WO2010015535A1 - Predictive marker for egfr inhibitor treatment - Google Patents

Predictive marker for egfr inhibitor treatment Download PDF

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WO2010015535A1
WO2010015535A1 PCT/EP2009/059633 EP2009059633W WO2010015535A1 WO 2010015535 A1 WO2010015535 A1 WO 2010015535A1 EP 2009059633 W EP2009059633 W EP 2009059633W WO 2010015535 A1 WO2010015535 A1 WO 2010015535A1
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gene
patient
cancer
treatment
cinp
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Paul Delmar
Jean-Pierre Delord
Philippe Rochaix
Fabienne Thomas
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F. Hoffmann-La Roche Ag
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

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  • the present invention provides a biomarker that is predictive for the response to treatment with an EGFR inhibitor in cancer patients.
  • EGF epidermal growth factor receptor
  • TGF- ⁇ transforming growth factor ⁇
  • TGF- ⁇ transforming growth factor ⁇
  • tumour cell proliferation A variety of intracellular pathways are subsequently activated, and these downstream events result in tumour cell proliferation in vitro. It has been postulated that stimulation of tumour cells via the EGFR may be important for both tumour growth and tumour survival in vivo.
  • Tarceva an inhibitor of the EGFR tyrosine kinase
  • Clinical phase I and II trials in patients with advanced disease have demonstrated that Tarceva has promising clinical activity in a range of epithelial tumours. Indeed, Tarceva has been shown to be capable of inducing durable partial remissions in previously treated patients with head and neck cancer, and NSCLC (Non small cell lung cancer) of a similar order to established second line chemotherapy, but with the added benefit of a better safety profile than chemo therapy and improved convenience (tablet instead of intravenous [i.v.] administration).
  • a recently completed, randomised, double-blind, placebo- controlled trial (BR.21) has shown that single agent Tarceva significantly prolongs and improves the survival of NSCLC patients for whom standard therapy for advanced disease has failed.
  • Tarceva is a small chemical molecule; it is an orally active, potent, selective inhibitor of the EGFR tyrosine kinase (EGFR-TKI).
  • the human epidermal growth factor receptor is a tyrosine-kinase (TK) receptor that plays an important role in several cellular signaling pathways, including those involved in proliferation and survival.
  • EGFR has a well established role in several solid tumor types and constitutes a clinically validated target for anticancer therapies.
  • Erlotinib (OSI-774, Tarceva®) is a potent, orally available EGFR tyrosine-kinase inhibitor (TKI) that blocks EGFR-mediated intracellular signaling and induces tumor cell cycle arrest.
  • Erlotinib is approved by the US Food and Drug Administration (FDA) and the European Medicines Agency for treatment of patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) after failure of at least one prior chemotherapy regimen. It is also approved by the US FDA for treatment, in combination with gemcitabine, of locally advanced unresectable or metastatic pancreatic cancer.
  • FDA US Food and Drug Administration
  • NSCLC non-small-cell lung cancer
  • erlotinib and gefitinib Iressa®; another EGFR TKI
  • EGFR has been implicated in the tumorigenesis of head and neck squamous-cell carcinoma (FINSCC).
  • erlotinib produced stable disease lasting for 15 months in one patient with FINSCC.
  • phase II study erlotinib was well tolerated in a heavily-pretreated population of patients with FINSCC and produced disease stabilization in 38% of cases, with a median duration of 16.1 weeks.
  • cetuximab an antibody targeting EGFR
  • EGFR-targeted molecules are likely to become a therapeutic option in FINSCC; however there is a clear medical need to identify which patients are most likely to benefit from therapy with EGFR inhibitors. Contrary to NSCLC, few factors predictive of response have been identified in FINSCC.
  • EGFR TK mutations Numerous teams have assessed the existence of EGFR TK mutations in this disease but they seem to be rare at least in Caucasian patients. Development and intensity of skin rash caused by anti-EGFR therapies have been correlated with improved survival. Recently, Agulnik et al investigated tumor and skin tissue samples to identify biomarkers correlated with response to treatment with erlotinib and cisplatin. Their results suggest that FINSCC patients with high gene copy number of EGFR gene may have higher response rate. Among the EGFR signaling proteins investigated before and after treatment, the decrease of phosphorylated EGFR (p-EGFR) in both normal and tumor tissue was linked with increased overall survival indicating that decrease in p-EGFR may represent a potential surrogate marker for outcome.
  • p-EGFR phosphorylated EGFR
  • It is an object of the present invention to provide an in vitro method of predicting the response of a cancer patient to treatment with an EGFR inhibitor comprising: determining an expression level of a gene CINP (cyclin-dependent kinase 2-interacting protein ) in a tumour sample of a patient and comparing the expression level of the gene CINP to a value representative of the gene CINP expression level in tumours of a non responding patient population, wherein a lower expression level of the gene CINP in the tumour sample of the patient is indicative for a patient who will respond to the treatment.
  • CINP cyclin-dependent kinase 2-interacting protein
  • the marker gene CINP shows typically between 1.2 and 1.6 ore more fold lower expression in the tumour sample of the responding patient compared to a value representative of the gene CINP expression level in tumours of a non responding patient population.
  • a value representative of an expression level of the at least one gene in tumours of a non responding patient population refers to an estimate of the mean expression level of the marker gene in tumours of a population of non responding patients.
  • the expression level of the gene CINP is determined by microarray technology or other technologies that assess RNA expression levels like quantitative RT-PCR, or by any method looking at the expression level of the respective protein, e.g. immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the gene expression level can be determined by other methods that are known to a person skilled in the art such as e.g. northern blots, RT-PCR, real time quantitative PCR, primer extension, RNase protection, RNA expression profiling.
  • the marker gene of the present invention can be combined with other biomarkers to biomarker sets.
  • Biomarker sets can be built from any combination of predictive biomarkers to make predictions about the effect of EGFR inhibitor treatment in cancer patients.
  • the various biomarkers and biomarkers sets described herein can be used, for example, to predict how patients with cancer will respond to therapeutic intervention with an EGFR inhibitor.
  • gene as used herein comprises variants of the gene.
  • variant relates to nucleic acid sequences which are substantially similar to the nucleic acid sequences given by the GenBank accession number.
  • substantially similar is well understood by a person skilled in the art.
  • a gene variant may be an allele which shows nucleotide exchanges compared to the nucleic acid sequence of the most prevalent allele in the human population.
  • a substantially similar nucleic acid sequence has a sequence similarity to the most prevalent allele of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.
  • variants is also meant to relate to splice variants.
  • the EGFR inhibitor can be selected from the group consisting of gef ⁇ tinib, erlotinib, PKI- 166, EKB-569, GW2016, CI- 1033 and an anti-erbB antibody such as trastuzumab and cetuximab.
  • the EGFR inhibitor is erlotinib.
  • the cancer is head and neck squamous-cell carcinoma (HNSCC).
  • HNSCC head and neck squamous-cell carcinoma
  • Techniques for the detection and quantitation of gene expression of the genes described by this invention include, but are not limited to northern blots, RT-PCR, real time quantitative PCR, primer extension, RNase protection, RNA expression profiling and related techniques. These techniques are well known to those of skill in the art see e.g. Sambrook J et al, Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Press, Cold Spring Harbor, 2000).
  • Techniques for the detection of protein expression of the respective genes described by this invention include, but are not limited to immunohistochemistry (IHC).
  • cells from a patient tissue sample e.g. a tumour or cancer biopsy can be assayed to determine the expression pattern of one or more biomarkers. Success or failure of a cancer treatment can be determined based on the biomarker expression pattern of the cells from the test tissue (test cells), e.g., tumour or cancer biopsy, as being relatively similar or different from the expression pattern of a control set of the one or more biomarkers.
  • test cells e.g., tumour or cancer biopsy
  • the genes listed in table 2 are up- regulated i.e. show a higher expression level, in tumours of patients who respond to the EGFR inhibitor treatment compared to tumours of patients who do not respond to the EGFR inhibitor treatment.
  • test cells show a biomarker expression profile which corresponds to that of a patient who responded to cancer treatment, it is highly likely or predicted that the individual's cancer or tumour will respond favourably to treatment with the EGFR inhibitor.
  • test cells show a biomarker expression pattern corresponding to that of a patient who did not respond to cancer treatment, it is highly likely or predicted that the individual's cancer or tumour will not respond to treatment with the EGFR inhibitor.
  • the biomarkers of the present invention i.e. the genes listed in table 2 are a first step towards an individualized therapy for patients with cancer, in particular patients with head and neck cancer.
  • This individualized therapy will allow treating physicians to select the most appropriate agent out of the existing drugs for cancer therapy, in particular head and neck cancer.
  • the benefit of individualized therapy for each future patient are: response rates / number of benefiting patients will increase and the risk of adverse side effects due to ineffective treatment will be reduced.
  • the present invention provides a therapeutic method of treating a cancer patient identified by the in vitro method of the present invention.
  • Said therapeutic method comprises administering an EGFR inhibitor to the patient who has been selected for treatment based on the predictive expression pattern of at least one of the genes listed in table 2.
  • a preferred EGFR inhibitor is erlotinib and a preferred cancer to be treated is head and neck squamous-cell carcinoma (FINSCC).
  • pan-endoscopy After diagnosis, patients underwent routine pan-endoscopy. Treatment with oral erlotinib 150 mg/day (F. Hoffmann-La Roche, Basel, Switzerland) was started the following day. Patients were treated for a variable period ranged from 18 to 30 days, corresponding to the time between pan-endoscopy and surgical resection (see figure 1). In the event of grade 2 diarrhea or skin rash that was symptomatically unacceptable to the patient, treatment was withheld until resolution to grade 1, and then erlotinib was restarted at a dose of 100 mg/day. If toxicity reoccurred, erlotinib was stopped.
  • oral erlotinib 150 mg/day F. Hoffmann-La Roche, Basel, Switzerland
  • Tissue Biopsies Tumor tissue biopsies were collected both before and after treatment. Samples were snap frozen in liquid nitrogen and kept at -80 0 C.
  • Table 1 shows the clinical characteristics of the 26 patients analyzed for the "before/after" analysis.
  • log-ratio log-ratio
  • a statistical model was fitted independently to each of the 16810 probe-set.
  • the model is a linear model that takes "log-ratio" as outcome variable and Response status as predictor.
  • the corresponding p-value evaluates the hypothesis that there is no difference in mean "log-ratio" between the responder and non-responder groups.
  • the probe-set the lowest outstandingly low p-value was identified as a marker.
  • Table 2 Marker based on comparing "Responders" to “Non Responders”.
  • Column 1 is the Affymetrix identifier for the probe-set.
  • Column 2 is the GenBank accession number of the corresponding gene sequence.
  • Column 3 is the corresponding official gene name.
  • Column 4 is the corresponding adjusted mean fold change in ratio (after/before) level between “responder” and “non responder”.
  • Column 5 is the p-value for the test of difference in expression level between "responders” and “non responders”.
  • Tumor tissue biopsies were collected both before and after treatment and were snap frozen in liquid nitrogen. The biopsy content was checked on slides stained with hemalun eosin. If the biopsy contained only tumor cells, the biopsy was directly dissolved in lysis buffer and RNA was extracted with RNeasy Mini Kit (Qiagen®). If the biopsy contained also normal cells, then 10 slides of 10 ⁇ m thick were prepared and stained and tumoral cells were scraped and dissolved in lysis buffer as previously described. The quantity and quality of RNA were checked with a bioanalyser Agilent 2100 Expert.
  • Target preparation and microarray hybridization l ⁇ g total RNA, prepared from each of the biopsy samples (table xx), was used to generate biotinylated cRNA following the Affymetrix standard protocol using their single cycle amplification kit (Part Number 900493; Affymetrix, Inc.; Santa Clara, CA, http ://www. affymetrix. co m/support/technical/manual/expression_manual. affx) . 15 ⁇ g cRNA was hybridized for 16 h at 45oC to Human Genome Ul 33 A GeneChip® oligonucleotide arrays, which carry probes representing >22,000 well-characterized transcripts, from the Human Unigene database (Build 133).
  • arrays were washed and stained with streptavidin-phycoerythrin and thereafter scanned using an Affymetrix GeneChip Scanner 3000 according to the manufacturer's protocols (Affymetrix); signal intensities were calculated automatically by GCOS.

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Abstract

The present invention provides the gene CINP (cyclin-dependent kinase 2-interacting protein) as marker for predicting the response of a cancer patient to EGFR inhibitor treatment.

Description

PREDICTIVE MARKER FOR EGFR INHIBITOR TREATMENT
The present invention provides a biomarker that is predictive for the response to treatment with an EGFR inhibitor in cancer patients.
A number of human malignancies are associated with aberrant or over-expression of the epidermal growth factor receptor (EGFR). EGF, transforming growth factor α (TGF-α), and a number of other ligands bind to the EGFR, stimulating autophosphorylation of the intracellular tyrosine kinase domain of the receptor. A variety of intracellular pathways are subsequently activated, and these downstream events result in tumour cell proliferation in vitro. It has been postulated that stimulation of tumour cells via the EGFR may be important for both tumour growth and tumour survival in vivo. Early clinical data with Tarceva, an inhibitor of the EGFR tyrosine kinase, indicate that the compound is safe and generally well tolerated at doses that provide the targeted effective concentration (as determined by preclinical data). Clinical phase I and II trials in patients with advanced disease have demonstrated that Tarceva has promising clinical activity in a range of epithelial tumours. Indeed, Tarceva has been shown to be capable of inducing durable partial remissions in previously treated patients with head and neck cancer, and NSCLC (Non small cell lung cancer) of a similar order to established second line chemotherapy, but with the added benefit of a better safety profile than chemo therapy and improved convenience (tablet instead of intravenous [i.v.] administration). A recently completed, randomised, double-blind, placebo- controlled trial (BR.21) has shown that single agent Tarceva significantly prolongs and improves the survival of NSCLC patients for whom standard therapy for advanced disease has failed.
Tarceva (erlotinib) is a small chemical molecule; it is an orally active, potent, selective inhibitor of the EGFR tyrosine kinase (EGFR-TKI).
The human epidermal growth factor receptor (EGFR) is a tyrosine-kinase (TK) receptor that plays an important role in several cellular signaling pathways, including those involved in proliferation and survival. EGFR has a well established role in several solid tumor types and constitutes a clinically validated target for anticancer therapies. Erlotinib (OSI-774, Tarceva®) is a potent, orally available EGFR tyrosine-kinase inhibitor (TKI) that blocks EGFR-mediated intracellular signaling and induces tumor cell cycle arrest. Erlotinib is approved by the US Food and Drug Administration (FDA) and the European Medicines Agency for treatment of patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) after failure of at least one prior chemotherapy regimen. It is also approved by the US FDA for treatment, in combination with gemcitabine, of locally advanced unresectable or metastatic pancreatic cancer. Several studies in NSCLC have shown that erlotinib and gefitinib (Iressa®; another EGFR TKI) produce radiographic responses in approximately 10% of patients treated in the second- or third- line setting. Clinical characteristics associated with tumor response have been extensively described and include: female gender, never-smoking status, adenocarcinoma histology, Asian ethnic origin, EGFR gene amplification and the presence of specific mutations in the EGFR TK domain. The study of molecular biomarkers of erlotinib response showed that the incidence of EGFR mutations in lung cancer was 22%.
EGFR has been implicated in the tumorigenesis of head and neck squamous-cell carcinoma (FINSCC). The antitumor activity of erlotinib, alone or in combination with cisplatin, has been demonstrated in vivo using murine xenografts of a human FINSCC cell line. Furthermore, in a phase I study, erlotinib produced stable disease lasting for 15 months in one patient with FINSCC. In a subsequent phase II study, erlotinib was well tolerated in a heavily-pretreated population of patients with FINSCC and produced disease stabilization in 38% of cases, with a median duration of 16.1 weeks. Recently, cetuximab (Erbitux®), an antibody targeting EGFR, showed encouraging results in combination with radiotherapy for treatment of locally advanced head and neck squamous cell carcinomas that led to its approval in this indication. A phase III study published by Burtness et al. demonstrated that the combination of cisplatin and cetuximab was active in the first line treatment of recurrent FINSCC. EGFR-targeted molecules are likely to become a therapeutic option in FINSCC; however there is a clear medical need to identify which patients are most likely to benefit from therapy with EGFR inhibitors. Contrary to NSCLC, few factors predictive of response have been identified in FINSCC. Numerous teams have assessed the existence of EGFR TK mutations in this disease but they seem to be rare at least in Caucasian patients. Development and intensity of skin rash caused by anti-EGFR therapies have been correlated with improved survival. Recently, Agulnik et al investigated tumor and skin tissue samples to identify biomarkers correlated with response to treatment with erlotinib and cisplatin. Their results suggest that FINSCC patients with high gene copy number of EGFR gene may have higher response rate. Among the EGFR signaling proteins investigated before and after treatment, the decrease of phosphorylated EGFR (p-EGFR) in both normal and tumor tissue was linked with increased overall survival indicating that decrease in p-EGFR may represent a potential surrogate marker for outcome. However, the results were obtained for erlotinib in combination with cisplatin in patients participating to phases I and II who were already heavily pretreated. Such biomarkers should be examined in patients treated with erlotinib as a single agent in order to characterize the clinical response to this particular therapy.
It has long been acknowledged that there is a need to develop methods of individualising cancer treatment. With the development of targeted cancer treatments, there is a particular interest in methodologies which could provide a molecular profile of the tumour target, (i.e. those that are predictive for clinical benefit). Proof of principle for gene expression profiling in cancer has already been established with the molecular classification of tumour types which are not apparent on the basis of current morphological and immunohistochemical tests.
Therefore, it is an aim of the present invention to provide an expression bio marker that is predictive for response to EGFR inhibitor treatment in cancer patients.
It is an object of the present invention to provide an in vitro method of predicting the response of a cancer patient to treatment with an EGFR inhibitor comprising: determining an expression level of a gene CINP (cyclin-dependent kinase 2-interacting protein ) in a tumour sample of a patient and comparing the expression level of the gene CINP to a value representative of the gene CINP expression level in tumours of a non responding patient population, wherein a lower expression level of the gene CINP in the tumour sample of the patient is indicative for a patient who will respond to the treatment.
In a preferred embodiment, the marker gene CINP shows typically between 1.2 and 1.6 ore more fold lower expression in the tumour sample of the responding patient compared to a value representative of the gene CINP expression level in tumours of a non responding patient population. The term "a value representative of an expression level of the at least one gene in tumours of a non responding patient population" refers to an estimate of the mean expression level of the marker gene in tumours of a population of non responding patients.
In a preferred embodiment, the expression level of the gene CINP is determined by microarray technology or other technologies that assess RNA expression levels like quantitative RT-PCR, or by any method looking at the expression level of the respective protein, e.g. immunohistochemistry (IHC). The construction and use of gene chips are well known in the art. see, U. S. Pat Nos. 5,202,231; 5,445,934; 5,525,464; 5,695,940; 5,744,305; 5,795, 716 and 1 5,800,992. See also, Johnston, M. Curr. Biol. 8:R171-174 (1998); Iyer VR et al, Science 283:83- -A-
87 (1999). Of course, the gene expression level can be determined by other methods that are known to a person skilled in the art such as e.g. northern blots, RT-PCR, real time quantitative PCR, primer extension, RNase protection, RNA expression profiling.
The marker gene of the present invention can be combined with other biomarkers to biomarker sets. Biomarker sets can be built from any combination of predictive biomarkers to make predictions about the effect of EGFR inhibitor treatment in cancer patients. The various biomarkers and biomarkers sets described herein can be used, for example, to predict how patients with cancer will respond to therapeutic intervention with an EGFR inhibitor.
The term "gene" as used herein comprises variants of the gene. The term "variant" relates to nucleic acid sequences which are substantially similar to the nucleic acid sequences given by the GenBank accession number. The term "substantially similar" is well understood by a person skilled in the art. In particular, a gene variant may be an allele which shows nucleotide exchanges compared to the nucleic acid sequence of the most prevalent allele in the human population. Preferably, such a substantially similar nucleic acid sequence has a sequence similarity to the most prevalent allele of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%. The term "variants" is also meant to relate to splice variants.
The EGFR inhibitor can be selected from the group consisting of gefϊtinib, erlotinib, PKI- 166, EKB-569, GW2016, CI- 1033 and an anti-erbB antibody such as trastuzumab and cetuximab. In another embodiment, the EGFR inhibitor is erlotinib.
In yet another embodiment, the cancer is head and neck squamous-cell carcinoma (HNSCC).
Techniques for the detection and quantitation of gene expression of the genes described by this invention include, but are not limited to northern blots, RT-PCR, real time quantitative PCR, primer extension, RNase protection, RNA expression profiling and related techniques. These techniques are well known to those of skill in the art see e.g. Sambrook J et al, Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Press, Cold Spring Harbor, 2000).
Techniques for the detection of protein expression of the respective genes described by this invention include, but are not limited to immunohistochemistry (IHC).
In accordance with the invention, cells from a patient tissue sample, e.g. a tumour or cancer biopsy can be assayed to determine the expression pattern of one or more biomarkers. Success or failure of a cancer treatment can be determined based on the biomarker expression pattern of the cells from the test tissue (test cells), e.g., tumour or cancer biopsy, as being relatively similar or different from the expression pattern of a control set of the one or more biomarkers. In the context of this invention, it was found that the genes listed in table 2 are up- regulated i.e. show a higher expression level, in tumours of patients who respond to the EGFR inhibitor treatment compared to tumours of patients who do not respond to the EGFR inhibitor treatment. Thus, if the test cells show a biomarker expression profile which corresponds to that of a patient who responded to cancer treatment, it is highly likely or predicted that the individual's cancer or tumour will respond favourably to treatment with the EGFR inhibitor. By contrast, if the test cells show a biomarker expression pattern corresponding to that of a patient who did not respond to cancer treatment, it is highly likely or predicted that the individual's cancer or tumour will not respond to treatment with the EGFR inhibitor.
The biomarkers of the present invention i.e. the genes listed in table 2 are a first step towards an individualized therapy for patients with cancer, in particular patients with head and neck cancer. This individualized therapy will allow treating physicians to select the most appropriate agent out of the existing drugs for cancer therapy, in particular head and neck cancer. The benefit of individualized therapy for each future patient are: response rates / number of benefiting patients will increase and the risk of adverse side effects due to ineffective treatment will be reduced.
In a further object the present invention provides a therapeutic method of treating a cancer patient identified by the in vitro method of the present invention. Said therapeutic method comprises administering an EGFR inhibitor to the patient who has been selected for treatment based on the predictive expression pattern of at least one of the genes listed in table 2. A preferred EGFR inhibitor is erlotinib and a preferred cancer to be treated is head and neck squamous-cell carcinoma (FINSCC).
Short description of the figures Figure 1 shows the study design.
Experimental part Rationale for the Study and Study Design
Patients
Patients were eligible if they had non-metastatic, histo logically-confirmed FINSCC (stage ? T2N x MO), and were candidates for first-line curative surgical treatment or had been scheduled for surgery by necessity (neck nodes dissection for bulky lymphadenopathies prior to radiotherapy). Other eligibility criteria included WHO performance status 2; able to swallow food; aged > 18 years; and provision of written informed consent.
Patients were not eligible if they had relapsed after radiotherapy, or if they had recent massive gastrointestinal hemorrhage, the presence of a medical contra-indication in the form of a major impairment of general condition, or an ongoing unmanaged serious infectious disease or major metabolic disorder. Other exclusion criteria included: neutrophil count < 1 x 109/L or platelet count < 75 x 109/L at study entry; bilirubin > 1.5-fold above the upper limit of normal; and kidney failure (glomerular filtration rate, calculated using Cockcroft's formula, of < 40mL/min). Pregnant women and women of childbearing potential were also excluded.
Treatment Plan
After diagnosis, patients underwent routine pan-endoscopy. Treatment with oral erlotinib 150 mg/day (F. Hoffmann-La Roche, Basel, Switzerland) was started the following day. Patients were treated for a variable period ranged from 18 to 30 days, corresponding to the time between pan-endoscopy and surgical resection (see figure 1). In the event of grade 2 diarrhea or skin rash that was symptomatically unacceptable to the patient, treatment was withheld until resolution to grade 1, and then erlotinib was restarted at a dose of 100 mg/day. If toxicity reoccurred, erlotinib was stopped.
Clinical Evaluation
Collection of a full medical history, physical examination, electrocardiogram, and laboratory tests were performed at baseline. Computed tomography imaging of the involved site and systematic radiologic chest evaluation were performed within 1 month of enrolment to ensure that no lung metastases were present. Toxicities were evaluated at each visit and graded using the National Cancer Institute Common Toxicity Criteria, version 2.0. Tumor response was assessed using CT scans taken before and after treatment (on the day before surgery). CT scans to confirm response were not possible as the patients were operated upon. Due to the very short treatment period, patients with tumor shrinkage of more than 25% were arbitrarily considered as responders.
Tissue Biopsies Tumor tissue biopsies were collected both before and after treatment. Samples were snap frozen in liquid nitrogen and kept at -800C.
Aim of the expression profiling study with Affymetrix chips.
To study genomic expression of tumors before and after treatment with Affymetrix Human Genome U133A GeneChip®.
Results
We compared the modification of expression during the treatment (expression after treatment compared to expression before treatment) in responders and non-responders. For this particular analysis, only patients that followed the complete treatment were analyzed i.e. 29 patients. Moreover, tumor biopsies from 3 others patients were contaminated with muscle cells and display aberrant results. For this reason, these 3 patients were also discarded. The "before/after" analysis was the performed in 26 patients.
Table 1 shows the clinical characteristics of the 26 patients analyzed for the "before/after" analysis.
Figure imgf000008_0001
Data Analysis
Signal intensities were normalized using a quantile-quantile method [I]. All normalized data were Iog2 -transformed prior to analysis to down-weight the influence of high expression values. Probe-sets called "absent" or "marginal" by the Affymetrix MAS5 algorithm in all 39 samples were removed from further analysis. In total 5473 probe-sets out of 22283 (25,1%) were removed.
For each patient, the difference between the vectors of normalized expression intensity after and before treatment was formed and used in subsequent analyses. This quantity will be referred to as "log-ratio".
A statistical model was fitted independently to each of the 16810 probe-set. The model is a linear model that takes "log-ratio" as outcome variable and Response status as predictor. The corresponding p-value evaluates the hypothesis that there is no difference in mean "log-ratio" between the responder and non-responder groups. The probe-set the lowest outstandingly low p-value was identified as a marker.
The expression of the following gene was decreased by treatment (i.e., ratio after/before <0) in responders patients and did not decrease in non-responders patients. For this reason, we think that this gene (coding for cyclin-dependent kinase 2-interacting protein CINP) is a good pharmacodynamic marker. The function of this gene has not been reported but CINP seems to interact with cell cycle proteins.
Table 2: Marker based on comparing "Responders" to "Non Responders".
Responders were defined as patients with tumor shrinkage of more than 25%.
Column 1 is the Affymetrix identifier for the probe-set. Column 2 is the GenBank accession number of the corresponding gene sequence. Column 3 is the corresponding official gene name. Column 4 is the corresponding adjusted mean fold change in ratio (after/before) level between "responder" and "non responder". Column 5 is the p-value for the test of difference in expression level between "responders" and "non responders".
Figure imgf000009_0001
Materiels and Methods
RNA extraction
Tumor tissue biopsies were collected both before and after treatment and were snap frozen in liquid nitrogen. The biopsy content was checked on slides stained with hemalun eosin. If the biopsy contained only tumor cells, the biopsy was directly dissolved in lysis buffer and RNA was extracted with RNeasy Mini Kit (Qiagen®). If the biopsy contained also normal cells, then 10 slides of 10 μm thick were prepared and stained and tumoral cells were scraped and dissolved in lysis buffer as previously described. The quantity and quality of RNA were checked with a bioanalyser Agilent 2100 Expert.
Target preparation and microarray hybridization lμg total RNA, prepared from each of the biopsy samples (table xx), was used to generate biotinylated cRNA following the Affymetrix standard protocol using their single cycle amplification kit (Part Number 900493; Affymetrix, Inc.; Santa Clara, CA, http ://www. affymetrix. co m/support/technical/manual/expression_manual. affx) . 15 μg cRNA was hybridized for 16 h at 45oC to Human Genome Ul 33 A GeneChip® oligonucleotide arrays, which carry probes representing >22,000 well-characterized transcripts, from the Human Unigene database (Build 133). Following hybridization, arrays were washed and stained with streptavidin-phycoerythrin and thereafter scanned using an Affymetrix GeneChip Scanner 3000 according to the manufacturer's protocols (Affymetrix); signal intensities were calculated automatically by GCOS.

Claims

Claims
1. An in vitro method of predicting the response of a cancer patient to treatment with an EGFR inhibitor comprising: determining an expression level of a gene CINP in a tumour sample of a patient and comparing the expression level of the gene CINP to a value representative of the gene CINP expression level in tumours of a non responding patient population, wherein a lower expression level of the gene CINP in the tumour sample of the patient is indicative for a patient who will respond to the treatment.
2. The method of claim 1, wherein the marker gene CINP shows typically between 1.2 and
1.6 or more fold lower expression in the tumour sample of the responding patient compared to a value representative of the gene CINP expression level in tumours of a non responding patient population.
3. The method of claims 1 to 2, wherein the expression level is determined by microarray technology.
4. The method of claims 1 to 5, wherein the EGFR inhibitor is erlotinib.
5. The method of claims 1 to 6, wherein the cancer is head and neck squamous-cell carcinoma (HNSCC).
6. Use of a CINP gene for predicting the response of a cancer patient to EGFR inhibitor treatment.
7. The use of claim 6, wherein the cancer is head and neck squamous-cell carcinoma (HNSCC).
8. The use of claim 7, wherein the EGFR inhibitor is erlotinib.
9. A method of treating a cancer patient identified by a method of claims 1 to 5 comprising administering an EGFR inhibitor to the patient.
10. The method of claim 9, wherein the EGFR inhibitor is erlotinib.
11. The method of claim 9 or 10, wherein the cancer is head and neck squamous-cell carcinoma (HNSCC).
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