U.S. patent application number 12/826386 was filed with the patent office on 2010-11-04 for method for predicting response to epidermal growth factor receptor-directed therapy.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Sarah S. BACUS, Pamela LOCKBAUM, David Haskett LYNCH, Gisela SCHWAB, Xiao-dong YANG.
Application Number | 20100279323 12/826386 |
Document ID | / |
Family ID | 30000467 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279323 |
Kind Code |
A1 |
BACUS; Sarah S. ; et
al. |
November 4, 2010 |
METHOD FOR PREDICTING RESPONSE TO EPIDERMAL GROWTH FACTOR
RECEPTOR-DIRECTED THERAPY
Abstract
This invention provides methods for determining or predicting
response to cancer therapy in an individual.
Inventors: |
BACUS; Sarah S.; (Hinsdale,
IL) ; LYNCH; David Haskett; (Bainsbridge Island,
WA) ; LOCKBAUM; Pamela; (Moss Beach, CA) ;
SCHWAB; Gisela; (Hayward, CA) ; YANG; Xiao-dong;
(Palo Alto, CA) |
Correspondence
Address: |
AMGEN INC.
MAIL STOP 28-2-C, ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
|
Family ID: |
30000467 |
Appl. No.: |
12/826386 |
Filed: |
June 29, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11548386 |
Oct 11, 2006 |
7771958 |
|
|
12826386 |
|
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Current U.S.
Class: |
435/7.23 ;
435/7.4 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/2863 20130101; A61K 38/1808 20130101 |
Class at
Publication: |
435/7.23 ;
435/7.4 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/573 20060101 G01N033/573 |
Claims
1. A method for predicting a response to an epidermal growth factor
receptor-directed therapy in a human subject, the method comprising
the step of assaying a tumor sample from the human subject before
therapy with one or a plurality of reagents that detect expression
and/or activation of predictive biomarkers for cancer; and
determining a pattern of expression and/or activation of at least
two of said predictive biomarkers, wherein the pattern predicts the
human subject's response to the epidermal growth factor
receptor-directed therapy.
2. The method of claim 1, wherein the predictive biomarker is a
growth factor receptor, or a growth factor receptor-related
downstream signaling molecule.
3. The method of claim 2, wherein the growth factor receptor is
HER1 (EGFR), pHER1, HER2/neu, HER3, or any combination thereof.
4. The method of claim 2, wherein the growth factor
receptor-related downstream signaling molecules is pERK.
5. The method of claim 2, wherein the growth factor receptor is
HER1 (EGFR), pHER1, HER2/neu, HER3, or any combination thereof, and
the growth factor receptor-related downstream signaling molecules
is pERK.
6. The method of claim 1, where in the predictive biomarkers are
HER1 (EGFR) and HER3.
7. The method of claim 6, wherein when HER1 (EGFR) is undetectable
is predictive of the human subject not responding to the epidermal
growth factor receptor-directed therapy.
8. The method of claim 6, wherein when HER3 is undetectable is
predictive of the human subject responding to the epidermal growth
factor receptor-directed therapy.
9. The method of claim 1, where in the predictive biomarkers are
HER1 (EGFR) and pERK.
10. The method of claim 1, where in the predictive biomarkers are
pERK and HER3.
11. The method of claim 1, where in the predictive biomarkers are
HER1 (EGFR), HER3, and pERK.
12. A method of selecting a subject with cancer for treatment with
a molecule targeting epidermal growth factor receptor (EGFR),
comprising determining the level of expression of HER3 in a cell or
tissue sample from the subject, wherein if the level of HER3
expression is low in the cells, the subject is selected.
13. The method of claim 12, wherein the molecule is an anti-EGFR
antibody.
14. The method of claim 13, wherein the antibody is ABX-0303.
15. The method of claim 12, wherein the determining step further
comprises determining expression of one or more of HER1 (EGFR),
pHER1, HER2/neu, and pERK.
16. A method of predicting the likely response rate to a molecule
targeting epidermal growth factor receptor (EGFR) of a subject
having a cancer that overexpresses EGFR, comprising the step of
determining the level of expression of HER3 in a cell or tissue
sample from the subject, wherein if the level of HER3 expression is
low in the cells, the subject is likely to respond to the molecule
targeting EGFR.
17. The method of claim 16, wherein the molecule is an anti-EGFR
antibody.
18. The method of claim 17, wherein the antibody is ABX-0303.
19. The method of claim 16, wherein the determining step further
comprises determining expression of one or more of HER1 (EGFR),
pHER1, HER2/neu, and pERK.
20. A method of selecting a subject with cancer for treatment with
a molecule targeting epidermal growth factor receptor (EGFR), the
method comprising: a) determining an expression and/or activation
profile of two or more growth factor receptors in cells and/or
tissues of the subject; and b) selecting the subject based on the
expression and/or activation profile, wherein the subject is
selected when the level of expression of HER3 is low, the level of
expression of the HER1 is high, and/or the level of the pERK index
is high.
21. The method of claim 20, wherein the molecule is an anti-EGFR
antibody.
22. The method of claim 21, wherein the antibody is ABX-0303.
23. The method of claim 22, wherein the growth factor receptors
comprise one or more of HER1 (EGFR), pHER1, HER2/neu, and HER3.
Description
[0001] This application is a divisional application of U.S.
Divisional application Ser. No. 11/548,386, Oct. 11, 2006, which
claims the benefit of U.S. application Ser. No. 10/600,129, Jun.
19, 2003, which claims the benefit of U.S. Provisional Application
Ser. No. 60/389,796, filed Jun. 19, 2002.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to methods for predicting the
response to cancer therapy in an individual.
