U.S. patent application number 14/334855 was filed with the patent office on 2015-01-22 for molecular diagnosis and typing of lung cancer variants.
The applicant listed for this patent is University of North Carolina at Chapel Hill, University of Utah Research Foundation. Invention is credited to PHILIP BERNARD, DAVID N. HAYES, CHARLES M. PEROU.
Application Number | 20150024399 14/334855 |
Document ID | / |
Family ID | 39687112 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150024399 |
Kind Code |
A1 |
HAYES; DAVID N. ; et
al. |
January 22, 2015 |
MOLECULAR DIAGNOSIS AND TYPING OF LUNG CANCER VARIANTS
Abstract
Compositions and methods useful in determining the major
morphological types of lung cancer are provided. The methods
include detecting expression of at least one gene or biomarker in a
sample. The expression of the gene or biomarker is indicative of
the lung tumor subtype. The compositions include subsets of genes
that are monitored for gene expression. The gene expression is
capable of distinguishing between normal lung parenchyma and the
major morphological types of lung cancer. The gene expression and
somatic mutation data are useful in developing a complete
classification of lung cancer that is prognostic and predictive for
therapeutic response. The methods are suited for analysis of
paraffin-embedded tissues. Methods of the invention include means
for monitoring gene or biomarker expression including PCR and
antibody-based detection. The biomarkers of the invention are genes
and/or proteins that are selectively expressed at a high or low
level in certain tumor subtypes. Biomarker expression can be
assessed at the protein or nucleic acid level.
Inventors: |
HAYES; DAVID N.; (CHAPEL
HILL, NC) ; PEROU; CHARLES M.; (CHAPEL HILL, NC)
; BERNARD; PHILIP; (SALT LAKE CITY, UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of North Carolina at Chapel Hill
University of Utah Research Foundation |
Chapel Hill
Salt Lake City |
NC
UT |
US
US |
|
|
Family ID: |
39687112 |
Appl. No.: |
14/334855 |
Filed: |
July 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12602649 |
May 26, 2010 |
8822153 |
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PCT/US08/65489 |
Jun 2, 2008 |
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14334855 |
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60941520 |
Jun 1, 2007 |
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Current U.S.
Class: |
435/6.12 ;
435/7.1 |
Current CPC
Class: |
C12Q 2600/118 20130101;
C12Q 1/686 20130101; C12Q 1/6886 20130101; C12Q 2600/112 20130101;
C12Q 2600/156 20130101; G01N 33/57423 20130101; C12Q 2600/106
20130101 |
Class at
Publication: |
435/6.12 ;
435/7.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method for diagnosing lung cancer in a patient, said method
comprising detecting expression of at least one nuclear biomarker
in a body sample from the patient, wherein the detection of
expression of said nuclear biomarker specifically identifies
samples that are indicative of a lung cancer subtype and wherein
said sample is paraffin embedded.
2. The method of claim 1, wherein detecting expression comprises
using at least one antibody to detect biomarker protein
expression.
3. The method of claim 2, wherein the method comprises performing
immunocytochemistry.
4. The method of claim 1, wherein expression of the nuclear
biomarker is detected at the nucleic acid level.
5. The method of claim 4, wherein detecting expression comprises
performing quantitative RT-PCR.
6. The method of claim 1, wherein the sample comprises lung cells
embedded in paraffin.
7. A method for diagnosing lung cancer in a patient, said method
comprising detecting expression of at least two biomarkers in a
body sample from the patient, wherein the detection of expression
of said biomarkers specifically identifies samples that are
indicative of a lung cancer subtype.
8. The method of claim 7, wherein said nuclear biomarker is a gene
set forth in Table 1.
9. A method for diagnosing lung cancer in a patient, said method
comprising: a) obtaining a lung biopsy sample from said patient; b)
contacting said sample with at least two antibodies, wherein each
of said antibodies specifically binds to a distinct biomarker
protein that is selectively expressed in lung cancer subtypes; and,
c) detecting binding of said antibodies to said biomarker
proteins.
10. A kit comprising at least two antibodies, wherein each of said
antibodies specifically binds to a distinct biomarker protein that
is selectively expressed in lung cancer cells.
11. A method for diagnosing lung cancer in a patient comprising
detecting expression of at least one nucleic acid molecule that
encodes a biomarker that is selectively expressed in lung cancer
cells, wherein the nuclear biomarker is selected from the group
consisting of the biomarker genes set forth in Table 1, the method
comprising: a) obtaining a lung sample from the patient; b)
isolating nucleic acid material from the sample; c) mixing said
nucleic acid material with at least one pair of oligonucleotide
primers specific for the biomarker and a thermostable DNA
polymerase under conditions that are suitable for amplification by
polymerase chain reaction (PCR); d) performing PCR; and, e)
detecting PCR amplification products.
12. The method of claim 11, wherein performing PCR comprises
performing RT-PCR.
13. A kit comprising at least one pair of oligonucleotide primers
specific for a nuclear biomarker that is selectively expressed in
lung cancer.
14. A kit comprising at least two pairs of oligonucleotide primers,
wherein each pair of oligonucleotide primers is specific for a
biomarker that is selectively expressed in a lung cancer subtype.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for characterizing
lung cancer subtypes and for evaluating the prognosis of a subject
inflicted with lung cancer.
BACKGROUND OF THE INVENTION
[0002] Lung cancer is the leading cause of cancer deaths both in
the United States and worldwide. Despite many classification
schemes and ongoing clinical trials, there has been overall
disappointing progress in the field of clinical diagnostics and
therapeutics. Approximately 172,000 tumors of the lung were
diagnosed in 2005 with an estimated 163,000 deaths, more than
colon, breast, and prostate combined. At least 75% of patients
present with locally advanced disease. Although there has been much
effort to improve screening using technology such as
high-resolution CT, these methods often produce false positive
results and usually do not change outcome. Thus, even small tumors
detected early present a significant threat to patients with
postoperative 5-year survival rates for stage I lung cancer
estimated between 47 to 63 percent. For patients with advanced
disease the prognosis is worse with median survivals well under a
year. In general, palliative therapy is effective but not
sustainable and the average impact on overall survival is
approximately 3 months. At the population level the underlying
cause of lung cancer is clearly tobacco use, with 90% of all lung
cancers attributed directly to smoking. Smoking is so tightly
correlated with lung cancer that it confounds definitive
association with most other risk factors; although asbestos, radon,
and a number of lung irritants are generally accepted as lung
cancer risk factors. A genetic association is strongly suspected,
however, the exact mechanism remains to be determined outside of a
select group of rare Mendelian cancer syndromes.
SUMMARY OF THE INVENTION
[0003] Compositions and methods useful in determining the major
morphological types of lung cancer are provided. The methods
comprise detecting expression of at least one gene or biomarker in
a sample. The expression of the gene or biomarker is indicative of
the lung tumor subtype. The compositions comprise subsets of genes
that are monitored for gene expression. The gene expression is
capable of distinguishing between normal lung parenchyma and the
major morphological types of lung cancer. The gene expression and
somatic mutation data are useful in developing a complete
classification of lung cancer that is prognostic and predictive for
therapeutic response. The methods are suited for analysis of
paraffin-embedded tissues.
[0004] Methods of the invention include means for monitoring gene
or biomarker expression including PCR and antibody-based detection.
The biomarkers of the invention are genes and/or proteins that are
selectively expressed at a high or low level in certain tumor
subtypes. Biomarker expression can be assessed at the protein or
nucleic acid level. In some embodiments, immunocytochemistry
techniques are provided that utilize antibodies to detect the
expression of biomarker proteins in samples. In this aspect of the
invention, at least one antibody directed to a specific biomarker
of interest is used. Expression can also be detected by nucleic
acid-based techniques, including, for example, hybridization and
RT-PCR. Kits comprising reagents for practicing the methods of the
invention are further provided.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0005] Having thus described the invention in general terms,
reference will now be made to the accompanying drawings, which are
not necessarily drawn to scale, and wherein:
[0006] FIG. 1 shows protein clustering of the entire set of 152
evaluable NSCLC.
[0007] FIG. 2 shows protein clustering of the adenocarcinoma subset
(n=85).
[0008] FIG. 3 shows protein clustering of the squamous carcinoma
subset (n=53).
[0009] FIG. 4 shows protein clustering of the adenocarcinoma subset
(n=85). 152 of 187 samples were evaluable. 16 samples were excluded
because the tumor or the core was absent, and 19 were excluded due
to <10% cells staining for the marker. Of the 152 evaluable
samples, 85 were adenocarcinoma. The staining patterns of the 85
samples are ordered by hierarchical agglomerative clustering, and
show distinct clustering into three groups (FIG. 1). The Chi square
test p value for this distribution of staining is
p<2.2.times.10.sup.-16.
[0010] FIG. 5 shows survival by tumor subtype. The clinical
phenotype associated with the 3 molecular subtypes of lung
adenocarcinoma reproduces previous work. Most notably, there are
clear differences in the frequency and pattern of recurrence (table
1) by tumor subtype and survival.
[0011] FIG. 6 shows the classification of lung cancer using a
minimized qRT-PCR assay. 52-classifier genes were identified to
determine histological and molecular subtypes of lung cancer.
Up-regulated and down-regulated genes were selected for determining
histological subtypes (A-C), and additional molecular subtypes of
AC comprised of bronchioid, squamoid, and magnoid.
[0012] FIG. 7 shows a consensus clustering for the HCl (A) and UNC
(B) adenocarcinoma samples. The adjacent CDF curves demonstrate the
largest change in area under the curve from two to three clusters,
indicating three as the optimal number of clusters. This parallels
clustering results from microarray data.
[0013] FIG. 8 shows survival by adenocarcinoma subtype. The
clinical phenotype associated with the 3 molecular subtypes of lung
adenocarcinoma reproduces previous work.
[0014] FIG. 9 shows the classification of bronchoid, squamoid and
magnoid subtypes. Each sample was assigned uniquely to one of 3
adenocarcinoma subtypes as described in the text. Nine centroids
were prepared from these data (3 tumor subtypes.times.3 studies).
Additional centroids were prepared for the major tumor histologies
present in the cohorts for a total of 19 centroids. Hierarchical
agglomerative clustering was performed using a distance measure of
1-pearsons correlation across all reliable 2553 gene correlations.
The results of clustering are shown as the branched tree dendrogram
at the top of the figure. Below the dendrogram, the pair-wise
relationships between each of the centroids are displayed
graphically as a series of shaded pixels. Each pixel represents a
pair-wise correlation between centroids named by the row and column
with darker shading corresponding to higher correlation. The figure
shows that the tightest correlations are between histologic groups,
indicated by groups of circled pixels. Correlations of similar
strength are indicated between adenocarcinoma subtypes in
essentially every case. Additionally, the figure demonstrates
interesting patterns of low centroid correlation. The squamoid and
bronchioid subtypes have low pair-wises correlations, and in fact
the squamoid subtype is more correlated with centroids derived from
squamous cell carcinoma. The single centroid with an overall
ambiguous set of pair-wise correlations is the Magnoid centroid
derived from the Michigan cohort which nonetheless was grouped at a
position intermediated to the Magnoids and squamoids in the
dendrogram. Abbreviations: DF=Dana Farber, SU=Stanford University,
UM=University of Michigan.
[0015] FIG. 10 shows survival by SCC subtype in two independent
cohorts of lung cancer patients.
[0016] FIG. 11 shows the correlation of tumor morphology as
determined by light microscopy compared to that determined by gene
expression profiling from FFPE samples.
[0017] FIG. 12 is a Diagnostic Decision Tree for Lung Cancer. The
Calibrated Classifier (CC), derived from the 500 gene set (PDC), is
used in the qRT-PCR assay to first distinguish the major
histological types of lung cancer and then further subtype based on
the molecular classification. The numbers next to each histological
type show the distribution of samples, with the expected
frequencies of subtypes within each class provided below in
parentheses.
[0018] FIG. 13 shows a survival plot for Class I, Class II, and
Class III in the Duke samples.
[0019] FIG. 14 shows a survival plot for Class I, Class II, and
Class III in the Veridex samples.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides compositions and methods for
identifying or diagnosing lung cancer. That is, the methods are
useful for molecularly defining subsets of lung cancer. The methods
provide a classification of lung cancer that is prognostic and
predictive for therapeutic response. While a useful term for
epidemiologic purposes, "lung cancer" does not refer to a specific
disease, but rather represents a heterogeneous collection of tumors
of the lung, bronchus, and pleura. For practical purposes, lung
cancer is generally divided into two histological subtypes--small
cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).
These main tumor types present at different frequencies, different
anatomic locations, have different predilections for metastasis,
respond differently to therapy, and are likely derived from
different cell progenitors. For instance, SCLC often presents near
the central bronchus, has neuroendocrine features, and responds
initially to therapy but often recurs. Most lung cancers are
classified as NSCLC (>85%), which is a diverse group with
subtypes occurring throughout the respiratory tract. Adenocarcinoma
(AD) and squamous cell carcinomas (SCC), the 2 main subtypes of
NSCLC, are diagnosed at near equal frequency but are often found at
different locations with SCC occurring more centrally. The 6th
edition of the consensus classification of lung cancers developed
by the World Health Organization (WHO) describes no fewer than 90
malignant morphologic classes and variants. There is often
heterogeneity, especially in larger tumors >1.5 cm, making
morphological classification more difficult and leading to
designations such as adeno-squamous carcinoma. The methods of the
invention provide a means for determining the cellular and
molecular origins of lung cancer and thus, provide for more
accurate diagnoses and treatments.
[0021] In one embodiment, the expression profile associated with
the gene cassettes described herein is useful for distinguishing
between normal and tumor samples. In another embodiment, the
expression profile can distinguish between small cell lung
carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). In
another embodiment, NSCLC can be further classified as carcinoid,
adenocarcinoma, or squamous cell carcinoma. In yet another
embodiment, adenocarcinomas can be characterized as bronchoid,
squamoid, or magnoid. The characterization of bronchoid, squamoid,
and magnoid adenocarcinomas using tumor biopsy tissue has been
described in Hayes et al. (2006) J. Clin Oncol. 24(31):5079-90.
[0022] "Diagnosing lung tumor subtypes" or "determining subsets of
lung cancer" is intended to include, for example, diagnosing or
detecting the presence and type of lung cancer, monitoring the
progression of the disease, and identifying or detecting cells or
samples that are indicative of subtypes. The terms diagnosing,
detecting, and identifying lung cancer subtypes are used
interchangeably herein.
[0023] The methods of the invention also find use in predicting
response to chemotherapy, particularly first-line chemotherapy.
Chemotherapeutic response is improved by more accurately assigning
tumor subtypes. Likewise, treatment regimens can be formulated
based on the tumor subtype. For example, clinical trials have shown
convincing evidence that the VEGF inhibitor, bevacizumab, is
effective in the treatment of NSCLC. The drug carries a concerning
toxicity of tumor necrosis and associated fatal pulmonary
hemorrhage, an event overwhelmingly associated with tumors of
squamous cell histology. Accordingly, there has been an active
attempt to exclude such patients from treatment with the drug. As
drugs of this class increasingly enter clinical practice, the
methods of the invention will be useful to more accurately diagnose
squamous cell carcinoma even from limited tissue samples.
Overview
[0024] Expression profiles using the discriminative genes disclosed
herein provide valuable molecular tools for specifically
classifying lung cancer types and subtypes, and for evaluating
therapeutic efficacy in treating lung cancer. Accordingly, the
invention provides methods for screening a subject for lung cancer
(diagnostic) including classification into molecular subtypes,
methods for monitoring the progression of lung cancer in a subject,
and methods for monitoring the efficacy of certain therapeutic
treatments for lung cancer.
[0025] In some instances, a single discriminating gene is capable
of classifying types and subtypes of lung cancer with a predictive
success of at least about 70%, at least about 71%, at least about
72%, about 73%, about 74%, about 75%, about 76%, about 77%, about
78%, about 79%, about 80%, about 81%, about 82%, about 83%, about
84%, about 85%, about 86%, about 87%, about 88%, about 89%, about
90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, about 99%, up to 100%, whereas, in other
instances, a combination of predictive genes is used to obtain a
predictive success of at least about 70%, at least about 71%, at
least about 72%, about 73%, about 74%, about 75%, about 76%, about
77%, about 78%, about 79%, about 80%, about 81%, about 82%, about
83%, about 84%, about 85%, about 86%, about 87%, about 88%, about
89%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, about 99%, up to 100%.
[0026] In some instances, a single predictive gene is capable of
classifying lung cancer types or subtypes with a sensitivity or
specificity of at least about 70%, at least about 71%, at least
about 72%, about 73%, about 74%, about 75%, about 76%, about 77%,
about 78%, about 79%, about 80%, about 81%, about 82%, about 83%,
about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,
about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,
about 96%, about 97%, about 98%, about 99%, up to 100%, whereas, in
other instances, a combination or plurality of discriminative genes
is used to obtain a sensitivity or specificity of at least about
70%, at least about 71%, at least about 72%, about 73%, about 74%,
about 75%, about 76%, about 77%, about 78%, about 79%, about 80%,
about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,
about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,
about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,
about 99%, up to 100%.
[0027] The present invention also encompasses a system capable of
classifying subjects according to lung cancer status, including
distinguishing between various subtypes of lung cancer not
detectable using current methods. This system is capable of
processing a large number of subjects and subject variables such as
expression profiles and other diagnostic criteria. The methods
described herein can also be used for "pharmacometabonomics," in
analogy to pharmacogenomics, e.g., predictive of response to
therapy. In this embodiment, subjects could be divided into
"responders" and "nonresponders" using the expression profile as
evidence of "response," and features of the expression profile
could then be used to target future subjects who would likely
respond to a particular therapeutic course.
[0028] The methods are also useful for evaluating clinical response
to therapy, as well as for endpoints in clinical trials for
efficacy of new therapies. The extent to which sequential
diagnostic expression profiles move towards normal can be used as
one measure of the efficacy of the candidate therapy.
[0029] The expression profile can be used in combination with other
diagnostic methods including histochemical, immunohistochemical,
cytologic, immunocytologic, and visual diagnostic methods including
histologic or morphometric evaluation of lung tissue.
