U.S. patent application number 10/567012 was filed with the patent office on 2006-09-14 for treatment of cancers expressing p95 erbb2.
Invention is credited to Neil Lee Spector, Wenle Xia.
Application Number | 20060204966 10/567012 |
Document ID | / |
Family ID | 34115547 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060204966 |
Kind Code |
A1 |
Spector; Neil Lee ; et
al. |
September 14, 2006 |
Treatment of cancers expressing p95 erbb2
Abstract
The truncated ErbB2 receptor (p95.sup.ErbB2) is shown to differ
from the full-length ErbB2 receptor in its association with other
ErbB receptors. The truncated receptor preferentially associated
with ErbB3, whereas full length ErbB2 heterodimerizes with either
EGFR or ErbB3. Consistent with p95.sup.ErbB2 heterodimerization
with ErbB3, it is shown that heregulin (an ErbB3 ligand) stimulates
p95.sup.ErbB2 phosphorylation in breast cancer cell lines.
Described herein are methods of identifying patients suitable for
treatment with a p95.sup.ErbB2 inhibitor, and methods of treating
such patients.
Inventors: |
Spector; Neil Lee; (Durham,
NC) ; Xia; Wenle; (Durham, NC) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B475
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
34115547 |
Appl. No.: |
10/567012 |
Filed: |
August 2, 2004 |
PCT Filed: |
August 2, 2004 |
PCT NO: |
PCT/US04/24888 |
371 Date: |
February 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60491752 |
Aug 1, 2003 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
G01N 2333/71 20130101;
A61K 2039/545 20130101; C12Q 1/6886 20130101; G01N 2333/91205
20130101; A61P 35/00 20180101; A61K 31/517 20130101; G01N 33/57492
20130101; G01N 33/57415 20130101; A61K 39/39558 20130101; G01N
33/57407 20130101; A61K 31/519 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method of screening a human subject in need of treatment for a
solid epithelial tumor that overexpresses p185.sup.ErbB2, as an aid
in selecting therapy, comprising determining whether the tumor
expresses p95.sup.ErbB2, where expression of p95.sup.ErbB2
indicates said subject is more likely to exhibit a favorable
clinical response to treatment that includes GW572016, than to
treatment that does not include GW572016.
2. A method according to claim 1 where expression of p95.sup.ErbB2
in tumor tissue is assessed by immunohistochemical methods.
3. A method according to claim 1 where expression of p95.sup.ErbB2
is assessed by measuring ErbB2 extracellular domain (ECD) in a
sample of the subject's serum.
4-5. (canceled)
6. A method according to claim 1 where said tumor is selected from
breast, ovarian, colon, head and neck, bladder, renal cell and lung
tumors.
7. A method according to claim 1 where said subject has breast
cancer.
8. A method according to claim 1 where said subject has previously
been treated with a p185.sup.ErbB2 inhibitor.
9. A method according to claim 1 where said subject has previously
been treated with trastuzumab.
10. A method of treating a subject with a solid epithelial tumor
that overexpresses p185.sup.ErbB2, comprising determining whether
the tumor expresses p95.sup.ErbB2, and treating said subject with
GW572016 if said tumor expresses p95.sup.ErbB2.
11. A method according to claim 10 where expression of
p95.sup.ErbB2 in tumor tissue is assessed by immunohistochemical
methods.
12. A method according to claim 10 where expression of
p95.sup.ErbB2 is assessed by measuring ErbB2 extracellular domain
(ECD) in the subject's serum.
13.-14. (canceled)
15. A method according to claim 10 where said tumor is selected
from breast, ovarian, colon, head and neck, bladder, renal cell and
lung tumors.
16. A method according to claim 10 where said subject has breast
cancer.
17. A method according to claim 10 where said subject has
previously been treated with a p185.sup.ErbB2 inhibitor.
18. A method according to claim 10 where said subject has
previously been treated with trastuzumab.
19. (canceled)
20. A method of treating a subject with a solid epithelial tumor
that expresses p95.sup.ErbB2, comprising administering a
therapeutically effective amount of GW572016 to said subject.
21. A method according to claim 20, where said subject has breast
cancer.
22. (canceled)
23. A method according to claim 20 where said subject has
previously been treated with trastuzumab.
24. A method of treating a subject with breast cancer that is
resistant to treatment with a p185.sup.ErbB2 inhibitor that binds
to the extracellular domain of ErbB2 and whose tumor expresses
p95.sup.ErbB2, comprising administering a therapeutically effective
amount of a p95.sup.ErbB2 inhibitor to said subject.
25. (canceled)
26. A method according to claim 24 where said subject has
previously been treated with trastuzumab.
27. A method of screening a subject in need of treatment for breast
cancer to determine suitability for treatment with a p185.sup.ErbB2
inhibitor that binds to the extracellular domain of ErbB2,
comprising determining whether said tumor expresses an elevated
level of p95.sup.ErbB2, where an elevated level of p95.sup.ErbB2
indicates that said subject should not be treated with a
p185.sup.ErbB2 inhibitor in the absence of treatment with a
p95.sup.ErbB2 inhibitor.
28. A method according to claim 27, further comprising treating
said subject with a treatment selected from (a) a p185.sup.ErbB2
inhibitor, (b) a combined a p185.sup.ErbB2 inhibitor and GW572016.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of treating solid
tumors that express p95.sup.ErbB2, and methods for selecting
subjects suitable for such treatment.
BACKGROUND
[0002] Many existing anti-cancer chemotherapeutics are
non-specific, in that they typically damage or kill normal cells as
well as malignant cells. Research in oncology is increasingly
focused on targeted therapies, in which a therapeutic compound
interacts with a specific molecule to interfere with a particular
molecular pathway. Tumors in different individuals, even when found
at the same anatomic location, can differ in their molecular
signalling pathways. Accordingly, it is important to know which
molecules and pathways are targeted by a therapeutic compound, so
that the treatment can be provided to the appropriate patients.
Determining which molecules and pathways are affected by a
therapeutic compound also provides diagnostic techniques to
identify those patients suitable for treatment with that
therapeutic.
ErbB Receptors
[0003] The ErbB family of type I receptor tyrosine kinases includes
ErbB1 (also known as the epidermal growth factor receptor (EGFR or
HER1)), ErbB2 (also known as Her2), ErbB3, and ErbB4. These
receptor tyrosine kinases are widely expressed in epithelial,
mesenchymal, and neuronal tissues where they play a role in
regulating cell proliferation, survival, and differentiation
(Sibilia and Wagner, Science, 269: 234 (1995); Threadgill et al.,
Science, 269: 230 (1995)). Increased expression of wild-type ErbB2
or EGFR, or expression of constitutively activated receptor
mutants, transforms cells in vitro (Di Fiore et al., 1987; DiMarco
et al, Oncogene, 4: 831 (1989); Hudziak et al., Proc. Natl. Acad.
Sci. USA., 84:7159 (1987); Qian et al., Oncogene, 10:211 (1995)).
Increased expression of ErbB2 or EGFR has been correlated with a
poorer clinical outcome in some breast cancers and a variety of
other malignancies (Slamon et al., Science, 235: 177 (1987); Slamon
et al., Science, 244:707 (1989); Bacus et al, Am. J. Clin. Path.,
102:S13 (1994)).
[0004] A family of peptide ligands binds to and activates ErbB
receptor signaling, and includes epidermal growth factor (EGF) and
transforming growth factor .alpha. (TGF-.alpha.), each of which
binds to EGFR (Reise and Stern, Bioessays, 20:41 (1998); Salomon et
al., Crit. Rev. Oncol. Hematol., 19: 183 (1995)). Ligand-receptor
interactions are selective in that epidermal growth factor (EGF)
and transforming growth factor alpha (TGF.alpha.) bind EGFR while
heregulin binds ErbB3 and ErbB4. Ligand binding induces ErbB
receptor phosphorylation (activation) with subsequent formation of
homo- and heterodimers. ErbB2 is the preferred heterodimeric
partner for EGFR, ErbB3, and ErbB4 (Graus-Porta et al., EMBO J.,
16:1647 (1997); Tzahar et al., Mol. Cell. Biol., 16: 5276 (1996)).
A number of soluble ligands have been identified for EGFR, ErbB3,
and ErbB4, but none have been identified for ErbB2, which seems to
be transactivated following heterodimerization (Ullrich and
Schlessinger, Cell, 61: 203 (1990); Wada et al., Cell, 61: 1339
(1990); Karunagaran et al., EMBO J., 15:254 (1996); Stem and Kamps,
EMBO J., 7: 995 (1988)).
Truncated ErbB2
[0005] The ErbB2 gene encodes a Mr 185,000 member of the ErbB
family. The full-length ErbB2 receptor (p185.sup.ErbB2) undergoes
proteolytic cleavage releasing its extracellular domain (ECD),
which can be detected in cell culture medium and in patient's sera.
(Lin and Clinton, Oncogene 6:639 (1991); Zabrecky et al., J. Biol.
Chem. 266:1716 (1991); Pupa et al., Oncogene 8:2917 (1993)).
Cleavage of ErbB2 appears to be mediated by a member of the matrix
metalloprotease (MMP) family (Codony-Servat et al., Cancer Res.
59:1196 (1999)). The truncated ErbB2 receptor (p95.sup.ErbB2) that
remains after proteolytic cleavage exhibits increased autokinase
activity and transforming efficiency compared with the full-length
receptor, inplicating the ErbB2 ECD as a negative regulator of
ErbB2 kinase and oncogenic activity. (Di Fiore et al., Science
237:178 (1987); Bargmann and Weinberg, EMBO J 7:2043 (1988);
Segatto et al., Mol. Cell. Biol. 8:5570 (1988)).
