser Trp Pro Gly Tyr Val Arg Pro Leu Pro Pro Ala Ala Ile Glu 50 55 60 Ser Pro Ala Val Ala Ala Pro Ala Tyr Ser Arg Ala Leu Ser Arg Gln 65 70 75 80 Leu Ser Ser Gly Val Ser Glu Ile Arg His Thr Ala Asp Arg Trp Arg 85 90 95 Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys 100 105 110 Thr Lys Asp Gly Val Val Glu Ile Thr Gly Lys His Glu Glu Arg Gln 115 120 125 Asp Glu His Gly Tyr Ile Ser Arg Cys Phe Thr Arg Lys Tyr Thr Leu 130 135 140 Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser Leu Ser Pro Glu 145 150 155 160 Gly Thr Leu Thr Val Glu Ala Pro Met Pro Lys Leu Ala Thr Gln Ser 165 170 175 Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser Arg Ala Gln Leu Gly 180 185 190 Gly Pro Glu Ala Ala Lys Ser Asp Glu Thr Ala Ala Lys 195 200 205 36166PRTHUMAN 36Met Ala Ser Gly Val Ala Val Ser Asp Gly Val Ile Lys Val Phe Asn 1 5 10 15 Asp Met Lys Val Arg Lys Ser Ser Thr Pro Glu Glu Val Lys Lys Arg 20 25 30 Lys Lys Ala Val Leu Phe Cys Leu Ser Glu Asp Lys Lys Asn Ile Ile 35 40 45 Leu Glu Glu Gly Lys Glu Ile Leu Val Gly Asp Val Gly Gln Thr Val 50 55 60 Asp Asp Pro Tyr Ala Thr Phe Val Lys Met Leu Pro Asp Lys Asp Cys 65 70 75 80 Arg Tyr Ala Leu Tyr Asp Ala Thr Tyr Glu Thr Lys Glu Ser Lys Lys 85 90 95 Glu Asp Leu Val Phe Ile Phe Trp Ala Pro Glu Ser Ala Pro Leu Lys 100 105 110 Ser Lys Met Ile Tyr Ala Ser Ser Lys Asp Ala Ile Lys Lys Lys Leu 115 120 125 Thr Gly Ile Lys His Glu Leu Gln Ala Asn Cys Tyr Glu Glu Val Lys 130 135 140 Asp Arg Cys Thr Leu Ala Glu Lys Leu Gly Gly Ser Ala Val Ile Ser 145 150 155 160 Leu Glu Gly Lys Pro Leu 165 37335PRTHUMAN 37Met Gly Lys Val Lys Val Gly Val Asn Gly Phe Gly Arg Ile Gly Arg 1 5 10 15 Leu Val Thr Arg Ala Ala Phe Asn Ser Gly Lys Val Asp Ile Val Ala 20 25 30 Ile Asn Asp Pro Phe Ile Asp Leu Asn Tyr Met Val Tyr Met Phe Gln 35 40 45 Tyr Asp Ser Thr His Gly Lys Phe His Gly Thr Val Lys Ala Glu Asn 50 55 60 Gly Lys Leu Val Ile Asn Gly Asn Pro Ile Thr Ile Phe Gln Glu Arg 65 70 75 80 Asp Pro Ser Lys Ile Lys Trp Gly Asp Ala Gly Ala Glu Tyr Val Val 85 90 95 Glu Ser Thr Gly Val Phe Thr Thr Met Glu Lys Ala Gly Ala His Leu 100 105 110 Gln Gly Gly Ala Lys Arg Val Ile Ile Ser Ala Pro Ser Ala Asp Ala 115 120 125 Pro Met Phe Val Met Gly Val Asn His Glu Lys Tyr Asp Asn Ser Leu 130 135 140 Lys Ile Ile Ser Asn Ala Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu 145 150 155 160 Ala Lys Val Ile His Asp Asn Phe Gly Ile Val Glu Gly Leu Met Thr 165 170 175 Thr Val His Ala Ile Thr Ala Thr Gln Lys Thr Val Asp Gly Pro Ser 180 185 190 Gly Lys Leu Trp Arg Asp Gly Arg Gly Ala Leu Gln Asn Ile Ile Pro 195 200 205 Ala Ser Thr Gly Ala Ala Lys Ala Val Gly Lys Val Ile Pro Glu Leu 210 215 220 Asp Gly Lys Leu Thr Gly Met Ala Phe Arg Val Pro Thr Ala Asn Val 225 230 235 240 Ser Val Val Asp Leu Thr Cys Arg Leu Glu Lys Pro Ala Lys Tyr Asp 245 250 255 Asp Ile Lys Lys Val Val Lys Gln Ala Ser Glu Gly Pro Leu Lys Gly 260 265 270 Ile Leu Gly Tyr Thr Glu His Gln Val Val Ser Ser Asp Phe Asn Ser 275 280 285 Asp Thr His Ser Ser Thr Phe Asp Ala Gly Ala Gly Ile Ala Leu Asn 290 295 300 Asp His Phe Val Lys Leu Ile Ser Trp Tyr Asp Asn Glu Phe Gly Tyr 305 310 315 320 Ser Asn Arg Val Val Asp Leu Met Ala His Met Ala Ser Lys Glu 325 330 335 38152PRTHUMAN 38 Met Ala Asn Leu Glu Arg Thr Phe Ile Ala Ile Lys Pro Asp Gly Val 1 5 10 15 Gln Arg Gly Leu Val Gly Glu Ile Ile Lys Arg Phe Glu Gln Lys Gly 20 25 30 Phe Arg Leu Val Ala Met Lys Phe Leu Arg Ala Ser Glu Glu His Leu 35 40 45 Lys Gln His Tyr Ile Asp Leu Lys Asp Arg Pro Phe Phe Pro Gly Leu 50 55 60 Val Lys Tyr Met Asn Ser Gly Pro Val Val Ala Met Val Trp Glu Gly 65 70 75 80 Leu Asn Val Val Lys Thr Gly Arg Val Met Leu Gly Glu Thr Asn Pro 85 90 95 Ala Asp Ser Lys Pro Gly Thr Ile Arg Gly Asp Phe Cys Ile Gln Val 100 105 110 Gly Arg Asn Ile Ile His Gly Ser Asp Ser Val Lys Ser Ala Glu Lys 115 120 125 Glu Ile Ser Leu Trp Phe Lys Pro Glu Glu Leu Val Asp Tyr Lys Ser 130 135 140 Cys Ala His Asp Trp Val Tyr Glu 145 150 39858PRTHUMAN 39Met Val Asn Phe Thr Val Asp Gln Ile Arg Ala Ile Met Asp Lys Lys 1 5 10

15 Ala Asn Ile Arg Asn Met Ser Val Ile Ala His Val Asp His Gly Lys 20 25 30 Ser Thr Leu Thr Asp Ser Leu Val Cys Lys Ala Gly Ile Ile Ala Ser 35 40 45 Ala Arg Ala Gly Glu Thr Arg Phe Thr Asp Thr Arg Lys Asp Glu Gln 50 55 60 Glu Arg Cys Ile Thr Ile Lys Ser Thr Ala Ile Ser Leu Phe Tyr Glu 65 70 75 80 Leu Ser Glu Asn Asp Leu Asn Phe Ile Lys Gln Ser Lys Asp Gly Ala 85 90 95 Gly Phe Leu Ile Asn Leu Ile Asp Ser Pro Gly His Val Asp Phe Ser 100 105 110 Ser Glu Val Thr Ala Ala Leu Arg Val Thr Asp Gly Ala Leu Val Val 115 120 125 Val Asp Cys Val Ser Gly Val Cys Val Gln Thr Glu Thr Val Leu Arg 130 135 140 Gln Ala Ile Ala Glu Arg Ile Lys Pro Val Leu Met Met Asn Lys Met 145 150 155 160 Asp Arg Ala Leu Leu Glu Leu Gln Leu Glu Pro Glu Glu Leu Tyr Gln 165 170 175 Thr Phe Gln Arg Ile Val Glu Asn Val Asn Val Ile Ile Ser Thr Tyr 180 185 190 Gly Glu Gly Glu Ser Gly Pro Met Gly Asn Ile Met Ile Asp Pro Val 195 200 205 Leu Gly Thr Val Gly Phe Gly Ser Gly Leu His Gly Trp Ala Phe Thr 210 215 220 Leu Lys Gln Phe Ala Glu Met Tyr Val Ala Lys Phe Ala Ala Lys Gly 225 230 235 240 Glu Gly Gln Leu Gly Pro Ala Glu Arg Ala Lys Lys Val Glu Asp Met 245 250 255 Met Lys Lys Leu Trp Gly Asp Arg Tyr Phe Asp Pro Ala Asn Gly Lys 260 265 270 Phe Ser Lys Ser Ala Thr Ser Pro Glu Gly Lys Lys Leu Pro Arg Thr 275 280 285 Phe Cys Gln Leu Ile Leu Asp Pro Ile Phe Lys Val Phe Asp Ala Ile 290 295 300 Met Asn Phe Lys Lys Glu Glu Thr Ala Lys Leu Ile Glu Lys Leu Asp 305 310 315 320 Ile Lys Leu Asp Ser Glu Asp Lys Asp Lys Glu Gly Lys Pro Leu Leu 325 330 335 Lys Ala Val Met Arg Arg Trp Leu Pro Ala Gly Asp Ala Leu Leu Gln 340 345 350 Met Ile Thr Ile His Leu Pro Ser Pro Val Thr Ala Gln Lys Tyr Arg 355 360 365 Cys Glu Leu Leu Tyr Glu Gly Pro Pro Asp Asp Glu Ala Ala Met Gly 370 375 380 Ile Lys Ser Cys Asp Pro Lys Gly Pro Leu Met Met Tyr Ile Ser Lys 385 390 395 400 Met Val Pro Thr Ser Asp Lys Gly Arg Phe Tyr Ala Phe Gly Arg Val 405 410 415 Phe Ser Gly Leu Val Ser Thr Gly Leu Lys Val Arg Ile Met Gly Pro 420 425 430 Asn Tyr Thr Pro Gly Lys Lys Glu Asp Leu Tyr Leu Lys Pro Ile Gln 435 440 445 Arg Thr Ile Leu Met Met Gly Arg Tyr Val Glu Pro Ile Glu Asp Val 450 455 460 Pro Cys Gly Asn Ile Val Gly Leu Val Gly Val Asp Gln Phe Leu Val 465 470 475 480 Lys Thr Gly Thr Ile Thr Thr Phe Glu His Ala His Asn Met Arg Val 485 490 495 Met Lys Phe Ser Val Ser Pro Val Val Arg Val Ala Val Glu Ala Lys 500 505 510 Asn Pro Ala Asp Leu Pro Lys Leu Val Glu Gly Leu Lys Arg Leu Ala 515 520 525 Lys Ser Asp Pro Met Val Gln Cys Ile Ile Glu Glu Ser Gly Glu His 530 535 540 Ile Ile Ala Gly Ala Gly Glu Leu His Leu Glu Ile Cys Leu Lys Asp 545 550 555 560 Leu Glu Glu Asp His Ala Cys Ile Pro Ile Lys Lys Ser Asp Pro Val 565 570 575 Val Ser Tyr Arg Glu Thr Val Ser Glu Glu Ser Asn Val Leu Cys Leu 580 585 590 Ser Lys Ser Pro Asn Lys His Asn Arg Leu Tyr Met Lys Ala Arg Pro 595 600 605 Phe Pro Asp Gly Leu Ala Glu Asp Ile Asp Lys Gly Glu Val Ser Ala 610 615 620 Arg Gln Glu Leu Lys Gln Arg Ala Arg Tyr Leu Ala Glu Lys Tyr Glu 625 630 635 640 Trp Asp Val Ala Glu Ala Arg Lys Ile Trp Cys Phe Gly Pro Asp Gly 645 650 655 Thr Gly Pro Asn Ile Leu Thr Asp Ile Thr Lys Gly Val Gln Tyr Leu 660 665 670 Asn Glu Ile Lys Asp Ser Val Val Ala Gly Phe Gln Trp Ala Thr Lys 675 680 685 Glu Gly Ala Leu Cys Glu Glu Asn Met Arg Gly Val Arg Phe Asp Val 690 695 700 His Asp Val Thr Leu His Ala Asp Ala Ile His Arg Gly Gly Gly Gln 705 710 715 720 Ile Ile Pro Thr Ala Arg Arg Cys Leu Tyr Ala Ser Val Leu Thr Ala 725 730 735 Gln Pro Arg Leu Met Glu Pro Ile Tyr Leu Val Glu Ile Gln Cys Pro 740 745 750 Glu Gln Val Val Gly Gly Ile Tyr Gly Val Leu Asn Arg Lys Arg Gly 755 760 765 His Val Phe Glu Glu Ser Gln Val Ala Gly Thr Pro Met Phe Val Val 770 775 780 Lys Ala Tyr Leu Pro Val Asn Glu Ser Phe Gly Phe Thr Ala Asp Leu 785 790 795 800 Arg Ser Asn Thr Gly Gly Gln Ala Phe Pro Gln Cys Val Phe Asp His 805 810 815 Trp Gln Ile Leu Pro Gly Asp Pro Phe Asp Asn Ser Ser Arg Pro Ser 820 825 830 Gln Val Val Ala Glu Thr Arg Lys Arg Lys Gly Leu Lys Glu Gly Ile 835 840 845 Pro Ala Leu Asp Asn Phe Leu Asp Lys Leu 850 855 40187PRTHUMAN 40Met Pro Val Asp Leu Ser Lys Trp Ser Gly Pro Leu Ser Leu Gln Glu 1 5 10 15 Val Asp Glu Gln Pro Gln His Pro Leu His Val Thr Tyr Ala Gly Ala 20 25 30 Ala Val Asp Glu Leu Gly Lys Val Leu Thr Pro Thr Gln Val Lys Asn 35 40 45 Arg Pro Thr Ser Ile Ser Trp Asp Gly Leu Asp Ser Gly Lys Leu Tyr 50 55 60 Thr Leu Val Leu Thr Asp Pro Asp Ala Pro Ser Arg Lys Asp Pro Lys 65 70 75 80 Tyr Arg Glu Trp His His Phe Leu Val Val Asn Met Lys Gly Asn Asp 85 90 95 Ile Ser Ser Gly Thr Val Leu Ser Asp Tyr Val Gly Ser Gly Pro Pro 100 105 110 Lys Gly Thr Gly Leu His Arg Tyr Val Trp Leu Val Tyr Glu Gln Asp 115 120 125 Arg Pro Leu Lys Cys Asp Glu Pro Ile Leu Ser Asn Arg Ser Gly Asp 130 135 140 His Arg Gly Lys Phe Lys Val Ala Ser Phe Arg Lys Lys Tyr Glu Leu 145 150 155 160 Arg Ala Pro Val Ala Gly Thr Cys Tyr Gln Ala Glu Trp Asp Asp Tyr 165 170 175 Val Pro Lys Leu Tyr Glu Gln Leu Ser Gly Lys 180 185 41149PRTHUMAN 41Met Ala Asp Gln Leu Thr Glu Glu Gln Val Thr Glu Phe Lys Glu Ala 1 5 10 15 Phe Ser Leu Phe Asp Lys Asp Gly Asp Gly Cys Ile Thr Thr Arg Glu 20 25 30 Leu Gly Thr Val Met Arg Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu 35 40 45 Leu Arg Asp Met Met Ser Glu Ile Asp Arg Asp Gly Asn Gly Thr Val 50 55 60 Asp Phe Pro Glu Phe Leu Gly Met Met Ala Arg Lys Met Lys Asp Thr 65 70 75 80 Asp Asn Glu Glu Glu Ile Arg Glu Ala Phe Arg Val Phe Asp Lys Asp 85 90 95 Gly Asn Gly Phe Val Ser Ala Ala Glu Leu Arg His Val Met Thr Arg 100 105 110 Leu Gly Glu Lys Leu Ser Asp Glu Glu Val Asp Glu Met Ile Arg Ala 115 120 125 Ala Asp Thr Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val Arg 130 135 140 Val Leu Val Ser Lys 145 4219PRTHUMAN 42Ala Phe Leu Ala Ser Pro Glu Tyr Val Asn Leu Pro Ile Asn Gly Asn 1 5 10 15 Gly Lys Gln 4319PRTHUMAN 43Asp Gln Gln Glu Ala Ala Leu Val Asp Met Val Asn Asp Gly Val Glu 1 5 10 15 Asp Leu Arg 4416PRTHUMAN 44Phe Gln Asp Gly Asp Leu Thr Leu Tyr Gln Ser Asn Thr Ile Leu Arg 1 5 10 15 4511PRTHUMAN 45Met Leu Leu Ala Asp Gln Gly Gln Ser Trp Lys 1 5 10 4611PRTHUMAN 46Pro Pro Tyr Thr Val Val Tyr Phe Pro Val Arg 1 5 10 4726PRTHUMAN 47Thr Leu Gly Leu Tyr Gly Lys Asp Gln Gln Glu Ala Ala Leu Val Asp 1 5 10 15 Met Val Asn Asp Gly Val Glu Asp Leu Arg 20 25 489PRTHUMAN 48Asp Gln Leu Ile Tyr Asn Leu Leu Lys 1 5 498PRTHUMAN 49Phe Ile Ile Pro Asn Val Val Lys 1 5 5014PRTHUMAN 50Gly Glu Met Met Asp Leu Gln His Gly Ser Leu Phe Leu Arg 1 5 10 519PRTHUMAN 51Leu Val Ile Ile Thr Ala Gly Ala Arg 1 5 5211PRTHUMAN 52Gln Val Val Glu Ser Ala Tyr Glu Val Ile Lys 1 5 10 5312PRTHUMAN 53Val Ile Gly Ser Gly Cys Asn Leu Asp Ser Ala Arg 1 5 10 5410PRTHUMAN 54Val Thr Leu Thr Ser Glu Glu Glu Ala Arg 1 5 10 558PRTHUMAN 55Ala Val Leu Thr Ile Asp Glu Lys 1 5 5616PRTHUMAN 56Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val Lys 1 5 10 15 578PRTHUMAN 57Phe Leu Glu Asn Glu Asp Arg Arg 1 5 5815PRTHUMAN 58Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met 1 5 10 15 5922PRTHUMAN 59Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met Ser 1 5 10 15 Ile Pro Pro Glu Val Lys 20 6015PRTHUMAN 60Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 1 5 10 15 6110PRTHUMAN 61Ser Val Leu Gly Gln Leu Gly Ile Thr Lys 1 5 10 6215PRTHUMAN 62Thr Asp Thr Ser His His Asp Gln Asp His Pro Thr Phe Asn Lys 1 5 10 15 6318PRTHUMAN 63Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro 1 5 10 15 Leu Lys 6422PRTHUMAN 64Cys Gly Ala Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr Gln His 1 5 10 15 Ile Ala Asp Gln Val Arg 20 6521PRTHUMAN 65Gly Ala Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr Gln His Ile 1 5 10 15 Ala Asp Gln Val Arg 20 6624PRTHUMAN 66Met Met Cys Gly Ala Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr 1 5 10 15 Gln His Ile Ala Asp Gln Val Arg 20 6719PRTHUMAN 67Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr Gln His Ile Ala Asp 1 5 10 15 Gln Val Arg 6812PRTHUMAN 68Ser Gln Val Val Ala Gly Thr Asn Tyr Phe Ile Lys 1 5 10 6912PRTHUMAN 69Val His Val Gly Asp Glu Asp Phe Val His Leu Arg 1 5 10 708PRTHUMAN 70Ala Leu Tyr Glu Ala Gly Glu Arg 1 5 7111PRTHUMAN 71Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe Arg 1 5 10 7216PRTHUMAN 72Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg 1 5 10 15 7314PRTHUMAN 73Gly Thr Asp Val Asn Val Phe Asn Thr Ile Leu Thr Thr Arg 1 5 10 7413PRTHUMAN 74Gly Val Asp Glu Ala Thr Ile Ile Asp Ile Leu Thr Lys 1 5 10 7515PRTHUMAN 75Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile Leu Thr Thr Arg 1 5 10 15 768PRTHUMAN 76Asn Ala Leu Leu Ser Leu Ala Lys 1 5 7717PRTHUMAN 77Gln Ala Trp Phe Ile Glu Asn Glu Glu Gln Glu Tyr Val Gln Thr Val 1 5 10 15 Lys 7811PRTHUMAN 78Thr Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg 1 5 10 7913PRTHUMAN 79Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys 1 5 10 8014PRTHUMAN 80Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys Lys 1 5 10 8110PRTHUMAN 81Glu Val Val Glu Glu Ala Glu Asn Gly Arg 1 5 10 8211PRTHUMAN 82Lys Glu Val Val Glu Glu Ala Glu Asn Gly Arg 1 5 10 8314PRTHUMAN 83Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys 1 5 10 8413PRTHUMAN 84Asp Phe Glu Pro Ser Leu Gly Pro Val Cys Pro Phe Arg 1 5 10 8515PRTHUMAN 85Asp Leu Pro Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys 1 5 10 15 868PRTHUMAN 86Glu Leu His Leu Asp Asn Asn Lys 1 5 8711PRTHUMAN 87Val Ser Pro Gly Ala Phe Thr Pro Leu Val Lys 1 5 10 889PRTHUMAN 88Ala Asp Leu Ser Glu Ala Ala Asn Arg 1 5 8916PRTHUMAN 89Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp Leu Glu 1 5 10 15 909PRTHUMAN 90Asp Asn Leu Ala Glu Asp Ile Met Arg 1 5 9111PRTHUMAN 91Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg 1 5 10 9210PRTHUMAN 92Glu Lys Leu Gln Glu Glu Met Leu Gln Arg 1 5 10 9319PRTHUMAN 93Glu Met Glu Glu Asn Phe Ala Val Glu Ala Ala Asn Tyr Gln Asp Thr 1 5 10 15 Ile Gly Arg 9415PRTHUMAN 94Glu Thr Asn Leu Asp Ser Leu Pro Leu Val Asp Thr His Ser Lys 1 5 10 15 959PRTHUMAN 95Glu Tyr Gln Asp Leu Leu Asn Val Lys 1 5 9610PRTHUMAN 96Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 1 5 10 9710PRTHUMAN 97Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys 1 5 10 9814PRTHUMAN 98Ile Ser Leu Pro Leu Pro Asn Phe Ser Ser Leu Asn Leu Arg 1 5 10 999PRTHUMAN 99Lys Leu Leu Glu Gly Glu Glu Ser Arg 1 5 10010PRTHUMAN 100Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg 1 5 10 1018PRTHUMAN 101Leu Leu Glu Gly Glu Glu Ser Arg 1 5 10219PRTHUMAN 102Leu Leu Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr 1 5 10 15 Glu Phe Lys 1039PRTHUMAN 103Leu Gln Asp Glu Ile Gln Asn Met Lys 1 5 10414PRTHUMAN 104Leu Gln Asp Glu Ile Gln Asn Met Lys Glu Glu Met Ala Arg 1 5 10 1058PRTHUMAN 105Leu Gln Glu Glu Met Leu Gln Arg 1 5 10613PRTHUMAN 106Asn Leu Asp Ser Leu Pro Leu Val Asp Thr His Ser Lys 1 5 10 10710PRTHUMAN 107Asn Leu Gln Glu Ala Glu Glu Trp Tyr Lys 1 5 10 10810PRTHUMAN 108Gln Asp Val Asp Asn Ala Ser Leu Ala Arg 1 5 10 1099PRTHUMAN 109Gln Gln Tyr Glu Ser Val Ala Ala Lys 1 5 1109PRTHUMAN 110Gln Val Asp Gln Leu Thr Asn Asp Lys 1 5 11113PRTHUMAN 111Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys 1 5 10 11210PRTHUMAN 112Arg Gln Val Asp Gln Leu Thr Asn Asp Lys 1 5 10 11313PRTHUMAN 113Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp Arg 1 5 10 1149PRTHUMAN 114Val Glu Leu Gln Glu Leu Asn Asp Arg 1 5 11514PRTHUMAN 115Val Glu Val Glu Arg Asp Asn Leu Ala Glu Asp Ile Met Arg 1 5 10 11624PRTHUMAN 116Ala Tyr Glu Glu Asp Tyr Gly Ser Ser Leu Glu Glu Asp Ile Gln Ala 1 5 10 15