[0004] 2. Background of the Invention
[0005] Cellular growth and differentiation processes involve growth
factors that exert their actions through specific receptors
expressed in the surfaces of responsive cells. Ligands binding to
surface receptors, such as those that carry an intrinsic tyrosine
kinase activity, trigger a cascade of events that eventually lead
to cellular proliferation and differentiation (Carpenter et al.,
Biochem., 48: 193-216, 1979; Sachs et al., Cancer Res., 47:
1981-1986, 1987). Receptor tyrosine kinases can be classified into
several groups on the basis of sequence similarity and distinct
features. One of these groups includes the epidermal growth factor
receptor family, which included erbB-1 (EGFR or HER-1) (Carpenter
et al., Biochem., 48: 193-216, 1979); erbB-2 (HER-2/neu) (Semba et
al., Proc. Natl. Acad. Sci., 82: 6497-6501, 1985; Coussens et al.,
Science, 230: 1130-1139, 1985, Bargmann et al., Cell, Vol. 45,
649-657, 1986); erbB-3 (HER-3) (Kraus et al., Proc. Natl. Acad.
Sci., 86: 9193-9197, 1989; Carraway et al., R. A. J. Biol. Chem.,
269: 14303-14306, 1994), and erbB-4 (HER-4) (Plowman et al.,
Nature, 366: 473-475, 1993; Tzahar et al., Biol. Chem., 269:
25226-25233, 1994).
[0006] As an example of a ligand that can bind to surface
receptors, NDF (neu differentiation factor)/Heregulin is a receptor
tyrosine kinase ligand that can stimulate the tyrosine
phosphorylation of erbB-2 through heterodimerization with its
receptors erbB-3 or erbB-4 (Peles, et al., Cell, 69:205-216, 1992,
Peles, et al., EMBO J. Mar; 12(3):961-71. 1993; Holmes et al,
Science, 256:1205-1210, 1992. Tzahar et al., Biol. Chem., 269:
25226-25233, 1994; Plowman et al., Nature, 366: 473-475, 1993;
Pinkas-Kramarski et al., Proc. Natl. Acad. Sci., 91:9387-9391,
1994; Pinkas-Kramarski et al., The Journal of Biological Chemistry,
Vol. 271, No. 32: 19029-19032, 1996; Pinkas-Kramarski et al.,
Oncogene, 16, 1249-1258, 1998.). Depending on the cell line
studied, NDF/Heregulin can either elicit a growth arrest and
differentiation phenotype, resulting in morphological changes,
induction of lipids, and expression of intracellular adhesion
molecule-1, or induce a mitogenic response (Holmes et al., Science,
256:1205-1210, 1992; Peles et al., Cell, 69:205-216, 1992; Bacus et
al., Cancer Res. 53:5251-5261, 1993).
[0007] Activation of erbB receptor heterodimers is coupled to and
stimulates downstream MAPK-Erk1/2 and PI3K-AKT growth and survival
pathways whose deregulation in cancer has been linked to disease
progression and refractoriness to therapy (Olayioye, M. A., et al.,
Mol. Cell. Biol. 18, 5042-5051 (1998), Fukazawa, T., et al., J.
Biol. Chem. 271, 14554-14559 (1996), Hackel, P. O., et al., Curr.
Opin. Cell Biol. 11, 184-189 (1999); Tzahar, E., et al., Mol. Cell.
Biol. 16, 5276-5287 (1996); Lange, C. A., et al., J. Biol. Chem.
273, 31308-31316 (1998). For example, HER-3 is a major docking site
for phoshoinositide-3-kinase (PI3K). In addition, NDF/Heregulin
stimulation causes activation of the PI3K pathway and
phosphorylation of AKT (Altiok et al., J. Biol. Chem., 274,
32274-32278, 1999; Liu et al., Res. Comm., 261, 897-903, 1999; Xing
et al., Nature Med., 6, 189-195, 2000). These observations
implicate PI3K/AKT in the signaling cascade that results from HER-3
heterodimerization with overexpressed HER-2/neu receptors in breast
cancer cells; activation of PI3K/AKT promote cell survival and
enhanced tumor aggressiveness (Shak, Semin. Oncol., Suppl 12:71-77,
1999; Huang et al., Clinical Cancer Res., Vol. 7: 2166-2174, 2000).
In addition, AKT2 was reported to be activated and overexpressed in
HER-2/neu-overexpressing breast cancers (Bacus et al., Oncogene,
21: 3532-3540, 2002).
[0008] Most tumors of epithelial origin express multiple erbB (HER)
receptors and co-express one or more EGF-related ligands suggesting
that autocrine receptor activation plays a role in tumor cell
proliferation. Because these ligands activate different erbB/HER
receptors, it is possible that multiple erbB receptor combinations
might be active in a tumor, a characteristic that could influence
its response to an erbB-targeted therapeutic. For example,
erbB-2/HER-2 is overexpressed in 20 to 30% of all breast cancers,
and its overexpression is associated with poor prognosis,
suggesting that it could be used as a target for anti-tumor agents
(Slamon et al., Science, 235: 177-182, 1987; Tagliabue et al., Int.
J. Cancer, 47: 933-937, 1991; Hudziak et al., Mol. Cell. Biol., 9:
1165-1172, 1989). Studies have shown that in erbB-2 overexpressing
breast cancer cells, treatment with antibodies specific to
HER-2/erbB-2 in combination with chemotherapeutic agents (e.g.,
cisplatin, doxoubicin, taxol) elicits a higher cytotoxic response
than treatment with chemotherapy alone (Hancock et al., Cancer
Res., 51: 4575-4580, 1991; Arteaga et al., Cancer, 54:3758-3765,
1994; Pietras et al., Oncogene, 9: 1829-1838, 1994). One possible
mechanism by which HER-2/erbB-2 antibodies might enhance
cytotoxicity to chemotherapeutic agents is through the modulation
of the HER-2/erbB-2 protein expression, (Bacus et al., Cell Growth
& Diff., 3: 401-411, 1992, Bacus et al., Cancer Res.