[0006] Expression of the p95.sup.ErbB2 truncated ErbB2 receptors in
breast cancer cells has been correlated with positive lymph node
metastasis in ErbB2 overexpressing tumors. (Christianson et al.,
Cancer Res. 58:5123 (1998); Molina et al., Clin. Cancer Res. 8:347
(2002)). Elevated serum levels of ErbB2 ECD in women with breast
cancer has also been correlated with a poorer response to therapy.
(Brandt-Rauf, Mutat. Research 333:203 (1995); Kandl et al., Br. J.
Cancer 70:739 (1994); Yamauchi et al., J. Clin. Oncol. 15:2518
(1996); Colomer et al., Clin. Cancer Research 6:2356 (2000)).
Therapeutics and ErbB2
[0007] Trastuzumab (Herceptin.TM.), a humanized anti-ErbB2
monoclonal antibody has been approved for the treatment of breast
cancers that either overexpress ErbB2, or that demonstrate ErbB2
gene amplification (Cobleigh et al, J. Clin. Oncol., 17:2639
(1999)). Trastuzumab binds to the extracellular domain of the ErbB2
receptor, and has been reported to exert its antitumor effects
through several mechanisms. See e.g., Sliwkowski et al., Semin.
Oncol. 26(Suppl 12):60 (1999). In ErbB2 over-expressing cells,
trastuzumab has been reported to down-regulate ErbB2 expression
(Sarup et al., Growth Reg 1:72 (1991); Lane et al., Mol. Cell Biol.
20:3210 (2000)). In animal models, trastuzumab has been reported to
induce antibody-dependent cell-mediated cytotoxicity against ErbB2
expressing tumor cells (Clynes et al., Nat. Med. 6:443 (2000)).
Molina et al., Cancer Research 61:4744 (2001) report that
trastuzumab reduced ECD shedding from two breast adenocarcinoma
cell lines, whereas another antibody (2C4) directed against the
ErbB2 ectodomain did not.
[0008] Combination therapy with trastuzumab and chemotherapy has
been associated with a longer time to disease progression in breast
cancer, a longer duration of response, and longer survival,
compared to chemotherapy alone. Slamon et al., NEJM 344:783 (2001).
However, resistance to trastuzumab frequently occurs within the
first year of treatment. See e.g., Baselga et al. Eur J Cancer, 37
Suppl 1:18 (2001). Strategies to reduce or prevent resistance to
trastuzumab are needed; one such proposed strategy is to target
insulin-like growth factor I receptor (IGF-IR) signaling to delay
development of trastuzumab resistance. See, e.g., Lu et al., J Natl
Cancer Inst. Dec. 19, 2001;93(24):1852-7; Camirand et al., Med Sci
Monit. 8:BR521 (2002).
[0009] Because heterodimers of ErbB2 and EGFR can elicit potent
mitogenic signals, interrupting both ErbB2 and EGFR simultaneously
is a potential therapeutic strategy (Earp et al., Breast Cancer
Res. Treat., 35:115 (1995)). Small molecule, dual EGFR-ErbB2
tyrosine kinase inhibitors have been identified and their
pre-clinical anti-tumor activities reported (Fry et al., Proc.
Natl. Acad. Sci. USA., 95:12022 (1998); Cockerill et al.,
Bioorganic Med. Chem. Letts., 11:1401 (2001); Rusnak et al., Cancer
Res., 61:7196 (2001); Rusnak et al., Mol. Cancer Therap., 1:85
(2001)).
[0010] GW572016 (lapatinib) is a potent reversible, dual inhibitor
of the tyrosine kinase domains of both EGFR and ErbB2, with
IC.sub.50 values against purified EGFR and ErbB2 of 10.2 and 9.8
nM, respectively (Rusnak et al., Mol. Cancer Therap., 1:85 (2001)).
Recent reports have demonstrated that GW572016 inhibits EGFR and
ErbB2 autophosphorylation in tumor cell lines that overexpress
these receptors (Rusnak et al., Mol. Cancer Therap., 1:85 (2001)),
an effect that was primarily associated with tumor cell growth
arrest. The chemical name of GW572016 is
N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-methylsulfonyl)ethyl-
]amino}methyl)-2-furyl]-4-quinazolinamine (WO 99 35146, Carter et
al.); a ditosylate form is disclosed in WO 02 02552 (McClure et
al); methods of treating cancer are disclosed in WO 02/056912, and
PCT/US03/10747.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIG. 1 shows Western Blots indicating the effects of
GW572016 on the expression of phosphorylated ErbB2 (p185),
phosphorylated EGFR (p170) and p95 phosphotyrosine protein in HN5
(lanes 1-2), BT474 (lanes 3-4), S1 (lanes 5-6) and Hb4a (lanes 7-8)
cell lines. Western blot analysis was performed using equal amounts
of protein from whole cell extracts, and using anti-pTyr monoclonal
antibody. Steady state protein levels of phosphorylated
p185.sup.ErbB2 (upper arrow), p170.sup.EGFR (lower arrow) and p95
(arrowhead) are shown. Cells were treated with vehicle alone (DMSO
at a final concentration of 0.1%) (-) or GW572106 (1 .mu.M or 5
.mu.M as indicated (+) in the figure) for 24 hours).
[0012] FIG. 2 shows Western Blots indicating the effects of
GW572016 or trastuzumab on ErbB2, p95.sup.ErbB2,
pTyr/p95.sup.ErbB2, and pTyr/ErbB2 in BT474 cells. Exponentially
growing BT474 cells were co-cultured for 24 hours with either 0.5
.mu.M GW572016 (lanes 2,5,8; "016"), 10 ug/ml trastuzumab (lanes
3,6,9; "Trast."), or control vehicle (0.1% DMSO; lanes 1, 4, 7,
"cont."). Equal amounts of protein were separated by SDS-PAGE and
then ErbB2, p95.sup.ErbB2, pTyr/p95.sup.ErbB2, and pTyr/ErbB2
steady state protein levels were assessed by Western blot. Blots
were probed with the following antibodies: anti-ErbB2 ECD (lanes
1-3), anti-intracytoplasmic ErbB2 peptide (aa 1243-1255) (lanes
4-6), and anti-pTyr (lanes 7-9). Along the left sides of the lanes,
the uppermost arrow indicates p185.sup.ErbB2; lower arrowhead
indicates p95.sup.ErbB2. Molecular weights are indicated on the
right side of the figure.
[0013] FIG. 3a shows Western Blots indicating the effects of
GW572016 or trastuzumab on p95.sup.ErbB2 and p185.sup.ErbB2 protein
levels in BT474 tumor xenografts established in CD-1 nude mice.
When tumors were palpable, animals were administered either vehicle
alone (lanes 1-3), GW572016 (lanes 4-6), or trastuzumab (lanes
7-9). Total p185.sup.ErbB2 and p95.sup.ErbB2 steady state protein
levels were assessed by Western blot using an antibody recognizing
an intracytoplasmic peptide (aa 1243-1255) of ErbB2.
[0014] FIG. 3b shows Western Blots indicating the effects of
GW572016 or trastuzumab on phosphorylated p95.sup.ErbB2 and
phosphorylated p185.sup.ErbB2 protein levels in BT474 tumor
xenografts established in CD-1 nude mice. When tumors were
palpable, animals were administered either vehicle alone (lanes
1-3), GW572016 (lanes 4-6), or trastuzumab (lanes 7-9). Activated
p185.sup.ErbB2 (pTyr/p185.sup.ErbB2) and p95.sup.ErbB2
(pTyr/p95.sup.ErbB2) were assessed using an anti-pTyr mAb.
[0015] FIG. 3c shows Western Blots indicating the effects of
GW572016 or trastuzumab on phosphorylated p95.sup.ErbB2 and
phosphorylated p185.sup.ErbB2 protein levels in BT474 tumor
xenografts established in CD-1 nude mice. When tumors were
palpable, animals were administered either vehicle alone (lanes
1-3), GW572016 (lanes 4-6), or trastuzumab (lanes 7-9).
Phosphorylated p95.sup.ErbB2 and p185.sup.ErbB2 were assessed using
an anti-phospho-tyrosine specific monoclonal antibody recognizing
Y1248.
[0016] FIG. 4 shows Western Blots indicating the effects of
GW572016 on activation of MAPK-Erk1/2 and PI3K-AKT pathways, and on
cyclin D1/2 total steady state protein levels in BT474 xenografts.
Animals were administered either vehicle alone (lanes 1-3),
GW572016 (lanes 4-6), or trastuzumab (lanes 7-9). Equal amounts of
protein from tumor xenograft whole cell extracts were separated by
SDS-PAGE and steady state protein levels of total and
phosphorylated forms of Erk1/2 and AKT were assessed by Western
blot. Cyclin D1/2 steady state protein levels were also determined.
Steady state protein levels of actin were used to confirm equal
loading of protein.
[0017] FIG. 5 shows Western Blots indicating the effect of GW572016
on EGF- or Heregulin-induced phosphorylation of p95.sup.ErbB2
p170.sup.EGFR, ErbB3, and p185.sup.ErbB2 in BT474 cells. Cells were
cultured in serum-free medium containing 1.5% BSA with (+) or
without (-) 1 .mu.M GW572016 for 16 hours and then exposed to EGFR
(50 ng/ml) or heregulin (5 nM) for 15 min prior to harvesting. In
the uppermost panel of FIG. 5, EGFR immunoprecipitation was
followed by Western blot using anti-pTyr monoclonal antibody, and
indicated that EGF increased EGFR phosphorylation (compare lanes 1
and 2). GW572016 (1 .mu.M) inhibited baseline phosphorylation of
EGFR (compare lanes 1 and 4), and partially blocked EGF induced
phosphorylation of EGFR (compare lanes 2 and 5). In contrast,
addition of heregulin (HRG) did not exhibit much effect on
phosphorylation of EGFR (compare lanes 1 and 3). Moreover,
phospho-p95.sup.ErbB2 did not co-precipitate with EGFR (FIG. 5,
upper panel). As shown in the middle panel of FIG. 5, GW572016
inhibited phosphorylation of p185.sup.ErbB2 and p95.sup.ErbB2 in
both the presence and absence of HRG and EGF (FIG. 5, middle
panel). ErbB2 immunoprecipitation was followed by Western blot
using anti-pTyr monoclonal antibody. As shown in the lower panel of
FIG. 5, HRG, but not EGF, induced ErbB3 and p95.sup.ErbB2
phosphorylation. GW572016 partially blocked HRG-induced
p95.sup.ErbB2 phosphorylation. Western blot analysis of ErbB3 IP
revealed that phospho-p95.sup.ErbB2 associated with ErbB3.
