U.S. patent application number 13/372824 was filed with the patent office on 2012-08-02 for biomarkers and methods for determining sensitivity to epidermal growth factor receptor modulators.
This patent application is currently assigned to Bristol-Myers Squibb Company. Invention is credited to Edwin A. Clark, Shirin K. Ford, Xin Huang.
Application Number | 20120195889 13/372824 |
Document ID | / |
Family ID | 37772388 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120195889 |
Kind Code |
A1 |
Ford; Shirin K. ; et
al. |
August 2, 2012 |
BIOMARKERS AND METHODS FOR DETERMINING SENSITIVITY TO EPIDERMAL
GROWTH FACTOR RECEPTOR MODULATORS
Abstract
EGFR biomarkers useful in a method for predicting the likelihood
that a mammal that will respond therapeutically to a method of
treating cancer comprising administering an EGFR modulator, wherein
the method comprises (a) measuring in the mammal the level of at
least one biomarker selected from epiregulin and amphiregulin, (b)
exposing a biological sample from the mammal to the EGFR modulator,
and (c) following the exposing of step (b), measuring in the
biological sample the level of the at least one biomarker, wherein
an increase in the level of the at least one biomarker measured in
step (c) compared to the level of the at least one biomarker
measured in step (a) indicates an increased likelihood that the
mammal will respond therapeutically to the method of treating
cancer.
Inventors: |
Ford; Shirin K.; (Princeton,
NJ) ; Clark; Edwin A.; (Hopkinton, MA) ;
Huang; Xin; (Princeton, NJ) |
Assignee: |
Bristol-Myers Squibb
Company
|
Family ID: |
37772388 |
Appl. No.: |
13/372824 |
Filed: |
February 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11990713 |
Feb 20, 2008 |
8129114 |
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PCT/US2006/033073 |
Aug 24, 2006 |
|
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13372824 |
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60711054 |
Aug 24, 2005 |
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Current U.S.
Class: |
424/133.1 ;
424/138.1; 435/6.11; 506/9 |
Current CPC
Class: |
C12Q 2600/136 20130101;
C12Q 2600/106 20130101; A61P 35/00 20180101; G01N 2333/485
20130101; G01N 2333/71 20130101; G01N 33/57419 20130101; G01N
2333/70596 20130101; G01N 2800/52 20130101; C12Q 1/6886
20130101 |
Class at
Publication: |
424/133.1 ;
506/9; 435/6.11; 424/138.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/68 20060101 C12Q001/68; A61P 35/00 20060101
A61P035/00; C40B 30/04 20060101 C40B030/04 |
Claims
1-10. (canceled)
11. A method for predicting the likelihood a colorectal cancer
patient will respond therapeutically to a method of treating
colorectal cancer with a therapy that comprises administering an
anti-EGFR antibody that inhibits binding of EGF to EGFR, wherein
the method for predicting comprises measuring the mRNA expression
level of both epiregulin and amphiregulin biomarkers in a
colorectal cancer sample of said patient, wherein an elevated level
of said biomarkers in a colorectal cancer sample relative to a
predetermined level of said biomarkers indicates an increased
likelihood said patient will respond therapeutically to said method
of treating colorectal cancer.
12. The method of claim 11 further comprising the step of measuring
at least one additional biomarker selected from Table 1.
13. The method of claim 11 wherein said colorectal cancer sample is
a tissue sample comprising colorectal cancer cells and said tissue
is fixed; paraffin-embedded; fixed and paraffin-embedded;
formalin-fixed and paraffin-embedded; formaldehyde-fixed and
paraffin-embedded; in fresh, or frozen.
14. The method according to claim 11 wherein said mRNA expression
measurement is performed using a method selected from the group
consisting of: (a) PCR; (b) RT-PCR; (c) microarray; (d)
immunohistochemistry; (e) in situ hybridization; (f) array
hybridization; (g) Northern blot; (h) dot-blot; and (i) RNAse
protection assay.
15. The method of claim 11 further comprising the step of
determining whether said colorectal cancer sample has the presence
of a mutated K-RAS, wherein detection of a mutated K-RAS indicates
a decreased likelihood said patient will respond therapeutically to
said method of treating colorectal cancer.
16. The method of claim 11 further comprising the step of
determining whether said colorectal cancer sample has the presence
of wild-type K-RAS, wherein detection of wild-type K-RAS indicates
an increased likelihood said patient will respond therapeutically
to said method of treating colorectal cancer.
17. The method according to claim 11, wherein said anti-EGFR
antibody is selected from the group consisting of: a monoclonal,
polyclonal or single chain antibody.
18. The method of claim 11, wherein said anti-EGFR antibody is
cetuximab.
19. The method according to claim 11, wherein said anti-EGFR
antibody is panitumumab.
20. The method according to claim 11, further comprising the step
of administering said anti-EGFR antibody to said patient if the
level of said biomarkers is increased relative to a predetermined
level of said biomarkers in a colorectal cancer sample, wherein
said anti-EGFR antibody inhibits binding of EGF to EGFR.
21. The method according to claim 18, further comprising the step
of administering said anti-EGFR antibody to said patient if the
level of said biomarkers is increased relative to a predetermined
level of said biomarkers in a colorectal cancer sample, wherein
said anti-EGFR antibody inhibits binding of EGF to EGFR.
22. The method according to claim 19, further comprising the step
of administering said anti-EGFR antibody to said patient if the
level of said biomarkers is increased relative to a predetermined
level of said biomarkers in a colorectal cancer sample, wherein
said anti-EGFR antibody inhibits binding of EGF to EGFR.
23. A method for predicting the likelihood a colorectal cancer
patient will respond therapeutically to a method of treating
colorectal cancer with an EGFR modulator that inhibits binding of
EGF to EGFR, comprising: (a) measuring the mRNA expression level of
both epiregulin and amphiregulin biomarkers in a colorectal cancer
sample of said patient; and (b) administering a therapy comprising
cetuximab to said patient if said measuring step indicates said
patient has an elevated level of said biomarkers in said colorectal
cancer sample relative to a predetermined level.
24. A method for predicting the likelihood a colorectal cancer
patient will respond therapeutically to a method of treating
colorectal cancer with an EGFR modulator that inhibits binding of
EGF to EGFR, comprising: (a) measuring the mRNA expression level of
both epiregulin and amphiregulin biomarkers in a colorectal cancer
sample of said patient; and (b) administering a therapy comprising
panitumumab to said patient if said measuring step indicates said
patient has an elevated level of said biomarkers in said colorectal
cancer sample relative to a predetermined level.
Description
SEQUENCE LISTING
[0001] A compact disc labeled "Copy 1" contains the Sequence
Listing as 10646 PCT.ST25.txt. The Sequence Listing is 1241 KB in
size and was recorded Aug. 24, 2006. The compact disk is 1 of 2
compact disks. A duplicate copy of the compact disc is labeled
"Copy 2" and is 2 of 2 compact discs.
[0002] The compact disc and duplicate copy are identical and are
hereby incorporated by reference into the present application.
FIELD OF THE INVENTION
[0003] The present invention relates generally to the field of
pharmacogenomics, and more specifically to methods and procedures
to determine drug sensitivity in patients to allow the
identification of individualized genetic profiles which will aid in
treating diseases and disorders.
BACKGROUND OF THE INVENTION
[0004] Cancer is a disease with extensive histoclinical
heterogeneity. Although conventional histological and clinical
features have been correlated to prognosis, the same apparent
prognostic type of tumors varies widely in its responsiveness to
therapy and consequent survival of the patient.
[0005] New prognostic and predictive markers, which would
facilitate an individualization of therapy for each patient, are
needed to accurately predict patient response to treatments, such
as small molecule or biological molecule drugs, in the clinic. The
problem may be solved by the identification of new parameters that
could better predict the patient's sensitivity to treatment. The
classification of patient samples is a crucial aspect of cancer
diagnosis and treatment. The association of a patient's response to
a treatment with molecular and genetic markers can open up new
opportunities for treatment development in non-responding patients,
or distinguish a treatment's indication among other treatment
choices because of higher confidence in the efficacy. Further, the
pre-selection of patients who are likely to respond well to a
medicine, drug, or combination therapy may reduce the number of
patients needed in a clinical study or accelerate the time needed
to complete a clinical development program (Cockett et al., Current
Opinion in Biotechnology, 11:602-609 (2000)).
[0006] The ability to predict drug sensitivity in patients is
particularly challenging because drug responses reflect not only
properties intrinsic to the target cells, but also a host's
metabolic properties. Efforts to use genetic information to predict
drug sensitivity have primarily focused on individual genes that
have broad effects, such as the multidrug resistance genes, mdr1
and mrp1 (Sonneveld, J. Intern. Med., 247:521-534 (2000)).
[0007] The development of microarray technologies for large scale
characterization of gene mRNA expression pattern has made it
possible to systematically search for molecular markers and to
categorize cancers into distinct subgroups not evident by
traditional histopathological methods (Khan et al., Cancer Res.,
58:5009-5013 (1998); Alizadeh et al., Nature, 403:503-511 (2000);
Bittner et al., Nature, 406:536-540 (2000); Khan et al., Nature
Medicine, 7(6):673-679 (2001); and Golub et al., Science,
286:531-537 (1999); Alon et al., P. N. A. S. USA, 96:6745-6750
(1999)). Such technologies and molecular tools have made it
possible to monitor the expression level of a large number of
transcripts within a cell population at any given time (see, e.g.,
Schena et al., Science, 270:467-470 (1995); Lockhart et al., Nature
Biotechnology, 14:1675-1680 (1996); Blanchard et al., Nature
Biotechnology, 14:1649 (1996); U.S. Pat. No. 5,569,588).
[0008] Recent studies demonstrate that gene expression information
generated by microarray analysis of human tumors can predict
clinical outcome (van't Veer et al., Nature, 415:530-536 (2002);
Sorlie et al., P. N. A. S. USA, 98:10869-10874 (2001); M. Shipp et
al., Nature Medicine, 8(1):68-74 (2002): Glinsky et al., The
Journal of Clin. Invest., 113(6):913-923 (2004)). These findings
bring hope that cancer treatment will be vastly improved by better
predicting the response of individual tumors to therapy.
[0009] The epidermal growth factor receptor (EGFR) and its
downstream signaling effectors, notably members of the Ras/Raf/MAP
kinase pathway, play an important role in both normal and malignant
epithelial cell biology (Normanno et al., Gene 366, 2-16 (2006))
and have therefore become established targets for therapeutic
development. Whereas the anti-EGFR antibody cetuximab and the EGFR
small molecular tyrosine kinase inhibitors (TKIs) gefitinib and
erlotinib have demonstrated activity in a subset of patients
(Baselga and Arteaga, J. Clin. Oncol. 23, 2445-2459 (2005)), their
initial clinical development has not benefited from an accompanying
strategy for identifying the patient populations that would most
likely derive benefit. The hypothesis that only a relatively small
number of tumors are "EGFR-pathway dependent" and therefore likely
to respond to EGFR inhibitors might explain the limited clinical
activity that is observed with this class of therapeutics. For
example, in patients with refractory metastatic colorectal cancer
clinical response rates with cetuximab consistently range from 11%
in a monotherapy setting to 23% in a combination setting with
chemotherapy (Cunningham et al., N. Engl. J. Med 351, 337-345
(2004)). To date, significant efforts have been focused on
elucidating the mechanisms of sensitivity or resistance to EGFR
inhibition, particularly through evaluation of EGFR protein
expression, kinase domain mutations, and gene copy number.
[0010] While relative protein expression of the EGFR as measured by
immunohistochemistry (IHC) has been demonstrated in many solid
tumors (Ciardiello and Tortora, Eur. J. Cancer 39, 1348-1354
(2003)), no consistent association between EGFR expression and
response to EGFR inhibitors has been established. Clinical studies
of cetuximab in a monotherapy setting and in combination with
irinotecan in patients with mCRC failed to reveal an association
between radiographic response and EGFR protein expression as
measured by IHC (Cunningham et al., N. Engl. J. Med 351, 337-345
(2004); Saltz et al., J. Clin. Oncol. 22, 1201-1208 (2004)).
Furthermore, clinical responses have been demonstrated in patients
with undetectable EGFR protein expression (Chung et al., J. Clin.
Oncol., 23, 1803-1810 (2005); Lenz et al., Activity of cetuximab in
patients with colorectal cancer refractory to both irinotecan and
oxaliplatin. Paper presented at: 2004 ASCO Annual Meeting
Proceedings; Saltz, Clin Colorectal Cancer, 5 Suppl. 2, S98-100
(2005)). In comparison, clinical studies of erlotinib in NSCLC
patients and gefitinib in ovarian cancer did demonstrate an
association between EGFR expression, response, and survival
(Schilder et al., Clin. Cancer Res., 11, 5539-5548 (2005); Tsao et
al., N. Engl. J. Med., 353, 133-144 (2005)). The presence of
somatic mutations in the tyrosine kinase domain, particularly in
NSCLC has been extensively described (Janne et al., J. Clin.
Oncol., 23, 3227-3234 (2005)). In both preclinical and clinical
settings, these mutations are found to correlate with sensitivity
to gefitinib and erlotinib but not to cetuximab (Janne et al., J.
Clin. Oncol., 23, 3227-3234 (2005); Tsuchihashi et al., N. Engl. J.
Med., 353, 208-209 (2005)). In addition, the lack of EGFR kinase
domain mutations in CRC patients suggests that such mutations do
not underlie the response to cetuximab. EGFR gene copy number has
also been evaluated as a potential predictor of response to EGFR
inhibitors. Clinical studies of gefitinib demonstrated an
association between increased EGFR copy number, mutational status,
and clinical response (Cappuzzo et al., J. Natl. Cancer Inst., 97,
643-655 (2005)). A similar association was identified in a small
number of patients treated with the anti-EGFR monoclonal antibodies
cetuximab and panitumumab (Moroni et al., Lancet Oncol., 6, 279-286
(2005)). Additional potential predictive biomarkers have also been
evaluated. For example, in glioblastoma patients, a significant
association between co-expression of EGFRvIII and PTEN and response
to EGFR small molecule inhibitors was found (Mellinghoff et al., N.
Engl. J. Med., 353, 2012-2024 (2005)).
[0011] The anti-tumor activity of cetuximab has been attributed to
its ability to block EGFR ligand binding and ligand-dependent EGFR
activation. Clinical activity of cetuximab has been shown in
multiple epithelial tumor types (Bonner et al., N. Engl. J. Med.,
354, 567-578 (2006); Cunningham et al., N. Engl. J. Med., 351,
337-345 (2004)), however responses continue to be seen in only a
fraction of patients. Previous attempts to identify predictors of
sensitivity or resistance as described above have focused on
specific biomarkers rather than using genomic discovery approaches.
In addition, RNA-, DNA- and protein-based markers have rarely been
examined in the same patient population in a single study, making
comparisons challenging.
[0012] Biomarkers useful for determining sensitivity to EGFR
modulators have been described in published PCT applications
WO2004/063709, WO2005/067667, and WO2005/094332.
[0013] Needed are new and alternative methods and procedures to
determine drug sensitivity in patients to allow the development of
individualized genetic profiles which are necessary to treat
diseases and disorders based on patient response at a molecular
level.
SUMMARY OF THE INVENTION
[0014] The invention provides methods and procedures for
determining patient sensitivity to one or more Epidermal Growth
Factor Receptor (EGFR) modulators. The invention also provides
methods of determining or predicting whether an individual
requiring therapy for a disease state such as cancer will or will
not respond to treatment, prior to administration of the treatment,
wherein the treatment comprises administration of one or more EGFR
modulators. The one or more EGFR modulators are compounds that can
be selected from, for example, one or more EGFR-specific ligands,
one or more small molecule EGFR inhibitors, or one or more EGFR
binding monoclonal antibodies.
[0015] In one aspect, the invention provides a method for
predicting the likelihood a mammal will respond therapeutically to
a method of treating cancer comprising administering an EGFR
modulator, wherein the method comprises: (a) measuring in the
mammal the level of at least one biomarker selected from epiregulin
and amphiregulin; (b) exposing a biological sample from the mammal
to the EGFR modulator; (c) following the exposing of step (b),
measuring in the biological sample the level of the at least one
biomarker, wherein an increase in the level of the at least one
biomarker measured in step (c) compared to the level of the at
least one biomarker measured in step (a) indicates an increased
likelihood that the mammal will respond therapeutically to the
method of treating cancer. In one aspect, the at least one
biomarker comprises epiregulin and amphiregulin. In yet another
aspect, the at least one biomarker further comprises at least one
additional biomarker selected from Table 1. In another aspect, the
biological sample is a tissue sample comprising cancer cells and
the method further comprises the step of determining whether the
cancer cells have the presence of a mutated K-RAS, wherein
detection of a mutated K-RAS indicates a decreased likelihood that
that the mammal will respond therapeutically to the method of
treating cancer.
[0016] The biological sample can be, for example, a tissue sample
comprising cancer cells and the tissue is fixed, paraffin-embedded,
fresh, or frozen.
[0017] In another aspect, the EGFR modulator is cetuximab and the
cancer is colorectal cancer.
[0018] In another aspect, the invention is a method for predicting
the likelihood a mammal will respond therapeutically to a method of
treating cancer comprising administering an EGFR modulator, wherein
the method comprises: (a) measuring in the mammal the level of at
least one biomarker that comprises CD73; (b) exposing a biological
sample from the mammal to the EGFR modulator; (c) following the
exposing of step (b), measuring in the biological sample the level
of the at least one biomarker, wherein an increase in the level of
the at least one biomarker measured in step (c) compared to the
level of the at least one biomarker measured in step (a) indicates
a decreased likelihood that the mammal will respond therapeutically
to the method of treating cancer. In another aspect, the at least
one biomarker further comprises at least one additional biomarker
selected from Table 1. In another aspect, the method further
comprises the step of determining whether the cancer cells have the
presence of a mutated K-RAS, wherein detection of a mutated K-RAS
indicates a decreased likelihood that that the mammal will respond
therapeutically to the method of treating cancer.
[0019] A difference in the level of the biomarker that is
sufficient to predict the likelihood that the mammal will or will
not respond therapeutically to the method of treating cancer can be
readily determined by one of skill in the art using known
techniques. The increase or decrease in the level of the biomarker
can be correlated to determine whether the difference is sufficient
to predict the likelihood that a mammal will respond
therapeutically. The difference in the level of the biomarker that
is sufficient can, in one aspect, be predetermined prior to
predicting the likelihood that the mammal will respond
therapeutically to the treatment. In one aspect, the difference in
the level of the biomarker is a difference in the mRNA level
(measured, for example, by RT-PCR or a microarray), such as at
least a two-fold difference, at least a three-fold difference, or
at least a four-fold difference in the level of expression. In
another aspect, the difference in the level of the biomarker is
determined by IHC. In another aspect, the difference in the level
of the biomarker refers to a p-value of <0.05 in Anova (t test)
analysis. In yet another aspect, the difference is determined in an
ELISA assay.
[0020] As used herein, respond therapeutically refers to the
alleviation or abrogation of the cancer. This means that the life
expectancy of an individual affected with the cancer will be
increased or that one or more of the symptoms of the cancer will be
reduced or ameliorated. The term encompasses a reduction in
cancerous cell growth or tumor volume. Whether a mammal responds
therapeutically can be measured by many methods well known in the
art, such as PET imaging.
[0021] The mammal can be, for example, a human, rat, mouse, dog,
rabbit, pig sheep, cow, horse, cat, primate, or monkey.
[0022] The method of the invention can be, for example, an in vitro
method wherein the step of measuring in the mammal the level of at
least one biomarker comprises taking a biological sample from the
mammal and then measuring the level of the biomarker(s) in the
biological sample. The biological sample can comprise, for example,
at least one of serum, whole fresh blood, peripheral blood
mononuclear cells, frozen whole blood, fresh plasma, frozen plasma,
urine, saliva, skin, hair follicle, bone marrow, or tumor
tissue.
[0023] The level of the at least one biomarker can be, for example,
the level of protein and/or mRNA transcript of the biomarker. The
level of the biomarker can be determined, for example, by RT-PCR or
another PCR-based method, immunohistochemistry, proteomics
techniques, or any other methods known in the art, or their
combination.
[0024] In another aspect, the invention provides a method for
identifying a mammal that will respond therapeutically to a method
of treating cancer comprising administering of an EGFR modulator,
wherein the method comprises: (a) measuring in the mammal the level
of at least one biomarker selected from the biomarkers of Table 1;
(b) exposing a biological sample from the mammal to the EGFR
modulator; (c) following the exposing in step (b), measuring in
said biological sample the level of the at least one biomarker,
wherein a difference in the level of the at least one biomarker
measured in step (c) compared to the level of the at least one
biomarker measured in step (a) indicates that the mammal will
respond therapeutically to the said method of treating cancer.
[0025] In another aspect, the invention provides a method for
identifying a mammal that will respond therapeutically to a method
of treating cancer comprising administering an EGFR modulator,
wherein the method comprises: (a) exposing a biological sample from
the mammal to the EGFR modulator; (b) following the exposing of
step (a), measuring in said biological sample the level of at least
one biomarker selected from the biomarkers of Table 1, wherein a
difference in the level of the at least one biomarker measured in
step (b), compared to the level of the at least one biomarker in a
mammal that has not been exposed to said EGFR modulator, indicates
that the mammal will respond therapeutically to said method of
treating cancer.
[0026] In yet another aspect, the invention provides a method for
testing or predicting whether a mammal will respond therapeutically
to a method of treating cancer comprising administering an EGFR
modulator, wherein the method comprises: (a) measuring in the
mammal the level of at least one biomarker selected from the
biomarkers of Table 1; (b) exposing the mammal to the EGFR
modulator; (c) following the exposing of step (b), measuring in the
mammal the level of the at least one biomarker, wherein a
difference in the level of the at least one biomarker measured in
step (c) compared to the level of the at least one biomarker
measured in step (a) indicates that the mammal will respond
therapeutically to said method of treating cancer.
[0027] In another aspect, the invention provides a method for
determining whether a compound inhibits EGFR activity in a mammal,
comprising: (a) exposing the mammal to the compound; and (b)
following the exposing of step (a), measuring in the mammal the
level of at least one biomarker selected from the biomarkers of
Table 1, wherein a difference in the level of said biomarker
measured in step (b), compared to the level of the biomarker in a
mammal that has not been exposed to said compound, indicates that
the compound inhibits EGFR activity in the mammal.