53:5251-5261, 1993; Stancovski et al., Proc Natl Acad Sci USA 88:
8691-8695, 1991; Klapper et al., Oncogene 14, 2099-2109, 1997, and
Klapper et al., Cancer Res., 60: 3384-3388, 2000), or by
interfering with DNA repair (Arteaga et al., Cancer, 54:3758-3765,
1994, and Arteaga et al., J Clinical Oncology, Vol. 19, No 18 s, 32
s-40 s, 2001; Pietras et al., Oncogene, 9: 1829-1838, 1994).
[0009] Because of the effect of anti-HER-2/erbB-2 antibodies on
cellular growth, a number of approaches have been used to
therapeutically target HER-2/erbB-2 or EGFR overexpressing cancers.
For clinical use, one approach is to interfere with the kinase
activity of the receptor by using inhibitors that block the
nucleotide binding site of HER-2/neu or EGFR (Bruns, et al., Cancer
Research, 60, 2926-2935, (2000); Christensen, et al, Clinical
Cancer Research, Vol. 7, 4230-4238, 2001, Erlichman, et al., Cancer
Research 61, 739-748, 2001, Fujimura, et al., Clinical Cancer
Research, Vol. 8, 2448-2454, 2002; Herbst, et al., Journal of
Clincal Oncology, Vol. 20, No. 18, 3815-3825, 2002; Hidalgo, et al,
J. Clinical Oncology, Vol 19, No 13: pp 3267-3279, 2001; Moasser,
et al, Cancer Res., 61: 7184-7188, 2001; Normanno, et al, Ann. of
Oncol., 13: 65-72, 2002). A second approach is using ansamycins to
influence the stability of HER2/neu receptors (Munster, et al.,
Cancer Research 62, 3132-3137, 2002; Basso et al, Oncogene, 21:
1159-1166, 2002). Another approach is the use of antibodies
directed to various erbB receptors specifically EGFR or HER-2/neu
(Alaoui-Jamali, et al Biochem. Cell. Biol., 75:315-325, 1997;
Albanell, et al., J. National Cancer Institute, Vol 93, No. 24,
1830-31, 2001; Baselga, et al., Pharmacol Ther 64: 127-154, 1994
and Baselga, et al., Annuals of Oncology 13: 8-9, 2002; Mendelsohn,
Seminars in Cancer Biology, Vol. 1, pp. 339-344, 1990). A number of
monoclonal antibodies and small molecule, tyrosine kinase
inhibitors targeting EGFR or erbB-2 have been developed. For
example, HERCEPTIN.RTM. is approved for treating the 25% of women
whose breast cancers overexpress erbB-2 protein or demonstrate
erbB-2 gene amplification (Cobleigh, M. A., et al., J. Clin. Oncol.
17, 2639-2648 (1999)). Analysis of various antibodies to HER-2/neu
has led to the identification of the murine monoclonal, 4D5. This
antibody recognizes an extracellular epitope (amino acids 529 to
627) in the cysteine-rich II domain that resides very close to the
transmembrane region. Treatment of breast cancer cells with 4D5
partially blocks NDF/heregulin activation of HER-2-HER-3 complexes,
as measured by receptor phosphorylation assays. To allow for
chronic human administration, murine 4D5 was humanized to generate
HERCEPTIN.RTM. (trastuzumab) (Sliwkowski et al, Sem. in Oncol.,
26:60-70, 1999; Ye et al., Oncogene, 18: 731-738, 1999; Carter et
al, Proc. Natl. Acad Sci USA 89:4285-4289, 1992; Fujimoto-Ouchi et
al, Cancer Chemother Pharmacol, 49: 211-216, 2002; Vogel, et al.,
Oncology, 61(suppl 2):37-42, 2001; Vogel, et al., Journal of
Clinical Oncology, Vol 20, No. 3:719-726, 2002). In addition,
several EGFR-targeted therapies are currently under clinical
investigation (Mendelsohn, J., & Baselga, J., Oncogene 19,
6550-6565 (2000); Xia, W., et al. Oncogene 21, 6255-6263 (2002)).
In particular, a human anti-EGFr monoclonal antibody, designated
ABX-EGF (and also referred to herein as ABX-0303, as described in
detail in U.S. Pat. No. 6,235,883; the disclosure of which is
hereby incorporated by reference), is being developed by Abgenix,
Inc. and Immunex Corporation (Yang X et al. Development of ABX-EGF,
a fully human anti-EGF receptor monoclonal antibody, for cancer
therapy. Crit. Rev Oncol Hemato 38(1):17-23 (2001); Yang X-D et al.
Eradication of Established Tumors by a Fully Human Monoclonal
Antibody to the Epidermal Growth Factor Receptor without
Concomitant Chemotherapy. Cancer Research 59(6):1236-1243
(1999)).
[0010] Historically, cytotoxic cancer therapies have been developed
based on maximum tolerated doses (MTD), treating patients without
understanding the tumor profile for likely responders. Hence,
patients were often subjected to toxic therapies with limited
therapeutic benefit. Recently, elucidating tumor growth and
survival pathways has led to the development of tumor-targeted
therapies. An example of this approach is Gleevec.TM. an inhibitor
of the c-abl family of tyrosine kinases approved for treating
chronic myeloid leukemia and gastrointestinal stromal tumors
(Druker, B. J. et al., N Engl. J. Med. 344, 1031-1037 (2001);
Demitri, G. D., et al.; N. Engl. J. Med. 347, 472-480 (2002)).