[0018] FIG. 6a illustrates the activation-state of p185.sup.ErbB2
and p95.sup.ErbB2 in Bt474 tumor xenografts, as assessed by Western
blot analysis of anti-pTyr immunoprecipitation probed with
anti-ErbB2 monoclonal antibody recognizing the cytoplasmic peptide
1243-1255 (upper panel); and ErbB2 (aa 1243-1255)
immunoprecipitation probed with anti-ErbB2 (aa 1243-1255)
monoclonal antibody (lower panel). Tumor bearing mice were
administered vehicle alone or GW572016, and tumor cell extracts
from the animals were compared.
[0019] FIG. 6b illustrates the activation-state of ErbB3 and
p95.sup.ErbB2 in Bt474 tumor xenografts, as assessed by Western
blot analysis of anti-ErbB3 immunoprecipitation probed with
anti-pTyr monoclonal antibody (upper panel); and ErbB3
immunoprecipitation probed with .alpha. ErbB2 (aa 1243-1255) Ab
(lower panel). Tumor bearing mice were administered vehicle alone
or GW572016, and tumor cell extracts from the animals were
compared.
[0020] FIG. 6c illustrates the activation state of EGFR in Bt474
tumor xenografts, as assessed by Western blot analysis of an EGFR
immunoprecipitate probed with anti-pTyr monoclonal antibody (upper
panel); and EGFR immunoprecipitate probed with anti-ErbB2 (aa
1243-1255) monoclonal antibody (lower panel). Tumor bearing mice
were administered vehicle alone or GW572016, and tumor cell
extracts from the animals were compared.
[0021] FIG. 7 illustrates that GW572016 inhibits activation of
Erk1/2 and AKT by EGF and heregulin in BT474 cells. Addition of
GW572016 (1 .mu.M), EGF (50 ng/ml) and Heregulin (5 nM) is
indicated by (+). Equal amounts of cellular protein were analyzed
by Western blot for steady state protein levels of total and
phosphorylated forms of Erk1/2 and AKT. Proteins were visualized
using fluorescent-labeled secondary antibodies and quantified by
Odyssey Infrared Imaging System.
SUMMARY
[0022] A first aspect of the present invention is a method of
screening a subject needing treatment for a solid tumor, as an aid
in selecting therapy. The method comprises determining whether the
tumor expresses p95.sup.ErbB2, where expression of p95.sup.ErbB2
indicates that the subject is more likely to exhibit a favorable
clinical response to treatment that includes a p95.sup.ErbB2
inhibitor, than to treatment that does not include a p95.sup.ErbB2
inhibitor.
[0023] A further aspect of the present invention is a method of
treating a subject with a solid tumor, comprising determining
whether the tumor expresses p95.sup.ErbB2 and treating the subject
with a p95.sup.ErbB2 inhibitor if expression p95.sup.ErbB2 is
found.
[0024] A further aspect of the present invention is a method of
treating a subject with a solid tumor whose tumor expresses
p95.sup.ErbB2, by administering a therapeutically effective amount
of a p95.sup.ErbB2 inhibitor to the subject.
[0025] A further aspect of the present invention is a method of
treating a subject with breast cancer who has shown resistance to a
p185.sup.ErbB2 inhibitor and whose tumor expresses p95.sup.ErbB2,
by administering a therapeutically effective amount of a
p95.sup.ErbB2 inhibitor to the subject.
[0026] A further aspect of the present invention is a method of
treating a subject with breast cancer that overexpresses ErbB2,
where the subject has previously been treated with an ErbB2
inhibitor that interacts with the extracellular domain of ErbB2 and
is now showing resistance to treatment with that ErbB2 inhibitor.
The method comprises screening the subject to determine if the
breast cancer expresses p95.sup.ErbB2 and, where expression of
p95.sup.ErbB2 is found, treating the subject with a p95.sup.ErbB2
inhibitor.
DETAILED DESCRIPTION
[0027] As shown by the examples provided herein, GW572016, a
reversible, dual tyrosine kinase inhibitor of both ErbB2 and EGFR,
inhibits phosphorylation of p95.sup.ErbB2 in breast cancer cells
and tumor xenografts. In contrast to GW572016, trastuzumab is shown
to have limited effect on p95.sup.ErbB2 phosphorylation. Also shown
is that p95.sup.ErbB2 differs from the full-length ErbB2 receptor
in its association with other ErbB receptors. The truncated
receptor (p95.sup.ErbB2) preferentially associated with ErbB3,
whereas full length p185.sup.ErbB2 heterodimerizes with either EGFR
or ErbB3. Consistent with p95.sup.ErbB2 heterodimerization with
ErbB3, it is shown that heregulin (an ErbB3 ligand) stimulates
p95.sup.ErbB2 phosphorylation in breast cancer cell lines.
[0028] Expression of the NH.sub.2-terminally truncated ErbB2
receptor (p95.sup.ErbB2) in breast cancer has been correlated with
metastatic disease progression (compared to progression in disease
primarily expressing full-length p185.sup.ErbB2). GW572016 is shown
to inhibit baseline p95.sup.ErbB2 phosphorylation in breast cancer
cell lines and tumor xenografts; in contrast, trastuzumab was not
shown to significantly affect p95.sup.ErbB2. Thus, p95.sup.ErbB2
represents a target for therapeutic intervention, and is shown to
be sensitive to GW572016 inhibition. Accordingly, it would be
clinically beneficial to identify patients whose tumors contain
elevated p95.sup.ErbB2 levels, as an aid in providing therapy
appropriately targeted to the molecular pathways active in their
tumor.
The ErbB2 Receptor
[0029] Proteolytic cleavage of the ErbB2 extracellular domain,
results in the expression of a truncated ErbB2 receptor
(p95.sup.ErbB2) that exerts potent oncogenic signals in preclinical
models, and has been linked to metastatic disease progression in
ErbB2 overexpressing breast cancers (Christianson et al., Cancer
Res. 58:5123 (1998); Molina et al., Clin. Cancer Res. 8:347
(2002)). Whereas the role of p185.sup.ErbB2 in regulating breast
cancer cell growth and survival has been extensively studied,
relatively little is known about p95.sup.ErbB2. Here it is shown
that p95.sup.ErbB2 is constitutively activated in breast cancer
cell lines and tumor xenografts. Since ErbB2 tyrosine
autophosphorylation is a biochemical marker of increased
transforming activity, these data implicate activated,
phospho-p95.sup.ErbB2 as playing a role in the pathophysiology of
breast cancer.
[0030] Engineered truncated ErbB2 receptors possess enhanced cell
transformation activity compared with p185.sup.ErbB2 (Di Fiore et
al., Science 237:178 (1987); Bargmann and Weinberg, EMBO J. 7:2043
(1988); Segatto et al., Mol. Cell. Biol. 8:5570 (1988)). Deletions
within the ECD of ErbB2 increase ErbB2 autokinase and
transformation activities, implicating sites within the ErbB2 ECD
as exerting repressive effects on ErbB2 activity. The increased
oncogenic properties of p95.sup.ErbB2 are consistent with the
association of p95.sup.ErbB2 tumor expression and metastatic
disease progression in ErbB2 overexpressing breast cancers.
[0031] ErbB receptors optimally signal through heterodimeric
complexes, reported to be mediated through ECD interactions.
(Schaefer et al., J. Biol. Chem. 274:859 (1999); Fitzpatrick et
al., FEBS Ltt. 431:102 (1998)). ErbB3 and p185.sup.ErbB2 are
frequently co-expressed in breast cancers (Lemoine et al., Br. J.
Cancer 66:1116 (1992); Siegel et al., EMBO J. 18:2149 (1999);
Alimandi et al., Oncogene 10:1813 (1995); Rajkumar et al., Breast
Cancer Res. Treatment 29:3 (1995)). ErbB3-p185.sup.ErbB2
heterodimers represent a potent mitogenic, transforming receptor
complex. Since ErbB3 contains multiple binding sites for the SH2
domain of the p85 subunit of PI3K, ErbB3-containing heterodimers
are potent activators of the PI3K-AKT growth and survival pathway
(Soltoff et al., Mol. Cell. Biol. 14:3550 (1994); Prigent and
Gullick, EMBO J. 13:2831 (1994)). In addition, formation of
p185.sup.ErbB2-ErbB3 heterodimers enhances the binding affinity of
heregulin for ErbB3. Importantly, aberrant activation of the
PI3K-AKT pathway in breast and other carcinomas predicts for a
poorer clinical outcome (Vivanco and Sawyers, Nature Reviews/Cancer
2:489 (2002); Yakes et al., Cancer Res. 62:4132 (2002); Brognard et
al., Cancer Res. 61:3986 (2001); Cheng et al., Proc. Natl. Acad.
Sci. 89:9267 (1992)).