Expression Profiling
[0030] In one embodiment of the present invention, lung cancer
status is assessed through the evaluation of expression patterns,
or profiles, of a plurality of discriminative genes or biomarkers
in one or more subject samples. For the purpose of discussion, the
term subject, or subject sample, refers to an individual regardless
of health and/or disease status. A subject can be a subject, a
study participant, a control subject, a screening subject, or any
other class of individual from whom a sample is obtained and
assessed in the context of the invention. Accordingly, a subject
can be diagnosed with lung cancer (including various types,
subtypes, or grades thereof), can present with one or more symptoms
of lung cancer, or a predisposing factor, such as a family
(genetic) or medical history (medical) factor, for lung cancer, can
be undergoing treatment or therapy for lung cancer, or the like.
Alternatively, a subject can be healthy with respect to any of the
aforementioned factors or criteria. It will be appreciated that the
term "healthy" as used herein, is relative to lung cancer status,
as the term "healthy" cannot be defined to correspond to any
absolute evaluation or status. Thus, an individual defined as
healthy with reference to any specified disease or disease
criterion, can in fact be diagnosed with any other one or more
diseases, or exhibit any other one or more disease criterion,
including one or more other cancers.
[0031] As used herein, an "expression profile" comprises one or
more values corresponding to a measurement of the relative
abundance, presence, or absence of expression of a discriminative
gene. An expression profile can be derived from a subject prior to
or subsequent to a diagnosis of lung cancer, can be derived from a
biological sample collected from a subject at one or more time
points prior to or following treatment or therapy, can be derived
from a biological sample collected from a subject at one or more
time points during which there is no treatment or therapy (e.g., to
monitor progression of disease or to assess development of disease
in a subject diagnosed with or at risk for lung cancer), or can be
collected from a healthy subject.
[0032] In various embodiments of the present invention, the
expression profile derived from a subject is compared to a
reference expression profile. A "reference expression profile" can
be a profile derived from the subject prior to treatment or
therapy; can be a profile produced from the subject sample at a
particular time point (usually prior to or following treatment or
therapy, but can also include a particular time point prior to or
following diagnosis of lung cancer); or can be derived from a
healthy individual or a pooled reference from healthy individuals.
A reference expression profile can be generic for lung cancer, or
can be specific to different types or subtypes of lung cancer.
[0033] The reference expression profile can be compared to a test
expression profile. A "test expression profile" can be derived from
the same subject as the reference expression profile except at a
subsequent time point (e.g., one or more days, weeks or months
following collection of the reference expression profile) or can be
derived from a different subject. In summary, any test expression
profile of a subject can be compared to a previously collected
profile from the same subject or to a profile obtained from a
healthy individual.
[0034] The biomarkers of the invention include genes and proteins,
and variants and fragments thereof. Such biomarkers include DNA
comprising the entire or partial sequence of the nucleic acid
sequence encoding the biomarker, or the complement of such a
sequence. The biomarker nucleic acids also include any expression
product or portion thereof of the nucleic acid sequences of
interest. A biomarker protein is a protein encoded by or
corresponding to a DNA biomarker of the invention. A biomarker
protein comprises the entire or partial amino acid sequence of any
of the biomarker proteins or polypeptides.
[0035] A "biomarker" is any gene or protein whose level of
expression in a tissue or cell is altered compared to that of a
normal or healthy cell or tissue. The detection, and in some cases
the level, of the biomarkers of the invention permits the
differentiation of samples.
[0036] The biomarkers of the invention include any gene or protein
that is selectively expressed in lung cancer cells, as defined
herein above. Sample biomarker genes are listed in Table 1, below.
The first portion of the table represents the gene lists for
distinguishing types and subtypes. The middle portion of the table
represents genes that are useful for distinguishing the three
subtypes of squamous cell carcinoma, as well as the relative "up"
or "down" levels of expression for each gene. The last portion of
the table lists the same genes as the first portion, but with the
relative "up" or "down" indications for expression levels. The
comparisons are depicted in FIG. 6. Thus, the level of expression
of (for example) CAPG is said to be "down" relative to the
expression of CAPG in ACC/SCC lung cancer types. Although the
methods of the invention require the detection of at least one
biomarker in a patient sample, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
biomarkers may be used to practice the present invention.
TABLE-US-00001 TABLE 1 Classification, Housekeeping Genes,
Corresponding Primer Sets, and Relative Expression Levels Gene
symbol Gene name Forward primer SEQ ID Reverse primer SEQ ID Normal
classifier gene list CDH5 cadherin 5, type 2, AAGAGAGATTG 1
TTCTTGCGACTCA 58 VE-cadherin GATTTGGAACC CGCT (vascular epithelium)
CLEC3B C-type lectin CCAGAAGCCCA 2 GCTCCTCAAACAT 59 domain family
3, AGAAGATTGTA CTTTGTGTTCA member B PAICS phosphoribosylami
AATCCTGGTGT 3 GACCACTGTGGG 60 noimidazole CAAGGAAG TCATTATT
carboxylase, phosphoribosylami noimidazolc succinocarboxamide
synthetase PAK1 p21/Cdc42/Rac1- GGACCGATTTT 4 GAAATCTCTGGC 61
activated kinase 1 ACCGATCC CGCTCC (STE20 homolog, yeast) PECAM1
platelet/endothelial ACAGTCCAGAT 5 ACTGGGCATCAT 62 cell adhesion
AGTCGTATGT AAGAAATCC molecule (CD31 antigen) TFAP2A transcription
factor GTCTCCGCCATC 6 ACTGAACAGAAG 63 AP-2 alpha CCTAT ACTTCGT
(activating enhancer binding protein 2 alpha) SCLC classifier gene
list ACVR1 activin A receptor, ACTGGTGTAAC 7 AACCTCCAAGTG 64 type I
AGGAACAT GAAATTCT CDKN2C cyclin-dependent TTTGGAAGGAC 8
TCGGTCTTTCAAA 65 kinase inhibitor 2C TGCGCT TCGGGATTA (p18,
inhibits CDK4) CIB1 calcium and CACGTCATCTCC 9 CTGCTGTCACAG 66
integrin binding 1 CGTTC GACAAT (calmyrin) INSM1 insulinoma-
ATTGAACTTCCC 10 AAGGTAAAGCCA 67 associated 1 ACACGA GACTCCA LRP10
low density GGAACAGACTG 11 GGGAGCGTAGGG 68 lipoprotein TCACCAT
TTAAG receptor-related protein 10 STMN1 stathmin 1/ TCAGACTTCTTGG
12 CAGTGTATTCTGC 69 oncoprotein 18 TCAGGC ACAATCAAC Carcinoid
classifiers gene list CAPG capping protein GGGACAGCTTC 13
GTTCCAGGATGTT 70 (actin filament), AACACT GGACTTTC gelsolin-like
CHGA chromogranin A CCTGTGAACAG 14 GGAAAGTGTGTC 71 (parathyroid
CCCTATG GGAGAT secretory protein 1) LGALS3 lectin, galactoside-
TTCTGGGCACG 15 AGGCAACATCAT 72 binding, soluble, 3 GTGAAG TCCCTC
(galectin 3) MAPRE3 microtubule- GGCCAAACTAG 16 GTCAACACCCAT 73
associated protein, AGCACGAATA CTTCTTGAAA RP/EB family, member 3
SFN stratifin TCAGCAAGAAG 17 CGTAGTGGAAGA 74 GAGATGCC CGGAAA SNAP91
synaptosomal- GTGCTCCCTCTC 18 CTGGTGTAGAATT 75 associated protein,
CATTAAGTA AGGAGACGTA 91 kDa homolog (mouse) AC/SCC classifier gene
list ABCC5 ATP-binding CAAGTTCAGGA 19 GGCATCAAGAGA 76 cassette,
sub- GAACTCGAC GAGGC family C (CFTR/MRP), member 5 ALDH3B1 aldehyde
GGCTGTGGTTA 20 GATAAAGAGTTA 77 dehydrogenase 3 TGCGATAG
CAAGCTCCTCTG family, member B1 ANTXR1 anthrax toxin ACCCGAGGAAC 21
TCTAGGCCTTGAC 78 receptor 1 AACCTTA GGAT BMP7 bone CCCTCTCCATTC 22
TTTGGGCAAACCT 79 morphogenetic CCTACA CGGTAA protein 7 (osteogenic
protein 1) CACNB1 calcium channel, CAGAGCGCCAG 23 GCACAGCAAATG 80
voltage-dependent, GCATTA CCACT beta 1 subunit CBX1 chromobox
CCACTGGCTGA 24 CTTGTCTTTCCCT 81 homolog 1 (HP1 GGTGTTA ACTGTCTTAC
beta homolog Drosophila) CYB5B cytochrome b5 TGGGCGAGTCT 25
CTTGTTCCAGCAG 82 type B (outer ACGATG AACCT mitochondrial membrane)
DOK1 docking protein 1, CTTTCTGCCCTG 26 CAGTCCTCTGCAC 83 62 kDa
GAGATG CGTTA (downstream of tyrosine kinase 1) DSC3 desmocollin 3
GCGCCATTTGCT 27 CATCCAGATCCCT 84 AGAGATA CACAT FEN1 flap structure-
AGAGAAGATGG 28 CCAAGACACAGC 85 specific GCAGAAAG CAGTAAT
endonuclease 1 FOXH1 forkhead box H1 GCCCAGATCAT 29 TTTCCAGCCCTCG
86 CCGTCA TAGTC GJB5 gap junction ACCACAAGGAC 30 GGGACACAGGGA 87
protein, beta 5 TTCGAC AGAAC (connexin 31.1 ) HOXD1 homeobox D1
GCTCCGCTGCT 31 GTCTGCCACTCTG 88 ATCTTT CAAC HPN hepsin AGCGGCCAGGT
32 GTCGGCTGACGC 89 (transmembrane GGATTA TTTGA protease, serine 1)
HYAI2 hyaluronoglucosam ATGGGCTTTGG 33 GAACAAGTCAGT 90 inidase 2
GAGCATA CTAGGGAATAC ICA1 islet cell GACCTGGATGC 34 TGCTTTCGATAAG 91
autoantigen 1, CA AGCTA TCCAGACA 69 kDa ICAM5 intercellular
CCGGCTCTTGG 35 CCTCTGAGGCTG 92 adhesion molecule AAGTTG GAAACA 5,
telencephalin ITGA6 integrin, alpha 6 ACGCGGATCGA 36 ATCCACTGATCTT
93 GTTTGATAA CCTTGC LIPE lipase, hormone- CGCAAGTCCCA 37
CAGTGCTGCTTCA 94 sensitive GAAGAT GACACA ME3 malic enzyme 3,
CGCGGATACGA 38 CCTTTCTTCAAGG 95 NADP(+)- TGTCAC GTAAAGGC dependent,
mitochondrial MGRN1 mahogunin, ring GAACTCGGCCT 39 TCGAATTTCTCTC 96
finger 1 ATCGCT CTCCCAT MYBPH myosin binding TCTGACCTCATC 40
CTGAGTCCACAC 97 protein H ATCGGCAA AGGTTT MYO7A myosin VIIA
GAGGTGAAGCA 41 CCCATACTTGTTG 98 AACTACGGA ATGGCAATTA NFIL3 nuclear
factor, ACTCTCCACAA 42 TTTCCTGCGTGTG 99 interleukin 3 AGCTCG CTACT
regulated PIK3C2A phosphoinositide-3- GGATTTCAGCT 43 AGTCATCATGTAC
100 kinase, class 2, ACCAGTTACTT CCAGCA alpha polypeptide PLEKHA6
pleckstrin TTCGTCCTGGTG 44 CCCAGGATACTCT 101 homology domain GATCG
CTTCCTT containing, family A member 6 PSMD14 proteasome AGTGATTGATG
45 CACTGGATCAAC 102 (prosome, TGTTTGCTATG TGCCTC macropain) 26S
subunit, non- ATPase, 14 SCD5 stearoyl-CoA CAAAGCCAAGC 46
CAGCTGTCACAC 103 desaturase 5 CACTCACTC CCAGAGC SIAH2 seven in
absentia CTCGGCAGTCC 47 CGTATGGTGCAG 104 homolog 2 TGTTTC GGTCA
(Drosophila) TCF2 transcription factor ACACCTGGTAC 48 TCTGGACTGTCTG
105 2, hepatic; LF-B3; GTCAGAA GTTGAAT variant hepatic nuclear
factor TCP1 t-complex 1 ATGCCCAAGAG 49 CCTGTACACCAA 106 AATCGTAAA
GCTTCAT TTF1 thyroid ATGAGTCCAAA 50 CCATGCCCACTTT 107 transcription
GCACACGA CTTGTA factor 1 TRIM29 tripartite motif- TGAGATTGAGG 51
CATTGGTGGTGA 108 containing 29 ATGAAGCTGAG AGCTCTTG TUBA1 tubulin,
alpha 1 CCGACTCAACG 52 CGTGGACTGAGA 109 TGAGAC TGCATT Housekeeper
gene list CFL1 cofilin 1 non- GTGCCCTCTCCT 53 TTCATGTCGTTGA 110
muscle) TTTCCG ACACCTTG EEF1A1 eukaryotic CGTTCTTTTTCG 54
CATTTTGGCTTTT 111 translation CAACGG AGGGGTAG elongation factor 1
alpha 1 RPL10 ribosomal protein GGTGTGCCACT 55 GGCAGAAGCGAG 112 L10
GAAGAT ACTTT RPL28 ribosomal protein GTGTCGTGGTG 56 GCACATAGGAGG
113 L28 GTCATT TGGCA RPL37A ribosomal protein GCATGAAGACA 57
GCGGACTTTACC 114 L37a GTGGCT GTGAC SCC subtype genes Gene ''Group
Identified'' and Symbol Gene Name Level of Expression CYP4F11
cytochrome P450, family 4, subfamily F, ''Keratin''-High
polypeptide 11
UPK1B uroplakin 1B ''Keratin''-High NRXN3 neurexin 3
''Keratin''-High RHCG Rh family, C glycoprotein ''Keratin''-High
PAD13 peptidyl arginine deiminase, type III ''Keratin''-High IL32
interleukin 32 ''Keratin''-High PRRX2 paired related homeobox 2
''Keratin''-High RAB6B RAB6B, member RAS oncogene family
''Keratin''-High CALB1 calbindin 1, 28 kDa ''Keratin''-High G6PD
glucose-6-phosphate dehydrogenase ''Keratin''-High PITX1
paired-like homeodomain 1 ''Keratin''-Low SERPINB5 serpin peptidase
inhibitor, clade B ''Keratin''-Low (ovalbumin), member 5 KRT6B
keratin 6B ''Keratin''-Low KRT17 keratin 17 ''Keratin''-Low DSG3
desmoglein 3 (pemphigus vulgaris antigen) ''Keratin''-Low TRIM29
tripartite motif-containing 29 ''Keratin''-Low CALML3
calmodulin-like 3 ''Keratin''-Low PTPRZ1 protein tyrosine
phosphatase, receptor-type, ''Keratin''-Low Z polypeptide 1 KRT19
keratin 19 ''Keratin''-Low CD3G CD3g molecule, gamma (CD3-TCR
complex) ''Keratin''-Low S100A2 S100 calcium binding protein A2
''RAS-High''-High KRT17 keratin 17 ''RAS-High''-High RAB6B RAB6B,
member RAS oncogene family ''RAS-High''-High CYP4F11 cytochrome
P450, family 4, subfamily F, ''RAS-High''-High polypeptide 11 SYT17
synaptotagmin XVII ''RAS-High''-High KRT19 keratin 19
''RAS-High''-High CCL19 chemokine (C-C motif) ligand 19
''RAS-High''-High MS4A4A membrane-spanning 4-domains, subfamily A,
''RAS-High''-High member 4 TRIM29 tripartite motif-containing 29
''RAS-High''-High FXYD3 FXYD domain containing ion transport
''RAS-High''-High regulator 3 PADI3 peptidyl arginine deiminase,
type III ''RAS-High''-Low CXCL6 chemokine (C-X-C motif) ligand 6
(granulocyte ''RAS-High''-Low chemotactic protein 2) UPK1B
uroplakin 1B ''RAS-High''-Low SLC6A15 solute carrier family 6,
member 15 ''RAS-High''-Low PLAT plasminogen activator, tissue
''RAS-High''-Low MMP10 matrix metallopeptidase 10 (stromelysin 2)
''RAS-High''-Low TTC9 tetratricopeptide repeat domain 9
''RAS-High''-Low CXCL1 chemokine (C-X-C motif) ligand 1 (melanoma
''RAS-High''-Low growth stimulating activity, alpha) LAMC2 laminin,
gamma 2 ''RAS-High''-Low MMP13 matrix metallopeptidase 13
(collagenase 3) ''RAS-High''-Low MUC16 mucin 16, cell surface
associated ''Mucin-High''-High TTC9 tetratricopeptide repeat domain
9 ''Mucin-High''-High CD3G CD3g molecule, gamma (CD3-TCR complex)
''Mucin-High''-High PITX1 paired-like homeodomain 1
''Mucin-High''-High PTPRZ1 protein tyrosine phosphatase,
receptor-type, Z ''Mucin-High''-High polypeptide 1 CLCA2 chloride
channel, calcium activated, family ''Mucin-High''-High member 2
PLAU plasminogen activator, urokinase ''Mucin-High''-High ICAM1
intercellular adhesion molecule 1 (CD54), ''Mucin-High''-High human
rhinovirus receptor ABCC6 ATP-binding cassette, sub-family C
(CFTR/MRP), ''Mucin-High''-High member 6 WISP2 WNT1 inducible
signaling pathway protein 2 ''Mucin-High''-High CYP4F11 cytochrome
P450, family 4, subfamily F, ''Mucin-High''-Low polypeptide 11
RAB6B RAB6B, member RAS oncogene family ''Mucin-High''-Low IL32
interleukin 32 ''Mucin-High''-Low CALB1 calbindin 1, 28 kDa
''Mucin-High''-Low ODC1 ornithine decarboxylase 1
''Mucin-High''-Low ADAM23 ADAM metallopeptidase domain 23
''Mucin-High''-Low NRXN3 neurexin 3 ''Mucin-High''-Low PRKX protein
kinase, X-linked ''Mucin-High''-Low MYO1F myosin 1F
''Mucin-High''-Low NINJ2 ninjurin 2 ''Mucin-High''-Low Lung Cancer
Gene List excluding Squamous Cell subtypes GENE ''Distinguisher''
and Level SYMBOL GENE NAME of Expression SFN stratifin ''CARC vs
ALL''-down LGALS3 lectin, galactoside-binding, soluble, 3 ''CARC vs
ALL''-down (galectin 3) CAPG capping protein (actin filament),
''CARC vs ALL''-down gelsolin-like CHGA chromogranin A (parathyroid
secretory ''CARC vs ALL''-up protein 1) MAPRE3
microtubule-associated protein, RP/EB family, ''CARC vs ALL''-up
member 3 SNAP91 synaptosomal-associated protein, 91 kDa ''CARC vs
ALL''-up homolog (mouse) PSMD14 proteasome (prosome, rnacropain)
26S subunit, ''FOUR''-down non-ATPase, 14 CBX1 chromobox homolog 1
(HP1 beta homolog ''FOUR''-down Drosophila) NFIL3 nuclear factor,
interleukin 3 regulated ''FOUR''-down SCD5 stearoyl-CoA desaturase
5 ''FOUR''-up HOXD1 homeobox D1 ''FOUR''-up ICAM5 intercellular
adhesion molecule 5, ''FOUR''-up telencephalin FOXH1 forkhead box
H1 ''FOUR''-down CACNB1 calcium channel, voltage-dependent, beta 1
''FOUR''-down subunit TCF2 transcription factor 2, hepatic
''FOUR''-down TCP1 t-complex 1 ''FOUR''-up FEN1 flap
structure-specific endonuclease 1 ''FOUR''-up TUBA1 tubulin, alpha
1 (testis specific) ''FOUR''-up CYB5B cytochrome b5 type B (outer
mitochondrial ''FOUR''-down membrane) PIK3C2A
phosphoinositide-3-kinase, class 2, alpha ''FOUR''-down polypeptide
ANTXR1 anthrax toxin receptor 1 ''FOUR''-down LIPE lipase,
hormone-sensitive ''FOUR''-up MYBPH myosin binding protein H
''FOUR''-up DOK1 docking protein 1, 62 kDa (downstream of
''FOUR''-up tyrosine kinase 1) SIAH2 seven in absentia homolog 2
(Drosophila) ''FOUR''-down ITGA6 integrin, alpha 6 ''FOUR''-down
ICA1 islet cell autoantigen 1, 69 kDa ''FOUR''-up TITF1 thyroid
transcription factor 1 ''FOUR''-up HPN hepsin (transmembrane
protease, serine 1) ''FOUR''-up TRIM29 tripartite motif-containing
29 ''FOUR''-down DSC3 desmocollin 3 ''FOUR''-down BMP7 bone
morphogenetic protein 7 ''FOUR''-down (osteogenic protein 1) MGRN1
mahogunin, ring finger 1 ''FOUR''-up HYAL2 hyaluronoglueosaminidase