Asp Thr Ser Gly Tyr Leu Glu Arg 20 11711PRTHUMAN 117Glu Gly Val Ile Ile Glu Ile Leu Ala Ser Arg 1 5 10 11815PRTHUMAN 118Gly Ile Gly Thr Asn Glu Gln Ala Ile Ile Asp Val Leu Thr Lys 1 5 10 15 11911PRTHUMAN 119Asn Ala Leu Leu Ser Leu Val Gly Ser Asp Pro 1 5 10 12016PRTHUMAN 120Ser Ser Ser His Phe Asn Pro Asp Pro Asp Ala Glu Thr Leu Tyr Lys 1 5 10 15 12115PRTHUMAN 121Thr Leu Ser Ser Met Ile Met Glu Asp Thr Ser Gly Asp Tyr Lys 1 5 10 15 12225PRTHUMAN 122Ala Glu Gly Ser Asp Val Ala Asn Ala Val Leu Asp Gly Ala Asp Cys 1 5 10 15 Ile Met Leu Ser Gly Glu Thr Ala Lys 20 25 1238PRTHUMAN 123Ala Pro Ile Ile Ala Val Thr Arg 1 5 12416PRTHUMAN 124Glu Ala Glu Ala Ala Ile Tyr His Leu Gln Leu Phe Glu Glu Leu Arg 1 5 10 15 12517PRTHUMAN 125Glu Ala Glu Ala Ala Ile Tyr His Leu Gln Leu Phe Glu Glu Leu Arg 1 5 10 15 Arg 12615PRTHUMAN 126Phe Asp Glu Ile Leu Glu Ala Ser Asp Gly Ile Met Val Ala Arg 1 5 10 15 12718PRTHUMAN 127Gly Ala Asp Phe Leu Val Thr Glu Val Glu Asn Gly Gly Ser Leu Gly 1 5 10 15 Ser Lys 12811PRTHUMAN 128Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu Lys 1 5 10 1299PRTHUMAN 129Gly Asp Tyr Pro Leu Glu Ala Val Arg 1 5 13010PRTHUMAN 130Gly Ser Gly Thr Ala Glu Val Glu Leu Lys 1 5 10 13117PRTHUMAN 131Gly Val Asn Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu 1 5 10 15 Lys 13213PRTHUMAN 132Ile Tyr Val Asp Asp Gly Leu Ile Ser Leu Gln Val Lys 1 5 10 1339PRTHUMAN 133Lys Ala Ser Asp Val His Glu Val Arg 1 5 13422PRTHUMAN 134Leu Ala Pro Ile Thr Ser Asp Pro Thr Glu Ala Thr Ala Val Gly Ala 1 5 10 15 Val Glu Ala Ser Phe Lys 20 13511PRTHUMAN 135Leu Asp Ile Asp Ser Pro Pro Ile Thr Ala Arg 1 5 10 13613PRTHUMAN 136Asn Thr Gly Ile Ile Cys Thr Ile Gly Pro Ala Ser Arg 1 5 10 13716PRTHUMAN 137Arg Phe Asp Glu Ile Leu Glu Ala Ser Asp Gly Ile Met Val Ala Arg 1 5 10 15 13823PRTHUMAN 138Thr Ala Thr Glu Ser Phe Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val 1 5 10 15 Ala Val Ala Leu Asp Thr Lys 20 1399PRTHUMAN 139Val Asn Phe Ala Met Asn Val Gly Lys 1 5 1409PRTHUMAN 140Phe Ala Phe Gln Ala Glu Val Asn Arg 1 5 14114PRTHUMAN 141Gly Val Val Asp Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 1 5 10 1429PRTHUMAN 142Ile Tyr Phe Met Ala Gly Ser Ser Arg 1 5 14310PRTHUMAN 143Leu Gly Val Ile Glu Asp His Ser Asn Arg 1 5 10 14419PRTHUMAN 144Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu 1 5 10 15 Thr Val Lys 14514PRTHUMAN 145Asn Leu Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg 1 5 10 1468PRTHUMAN 146Ala Leu Glu Leu Glu Gln Glu Arg 1 5 14710PRTHUMAN 147Ala Leu Thr Ser Glu Leu Ala Asn Ala Arg 1 5 10 14810PRTHUMAN 148Ala Pro Asp Phe Val Phe Tyr Ala Pro Arg 1 5 10 14910PRTHUMAN 149Ala Gln Gln Glu Leu Glu Glu Gln Thr Arg 1 5 10 1508PRTHUMAN 150Glu Ala Leu Leu Gln Ala Ser Arg 1 5 1518PRTHUMAN 151Glu Lys Glu Glu Leu Met Glu Arg 1 5 15217PRTHUMAN 152Phe Tyr Pro Glu Asp Val Ser Glu Glu Leu Ile Gln Asp Ile Thr Gln 1 5 10 15 Arg 1539PRTHUMAN 153Ile Gly Phe Pro Trp Ser Glu Ile Arg 1 5 1549PRTHUMAN 154Ile Gln Val Trp His Glu Glu His Arg 1 5 1558PRTHUMAN 155Ile Ser Gln Leu Glu Met Ala Arg 1 5 15611PRTHUMAN 156Lys Ala Gln Gln Glu Leu Glu Glu Gln Thr Arg 1 5 10 15716PRTHUMAN 157Lys Thr Gln Glu Gln Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg 1 5 10 15 1588PRTHUMAN 158Gln Leu Phe Asp Gln Val Val Lys 1 5 15927PRTHUMAN 159Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu Gln 1 5 10 15 Asp Glu Gln Asp Glu Asn Gly Ala Glu Ala Ser 20 25 16012PRTHUMAN 160Thr Ala Asn Asp Met Ile His Ala Glu Asn Met Arg 1 5 10 16115PRTHUMAN 161Thr Gln Glu Gln Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg 1 5 10 15 16219PRTHUMAN 162Val Thr Thr Met Asp Ala Glu Leu Glu Phe Ala Ile Gln Pro Asn Thr 1 5 10 15 Thr Gly Lys 1639PRTHUMAN 163Glu Leu Pro Ser Phe Val Gly Glu Lys 1 5 16416PRTHUMAN 164Glu Leu Pro Ser Phe Val Gly Glu Lys Val Asp Glu Glu Gly Leu Lys 1 5 10 15 16511PRTHUMAN 165Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys 1 5 10 16612PRTHUMAN 166Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys Lys 1 5 10 16711PRTHUMAN 167Ser Ile Ile Gly Met Ile Asp Met Phe His Lys 1 5 10 16812PRTHUMAN 168Ala Asp Leu His Thr Leu Ser Glu Asp Ser Tyr Lys 1 5 10 16924PRTHUMAN 169Asp Asn Leu Thr Leu Trp Thr Ala Asp Asn Ala Gly Glu Glu Gly Gly 1 5 10 15 Glu Ala Pro Gln Glu Pro Gln Ser 20 17010PRTHUMAN 170Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 10 1718PRTHUMAN 171Glu Glu Lys Gly Pro Glu Val Arg 1 5 1729PRTHUMAN 172Glu Met Pro Pro Thr Asn Pro Ile Arg 1 5 17314PRTHUMAN 173Gly Ala Val Glu Lys Gly Glu Glu Leu Ser Cys Glu Glu Arg 1 5 10 1749PRTHUMAN 174Gly Glu Glu Leu Ser Cys Glu Glu Arg 1 5 1757PRTHUMAN 175Leu Ala Glu Gln Ala Glu Arg 1 5 1768PRTHUMAN 176Asn Leu Leu Ser Val Ala Tyr Lys 1 5 17711PRTHUMAN 177Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys 1 5 10 17812PRTHUMAN 178Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys Lys 1 5 10 17914PRTHUMAN 179Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu Val Arg 1 5 10 18019PRTHUMAN 180Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu Ser Glu Asp 1 5 10 15 Ser Tyr Lys 1818PRTHUMAN 181Val Leu Ser Ser Ile Glu Gln Lys 1 5 1829PRTHUMAN 182Tyr Glu Asp Met Ala Ala Phe Met Lys 1 5 18311PRTHUMAN 183Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys 1 5 10 1849PRTHUMAN 184Ile Ser Ser Pro Thr Glu Thr Glu Arg 1 5 18516PRTHUMAN 185Thr Glu Phe Leu Ser Phe Met Asn Thr Glu Leu Ala Ala Phe Thr Lys 1 5 10 15 18617PRTHUMAN 186Gly Asp Thr Ala Asn Glu Ile Gly Gln Val Leu His Phe Glu Asn Val 1 5 10 15 Lys 18719PRTHUMAN 187Val Cys Leu Glu Ile Thr Glu Asp Gly Gly Asp Ser Ile Glu Val Pro 1 5 10 15 Gly Ala Arg 1888PRTHUMAN 188Phe Asn Ala Leu Gln Tyr Leu Arg 1 5 18910PRTHUMAN 189Ile Ser Asn Ile Pro Asp Glu Tyr Phe Lys 1 5 10 19021PRTHUMAN 190Asn Ile Pro Thr Val Asn Glu Asn Leu Glu Asn Tyr Tyr Leu Glu Val 1 5 10 15 Asn Gln Leu Glu Lys 20 19110PRTHUMAN 191Asn Asn Gln Ile Asp His Ile Asp Glu Lys 1 5 10 19211PRTHUMAN 192Ser Leu Glu Asp Leu Gln Leu Thr His Asn Lys 1 5 10 19314PRTHUMAN 193Ser Leu Glu Tyr Leu Asp Leu Ser Phe Asn Gln Ile Ala Arg 1 5 10 19413PRTHUMAN 194Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met Cys Lys 1 5 10 19510PRTHUMAN 195Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg 1 5 10 19619PRTHUMAN 196Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala Ser Ser Ser Ser 1 5 10 15 Leu Glu Lys 1979PRTHUMAN 197Ala Val Phe Pro Ser Ile Val Gly Arg 1 5 19811PRTHUMAN 198Ala Val Phe Pro Ser Ile Val Gly Arg Pro Arg 1 5 10 19917PRTHUMAN 199Asp Asp Asp Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met Cys 1 5 10 15 Lys 20010PRTHUMAN 200Asp Glu Ser Gly Pro Ser Ile Val His Arg 1 5 10 2018PRTHUMAN 201Asp Leu Thr Asp Tyr Leu Met Lys 1 5 20221PRTHUMAN 202Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr Met Tyr Pro 1 5 10 15 Gly Ile Ala Asp Arg 20 20311PRTHUMAN 203Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys 1 5 10 20412PRTHUMAN 204Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg 1 5 10 20511PRTHUMAN 205Glu Ile Thr Ala Leu Ala Pro Ser Thr Met Lys 1 5 10 2069PRTHUMAN 206Gly Phe Ala Gly Asp Asp Ala Pro Arg 1 5 20710PRTHUMAN 207Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg 1 5 10 20811PRTHUMAN 208His Gln Gly Val Met Val Gly Met Gly Gln Lys 1 5 10 20911PRTHUMAN 209Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg 1 5 10 21022PRTHUMAN 210Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr Met Tyr 1 5 10 15 Pro Gly Ile Ala Asp Arg 20 21123PRTHUMAN 211Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala 1 5 10 15 Ser Ser Ser Ser Leu Glu Lys 20 21210PRTHUMAN 212Leu Leu Thr Glu Ala Pro Leu Asn Pro Lys 1 5 10 21313PRTHUMAN 213Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg 1 5 10 21413PRTHUMAN 214Ser Gly Gly Thr Thr Met Tyr Pro Gly Ile Ala Asp Arg 1 5 10 21516PRTHUMAN 215Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg 1 5 10 15 2169PRTHUMAN 216Thr Ala Leu Ala Pro Ser Thr Met Lys 1 5 2178PRTHUMAN 217Thr Glu Ala Pro Leu Asn Pro Lys 1 5 21822PRTHUMAN 218Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr 20 21926PRTHUMAN 219Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His 20 25 22029PRTHUMAN 220Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 20 25 22120PRTHUMAN 221Val Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala Ser Ser Ser 1 5 10 15 Ser Leu Glu Lys 20 2228PRTHUMAN 222Val Ala Pro Glu Glu His Pro Val 1 5 2239PRTHUMAN 223Val Ala Pro Glu Glu His Pro Val Leu 1 5 22410PRTHUMAN 224Val Ala Pro Glu Glu His Pro Val Leu Leu 1 5 10 22518PRTHUMAN 225Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn 1 5 10 15 Pro Lys 22615PRTHUMAN 226Val Leu Ser Gly Gly Thr Thr Met Tyr Pro Gly Ile Ala Asp Arg 1 5 10 15 22716PRTHUMAN 227Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp Asp Met Glu Lys 1 5 10 15 22810PRTHUMAN 228Asp Ala Val Thr Tyr Thr Glu His Ala Lys 1 5 10 22912PRTHUMAN 229Asp Asn Ile Gln Gly Ile Thr Lys Pro Ala Ile Arg 1 5 10 23010PRTHUMAN 230Ile Ser Gly Leu Ile Tyr Glu Glu Thr Arg 1 5 10 23112PRTHUMAN 231Thr Val Thr Ala Met Asp Val Val Tyr Ala Leu Lys 1 5 10 2328PRTHUMAN 232Val Phe Leu Glu Asn Val Ile Arg 1 5 23316PRTHUMAN 233Ala Phe Asp Gln Asp Gly Asp Gly His Ile Thr Val Asp Glu Leu Arg 1 5 10 15 23423PRTHUMAN 234Ala Phe Ser Ala Val Asp Thr Asp Gly Asn Gly Thr Ile Asn Ala Gln 1 5 10 15 Glu Leu Gly Ala Ala Leu Lys 20 23511PRTHUMAN 235Ala Gly Leu Glu Asp Leu Gln Val Ala Phe Arg 1 5 10 23619PRTHUMAN 236Ala Met Ala Gly Leu Gly Gln Pro Leu Pro Gln Glu Glu Leu Asp Ala 1 5 10 15 Met Ile Arg 2378PRTHUMAN 237Asn Leu Ser Glu Ala Gln Leu Arg 1 5 2388PRTHUMAN 238Val Asn Tyr Glu Glu Phe Ala Arg 1 5 23911PRTHUMAN 239Glu Lys Leu Glu Ala Thr Ile Asn Glu Leu Val 1 5 10 24013PRTHUMAN 240Thr Ala Phe Gln Glu Ala Leu Asp Ala Ala Gly Asp Lys 1 5 10 2419PRTHUMAN 241Val Gly Glu Phe Ser Gly Ala Asn Lys 1 5 24211PRTHUMAN 242Glu Gly Met Asn Ile Val Glu Ala Met Glu Arg 1 5 10 2439PRTHUMAN 243Phe Glu Asp Glu Asn Phe Ile Leu Lys 1 5 24414PRTHUMAN 244Ile Ile Pro Gly Phe Met Cys Gln Gly Gly Asp Phe Thr Arg 1 5 10 24510PRTHUMAN 245Ile Thr Ile Ala Asp Cys Gly Gln Leu Glu 1 5 10 24611PRTHUMAN 246Lys Ile Thr Ile Ala Asp Cys Gly Gln Leu Glu 1 5 10 24715PRTHUMAN 247Ser Ile Tyr Gly Glu Lys Phe Glu Asp Glu Asn Phe Ile Leu Lys 1 5 10 15 24818PRTHUMAN 248Val Asn Pro Thr Val Phe Phe Asp Ile Ala Val Asp Gly Glu Pro Leu 1 5 10 15 Gly Arg 2499PRTHUMAN 249Val Ser Phe Glu Leu Phe Ala Asp Lys 1 5 25011PRTHUMAN 250Glu Ile Ala Gln Asp Phe Lys Thr Asp Leu Arg 1 5 10 2519PRTHUMAN 251Ser Ala Pro Ala Thr Gly Gly Val Lys 1 5 25223PRTHUMAN 252Asp Asn Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu 1 5 10 15 Ala Gly Glu Gly Gly Glu Asn 20 25310PRTHUMAN 253Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 10 2549PRTHUMAN 254Glu Met Gln Pro Thr His Pro Ile Arg 1 5 25518PRTHUMAN 255Gly Ile Val Asp Gln Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile 1 5 10 15 Ser Lys 25619PRTHUMAN 256Gly Ile Val Asp Gln Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile 1 5 10 15 Ser Lys Lys 2577PRTHUMAN 257Leu Ala Glu Gln Ala Glu Arg 1 5 2589PRTHUMAN 258Met Asp Lys Asn Glu Leu Val Gln Lys 1 5 2598PRTHUMAN 259Asn Leu Leu Ser Val Ala Tyr Lys 1 5 26014PRTHUMAN 260Ser Val Thr Glu Gln Gly Ala Glu Leu Ser Asn Glu Glu Arg 1 5 10 26119PRTHUMAN 261Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys 2628PRTHUMAN 262Val Val Ser Ser Ile Glu Gln Lys 1 5 2639PRTHUMAN 263Tyr Asp Asp Met Ala Ala Cys Met Lys 1 5 26412PRTHUMAN 264Tyr Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys 1 5 10 26513PRTHUMAN 265Glu Leu Ser Glu Leu Val Tyr Thr Asp Val Leu Asp Arg 1 5 10 2668PRTHUMAN 266Asn Trp Gln Asp Tyr Gly Val Arg 1 5 26722PRTHUMAN 267Thr Cys Leu His Tyr Leu Gly Glu Phe Gly Glu Asp Gln Ile Tyr Glu 1 5 10 15 Ala His Gln Gln Gly Arg 20 26814PRTHUMAN 268Ala Ser Ala Glu Thr Val