[0028] In yet another aspect, the invention provides a method for
determining whether a mammal has been exposed to a compound that
inhibits EGFR activity, comprising (a) exposing the mammal to the
compound; and (b) following the exposing of step (a), measuring in
the mammal the level of at least one biomarker selected from the
biomarkers of Table 1, wherein a difference in the level of said
biomarker measured in step (b), compared to the level of the
biomarker in a mammal that has not been exposed to said compound,
indicates that the mammal has been exposed to a compound that
inhibits EGFR activity.
[0029] In another aspect, the invention provides a method for
determining whether a mammal is responding to a compound that
inhibits EGFR activity, comprising (a) exposing the mammal to the
compound; and (b) following the exposing of step (a), measuring in
the mammal the level of at least one biomarker selected from the
biomarkers of Table 1, wherein a difference in the level of the at
least one biomarker measured in step (b), compared to the level of
the at least one biomarker in a mammal that has not been exposed to
said compound, indicates that the mammal is responding to the
compound that inhibits EGFR activity.
[0030] As used herein, "responding" encompasses responding by way
of a biological and cellular response, as well as a clinical
response (such as improved symptoms, a therapeutic effect, or an
adverse event), in a mammal.
[0031] The invention also provides an isolated biomarker selected
from the biomarkers of Table 1. The biomarkers of the invention
comprise sequences selected from the nucleotide and amino acid
sequences provided in Table 1 and the Sequence Listing, as well as
fragments and variants thereof.
[0032] The invention also provides a biomarker set comprising two
or more biomarkers selected from the biomarkers of Table 1.
[0033] The invention also provides kits for determining or
predicting whether a patient would be susceptible or resistant to a
treatment that comprises one or more EGFR modulators. The patient
may have a cancer or tumor such as, for example, colorectal cancer,
NSCLC, or head and neck cancer.
[0034] In one aspect, the kit comprises a suitable container that
comprises one or more specialized microarrays of the invention, one
or more EGFR modulators for use in testing cells from patient
tissue specimens or patient samples, and instructions for use. The
kit may further comprise reagents or materials for monitoring the
expression of a biomarker set at the level of mRNA or protein.
[0035] In another aspect, the invention provides a kit comprising
two or more biomarkers selected from the biomarkers of Table 1.
[0036] In yet another aspect, the invention provides a kit
comprising at least one of an antibody and a nucleic acid for
detecting the presence of at least one of the biomarkers selected
from the biomarkers of Table 1. In one aspect, the kit further
comprises instructions for determining whether or not a mammal will
respond therapeutically to a method of treating cancer comprising
administering a compound that inhibits EGFR activity. In another
aspect, the instructions comprise the steps of (a) measuring in the
mammal the level of at least one biomarker selected from the
biomarkers of Table 1, (b) exposing the mammal to the compound, (c)
following the exposing of step (b), measuring in the mammal the
level of the at least one biomarker, wherein a difference in the
level of the at least one biomarker measured in step (c) compared
to the level of the at least one biomarker measured in step (a)
indicates that the mammal will respond therapeutically to said
method of treating cancer.
[0037] The invention also provides screening assays for determining
if a patient will be susceptible or resistant to treatment with one
or more EGFR modulators.
[0038] The invention also provides a method of monitoring the
treatment of a patient having a disease, wherein said disease is
treated by a method comprising administering one or more EGFR
modulators.
[0039] The invention also provides individualized genetic profiles
which are necessary to treat diseases and disorders based on
patient response at a molecular level.
[0040] The invention also provides specialized microarrays, e.g.,
oligonucleotide microarrays or cDNA microarrays, comprising one or
more biomarkers having expression profiles that correlate with
either sensitivity or resistance to one or more EGFR
modulators.
[0041] The invention also provides antibodies, including polyclonal
or monoclonal, directed against one or more biomarkers of the
invention.
[0042] The invention will be better understood upon a reading of
the detailed description of the invention when considered in
connection with the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 illustrates a scheme used for identifying the
biomarkers described herein.
[0044] FIG. 2 illustrates the expression profiling of the
biomarkers described herein.
[0045] FIG. 3 (FIGS. 3A and 3B) illustrates the mRNA expression
profiles of epiregulin and amphiregulin in 30 patients.
[0046] FIG. 4 illustrates the biological relationship of biomarkers
described herein using Ingenuity Pathway Analysis.
[0047] FIG. 5 illustrates a comparison of a single biomarker model
to multiple biomarker models.
[0048] FIG. 6 illustrates the filtering of candidate markers for
cetuximab response. Expression data on 640 probe sets from 164
primary colorectal tumors was subjected to an unsupervised
hierarchical clustering. The 164 tumors were divided into 3 major
classes (Class 1, 2 and 3). The 640 probe sets were divided into 5
clusters (labeled A through E). Cluster A, which contains cancer
antigens such as CEACAM 6 and CD24, also contains EREG and AREG.
Cluster A is most highly expressed in Class 1a, which represents
approximately 25% of the 164 colorectal tumor specimens.
[0049] FIG. 7 (FIGS. 7A and 7B) illustrates the mRNA levels of
epiregulin and amphiregulin in 80 patients. Affymetrix mRNA levels
of epiregulin (EREG, 205767_at) and amphiregulin (AREG, 205239_at)
are plotted on the y axis. Subjects are ordered by best clinical
response. There is a statistically significant difference in gene
expression levels between the disease control group (CR, PR and SD)
and the non-responder group (EREG p=1.474e.sup.-05, AREG
p=2.489e.sup.-05).
[0050] FIG. 8 (FIGS. 8A and 8B) illustrates receiver operating
characteristic (ROC) curves for prediction of patient response.
FIG. 8A provides ROC using EREG to predict on test samples. EREG
was the top single gene predictor using the discriminant function
analysis, and has an area under the ROC curve (AUC) of 0.845 on the
test set, indicating a high performance for prediction. FIG. 8B
provides ROC using AREG to predict on the test set. The AREG gene,
which was found to be coordinately regulated with the EREG gene,
has an AUC of 0.815 on the test set, indicating that it too has a
good prediction power as a single gene predictor.
[0051] FIG. 9 illustrates the results obtained from validation of
AREG and EREG Affymetrix expression by qRT-PCR. A good correlation
between the two methods (Pearson>0.85, R2>0.7) was seen. High
expression on Affymetrix arrays (y axis) corresponds to low
.DELTA.Ct values from TaqMan qPCR assays for both AREG and EREG (x
axis).
DETAILED DESCRIPTION OF THE INVENTION:
[0052] Identification of biomarkers that provide rapid and
accessible readouts of efficacy, drug exposure, or clinical
response is increasingly important in the clinical development of
drug candidates. Embodiments of the invention include measuring
changes in the levels of secreted proteins, or plasma biomarkers,
which represent one category of biomarker. In one aspect, plasma
samples, which represent a readily accessible source of material,
serve as surrogate tissue for biomarker analysis.
[0053] The invention provides biomarkers that respond to the
modulation of a specific signal transduction pathway and also
correlate with EGFR modulator sensitivity or resistance. These
biomarkers can be employed for predicting response to one or more
EGFR modulators. In one aspect, the biomarkers of the invention are
those provided in Table 1 and the Sequence Listing, including both
polynucleotide and polypeptide sequences. The invention also
includes nucleotide sequences that hybridize to the polynucleotides
provided in Table 1.
TABLE-US-00001 TABLE 1 Biomarkers Affymetrix Unigene title and SEQ
ID NO: Affymetrix Description Probe Set NT5E: 5'-nucleotidase, ecto
gb: NM_002526.1 /DEF = Homo sapiens 203939_at (CD73) (LOC4907) 5
nucleotidase (CD73) (NT5), mRNA. SEQ ID NOS: 1 (DNA) and 129 /FEA =
mRNA /GEN = NT5 /PROD = 5 (amino acid) nucleotidase /DB_XREF = gi:
4505466 /UG = Hs.153952 5 nucleotidase (CD73) /FL = gb: NM_002526.1
EREG: epiregulin (LOC2069) gb: NM_001432.1 /DEF = Homo sapiens
205767_at SEQ ID NOS: 2 (DNA) and 130 epiregulin (EREG), mRNA.
(amino acid) /FEA = mRNA /GEN = EREG /PROD = epiregulin precursor
/DB_XREF = gi: 4557566 /UG = Hs.115263 epiregulin /FL = gb:
D30783.1 gb: NM_001432.1 AREG: amphiregulin gb: NM_001657.1 /DEF =
Homo sapiens 205239_at (schwannoma-derived growth amphiregulin
(schwannoma-derived factor) (LOC374) growth factor) (AREG), mRNA.
SEQ ID NOS: 3 (DNA) and 131 /FEA = mRNA /GEN = AREG (amino acid)
/PROD = amphiregulin (schwannoma- derived growth factor) /DB_XREF =
gi: 4502198 /UG = Hs.270833 amphiregulin (schwannoma-derived growth
factor) /FL = gb: M30704.1 gb: NM_001657.1 LYZ: lysozyme (renal
Consensus includes gb: AV711904 213975_s_at amyloidosis) (LOC4069)
/FEA = EST /DB_XREF = gi: 10731210 SEQ ID NOS: 4 (DNA) and 132
/DB_XREF = est: AV711904 (amino acid) /CLONE = DCAAIE08 /UG =
Hs.277431 Homo sapiens cDNA: FLJ23356 fis, clone HEP14919 BST2:
bone marrow stromal cell gb: NM_004335.2 /DEF = Homo sapiens
201641_at antigen 2 (LOC684) bone marrow stromal cell antigen 2 SEQ
ID NOS: 5 (DNA) and 133 (BST2), mRNA. /FEA = mRNA (amino acid) /GEN
= BST2 /PROD = bone marrow stromal cell antigen 2 /DB_XREF = gi:
7262372 /UG = Hs.118110 bone marrow stromal cell antigen 2 /FL =
gb: D28137.1 gb: NM_004335.2 DUSP6: dual specificity gb: BC005047.1
/DEF = Homo sapiens, 208893_s_at phosphatase 6 (LOC1848) clone MGC:
12852, mRNA, complete SEQ ID NOS: 6 (DNA) and 134 cds. /FEA = mRNA
/PROD = Unknown (amino acid) (protein for MGC: 12852) /DB_XREF =
gi: 13477170 /UG = Hs.180383 dual specificity phosphatase 6 /FL =
gb: BC003562.1 gb: BC003143.1 gb: BC005047.1 gb: AB013382.1 gb:
NM_001946.1 VAV3: vav 3 oncogene gb: NM_006113.2 /DEF = Homo
sapiens 218807_at (LOC10451) vav 3 oncogene (VAV3), mRNA. SEQ ID
NOS: 7 (DNA) and 135 /FEA = mRNA /GEN = VAV3 (amino acid) /PROD =
vav 3 oncogene /DB_XREF = gi: 7262390 /UG = Hs.267659 vav 3
oncogene /FL = gb: AF067817.1 gb: AF118887.1 gb: NM_006113.2 VAV3:
vav 3 oncogene gb: AF118887.1 /DEF = Homo sapiens 218806_s_at
(LOC10451) VAV-3 protein (VAV-3) mRNA, SEQ ID NOS: 8 (DNA) and 136
alternatively spliced, complete cds. (amino acid) /FEA = mRNA /GEN
= VAV-3 /PROD = VAV-3 protein /DB_XREF = gi: 4416407 /UG =
Hs.267659 vav 3 oncogene /FL = gb: AF067817.1 gb: AF118887.1 gb:
NM_006113.2 CCL2: chemokine (C-C motif) Consensus includes gb:
S69738.1 216598_s_at ligand 2 (LOC6347) /DEF = MCP-1 = monocyte
chemotactic SEQ ID NOS: 9 (DNA) and 137 protein human, aortic
endothelial cells, (amino acid) mRNA, 661 nt. /FEA = mRNA /GEN =
MCP-1 /PROD = MCP-1 /DB_XREF = gi: 545464 /UG = Hs.303649 small
inducible cytokine A2 (monocyte chemotactic protein 1, homologous
to mouse Sig-je) SATB2: SATB family member 2 Consensus includes gb:
AB028957.1 213435_at (LOC23314) /DEF = Homo sapiens mRNA for SEQ ID
NOS: 10 (DNA) and KIAA1034 protein, partial cds. 138 (amino acid)
/FEA = mRNA /GEN = KIAA1034 /PROD = KIAA1034 protein /DB_XREF = gi:
5689404 /UG = Hs.12896 KIAA1034 protein AKAP12: A kinase (PRKA) gb:
AB003476.1 /DEF = Homo sapiens 210517_s_at anchor protein (gravin)
12 mRNA for gravin, complete cds. (LOC9590) /FEA = mRNA /PROD =
gravin SEQ ID NOS: 11 (DNA) and /DB_XREF = gi: 2081606 /UG = Hs.788
139 (amino acid) A kinase (PRKA) anchor protein (gravin) 12 /FL =
gb: AB003476.1 GCNT3: glucosaminyl (N- gb: NM_004751.1 /DEF = Homo
sapiens 219508_at acetyl) transferase 3, mucin type glucosaminyl
(N-acetyl) transferase 3, (LOC9245) mucin type (GCNT3), mRNA. SEQ
ID NOS: 12 (DNA) and /FEA = mRNA /GEN = GCNT3 140 (amino acid)
/PROD = glucosaminyl (N-acetyl) transferase 3, mucintype /DB_XREF =
gi: 4758421 /UG = Hs.194710 glucosaminyl (N- acetyl) transferase 3,
mucin type /FL = gb: AF102542.1 gb: AF038650.1 gb: NM_004751.1
SCRN1: secernin 1 (LOC9805) gb: NM_014766.1 /DEF = Homo sapiens
201462_at SEQ ID NOS: 13 (DNA) and KIAA0193 gene product
(KIAA0193), 141 (amino acid) mRNA. /FEA = mRNA /GEN = KIAA0193
/PROD = KIAA0193 gene product /DB_XREF = gi: 7661983 /UG = Hs.75137
KIAA0193 gene product /FL = gb: D83777.1 gb: NM_014766.1 FGFR3:
fibroblast growth factor gb: NM_000142.2 /DEF = Homo sapiens
204379_s_at receptor 3 (achondroplasia, fibroblast growth factor
receptor 3 thanatophoric dwarfism) (achondroplasia, thanatophoric
(LOC2261) dwarfism) (FGFR3), transcript variant SEQ ID NOS: 14
(DNA) and 1, mRNA. /FEA = mRNA 142 (amino acid) /GEN = FGFR3 /PROD
= fibroblast growth factor receptor 3, isoform 1precursor /DB_XREF
= gi: 13112046 /UG = Hs.1420 fibroblast growth factor receptor 3
(achondroplasia, thanatophoric dwarfism) /FL = gb: NM_000142.2 gb:
M58051.1 LY96: lymphocyte antigen 96 gb: NM_015364.1 /DEF = Homo
sapiens 206584_at (LOC23643) MD-2 protein (MD-2), mRNA. SEQ ID NOS:
15 (DNA) and /FEA = mRNA /GEN = MD-2 143 (amino acid) /PROD = MD-2
protein /DB_XREF = gi: 7662503 /UG = Hs.69328 MD-2 protein /FL =
gb: AB018549.1 gb: NM_015364.1 gb: AF168121.1 CKB: creatine kinase,
brain gb: NM_001823.1 /DEF = Homo sapiens 200884_at (LOC1152)
creatine kinase, brain (CKB), mRNA. SEQ ID NOS: 16 (DNA) and /FEA =
mRNA /GEN = CKB 144 (amino acid) /PROD = creatine kinase, brain
/DB_XREF = gi: 4502850 /UG = Hs.173724 creatine kinase, brain /FL =
gb: L47647.1 gb: BC001190.1 gb: BC004914.1 gb: M16364.1 gb:
M16451.1 gb: NM_001823.1 IFI16: interferon, gamma- gb: NM_005531.1
/DEF = Homo sapiens 206332_s_at inducible protein 16 (LOC3428)
interferon, gamma-inducible protein 16 SEQ ID NOS: 17 (DNA) and
(IFI16), mRNA. /FEA = mRNA 145 (amino acid) /GEN = IFI16 /PROD =
interferon, gamma-inducible protein 16 /DB_XREF = gi: 5031778 /UG =
Hs.155530 interferon, gamma- inducible protein 16 /FL = gb:
M63838.1 gb: NM_005531.1 PRSS8: protease, serine, 8 gb: NM_002773.1
/DEF = Homo sapiens 202525_at (prostasin) (LOC5652) protease,
serine, 8 (prostasin) (PRSS8), SEQ ID NOS: 18 (DNA) and mRNA. /FEA
= mRNA /GEN = PRSS8 146 (amino acid) /PROD = protease, serine, 8
(prostasin) /DB_XREF = gi: 4506152 /UG = Hs.75799 protease, serine,
8 (prostasin) /FL = gb: BC001462.1 gb: NM_002773.1 gb: L41351.1
IL1R2: interleukin 1 receptor, gb: NM_004633.1 /DEF = Homo sapiens
205403_at type II (LOC7850) interleukin 1 receptor, type II
(IL1R2), SEQ ID NOS: 19 (DNA) and mRNA. /FEA = mRNA /GEN = IL1R2
147 (amino acid) /PROD = interleukin 1 receptor, type II /DB_XREF =
gi: 4758597 /UG = Hs.25333 interleukin 1 receptor, type II /FL =
gb: U74649.1 gb: NM_004633.1 BHLHB3: basic helix-loop-helix
Consensus includes gb: BE857425 221530_s_at domain containing,
class B, 3 /FEA = EST /DB_XREF = gi: 10371439 (LOC79365) /DB_XREF =
est: 7f97a11.x1 SEQ ID NOS: 20 (DNA) and /CLONE = IMAGE: 3304892
148 (amino acid) /UG = Hs.33829 bHLH protein DEC2 /FL = gb:
AB044088.1 HLA-DRB4: major gb: BC005312.1 /DEF = Homo sapiens,
209728_at histocompatibility complex, clone MGC: 12387, mRNA,
complete class II, DR beta 4 (LOC3126) cds. /FEA = mRNA /PROD =
Unknown SEQ ID NOS: 21 (DNA) and (protein for MGC: 12387) 149
(amino acid) /DB_XREF = gi: 13529055 /UG = Hs.318720 Homo sapiens,
clone MGC: 12387, mRNA, complete cds /FL = gb: BC005312.1 gb:
M16942.1 CD163: CD163 antigen Consensus includes gb: Z22969.1
215049_x_at (LOC9332) /DEF = H. sapiens mRNA for M130 SEQ ID NOS:
22 (DNA) and antigen cytoplasmic variant 1. 150 (amino acid) /FEA =
mRNA /PROD = M130 antigen cytoplasmic variant 1 /DB_XREF = gi:
312143 /UG = Hs.74076 CD163 antigen CD163: CD163 antigen gb:
NM_004244.1 /DEF = Homo sapiens 203645_s_at (LOC9332) CD163 antigen
(CD163), mRNA. SEQ ID NOS: 23 (DNA) and /FEA = mRNA /GEN = CD163
151 (amino acid) /PROD = CD163 antigen /DB_XREF = gi: 4758721 /UG =
Hs.74076 CD163 antigen /FL = gb: NM_004244.1 C13orf18: chromosome
13 open gb: NM_025113.1 /DEF = Homo sapiens 219471_at reading frame
18 (LOC80183) hypothetical protein FLJ21562 SEQ ID NOS: 24 (DNA)
and (FLJ21562), mRNA. /FEA = mRNA 152 (amino acid) /GEN = FLJ21562
/PROD = hypothetical protein FLJ21562 /DB_XREF = gi: 13376686 /UG =
Hs.288708 hypothetical protein FLJ21562 /FL = gb: NM_025113.1
CCL11: chemokine (C-C motif) gb: D49372.1 /DEF = Human mRNA for
210133_at ligand 11 (LOC6356) eotaxin, complete cds. /FEA = mRNA
SEQ ID NOS: 25 (DNA) and /PROD = eotaxin 153 (amino acid) /DB_XREF
= gi: 1552240 /UG = Hs.54460 small inducible cytokine subfamily A
(Cys-Cys), member 11 (eotaxin) /FL = gb: U46573.1 gb: D49372.1 gb:
NM_002986.1 SLC26A2: solute carrier family Consensus includes gb:
AI025519 205097_at 26 (sulfate transporter), member /FEA = EST
/DB_XREF = gi: 3241132 2 (LOC1836) /DB_XREF = est: ov75c04.x1 SEQ
ID NOS: 26 (DNA) and /CLONE = IMAGE: 1643142 154 (amino acid) /UG =
Hs.29981 solute carrier family 26 (sulfate transporter), member 2
/FL = gb: NM_000112.1 gb: U14528.1 HLA-DQB1: major gb: M32577.1
/DEF = Human MHC 211656_x_at histocompatibility complex, HLA-DQ
beta mRNA, complete cds. class II, DQ beta 1 (LOC3119) /FEA = mRNA
/GEN = HLA-DQB1 SEQ ID NOS: 27 (DNA) and /DB_XREF = gi: 188194 155
(amino acid) /FL = gb: M32577.1 ENPP2: ectonucleotide gb: L35594.1
/DEF = Human autotaxin 209392_at pyrophosphatase/phosphodiesterase
mRNA, complete cds. /FEA = mRNA 2 (autotaxin) (LOC5168) /PROD =
autotaxin SEQ ID NOS: 28 (DNA) and /DB_XREF = gi: 537905 156 (amino
acid) /UG = Hs.174185 ectonucleotide
pyrophosphatasephosphodiesterase 2 (autotaxin) /FL = gb: L35594.1
PRSS3: protease, serine, 3 gb: NM_002770.1 /DEF = Homo sapiens
205402_x_at (mesotrypsin) (LOC5646) protease, serine, 2 (trypsin 2)
(PRSS2), SEQ ID NOS: 29 (DNA) and mRNA. /FEA = mRNA /GEN = PRSS2
157 (amino acid) /PROD = protease, serine, 2 (trypsin 2) /DB_XREF =
gi: 4506146 /UG = Hs.241561 protease, serine, 2 (trypsin 2) /FL =
gb: NM_002770.1 gb: M27602.1 CXCR4: chemokine (C--X--C Consensus
includes gb: AJ224869 217028_at motif) receptor 4 (LOC7852) /DEF =
Homo sapiens CXCR4 gene SEQ ID NOS: 30 (DNA) and encoding receptor
CXCR4 158 (amino acid) /FEA = mRNA /DB_XREF = gi: 3059119 /UG =
Hs.89414 chemokine (C--X--C motif), receptor 4 (fusin) SERPINB5:
serine (or cysteine) gb: NM_002639.1 /DEF = Homo sapiens
204855_at
proteinase inhibitor, clade B serine (or cysteine) proteinase
inhibitor, (ovalbumin), member 5 clade B (ovalbumin), member 5
(LOC5268) (SERPINB5), mRNA. /FEA = mRNA SEQ ID NOS: 31 (DNA) and
/GEN = SERPINB5 /PROD = serine (or 159 (amino acid) cysteine)
proteinase inhibitor, cladeB (ovalbumin), member 5 /DB_XREF = gi:
4505788 /UG = Hs.55279 serine (or cysteine) proteinase inhibitor,
clade B (ovalbumin), member 5 /FL = gb: NM_002639.1 gb: U04313.1
HLA-DPB1: major gb: NM_002121.1 /DEF = Homo sapiens 201137_s_at
histocompatibility complex, major histocompatibility complex, class
class II, DP beta 1 (LOC3115) II, DP beta 1 (HLA-DPB1), mRNA. SEQ
ID NOS: 32 (DNA) and /FEA = mRNA /GEN = HLA-DPB1 160 (amino acid)
/PROD = major histocompatibility complex, class II, DPbeta 1
/DB_XREF = gi: 4504404 /UG = Hs.814 major histocompatibility
complex, class II, DP beta 1 /FL = gb: J03041.1 gb: M57466.1 gb:
M83664.1 gb: NM_002121.1 gb: M28200.1 gb: M28202.1 AIF1: allograft
inflammatory Consensus includes gb: BF213829 215051_x_at factor 1
(LOC199) /FEA = EST /DB_XREF = gi: 11107415 SEQ ID NOS: 33 (DNA)
and /DB_XREF = est: 601848003F1 161 (amino acid) /CLONE = IMAGE:
4078849 /UG = Hs.76364 allograft inflammatory factor 1 IL8:
interleukin 8 (LOC3576) gb: NM_000584.1 /DEF = Homo sapiens
202859_x_at SEQ ID NOS: 34 (DNA) and interleukin 8 (IL8), mRNA. 162
(amino acid) /FEA = mRNA /GEN = IL8 /PROD = interleukin 8 /DB_XREF
= gi: 10834977 /UG = Hs.624 interleukin 8 /FL = gb: NM_000584.1 gb:
M17017.1 gb: M26383.1 IL8: interleukin 8 (LOC3576) gb: AF043337.1
/DEF = Homo sapiens 211506_s_at SEQ ID NOS: 35 (DNA) and
interleukin 8 C-terminal variant (IL8) 163 (amino acid) mRNA,
complete cds. /FEA = mRNA /GEN = IL8 /PROD = interleukin 8 C-
terminal variant /DB_XREF = gi: 12641914 /UG = Hs.624 interleukin 8
/FL = gb: AF043337.1 LY6G6D: lymphocyte antigen 6 gb: NM_021246.1
/DEF = Homo sapiens 207457_s_at complex, locus G6D
megakaryocyte-enhanced gene (LOC58530) transcript 1 protein
(MEGT1), mRNA. SEQ ID NOS: 36 (DNA) and /FEA = mRNA /GEN = MEGT1
164 (amino acid) /PROD = megakaryocyte-enhanced gene transcript
1protein /DB_XREF = gi: 10864054 /UG = Hs.241587 megakaryocyte-
enhanced gene transcript 1 protein /FL = gb: NM_021246.1 gb:
AF195764.1 CYP3A5: cytochrome P450, gb: NM_000777.1 /DEF = Homo
sapiens 205765_at family 3, subfamily A, cytochrome P450, subfamily
IIIA polypeptide 5 (LOC1577) (niphedipine oxidase), polypeptide 5
SEQ ID NOS: 37 (DNA) and (CYP3A5), mRNA. /FEA = mRNA 165 (amino
acid) /GEN = CYP3A5 /PROD = cytochrome P450, subfamily IIIA,
polypeptide 5 /DB_XREF = gi: 4503230 /UG = Hs.104117 cytochrome
P450, subfamily IIIA (niphedipine oxidase), polypeptide 5 /FL = gb:
J04813.1 gb: NM_000777.1 CSPG2: chondroitin sulfate Consensus
includes gb: BF590263 204619_s_at proteoglycan 2 (versican) /FEA =
EST /DB_XREF = gi: 11682587 (LOC1462) /DB_XREF = est: nab22b12.x1
SEQ ID NOS: 38 (DNA) and /CLONE = IMAGE: 3266638 166 (amino acid)
/UG = Hs.81800 chondroitin sulfate proteoglycan 2 (versican) /FL =
gb: NM_004385.1 CA9: carbonic anhydrase IX gb: NM_001216.1 /DEF =
Homo sapiens 205199_at (LOC768) carbonic anhydrase IX (CA9), mRNA.
