WO2004000101A2 - Methode de prevision d'une reponse a une therapie dirigee contre les recepteurs her1/her2 - Google Patents

Methode de prevision d'une reponse a une therapie dirigee contre les recepteurs her1/her2 Download PDF

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WO2004000101A2
WO2004000101A2 PCT/US2003/019697 US0319697W WO2004000101A2 WO 2004000101 A2 WO2004000101 A2 WO 2004000101A2 US 0319697 W US0319697 W US 0319697W WO 2004000101 A2 WO2004000101 A2 WO 2004000101A2
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growth factor
cancer therapy
erbb
cancer
her2
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PCT/US2003/019697
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WO2004000101A3 (fr
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Sarah S. Bacus
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Smithkline Beecham Corporation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to methods for predicting the response to cancer therapy in an individual.
  • 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.
  • erbB-1 epidermal growth factor receptor family
  • erbB-2 HER-2/neu
  • erbB-3 HER-3
  • NDF nerve differentiation factor
  • Heregulin is a receptor tyrosine kinase ligand that can stimulate the tyrosine phosphorylation of erB-2 through heterodimerization with its receptors erbB-3 or erbB-4.
  • 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).
  • Activation of erbB receptor heterodimers is coupled to and stimulates downstream MAPK-Erkl/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.
  • HER-3 is a major docking site for phoshoinositide-3- kinase (PI3K).
  • PI3K phoshoinositide-3- kinase
  • NDF/Heregulin stimulation causes activation of the PI3K pathway and phosphor ylation of AKT (Altiok et al, J. Biol. Chem., 274, 32274-32278, 1999; Liu et al, Res.
  • 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).
  • AKT2 was reported to be activated and overexpressed in HER-2/neu-overexpressing breast cancers (Bacus et al, Oncogene, 21 : 3532-3540, 2002).
  • HER erbB
  • EGF-related 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.
  • 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: 1 165-1172, 1989).
  • chemotherapeutic agents e.g., cisplatin, doxoubicin, taxol
  • 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.
  • 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.
  • HERCEPTIN 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)).
  • This antibody recognizes an extracellular epitope (amino acids 529 to 627) in the cysteine-rich II domain that resides very close to the
  • 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.
  • GleevecTM 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)).
  • This invention provides methods for predicting a response of an individual to a particular cancer treatment regimen.
  • the invention provides methods for predicting a response to a
  • 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 cancer therapy.
  • the predictive biomarkers consist of growth factor receptors, growth factor receptor ligands, and growth factor receptor-related downstream signaling molecules.
  • the growth factor receptors can be HER1 (EGFR), HER2/neu, HER3, and IGFR.
  • the growth factor receptor ligands can be NDF (Heregulin) and TGF- ⁇ .
  • the growth factor receptor-related downstream signaling molecules can be pAKT, pERK, and cyclin D.
  • the cancer therapy comprises a dual HER1/HER2 kinase inhibitor.
  • the cancer therapy comprises GW572016, obtained from Glaxo Smith-Kline.
  • the invention provides methods for predicting a response to a HERl/HER2-directed cancer 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 one of said predictive biomarkers, wherein the pattern predicts the human subject's response to the cancer therapy.
  • the predictive biomarkers consist of growth factor receptors, growth factor receptor ligands, and growth factor receptor-related downstream signaling molecules.
  • the growth factor receptors can be HER1 (EGFR), HER2/neu, HER3, and IGFR; the growth factor receptor ligands can be NDF (Heregulin) and TGF- ⁇ , and the growth factor receptor-related downstream signaling molecules can be pAKT, pERK, and cyclin D.
  • the predictive biomarker is pERK.
  • the pattern of expression and/or activation is the cellular distribution of total ERK within the cell.
  • the presence of ERK in s the nucleus is predictive of a response to a HERl/HER2-directed cancer therapy in the human subject.
  • FIGS 1A and IB illustrate inhibition of growth and survival pathways by
  • Figure 1A represents a Western blot analysis of GW572016-treated HN5, an EGFR-overexpressing tumor cell line, and BT474, an erbB-2 overexpressing tumor cell line.