2 ''FOUR''-up MYO7A myosin VIIA ''FOUR''-up ABCC5 ATP-binding
cassette, sub-family C (CFTR/MRP), ''FOUR''-down member 5 GJB5 gap
junction protein, beta 5 (connexin 31.1) ''FOUR''-down PLEKHA6
pleckstrin homology domain containing, ''FOUR''-up family A member
6 ME3 malic enzyme 3, NADP(+)-dependent, ''FOUR''-up mitochondrial
ALDH3B1 aldehyde dehydrogenase 3 family, member B1 ''FOUR''-up
RPL10 ribosomal protein L10 ''HK''-NA RPL28 ribosomal protein L28
''HK''-NA RNU31P2 RNA, U3 small nucleolar interacting protein 2
''HK''-NA RPL37A ribosomal protein L37a ''HK''-NA CFL1 cofilin 1
(non-muscle) ''HK''-NA MED6 mediator of RNA polymerase II
transcription, ''HK''-NA subunit 6 homolog (yeast) ST5 suppression
of tumorigenicity 5 ''HK''-NA EEF1A1 eukaryotic translation
elongation factor 1 ''HK''-NA alpha 1 CIB1 calcium and integrin
binding 1 (calmyrin) ''SCLC vs ALL''-down LRP10 low density
lipoprotein receptor-related ''SCLC vs ALL''-down
protein 10 ACVR1 activin A receptor, type I ''SCLC vs ALL''-down
STMN1 stathmin 1/oncoprotein 18 ''SCLC vs ALL''-up INSM1
insulinoma-associated 1 ''SCLC vs ALL''-up CDKN2C cyclin-dependent
kinase inhibitor 2C ''SCLC vs ALL''-up (p18, inhibits CDK4) PAK1
p21/Cdc42/Rac1-activated kinasc 1 (STE20 ''TUMOR vs NML''-down
homolog, yeast) TFAP2A transcription factor AP-2 alpha (activating
''TUMOR vs NML''-down enhancer binding protein 2 alpha) PAICS
phosphoribosylaminoimidazole carboxylase, ''TUMOR vs NML''-down
phosphoribosylaminoimidazole succinocarboxamide synthetase CLEC3B
C-type lectin domain family 3, member B ''TUMOR vs NML''-up CDH5
cadherin 5, type 2, VE-cadherin (vascular ''TUMOR vs NML''-up
epithelium) PECAM1 platelet/endothelial cell adhesion molecule
''TUMOR vs NML''-up (CD31 antigen) HK = Housekeeping SCLC = Small
cell lung carcinoma distinguishers NML = normal distinguishers CARC
= Carcinoid distinguishers FOUR = AC/SCC subtype distinguishers
[0037] It is recognized that additional genes/proteins can be used
in the practice of the invention. For example, vimentin, a member
of the intermediate filament family of proteins can be used to
identify the adenocarcinoma subtype magnoid, and SMA can be used to
identify squamoid subtype. In general, genes useful in classifying
various classes, subclasses, and grades of lung cancer include
those that are independently capable of distinguishing between
normal versus tumor, or between different classes or grades of lung
cancer. A gene is considered to be capable of reliably
distinguishing between classes if the area under the receiver
operator characteristic (ROC) curve is approximately 1.
[0038] A gene capable of reliable classification (herein referred
to as a "discriminating gene") may be one that is upregulated
(e.g., expression is increased) or downregulated (e.g., expression
is decreased) relative to the control. The expression values of
genes that are upregulated in a particular class or grade of lung
cancer can be pooled into one gene cassette, and the expression
values of genes that are downregulated in a particular class or
grade of lung cancer can be pooled into a separate gene cassette.
The overall expression level in each gene cassette is referred to
herein as the "expression profile" and is used to classify a test
sample according to class, subclass, or grade of lung cancer.
However, it is understood that independent evaluation of expression
for each of the genes disclosed herein can be used to classify
tumor types without the need to group upregulated and downregulated
genes into one or more gene cassettes.
[0039] In one embodiment, genes useful in classifying different
types and subtypes of lung cancer are set forth in Table 1. It is
understood that the expression level of any gene that is capable of
reliable classification of different subtypes can be utilized in
the methods described herein.
Measurement of Gene Expression
[0040] In one embodiment, the expression profile can comprise
values representing the measurement of the transcriptional state.
The transcriptional state of a sample includes the identities and
relative abundance of the RNA species encoded by the discriminative
genes disclosed herein. The transcriptional state can be
conveniently determined by measuring transcript presence or absence
by any of several existing gene expression technologies such as
reverse transcription-polymerase chain reaction (RT-PCR),
particularly quantitative RT-PCR (qRT-PCR). Methods for determining
the level of biomarker mRNA in a sample may involve the process of
nucleic acid amplification, e.g., by RT-PCR (the experimental
embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202),
ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA
88:189-193), self sustained sequence replication (Guatelli et al.
(1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional
amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA
86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988)
Bio/Technology 6:1197), rolling circle replication (Lizardi et al.,
U.S. Pat. No. 5,854,033) or any other nucleic acid amplification
Method, followed by the detection of the amplified molecules using
techniques well known to those of skill in the art.
[0041] This PCR-based analysis is suitable for use on paraffin
embedded tissues. Such tissues can be analyzed by molecular
diagnosis of lung cancer by gene expression. The method comprises a
novel and high-throughput approach to independently validate the
genes. The method utilizes a robust novel self-normalizing method
of classification which allows for a modular predictor. In the
methods of the invention predictions are made in two ways, using a
one versus all and all pairwise approach such that many classes can
be predicted using small gene cassettes. Since each of the gene
cassettes is independent, new cassettes can be added to the
existing predictor without changing the overall method of
prediction. This allows for the addition of new classes as needed.
For example, the current predictor distinguishes a total of 9
classes and subclasses of lung cancer. As new groups of lung cancer
are described or as new features of tumor behavior are identified,
these can be added to the current predictor as cassettes without
changing the existing structure of the classifier.
[0042] Gene selection for gene cassettes that are indicative of
lung cancer subtypes is performed in the following manner. A set of
gene expression cohorts are selected from among all published
reports of lung cancers assayed by gene expression data due to
their relatively large size and inclusion of a representative
spectrum of tumor variants. Expression datasets are transformed
using genomic meta-analysis methods that have been previously
reported. Briefly, all arrays are evaluated for the quality of the
scanned image. Probes are mapped to genes using Unigene
identifiers. In cases where multiple probes map to the same Unigene
identifier, these are averaged. Genes are evaluated for
cross-platform reliability using integrative correlations. Genes
with integrative correlations twice that observed by random chance
are considered reliable and retained across datasets. Reliable
genes are then ranked for each gene for its ability to distinguish
each of the morphologic variants in both the 1 versus all (i.e.,
tumor versus normal) and the all-pairwise (i.e., normal versus
small cell carcinoma) case. The statistic used for the ranking is
the area under the receiver operator characteristic (ROC) curve (a
plot of sensitivity versus (1-specificity)). Although genes are
evaluated for reliability across datasets, the independent sample
sets are not combined for the purposes of the ROC ranking. As a
result, multiple independent analyses are performed and multiple
independent rankings are obtained for each gene's ability to
distinguish groups of interest. A gene is considered reliable if
its area under the ROC curve is close to 1 for each of the
independent datasets. In cases where a gene's ROC is close to 1 in
some but not all datasets, the genes overall reliability is
considered as measured by integrative correlations. In one
embodiment, the genes are limited to approximately 100 genes. See
example 4 for more details.
[0043] Numerous different PCR or qRT-PCR protocols are known in the
art and can be directly applied or adapted for use using the
presently described compositions for the detection and/or
quantification of expression of discriminative genes in a sample.
See, for example, Fan et al. (2004) Genome Res. 14:878-885, herein
incorporated by reference. Generally, in PCR, a target
polynucleotide sequence is amplified by reaction with at least one
oligonucleotide primer or pair of oligonucleotide primers. The
primer(s) hybridize to a complementary region of the target nucleic
acid and a DNA polymerase extends the primer(s) to amplify the
target sequence. Under conditions sufficient to provide
polymerase-based nucleic acid amplification products, a nucleic
acid fragment of one size dominates the reaction products (the
target polynucleotide sequence which is the amplification product).
The amplification cycle is repeated to increase the concentration
of the single target polynucleotide sequence. The reaction can be
performed in any thermocycler commonly used for PCR. However,
preferred are cyclers with real-time fluorescence measurement
capabilities, for example, SMARTCYCLER.RTM. (Cepheid, Sunnyvale,
Calif.), ABI PRISM 7700.RTM. (Applied Biosystems, Foster City,
Calif.), ROTOR-GENE.TM. (Corbett Research, Sydney, Australia),
LIGHTCYCLER.RTM. (Roche Diagnostics Corp, Indianapolis, Ind.),
ICYCLER.RTM. (Biorad Laboratories, Hercules, Calif.) and
MX4000.RTM. (Stratagene, La Jolla, Calif.).
[0044] Quantitative RT-PCR (qRT-PCR) (also referred as real-time
RT-PCR) is preferred under some circumstances because it provides
not only a quantitative measurement, but also reduced time and
contamination. As used herein, "quantitative PCR (or "real time
qRT-PCR") refers to the direct monitoring of the progress of a PCR
amplification as it is occurring without the need for repeated
sampling of the reaction products. In quantitative PCR, the
reaction products may be monitored via a signaling mechanism (e.g.,
fluorescence) as they are generated and are tracked after the
signal rises above a background level but before the reaction
reaches a plateau. The number of cycles required to achieve a
detectable or "threshold" level of fluorescence varies directly
with the concentration of amplifiable targets at the beginning of
the PCR process, enabling a measure of signal intensity to provide
a measure of the amount of target nucleic acid in a sample in real
time. A labeled probe can be used to detect the extension product
generated by PCR amplification. Any probe format utilizing a
labeled probe comprising the sequences of the invention may be
used, e.g., such as SCORPIONS.TM. probes, sunrise probes,
TAQMAN.RTM. probes, or molecular beacon probes as is known in the
art or described elsewhere herein.
[0045] Methods for setting up a PCR reaction are well known to
those skilled in the art. The reaction mixture minimally comprises
template nucleic acid (except in the case of a negative control as
described below) and oligonucleotide primers and/or probes in
combination with suitable buffers, salts, and the like, and an
appropriate concentration of a nucleic acid polymerase. As used
herein, "nucleic acid polymerase" refers to an enzyme that
catalyzes the polymerization of nucleoside triphosphates.
Generally, the enzyme will initiate synthesis at the 3'-end of the
primer annealed to the target sequence, and will proceed in the
5'-direction along the template until synthesis terminates. An
appropriate concentration includes one which catalyzes this
reaction in the presently described methods. Known DNA polymerases
include, for example, E. coli DNA polymerase I, T7 DNA polymerase,
Thermus thermophilus (Tth) DNA polymerase, Bacillus
stearothermophilus DNA polymerase, Thermococcus litoralis DNA
polymerase, Thermus aquaticus (Taq) DNA polymerase and Pyrococcus
furiosus (Pfu) DNA polymerase.
[0046] Usually the reaction mixture will further comprise four
different types of dNTPs corresponding to the four-naturally
occurring nucleoside bases, i.e., dATP, dTTP, dCTP and dGTP. In the
subject methods, each dNTP will typically be present in an amount
ranging from about 10 to 5000 .mu.M, usually from about 20 to 1000
.mu.M, about 100 to 800 .mu.M, or about 300 to 600 .mu.M.
[0047] The reaction mixture prepared in the first step of the
subject methods further includes an aqueous buffer medium that
includes a source of monovalent ions, a source of divalent cations,
and a buffering agent. Any convenient source of monovalent ions,
such as potassium chloride, potassium acetate, ammonium acetate,
potassium glutamate, ammonium chloride, ammonium sulfate, and the
like may be employed. The divalent cation may be magnesium,
manganese, zinc and the like, where the cation will typically be
magnesium. Any convenient source of magnesium cation may be
employed, including magnesium chloride, magnesium acetate, and the
like. The amount of magnesium present in the buffer may range from
0.5 to 10 mM, but will preferably range from about 1 to about 6 mM,
or about 3 to about 5 mM. Representative buffering agents or salts
that may be present in the buffer include Tris, Tricine, HEPES,
MOPS and the like, where the amount of buffering agent will
typically range from about 5 to 150 mM, usually from about 10 to
100 mM, and more usually from about 20 to 50 mM, where in certain
preferred embodiments the buffering agent will be present in an
amount sufficient to provide a pH ranging from about 6.0 to 9.5.
Other agents which may be present in the buffer medium include
chelating agents, such as EDTA, EGTA and the like.
[0048] In preparing the reaction mixture, the various constituent
components may be combined in any convenient order. For example,
the buffer may be combined with primer, polymerase and then
template nucleic acid, or all of the various constituent components
may be combined at the same time to produce the reaction
mixture.
[0049] Alternatively, commercially available premixed reagents can
be utilized in the methods of the invention according to the
manufacturer's instructions, or modified to improve reaction
conditions (e.g., modification of buffer concentration, cation
concentration, or dNTP concentration, as necessary), including, for
example, TAQMAN.RTM. Universal PCR Master Mix (Applied Biosystems),
OMNIMIX.RTM. or SMARTMIX.RTM. (Cepheid), IQ.TM. Supermix (Bio-Rad
Laboratories), LIGHTCYCLER.RTM. FastStart (Roche Applied Science,
Indianapolis, Ind.), or BRILLIANT.RTM. qRT-PCR Master Mix
(Stratagene, La Jolla, Calif.).
[0050] Following preparation of the reaction mixture, the reaction
mixture is subjected to primer extension reaction conditions
("conditions sufficient to provide polymerase-based nucleic acid
amplification products"), i.e., conditions that permit for
polymerase mediated primer extension by addition of nucleotides to
the end of the primer molecule using the template strand as a
template. In many embodiments, the primer extension reaction
conditions are amplification conditions, which conditions include a
plurality of reaction cycles, where each reaction cycle comprises:
(1) a denaturation step, (2) an annealing step, and (3) a
polymerization step. The number of reaction cycles will vary
depending on the application being performed, but will usually be
at least 15, more usually at least 20 and may be as high as 60 or
higher, where the number of different cycles will typically range
from about 20 to 40. For methods where more than about 25, usually
more than about 30 cycles are performed, it may be convenient or
desirable to introduce additional polymerase into the reaction
mixture such that conditions suitable for enzymatic primer
extension are maintained.
[0051] The denaturation step comprises heating the reaction mixture
to an elevated temperature and maintaining the mixture at the
elevated temperature for a period of time sufficient for any double
stranded or hybridized nucleic acid present in the reaction mixture
to dissociate. For denaturation, the temperature of the reaction
mixture will usually be raised to, and maintained at, a temperature
ranging from about 85 to 100, usually from about 90 to 98.degree.
C. and more usually from about 93 to 96.degree. C., for a period of
time ranging from about 3 to 120 sec, usually from about 5 to 30
sec.