[0011] In contrast, most erbB-receptor targeted therapies primarily
exert cytostatic anti-tumor effects, necessitating their chronic
administration. Identification of biologically effective doses
(BED), the dose or dose range that maximally inhibits the intended
target, beyond which dose escalation is likely to add toxicity
without benefit, is therefore essential. Moreover, many of these
agents will be used in combination with cytotoxic therapies, where
added toxicity may not be tolerable, further supporting BED-based
dosing. Targeted-therapy implies that populations of likely
responders exists, and can be identified.
[0012] In view of the severe and deleterious consequences of
administering an inappropriate or ineffective therapy to a human
cancer patient, there exists a need in the art for predicting the
response to cancer therapy in an individual.
SUMMARY OF THE INVENTION
[0013] This invention provides methods for predicting a response of
an individual to a particular cancer treatment regimen.
[0014] In a first aspect, the invention provides methods for
predicting a response to an epidermal growth factor
receptor-directed therapy in a human subject, the method comprising
the step of assaying a tumor sample from the human subject before
therapy with one or a plurality of reagents that detect expression
and/or activation of predictive biomarkers for cancer; and
determining a pattern of expression and/or activation of at least
two of said predictive biomarkers, wherein the pattern predicts the
human subject's response to the epidermal growth factor
receptor-directed therapy. In certain embodiments, the predictive
biomarker is a growth factor receptor, or a growth factor
receptor-related downstream signaling molecule. The growth factor
receptors can be HER1 (EGFR), pHER1, HER2/neu, HER3, or any
combination thereof. The growth factor receptor-related downstream
signaling molecules can be pERK. In further embodiments, the
predictive biomarkers are HER1 (EGFR), pHER1, HER2/neu, HER3, or
pERK, or any combination thereof.
[0015] In further embodiments, the predictive biomarkers are HER1
(EGFR) and HER3. In other embodiments, when HER1 (EGFR) is
undetectable, it is predictive of the human subject not responding
to the epidermal growth factor receptor-directed therapy. In still
other embodiments, wherein when HER3 is undetectable, it is
predictive of the human subject responding to the epidermal growth
factor receptor-directed therapy. In further embodiments, the
predictive biomarkers are HER1 (EGFR) and pERK; or the predictive
biomarkers are pERK and HER3, or the predictive biomarkers are HER1
(EGFR), HER3, and pERK.
[0016] In a second aspect, the invention provides a kit for the
determining a response to an epidermal growth factor
receptor-directed therapy in a subject, wherein the kit comprises
at least two reagents that detect expression and/or activation of
predictive biomarkers for cancer. In certain embodiments, the kit
comprises three reagents. In other embodiments, the predictive
biomarkers are HER1, HER3, or pERK, or any combination thereof.
[0017] In a third aspect, the invention provides methods for
predicting a response to a cancer therapy in a human subject, the
method comprising the step of assaying a cell or tissue sample from
the human subject before therapy with one or a plurality of
reagents that detect expression and/or activation of predictive
biomarkers for cancer, wherein said predicative biomarkers consist
of growth factor receptor ligands; and determining a pattern of
expression and/or activation of at least two of said predictive
biomarkers, wherein the pattern predicts the human subject's
response to the cancer therapy. In other embodiments, the growth
factor receptors are HER1 (EGFR), pHER1, HER2/neu, HER3 or any
combination thereof. In still other embodiments, the cancer therapy
is an epidermal growth factor receptor-directed therapy. In further
embodiments, the cancer therapy is an anti-EGFR antibody. Further,
the antibody is ABX-0303.
[0018] In a fourth aspect, the invention provides methods of
selecting a subject with cancer for treatment with a molecule
targeting epidermal growth factor receptor (EGFR), comprising
determining the level of expression of HER3 in a cell or tissue
sample from the subject, wherein if the level of HER3 expression is
low in the cells, the subject is selected. In other embodiments,
the molecule is an anti-EGFR antibody. Further, the antibody is
ABX-0303. In still other embodiments, the determining step further
comprises determining expression of one or more of HER1 (EGFR),
pHER1, HER2/neu, and pERK.
[0019] In a fifth aspect, the invention provides method of
predicting the likely response rate to a molecule targeting
epidermal growth factor receptor (EGFR) of a subject having a
cancer that overexpresses EGFR, comprising the step of determining
the level of expression of HER3 in a cell or tissue sample from the
subject, wherein if the level of HER3 expression is low in the
cells, the subject is likely to respond to the molecule targeting
EGFR. In other embodiments, the molecule is an anti-EGFR antibody.
Further, the antibody is ABX-0303. In still other embodiments, the
determining step further comprises determining expression of one or
more of HER1 (EGFR), pHER1, HER2/neu, and pERK.
[0020] In a sixth aspect, the invention provides methods of
treating a subject with cancer, comprising determining the level of
expression of HER3 in the cells from the subject, and treating the
subject with an anti-EGFR antibody when HER3 expression levels in
the cell are low. In further embodiments, the antibody is ABX-0303.
In other embodiments, the determining step further comprises
determining expression of one or more of HER1 (EGFR), pHER1,
HER2/neu, and pERK. Further, the antibody is ABX-0303. In still
other embodiments, the level of expression of HER3 is undetectable.
Further, the antibody is ABX-0303.
[0021] In a seventh aspect, the invention provides methods of
selecting a subject with cancer for treatment with a molecule
targeting epidermal growth factor receptor (EGFR), the method
comprising: [0022] a) determining an expression and/or activation
profile of two or more growth factor receptors in cells and/or
tissues of the subject; and [0023] b) selecting the subject based
on the expression and/or activation profile, wherein the subject is
selected when the level of expression of HER3 is low, the level of
expression of the HER1 is high, and/or the level of the pERK index
is high. In other embodiments, the molecule is an anti-EGFR
antibody. Further, the antibody is ABX-0303. In another aspect, the
growth factor receptors comprise one or more of HER1 (EGFR), pHER1,
HER2/neu, and HER3.