The Present Studies
[0032] The present studies sought to determine whether
p95.sup.ErbB2 forms heterodimers, and if so, with what
heterodimeric partner. In breast cancer cell lines and BT474 tumor
xenografts, it was found that p95.sup.ErbB2 preferentially
associated with ErbB3, whereas p185.sup.ErbB2 formed heterodimers
with both EGFR and ErbB3. While not wishing to be limited by a
single theory, the present inventors note that these data provide
an explanation as to why an ErbB3 ligand such as heregulin, but not
EGF, regulates p95.sup.ErbB2 tyrosine phosphorylation. Similarly,
the fact that ErbB3 containing heterodimers potently activate the
PI3K-AKT pathway provides an explanation as to why heregulin
increases steady state protein levels of activated, p-AKT in breast
cancer cell lines and tumor xenografts.
[0033] GW572016 is reported to inhibit the activation of EGFR,
p185.sup.ErbB2, Erk1/2, and AKT as well as reduces cyclin D protein
in human tumor cell lines and xenografts (Xia et al., Oncogene
21:6255 (2002); Rusnak et al., Cancer Res. 61:7196 (2001); Rusnak
et al., Cancer Therap. 1:85 (2001)). Currently in clinical trials,
GW572016 has shown preliminary clinical activity in pre-treated
patients with metastatic cancers, most notably breast cancer
(Burris, Oncologist 9 Suppl 3:10 (2004) and unpublished data). In
this context the present studies indicate that GW572016 inhibits
p95.sup.ErbB2 phosphorylation; in contrast, trastuzumab, which
binds to the ECD of ErbB2, did not block p95.sup.ErbB2 activation.
While not wishing to be limited by a single hypothesis, the present
inventors note that the ability of a tumor cell to signal through
p95.sup.ErbB2-containing heterodimers may contribute to trastuzumab
resistance, as p95.sup.ErbB2 would not be affected by a monoclonal
antibody such as trastuzumab that is directed against the ErbB2
ECD.
[0034] The present data shed light on the role of p95.sup.ErbB2 in
the progression of breast cancer. Trastuzumab is reported to reduce
ErbB2 cleavage by binding to the ErbB2 ECD (Molina et al., Cancer
Res. 61:4744 (2001)). GW572016 therapy (or combined GW572016 and
trastuzumab therapy), may offer a clinical advantage over therapy
with trastuzumab in the absence of a p95.sup.ErbB2 inhibitor, by
inhibiting activation of p95.sup.ErbB2. Accordingly, in subjects
who (a) exhibit trastuzumab resistance, (b) express p95.sup.ErbB2
in tumor tissue, and/or (c) have elevated serum levels of ErbB2
ECD, treatment with GW572016 (or another p95.sup.ErbB2 inhibitor)
is indicated.
[0035] Identification of tumors expressing p95.sup.ErbB2 may be
used therefore to identify patients more likely to respond
clinically to treatment that includes a p95.sup.ErbB2 inhibitor,
compared to the clinical response that would be achieved by
treatment solely with an ErbB2 inhibitor that primarily inhibits
full-length p185.sup.ErbB2. ErbB2 inhibitors that act on sites
retained by the truncated (p95) ErbB2 receptor would, in general,
be expected to inhibit phosphorylation of p95.sup.ErbB2, compared
to ErbB2 inhibitors that act at a site on the extracellular domain
of the ErbB2 receptor. A particular ErbB2 inhibitor that acts at a
site retained by the truncated ErbB2 receptor is GW572016.
Identification of tumors expressing p95.sup.ErbB2 may be used to
identify patients more suitable for treatment that includes a
p95.sup.ErbB2 inhibitor (such as GW572016), compared to treatment
with an ErbB2 inhibitor that primarily inhibits the full-length
ErbB2 receptor (p185.sup.ErbB2). One such ErbB2 inhibitor that
appears to preferentially inhibit the full-length ErbB2 receptor is
the monoclonal antibody trastuzumab.
[0036] The present inventors propose that increased p95.sup.ErbB2
mediated signaling contributes to trastuzumab resistance observed
in some subjects with tumors (particularly breast cancers) that
show increased serum levels of shed ErbB2 ECD, and/or that display
tumor expression of p95.sup.ErbB2. As reported herein, GW572016
inhibited p95.sup.ErbB2 phosphorylation in both breast cancer cell
lines and xenografts. Trastuzumab did not have a similar effect on
p95.sup.ErbB2, presumably because the truncated receptor protein
lacks the antigen recognition site for this monoclonal
antibody.
[0037] The present data show that GW572016 inhibited
phosphorylation of ErbB2 (both p185.sup.ErbB2 and p95.sup.ErbB2),
leading to downstream inhibition of p-Erk1/2, p-AKT, and cyclin D
in both breast cancer cell lines and xenografts. In contrast,
trastuzumab was not found to greatly affect steady state levels of
phospho-ErbB2, nor did it measurably impact downstream p-Erk1/2,
p-AKT, or cyclin D. GW572016 inhibited the proliferation of BT474
cells, and this effect was not completely reversed in the presence
of EGF. Addition of heregulin partially reversed the inhibitory
effects of GW572016 in a 72 hour assay, highlighting the importance
of the ErbB2-ErbB3 signaling complex to tumor cell survival.
However, prolonged incubation of BT474 cells in the presence of 1
.mu.M GW572016 abrogated the protective effects of heregulin (data
not shown), an important consideration since GW572016 is currently
being administered on a daily schedule designed to achieve chronic
systemic exposures of 1 .mu.M or more in patients.
[0038] These data provide support for p95.sup.ErbB2 playing a key
role in the progression of ErbB2 overexpressing breast cancers. It
is shown that a truncated p95.sup.ErbB2 can preferentially
associate with ErbB3, providing a potent signaling complex coupled
to the PI3K-AKT survival pathway. In addition, the present data
indicate that overexpression of heregulin or other ErbB3 ligands in
the tumor microenvironment may further activate p95.sup.ErbB2-ErbB3
heterodimers, thereby contributing to resistance to hormonal and
chemotherapeutic agents through deregulation of the PI3K-AKT
survival pathway. The effects of heregulin on cell signaling and
the requirement of those effects on ErbB2 may be dependent upon the
cell type such that the associations demonstrated in the current
study may have particular relevance to breast cancer.
[0039] Trastuzumab targets the ErbB2 extracellular domain. It has
been shown to reduce ErbB2 cleavage but does not appear to affect
p95.sup.ErbB2 activation-state or signaling. Identification of
tumors that express p95.sup.ErbB2 may therefore be used to select
patients who should receive therapy with a p95.sup.ErbB2 inhibitor,
such as GW572016; such patients may benefit from therapy that
inhibits both p95.sup.ErbB2 and p185.sup.ErbB2 (e.g., by using a
therapeutic that inhibits both p95.sup.ErbB2 and p185.sup.ErbB2, or
a combination of p95.sup.ErbB2 and p185.sup.ErbB2 inhibitors, such
as combined GW572016 and trastuzumab therapy). The combination of
trastuzumab and GW572016 represents a therapeutic strategy to
reduce cleavage of ErbB2 via trastuzumab with concomitant
inhibition of p95.sup.ErbB2 activation and signaling by
GW572016.
Definitions
[0040] As used herein, a method of screening or assessing a subject
as an aid in predicting the subject's response to a therapeutic
treatment, or in identifying a subject as suitable for a particular
therapy, should not be confused with the use of disease prognosis
markers. Certain molecular markers are known as indicators of more
aggressive cancers and are associated with decreased average
survival time (compared to subjects whose tumors do not express
such markers). The present invention is not directed to general
disease prognosis markers, but to the use of specified biological
markers to assess an individual's potential for response to a
therapeutic treatment, and to select treatment suitable for that
individual's disease.
[0041] Methods of the present invention are directed to the
identification and selection of subjects with solid tumors who are
likely to respond more favorably to treatment with a p95.sup.ErbB2
inhibitor, compared to the response that would be expected from
treatment without a p95.sup.ErbB2 inhibitor. More specifically, the
methods of the present invention are directed to the identification
and selection of subjects likely to respond more favorably to
treatment with an ErbB2 inhibitor that is capable of inhibiting the
activation of the truncated p95.sup.ErbB2 receptor (such as
GW572016), compared to the response that would be expected from
treatment with an ErbB2 inhibitor that was unable to inhibit
p95.sup.ErbB2, or that primarily inhibited p185.sup.ErbB2 and did
not significantly inhibit p95.sup.ErbB2.
[0042] More specifically, methods of the present invention are
directed to assessing levels of p95.sup.ErbB2 expression in a
subject's tumor, or levels of ErbB2 ECD in the serum of a subject,
where that subject is being considered for treatment of a solid
tumor (particularly breast cancer) with an ErbB2 inhibitor.
Subjects having elevated levels of serum ErbB2 ECD, or whose tumors
express p95.sup.ErbB2 are considered to be more likely to exhibit a
favorable clinical response to treatment with a therapeutic regime
that includes a p95.sup.ErbB2 inhibitor, compared to a treatment
regime that does not. More particularly, such subjects are
considered to be more likely to exhibit a favorable clinical
response to treatment with GW572016 (or combined GW572016 and
trastuzumab treatment), compared to treatment with trastuzumab
alone. In one embodiment of the present invention, the subject
being assessed has previously been treated with trastuzumab or
another p185.sup.ErbB2 inhibitor, and is either has not responded
clinically or has evidence of progressive disease after an initial
period of clinical response (i.e., shows resistance to the
therapy).
[0043] As used herein, methods to "predict" a favorable clinical
response, or to "identify" suitable subjects, is not meant to imply
a 100% predictive ability, but to indicate that subjects with
certain characteristics are more likely to experience a favorable
clinical response to a specified therapy than subjects who lack
such characteristics. However, as will be apparent to one skilled
in the art, some individuals identified as more likely to
experience a favorable clinical response will nonetheless fail to
demonstrate measurable clinical response to the treatment.
