U.S. patent application number 14/602916 was filed with the patent office on 2016-01-21 for identification of cancer protein biomarkers using proteomic techniques.
This patent application is currently assigned to Yale University. The applicant listed for this patent is Patricia Bray-Ward, Gil G. Mor, David C. Ward. Invention is credited to Patricia Bray-Ward, Gil G. Mor, David C. Ward.
Application Number | 20160018402 14/602916 |
Document ID | / |
Family ID | 34915557 |
Filed Date | 2016-01-21 |
United States Patent
Application |
20160018402 |
Kind Code |
A2 |
Mor; Gil G. ; et
al. |
January 21, 2016 |
IDENTIFICATION OF CANCER PROTEIN BIOMARKERS USING PROTEOMIC
TECHNIQUES
Abstract
The claimed invention describes methods to diagnose or aid in
the diagnosis of cancer. The claimed methods are based on the
identification of biomarkers which are particularly well suited to
discriminate between cancer subjects and healthy subjects. These
biomarkers were identified using a unique and novel screening
method described herein. The biomarkers identified herein can also
be used in the prognosis and monitoring of cancer. The invention
comprises the use of leptin, prolactin, OPN and IGF-II for
diagnosing, prognosis and monitoring of ovarian cancer.
Inventors: |
Mor; Gil G.; (Cheshire,
CT) ; Ward; David C.; (Las Vegas, NV) ;
Bray-Ward; Patricia; (Las Vegas, NV) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mor; Gil G.
Ward; David C.
Bray-Ward; Patricia |
Cheshire
Las Vegas
Las Vegas |
CT
NV
NV |
US
US
US |
|
|
Assignee: |
Yale University
New Haven
CT
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20150233929 A1 |
August 20, 2015 |
|
|
Family ID: |
34915557 |
Appl. No.: |
14/602916 |
Filed: |
January 22, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12644677 |
Mar 10, 2015 |
8975379 |
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14602916 |
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11037889 |
Feb 23, 2010 |
7666583 |
|
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12644677 |
|
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60545900 |
Feb 20, 2004 |
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60545581 |
Feb 19, 2004 |
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Current U.S.
Class: |
506/18 ;
435/7.92 |
Current CPC
Class: |
G06F 19/00 20130101;
C12Q 2600/158 20130101; C12Q 2600/136 20130101; G01N 2333/65
20130101; G01N 2333/5756 20130101; G01N 2500/04 20130101; G01N
33/57484 20130101; G01N 33/57449 20130101; G01N 2333/575 20130101;
C12Q 1/6886 20130101; G01N 2800/52 20130101; G01N 2333/4703
20130101; C12Q 2600/112 20130101; G01N 33/57419 20130101; G01N
33/57415 20130101; G01N 2333/99 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574 |
Goverment Interests
FUNDING
[0002] This invention was made with government support under grant
number DE-FG02-ER63462 awarded by the United States Department of
Energy. The government has certain rights in the invention.
Claims
1-58. (canceled)
59. A kit for diagnosing cancer comprising reagents that consist
essentially of primary antibodies or antibody fragments that
specifically detect biomarker proteins leptin, prolactin,
osteopontin (OPN) and at least one additional biomarker protein
selected from insulin-like growth factor II (IGF-II), 6Ckine,
angiotensin converting enzyme (ACE), brain-derived neurotrophic
factor (BDNF), E-Selectin, epidermal growth factor (EGF), eotaxin-2
(Eot-2), epidermal growth factor receptor 1 (ErbB1), follistatin,
hemofiltrate CC chemokine 4 (HCC4), herpes virus entry mediator
(HVEM), insulin-like growth factor binding protein 2 (IGFBP-2),
interleukin-17 (IL-17), interleukin 1 soluble receptor II (IL-1
sRII), interleukin 2 soluble receptor alpha (IL-2 sRa), macrophage
colony stimulating factor receptor (M-CSF R), macrophage migration
inhibitory factor (MIF), macrophage inflammatory protein 1 alpha
(MIP-1a), macrophage inflammatory protein 3 beta (MIP3.beta.),
matrix Metalloproteinase-8 (MMP-8), matrix metalloproteinase 7
(MMP-7), myeloid progenitor inhibitory factor 1 (MPIF-1), pulmonary
and activation-regulated chemokine (PARC), platelet-derived growth
factor receptor beta (PDGF R(3), protein C, tumor necrosis factor
receptor I (TNF-RI), tumor necrosis factor alpha (TNF-.alpha.),
soluble Vascular Adhesion Protein-1 (sVAP-1), vascular endothelial
growth factor receptor 2 (VEGF R2), VEGF receptor 3 (VEGF R3),
human stratum corneum chymotryptic enzyme (HSCCE), kallikrein 4,
kallikrein 5, kallikrein 6 (protease M), kallikrein 8, kallikrein
9, kallikrein 10, cancer antigen 125 (CA125), CA15-3, CA19-9, OVX1,
lysophosphatidic acid (LPA), carcinoembryonic antigen (CEA),
macrophage colony-stimulating factor (M-CSF), prostasin, CA54-61,
CA72, HMFG2, interleukin-6 (IL-6), interleukin-10 (IL-10), LSA,
NB70K, PLAP, TAG72, TPA, UGTF, WAP four-disulfide core domain 2
(HE4), matrix metalloprotease 2, tetranectin, inhibin, mesothelin,
MUC1, vascular endothelial growth factor (VEGF) NOTCH3, E2F
transcription factor 3 (E2F3), GTPase activating protein (RACGAP1),
hemotological and neurological expressed 1 (HN1), apolipoprotein
A1, laminin, claudin 3 (CLDN3), claudin 4, tumor-associated calcium
signal transducer 1 (TROP-1/Ep-CAM), tumor-associated calcium
signal transducer 2 (TROP-2), ladinin 1, S100A2, SERPIN2 (PAI-2),
CD24, lipocalin 2, matriptase (TADG-15), stratifin, transforming
growth factor-beta receptor III (TGF-.beta.-RIII), platelet-derived
growth factor receptor alpha, SEMACAP3, ras homology gene family
member I (ARHI), thrombospondin 2, disabled-2/differentially
expressed in ovarian carcinoma 2 (Dab2/DOC2) and haptoglobin-alpha
subunit, and secondary antibodies that bind to the primary
antibodies or primary antibody fragments and are conjugated to a
detectable label.
60. The kit of claim 59, wherein the at least one additional
biomarker protein is CA125, MIF, HE4, mesothelin, or a combination
of CA125, MIF, HE4 and mesothelin.
61. The kit of claim 59, further comprising a reference sample or a
predetermined standard.
62. The kit of claim 59, wherein the primary antibodies or primary
antibody fragments are attached to a solid support.
63. The kit of claim 59, wherein the primary antibodies or primary
antibody fragments are synthetic.
64. The kit of claim 59, wherein the secondary antibodies are
synthetic.
65. A kit for diagnosing cancer comprising reagents that consist
essentially of primary antibodies or primary antibody fragments
that specifically detect biomarker proteins leptin, prolactin,
osteopontin (OPN) and at least one additional biomarker protein
selected from insulin-like growth factor II (IGF-II), 6Ckine,
angiotensin converting enzyme (ACE), brain-derived neurotrophic
factor (BDNF), E-Selectin, epidermal growth factor (EGF), eotaxin-2
(Eot-2), epidermal growth factor receptor 1 (ErbB1), follistatin,
hemofiltrate CC chemokine 4 (HCC4), herpes virus entry mediator
(HVEM), insulin-like growth factor binding protein 2 (IGFBP-2),
interleukin-17 (IL-17), interleukin 1 soluble receptor II (IL-1
sRII), interleukin 2 soluble receptor alpha (IL-2 sRa), macrophage
colony stimulating factor receptor (M-CSF R), macrophage migration
inhibitory factor (MIF), macrophage inflammatory protein 1 alpha
(MIP-1.alpha.), macrophage inflammatory protein 3 beta
(MIP3.beta.), matrix Metalloproteinase-8 (MMP-8), matrix
metalloproteinase 7 (MMP-7), myeloid progenitor inhibitory factor 1
(MPIF-1), pulmonary and activation-regulated chemokine (PARC),
platelet-derived growth factor receptor beta (PDGF R(3), protein C,
tumor necrosis factor receptor I (TNF-RI), tumor necrosis factor
alpha (TNF-.alpha.), soluble Vascular Adhesion Protein-1 (sVAP-1),
vascular endothelial growth factor receptor 2 (VEGF R2), VEGF
receptor 3 (VEGF R3), human stratum corneum chymotryptic enzyme
(HSCCE), kallikrein 4, kallikrein 5, kallikrein 6 (protease M),
kallikrein 8, kallikrein 9, kallikrein 10, cancer antigen 125
(CA125), CA15-3, CA19-9, OVX1, lysophosphatidic acid (LPA),
carcinoembryonic antigen (CEA), macrophage colony-stimulating
factor (M-CSF), prostasin, CA54-61, CA72, HMFG2, interleukin-6
(IL-6), interleukin-10 (IL-10), LSA, NB70K, PLAP, TAG72, TPA, UGTF,
WAP four-disulfide core domain 2 (HE4), matrix metalloprotease 2,
tetranectin, inhibin, mesothelin, MUC1, vascular endothelial growth
factor (VEGF) NOTCH3, E2F transcription factor 3 (E2F3), GTPase
activating protein (RACGAP1), hemotological and neurological
expressed 1 (HN1), apolipoprotein A1, laminin, claudin 3 (CLDN3),
claudin 4, tumor-associated calcium signal transducer 1
(TROP-1/Ep-CAM), tumor-associated calcium signal transducer 2
(TROP-2), ladinin 1, S100A2, SERPIN2 (PAI-2), CD24, lipocalin 2,
matriptase (TADG-15), stratifin, transforming growth factor-beta
receptor III (TGF-.beta.-RIII), platelet-derived growth factor
receptor alpha, SEMACAP3, ras homology gene family member I (ARHI),
thrombospondin 2, disabled-2/differentially expressed in ovarian
carcinoma 2 (Dab2/DOC2) and haptoglobin-alpha subunit, wherein the
primary antibodies or primary antibody fragments are conjugated to
a detectable label.
66. The kit of claim 65, wherein the at least one additional
biomarker protein is CA125, MIF, HE4, mesothelin, or a combination
of CA125, MIF, HE4 and mesothelin.
67. The kit of claim 65, further comprising a reference sample or a
predetermined standard.
68. The kit of claim 65, wherein the primary antibodies or primary
antibody fragments are attached to a solid support.
69. The kit of claim 65, wherein the primary antibodies or primary
antibody fragments are synthetic.
70. A kit for diagnosing cancer comprising reagents that consist
essentially of primary monoclonal antibodies or primary monoclonal
antibody fragments that specifically detect biomarker proteins
leptin, prolactin, osteopontin (OPN) and at least one additional
biomarker protein selected from insulin-like growth factor II
(IGF-II), 6Ckine, angiotensin converting enzyme (ACE),
brain-derived neurotrophic factor (BDNF), E-Selectin, epidermal
growth factor (EGF), eotaxin-2 (Eot-2), epidermal growth factor
receptor 1 (ErbB1), follistatin, hemofiltrate CC chemokine 4
(HCC4), herpes virus entry mediator (HVEM), insulin-like growth
factor binding protein 2 (IGFBP-2), interleukin-17 (IL-17),
interleukin 1 soluble receptor II (IL-1 sRII), interleukin 2
soluble receptor alpha (IL-2 sRa), macrophage colony stimulating
factor receptor (M-CSF R), macrophage migration inhibitory factor
(MIF), macrophage inflammatory protein 1 alpha (MIP-1.alpha.),
macrophage inflammatory protein 3 beta (MIP3.beta.), matrix
Metalloproteinase-8 (MMP-8), matrix metalloproteinase 7 (MMP-7),
myeloid progenitor inhibitory factor 1 (MPIF-1), pulmonary and
activation-regulated chemokine (PARC), platelet-derived growth
factor receptor beta (PDGF R(3), protein C, tumor necrosis factor
receptor I (TNF-RI), tumor necrosis factor alpha (TNF-.alpha.),
soluble Vascular Adhesion Protein-1 (sVAP-1), vascular endothelial
growth factor receptor 2 (VEGF R2), VEGF receptor 3 (VEGF R3),
human stratum corneum chymotryptic enzyme (HSCCE), kallikrein 4,
kallikrein 5, kallikrein 6 (protease M), kallikrein 8, kallikrein
9, kallikrein 10, cancer antigen 125 (CA125), CA15-3, CA19-9, OVX1,
lysophosphatidic acid (LPA), carcinoembryonic antigen (CEA),
macrophage colony-stimulating factor (M-CSF), prostasin, CA54-61,
CA72, HMFG2, interleukin-6 (IL-6), interleukin-10 (IL-10), LSA,
NB70K, PLAP, TAG72, TPA, UGTF, WAP four-disulfide core domain 2
(HE4), matrix metalloprotease 2, tetranectin, inhibin, mesothelin,
MUC1, vascular endothelial growth factor (VEGF) NOTCH3, E2F
transcription factor 3 (E2F3), GTPase activating protein (RACGAP1),
hemotological and neurological expressed 1 (HN1), apolipoprotein
A1, laminin, claudin 3 (CLDN3), claudin 4, tumor-associated calcium
signal transducer 1 (TROP-1/Ep-CAM), tumor-associated calcium
signal transducer 2 (TROP-2), ladinin 1, S100A2, SERPIN2 (PAI-2),
CD24, lipocalin 2, matriptase (TADG-15), stratifin, transforming
growth factor-beta receptor III (TGF-.beta.-RIII), platelet-derived
growth factor receptor alpha, SEMACAP3, ras homology gene family
member I (ARHI), thrombospondin 2, disabled-2/differentially
expressed in ovarian carcinoma 2 (Dab2/DOC2) and haptoglobin-alpha
subunit.