  • Figure IB represents quantitative immunohistochemical analysis of p-erbB-2, p-EGFR, p-Erkl/2, p-AKT, and cyclin Dl from BT474 xenografts obtained from tumor-bearing C.B-17 SCID mice treated with either GW572016 (100 mg/kg) or vehicle alone for 5 doses.
  • the optical density (OD) values assigned to each specimen are shown. OD values of ⁇ 10, 10-15, >15 roughly correlate with 1, 2, 3+ in the HercepTest standards, respectively.
  • Figures 2A through 2D illustrates that inhibition of the pro-survival factor, p- AKT, in response to GW572016 therapy correlates with tumor cell apoptosis.
  • Figure 2 A represents an immunohistochemical analysis of EGFR and erbB-2 in a metastatic breast cancer nodule from patient 361.
  • Figure 2B shows reduced expression of intra-tumor p- AKT by day 21 (d 21) of therapy in a woman (patient 372) with metastatic breast cancer overexpressing erbB-2 and erbB-3, thereby demonstrating that GW572016 leads to the inhibition of p-AKT.
  • Figure 2C demonstrates inhibition of intra-tumor p-AKT in Patients 372 and 369.
  • Figures 3A and 3B illustrates that GW572016 therapy inhibits p-Erkl/2 expression in tumors that overexpress both EGFR and erbB-2.
  • Figure 3A shows that patients 361 and 364 had markedly elevated p-Erk indices prior to therapy.
  • Figure 4 shows that Cyclin Dl immunoreactivity is reduced in response to GW572016 therapy. Inhibition of cyclin Dl immunoreactivity is shown in sequential tumor biopsies obtained prior to (d 0) and after 21 days of GW572016 therapy from patients 361 and 364.
  • This invention provides methods for predicting response in cancer subjects to cancer therapy, including human cancer patients.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • HER-2/neu HER-2/neu
  • HERCEPTD ® HERCEPTD ®
  • tumor cells respond to inhibition of ErbB receptors, despite exhibiting aberrant ErbB-1 and/or ErbB-2 expression.
  • MKN7 and BT474 ErbB receptor-overexpressing carcinoma cell lines wherein BT474 cells respond to HERCEPTIN ® but MKN7 cells do not.
  • % consequence of decreased ErbB-1 or ErbB-2 receptor activity can be overcome by the presence of EGF-related ligands such as EGF or NDF/Heregulin (Lane et al).
  • EGF-related ligands such as EGF or NDF/Heregulin (Lane et al).
  • This phenomenon can be attenuated using a bispecific ErbB-1 /ErbB-2 inhibitor, thus supporting increased efficacy through simultaneous inhibition of multiple ErbB receptors.
  • a combination of the ErbB-1 -directed mAb 225 and mAb 4D5 inhibited proliferation of an ovarian tumor cell line more strongly than either mAb alone.
  • mAb 4D5 the mAb from which HERCEPTIN ® was derived
  • dual EGFR/erbB-2 kinase inhibitors also referred to as dual EGFR/erbB-2 kinase inhibitors.
  • GW572016 is a non-limiting example of a dual EGFR/erbB-2 kinase inhibitor obtained from Glaxo Smith-Kline.
  • HER-2/neu overexpression alone was not the only predictor of response to molecules such as HERCEPTIN ® .
  • HER-2/neu is a ligandless orphan receptor in need of its partner HER-1 and HER-3 in order to exert its activity.
  • HER-1 and HER-3 heterodimerization with HER-2 is enhanced by the presence of EGF or NDF, and correspondingly HER-2 activity is dependant on HER-1 or HER-3.
  • NDF/Heregulin or TGF are expressed to provide an autocrine loop of HER-2/HER-1 heterodimerization. Downregulation of HER-2/neu is an important pathway for inhibition of signals generated by these heterodimers.
  • HERCEPTESr which affects the HER-2/neu receptor downregulation by receptor endocytosis.
  • high levels of phosphorylated ERK are a negative predictor, in conjunction with the expression of HER-1 and the presence of NDF, pointing to other pathways that might promote proliferation the tumor cellular growth.