SEQ ID NOS: 39 (DNA) and /FEA = mRNA /GEN = CA9 167 (amino acid)
/PROD = carbonic anhydrase IX precursor /DB_XREF = gi: 9955947 /UG
= Hs.63287 carbonic anhydrase IX /FL = gb: NM_001216.1 ACE2:
angiotensin I converting gb: NM_021804.1 /DEF = Homo sapiens
219962_at enzyme (peptidyl-dipeptidase A) angiotensin I converting
enzyme 2 (LOC59272) (peptidyl-dipeptidase A) 2 (ACE2), SEQ ID NOS:
40 (DNA) and mRNA. /FEA = mRNA /GEN = ACE2 168 (amino acid) /PROD =
angiotensin I converting enzyme(peptidyl-dipeptidase A) 2 /DB_XREF
= gi: 11225608 /UG = Hs.178098 angiotensin I converting enzyme
(peptidyl- dipeptidase A) 2 /FL = gb: NM_021804.1 gb: AB046569.1
gb: AF241254.1 gb: AF291820.1 CXCL13: chemokine (C--X--C gb:
NM_006419.1 /DEF = Homo sapiens 205242_at motif) ligand 13 (B-cell
small inducible cytokine B subfamily chemoattractant) (LOC10563)
(Cys-X-Cys motif), member 13 (B-cell SEQ ID NOS: 41 (DNA) and
chemoattractant) (SCYB13), mRNA. 169 (amino acid) /FEA = mRNA /GEN
= SCYB13 /PROD = small inducible cytokine B subfamily
(Cys-X-Cysmotif), member 13 (B-cell chemoattractant) /DB_XREF = gi:
5453576 /UG = Hs.100431 small inducible cytokine B subfamily
(Cys-X-Cys motif), member 13 (B-cell chemoattractant) /FL = gb:
AF044197.1 gb: AF029894.1 gb: NM_006419.1 COL10A1: collagen, type
X, Consensus includes gb: X98568 217428_s_at alpha 1(Schmid
metaphyseal /DEF = H. sapiens type X collagen gene
chondrodysplasia) (LOC1300) /FEA = mRNA /DB_XREF = gi: 1405722 SEQ
ID NOS: 42 (DNA) and /UG = Hs.179729 collagen, type X, 170 (amino
acid) alpha 1 (Schmid metaphyseal chondrodysplasia) CPNE1: copine I
(LOC8904) gb: NM_003915.1 /DEF = Homo sapiens 206918_s_at SEQ ID
NOS: 43 (DNA) and copine I (CPNE1), mRNA. 171 (amino acid) /FEA =
mRNA /GEN = CPNE1 /PROD = copine I /DB_XREF = gi: 4503012 /UG =
Hs.166887 copine I /FL = gb: U83246.1 gb: NM_003915.1 C13orf18:
chromosome 13 open Cluster Incl. AI129310: qc48a05.x1 44790_s_at
reading frame 18 (LOC80183) Homo sapiens cDNA, 3 end SEQ ID NOS: 44
(DNA) and /clone = IMAGE-1712816 /clone_end = 3' 172 (amino acid)
/gb = AI129310 /gi = 3597824 /ug = Hs.234923 /len = 811 GREM1:
gremlin 1 homolog, gb: NM_013372.1 /DEF = Homo sapiens 218469_at
cysteine knot superfamily cysteine knot superfamily 1, BMP (Xenopus
laevis) (LOC26585) antagonist 1 (CKTSF1B1), mRNA. SEQ ID NOS: 45
(DNA) and /FEA = mRNA /GEN = CKTSF1B1 173 (amino acid) /PROD =
cysteine knot superfamily 1, BMP antagonist 1 /DB_XREF = gi:
7019348 /UG = Hs.40098 cysteine knot superfamily 1, BMP antagonist
1 /FL = gb: AF154054.1 gb: AF045800.1 gb: AF110137.2 gb:
NM_013372.1 HLA-DQB1: major gb: M17955.1 /DEF = Human MHC
209823_x_at histocompatibility complex, class II HLA-DQ-beta mRNA,
class II, DQ beta 1 (LOC3119) complete cds. /FEA = mRNA SEQ ID NOS:
46 (DNA) and /DB_XREF = gi: 188178 /UG = Hs.73931 174 (amino acid)
major histocompatibility complex, class II, DQ beta 1 /FL = gb:
M33907.1 gb: M17955.1 gb: M17563.1 gb: M26042.1 gb: M20432.1 gb:
M16996.1 TCN1: transcobalamin I gb: NM_001062.1 /DEF = Homo sapiens
205513_at (vitamin B12 binding protein, R transcobalamin I (vitamin
B12 binding binder family) (LOC6947) protein, R binder family)
(TCN1), SEQ ID NOS: 47 (DNA) and mRNA. /FEA = mRNA /GEN = TCN1 175
(amino acid) /PROD = transcobalamin I (vitamin B12 binding protein,
Rbinder family) /DB_XREF = gi: 4507406 /UG = Hs.2012 transcobalamin
I (vitamin B12 binding protein, R binder family) /FL = gb: J05068.1
gb: NM_001062.1 PIGR: polymeric gb: NM_002644.1 /DEF = Homo sapiens
204213_at immunoglobulin receptor polymeric immunoglobulin receptor
(LOC5284) (PIGR), mRNA. /FEA = mRNA SEQ ID NOS: 48 (DNA) and /GEN =
PIGR /PROD = polymeric 176 (amino acid) immunoglobulin receptor
/DB_XREF = gi: 11342673 /UG = Hs.288579 polymeric immunoglobulin
receptor /FL = gb: NM_002644.1 COL10A1: collagen, type X, Consensus
includes gb: AI376003 205941_s_at alpha 1(Schmid metaphyseal /FEA =
EST /DB_XREF = gi: 4175993 chondrodysplasia) (LOC1300) /DB_XREF =
est: tc30d11.x1 SEQ ID NOS: 49 (DNA) and /CLONE = IMAGE: 2066133
177 (amino acid) /UG = Hs.179729 collagen, type X, alpha 1 (Schmid
metaphyseal chondrodysplasia) /FL = gb: NM_000493.1 KCTD12:
potassium channel Consensus includes gb: AI718937 212192_at
tetramerisation domain /FEA = EST /DB_XREF = gi: 5036193 containing
12 (LOC115207) /DB_XREF = est: as50b04.x1 SEQ ID NOS: 50 (DNA) and
/CLONE = IMAGE: 2320591 178 (amino acid) /UG = Hs.109438 Homo
sapiens clone 24775 mRNA sequence LCK: lymphocyte-specific gb:
NM_005356.1 /DEF = Homo sapiens 204891_s_at protein tyrosine kinase
lymphocyte-specific protein tyrosine (LOC3932) kinase (LCK), mRNA.
/FEA = mRNA SEQ ID NOS: 51 (DNA) and /GEN = LCK /PROD = lymphocyte-
179 (amino acid) specific protein tyrosine kinase /DB_XREF = gi:
4885448 /UG = Hs.1765 lymphocyte-specific protein tyrosine kinase
/FL = gb: M36881.1 gb: U07236.1 gb: NM_005356.1 LAPTM4B: lysosomal
gb: NM_018407.1 /DEF = Homo sapiens 208029_s_at associated protein
putative integral membrane transporter transmembrane 4 beta (LC27),
mRNA. /FEA = mRNA (LOC55353) /GEN = LC27 /PROD = putative integral
SEQ ID NOS: 52 (DNA) and membrane transporter 180 (amino acid)
/DB_XREF = gi: 8923827 /FL = gb: NM_018407.1 CEACAM5:
carcinoembryonic gb: NM_004363.1 /DEF = Homo sapiens 201884_at
antigen-related cell adhesion carcinoembryonic antigen-related cell
molecule 5 (LOC1048) adhesion molecule 5 (CEACAM5), SEQ ID NOS: 53
(DNA) and mRNA. /FEA = mRNA 181 (amino acid) /GEN = CEACAM5 /PROD =
carcinoembryonic antigen- related cell adhesionmolecule 5 /DB_XREF
= gi: 11386170 /UG = Hs.220529 carcinoembryonic antigen-related
cell adhesion molecule 5 /FL = gb: NM_004363.1 gb: M29540.1 LDHB:
lactate dehydrogenase B gb: NM_002300.1 /DEF = Homo sapiens
201030_x_at (LOC3945) lactate dehydrogenase B (LDHB), SEQ ID NOS:
54 (DNA) and mRNA. /FEA = mRNA /GEN = LDHB 182 (amino acid) /PROD =
lactate dehydrogenase B /DB_XREF = gi: 4557031 /UG = Hs.234489
lactate dehydrogenase B /FL = gb: BC002362.1 gb: NM_002300.1 IFI27:
interferon, alpha- gb: NM_005532.1 /DEF = Homo sapiens 202411_at
inducible protein 27 (LOC3429) interferon, alpha-inducible protein
27 SEQ ID NOS: 55 (DNA) and (IFI27), mRNA. /FEA = mRNA 183 (amino
acid) /GEN = IFI27 /PROD = interferon, alpha- inducible protein 27
/DB_XREF = gi: 5031780 /UG = Hs.278613 interferon, alpha- inducible
protein 27 /FL = gb: NM_005532.1 EPHB2: EphB2 (LOC2048) gb:
D31661.1 /DEF = Human mRNA for 211165_x_at SEQ ID NOS: 56 (DNA) and
tyrosine kinase, complete cds. 184 (amino acid) /FEA = mRNA /GEN =
ERK /PROD = tyrosine kinase precursor /DB_XREF = gi: 495677 /UG =
Hs.125124 EphB2 /FL = gb: D31661.1 ACACA: acetyl-Coenzyme A
Consensus includes gb: BE855983 212186_at carboxylase alpha (LOC31)
/FEA = EST /DB_XREF = gi: 10368561 SEQ ID NOS: 57 (DNA) and
/DB_XREF = est: 7f85g11.x1 185 (amino acid) /CLONE = IMAGE: 3303812
/UG = Hs.7232 acetyl-Coenzyme A carboxylase alpha /FL = gb:
NM_000664.1 gb: U19822.1 CD14: CD14 antigen (LOC929) gb:
NM_000591.1 /DEF = Homo sapiens 201743_at SEQ ID NOS: 58 (DNA) and
CD14 antigen (CD14), mRNA. 186 (amino acid) /FEA = mRNA /GEN = CD14
/PROD = CD14 antigen precursor /DB_XREF = gi: 4557416 /UG =
Hs.75627 CD14 antigen /FL = gb: M86511.1 gb: AF097942.1 gb:
NM_000591.1 ABHD2: abhydrolase domain Cluster Incl. AI832249:
td14g10.x1 87100_at containing 2 (LOC11057) Homo sapiens cDNA, 3
end SEQ ID NOS: 59 (DNA) and /clone = IMAGE-2075682 /clone_end = 3'
187 (amino acid) /gb = AI832249 /gi = 5452920
/ug = Hs.211522 /len = 545 TNFRSF6B: tumor necrosis gb: NM_003823.1
/DEF = Homo sapiens 206467_x_at factor receptor superfamily, tumor
necrosis factor receptor member 6b, decoy (LOC8771) superfamily,
member 6b, decoy SEQ ID NOS: 60 (DNA) and (TNFRSF6B), mRNA. /FEA =
mRNA 188 (amino acid) /GEN = TNFRSF6B /PROD = decoy receptor 3
/DB_XREF = gi: 4507584 /UG = Hs.278556 tumor necrosis factor
receptor superfamily, member 6b, decoy /FL = gb: AF104419.1 gb:
NM_003823.1 gb: AF134240.1 gb: AF217794.1 GREM1: gremlin 1 homolog,
gb: AF154054.1 /DEF = Homo sapiens 218468_s_at cysteine knot
superfamily DRM (DRM) mRNA, complete cds. (Xenopus laevis)
(LOC26585) /FEA = mRNA /GEN = DRM SEQ ID NOS: 61 (DNA) and /PROD =
DRM 189 (amino acid) /DB_XREF = gi: 10863087 /UG = Hs.40098
cysteine knot superfamily 1, BMP antagonist 1 /FL = gb: AF154054.1
gb: AF045800.1 gb: AF110137.2 gb: NM_013372.1 ACE2: angiotensin I
converting Consensus includes gb: AK026461.1 222257_s_at enzyme
(peptidyl-dipeptidase A) /DEF = Homo sapiens cDNA: FLJ22808 2
(LOC59272) fis, clone KAIA2925. /FEA = mRNA SEQ ID NOS: 62 (DNA)
and /DB_XREF = gi: 10439331 190 (amino acid) /UG = Hs.178098
angiotensin I converting enzyme (peptidyl- dipeptidase A) 2 COL5A2:
collagen, type V, Consensus includes gb: NM_000393.1 221730_at
alpha 2 (LOC1290) /DEF = Homo sapiens collagen, type V, SEQ ID NOS:
63 (DNA) and alpha 2 (COL5A2), mRNA. 191 (amino acid) /FEA = CDS
/GEN = COL5A2 /PROD = collagen, type V, alpha 2 /DB_XREF = gi:
4502958 /UG = Hs.82985 collagen, type V, alpha 2 /FL = gb:
NM_000393.1 CXCL9: chemokine (C--X--C gb: NM_002416.1 /DEF = Homo
sapiens 203915_at motif) ligand 9 (LOC4283) monokine induced by
gamma SEQ ID NOS: 64 (DNA) and interferon (MIG), mRNA. 192 (amino
acid) /FEA = mRNA /GEN = MIG /PROD = monokine induced by gamma
interferon /DB_XREF = gi: 4505186 /UG = Hs.77367 monokine induced
by gamma interferon /FL = gb: NM_002416.1 HOXC6: homeo box C6 gb:
NM_004503.1 /DEF = Homo sapiens 206858_s_at (LOC3223) homeo box C6
(HOXC6), mRNA. SEQ ID NOS: 65 (DNA) and /FEA = mRNA /GEN = HOXC6
193 (amino acid) /PROD = homeo box C6 /DB_XREF = gi: 4758553 /UG =
Hs.820 homeo box C6 /FL = gb: NM_004503.1 KCNMA1: potassium large
gb: U11058.2 /DEF = Homo sapiens 221584_s_at conductance
calcium-activated large conductance calcium- and channel, subfamily
M, alpha voltage-dependent potassium channel member 1 (LOC3778)
alpha subunit (MaxiK) mRNA, SEQ ID NOS: 66 (DNA) and complete cds.
/FEA = mRNA 194 (amino acid) /GEN = MaxiK /PROD = large conductance
calcium- and voltage- dependentpotassium channel alpha subunit
/DB_XREF = gi: 7914977 /UG = Hs.89463 potassium large conductance
calcium-activated channel, subfamily M, alpha member 1 /FL = gb:
AF025999.1 gb: NM_002247.1 gb: AF118141.1 gb: U13913.1 gb: U11717.1
gb: U23767.1 gb: U11058.2 MMP1: matrix metalloproteinase gb:
NM_002421.2 /DEF = Homo sapiens 204475_at 1 (interstitial
collagenase) matrix metalloproteinase 1 (interstitial (LOC4312)
collagenase) (MMP1), mRNA. SEQ ID NOS: 67 (DNA) and /FEA = mRNA
/GEN = MMP1 195 (amino acid) /PROD = matrix metalloproteinase 1
preproprotein /DB_XREF = gi: 13027798 /UG = Hs.83169 matrix
metalloproteinase 1 (interstitial collagenase) /FL = gb:
NM_002421.2 gb: M13509.1 PLCB4: phospholipase C, beta 4 Consensus
includes gb: AL535113 203895_at (LOC5332) /FEA = EST /DB_XREF = gi:
12798606 SEQ ID NOS: 68 (DNA) and /DB_XREF = est: AL535113 196
(amino acid) /CLONE = CS0DF008YC23 (3 prime) /UG = Hs.283006
phospholipase C, beta 4 /FL = gb: NM_000933.1 gb: L41349.1 PTPRD:
protein tyrosine Consensus includes gb: BF062299 214043_at
phosphatase, receptor type, D /FEA = EST /DB_XREF = gi: 10821197
(LOC5789) /DB_XREF = est: 7k76c03.x1 SEQ ID NOS: 69 (DNA) and
/CLONE = IMAGE: 3481325 197 (amino acid) /UG = Hs.323079 Homo
sapiens mRNA; cDNA DKFZp564P116 (from clone DKFZp564P116) KCNK1:
potassium channel, gb: U90065.1 /DEF = Human potassium 204678_s_at
subfamily K, member 1 channel KCNO1 mRNA, complete cds. (LOC3775)
/FEA = mRNA /PROD = potassium SEQ ID NOS: 70 (DNA) and channel
KCNO1 198 (amino acid) /DB_XREF = gi: 1916294 /UG = Hs.79351
potassium channel, subfamily K, member 1 (TWIK-1) /FL = gb:
U33632.1 gb: U90065.1 gb: U76996.1 gb: NM_002245.1 ALOX5:
arachidonate 5- gb: NM_000698.1 /DEF = Homo sapiens 204446_s_at
lipoxygenase (LOC240) arachidonate 5-lipoxygenase (ALOX5), SEQ ID
NOS: 71 (DNA) and mRNA. /FEA = mRNA /GEN = ALOX5 199 (amino acid)
/PROD = arachidonate 5-lipoxygenase /DB_XREF = gi: 4502056 /UG =
Hs.89499 arachidonate 5- lipoxygenase /FL = gb: J03600.1 gb:
J03571.1 gb: NM_000698.1 CXCL10: chemokine (C--X--C gb: NM_001565.1
/DEF = Homo sapiens 204533_at motif) ligand 10 (LOC3627) small
inducible cytokine subfamily B SEQ ID NOS: 72 (DNA) and
(Cys-X-Cys), member 10 (SCYB10), 200 (amino acid) mRNA. /FEA = mRNA
/GEN = SCYB10 /PROD = interferon gamma-induced precursor /DB_XREF =
gi: 4504700 /UG = Hs.2248 small inducible cytokine subfamily B
(Cys-X-Cys), member 10 /FL = gb: NM_001565.1 TMPRSS2: transmembrane
gb: AF270487.1 /DEF = Homo sapiens 211689_s_at protease, serine 2
(LOC7113) androgen-regulated serine protease SEQ ID NOS: 73 (DNA)
and TMPRSS2 precursor (TMPRSS2) 201 (amino acid) mRNA, complete
cds. /FEA = mRNA /GEN = TMPRSS2 /PROD = androgen- regulated serine
protease TMPRSS2precursor /DB_XREF = gi: 13540003 /FL = gb:
AF270487.1 PRG1: proteoglycan 1, secretory gb: J03223.1 /DEF =
Human secretory 201858_s_at granule (LOC5552) granule proteoglycan
peptide core SEQ ID NOS: 74 (DNA) and mRNA, complete cds. /FEA =
mRNA 202 (amino acid) /GEN = PRG1 /DB_XREF = gi: 190419 /UG =
Hs.1908 proteoglycan 1, secretory granule /FL = gb: J03223.1 gb:
NM_002727.1 HLA-DQA1: major Consensus includes gb: BG397856
212671_s_at histocompatibility complex, /FEA = EST /DB_XREF = gi:
13291304 class II, DQ alpha 1 (LOC3117) /DB_XREF = est: 602438950F1
SEQ ID NOS: 75 (DNA) and /CLONE = IMAGE: 4564956 203 (amino acid)
/UG = Hs.198253 major histocompatibility complex, class II, DQ
alpha 1 NR4A2: nuclear receptor Consensus includes gb: S77154.1
216248_s_at subfamily 4, group A, member 2 /DEF = TINUR =
NGFI-Bnur77 beta- (LOC4929) type transcription factor homolog SEQ
ID NOS: 76 (DNA) and human, T lymphoid cell line, PEER, 204 (amino
acid) mRNA, 2469 nt. /FEA = mRNA /GEN = TINUR /DB_XREF = gi: 913966
/UG = Hs.82120 nuclear receptor subfamily 4, group A, member 2
KCTD12: potassium channel Consensus includes gb: AA551075 212188_at
tetramerisation domain /FEA = EST /DB_XREF = gi: 2321327 containing
12 (LOC115207) /DB_XREF = est: nk74h06.s1 SEQ ID NOS: 77 (DNA) and
/CLONE = IMAGE: 1019291 205 (amino acid) /UG = Hs.109438 Homo
sapiens clone 24775 mRNA sequence RARRES3: retinoic acid gb:
NM_004585.2 /DEF = Homo sapiens 204070_at receptor responder
(tazarotene retinoic acid receptor responder induced) 3 (LOC5920)
(tazarotene induced) 3 (RARRES3), SEQ ID NOS: 78 (DNA) and mRNA.