[0024] Specific embodiments of the present invention will become
evident from the following more detailed description of certain
preferred embodiments and the claims.
DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 illustrates the response to ABX-0303 by a patient
with elevated HER1 and pERK, and decreased levels of HER3. The
figure represents quantitative immunohistochemical analysis of
EGFR, pEGFR, HER2, HER3, and pERK.
[0026] FIG. 2 illustrates the response to ABX-0303 by a patient
with elevated HER1, HER3, and pERK. The figure represents
quantitative immunohistochemical analysis of EGFR, pEGFR, HER2,
HER3, and pERK.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0027] This invention provides methods for predicting response in
cancer subjects to cancer therapy, including human cancer
patients.
[0028] In contrast to traditional anticancer methods, where
chemotherapeutic drug treatment is undertaken as an adjunct to and
after surgical intervention, neoadjuvant (or primary) chemotherapy
consists of administering drugs as an initial treatment in cancer
patients. One advantage of such an approach is that, for primary
tumors of more than 3 cm, it permits the use of conservative
surgical procedures (as opposed to, e.g., radical mastectomy in
breast cancer patients) for the majority of patients, due to the
tumor-shrinking effect of the chemotherapy. Another advantage is
that for many cancers, a partial and/or complete response is
achieved in about two-thirds of all cases. Finally, because the
majority of patients are responsive after two to three cycles of
chemotherapeutic treatment, it is possible to monitor the in vivo
efficacy of the chemotherapeutic regimen employed, which is
important for a timely identification of those cancers which are
non-responsive to chemotherapeutic treatment. Timely identification
of non-responsive tumors, in turn, allows the clinician to limit
the cancer patient's exposure to unnecessary side-effects of
treatment and to institute alternative treatments. However, the
methods present in the art, including histological examination, are
insufficient for such a timely and accurate identification. The
present invention provides methods by which a more informed and
effective regime of therapy can be administered.
[0029] A cancer diagnosis, both an initial diagnosis of disease and
subsequent monitoring of the disease course (before, during, or
after treatment) is conventionally confirmed through histological
examination of cell or tissue samples removed from a patient.
Clinical pathologists need to be able to accurately determine
whether such samples are benign or malignant and to classify the
aggressiveness of tumor samples deemed to be malignant, because
these determinations often form the basis for selecting a suitable
course of patient treatment. Similarly, the pathologist needs to be
able to detect the extent to which a cancer has grown or gone into
remission, particularly as a result of or consequent to treatment,
most particularly treatment with chemotherapeutic or biological
agents.
[0030] Histological examination traditionally entails
tissue-staining procedures that permit morphological features of a
sample to be readily observed under a light microscope. A
pathologist, after examining the stained sample, typically makes a
qualitative determination of whether the tumor sample is malignant.
It is difficult, however, to ascertain a tumor's aggressiveness
merely through histological examination of the sample, because a
tumor's aggressiveness is often a result of the biochemistry of the
cells within the tumor, such as protein expression or suppression
and protein activation, which may or may not be reflected by the
morphology of the sample. Therefore, it is important to be able to
assess the biochemistry of the cells within a tumor sample.
Further, it is desirable to observe and quantitate both gene
expression and protein activation of tumor related genes or
proteins, or more specifically cellular components of a
tumor-related signally pathway.
[0031] Cancer therapy can be based on molecular profiling of tumors
rather than histology or site of disease. Elucidating the
biological effects of targeted-therapies in tumor tissue and
correlating these effects with clinical response helps identify the
predominant growth and survival pathways operative in tumors,
thereby establishing a profile of likely responders and conversely
providing a rational for designing strategies to overcoming
resistance.
[0032] It is necessary to consider additional biomarkers beyond the
presence of the target, such as EGFR, for subjects who are
considered for treatment with, for example, biomolecules that
modulate EGFR. Not all tumor cells respond to inhibition of ErbB
receptors, despite exhibiting aberrant ErbB-1 and/or ErbB-2
expression. Examples include MKN7 and BT474 ErbB
receptor-overexpressing carcinoma cell lines, wherein BT474 cells
respond to HERCEPTIN.RTM. but MKN7 cells do not. These observations
have clear implications for erbB-directed therapeutics and the
consideration of the expression of multiple erbB receptors and in
tumors.
[0033] For example, ABX-0303 (as referred to herein as ABX-EGF), an
epidermal growth factor receptor-directed therapy sponsored by
Abgenix and Immunex Corporation, effectively targets HER1 to
prevent the growth of renal cell cancers. Based on the positive
correlation between pERK expression and response to ABX-0303, it is
likely that HER1 is acting through the MAPK pathway. In addition,
HER3 was found to be elevated in a large percentage of renal
biopsies analyzed from non-responders. One possibility is that HER3
is interacting with HER2 to confound the action of the drug.
[0034] Automated (computer-aided) image analysis systems known in
the art can augment visual examination of samples. In a
representative system, the cell or tissue sample is exposed to
detectably labeled reagents specific for a particular biological
marker, and the magnified image of the cell is then processed by a
computer that receives the image from a charge-coupled device (CCD)
or camera such as a television camera. Such a system can be used,
for example, to detect and measure expression and activation levels
of Herl, pHER1HER2, HER3, and pERK in a sample. Additional
biomarkers are also contemplated by this invention. This
methodology provides more accurate diagnosis of cancer and a better
characterization of gene expression in histologically identified
cancer cells, most particularly with regard to expression of tumor
marker genes or genes known to be expressed in particular cancer
types and subtypes (i.e., different degrees of malignancy). This
information permits a more informed and effective regimen of
therapy to be administered, because drugs with clinical efficacy
for certain tumor types or subtypes can be administered to patients
whose cells are so identified.