  • High pERK was associated with resistance to HERCEPTIN ® through downregulation of p27, and may indicate that other signals such as the estrogen receptor's cross activation of the MAPK and AKT pathways may contribute to high levels of pERK and to tumor cell growth or survival in a manner independent of the tumor growth for the erbB receptor.
  • HERCEPTIN ® patients bearing tumors that expressed elevated levels of phosphorylated AKT showed a poor response to HERCEPTIN ® ; this effect is accompanied by and may be related to high level expression of other growth factor receptors in such cells, including insulin-like growth factor receptor (IGFR-1) and platelet-derived growth factor receptor (PDGFR).
  • IGFR-1 insulin-like growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • Another important predictor of response to HERCEPTIN ® is upregulation of the AKT/mTOR pathway by Heregulin/NDF. Patients having this phenotype showed tumor cell growth inhibition after HERCEPTIN ® treatment associated with upregulation of p27.
  • the invention provides methods for using analysis of diagnostic tumor markers, specifically related to the erbB receptor family, to predict response to erbB-directed therapies.
  • HERCEPTIN ® treatment is effective when a patient's tumor cell growth is dependent on and regulated by a cellular pathway involving AKT/mTOR that is driven by erbB receptor and not by other tyrosine kinases, like IGFR-1 and PDGFR.
  • a cellular pathway involving AKT/mTOR that is driven by erbB receptor and not by other tyrosine kinases, like IGFR-1 and PDGFR.
  • HERCEPTIN ® treatment was found to be ineffective.
  • the inventive methods provide a diagnostic basis for determining prior to treatment whether erbB-directed therapies, such as HERCEPTIN ® are likely to be effective, thus minimizing the risk of administering ultimately ineffective treatments to cancer patients.
  • Automated (computer-aided) image analysis systems can augment visual examination of samples.
  • 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.
  • CCD charge-coupled device
  • Such a system can be used, for example, to detect and measure expression and activation levels of Herl, HER2, HER3, HER4, ERK, pERK, NDF, TGF , IGFR, c-kit, SCF, pc- kit, and pAKT 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 t ⁇ administered, because drugs with clinical efficacy for certain tumor types or subtypes can be administered to patients whose cells are so identified.
  • Another drawback of conventional anticancer therapies is that the efficacy of specific chemotherapeutic agents in treating a particular cancer in an individual human patient is unpredictable.
  • the art is unable to determine, prior to starting therapy, whether one or more selected agents would be active as anti- tumor agents or to render an accurate prognosis of course of treatment in an individual patient. This is especially important because the same clinical cancer may present the clinician with a choice of treatment regimens, without any current way of assessing which regimen will be most efficacious for a particular individual. It is an advantage of the methods of this invention that they are able to better assess the expected efficacy of a proposed therapeutic agent (or combination of agents) in an individual patient. Additional advantageous features of the claimed methods for assessing the efficacy of chemotherapeutic regimens are that they are both time- and cost-effective and minimally traumatic to cancer patients.
  • proteins associated with breast 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, p-Her-2, p-ERK, or p-AKT, 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").
  • a suitable primary antibody against biomarkers such as, but not limited to, Her-1, Her-2, p-Her-1, p-Her-2, p-ERK, or p-AKT
  • a secondary antibody such as rabbit anti-mouse IgG when using mouse primary antibodies
  • a tertiary avidin (or Strepavidin) biotin complex such as a tertiary avidin (or Strepavidin) biotin complex
  • reagents examples include a rabbit polyclonal antibody specific for pAKT and obtained from Cell Signaling Technology (Beverly, MA, Cat. No. 9277); anti-SCF antibody, obtained from Santa Cruz Biotechnology (Santa Cruz, CA, Cat. No. SC-9132); and a polyclonal anti-c-t ⁇ antibody obtained from Neomarkers, Inc. (Fremont, CA, Cat. No. RB-1518).
  • 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.
  • 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.
  • determining a pattern of expression and/or activation of a biomarker is understood broadly to mean merely obtaining gene expression and/or gene product activation information on such biomarkers.
  • Human tumor tissue samples were stained as follows. Tumor tissue in 10% Neutral Buffered Formalin Paraffin blocks were sectioned at 4 microns and the sections placed onto coated slides. Her-1 and Her-2 immunostaining was performed by using the pre-diluted Her-1 and Her-2 antibodies from Ventana Medical Instruments, Inc. (VMSI, Arlington, AZ.). Her-3 (1 :10) and Heregulin (1 :25) antibodies were obtained from NeoMarkers (Fremont, CA.). Her-1, Her-2, Her-3, and Heregulin were immunostained using the "BenchMark” (VMSI) with I- VIEW (VMSI) detection chemistry.
  • p-ERK (1 :100) and p-AKT (1 :75) were obtained from Cell Signaling Technology (Beverly, MA), and immunostained using a labeled streptavidin peroxidase technique.
  • Slides for p-ERK (1 :100) and p-AKT (1 :100) were 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 °C. The next day, the slides for pERK and pAKT, were placed onto the Autostainer (Dako Corp.) and the "LSAB2" kit (Dako) was employed as the detection chemistry.
  • Tris-HCl pH 7.5
  • 137 mM NaCl 137 mM NaCl
  • ImM EDTA 1% Nonidet P-40
  • Triton X-100 0.2%
  • glycerol 0.1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM ⁇ - glycerophosphate, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 2 ⁇ M leupeptin, and 2 ⁇ g/ml aprotinin).
  • Total protein concentration was determined with a BioRad Protein Assay Kit (BioRad Laboratories, Hercules, CA). Equal amounts of protein, such as 15 ⁇ g protein per lane, were separated by gel electrophoresis, such as pre-cast 4-12%
  • Antibodies utilized for Western blotting include anti-phosphotyrosine purchased from Sigma Chemical (St. Louis, MO); anti-EGFR (Ab-12) and anti-erbB-2 (Ab-11) from NeoMarkers; antibodies to p-AKT (Ser 437), p-Erkl/2, Erkl/2 purchased from Santa Cruz Biotechnology; and anti-cyclin Dl and 2 from Upstate (Lake Placid, NY).
  • EGFR, erbB-2, and cyclin Dl immunostaining was performed using the pre-diluted EGFR, erbB-2, and cyclin Dl antibodies from Ventana on the VMSI automated "BenchMark” staining module as described.
  • the VMSI "I- View” detection kit was used for all three of the
  • VMSI pre-diluted primary antibodies Erkl/2 (1 : 1200), erbB-3 (1 :10), Heregulin (1 :25), and TGF ⁇ (1 :20), were also immunostained using the "BenchMark" with I- VIEW detection chemistry.
  • Phospho-Erkl/2 and p-AKT slides were antigen retrieved as described (Bacus, S., et al, Analyt. Quant. Cytol istol. 19, 316-328 (1997)).
  • EGFR, erbB-2, erbB-3, heregulin, Erkl/2, p-AKT, p-Erkl/2, p-EGFR, p-erbB-2, cyclin Dl, IGFR-1, and TGF ⁇ were counterstained manually with 4% ethyl green (Sigma).
  • TUNEL assay (Roche Diagnostics, Indianapolis) was performed according to the manufacturer's instructions, investigators preparing and analyzing tissue sections were blinded to both patient tumor type and response to therapy.
  • antibodies to EGFR, erbB-2, and cyclin D antibodies were from Ventana Medical Scientific instruments (VMSI) (Tucson, AZ); anti- p-AKT (Ser 437) and p- Erkl/2 were from Cell Signaling Technology Inc. (Beverly, MA); anti p-EGFR and p- erbB2 were from Chemicon (Temecula, CA) and NeoMarkers (Fremont, CA), respectively; antibodies to TGF ⁇ , erbB3, heregulin, IGFR-1, and phospho-erbB2 were from NeoMarkers; anti-Erkl/2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
  • the HER1/HER2 combination inhibitor GW572016 (obtained from Glaxo Smith- Kline) reversibly inhibits both EGFR and erbB-2 and also inhibits steady state protein levels of pErkl/2, pAKT, and cyclin D in erbB-2- (BT474) and EGFR- (HN5) overexpressing tumor cell lines.
  • the erbB-2 overexpressing human breast adenocarcinoma line, BT474 was obtained from the American Type Culture Collection (Rockville, MD) and the EGFR-expressing LICR-LON-HN5 head and neck carcinoma cell line (HN5) was kindly provided by Helmout Modjtahedi at the Institute of Cancer Research, Surrey, UK.
  • Quantitative IHC offers advantages over Western-blot by enabling direct visualization of the biological effects of therapy on tumor cells interspersed amongst surrounding fibrotic tissue, normal cells, and stroma using limited amounts of tissue. Inhibitory effects similar to that observed by Western-blot were demonstrated by qlHC in tumor biopsies from mice bearing established BT474 tumor xenografts that had been treated with either GW572016 or vehicle alone.
  • Figure IB shows the results of quantitative IHC analysis of p-erbB2, p-EGFR, p-Erkl/2, p-AKT, and cyclin Dl from BT474 xenografts obtained from tumor-bearing C.B-17 SCID mice treated with either the duel EGFR/erbB-2 kinase inhibitor (100 mg/kg) or vehicle alone for 5 doses.
  • the optical density (OD) values assigned to the specimen are shown. OD values of ⁇ 10, 10-15, >15 roughly correlate with 1, 2, 3+ in the HercepTest standards, respectively.
  • patients were enrolled in an open-labeled, randomized trial.
  • GW572016 in dosages of 500, 650, 900, 1200, or 1600 mg was administered orally once a day.
  • Patients whose tumors (i) overexpressed EGFR or erbB-2 (2-3+ IHC staining in >10% of tumor cells), (ii) demonstrated erbB-2 gene amplification by FISH, or (iii) expressed activated, p-EGFR or erbB-2 by IHC (2-3+ staining in >10% of tumor cells) were enrolled if they satisfied other eligibility criteria.
  • Tumor biopsies were obtained immediately prior to initiating the duel EGFR/erbB-2 kinase inhibitor therapy and again on day 21, within 12 hr of receiving drug. Biopsies were fixed in 10% Neutral Buffered Formalin (NBF) and paraffin-embedded sections prepared. Patients were monitored by physical exams, clinical chemistry and hematology blood tests, and formally re-staging with imaging modalities after 8 weeks of therapy. RECIST criteria were used to assess clinical response ( Therasse, P., et al, J. Natl. Cancer Inst. 92, 205-216 (2000)).
  • NNF Neutral Buffered Formalin
  • FIG. 1 Two patients (361, 372) achieved partial remissions following therapy (Table 1). Both have metastatic breast cancer that was resistant to HERCEPTIN ® and multiple prior chemotherapy regimens.
  • Figure 2A represents a sample from patient 361 stained for EGFR and erbB-2. This analysis of EGFR and erbB-2 immunoreactivity in a metastatic breast cancer nodule from patient 361 showed overexpression of both receptors.
  • Pre- (d 0) and post-treatment (d 21) biopsies show that expression of total EGFR/erbB-2 protein is unchanged in response to GW572016.
  • Patient 372 overexpressed erbB-2 and erbB-3 (Table 1).
  • FIG. 2B represents a sample from patient 372 stained for pAKT. Reduced expression of intra-tumor pAKT was observed by d 21 of therapy in this woman (patient 372) with metastatic breast cancer overexpressing erbB-2 and erbB-3.
  • Activated pAKT expression was also inhibited in d 21 tumor biopsies from patients 361 (Table 1).
  • patient 369 with head and neck carcinoma overexpressing EGFR and erbB-2 demonstrated 70% inhibition of pAKT following therapy, and experienced disease stabilization (Table 1).
  • the effect of pAKT inhibition on tumor cell survival using TUNEL assay as an indicator of apoptosis was studied as a pro-survival factor (Vivanco, I., & Sawyers, C.L., Nature Reviews/Cancer 2, 489-501 (2002); Tzahar, E., et al, Mol. Cell. Biol.
  • Patients 372 and 369 had favorable clinical responses to therapy, with 372 achieving a partial remission and 369 experiencing disease stabilization.
  • Day 21 tumor biopsies from both patients revealed a 9- to 10-fold increase in tumor cell apoptosis as quantified by TUNEL assay.
  • Figure 2D shows that patient 361 also achieved a partial remission in response to GW572016 therapy.
  • TUNEL staining reveals increased tumor cell apoptosis.
  • Results of TUNEL assay are shown using 10X and 40X magnification. The percentage of TUNEL positive cells observed prior to (d 0), and at d 21 of therapy are indicated.
  • FIG. 3A represents staining tumor specimens for pERKl/2.
  • the pErk index in patient 361 was markedly elevated (4015) at baseline (Table 1 and Figure 3 A). By d 21, it was reduced to 0, with intra-tumor p-Erkl/2 immunoreactivity completely inhibited (Fig. 3A).
  • patient 364 had the markedly elevated pERK index of 1634 prior to therapy (Table 1 and Figure 3A).
  • pErkl/2 was inhibited 92% in patient 364 after treatment with GW572016. In both cases, p-Erkl/2 inhibition was associated with a favorable clinical response to therapy.
  • Activated Erkl/2 resides in the nucleus, where it regulates gene transcription promoting tumor growth and survival (Albanell, J., et al, Cancer Res. 61, 6500-6510 (2001); Kha ⁇ tonenkov, A., et al; Nature 386, 181-186 (1997)). Consistent with inhibition of pErkl/2, total Erkl/2 cellular distribution shifted following therapy.
  • Figure 3B represents staining tumor specimens for pERKl/2. Sequential tumor biopsies from a metastatic nodule (patient 361) were analyzed for total Erkl/2 protein expression prior to (d 0) and after 21 days of therapy (d 21). Whereas Erkl/2 was intra-nuclear prior to therapy, it was exclusively cytoplasmic at d 21. Two fields representative of the entire biopsy are shown. The shift in the intra-cellular localization of total Erkl/2 was consistent with the inhibition of p-Erkl/2, as seen in Figure 3 A. GW572016-treated tumor cell lines undergo Gl cell cycle arrest (Rusnak, D.W., et al., Mol. Cancer Therap. 1, 85-94 (2001)).
  • cyclin D protein a regulator of the Gl/S phase transition
  • Deregulation of cyclin D has been implicated in the pathogenesis of breast cancer, particularly tumors overexpressing erbB-2 (Lee, R.I., et al, Mol Cell. Biol. 20, 672-683 (2000); Zwijsen, R.M.L., et al Mol. Cell Biol.
  • Cyclin D protein was inhibited in several patients treated with GW572016 (Table 1).
  • Figure 4 represents staining of sequential tumor biopsies for cyclin D. Inhibition of cyclin Dl is shown in sequential tumor biopsies obtained prior to (d 0) and after 21 days of therapy with GW572016 from patients 361 and 364.
  • TGF- and heregulin ligands for EGFR and erbB-3/erbB-4, respectively, (Reese, D.J.I., & Stern, D.F.; Bioassays 20, 41-48 (1998)) was examined in biopsies to determine if the presence of these ligands influenced biological and clinical response to therapy.
  • Baseline elevated TGF- and heregulin immunoreactivity was increased in two patients achieving partial response (PR).
  • PR partial response
  • therapy appeared to reduce tumor immunoreactivity of TGF- and/or heregulin, which occurred more frequently in responding patients, otherwise there was no apparent correlation with response (Table 1).
  • IGFR-1 insulin-like growth factor receptor 1
  • HerceptinTM insulin-like growth factor receptor 1
  • Patient 371 is a woman with metastatic breast cancer overexpressing EGFR and erbB2 who had a "beneficial" biological response to therapy, yet still had disease progression (Table 1).
  • Phospho-Erkl/2 was completely inhibited following therapy, and cyclin D protein inhibited by 75% (Table 1).

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Abstract

Cette invention concerne des méthodes permettant de prédire réponse à un traitement anticancéreux chez un individu.
PCT/US2003/019697 2002-06-19 2003-06-19 Methode de prevision d'une reponse a une therapie dirigee contre les recepteurs her1/her2 WO2004000101A2 (fr)

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XIA W. ET AL.: 'Anti-tumor activity of GW572016: a dual tyrosine kinase inhibitor blocks EGF activation of EGRFR/erbB2 and downstream Erk 1/2 and AKT' ONCOGENE vol. 21, 12 September 2002, pages 6255 - 6263, XP002302016 *

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