71. The kit of claim 70, wherein the at least one additional
biomarker protein is CA125, MIF, HE4, mesothelin, or a combination
of CA125, MIF, HE4 and mesothelin.
72. The kit of claim 70, further comprising a reference sample or a
predetermined standard.
73. The kit of claim 70, wherein the primary monoclonal antibodies
or primary monoclonal antibody fragments are attached to a solid
support.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/644,677, filed Dec. 22, 2009, which is a continuation of
U.S. application Ser. No. 11/037,889, filed Jan. 18, 2005, which
claims the benefit of U.S. Provisional Application No. 60/545,581,
filed Feb. 19, 2004, and U.S. Provisional Application No.
60/545,900, filed Feb. 20, 2004. The teachings of each referenced
application are incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] Epithelial Ovarian Cancer (EOC) is the fourth leading cause
of cancer-related death in women in the United States and the
leading cause of gynecologic cancer death. EOC is characterized by
few early symptoms, presentation at an advanced stage, and poor
survival. This year approximately 25,000 women will be newly
diagnosed with ovarian cancer and 13,500 will die from the disease.
The major limitations in the treatment of ovarian cancer are: i)
the lack of an early detection tumor marker, ii) the resistance to
chemotherapeutic agents, and iii) the lack of obvious early warning
symptoms. The high mortality rate is related to the inability to
detect early disease, as approximately 70% of patients are
diagnosed at an advanced stage. In patients diagnosed with early
(stage I or II) disease, the five-year survival rate ranges from 60
to 90% depending on the degree of tumor differentiation. Although
the clinical presentation of heritable cancer is similar to the
high-risk population, the onset of ovarian cancer in this group
tends to occur 10-15 years earlier than that of the general
population (early 40's rather than 60's). One of the most promising
approaches to management of EOC is early detection. The most
commonly used test, CA125 identifies a group of cell surface
glycoproteins that have uncertain biological behavior and very
limited clinical application for the detection of early stage
disease. As a single marker, CA125 has a predictive value of less
than 10% in Stage I. Even the addition of ultrasound screening to
CA125 measurement improves the positive prediction value to only
about 20%. The lack of specific markers for ovarian cancer makes it
difficult to achieve the clinical objective of screening and early
detection.
[0004] Presently there is no commercially available test that can
be used to diagnose either early or advanced stage ovarian cancer.
Thus, the identification of a test that can be used to diagnose
early or advance stage ovarian cancer is required.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention comprises a method for diagnosing or aiding in
the diagnosis of cancer in a subject comprising comparing the
expression of one or more biomarkers in a sample from a subject to
a predetermined standard for each said one or more biomarkers;
wherein said one or more biomarkers are selected from the group
consisting of: 6Ckine, ACE, BDNF, CA125, E-Selectin, EGF, Eot2,
ErbB1, follistatin, HCC4, HVEM, IGF-II, IGFBP-1, IL-17, IL-1srII,
IL-2sRa, leptin, M-CSF R, MIF, MIP-1a, MIP3b, MMP-8, MMP7, MPIF-1,
OPN, PARC, PDGF Rb, prolactin, ProteinC, TGF-b RIII, TNF-R1, TNF-a,
VAP-1, VEGF R2 and VEGF R3; and wherein a significant difference in
the expression of said one or more biomarkers in said sample as
compared to a predetermined standard of each said one or more
biomarkers diagnoses or aids in the diagnosis of cancer.
[0006] In one embodiment, the predetermined standard corresponds
to: (a) the expression level of said biomarker in healthy subjects,
or (b) the expression level of said biomarker in non-cancerous
tissue from the same subject.
[0007] In one embodiment, the method further comprises comparing
the expression of two or more biomarkers, wherein the diagnosis of
cancer is based on a score-based classification method. In one
embodiment, the method comprises comparing the expression of m
different biomarkers; wherein each biomarker is assigned a score of
0 or 1, wherein a biomarker is assigned a score of 0 if the
expression of said biomarker is not significantly different from
the expression of said biomarker in a predetermined standard and
wherein a biomarker is assigned a score of 1 if the expression of
said biomarker is significantly different from the expression of
said biomarker in a predetermined standard; wherein the subject is
assigned an overall score which corresponds to the sum of the
assigned scores from m different markers; and wherein a given
threshold (t) is used to diagnose or aid in the diagnosis of
cancer.
[0008] In another embodiment, the method comprises comparing the
expression of two or more biomarkers, wherein the diagnosis of
cancer is made by comparing the expression profile of said two or
more biomarkers to a predetermined standard profile for said
biomarkers, and wherein a difference in the profiles diagnoses or
aids in the diagnosis of cancer. In one embodiment, the
predetermined standard profile is determined by comparing the
expression of said two or more biomarkers in cancer subjects to the
expression of said two or more biomarkers in healthy subjects using
a machine learning technique. In one embodiment, the predetermined
standard profile is determined by comparing the expression of said
two or more biomarkers in cancer subjects and in healthy subjects
using support vector machines, K-nearest neighbor classifier, or
classification tree analysis.
[0009] In one embodiment, the method is for the diagnosis for
ovarian cancer, and the method further comprises detecting an
additional biomarker for ovarian cancer which is not identified in
Table 2. In one embodiment, the additional biomarker for ovarian
cancer may be selected from the group consisting of: human stratum
corneum chymotryptic enzyme (HSCCE), kallikrein 4, kallikrein 5,
kallikrein 6 (protease M), kallikrein 8, kallikrein 9, kallikrein
10, CA125, CA15-3, CA19-9, OVX1, lysophosphatidic acid (LPA),
carcinoebryonic antigen (CEA), macrophage colony-stimulating factor
(M-CSF), prostasin, CA54-61, CA72, HMFG2, IL-6, IL-10, LSA, M-CSF,
NB70K, PLAP, TAG72, TNF, TPA, UGTF, WAP four-disulfide core domain
2 (HE4), matrix metalloprotease 2, tetranectin, inhibin,
mesothelyn, MUC1, VEGF, CLDN3, NOTCH3, E2F transcription factor 3
(E2F3), GTPase activating protein (RACGAP1), hemotological and
neurological expressed 1 (HN1), apolipoprotein A1, laminin, claudin
3, claudin 4, tumor-associated calcium signal transducer 1
(TROP-1/Ep-CAM), tumor-associated calcium signal transducer 2
(TROP-2), ladinin 1, S100A2, SERPIN2 (PAI-2), CD24, lipocalin 2,
matriptase (TADG-15), stratifin, transforming growth factor-beta
receptor III, platelet-derived growth factor receptor alpha,
SEMACAP3, ras homology gene family member I (ARHI), thrombospondin
2, disabled-2/differentially expressed in ovarian carcinoma 2
(Dab2/DOC2), and haptoglobin-alpha subunit. In another embodiment,
the additional biomarker for ovarian cancer is the truncated form
of transthyretin or the cleavage fragment of inter-alpha-trypsin
inhibitor heavy chain H4 identified by Zhang et al., Cancer Res.
64(16):5882-90 (2004). In one embodiment, the additional biomarker
for ovarian cancer is CA125.
[0010] The above described methods of diagnosing or aiding in the
diagnosis of cancer can be applied to diagnose or aid in the
diagnosis of any cancer or tumor. In one embodiment, the method is
for the diagnosis of breast cancer. In one embodiment, the method
is for the diagnosis of colon cancer. In another embodiment, the
method is for the diagnosis of cervical cancer.
[0011] The invention also comprises a method for diagnosing or
aiding in the diagnosis of cancer in a subject comprising comparing
the expression of one or more biomarkers in a sample from a subject
to a predetermined standard for each said one or more biomarkers;
wherein said one or more biomarkers are selected from the group
consisting of: prolactin, MIF, OPN, IGF-II, E-Selectin, leptin,
EGF, IL-17, MPIF-1, and IL-2sRa; and wherein a significant
difference in the expression of said one or more biomarkers in said
sample as compared to a predetermined standard of each said one or
more biomarkers diagnoses or aids in the diagnosis of cancer.
[0012] The invention also comprises a method for diagnosing or
aiding in the diagnosis of cancer in a subject comprising comparing
the expression of one or more biomarkers in a sample from a subject
to a predetermined standard for each said one or more biomarkers;
wherein said one or more biomarkers are selected from the group
consisting of: leptin, prolactin, OPN and IGF-II; and wherein a
significant difference in the expression of one or more biomarkers
in said sample as compared to a predetermined standard of each said
one or more biomarkers diagnoses or aids in the diagnosis of
cancer.
[0013] The invention also comprises a method for diagnosing or
aiding in the diagnosis of cancer in a subject comprising comparing
the expression of the following four biomarkers: Ieptin, prolactin,
OPN and IGF-II, in a sample from a subject to a predetermined
standard for each said biomarkers; wherein a significant difference
in the expression of two or more of said biomarkers in said sample
as compared to a predetermined standard of each said one or more
biomarkers diagnoses or aids in the diagnosis of cancer.
[0014] The invention also comprises a method for diagnosing or
aiding in the diagnosis of ovarian cancer in a subject comprising
comparing the expression of the following four biomarkers: Ieptin,
prolactin, OPN and IGF-II, in a sample from a subject to a
predetermined standard for each said biomarkers; wherein a
significant difference in the expression of two or more of said
biomarkers in said sample as compared to a predetermined standard
of each said one or more biomarkers diagnoses or aids in the
diagnosis of cancer.
[0015] The invention also comprises a method for diagnosing or
aiding in the diagnosis of breast cancer in a subject comprising
comparing the expression of the following four biomarkers: Ieptin,
prolactin, OPN and IGF-II, in a sample from a subject to a
predetermined standard for each said biomarkers; wherein a
significant difference in the expression of two or more of said
biomarkers in said sample as compared to a predetermined standard
of each said one or more biomarkers diagnoses or aids in the
diagnosis of cancer.
[0016] The invention also comprises a method for diagnosing or
aiding in the diagnosis of colon cancer in a subject comprising
comparing the expression of the following four biomarkers: Ieptin,
prolactin, OPN and IGF-II, in a sample from a subject to a
predetermined standard for each said biomarkers; wherein a
significant difference in the expression of two or more of said
biomarkers in said sample as compared to a predetermined standard
of each said one or more biomarkers diagnoses or aids in the
diagnosis of cancer.
[0017] In one embodiment, the above described methods comprise
comparing the expression of prolactin and/or OPN to a predetermined
standard of said biomarker, wherein an increase in the expression
of said biomarker as compared to the predetermined standard for
said biomarker diagnoses or aids in the diagnosis of cancer.
[0018] In one embodiment, the above described methods comprise
comparing the expression of leptin and/or IGF-II to a predetermined
standard of said biomarker, and wherein a decrease in the
expression of said biomarker as compared to the predetermined
standard for said biomarker diagnoses or aids in the diagnosis of
cancer.
[0019] In one embodiment, the above described methods of diagnosing
or aiding in the diagnosis of cancer comprises detecting the
expression of two or more biomarkers. In one embodiment, said two
or more biomarkers are selected from the group consisting of:
prolactin, MIF, OPN, IGF-II, E-Selectin, leptin, EGF, IL-17,
MPIF-1, and IL-2sRa. In one embodiment, said two or more biomarkers
are selected from the group consisting of: leptin, prolactin, OPN
and IGF-II. In one embodiment, a significant difference in the
expression of at least two or said two or more biomarkers diagnoses
or aids in the diagnosis of cancer.
[0020] In one embodiment, the above described methods of diagnosing
or aiding in the diagnosis of cancer comprises comparing the
expression of three or more biomarkers. In one embodiment, said
three or more biomarkers are selected from the group consisting of:
leptin, prolactin, OPN and IGF-II. In one embodiment, a significant
difference in the expression of said three or more biomarkers
diagnoses or aids in the diagnosis of cancer.
[0021] In one embodiment, the above described methods of diagnosing
or aiding in the diagnosis of cancer comprises comparing the
expression of four or more biomarkers. In one embodiment, said four
or more biomarkers include leptin, prolactin, OPN and IGF-II. In
one embodiment, a significant difference in the expression of four
or more biomarkers diagnoses or aids in the diagnosis of
cancer.
[0022] In one embodiment, the expression of a biomarker is detected
or measured using a reagent that detects said one or more
biomarkers. In one embodiment, the reagent is an antibody or
fragment thereof specific for said one or more biomarkers. In one
embodiment, the reagent is directly or indirectly labeled with a
detectable substance. In another embodiment, the expression of said
one or more biomarker is detected using mass spectroscopy. In
another embodiment, the expression of said one or more biomarker is
detected by measuring the mRNA transcription levels of the gene
encoding said one or more biomarker.
[0023] In another embodiment, the expression of said one or more
biomarker is detected by: (a) detecting the expression of a
polypeptide which is regulated by said one or more biomarker; (b)
detecting the expression of a polypeptide which regulates said
biomarker; or (c) detecting the expression of a metabolite of said
biomarker.
[0024] In one embodiment, the sample used in the above described
methods is a body fluid sample. In one embodiment, the body fluid
sample is blood or serum.
[0025] The invention also comprises methods for monitoring the
progression of cancer in a subject. In one embodiment, the
invention comprises a method of monitoring the progression of
cancer in a subject comprising comparing the expression of one or
more biomarkers in a sample from a subject to the expression of
said one or more biomarkers in a sample obtained from the subject
at a subsequent point in time; wherein said one or more biomarkers
are selected from the group consisting of: 6Ckine, ACE, BDNF,
CA125, E-Selectin, EGF, Eot2, ErbB1, follistatin, HCC4, HVEM,
IGF-II, IGFBP-1, IL-17, IL-1srII, IL-2sRa, leptin, M-CSF R, MIF,
MIP-1a, MIP3b, MMP-8, MMPI, MPIF-1, OPN, PARC, PDGF Rb, prolactin,
ProteinC, TGF-b RIII, TNF-R1, TNF-.alpha., VAP-1, VEGF R2 and VEGF
R3; and wherein a difference in the expression of said one or more
biomarker diagnoses or aids in the diagnosis of the progression of
the cancer in the subject. In one embodiment, said one or more
biomarkers are selected from the group consisting of: prolactin,
MIF, OPN, IGF-II, E-Selectin, leptin, EGF, IL-17, MPIF-1, and
IL-2sRa. In one embodiment, said one or more biomarkers are
selected from the group consisting of: leptin, prolactin, OPN and
IGF-II.