/FEA = mRNA 206 (amino acid) /GEN = RARRES3 /PROD = retinoic acid
receptor responder (tazaroteneinduced) 3 /DB_XREF = gi: 8051633 /UG
= Hs.17466 retinoic acid receptor responder (tazarotene induced) 3
/FL = gb: AF060228.1 gb: AF092922.1 gb: NM_004585.2 gb: AB030815.1
LDHB: lactate dehydrogenase B Consensus includes gb: BE042354
213564_x_at (LOC3945) /FEA = EST /DB_XREF = gi: 8359407 SEQ ID NOS:
79 (DNA) and /DB_XREF = est: ho19b09.x1 207 (amino acid) /CLONE =
IMAGE: 3037817 /UG = Hs.234489 lactate dehydrogenase B CLECSF2:
C-type (calcium gb: BC005254.1 /DEF = Homo sapiens, 209732_at
dependent, carbohydrate- Similar to C-type (calcium dependent,
recognition domain) lectin, carbohydrate-recognition domain)
superfamily member 2 lectin, superfamily member 2
(activation-induced) (LOC9976) (activation-induced), clone SEQ ID
NOS: 80 (DNA) and MGC: 12289, mRNA, complete cds. 208 (amino acid)
/FEA = mRNA /PROD = Similar to C- type (calcium dependent,
carbohydrate- recognition domain) lectin, superfamilymember 2
(activation- induced) /DB_XREF = gi: 13528920 /UG = Hs.85201 C-type
(calcium dependent, carbohydrate-recognition domain) lectin,
superfamily member 2 (activation-induced) /FL = gb: BC005254.1 gb:
AB015628.1 gb: NM_005127.1 FLNA: filamin A, alpha (actin Consensus
includes gb: AW051856 213746_s_at binding protein 280) (LOC2316)
/FEA = EST /DB_XREF = gi: 5914215 SEQ ID NOS: 81 (DNA) and /DB_XREF
= est: wz04a05.x1 209 (amino acid) /CLONE = IMAGE: 2557040 /UG =
Hs.195464 filamin A, alpha (actin-binding protein-280) CXCL5:
chemokine (C--X--C Consensus includes gb: AK026546.1 214974_x_at
motif) ligand 5 (LOC6374) /DEF = Homo sapiens cDNA: FLJ22893 SEQ ID
NOS: 82 (DNA) and fis, clone KAT04792. /FEA = mRNA 210 (amino acid)
/DB_XREF = gi: 10439427 /UG = Hs.287716 Homo sapiens cDNA: FLJ22893
fis, clone KAT04792 AEBP1: AE binding protein 1 gb: NM_001129.2
/DEF = Homo sapiens 201792_at (LOC165) AE-binding protein 1
(AEBP1), SEQ ID NOS: 83 (DNA) and mRNA. /FEA = mRNA /GEN = AEBP1
211 (amino acid) /PROD = adipocyte enhancer binding protein 1
precursor /DB_XREF = gi: 4755145 /UG = Hs.118397 AE-binding protein
1 /FL = gb: D86479.1 gb: AF053944.1 gb: NM_001129.2 BGN: biglycan
(LOC633) Consensus includes gb: AA845258 213905_x_at SEQ ID NOS: 84
(DNA) and /FEA = EST /DB_XREF = gi: 2931709 212 (amino acid)
/DB_XREF = est: ak84a11.s1 /CLONE = IMAGE: 1414556 /UG = Hs.821
biglycan SULF1: sulfatase 1 (LOC23213) Consensus includes gb:
AI479175 212353_at SEQ ID NOS: 85 (DNA) and /FEA = EST /DB_XREF =
gi: 4372343 213 (amino acid) /DB_XREF = est: tm55c05.x1 /CLONE =
IMAGE: 2162024 /UG = Hs.70823 KIAA1077 protein COL6A2: collagen,
type VI, gb: AY029208.1 /DEF = Homo sapiens 209156_s_at alpha 2
(LOC1292) type VI collagen alpha 2 chain SEQ ID NOS: 86 (DNA) and
precursor (COL6A2) mRNA, complete 214 (amino acid) cds,
alternatively spliced. /FEA = mRNA /GEN = COL6A2 /PROD = type VI
collagen alpha 2 chain precursor /DB_XREF = gi: 13603393 /UG =
Hs.159263 collagen, type VI, alpha 2 /FL = gb: AY029208.1 THBS2:
thrombospondin 2 gb: NM_003247.1 /DEF = Homo sapiens 203083_at
(LOC7058) thrombospondin 2 (THBS2), mRNA. SEQ ID NOS: 87 (DNA) and
/FEA = mRNA /GEN = THBS2 215 (amino acid) /PROD = thrombospondin 2
/DB_XREF = gi: 4507486 /UG = Hs.108623 thrombospondin 2 /FL = gb:
L12350.1 gb: NM_003247.1 PLCB4: phospholipase C, beta 4 gb:
NM_000933.1 /DEF = Homo sapiens 203896_s_at (LOC5332) phospholipase
C, beta 4 (PLCB4), SEQ ID NOS: 88 (DNA) and mRNA. /FEA = mRNA /GEN
= PLCB4 216 (amino acid) /PROD = phospholipase C, beta 4 /DB_XREF =
gi: 4505866 /UG = Hs.283006 phospholipase C, beta 4 /FL = gb:
NM_000933.1 gb: L41349.1 CALD1: caldesmon 1 (LOC800) gb:
NM_004342.2 /DEF = Homo sapiens 201617_x_at SEQ ID NOS: 89 (DNA)
and caldesmon 1 (CALD1), mRNA. 217 (amino acid) /FEA = mRNA /GEN =
CALD1
/PROD = caldesmon 1 /DB_XREF = gi: 11091984 /UG = Hs.325474
caldesmon 1 /FL = gb: NM_004342.2 gb: M64110.1 NGFRAP1: nerve
growth factor gb: NM_014380.1 /DEF = Homo sapiens 217963_s_at
receptor (TNFRSF16) associated p75NTR-associated cell death protein
1 (LOC27018) executor; ovarian granulosa cell protein SEQ ID NOS:
90 (DNA) and (13 kD) (DXS6984E), mRNA. 218 (amino acid) /FEA = mRNA
/GEN = DXS6984E /PROD = p75NTR-associated cell death executor;
ovariangranulosa cell protein (13 kD) /DB_XREF = gi: 7657043 /UG =
Hs.17775 p75NTR-associated cell death executor; ovarian granulosa
cell protein (13 kD) /FL = gb: NM_014380.1 gb: AF187064.1 IFI16:
interferon, gamma- Consensus includes gb: BG256677 208965_s_at
inducible protein 16 (LOC3428) /FEA = EST /DB_XREF = gi: 12766493
SEQ ID NOS: 91 (DNA) and /DB_XREF = est: 602370865F1 219 (amino
acid) /CLONE = IMAGE: 4478872 /UG = Hs.155530 interferon, gamma-
inducible protein 16 /FL = gb: AF208043.1 RAB31: RAB31, member RAS
gb: NM_006868.1 /DEF = Homo sapiens 217763_s_at oncogene family
(LOC11031) RAB31, member RAS oncogene family SEQ ID NOS: 92 (DNA)
and (RAB31), mRNA. /FEA = mRNA 220 (amino acid) /GEN = RAB31 /PROD
= RAB31, member RAS oncogene family /DB_XREF = gi: 5803130 /UG =
Hs.223025 RAB31, member RAS oncogene family /FL = gb: AF234995.1
gb: BC001148.1 gb: U59877.1 gb: U57091.1 gb: NM_006868.1 gb:
AF183421.1 COL5A1: collagen, type V, Consensus includes gb:
AI130969 203325_s_at alpha 1 (LOC1289) /FEA = EST /DB_XREF = gi:
3600985 SEQ ID NOS: 93 (DNA) and /DB_XREF = est: qc15e01.x1 221
(amino acid) /CLONE = IMAGE: 1709688 /UG = Hs.146428 collagen, type
V, alpha 1 /FL = gb: M76729.1 gb: D90279.1 gb: NM_000093.1 KLK10:
kallikrein 10 gb: BC002710.1 /DEF = Homo sapiens, 209792_s_at
(LOC5655) kallikrein 10, clone MGC: 3667, SEQ ID NOS: 94 (DNA) and
mRNA, complete cds. /FEA = mRNA 222 (amino acid) /PROD = kallikrein
10 /DB_XREF = gi: 12803744 /UG = Hs.69423 kallikrein 10 /FL = gb:
BC002710.1 PCP4: Purkinje cell protein 4 gb: NM_006198.1 /DEF =
Homo sapiens 205549_at (LOC5121) Purkinje cell protein 4 (PCP4),
mRNA. SEQ ID NOS: 95 (DNA) and /FEA = mRNA /GEN = PCP4 223 (amino
acid) /PROD = Purkinje cell protein 4 /DB_XREF = gi: 5453857 /UG =
Hs.80296 Purkinje cell protein 4 /FL = gb: U52969.1 gb: NM_006198.1
NR4A2: nuclear receptor gb: NM_006186.1 /DEF = Homo sapiens
204622_x_at subfamily 4, group A, member 2 nuclear receptor
subfamily 4, group A, (LOC4929) member 2 (NR4A2), mRNA. SEQ ID NOS:
96 (DNA) and /FEA = mRNA /GEN = NR4A2 224 (amino acid) /PROD =
nuclear receptor subfamily 4, group A, member 2 /DB_XREF = gi:
5453821 /UG = Hs.82120 nuclear receptor subfamily 4, group A,
member 2 /FL = gb: NM_006186.1 IGFBP3: insulin-like growth gb:
M31159.1 /DEF = Human growth 210095_s_at factor binding protein 3
hormone-dependent insulin-like growth (LOC3486) factor-binding
protein mRNA, SEQ ID NOS: 97 (DNA) and complete cds. /FEA = mRNA
225 (amino acid) /GEN = IGFBP1 /DB_XREF = gi: 183115 /UG = Hs.77326
insulin-like growth factor binding protein 3 /FL = gb: BC000013.1
gb: M31159.1 STAT1: signal transducer and gb: BC002704.1 /DEF =
Homo sapiens, 209969_s_at activator of transcription 1, Similar to
signal transducer and 91 kDa (LOC6772) activator of transcription
1, 91 kD, SEQ ID NOS: 98 (DNA) and clone MGC: 3493, mRNA, complete
226 (amino acid) cds. /FEA = mRNA /PROD = Similar to signal
transducer and activator oftranscription 1, 91 kD /DB_XREF = gi:
12803734 /UG = Hs.21486 signal transducer and activator of
transcription 1, 91 kD /FL = gb: BC002704.1 CYP1B1: cytochrome
P450, Consensus includes gb: AU144855 202436_s_at family 1,
subfamily B, /FEA = EST /DB_XREF = gi: 11006376 polypeptide 1
(LOC1545) /DB_XREF = est: AU144855 SEQ ID NOS: 99 (DNA) and /CLONE
= HEMBA1003161 227 (amino acid) /UG = Hs.154654 cytochrome P450,
subfamily I (dioxin-inducible), polypeptide 1 (glaucoma 3, primary
infantile) /FL = gb: NM_000104.2 gb: U03688.1 COL1A1: collagen,
type I, alpha Consensus includes gb: AI743621 202311_s_at 1
(LOC1277) /FEA = EST /DB_XREF = gi: 5111909 SEQ ID NOS: 100 (DNA)
and /DB_XREF = est: wg51h09.x1 228 (amino acid) /CLONE = IMAGE:
2368673 /UG = Hs.172928 collagen, type I, alpha 1 /FL = gb:
NM_000088.1 DKFZP434F0318: hypothetical gb: NM_030817.1 /DEF = Homo
sapiens 221031_s_at protein DKFZp434F0318 hypothetical protein
DKFZp434F0318 (LOC81575) (DKFZP434F0318), mRNA. SEQ ID NOS: 101
(DNA) and /FEA = mRNA /GEN = DKFZP434F0318 229 (amino acid) /PROD =
hypothetical protein DKFZp434F0318 /DB_XREF = gi: 13540611 /FL =
gb: NM_030817.1 TUBA3: tubulin, alpha 3 gb: AF141347.1 /DEF = Homo
sapiens 209118_s_at (LOC7846) hum-a-tub2 alpha-tubulin mRNA, SEQ ID
NOS: 102 (DNA) and complete cds. /FEA = mRNA 230 (amino acid) /PROD
= alpha-tubulin /DB_XREF = gi: 4929133 /UG = Hs.272897 Tubulin,
alpha, brain- specific /FL = gb: AF141347.1 gb: NM_006009.1 GZMB:
granzyme B (granzyme gb: J03189.1 /DEF = Human proteolytic
210164_at 2, cytotoxic T-lymphocyte- serine esterase-like protein
(SECT) associated serine esterase 1) gene, complete cds. /FEA =
mRNA (LOC3002) /DB_XREF = gi: 338010 /UG = Hs.1051 SEQ ID NOS: 103
(DNA) and granzyme B (granzyme 2, cytotoxic T- 231 (amino acid)
lymphocyte-associated serine esterase 1) /FL = gb: J04071.1 gb:
J03189.1 gb: M17016.1 gb: NM_004131.2 ROBO1: roundabout, axon
Consensus includes gb: BF059159 213194_at guidance receptor,
homolog 1 /FEA = EST /DB_XREF = gi: 10813055 (Drosophila) (LOC6091)
/DB_XREF = est: 7k66g04.x1 SEQ ID NOS: 104 (DNA) and /CLONE =
IMAGE: 3480391 232 (amino acid) /UG = Hs.301198 roundabout (axon
guidance receptor, Drosophila) homolog 1 /FL = gb: AF040990.1 gb:
NM_002941.1 CHGA: chromogranin A gb: NM_001275.2 /DEF = Homo
sapiens 204697_s_at (parathyroid secretory protein 1) chromogranin
A (parathyroid secretory (LOC1113) protein 1) (CHGA), mRNA. SEQ ID
NOS: 105 (DNA) and /FEA = mRNA /GEN = CHGA 233 (amino acid) /PROD =
chromogranin A /DB_XREF = gi: 10800418 /UG = Hs.172216 chromogranin
A (parathyroid secretory protein 1) /FL = gb: NM_001275.2 gb:
BC001059.1 gb: J03483.1 gb: J03915.1 SLC7A8: solute carrier family
7 gb: NM_012244.1 /DEF = Homo sapiens 202752_x_at (cationic amino
acid transporter, solute carrier family 7 (cationic amino y+
system), member 8 acid transporter, y+ system), member 8 (LOC23428)
(SLC7A8), mRNA. /FEA = mRNA SEQ ID NOS: 106 (DNA) and /GEN = SLC7A8
/PROD = solute carrier 234 (amino acid) family 7 (cationic amino
acidtransporter, y+ system), member 8 /DB_XREF = gi: 6912669 /UG =
Hs.22891 solute carrier family 7 (cationic amino acid transporter,
y+ system), member 8 /FL = gb: AB037669.1 gb: AF171669.1 gb:
NM_012244.1 GPA33: glycoprotein A33 gb: NM_005814.1 /DEF = Homo
sapiens 205929_at (transmembrane) (LOC10223) glycoprotein A33
(transmembrane) SEQ ID NOS: 107 (DNA) and (GPA33), mRNA. /FEA =
mRNA 235 (amino acid) /GEN = GPA33 /PROD = transmembrane
glycoprotein A33 precursor /DB_XREF = gi: 5031560 /UG = Hs.143131
glycoprotein A33 (transmembrane) /FL = gb: U79725.1 gb: NM_005814.1
QPRT: quinolinate gb: NM_014298.2 /DEF = Homo sapiens 204044_at
phosphoribosyltransferase quinolinate phosphoribosyltransferase
(nicotinate-nucleotide (nicotinate-nucleotide pyrophosphorylase
pyrophosphorylase (carboxylating)) (carboxylating)) (LOC23475)
(QPRT), mRNA. /FEA = mRNA SEQ ID NOS: 108 (DNA) and /GEN = QPRT
/PROD = quinolinate 236 (amino acid) phosphoribosyltransferase
/DB_XREF = gi: 9257236 /UG = Hs.8935 quinolinate
phosphoribosyltransferase (nicotinate-nucleotide pyrophosphorylase
(carboxylating)) /FL = gb: D78177.1 gb: BC005060.1 gb: NM_014298.2
DDC: dopa decarboxylase gb: NM_000790.1 /DEF = Homo sapiens
205311_at (aromatic L-amino acid dopa decarboxylase (aromatic
L-amino decarboxylase) (LOC1644) acid decarboxylase) (DDC), mRNA.
SEQ ID NOS: 109 (DNA) and /FEA = mRNA /GEN = DDC 237 (amino acid)
/PROD = dopa decarboxylase (aromatic L-amino aciddecarboxylase)
/DB_XREF = gi: 4503280 /UG = Hs.150403 dopa decarboxylase (aromatic
L-amino acid decarboxylase) /FL = gb: BC000485.1 gb: M76180.1 gb:
M88700.1 gb: NM_000790.1 COL11A1: collagen, type XI, gb:
NM_001854.1 /DEF = Homo sapiens 204320_at alpha 1 (LOC1301)
collagen, type XI, alpha 1 (COL11A1), SEQ ID NOS: 110 (DNA) and
mRNA. /FEA = mRNA 238 (amino acid) /GEN = COL11A1 /PROD = collagen,
type XI, alpha 1 /DB_XREF = gi: 4502938 /UG = Hs.82772 collagen,
type XI, alpha 1 /FL = gb: J04177.1 gb: NM_001854.1 C2orf23:
chromosome 2 open Consensus includes gb: BE535746 204364_s_at
reading frame 23 (LOC65055) /FEA = EST /DB_XREF = gi: 9764391 SEQ
ID NOS: 111 (DNA) and /DB_XREF = est: 601060419F1 239 (amino acid)
/CLONE = IMAGE: 3446788 /UG = Hs.7358 hypothetical protein FLJ13110
/FL = gb: NM_022912.1 SULF1: sulfatase 1 (LOC23213) Consensus
includes gb: BE500977 212354_at SEQ ID NOS: 112 (DNA) and /FEA =
EST /DB_XREF = gi: 9703385 240 (amino acid) /DB_XREF = est:
7a33h02.x1 /CLONE = IMAGE: 3220563 /UG = Hs.70823 KIAA1077 protein
PCOLCE: procollagen C- gb: NM_002593.2 /DEF = Homo sapiens
202465_at endopeptidase enhancer procollagen C-endopeptidase
enhancer (LOC5118) (PCOLCE), mRNA. /FEA = mRNA SEQ ID NOS: 113
(DNA) and /GEN = PCOLCE /PROD = procollagen 241 (amino acid)
C-endopeptidase enhancer /DB_XREF = gi: 7262388 /UG = Hs.202097
procollagen C- endopeptidase enhancer /FL = gb: BC000574.1 gb:
AB008549.1 gb: L33799.1 gb: NM_002593.2 C14orf78: chromosome 14
open Consensus includes gb: AI935123 212992_at reading frame 78
(LOC113146) /FEA = EST /DB_XREF = gi: 5673993 SEQ ID NOS: 114 (DNA)
and /DB_XREF = est: wp13h09.x1 242 (amino acid) /CLONE = IMAGE:
2464769 /UG = Hs.57548 ESTs CXCR4: chemokine (C--X--C gb: L01639.1
/DEF = Human (clone 209201_x_at motif) receptor 4 (LOC7852) HSY3RR)
neuropeptide Y receptor SEQ ID NOS: 115 (DNA) and (NPYR) mRNA,
complete cds. 243 (amino acid) /FEA = mRNA /GEN = NPYR /PROD =
neuropeptide Y receptor /DB_XREF = gi: 189313 /UG = Hs.89414
chemokine (C--X--C motif), receptor 4 (fusin) /FL = gb: L01639.1
gb: AF025375.1 gb: M99293.1 gb: L06797.1 gb: NM_003467.1 gb:
AF147204.1 CSPG2: chondroitin sulfate Consensus includes gb: R94644
215646_s_at proteoglycan 2 (versican) /FEA = EST /DB_XREF = gi:
970039 (LOC1462) /DB_XREF = est: yq42a12.r1 SEQ ID NOS: 116 (DNA)
and /CLONE = IMAGE: 198430 244 (amino acid) /UG = Hs.306542 Homo
sapiens versican Vint isoform, mRNA, partial cds SERPINF1: serine
(or cysteine) gb: NM_002615.1 /DEF = Homo sapiens 202283_at
proteinase inhibitor, clade F serine (or cysteine) proteinase
inhibitor, (alpha-2 antiplasmin, pigment clade F (alpha-2
antiplasmin, pigment epithelium derived factor), epithelium derived
factor), member 1
member 1 (LOC5176) (SERPINF1), mRNA. /FEA = mRNA SEQ ID NOS: 117
(DNA) and /GEN = SERPINF1 /PROD = serine (or 245 (amino acid)
cysteine) proteinase inhibitor, cladeF (alpha-2 antiplasmin,
pigment epithelium derivedfactor), member 1 /DB_XREF = gi: 4505708
/UG = Hs.173594 serine (or cysteine) proteinase inhibitor, clade F
(alpha-2 antiplasmin, pigment epithelium derived factor), member 1
/FL = gb: M90439.1 gb: BC000522.1 gb: M76979.1 gb: NM_002615.1
SPON1: spondin 1, extracellular Consensus includes gb: AB018305.1
209436_at matrix protein (LOC10418) /DEF = Homo sapiens mRNA for
SEQ ID NOS: 118 (DNA) and KIAA0762 protein, partial cds. 246 (amino
acid) /FEA = mRNA /GEN = KIAA0762 /PROD = KIAA0762 protein /DB_XREF
= gi: 3882244 /UG = Hs.5378 spondin 1, (f-spondin) extracellular
matrix protein /FL = gb: AB051390.1 COL11A1: collagen, type XI,
Cluster Incl. J04177: Human alpha-1 37892_at alpha 1 (LOC1301) type
XI collagen (COL11A1) mRNA, SEQ ID NOS: 119 (DNA) and complete cds
/cds = (161,5581) 247 (amino acid) /gb = J04177 /gi = 179729 /ug =
Hs.82772 /len = 6158 MAFB: v-maf gb: NM_005461.1 /DEF = Homo
sapiens 218559_s_at musculoaponeurotic Kreisler (mouse) maf-related
leucine fibrosarcoma oncogene homolog zipper homolog (KRML), mRNA.