[0044] As used herein, a subject refers to a mammal, including
humans, canines and felines. Preferably subjects treated with the
present methods are humans.
[0045] As used herein, a `favorable response` (or `favorable
clinical response`) to an anticancer treatment refers to a
biological or physical response that is recognized by those skilled
in the art as indicating a decreased rate of tumor growth, compared
to tumor growth that would occur with an alternate treatment or the
absence of any treatment. "Favorable clinical response" as used
herein is not meant to indicate a cure. A favorable clinical
response to therapy may include a lessening of symptoms experienced
by the subject, an increase in the expected or achieved survival
time, a decreased rate of tumor growth, cessation of tumor growth
(stable disease), regression in the number or mass of metastatic
lesions, and/or regression of the overall tumor mass (each as
compared to that which would occur in the absence of therapy).
[0046] As is well known in the art, tumors are frequently
metastatic, in that a first (primary) locus of tumor growth spreads
to one or more anatomically separate sites. As used herein,
reference to "a tumor" in a subject includes not only the primary
tumor, but metastatic tumor growth as well.
[0047] As used herein, an ErbB2 inhibitor is an agent that inhibits
or reduces the formation of p-Tyr/ErbB2 (activated ErbB2), compared
to the formation of p-Tyr/ErbB2 that would occur in the absence of
the ErbB2 inhibitor. Such inhibitors include small chemical
molecules and biologic agents such as monoclonal antibodies. As
used herein, a p95.sup.ErbB2 inhibitor is an agent that inhibits or
reduces the formation of p-Tyr/p95.sup.ErbB2 (activated
p95.sup.ErbB2), compared to the formation of p-Tyr/p95.sup.ErbB2
that would be formed in the absence of the agent. As used herein, a
p185.sup.ErbB2 inhibitor is an agent that inhibits or reduces the
formation of p-Tyr/p185.sup.ErbB2 (activated p185.sup.ErbB2),
compared to the formation of p-Tyr/p185.sup.ErbB2 that would be
formed in the absence of the agent. An agent may be both a
p95.sup.ErbB2 inhibitor and a p185.sup.ErbB2 inhibitor.
[0048] As used herein, a cell "overexpressing" ErbB2 refers to a
cell having a significantly increased number of functional ErbB2
receptors, compared to the average number of receptors that would
be found on a cell of that same type. Overexpression of ErbB2 has
been documented in various cancer types, including breast (Verbeek
et al., FEBS Letters 425:145 (1998); colon (Gross et al., Cancer
Research 51:1451 (1991)); lung (Damstrup et al., Cancer Research
52:3089 (1992), renal cell (Stumm et al, Int. J. Cancer 69:17
(1996), Sargent et al., J. Urology 142: 1364 (1989)) and bladder
(Chow et al., Clin. Cancer Res. 7:1957 (2001); Bue et al., Int. J.
Cancer, 76:189 (1998); Turkeri et al., Urology 51: 645 (1998)).
Overexpression of ErbB2 may be assessed by any suitable method as
is known in the art, including but not limited to imaging, gene
amplification, number of cell surface receptors present, protein
expression, and mRNA expression. See e.g., Piffanelli et al.,
Breast Cancer Res. Treatment 37:267 (1996).
[0049] The DAKO HercepTest.RTM. (DakoCytomation, Denmark), is an
FDA approved IHC assay for the evaluation of ErbB2 protein
overexpression, and provides semi-quantitative results of
p185.sup.ErbB2 overexpression by light microscopy. Samples are
scored as from 0 (no staining, negative), 1+ (weak staining,
negative), 2++ (weakly positive) and 3+++ (strongly positive).
Typically patients with 2++ or 3+++ results are considered to be
overexpressing ErbB2 and thus suitable for treatment with
trastuzumab. Accordingly, a cell that `overexpresses`
p185.sup.ErbB2 is one that would score 2++ or 3+++ on the
HercepTest.RTM., or achieve a comparable score using another
assay.
[0050] As used herein, "solid tumor" does not include leukemia or
other hematologic cancers.
[0051] As used herein, an "epithelial tumor" is one arising from
epithelial tissue.
[0052] Inhibitors of ErbB2 used in the present methods should
preferentially inhibit phosphorylation of tyrosine residues within
the kinase domain, which are the residues implicated in regulating
downstream MAPK/Erk and PI3K/AKT pathways.
[0053] Non-ErbB transactivating factors (such as growth hormone,
which is increased in many cancer patients) regulate
phosphorylation of tyrosine residues external to the catalytic
kinase domain (e.g., Y992, Y1068, Y1148, and Y1173). When
conducting immunohistochemistry (IHC) to assess the phosphorylation
state of ErbB2, the use of anti-receptor antibodies that are not
domain specific will not distinguish between phosphorylation events
in tyrosine residues in the kinase domain and those external to the
kinase domain; in this situation the overall phosphorylation state
of EGFR and ErbB2 may appear unchanged even when key residues
within the kinase domain that regulate downstream Erk and AKT
pathways have been inhibited. Accordingly, the use of antibodies
that are domain specific is preferred when IHC is utilized in the
methods of the present invention.
Methods of Measuring ECD and p95
[0054] The identification of subjects whose tumors express p95
(truncated ErbB2) may be achieved using any suitable means as is
known in the art. One such method is to assess the levels of
circulating ErbB2 ECD in serum of subjects, where an elevated level
of ECD indicates that the subject's tumor contains truncated ErbB2,
and thus treatment that includes a p95.sup.ErbB2 inhibitor such as
GW572016 is preferred to a treatment regime that does not include a
p95.sup.ErbB2 inhibitor. Alternatively, the presence of truncated
ErbB2 in the tumor tissue may be detected directly, e.g., by using
Western blot techniques as described herein.
[0055] ErbB2 ECD may be detected in the serum of subjects using any
suitable technique known in the art. Serum ECD may be measured, for
example, using an enzyme immunoassay. Various groups have developed
assays that detect the ECD of ErbB2, using monoclonal antibodies
that react with the external domain of ErbB2. See, e.g., Hayes, et
al., Clin. Cancer Res. 7:2703 (2001); Hayes et al., Breast Cancer
Res. Treat., 14:135a 1989; Carney et al., J. Tumor Marker Oncol.,
6:53 (1991); Leitzel et al., J. Clin. Oncol., 10:1436, (1992);
Yamauchi et al., J. Clin. Oncol., 15: 2518 (1997).
[0056] Harris et al. (J. Clin. Oncol. 19:1698 (2001)), in a study
of 425 patients with measurable, metastatic breast cancer, assayed
serum samples for ErbB2 ECD using an enzyme-linked immunoassay kit
(Bayer Diagnostics, Walpole, Mass.). Colomer et al. (Clin. Cancer
Research, 6:2356 (2000) used a sandwich enzyme immunoassay (Human
neu quantitative ELISA, Calbiochem; Carney et al., J. Tumor Mark.
Oncol. 6:53 (1991)), and found circulating ECD levels of from 155
to 38,871 fmol/ml (median 427 fmol/ml) in samples from 58 patients
with metastatic breast cancer. Using 450 fmol/ml as the cut-off for
"elevated" circulating ECD levels, Colomer et al. found that 41% of
their subjects had elevated ECD levels.
[0057] Hayes et al., (Clin. Canc. Res. 7:2703 (2001)) assayed
plasma samples from 242 metastatic breast cancer patients for
circulating ErbB2 ECD levels, using a sandwich enzyme immunoassay.
The assay utilized MAb NB3 bound to 96-well plate and enzyme-linked
MAb TA1 as a tracer. On the basis of previous studies, Hayes et al.
utilized a cutoff of 10.5 ng/ml (mean+2SD in healthy subjects) to
distinguish elevated from nonelevated levels (Carney et al., J.
Tumor Marker Oncol., 6:53 (1991)). Eighty-nine (37%) of the 242
patients had elevated ErbB2 ECD levels.
[0058] Accordingly, an "elevated" level of serum ErbB2 ECD for a
subject with a given disease may be defined as a level greater than
the median level seen in subjects with the same gross disease; as a
level which is greater than or equal to the mean plus two standard
deviations found in healthy subjects; or alternatively as a level
that has been associated with improved response to treatment that
includes a p95.sup.ErbB2 inhibitor (compared to the response that
would be obtained in the absence of a p95.sup.ErbB2 inhibitor). For
any particular disease, the level of serum ErbB2 ECD that is
correlated with such an improved response to p95.sup.ErbB2
inhibitor treatment can be determined by one skilled in the art,
using methods known in the art.
[0059] Alternatively or in addition to assessing the levels of
ErbB2 ECD in serum of subjects, the presence of truncated ErbB2 in
the tumor tissue may be assessed. The relative level of truncated
ErbB2 may be assessed by comparing whether truncated receptor is
expressed at a certain level relative to the expression of the
entire (p185) ErbB2 receptor. See, e.g.,. Christianson et al.,
Cancer Res. 15:5123 (1998); Molina et al., Cancer Res. 61:4744
(2001). Accordingly, cells that express (or `overexpress`)
p95.sup.ErbB2 can be defined as those in which the truncated ErbB2
receptor is expressed at a level at least equal to about 5%, 10%,
20%, 25% or more of full-length ErbB2 receptor expression.
[0060] One method of detecting p95.sup.ErbB2 levels in tissue
samples utilizes immunohistochemistry, a staining method based on
immunoenzymatic reactions using monoclonal or polyclonal antibodies
to detect cells or specific proteins such as tissue antigens.
Typically, immunohistochemistry protocols include detection systems
that make the presence of the markers visible (to either the human
eye or an automated scanning system), for qualitative or
quantitative analyses. Various immunoenzymatic staining methods are
known in the art for detecting a protein of interest. For example,
immunoenzymatic interactions can be visualized using different
enzymes such as peroxidase, alkaline phosphatase, or different
chromogens such as DAB, AEC or Fast Red.