[0026] In one embodiment, the above described methods of monitoring
the progression of cancer comprises comparing the expression of two
or more biomarkers. In one embodiment, said two or more biomarkers
are selected from the group consisting of: prolactin, MIF, OPN,
IGF-II, E-Selectin, leptin, EGF, IL-17, MPIF-1, and IL-2sRa. In
another embodiment, said two or more biomarkers are selected from
the group consisting of: leptin, prolactin, OPN and IGF-II.
[0027] In one embodiment, the above described methods of monitoring
the progression of cancer comprises comparing the expression of
three or more biomarkers. In one embodiment, the above described
methods of monitoring the progression of cancer comprises comparing
the expression of four or more biomarkers. In one embodiment, the
above described methods of monitoring the progression of cancer
comprises comparing the expression of four or more biomarkers,
wherein said four or more biomarkers include leptin, prolactin, OPN
and IGF-II. In another embodiment, the above described method of
monitoring the progression of cancer comprises comparing the
expression of four biomarkers, wherein the four biomarkers are
leptin, prolactin, OPN and IGF-II.
[0028] The invention also comprises methods for monitoring the
effectiveness of a treatment against cancer. In one embodiment, the
invention comprise a method for monitoring the effectiveness of a
treatment against cancer comprising comparing the expression of one
or more biomarkers in a sample from a subject prior to providing at
least a portion of a treatment to the expression of said one or
more biomarkers in a sample obtained from the subject after the
subject has received at least a portion of the treatment; wherein
said one or more biomarkers are selected from the group consisting
of: 6Ckine, ACE, BDNF, CA125, E-Selectin, EGF, Eot2, ErbB1,
follistatin, HCC4, HVEM, IGF-II, IGFBP-1, IL-17, IL-1srII, IL-2sRa,
leptin, M-CSF R, MIF, MIP-1a, MIP3b, MMP-8, MMPI, MPIF-1, OPN,
PARC, PDGF Rb, prolactin, ProteinC, TGF-b RIII, TNF-R1, TNF-a,
VAP-1, VEGF R2 and VEGF R3; and wherein a difference in the
expression of said one or more biomarker diagnoses or aids in the
diagnosis of the efficacy of the treatment. In one embodiment, said
one or more biomarkers are selected from the group consisting of:
prolactin, MIF, OPN, IGF-II, E-Selectin, leptin, EGF, IL-17,
MPIF-1, and IL-2sRa. In one embodiment, said one or more biomarkers
are selected from the group consisting of: leptin, prolactin, OPN
and IGF-II.
[0029] In one embodiment, the above described methods of monitoring
the effectiveness of a treatment against cancer comprises comparing
the expression of two or more biomarkers. In one embodiment, said
two or more biomarkers are selected from the group consisting of:
prolactin, MIF, OPN, IGF-II, E-Selectin, leptin, EGF, IL-17,
MPIF-1, and IL-2sRa. In another embodiment, said two or more
biomarkers are selected from the group consisting of: leptin,
prolactin, OPN and IGF-II.
[0030] In one embodiment, the above described methods of monitoring
the effectiveness of a treatment against cancer comprises comparing
the expression of three or more biomarkers. In one embodiment, the
above described methods of monitoring the effectiveness of a
treatment against cancer comprises comparing the expression of four
or more biomarkers. In one embodiment, the above described methods
of monitoring the effectiveness of a treatment against cancer
comprises comparing the expression of four or more biomarkers,
wherein said four or more biomarkers include leptin, prolactin, OPN
and IGF-II. In another embodiment, the above described method of
monitoring the effectiveness of a treatment against cancer
comprises comparing the expression of four biomarkers, wherein the
four biomarkers are leptin, prolactin, OPN and IGF-II.
[0031] The invention also comprises kits for diagnosing or aiding
in the diagnosis of cancer and kits for monitoring cancer. In one
embodiment, the kit comprises: (i) a receptacle for receiving a
sample; (ii) one or more reagents for detecting one or more
biomarkers selected from the group consisting of: 6Ckine, ACE,
BDNF, CA125, E-Selectin, EGF, Eot2, ErbB1, follistatin, HCC4, HVEM,
IGF-II, IGFBP-1, IL-17, IL-1srII, IL-2sRa, leptin, M-CSF R, MIF,
MIP-1a, MIP3b, MMP-8, MMP7, MPIF-1, OPN, PARC, PDGF Rb, prolactin,
ProteinC, TGF-b RIII, TNF-R1, TNF-.alpha., VAP-1, VEGF R2 and VEGF
R3; and (iii) a reference sample. In one embodiment, the kit
comprises one or more reagents for the detection of leptin,
prolactin, OPN and IGF-II.
[0032] The invention also comprises a method to screen for a
candidate compound useful to treat cancer. In one embodiment, the
invention comprises a method to screen for a candidate compound
useful to treat cancer comprising: (i) identifying a candidate
compound which regulates the expression of at least one biomarker
selected from the group consisting of: 6Ckine, ACE, BDNF, CA125,
E-Selectin, EGF, Eot2, ErbB1, follistatin, HCC4, HVEM, IGF-II,
IGFBP-1, IL-17, IL-1srII, IL-2sRa, leptin, M-CSF R, MIF, MIP-1a,
MIP3b, MMP-8, MMP7, MPIF-1, OPN, PARC, PDGF Rb, prolactin,
ProteinC, TGF-b RIII, TNF-R1, TNF-.alpha., VAP-1, VEGF R2 and VEGF
R3; and (ii) determining whether such candidate compound is
effective to treat cancer. In one embodiment, the method comprises
identifying a candidate compound which regulates the expression of
at least one biomarkers selected from the group consisting of:
prolactin, MIF, OPN, IGF-II, E-Selectin, leptin, EGF, IL-17,
MPIF-1, and IL-2sRa. In one embodiment, the method comprises
identifying a candidate compound which regulates the expression of
at least one biomarkers selected from the group consisting of
leptin, prolactin, OPN and IGF-II.
[0033] The invention also comprises a method of conducting a
business. In one embodiment, the method of conducting a business
comprises: (i) obtaining a sample; (ii) detecting the expression of
one or more biomarker in the sample, wherein said one or more
biomarkers are selected from the group consisting of: 6Ckine, ACE,
BDNF, CA125, E-Selectin, EGF, Eot2, ErbB1, follistatin, HCC4, HVEM,
IGF-II, IGFBP-1, IL-17, IL-1srII, IL-2sRa, leptin, M-CSF R, MIF,
MIP-1a, MIP3b, MMP-8, MMPI, MPIF-1, OPN, PARC, PDGF Rb, prolactin,
ProteinC, TGF-b RIII, TNF-R1, TNF-.alpha., VAP-1, VEGF R2 and VEGF
R3; and (iii) reporting the results of such detection. In one
embodiment, said one or more biomarkers are selected from the group
consisting of: prolactin, MIF, OPN, IGF-II, E-Selectin, leptin,
EGF, IL-17, MPIF-1, and IL-2sRa. In another embodiment, said one or
more biomarkers are selected from the group consisting of: leptin,
prolactin, OPN and IGF-II.
[0034] In one embodiment, the invention comprises a method of
conducting a business comprising: (i) obtaining a sample; (ii)
detecting the expression of four biomarkers in the sample, wherein
said four biomarkers leptin, prolactin, OPN and IGF-II; and (iii)
reporting the results of such detection.
[0035] The invention also comprises methods to screen for candidate
cancer biomarkers. In one embodiment, the invention comprises a
method to screen for candidate cancer biomarkers comprising: (i)
identifying a group of biomarkers that are potentially associated
with cancer; (ii) comparing the level of expression of the
biomarkers identified in step (i) in a first population of cancer
subjects and in healthy subjects; (iii) selecting biomarkers
exhibiting a significant difference in expression in said first
population of cancer subjects; (iv) comparing the level of
expression of the biomarkers identified in step (iii) in a second
population of cancer subjects and in healthy subjects; and (v)
selecting biomarkers exhibiting a significant difference in
expression in said second population of cancer subjects; wherein
the biomarkers identified in step (v) are candidate cancer
biomarkers. In one embodiment, said first population of cancer
subjects have newly diagnosed cancer, and said second population of
cancer subjects have recurrent cancer. In one embodiment, said
first population of cancer subjects have recurrent cancer and said
second population of cancer subjects have newly diagnosed cancer.
In another embodiment, wherein said first population of cancer
subjects have late stage cancer and said second population of
cancer subjects have early stage cancer. In another embodiment,
said first population of cancer patients have early stage cancer
and said second population of cancer subjects have later stage
cancer. In another embodiment, said method further comprises: (vi)
comparing the level of expression of the biomarkers identified in
step (v) in a third population of cancer subjects and in healthy
subjects, wherein the expression of said biomarkers is detected by
using a different assay format; and (vii) selecting biomarkers
exhibiting a significant different in expression in said third
population of cancer subjects; wherein the biomarkers identified in
step (vii) are candidate biomarkers for cancer. In one embodiment,
said method further comprises determining whether the biomarkers
identified in step (v) or (vii) could distinguish between cancer
and healthy subjects in a blind study.
[0036] The invention also comprises a method to screen for
candidate cancer biomarkers comprising: (i) identifying a cancer
biomarker; (ii) selecting polypeptides which regulate or are
regulated by the biomarker identified in step (i); and (iii)
measuring the expression of the polypeptides identified in step
(ii) in cancer subjects and in healthy subjects, wherein a
polypeptide which is differentially expressed in cancer subjects
and in healthy subjects is a candidate cancer biomarker.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 is a schematic representation of the screening
process used to identify biomarkers which could discriminate
between subjects with cancer and healthy subjects.
[0038] FIG. 2 is a schematic representation of a sample protein
microarray slide with 16 subarrays. Subarrays refer to the 16
wells, or circular analysis sites, on the slide. Array refers to
the antibody content printed in a well. Each microarray slide
contains only one type of array.
[0039] FIG. 3-FIG. 3 (cont.) shows the difference in expression of
four proteins (leptin, prolactin, OPN and IGF-II) in subjects with
ovarian cancer and in healthy subjects using two different assays:
RCA microarray immunoassay and ELISA.
[0040] FIG. 4 shows results of analysis of the expression data of
four proteins (leptin (identified as "1"), prolactin (identified as
"2"), OPN (identified as "3") and IGF-II (identified as "4")) in
206 subjects, using the least square fit in a traditional binary
data set analysis. The protein levels of healthy subjects are shown
in black (left) and those for subjects with ovarian cancer are
shown in gray (right)
[0041] FIG. 5 shows results of analysis of the expression data of
four proteins (leptin (identified as "1"), prolactin (identified as
"2"), OPN (identified as "3") and IGF-II (identified as "4")) in
206 subjects, using pair plots. The data points derived from
healthy subjects are in black and the data points derived from
subjects with ovarian cancer are in gray.
[0042] FIG. 6 shows the scores assigned to 206 subjects including
106 healthy subjects and 100 subjects with ovarian cancer based on
the score-based classification system described herein. Subjects
having a score greater than or equal to 2 can be diagnosed with
ovarian cancer, while subjects with score less than or equal to 1
can be diagnosed as free of ovarian cancer. The data points derived
from healthy subjects are in light gray and the data points derived
from subjects with ovarian cancer are in dark gray.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[0043] Described herein is a method which can be used to
discriminate between cancer subjects (including subjects diagnosed
with early stage (stage I-II) disease) and healthy subjects. This
method is based on the identification of biomarkers which are
particularly well suited to discriminate between cancer subjects
and healthy subjects. These biomarkers were identified using a
unique and novel screening method described herein involving
several different screening steps using samples from different
subjects in each step and validation with different techniques. The
biomarkers disclosed herein can be used in the diagnosis, prognosis
and monitoring of cancer.
[0044] In one particular embodiment, the invention disclosed herein
refers to a new test based on four biomarkers: leptin, prolactin,
ORN and IGF II, which discriminate between cancer subjects and
healthy subjects, particularly between ovarian cancer subjects and
healthy subjects. In one embodiment, these four biomarkers can be
used in a blood test for the diagnosis, prognosis and monitoring of
ovarian cancer.
[0045] These biomarkers identified herein can be used in
combination with additional known biomarkers. For example, a known
biomarker of ovarian cancer is CA125. The use of CA125 in
conjunction with the biomarkers identified herein presents a novel
approach for the early detection of ovarian cancer and may
significantly improve our ability to accurately detect
pre-malignant change or early stage ovarian cancer in asymptomatic
women at increased risk for the development of ovarian cancer.
Further, the biomarkers identified in this application can be used
in conjunction with other diagnostic techniques. For example, for
the diagnosis of ovarian cancer, the biomarkers identified in this
application can be used in conjunction with vaginal examination,
ultrasound or MRI to diagnose ovarian cancer.
[0046] The articles "a," "an" and "the" are used herein to refer to
one or to more than one (i.e., to at least one) of the grammatical
object of the article.
[0047] The term "including" is used herein to mean, and is used
interchangeably with, the phrase "including but not limited
to".
[0048] The term "or" is used herein to mean, and is used
interchangeably with, the term "and/or," unless context clearly
indicates otherwise.
[0049] The term "such as" is used herein to mean, and is used
interchangeably with, the phrase "such as but not limited to".