[0035] For example, expression and activation of proteins expressed
from tumor-related genes can be detected and quantitated using
methods of the present invention. Further, expression and
activation of proteins that are cellular components of a
tumor-related signaling pathway can be detected and quantitated
using methods of the present invention. Further, proteins
associated with cancer can be quantified by image analysis using a
suitable primary antibody against biomarkers, such as, but not
limited to, Her-1, Her-2, p-Her-1, Her-3, or p-ERK, and a secondary
antibody (such as rabbit anti-mouse IgG when using mouse primary
antibodies) and/or a tertiary avidin (or Strepavidin) biotin
complex ("ABC").
[0036] In practicing the method of the present invention, staining
procedures can be carried out by a technician in the laboratory.
Alternatively, the staining procedures can be carried out using
automated systems. In either case, staining procedures for use
according to the methods of this invention are performed according
to standard techniques and protocols well-established in the
art.
[0037] By "cell or tissue sample" is meant biological samples
comprising cells, most preferably tumor cells, that are isolated
from body samples, such as, but not limited to, smears, sputum,
biopsies, secretions, cerebrospinal fluid, bile, blood, lymph
fluid, urine and faeces, or tissue which has been removed from
organs, such as breast, lung, intestine, skin, cervix, prostate,
and stomach. For example, a tissue sample can comprise a region of
functionally related cells or adjacent cells.
[0038] The amount of target protein can then be quantitated by the
average optical density of the stained antigens. Also, the
proportion or percentage of total tissue area stained may be
readily calculated, as the area stained above an antibody threshold
level in the second image. Following visualization of nuclei
containing biomarkers, the percentage or amount of such cells in
tissue derived from patients after treatment may be compared to the
percentage or amount of such cells in untreated tissue or said
tissue prior to treatment. For purposes of the invention herein,
"determining" a pattern of expression and/or activation of a
biomarker is understood broadly to mean merely obtaining the
information on such biomarker(s), either through direct examination
or indirectly from, for example, a contract diagnostic service.
[0039] Thus, the level of expression and/or activation in a cell
can be determined by, for example, quantitative
immunohistochemistry. In this case, the level of expression of
HER1, HER2, and/or HER3 is considered to be low if the OD is less
than 9. Further, the level of expression is also considered to be
low if the OD is less than 5, or less than 3, or if the OD is 0
(undetectable). In addition, the level of expression of HER1, HER2,
and/or HER3 is considered to be high is the OD is greater than 9.
Further, the level of expression can be considered high for pERK
when the pERK index is greater than 99.
[0040] Particularly useful embodiments of the present invention and
the advantages thereof can be understood by referring to Examples
1-7. These Examples are illustrative of specific embodiments of the
invention, and various uses thereof. They are set forth for
explanatory purposes only, and are not to be taken as limiting the
invention.
Example 1
Staining Procedure for Biomarkers
[0041] Human tumor tissue samples were stained as follows. Tumor
tissue in 10% Neutral Buffered Formalin Paraffin blocks are
sectioned at 4 microns and the sections placed onto coated slides.
EGFR immunostaining is preformed by using Ventana Medical
Instruments, Inc. monoclonal 111.6; HER-2 immunostaining is
performed by using Ventana Medical Instruments, Inc. monoclonal
CB11, and HER-3 immunostaining is performed by using Ventana
Medical Instruments, Inc. monoclonal SGP1. Her-1, Her-2, and Her-3
are immunostained using, for example, the "BenchMark" (VMSI) with
I-VIEW (VMSI) detection chemistry. pEGFR immunostaining is
performed by using Chemicon monoclonal MB3052. p-ERK (1:100) is
obtained from Cell Signaling Technology (Beverly, Mass.) and
immunostained using a labeled streptavidin peroxidase
technique.
[0042] For example, slides for p-ERK (1:100) are antigen retrieved
using 0.1 M citrate buffer, pH 6.0 in the "decloaker" (Biocare
Corp.) and the sections incubated overnight with the primaries at
4.degree. C. The next day, the slides for pERK and pAKT are placed
onto the Autostainer (Dako Corp.) and the "LSAB2" kit (Dako) is
employed as the detection chemistry. DAB (Dako) is used as the
chromogen. After immunostaining, all immunomarkers, are
counterstained manually with 4% ethyl green (Sigma).
Example 2
Procedure for Immunohistochemistry
[0043] Quantitative immunohistochemistry (IHC) is performed as
previously described (Bacus, S., et al., Analyt. Quant. Cytol.
Histol. 19, 316-328 (1997)). EGFR, and erbB-2 immunostaining is
performed using the pre-diluted EGFR (Ventana monoclonal 111.6) and
erbB-2 (Ventana monoclonal CB11) antibodies from Ventana on the
VMSI automated "BenchMark" staining module as described. The VMSI
"I-View" detection kit is used for all of the VMSI pre-diluted
primary antibodies. HER-3 is also immunostained using the
"BenchMark" with I-VIEW detection chemistry. pErk is immunostained
using a labeled streptavidin peroxidase technique. Phospho-Erk1/2
slides are antigen retrieved as described (Bacus, S., et al.,
Analyt. Quant. Cytol. Histol. 19, 316-328 (1997)). Slides are
placed onto the Autostainer (Dako Corp.) and the "LSAB2" kit (Dako)
employed as the detection chemistry. pEGFR is immunostained in a
similar labeled streptavidin peroxidase technique. pEGFR slides are
antigen retrieved with 1 mM EDTA and slides for p-erbB-2 with 0.1M
citrate buffer, pH 6.0, in the "decloaker". After staining, EGFR,
HER2, HER3, pErk, and pEGFR, are counterstained manually with 4%
ethyl green (Sigma). TUNEL assay (Roche Diagnostics, Indianapolis)
is performed according to the manufacturer's instructions.