[0061] The methods of the present invention may be accomplished
using any suitable method or system of immunohistochemistry, as
will be apparent to one skilled in the art, including automated
systems, quantitative IHC, semi-quantitative IHC, and manual
methods.
[0062] As used herein, "quantitative" immunohistochemistry refers
to an automated method of scanning and scoring samples that have
undergone immunohistochemistry, to identify and quantitate the
presence of a specified biomarker, such as an antigen or other
protein. The score given to the sample is a numerical
representation of the intensity of the immunohistochemical staining
of the sample, and represents the amount of target biomarker
present in the sample. As used herein, Optical Density (OD) is a
numerical score that represents intensity of staining. As used
herein, semi-quantitative immunohistochemistry refers to scoring of
immunohistochemical results by human eye, where a trained operator
ranks results numerically (e.g., as 1, 2 or 3).
[0063] Various automated sample processing, scanning and analysis
systems suitable for use with immunohistochemistry are available in
the art. Such systems may include automated staining and
microscopic scanning, computerized image analysis, serial section
comparison (to control for variation in the orientation and size of
a sample), digital report generation, and archiving and tracking of
samples (such as slides on which tissue sections are placed).
Cellular imaging systems are commercially available that combine
conventional light microscopes with digital image processing
systems to perform quantitative analysis on cells and tissues,
including immunostained samples. See, e.g., the CAS-200 system
(Becton, Dickinson & Co.).
[0064] Where phosphorylated proteins are being assayed, tissue must
be processed in a manner that allows accurate detection of
phosphorylated proteins. E.g., if the tissue sample is
paraffin-embedded, it may be fixed in the presence of phosphatase
inhibitors and in a neutralized buffered formalin solution.
Treatment of Subjects
[0065] The present invention provides a method of screening
subjects who are being considered for treatment with an ErbB2
inhibitor for a solid tumor, to identify those subjects who are
likely to respond more favorably to therapy that includes a
p95.sup.ErbB2 inhibitor (such as GW572016), compared to therapy
that does not include such an inhibitor. Stated another way, the
present invention provides a method of screening an individual
subject in need of such treatment for a solid tumor, to identify
whether the subject is likely to respond favorably to treatment
with a p95.sup.ErbB2 inhibitor, as an aid in clinical
decision-making.
[0066] The methods of the present invention are suitable for use in
subjects afflicted with a solid tumor, particularly one of
epithelial origin, that expresses ErbB2. In one embodiment of the
present invention, the subject is afflicted with a solid tumor of
epithelial origin that overexpresses ErbB2. In on preferred
embodiment, the subject is afflicted with breast cancer, where the
breast cancer cells overexpress p185.sup.ErbB2 and express
p95.sup.ErbB2.
[0067] The methods of the present invention comprise determining
prior to treatment whether a subject's tumor expresses
p95.sup.ErbB2, e.g., by measuring either circulating ErbB2 ECD or
measuring p95.sup.ErbB2 expression in tumor tissue. Any suitable
method of determining the level of truncated ErbB2 (and/or
circulating ErbB2 ECD) may be utilized in the present methods.
[0068] According to one embodiment of the present methods, the
pre-treatment level of p95.sup.ErbB2 (or ErbB2 ECD) is assessed
immediately before the subject begins a course of anti-neoplastic
therapeutic treatment. As used herein, `immediately` before
treatment refers to a biologically relevant time frame. Preferably
the assessment is done within about three months, two months, or
six weeks prior to treatment, more preferably within about four
weeks, three weeks, two weeks, ten days or less prior to treatment.
Alternatively in the methods of the present invention, the level of
the specified marker may be assessed after treatment has begun, to
ascertain whether the appropriate treatment is being used.
[0069] As is known in the art, clinical use of an antineoplastic
agent typically involves repeated administration of the agent to a
subject over a set time period, on a pre-established schedule.
Therapeutic agents may be administered in any suitable method,
including but not limited to intravenously (intermittently or
continuously) or orally. For example, a `course` of a certain
therapeutic agent may require daily administration of the agent for
two weeks; a course of therapy using a different therapeutic agent
or for a different tumor type may involve once weekly
administration for six weeks. As used herein, a "course" of therapy
refers to a therapeutic schedule (dosage, timing of administration,
and duration of therapy) that is specific to the therapeutic agent
being used and/or the tumor type being treated, and that is
accepted in the art as therapeutically effective. Such schedules
are developed using pharmacologic and clinical data, as is known in
the art. A subject may undergo multiple courses of treatment over
time, using the same or different therapeutic agents, depending on
whether disease progression occurs.
[0070] The present methods are suitable for use in subjects
undergoing their first course of antineoplastic treatment, or
subjects who have previously received a course of antineoplastic
treatment for a tumor.
[0071] The present methods are suited for use with any form of
ErbB2 inhibitors, including organic molecules such as GW572016,
monoclonal antibodies, or other chemical or biological therapeutic
agents. Specific inhibitors, as well as processes of making
thereof, are provided in U.S. Pat. No. 6,169,091; U.S. Pat. No.
6,174,889; U.S. Pat. No. 6,207,669; U.S. Pat. No. 6,391,874; WO
99/35146; WO 01/04111.
EXAMPLE 1
Materials and Methods
Materials
[0072] HN5, an EGFR overexpressing LICR-LON-HN5 head and neck
carcinoma cell line was kindly provided by Helmout Modjtahedi at
the Institute of Cancer Research, Surrey, U.K. The ErbB2
overexpressing human breast adenocarcinoma cell line, BT474 was
obtained from the American Type Culture Collection (Manassas, Va.,
USA). HB4a cells are derived from human mammary luminal tissue;
ErbB2 transfection of parental HB4a cells yielded the HB4a C5.2
cell line (Harris et al., Int J Cancer. 80:477 1999). S1 cells,
which express elevated levels of p-ErbB2 were established by
sub-cloning HB4a C5.2 (Xia et al., Oncogene, 21:6255 (2002)). EGF
was purchased from Sigma Chemical (St. Louis, Mo., USA).
Recombinant human NRG-1-B1/HRGB1 EGFR domain (heregulin, HRG) was
from RD system (Minneapolis, Minn., USA). Anti-phosphotyrosine
antibody was purchased from Sigma and Upstate (Lake Placid, N.Y.,
USA). Anti-EGFR (Ab-12) and anti-c-ErbB2 (Ab-11) monoclonal
antibodies were from Neo Markers (Union City, Calif., USA).
Anti-ErbB2 (AA1243-1255), anti-phospho-ErbB2 (Y1248), and
anti-cyclin D1/2 were from Upstate. Anti-phospho-AKT (Ser 437)
monoclonal antibody was from Cell Signaling Technology, Inc.
(Beverly, Mass., USA). Anti-AKT1/2, anti-phospho-Erk1/2, anti-Erk1
and anti-Erk2 antibodies were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, Calif., USA). Trastuzumab was
purchased from Genentech, Inc. (South San Francisco, Calif., USA).
SuperSignal West Femto Maximum Sensitivity Substrate was from
Pierce (Rockford, Ill., USA). Protein G agarose was purchased from
Boehringer Mannheim (Germany). IRDye800 Conjugated Affinity
Purified Anti-Rabbit IgG and anti Mouse IgG were from Rockland
(Gilbertsville, Pa., USA). Alexa Fluor680 goat anti-rabbit IgG was
obtained from Molecular Probes (Eugene, Oreg., USA). GW572016,
N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methylsul-
fonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine, was
synthesized as previously described. GW572016 for cell culture work
was dissolved in DMSO.
Cell Cultures
[0073] BT474 cells were cultured in RPMI 1640 supplemented with
L-glutamine, 10% FBS (Hyclone) and 5.mu./ml insulin. HB4a cells
were cultured under identical conditions to BT474 cells, in
addition, with 10 .mu.g/ml hydrocortisone. S1 cells were cultured
in RPMI 1640 supplemented with L-glutamine, 10% FBS and 50 .mu.g/ml
hygromycin. HN5 cells were cultured in DMEM supplemented with high
glucose and 10% fetal bovine serum (FBS). All cell cultures were
maintained in a humidified atmosphere of 5% CO.sub.2 at 37.degree.
C.
EGF and HRG Stimulation Experiments
[0074] Cells were seeded at low density in serum free-medium
supplemented with 1.5% BSA, and then exposed for 6-24 h to GW572016
at various concentrations indicated in the figure legends, or 10
.mu.g/ml trastuzumab. Cells were stimulated with 50 ng/ml EGF or 5
nM HRG for 15 minutes, harvested on ice, and then lysed in RIPA
buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25% (w/v)
deoxycholate, 1% NP-40, 5 mM sodium orthovanadate, 2 mM sodium
fluoride, and a protease inhibitor cocktail).
Immunoprecipitation and Western Blots
[0075] Immunoprecipitations and Western blots were performed as
previously described (Xia et al., Oncogene, 21:6255 (2002)).
Briefly, protein concentrations were determined using a
modification of the Bradford method (Bio-Rad Laboratory) and equal
amounts of protein subjected to immunoprecipitation and Western
blot. Efficiency and equal loading of proteins was evaluated by
Ponceau S staining. Membranes were blocked for 1 h in TBS (25 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 2.7 mM KCl) containing 4% (w/v)
lowfat milk or 3% BSA (w/v). Membranes were then probed with
specific antibodies recognizing target proteins, which were
visualized with the SuperSignal West Femto Maximum sensitivity
substrate kit (Pierce). Other blots were visualized using the
Odyssey Infrared Imaging System (LI-COR, Inc., Lincoln, Nev., USA).
For the Odyssey, membranes were incubated with fluorescent-labeled
secondary antibody at 1:10000 dilution with 3% BSA in PBS for 60
min protected from light. After washing in PBS+0.1% tween-20, the
membranes were scanned using an Odyssey imaging system.