B (avian) (LOC9935) /FEA = mRNA /GEN = KRML SEQ ID NOS: 120 (DNA)
and /PROD = Kreisler (mouse) maf-related 248 (amino acid) leucine
zipperhomolog /DB_XREF = gi: 4885446 /UG = Hs.169487 Kreisler
(mouse) maf- related leucine zipper homolog /FL = gb: AF134157.1
gb: NM_005461.1 DDX17: DEAD (Asp-Glu-Ala- Consensus includes gb:
AW188131 213998_s_at Asp) box polypeptide 17 /FEA = EST /DB_XREF =
gi: 6462567 (LOC10521) /DB_XREF = est: xj92f11.x1 SEQ ID NOS: 121
(DNA) and /CLONE = IMAGE: 2664717 249 (amino acid) /UG = Hs.6179
DEADH (Asp-Glu-Ala- AspHis) box polypeptide 17 (72 kD) PHLDA1:
pleckstrin homology- Consensus includes gb: NM_007350.1 217999_s_at
like domain, family A, member /DEF = Homo sapiens pleckstrin 1
(LOC22822) homology-like domain, family A, SEQ ID NOS: 122 (DNA)
and member 1 (PHLDA1), mRNA. 250 (amino acid) /FEA = mRNA /GEN =
PHLDA1 /PROD = pleckstrin homology-like domain, family A, member 1
/DB_XREF = gi: 6679302 /UG = Hs.82101 pleckstrin homology- like
domain, family A, member 1 /FL = gb: NM_007350.1 ETV5: ets variant
gene 5 (ets- gb: NM_004454.1 /DEF = Homo sapiens 203349_s_at
related molecule) (LOC2119) ets variant gene 5 (ets-related SEQ ID
NOS: 123 (DNA) and molecule) (ETV5), mRNA. 251 (amino acid) /FEA =
mRNA /GEN = ETV5 /PROD = ets variant gene 5 (ets-related molecule)
/DB_XREF = gi: 4758315 /UG = Hs.43697 ets variant gene 5 (ets-
related molecule) /FL = gb: NM_004454.1 DUSP4: dual specificity gb:
BC002671.1 /DEF = Homo sapiens, 204015_s_at phosphatase 4 (LOC1846)
dual specificity phosphatase 4, clone SEQ ID NOS: 124 (DNA) and
MGC: 3713, mRNA, complete cds. 252 (amino acid) /FEA = mRNA /PROD =
dual specificity phosphatase 4 /DB_XREF = gi: 12803670 /UG =
Hs.2359 dual specificity phosphatase 4 /FL = gb: U48807.1 gb:
NM_001394.2 gb: BC002671.1 gb: U21108.1 DUSP4: dual specificity gb:
NM_001394.2 /DEF = Homo sapiens 204014_at phosphatase 4 (LOC1846)
dual specificity phosphatase 4 SEQ ID NOS: 125 (DNA) and (DUSP4),
mRNA. /FEA = mRNA 253 (amino acid) /GEN = DUSP4 /PROD = dual
specificity phosphatase 4 /DB_XREF = gi: 12707552 /UG = Hs.2359
dual specificity phosphatase 4 /FL = gb: U48807.1 gb: NM_001394.2
gb: BC002671.1 gb: U21108.1 POFUT1: protein O- Consensus includes
gb: AL045513 212349_at fucosyltransferase 1 /FEA = EST /DB_XREF =
gi: 5433649 (LOC23509) /DB_XREF = est: DKFZp434J015_r1 SEQ ID NOS:
126 (DNA) and /CLONE = DKFZp434J015 254 (amino acid) /UG =
Hs.178292 KIAA0180 protein TBXAS1: thromboxane A gb: NM_030984.1
/DEF = Homo sapiens 208130_s_at synthase 1 (platelet, cytochrome
thromboxane A synthase 1 (platelet, P450, family 5, subfamily A)
cytochrome P450, subfamily V) (LOC6916) (TBXAS1), transcript
variant TXS-II, SEQ ID NOS: 127 (DNA) and mRNA. /FEA = mRNA 255
(amino acid) /GEN = TBXAS1 /PROD = thromboxane A synthase 1
(platelet, cytochromeP450, subfamily V), isoform TXS-II /DB_XREF =
gi: 13699839 /FL = gb: NM_030984.1 KCNK5: potassium channel, gb:
NM_003740.1 /DEF = Homo sapiens 219615_s_at subfamily K, member 5
potassium channel, subfamily K, (LOC8645) member 5 (TASK-2)
(KCNK5), SEQ ID NOS: 128 (DNA) and mRNA. /FEA = mRNA /GEN = KCNK5
256 (amino acid) /PROD = potassium channel, subfamily K, member
5(TASK-2) /DB_XREF = gi: 4504850 /UG = Hs.127007 potassium channel,
subfamily K, member 5 (TASK-2) /FL = gb: AF084830.1 gb:
NM_003740.1
[0054] The biomarkers provided in Table 1, which include the
nucleotide sequences of SEQ ID NOS:1-128 and the amino acid
sequences of SEQ ID NOS:129-256, are referred to herein as a total
of 128 biomarkers with reference to the Unigene Title.
[0055] The biomarkers have expression levels in cells that may be
dependent on the activity of the EGFR signal transduction pathway,
and that are also highly correlated with EGFR modulator sensitivity
exhibited by the cells. Biomarkers serve as useful molecular tools
for predicting the likelihood of a response to EGFR modulators,
preferably biological molecules, small molecules, and the like that
affect EGFR kinase activity via direct or indirect inhibition or
antagonism of EGFR kinase function or activity.
Wild Type K-Ras and Mutated K-Ras
[0056] As used herein, wild type K-Ras can be selected from the
K-Ras variant a and variant b nucleotide and amino acid sequences.
Wild type K-Ras variant a has a nucleotide sequence that is 5436
nucleotides (GenBank Accession No. NM.sub.--033360.2) and encodes a
protein that is 189 amino acids (GenBank Accession No.
NP.sub.--203524.1). Wild type K-Ras variant b has a nucleotide
sequence that is 5312 nucleotides (GenBank Accession No.
NM.sub.--004985.3) and encodes a protein that is 188 amino acids
(GenBank Accession No. NP.sub.--004976.2).
[0057] A mutated form of K-Ras is a nucleotide or amino acid
sequence that differs from wild type K-Ras at least at one
position, preferably at least one nucleotide position that encodes
an amino acid that differs from wild type K-Ras. In one aspect, the
mutated form of K-Ras includes at least one mutation in exon 2. In
another aspect, the mutated form of K-RAS includes at least one of
the following mutations in exon 2 (base change (amino acid
change)): 200G>A (V7M); 216G>C (G12A); 215G>T (G12C);
216G>A (G12D); 215G>C (G12R); 215G>A (G12S); 216G>T
(G12V); 218G>T (G13C); 219G>A (G13D).
[0058] Methods for detecting K-Ras mutations are well known in the
art and include, for example, the methods described in PCT
Publication No. Wo2005/118876.
EGFR Modulators
[0059] As used herein, the term "EGFR modulator" is intended to
mean a compound or drug that is a biological molecule or a small
molecule that directly or indirectly modulates EGFR activity or the
EGFR signal transduction pathway. Thus, compounds or drugs as used
herein is intended to include both small molecules and biological
molecules. Direct or indirect modulation includes activation or
inhibition of EGFR activity or the EGFR signal transduction
pathway. In one aspect, inhibition refers to inhibition of the
binding of EGFR to an EGFR ligand such as, for example, EGF. In
another aspect, inhibition refers to inhibition of the kinase
activity of EGFR.
[0060] EGFR modulators include, for example, EGFR-specific ligands,
small molecule EGFR inhibitors, and EGFR monoclonal antibodies. In
one aspect, the EGFR modulator inhibits EGFR activity and/or
inhibits the EGFR signal transduction pathway. In another aspect,
the EGFR modulator is an EGFR monoclonal antibody that inhibits
EGFR activity and/or inhibits the EGFR signal transduction
pathway.
[0061] EGFR modulators include biological molecules or small
molecules. Biological molecules include all lipids and polymers of
monosaccharides, amino acids, and nucleotides having a molecular
weight greater than 450. Thus, biological molecules include, for
example, oligosaccharides and polysaccharides; oligopeptides,
polypeptides, peptides, and proteins; and oligonucleotides and
polynucleotides. Oligonucleotides and polynucleotides include, for
example, DNA and RNA.
[0062] Biological molecules further include derivatives of any of
the molecules described above. For example, derivatives of
biological molecules include lipid and glycosylation derivatives of
oligopeptides, polypeptides, peptides, and proteins.
[0063] Derivatives of biological molecules further include lipid
derivatives of oligosaccharides and polysaccharides, e.g.,
lipopolysaccharides. Most typically, biological molecules are
antibodies, or functional equivalents of antibodies. Functional
equivalents of antibodies have binding characteristics comparable
to those of antibodies, and inhibit the growth of cells that
express EGFR. Such functional equivalents include, for example,
chimerized, humanized, and single chain antibodies as well as
fragments thereof.
[0064] Functional equivalents of antibodies also include
polypeptides with amino acid sequences substantially the same as
the amino acid sequence of the variable or hypervariable regions of
the antibodies. An amino acid sequence that is substantially the
same as another sequence, but that differs from the other sequence
by means of one or more substitutions, additions, and/or deletions,
is considered to be an equivalent sequence. Preferably, less than
50%, more preferably less than 25%, and still more preferably less
than 10%, of the number of amino acid residues in a sequence are
substituted for, added to, or deleted from the protein.
[0065] The functional equivalent of an antibody is preferably a
chimerized or humanized antibody. A chimerized antibody comprises
the variable region of a non-human antibody and the constant region
of a human antibody. A humanized antibody comprises the
hypervariable region (CDRs) of a non-human antibody. The variable
region other than the hypervariable region, e.g., the framework
variable region, and the constant region of a humanized antibody
are those of a human antibody.
[0066] Suitable variable and hypervariable regions of non-human
antibodies may be derived from antibodies produced by any non-human
mammal in which monoclonal antibodies are made. Suitable examples
of mammals other than humans include, for example, rabbits, rats,
mice, horses, goats, or primates.
[0067] Functional equivalents further include fragments of
antibodies that have binding characteristics that are the same as,
or are comparable to, those of the whole antibody. Suitable
fragments of the antibody include any fragment that comprises a
sufficient portion of the hypervariable (i.e., complementarity
determining) region to bind specifically, and with sufficient
affinity, to EGFR tyrosine kinase to inhibit growth of cells that
express such receptors.
[0068] Such fragments may, for example, contain one or both Fab
fragments or the F(ab').sub.2 fragment. Preferably, the antibody
fragments contain all six complementarity determining regions of
the whole antibody, although functional fragments containing fewer
than all of such regions, such as three, four, or five CDRs, are
also included.
[0069] In one aspect, the fragments are single chain antibodies, or
Fv fragments. Single chain antibodies are polypeptides that
comprise at least the variable region of the heavy chain of the
antibody linked to the variable region of the light chain, with or
without an interconnecting linker. Thus, Fv fragment comprises the
entire antibody combining site. These chains may be produced in
bacteria or in eukaryotic cells.
[0070] The antibodies and functional equivalents may be members of
any class of immunoglobulins, such as IgG, IgM, IgA, IgD, or IgE,
and the subclasses thereof. In one aspect, the antibodies are
members of the IgG1 subclass. The functional equivalents may also
be equivalents of combinations of any of the above classes and
subclasses.
[0071] In one aspect, EGFR antibodies can be selected from
chimerized, humanized, fully human, and single chain antibodies
derived from the murine antibody 225 described in U.S. Pat. No.
4,943,533.
[0072] In another aspect, the EGFR antibody is cetuximab (IMC-C225)
which is a chimeric (human/mouse) IgG monoclonal antibody, also
known under the tradename ERBITUX. Cetuximab Fab contains the Fab
fragment of cetuximab, i.e., the heavy and light chain variable
region sequences of murine antibody M225 (U.S. Application No.
2004/0006212, incorporated herein by reference) with human IgG1
C.sub.H1 heavy and kappa light chain constant domains. Cetuximab
includes all three IgG1 heavy chain constant domains.
[0073] In another aspect, the EGFR antibody can be selected from
the antibodies described in U.S. Pat. No. 6,235,883, U.S. Pat. No.
5,558,864, and U.S. Pat. No. 5,891,996. The EGFR antibody can be,
for example, AGX-EGF (Amgen Inc.) (also known as panitumumab) which
is a fully human IgG2 monoclonal antibody. The sequence and
characterization of ABX-EGF, which was formerly known as clone
E7.6.3, is disclosed in U.S. Pat. No. 6,235,883 at column 28, line
62 through column 29, line 36 and FIGS. 29-34, which is
incorporated by reference herein. The EGFR antibody can also be,
for example, EMD72000 (Merck KGaA), which is a humanized version of
the murine EGFR antibody EMD 55900. The EGFR antibody can also be,
for example: h-R3 (TheraCIM), which is a humanized EGFR monoclonal
antibody; Y10 which is a murine monoclonal antibody raised against
a murine homologue of the human EGFRvIII mutation; or MDX-447
(Medarex Inc.).
[0074] In addition to the biological molecules discussed above, the
EGFR modulators useful in the invention may also be small
molecules. Any molecule that is not a biological molecule is
considered herein to be a small molecule. Some examples of small
molecules include organic compounds, organometallic compounds,
salts of organic and organometallic compounds, saccharides, amino
acids, and nucleotides. Small molecules further include molecules
that would otherwise be considered biological molecules, except
their molecular weight is not greater than 450. Thus, small
molecules may be lipids, oligosaccharides, oligopeptides, and
oligonucleotides and their derivatives, having a molecular weight
of 450 or less.
[0075] It is emphasized that small molecules can have any molecular
weight. They are merely called small molecules because they
typically have molecular weights less than 450. Small molecules
include compounds that are found in nature as well as synthetic
compounds. In one embodiment, the EGFR modulator is a small
molecule that inhibits the growth of tumor cells that express EGFR.
In another embodiment, the EGFR modulator is a small molecule that
inhibits the growth of refractory tumor cells that express
EGFR.
[0076] Numerous small molecules have been described as being useful
to inhibit EGFR.
[0077] One example of a small molecule EGFR antagonist is IRESSA
(ZD1939), which is a quinozaline derivative that functions as an
ATP-mimetic to inhibit EGFR. See, U.S. Pat. No. 5,616,582; WO
96/33980 at page 4. Another example of a small molecule EGFR
antagonist is TARCEVA (OSI-774), which is a 4-(substituted
phenylamino)quinozaline derivative
[6,7-Bis(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-1-phenyl)amine
hydrochloride] EGFR inhibitor. See WO 96/30347 (Pfizer Inc.) at,
for example, page 2, line 12 through page 4, line 34 and page 19,
lines 14-17. TARCEVA may function by inhibiting phosphorylation of
EGFR and its downstream PI3/Akt and MAP (mitogen activated protein)
kinase signal transduction pathways resulting in p27-mediated
cell-cycle arrest. See Hidalgo et al., Abstract 281 presented at
the 37th Annual Meeting of ASCO, San Francisco, Calif., 12-15 May
2001.
[0078] Other small molecules are also reported to inhibit EGFR,
many of which are thought to be specific to the tyrosine kinase
domain of an EGFR. Some examples of such small molecule EGFR
antagonists are described in WO 91/116051, WO96/30347, WO96/33980,
WO97/27199. WO97/30034, WO97/42187, WO97/49688, WO98/33798,
WO00/18761, and WO00/31048. Examples of specific small molecule
EGFR antagonists include C1-1033 (Pfizer Inc.), which is a
quinozaline
(N-[4-(3-chloro-4-fluoro-phenylamino)-7-(3-mprpholin-4-yl-propoxy)-quinaz-
olin-6-yl]-acrylamide) inhibitor of tyrosine kinases, particularly
EGFR and is described in WO00/31048 at page 8, lines 22-6; PKI166
(Novartis), which is a pyrrolopyrimidine inhibitor of EGFR and is
described in WO97/27199 at pages 10-12; GW2016 (GlaxoSmitbKline),
which is an inhibitor of EGFR and HER2; EKB569 (Wyeth), which is
reported to inhibit the growth of tumor cells that overexpress EGFR
or HER2 in vitro and in vivo; AG-1478 (Tryphostin), which is a
quinazoline small molecule that inhibits signaling from both EGFR
and erbB-2; AG-1478 (Sugen), which is a bisubstrate inhibitor that
also inhibits protein kinase CK2; PD 153035 (Parke-Davis) which is
reported to inhibit EGFR kinase activity and tumor growth, induce
apoptosis in cells in culture, and enhance the cytotoxicity of
cytotoxic chemotherapeutic agents; SPM-924 (Schwarz Pharma), which
is a tyrosine kinase inhibitor targeted for treatment of prostrate
cancer; CP-546,989 (OSI Pharmaceuticals), which is reportedly an
inhibitor of angiogenesis for treatment of solid tumors; ADL-681,
which is a EGFR kinase inhibitor targeted for treatment of cancer;
PD 158780, which is a pyridopyrimidine that is reported to inhibit
the tumor growth rate of A4431 xenografts in mice; CP-358,774,
which is a quinzoline that is reported to inhibit
autophosphorylation in HN5 xenografts in mice; ZD1839, which is a
quinzoline that is reported to have antitumor activity in mouse
xenograft models including vulvar, NSCLC, prostrate, ovarian, and
colorectal cancers; CGP 59326A, which is a pyrrolopyrimidine that
is reported to inhibit growth of EGFR-positive xenografts in mice;
PD 165557 (Pfizer); CGP54211 and CGP53353 (Novartis), which are
dianilnophthalimides. Naturally derived EGFR tyrosine kinase
inhibitors include genistein, herbimycin A, quercetin, and
erbstatin.
[0079] Further small molecules reported to inhibit EGFR and that
are therefore within the scope of the present invention are
tricyclic compounds such as the compounds described in U.S. Pat.
No. 5,679,683; quinazoline derivatives such as the derivatives
described in U.S. Pat. No. 5,616,582; and indole compounds such as
the compounds described in U.S. Pat. No. 5,196,446.
[0080] Further small molecules reported to inhibit EGFR and that
are therefore within the scope of the present invention are styryl
substituted heteroaryl compounds such as the compounds described in
U.S. Pat. No. 5,656,655. The heteroaryl group is a monocyclic ring
with one or two heteroatoms, or a bicyclic ring with 1 to about 4
heteroatoms, the compound being optionally substituted or
polysubstituted.
[0081] Further small molecules reported to inhibit EGFR and that
are therefore within the scope of the present invention are bis
mono and/or bicyclic aryl heteroaryl, carbocyclic, and
heterocarbocyclic compounds described in U.S. Pat. No.
5,646,153.
[0082] Further small molecules reported to inhibit EGFR and that
are therefore within the scope of the present invention is the
compound provided FIG. 1 of Fry et al., Science 265,1093-1095
(1994) that inhibits EGFR.
[0083] Further small molecules reported to inhibit EGFR and that
are therefore within the scope of the present invention are
tyrphostins that inhibit EGFR/HER1 and HER 2, particularly those in
Tables I, II, III, and IV described in Osherov et al., J. Biol.
Chem., 25;268(15):11134-42 (1993).
[0084] Further small molecules reported to inhibit EGFR and that
are therefore within the scope of the present invention is a
compound identified as PD166285 that inhibits the EGFR, PDGFR, and
FGFR families of receptors. PD166285 is identified as
6-(2,6-dichlorophenyl)-2-(4-(2-diethylaminoethyoxy)phenylamino)-8-methyl--
8H-pyrido(2,3-d)pyrimidin-7-one having the structure shown in FIG.
1 on page 1436 of Panek et al., Journal of Pharmacology and
Experimental Therapeutics 283, 1433-1444 (1997).
[0085] It should be appreciated that useful small molecule to be
used in the invention are inhibitors of EGFR, but need not be
completely specific for EGFR.