Asp Pro Ala Ser Leu Trp Glu Tyr 1 5 10 26918PRTHUMAN 269Asp Val Pro Trp Gly Val Asp Ser Leu Ile Thr Leu Ala Phe Gln Asp 1 5 10 15 Gln Arg 27010PRTHUMAN 270Phe Leu Ile Val Ala His Asp Asp Gly Arg 1 5 10 27110PRTHUMAN 271Lys Val Thr Gly Thr Leu Asp Ala Asn Arg 1 5 10 27214PRTHUMAN 272Tyr Leu Ala Ala Asp Lys Asp Gly Asn Val Thr Cys Glu Arg 1 5 10 27310PRTHUMAN 273Asp Asp Met Leu Cys Ala Gly Asn Thr Arg 1 5 10 27415PRTHUMAN 274Glu Gln His Leu Tyr Tyr Gln Asp Gln Leu Leu Pro Val Ser Arg 1 5 10 15 2759PRTHUMAN 275Ile Val Gly Gly Gln Glu Ala Pro Arg 1 5 27612PRTHUMAN 276Val Pro Ile Met Glu Asn His Ile Cys Asp Ala Lys 1 5 10 27712PRTHUMAN 277Tyr His Leu Gly Ala Tyr Thr Gly Asp Asp Val Arg 1 5 10 27815PRTHUMAN 278Ala Met Gly Ile Met Asn Ser Phe Val Asn Asp Ile Phe Glu Arg 1 5 10 15 2799PRTHUMAN 279Leu Leu Leu Pro Gly Glu Leu Ala Lys 1 5 28011PRTHUMAN 280Gln Val His Pro Asp Thr Gly Ile Ser Ser Lys 1 5 10 28111PRTHUMAN 281Ala Ala Glu Glu Pro Ser Lys Val Glu Glu Lys 1 5 10 28227PRTHUMAN 282Glu Ala Pro Ala Glu Gly Glu Ala Ala Glu Pro Gly Ser Pro Thr Ala 1 5 10 15 Ala Glu Gly Glu Ala Ala Ser Ala Ala Ser Ser 20 25 2839PRTHUMAN 283Gly Pro Ser Ser Val Glu Asp Ile Lys 1 5 2847PRTHUMAN 284Val Thr Leu Ala Thr Leu Lys 1 5 2858PRTHUMAN 285Ala Leu Pro Ala Pro Ile Glu Lys 1 5 2869PRTHUMAN 286Asp Tyr Phe Pro Glu Pro Val Thr Val 1 5 28714PRTHUMAN 287Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 1 5 10 28822PRTHUMAN 288Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 1 5 10 15 Pro Glu Asn Asn Tyr Lys 20 28912PRTHUMAN 289Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 29010PRTHUMAN 290Asn Gln Val Ser Leu Thr Cys Leu Val Lys 1 5 10 29112PRTHUMAN 291Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys 1 5 10 29214PRTHUMAN 292Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 1 5 10 29326PRTHUMAN 293Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 20 25 29419PRTHUMAN 294Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 1 5 10 15 Glu Val Lys 29517PRTHUMAN 295Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 1 5 10 15 Lys 29616PRTHUMAN 296Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 1 5 10 15 2978PRTHUMAN 297Ala Val Leu Thr Ile Asp Glu Lys 1 5 29816PRTHUMAN 298Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val Lys 1 5 10 15 29910PRTHUMAN 299Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys 1 5 10 30021PRTHUMAN 300Gly Ala Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr Gln His Ile 1 5 10 15 Ala Asp Gln Val Arg 20 30124PRTHUMAN 301Met Met Cys Gly Ala Pro Ser Ala Thr Gln Pro Ala Thr Ala Glu Thr 1 5 10 15 Gln His Ile Ala Asp Gln Val Arg 20 30212PRTHUMAN 302Ser Gln Val Val Ala Gly Thr Asn Tyr Phe Ile Lys 1 5 10 30321PRTHUMAN 303Val Phe Gln Ser Leu Pro His Glu Asn Lys Pro Leu Thr Leu Ser Asn 1 5 10 15 Tyr Gln Thr Asn Lys 20 30413PRTHUMAN 304Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys 1 5 10 30514PRTHUMAN 305Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys Lys 1 5 10 30610PRTHUMAN 306Glu Val Val Glu Glu Ala Glu Asn Gly Arg 1 5 10 30714PRTHUMAN 307Arg Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys 1 5 10 30814PRTHUMAN 308Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys 1 5 10 30916PRTHUMAN 309Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly Lys 1 5 10 15 31010PRTHUMAN 310Glu Phe Glu Pro Leu Leu Asn Trp Met Lys 1 5 10 3118PRTHUMAN 311Glu Val Glu Glu Asp Glu Tyr Lys 1 5 3129PRTHUMAN 312Phe Ala Phe Gln Ala Glu Val Asn Arg 1 5 31311PRTHUMAN 313Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg 1 5 10 31410PRTHUMAN 314Leu Gly Val Ile Glu Asp His Ser Asn Arg 1 5 10 31519PRTHUMAN 315Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu 1 5 10 15 Thr Val Lys 31624PRTHUMAN 316Thr Asp Asp Glu Val Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp 1 5 10 15 Gly Leu Asn Ala Ser Gln Ile Arg 20 31716PRTHUMAN 317Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg 1 5 10 15 31813PRTHUMAN 318Val Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys 1 5 10 31917PRTHUMAN 319Ala Ala Asp Thr Asp Gly Asp Gly Gln Val Asn Tyr Glu Glu Phe Val 1 5 10 15 Arg 32013PRTHUMAN 320Ala Asp Gln Leu Thr Glu Glu Gln Val Thr Glu Phe Lys 1 5 10 32111PRTHUMAN 321Leu Ser Asp Glu Glu Val Asp Glu Met Ile Arg 1 5 10 32212PRTHUMAN 322Ser Leu Gly Gln Asn Pro Thr Glu Ala Glu Leu Arg 1 5 10 32316PRTHUMAN 323Val Phe Asp Lys Asp Gly Asn Gly Phe Val Ser Ala Ala Glu Leu Arg 1 5 10 15 32420PRTHUMAN 324Gly Asn Asp Ile Ser Ser Gly Thr Val Leu Ser Asp Tyr Val Gly Ser 1 5 10 15 Gly Pro Pro Lys 20 32514PRTHUMAN 325Leu Tyr Thr Leu Val Leu Thr Asp Pro Asp Ala Pro Ser Arg 1 5 10 3268PRTHUMAN 326Val Leu Thr Pro Thr Gln Val Lys 1 5 32711PRTHUMAN 327Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys 1 5 10 32820PRTHUMAN 328Lys Ile Asp Phe Ser Glu Phe Leu Ser Leu Leu Gly Asp Ile Ala Thr 1 5 10 15 Asp Tyr His Lys 20 32913PRTHUMAN 329Gln Ser His Gly Ala Ala Pro Cys Ser Gly Gly Ser Gln 1 5 10 33011PRTHUMAN 330Ser Ile Ile Gly Met Ile Asp Met Phe His Lys 1 5 10 33110PRTHUMAN 331Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 10 33224PRTHUMAN 332Glu Lys Val Glu Thr Glu Leu Gln Gly Val Cys Asp Thr Val Leu Gly 1 5 10 15 Leu Leu Asp Ser His Leu Ile Lys 20 33328PRTHUMAN 333Glu Lys Val Glu Thr Glu Leu Gln Gly Val Cys Asp Thr Val Leu Gly 1 5 10 15 Leu Leu Asp Ser His Leu Ile Lys Glu Ala Gly Asp 20 25 3349PRTHUMAN 334Glu Met Pro Pro Thr Asn Pro Ile Arg 1 5 33514PRTHUMAN 335Gly Ala Val Glu Lys Gly Glu Glu Leu Ser Cys Glu Glu Arg 1 5 10 3369PRTHUMAN 336Gly Glu Glu Leu Ser Cys Glu Glu Arg 1 5 33710PRTHUMAN 337Gly Ser Glu Glu Lys Gly Pro Glu Val Arg 1 5 10 3387PRTHUMAN 338Leu Ala Glu Gln Ala Glu Arg 1 5 3398PRTHUMAN 339Asn Leu Leu Ser Val Ala Tyr Lys 1 5 34012PRTHUMAN 340Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys Lys 1 5 10 34114PRTHUMAN 341Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu Val Arg 1 5 10 34219PRTHUMAN 342Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu Ser Glu Asp 1 5 10 15 Ser Tyr Lys 34322PRTHUMAN 343Val Glu Thr Glu Leu Gln Gly Val Cys Asp Thr Val Leu Gly Leu Leu 1 5 10 15 Asp Ser His Leu Ile Lys 20 3448PRTHUMAN 344Val Leu Ser Ser Ile Glu Gln Lys 1 5 3459PRTHUMAN 345Tyr Glu Asp Met Ala Ala Phe Met Lys 1 5 34611PRTHUMAN 346Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys 1 5 10 34710PRTHUMAN 347Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg 1 5 10 3489PRTHUMAN 348Ala Val Phe Pro Ser Ile Val Gly Arg 1 5 34911PRTHUMAN 349Ala Val Phe Pro Ser Ile Val Gly Arg Pro Arg 1 5 10 35017PRTHUMAN 350Asp Asp Asp Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met Cys 1 5 10 15 Lys 35121PRTHUMAN 351Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr Met Tyr Pro 1 5 10 15 Gly Ile Ala Asp Arg 20 35211PRTHUMAN 352Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys 1 5 10 35312PRTHUMAN 353Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg 1 5 10 35411PRTHUMAN 354Glu Ile Thr Ala Leu Ala Pro Ser Thr Met Lys 1 5 10 35510PRTHUMAN 355Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg 1 5 10 35611PRTHUMAN 356His Gln Gly Val Met Val Gly Met Gly Gln Lys 1 5 10 3577PRTHUMAN 357Ile Ile Ala Pro Pro Glu Arg 1 5 35811PRTHUMAN 358Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg 1 5 10 35923PRTHUMAN 359Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala 1 5 10 15 Ser Ser Ser Ser Leu Glu Lys 20 36029PRTHUMAN 360Leu Asp Leu Ala Gly Arg Asp Leu Thr Asp Tyr Leu Met Lys Ile Leu 1 5 10 15 Thr Glu Arg Gly Tyr Ser Phe Thr Thr Thr Ala Glu Phe 20 25 36113PRTHUMAN 361Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg 1 5 10 36216PRTHUMAN 362Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg 1 5 10 15 3638PRTHUMAN 363Thr Glu Ala Pro Leu Asn Pro Lys 1 5 36429PRTHUMAN 364Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 20 25 36510PRTHUMAN 365Val Ala Pro Glu Glu His Pro Val Leu Leu 1 5 10 36618PRTHUMAN 366Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn 1 5 10 15 Pro Lys 36716PRTHUMAN 367Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp Asp Met Glu Lys 1 5 10 15 3689PRTHUMAN 368Asp Leu Ala Gly Ser Ile Ile Gly Lys 1 5 36911PRTHUMAN 369Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp Arg 1 5 10 37016PRTHUMAN 370Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr Ile Gly Glu Ile Leu Lys 1 5 10 15 3719PRTHUMAN 371Ala Thr Val Gln Gln Leu Glu Gly Arg 1 5 3729PRTHUMAN 372Glu Leu Gly Val Gly Ile Ala Leu Arg 1 5 3739PRTHUMAN 373Phe Glu Glu Thr Thr Ala Asp Gly Arg 1 5 3747PRTHUMAN 374Gly Phe Asp Glu Tyr Met Lys 1 5 37520PRTHUMAN 375Thr Thr Gln Phe Ser Cys Thr Leu Gly Glu Lys Phe Glu Glu Thr Thr 1 5 10 15 Ala Asp Gly Arg 20 37612PRTHUMAN 376Ala Ala Glu Glu Glu Asp Glu Ala Asp Pro Lys Arg 1 5 10 3778PRTHUMAN 377Gly Phe Gly Phe Val Leu Phe Lys 1 5 3789PRTHUMAN 378Val Gly Tyr Val Ser Gly Trp Gly Arg 1 5 37910PRTHUMAN 379Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 10 38018PRTHUMAN 380Gly Ile Val Asp Gln Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile 1 5 10 15 Ser Lys 3817PRTHUMAN 381Leu Ala Glu Gln Ala Glu Arg 1 5 3828PRTHUMAN 382Asn Leu Leu Ser Val Ala Tyr Lys 1 5 38314PRTHUMAN 383Ser Val Thr Glu Gln Gly Ala Glu Leu Ser Asn Glu Glu Arg 1 5 10 38419PRTHUMAN 384Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys 3858PRTHUMAN 385Val Val Ser Ser Ile Glu Gln Lys 1 5 38612PRTHUMAN 386Tyr Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys 1 5 10 38710PRTHUMAN 387Ala Gly Val Glu Thr Thr Thr Pro Ser Lys 1 5 10 38811PRTHUMAN 388Asp Thr Glu Arg Pro Ser Gly Ile Pro Glu Arg 1 5 10 38919PRTHUMAN 389Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg 39015PRTHUMAN 390Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 1 5 10 15 39122PRTHUMAN 391Ala Gln Pro Val Gln Val Ala Glu Gly Ser Glu Pro Asp Gly Phe Trp 1 5 10 15 Glu Ala Leu Gly Gly Lys 20 39218PRTHUMAN 392Thr Pro Ser Ala Ala Tyr Leu Trp Val Gly Thr Gly Ala Ser Glu Ala 1 5 10 15 Glu Lys 39310PRTHUMAN 393Ala Gly Ala His Leu Gln Gly Gly Ala Lys 1 5 10 39415PRTHUMAN 394Gly Ala Leu Gln Asn Ile Ile Pro Ala Ser Thr Gly Ala Ala Lys 1 5 10 15 3957PRTHUMAN 395Leu Thr Gly Met Ala Phe Arg 1 5 3968PRTHUMAN 396Gln Ala Ser Glu Gly Pro Leu Lys 1 5 3978PRTHUMAN 397Thr Val Asp Gly Pro Ser Gly Lys 1 5 3988PRTHUMAN 398Val Ile Pro Glu Leu Asp Gly Lys 1 5 39914PRTHUMAN 399Val Pro Thr Ala Asn Val Ser Val Val Asp Leu Thr Cys Arg 1 5 10 40012PRTHUMAN 400Val Val Asp Leu Met Ala His Met Ala Ser Lys Glu 1 5 10 40118PRTHUMAN 401Gly Ala Asp Phe Leu Val Thr Glu Val Glu Asn Gly Gly Ser Leu Gly 1 5 10 15 Ser Lys 4028PRTHUMAN 402Ala Pro Ile Ile Ala Val Thr Arg 1 5 40311PRTHUMAN 403Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu Lys 1 5 10 4049PRTHUMAN 404Gly Asp Tyr Pro Leu Glu Ala Val Arg 1 5 4058PRTHUMAN 405Ile Glu Asn His Glu Gly Val Arg 1 5 4069PRTHUMAN 406Lys Ala Ser Asp Val His Glu Val Arg 1 5 40711PRTHUMAN 407Leu Asp Ile Asp Ser Pro Pro Ile Thr Ala Arg 1 5 10 40813PRTHUMAN 408Asn Thr Gly Ile Ile Cys Thr Ile Gly Pro Ala Ser Arg 1 5 10 40910PRTHUMAN 409Ala Gln Leu Gly Gly Pro Glu Ala Ala Lys 1 5 10 41017PRTHUMAN 410Leu Ala Thr Gln Ser Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser 1 5 10 15 Arg 4119PRTHUMAN 411Gln Asp Glu His Gly Tyr Ile Ser Arg 1 5 41210PRTHUMAN 412Gln Leu Ser Ser Gly Val Ser Glu Ile Arg 1 5 10 41316PRTHUMAN 413Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys 1 5 10 15 41413PRTHUMAN 414Glu Asp Ser Gln Arg Pro Gly Ala His Leu Thr Val Lys 1 5 10 41511PRTHUMAN 415Ser Glu Ser Pro Lys Glu Pro Glu Gln Leu Arg 1 5 10 41610PRTHUMAN 416Gly Glu Gly Gln Leu Gly Pro Ala Glu Arg 1 5 10 4178PRTHUMAN 417Ser Asp Pro Val Val Ser Tyr Arg 1 5 41816PRTHUMAN 418Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu

Gly Lys 1 5 10 15 41910PRTHUMAN 419Glu Phe Glu Pro Leu Leu Asn Trp Met Lys 1 5 10 4208PRTHUMAN 420Glu Val Glu Glu Asp Glu Tyr Lys 1 5 4219PRTHUMAN 421Phe Ala Phe Gln Ala Glu Val Asn Arg 1 5 42211PRTHUMAN 422Lys Glu Ala Glu Ser Ser Pro Phe Val Glu Arg 1 5 10 42310PRTHUMAN 423Leu Gly Val Ile Glu Asp His Ser Asn Arg 1 5 10 42419PRTHUMAN 424Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu 1 5 10 15 Thr Val Lys 42524PRTHUMAN 425Thr Asp Asp Glu Val Val Gln Arg Glu Glu Glu Ala Ile Gln Leu Asp 1 5 10 15 Gly Leu Asn Ala Ser Gln Ile Arg 20 42616PRTHUMAN 426Thr Val Leu Asp Leu Ala Val Val Leu Phe Glu Thr Ala Thr Leu Arg 1 5 10 15 42713PRTHUMAN 427Val Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys 1 5 10 4288PRTHUMAN 428Gly Leu Val Gly Glu Ile Ile Lys 1 5 42910PRTHUMAN 429Asn Ile Ile His Gly Ser Asp Ser Val Lys 1 5 10 43012PRTHUMAN 430Ala Ser Gly Val Ala Val Ser Asp Gly Val Ile Lys 1 5 10 4319PRTHUMAN 431Ile Ser Ser Pro Thr Glu Thr Glu Arg 1 5 43210PRTHUMAN 432Asn Gln Lys Asp Pro Gly Val Leu Asp Arg 1 5 10 43310PRTHUMAN 433Ala Thr Ala Val Val Asp Gly Ala Phe Lys 1 5 10 4348PRTHUMAN 434Gly Leu Phe Ile Leu Asp Gly Lys 1 5 43518PRTHUMAN 435Lys Glu Gly Gly Leu Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val 1 5 10 15 Thr Arg 4369PRTHUMAN 436Leu Ser Glu Asp Tyr Gly Val Leu Lys 1 5 43711PRTHUMAN 437Gln Ile Thr Val Asn Asp Leu Pro Val Gly Arg 1 5 10 4388PRTHUMAN 438Thr Asp Glu Gly Ile Ala Tyr Arg 1 5 4397PRTHUMAN 439Thr Leu Val Val His Glu Lys 1 5 44015PRTHUMAN 440Ala Met Gly Ile Met Asn Ser Phe Val Asn Asp Ile Phe Glu Arg 1 5 10 15 4419PRTHUMAN 441Leu Leu Leu Pro Gly Glu Leu Ala Lys 1 5 44211PRTHUMAN 442Gln Val His Pro Asp Thr Gly Ile Ser Ser Lys 1 5 10 44311PRTHUMAN 443Ala Ala Glu Glu Pro Ser Lys Val Glu Glu Lys 1 5 10 44415PRTHUMAN 444Ala Glu Asp Gly Ala Thr Pro Ser Pro Ser Asn Glu Thr Pro Lys 1 5 10 15 44516PRTHUMAN 445Ala Glu Asp Gly Ala Thr Pro Ser Pro Ser Asn Glu Thr Pro Lys Lys 1 5 10 15 44614PRTHUMAN 446Ala Val Ala Pro Glu Lys Pro Pro Ala Ser Asp Glu Thr Lys 1 5 10 44717PRTHUMAN 447Glu Ala Gly Glu Gly Gly Glu Ala Glu Ala Pro Ala Ala Glu Gly Gly 1 5 10 15 Lys 44827PRTHUMAN 448Glu Ala Pro Ala Glu Gly Glu Ala Ala Glu Pro Gly Ser Pro Thr Ala 1 5 10 15 Ala Glu Gly Glu Ala Ala Ser Ala Ala Ser Ser 20 25 44927PRTHUMAN 449Glu Glu Leu Gln Ala Asn Gly Ser Ala Pro Ala Ala Asp Lys Glu Glu 1 5 10 15 Pro Ala Ala Ala Gly Ser Gly Ala Ala Ser Pro 20 25 45019PRTHUMAN 450Gly Glu Ala Ala Ala Glu Arg Pro Gly Glu Ala Ala Val Ala Ser Ser 1 5 10 15 Pro Ser Lys 45118PRTHUMAN 451Gly Glu Pro Ala Ala Ala Ala Ala Pro Glu Ala Gly Ala Ser Pro Val 1 5 10 15 Glu Lys 45226PRTHUMAN 452Gly Ser Ala Pro Ala Ala Asp Lys Glu Glu Pro Ala Ala Ala Gly Ser 1 5 10 15 Gly Ala Ala Ser Pro Ser Ala Ala Glu Lys 20 25 45315PRTHUMAN 453Val Asn Gly Asp Ala Ser Pro Ala Ala Ala Glu Ser Gly Ala Lys 1 5 10 15 45419PRTHUMAN 454Ala Phe Leu Ala Ser Pro Glu Tyr Val Asn Leu Pro Ile Asn Gly Asn 1 5 10 15 Gly Lys Gln 45520PRTHUMAN 455Ala Leu Pro Gly Gln Leu Lys Pro Phe Glu Thr Leu Leu Ser Gln Asn 1 5 10 15 Gln Gly Gly Lys 20 45610PRTHUMAN 456Ala Ser Cys Leu Tyr Gly Gln Leu Pro Lys 1 5 10 45716PRTHUMAN 457Phe Gln Asp Gly Asp Leu Thr Leu Tyr Gln Ser Asn Thr Ile Leu Arg 1 5 10 15 4589PRTHUMAN 458Gly Pro Ser Ser Val Glu Asp Ile Lys 1 5 45921PRTHUMAN 459Met Ser Val Gln Pro Thr Val Ser Leu Gly Gly Phe Glu Ile Thr Pro 1 5 10 15 Pro Val Val Leu Arg 20 46014PRTHUMAN 460Met Thr Asp Gln Glu Ala Ile Gln Asp Leu Trp Gln Trp Arg 1 5 10 46111PRTHUMAN 461Ala Ser Leu Glu Asn Ser Leu Glu Glu Thr Lys 1 5 10 4629PRTHUMAN 462Asp Ala Glu Glu Trp Phe Phe Thr Lys 1 5 46319PRTHUMAN 463Ile Leu Thr Ala Thr Val Asp Asn Ala Asn Val Leu Leu Gln Ile Asp 1 5 10 15 Asn Ala Arg 46411PRTHUMAN 464Lys Val Val Ser Thr His Glu Gln Val Leu Arg 1 5 10 46510PRTHUMAN 465Val Val Ser Thr His Glu Gln Val Leu Arg 1 5 10 46621PRTHUMAN 466Ala Thr Leu Lys Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu Glu Gln 1 5 10 15 Thr Pro Gln Asn Lys 20 4679PRTHUMAN 467Asp Gln Leu Ile Tyr Asn Leu Leu Lys 1 5 46817PRTHUMAN 468Asp Gln Leu Ile Tyr Asn Leu Leu Lys Glu Glu Gln Thr Pro Gln Asn 1 5 10 15 Lys 4697PRTHUMAN 469Phe Ile Ile Pro Asn Trp Lys 1 5 47014PRTHUMAN 470Gly Glu Met Met Asp Leu Gln His Gly Ser Leu Phe Leu Arg 1 5 10 47111PRTHUMAN 471Lys Ser Ala Asp Thr Leu Trp Gly Ile Gln Lys 1 5 10 4729PRTHUMAN 472Leu Val Ile Ile Thr Ala Gly Ala Arg 1 5 47311PRTHUMAN 473Gln Val Val Glu Ser Ala Tyr Glu Val Ile Lys 1 5 10 47410PRTHUMAN 474Arg Val His Pro Val Ser Thr Met Ile Lys 1 5 10 47510PRTHUMAN 475Ser Ala Asp Thr Leu Trp Gly Ile Gln Lys 1 5 10 47616PRTHUMAN 476Thr Leu His Pro Asp Leu Gly Thr Asp Lys Asp Lys Glu Gln Trp Lys 1 5 10 15 4779PRTHUMAN 477Val His Pro Val Ser Thr Met Ile Lys 1 5 47812PRTHUMAN 478Val Ile Gly Ser Gly Cys Asn Leu Asp Ser Ala Arg 1 5 10 47910PRTHUMAN 479Val Thr Leu Thr Ser Glu Glu Glu Ala Arg 1 5 10 48010PRTHUMAN 480Ala Ser Pro Asp Trp Gly Tyr Asp Asp Lys 1 5 10 48118PRTHUMAN 481Ala Ser Pro Asp Trp Gly Tyr Asp Asp Lys Asn Gly Pro Glu Gln Trp 1 5 10 15 Ser Lys 48219PRTHUMAN 482Glu Ile Ile Asn Val Gly His Ser Phe His Val Asn Phe Glu Asp Asn 1 5 10 15 Asp Asn Arg 4839PRTHUMAN 483Gly Gly Pro Phe Ser Asp Ser Tyr Arg 1 5 48410PRTHUMAN 484His Asp Thr Ser Leu Lys Pro Ile Ser Val 1 5 10 48518PRTHUMAN 485His Asp Thr Ser Leu Lys Pro Ile Ser Val Ser Tyr Asn Pro Ala Thr 1 5 10 15 Ala Lys 48616PRTHUMAN 486Leu Tyr Pro Ile Ala Asn Gly Asn Asn Gln Ser Pro Val Asp Ile Lys 1 5 10 15 48713PRTHUMAN 487Ser Ala Glu Leu His Val Ala His Trp Asn Ser Ala Lys 1 5 10 48825PRTHUMAN 488Ser Leu Leu Ser Asn Val Glu Gly Asp Asn Ala Val Pro Met Gln His 1 5 10 15 Asn Asn Arg Pro Thr Gln Pro Leu Lys 20 25 4899PRTHUMAN 489Ser Ser Glu Gln Leu Ala Gln Phe Arg 1 5 4909PRTHUMAN 490Val Leu Asp Ala Leu Gln Ala Ile Lys 1 5 49114PRTHUMAN 491Tyr Ser Ala Glu Leu His Val Ala His Trp Asn Ser Ala Lys 1 5 10 49210PRTHUMAN 492Tyr Ser Ser Leu Ala Glu Ala Ala Ser Lys 1 5 10 49316PRTHUMAN 493Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val Thr Thr Val Lys 1 5 10 15 49410PRTHUMAN 494Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys 1 5 10 49515PRTHUMAN 495Gly Thr Glu Ala Ala Gly Ala Met Phe Leu Glu Ala Ile Pro Met 1 5 10 15 49610PRTHUMAN 496Lys Leu Ser Ser Trp Val Leu Leu Met Lys 1 5 10 49710PRTHUMAN 497Leu Gly Met Phe Asn Ile Gln His Cys Lys 1 5 10 49815PRTHUMAN 498Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys 1 5 10 15 49910PRTHUMAN 499Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys 1 5 10 50010PRTHUMAN 500Leu Val Asp Lys Phe Leu Glu Asp Val Lys 1 5 10 50118PRTHUMAN 501Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala Pro 1 5 10 15 Leu Lys 50216PRTHUMAN 502Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu Lys 1 5 10 15 50317PRTHUMAN 503Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu Lys 1 5 10 15 Lys 5048PRTHUMAN 504Ala Leu Tyr Glu Ala Gly Glu Arg 1 5 50512PRTHUMAN 505Cys Ala Thr Ser Lys Pro Ala Phe Phe Ala Glu Lys 1 5 10 50611PRTHUMAN 506Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe Arg 1 5 10 50724PRTHUMAN 507Gly Gly Pro Gly Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro Ser 1 5 10 15 Ser Asp Val Ala Ala Leu His Lys 20 50816PRTHUMAN 508Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg 1 5 10 15 50914PRTHUMAN 509Gly Thr Asp Val Asn Val Phe Asn Thr Ile Leu Thr Thr Arg 1 5 10 51013PRTHUMAN 510Gly Val Asp Glu Ala Thr Ile Ile Asp Ile Leu Thr Lys 1 5 10 51115PRTHUMAN 511Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile Leu Thr Thr Arg 1 5 10 15 5128PRTHUMAN 512Asn Ala Leu Leu Ser Leu Ala Lys 1 5 51316PRTHUMAN 513Ser Glu Asp Phe Gly Val Asn Glu Asp Leu Ala Asp Ser Asp Ala Arg 1 5 10 15 5149PRTHUMAN 514Ser Glu Ile Asp Met Asn Asp Ile Lys 1 5 51511PRTHUMAN 515Thr Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg 1 5 10 5169PRTHUMAN 516Asp Gly Val Val Glu Ile Thr Gly Lys 1 5 51728PRTHUMAN 517Lys Tyr Thr Leu Pro Pro Gly Val Asp Pro Thr Gln Val Ser Ser Ser 1 5 10 15 Leu Ser Pro Glu Gly Thr Leu Thr Val Glu Ala Pro 20 25 51817PRTHUMAN 518Leu Ala Thr Gln Ser Asn Glu Ile Thr Ile Pro Val Thr Phe Glu Ser 1 5 10 15 Arg 5199PRTHUMAN 519Gln Asp Glu His Gly Tyr Ile Ser Arg 1 5 52010PRTHUMAN 520Gln Leu Ser Ser Gly Val Ser Glu Ile Arg 1 5 10 52111PRTHUMAN 521Thr Lys Asp Gly Val Val Glu Ile Thr Gly Lys 1 5 10 52216PRTHUMAN 522Val Ser Leu Asp Val Asn His Phe Ala Pro Asp Glu Leu Thr Val Lys 1 5 10 15 52313PRTHUMAN 523Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys 1 5 10 52414PRTHUMAN 524Ala Ala Glu Asp Asp Glu Asp Asp Asp Val Asp Thr Lys Lys 1 5 10 52510PRTHUMAN 525Glu Val Val Glu Glu Ala Glu Asn Gly Arg 1 5 10 52615PRTHUMAN 526Glu Val Val Glu Glu Ala Glu Asn Gly Arg Asp Ala Pro Ala Asn 1 5 10 15 52711PRTHUMAN 527Lys Glu Val Val Glu Glu Ala Glu Asn Gly Arg 1 5 10 52814PRTHUMAN 528Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys 1 5 10 52915PRTHUMAN 529Asp Leu Pro Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys 1 5 10 15 53011PRTHUMAN 530Asn Leu His Ala Leu Ile Leu Val Asn Asn Lys 1 5 10 53113PRTHUMAN 531Ser Ser Gly Ile Glu Asn Gly Ala Phe Gln Gly Met Lys 1 5 10 53211PRTHUMAN 532Val Ser Pro Gly Ala Phe Thr Pro Leu Val Lys 1 5 10 53314PRTHUMAN 533Val Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys 1 5 10 5349PRTHUMAN 534Ala Asp Leu Ser Glu Ala Ala Asn Arg 1 5 53516PRTHUMAN 535Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp Leu Glu 1 5 10 15 5369PRTHUMAN 536Asp Asn Leu Ala Glu Asp Ile Met Arg 1 5 53711PRTHUMAN 537Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg 1 5 10 53810PRTHUMAN 538Glu Lys Leu Gln Glu Glu Met Leu Gln Arg 1 5 10 53919PRTHUMAN 539Glu Met Glu Glu Asn Phe Ala Val Glu Ala Ala Asn Tyr Gln Asp Thr 1 5 10 15 Ile Gly Arg 54015PRTHUMAN 540Glu Thr Asn Leu Asp Ser Leu Pro Leu Val Asp Thr His Ser Lys 1 5 10 15 5419PRTHUMAN 541Glu Tyr Gln Asp Leu Leu Asn Val Lys 1 5 54210PRTHUMAN 542Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 1 5 10 54310PRTHUMAN 543Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys 1 5 10 54414PRTHUMAN 544Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys 1 5 10 54514PRTHUMAN 545Ile Ser Leu Pro Leu Pro Asn Phe Ser Ser Leu Asn Leu Arg 1 5 10 5469PRTHUMAN 546Lys Leu Leu Glu Gly Glu Glu Ser Arg 1 5 54713PRTHUMAN 547Lys Val Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys 1 5 10 54814PRTHUMAN 548Lys Val Glu Ser Leu Gln Glu Glu Ile Ala Phe Leu Lys Lys 1 5 10 54910PRTHUMAN 549Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg 1 5 10 55019PRTHUMAN 550Leu Leu Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr 1 5 10 15 Glu Phe Lys 55114PRTHUMAN 551Leu Gln Asp Glu Ile Gln Asn Met Lys Glu Glu Met Ala Arg 1 5 10 5528PRTHUMAN 552Leu Gln Glu Glu Met Leu Gln Arg 1 5 55313PRTHUMAN 553Asn Leu Asp Ser Leu Pro Leu Val Asp Thr His Ser Lys 1 5 10 55410PRTHUMAN 554Asn Leu Gln Glu Ala Glu Glu Trp Tyr Lys 1 5 10 55510PRTHUMAN 555Gln Asp Val Asp Asn Ala Ser Leu Ala Arg 1 5 10 5569PRTHUMAN 556Gln Gln Tyr Glu Ser Val Ala Ala Lys 1 5 5579PRTHUMAN 557Gln Val Asp Gln Leu Thr Asn Asp Lys 1 5 55813PRTHUMAN 558Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys 1 5 10 55910PRTHUMAN 559Arg Gln Val Asp Gln Leu Thr Asn Asp Lys 1 5 10 56021PRTHUMAN 560Thr Val Glu Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His 1 5 10 15 His Asp Asp Leu Glu 20 5619PRTHUMAN 561Val Glu Leu Gln Glu Leu Asn Asp Arg 1 5 56214PRTHUMAN 562Val Glu Val Glu Arg Asp Asn Leu Ala Glu Asp Ile Met Arg 1 5 10 56316PRTHUMAN 563Asp Asp Glu Val Asp Val Asp Gly Thr Val Glu Glu Asp Leu Gly Lys 1 5 10 15 56410PRTHUMAN 564Glu Phe Glu Pro Leu Leu Asn Trp Met Lys 1 5 10 56510PRTHUMAN 565Glu Gly Val Lys Phe Asp Glu Ser Glu Lys 1 5 10 56612PRTHUMAN 566Glu Leu Ile Ser Asn Ala Ser Asp Ala Leu Asp Lys 1 5 10 56719PRTHUMAN 567Phe Gln Ser Ser His His Pro Thr Asp Ile Thr Ser Leu Asp Gln Tyr 1 5 10 15 Val Glu Arg 5689PRTHUMAN 568Gly Leu Phe Asp Glu Tyr Gly Ser Lys 1 5 56914PRTHUMAN 569Gly Val Val Asp