II. Methods of Diagnosis
[0050] In one embodiment, the invention refers to a method for
diagnosing or aiding in the diagnosis of cancer in a subject. In
one embodiment, the method comprises comparing the expression of
one or more biomarkers selected from the group consisting of the
biomarkers identified in Table 2 in a sample from a subject to a
predetermined standard for each said one or more biomarkers,
wherein a significant difference in the expression of said one or
more biomarkers in said sample as compared to a predetermined
standard of each said one or more biomarkers diagnoses or aids in
the diagnosis of cancer. In one embodiment, said one or more
biomarkers are selected from the group consisting of the biomarkers
identified in Table 3. In another embodiment, said one or more
biomarkers are selected from the group consisting of: leptin,
prolactin, OPN and IGF-II.
[0051] When the biomarkers are prolactin and/or OPN, an increase in
the expression of said biomarkers as compared to the predetermined
standard for said biomarker diagnoses or aids in the diagnosis of
cancer. When the biomarkers is leptin and/or IGF-II, a decrease in
the expression of said biomarker as compared to the predetermined
standard for said biomarker diagnoses or aids in the diagnoses of
cancer. As used herein, an increase or decrease in expression
refers to the fact that level of a gene expression product is made
higher or lower, or to the fact that the activity of the gene
expression product is enhanced or lowered.
[0052] The above described methods can be used to diagnose any
cancer or tumor. In one embodiment, the cancer is ovarian cancer.
In another embodiment, the cancer is breast cancer. In another
embodiment, the cancer is colon cancer. In another embodiment, the
cancer is prostate cancer. In another embodiment, the cancer is
cervical cancer.
[0053] As used herein, the term "biomarker" refers to one or more
polypeptides that can be used to: diagnose, or to aid in the
diagnosis or prognosis of, cancer either alone or as combination of
multiple polypeptides; monitor the progression of cancer; and/or
monitor the effectiveness of a cancer treatment. As used herein,
the term "polypeptide" refers to a polymer of amino acids, and not
to a specific length. Thus, peptides, oligopeptides and proteins
are included within the definition of polypeptide.
[0054] As used herein, the term "leptin" includes all homologs,
naturally occurring allelic variants, isoforms and precursors of
leptin. Leptin is also known as HGNC:6553, OB, OBS, obesity, or
murine obesity homolog. In one embodiment, leptin comprises the
amino acid sequence of GenBank Accession No. NP.sub.--000221.
[0055] As used herein, the term "prolactin" includes all homologs,
naturally occurring allelic variants, isoforms and precursors of
prolactin. Prolactin is also known as PRL or HGNC:9445. In one
embodiment, prolactin comprises the amino acid sequence of GenBank
Accession No. NP.sub.--000939.
[0056] As used herein, the term "OPN" includes all homologs,
naturally occurring allelic variants, isoforms and precursors of
OPN. OPN is also known as HGNC:11255, BNSP, BSPI, ETA-1, secreted
phosphoprotein-1 or osteopontin. In one embodiment, OPN comprises
the amino acid sequence of GenBank Accession No.
NP.sub.--000573.
[0057] As used herein, the term "IGF-II" includes all homologs,
naturally occurring allelic variants, isoforms and precursors of
IGF-II. IGF-II is also known as HGNC:5466, insulin-like growth
factor 2, insulin-like growth factor II or somatomedin A. In one
embodiment, IGF-II comprises the amino acid sequence of GenBank
Accession No. NP.sub.--000603.
[0058] As used herein, the term "subject" or "patient" includes all
warm-blooded animals. In one embodiment the subject is a human. In
one embodiment, the subject is a subject with an enhanced risk of
developing cancer.
[0059] In one embodiment, when the method relates to ovarian
cancer, the subject is a female (such as a woman) suspected of
having or known to have ovarian cancer, or with an enhanced risk of
developing ovarian cancer. For example, for ovarian cancer subjects
having a familial history of ovarian cancer, subjects identified as
having a mutant oncogene, and subjects at least about 50 years of
age have an enhanced risk of developing ovarian cancer.
[0060] As used herein, the term "sample" refers to a material
obtained from a subject. The sample can be derived from any
biological source, including all body fluids (such as, for example,
whole blood, plasma, serum, saliva, ocular lens fluid, sweat,
urine, milk, etc.), tissue or extracts, cells, etc. Examples of
ovary-associated body fluids include blood fluids (e.g. whole
blood, blood serum, blood having platelets removed therefrom,
etc.), lymph, ascitic fluids, gynecological fluids (e.g. ovarian,
fallopian, and uterine secretions, menses, vaginal douching fluids,
fluids used to rinse ovarian cell samples, etc.), cystic fluid,
urine, and fluids collected by peritoneal rinsing (e.g. fluids
applied and collected during laparoscopy or fluids instilled into
and withdrawn from the peritoneal cavity of a human patient).
[0061] The term "expression" is used herein to mean the process by
which a polypeptide is produced from DNA. The process involves the
transcription of the gene into mRNA and the translation of this
mRNA into a polypeptide. Depending on the context in which used,
"expression" may refer to the production of RNA, protein or
both.
[0062] Expression of a biomarker of the invention may be assessed
by any of a wide variety of well known methods for detecting
expression of a transcribed molecule or its corresponding protein.
Non-limiting examples of such methods include immunological methods
for detection of secreted proteins, protein purification methods,
protein function or activity assays, nucleic acid hybridization
methods, nucleic acid reverse transcription methods, and nucleic
acid amplification methods. In a preferred embodiment, expression
of a marker gene is assessed using an antibody (e.g. a
radio-labeled, chromophore-labeled, fluorophore-labeled, or
enzyme-labeled antibody), an antibody derivative (e.g. an antibody
conjugated with a substrate or with the protein or ligand of a
protein-ligand pair {e.g. biotin-streptavidin}), or an antibody
fragment (e.g. a single-chain antibody, an isolated antibody
hypervariable domain, etc.) which binds specifically with a protein
corresponding to the marker gene, such as the protein encoded by
the open reading frame corresponding to the marker gene or such a
protein which has undergone all or a portion of its normal
post-translational modification. In another preferred embodiment,
expression of a marker gene is assessed by preparing mRNA/cDNA
(i.e. a transcribed polynucleotide) from cells in a patient sample,
and by hybridizing the mRNA/cDNA with a reference polynucleotide
which is a complement of a polynucleotide comprising the marker
gene, and fragments thereof. cDNA can, optionally, be amplified
using any of a variety of polymerase chain reaction methods prior
to hybridization with the reference polynucleotide; preferably, it
is not amplified.
[0063] As used herein, a "predetermined standard" for a biomarker
refers to the levels of expression of said biomarker in healthy
subjects or the expression levels of said biomarker in
non-cancerous tissue from the same subject. The predetermined
standard expression levels for a given biomarker can be established
by prospective and/or retrospective statistical studies using only
routine experimentation. Said predetermined standard expression
levels can be determined by a person having ordinary skill in the
art using well known methods.
[0064] The term "healthy subject" refers to a subject has not been
diagnosed with cancer or who has not been diagnosed with cancer of
the type which is being analyzed. Thus, for example, in a method to
diagnose ovarian cancer, a "healthy subject" refers to a subject
who does cancer or who does not have ovarian cancer.
[0065] As used herein, the term "significant difference" is well
within the knowledge of a skilled artisan and will be determined
empirically with reference to each particular biomarker. For
example, a significant difference in the expression of a biomarker
in a subject with cancer as compared to a healthy subject is any
difference in expression which is statistically significant.
[0066] In one embodiment, the method comprises comparing the
expression of two or more biomarkers and the diagnosis of cancer is
based on a score-based classification method. In one embodiment,
the score-based classification system is a based on binary numbers.
In one embodiment, the score-based classification system comprises
determining the expression of m different biomarkers; wherein each
biomarker is assigned a score of 0 or 1, wherein a biomarker is
assigned a score of 0 if the expression of said biomarker is not
significantly different from the expression of said biomarker in a
predetermined standard and wherein a biomarker is assigned a score
of 1 if the expression of said biomarker is significantly different
from the expression of said biomarker in a predetermined standard;
wherein the subject is assigned an overall score which corresponds
to the sum of the assigned scores from m different markers; and
wherein a given threshold (t) is used to diagnose or aid in the
diagnosis of cancer.
[0067] In one embodiment, the score-based classification system
comprises comparing the expression of four (4) different
biomarkers; wherein each biomarker is assigned a score of 0 or 1,
wherein a biomarker is assigned a score of 0 if the expression of
said biomarker is not significantly different from the expression
of said biomarker in a predetermined standard and wherein a
biomarker is assigned a score of 1 if the expression of said
biomarker is significantly different from the expression of said
biomarker in a predetermined standard; wherein the subject is
assigned an overall score which corresponds to the sum of the
assigned scores from four (4) different markers; and wherein a
score or 2 or more diagnoses or aids in the diagnosis of cancer. In
one embodiment, the four biomarkers are leptin, prolactin, OPN and
IGF-II.
[0068] In one embodiment, the method comprises comparing the
expression of two or more biomarkers, wherein the diagnosis of
cancer is made by comparing the expression profile of said two or
more biomarkers to a predetermined standard profile for said
biomarkers, and wherein a difference in the profiles diagnoses or
aids in the diagnosis of cancer. As used herein, an "expression
profile" is a representation of the levels of expression of one or
more biomarkers in a given sample.
[0069] In one embodiment, the predetermined standard profile is
determined by comparing the expression of said two or more
biomarkers in cancer subjects to the expression of said two or more
biomarkers in healthy subjects using a machine learning technique.
In one embodiment, the predetermined standard profile is determined
by comparing the expression of said two or more biomarkers in
cancer subjects and in healthy subjects using support vector
machines, K-nearest neighbor classifier, or classification tree
analysis.
[0070] In one embodiment, the method comprises detecting an
additional known biomarker which is not identified in Table 2 and
comparing the expression of said additional known biomarker to a
predetermined standard for said additional known biomarker.
Additional biomarkers for cancer can be identified by a person
having ordinary skill in the art by reference to the published
literature. In one embodiment, the cancer is ovarian cancer, and
the additional biomarker for ovarian cancer is selected from the
group consisting of human stratum corneum chymotryptic enzyme
(HSCCE), kallikrein 4, kallikrein 5, kallikrein 6 (protease M),
kallikrein 8, kallikrein 9, kallikrein 10, CA125, CA15-3, CA19-9,
OVX1, lysophosphatidic acid (LPA), carcinoebryonic antigen (CEA),
macrophage colony-stimulating factor (M-CSF), prostasin, CA54-61,
CA72, HMFG2, IL-6, IL-10, LSA, M-CSF, NB70K, PLAP, TAG72, TNF, TPA,
UGTF, WAP four-disulfide core domain 2 (HE4), matrix
metalloprotease 2, tetranectin, inhibin, mesothelyn, MUC1, VEGF,
CLDN3, NOTCH3, E2F transcription factor 3 (E2F3), GTPase activating
protein (RACGAP1), hemotological and neurological expressed 1
(HN1), apolipoprotein A1, laminin, claudin 3, claudin 4,
tumor-associated calcium signal transducer 1 (TROP-1/Ep-CAM),
tumor-associated calcium signal transducer 2 (TROP-2), ladinin 1,
S100A2, SERPIN2 (PAI-2), CD24, lipocalin 2, matriptase (TADG-15),
stratifin, transforming growth factor-beta receptor III,
platelet-derived growth factor receptor alpha, SEMACAP3, ras
homology gene family member I (ARHI), thrombospondin 2,
disabled-2/differentially expressed in ovarian carcinoma 2
(Dab2/DOC2), and haptoglobin-alpha subunit. In another embodiment,
the additional biomarker for ovarian cancer is the truncated form
of transthyretin or the cleavage fragment of inter-alpha-trypsin
inhibitor heavy chain H4 identified by Zhang et al., Cancer Res.
64(16):5882-90 (2004). In a preferred embodiment, the additional
biomarker for ovarian cancer is CA125.
[0071] In one embodiment, the invention refers to a method for
diagnosing or aiding in the diagnosis of cancer in a subject
comprising comparing the expression of two or more biomarkers
selected from the group consisting of the biomarkers identified in
Table 2 in a sample from a subject to a predetermined standard for
each said biomarker, wherein a significant difference in the
expression of one or more biomarkers in said sample as compared to
a predetermined standard of each biomarker diagnoses or aids in the
diagnosis of cancer. In one embodiment, said two or more biomarkers
are selected from the group consisting of the biomarkers identified
in Table 3. In another embodiment, said two or more biomarkers are
selected from the group consisting of: leptin, prolactin, OPN and
IGF-II. In one embodiment, a significant difference in the
expression of at least two of said two or more biomarkers diagnoses
or aids in the diagnosis of cancer.
[0072] In one embodiment, the invention comprises to a method for
diagnosing or aiding in the diagnosis of cancer in a subject
comprising comparing the expression of three or more biomarkers
selected from the group consisting of the biomarkers identified in
Table 2 in a sample from a subject to a predetermined standard for
each biomarker, wherein a significant difference in the expression
of one or more biomarkers in said sample as compared to a
predetermined standard of each said biomarker diagnoses or aids in
the diagnosis of cancer. In one embodiment, said three or more
biomarkers are selected from the group consisting of the biomarkers
identified in Table 3. In another embodiment, said three or more
biomarkers are selected from the group consisting of: leptin,
prolactin, OPN and IGF-II. In one embodiment, a significant
difference in the expression of at least two or said two or more
biomarkers diagnoses or aids in the diagnosis of cancer.
[0073] In one embodiment, the invention refers to a method for
diagnosing or aiding in the diagnosis of cancer in a subject
comprising comparing the expression of four or more biomarkers
selected from the group consisting of the biomarkers identified in
Table 2 in a sample from a subject to a predetermined standard for
each biomarker, wherein a significant difference in the expression
of one or more biomarkers in said sample as compared to a
predetermined standard of each said biomarker diagnoses or aids in
the diagnosis of cancer. In one embodiment, said four or more
biomarkers are selected from the group consisting of the biomarkers
identified in Table 3. In another embodiment, said four or more
biomarkers are selected from the group consisting of: leptin,
prolactin, OPN and IGF-II. In one embodiment, a significant
difference in the expression of at least two of said two or more
biomarkers diagnoses or aids in the diagnosis of cancer.