Biomarkers and Biomarker Sets
[0086] The invention includes individual biomarkers and biomarker
sets having both diagnostic and prognostic value in disease areas
in which signaling through EGFR or the EGFR pathway is of
importance, e.g., in cancers or tumors, in immunological disorders,
conditions or dysfunctions, or in disease states in which cell
signaling and/or cellular proliferation controls are abnormal or
aberrant. The biomarker sets comprise a plurality of biomarkers
such as, for example, a plurality of the biomarkers provided in
Table 1, that highly correlate with resistance or sensitivity to
one or more EGFR modulators.
[0087] The biomarkers and biomarker sets of the invention enable
one to predict or reasonably foretell the likely effect of one or
more EGFR modulators in different biological systems or for
cellular responses. The biomarkers and biomarker sets can be used
in in vitro assays of EGFR modulator response by test cells to
predict in vivo outcome. In accordance with the invention, the
various biomarkers and biomarker sets described herein, or the
combination of these biomarker sets with other biomarkers or
markers, can be used, for example, to predict how patients with
cancer might respond to therapeutic intervention with one or more
EGFR modulators.
[0088] A biomarker and biomarker set of cellular gene expression
patterns correlating with sensitivity or resistance of cells
following exposure of the cells to one or more EGFR modulators
provides a useful tool for screening one or more tumor samples
before treatment with the EGFR modulator. The screening allows a
prediction of cells of a tumor sample exposed to one or more EGFR
modulators, based on the expression results of the biomarker and
biomarker set, as to whether or not the tumor, and hence a patient
harboring the tumor, will or will not respond to treatment with the
EGFR modulator.
[0089] The biomarker or biomarker set can also be used as described
herein for monitoring the progress of disease treatment or therapy
in those patients undergoing treatment for a disease involving an
EGFR modulator.
[0090] The biomarkers also serve as targets for the development of
therapies for disease treatment. Such targets may be particularly
applicable to treatment of colorectal cancer. Indeed, because these
biomarkers are differentially expressed in sensitive and resistant
cells, their expression patterns are correlated with relative
intrinsic sensitivity of cells to treatment with EGFR modulators.
Accordingly, the biomarkers highly expressed in resistant cells may
serve as targets for the development of new therapies for the
tumors which are resistant to EGFR modulators, particularly EGFR
inhibitors.
[0091] The level of biomarker protein and/or mRNA can be determined
using methods well known to those skilled in the art. For example,
quantification of protein can be carried out using methods such as
ELISA, 2-dimensional SDS PAGE, Western blot, immunopreciptation,
immunohistochemistry, fluorescence activated cell sorting (FACS),
or flow cytometry. Quantification of mRNA can be carried out using
methods such as PCR, array hybridization, Northern blot, in-situ
hybridization, dot-blot, Taqman, or RNAse protection assay.
Microarrays
[0092] The invention also includes specialized microarrays, e.g.,
oligonucleotide microarrays or cDNA microarrays, comprising one or
more biomarkers, showing expression profiles that correlate with
either sensitivity or resistance to one or more EGFR modulators.
Such microarrays can be employed in in vitro assays for assessing
the expression level of the biomarkers in the test cells from tumor
biopsies, and determining whether these test cells are likely to be
resistant or sensitive to EGFR modulators. For example, a
specialized microarray can be prepared using all the biomarkers, or
subsets thereof, as described herein and shown in Table 1. Cells
from a tissue or organ biopsy can be isolated and exposed to one or
more of the EGFR modulators. In one aspect, following application
of nucleic acids isolated from both untreated and treated cells to
one or more of the specialized microarrays, the pattern of gene
expression of the tested cells can be determined and compared with
that of the biomarker pattern from the control panel of cells used
to create the biomarker set on the microarray. Based upon the gene
expression pattern results from the cells that underwent testing,
it can be determined if the cells show a resistant or a sensitive
profile of gene expression. Whether or not the tested cells from a
tissue or organ biopsy will respond to one or more of the EGFR
modulators and the course of treatment or therapy can then be
determined or evaluated based on the information gleaned from the
results of the specialized microarray analysis.
Antibodies
[0093] The invention also includes antibodies, including polyclonal
or monoclonal, directed against one or more of the polypeptide
biomarkers. Such antibodies can be used in a variety of ways, for
example, to purify, detect, and target the biomarkers of the
invention, including both in vitro and in vivo diagnostic,
detection, screening, and/or therapeutic methods.
Kits
[0094] The invention also includes kits for determining or
predicting whether a patient would be susceptible or resistant to a
treatment that comprises one or more EGFR modulators. The patient
may have a cancer or tumor such as, for example, colorectal cancer.
Such kits would be useful in a clinical setting for use in testing
a patient's biopsied tumor or other cancer samples, for example, to
determine or predict if the patient's tumor or cancer will be
resistant or sensitive to a given treatment or therapy with an EGFR
modulator. The kit comprises a suitable container that comprises:
one or more microarrays, e.g., oligonucleotide microarrays or cDNA
microarrays, that comprise those biomarkers that correlate with
resistance and sensitivity to EGFR modulators, particularly EGFR
inhibitors; one or more EGFR modulators for use in testing cells
from patient tissue specimens or patient samples; and instructions
for use. In addition, kits contemplated by the invention can
further include, for example, reagents or materials for monitoring
the expression of biomarkers of the invention at the level of mRNA
or protein, using other techniques and systems practiced in the art
such as, for example, RT-PCR assays, which employ primers designed
on the basis of one or more of the biomarkers described herein,
immunoassays, such as enzyme linked immunosorbent assays (ELISAs),
immunoblotting, e.g., Western blots, or in situ hybridization, and
the like.
Application of Biomarkers and Biomarker Sets
[0095] The biomarkers and biomarker sets may be used in different
applications. Biomarker sets can be built from any combination of
biomarkers listed in Table 1 to make predictions about the effect
of an EGFR modulator in different biological systems. The various
biomarkers and biomarkers sets described herein can be used, for
example, as diagnostic or prognostic indicators in disease
management, to predict how patients with cancer might respond to
therapeutic intervention with compounds that modulate the EGFR, and
to predict how patients might respond to therapeutic intervention
that modulates signaling through the entire EGFR regulatory
pathway.
[0096] The biomarkers have both diagnostic and prognostic value in
diseases areas in which signaling through EGFR or the EGFR pathway
is of importance, e.g., in immunology, or in cancers or tumors in
which cell signaling and/or proliferation controls have gone
awry.
[0097] In one aspect, cells from a patient tissue sample, e.g., a
tumor or cancer biopsy, can be assayed to determine the expression
pattern of one or more biomarkers prior to treatment with one or
more EGFR modulators. In one aspect, the tumor or cancer is
colorectal. Success or failure of a treatment can be determined
based on the biomarker expression pattern of the cells from the
test tissue (test cells), e.g., tumor or cancer biopsy, as being
relatively similar or different from the expression pattern of a
control set of the one or more biomarkers. Thus, if the test cells
show a biomarker expression profile which corresponds to that of
the biomarkers in the control panel of cells which are sensitive to
the EGFR modulator, it is highly likely or predicted that the
individual's cancer or tumor will respond favorably to treatment
with the EGFR modulator. By contrast, if the test cells show a
biomarker expression pattern corresponding to that of the
biomarkers of the control panel of cells which are resistant to the
EGFR modulator, it is highly likely or predicted that the
individual's cancer or tumor will not respond to treatment with the
EGFR modulator.
[0098] The invention also provides a method of monitoring the
treatment of a patient having a disease treatable by one or more
EGFR modulators. The isolated test cells from the patient's tissue
sample, e.g., a tumor biopsy or tumor sample, can be assayed to
determine the expression pattern of one or more biomarkers before
and after exposure to an EGFR modulator wherein, preferably, the
EGFR modulator is an EGFR inhibitor. The resulting biomarker
expression profile of the test cells before and after treatment is
compared with that of one or more biomarkers as described and shown
herein to be highly expressed in the control panel of cells that
are either resistant or sensitive to an EGFR modulator. Thus, if a
patient's response is sensitive to treatment by an EGFR modulator,
based on correlation of the expression profile of the one or
biomarkers, the patient's treatment prognosis can be qualified as
favorable and treatment can continue. Also, if, after treatment
with an EGFR modulator, the test cells don't show a change in the
biomarker expression profile corresponding to the control panel of
cells that are sensitive to the EGFR modulator, it can serve as an
indicator that the current treatment should be modified, changed,
or even discontinued. This monitoring process can indicate success
or failure of a patient's treatment with an EGFR modulator and such
monitoring processes can be repeated as necessary or desired.
EXAMPLES
Example 1
Interim Analysis Identification of Biomarkers
[0099] The CA225-045 pharmacogenomics trial is a phase II
randomized exploratory study of ERBITUX (cetuximab) monotherapy in
patients with refractory metastatic colorectal cancer (mCRC). An
interim analysis of data from samples obtained from this trial was
performed to examine the preclinically discovered markers in the
clinical samples and to identify response prediction markers de
novo.
Clinical Samples:
[0100] 49 RNA patient samples isolated from pre-treatment tumor
biopsies of the metastatic site were randomized into five blocks
and profiled on U133A v2.0 chips (Affymetrix, Santa Clara, Calif.).
Profiling data from 30/49 patients were included in the analysis
based on meeting the following criteria: completion of at least two
cycles of therapy; availability of sufficient clinical data to
evaluate response; presence of tumor cells in biopsy sample; and
good quality profiling data from chip.
[0101] The 30 patient expression profiles consisted of 24 liver
metastases and 6 other tissue types. The Best Clinical Response
information from the 30 patients identified 4 partial responders
(PR), 5 stable disease (SD) and 21 progressive disease (PD)
patients. Assessment of response was performed according to a
modified version of the World Health Organization (WHO) criteria
(Miller et al., Cancer, 47: 207-214 (1981)). Overall response was
determined based on evaluation of target, non-target, and new
lesions. Partial response (PR) was defined as at least a 50%
decrease in the sum of the product of diameters (SPD) of target
lesions, taking as reference the baseline SPD. Progressive disease
(PD) was defined as a 25% or greater increase in the SPD of target
lesions, taking as reference the smallest SPD recorded since the
treatment started or the appearance of new lesions. Stable disease
(SD) was defined as neither sufficient shrinkage to qualify for PR
nor sufficient increase to qualify for PD.
Gene Expression Profiling:
[0102] Pre-treatment biopsies were obtained from the metastatic
site for RNA isolation. RNA was isolated from the pre-treatment
biopsies using the RNeasy mini kit (Qiagen, Valencia, Calif.). The
quality of RNA was checked by measuring the 28S:18S ribosomal RNA
ratio using an Agilent 2100 Bioanalyzer (Agilent Technologies,
Rockville, Md.). Concentration of total RNA was determined
spectrophotometrically. 1 .mu.g of total RNA was used to prepare
biotinylated probes according to the Affymetrix Genechip Expression
Analysis Technical Manual. Targets were hybridized to human
HG-U133A v2.0 gene chips according to the manufacturer's
instructions. Data were preprocessed using the MAS 5.0 software
(Affymetrix, Santa Clara, Calif.).
Data Analysis:
[0103] Of the 22,215 probesets present on the U133A v2.0 chip,
17,261 probesets that had present calls in at least two liver
metastatic tissues were included for data analysis. Data was
analyzed by performing a two-sided unequal variance t test with
Microsoft Excel or Anova analysis using PartekPro Pattern
Recognition Software (Partek, St. Charles, Mo.). The statistical
analyses were performed using MAS 5.0 quantile normalized values
for signal intensity for 17,261 probe sets.
Analysis of Biomarkers Using t Test and ANOVA Analysis:
[0104] The first step was to examine 42 probesets that were
identified preclinically (FIG. 1) in the transcriptional profiles
of 30 metastatic tumors. This was done to examine whether the
preclinical markers are differentially expressed between patients
who derive clinical benefit (PR and SD) from ERBITUX treatment and
those who do not (PD).
[0105] A two-sided unequal variance t test was performed between
the 9 patients who derive clinical benefit and the 21 patients who
have progressive disease. Three probesets out of 42 are
differentially expressed between 9 (PR+SD) patients and 21 (PD)
patients (p<0.05). These probesets represent the mRNA expression
of Annexin A1 (ANXA1 201012_at), serine proteinase inhibitor clade
B member 5 (SERPINB5 204855_at), and fibroblast growth factor
receptor 3 (FGFR3 204379_s_at).
[0106] Next, a broader list of 640 genes from which the 42 probe
set list had been derived (FIG. 1) was examined. 635 out of the 640
probesets were present in the 17,261 probe sets that are included
in the analysis. The 635 probesets were identified as being highly
variably expressed in transcriptional profiles of 164 primary
untreated CRC tumors. They expressed at a moderate to high level in
colon tumors (at least one expression value of two times the mean
value for the array, i.e., 3000 expression units) and with a
population variance value of >0.1.
[0107] The 635 probe sets were examined in transcriptional profiles
of 30 metastatic tumors from the CA225-045 trial. 39 out of 635
probesets were found to be differentially expressed between 9
(PR+SD) and 21 (PD), p<0.05 and are described in Table 2. 19 of
the 39 probe sets are resistance markers for ERBITUX and 20 of
these are sensitivity markers for ERBITUX (FIG. 2).
TABLE-US-00002 TABLE 2 39 Markers for Response Prediction to
ERBITUX Affymetrix ID p value Gene name Symbol 1 205767_at 0.0002
epiregulin EREG 2 201012_at 0.006 annexin A1 ANXA1 3 205239_at
0.0068 amphiregulin AREG 4 213435_at 0.0098 SATB family member 2
SATB2 5 209260_at 0.0122 stratifin SFN 6 204379_s_at 0.0129
fibroblast growth factor receptor 3 FGFR3 7 205295_at 0.0143
creatine kinase, mitochondrial 2 CKMT2 8 204678_s_at 0.0148
potassium channel, subfamily K, memb. 1 KCNK1 9 204044_at 0.0151
quinolinate phosphoribosyltransferase QPRT 10 203726_s_at 0.0154
laminin, alpha 3 LAMA3 11 219555_s_at 0.0165 uncharacterized bone
marrow prtn BM039 BM039 12 216598_s_at 0.0188 chemokine (C-C motif)
ligand 2 CCL2 13 209425_at 0.0195 alpha-methylacyl-CoA racemase
AMACR 14 204855_at 0.0207 serine proteinase inhibitor, clade B,
memb. 5 SERPINB5 15 218807_at 0.0213 vav 3 oncogene VAV3 16
210764_s_at 0.0261 cysteine-rich, angiogenic inducer, 61 CYR61 17
210511_s_at 0.0265 inhibin, beta A INHBA 18 220834_at 0.0266
membrane-spanning 4-domains, subfly A, 12 MS4A12 19 210809_s_at
0.0268 periostin, osteoblast specific factor POSTN 20 213385_at
0.0304 chimerin 2 CHN2 21 218468_s_at 0.0323 gremlin 1 homolog,
cysteine knot GREM1 superfamily 22 202859_x_at 0.033 interleukin 8
IL8 23 206754_s_at 0.0337 cytochrome P450, 2B6 CYP2B6 24
218806_s_at 0.034 vav 3 oncogene VAV3 25 218469_at 0.0342 gremlin 1
homolog, cysteine knot GREM1 superfamily 26 219508_at 0.0347
glucosaminyl (N-acetyl) transferase 3, GCNT3 mucin type 27
204364_s_at 0.0367 chromosome 2 open reading frame 23 C2orf23 28
219471_at 0.0376 chromosome 13 open reading frame 18 C13orf18 29
219014_at 0.0396 placenta-specific 8 PLAC8 30 203939_at 0.04
5'-nucleotidase, ecto (CD73) NT5E 31 211506_s_at 0.0401 interleukin
8 IL8 32 206143_at 0.0404 solute carrier family 26, member 3
SLC26A3 33 44790_s_at 0.0425 chromosome 13 open reading frame 18
C13orf18 34 202075_s_at 0.0427 phospholipid transfer protein PLTP
35 201650_at 0.0436 keratin 19 KRT19 36 205259_at 0.046 nuclear
receptor subfamily 3, C2 NR3C2 37 208893_s_at 0.0466 dual
specificity phosphatase 6 DUSP6 38 209436_at 0.048 spondin 1,
extracellular matrix protein SPON1 39 218087_s_at 0.0496 sorbin and
SH3 domain containing 1 SORBS1
[0108] The top 3 markers based on lowest p value were epiregulin
(EREG, 205767_at), annexin Al (ANXA1 201012_at), and amphiregulin
(AREG, 205239_at). Interestingly, epiregulin and amphiregulin are
ligands for EGFR. Examination of their individual mRNA expression
profiles indicates that they appear to be more highly expressed in
patients who derive clinical benefit from ERBITUX treatment (FIGS.
3A and 3B). This suggests that patients who have high levels of
epiregulin and amphiregulin have tumors that are addicted to the
EGFR signaling pathway that is being driven by these two
ligands.
[0109] The expression levels of epidermal growth factor (EGF,
206254_at), transforming growth factor alpha (TGF.alpha.,
205016_at), betacellulin (BTC, 207326_at), and heparin binding-EGF
(HB-EGF, 203821_at), which are the other known ligands for EGFR,
were also examined. Their expression levels showed no correlation
with response to ERBITUX.
Determination of Biological Relationships Between 39
Biomarkers:
[0110] The Ingenuity Pathway Analysis web-based application
(Ingenuity Systems Inc., Mountain View, Calif.) was used to test
the biological relationship between the 39 biomarkers of Table 2.
This application makes use of the Ingenuity Knowledge Base, a
curated database consisting of millions of individually modeled
relationships between proteins, genes, complexes, cells, tissues,
drugs, and diseases. The 39 genes were inputted into the Pathway
Analysis application. The Ingenuity Knowledge base had information
on 25 of the 39 genes. Strikingly, of the 25 "network eligible"
genes, 17 mapped to the EGFR network (FIG. 4, 17 genes are shaded)
indicating a strong link between the EGFR signaling status in the
tumors and response to ERBITUX. No other network emerged from the
analysis of the 39 genes. Of the 17 genes, DUSP6 is a member of the
ERK/MAPK signaling pathway and SFN is a member of the PI3K/AKT
signaling pathway, which are the two key pathways downstream of
EGFR signaling.
Multivariate Analysis:
[0111] The t test and ANOVA analysis was used to assess the ability
of individual biomarkers to separate PR/SD patients from PD
patients. Multivariate discriminant analysis was used to assess the
prediction power of the 39 markers on patient response, and
identify the set of variables/biomarkers that would be the best
predictors of response to ERBITUX treatment.
[0112] SAS discriminant function analysis (SAS Scientific Discovery
Solutions, release 8.02, SAS Institute Inc., Cary, N.C.) was
applied to the data set of 39 markers. Discriminant function
analysis was broken into a 2-step process: (1) testing the
significance of a set of discriminant functions; and (2) using
these functions to classify the sample objects to the corresponding
response groups. The first step was accomplished by a SAS
"stepwise" procedure using the forward variable selection method.
The derived discriminant functions were passed on to the second SAS
procedure, called the "discrim" procedure, for classification of
the given samples.
[0113] Given the small sample size of 30 patients, the samples were
not partitioned into separate training and test data sets. Instead
a single data set was used, and the leave-one-out cross-validation
method was applied to test the prediction power of the identified
biomarker predictors. A SAS cross-validation protocol was
developed, which implemented leave-one-out cross-validation method
in a SAS program, and was run on this data set to define the number
of predictors that could be used for building the discriminant
function models. This method allowed a comparison of a single
biomarker model to multiple biomarker models (up to 15 biomarkers)
(FIG. 5). The single gene predictor model was found to have 0.7037
prediction power as measured by AUC coverage (area under the
Receiver Operating Characteristic (ROC) curve which shows the
tradeoff between sensitivity and specificity). An area of 1
represents completely accurate prediction. When the number of
predictors included in the model goes up to three biomarkers, the
prediction power increases to 0.9. When the number of predictors
included in the model exceeds three, there tends to be a decrease
in prediction power. These results indicate that the best
prediction power is achieved by building a discriminant function
model with 3 out of the 39 biomarkers.
Correlation of the 39 Biomarkers:
[0114] Ingenuity Pathway analysis suggested that at least 17 of the
39 biomarkers identified belong to a single interaction network. A
correlation analysis using SAS "corr" procedure was applied to
investigate the correlation of genes identified from the
discriminant analysis. Table 3 shows an example of a correlation
matrix of some of the top predictors selected by the SAS procedure.
Some of the genes show very high correlation coefficient values
which suggests they are highly correlated. For example, 205767_at
(EREG) and 205239_at (AREG), or 205767_at (EREG) and 218807_at
(VAV3), or 206754_s_at (CYP2B6) and 209260_at (SFN) were found to
be highly correlated. The highly correlated genes could replace
each other to explain a certain proportion of the variation between
the groups of patients who derive clinical benefit and those that
do not. These results show excellent agreement between the possible
biological mechanism as elucidated by Ingenuity Pathway Analysis
and literature, and the statistical prediction as determined by the
SAS procedure.
TABLE-US-00003 TABLE 3 Pearson Correlation Co-Efficients on 7 Most
Frequent Probesets That Were Identified As Top Variables For
Discriminant Analysis Affymetrix ID 205767_at 201012_at 205239_at
206754_at 209260_at 205259_at 2188 205767_at 1 -0.28587 0.84089
-0.16409 -0.04261 -0.02338 0.64 201012_at -0.28587 1 -0.16652
-0.41722 0.31615 -0.45851 0.28 205239_at 0.84089 -0.16652 1
-0.21894 0.07064 -0.19815 0.60 206754_s_at -0.16409 -0.41722
-0.21894 1 -0.47769 0.53511 -0.21 209260_at -0.04261 0.31615
0.07064 -0.47769 1 -0.26621 0.26 205259_at -0.02338 -0.45851
-0.19815 0.53511 -0.26621 1 -0.02 218807_at 0.64133 -0.28141
0.60752 -0.21663 0.26204 -0.02668 1 indicates data missing or
illegible when filed
Best Prediction Models:
[0115] The best prediction models were determined using the SAS
stepwise procedure. 205767_at (EREG) was always picked first. This
suggests that the expression of the EGFR ligand epiregulin can
explain most of the variation that exists between the group of
patients that are PR/SD and the group of patients who are PD. The
second predictor aids in picking up the largest proportion of the
unexplained variation from the first variable function (predictor)
and so on. The misclassification rates of the best SAS selected
models were:
TABLE-US-00004 Model Error rate 205767_at (EREG) 0.2143 205767_at
(EREG), 206754_s_at (CYP2B6) 0.127 205767_at (EREG), 206754_s_at
(CYP2B6), 201650_at 0.1032 (KRT19) 205767_at (EREG), 206754_s_at
(CYP2B6), 201650_at 0.1032 (KRT19), 204678_at (KCNK1)
Biomarkers were also selected based on their biological,
functional, and co-regulation information, and the derived
prediction functions were used to classify the 30 sample data set
using the SAS "discrim" procedure. Using this approach, some
optimal combinations of biomarker variables and their corresponding
misclassification rates were identified, such as:
TABLE-US-00005 Model Error rate 205767_at (EREG), 206754_s_at
(CYP2B6) 201650_at 0.1032 (KRT19) 205767_at (EREG), 209260_at
(SFN), 205259_at 0.079 (NR3C2) 201012_at (ANXA1), 205239_at (AREG),
209260_at 0.07 (SFN), 205259_at (NR3C2), 218807_at (VAV3) 209260_at
(SFN), 218807_at (VAV3) 0.1270
Example 2
Identification of Biomarkers Following Interim Analysis
[0116] As mentioned above, the CA225-045 pharmacogenomics trial is
a phase II randomized exploratory study of ERBITUX (cetuximab)
monotherapy in patients with refractory metastatic colorectal
cancer (mCRC). This trial enrolled 111 patients. A standard
cetuximab dosing regimen was followed for the first 3 weeks of
therapy, thereafter patients were eligible for dose escalation
every 3 weeks to a maximum dose of 400 mg/m.sup.2 provided they had
not experienced a >grade 2 skin rash. During the pre-treatment
phase, all patients underwent a tumor biopsy procedure involving
three passes with an 18-gauge needle of a single metastatic lesion.