Investigators preparing and analyzing tissue sections are blinded
to both patient tumor type and response to therapy.
[0044] For IHC, antibodies to EGFR, HER2 and HER3 were from Ventana
Medical Scientific Instruments (VMSI) (Tucson, Ariz.); pERK was
from Cell Signaling Technology Inc. (Beverly, Mass.); anti pEGFR
and from Chemicon (Temecula, Calif.).
Example 3
Analysis of Treatment with an Epidermal Growth Factor-Directed
Therapy
[0045] 53 samples from renal cancer patients enrolled in a clinical
trial sponsored by Abgenix and Immunex Corporation for an
investigational drug directed to EGFR were analyzed for expression
of various biomarkers. The sample slides were obtained from Impath
Laboratories, Inc.
[0046] Immunohistochemical (IHC) analyses were carried out using
the automated staining devices as described above. The antibodies
used for the specific biomarkers included: Ventana monoclonal 111.6
for EGFR, Chemicon monoclonal MB3052 for pEGFR, polyclonal pERK
from Cell Signaling Technology for pERK, Ventana monoclonal SGP1
for HER3, and Ventana monoclonal CB11 for HER2. For each specimen,
a slide was stained with control mouse immunoglobulins to establish
the existence and localization of background staining. In addition,
appropriate positive controls were run for each IHC stain.
Following counterstaining with ethyl green, the slides were
permanently mounted and analyzed using interactive image analysis
to establish the optical density of peroxidase stained cytoplasmic
and membrane staining. In the case of pERK, the fraction of cells
expressing nuclear pERK, and the intensity of the stain were
measured using a CAS system, and the results were expressed as the
pERK index (product of OD.times.percent positive nuclear area). The
technician quantifying the results observed areas of tumor that
were not adjacent to normal renal tubules to avoid confounding the
quantification. In all cases, the stained slides were viewed by at
least two people, including a pathologist and a senior scientist,
to establish that the quantification results were representative of
the stained sections.
[0047] Immunohistochemical analyses, quantification, and
correlation with response data were completed for twenty-nine (29)
of the specimens. Partial data, representing analysis of only a
subset of the selected biomarkers, was available for an additional
twelve (12) samples. No data was obtained on the remaining
specimens because of questions as to the identity of the slides, or
the absence of information concerning the patient's response to
ABX-0303. The conclusions that can be drawn from the analysis
include, but are not limited to, that response to ABX-0303 is
related to the expression of HER1, and that elevated expression of
HER3 compromises the action of the drug.
[0048] Results of the IHC analysis of the renal cancer biopsies,
for which at least HER1 IHC results and clinical response
information was available, is presented in Table 1.
TABLE-US-00001 TABLE I IHC ANALYSIS HER1 pERK HER3- HER2- HER2
Treatment Pt # HER1 "score" pHER1 Index st st cocktail Group
Histologic Type Response 3001 8 0 36 17 3 1.0 mg/kg Clear Cell
Carcinoma PD 3002 19 +2 20 7 27 5 +2 1.0 mg/kg Clear Cell Carcinoma
S 3003 8 2 1.0 mg/kg Other MR 3006 6 +1 0 896 32 0 +1 Clear Cell
Carcinoma PD (focal) 3007 18 +2 6 1127 27 0 0 1.0 mg/kg Clear Cell
Carcinoma S 3008 19 +2 0 12 15 3 1.0 mg/kg Clear Cell Carcinoma PD
3009 0 +2 0 96 15 0 0 1.0 mg/kg Clear Cell Carcinoma PD (focal)
3010 21 +3 0 1100 33 10 PD 3011 5 0 1.0 mg/kg Clear Cell Carcinoma
S 3012 8 +1 3 40 15 1 0 1.0 mg/kg Clear Cell Carcinoma S 3014 17 +2
to +3 132 0 1.0 mg/kg Other S 3018 20 +3 9 90 16 0 0 1.0 mg/kg
Papillary Carcinoma PD 3019 18 +2 14 924 2 0 1.0 mg/kg Other PR
3020 10 +3 3 1176 14 13 1.0 mg/kg Clear Cell Carcinoma S 3031 0 0 5
0 0 0 +1 1.5 mg/kg Clear Cell Carcinoma PD 3032 0 +1 1 99 2 0 +1
1.5 mg/kg Clear Cell Carcinoma S (very weak) 3033 18 +2 to +3 14
390 50 2 +2 PD 3036 4 +1 0 540 0 0 +1 S 3037 8 1.5 mg/kg Clear Cell
Carcinoma S 3039 16 +2 0 208 21 1.5 mg/kg Clear Cell Carcinoma S
3043 15 +2 0 143 40 0 +2 1.5 mg/kg Papillary Carcinoma PD 3047 11
+1 to +2 0 64 37 0 +1 1.5 mg/kg Clear Cell Carcinoma S (focal) 3051
18 +2 5 247 1.5 mg/kg Clear Cell Carcinoma S 3053 7 +1 26 0 45 2
1.5 mg/kg Other PD 3065 23 +3 15 108 22 2.0 mg/kg Clear Cell
Carcinoma S 3068 16 +2 10 221 4 9 +1 2.0 mg/kg Clear Cell Carcinoma
S 3070 33 +3 0 465 38 15 2.0 mg/kg Clear Cell Carcinoma PD 3073 2 0
0 126 23 0 2.0 mg/kg Papillary Carcinoma PD 3075 22 +3 4 77 28 13
2.0 mg/kg Clear Cell Carcinoma PD 3077 25 0 2.0 mg/kg Clear Cell
Carcinoma S 3078 6 0 1 90 11 2.0 mg/kg Clear Cell Carcinoma PD 3080
8 +1 21 8 44 11 2.0 mg/kg Clear Cell Carcinoma PD (weak) 3084 20 +3
0 8 34 5 2.0 mg/kg Clear Cell Carcinoma S 3092 6 +1 5 576 2.5 mg/kg
Clear Cell Carcinoma S 3095 10 +1 1 0 9 0 +2 MR 3099 13 +2 34 90 13
6 2.5 mg/kg Clear Cell Carcinoma S 3101 11 +2 to +3 0 6 25 0 0 2.5
mg/kg Other PD 3103 20 +3 0 1134 30 5 +2 2.5 mg/kg Clear Cell
Carcinoma PD 3105 15 2.5 mg/kg Papillary Carcinoma PD 3108 11 2.5
mg/kg Clear Cell Carcinoma S Results are presented as OD unless
otherwise indicated.