Tumor Xenografts
[0076] BT474 tumors were maintained by serial passage of fragments
into female C.B-17 SCID mice, for up to 10 passages. Once tumor
implants were palpable, mice were administered either vehicle (0.5%
hydroxypropylmethylcellulose/0.1% Tween 80) given orally (PO), five
doses of GW572016 at 100 mg/lkg (PO) twice daily at 8 hour
intervals, or trastuzumab at 100 mg/kg given intraperitoneally (IP)
daily for 3 days. Tumors were removed 4 hours after the last dose,
frozen in liquid Nitrogen, and stored at -80.degree. C. until
analysis. For the terminal biopsy, mice were euthanized with
CO.sub.2 inhalation. Cell extracts were prepared by homogenization
in RIPA buffer at 4.degree. C.
EXAMPLE 2
Inhibition of p95 by GW572016
[0077] FIG. 1 demonstrates the effects of GW572016 on the
expression of phospho-ErbB2 (p185), EGFR (p170), and p95 in BT474,
HN5, S1 and Hb4a cell lines. Western blot analysis was performed
using equal amounts of protein from whole cell extracts using
anti-pTyr monoclonal antibody. Steady state protein levels of
phosphorylated p185.sup.ErbB2 (top arrow), p170.sup.EGFR (lower
arrow) and p95 (arrowhead) are shown in FIG. 1. Cells were treated
with vehicle alone (DMSO at a final concentraton of 0.1%; indicated
by "-") or GW572016 (1 .mu.M for BT474; 5 .mu.M for other cell
lines, indicated by "+") for 24 hours.
[0078] As shown in FIG. 1 (compare lanes 3 and 4), treating ErbB2
overexpressing BT474 breast cancer cells with 1 .mu.M GW572016
inhibited not only p185.sup.ErbB2 phosphorylation (top arrow) but
also inhibited a 95 kDa phosphotyrosine protein (p95,
arrowhead).
[0079] In S1 cells, treatment with 5 .mu.M GW572016 inhibited
p185.sup.ErbB2 and p95 phosphorylation (FIG. 1, compare lanes 5 and
6). S1 cells are a cell line established by single cell cloning of
Hb4ac5.2 cells, a non-malignant mammary epithelial line stably
transfected with ErbB2 (Xia et al., 2002).
[0080] In contrast, p95 was not identified in the
EGFR-overexpressing head and neck squamous cell carcinoma line HN5
(FIG. 1, lanes 1 and 2) or in parental Hb4a cells (lanes 7 and 8).
However, 5 .mu.M GW572016 inhibited phosphorylation of
p170.sup.EGFR and p185.sup.ErbB2, respectively, in these cells
(FIG. 1).
EXAMPLE 3
Identification of p95 as the Truncated ErbB2 Receptor
(p95.sup.ErbB2)
[0081] Proteolytic cleavage of the extracellular domain of
p185.sup.ErbB2 leads to the appearance of the truncated 95 kDa
ErbB2 receptor (p95.sup.ErbB2), which is highly phosphorylated
(Christianson et al., Cancer Res. 58:5123 (1998)). To determine
whether the 95 kDa phosphotyrosine protein as identified in the
Example 1 was p95.sup.ErbB2, equal amounts of protein from total
cell extracts were subjected to Western blot analysis using
anti-ErbB2 mAbs that recognized distinct epitopes.
[0082] As shown in FIG. 2, exponentially growing BT474 cells were
co-cultured for 24 hours with vehicle alone (0.1% DMSO; lanes 1, 4
and 7), 0.5 .mu.M GW572016 (lanes 2, 5 and 8), or 10 .mu.g/ml
trastuzumab (lanes 3, 6 and 9). Equal amounts of protein were
separated by SDS-PAGE and then ErbB2, p95.sup.ErbB2,
pTyr/p95.sup.ErbB2, and pTyr/ErbB2 steady state protein levels were
assessed by Western blot. Blots were probed with the following
monoclonal antibodies: (a) anti-ErbB2 ECD (lanes 1-3), (b)
anti-intracytoplasmic ErbB2 peptide (amino acids 1243-1255, an
intra-cytoplasmic ErbB2 sequence distinct from EGFR or ErbB3)
(lanes 4-6), or (c) anti-pTyr (lanes 7-9). Along the left side of
the lanes, the uppermost arrow indicates p185; lower arrowhead
indicates p95. Molecular weights are indicated on the right side of
the figure.
[0083] As shown in FIG. 2 (lanes 1-3), using the anti-ErbB2 ECD mab
indicated that steady-state p185.sup.ErbB2 protein levels (arrow)
did not vary among BT474 control cells (lane 1), cells treated with
0.5 .mu.M GW572016 (lane 2), and cells treated with 10 .mu.g/ml
trastuzumab (lane 3). However, p95 was not identified using this
particular mAb.
[0084] As shown in FIG. 2 (lanes 4-6), using the anti-ErbB2 mab
recognizing peptide 1243-1255, both p185.sup.ErbB2 (arrow) and p95
(arrowhead) were identified in Western blots from BT474 whole cell
extracts. These results indicate that p95 is the truncated ErbB2
receptor, p95.sup.ErbB2. BT474 cells treated with trastuzumab (10
.mu.g/ml; lane 6) exhibited reduced p95.sup.ErbB2 steady state
protein levels (lane 6).
[0085] As shown in FIG. 2 (lanes 7-9), using the anti-pTyr mab
indicates that whereas tyrosine phosphorylation of both
p185.sup.ErbB2 and p95.sup.ErbB2 was inhibited by GW572016 (0.5
.mu.M) (compare lanes 7 and lane 8), trastuzumab did not have a
similar inhibitory effect (compare lanes 7 and 9).
[0086] The above data indicate that the 95 kDa phosphotyrosine
protein shown to be GW572016-sensitive is the truncated ErbB2
receptor, p95.sup.ErbB2.
EXAMPLE 4
GW572016 Inhibits Both p95.sup.ErbB2 and p185.sup.ErbB2 in Breast
Cancer Xenografts
[0087] As shown in FIGS. 3a-3c, the in vivo effects of GW572016 on
p95.sup.ErbB2 were examined in mice bearing established BT474 tumor
implants. BT474 tumor xenografts were established in CD-1 nude
mice. When tumors were palpable, animals were administered vehicle
alone (lanes 1-3), GW572016 ((100 mg/kg/dose; lanes 4-6), or
trastuzumab (lanes 7-9). Total p185.sup.ErbB2 and p95.sup.ErbB2
steady state protein levels were assessed by Western blot using
three different monoclonal antibodies: in FIG. 3a, the mab
recognized an intracytoplasmic peptide (aa 1243-1255) of ErbB2; in
FIG. 3b, anti-phosphotyrosine mab was used to assess activated
p185.sup.ErbB2 (pTyr/p185.sup.ErbB2) and p95.sup.ErbB2
(pTyr/p95.sup.ErbB2); in FIG. 3C, phosphorylated p95.sup.ErbB2 and
p185.sup.ErbB2 were assessed using an anti-phosphotyrosine specific
mAb recognizing Y1248.
[0088] As shown in FIG. 3a, steady state protein levels of total
p185.sup.ErbB2 and p95.sup.ErbB2 were unchanged in tumor xenografts
from vehicle (lanes 1-3), GW572016 (lanes 4-6), or trastuzumab
(lanes 7-9) treated mice.
[0089] As shown in FIG. 3b, phospho-p185.sup.ErbB2 steady state
protein levels were inhibited in BT474 tumor xenografts from
GW572016 treated mice (compare lanes 4-6 to other lanes).
Similarly, GW572016 inhibited phospho-p95.sup.ErbB2 steady state
protein levels (compare lanes 4-6 to other lanes). Trastuzumab did
not appear to inhibit phospho-p185.sup.ErbB2 expression (compare
lanes 7-9 to control lanes 1-3).
[0090] As shown in FIG. 3c, the effect of GW572016 on tyrosine 1248
(Y1248), a key ErbB2 autophosphorylation site linked to downstream
MAPK-Erk1/2 signaling, was also investigated. GW572016 (100
mg/kg/dose) inhibited Y1248 phosphorylation in p95.sup.ErbB2
(compare lanes 4-6 to other lanes), whereas trastuzumab did not
appear to have any marked effect (compare lanes 7-9 to control
lanes 1-3).
[0091] In BT474 tumor cell xenografts, inhibition of p185.sup.ErbB2
and p95.sup.ErbB2 phosphorylation by GW572016 also blocked
activation of downstream signaling pathways involved in tumor cell
growth and survival (MAPK-Erk1/2 and PI3K-AKT), and reduced cyclin
D1/2 total steady state protein levels. Equal amounts of protein
from tumor xenograft whole cell extracts were separated by SDS-PAGE
and steady state protein levels of total and phosphorylated forms
Erk1/2 and AKT were assessed by Western blot. Cyclin D1/2 steady
state protein levels were also determined. Steady state protein
levels of actin confirmed equal loading of protein.
[0092] As shown in FIG. 4, GW572016 (lanes 4-6) inhibited p-Erk1/2
and p-AKT steady state protein levels without affecting total
steady state protein levels of either molecule. In addition, cyclin
D steady state protein levels were inhibited by GW572016.
Conversely, trastuzumab (10 mg/kg; lanes 7-9) had little effect on
p-Erk1/2, p-AKT, or cyclin D protein expression.
EXAMPLE 5
p95.sup.ErbB2 Preferentially Associates with ErbB3
[0093] In addition to ErbB2, BT474 cells express EGFR and ErbB3. To
determine whether truncated p95.sup.ErbB2 forms heterodimers, BT474
whole cell extracts were subjected to immunoprecipitation (IP) with
either anti-EGFR, anti-ErbB2 or anti-ErbB3 monoclonal antibodies,
followed by Western blot using anti-pTyr mAb.