[0052] Following denaturation, the reaction mixture will be
subjected to conditions sufficient for primer annealing to template
nucleic acid present in the mixture (if present), and for
polymerization of nucleotides to the primer ends in a manner such
that the primer is extended in a 5' to 3' direction using the
nucleic acid to which it is hybridized as a template, i.e.,
conditions sufficient for enzymatic production of primer extension
product. The temperature to which the reaction mixture is lowered
to achieve these conditions will usually be chosen to provide
optimal efficiency and specificity, and will generally range from
about 50 to 75, usually from about 55 to 70 and more usually from
about 60 to 68.degree. C., more particularly around 62.degree. C.
Annealing conditions will be maintained for a period of time
ranging from about 15 sec to 30 min, usually from about 20 sec to 5
min, or about 30 sec to 1 minute, or about 43 seconds.
[0053] This step can optionally comprise one of each of an
annealing step and an extension step with variation and
optimization of the temperature and length of time for each step.
In a 2-step annealing and extension, the annealing step is allowed
to proceed as above. Following annealing of primer to template
nucleic acid, the reaction mixture will be further subjected to
conditions sufficient to provide for polymerization of nucleotides
to the primer ends as above. To achieve polymerization conditions,
the temperature of the reaction mixture will typically be raised to
or maintained at a temperature ranging from about 65 to 75, usually
from about 67 to 73.degree. C. and maintained for a period of time
ranging from about 15 sec to 20 min, usually from about 30 sec to 5
min.
[0054] The above cycles of denaturation, annealing and
polymerization may be performed using an automated device,
typically known as a thermal cycler. Thermal cyclers that may be
employed are described elsewhere herein as well as in U.S. Pat.
Nos. 5,612,473; 5,602,756; 5,538,871; and 5,475,610, the
disclosures of which are herein incorporated by reference.
[0055] The methods of the invention can also be used in non-PCR
based applications to detect a target nucleic acid sequence, where
such target that may be immobilized on a solid support. Methods of
immobilizing a nucleic acid sequence on a solid support are known
in the art and are described in Ausubel et al. Current Protocols in
Molecular Biology, John Wiley and Sons, Inc. and in protocols
provided by the manufacturers, e.g. for membranes: Pall
Corporation, Schleicher & Schuell, for magnetic beads: Dynal,
for culture plates: Costar, Nalgenunc, and for other supports
useful according to the invention, CPG, Inc.
[0056] The person skilled in the art of nucleic acid amplification
knows the existence of other rapid amplification procedures such as
ligase chain reaction (LCR), transcription-based amplification
systems (TAS), self-sustained sequence replication (3SR), nucleic
acid sequence-based amplification (NASBA), strand displacement
amplification (SDA) and branched DNA (bDNA) (Persing et al, 1993.
Diagnostic Molecular Microbiology Principles and Applications,
American Society for Microbiology, Washington, D.C.). The scope of
this invention is not limited to the use of amplification by PCR,
but rather includes the use of any rapid nucleic acid amplification
methods or any other procedures which may be useful with the
sequences of the invention for the detection and/or quantification
of expression of one or more of the discriminative genes disclosed
herein.
[0057] Further, variations on the exact amounts of the various
reagents and on the conditions for the PCR or other suitable
amplification procedure (e.g., buffer conditions, cycling times,
etc.) that lead to similar amplification or
detection/quantification results are known to those of skill in the
art and are considered to be equivalents. In one embodiment, the
subject qRT-PCR detection has a sensitivity of detecting fewer than
50 copies (preferably fewer than 25 copies, more preferably fewer
than 15 copies, still more preferably fewer than 10 copies) of
target nucleic acid (e.g., genomic or cDNA) in a sample. In one
embodiment, a hot-start PCR reaction is performed (e.g., using a
hot start Taq DNA polymerase) so as to improve PCR reaction by
decreasing background from non-specific amplification and to
increase amplification of the desired extension product.
Evaluation of Protein Expression
[0058] In one embodiment, lung cancer status is evaluated using
levels of protein expression of one or more of the discriminative
genes listed in Table 1. The level of protein expression can be
measured using an immunological detection method.
[0059] Immunological detection methods which can be used herein
include, but are not limited to, competitive and non-competitive
assay systems using techniques such as Western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement-fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays, and the like. Such assays are routine and well known
in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols
in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York, which is incorporated by reference herein in its
entirety).
[0060] In one embodiment, antibodies specific for biomarker
proteins are utilized to detect the expression of a biomarker
protein in a body sample. The method comprises obtaining a body
sample from a patient, contacting the body sample with at least one
antibody directed to a biomarker that is selectively expressed in
lung cancer cells, and detecting antibody binding to determine if
the biomarker is expressed in the patient sample. A preferred
aspect of the present invention provides an immunocytochemistry
technique for diagnosing lung cancer subtypes. One of skill in the
art will recognize that the immunocytochemistry method described
herein below may be performed manually or in an automated
fashion.
[0061] The terms "antibody" and "antibodies" broadly encompass
naturally occurring forms of antibodies and recombinant antibodies
such as single-chain antibodies, chimeric and humanized antibodies
and multi-specific antibodies as well as fragments and derivatives
of all of the foregoing, which fragments and derivatives have at
least an antigenic binding site. Antibody derivatives may comprise
a protein or chemical moiety conjugated to the antibody.
[0062] "Antibodies" and "immunoglobulins" (Igs) are glycoproteins
having the same structural characteristics. While antibodies
exhibit binding specificity to an antigen, immunoglobulins include
both antibodies and other antibody-like molecules that lack antigen
specificity. The term "antibody" is used in the broadest sense and
covers fully assembled antibodies, antibody fragments that can bind
antigen (e.g., Fab', F'(ab).sub.2, Fv, single chain antibodies,
diabodies), and recombinant peptides comprising the foregoing.
[0063] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally-occurring
mutations that may be present in minor amounts.
[0064] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen-binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et
al. (1995) Protein Eng. 8(10):1057-1062); single-chain antibody
molecules; and multispecific antibodies formed from antibody
fragments. Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, whose
name reflects its ability to crystallize readily. Pepsin treatment
yields an F(ab')2 fragment that has two antigen-combining sites and
is still capable of cross-linking antigen.
[0065] "Fv" is the minimum antibody fragment that contains a
complete antigen recognition and binding site. In a two-chain Fv
species, this region consists of a dimer of one heavy- and one
light-chain variable domain in tight, non-covalent association. In
a single-chain Fv species, one heavy- and one light-chain variable
domain can be covalently linked by flexible peptide linker such
that the light and heavy chains can associate in a "dimeric"
structure analogous to that in a two-chain Fv species. It is in
this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0066] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (C.sub.H1) of the heavy
chain. Fab fragments differ from Fab' fragments by the addition of
a few residues at the carboxy terminus of the heavy-chain C.sub.H1
domain including one or more cysteines from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol
group. F(ab')2 antibody fragments originally were produced as pairs
of Fab' fragments that have hinge cysteines between them.
[0067] Polyclonal antibodies can be prepared by immunizing a
suitable subject (e.g., rabbit, goat, mouse, or other mammal) with
a biomarker protein immunogen. The antibody titer in the immunized
subject can be monitored over time by standard techniques, such as
with an enzyme linked immunosorbent assay (ELISA) using immobilized
biomarker protein. At an appropriate time after immunization, e.g.,
when the antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497, the human B cell hybridoma technique (Kozbor et al.
(1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et
al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed.
Reisfeld and Sell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96)
or trioma techniques. The technology for producing hybridomas is
well known (see generally Coligan et al., eds. (1994) Current
Protocols in Immunology (John Wiley & Sons, Inc., New York,
N.Y.); Galfre et al. (1977) Nature 266:550-52; Kenneth (1980) in
Monoclonal Antibodies: A New Dimension In Biological Analyses
(Plenum Publishing Corp., NY); and Lerner (1981) Yale J. Biol.
Med., 54:387-402).
[0068] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody can be identified and isolated by
screening a recombinant combinatorial immunoglobulin library (e.g.,
an antibody phage display library) with a biomarker protein to
thereby isolate immunoglobulin library members that bind the
biomarker protein. Kits for generating and screening phage display
libraries are commercially available (e.g., the Pharmacia
Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the
Stratagene SurfZAP 9 Phage Display Kit, Catalog No. 240612).
Additionally, examples of methods and reagents particularly
amenable for use in generating and screening antibody display
library can be found in, for example, U.S. Pat. No. 5,223,409; PCT
Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO
92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et
al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.
[0069] Techniques for detecting antibody binding are well known in
the art. Antibody binding to a biomarker of interest may be
detected through the use of chemical reagents that generate a
detectable signal that corresponds to the level of antibody binding
and, accordingly, to the level of biomarker protein expression. In
one method, antibody binding can be detected through the use of a
secondary antibody that is conjugated to a labeled polymer.
Examples of labeled polymers include but are not limited to
polymer-enzyme conjugates. The enzymes in these complexes are
typically used to catalyze the deposition of a chromogen at the
antigen-antibody binding site, thereby resulting in cell staining
that corresponds to expression level of the biomarker of interest.
Enzymes of particular interest include horseradish peroxidase (HRP)
and alkaline phosphatase (AP). Samples may be reviewed via
automated microscopy or by personnel with the assistance of
computer software that facilitates the identification of positive
staining cells.
[0070] Detection of antibody binding can be facilitated by coupling
the antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, or .sup.3H.
[0071] In regard to detection of antibody staining in the
immunocytochemistry methods of the invention, there also exist in
the art, video-microscopy and software methods for the quantitative
determination of an amount of multiple molecular species (e.g.,
biomarker proteins) in a biological sample wherein each molecular
species present is indicated by a representative dye marker having
a specific color. Such methods are also known in the art as a
colorimetric analysis methods. In these methods, video-microscopy
is used to provide an image of the biological sample after it has
been stained to visually indicate the presence of a particular
biomarker of interest. Some of these methods, such as those
disclosed in U.S. patent application Ser. No. 09/957,446 to
Marcelpoil et al. and U.S. patent application Ser. No. 10/057,729
to Marcelpoil et al., incorporated herein by reference, disclose
the use of an imaging system and associated software to determine
the relative amounts of each molecular species present based on the
presence of representative color dye markers as indicated by those
color dye markers' optical density or transmittance value,
respectively, as determined by an imaging system and associated
software. These techniques provide quantitative determinations of
the relative amounts of each molecular species in a stained
biological sample using a single video image that is
"deconstructed" into its component color parts.
[0072] The antibodies used to practice the invention are selected
to have high specificity for the biomarker proteins of interest.
Methods for making antibodies and for selecting appropriate
antibodies are known in the art. See, for example, Celis, ed. (in
press) Cell Biology & Laboratory Handbook, 3rd edition
(Academic Press, New York), which is herein incorporated in its
entirety by reference. In some embodiments, commercial antibodies
directed to specific biomarker proteins may be used to practice the
invention.
[0073] Furthermore, one of skill in the art will recognize that the
concentration of a particular antibody used to practice the methods
of the invention will vary depending on such factors as time for
binding, level of specificity of the antibody for the biomarker
protein, and method of body sample preparation. Moreover, when
multiple antibodies are used, the required concentration may be
affected by the order in which the antibodies are applied to the
sample, i.e., simultaneously as a cocktail or sequentially as
individual antibody reagents. Furthermore, the detection chemistry
used to visualize antibody binding to a biomarker of interest must
also be optimized to produce the desired signal to noise ratio.
[0074] Monoclonal and polyclonal antibodies available in the art
can be used in the practice of the invention. In particular,
antibodies against any of the biomarkers in Table 1 can be used in
the practice of the invention. Monoclonal and polyclonal antibodies
have been developed against peptide hormones (ACTH, GGRP,
chromogranin A, serotonin, etc), specific enzymes (NSE, CK-BB,
chromogranin A), cell adhesion molecules (NCAM), altered sugar
chains (mucin), and markers of proliferation (ki-67, PCNA). Many of
these antibodies can be used to identify the different types of
normal cells populating the respiratory tract based on their
function. For instance, the distinction between SCLC and NSCLC can
be defined relatively well with neuroendocrine markers, such as
neuron-specific enolase (NSE), neural cell adhesion molecule
(NCAM), and peptide hormones. Approximately 80% of SCLC will show a
neuroendocrine feature, but these markers are not specific since
they are also exhibited by carcinoid tumors and a small percent of
adenocarcinomas. Carcinoid tumors are believed to arise from
Kulchitsky's cells, which also have an endocrine function. Another
example of exploiting the specialized functions of cells to
diagnose lung tumors is in the use of antibodies against surfactant
apoprotein. This antibody reacts with type II alveolar cells and
with non-mucinous BAC, potentially due to a common progenitor. In
contrast to using markers of neuronal differentiation to diagnose
neuroendocrine tumors, surfactant apoprotein is specific for lung
adenocarcinoma but has poor sensitivity with only 50% of
adenocarcinomas staining positive. In some embodiments, surfactant
expressing adenocarcinomas are a definable entity by genomics and
this group has a distinct prognosis separate from other
adenocarcinomas. In addition to standard molecular techniques in
clinical pathology, tremendous effort has been devoted to the
development of novel biomarkers to improve lung cancer diagnostics.
Compared to other malignancies, particularly lymphoma and leukemia,
little of this work has born fruit in terms of clinically useful
tests. While numerous authors have documented that markers of poor
prognosis can be reliably identified, these do not appear to have
garnered much clinical interest (Gordon et al. (2003) Cancer
Epidemiol Biomarkers Prey 12(9):905-10). Finally, markers
associated with specific outcomes such as site-specific metastasis
and response to treatment (with the exception of EGFR) have been
too preliminary to be medically actionable.
Subjects
[0075] Lung cancer status can be assessed using the biomarker
proteins described herein in human as well as in non-human subjects
(e.g., non-human animals, such as laboratory animals, e.g., mice,
rats, guinea pigs, rabbits; domesticated livestock, e.g., cows,
horses, goats, sheep, chicken, etc.; and companion animals, e.g.,
dogs, cats, etc.). Suitable controls are usually selected on the
basis of the subject under study, and the nature of the study
(e.g., type of sample, type of spectra, etc.). Usually, controls
are selected to represent the state of "normality." As described
herein, deviations from normality (e.g., higher than normal, lower
than normal) in test data, test samples, test subjects, etc. are
used in classification, diagnosis, etc.
[0076] For example, in most cases, control subjects are the same
species as the test subject and are chosen to be representative of
the equivalent normal (e.g., healthy) subject. A control population
is a population of control subjects. If appropriate, control
subjects may have characteristics in common (e.g., sex, ethnicity,
age group, etc.) with the test subject. If appropriate, control
subjects may have characteristics (e.g., age group, etc.) which
differ from those of the test subject. For example, it may be
desirable to choose healthy 20-year olds of the same sex and
ethnicity as the study subject as control subjects.
[0077] In most cases, control samples are taken from control
subjects. Usually, control samples are of the same sample type
(e.g., fresh biopsy, paraffin embedded tissue, cryogenically
preserved tissue, etc.), and are collected and handled under the
same or similar conditions as the sample under study. Likewise,
control data (e.g., control values) are usually obtained from
control samples which are taken from control subjects. Usually,
control data are of the same type and are collected and handled
(e.g., recorded, processed) under the same or similar conditions as
the test data.
[0078] A control sample for use in the invention may be derived
from non-malignant cells or tissue (including "normal" cells or
tissue, or cells or tissue displaying benign lesions), or may be
derived from cells or tissue displaying a different type or grade
of lung cancer as the test sample.
Class Prediction
[0079] The present invention provides a method for classifying lung
cancer classes and subclasses using gene expression analysis. In
one embodiment, the method utilizes a classifier mechanism in which
the expression profiles of each group (e.g., one versus all, such
as tumor vs. normal, or each pairwise, such as normal vs. small
cell, or small cell vs. non-small cell, etc.) are self-normalized.
For each one versus all and each pairwise classification, the gene
cassette includes genes which are expressed at high levels relative
to the alternative class and low levels relative to the alternative
class. It is the ratio of each that is then used to make the class
distinction, as well as the gene expression values themselves.
[0080] The data obtained from the expression profiles, and
optionally from demographic information, can be evaluated using one
or more pattern recognition algorithms. In one embodiment, the
expression of a plurality of genes listed in Table 1 is used to
classify tumor types, subtypes, and class. It is to be understood
that other genes and/or diagnostic criteria may be used in this
invention. For example, the results of imaging tests or
histological evaluation may optionally be combined with expression
profiles generated using the genes disclosed herein.
[0081] Such analysis methods may be used to form a predictive
model, and then use that model to classify test data. For example,
one convenient and particularly effective method of classification
employs multivariate statistical analysis modeling, first to form a
model (a "predictive mathematical model") using data ("modeling
data") from samples of known class (e.g., from subjects known to
have, or not have, a particular class, subclass or grade of lung
cancer), and second to classify an unknown sample (e.g., "test
data"), according to lung cancer status.
[0082] Pattern recognition (PR) methods have been used widely to
characterize many different types of problems ranging for example
over linguistics, fingerprinting, chemistry and psychology. In the
context of the methods described herein, pattern recognition is the
use of multivariate statistics, both parametric and non-parametric,
to analyze spectroscopic data, and hence to classify samples and to
predict the value of some dependent variable based on a range of
observed measurements. There are two main approaches. One set of
methods is termed "unsupervised" and these simply reduce data
complexity in a rational way and also produce display plots which
can be interpreted by the human eye. The other approach is termed
"supervised" whereby a training set of samples with known class or
outcome is used to produce a mathematical model and is then
evaluated with independent validation data sets.
[0083] Unsupervised PR methods are used to analyze data without
reference to any other independent knowledge. Examples of
unsupervised pattern recognition methods include principal
component analysis (PCA), hierarchical cluster analysis (HCA), and
non-linear mapping (NLM).
[0084] Alternatively, and in order to develop automatic
classification methods, it has proved efficient to use a
"supervised" approach to data analysis. Here, a "training set" of
biomarker expression data is used to construct a statistical model
that predicts correctly the "class" of each sample. This training
set is then tested with independent data (referred to as a test or
validation set) to determine the robustness of the computer-based
model. These models are sometimes termed "expert systems," but may
be based on a range of different mathematical procedures.