Two pre-treatment core needle biopsies were stored in a single tube
of RNALater at room temperature and one core needle biopsy was
formalin-fixed and embedded in paraffin for subsequent analyses.
All subjects also underwent a pre-treatment blood draw. All
specimens were obtained from patients with appropriate informed
consent and IRB approval.
[0117] Tumor response was evaluated every nine weeks (one cycle of
therapy) according to the modified World Health Organization
criteria (Miller et al., Cancer, 47, 207-214 (1981)). Overall
response was determined based on evaluation of target, non-target
and new lesions. For this analysis, subjects experiencing a
complete (CR) or partial response (PR), or stable disease (SD),
were grouped as the disease control group; progressive disease (PD)
and select unable to determine (UTD) subjects were grouped as
non-responders. The UTD subjects that were included in the
non-responder group for analysis were those that died prior to the
response assessment. All other UTD subjects were excluded from the
analysis.
RNA and DNA Extraction:
[0118] For each subject's tumor sample, RNA and DNA were isolated
from two pre-treatment core needle biopsies provided in a single
tube of RNA Later at room temperature within seven days from the
date of the biopsy procedure. RNA was isolated using the RNeasy
mini kit (Qiagen, Valencia, Calif.). The quality of RNA was checked
by measuring the 28S:18S ribosomal RNA ratio using an Agilent 2100
Bioanalyzer (Agilent Technologies, Rockville, Md.). DNA was
isolated from the flow-through collected during the RNA isolation
procedure using the DNeasy mini kit (Qiagen). Concentration of RNA
and DNA was determined spectrophotometrically.
Gene Expression Profiling and Statistical Analysis:
[0119] For each sample from which sufficient RNA was available, 1
.mu.g of total RNA was used to prepare biotinylated probes
according to the Affymetrix GeneChip Expression Analysis Technical
Manual. Targets were hybridized to human HG-U133A v2.0 GeneChips
according to the manufacturer's instructions. Data were
preprocessed using the MAS 5.0 software (Affymetrix, Santa Clara,
Calif.) and statistical analyses were performed using quantile
normalized values for signal intensity. Univariate analysis was
done by using a two-sided unequal variance t-test. For multivariate
analysis samples were randomly partitioned 50-50 into a training
set and a test set. Top candidate predictors were selected from the
training set using a t-test. This was followed by model
construction using stepwise discriminant analysis (v8.2, SAS, Cary,
N.C.). Class prediction was assessed using 10-fold cross
validation. The models developed from the training set were
evaluated using a test set.
[0120] In addition to the profiling of RNA from the clinical study,
an expression database of 164 primary colorectal tumors (Banerjea
et al., Mol. Cancer, 3, 21 (2004)) was examined to identify
potential predictive markers. Data from the 640 probe sets that
passed the filtering steps described above in the results were then
subjected to an unsupervised average linkage hierarchical
clustering using CLUSTER and the results were displayed by using
TREEVIEW.
RT-qPCR for Gene Expression Analysis:
[0121] For each sample from which RNA was available, approximately
100 ng RNA was converted into cDNA by the random priming method
using Multi Scribe Reverse Transcriptase according to the
manufacturer's instructions (TaqMan Reverse Transcription Reagents,
Applied Biosystems Inc. ((ABI), Foster City, Calif.). The resulting
cDNA was measured on the ABI 7900HT Sequence Detection System using
ABI Assay-on-Demand primer/probe sets directed against the
amphiregulin (Hs00155832_ml) and epiregulin (Hs00154995_ml) genes.
Relative expression levels were calculated using the .DELTA.Ct
method in which average values of duplicate reactions were
compared, with GAPDH (Hs001266705_gl) serving as the internal
reference. In this experimental design, low .DELTA.Ct values
correspond to high levels of expression.
Nucleotide Sequence Analysis:
[0122] Mutational analyses of EGFR, K-RAS, and BRAF were performed
using available genomic DNAs isolated from tumor specimens. Primers
used for EGFR exons 18-21, coding for the TK domain, were published
previously (Lynch et al., N. Engl. J. Med., 350, 2129-2139 (2004)).
The primers used to evaluate exon 2 of K-RAS and exon 15 of BRAF
were as follows: K-RAS F 5'-TAAGGCCTGCTGAAAATGACTG-3' (SEQ ID
NO:257) and K-RAS R 5'-TGGTCCTGCACCAGTAA TATGC-3' (SEQ ID NO:258);
BRAF F 5'-TCATAATGCTTGCTCTGATAGGA-3' (SEQ ID NO:259) and BRAF R
5'-GGCCAAAAATTTAATCAGTGGA-3' (SEQ ID NO:260). PCR was performed
using conditions as previously described (Chen et al., Hum. Mutat.,
27, 427-435 (2006)). PCR fragments were cleaned with QIAquick PCR
Purification Kit (Qiagen), sequenced on an ABI 3100A Capillary
Genetic Analyzer (Applied Biosystems Inc.) and analyzed in both
sense and antisense directions for the presence of heterozygous
mutations. Analysis of the DNA sequence was performed using
SEQUENCHER v4.2 (Gene Codes, Ann Arbor, Mich.) followed by visual
analysis of each electropherogram by two independent reviewers.
Appropriate positive and negative controls were included for each
of the exons evaluated. Mutational analyses were done without
knowledge of clinical outcome including tumor response.
Results
Patients' Characteristics and Clinical Outcome:
[0123] The primary objective of this study was to identify
predictive markers of response to cetuximab therapy in CRC.
Evaluable RNA and/or DNA and/or plasma samples were available for
103 out of 111 subjects. The objective response determination for
these 103 subjects were: one complete response (CR), six partial
response (PR), twenty-eight stable disease (SD), fifty-six
progressive disease (PD), and twelve patients who died prior to
their first radiographic assessment and are therefore unable to
determine (UTD). Thirty-four percent of the subjects either
responded or had disease stabilization whereas the remaining 66%
were classified as non-responders.
Genomic Analysis of Tumor-Derived RNAs:
[0124] In order to identify genes that were differentially
expressed between the disease control and non-responder groups,
gene expression profiling was carried out on RNA isolated from 95
pre-treatment biopsies. Seventy percent of the biopsies were taken
from the liver metastatic tissue, and 30% of the biopsies were
taken from non-hepatic tissue sites. 91 out of the 95 samples
yielded >500 ng RNA and were randomized into ten blocks and
profiled on U133A v2.0 chips (Affymetrix). High quality
transcriptional profiling data were obtained from 87 patients.
Seven patients were excluded from further analysis either because
they withdrew from the study prior to the first assessment,
experienced hypersensitivity or withdrew their consent. Final data
analysis was carried out using best clinical response assessments
for the remaining 80 patients and expression profiles from these
patients were included in the statistical analysis. These 80
patients included 1 CR, 5 PR, 19 SD, 43 PD, and 12 UTD.
[0125] An initial candidate set of genes was identified that were
variably expressed in an independent set of 164 primary colorectal
tumors by filtering transcriptional data from all 22,215 probe
sets. This filtering yielded 640 probe sets that were expressed at
a moderate to high level in colon tumors (at least one expression
value of two times the mean value for the array i.e. 3000
expression units) and with a population variance value of >0.1.
It was proposed that these 640 probe sets that had a highly dynamic
range of expression across a population of CRC tumors were most
likely to yield markers that would be useful for patient selection.
Unsupervised hierarchical clustering of the 640 probe sets across
the 164 primary colon tumors showed that biologically interesting
genes that might be predictive of response to cetuximab were
preferentially expressed in a subset of colorectal tumors (FIG. 6).
In FIG. 6, the 164 tumors were divided into 3 major classes (Class
1, 2 and 3). The 640 probe sets were divided into 5 clusters
(labeled A through E). Cluster A, which contains cancer antigens
such as CEACAM 6 and CD24, also contains the EGFR ligands EREG and
AREG. Cluster A is most highly expressed in Class 1a, which
represents approximately 25% of the 164 colorectal tumor
specimens.
[0126] Out of 22,215 probe sets, data analysis was conducted on
17,137 probe sets that were found to be expressed in at least 10%
of the liver metastases patient samples. 629 of the previously
identified 640 probe sets were present in the 17,137 probe set
list. Their gene expression profiles were examined in the data from
80 patients and were correlated with response assessments. 121 out
of the 629 probe sets were found to be differentially expressed
between 25 patients with disease control and 55 non-responders,
p<0.05 (t test of the disease group (CR, PR, SD) vs.
non-responders) as shown in Table 4.
TABLE-US-00006 TABLE 4 121 Probe Sets Differentially Expressed
Between 25 patients with disease control and 55 non-responders, p
< 0.05 Affymetrix ID p value Gene name Symbol 203939_at
3.787E-07 5'-nucleotidase, ecto (CD73) NT5E 205767_at 1.474E-05
epiregulin EREG 205239_at 2.489E-05 amphiregulin (schwannoma- AREG
derived growth factor) 213975_s_at 3.617E-05 lysozyme (renal
amyloidosis) /// LYZ /// LILRB1 leukocyte immunoglobulin-like
receptor, subfamily B (with TM and ITIM domains), member 1
201641_at 9.146E-05 bone marrow stromal cell antigen 2 BST2
208893_s_at 0.000257 dual specificity phosphatase 6 DUSP6 218807_at
0.000507 vav 3 oncogene VAV3 218806_s_at 0.000513 vav 3 oncogene
VAV3 216598_s_at 0.000680 chemokine (C-C motif) ligand 2 CCL2
213435_at 0.000909 SATB family member 2 SATB2 210517_s_at 0.001636
A kinase (PRKA) anchor protein AKAP12 (gravin) 12 219508_at
0.001935 glucosaminyl (N-acetyl) transferase GCNT3 3, mucin type
201462_at 0.001937 secernin 1 SCRN1 204379_s_at 0.002008 fibroblast
growth factor receptor 3 FGFR3 (achondroplasia, thanatophoric
dwarfism) 206584_at 0.002018 lymphocyte antigen 96 LY96 200884_at
0.002042 creatine kinase, brain CKB 206332_s_at 0.002612
interferon, gamma-inducible IFI16 protein 16 202525_at 0.002630
protease, serine, 8 (prostasin) PRSS8 205403_at 0.002869
interleukin 1 receptor, type II IL1R2 221530_s_at 0.002881 basic
helix-loop-helix domain BHLHB3 containing, class B, 3 209728_at
0.003260 major histocompatibility complex, HLA-DRB4 class II, DR
beta 4 /// major histocompatibility complex, class II, DR beta 4
215049_x_at 0.004039 CD 163 antigen CD163 203645_s_at 0.004182 CD
163 antigen CD163 219471_at 0.004627 chromosome 13 open reading
C13orf18 frame 18 210133_at 0.004790 chemokine (C-C motif) ligand
11 CCL11 205097_at 0.005553 solute carrier family 26 (sulfate
SLC26A2 transporter), member 2 211656_x_at 0.006050 major
histocompatibility complex, HLA-DQB1 class II, DQ beta 1 /// major
histocompatibility complex, class II, DQ beta 1 209392_at 0.006150
ectonucleotide ENPP2 pyrophosphatase/phosphodiesterase 2
(autotaxin) 205402_x_at 0.006181 protease, serine, 2 (trypsin 2)
PRSS2 217028_at 0.006582 chemokine (C-X-C motif) receptor CXCR4 4
204855_at 0.006615 serpin peptidase inhibitor, clade B SERPINB5
(ovalbumin), member 5 201137_s_at 0.007369 major histocompatibility
complex, HLA-DPB1 class II, DP beta 1 215051_x_at 0.007563
allograft inflammatory factor 1 AIF1 202859_x_at 0.007872
interleukin 8 IL8 211506_s_at 0.008119 interleukin 8 IL8
207457_s_at 0.008600 lymphocyte antigen 6 complex, LY6G6D locus G6D
205765_at 0.009101 cytochrome P450, family 3, CYP3A5 subfamily A,
polypeptide 5 204619_s_at 0.009733 chondroitin sulfate proteoglycan
2 CSPG2 (versican) 205199_at 0.010621 carbonic anhydrase IX CA9
219962_at 0.010751 angiotensin I converting enzyme ACE2
(peptidyl-dipeptidase A) 2 205242_at 0.011022 chemokine (C-X-C
motif) ligand CXCL13 13 (B-cell chemoattractant) 217428_s_at
0.011274 collagen, type X, alpha 1(Schmid COL10A1 metaphyseal
chondrodysplasia) 206918_s_at 0.011540 copine I CPNE1 44790_s_at
0.011645 chromosome 13 open reading C13orf18 frame 18 218469_at
0.011704 gremlin 1, cysteine knot GREM1 superfamily, homolog
(Xenopus laevis) 209823_x_at 0.011862 major histocompatibility
complex, HLA-DQB1 class II, DQ beta 1 205513_at 0.011867
transcobalamin I (vitamin B12 TCN1 binding protein, R binder
family) 204213_at 0.012198 polymeric immunoglobulin PIGR receptor
205941_s_at 0.012335 collagen, type X, alpha 1(Schmid COL10A1
metaphyseal chondrodysplasia) 212192_at 0.012522 potassium channel
tetramerisation KCTD12 domain containing 12 204891_s_at 0.012755
lymphocyte-specific protein LCK tyrosine kinase 208029_s_at
0.012800 lysosomal associated protein LAPTM4B transmembrane 4 beta
/// lysosomal associated protein transmembrane 4 beta 201884_at
0.013032 carcinoembryonic antigen-related CEACAM5 cell adhesion
molecule 5 201030_x_at 0.013074 lactate dehydrogenase B LDHB
202411_at 0.013302 interferon, alpha-inducible protein 27 IFI27
211165_x_at 0.013671 EPH receptor B2 EPHB2 212186_at 0.014902
acetyl-Coenzyme A carboxylase ACACA alpha 201743_at 0.015156 CD14
antigen /// CD14 antigen CD14 87100_at 0.015861 -- -- 206467_x_at
0.015975 tumor necrosis factor receptor TNFRSF6B /// superfamily,
member 6b, decoy /// RTEL1 regulator of telomere elongation
helicase 1 218468_s_at 0.016329 gremlin 1, cysteine knot GREM1
superfamily, homolog (Xenopus laevis) 222257_s_at 0.016397
angiotensin I converting enzyme ACE2 (peptidyl-dipeptidase A) 2
221730_at 0.016992 collagen, type V, alpha 2 COL5A2 203915_at
0.017412 chemokine (C-X-C motif) ligand 9 CXCL9 206858_s_at
0.017492 homeo box C6 HOXC6 221584_s_at 0.017554 potassium large
conductance KCNMA1 calcium-activated channel, subfamily M, alpha
member 1 204475_at 0.018085 matrix metallopeptidase 1 MMP1
(interstitial collagenase) 203895_at 0.018353 phospholipase C, beta
4 PLCB4 214043_at 0.018926 Protein tyrosine phosphatase, PTPRD
receptor type, D 204678_s_at 0.019645 potassium channel, subfamily
K, KCNK1 member 1 204446_s_at 0.019912 arachidonate 5-lipoxygenase
ALOX5 204533_at 0.020226 chemokine (C-X-C motif) ligand CXCL10 10
211689_s_at 0.020262 transmembrane protease, serine 2 /// TMPRSS2
transmembrane protease, serine 2 201858_s_at 0.020471 proteoglycan
1, secretory granule PRG1 212671_s_at 0.020852 major
histocompatibility complex, HLA-DQA1 /// class II, DQ alpha 1 ///
major HLA-DQA2 histocompatibility complex, class II, DQ alpha 2
216248_s_at 0.021062 nuclear receptor subfamily 4, group NR4A2 A,
member 2 212188_at 0.021225 potassium channel tetramerisation
KCTD12 domain containing 12 /// potassium channel tetramerisation
domain containing 12 204070_at 0.021833 retinoic acid receptor
responder RARRES3 (tazarotene induced) 3 213564_x_at 0.022061
lactate dehydrogenase B LDHB 209732_at 0.022699 C-type lectin
domain family 2, CLEC2B member B 213746_s_at 0.023141 filamin A,
alpha (actin binding FLNA protein 280) 214974_x_at 0.023351
chemokine (C-X-C motif) ligand 5 CXCL5 201792_at 0.023592 AE
binding protein 1 AEBP1 213905_x_at 0.023638 biglycan ///
serologically defined BGN /// colon cancer antigen 33 SDCCAG33
212353_at 0.024175 sulfatase 1 SULF1 209156_s_at 0.024926 collagen,
type VI, alpha 2 COL6A2 203083_at 0.025140 thrombospondin 2 THBS2
203896_s_at 0.025311 phospholipase C, beta 4 PLCB4 201617_x_at
0.025316 caldesmon 1 CALD1 217963_s_at 0.025667 nerve growth factor
receptor NGFRAP1 (TNFRSF16) associated protein 1 208965_s_at
0.025706 interferon, gamma-inducible IFI16 protein 16 217763_s_at
0.026315 RAB31, member RAS oncogene RAB31 family 203325_s_at
0.026698 collagen, type V, alpha 1 COL5A1 209792_s_at 0.026893
kallikrein 10 KLK10 205549_at 0.027028 Purkinje cell protein 4 PCP4
204622_x_at 0.028026 nuclear receptor subfamily 4, group NR4A2 A,
member 2 210095_s_at 0.030712 insulin-like growth factor binding
IGFBP3 protein 3 209969_s_at 0.031010 signal transducer and
activator of STAT1 transcription 1, 91 kDa 202436_s_at 0.031792
cytochrome P450, family 1, CYP1B1 subfamily B, polypeptide 1
202311_s_at 0.032306 collagen, type I, alpha 1 COL1A1 221031_s_at
0.032415 hypothetical protein DKFZP434F0318 DKFZp434F0318 ///
hypothetical protein DKFZp434F0318 209118_s_at 0.032949 tubulin,
alpha 3 TUBA3 210164_at 0.033266 granzyme B (granzyme 2, GZMB
cytotoxic T-lymphocyte-associated serine esterase 1) /// granzyme B
(granzyme 2, cytotoxic T- lymphocyte-associated serine esterase 1)
213194_at 0.034686 roundabout, axon guidance ROBO1 receptor,
homolog 1 (Drosophila) 204697_s_at 0.034934 chromogranin A
(parathyroid CHGA secretory protein 1) 202752_x_at 0.035921 solute
carrier family 7 (cationic SLC7A8 amino acid transporter, y+
system), member 8 205929_at 0.037216 glycoprotein A33
(transmembrane) GPA33 204044_at 0.037293 quinolinate QPRT
phosphoribosyltransferase (nicotinate-nucleotide pyrophosphorylase
(carboxylating)) 205311_at 0.037673 dopa decarboxylase (aromatic L-
DDC amino acid decarboxylase) 204320_at 0.038710 collagen, type XI,
alpha 1 COL11A1 204364_s_at 0.040104 chromosome 2 open reading
frame 23 C2orf23 212354_at 0.040347 sulfatase 1 SULF1 202465_at
0.040639 procollagen C-endopeptidase PCOLCE enhancer 212992_at
0.041178 chromosome 14 open reading C14orf78 frame 78 209201_x_at
0.042126 chemokine (C-X-C motif) receptor 4 CXCR4 215646_s_at
0.043050 chondroitin sulfate proteoglycan 2 CSPG2 (versican) ///
chondroitin sulfate proteoglycan 2 (versican) 202283_at 0.045795
serpin peptidase inhibitor, clade F SERPINF1 (alpha-2 antiplasmin,
pigment epithelium derived factor), member 1 209436_at 0.046099
spondin 1, extracellular matrix SPON1 protein 37892_at 0.048675
collagen, type XI, alpha 1 COL11A1 218559_s_at 0.048679 v-maf
musculoaponeurotic MAFB fibrosarcoma oncogene homolog B (avian)
213998_s_at 0.049742 DEAD (Asp-Glu-Ala-Asp) box DDX17 polypeptide
17
[0127] The top three candidate markers based on lowest p value were
5'nucleotidase ecto (CD73, 203939_at), epiregulin (EREG, 205767_at)
and amphiregulin (AREG, 205239_at). CD73 is a purine metabolizing
enzyme that may have prognostic value in colorectal and pancreatic
cancer (Eroglu et al., Med. Oncol., 17, 319-324 (2000); Giovannetti
et al., Cancer Res., 66, 3928-3935 (2006)). Examination of its mRNA
profile showed that it is expressed at higher levels in the
non-responder group. Epiregulin and amphiregulin are ligands for
EGFR (Singh and Harris, Cell Signal, 17, 1183-1193 (2005)).
Examination of their individual mRNA expression profiles revealed
that they were more highly expressed in patients in the disease
control group (FIGS. 7A and 7B). FIGS. 7A and 7B provide mRNA
levels of EGFR ligands epiregulin and amphiregulin. Affymetrix mRNA
levels of Epiregulin (EREG, 205767_at) and Amphiregulin (AREG,
205239_at) are plotted on the y axis. There is a statistically
significant difference in gene expression levels between the
disease control group (CR, PR and SD) and the non-responder group
(EREG p=1.474e.sup.-05, AREG p=2.489e.sup.-05). These results
suggest that patients who have high levels of EREG and AREG have
tumors that are addicted to the EGFR signaling pathway and are
therefore most likely to experience disease control on treatment
with cetuximab.