Based on this analysis, in which the positive and negative
predictive values were calculated as a function of the optical
density, or fraction positivity, values were determined to stratify
samples based upon expression of the biomarkers analyzed. The
results of the analysis of using these stratification criteria is
presented in Table II.
TABLE-US-00002 TABLE II DATA ANALYSIS group of samples (n)
RESPONDERS NONRESPONDERS all reported in study (41) 56% 44% HER1 OD
> 9 (25) 60% 40% HER1 OD < 10 (16) 44% 56% HER1 visual score
of +1 or 60% 40% greater (30) pERK index > 99 (19) 63% 37% pERK
index < 100 (16) 38% 62% HER1+/perk- (8)* 50% 50% HER1+/perk+
(12) 64% 36% HER3 OD > 9 (26) 38% 62% HER3 OD < 10 (7) 86%
14% HER3+/HER1+ (17) 47% 53% HER3+HER1- (9) 22% 78% HER3+/perk+
(13) 46% 54% HER3+/perk- (13) 31% 69% HER2 OD > 9 (6) 33% 67%
HER2 OD < 10 (23) 48% 52% *for purposes of this analysis "+"
refers to OD greater than 9 upon quantification of HER1, HER2, or
HER3; or pERK index of greater than 99.
Overall, there was no single marker that, when quantified,
absolutely correlated with response to ABX-0303. This data
indicates, however, that expression of HER1 and pERK predict
response to the drug, while samples expressing HER3 are less likely
to respond well. The quantitative analysis presented assumes that
any expression of these markers that gives an optical density
reading of 10 or greater was significant. It is interesting to note
that visual assessment of HER1 staining, where any intensity of 1+
or greater is considered positive, agrees with the quantification
of this marker. Also of interest, only three of the thirty-three
samples examined by a pathologist were scored as "0" for HER1
staining intensity, and all three samples were from patients who
failed to respond to ABX-0303. Thus, the absence of detectable HER1
(staining intensity "0"), can also be a predictor of response to
ABX-0303.
[0049] The presence of HER3 seems to be a negative predictor of
response. Patients whose specimens lacked HER3 by the criterion
used here were more likely to respond than those that had HER3 (86%
vs. 38%). There was no significant correlation between the presence
of the phosphorylated form of HER1 and response to ABX-0303. The
lack of pHER1 expression, however, even in samples with
significantly elevated levels of HER1, may have been a result of a
failure to preserve the phosphorylated form of this protein during
the collection and processing of biopsies. Only 6 of the samples
analyzed by quantitative IHC were HER2 "positive" by the criterion
of having on OD of 10 or greater. As shown in Table II, these were
predominantly poor responders to the drug. Interestingly, all six
of these samples had elevated levels of HER3. HER2 expression,
quantified using a monoclonal antibody directed against the
external domain of HER2, was further determined using a cocktail of
antibodies that recognize both the internal and external domains of
the protein. While some additional samples appeared to be positive
using this alternate approach, these observations were not
sufficient to confirm the correlation between HER2 and HER3
expression with lack of response to ABX-0303.
[0050] The expression and co-expression of HER1, HER3, and pERK
indicates that HER1, acting through pERK, was critical to ABX-0303
response, and that the action of the drug is compromised in some
manner by the presence of HER3. Notably, biopsies that showed HER3
but not HER1 expression were less likely to respond to ABX-0303
(22% response rate) than patients whose tumors expressed both
proteins (47% response rate). Dramatically, samples from patients
that had low levels of HER3 but expressed HER1 and/or pERK at
levels of greater than 9, had a 100% response rate to ABX-0303. An
analysis with a greater number of samples will help confirm any of
the conclusions drawn from this analysis. Examples of tumors with
high and low levels of HER3 are provided in FIGS. 1 and 2.
[0051] This data indicates that ABX-0303 effectively targets HER1
to prevent the growth of renal cell cancers. It is not surprising
that HER1 seems to be acting through the MAPK pathway, as shown by
the positive correlation between pERK expression and response. Of
interest is the role of HER3, which was found elevated in 79% of
the renal biopsies analyzed.
[0052] As will be appreciated, the above findings provide useful
methods for the selection of patients for treatment with molecules
that target EGFR and predictors of the response of patients. In
addition, the above findings provide useful methods for the use of
ABX-0303. ABX-0303 is described in detail in U.S. Pat. No.
6,235,883 (the disclosure of which is hereby incorporated by
reference) and referred to therein in connection with the
discussions related to hybridoma E7.6.3.
[0053] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims. All references discussed herein are hereby incorporated by
reference in their entirety.
* * * * *