Supervised methods can use a data set with reduced dimensionality
(for example, the first few principal components), but typically
use unreduced data, with all dimensionality. In all cases the
methods allow the quantitative description of the multivariate
boundaries that characterize and separate each class, for example,
each class of lung cancer in terms of its biomarker expression
profile. It is also possible to obtain confidence limits on any
predictions, for example, a level of probability to be placed on
the goodness of fit (see, for example, Kowalski et al., 1986). The
robustness of the predictive models can also be checked using
cross-validation, by leaving out selected samples from the
analysis.
[0085] Examples of supervised pattern recognition methods include
the following nearest centroid methods (Dabney (2005)
Bioinfommatics 21(22):4148-4154 and Tibshirani et al. (2002) Proc.
Natl. Acad. Sci. USA 99(10):6576-6572); soft independent modeling
of class analysis (SIMCA) (see, for example, Wold, 1976); partial
least squares analysis (PLS) (see, for example, Wold, 1966;
Joreskog, 1982; Frank, 1984; Bro, R., 1997); linear descriminant
analysis (LDA) (see, for example, Nillson, 1965); K-nearest
neighbour analysis (KNN) (see, for example, Brown et al., 1996);
artificial neural networks (ANN) (see, for example, Wasserman,
1989; Anker et al., 1992; Hare, 1994); probabilistic neural
networks (PNNs) (see, for example, Parzen, 1962; Bishop, 1995;
Speckt, 1990; Broomhead et al., 1988; Patterson, 1996); rule
induction (RI) (see, for example, Quinlan, 1986); and, Bayesian
methods (see, for example, Bretthorst, 1990a, 1990b, 1988). In one
embodiment, the classifier for identifying tumor subtypes based on
gene expression data is the centroid based method described in
Mullins et al. (2007) Clin Chem. 53(7):1273-9, which is herein
incorporated by reference in its entirety for its teachings
regarding tumor classification.
[0086] It is often useful to pre-process data, for example, by
addressing missing data, translation, scaling, weighting, etc.
Multivariate projection methods, such as principal component
analysis (PCA) and partial least squares analysis (PLS), are
so-called scaling sensitive methods. By using prior knowledge and
experience about the type of data studied, the quality of the data
prior to multivariate modeling can be enhanced by scaling and/or
weighting. Adequate scaling and/or weighting can reveal important
and interesting variation hidden within the data, and therefore
make subsequent multivariate modeling more efficient. Scaling and
weighting may be used to place the data in the correct metric,
based on knowledge and experience of the studied system, and
therefore reveal patterns already inherently present in the
data.
[0087] If possible, missing data, for example gaps in column
values, should be avoided. However, if necessary, such missing data
may replaced or "filled" with, for example, the mean value of a
column ("mean fill"); a random value ("random fill"); or a value
based on a principal component analysis ("principal component
fill"). Each of these different approaches will have a different
effect on subsequent PR analysis.
[0088] "Translation" of the descriptor coordinate axes can be
useful. Examples of such translation include normalization and mean
centering. "Normalization" may be used to remove sample-to-sample
variation. Many normalization approaches are possible, and they can
often be applied at any of several points in the analysis. "Mean
centering" may be used to simplify interpretation. Usually, for
each descriptor, the average value of that descriptor for all
samples is subtracted. In this way, the mean of a descriptor
coincides with the origin, and all descriptors are "centered" at
zero. In "unit variance scaling," data can be scaled to equal
variance. Usually, the value of each descriptor is scaled by
1/StDev, where StDev is the standard deviation for that descriptor
for all samples. "Pareto scaling" is, in some sense, intermediate
between mean centering and unit variance scaling. In pareto
scaling, the value of each descriptor is scaled by 1/sqrt(StDev),
where StDev is the standard deviation for that descriptor for all
samples. In this way, each descriptor has a variance numerically
equal to its initial standard deviation. The pareto scaling may be
performed, for example, on raw data or mean centered data.
[0089] "Logarithmic scaling" may be used to assist interpretation
when data have a positive skew and/or when data spans a large
range, e.g., several orders of magnitude. Usually, for each
descriptor, the value is replaced by the logarithm of that value.
In "equal range scaling," each descriptor is divided by the range
of that descriptor for all samples. In this way, all descriptors
have the same range, that is, 1. However, this method is sensitive
to presence of outlier points. In "autoscaling," each data vector
is mean centred and unit variance scaled. This technique is a very
useful because each descriptor is then weighted equally and large
and small values are treated with equal emphasis. This can be
important for analytes present at very low, but still detectable,
levels.
[0090] Several supervised methods of scaling data are also known.
Some of these can provide a measure of the ability of a parameter
(e.g., a descriptor) to discriminate between classes, and can be
used to improve classification by stretching a separation. For
example, in "variance weighting," the variance weight of a single
parameter (e.g., a descriptor) is calculated as the ratio of the
inter-class variances to the sum of the intra-class variances. A
large value means that this variable is discriminating between the
classes. For example, if the samples are known to fall into two
classes (e.g., a training set), it is possible to examine the mean
and variance of each descriptor. If a descriptor has very different
mean values and a small variance, then it will be good at
separating the classes. "Feature weighting" is a more general
description of variance weighting, where not only the mean and
standard deviation of each descriptor is calculated, but other well
known weighting factors, such as the Fisher weight, are used.
[0091] The methods described herein may be implemented and/or the
results recorded using any device capable of implementing the
methods and/or recording the results. Examples of devices that may
be used include but are not limited to electronic computational
devices, including computers of all types. When the methods
described herein are implemented and/or recorded in a computer, the
computer program that may be used to configure the computer to
carry out the steps of the methods may be contained in any computer
readable medium capable of containing the computer program.
Examples of computer readable medium that may be used include but
are not limited to diskettes, CD-ROMs, DVDs, ROM, RAM, and other
memory and computer storage devices. The computer program that may
be used to configure the computer to carry out the steps of the
methods and/or record the results may also be provided over an
electronic network, for example, over the Internet, an intranet, or
other network.
[0092] The process of comparing a measured value and a reference
value can be carried out in any convenient manner appropriate to
the type of measured value and reference value for the
discriminative gene at issue. "Measuring" can be performed using
quantitative or qualitative measurement techniques, and the mode of
comparing a measured value and a reference value can vary depending
on the measurement technology employed. For example, when a
qualitative colorimetric assay is used to measure expression
levels, the levels may be compared by visually comparing the
intensity of the colored reaction product, or by comparing data
from densitometric or spectrometric measurements of the colored
reaction product (e.g., comparing numerical data or graphical data,
such as bar charts, derived from the measuring device). However, it
is expected that the measured values used in the methods of the
invention will most commonly be quantitative values. In other
examples, measured values are qualitative. As with qualitative
measurements, the comparison can be made by inspecting the
numerical data, or by inspecting representations of the data (e.g.,
inspecting graphical representations such as bar or line
graphs).
[0093] The process of comparing may be manual (such as visual
inspection by the practitioner of the method) or it may be
automated. For example, an assay device (such as a luminometer for
measuring chemiluminescent signals) may include circuitry and
software enabling it to compare a measured value with a reference
value for a biomarker protein. Alternately, a separate device
(e.g., a digital computer) may be used to compare the measured
value(s) and the reference value(s). Automated devices for
comparison may include stored reference values for the biomarker
protein(s) being measured, or they may compare the measured
value(s) with reference values that are derived from
contemporaneously measured reference samples (e.g., samples from
control subjects).
[0094] As will be apparent to those of skill in the art, when
replicate measurements are taken, the measured value that is
compared with the reference value is a value that takes into
account the replicate measurements. The replicate measurements may
be taken into account by using either the mean or median of the
measured values as the "measured value."
FFPE
[0095] In one aspect of the invention, the analysis is performed on
lung biopsies that are embedded in paraffin wax. This aspect of the
invention provides a means to improve current diagnostics by
accurately identifying the major histological types, even from
small biopsies. The methods of the invention, including the RT-PCR
methods, are sensitive, precise and have multianalyte capability
for use with paraffin embedded samples. See, for example, Cronin et
al. (2004) Am. J Pathol. 164(1):35-42, herein incorporated by
reference.
[0096] Formalin fixation and tissue embedding in paraffin wax is a
universal approach for tissue processing prior to light microscopic
evaluation. A major advantage afforded by formalin-fixed
paraffin-embedded (FFPE) specimens is the preservation of cellular
and architectural morphologic detail in tissue sections. (Fox et
al. (1985)J Histochem Cytochem 33:845-853). The standard buffered
formalin fixative in which biopsy specimens are processed is
typically an aqueous solution containing 37% formaldehyde and
10-15% methyl alcohol. Formaldehyde is a highly reactive dipolar
compound that results in the formation of protein-nucleic acid and
protein-protein crosslinks in vitro (Clark et al. (1986) J
Histochem Cytochem 34:1509-1512; McGhee and von Hippel (1975)
Biochemistry 14:1281-1296).
[0097] The ability to analyze gene expression patterns in these
archived tissues would greatly facilitate retrospective studies to
correlate gene expression patterns with given disease states, or
histological and clinical phenotypes. This approach could be used
to discover biomarkers for therapeutic decision making and also to
develop clinical tests, as FFPE sample collection and storage is a
routine practice in pathology laboratories.
[0098] Methods are known in the art for the isolation of RNA from
FFPE tissue. In one embodiment, total RNA can be isolated from FFPE
tissues as described by Bibikova et al. (2004) American Journal of
Pathology 165:1799-1807, herein incorporated by reference.
Likewise, the High Pure RNA Paraffin Kit (Roche) can be used.
Paraffin is removed by xylene extraction followed by ethanol wash.
RNA can be isolated from sectioned tissue blocks using the
MasterPure Purification kit (Epicenter, Madison, Wis.); a DNase I
treatment step is included. RNA can be extracted from frozen
samples using Trizol reagent according to the supplier's
instructions (Invitrogen Life Technologies, Carlsbad, Calif.).
Samples with measurable residual genomic DNA can be resubjected to
DNaseI treatment and assayed for DNA contamination. All
purification, DNase treatment, and other steps can be performed
according to the manufacturer's protocol. After total RNA
isolation, samples can be stored at -80.degree. C. until use.
Kits
[0099] Kits for practicing the methods of the invention are further
provided. By "kit" is intended any manufacture (e.g., a package or
a container) comprising at least one reagent, e.g., an antibody, a
nucleic acid probe or primer, etc., for specifically detecting the
expression of a biomarker of the invention. The kit may be
promoted, distributed, or sold as a unit for performing the methods
of the present invention. Additionally, the kits may contain a
package insert describing the kit and methods for its use.
[0100] In one embodiment, kits for practicing the methods of the
invention are provided. Such kits are compatible with both manual
and automated immunocytochemistry techniques (e.g., cell staining).
These kits comprise at least one antibody directed to a biomarker
of interest, chemicals for the detection of antibody binding to the
biomarker, a counterstain, and, optionally, a bluing agent to
facilitate identification of positive staining cells. Any chemicals
that detect antigen-antibody binding may be used in the practice of
the invention. The kits may comprise at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10, or more antibodies for use in the methods of the
invention.
[0101] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Example 1
Immunohistochemical Detection of Gene Expression Subsets of
Non-Small Cell Carcinoma
Background:
[0102] Messenger RNA abundance studies using nucleic acid
microarrays allow cluster separation of morphologically similar
diseases into molecular subsets. Outcome studies in breast
carcinomas and diffuse large cell lymphomas show the relevance of
this approach. More recently, lung adenocarcinomas have been
subdivided at the mRNA cluster level into magnoid, squamoid, and
bronchioid types (1). The purpose of this project was to pick
single representative loci from each cluster, and to screen for
distinct clustering at the protein level within an unselected set
of non-small cell lung carcinoma (NSCLC).
Design:
[0103] Duplicate-core tissue microarrays were manufactured from 187
surgically resected primary NSCLC. Cases were unselected for
morphology, stage, demographics, risk factors, or outcomes.
Screening loci were selected using a rational approach based on
gene expression profiling. Loci had to segregate the 3 reported
adenocarcinoma subtypes (bronchioid, magnoid, and squamoid), and
had to be commercially available. Smooth muscle actin, vimentin,
and TTF-1 were chosen. Paraffin immunostains were scored by one
Pathologist for signal strength and carcinoma percent positivity.
Sample data were included if cores stained for at least one of the
3 markers in at least 10% of cells in at least one of 2 replicate
stains. If neither replicate sample stained for any of the 3
markers, it was excluded.
Results:
[0104] 152 of 187 samples were evaluable. 16 samples were excluded
because the tumor or the core was absent, and 19 were excluded due
to <10% cells staining for the marker. The staining patterns of
the 152 samples are ordered by hierarchical agglomerative
clustering, and show distinct clustering into three groups. The Chi
square test p value for this distribution of staining is
p<2.2e-16.
Conclusions:
[0105] This study shows that molecular subsets exist within
unselected NSCLC, and that these distinguish distinct clusters
similar to those seen with mRNA abundance analyses. The RNA and
protein expression in NSCLC is useful to correlate with recognized
morphologic subgroups, and with treatment response and survival.
The study identified:
1) Representative markers from the adenocarcinoma mRNA clusters to
distinguish histologically diagnostic types of NSCLC at the protein
level. 2) Representative markers from the adenocarcinoma mRNA
clusters to screen for the 3 subtypes of adenocarcinoma at the
protein level.
Methods
[0106] Tissue microarrays were made from duplicate 1 mm cores taken
from 187 unselected UNC non-small cell lung carcinoma (NSCLC)
resection specimens.
[0107] To select markers distinguishing reported tumor subtypes,
the following approach was used. A list of all commercially
available antibodies for which protocols are established was
prepared (approximately 120). The gene targets of these antibodies
were identified using data provided by the NCBI and Genbank.
Existing gene expression data from the published record (1) were
reviewed for those genes which distinguish established lung
adenocarcinoma subtypes, and cross-referenced with the available
antibody list. Approximately 50 genes with antibodies readily
available were also statistically associated with one or more of
the adenocarcinoma subtypes. Three were selected, one for each of
the subtypes, accounting for experience with the antibodies to aid
in those most likely to be useful. TTF-1 was chosen to represent
the "bronchioid" cluster, and was detected with Ventana clone
867G34-1 after EDTA pH8 HIER. SMA was chosen to represent the
"squamoid" cluster, and was detected with Ventana clone 1A4 after
EDTA pH8 HIER. Vimentin was chosen to represent the "magnoid"
cluster, and was detected with Ventana clone V9 after EDTA pH8
HIER. Stained sections were scored for average signal strength
(0-3+) and percentage of tumor cells which were positive to any
degree. Data from each of two cores were collated, and the %
positivity data were averaged. Sample data were included if cores
stained for at least one of the markers in >10% of the tumor
cells.
Results
[0108] Of the 187 samples, 152 were evaluable. 16 samples were
excluded because of mechanical core loss in the stained TMA
section, and 19 samples were excluded because of <10% staining
for each of the three markers. Of the 152 evaluable cases, 85 (56%)
were adenocarcinoma, 53 (35%) were squamous carcinoma, and 14 (9%)
were NSCLC, subtype not specified.
[0109] Immunoreactivities for each of the three proteins were
clustered for the total set of 152 cases, and show three major
clusters (FIG. 1). These clusters are distinct by chi-square
analysis (p<2.2e-16).
[0110] Immunoreactivities for each of the three proteins were
clustered for the histologically diagnosed adenocarcinomas (n=85),
and show three major clusters (FIG. 2).
[0111] Immunoreactivities for each of the three proteins were
clustered for the histologically diagnosed squamous carcinomas
(n=53), and show three major clusters (FIG. 3).
[0112] In recent experiments (data not shown), there is good
correlation between TTF-1 mRNA abundance and protein abundance.
Conclusions
[0113] 1. The rational antibody selection process described here
was successful at leveraging existing antibody resources for novel
genomic analysis. 2. The first hypothesis of antibody selection
clearly distinguished 3 groups of lung adenocarcinomas, as expected
based on the genomic results. 3. The genomic analysis is useful for
predicting the clinical outcomes of these adenocarcinoma
subtypes.
REFERENCES
[0114] 1. DN Hayes et al. Gene expression profiling reveals
reproducible human lung adenocarcinoma subtypes in multiple
independent patient cohorts. J Clin Onc 24:5079, 2006. [0115] 2. A
Bhattacharjee et al. Classification of human lung carcinomas by
mRNA expression profiling reveals distinct adenocarcinoma
subclasses. PNAS 98:13790, 2001. [0116] 3. ME Garber et al.
Diversity of gene expression in adenocacnoma of the lung. PNAS
98-13784, 2001. [0117] 4. DG Beer et al. Gene-expression profiles
predict survival of patients with lung adenocarcinoma. Nat Med
8:816, 2002. [0118] 5. AC Borczuk, C A Powell. Expression profiling
and lung cancer development. Proc Am Thor Soc 4:127, 2007. [0119]
6. TA D'Amico et al. A biologic risk model for stage 1 lung cancer:
Immunohistochemical analysis of 408 patients with the use of ten
molecular markers. Thorac Cardiovasc Surg 117:736, 1999.
Example 2
Subtypes of Lung Adenocarcinoma Derived from Gene Expression
Patterns are Recapitulated Using a Tissue Microarray System and
Immunohistochemistry
Background
[0120] Messenger RNA abundance studies using nucleic acid
microarrays allow cluster separation of morphologically similar
diseases into molecular subsets. Outcome studies in breast
carcinomas and diffuse large cell lymphomas show the relevance of
this approach. In the field of lung cancer, multiple groups have
shown that differences in mRNA abundance distinguish the
morphologic variants, including squamous cell carcinoma, small cell
carcinoma, and adenocarcinoma. More recently, lung adenocarcinomas
have been subdivided at the mRNA cluster level into magnoid,
squamoid, and bronchioid types using gene expression array-based
technology (Hayes et al. J Clin Onc 24:5079, 2006). The goal was to
translate these findings into more conventional paraffin-based
diagnostic tests.
[0121] The purpose of this project was to pick single
representative markers from each cluster, and to screen for
distinct clustering at the protein level within a set of lung
adenocarcinomas. The goal was to identify the three distinct mRNA
clusters in a set of paraffin-embedded adenocarcinomas which would
not otherwise be subdivided in routine clinical practice. The
clinical phenotypes and survival outcomes of these molecular
subtypes are also reported.
[0122] Finally, another goal was to expedite marker identification,
using existing immunohistochemical reagents. It was determined that
clinically useful subtypes of lung adenocarcinomas previously
identified using gene expression can also be identified using
immunohistochemistry (IHC) in clinically obtained paraffin
specimens.
Methods
[0123] Duplicate-core tissue microarrays were manufactured from 187
surgically resected primary NSCLC. Only adenocarcinoma histology
was considered for the current study. Cases were unselected for
morphology, stage, demographics, risk factors, or outcomes.
Screening markers were selected using a rational approach based on
gene expression profiling and only commercially available reagents
were considered using the following approach. A list of all
commercially available antibodies for which protocols are
established was prepared (approximately 120). The gene targets of
these antibodies were identified using data provided by the NCBI
and Genbank. Existing gene expression data from the published
record was reviewed for those genes which distinguish established
lung adenocarcinoma subtypes, and cross-referenced with the
available antibody list.
[0124] Approximately 50 genes with antibodies readily available
were also statistically associated with one or more of the
adenocarcinoma subtypes. From the set of 50 likely candidates, 3
markers were selected (one for each of the subtypes--bronchioid,
magnoid, and squamoid) based on the area under the receiver
operator characteristic curve (ROC) (see Results). Experience with
the antibodies was further considered to aid in those most likely
to be useful. Thyroid transcription factor 1 (TTF-1) was chosen to
represent the "bronchioid" cluster, and was detected with Ventana
clone 867G34-1 after EDTA pH8 HIER. Smooth muscle actin (SMA) was
chosen to represent the "squamoid" cluster, and was detected with
Ventana clone 1A4 after EDTA pH8 HIER. Vimentin was chosen to
represent the "magnoid" cluster, and was detected with Ventana
clone V9 after EDTA pH8 HIER. Stained sections were scored for
average signal strength (0-3+) and percentage of tumor cells which
were positive to any degree. Data from each of two cores were
collated, and the % positivity data were averaged. Sample data were
included if cores stained for at least one of the markers in
>10% of the tumor cells. If neither replicate sample stained for
any of the 3 markers, it was excluded. The gene-protein concordance
of the targets was evaluated in an unbiased manner using the
integrative correlations method (IC). Clinical histories were
abstracted from the medical record including staging, surgical
treatment, survival, and site of relapse.
Results
[0125] 152 of 187 samples were evaluable. 16 samples were excluded
because the tumor or the core was absent, and 19 were excluded due
to <10% cells staining for the marker. Of the 152 evaluable
samples, 85 were adenocarcinoma. The staining patterns of the 85
samples are ordered by hierarchical agglomerative clustering, and
show distinct clustering into three groups (FIG. 4). The Chi square
test p value for this distribution of staining is
p<2.2.times.10-16.
[0126] Approximately 125 antibodies were screened and associated
with approximately 200 transcripts from the gene expression array
repository. Of these, 35% were statistically significantly
associated with at least one tumor subtype in keeping with previous
reports. The following targets each demonstrated area under the
ROC>0.9 for distinguishing adenocarcinoma subtypes and were
selected for IHC confirmation in the clinical TMA system:
vimentin-magnoid subtype; SMA-squamoid subtype; and TTF1-bronchioid
subtype.
TABLE-US-00002 TABLE 2 Patient Characteristics by Molecular Subtype
of Adenocarcinoma The p value for the IC coefficient for each of
these 3 markers was <0.001 suggesting that the IHC markers were
reliable representations the gene expression. Importantly, staining
positive for one marker was associated with negative staining for
the others (chi squared p value <0.02). TTF1/ Vimentin/
Bronchioid Magnoid SMA/Squamoid N = 85 45(53%) 13(15%) 27(32%)
Smoking status Current 20 3 9 Former 19 10 9 Never 2 0 2 Unknown 4
0 7 Gender Male 22 7 12 Female 23 6 15 Race African American 14 4 1
Caucasian 30 9 26 Grade Unknown 4 0 1 Well 5 1 5 Well-Moderate 4 0
2 Moderate 21 4 9 Moderate-Poor 9 1 2 Poor 5 5 7 Bronchioalveolar 3
2 0 Morphology Clinical Stage IA 16 7 13 IB 11 4 6 IIA 0 0 1 IIB 5
0 2 IIIA 4 0 0 IIIB 0 1 0 UNK 9 1 5 Pathologic Stage IA 18 5 14 IB
5 2 4 IIA 3 1 0 IIB 8 1 6 IIIA 8 2 1 IIIB 0 1 0 UNK 3 1 2 Site of
1st recurrence 12(26%) 5(38%) 9(33%) 26(31%) with metastasis in
study period Bone 3 0 0 Brain 3 3 4 Lung 6 2 4 Node 0 0 1
[0127] The clinical phenotype associated with the 3 molecular
subtypes of lung adenocarcinoma reproduces previous work. Most
notably, there are clear differences in the frequency and pattern
of recurrence (Table 2) by tumor subtype and survival. FIG. 5
demonstrates the survival by tumor subtype.
Conclusions
[0128] 1. The rational antibody selection process described here
was successful at leveraging existing antibody resources for novel
genomic analysis. 2. The first hypothesis of antibody selection
clearly distinguished 3 groups of lung adenocarcinomas, as expected
based on the genomic results. 3. Dramatic patterns of tumor
behavior were observed that were associated with tumor subtypes
potentially vital to patients and clinicians. These include
differences in the frequency and pattern of relapse which may allow
targeted interventions.
Example 3
Paraffin-Based Molecular Diagnosis of Lung Cancer Reproduces
Morphologic and Molecular Subtypes of Lung Cancer
Background
[0129] Gene expression classifications of lung cancer by microarray
have shown potential for guiding therapy (1-2). However,
high-quality RNA from fresh tissue for microarray is generally
unavailable in clinical practice. A real-time quantitative reverse
transcription PCR (qRT-PCR) assay and analytic method for
identifying morphological subtypes of lung cancer from clinically
obtained formalin-fixed, paraffin-embedded (FFPE) tissues are
introduced.
Methods
[0130] Approximately 700 DNA microarrays were analyzed to select
genes distinguishing the major histological variants of NSCLC
carcinoma and SCLC carcinoma. Previous work has shown that NSCLC is
a molecularly diverse group, especially among adenocarcinomas
(AC)(3). A 57-gene qRT-PCR assay (52 classifiers and 5 control
genes) that molecularly identifies histological subtypes of lung
cancer and molecular subtypes of adenocarcinoma (FIG. 6) was
developed. This assay was used to profile RNA extracted from a
cohort of 257 surgically treated NSCLC patients (Table 3) and 2
SCLC cell cultures. Samples were procured as fresh frozen (FF) and
FFPE tissues archived between 1-15 years.
Results
[0131] The cohort profiled by this qRT-PCR assay of 52 classifiers
and 5 control (i.e. housekeeper) genes (Table 1) contained a broad
spectrum of tumors in proportions consistent with clinical practice
(Table 3). Gene amplification was successful in 238 of 257 (92.6%)
lung cancer, normal, and cell culture samples. Matched FF and FFPE
had Pearson correlations of approximately 70%. Linear discriminant
analysis of gene expression data agreed with morphologic
classification by light microscopy with accuracies of 94-100%
(Table 3). More importantly, the method successfully re-identified
molecular subtypes of lung cancer using a FFPE tissue assay (FIG.
7). Clinical outcomes previously associated with molecular tumor
subtypes, including differential survival, were again seen in this
cohort (FIG. 8).
TABLE-US-00003 TABLE 3 Clinical and pathological parameters of lung
samples used in qRT-PCR. HCI FF HCI FFPE UNC FFPE Total 29 60 168
257 Gender Male 12 21 92 125 Female 15 37 73 125 Cell Cx 2 2 0 4
Age <60 8 18 48 74 60-75 17 35 92 144 >75 2 5 23 30 Race
Caucasian 24 57 128 209 African 1 1 37 39 American Native 1 1 0 2
American Smoking status .ltoreq.20 pkyrs 1 5 16 22 >20 pkyrs 14
30 100 144 Positive 4 10 26 40 Negative 2 5 7 14 Unknown 6 8 19 33
Grade Well 1 2 14 17 Well-Moderate 2 5 8 15 Moderate 5 12 88 105
Moderate-Poor 6 10 14 30 Poor 6 10 33 49 Atypical 4 4 0 8 Typical 1
1 0 2 Unknown 2 4 11 17 Histology Normal 0 10 0 10 SCLC 2 2 0 4
Carcinoid 7 9 0 16 SCC 5 12 63 80 AC 15 27 105 147 Stage IA 11 19
61 91 IB 3 11 37 51 IIA 2 2 4 8 IIB 2 3 21 26 IIIA 6 9 12 27 IIIB 2
3 3 8 IV 1 1 0 2 Unknown 0 0 30 30 qRT-PCR Prediction ADENO COID
NRML SCC SCLC TOTAL Histology ADENO 130 1 6 137 COID 16 1 17 NRML 8
8 SCC 6 1 67 74 SCLC 2 2 TOTAL 136 16 11 73 2 238
Conclusions
[0132] For the first time, a clinically meaningful and robust
molecular diagnosis of a cohort of lung cancer patients which is
complementary to morphologic cancer diagnosis is described here.
This assay is easily implemented using specimens routinely
collected in current patient care.
REFERENCES
[0133] 1) Potti A, Mukherjee S, Petersen R, et al: A Genomic
Strategy to Refine Prognosis in Early-Stage Non-Small-Cell Lung
Cancer. N Engl J Med 355:570-580, 2006 [0134] 2) Beer D G, Kardia S
L, Huang C C, et al: Gene-expression profiles predict survival of
patients with lung adenocarcinoma. Nat Med 8:816-824, 2002 [0135]
3) Hayes D N, Monti S, Parmigiani G, et al: Gene Expression
Profiling Reveals Reproducible Human Lung Adenocarcinoma Subtypes
in Multiple Independent Patient Cohorts. J Clin Oncol 24:5079-5090,
2006
Example 4
[0136] Although the differentiation of malignant from normal lung
tissue is a very reliable diagnostic distinction, the accurate and
reproducible classification of variants of lung cancer has never
been described. Although over 100 morphologic variants of lung
tumors have been described, for all practical purposes only 2
distinctions are generally made with confidence, the distinction of
small cell lung cancer from non-small cell lung cancer (a diagnosis
of exclusion). The reason for the unreliability of lung cancer
diagnosis includes the general small volumes of tissues and
intra-tumor morphologic variation.
[0137] New and developing technologies such DNA microarrays have
demonstrated that gene expression can reliably distinguish known
morphologic variants of lung cancer as well as novel subtypes not
otherwise distinguished by any clinically available technology. The
technologies currently in place are not appropriate for diagnostic
and clinical use due to their cost and technical limitations. There
is no current technology for making molecular diagnosis of lung
cancer morphologic variants using gene expression measures. There
is no current technology for diagnosing molecular subtypes of lung
cancer.
[0138] Provided herein is the first application of a technology
capable of diagnosing both commonly accepted morphologic variants
of lung tumors, as well as newly described molecular variants and
clinically meaningful behavior of lung cancer such as survival. The
approach has a number of novel components. Firstly, its application
to lung tumors is new. Secondly, the use of paraffin embedded
tissues for molecular diagnosis of lung cancer by gene expression
is new. Third, a novel and high-throughput approach was used to
independently validate the genes in the classifier.
[0139] Fourth, a robust novel self-normalizing method of
classification was used which allows for a modular predictor. In
this approach, predictions are made in 2 ways, using a one versus
all and all pairwise approach such that many classes can be
predicted using small gene cassettes. Since each of the gene
cassettes is independent, new cassettes can be added to the
existing predictor without changing the overall method of
prediction. This allows the user to add new classes as desired. For
example, the current predictor distinguished a total of 9 classes
and subclasses of lung cancer (FIG. 1). As new groups of lung
cancer are described or as new features of tumor behavior are
identified, these can be added to the current predictor as
cassettes without changing the existing structure of the
classifier.
[0140] Gene selection for gene cassettes is in the following
manner. A set of gene expression cohorts was selected from among
all published reports of lung cancers assayed by gene expression
data due to their relatively large size and inclusion of a
representative spectrum of tumor variants. Expression datasets were
transformed using genomic meta-analysis methods that have been
previously reported. Briefly, all arrays were evaluated for the
quality of the scanned image. Probes were mapped to genes using
Unigene identifiers. In cases where multiple probes mapped to the
same Unigene identifier, these were averaged. Genes were evaluated
for cross-platform reliability using integrative correlations.
Genes with integrative correlations twice that observed by random
chance were considered reliable and retained across datasets.
Reliable genes were then ranked for each gene for their ability to
distinguish each of the morphologic variants in both the 1 versus
all (i.e. tumor versus normal) and the all-pairwise (i.e. normal
versus small cell carcinoma) case. The statistic used for the
ranking was the area under the receiver operator characteristic
(ROC) curve (a plot of sensitivity versus (1-specificity)).
Although genes were evaluated for reliability across datasets, the
independent sample sets were not combined for the purposes of the
ROC ranking. As a result, multiple independent analyses were
performed and obtained multiple independent rankings for each
gene's ability to distinguish groups of interest. A gene was
considered reliable if its area under the ROC curve was close to 1
for each of the independent datasets. In cases were a gene's ROC
was close to 1 in some but not all datasets, the gene's overall
reliability as measured by integrative correlations was considered.
The gene selection process was pruned to approximately 100 genes
based on prior experience that this was the number that could be
reliably obtained from paraffin samples using current
techniques.
[0141] Using previously described techniques, tumors from the
existing cohorts were divided into their respective gene expression
subtypes (3 subtypes of lung adenocarcinoma). A similar ROC-based
approach to that described above was used to select gene cassettes
capable of distinguishing these expression subtypes. Additionally,
gene cassettes were selected to identify genes associated with
survival and metastasis.
Selecting Housekeeper Genes
[0142] Housekeeper (HK) genes were used to control for variation in
RNA quality and cDNA input. Nine housekeeper genes that had among
the highest average signal/noise ratio across histological types in
the preliminary microarray data set were selected. The high average
signal/noise ratio suggests relatively high levels of expression
and lower variation across the lung sample types. After qRT-PCR
profiling across these lung samples, five genes were selected
(CFL1, EEF1A1, RPL10, RPL28, and RPL37A) as housekeepers using the
statistical methods described in previous reports.
Designing Primers
[0143] Genbank sequences were downloaded from Ensembl (which can be
found on the internet at www.ensembl.org/Homo_sapiens/index.html,
release 42-December 2006) into LightTyper Probe Design software
(version: 2.0.B.22) (Roche Applied Science, Indianapolis, Ind.).
Primer sets were designed and performance was assessed using
criteria described elsewhere. Primers were tested on lung cDNA from
FFPE tissue prior to utilization for qRT-PCR. Gene references and
primer sequences are listed in Table 1.
qRT-PCR
[0144] Each 5 .mu.l PCR reaction included 2-fold concentrated
LightCycler 480 SYBR Green I Master Mix (Roche Applied Science,
Indianapolis, Ind.) and 2.5 ng (1.25 ng/uL) cDNA. Liquid handling
for loading 384-well plates was done using the Evolution P3
Precision Pipetting Platform (PerkinElmer Ltd, Shelton, Conn.).
Each run contained an internal 10 ng calibrator reference using
cDNA made from Human Reference Total RNA (catalogue number #750500;
Stratagene, La Jolla, Calif.) and two small cell lung cultures (DMS
53 and DMS 114). Samples and calibrator were present in
duplicate.
[0145] PCR amplification was performed in the LC480 (Roche Applied
Science, Indianapolis, Ind.) using an initial denaturation step
(95.degree. C., 8 minutes) followed by 45 cycles of denaturation
(95.degree. C., 4 seconds), annealing (56.degree. C., 6 seconds
with 2.5.degree. C./s transition), and extension (72.degree. C., 6
seconds with 2.degree. C./sec transition). Fluorescence (530 nm)
from the dsDNA dye SYBR Green I was acquired for each cycle after
the extension step. The specificity of the PCR was determined by
post-amplification melting curve analysis. Reactions were
automatically cooled to 65.degree. C. and slowly heated at
2.degree. C./s to 99.degree. C. while continuously monitoring
fluorescence (10 acquisitions/1.degree. C.).
Data Analysis for qRT-PCR
[0146] Quantification was performed using the LC480 software
(version: 1.2.0.169) (Roche Applied Science, Indianapolis, Ind.).
Relative copy numbers were calculated by importing an external
standard curve made from a serial 10-fold dilution of GAPDH
(efficiency 1.8) and correcting to the Cp of the 10 ng calibrator.
The copy number for each classifier gene was normalized to the
average of all 5 housekeepers. Samples missing more that 10% of
genes were excluded from further analysis. Missing data in
remaining samples were imputed using k-nearest neighbor.
Lung Sample Class Prediction
[0147] Using the genes selected and measured as described above, a
robust self-normalizing classifier was used to predict tumor
classes and subclasses. Briefly the self normalizing classifier
works in the following manner. For each one versus all and each
pairwise classification being made, the gene cassette includes
genes which are expressed at high levels relative to the
alternative class and low levels relative to the alternative class.
It is the ratios that are then used to make the class distinctions,
as well as the gene expression values themselves. The use of
unitless (normalized) ratios is less sensitive to technical
artifacts that occur in the measurement of gene expression and
allows more reliable classification. A second set of class calls is
made for validation purposes using linear discriminant analysis on
housekeeper-normalized gene expression data.