[0074] The expression of said one or more biomarkers can be
detected using any method known to a person having ordinary skill
in the art. In one embodiment, the expression of said one or more
biomarkers can be detected using a reagent that detects said one or
more biomarkers. Said reagent can be any reagent that specifically
detects said one or more biomarkers. Said reagent can be an
antibody (natural or synthetic) or a fragment thereof specific for
the biomarker, a peptide, a nucleic acid, or any other reagent that
can specifically detect a biomarker. As used herein, the term
"antibody" includes chimeric and synthetic antibodies, e.g.,
generated by combinatorial mutagenesis and phage display. The term
"antibody" includes mimetics or peptidomimetics of antibodies.
Peptidomimetics are compounds based on, or derived from, peptides
and proteins. The peptidomimetics of the present invention
typically can be obtained by structural modification of a known
peptide sequence using unnatural amino acids, conformational
restraints, isosteric replacement, and the like. The subject
peptidomimetics constitute the continuum of structural space
between peptides and non-peptide synthetic structures;
peptidomimetics may be useful, therefore, in delineating
pharmacophores and in helping to translate peptides into
non-peptide compounds with the activity of the parent peptides. For
illustrative purposes, peptide analogs of the antibodies can be
generated using, for example, benzodiazepines (e.g., see Freidinger
et al. in Peptides: Chemistry and Biology, G. R. Marshall ed.,
ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma
lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.
R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p
123), C-7 mimics (Huffman et al. in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands,
1988, p. 105), keto-methylene pseudopeptides (Ewenson et al. (1986)
J Med Chem 29:295; and Ewenson et al. in Peptides: Structure and
Function (Proceedings of the 9.sup.th American Peptide Symposium)
Pierce Chemical Co. Rockland, I L, 1985), .beta.-turn dipeptide
cores (Nagai et al. (1985) Tetrahedron Lett 26:647; and Sato et al.
(1986) J Chem Soc Perkin Trans 1:1231), .beta.-aminoalcohols
(Gordon et al. (1985) Biochem Biophys Res Commun 126:419; and Dann
et al. (1986) Biochem Biophys Res Commun 134:71), diaminoketones
(Nataraj an et al. (1984) Biochem Biophys Res Commun 124:141), and
methyleneamino-modified (Roark et al. in Peptides: Chemistry and
Biology, G. R. Marshall ed., ESCOM Publsiher: Leiden, Netherlands,
1988, p 134). Also, see generally, Session III: Analytic and
synthetic methods, in Peptides: Chemistry and Biology, G. R.
Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988).
[0075] In another embodiment, said reagent is directly or
indirectly labeled with a detectable substance. The detectable
substance may be, for example, selected, e.g., from a group
consisting of radioisotopes, fluorescent compounds, enzymes, and
enzyme co-factor. Methods of labeling antibodies are well known in
the art.
[0076] As used herein, the term "detect", "detected" or "detecting"
includes measure, measured or measuring.
[0077] The above described methods can be performed using any
sample. In one embodiment, the sample is a body fluid sample. In
one embodiment, the body fluid sample is blood or serum.
[0078] In another embodiment, the expression of said one or more
biomarkers are detected using mass spectroscopy.
[0079] In yet another embodiment, the expression of said one or
more biomarkers is detected by detecting the mRNA transcription
levels of the gene encoding said at one or more biomarker.
[0080] In yet another embodiment, the expression of said one or
more biomarkers can be detected by ELISA, RCA immunoassay,
chemiluminescence, thin-film optical biosensor, proton resonance
technology, protein microarray assay or any other detection method
known in the art.
[0081] In one embodiment, the expression of said one or more
biomarkers are detected by: (a) detecting the expression of a
polypeptide which is regulated by said one or more biomarker; (b)
detecting the expression of a polypeptide which regulates said
biomarker; or (c) detecting the expression of a metabolite of said
biomarker. A person of skill in the art would be able to identify
polypeptides which regulate or are regulated by a biomarker, and
metabolites of a biomarker, using only routine experimentation.
[0082] The above described methods to diagnose or aid in the
diagnosis of cancer may be used in conjunction with other methods
to validate the results (i.e. to more conclusively determine
whether a subject has cancer). In one embodiment, the cancer is
ovarian cancer and the above described methods further comprise:
physical examination, ultrasound examination, x-ray examination,
MRI examination, laparotomy and/or hematological tests.
Hematological tests which may be indicative of ovarian cancer in a
patient include analyses of serum levels of additional biomarkers
of ovarian cancer.
III. Methods of Monitoring
[0083] In one embodiment, the invention comprises a method of
monitoring the progression of cancer in a subject comprising
comparing the expression of one or more biomarkers selected from
the group consisting of the biomarkers identified in Table 2 in a
sample from a subject; to the expression of said one or more
biomarkers in a sample obtained from the subject at a subsequent
point in time, wherein a difference in the expression of said one
or more biomarkers are indicative of the progression of the cancer
in the subject. In one embodiment, said one or more biomarkers are
selected from the group consisting of the biomarkers identified in
Table 3. In another embodiment, said one or more biomarkers are
selected from the group consisting of leptin, prolactin, OPN and
IGF-II.
[0084] In one embodiment, the method comprises comparing the
expression of two or more biomarkers. In another embodiment, the
method comprises comparing the expression of three or more
biomarkers. In another embodiment, the method comprises comparing
the expression of four or more biomarkers. In one embodiment, the
method comprises comparing the expression of four or more
biomarkers, wherein said four or more biomarkers include leptin,
prolactin, OPN and IGF-II. In yet another embodiment, the method
comprises comparing the expression of four biomarkers: leptin,
prolactin, OPN and IGF-II.
[0085] In one embodiment, the method is used to monitor the
progression of cancer after the subject has received a treatment
for cancer.
[0086] The invention also comprises a method for monitoring the
effectiveness of a treatment against cancer, comprising comparing
the expression of one or more biomarkers selected from the group
consisting of the biomarkers identified in Table 3 in a sample from
a subject prior to providing at least a portion of a treatment to
the expression of said one or more biomarkers in a sample obtained
from the subject after the subject has received at least a portion
of the treatment, wherein a difference in the expression of said
one or more biomarkers are indicative of the efficacy of the
treatment.
[0087] In one embodiment, said one or more biomarkers are selected
from the group consisting of the biomarkers identified in Table 3.
In another embodiment, said one or more biomarkers are selected
from the group consisting of leptin, prolactin, OPN and IGF-II.
[0088] In one embodiment, the method comprises comparing the
expression of two or more biomarkers. In another embodiment, the
method comprises comparing the expression or three or more
biomarkers. In another embodiment, the method comprises comparing
the expression of four or more biomarkers. In one embodiment, the
method comprises comparing the expression of four or more
biomarkers, wherein said four or more biomarkers include leptin,
prolactin, OPN and IGF-II. In yet another embodiment, the method
comprises comparing the expression of four biomarkers: leptin,
prolactin, OPN and IGF-II.
[0089] It will be appreciated that as used herein, the "treatment"
may be any treatment for treating ovarian cancer including, but not
limited to, chemotherapy, immunotherapy, gene therapy, radiation
therapy and surgical removal of tissue. As used herein, "a portion
of a treatment" refers to any portion of a treatment for cancer,
such as a dose of a compound used to treat cancer, or a portion of
a treatment such as chemotherapy.
[0090] The above described methods of monitoring cancer are
applicable to any cancer or tumor. In one embodiment, the method is
for monitoring ovarian cancer. In one embodiment, the method is for
the monitoring breast cancer. In one embodiment, the method is for
monitoring colon cancer. In another embodiment, the method is for
monitoring cervical cancer.
IV. Kits
[0091] The invention also comprises kits for diagnosing or aiding
in the diagnosis of cancer or for monitoring cancer. The kits can
be used to diagnose or monitor any cancer. In one embodiment, the
kit is for the diagnosis or monitoring of ovarian cancer. In one
embodiment, the kit is for the diagnosis or monitoring of breast
cancer. In one embodiment, the kit is for the diagnosis or
monitoring of colon cancer. In one embodiment, the kit is for the
diagnosis or monitoring of cervical cancer.
[0092] In one embodiment, the kit comprises: (i) a receptacle for
receiving a sample; (ii) one or more reagents for detecting one or
more biomarkers selected from the group consisting of the
biomarkers identified in Table 2; and (iii) a reference sample. In
one embodiment, the kit comprises one or more reagents for
detecting one or more biomarkers selected from the group consisting
of the biomarkers identified in Table 3. In one embodiment, the kit
comprises one or more reagents for detecting one or more biomarkers
selected from the group consisting of leptin, prolactin, OPN and
IGF-II.
[0093] In one embodiment, the kit comprises reagents for detecting
two or more biomarkers. In one embodiment, said two or more
biomarkers are selected from the group consisting of: leptin,
prolactin, OPN and IGF-II.
[0094] In another embodiment, said kit comprises reagents for
detecting three or more biomarkers.
[0095] In one embodiment, the kit comprises reagents for detecting
four or more biomarkers. In one embodiment, said four or more
biomarkers include leptin, prolactin, OPN and IGF-II.
[0096] The reagents may be labeled compounds or agents capable of
detecting a polypeptide or an mRNA encoding a polypeptide
corresponding to a marker gene of the invention in a biological
sample and means for determining the amount of the polypeptide or
mRNA in the sample (e.g., an antibody which binds the polypeptide
or an oligonucleotide probe which binds to DNA or mRNA encoding the
polypeptide). Suitable reagents for binding with a polypeptide
corresponding to a marker gene of the invention include antibodies,
antibody derivatives, antibody fragments, and the like. Suitable
reagents for binding with a nucleic acid (e.g. a genomic DNA, an
mRNA, a spliced mRNA, a cDNA, or the like) include complementary
nucleic acids.
[0097] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a polypeptide corresponding to a marker gene of the
invention; and, optionally, (2) a second, different antibody which
binds to either the polypeptide or the first antibody and is
conjugated to a detectable label.
[0098] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding a polypeptide corresponding to a marker gene of the
invention or (2) a pair of primers useful for amplifying a nucleic
acid molecule corresponding to a marker gene of the invention.
[0099] The reference sample is used to compare the results obtained
from the sample being tested.
[0100] The kit can also comprise other components such as a
buffering agent, a preservative, or a protein stabilizing agent.
The kit can further comprise components necessary for detecting the
detectable label (e.g., an enzyme or a substrate).
[0101] Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package, along with instructions for interpreting
the results of the assays performed using the kit.
V. Screening methods
[0102] The present invention also comprises methods to screen for
candidate compounds useful to treat cancer. In one embodiment, the
invention comprises a method to screen for a candidate compound
useful to treat cancer comprising: (i) identifying a candidate
compound which regulates the expression of one or more biomarkers
selected from the group consisting of the biomarkers identified in
Table 2; and (ii) determining whether such candidate compound is
effective to treat cancer. In one embodiment, said one or more
biomarkers are selected from the group consisting of the biomarkers
identified in Table 3. In another embodiment, said one or more
biomarkers are selected from the group consisting of leptin,
prolactin, OPN and IGF-II.
[0103] In one embodiment, the invention comprises a method to
screen for a candidate compound useful to treat cancer comprising:
(i) identifying a candidate compound which regulates the expression
of two or more biomarkers selected from the group consisting of the
biomarkers identified in Table 2; and (ii) determining whether such
candidate compound is effective to treat cancer. In one embodiment,
said two or more biomarkers are selected from the group consisting
of the biomarkers identified in Table 3. In another embodiment,
said two or more biomarkers are selected from the group consisting
of leptin, prolactin, OPN and IGF-II.
[0104] The present invention also comprises methods to screen for
candidate compounds useful to treat cancer. In one embodiment, the
invention comprises a method to screen for a candidate compound
useful to treat cancer comprising: (i) identifying a candidate
compound which regulates the expression of three or more biomarkers
selected from the group consisting of the biomarkers identified in
Table 2; and (ii) determining whether such candidate compound is
effective to treat cancer. In one embodiment, said three or more
biomarkers are selected from the group consisting of the biomarkers
identified in Table 3. In another embodiment, said three or more
biomarkers are selected from the group consisting of leptin,
prolactin, OPN and IGF-II.
[0105] The present invention also comprises methods to screen for
candidate compounds useful to treat cancer. In one embodiment, the
invention comprises a method to screen for a candidate compound
useful to treat cancer comprising: (i) identifying a candidate
compound which regulates the expression of four or more biomarkers
selected from the group consisting of the biomarkers identified in
Table 2; and (ii) determining whether such candidate compound is
effective to treat cancer. In one embodiment, said four or more
biomarkers are selected from the group consisting of the biomarkers
identified in Table 3. In another embodiment, said four or more
biomarkers include leptin, prolactin, OPN and IGF-II.
[0106] As used herein, the term "compound" refers to any chemical
entity, pharmaceutical, drug, and the like that can be used to
treat or prevent a disease, illness, conditions, or disorder of
bodily function. Compounds comprise both known and potential
therapeutic compounds. A compound can be determined to be
therapeutic by screening using the screening methods of the present
invention. Examples of test compounds include, but are not limited
to peptides, polypeptides, synthetic organic molecules, naturally
occurring organic molecules, nucleic acid molecules, and
combinations thereof.
[0107] The above described screening methods can be used to screen
for candidate compounds useful to treat any cancer. In one
embodiment, the method is to screen for candidate compounds useful
to treat ovarian cancer. In another embodiment, the method is to
screen for candidate compounds useful to treat breast cancer. In
another embodiment, the method is to screen for candidate compounds
useful to treat colon cancer. In another embodiment, the method is
to screen for candidate compounds useful to treat cervical
cancer.