[0094] FIG. 5 demonstrates the effect of GW572016 on EGF- and
HRG-induced phosphorylation of p95.sup.ErbB2 and p185.sup.ErbB2 in
BT474 cells. Cells were cultured in serum-free medium containing
1.5% BSA with or without 1 .mu.MGW572016 for 16 hours and then
exposed to EGFR (50 ng/ml) or HRG (5 nM) for 15 minutes prior to
harvesting. Phospho-p95.sup.ErbB2 and p185.sup.ErbB2 were assessed
by IP Western blot using anti-pTyr monoclonal antibody.
[0095] As shown in the uppermost panel of FIG. 5, EGFR IP followed
by Western blot using anti-pTyr mAb showed that addition of EGF (50
ng/ml) increased EGFR phosphorylation in BT474 cells (compare lanes
1 and 2). GW572016 (1 .mu.M) inhibited baseline phosphorylation of
EGFR (compare lanes 1 and 4), and partially blocked EGF induced
phosphorylation of EGFR (compare lanes 2 and 5). In contrast,
addition of heregulin (HRG) did not exhibit much effect on
phosphorylation of EGFR (compare lanes 1 and 3). Moreover,
phospho-p95.sup.ErbB2 did not co-precipitate with EGFR (FIG. 5,
upper panel).
[0096] As shown in the middle panel of FIG. 5, GW572016 inhibited
phosphorylation of p185.sup.ErbB2 and p95.sup.ErbB2 in both the
presence and absence of HRG and EGF (FIG. 5, middle panel).
[0097] As shown in the lower panel of FIG. 5, HRG, but not EGF,
induced ErbB3 and p95.sup.ErbB2 phosphorylation in BT474 cells.
GW572016 partially blocked HRG-induced p95.sup.ErbB2
phosphorylation. Western blot analysis of ErbB3 P revealed that
phospho-p95.sup.ErbB2 associated with ErbB3. We were unable to
demonstrate ErbB4 in these cells (data not shown).
[0098] ErbB3-p95.sup.ErbB2 heterodimers were also identified in
BT474 tumor xenografts by comparing tumor cell extracts from
animals treated with vehicle alone or GW572016 (100 mg/kg/dose).
Tumor bearing mice were administered vehicle alone or GW572016 (see
Example 1, Material and Methods). As shown in FIG. 6a-6c, the
activation-state of EGFR, p185.sup.ErbB2, p95.sup.ErbB2, and ErbB3
was assessed by IP Western blot. FIG. 6a shows (upper panel)
Western blot analysis of anti-pTyr immunoprecipitated proteins
probed with anti-ErbB2 mAb recognizing the cytoplasmic peptide
1243-1255, and in the lower panel shows anti-ErbB2 (peptide
1243-1255) immunoprecipitated proteins probed with anti-pTyr mAb.
FIG. 6b shows (upper panel) Western blot analysis of anti-ErbB3
immunoprecipitated proteins probed with anti-pTyr mAb; and in the
lower panel ErbB3 immunoprecipitated proteins probed with
anti-ErbB2 (peptide 1243-1255) Ab. FIG. 6c shows (upper panel)
Western blot analysis of EGFR immunoprecipitated proteins probed
with anti-pTyr mAb; and in the lower panel EGFR immunoprecipitated
proteins probed with anti-ErbB2 (peptide 1243-1255) Ab.
[0099] The presence of p95.sup.ErbB2 in ErbB3 immunoprecipitations
was analyzed by Western blot using anti-pTyr or anti-ErbB2 (peptide
1243-1253) Abs. GW572016 treatment markedly inhibited ErbB3 and
p95.sup.ErbB2 tyrosine phosphorylation compared with vehicle
treatment alone (FIG. 6a, upper panel). Similar to in vitro
studies, ErbB3-p95.sup.ErbB2 heterodimers were identified in BT474
tumor xenografts (FIG. 6b, upper and lower panels). Whereas
p95.sup.ErbB2 was markedly phosphorylated in vehicle-treated
animals, GW572016 inhibited both p95.sup.ErbB2 and ErbB3 tyrosine
phosphorylation (FIG. 6b, upper panel).
[0100] In contrast, p95.sup.ErbB2 did not co-precipitate with EGFR
(FIG. 6c, upper and lower panels). Full-length p185.sup.ErbB2
formed heterodimers with EGFR (lower panel). GW572016, but not
vehicle inhibited tyrosine phosphorylation of p95.sup.ErbB2 and
p185.sup.ErbB2 in tumor xenografts (FIG. 6c).
[0101] The cytoplasmic domain of ErbB3 contains at least seven
tyrosine residues, which serve as docking sites for the p85 subunit
of PI3K (Soltoff et al., 1994; Prigent and Gullick, 1994).
Phosphorylation of these tyrosine residues leads to AKT
phosphorylation and activation. Since p95.sup.ErbB2 preferentially
associates with ErbB3 in BT474 breast cancer cells, the effects of
HRG (an ErbB3 ligand) on MAPK-Erk1/2 and PI3K-AKT were examined.
Exponentially growing cells were treated as discussed in Example 1
(Material and methods). GW572016 was added for 24 hours. Equal
amounts of protein were analyzed by Western blot for steady state
protein levels of total and phosphorylated forms of Erk1/2 and AKT.
Proteins were visualized using fluorescent-labeled secondary
antibodies and quantified by Odyssey Infrared Imaging System
(LI-COR Biosciences, Lincoln, Neb.).
[0102] As shown in FIG. 7, treating BT474 cells with EGF (50 ng/ml)
for 15 minutes increased p-Erk1/2 steady state protein levels,
which was blocked by GW572016 (1 .mu.M). AKT phosphorylation also
increased in response to HRG (5 .mu.M), and was partially inhibited
by GW572016 (1 .mu.M). These results indicate that GW572016
inhibits activation of Erk1/2 and AKT by EGF and heregulin,
respectively, in BT474 cells.
EXAMPLE 6
Clinical Use of GW572016 in Trastuzumab Refractory Patients
[0103] An open label, multi-dose clinical trial of GW572016
administered to subjects with cancer was carried out to evaluate
pharmacokinetic and safety profiles of the compound, and to study
the biological effects of the compound on the expression of the
activated forms of ErbB2. Patients were randomized to receive one
of five different oral doses of GW572016, administered as GW572016
ditosylate salt, in a series of 21 daily doses. Upon completion of
the study, patients were able to continue on therapy until disease
progression, treatment-emergent toxicities, maximum response, or
withdrawal of consent. Male and female subjects with histological
confirmation of a solid tumor (non-site specific) that (i)
overexpressed total EGFR by IHC or ErbB2 by IHC or FISH; (ii) or
expressed activated EGFR or ErbB2, (iii) that could be readily
biopsied, and (iv) are amenable to treatment with GW572016, were
included in the protocol.
[0104] In this clinical trial, of the initial 33 subjects whose
responses have been validated, 11 had breast cancer (all previously
treated with trastuzumab) with seven of the eleven exhibiting
responses (three Partial Responses, four Stable Disease).
Additional stable disease was observed in other tumor types
including ovarian cancer (2), colorectal cancer (2), lung cancer
(1), head and neck cancer (1), adenocarcinoma of unknown primary
origin (1), and granular cell cancer (1), providing a total of 15
responses out of 33 subjects (including the seven breast cancer
responders).
[0105] Thus, several patients in this clinical trial had
p185.sup.ErbB2 overexpressing breast cancer that had previously
been treated with trastuzumab. The following summaries are two
examples of such patients; detailed results of the each subject in
the clinical trial are not provided herein.
[0106] Patient No. 372 was a 53 year old female who underwent a
mastectomy in January 2001 for estrogen receptor negative,
progesterone receptor negative, ErbB2++ breast cancer. Eight of 23
lymph nodes sampled were positive for disease, and there were
supraclavicular node metastases and bone metastases. A partial
response was seen with paclitaxel/carboplatin/trastuzumab. In
February 2002, progressive disease was found and treatment with
gemcitabine/trastuzumab was started. In April 2002, progressive
disease was found, and treatment with hyperthermia, radiation
therapy, and trastuzumab was started. In May 2002, due to
progressive disease, treatment with vinorelbine and trastuzumab was
started. In August 2002, due to progressive disease, treatment with
GW572016 (1200 mg/day) was started. In October 2002, a 70% decrease
in two right breast masses was noted, and anterior abdominal wall
nodules also improved. Platelets increased from 30,000 to 95,000.
However, in December 2002, there was progressive disease noted in
the right breast mass upon computerized tomography scan.
[0107] Patient No. 361 was a 46 year old female who underwent a
right mastectomy in November 1994 for estrogen receptor negative,
progesterone receptor negative, ErbB2+++ breast cancer. Thirteen of
13 lymph nodes sampled were positive for disease. She received
adjuvant CAF (cytoxan, adriamycin and 5-fluorouracil) and radiation
therapy. In July of 1997, the disease recurred and from fall of
1997 through 1999 she was treated with paclitaxel, doxorubicin,
cyclophosphamide/zinecard, 5-fluorouracil, and eniluracil. From
January to July 2000 she was treated with trastuzumab, with
complete remission of subcutaneous nodules. In November 2000 she
began treatment with trastuzumab and vinorelbine, and from July to
October 2001 she received paclitaxel. From October 2001 to February
2002 she received trastuzumab and capecitabine. In March 2002 she
exhibited subcutaneous nodules and bone metastases, and was started
on GW572016 (1200 mg/day). All subcutaneous nodules disappeared
within 28 days of starting GW572016; bone metastases did not
respond. In June 2002 the subcutaneous nodules returned.
* * * * *