Ser Asp Asp Leu Pro Leu Asn Val Ser Arg 1 5 10 57013PRTHUMAN 570Lys Ile Ala Asp Asp Lys Tyr Asn Asp Thr Phe Trp Lys 1 5 10 57110PRTHUMAN 571Leu Gly Val Ile Glu Asp His Ser Asn Arg 1 5 10 57219PRTHUMAN 572Leu Ile Ser Leu Thr Asp Glu Asn Ala Leu Ser Gly Asn Glu Glu Leu 1 5 10 15 Thr Val Lys 57314PRTHUMAN 573Asn Leu Leu His Val Thr Asp Thr Gly Val Gly Met Thr Arg 1 5 10 57414PRTHUMAN 574Arg Val Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys 1 5 10 57511PRTHUMAN 575Ser Ile Leu Phe Val Pro Thr Ser Ala Pro Arg 1 5 10 57613PRTHUMAN 576Val Phe Ile Thr Asp Asp Phe His Asp Met Met Pro Lys 1 5 10 5779PRTHUMAN 577Glu Leu Pro Ser Phe Val Gly Glu Lys 1 5 57816PRTHUMAN 578Glu Leu Pro Ser Phe Val Gly Glu Lys Val Asp Glu Glu Gly Leu Lys 1 5 10 15 57917PRTHUMAN 579Glu Leu Pro Ser Phe Val Gly Glu Lys Val Asp Glu Glu Gly Leu Lys 1 5 10 15 Lys 58010PRTHUMAN 580Gly Glu Lys Val Asp Glu Glu Gly Leu Lys 1 5 10 5818PRTHUMAN 581Val Asp Glu Glu Gly Leu Lys Lys 1 5 58210PRTHUMAN 582Tyr Ser Cys Gln Glu Gly Asp Lys Phe Lys 1 5 10 58319PRTHUMAN 583Cys Leu Thr Thr Asp Glu Tyr Asp Gly His Ser Thr Tyr Pro Ser His 1 5 10 15 Gln Tyr Gln 5849PRTHUMAN 584His Asp Leu Gly His Phe Met Leu Arg 1 5 58524PRTHUMAN 585Leu Pro Ser Glu Gly Pro Arg Pro Ala His Val Val Val Gly Asp Val 1 5 10 15 Leu Gln Ala Ala Asp Val Asp Lys 20 58610PRTHUMAN 586Leu Gln Ala Val Thr Asp Asp His Ile Arg 1 5 10 58714PRTHUMAN 587Asn Asp Leu Ser Pro Thr Thr Val Met Ser Glu Gly Ala Arg 1 5 10 58817PRTHUMAN 588Pro Ala His Val Val Val Gly Asp Val Leu Gln Ala Ala Asp Val Asp 1 5 10 15 Lys 58915PRTHUMAN 589Thr Val Ala Gly Gln Asp Ala Val Ile Val Leu Leu Gly Thr Arg 1 5 10 15 59013PRTHUMAN 590Glu Asn Phe Pro Asn Phe Leu Ser Ala Cys Asp Lys Lys 1 5 10 59111PRTHUMAN 591Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys 1 5 10 59212PRTHUMAN 592Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys Lys 1 5 10 59311PRTHUMAN 593Ile Asp Lys Pro Ser Leu Leu Thr Met Met Lys 1 5 10 59413PRTHUMAN 594Lys Gly Thr Asn Tyr Leu Ala Asp Val Phe Glu Lys Lys 1 5 10 59513PRTHUMAN 595Gln Ser His Gly Ala Ala Pro Cys Ser Gly Gly Ser Gln 1 5 10 59611PRTHUMAN 596Ser Ile Ile Gly Met Ile Asp Met Phe His Lys 1 5 10 59717PRTHUMAN 597Ala Asp Asn Ala Gly Glu Glu Gly Gly Glu Ala Pro Gln Glu Pro Gln 1 5 10 15 Ser 59823PRTHUMAN 598Asp Asn Leu Thr Leu Trp Gly Ala Asp Asn Ala Gly Glu Glu Gly Gly 1 5 10 15 Glu Ala Pro Gln Glu Pro Gln 20 59924PRTHUMAN 599Asp Asn Leu Thr Leu Trp Gly Ala Asp Asn Ala Gly Glu Glu Gly Gly 1 5 10 15 Glu Ala Pro Gln Glu Pro Gln Ser 20 60010PRTHUMAN 600Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 10 6019PRTHUMAN 601Glu Met Pro Pro Thr Asn Pro Ile Arg 1 5 60214PRTHUMAN 602Gly Ala Val Glu Lys Gly Glu Glu Leu Ser Cys Glu Glu Arg 1 5 10 6039PRTHUMAN 603Gly Glu Glu Leu Ser Cys Glu Glu Arg 1 5 6047PRTHUMAN 604Leu Ala Glu Gln Ala Glu Arg 1 5 6058PRTHUMAN 605Asn Leu Leu Ser Val Ala Tyr Lys 1 5 60612PRTHUMAN 606Arg Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys 1 5 10 60711PRTHUMAN 607Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys 1 5 10 60812PRTHUMAN 608Ser Ala Tyr Gln Glu Ala Met Asp Ile Ser Lys Lys 1 5 10 60914PRTHUMAN 609Ser Asn Glu Glu Gly Ser Glu Glu Lys Gly Pro Glu Val Arg 1 5 10 6109PRTHUMAN 610Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 61119PRTHUMAN 611Ser Val Phe His Tyr Glu Ile Ala Asn Ser Pro Glu Glu Ala Ile Ser 1 5 10 15 Leu Ala Lys 61218PRTHUMAN 612Thr Ala Asp Asn Ala Gly Glu Glu Gly Gly Glu Ala Pro Gln Glu Pro 1 5 10 15 Gln Ser 61317PRTHUMAN 613Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu Ser Glu Asp 1 5 10 15 Ser 61419PRTHUMAN 614Thr Thr Phe Asp Glu Ala Met Ala Asp Leu His Thr Leu Ser Glu Asp 1 5 10 15 Ser Tyr Lys 6158PRTHUMAN 615Val Leu Ser Ser Ile Glu Gln Lys 1 5 6169PRTHUMAN 616Tyr Glu Asp Met Ala Ala Phe Met Lys 1 5 61711PRTHUMAN 617Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys 1 5 10 61812PRTHUMAN 618Tyr Leu Ala Glu Val Ala Thr Gly Asp Asp Lys Lys 1 5 10 6199PRTHUMAN 619Asp Gly Tyr Asn Tyr Thr Leu Ser Lys 1 5 6209PRTHUMAN 620Ile Ser Ser Pro Thr Glu Thr Glu Arg 1 5 62116PRTHUMAN 621Thr Glu Phe Leu Ser Phe Met Asn Thr Glu Leu Ala Ala Phe Thr Lys 1 5 10 15 62210PRTHUMAN 622Ala Thr Ala Val Val Asp Gly Ala Phe Lys 1 5 10 62317PRTHUMAN 623Glu Gly Gly Leu Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr 1 5 10 15 Arg 6248PRTHUMAN 624Gly Leu Phe Ile Ile Asp Gly Lys 1 5 6259PRTHUMAN 625Ile Gly Lys Pro Ala Pro Asp Phe Lys 1 5 62618PRTHUMAN 626Lys Glu Gly Gly Leu Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val 1 5 10 15 Thr Arg 6279PRTHUMAN 627Leu Ser Glu Asp Tyr Gly Val Leu Lys 1 5 62827PRTHUMAN 628Leu Val Gln Ala Phe Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro 1 5 10 15 Ala Gly Trp Lys Pro Gly Ser Asp Thr Ile Lys 20 25 62911PRTHUMAN 629Gln Ile Thr Val Asn Asp Leu Pro Val Gly Arg 1 5 10 63027PRTHUMAN 630Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp Lys Pro 1 5 10 15 Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Cys 20 25 63110PRTHUMAN 631Arg Leu Ser Glu Asp Tyr Gly Val Leu Lys 1 5 10 6328PRTHUMAN 632Thr Asp Glu Gly Ile Ala Tyr Arg 1 5 6338PRTHUMAN 633Phe Asn Ala Leu Gln Tyr Leu Arg 1 5 6349PRTHUMAN 634Ile Leu Gly Pro Leu Ser Tyr Ser Lys 1 5 63515PRTHUMAN 635Ile Ser Glu Thr Ser Leu Pro Pro Asp Met Tyr Glu Cys Leu Arg 1 5 10 15 63610PRTHUMAN 636Ile Ser Asn Ile Pro Asp Glu Tyr Phe Lys 1 5 10 63711PRTHUMAN 637Leu Lys Glu Asp Ala Val Ser Ala Ala Phe Lys 1 5 10 63818PRTHUMAN 638Leu Pro Ser Gly Leu Pro Val Ser Leu Leu Thr Leu Tyr Leu Asp Asn 1 5 10 15 Asn Lys 63921PRTHUMAN 639Asn Ile Pro Thr Val Asn Glu Asn Leu Glu Asn Tyr Tyr Leu Glu Val 1 5 10 15 Asn Gln Leu Glu Lys 20 64010PRTHUMAN 640Asn Asn Gln Ile Asp His Ile Asp Glu Lys 1 5 10 6419PRTHUMAN 641Arg Phe Asn Ala Leu Gln Tyr Leu Arg 1 5 64211PRTHUMAN 642Ser Leu Glu Asp Leu Gln Leu Thr His Asn Lys 1 5 10 64314PRTHUMAN 643Ser Leu Glu Tyr Leu Asp Leu Ser Phe Asn Gln Ile Ala Arg 1 5 10 64410PRTHUMAN 644Ser Val Pro Met Val Pro Pro Gly Ile Lys 1 5 10 6458PRTHUMAN 645Val Ala Asn Glu Val Thr Leu Asn 1 5 64610PRTHUMAN 646Ala Gly Phe Ala Gly Asp Asp Ala Pro Arg 1 5 10 64719PRTHUMAN 647Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala Ser Ser Ser Ser 1 5 10 15 Leu Glu Lys 6489PRTHUMAN 648Ala Val Phe Pro Ser Ile Val Gly Arg 1 5 64911PRTHUMAN 649Ala Val Phe Pro Ser Ile Val Gly Arg Pro Arg 1 5 10 65028PRTHUMAN 650Cys Pro Glu Ala Leu Phe Gln Pro Ser Phe Leu Gly Met Glu Ser Cys 1 5 10 15 Gly Ile His Glu Thr Thr Phe Asn Ser Ile Met Lys 20 25 65117PRTHUMAN 651Asp Asp Asp Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met Cys 1 5 10 15 Lys 65216PRTHUMAN 652Asp Asp Ile Ala Ala Leu Val Val Asp Asn Gly Ser Gly Met Cys Lys 1 5 10 15 6538PRTHUMAN 653Asp Leu Thr Asp Tyr Leu Met Lys 1 5 65421PRTHUMAN 654Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr Met Tyr Pro 1 5 10 15 Gly Ile Ala Asp Arg 20 65511PRTHUMAN 655Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys 1 5 10 65612PRTHUMAN 656Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys Arg 1 5 10 65711PRTHUMAN 657Glu Ile Thr Ala Leu Ala Pro Ser Thr Met Lys 1 5 10 6589PRTHUMAN 658Gly Phe Ala Gly Asp Asp Ala Pro Arg 1 5 65912PRTHUMAN 659Gly Ile His Glu Thr Thr Phe Asn Ser Ile Met Lys 1 5 10 66011PRTHUMAN 660Gly Ile Val Thr Asn Trp Asp Asp Met Glu Lys 1 5 10 66114PRTHUMAN 661Gly Gln Lys Asp Ser Tyr Val Gly Asp Glu Ala Gln Ser Lys 1 5 10 66210PRTHUMAN 662Gly Tyr Ser Phe Thr Thr Thr Ala Glu Arg 1 5 10 66311PRTHUMAN 663His Gln Gly Val Met Val Gly Met Gly Gln Lys 1 5 10 66422PRTHUMAN 664His Gln Gly Val Met Val Gly Met Gly Gln Lys Asp Ser Tyr Val Gly 1 5 10 15 Asp Glu Ala Gln Ser Lys 20 6657PRTHUMAN 665Ile Ile Ala Pro Pro Glu Arg 1 5 6668PRTHUMAN 666Ile Ile Ala Pro Pro Glu Arg Lys 1 5 66711PRTHUMAN 667Ile Trp His His Thr Phe Tyr Asn Glu Leu Arg 1 5 10 66822PRTHUMAN 668Lys Asp Leu Tyr Ala Asn Thr Val Leu Ser Gly Gly Thr Thr Met Tyr 1 5 10 15 Pro Gly Ile Ala Asp Arg 20 66914PRTHUMAN 669Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg 1 5 10 67017PRTHUMAN 670Lys Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp Asp Met Glu 1 5 10 15 Lys 67123PRTHUMAN 671Leu Cys Tyr Val Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala 1 5 10 15 Ser Ser Ser Ser Leu Glu Lys 20 67210PRTHUMAN 672Leu Leu Thr Glu Ala Pro Leu Asn Pro Lys 1 5 10 67315PRTHUMAN 673Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp Asp Met Glu Lys 1 5 10 15 67413PRTHUMAN 674Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg 1 5 10 67519PRTHUMAN 675Arg Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu 1 5 10 15 Asn Pro Lys 67613PRTHUMAN 676Ser Gly Gly Thr Thr Met Tyr Pro Gly Ile Ala Asp Arg 1 5 10 67715PRTHUMAN 677Ser Lys Gln Glu Tyr Asp Glu Ser Gly Pro Ser Ile Val His Arg 1 5 10 15 67810PRTHUMAN 678Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile 1 5 10 67912PRTHUMAN 679Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile 1 5 10 68016PRTHUMAN 680Ser Tyr Glu Leu Pro Asp Gly Gln Val Ile Thr Ile Gly Asn Glu Arg 1 5 10 15 68114PRTHUMAN 681Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His 1 5 10 68222PRTHUMAN 682Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr 20 68326PRTHUMAN 683Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His 20 25 68429PRTHUMAN 684Thr Thr Gly Ile Val Met Asp Ser Gly Asp Gly Val Thr His Thr Val 1 5 10 15 Pro Ile Tyr Glu Gly Tyr Ala Leu Pro His Ala Ile Leu 20 25 68516PRTHUMAN 685Thr Val Leu Ser Gly Gly Thr Thr Met Tyr Pro Gly Ile Ala Asp Arg 1 5 10 15 6868PRTHUMAN 686Val Ala Pro Glu Glu His Pro Val 1 5 6879PRTHUMAN 687Val Ala Pro Glu Glu His Pro Val Leu 1 5 68810PRTHUMAN 688Val Ala Pro Glu Glu His Pro Val Leu Leu 1 5 10 68913PRTHUMAN 689Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala 1 5 10 69016PRTHUMAN 690Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn 1 5 10 15 69118PRTHUMAN 691Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn 1 5 10 15 Pro Lys 69221PRTHUMAN 692Val Ala Pro Glu Glu His Pro Val Leu Leu Thr Glu Ala Pro Leu Asn 1 5 10 15 Pro Lys Ala Asn Arg 20 69316PRTHUMAN 693Tyr Pro Ile Glu His Gly Ile Val Thr Asn Trp Asp Asp Met Glu Lys 1 5 10 15 69421PRTHUMAN 694Tyr Val Ala Leu Asp Phe Glu Gln Glu Met Ala Thr Ala Ala Ser Ser 1 5 10 15 Ser Ser Leu Glu Lys 20 69518PRTHUMAN 695Lys Pro Leu Thr Ser Ser Ser Ala Ala Pro Gln Arg Pro Ile Ser Thr 1 5 10 15 Gln Arg 69612PRTHUMAN 696Ala Ala Gln Gln Gln Gln Pro Ser Ala Ser Pro Arg 1 5 10 69712PRTHUMAN 697Arg Pro Asn Glu Thr Phe His Leu Ala Thr Arg Lys 1 5 10 69823PRTHUMAN 698Asp Asn Leu Thr Leu Trp Thr Ser Asp Thr Gln Gly Asp Glu Ala Glu 1 5 10 15 Ala Gly Glu Gly Gly Glu Asn 20 69910PRTHUMAN 699Asp Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 10 7008PRTHUMAN 700Glu Lys Ile Glu Thr Glu Leu Arg 1 5 70118PRTHUMAN 701Gly Ile Val Asp Gln Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile 1 5 10 15 Ser Lys 70219PRTHUMAN 702Gly Ile Val Asp Gln Ser Gln Gln Ala Tyr Gln Glu Ala Phe Glu Ile 1 5 10 15 Ser Lys Lys 7037PRTHUMAN 703Leu Ala Glu Gln Ala Glu Arg 1 5 7049PRTHUMAN 704Met Asp Lys Asn Glu Leu Val Gln Lys 1 5 7058PRTHUMAN 705Asn Leu Leu Ser Val Ala Tyr Lys 1 5 70616PRTHUMAN 706Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly Glu Gly Gly Glu Asn 1 5 10 15 7079PRTHUMAN 707Ser Thr Leu Ile Met Gln Leu Leu Arg 1 5 70814PRTHUMAN 708Ser Val Thr Glu Gln Gly Ala Glu Leu Ser Asn Glu Glu Arg 1 5 10 70919PRTHUMAN 709Thr Ala Phe Asp Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu 1 5 10 15 Ser Tyr Lys 7108PRTHUMAN 710Val Val Ser Ser Ile Glu Gln Lys 1 5 7119PRTHUMAN 711Tyr Asp Asp Met Ala Ala Cys Met Lys 1 5 71212PRTHUMAN 712Tyr Leu Ala Glu Val Ala Ala Gly Asp Asp Lys Lys 1 5 10 71310PRTHUMAN 713Ala Glu Ser Met Leu Gln Gln Ala Asp Lys 1 5 10 71414PRTHUMAN 714His Val Ile Pro Met Asn Pro Asn Thr Asp Asp Leu Phe Lys 1 5 10 71516PRTHUMAN 715Ile Asp Ile Asn Met Ser Gly Phe Asn Glu Thr Asp Asp Leu Lys Arg 1