VI. Business Methods
[0108] The invention further comprises a method of conducting a
business comprising: (i) obtaining a sample; (ii) detecting the
expression of at least one biomarker in the sample, wherein said
one or more biomarker is selected from the group consisting of the
biomarkers identified in Table 2; and (iii) reporting the results
of such detection. In one embodiment, said one or more biomarkers
are selected from the group consisting of the biomarkers identified
in Table 3. In another embodiment, said one or more biomarkers are
selected from the group consisting of leptin, prolactin, OPN and
IGF-II.
[0109] The invention further comprises a method of conducting a
business comprising: (i) obtaining a sample; (ii) detecting the
expression of leptin, prolactin, OPN and IGF-II; and (iii)
reporting the results of such detection.
VII. General Screening Methods
[0110] The invention also comprises a method to screen for
candidate cancer biomarkers comprising: (i) identifying a group of
biomarkers that are potentially associated with cancer (such as
oncogenes, tumor suppressor genes, growth factor-like genes,
protease-like genes, and protein kinase-like genes); (ii) comparing
the level of expression of the biomarkers identified in step (i) in
a first population of cancer subjects and in healthy subjects;
(iii) selecting biomarkers exhibiting a significant difference in
expression in said first population of cancer subjects; (iv)
comparing the level of expression of the biomarkers identified in
step (iii) in a second population of cancer subjects and in healthy
subjects; and (v) selecting biomarkers exhibiting a significant
difference in expression in said second population of cancer
subjects; wherein the biomarkers identified in step (v) are
candidate cancer biomarkers. The first population of cancer
subjects and the second population of cancer subjects may be any
two cancer populations so long as the two populations are
different. In one embodiment, said first population of cancer
subjects consists of subjects newly diagnosed with cancer, and said
second population of cancer subjects consists of subjects having
recurrent cancer. In another embodiment, said first population of
cancer subjects consists of subjects having later stage cancer and
said second population of cancer subjects consists of subjects
having early stage cancer; or where said first population of cancer
patients consists of subjects having early stage cancer and said
second population of cancer subjects consists of subjects having
later stage cancer.
[0111] A person of skill in the art would be able to identify
biomarkers which are potentially associated with cancer. Such
biomarkers can be selected from the group consisting of as
oncogenes, tumor suppressor genes, growth factor-like genes,
protease-like genes, and protein kinase-like genes.
[0112] In one embodiment, the method further comprises: (vi)
comparing the level of expression of the biomarkers identified in
step (v) in a third population of cancer subjects and in healthy
subjects, wherein the expression of said biomarkers is detected by
using a different assay format; and (vi) selecting biomarkers
exhibiting a significant different in expression in said third
population of cancer patients; wherein the biomarkers identified in
step (vii) are candidate biomarkers for cancer. Thus, for example,
in one embodiment, the expression of said biomarker is first
detected using a high throughput assay, and then detected using an
assay that is specific for the protein in question. For example, in
one embodiment, the expression of said biomarker is first detected
by using RCA microarray immunoassay and then detected by ELISA
assay. The third population of cancer subjects may be the same or
different from the first and second population of cancer
subjects.
[0113] In one embodiment, the method further comprises determining
whether the biomarkers identified in step (v) or (vii) could
distinguish between cancer and healthy subjects in a blind study.
The results of the blind assay can be analyzed using well known
statistical methods.
[0114] The expression of said biomarkers can be compared using any
method known in the art. In one embodiment, the expression of the
biomarkers is detected using protein array, mass spectroscopy, gel
electrophoresis or an immunoassay. In one embodiment, the
expression of the biomarkers is detected using RCA microarray
immunoassay. In another embodiment, the expression of the
biomarkers is measured using ELISA. These methods are well known in
the art.
[0115] The invention also comprise a method to screen for candidate
cancer biomarkers comprising: (i) identifying a cancer biomarker;
(ii) selecting polypeptides which regulate or are regulated by the
biomarker identified in step (i); and (iii) measuring the
expression of the polypeptides identified in step (ii) in cancer
subjects and in healthy subjects, wherein a polypeptide which is
differentially expressed in cancer subjects and in healthy subjects
is a candidate cancer biomarker.
[0116] The above described screening methods can be used to screen
for candidate biomarkers of any cancer. In one embodiment, the
method is to screen for candidate compounds useful to treat ovarian
cancer. In another embodiment, the method is to screen for
candidate biomarkers of breast cancer. In another embodiment, the
method is to screen for candidate biomarkers of colon cancer. In
another embodiment, the method is to screen for candidate
biomarkers of cervical cancer.
EXEMPLIFICATION
Example 1
The Identification of Biomarkers of Ovarian Cancer
[0117] FIG. 1 is a schematic representation of the novel screening
assay used to identify biomarkers of ovarian cancer which can be
used to distinguish subjects with ovarian cancer and healthy
subjects. As shown in FIG. 1, during Phase I of the screening
method, the levels of expression of 169 proteins were measured in
46 serum samples (18 samples were obtained from subjects with
ovarian cancer and 28 samples were obtained from healthy,
age-matched controls) via RCA immunoassay microarray in order to
identify proteins that are differentially expressed in subjects
with ovarian cancer and in healthy subjects.
TABLE-US-00001 TABLE 1 Proteins (analytes) used to screen for
biomarkers of ovarian cancer. (As used herein, the tern "analyte"
refers to a molecule or compound, such as a polypeptide or nucleic
acid, whose presence is to be identified in a sample.) Protein
(abbr.) Protein (full name) Array 1 analytes 1 ANG Angiogenin 2 BLC
(BCA-1) B-lymphocyte chemoattractant 3 EGF Epidermal growth factor
4 ENA-78 Epithelial cell-derived neutrophil-activating peptide 5
Eot Eotaxin 6 Eot-2 Eotaxin-2 7 Fas Fas (CD95) 8 FGF-7 Fibroblast
growth factor-7 9 FGF-9 Fibroblast growth factor-9 10 GDNF Glial
cell line derived neurotrophic factor 11 GM-CSF Granulocyte
macrophage colony stimulating factor 12 IL-1ra Interleukin 1
receptor antagonist 13 IL-2 sR.alpha. Interleukin 2 soluble
receptor alpha 14 IL-3 Interleukin 3 15 IL-4 Interleukin 4 16 IL-5
Interleukin 5 17 IL-6 Interleukin 6 18 IL-7 Interleukin 7 19 IL-8
Interleukin 8 20 IL-13 Interleukin 13 21 IL-15 Interleukin 15 22
MCP-2 Monocyte chemotactic protein 2 23 MCP-3 Monocyte chemotactic
protein 3 24 MIP-1.alpha. Macrophage inflammatory protein 1 alpha
25 MPIF Myeloid progenitor inhibitory factor 1 26 OSM Oncostatin M
27 PlGF Placental growth factor Array 2 analytes 1 AR Amphiregulin
2 BDNF Brain-derived neurotrophic factor 3 Flt-3 Lig fms-like
tyrosine kinase-3 ligand 4 GCP-2 Granulocyte chemotactic protein 2
5 HCC4 (NCC4) Hemofiltrate CC chemokine 4 6 I-309 I-309 7
IL-1.alpha. Interleukin 1 alpha 8 IL-1.beta. Interleukin 1 beta 9
IL-2 Interleukin 2 10 IL-17 Interleukin 17 11 MCP-1 Monocyte
chemotactic protein 1 12 M-CSF Macrophage colony stimulating factor
13 MIG Monokine induced by interferon gamma 14 MIP-1.beta.
Macrophage inflammatory protein 1 beta 15 MIP-1.delta. Macrophage
inflammatory protein 1 delta 16 NT-3 Neurotrophin 3 17 NT-4
Neurotrophin 4 18 PARC Pulmonary and activation-regulated chemokine
19 RANTES Regulated upon activation, normal T expressed and
presumably secreted 20 SCF Stem cell factor 21 sgp130 Soluble
glycoprotein 130 22 TARC Thymus and activation regulated chemokine
23 TNF-RI Tumor necrosis factor receptor I 24 TNF-.alpha. Tumor
necrosis factor alpha 25 TNF-.beta. Tumor necrosis factor beta 26
VEGF Vascular endothelial growth factor Array 3 analytes 1 BTC
Betacellulin 2 DR6 Death receptor 6 3 Fas Lig Fas ligand 4 FGF acid
(FGF-1) Fibroblast growth factor acidic 5 Fractalkine Fractalkine 6
GRO-.beta. Growth related oncogene beta 7 HCC-1 Hemofiltrate CC
chemokine 1 8 HGF Hepatocyte growth factor 9 HVEM Herpes virus
entry mediator 10 ICAM-3 (CD50) Intercellular adhesion molecule 3
11 IGFBP-2 Insulin-like growth factor binding protein 2 12 IL-2
R.gamma. Interleukin 2 receptor gamma 13 IL-5 R.alpha. (CD125)
Interleukin 5 receptor alpha 14 IL-9 Interleukin 9 15 Leptin/OB
Leptin 16 L-Selectin (CD62L) Leukocyte selectin 17 MCP-4 Monocyte
chemotactic protein 4 18 MIP-3.beta. Macrophage inflammatory
protein 3 beta 19 MMP-7 (total) Matrix metalloproteinase 7 20 MMP-9
Matrix metalloproteinase 9 21 PECAM-1 (CD31) Platelet endothelial
cell adhesion molecule-1 22 RANK Receptor activator of NF-kappa-B
23 SCF R Stem cell factor receptor 24 TIMP-1 Tissue inhibitors of
metalloproteinases 1 25 TRAIL R4 TNF-related apoptosis-inducing
ligand receptor 4 26 VEGF-R2 (Flk-1/KDR) Vascular endothelial
growth factor receptor 2 27 ST2 Interleukin 1 receptor 4 Array 4
analytes 1 ALCAM Activated leukocyte cell adhesion molecule 2
.beta.-NGF beta-nerve growth factor 3 CD27 CD27 4 CTACK Cutaneous
T-cell attracting chemokine 5 CD30 CD30 6 Eot-3 Eotaxin-3 7 FGF-2
Fibroblast growth factor-2 (FGF-basic) 8 FGF-4 Fibroblast growth
factor-4 9 Follistatin Follistatin 10 GRO-.gamma. Growth related
oncogene gamma 11 ICAM-1 Intercellular adhesion molecule 1 12
IFN-.gamma. Interferon gamma 13 IFN-.omega. Interferon omega 14
IGF-1R Insulin-like growth factor I receptor 15 IGFBP-1
Insulin-like growth factor binding protein 1 16 IGFBP-3
Insulin-like growth factor binding protein 3 17 IGFBP-4
Insulin-like growth factor binding protein 4 18 IGF-II Insulin-like
growth factor II 19 IL-1 sR1 Interleukin 1 soluble receptor I 20
IL-1 sRII Interleukin 1 soluble receptor II 21 IL-10 R.beta.
Interleukin 10 receptor beta 22 IL-16 Interleukin 16 23 IL-2
R.beta. Interleukin 2 receptor beta 24 I-TAC Interferon
gamma-inducible T cell alpha chemoattractant 25 Lptn Lymphotactin
26 LT .beta.R lymphotoxin-beta receptor 27 M-CSF R Macrophage
colony stimulating factor receptor 28 MIP-3.alpha. Macrophage
inflammatory protein 3 alpha 29 MMP-10 Matrix metalloproteinase 10
30 PDGF R.alpha. Platelet-derived growth factor receptor alpha 31
PF4 Platelet factor-4 32 sVAP-1 Soluble Vascular Adhesion Protein-1
33 TGF-.alpha. Transforming growth factor alpha 34 TIMP-2 Tissue
inhibitors of metalloproteinases 2 35 TRAIL R1 TNF-related
apoptosis-inducing ligand receptor 1 36 VE-cadherin Vascular
Endothelial Cadherin 37 VEGF-D Vascular endothelial growth factor-D
Array 5 analytes 1 4-1BB (CD137) 4-1BB 2 ACE-2 Angiotensin I
converting enzyme-2 3 AFP Alpha fetoprotein 4 AgRP Agouti-related
protein 5 CD141 Thrombomodulin/CD141 6 CD40 CD40 7 CNTF R.alpha.
Ciliary neurotrophic factor receptor alpha 8 CRP C-reactive protein
9 D-Dimer D-Dimer 10 E-Selectin E-selectin 11 HCG Human chorionic
gonadotrophin 12 IGFBP-6 Insulin-like Growth Factor Binding Protein
6 13 IL-12 (p40) Interleukin 12 p40 14 IL-18 Interleukin 18 15 LIF
R.alpha. (gp190) Leukemia inhibitory factor soluble receptor alpha
16 MIF Macrophage migration inhibitory factor 17 MMP-8 (total)
Matrix Metalloproteinase-8 18 NAP-2 Neutrophil Activating Peptide 2
19 Neutrophil elastase Neutrophil elastase 20 PAI-II Plasminogen
activator inhibitor-II 21 Prolactin Prolactin 22 Protein C Human
Protein C 23 Protein S Human Protein S 24 P-Selectin P-Selectin 25
TSH Thyroid stimulating hormone Array 6 analytes 1 6Ckine 6Ckine 2
ACE Angiotensin converting enzyme 3 CA 125 Cancer antigen 125 4
CNTF Ciliary neurotrophic factor 5 Endostatin Endostatin 6
Endothelin 3 Endothelin 3 7 ErbB1 Epidermal growth factor receptor
1 8 ErbB2 Epidermal growth factor receptor 2 9 FGF R3 (IIIc)
Fibroblast growth factor receptor 3 IIIc isoform 10 FGF-6
Fibroblast growth factor-6 11 FGF-R3 (IIIb) Fibroblast growth
factor receptor 3 IIIb isoform 12 G-CSF Granulocyte colony
stimulating factor 13 HB-EGF Heparin-Binding EGF-like Growth Factor
14 IFN-a Interferon alpha 15 LIF Leukemia inhibitory factor 16
MMP-1 Matrix metalloproteinase 1 17 MMP-2 Matrix metalloproteinase
2 18 Osteopontin Osteopontin 19 PAI-1 Plasminogen activator
inhibitor type 1 20 PDGF Rb Platelet-derived growth factor receptor
beta 21 PEDF Pigment epithelium-derived factor 22 sVCAM-1 Soluble
VCAM-1 23 TGF-b RIII Transforming growth factor beta receptor III
24 Tie-2 Tyrosine kinase with Ig and EGF homology domains 2 25 uPA
Urokinase plasminogen activator 26 uPAR Urokinase plasminogen
activator receptor 27 VEGF R3 VEGF receptor 3
[0118] From this group of 169 proteins, 35 proteins were identified
which were differentially expressed between healthy subjects and
subjects with ovarian cancer (p-values less than 0.05 based on
analysis of variance tests (ANOVA)). This data is identified in
Table 2.