[0148] In summary, the present invention provides the first
paraffin-based gene-expression-based molecular diagnostic for lung
cancer capable of distinguishing a range of biologically and
phenotypically relevant tumor classes. This real-time qRT-PCR assay
for approximately 100 genes is useful for making accurate lung
cancer classifications from either formalin-fixed-paraffin-embedded
or fresh tumor biopsy specimens. This assay is appropriate either
for grossly resected and small volume biopsies such as fine needle
aspirates or cytologic specimens. See FIG. 6.
[0149] As noted in FIG. 6, in normal tissue the following genes are
upregulated: CDH5, CLE3B, and PECAM 1; and the following genes are
downregulated: PAICS, PAK1, TFAP2A. In small cell lung carcinoma
(SCLC) CDKN2C, INSM1, and STMN1 are upregulated and ACVR1, CIB1,
and LRP10 are downregulated. In carcinoid cells (COID) CHGA,
MAPRE3, and SNAP91 are upregulated and CAPG, LGALS3, and SFN are
downregulated. Table 1 lists additional classifier genes according
to class and subclass.
Example 5
Background
[0150] Lung cancer is the leading cause of cancer deaths both in
the United States and worldwide (1, 2). The irony of clinical lung
cancer management is that despite major advances in recent years
documenting the effectiveness of a host of interventions (adjuvant
chemotherapy, combined modality chemotherapy and radiation,
palliative chemotherapy, targeted treatments), more than 85% of
patients diagnosed with lung cancer will die of their disease,
often in less than a year. (3-7). A compelling argument can be made
that a single fact explains the striking disconnect between the
number of available effective therapies and the poor outcomes for
lung cancer patients. There are very limited tools to assist
practitioners in matching the correct therapies to the patients
likely to benefit from them. In current practice, two pieces of
information are generally all that is considered when prescribing
therapy to lung cancer patients: small-cell versus non-small cell
morphology and clinical stage.
Lung Cancer Genomics
[0151] Previous clinical work has demonstrated the probable success
in predicting surgically-treated patients at high risk for
recurrence and death. The ability of microarrays to reliably
distinguish gross histologic subtypes, such as tumor versus normal
lung, has been demonstrated by a number of authors (34, 36, 37).
Genomic meta-analysis has been developed, documenting for the first
time the reproducibility of subtypes of lung adenocarcinoma. Using
5 independent cohorts totaling approximately 500 patients, it has
been demonstrated that morphologically indistinguishable tumors not
otherwise classifiable by existing diagnostics can be reliably
subdivided into 3 subgroups (coined bronchioid, squamoid, and
magnoid) based on differences in gene expression (FIG. 9). These
differences are not a subtle statistical phenomenon, but involve
differential co-expression within the subgroups of as many as 1/3
of all expressed genes in the tumors. Accordingly, these novel
adenocarcinoma subtypes appear to account for a host of clinical
patterns of lung cancer, including probability of recurrence,
survival, and metastatic pattern (43). Although the technologies
that allow expression profiling are barely a decade old, findings
parallel to those described above have been successfully validated
in a range of tumors, perhaps most definitively in breast cancer,
lymphoma, and leukemia (45-47).
Squamous Cell Carcinoma
[0152] Building on work with adenocarcinomas, risk stratification
was also performed for SCC, the other major histological types of
NSCLC using expression profiling. There is only one report of
different tumor subtypes of SCC by DNA microarrays (44). As with
the AD subtypes, the reported subtypes differ dramatically in terms
of survival, in this case for 2 subtypes of SCC, with six year
survival 80% in the favorable group compared to 40% in the
unfavorable (p=01). Hayes et al. have been able to validate the 2
distinct subtypes of SCC based on gene expression patterns (43).
Additionally, the magnitude of the survival difference in SCC
subtypes has been documented, as in the original report by Inamura,
although this did not meet a significance level of p=0.05 due to
small numbers (FIG. 10. hazard ratio 0.55 p=0.18, 6 year survival
60% versus 20% p=0.13).
Data Sources
[0153] Microarray gene expression data of squamous lung tumors and
clinical information corresponding to two independent published
studies, referred to as veridex (1) and duke (2), were downloaded
from publicly available websites (3, 4). Veridex gene expression
data was derived from the Affymetrix U133A array and contains
probeset expression values generated by the MASS algorithm. Duke
gene expression data was derived from the Affymetrix U133 Plus 2
array and provides probe-level expression values in the Affymetrix
CEL format. The veridex set consists of 130 squamous lung tumor
samples. The duke set consists of 53 squamous samples.
Two-Study Gene Annotation Mapping
[0154] For this study, gene annotation space is defined by HUGO
gene symbols. Veridex probeset expression values were averaged by
their HUGO identifier. For the duke set, a custom probe to probeset
mapping (CDF) was built using blat (5) alignment of the probe
sequences to mRNA transcript sequences downloaded from the
SpliceMiner website (genepattern.broad.mit.edu/gp/pages/index.jsf).
This custom CDF was used to create MASS gene expression values.
Lung Tumor Squamous Subtypes Defined by Unsupervised Clustering in
Independent Data Sets
[0155] In order to effectively cluster tumor samples, a gene list
was constructed on the basis of statistical variability. For this
list, genes with a median absolute deviation in the veridex set
exceeding and also present in duke set were selected for a total of
2,361 genes. Consensus hierarchical clustering (6, 7) was executed
independently for the veridex and duke sets to define the number of
lung tumor squamous subtypes. The associated consensus distribution
graphs and graphics support 3 distinct clusters in both datasets.
Correlations of cluster medoids were calculated to identify
corresponding clusters between the data sets. (Table 4) These
correlations show a clear one-to-one cluster mapping between the
data sets. These correlations show that these clusters are
detectable in independent tumor sample sets by different gene
expression assays and provide evidence that these clusters are the
result of biological processes driving gene expression rather than
a result of technical artifacts. In order to refer to these
clusters, a name was assigned to each cluster based on
manually-selected genes that exhibited differential expression
relative to the two other clusters. These clusters are named as the
keratin-low subtype, the ms-high subtype, and the mucin-high
subtype.
TABLE-US-00004 TABLE 4 Correlation of cluster medoids between duke
and veridex data sets. duke veridex cluster cluster 1 2 3 1
0.65669834 -0.42840407 -0.20990321 2 0.18027364 -0.23557283
0.37177577 3 -0.39141677 0.58898719 0.14150200
Genes Significantly Associated with Subtype
[0156] To identify genes associated with each subtype, the Rank
Product method (8) was executed to identify statistically
significant genes over-expressed and under-expressed. The top 20
over-expressed and top 20 under-expressed genes as ranked by lowest
percent false positive were extracted from the following
comparisons: keratin-low versus ras-high and mucin-high; ras-high
versus versus keratin-low and mucin-high; mucin-high versus
keratin-low and ras-high). Of these 60 genes, there are
non-redundant genes that are associated with these subtypes, which
are listed in Table 1 under the subheading "SCC subtype genes."
Survival of Subtypes
[0157] Veridex tumor samples were analyzed for survival as a
function of subtype. Kaplan-Meier survival curves in the interval 0
to 36 months (FIG. 14) suggest some differences in survival. The
mucin-high versus keratin-low and rash-high versus keratin-low
survival curves approach statistical significance, with p=0.1485
and p=0.165 respectively.
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Predicting Prognosis of Squamous Cell and Adenocarcinomas of the
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7. Monti, S., et al. (2003) Consensus Clustering: A
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Small Cell Carcinoma
[0166] Tumor subtypes have been described for SCLC carcinoma in a
single large case series, high-grade neuroendocrine tumors type I
and II (HGNET1 and HGNET2) (20). Patients with SCLC rarely undergo
surgery, and independent data are too sparse to make confirmatory
statements on this finding.
Translation to Clinical Medicine
[0167] While the interest in expression profiling both for clinical
and research purposes continues to grow, there are limitations
which must be addressed to bring the promise of this technology to
routine patient care. Chief among these is that, gene expression
arrays rely on the availability of relatively large amounts of
fresh tissue which is commonly available for patients with lung
cancer. While technological advances or changes in clinical
practice might ultimately allow expression arrays to enter routine
patient care, most agree that an assay targeted to the routinely
prepared formalin-fixed paraffin-embedded tissue would be strongly
preferred. Methods of real-time PCR techniques for genetic testing
and tumor profiling have developed and implemented.
[0168] To maximize the chances of success, this project was
designed around the concept of genomic classifier validation. A
diagnostic test or "classifier" is validated if genes derived from
one data set and used for diagnosis or "classification" in an
independent data set (Test Set) produce the same results. If a
classifier can pass this test, then it is strong enough to be
independent of technical peculiarities specific to one platform, or
to sampling bias.
Selection of Valid Genes To ensure that genes selected for the
current proposal are most likely to be valid, a cohort was
assembled of all available lung cancer arrays (11, 13, 14, 16-18,
20, 31, 32, 34, 35, 59). In all, this totals over 1000 patient
samples from diverse backgrounds (ethnicity and geographic
location) and different environmental exposures (smokers and
non-smokers). These independent cohorts allow pre-selection and
validation of genes with properties most favorable for further
testing using a PCR-based assay in paraffin samples. Although the
focus of the current work is on novel methods of classification of
lung cancer, the most conservative approach may be a proof of
principle using conventional morphologic classes of lung cancer
(i.e. SCC, AD, etc). To accomplish this goal, an independent
validation of a classifier that detects the different histological
types of lung cancer was performed using methods of combining
genomic data which have previously been reported (43). Once the
data were merged into a single cohort, samples were divided into
the training and testing sets. Genes were selected in the training
set that distinguished each morphologic lung cancer class from the
others using the relative difference statistic as implemented in
the statistical analysis of microarrays algorithm, a method which
accounts for the probability of false discovery of spurious
associations. Based on experience derived from work in breast
cancer, the list of several thousand highly significant candidate
genes was culled to the top 500, a group called the Preliminary
Data Classifier (PDC). These genes were used to prove an upper
bound on the expected performance of a clinical assay. Using a
nearest centroid classifier, expression based diagnosis agreed with
the pathologist's histological diagnosis in 93% of cases (60, 61).
This level of agreement compares very favorably to historic reports
of the reliability of lung cancer morphologic classification which
can fall between 70-90%, but which generally have been lower
(62-64). All of the cases in which there was a disagreement between
genomic diagnosis and morphologic diagnosis were reviewed.
One-third of discrepant cases were from samples with less that 40%
tumor nuclei, and 1/3 cases were poorly differentiated tumors
without a consensus of the diagnosis after review by multiple
pathologists. In brief, accounting for poor quality samples and
samples in which the underlying diagnosis was unclear, the
agreement between genomic classifier and morphologic classifier was
nearly perfect.
[0169] Perfect agreement between "truth" and "prediction" in a
genomic study such as this would generally be a sign of data
over-fitting and a cause for concern. To evaluate the validity of
the 500 gene list, the exact classifier was applied to an
independent test set where agreement between genomic prediction and
histological diagnosis was 91%. Importantly, all non-neoplastic
samples were correctly identified as normal lung by the classifier.
Eighty percent of the cases where the classifier was discordant
with the histological diagnosis were from small volume samples.
Interestingly, the test set contained a set of samples given only
nonspecific diagnosis by the pathologist of either NSCLC or simply
"carcinoma" where the expression-based classifier was able to make
unambiguous calls for >75%.
Use of Paraffin-Embedded Tissue
[0170] To test the feasibility of gene expression profiling from
paraffin-embedded lung cancer samples, cohort of 157 lung cancer
and normal samples was assembled from banked paraffin tissues at
the University of North Carolina and the Huntsman Cancer Institute
at the University of Utah. From the 500 genes of the PDC, a
representative set of 62 genes (8 housekeeper and 54 tumor
classifying genes) was selected for PCR analysis using methods
previously reported (58). Primer pairs were designed using
commercially available software and evaluated using model cell
lines representative of the dominant lung cancer morphologic
variants. RNA was extracted using the High Pure Paraffin RNA
Extraction Kit (Roche) from 4 full face 10 micron sections, PCR
amplified and quantified using standard methods. Using a
modification of the .DELTA..DELTA.Cp method, relative copy number
for each gene of interest in these lung cancer samples was
calculated. The copy number for each control and test gene in an
experimental sample is first normalized to the same genes in a
common reference sample. This copy number for each test gene is
then corrected for differences in starting material by dividing by
the average copy number of several control (housekeeper) genes. The
results demonstrate that of the 62 selected targets, 61 primer
pairs passed quality control in the cell line system. Fifty-eight
genes were successfully amplified in at least 90% of clinical
samples. RNA was amplified for 90% of gene targets in 98% of the
samples tested. Twenty samples were assayed in duplicate or
triplicate with the mean Pearson's correlation coefficient of gene
expression data across the replicate pairs of 0.94. Five percent of
samples had both frozen and paraffin-embedded tissue for
confirmation with mean Pearson's correlation coefficient of
>80%. This cohort included patients whose paraffin tumors were
banked between 1-15 years ago, with a trend towards more failed
amplifications in the oldest samples. Overall, however, the yields
were considered good to excellent. To test the ability of these
gene expression data to correctly identify the morphologic lung
cancer variants, linear discriminant analysis was used and the
results are shown in FIG. 11. Remarkably, in 93% of cases, the
morphologic diagnosis and gene expression diagnosis agreed.
Discordant cases were of 3 varieties: adenocarcinoma called
squamous cell carcinoma, squamous cell carcinoma called
adenocarcinoma, and tumor identified as normal. Review of the cases
where tumor was misidentified as normal revealed samples of either
low tumor content or poor RNA amplification.
Summary
[0171] The ability to select a robust set of genes which can be
profiled using routinely available clinical samples for the
purposes of gene expression profiling was successfully
demonstrated. In this proof of principle example, it was shown that
this system can be used to make clinically meaningful distinctions
and open the possibility for a range of novel diagnostics in lung
cancer.
Development and Validation of an Expression-Based Classification of
Lung Cancer Using Real-Time qRT-PCR from Formalin-Fixed
Paraffin-Embedded (FFPE) Tissues
Tumor Subtypes and Sample Selection
[0172] The only current means of distinguishing subtypes of lung
AD, SCC, and SCLC is through the use of gene expression arrays.
Therefore, in order to assess the accuracy of the paraffin assay
described herein, a set of lung cancer samples is assayed in the
following parallel manner: fresh frozen tissue is hybridized
against gene expression arrays and FFPE tissue is assayed using
real-time qRT-PCR. Additionally, as part of the quality control
evaluation of gene targets, real-time qRT-PCR on RNA isolated from
fresh tissue taken in parallel with matched FFPE tissue is
performed. To ensure sufficient power to validate the diagnostic
ability of this assay, a new cohort of lung cancer patients is
assembled with both frozen and matched FFPE tissue available,
herein referred to as the UNC cohort. Tumors are collected
sequentially, and to ensure an appropriate mix of tumors for
classification, patients are sampled with the following
distribution of morphologic diagnoses: SCLC 15 (50/50), SCC 25
(60/30), AD 40(40/40/20), carcinoid 10, normal lung 10. The values
in parentheses represent the relative proportions of tumor subtypes
for each morphologic diagnosis based on previous reports. In the
case of SCC, the preliminary data suggest either 60/30 or 50/50
split of tumor subtypes. The relative proportions of each tumor
type were selected to ensure at least 7 samples per tumor subtype.
The selection of 7 as a minimum comes from the experience that
multiple existing datasets in this collection contained 7 or fewer
small cell carcinomas, yet still provided interpretable data for
genomic analysis. The total sample size was estimated based on the
finding that agreement between morphologic and molecular diagnosis
was 93%, a value at the upper end of that reported for the
inter-observer agreement between pathologists (63). Under the null
hypothesis of 93% agreement and alternate hypothesis of 85%
agreement with power of 80% and alpha of 0.05, a minimum of 90
total samples is required. Therefore, the reproducibility of
expression based classification on an order of magnitude that
allows comparison to morphologic classification as determined by
traditional light microscopy. For the current analysis there is no
attempt to differentiate large cell lung cancer (LCLC) as an entity
separate from adenocarcinoma. There are few published reports
documenting the reliability of a LCLC diagnosis, and in previous
publications of microarray data it tended to cluster most closely
with a subtype of adenocarcinoma.
[0173] Starting with the cohort of 1000 lung cancer microarray
samples described above, a process similar to that described in the
proof of principle analysis is used. Using the nearest centroid
methods, all SCC and AD NSCLC are assigned to their respective
tumor subtypes (43, 60). The PDC is reconstructed by first
selecting only those genes which are reliably measured across the
independent datasets in the cohorts using the integrative
correlations method (37). Using only these highly reliable genes,
the 500 genes most associated with each of the following categories
of tumor are selected: normal lung parenchyma, SCLC, SCC, AD, and
carcinoid. The 500 genes will also include a set which
distinguishes the 3 known subtypes of AD (bronchioid, squamoid, and
magnoid), 2 reported subtypes of SCC (A and B), and the reported
subtypes of small cell carcinoma (HGNET1 and HGNET2). Gene
pre-selection for the PDC in this rich collection of multiple
independent datasets ensure that genes ultimately selected for
validation in the following steps are highly likely to be
associated with the diagnostic groups of interest and not spurious.
The performance of the PDC is evaluated as described in the section
tiled selection of valid genes above. Samples from the UNC cohort
constitute the test set, and as such, are assigned to tumor
subtypes but not used in selection of genes for the PDC.
Gene and Primer Validation
[0174] The performance of the PDC is evaluated in the 1000 sample
cohort to estimate the upper bound on reliability on tumor
classification. The 500 gene PDC is reduced to 100 genes by the
nearest shrunken centroid method (65). The 100 most predictive
genes, called the calibrated classifier (CC), are then be evaluated
for their predictive power in the UNC cohort of expression arrays
to estimate the upper limit on the predictive power of the FFPE
assay. If the CC underperforms with regard to expected diagnostic
power, the PDC is repeated to refine a more predictive gene set.