5 10 15 7169PRTHUMAN 716Ile Lys Val Pro Val Asp Trp Ser Lys 1 5 71713PRTHUMAN 717Met Ile Asn Leu Ser Val Pro Asp Thr Ile Asp Glu Arg 1 5 10 71814PRTHUMAN 718Ser Gly Asn Leu Thr Glu Asp Asp Lys His Asn Asn Ala Lys 1 5 10 71925PRTHUMAN 719Thr Ile Ser Ser Ser Leu Ala Trp Asp Leu Ile Asp Ala Ile Gln Pro 1 5 10 15 Gly Cys Ile Asn Tyr Asp Leu Val Lys 20 25 7208PRTHUMAN 720Val Asn Lys Pro Pro Tyr Pro Lys 1 5 72114PRTHUMAN 721Val Tyr Ala Leu Pro Glu Asp Leu Val Glu Val Lys Pro Lys 1 5 10 72215PRTHUMAN 722Ala Leu Thr Val Pro Glu Leu Thr Gln Gln Met Phe Asp Ala Lys 1 5 10 15 72315PRTHUMAN 723Ala Val Leu Val Asp Leu Glu Pro Gly Thr Met Asp Ser Val Arg 1 5 10 15 72417PRTHUMAN 724Glu Ile Val His Leu Gln Ala Gly Gln Cys Gly Asn Gln Ile Gly Ala 1 5 10 15 Lys 72518PRTHUMAN 725Gly His Tyr Thr Glu Gly Ala Glu Leu Val Asp Ser Val Leu Asp Val 1 5 10 15 Val Arg 72612PRTHUMAN 726Ile Met Asn Thr Phe Ser Val Val Pro Ser Pro Lys 1 5 10 7278PRTHUMAN 727Ile Arg Glu Glu Tyr Pro Asp Arg 1 5 72814PRTHUMAN 728Leu His Phe Phe Met Pro Gly Phe Ala Pro Leu Thr Ser Arg 1 5 10 72925PRTHUMAN 729Leu Thr Thr Pro Thr Tyr Gly Asp Leu Asn His Leu Val Ser Ala Thr 1 5 10 15 Met Ser Gly Val Thr Thr Cys Leu Arg 20 25 73026PRTHUMAN 730Ser Gly Pro Phe Gly Gln Ile Phe Arg Pro Asp Asn Phe Val Phe Gly 1 5 10 15 Gln Ser Gly Ala Gly Asn Asn Trp Ala Lys 20 25 7319PRTHUMAN 731Thr Ala Val Cys Asp Ile Pro Pro Arg 1 5 73215PRTHUMAN 732His His Ala Ala Tyr Val Asn Asn Leu Asn Val Thr Glu Glu Lys 1 5 10 15 73314PRTHUMAN 733Ala Ser Ala Glu Thr Val Asp Pro Ala Ser Leu Trp Glu Tyr 1 5 10 73418PRTHUMAN 734Asp Val Pro Trp Gly Val Asp Ser Leu Ile Thr Leu Ala Phe Gln Asp 1 5 10 15 Gln Arg 7358PRTHUMAN 735Glu Val Pro Gly Pro Asp Cys Arg 1 5 73610PRTHUMAN 736Phe Leu Ile Val Ala His Asp Asp Gly Arg 1 5 10 73710PRTHUMAN 737Lys Val Thr Gly Thr Leu Asp Ala Asn Arg 1 5 10 73816PRTHUMAN 738Leu Val Ala Arg Pro Glu Pro Ala Thr Gly Tyr Thr Leu Glu Phe Arg 1 5 10 15 7399PRTHUMAN 739Trp Ser Leu Gln Ser Glu Ala His Arg 1 5 74014PRTHUMAN 740Tyr Leu Ala Ala Asp Lys Asp Gly Asn Val Thr Cys Glu Arg 1 5 10 74120DNAHUMAN 741atgtacttgg aaaaaggccg 2074220DNAHUMAN 742ccctgctctt gaggagctta 2074323DNAHUMAN 743agagacacag agtccggcat tgg 2374424DNAHUMAN 744tccaccttct cccggtactc acgc 2474524DNAHUMAN 745cttccttagt gcctgtgaca aaaa 2474628DNAHUMAN 746aaggacagaa actcagaaaa atcaatct 2874725DNAHUMAN 747atgtcagacg cagccgtaga cacca 2574824DNAHUMAN 748ctagtcatcc tcgtcggtct tctg 2474924DNAHUMAN 749agcagagctc ggaatcttcc tctt 2475024DNAHUMAN 750atcagcactg aaaccaacca tgcc 2475123DNAHUMAN 751cagccatgta cgttgctatc cag 2375221DNAHUMAN 752gtttcgtgga tgccacagga c 2175398PRTHUMAN 753Met Met Cys Ser Ser Leu Glu Gln Ala Leu Ala Val Leu Val Thr Thr 1 5 10 15 Phe His Lys Tyr Ser Cys Gln Glu Gly Asp Lys Phe Lys Leu Ser Lys 20 25 30 Gly Glu Met Lys Glu Leu Leu His Lys Glu Leu Pro Ser Phe Val Gly 35 40 45 Glu Lys Val Asp Glu Glu Gly Leu Lys Lys Leu Met Gly Ser Leu Asp 50 55 60 Glu Asn Ser Asp Gln Gln Val Asp Phe Gln Glu Tyr Ala Val Phe Leu 65 70 75 80 Ala Leu Ile Thr Val Met Cys Asn Asp Phe Phe Gln Gly Cys Pro Asp 85 90 95 Arg Pro 754500PRTHUMAN 754Ser Ala Ser Ser Thr Ala Thr Met Thr Ala Asn Gly Thr Ala Glu Ala 1 5 10 15 Val Gln Ile Gln Phe Gly Leu Ile Asn Cys Gly Asn Lys Tyr Leu Thr 20 25 30 Ala Glu Ala Phe Gly Phe Lys Val Asn Ala Ser Ala Ser Ser Leu Lys 35 40 45 Lys Lys Gln Ile Trp Thr Leu Glu Gln Pro Pro Asp Glu Ala Gly Ser 50 55 60 Ala Ala Val Cys Leu Arg Ser His Leu Gly Arg Tyr Leu Ala Ala Asp 65 70 75 80 Lys Asp Gly Asn Val Thr Cys Glu Arg Glu Val Pro Gly Pro Asp Cys 85 90 95 Arg Phe Leu Ile Val Ala His Asp Asp Gly Arg Trp Ser Leu Gln Ser 100 105 110 Glu Ala His Arg Arg Tyr Phe Gly Gly Thr Glu Asp Arg Leu Ser Cys 115 120 125 Phe Ala Gln Thr Val Ser Pro Ala Glu Lys Trp Ser Val His Ile Ala 130 135 140 Met His Pro Gln Val Asn Ile Tyr Ser Val Thr Arg Lys Arg Tyr Ala 145 150 155 160 His Leu Ser Ala Arg Pro Ala Asp Glu Ile Ala Val Asp Arg Asp Val 165 170 175 Pro Trp Gly Val Asp Ser Leu Ile Thr Leu Ala Phe Gln Asp Gln Arg 180 185 190 Tyr Ser Val Gln Thr Ala Asp His Arg Phe Leu Arg His Asp Gly Arg 195 200 205 Leu Val Ala Arg Pro Glu Pro Ala Thr Gly Tyr Thr Leu Glu Phe Arg 210 215 220 Ser Gly Lys Val Ala Phe Arg Asp Cys Glu Gly Arg Tyr Leu Ala Pro 225 230 235 240 Ser Gly Pro Ser Gly Thr Leu Lys Ala Gly Lys Ala Thr Lys Val Gly 245 250 255 Lys Asp Glu Leu Phe Ala Leu Glu Gln Ser Cys Ala Gln Val Val Leu 260 265 270 Gln Ala Ala Asn Glu Arg Asn Val Ser Thr Arg Gln Gly Met Asp Leu 275 280 285 Ser Ala Asn Gln Asp Glu Glu Thr Asp Gln Glu Thr Phe Gln Leu Glu 290 295 300 Ile Asp Arg Asp Thr Lys Lys Cys Ala Phe Arg Thr His Thr Gly Lys 305 310 315 320 Tyr Trp Thr Leu Thr Ala Thr Gly Gly Val Gln Ser Thr Ala Ser Ser 325 330 335 Lys Asn Ala Ser Cys Tyr Phe Asp Ile Glu Trp Arg Asp Arg Arg Ile 340 345 350 Thr Leu Arg Ala Ser Asn Gly Lys Phe Val Thr Ser Lys Lys Asn Gly 355 360 365 Gln Leu Ala Ala Ser Val Glu Thr Ala Gly Asp Ser Glu Leu Phe Leu 370 375 380 Met Lys Leu Ile Asn Arg Pro Ile Ile Val Phe Arg Gly Glu His Gly 385 390 395 400 Phe Ile Gly Cys Arg Lys Val Thr Gly Thr Leu Asp Ala Asn Arg Ser 405 410 415 Ser Tyr Asp Val Phe Gln Leu Glu Phe Asn Asp Gly Ala Tyr Asn Ile 420 425 430 Lys Asp Ser Thr Gly Lys Tyr Trp Thr Val Gly Ser Asp Ser Ala Val 435 440 445 Thr Ser Ser Gly Asp Thr Pro Val Asp Phe Phe Phe Glu Phe Cys Asp 450 455 460 Tyr Asn Lys Val Ala Ile Lys Val Gly Gly Arg Tyr Leu Lys Gly Asp 465 470 475 480 His Ala Gly Val Leu Lys Ala Ser Ala Glu Thr Val Asp Pro Ala Ser 485 490 495 Leu Trp Glu Tyr 500 755375PRTHUMAN 755Met Asp Ala Leu Gln Leu Ala Asn Ser Ala Phe Ala Val Asp Leu Phe 1 5 10 15 Lys Gln Leu Cys Glu Lys Glu Pro Leu Gly Asn Val Leu Phe Ser Pro 20 25 30 Ile Cys Leu Ser Thr Ser Leu Ser Leu Ala Gln Val Gly Ala Lys Gly 35 40 45 Asp Thr Ala Asn Glu Ile Gly Gln Val Leu His Phe Glu Asn Val Lys 50 55 60 Asp Ile Pro Phe Gly Phe Gln Thr Val Thr Ser Asp Val Asn Lys Leu 65 70 75 80 Ser Ser Phe Tyr Ser Leu Lys Leu Ile Lys Arg Leu Tyr Val Asp Lys 85 90 95 Ser Leu Asn Leu Ser Thr Glu Phe Ile Ser Ser Thr Lys Arg Pro Tyr 100 105 110 Ala Lys Glu Leu Glu Thr Val Asp Phe Lys Asp Lys Leu Glu Glu Thr 115 120 125 Lys Gly Gln Ile Asn Asn Ser Ile Lys Asp Leu Thr Asp Gly His Phe 130 135 140 Glu Asn Ile Leu Ala Asp Asn Ser Val Asn Asp Gln Thr Lys Ile Leu 145 150 155 160 Val Val Asn Ala Ala Tyr Phe Val Gly Lys Trp Met Lys Lys Phe Pro 165 170 175 Glu Ser Glu Thr Lys Glu Cys Pro Phe Arg Leu Asn Lys Thr Asp Thr 180 185 190 Lys Pro Val Gln Met Met Asn Met Glu Ala Thr Phe Cys Met Gly Asn 195 200 205 Ile Asp Ser Ile Asn Cys Lys Ile Ile Glu Leu Pro Phe Gln Asn Lys 210 215 220 His Leu Ser Met Phe Ile Leu Leu Pro Lys Asp Val Glu Asp Glu Ser 225 230 235 240 Thr Gly Leu Glu Lys Ile Glu Lys Gln Leu Asn Ser Glu Ser Leu Ser 245 250 255 Gln Trp Thr Asn Pro Ser Thr Met Ala Asn Ala Lys Val Lys Leu Ser 260 265 270 Ile Pro Lys Phe Lys Val Glu Lys Met Ile Asp Pro Lys Ala Cys Leu 275 280 285 Glu Asn Leu Gly Leu Lys His Ile Phe Ser Glu Asp Thr Ser Asp Phe 290 295 300 Ser Gly Met Ser Glu Thr Lys Gly Val Ala Leu Ser Asn Val Ile His 305 310 315 320 Lys Val Cys Leu Glu Ile Thr Glu Asp Gly Gly Asp Ser Ile Glu Val 325 330 335 Pro Gly Ala Arg Ile Leu Gln His Lys Asp Glu Leu Asn Ala Asp His 340 345 350 Pro Phe Ile Tyr Ile Ile Arg His Asn Lys Thr Arg Asn Ile Ile Phe 355 360 365 Phe Gly Lys Phe Cys Ser Pro 370 375 756327PRTHUMAN 756Met Ala Trp Trp Lys Ala Trp Ile Glu Gln Glu Gly Val Thr Val Lys 1 5 10 15 Ser Ser Ser His Phe Asn Pro Asp Pro Asp Ala Glu Thr Leu Tyr Lys 20 25 30 Ala Met Lys Gly Ile Gly Thr Asn Glu Gln Ala Ile Ile Asp Val Leu 35 40 45 Thr Lys Arg Ser Asn Thr Gln Arg Gln Gln Ile Ala Lys Ser Phe Lys 50 55 60 Ala Gln Phe Gly Lys Asp Leu Thr Glu Thr Leu Lys Ser Glu Leu Ser 65 70 75 80 Gly Lys Phe Glu Arg Leu Ile Val Ala Leu Met Tyr Pro Pro Tyr Arg 85 90 95 Tyr Glu Ala Lys Glu Leu His Asp Ala Met Lys Gly Leu Gly Thr Lys 100 105 110 Glu Gly Val Ile Ile Glu Ile Leu Ala Ser Arg Thr Lys Asn Gln Leu 115 120 125 Arg Glu Ile Met Lys Ala Tyr Glu Glu Asp Tyr Gly Ser Ser Leu Glu 130 135 140 Glu Asp Ile Gln Ala Asp Thr Ser Gly Tyr Leu Glu Arg Ile Leu Val 145 150 155 160 Cys Leu Leu Gln Gly Ser Arg Asp Asp Val Ser Ser Phe Val Asp Pro 165 170 175 Gly Leu Ala Leu Gln Asp Ala Gln Asp Leu Tyr Ala Ala Gly Glu Lys 180 185 190 Ile Arg Gly Thr Asp Glu Met Lys Phe Ile Thr Ile Leu Cys Thr Arg 195 200 205 Ser Ala Thr His Leu Leu Arg Val Phe Glu Glu Tyr Glu Lys Ile Ala 210 215 220 Asn Lys Ser Ile Glu Asp Ser Ile Lys Ser Glu Thr His Gly Ser Leu 225 230 235 240 Glu Glu Ala Met Leu Thr Val Val Lys Cys Thr Gln Asn Leu His Ser 245 250 255 Tyr Phe Ala Glu Arg Leu Tyr Tyr Ala Met Lys Gly Ala Gly Thr Arg 260 265 270 Asp Gly Thr Leu Ile Arg Asn Ile Val Ser Arg Ser Glu Ile Asp Leu 275 280 285 Asn Leu Ile Lys Cys His Phe Lys Lys Met Tyr Gly Lys Thr Leu Ser 290 295 300 Ser Met Ile Met Glu Asp Thr Ser Gly Asp Tyr Lys Asn Ala Leu Leu 305 310 315 320 Ser Leu Val Gly Ser Asp Pro 325 757210PRTHUMAN 757Met Pro Pro Tyr Thr Val Val Tyr Phe Pro Val Arg Gly Arg Cys Ala 1 5 10 15 Ala Leu Arg Met Leu Leu Ala Asp Gln Gly Gln Ser Trp Lys Glu Glu 20 25 30 Val Val Thr Val Glu Thr Trp Gln Glu Gly Ser Leu Lys Ala Ser Cys 35 40 45 Leu Tyr Gly Gln Leu Pro Lys Phe Gln Asp Gly Asp Leu Thr Leu Tyr 50 55 60 Gln Ser Asn Thr Ile Leu Arg His Leu Gly Arg Thr Leu Gly Leu Tyr 65 70 75 80 Gly Lys Asp Gln Gln Glu Ala Ala Leu Val Asp Met Val Asn Asp Gly 85 90 95 Val Glu Asp Leu Arg Cys Lys Tyr Val Ser Leu Ile Tyr Thr Asn Tyr 100 105 110 Glu Val Gly Lys Asp Asp Tyr Val Lys Ala Leu Pro Gly Gln Leu Lys 115 120 125 Pro Phe Glu Thr Leu Leu Ser Gln Asn Gln Gly Gly Lys Thr Phe Ile 130 135 140 Val Gly Asp Gln Ile Ser Phe Ala Asp Tyr Asn Leu Leu Asp Leu Leu 145 150 155 160 Leu Ile His Glu Val Leu Ala Pro Gly Cys Leu Asp Ala Phe Pro Leu 165 170 175 Leu Ser Ala Tyr Val Gly Arg Leu Ser Ala Arg Pro Lys Leu Lys Ala 180 185 190 Phe Leu Ala Ser Pro Glu Tyr Val Asn Leu Pro Ile Asn Gly Asn Gly 195 200 205 Lys Gln 210 758466PRTHUMAN 758Met Ser Thr Arg Ser Val Ser Ser Ser Ser Tyr Arg Arg Met Phe Gly 1 5 10 15 Gly Pro Gly Thr Ala Ser Arg Pro Ser Ser Ser Arg Ser Tyr Val Thr 20 25 30 Thr Ser Thr Arg Thr Tyr Ser Leu Gly Ser Ala Leu Arg Pro Ser Thr 35 40 45 Ser Arg Ser Leu Tyr Ala Ser Ser Pro Gly Gly Val Tyr Ala Thr Arg 50 55 60 Ser Ser Ala Val Arg Leu Arg Ser Ser Val Pro Gly Val Arg Leu Leu 65 70 75 80 Gln Asp Ser Val Asp Phe Ser Leu Ala Asp Ala Ile Asn Thr Glu Phe 85 90 95 Lys Asn Thr Arg Thr Asn Glu Lys Val Glu Leu Gln Glu Leu Asn Asp 100 105 110 Arg Phe Ala Asn Tyr Ile Asp Lys Val Arg Phe Leu Glu Gln Gln Asn 115 120 125 Lys Ile Leu Leu Ala Glu Leu Glu Gln Leu Lys Gly Gln Gly Lys Ser 130 135 140 Arg Leu Gly Asp Leu Tyr Glu Glu Glu Met Arg Glu Leu Arg Arg Gln 145 150 155 160 Val Asp Gln Leu Thr Asn Asp Lys Ala Arg Val Glu Val Glu Arg Asp 165 170 175 Asn Leu Ala Glu Asp Ile Met Arg Leu Arg Glu Lys Leu Gln Glu Glu 180 185 190 Met Leu Gln Arg Glu Glu Ala Glu Asn Thr Leu Gln Ser Phe Arg Gln 195 200 205 Asp Val Asp Asn Ala Ser Leu Ala Arg Leu Asp Leu Glu Arg Lys Val 210 215 220 Glu Ser Leu Gln Glu

Glu Ile Ala Phe Leu Lys Lys Leu His Glu Glu 225 230 235 240 Glu Ile Gln Glu Leu Gln Ala Gln Ile Gln Glu Gln His Val Gln Ile 245 250 255 Asp Val Asp Val Ser Lys Pro Asp Leu Thr Ala Ala Leu Arg Asp Val 260 265 270 Arg Gln Gln Tyr Glu Ser Val Ala Ala Lys Asn Leu Gln Glu Ala Glu 275 280 285 Glu Trp Tyr Lys Ser Lys Phe Ala Asp Leu Ser Glu Ala Ala Asn Arg 290 295 300 Asn Asn Asp Ala Leu Arg Gln Ala Lys Gln Glu Ser Thr Glu Tyr Arg 305 310 315 320 Arg Gln Val Gln Ser Leu Thr Cys Glu Val Asp Ala Leu Lys Gly Thr 325 330 335 Asn Glu Ser Leu Glu Arg Gln Met Arg Glu Met Glu Glu Asn Phe Ala 340 345 350 Val Glu Ala Ala Asn Tyr Gln Asp Thr Ile Gly Arg Leu Gln Asp Glu 355 360 365 Ile Gln Asn Met Lys Glu Glu Met Ala Arg His Leu Arg Glu Tyr Gln 370 375 380 Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile Glu Ile Ala Thr Tyr 385 390 395 400 Arg Lys Leu Leu Glu Gly Glu Glu Ser Arg Ile Ser Leu Pro Leu Pro 405 410 415 Asn Phe Ser Ser Leu Asn Leu Arg Glu Thr Asn Leu Asp Ser Leu Pro 420 425 430 Leu Val Asp Thr His Ser Lys Arg Thr Leu Leu Ile Lys Thr Val Glu 435 440 445 Thr Arg Asp Gly Gln Val Ile Asn Glu Thr Ser Gln His His Asp Asp 450 455 460 Leu Glu 465 759206PRTHUMAN 759Met Ala Val Lys Lys Ile Ala Ile Phe Gly Ala Thr Gly Gln Thr Gly 1 5 10 15 Leu Thr Thr Leu Ala Gln Ala Val Gln Ala Gly Tyr Glu Val Thr Val 20 25 30 Leu Val Arg Asp Ser Ser Arg Leu Pro Ser Glu Gly Pro Arg Pro Ala 35 40 45 His Val Val Val Gly Asp Val Leu Gln Ala Ala Asp Val Asp Lys Thr 50 55 60 Val Ala Gly Gln Asp Ala Val Ile Val Leu Leu Gly Thr Arg Asn Asp 65 70 75 80 Leu Ser Pro Thr Thr Val Met Ser Glu Gly Ala Arg Asn Ile Val Ala 85 90 95 Ala Met Lys Ala His Gly Val Asp Lys Val Val Ala Cys Thr Ser Ala 100 105 110 Phe Leu Leu Trp Asp Pro Thr Lys Val Pro Pro Arg Leu Gln Ala Val 115 120 125 Thr Asp Asp His Ile Arg Met His Lys Val Leu Arg Glu Ser Gly Leu 130 135 140 Lys Tyr Val Ala Val Met Pro Pro His Ile Gly Asp Gln Pro Leu Thr 145 150 155 160 Gly Ala Tyr Thr Val Thr Leu Asp Gly Arg Gly Pro Ser Arg Val Ile 165 170 175 Ser Lys His Asp Leu Gly His Phe Met Leu Arg Cys Leu Thr Thr Asp 180 185 190 Glu Tyr Asp Gly His Ser Thr Tyr Pro Ser His Gln Tyr Gln 195 200 205 760472PRTHUMAN 760Met Thr Thr Cys Ser Arg Gln Phe Thr Ser Ser Ser Ser Met Lys Gly 1 5 10 15 Ser Cys Gly Ile Gly Gly Gly Ile Gly Gly Gly Ser Ser Arg Ile Ser 20 25 30 Ser Val Leu Ala Gly Gly Ser Cys Arg Ala Pro Ser Thr Tyr Gly Gly 35 40 45 Gly Leu Ser Val Ser Ser Ser Arg Phe Ser Ser Gly Gly Ala Tyr Gly 50 55 60 Leu Gly Gly Gly Tyr Gly Gly Gly Phe Ser Ser Ser Ser Ser Ser Phe 65 70 75 80 Gly Ser Gly Phe Gly Gly Gly Tyr Gly Gly Gly Leu Gly Ala Gly Leu 85 90 95 Gly Gly Gly Phe Gly Gly Gly Phe Ala Gly Gly Asp Gly Leu Leu Val 100 105 110 Gly Ser Glu Lys Val Thr Met Gln Asn Leu Asn Asp Arg Leu Ala Ser 115 120 125 Tyr Leu Asp Lys Val Arg Ala Leu Glu Glu Ala Asn Ala Asp Leu Glu 130 135 140 Val Lys Ile Arg Asp Trp Tyr Gln Arg Gln Arg Pro Ala Glu Ile Lys 145 150 155 160 Asp Tyr Ser Pro Tyr Phe Lys Thr Ile Glu Asp Leu Arg Asn Lys Ile 165 170 175 Leu Thr Ala Thr Val Asp Asn Ala Asn Val Leu Leu Gln Ile Asp Asn 180 185 190 Ala Arg Leu Ala Ala Asp Asp Phe Arg Thr Lys Tyr Glu Thr Glu Leu 195 200 205 Asn Leu Arg Met Ser Val Glu Ala Asp Ile Asn Gly Leu Arg Arg Val 210 215 220 Leu Asp Glu Leu Thr Leu Ala Arg Ala Asp Leu Glu Met Gln Ile Glu 225 230 235 240 Ser Leu Lys Glu Glu Leu Ala Tyr Leu Lys Lys Asn His Glu Glu Glu 245 250 255 Met Asn Ala Leu Arg Gly Gln Val Gly Gly Asp Val Asn Val Glu Met 260 265 270 Asp Ala Ala Pro Gly Val Asp Leu Ser Arg Ile Leu Asn Glu Met Arg 275 280 285 Asp Gln Tyr Glu Lys Met Ala Glu Lys Asn Arg Lys Asp Ala Glu Glu 290 295 300 Trp Phe Phe Thr Lys Thr Glu Glu Leu Asn Arg Glu Val Ala Thr Asn 305 310 315 320 Ser Glu Leu Val Gln Ser Gly Lys Ser Glu Ile Ser Glu Leu Arg Arg 325 330 335 Thr Met Gln Asn Leu Glu Ile Glu Leu Gln Ser Gln Leu Ser Met Lys 340 345 350 Ala Ser Leu Glu Asn Ser Leu Glu Glu Thr Lys Gly Arg Tyr Cys Met 355 360 365 Gln Leu Ala Gln Ile Gln Glu Met Ile Gly Ser Val Glu Glu Gln Leu 370 375 380 Ala Gln Leu Arg Cys Glu Met Glu Gln Gln Asn Gln Glu Tyr Lys Ile 385 390 395 400 Leu Leu Asp Val Lys Thr Arg Leu Glu Gln Glu Ile Ala Thr Tyr Arg 405 410 415 Arg Leu Leu Glu Gly Glu Asp Ala His Leu Ser Ser Ser Gln Phe Ser 420 425 430 Ser Gly Ser Gln Ser Ser Arg Asp Val Thr Ser Ser Ser Arg Gln Ile 435 440 445 Arg Thr Lys Val Met Asp Val His Asp Gly Lys Val Val Ser Thr His 450 455 460 Glu Gln Val Leu Arg Thr Lys Asn 465 470 761346PRTHUMAN 761Met Ala Met Val Ser Glu Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn 1 5 10 15 Glu Glu Gln Glu Tyr Val Gln Thr Val Lys Ser Ser Lys Gly Gly Pro 20 25 30 Gly Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro Ser Ser Asp Val 35 40 45 Ala Ala Leu His Lys Ala Ile Met Val Lys Gly Val Asp Glu Ala Thr 50 55 60 Ile Ile Asp Ile Leu Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln Ile 65 70 75 80 Lys Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu 85 90 95 Lys Lys Ala Leu Thr Gly His Leu Glu Glu Val Val Leu Ala Leu Leu 100 105 110 Lys Thr Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala Ala Met Lys 115 120 125 Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg 130 135 140 Thr Asn Lys Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu 145 150 155 160 Lys Arg Asp Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe 165 170 175 Arg Asn Ala Leu Leu Ser Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe 180 185 190 Gly Val Asn Glu Asp Leu Ala Asp Ser Asp Ala Arg Ala Leu Tyr Glu 195 200 205 Ala Gly Glu Arg Arg Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile 210 215 220 Leu Thr Thr Arg Ser Tyr Pro Gln Leu Arg Arg Val Phe Gln Lys Tyr 225 230 235 240 Thr Lys Tyr Ser Lys His Asp Met Asn Lys Val Leu Asp Leu Glu Leu 245 250 255 Lys Gly Asp Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys Ala Thr 260 265 270 Ser Lys Pro Ala Phe Phe Ala Glu Lys Leu His Gln Ala Met Lys Gly 275 280 285 Val Gly Thr Arg His Lys Ala Leu Ile Arg Ile Met Val Ser Arg Ser 290 295 300 Glu Ile Asp Met Asn Asp Ile Lys Ala Phe Tyr Gln Lys Met Tyr Gly 305 310 315 320 Ile Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr Lys Gly Asp Tyr Glu 325 330 335 Lys Ile Leu Val Ala Leu Cys Gly Gly Asn 340 345 762153PRTHUMAN 762Ala Thr Lys Ala Val Cys Val Leu Lys Gly Asx Gly Pro Val Glx Gly 1 5 10 15 Ile Ile Asx Phe Glx Glx Lys Glu Ser Asn Gly Pro Val Lys Val Trp 20 25 30 Gly Ser Ile Lys Gly Leu Thr Glu Gly Leu His Gly Phe His Val His 35 40 45 Glu Phe Gly Asp Asn Thr Ala Gly Cys Thr Ser Ala Gly Pro His Phe 50 55 60 Asn Pro Leu Ser Arg Lys His Gly Gly Pro Lys Asp Glu Glu Arg His 65 70 75 80 Val Gly Asx Leu Gly Asx Val Thr Ala Asx Lys Asx Gly Val Ala Asx 85 90 95 Val Ser Ile Glx Asx Ser Val Ile Ser Leu Ser Gly Asx His Cys Ile 100 105 110 Ile Gly Arg Thr Leu Val Val His Glu Lys Ala Asp Asp Leu Gly Lys 115 120 125 Gly Gly Asn Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg Leu 130 135 140 Ala Cys Gly Val Ile Gly Ile Ala Gln 145 150 763320PRTHUMAN 763Arg Pro Leu Pro Ser Pro Ser Ala Ala Pro Ser Phe Pro Ser Pro Ser 1 5 10 15 Arg Ser Leu Arg Ala Gly Arg Cys Gly Gly Gly Thr Arg Pro Ser Pro 20 25 30 Thr Pro Gly Gly Gly Gly Ala Ala Gly Arg Gly Arg Gly Pro Arg Gly 35 40 45 Pro Lys Arg Glu Gly Lys Ala Trp Pro Arg Ala Gly Arg Ser Pro Gly 50 55 60 Leu Ala Arg Arg Arg Pro Pro Glu His Pro Val Met Asp Lys Asn Glu 65 70 75 80 Leu Val Gln Lys Ala Lys Leu Ala Glu Gln Ala Glu Arg Tyr Asp Asp 85 90 95 Met Ala Ala Cys Met Lys Ser Val Thr Glu Gln Gly Ala Glu Leu Ser 100 105 110 Asn Glu Glu Arg Asn Leu Leu Ser Val Ala Tyr Lys Asn Val Val Gly 115 120 125 Ala Arg Arg Ser Ser Trp Arg Val Val Ser Ser Ile Glu Gln Lys Thr 130 135 140 Glu Gly Ala Glu Lys Lys Gln Gln Met Ala Arg Glu Tyr Arg Glu Lys 145 150 155 160 Ile Glu Thr Glu Leu Arg Asp Ile Cys Asn Asp Val Leu Ser Leu Leu 165 170 175 Glu Lys Phe Leu Ile Pro Asn Ala Ser Gln Ala Glu Ser Lys Val Phe 180 185 190 Tyr Leu Lys Met Lys Gly Asp Tyr Tyr Arg Tyr Leu Ala Glu Val Ala 195 200 205 Ala Gly Asp Asp Lys Lys Gly Ile Val Asp Gln Ser Gln Gln Ala Tyr 210 215 220 Gln Glu Ala Phe Glu Ile Ser Lys Lys Glu Met Gln Pro Thr His Pro 225 230 235 240 Ile Arg Leu Gly Leu Ala Leu Asn Phe Ser Val Phe Tyr Tyr Glu Ile 245 250 255 Leu Asn Ser Pro Glu Lys Ala Cys Ser Leu Ala Lys Thr Ala Phe Asp 260 265 270 Glu Ala Ile Ala Glu Leu Asp Thr Leu Ser Glu Glu Ser Tyr Lys Asp 275 280 285 Ser Thr Leu Ile Met Gln Leu Leu Arg Asp Asn Leu Thr Leu Trp Thr 290 295 300 Ser Asp Thr Gln Gly Asp Glu Ala Glu Ala Gly Glu Gly Gly Glu Asn 305 310 315 320

Claims

1. A method for detecting a head-and-neck disease in a subject, the method comprising comparing: (a) levels of one or more OPL or head-and-neck cancer markers listed in Table 5 that are extracted from a sample from the subject; and (b) normal levels of expression of the OPL or head-and-neck cancer markers in a control sample, wherein a significant difference in levels of OPL or head-and-neck cancer markers, relative to the corresponding normal levels, is indicative of head-and-neck disease.

2. A method according to claim 1, comprising: (a) contacting a biological sample obtained from a subject with one or more binding agent that specifically binds to the OPL or head-and-neck cancer markers or parts thereof; and (b) detecting in the sample amounts of OPL or head-and-neck cancer markers that bind to the binding agents, relative to a predetermined standard or cut-off value, and thereby determining the presence or absence of the head-and-neck disease in the subject.

3. A method according to claim 2, wherein the binding agent is an antibody.

4. A method according to claim 1, wherein detection of a level of OPL or head- and-neck cancer markers that is significantly higher compared to the standard is indicative of OPL or head-and-neck cancer.

5. A method according to claim 1, wherein detection of a level of OPL or head- and-neck cancer markers that is significantly lower compared to the standard is indicative of OPL or head-and-neck cancer.

6. A method according to claim 1, wherein the sample is obtained from tissues, extracts, cell cultures, cell lysates, lavage fluid, or physiological fluids.

7. A method according to claim 6, wherein the sample is obtained from an OPL or tumor tissue.

8. A method of claim 1, wherein the one or more OPL or head-and-neck cancer markers comprises one or more of YWHAZ, S100A7, hnRNPK and stratifin.

9. A method for assessing the aggressiveness or indolence of OPL or head-and-neck cancer, comprising comparing: (a) levels of expression of one or more OPL or head-and-neck cancer markers or polynucleotides encoding the markers listed in Table 1 and Table 6 in a subject sample; and (b) normal levels of expression of the OPL or head-and-neck cancer markers or polynucleotides encoding the markers, in a control sample, wherein a significant difference between the levels in the subject sample and normal levels is an indication that the OPL or cancer is aggressive or indolent.

10. A method according to claim 9, comprising: (a) contacting a biological sample obtained from a subject with one or more binding agent that specifically binds to the OPL or head-and-neck cancer markers or parts thereof; and (b) detecting in the sample amounts of OPL or head-and-neck cancer markers that bind to the binding agents, relative to a predetermined standard or cut-off value, and thereby determining the presence or absence of the head-and-neck disease in the subject.

11. A method according to claim 10, wherein the binding agent is an antibody.

12. A method according to claim 9, wherein detection of a level of OPL or head- and-neck cancer markers that is significantly higher compared to the standard is indicative of OPL or head-and-neck cancer.

13. A method according to claim 9, wherein detection of a level of OPL or head- and-neck cancer markers that is significantly lower compared to the standard is indicative of OPL or head-and-neck cancer.

14. A method according to claim 9, wherein the sample is obtained from tissues, extracts, cell cultures, cell lysates, lavage fluid, or physiological fluids.

15. A method according to claim 7, wherein the sample is obtained from an OPL or tumor tissue.

16. The method of claim 9, wherein the one or more OPL or head-and-neck cancer markers or polynucleotides encoding the marker comprises one or more of YWHAZ, S100A7, hnRNPK and stratifin.

17. A diagnostic composition or kit comprising (a) an agent that binds to an OPL or head-and-neck cancer marker listed in Table 5 or hybridizes to a polynucleotide encoding such marker; or (b) a set of OPL or head-and-neck cancer markers, comprising a plurality of polypeptides comprising or consisting of at least 2, 3, 4, 5, or 6 of the markers listed in Table 5 or Table 2 or Table 6.

18. A diagnostic composition or kit according to claim 17, wherein the polypeptides comprise one or more of YWHAZ, S100A7, and stratifin.

19. A diagnostic composition or kit according to claim 17, wherein the polypeptides comprise one or more of YWHAZ, hnRNPK and stratifin.

20. A diagnostic composition or kit according to claim 17, wherein the binding agent comprises a detectable substance or binds directly or indirectly to a detectable substance.