TABLE-US-00002 TABLE 2 Proteins showing a significant (p < 0.05)
difference in expression between subjects with ovarian cancer and
healthy subjects. Healthy Ovarian Healthy-Ovarian Subjects Cancer
Cancer Protein Mean Std Dev N Mean Std Dev N Mean Std Dev EffSize
p-value 6Ckine 9.18 0.52 28 9.67 0.69 51 -0.49 0.64 -0.76 0.001813
ACE 12.09 0.43 28 11.67 0.61 51 0.42 0.56 0.76 0.001763 BDNF 13.7
0.92 28 12.82 1.21 51 0.88 1.12 0.79 0.001293 CA125 7.05 0.42 28
11.3 2.45 51 -4.25 1.99 -2.13 <.000001 E-Selectin 13.83 0.62 28
13.4 0.76 51 0.44 0.71 0.61 0.011176 EGF 8.41 1.63 28 10.14 1.57 51
-1.73 1.59 -1.09 0.000015 Eot2 13.12 1.11 28 12.55 1.2 51 0.57 1.17
0.49 0.04228 ErbB1 11.79 0.39 28 11.36 0.55 51 0.44 0.5 0.87
0.000383 Follistatin 10.26 0.63 28 10.76 1.02 51 -0.49 0.9 -0.55
0.0225 HCC4 13.93 0.59 28 14.17 0.45 51 -0.25 0.5 -0.49 0.04178
HVEM 8.33 0.67 28 8.75 0.7 51 -0.42 0.69 -0.61 0.011777 IGF-II
13.53 0.46 28 13.04 0.53 51 0.49 0.51 0.97 0.000094 IGFBP-1 13.24
1.58 28 13.97 1.34 51 -0.73 1.43 -0.51 0.033016 IL-17 8.78 0.56 28
8.24 0.55 51 0.53 0.55 0.96 0.000105 IL-1srII 9.96 0.6 28 9.48 0.69
51 0.48 0.66 0.72 0.002983 IL-2sRa 13.14 0.67 27 13.77 0.57 51
-0.63 0.6 -1.04 0.00004 Leptin 12.77 1.62 27 10.83 2.78 51 1.94
2.44 0.79 0.00134 M-CSF R 12.98 0.35 28 12.78 0.37 51 0.19 0.37
0.53 0.027136 MIF 10.75 0.75 28 11.82 0.75 51 -1.07 0.75 -1.42
<.000001 MIP-1a 6.85 0.69 28 6.45 0.73 51 0.4 0.71 0.56 0.020757
MIP3b 7.55 0.73 28 7.92 0.8 51 -0.37 0.77 -0.48 0.043303 MMP-8
13.92 1.03 28 14.53 0.82 51 -0.61 0.9 -0.68 0.004956 MMP7 11.57
0.48 28 12 0.58 51 -0.43 0.55 -0.79 0.001262 MPIF-1 9.27 0.6 28 9.9
0.7 51 -0.63 0.67 -0.94 0.000155 OPN 12.62 0.79 28 13.81 0.69 51
-1.2 0.73 -1.64 <.000001 PARC 14.21 0.2 28 14.38 0.23 51 -0.17
0.22 -0.78 0.001318 PDGF Rb 10.74 0.97 28 10.13 1.13 50 0.61 1.08
0.56 0.019795 Prolactin 11.01 0.51 28 11.75 1.12 51 -0.74 0.95
-0.78 0.001445 ProteinC 13.59 0.31 28 13.24 0.38 51 0.35 0.36 0.97
0.000089 TGF-b 10.46 1.15 28 11.46 1.12 51 -1 1.13 -0.88 0.000344
RIII TNF-R1 10.14 1.23 28 10.73 1.18 50 -0.59 1.2 -0.5 0.039197
TNF-a 7.06 0.97 28 6.3 0.7 51 0.75 0.8 0.94 0.000152 VAP-1 14.06
0.28 24 13.78 0.65 44 0.29 0.55 0.52 0.042888 VEGF R2 8.84 0.38 28
8.59 0.49 51 0.26 0.46 0.56 0.0189 VEGF R3 10 0.55 28 9.51 0.67 51
0.49 0.63 0.78 0.001388
[0119] The protein (or analytes) identified in Table 2 are also
known by other names, which can be identified by reference to the
full name of the protein as described in Table 1 and by reference
to the published literature. One way of identifying other names for
the proteins identified in Table 2 is by reference to the various
NCBI databases, which include GenBank.
[0120] These 35 proteins were selected for further characterization
with 40 serum samples obtained from subjects with recurrent ovarian
cancer. From this group of 35 proteins, ten (10) biomarkers
exhibited the greatest differences in protein expression between
subjects with recurrent ovarian cancer and healthy subjects. These
10 biomarkers are identified in Table 3.
TABLE-US-00003 TABLE 3 Proteins showing significant difference in
expression between subjects with recurrent ovarian cancer and
healthy subjects. Protein Bonferroni adjusted p-value Prolactin
3.69E-13 MIF 3.61E-06 OPN 0.00001 IGF-II 0.00009 E-Selectin 0.00155
Leptin 0.00249 EGF 0.00382 IL-17 0.00313 MPIF.1 0.00839 IL.2sRa
0.49340
[0121] Of these 10 proteins, some of the proteins that showed the
most potential for differentiating between not only healthy
subjects and subjects newly diagnosed ovarian cancer, but also
between healthy subjects and subjects with recurrent disease, were
assayed using sandwich Enzyme Linked ImmunoSorbent Assay (ELISA) on
a small cohort of 50 subjects (25 cancer subjects with Stage III/IV
ovarian cancer and having an average age of 63.4 years and 25
healthy subjects having an average age of 57 years). Based on ELISA
testing of the original sample set, EGF, TNFa, and IL-17 did not
provide consistent differentiation between the cancer and control
serum samples. MIF-1 was a promising marker but ELISA kits were not
reliably available to continue testing. As shown in FIG. 3, four
proteins showed perfect correlation between the RCA microarray
immunoassays and the ELISA assays. The average concentrations of
the four biomarkers determined for these samples are shown below in
Table 4.
TABLE-US-00004 TABLE 4 Average protein levels for each of the four
biomarkers (specific for the ELISA tests used). Comparison of
Average Protein Levels in Sera of Healthy vs. Ovarian Cancer
Patients Leptin IGF-II (ng/ml) Prolactin (ng/ml) OPN (ng/ml)
(ng/ml) Normal Range [7-50] [0-25] [0-19] [450-2500] Healthy 12 1
11 716 Ovarian Cancer 3 40 49 350
[0122] As determined experimentally above using a specific ELISA
test, the predetermined standard of leptin is 7-50 ng/ml; the
predetermined standard of prolactin is 0-10 pg/ml; the
predetermined standard of OPN is 0.5-19 pg/ml; and the
predetermined standard of IGF-II is 450-2500 ng/ml. A person of
skill in the art would understand that the predetermined standard
concentration of a biomarker may vary from assay to assay depending
on various factors.
[0123] A final panel of four biomarkers (leptin, prolactin, OPN and
IGF-II) were selected for assay in a blind study consisting of 206
serum samples which included samples from 106 healthy subjects and
100 ovarian cancer subjects Stages I-IV. The characteristics of the
subjects used in this blind study are described in Table 5. The
expression of these four biomarkers was determined by ELISA.
TABLE-US-00005 TABLE 5 Disease Status and Ages Of Patient
Population Participating in Blind Study Masked Group (n = 206)
Average Age Healthy Women Healthy 66 58.4 High-risk 40 57.6 Women
with Ovarian Cancer Stage I/II 24 59.5 Stage III/IV 76 63
[0124] To differentiate between subjects with ovarian cancer and
healthy subjects, statistical cluster analysis was performed.
Although none of the four markers could reliably separate the
normal and cancer groups using the least squares fit in a
traditional binary data set analysis (FIG. 4), pair plots of the
four markers showed better separation between subjects these groups
(FIG. 5).
[0125] The combined data for the four biomarkers was analyzed by
four different classifiers: support vector machines (SVM),
k-nearest neighbor classifiers (k-NN), classification trees, and a
score-based classification system to classify samples from healthy
subjects and samples from subjects with ovarian cancer. Support
vector machines (SVM), k-nearest neighbor classifiers (k-NN),
classification trees are hierarchical clustering models.
[0126] FIG. 6 shows the result of the score based classification
system. Particularly, FIG. 6 shows the scores assigned to the 206
subjects who participated in the phase of the screening assay. The
scores were assigned using the following method: For each marker,
the best split point to minimize the number of misclassified
subjects was found. The split point divides the sample space into
two intervals: one for healthy and another for cancer. A score 0 is
assigned to a subject if its related observation falls in the
normal interval; otherwise, a score 1 is assigned. Table 6 shows
the split point for each of the four biomarkers described above.
Overall, an individual is assigned a score as the sum of these
assigned scores from 4 different markers. Thus, in this instance
the range of such score is [0, 4]. FIG. 6 illustrates that subjects
having a score greater than or equal to 2 are likely to have
cancer; and subjects with a score less than or equal to 1 are
likely to be healthy.
TABLE-US-00006 TABLE 6 Scoring Criteria for Biomarkers Biomarker
Split point Left interval Right interval Leptin (1) 2.5 ng/ml
Cancer (1) Normal (0) Prolactin (2) 10 pg/ml Normal (0) Cancer (1)
OPN (3) 21 pg/ml Normal (0) Cancer (1) IGF-II (4) 491 ng/ml Cancer
(1) Normal (0)
[0127] Table 7 gives classification results based on 10-fold
cross-validation for all four classification methods considered.
The results indicated that all the classification methods can well
distinguish normal and cancer groups. The proposed score based
classification method performed better than the nearest neighbor
and classification tree methods. The results from the scoring
method are comparable to those of SVM. The sensitivity of the test
is 96%, specificity 97%, PPV 97% and NPV 96%. The "sensitivity" of
an assay refers to the probability that the test will yield a
positive result in an individual afflicted with ovarian cancer. The
"specificity" of an assay refers to the probability that the test
will yield a negative result in an individual not afflicted with
ovarian cancer. The "positive predictive value" (PPV) of an assay
is the ratio of true positive results (i.e. positive assay results
for patients afflicted with ovarian cancer) to all positive results
(i.e. positive assay results for patients afflicted with ovarian
cancer+positive assay results for patients not afflicted with
ovarian cancer).
TABLE-US-00007 TABLE 7 Classification results based on 10-fold
cross-validation. Classification Method False Positive False
Negative SVM 3/106 4/100 TREE 10/106 7/100 k-NN 6/106 10/100
Score-based 6/106 4/100
[0128] Finally, an additional validation blind study was performed
on forty (40) samples using the score-based classification system
discussed above. This method was able to accurately classify 38 out
of the 40 subjects as having ovarian cancer or not (one sample was
classified as a false positive and another sample was classified as
a false negative).
[0129] Table 8 summarizes the level of four biomarkers identified
herein (leptin, prolactin, ODN and IGF-II) and biomarker CA125 in
subjects having stage I and stage II ovarian cancer who
participated in the screening assays described above (phase IV and
V), as determined by the ELISA assays described herein. (The
patients in bold/italics participated in phase V of the screening
assay described herein.)
TABLE-US-00008 TABLE 8 Expression levels of biomarkers in patients
with Stage I and Stage II ovarian cancer. Patient Code Sample
Description Leptin Prolactin OPN IGF-II CA125 Stage I C4 Stage IC
Rec granulosa cell OVCA 11 45 10 547 14 C69 Stage I OVCA (Cellular
fibroma) 0.2 35 23 484 ND C113 Stage IA OVCA/endo CA 9 70 32 475 6
C114 Stage IA 2 24 22 319 ND Stage II C6 Stage II Bilateral
dysgerminoma(germ-cell 11 63 38 638 51.2 OVCA) C8 Stage II A/11C
OVCA 3 30 28 513 57.3 C9 Stage II C clear cell OVCA 3 81 40 553 981
C16 Stage II C malig steroid cell OVCA 1 36 106 381 15 C19 Stage II
C Papillary serous OVCA 2 10 86 193 C24 Stage II C Papillary serous
OVCA 0 37 21 364 122 C328 Stage II C Papillary serous OVCA 1 48 10
302 977 C48 Stage II Bilateral dysgerminoma(germ-cell 10 37 42 894
125 OVCA) C59 Stage II A/II C OVCA 1 12 16 421 57.3 C62 Stage II C
OVCA 0.2 35 23 484 ND C63 Stage II C OVCA 3 81 40 553 981 C70 Stage
II C borderline OVCA 1 84 21 543 119 C71 Stage II Small cell OVCA 0
28 217 303 ND C77 Stage II B serous cell OVCA 5 77 81 230 99.2 C89
Stage II A OVCA 12 16 13 431 405 C102 Stage II C OV Adenocarcinoma
9 1 32 634 C103 Stage II osteogenic sarcoma OVCA 9 27 51 371 46.9
C117 Stage II B/L Stage 11C Papillary serous 0 119 48 260 634 OVCA
C120 Stage II C OVCA 0 57 41 124 99 C135 Stage II C OVCA 0 78 29
501 173
Materials and Methods Used in Example 1
[0130] Microarray Manufacture: Microarrays were prepared according
to Schweitzer et al., Nat Biotech (2002) 20:359. In short, glass
slides were cleaned and derivatized with
3-cyanopropyltriethoxysilane. The slides were equipped with a
Teflon mask, which divided the slide into sixteen 0.65 cm diameter
wells or circular analysis sites called subarrays (FIG. 2).