[0128] In addition to the gene filtering approach described above,
a de novo analysis was performed on the transcriptional profiles of
the same 80 patients. A two-sided unequal-variance t-test was done
on all 17,137 probe sets. The top 10 genes are provided in Table
5.
TABLE-US-00007 TABLE 5 Top 10 Genes from De Novo Analysis
Affymetrix ID p value Gene name Symbol 203939_at 3.787E-07
5'-nucleotidase, ecto (CD73) NT5E 217999_s_at 7.056E-06 Pleckstrin
homology-like domain, family A, PHLDA1 member 1 205767_at 1.474E-05
epiregulin EREG 203349_s_at 1.704E-05 ets variant gene 5
(ets-related molecule) ETV5 204015_s_at 1.812E-05 dual specificity
phosphatase 4 DUSP4 204014_at 1.856E-05 dual specificity
phosphatase 4 DUSP4 212349_at 2.395E-05 protein
O-fucosyltransferase 1 POFUT1 205239_at 2.489E-05 amphiregulin
(schwannoma-derived growth AREG factor) 208130_s_at 2.646E-05
thromboxane A synthase 1 (platelet, TBXAS1 cytochrome P450, family
5, subfamily A) /// thromboxane A synthase 1 (platelet, cytochrome
P450, family 5, subfamily A) 219615_s_at 3.153E-05 potassium
channel, subfamily K, member 5 KCNK5
Examination of the top 10 genes with the lowest p value revealed
that EREG and AREG were once again found to be top sensitivity
markers. CD73, dual specificity phosphatase 4 (DUSP4, 204015_s_at
and 204014_at), and pleckstrin homology like domain A1 (PHLDA1,
217999_s_at) were found to be top resistance markers. The mRNA
expression levels of epidermal growth factor (EGF, 206254_at),
transforming growth factor alpha (TGF.alpha., 205016_at),
betacellulin (BTC, 207326_at) and heparin binding -EGF (HB-EGF,
203821_at), some of the other known ligands for EGFR, were also
reviewed. Their expression levels showed no correlation with
response to cetuximab. It is also worth noting that no correlation
was seen between EGFR (201983_s_at) mRNA levels and response to
cetuximab. These results suggest that a de novo analysis using only
the transcriptional profiling data gathered from this clinical
study could find the candidate markers EREG and AREG. However,
given the issue of multiple test comparisons, the identification of
EREG and AREG using an independent filtering approach described
above lends additional support to their being candidates for
predicting cetuximab response.
[0129] From the t-test analyses, the ability of individual
biomarkers to separate the disease control group from the
non-responders could be assessed. Using discriminant function
analysis, the prediction power of a set of the 100 top candidate
markers for patient response was assessed in order to identify the
set of variables that would be the best predictors of disease
control with cetuximab treatment. The AUC (area under the receiver
operating characteristic curve) values of the different multi-gene
models showed that as the number of genes in the model increased
from one to fifteen the predictive power of the model did not
improve. The AUC value of a single gene model was >0.8. An
independent test was done to assess the performance of the most
frequently identified gene, EREG, and also of AREG, as individual
predictors. EREG has an AUC value of 0.845, and AREG has an AUC
value of 0.815, indicating that they are both highly powerful
predictive markers for patient selection (FIGS. 8A and 8B).
Analysis of Candidate Markers Epiregulin and Amphiregulin:
[0130] In order to independently verify gene expression with a
different technology platform that may ultimately be more easily
transferable into a diagnostic test, AREG and EREG transcript
levels were measured using quantitative RT-PCR TaqMan assays.
Expression levels of these genes were obtained for tumor samples
from 73 of the subjects using both array-based and qRT-PCR methods
(Table 6).
TABLE-US-00008 TABLE 6 Expression Levels of Amphiregulin and
Epiregulin by quantitative RT- PCR TaqMan Assays KRAS KRAS Order
Best qRT- qRT- Mutation Mutation of Clinical AffyQ AffyQ PCR PCR
codon amino sample Response AREG EREG AREG EREG base acid on
Assessment expression expression dCt dCt change change FIG. 7 CR
2573.74 1659.91 5.80 5.32 1 PD 949.81 450.25 7.79 7.20 WT 36 SD
3353.93 2336.8 9.58 8.89 c.35G > T G12V 7 SD 105.82 89.23 9.35
9.31 WT 8 UTD 1581.54 603.27 6.48 6.20 c.35G > A G12D 73 SD PD
1626.87 668.84 5.40 5.48 c.35G > T G12V 32 PD 122.3 46.36 58 UTD
321.51 56.59 9.20 9.31 c.35G > A G12D 69 SD SD PD 177.95 128.85
9.01 8.76 c.35G > A G12D 67 PD 2550.49 655.04 4.57 5.64 WT 30 PR
3974.98 1108.91 3.23 4.38 WT 2 PD 1084.91 622.01 5.35 5.46 WT 26 PD
611.84 573.66 6.17 5.60 WT 47 SD 955.24 292.33 6.22 7.30 WT 11 PR
5083.12 1166.18 WT 5 PD SD 2481.22 1154.9 4.56 4.99 WT 12 SD
2527.86 1395.95 5.37 4.35 WT 13 SD WT PD c.35G > A G12D PD
402.53 419.27 9.34 6.14 c.35G > A G12D 62 PR 3395.09 1447.49
3.76 4.14 WT 3 PD 2134.23 906.03 7.11 6.45 c.35G > T G12V 37 PD
1163.17 100.48 6.39 9.52 c.35G > T G12V 27 UTD 1086.48 113.14
UTD UTD WT 70 UTD 301.36 241.05 8.82 8.30 WT 74 SD 4414.67 1331.61
3.77 4.67 WT 14 SD 609.57 62.96 c.35G > A G12D 15 PD WT PD
901.86 459.6 8.30 7.43 WT 68 PD WT PR 3332.21 2042.92 5.17 3.47 WT
6 PD 42.03 78.71 11.81 9.19 WT 48 SD WT PD c.35G > C G12A PR
1418.75 2411.15 4.91 3.40 WT 4 UTD 872.72 469.76 6.32 5.55 c.35G
> A G12D 71 SD 1384.71 632.61 5.75 5.60 na 9 PD 503.53 206.2
6.83 7.10 na 59 PD 75.64 50.98 10.33 9.52 61 PD 1879.09 587.4 7.50
7.25 na 41 PD 471.68 36.46 5.60 4.77 34 PD 39.27 8.15 12.33 13.18
WT 55 PD 111.94 107.83 10.02 8.30 WT 43 PD na PR na PD 1464.45
298.7 5.94 7.16 WT 51 SD 5533.18 2232.8 na 10 PD 236.8 42.59 8.96
UTD 54 SD 1416.68 819.85 WT 16 PD 719.16 550.72 6.38 5.90 c.35G
> A G12D 42 PD UTD 127.95 12.85 9.86 10.64 c.35G > A G12D 72
PD 331.54 307.55 8.22 6.83 WT 33 PD 936.71 64.49 8.28 10.95 WT 65
PD 132.01 28.72 10.55 12.04 c.35G > A G12D 35 UTD 760.08 221.16
6.27 8.55 75 PD 162.74 71.16 10.21 11.17 WT 28 UTD 865.02 258.5
7.95 8.94 c.34G > A G12S 76 PD 489.57 224.81 8.17 7.70 c.35G
> T G12V 46 PD 813.24 529.95 7.16 6.79 c.35G > A G12D 38 PD
PD PD 1556.84 703.23 5.70 5.40 c.35G > C G12A 60 SD PD 1646.55
1127.43 6.44 5.39 WT 57 PD PD 27.71 1.05 13.23 UTD WT 56 PD 1182.47
76.66 7.48 10.91 c.34G > A G12S 50 PD PD 532.55 171.22 8.87 8.79
c.35G > C G12A 45 PD 12.43 13.62 UTD 13.67 WT 63 SD 2809.16
804.93 6.13 5.20 WT 17 UTD 1656.76 665.01 6.14 5.07 c.38G > A
G13D 77 SD 18.88 2.2 10.67 12.31 WT 18 SD 1479.28 799.93 5.74 6.28
WT 19 PD 1034.32 384.07 6.64 7.29 WT 53 UTD 24.18 15.47 UTD UTD WT
78 UTD 54.13 11.49 9.44 11.32 WT 79 SD 1554.57 646.2 5.23 5.86 WT
20 SD 3536.88 1764.91 5.82 3.45 WT 21 SD WT SD 6390.33 3078.94 3.47
4.02 WT 22 PD PD 801.39 486.2 6.81 7.14 WT 40 SD c.35G > A G12D
UTD 1945.99 240.5 8.21 10.16 c.38G > A G13D 80 PD 1984.72 897.89
4.21 4.31 c.35G > T G12V 64 SD 5830.27 1980.37 2.58 3.11 WT 23
PD 2321 784.77 5.41 5.21 c.35G > T G12V 29 PD WT PD 1095.66
468.77 9.03 7.75 c.38G > A G13D 66 PD 442.29 77.8 9.84 10.39
c.35G > A G12D 49 SD 1610.75 442.09 5.25 6.21 WT 24 SD 2615.62
1113.89 5.67 7.03 WT 25 PD 1737.75 694.22 6.05 7.01 WT 44 SD WT PD
2271.37 634.05 5.32 5.61 c.35G > A G12D 39 PD 1858.06 870.14
6.27 6.34 c.35G > A G12D 52 PD 1018.25 859.41 8.08 5.91 WT
31
There was good correlation between the two methods (for
log.sub.2-transformed array data, Pearson>0.85, R.sup.2>0.7),
with high expression on Affymetrix arrays corresponding to low
.DELTA.Ct values from TaqMan assays for both amphiregulin and
epiregulin (FIG. 9). Genetic Analysis of DNA Isolated from Tumor
Biopsies and Whole Blood:
[0131] Somatic mutations in the EGFR tyrosine kinase domain are
found to be strongly associated with sensitivity to gefitinib and
erlotinib in NSCLC (Janne et al., J. Clin. Oncol., 23, 3227-3234
(2005)). It has been reported that somatic mutations in the EGFR TK
domain are not required for response to cetuximab, nor do they
appear to be predictive of response to cetuximab (Tsuchihashi et
al., N. Engl. J. Med., 353, 208-209 (2005)). Somatic mutations in
K-RAS are associated with a lack of sensitivity to gefitinib and
erlotinib in NSCLC but their role in cetuximab sensitivity in CRC
is unclear (Moroni et al., Lancet Oncol., 6, 279-286 (2005); Pao et
al., PLoS Med., 2, e17 (2005)). DNA from 80 tumor biopsies was
evaluated for mutations in EGFR, K-RAS and BRAF. Not a single
heterozygous mutation was detected in either the EGFR kinase domain
or in exon 15 of the BRAF gene. K-RAS exon 2 mutations affecting
codon 12 and 13 were detected in 30 out of 80 (38%) analyzed
samples (Table 6). K-RAS mutations were detected in only 3 Stable
Disease patients out of the 27 Disease Control Group (5 PR and 22
SD) patients tested (11%). On the other hand, K-RAS mutations were
detected in 27 out of 53 non-responders (51%). The data clearly
show that the presence of a K-RAS mutation correlates with a lack
of response to cetuximab therapy.
Discussion:
[0132] The key findings from the analysis of pre-treatment biopsies
are that patients whose tumors express high levels of the EGFR
ligands epiregulin and amphiregulin are most likely to benefit from
cetuximab therapy. In addition, it was found that patients whose
tumors do not have K-RAS mutations have a significantly higher
disease control rate than those with K-RAS mutations.
[0133] The genes for the EGFR ligands epiregulin and amphiregulin
are co-localized on chromosome 4q13.3 (Conti et al., Mol.
Endocrinol., 20, 715-723 (2006)). It was observed that the
expression of epiregulin and amphiregulin was coordinately
regulated (Pearson correlation=0.85). Epiregulin is known to bind
more weakly to EGFR and ERBB4 than the EGF ligand, but is a much
more potent mitogen than EGF and leads to a prolonged state of
receptor activation (Shelly et al., J. Biol. Chem., 273,
10496-10505 (1998)). Elevated expression of epiregulin and/or
amphiregulin may play an important role in tumor growth and
survival by stimulating an autocrine loop through EGFR. This may
characterize a tumor that is "EGFR-dependent" and therefore
sensitive to the ability of cetuximab to block ligand-receptor
interaction. The observations that constitutive epiregulin and
amphiregulin expression in L2987 cells is decreased upon EGFR
inhibitor treatment, is stimulated by EGF treatment, and that
cetuximab treatment blocks L2987 cell growth, support the
hypothesis that these EGFR ligands are beacons of an activated EGFR
pathway and perhaps autocrine stimulators. This hypothesis is also
supported by results in a lung cancer mouse model in which high
expression of epiregulin and amphiregulin, as well as ERBB3, was
dependent on EGFR activation (Fujimoto et al., Cancer Res., 65,
11478-11485 (2005)).
[0134] It is not surprising that the findings of epiregulin and
amphiregulin RNA expression was not translated into protein-based
assays. The mRNA transcripts may code for the membrane-anchored
precursor forms that are eventually cleaved to generate soluble
forms. In the case of amphiregulin, it has been shown that the
membrane-anchored isoform, as well as the soluble form, are
biologically active and may induce juxtacrine, autocrine or
paracrine signaling (Singh and Harris, Cell Signal, 17, 1183-1193
(2005)). It is interesting to note that in contrast to these
findings, elevated serum levels of amphiregulin and TGF.alpha. have
been reported to predict poor response to gefitinib in patients
with advanced NSCLC. (Ishikawa et al., Cancer Res., 65, 9176-9184
(2005)). It remains to be determined whether the tumors of the
patients with high serum levels of amphiregulin and TGF.alpha.
described in that study may have other genetic aberrations such as
K-RAS mutation that may allow by-pass of their dependence on EGFR
signaling for growth and survival.
[0135] Epiregulin and amphiregulin can be used to identify other
tumor types that might be sensitive to cetuximab. Epiregulin and
amphiregulin expression is increased in androgen-independent
prostate cancer cells and after castration in an androgen-sensitive
prostate cancer xenograft (Torring et al., Prostate, 64, 1-8
(2005); Toning et al., Anticancer Res., 20, 91-95 (2000)).
Epiregulin expression is higher in pancreatic cancer where it
stimulates cell growth (Zhu et al., Biochem. Biophys. Res. Commun.,
273, 1019-1024 (2000)) and in bladder cancer patients where it is
correlated with survival (Thogersen et al., Cancer Res., 61,
6227-6233 (2001)). The enhanced expression of amphiregulin is found
to be significantly correlated with overall survival in non-small
cell lung cancer (NSCLC) (Fontanini et al., Clin. Cancer Res., 4,
241-249 (1998)). Amphiregulin expression is higher in multiple
myeloma cells expressing ERBB receptors and promotes their growth
(Mahtouk et al., Oncogene, 24, 3512-3524 (2005)). Recently, it has
been found that high levels of lutenizing hormone may elevate the
risk of ovarian and breast cancers through the stimulation of
epiregulin and amphiregulin which in turn could stimulate mitogenic
EGFR signaling (Freimann et al., Biochem. Pharmacol., 68, 989-996
(2004)). Finally, the observation that EGFR and estrogen receptor
(ER.alpha.) mediate expression of amphiregulin (Britton et al.,
Breast Cancer Res. Treat., 96, 131-146 (2006)) suggests that a
subset of breast cancer patients (EGFR+, ER+, amphiregulin+) may
benefit from cetuximab therapy. It is notable that among metastatic
breast cancer patients treated with the EGFR inhibitor gefitinib in
combination with taxotere, significantly better response rates were
seen in ER positive than in ER negative tumors (Ciardiello et al.,
Br. J. Cancer, 94, 1604-1609 (2006)).
[0136] In addition to the observation that the two EGFR ligands are
predictive of response to cetuximab, it was found that patients
without K-RAS mutations have a higher disease control rate (48%)
than those with K-RAS mutations (10%). This result confirms
findings from a recently reported study that shows that patients
without K-RAS mutations have a higher disease control rate (76%)
than those with K-RAS mutations (31%) (Lievre et al., Cancer Res.,
66, 3992-3995 (2006)). Interestingly, a majority of the patients
described in the previous study were treated with a combination of
cetuximab and chemotherapy, suggesting that the K-RAS mutations are
predictive of disease progression in both the monotherapy and
combination therapy settings. K-RAS plays a crucial role in the
RAS/MAPK pathway, which is located downstream of EGFR and other
growth factor receptors, and is involved in cell proliferation. The
presence of activating mutations in K-RAS might be expected to
circumvent the inhibitory activity of cetuximab. K-RAS mutations
have also been found to be associated with resistance to gefitinib
and erlotinib in NSCLC (Pao et al., PLoS Med., 2, e17 (2005)).
These data consistently support the role of K-RAS mutations in
predicting response to cetuximab and/or other EGFR inhibitors, and
should continue to be evaluated in cancers where RAS mutations are
prevalent such as CRC, NSCLC and pancreatic cancer (Minamoto et
al., Cancer Detect. Prev., 24, 1-12 (2000)).
[0137] In contrast to what has been observed in patients with NSCLC
(Janne et al., J. Clin. Oncol., 23, 3227-3234 (2005)), mutations in
the EGFR gene (exons 18-21) in the patients enrolled in this CRC
study were not detected, confirming the paucity of mutations in
patients with CRC (Tsuchihashi et al., N. Engl. J. Med., 353,
208-209 (2005)). Mutations in BRAF (exon 15) were not detected,
though such mutations have been observed at a low frequency
(<5%) in other studies (Moroni et al., Lancet Oncol., 6, 279-286
(2005)). An increase in EGFR gene copy number was observed in less
than 10% of the patients evaluated in this study and while there
was a trend towards higher copy number in the patients with disease
control, the result was more in line with that of Lievre et al (10%
of patients had amplification) than with Moroni et al (31% of
patients had amplification). Assessment of the performance of a
model using the combination of K-RAS mutation status and epiregulin
mRNA expression levels showed excellent prediction power (AUC value
of 0.89).
Example 3
Production of Antibodies Against the Biomarkers
[0138] Antibodies against the biomarkers can be prepared by a
variety of methods. For example, cells expressing a biomarker
polypeptide can be administered to an animal to induce the
production of sera containing polyclonal antibodies directed to the
expressed polypeptides. In one aspect, the biomarker protein is
prepared and isolated or otherwise purified to render it
substantially free of natural contaminants, using techniques
commonly practiced in the art. Such a preparation is then
introduced into an animal in order to produce polyclonal antisera
of greater specific activity for the expressed and isolated
polypeptide.
[0139] In one aspect, the antibodies of the invention are
monoclonal antibodies (or protein binding fragments thereof). Cells
expressing the biomarker polypeptide can be cultured in any
suitable tissue culture medium, however, it is preferable to
culture cells in Earle's modified Eagle's medium supplemented to
contain 10% fetal bovine serum (inactivated at about 56.degree.
C.), and supplemented to contain about 10 g/l nonessential amino
acids, about 1.00 U/ml penicillin, and about 100 .mu.g/ml
streptomycin.
[0140] The splenocytes of immunized (and boosted) mice can be
extracted and fused with a suitable myeloma cell line. Any suitable
myeloma cell line can be employed in accordance with the invention,
however, it is preferable to employ the parent myeloma cell line
(SP2/0), available from the ATCC (Manassas, Va.). After fusion, the
resulting hybridoma cells are selectively maintained in HAT medium,
and then cloned by limiting dilution as described by Wands et al.
(1981, Gastroenterology, 80:225-232). The hybridoma cells obtained
through such a selection are then assayed to identify those cell
clones that secrete antibodies capable of binding to the
polypeptide immunogen, or a portion thereof.
[0141] Alternatively, additional antibodies capable of binding to
the biomarker polypeptide can be produced in a two-step procedure
using anti-idiotypic antibodies. Such a method makes use of the
fact that antibodies are themselves antigens and, therefore, it is
possible to obtain an antibody that binds to a second antibody. In
accordance with this method, protein specific antibodies can be
used to immunize an animal, preferably a mouse. The splenocytes of
such an immunized animal are then used to produce hybridoma cells,
and the hybridoma cells are screened to identify clones that
produce an antibody whose ability to bind to the protein-specific
antibody can be blocked by the polypeptide. Such antibodies
comprise anti-idiotypic antibodies to the protein-specific antibody
and can be used to immunize an animal to induce the formation of
further protein-specific antibodies.
Example 4
Immunofluorescence Assays
[0142] The following immunofluorescence protocol may be used, for
example, to verify EGFR biomarker protein expression on cells or,
for example, to check for the presence of one or more antibodies
that bind EGFR biomarkers expressed on the surface of cells.
Briefly, Lab-Tek II chamber slides are coated overnight at
4.degree. C. with 10 micrograms/milliliter (.mu.g/ml) of bovine
collagen Type II in DPBS containing calcium and magnesium (DPBS++).
The slides are then washed twice with cold DPBS++ and seeded with
8000 CHO-CCR5 or CHO pC4 transfected cells in a total volume of 125
.mu.l and incubated at 37.degree. C. in the presence of 95%
oxygen/5% carbon dioxide.
[0143] The culture medium is gently removed by aspiration and the
adherent cells are washed twice with DPBS++ at ambient temperature.
The slides are blocked with DPBS++ containing 0.2% BSA (blocker) at
0-4.degree. C. for one hour. The blocking solution is gently
removed by aspiration, and 125 .mu.l of antibody containing
solution (an antibody containing solution may be, for example, a
hybridoma culture supernatant which is usually used undiluted, or
serum/plasma which is usually diluted, e.g., a dilution of about
1/100 dilution). The slides are incubated for 1 hour at 0-4.degree.
C. Antibody solutions are then gently removed by aspiration and the
cells are washed five times with 400 .mu.l of ice cold blocking
solution. Next, 125 .mu.l of 1 .mu.g/ml rhodamine labeled secondary
antibody (e.g., anti-human IgG) in blocker solution is added to the
cells. Again, cells are incubated for 1 hour at 0-4.degree. C.
[0144] The secondary antibody solution is then gently removed by
aspiration and the cells are washed three times with 400 .mu.l of
ice cold blocking solution, and five times with cold DPBS++. The
cells are then fixed with 125 .mu.l of 3.7% formaldehyde in DPBS++
for 15 minutes at ambient temperature. Thereafter, the cells are
washed five times with 400 .mu.l of DPBS++ at ambient temperature.
Finally, the cells are mounted in 50% aqueous glycerol and viewed
in a fluorescence microscope using rhodamine filters.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120195889A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120195889A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References