This method allows for the subtle refinement in the training CC
without considering genes from the test set (the UNC cohort) where
the risk of selecting spurious genes is highest. With the CC
calibrated in this manner the real-time qRT-PCR is designed as
described above. Briefly, commercial software is used to design the
real-time qRT-PCR primer pairs, which are all evaluated for the
uniqueness of their targets in the genome by sequence searches. All
primer sets are designed to have a Tm.apprxeq.60.degree. C., GC
content.apprxeq.50% and to generate a PCR amplicon <100 bps. All
primer sets are tested using SYBR Green to assess efficiency of PCR
and the presence of primer-dimers. Melting curve analysis is used
to distinguish primer-dimer formation from specific product. Each
new primer set will first be tested for performance using the
following criteria: 1) Target Cp<30 using 10 ng reference cDNA,
2) PCR efficiency >1.7, 3) No primer-dimers in presence of
template, and 4) No primer-dimers in negative template control
before cycle 40.
[0175] Each target gene of the CC is evaluated first by real-time
qRT-PCR from fresh tissue to ensure good correlation with matched
expression measured by DNA microarray. Genes with high correlation
between expression array and PCR based quantification are included
in the final CC set for evaluation in paraffin. Primers which
generate poor correlation between the expression array and
real-time qRT-PCR as measured in fresh tissue are replaced. The
first attempt considers new primers for the same target. If these
fail then a new gene is selected from the PDC. In a similar manner,
the correlations of real-time qRT-PCR from paraffin to paired
expression from the microarray and real-time qRT-PCR from fresh
tissue is performed, replacing any genes with poor overall
correlation with alternates from the PDC.
[0176] In a final step the CC genes are used to classify the 100
samples with regard to both tumor morphology and tumor subtypes. If
there are systematic errors in the classification, this represents
an additional opportunity to return to the PDC to select alternate
candidates. The design is a 2 stage algorithm in which the first
prediction is of morphologic variant followed in a second step by
prediction of tumor subtype (FIG. 12).
Assessing Predictive and Prognostic Significance of Molecular
Subtypes of Non-Small Cell Lung Cancer:
[0177] This step documents the associations between lung cancer
subtypes and clinical outcomes including relapse-free survival,
overall survival, pattern of recurrence, and response to first line
chemotherapy.
Relapse-Free Survival and Overall Survival
[0178] Previous reports of AD, SCC, and SCLC tumor subtypes have
reported clinically meaningful differences in survival according to
tumor subtype as measured by expression microarrays. Both
retrospective and prospective cohorts of each of these tumors are
assembled to confirm these associations using the qRT-PCR assay.
Historical patients are restricted to early stage, surgically
treated individuals with 5 years of follow up. Recurrence pattern
and dates are recorded in a database. All surgically treated
incidental patients are followed prospectively for recurrence and
overall survival. Paraffin samples are obtained for both groups and
analyzed with the assay developed in specific aim 1. Relapse-free
and overall survival are assessed using standard Kaplan-Meier plots
as well as Cox proportional hazards modeling.
[0179] In samples identified as AD, the hypothesis is based on
repeated observation in preliminary data of a 25% absolute
improvement in survival at 5 years from 50% to 75% for the
bronchioid subtype. To detect this difference at an 80% power with
a one-sided alpha of 0.05, 58 samples per class are needed. Since
bronchioids and squamoids together comprise 80% of AD (40% each
with the remaining 20% being magnoids), a total of 145 AD samples
are needed. In samples identified as SCC, the hypothesis is that
there is a 30% absolute survival benefit from 80%-50% between the 2
subtypes, with subtype 2 having the favorable prognosis. Assuming
the differential survival above, with an alpha of 0.05 and 80%
power the total sample size is 76 SCC tumors. To ensure a minimum
of 76 SCC and 145 AD from an unselected cohort of NSCLC tumors, the
following calculation is made. Approximately 35% of NSCLC are SCC
with the remainder assumed to be AD. The maximum of 76/0.35 and
145/0.65 is 223. Therefore 223 NSCLC patients is expected to
produce a cohort sufficient to test the hypotheses above. In the
case of SCLC preliminary data for the use of tumor subtypes
suggests a dramatic survival differential between subtypes of 50%.
To detect a 50% difference in survival at 3 years with 80% power
and an alpha of 0.05, 28 patients are needed.
Chemotherapy Treatment Response
[0180] Most patients treated with chemotherapy are in the setting
of advanced and unresectable disease. To evaluate the association
between PCR-based diagnosis of tumor subtype and treatment
response, the following 2 categories of patients with gross tumor
available are used for analysis: (1) recurrence after surgical
resection, (2) patients with advanced disease who have gross tumor
resected, including mediastinoscopy or other open surgical
procedure. Of the 2500 patients with FFPE available in the
pathology banks, approximately 750 patients will have recurred and
have tissue available in the banks. The majority of these patients
will have received platinum-based chemotherapy in the first line
setting. Evidence from a variety of sources suggests that patients
of the bronchioid subtype are approximately half as likely to
respond to platinum-containing regimens, with the best estimate
coming from a clinical trial published in the Lancet (66). Based on
these data the hypothesized response rate for the bronchioid
subtype is 25% compared to 40% for all other NSCLC. With a
one-sided alpha of 0.05 and 80% power, 320 patients are required to
test this hypothesis.
Pattern of Recurrence
[0181] An estimated 30% of the 223 NSCLC patients from the Survival
cohort above (n=70) will be diagnosed with metastases by the end of
their follow-up. The majority of the 320 patients from Chemotherapy
cohort will either have a metastasis present at diagnosis or
develop metastasis during follow-up. The association between tumor
subtype and specific patterns of recurrence (bone, brain, local,
and visceral) is assessed using these two established cohorts as
well as the prospective cohort described above using logistic
regression models. Of particular interest are the reported
association between squamoid adenocarcinoma subtype and brain
recurrence, and the overall high rate of metastasis in this subtype
relative to the bronchioid subtype.
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Example 6
Molecular Markers Distinguish Patients at Differential Risk of
Brain Metastases in Lung Cancer by Immunohistochemistry
Background
[0248] Patients with surgically resected lung caner, even at the
earliest stages of disease still face a risk of recurrence between
30-50%, including a significant number of patients with brain only
metastases. Although evidence suggests brain metastases may be
preventable through prophylactic cranial radiation (PCR),
widespread acceptance of the therapy has not occurred due to
patient preference and other factors. Predictors of brain
metastasis might promote the use of PCR.
Methods
[0249] 150 surgically treated lung cancer patients treated at the
University of North Carolina were selected for analysis. A tissue
microarray was generated from primary tumor material and assayed
this using commercially available antibodies for 16 proteins
suspected or previously reported to be associated with brain
metastases (TTF1, Vimentin, SMA, BCL2, cyclin D1, UPA, AKT, pAKT,
ADAM9, ERK, pERK, EGFR, Her-2/neu, Ki67, p53, Rb). Samples were
assayed in triplicate at a minimum and associations with pattern of
metastasis were determined.
Results
[0250] The cohort contained 85 adenocarcinomas, 59 squamous cell
carcinomas, and 6 poorly differentiated carcinomas. There were 58
recurrence in total of which 38 were solitary recurrences (21 lung
only, 9 brain only, 5 liver only, 3 misc). A total of 13 brain
metastases occurred, 11 of which were in lung adenocarcinoma. The
following patterns were observed. Patients who were TTF1 (+) at any
level and vimentin (-), had a documented brain metastasis rate of
4%, compared to patients who were TTF1 (+), vimentin (+) where the
rate was 17% (p=0.06). All of the remaining patients with brain
metastases were negative for both TTF 1 and vimentin, but stained
at approximately 2 time background for SMA (p<0.05). Additional
antibodies correlated with brain metastases were UPA and ADAM9.
Conclusions
[0251] Patients at relatively high and low risk of brain metastasis
can be identified using a small number of immunohistochemical
markers.
[0252] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
[0253] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious that certain changes and
modifications may be practiced within the scope of the appended
embodiments.
Sequence CWU 1
1
114122DNAArtificial SequenceSynthetic sequence 1aagagagatt
ggatttggaa cc 22222DNAArtificial SequenceSynthetic sequence
2ccagaagccc aagaagattg ta 22319DNAArtificial SequenceSynthetic
sequence 3aatcctggtg tcaaggaag 19419DNAArtificial SequenceSynthetic
sequence 4ggaccgattt taccgatcc 19521DNAArtificial SequenceSynthetic
sequence 5acagtccaga tagtcgtatg t 21617DNAArtificial
SequenceSynthetic sequence 6gtctccgcca tccctat 17719DNAArtificial
SequenceSynthetic sequence 7actggtgtaa caggaacat 19817DNAArtificial
SequenceSynthetic sequence 8tttggaagga ctgcgct 17917DNAArtificial
SequenceSynthetic sequence 9cacgtcatct cccgttc 171018DNAArtificial
SequenceSynthetic sequence 10attgaacttc ccacacga
181118DNAArtificial SequenceSynthetic sequence 11ggaacagact
gtcaccat 181217DNAArtificial SequenceSynthetic sequence
12tcagagtgtg gtcaggc 171317DNAArtificial SequenceSynthetic sequence
13gggacagctt caacact 171418DNAArtificial SequenceSynthetic sequence
14cctgtgaaca gccctatg 181517DNAArtificial SequenceSynthetic
sequence 15ttctgggcac ggtgaag 171621DNAArtificial SequenceSynthetic
sequence 16ggccaaacta gagcacgaat a 211719DNAArtificial
SequenceSynthetic sequence 17tcagcaagaa ggagatgcc
191821DNAArtificial SequenceSynthetic sequence 18gtgctccctc
tccattaagt a 211920DNAArtificial SequenceSynthetic sequence
19caagttcagg agaactcgac 202019DNAArtificial SequenceSynthetic
sequence 20ggctgtggtt atgcgatag 192118DNAArtificial
SequenceSynthetic sequence 21acccgaggaa caacctta
182218DNAArtificial SequenceSynthetic sequence 22ccctctccat
tccctaca 182317DNAArtificial SequenceSynthetic sequence
23cagagcgcca ggcatta 172418DNAArtificial SequenceSynthetic sequence
24ccactggctg aggtgtta 182517DNAArtificial SequenceSynthetic
sequence 25tgggcgagtc tacgatg 172618DNAArtificial SequenceSynthetic
sequence 26ctttctgccc tggagatg 182719DNAArtificial
SequenceSynthetic sequence 27gcgccatttg ctagagata
192819DNAArtificial SequenceSynthetic sequence 28agagaagatg
ggcagaaag 192917DNAArtificial SequenceSynthetic sequence
29gcccagatca tccgtca 173017DNAArtificial SequenceSynthetic sequence
30accacaagga cttcgac 173117DNAArtificial SequenceSynthetic sequence
31gctccgctgc tatcttt 173217DNAArtificial SequenceSynthetic sequence
32agcggccagg tggatta 173318DNAArtificial SequenceSynthetic sequence
33atgggctttg ggagcata 183418DNAArtificial SequenceSynthetic
sequence 34gacctggatg ccaagcta 183517DNAArtificial
SequenceSynthetic sequence 35ccggctcttg gaagttg 173620DNAArtificial
SequenceSynthetic sequence 36acgcggatcg agtttgataa
203717DNAArtificial SequenceSynthetic sequence 37cgcaagtccc agaagat
173817DNAArtificial SequenceSynthetic sequence 38cgcggatacg atgtcac
173917DNAArtificial SequenceSynthetic sequence 39gaactcggcc tatcgct
174020DNAArtificial SequenceSynthetic sequence 40tctgacctca
tcatcggcaa 204120DNAArtificial SequenceSynthetic sequence
41gaggtgaagc aaactacgga 204217DNAArtificial SequenceSynthetic
sequence 42actctccaca aagctcg 174322DNAArtificial SequenceSynthetic
sequence 43ggatttcagc taccagttac tt 224417DNAArtificial
SequenceSynthetic sequence 44ttcgtcctgg tggatcg 174522DNAArtificial
SequenceSynthetic sequence 45agtgattgat gtgtttgcta tg
224620DNAArtificial SequenceSynthetic sequence 46caaagccaag
ccactcactc 204717DNAArtificial SequenceSynthetic sequence
47ctcggcagtc ctgtttc 174818DNAArtificial SequenceSynthetic sequence
48acacctggta cgtcagaa 184920DNAArtificial SequenceSynthetic
sequence 49atgcccaaga gaatcgtaaa 205019DNAArtificial
SequenceSynthetic sequence 50atgagtccaa agcacacga
195122DNAArtificial SequenceSynthetic sequence 51tgagattgag
gatgaagctg ag 225217DNAArtificial SequenceSynthetic sequence
52ccgactcaac gtgagac 175317DNAArtificial SequenceSynthetic sequence
53gtgccctctc cttttcg 175418DNAArtificial SequenceSynthetic sequence
54cgttcttttt cgcaacgg 185517DNAArtificial SequenceSynthetic
sequence 55ggtgtgccac tgaagat 175617DNAArtificial SequenceSynthetic
sequence 56gtgtcgtggt ggtcatt 175717DNAArtificial SequenceSynthetic
sequence 57gcatgaagac agtggct 175817DNAArtificial SequenceSynthetic
sequence 58ttcttgcgac tcacgct 175924DNAArtificial SequenceSynthetic
sequence 59gctcctcaaa catctttgtg ttca 246020DNAArtificial
SequenceSynthetic sequence 60gaccactgtg ggtcattatt
206117DNAArtificial SequenceSynthetic sequence 61gaaatctctg gccgctc
176221DNAArtificial SequenceSynthetic sequence 62actgggcatc
ataagaaatc c 216319DNAArtificial SequenceSynthetic sequence
63actgaacaga agacttcgt 196420DNAArtificial SequenceSynthetic
sequence 64aacctccaag tggaaattct 206522DNAArtificial
SequenceSynthetic sequence 65tcggtctttc aaatcgggat ta
226618DNAArtificial SequenceSynthetic sequence 66ctgctgtcac
aggacaat 186719DNAArtificial SequenceSynthetic sequence
67aaggtaaagc cagactcca 196817DNAArtificial SequenceSynthetic
sequence 68gggagcgtag ggttaag 176922DNAArtificial SequenceSynthetic
sequence 69cagtgtattc tgcacaatca ac 227021DNAArtificial
SequenceSynthetic sequence 70gttccaggat gttggacttt c
217118DNAArtificial SequenceSynthetic sequence 71ggaaagtgtg
tcggagat 187218DNAArtificial SequenceSynthetic sequence
72aggcaacatc attccctc 187322DNAArtificial SequenceSynthetic
sequence 73gtcaacaccc atcttcttga aa 227418DNAArtificial
SequenceSynthetic sequence 74cgtagtggaa gacggaaa
187523DNAArtificial SequenceSynthetic sequence 75ctggtgtaga
attaggagac gta 237617DNAArtificial SequenceSynthetic sequence
76ggcatcaaga gagaggc 177724DNAArtificial SequenceSynthetic sequence
77gataaagagt tacaagctcc tctg 247817DNAArtificial SequenceSynthetic
sequence 78tctaggcctt gacggat 177919DNAArtificial SequenceSynthetic
sequence 79tttgggcaaa cctcggtaa 198017DNAArtificial
SequenceSynthetic sequence 80gcacagcaaa tgccact 178123DNAArtificial
SequenceSynthetic sequence 81cttgtctttc cctactgtct tac
238218DNAArtificial SequenceSynthetic sequence 82cttgttccag
cagaacct 188318DNAArtificial SequenceSynthetic sequence
83cagtcctctg caccgtta 188418DNAArtificial SequenceSynthetic
sequence 84catccagatc cctcacat 188519DNAArtificial
SequenceSynthetic sequence 85ccaagacaca gccagtaat
198618DNAArtificial SequenceSynthetic sequence 86tttccagccc
tcgtagtc 188717DNAArtificial SequenceSynthetic sequence
87gggacacagg gaagaac 178817DNAArtificial SequenceSynthetic sequence
88gtctgccact ctgcaac 178917DNAArtificial SequenceSynthetic sequence
89gtcggctgac gctttga 179023DNAArtificial SequenceSynthetic sequence
90gaacaagtca gtctagggaa tac 239121DNAArtificial SequenceSynthetic
sequence 91tgctttcgat aagtccagac a 219218DNAArtificial
SequenceSynthetic sequence 92cctctgaggc tggaaaca
189319DNAArtificial SequenceSynthetic sequence 93atccactgat
cttccttgc 199419DNAArtificial SequenceSynthetic sequence
94cagtgctgct tcagacaca 199521DNAArtificial SequenceSynthetic
sequence 95cctttcttca agggtaaagg c 219620DNAArtificial
SequenceSynthetic sequence 96tcgaatttct ctcctcccat
209718DNAArtificial SequenceSynthetic sequence 97ctgagtccac
acaggttt 189823DNAArtificial SequenceSynthetic sequence
98cccatacttg ttgatggcaa tta 239918DNAArtificial SequenceSynthetic
sequence 99tcctgcgtgt gttctact 1810019DNAArtificial
SequenceSynthetic sequence 100agtcatcatg tacccagca
1910120DNAArtificial SequenceSynthetic sequence 101cccaggatac
tctcttcctt 2010218DNAArtificial SequenceSynthetic sequence
102cactggatca actgcctc 1810319DNAArtificial SequenceSynthetic
sequence 103cagctgtcac acccagagc 1910417DNAArtificial
SequenceSynthetic sequence 104cgtatggtgc agggtca
1710520DNAArtificial SequenceSynthetic sequence 105tctggactgt
ctggttgaat 2010619DNAArtificial SequenceSynthetic sequence
106cctgtacacc aagcttcat 1910719DNAArtificial SequenceSynthetic
sequence 107ccatgcccac tttcttgta 1910820DNAArtificial
SequenceSynthetic sequence 108cattggtggt gaagctcttg
2010918DNAArtificial SequenceSynthetic sequence 109cgtggactga
gatgcatt 1811021DNAArtificial SequenceSynthetic sequence
110ttcatgtcgt tgaacacctt g 2111121DNAArtificial SequenceSynthetic
sequence 111cattttggct tttaggggta g 2111217DNAArtificial
SequenceSynthetic sequence 112ggcagaagcg agacttt
1711317DNAArtificial SequenceSynthetic sequence 113gcacatagga
ggtggca 1711417DNAArtificial SequenceSynthetic sequence
114gcggacttta ccgtgac 17
* * * * *
References