Printing was performed with a Perkin-Elmer SpotArray Enterprise
non-contact arrayer equipped with piezoelectric tips, which
dispense a droplet (.about.350 pL) for each microarray spot.
Antibodies were applied at a concentration of 0.5 mg/mL at defined
positions. Each chip was printed with sixteen copies of one type of
array, either array 1, 2, 3, 4, 5 or 6. (See Table 1.) A set of
antibodies was printed with quadruplicate spots in each subarray.
After printing, chips were inspected using light microscopy. If the
percentage of missing spots observed was greater than 5%, then the
batch failed and the slides were discarded immediately. For all
print runs described herein, 100% of the antibody features and
>95% of the biotin calibrators were printed. Microarray chips
were validated in concert with a set of qualified reagents in two
ways. First, mixtures of 1-3 different cytokines were prepared so
as to provide a high intensity signal and applied to 14 wells on a
chip (each well treated with a different mixture up to the total
complement of detector antibodies) and two arrays were used as
blank controls. The chips were developed and scanned and the
resulting signals were compared to the positional map of the
particular array. Second, a titration QC for all analytes of a
specified array using known sample matrices was performed. Normal
human serum and heparinized plasma were assayed neat or spiked with
purified recombinant cytokines representing all analytes in the
array. Spiked mixtures were then titrated down the subarrays of a
slide from 9,000 pg/mL to 37 pg/mL of spiked cytokine
concentrations along with two subarrays for each un-spiked control
sample. The data was quantified and for every analyte in the array
a titration curve was generated to examine feature intensity
behavior as a function of concentration. Taken together, this data
was used to confirm the activity of array features and reagent
sets.
[0131] RCA Microarrray Immunoassay:
[0132] Prior to assay, the slides were removed from storage at room
temperature in sealed containers and opened in a humidity
controlled chamber (45-55%). Slides were blocked with Seablock
(Pierce Chemical Co.), diluted 1:1 with PBS for 1 h at 37.degree.
C. in a humidified chamber. Following removal of the blocking
solution, they were washed twice with 1.times.PBS/0.5% Brij 35
prior to application of sample. Four controls were included on each
sample slide with feature concentrations corresponding to four
anchor points on the full titration curve. The test samples were
assayed on the remaining 12 subarrays. Twenty .mu.L of the treated
sample were then applied to each subarray. The basics of performing
immunoassays with RCA signal amplification has been described
(Schweitzer et al., Nat. Biotechnol. (2002) 20:359-65) and we are
using SOPs derived from the protocols used in that study. Slides
were scanned using a LS200 scanner (PECAN). Scanned images were
analyzed using proprietary software. The fluorescence intensity of
microarray spots was analyzed for each feature and sample, and the
resulting mean intensity values were determined. Dose-response
curves for selected cytokines were examined, ensuring that feature
intensity is above background and exhibiting increasing intensity
with increasing analyte concentration.
[0133] Subject Population for RCA Microarray Immunoassay:
[0134] For the RCA microarray immunoassays the serum from 86
subjects was assayed. Of the 86 subjects, 28 were healthy and had
an average age of 60.8 years, 58 had Stage III/IV ovarian cancer
and had an average age of 57.1. Of the 58 patients with Stage
III/IV ovarian cancer, 18 were newly diagnosed and the remaining 40
were subjects with recurrent disease.
[0135] Subject Population for Blind Study (ELISA):
[0136] For the panel of ELISA, serum samples were collected from
100 subjects with ovarian cancer and 106 healthy/disease-free or
high-risk subjects (as part of the Yale New Haven Hospital Early
Detection program (HIC 10425). The normal group consisted of 66
healthy/disease-free (including the 28 healthy samples sent for the
arrays) and 40 women considered to be at high-risk. Of the 100
ovarian cancer patients, 24 women were diagnosed with Stage I/II
and 76 women with Stage III/IV EOC. Included in this group were the
18 newly diagnosed OVCA samples. Serum from the
healthy/disease-free group served as baseline values or "normal
range" values for the presence of carcinoma.
[0137] Sera Collection:
[0138] 10 ml of blood was collected, centrifuged at 1500 rpm for 10
min and the serum stored at -80.degree. C. in the OB/GYN Tissue
bank until further use. Collection, preparation and storage of the
blood samples were done utilizing guidelines set by the NCI
Intergroup Specimen Banking Committee. Consent for participation in
this study was obtained by qualified personnel. Before analyses,
the sera was thawed once and 8 (25-50 ul) aliquots stored at
-80.degree. C. to ensure unnecessary freezing and thawing.
[0139] ELISA Assay:
[0140] The leptin, prolactin and IGF-II kits were purchased from
Diagnostic Systems Laboratories, Inc. Webster, Tex. and the OPN
(Osteopontin) from Assay Designs, Inc. (Ann Arbor, Mich.). Assays
were performed following kit instructions, and the results were
read on a Spectra Max M2 Microplate Reader (Molecular Devices,
Sunnyvale Calif.) set to a dual wavelength of 405 nM with the
appropriate correction for each assay. Three classifiers: support
vector machines (SVM), K-nearest neighbor classifiers (k-NN) and
classification trees were used to analyze results (distinguish the
healthy/disease/free from the ovarian cancer patients).
[0141] Statistical Analysis:
[0142] Analysis of variance (ANOVA) was used to test the
significance of the protein expression differences detected by RCA
microarray immunoassays between subjects with ovarian cancer and
healthy subjects, using the GLM procedure of SAS. Reported effect
size measures the difference in mean between two groups, normalized
by within group standard deviation, and is independent of the
sample size:
Effect Size=(Mean_Group1-Mean_Group2)/Std_Group1_Group2
[0143] Effect size has a direct association with the predictive
ability of a particular variable. Table 9 shows conversions of
effect sizes (column 1) to probability (column 2). The example
presented in Table 9 is intended to demonstrate the relationship
between effect size and predictive ability. For example, with an
effect size of 0.3 observed between the two groups, the probability
of correctly identifying the groups is 0.56. With an effect size of
1, the probability increases to 0.69.
TABLE-US-00009 TABLE 9 Effect size as the measure of predictive
ability. Probability that grouping could be correctly Effect Size
assigned based on protein expression 0 0.5 0.1 0.52 0.2 0.54 0.3
0.56 0.4 0.58 0.5 0.6 0.6 0.62 0.7 0.64 0.8 0.66 0.9 0.67 1 0.69
1.2 0.73 1.4 0.76 1.6 0.79 1.8 0.82 2 0.84 2.5 0.89 3 0.93
[0144] To differentiate between normal/high risk and ovarian cancer
patients, statistical cluster analysis was performed on 4 protein
expression markers obtained from patient serum. Three commonly used
classification methods were used: support vector machine (SVM),
k-nearest neighbors (kNN), and classification trees (Hastie, et al.
2001). We used 10-fold cross validation to evaluate the
classification accuracy.
[0145] In addition to these three classification methods, we used a
score-based classification method that is more biologically
interpretable. The score-based classification system can be carried
out as follows: (i) For each marker, find the best split point to
minimize the number of misclassified subjects. The split point
defines two intervals: one for normal and another for cancer. A
score of 0 is assigned to a subject if its related observation
falls in the normal interval; otherwise, a score of 1 is assigned.
(ii) Overall, a subject is assigned a score as the sum of these
assigned scores from m different markers. Therefore, the range of
such score is between 0 and m. (iii) A given threshold (t) is used
to predict the disease status for a given subject, e.g. a given
subject with a total score equal or less than t is predicted to
have normal status, whereas a subject with a score higher than t
will be diagnosed to have disease.
[0146] The "split point" described above in connection to the
described score based classification system may be identified as
follows: Suppose there are n samples classified into two groups.
For each marker X, let x.sub.--1, x.sub.--2, . . . , x_n be the
observed measurements. We screen (n-1) split points y.sub.--1,
y.sub.--2, . . . , y_(n-1), where y_k=0.5*(x_k+x_(k+1)) for k=1, 2,
. . . , n-1. For each split point y_k, there are a.sub.--1 and
a.sub.--2 observed measurements less than y_k in the first and the
second groups, respectively; and there are b.sub.--1 and b.sub.--2
observed measurements greater than y_k in the first and the second
groups, respectively. If the left and the right sides of y_k are
assigned to the first and the second groups, respectively, then
there are a.sub.--2 and b.sub.--1 misclassified samples. If the
left and the right sides of y_k are assigned to the second and the
first groups, respectively, then there are a.sub.--1 and b.sub.--2
misclassified samples. We choose the assignment that minimizes the
number of misclassified samples.
Discussion
[0147] Ovarian cancer is a "relatively silent" disease with
intra-abdominal inaccessibility which makes the monitoring and
early detection of the disease utilizing a non-invasive approach
such as serum tumor markers an attractive idea. A simple, reliable,
reproducible and rapid screening strategy with adequate sensitivity
for early detection is needed to improve our ability to accurately
detect pre-malignant change or early stage ovarian cancer in
asymptomatic women at increased risk for the development of ovarian
cancer. It has been suggested that, in order to be acceptable, any
screening strategy for early detection must achieve a minimum of
99.6% specificity, hence the need for a combined regimen of tests
since it is unlikely that any single analyte test will be that
specific. In fact, given the rarity of ovarian cancer, very low
levels of false positive classification will result in a large
number of women being incorrectly classified as potentially ovarian
cancer patients if biomarker screening tests are used as the only
means of classification. We assert that initial serum screening for
a combination of analytes, followed by transvaginal ultrasound and
mammography or thermal breast imaging, should provide a
sufficiently low false positive rate to justify subsequent
laparoscopic surgery on individuals with detectable pelvic masses
to validate the results of the diagnostic assay. This approach is
supported by results of studies in which the combination of CA125
and transvaginal ultrasound detected a significant proportion of
preclinical ovarian cancers (Jacobs, Mol Cell Proteomics
3(4):355-366 (2004).
[0148] Our approach to identify serum biomarkers was based on a
strategy of screening multiple serum proteins by high-throughput
microarray analysis to identify biomarkers that had the potential
to accurately discriminate between healthy/high-risk and cancer and
still have the sensitivity to detect early stage I and II ovarian
cancers. Based on the microarray results, a promising subset of
biomarkers were selected for further analysis by ELISA. Four of the
biomarkers selected based on the microarray data were confirmed as
useful for early detection and a high level of
sensitivity/specificity using ELISA analysis. Initial confirmation
of the utility of biomarkers does not require many analyses if high
specificity and sensitivity is sought. Biomarkers can be eliminated
based on analysis of 15-20 normal and patient samples once quality
control of the ELISA in the hands of the technician performing the
assay is established. Analysis of a larger number of samples is
required for application of statistical techniques (such as SVM,
k-NN and Tree), to determine sensitivity/specificity, split-points
and combinatorial strategies. After validating the contribution
that each biomarker may contribute to a combined assay, split
points for each biomarker are defined and the utility of
combinations of two or more markers are explored statistically.
Using split points the status of each biomarker is assigned as a
binary result (normal versus abnormal levels). The number of
biomarkers classified as abnormal is used to define an individual
as having or not having cancer; in this case, individuals with
three or four biomarkers that are abnormal are classified as having
ovarian cancer while subjects with two or fewer biomarkers that are
abnormal are classified as not having cancer. The biomarkers as
individual analytes may not have sufficient sensitivity and
specificity, rather the combination of biomarkers may be required
for a diagnostic test.
Example 2
The Used of the Biomarkers Identified in Ovarian Cancer Patients to
Diagnose Breast Cancer and Colon Cancer
[0149] Certain samples were analyzed to determined whether the
biomarkers identified above (leptin, prolactin, OPN and IGF-II)
were differentially expressed in other types of cancer. The
results, shown in Table 9, indicate that the biomarkers identified
above can be used to diagnose other types of cancer including
breast cancer and colon cancer.
[0150] As shown in Table 10, samples corresponding to subjects with
cancer could be distinguished from samples from healthy subjects by
the differential expression of two or more biomarkers as compared
to their predetermined standard. In Table 10, the expression levels
indicated in italics and bold corresponds to expression levels
outside of the predetermined standard for said biomarker.
TABLE-US-00010 TABLE 10 Analysis of the expression of leptin,
prolactin, OPN and IGF-II in breast cancer and colon cancer.
Patient Code Sample Description Leptin (ng/ml) Prolactin (pg/ml)
OPN (pg/ml) IGF-II (ng/ml) CA125 Breast Cancer C51 Stage I Breast
Cancer 2/8/00 @ Age 42 540 ND C57 Fibrocystic breast mass (No
Breast Cancer) 3 0 8.3 C66 Mesothelial cysts/Breast Cancer (ductal)
2001/ 460 ND Tamox C92 Stage I Breast Cancer 2/8/00 @ Age 42 1 780
ND C29 Stage IV Breast Cancer 1 ND Colon Cancer C107 Stage 1V Colon
Cancer/OVCA ND C52 Stage 111 Colon Cancer/OV Cysts 592 ND
INCORPORATION BY REFERENCE
[0151] All of the publications cited herein are hereby incorporated
by reference in their entirety to describe more fully the art to
which the application pertains.
EQUIVALENTS
[0152] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *