HK1119205A - Anti-egfr antibody therapy based on an increased copy number of the egfr gene in tumor tissues - Google Patents

Description

anti-EGFR antibody treatment based on increased EGFR gene copy number in tumor tissue

Technical Field

The present invention relates to the diagnosis and treatment of tumors expressing high levels of Epithelial Growth Factor Receptor (EGFR) by anti-EGFR antibodies. The present invention further relates to the individualized and personalized diagnosis and treatment of EGFR expressing cancers based on specific molecular alterations that occur in specific tumor tissues of a specific tumor patient population. The treatment and diagnosis is based on the finding that for tumor tissues with a specific EGFR that exhibit an amplified copy number of the EGFR gene, the anti-EGFR antibody can inhibit proliferation and tumor growth of that tumor tissue, while other individual molecular changes that occur in the tumor tissue, such as specific gene mutations, are not affected by the same anti-EGFR antibody treatment.

Background

Biomolecules, such as monoclonal antibodies (mabs) or other proteins/polypeptides, as well as small chemical compounds directed against various receptors and other antigens located on the surface of tumor cells, have been considered suitable for tumor therapy for over 20 years. For antibody approaches, most of these mabs are chimeric or humanized to improve tolerance of the human immune system. Mabs, or the above-mentioned chemicals, specifically bind to their target structure on tumor cells and in most cases also to normal tissue, which binding may cause different effects depending on their epitope specificity and/or the functional characteristics of the specific antigen.

ErbB receptors are the typical receptor tyrosine kinases implicated in cancer in the 80's of the 20 th century. Tyrosine kinases are a class of enzymes that catalyze the transfer of the terminal phosphate group of adenosine triphosphate to tyrosine residues in protein substrates. Tyrosine kinases are thought to play a critical role in signal transduction for many cellular functions through substrate phosphorylation. Although the exact mechanism of signal transduction is not known, tyrosine kinases have been shown to be important contributing factors to cell proliferation, carcinogenesis, and cell differentiation. Receptor type tyrosine kinases have an extracellular, transmembrane and intracellular portion, whereas non-receptor type tyrosine kinases are all intracellular. Receptor-linked tyrosine kinases are transmembrane proteins comprising an extracellular ligand binding domain, a transmembrane sequence, and a cytoplasmic tyrosine kinase domain. Receptor-type tyrosine kinases are composed of a number of transmembrane receptors with diverse biological activities.

Different subfamilies of receptor-type tyrosine kinases have been identified. The tyrosine kinases involved include Fibroblast Growth Factor (FGF) receptors, Epithelial Growth Factor (EGF) receptors of the ErbB major family, and platelet-derived growth factor (PDGF) receptors. Also contemplated are Nerve Growth Factor (NGF) receptors, Brain Derived Neurotrophic Factor (BDNF) receptors and neurotrophin-3 (NT-3) receptors, as well as neurotrophin-4 (NT-4) receptors.

EGFR, encoded by the erbB1 gene, has thus been implicated in human malignancies. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck, stomach, and malignant gliomas. Increased EGFR receptor expression is often associated with increased production of the EGFR ligand, transforming growth factor alpha (TGF-. alpha.), and for the same reason, tumor cells produce receptor activation by an autocrine stimulatory pathway (Baselga and Mendelsohn, Pharmac. Ther.64: 127-. The EGF receptor is a transmembrane glycoprotein with a molecular weight of 170,000, present on a variety of epithelial cell types. It can be activated by at least three ligands: EGF, TGF-alpha (transforming growth factor alpha), and amphiregulin. Both Epithelial Growth Factor (EGF) and transforming growth factor-alpha (TGF-alpha) have been shown to bind to EGF receptors, resulting in cell proliferation and tumor growth.

It has been demonstrated that inhibition of tumor cell proliferation appears when antibodies against the EGF receptor block the binding of EGF and TGF- α to the receptor. In view of these findings, a number of murine and rat monoclonal antibodies against the EGF receptor have been developed and tested for their ability to inhibit tumor cell growth in vitro and in vivo (Modjtahedi and Dean, 1994, j. Humanized 425(hMAb 425, Matuzumab; US5,558,864; EP 0531472) and chimeric 225(cMAb 225) monoclonal antibodies against the EGF receptor have shown their efficacy in clinical trials. It was demonstrated that the C225 antibody (cetuximab) inhibits EGF-mediated tumor cell growth in vitro and human tumor formation in nude mice. First, the antibody, and generally all anti-EGFR antibodies, exhibit synergistic effects with certain chemotherapeutic agents (i.e., doxorubicin, adriamycin, paclitaxel, and cisplatin), eradicating human tumors in vivo in a xenografted mouse model (see, e.g., EP 0667165). Ye et al (1999, Oncogene 18, 731) have reported that the combination of chimeric mAb 225 and humanized mAb 4D5 against the HER2 receptor successfully treated human ovarian cancer cells. Furthermore, the combination of matuzumab and cetuximab also elicited a synergistic anti-tumor response (WO 04/32960). Another intact human anti-EGFR antibody is palimumab (mAb ABX) developed with XenoMouse ® technology (e.g., WO 98/50433, US 6,235,883).

Monoclonal antibodies against Epithelial Growth Factor Receptor (EGFR), such as the chimeric monoclonal antibody c225 (cetuximab) and the fully human antibody parlimumab, have shown significant clinical activity in about 10% of patients with chemotherapy-resistant metastatic colorectal cancer (mCRC). The molecular mechanisms of clinical responsiveness or resistance to these substances are not currently known.

For metastatic colorectal cancer (mCRC), the third most common cause of cancer death, monoclonal antibodies (mAbs) against the extracellular domain of the Epidermal Growth Factor Receptor (EGFR) have been used to boost the therapeutics against it (Erlichman and Sargent; 2004, N Engl J Med 351: 391) 392). Among anti-EGFR mabs, the chimeric antibody of cetuximab (Erbitux ®) and the fully human antibody parlimumab have been shown to exhibit significant clinical activity in about 10% of patients with chemotherapy-resistant mCRC, respectively, but the molecular mechanisms underlying their clinical responsiveness or resistance are still unclear at present. Neither the diagnostic characteristics nor the extent of tumor EGFR expression assessed by immunohistochemistry were correlated with Clinical response (Saltz et al, 2004, J Clin Oncol 22: 1201-. An understanding of the molecular basis of clinical sensitivity or resistance of moabs against EGFR may allow identification of patients who may benefit from cetuximab or pamabrin treatment. EGFR biology has been studied in detail using genetic and biochemical approaches (Ciardiello et al, 2003, Eur J Cancer 39: 1348-1354; Holbro et al, 2004, Annu Rev Pharmacol Toxicol 44: 195-217). The initial step of ligand binding to the extracellular portion of the receptor promotes receptor dimerization and activation of its enzymatic activity, thus leading to phosphorylation of the intracellular domain. Subsequently, cellular effectors bind to residues phosphorylated by the intracellular domain and are activated, mainly by their relocation on the plasma membrane. The small G proteins Ras, protein kinase Raf and lipid kinase PI3K play important roles as intracellular mediators of EGFR signaling. Genetic alterations in EGFR and its effectors have been previously found in a variety of cancers (Bardelli et al, 2003, Science 300: 949; Vogelstein et al, 2004, Nat Med 10: 789-.

Thus, the hypothesis can be derived that the clinical response to certain specific anti-EGFR antibodies, such as cetuximab, parlimumab or matuzumab, is related to molecular alterations that affect EGFR or its direct intracellular signal transduction.

In many cancers, such as mCRC, the degree of expression of EGFR, whether diagnostic of the tumor or assessed by immunohistochemistry, is not correlated with clinical response to EGFR antagonists, particularly anti-EGFR antibodies, such as cetuximab, matuzumab (hMab 425), or parlimumab. Thus, currently most patients being treated are exposed to the risk of ineffective treatment of undesirable side effects. The efficacy of treatment of mCRC patients with anti-EGFR mabs (e.g., cetuximab, matuzumab, or parlimumab) indicates a significant medical advance. However, in clinical studies involving patients with chemical resistance (chemo-reactivity), treatment with mabs against EGFR has produced a targeted response in only a subset of patients, and there is no diagnostic tool to identify who might benefit from the treatment. As a result, most treated patients are exposed to the risk of ineffective treatment of undesirable side effects. Non-personalized treatment also results in a huge financial burden on the hygiene system.

Thus, there is a need to demonstrate differential responses of patients to anti-EGFR monoclonal antibodies and to develop strategies to identify cancer patients (e.g., CRC patients) that may benefit from anti-EGFR antibody therapy. The molecular mechanism by which EGFR-expressing cancer cells respond or are refractory to anti-EGFR mabs is currently unknown. Therefore, there is an additional need to provide diagnostic tools to show whether the response to anti-EGFR mabs in cancer is associated with the following biological predictors (predictors) or markers, including (i) mutations affecting the catalytic domain of the EGFR gene, (ii) mutations affecting downstream signaling effectors of EGFR; or (iii) amplification of the EGFR locus.

Summary of The Invention

According to the present invention, it has now been found that for tumors in tumor patients, including patients with chemical resistance, the tumor cells show an increase in the copy number of the EGFR gene in about 89% of the patients who respond to the above-mentioned tumor targets, while in patients with stable or progressive disease, this is only 5.0%. Thus, the mutated state of the EGFR catalytic domain and its immediate downstream effectors PI3K, RAS, RAF is not relevant for the response.

According to the present invention, the same concentration of a particular anti-EGFR antibody (e.g., cetuximab, matuzumab or parlimumab) that can completely impair the proliferation of cells exhibiting EGFR gene copy number amplification does not affect cells that do not exhibit EGFR copy number amplification in a cell model of a particular cancer (e.g., colorectal cancer).

According to the present invention, the response to treatment with a particular anti-EGFR antibody, such as parlimumab, cetuximab or matuzumab (or any immunologically effective fragment or fusion protein thereof), in patients with a particular cancer, preferably mCRC, has a clear correlation with the presence of EGFR gene copy number amplification. In other words: patients who respond or are sensitive to anti-EGFR treatment have an increased EGFR gene copy number compared to those who do not respond to the same dose of alloantibody treatment. Furthermore, it can be observed that increased EGFR gene copy number is associated with tumor shrinkage and prolonged survival in patients by treatment with the mAb. In these patients, tumor growth may be driven primarily by the EGFR pathway.

The amplified EGFR gene copy number can be determined according to the present invention by determining the ratio of EGFR gene per nucleus and/or the ratio defined by the EGFR gene copy to the number of CEP7 (chromosome 7 centromeric probe). It has been found, according to the invention, that in a tumor sample the ratio is: EGFR gene copy number/nucleus > 4, preferably in the range of 5.7-7.1, and/or EGFR gene copy number/CEP 7 > 2, administration of anti-EGFR antibodies to patients from whom the tumor specimen was derived is more effective than in patients for which the defined copy number ratio is lower than indicated. Patients whose tumor cells exhibit a non-amplified or only slightly amplified EGFR gene copy number (ratio: 1 or < 2) do not respond at all or do not respond adequately to anti-EGFR antibody treatment.

This observation represents the first example of personalized targeted therapy of a particular cancer (e.g., colorectal cancer) based on specific molecular alterations. In order to administer the drug most effectively among patients, tools are now provided to identify those patients most likely to benefit.

In addition, it was found that there are new somatic mutations in the EGFR catalytic domain, and that there are multiple mutations in its immediate downstream effectors (e.g., KRAS and PI3KCA), which are not associated with responsiveness to anti-EGFR mabs. These findings have many clinical and biological implications. In cancers that express and overexpress EGFR, the response of anti-EGFR mabs is less correlated with EGFR gene mutation than with increased/amplified copy number of the gene. These results suggest that anti-EGFR antibody-based treatments may be more effective on amplified targets than targets affected by point mutations. However, genetic mutations such as point mutations may contribute to the efficacy and efficacy of anti-EGFR antibody treatments.

In particular for CRC; proliferation of CRC cells with amplified EGFR gene copy number can be abolished by anti-EGFR antibodies (e.g., cetuximab), but CRC cells with non-amplified EGFR copy number are not affected by the same dose of anti-EGFR monoclonal antibody. This indicates that cancer cells with amplified EGFR gene, especially CRC cells, are proliferation dependent or even inseparable from this molecular change.

The present data also indicate that the measurement of EGFR gene copy number by FISH (fluorescent in situ hybridization) can represent an experimental tool for identifying patients with mCRC and other cancers who may respond to anti-EGFR targeted mabs. Furthermore, in the case of EGFR protein overexpression and increased copy number of EGFR genes localized to discrete foci of the same tumor (fig. 3), FISH analysis is not affected by contamination with the presence of dimeric tumor cells or normal somatic cells, relative to semi-quantitative assays such as qPCR and Western blot. Therefore, in explaining the lack of correlation between IHC and mAb clinical responses, one should consider the possible isotype patterns of EGFR expression (fig. 3).

In other words: according to the present invention it was furthermore stated for the first time that cancer patients, preferably mCRC patients, who show a clinical response to administration of an anti-EGFR mAb (e.g. cetuximab, matuzumab or parlimumab) apparently on the basis of an increased EGFR gene copy number, can be selected and evaluated by analyzing individual tumor samples of said patients using FISH. In other words: patients who are FISH positive have higher gene copy numbers than patients who are FISH negative. Thus, it can be concluded that patients showing increased EGFR copy number by FISH analysis have a better survival expectation than those showing low gene copy number.

For a more complete summary, the invention relates to the following subject matter:

a method for treating a patient with a tumor expressing the EGF receptor (EGFR) by administering to said patient an amount of an anti-EGFR antibody sufficient to destroy the proliferation of said tumor cells with amplified copy number of the EGFR gene.

A corresponding method, wherein the treatment is more effective than the treatment of tumor cells that do not show EGFR gene copy number expansion with the same antibody at the same dose.

A corresponding method, wherein the tumor cells further exhibit a molecular alteration or a genetic mutation.

A corresponding method, wherein the amplified EGFR gene copy number is specific for the tumor.

A corresponding method, wherein the amplified EGFR gene copy number is specific for the individual cancer tissue profile (individual cancer tissue profile) of the patient.

A corresponding method, wherein the individual cancer tissue profile has a molecular alteration.

A corresponding method, wherein said EGFR expressing tumor is colorectal cancer (CRC).

A corresponding method, wherein said colorectal cancer is metastatic (mCRC).

A corresponding method, wherein the anti-EGFR antibody is Mab 225 and Mab 425 selected from murine, chimeric and humanized versions thereof.

Use of an anti-EGFR antibody in the manufacture of a medicament for the treatment of cancer, based on EGFR expressing tumor cells having an amplified EGFR gene copy number, wherein said treatment is more effective than treatment of tumor cells that do not exhibit an amplified EGFR gene copy number with the same dose of the same antibody.

Use of a corresponding anti-EGFR antibody, wherein said tumor cells further exhibit a molecular alteration or a genetic mutation.

A corresponding use, wherein said amplified EGFR gene copy number is specific for said tumor.

A corresponding use, wherein the amplified EGFR gene copy number is specific for the individual cancer tissue spectrum of the patient.

A corresponding use, wherein the individual has a molecular alteration in the cancer tissue profile.

A corresponding use, wherein the EGFR expressing tumor is colorectal cancer (CRC).

A corresponding use, wherein the colorectal cancer is metastatic (mCRC).

Corresponding use, wherein the anti-EGFR antibody is selected from Mab 225 and Mab 425 in its murine, chimeric and humanized forms.

A method for detecting and measuring the EGFR gene copy number of tumor tissue in vitro by using Fluorescence In Situ Hybridization (FISH).

Use of Fluorescence In Situ Hybridization (FISH) to identify in vitro patients with tumors that respond to anti-EGFR antibodies.

Use of Fluorescence In Situ Hybridization (FISH) to identify in vitro patients with tumors that exhibit increased EGFR gene copy number.

A corresponding use, wherein the tumour is colorectal cancer (CRC), preferably metastatic CRC.

The corresponding use, wherein the antibody is 225 or 425 in murine, chimeric or humanized form.

An in vitro method for detecting and analyzing whether a patient suffers from a cancer that overexpresses the EGF receptor (EGFR), that positively responds to the administration of an anti-EGFR antibody or an immunologically effective fragment thereof, comprising determining in vitro the EGFR gene copy number of a sample of tumor cells obtained from said patient, and selecting for administration of said anti-EGFR antibody to said patient if the tumor cells of said patient exhibit an amplified EGFR gene copy number.

Corresponding method, wherein the EGFR gene copy number is measured as a ratio of EGFR gene basis factors per nucleus.

The corresponding method, wherein the ratio is between 4.0 and 8.2.

The corresponding method, wherein the ratio is between 5.7 and 7.1.

Corresponding method, wherein the EGFR gene copy number is measured as a ratio of EGFR gene basis factors per CEP 7.

The corresponding method, wherein the ratio is > 2.

Corresponding methods, in which the EGFR gene copy number is measured by FISH analysis (fluorescence in situ hybridization).

A corresponding method, wherein the amplified EGFR gene copy number is specific for the tumor.

A corresponding method, wherein the amplified EGFR gene copy number is specific for the individual cancer tissue profile of the patient.

A corresponding method, wherein the individual cancer tissue profile further has a molecular alteration.

A corresponding method, wherein the molecular alteration is a point mutation within the EGFR gene.

A corresponding method, wherein said anti-EGFR antibody is selected from the group consisting of cetuximab (mAbc225), matuzumab (mAb h425) and parlimumab (mAb ABX) or specific murine, chimeric and humanized forms thereof.

Corresponding method, wherein the cancer is colorectal cancer (CRC), lung cancer, head and neck cancer and breast cancer.

Use of an anti-EGFR antibody or immunologically effective fragment thereof in the manufacture of a medicament for treating cancer in a patient, wherein the cancer overexpresses EGFR and exhibits amplified EGFR gene copy number.

The corresponding use, wherein the EGFR gene copy number is measured as a ratio of EGFR genes per nucleus, and the value of this ratio is between 4.0 and 8.2.

The corresponding use, wherein the value of said ratio is between 5.7 and 7.1.

A corresponding use, wherein the treatment of said cancer is more effective than the treatment of a cancer patient wherein the cancer cells do not show an expanded copy number of EGFR with the same antibody at the same dose.

A corresponding use, wherein said amplified EGFR gene copy number is specific for said tumor.

A corresponding use, wherein the amplified EGFR gene copy number is specific for the individual cancer tissue spectrum of the patient.

A corresponding use, wherein the individual has a cancer tissue profile with a genetic mutation.

A corresponding use, wherein the EGFR expressing tumor is colorectal cancer (CRC), lung cancer, breast cancer or head and neck cancer.

A corresponding use, wherein the anti-EGFR antibody is selected from cetuximab (mAbc225), matuzumab (mAb h425) and parlimumab (mAb ABX) or specific murine, chimeric and humanized forms thereof.

A method for detecting and measuring the EGFR gene copy number of EGFR overexpressing tumor tissue in vitro by using Fluorescence In Situ Hybridization (FISH) in an assay to determine the response of cancer patients to administration of anti-EGFR antibodies.

Brief Description of Drawings

FIG. 1 missense heterozygous mutations in exon 21(G857R) found in the tumor of patient 13 (see also Table 2). Mutations affect key residues located in the activation loop of the EGFR kinase domain. G857R is a single amino acid mutation in addition to the recently described L858R mutation found in gefitinib and erlotinib responders to non-small cell lung cancer (NSCLC) (Lynch et al, 2004, N Engl J Med 350: 2129-2139; Paez et al, 2004, Science 304: 1497-1500; Pao et al, 2004, Proc Natl Acad Sci USA 101: 13306-13311). Mutations affecting similar residues in the BRAF gene (G595R) have been previously detected in colorectal cancer (CRC) (Wiley, Diaz, 2004, Jama 291: 2019-2020).

FIG. 2. two-color fluorescence in situ hybridization assay for EGFR gene (red) and chromosome 7(CEP 7; green) probes. (A) Balanced disomy in normal colorectal mucosa; (B) balanced disomy in the tumor of patient 27; (C) balance polysomy in the tumor of patient 3; (D) expansion in tumors of patient 5.

FIG. 3 EGFR amplification and protein expression in tumors of patient 10. (A) Routine histology with hematoxylin and eosin staining. (B and C) EGFR gene amplification and protein overexpression in the corresponding regions of the same tumor by immunohistochemistry (Moroni et al, 2001, Clin Cancer Res 7: 2770-5).

Figure 4. molecular alterations in EGFR gene and clinical responses observed in patient 1. (A) Two-color fluorescence in situ hybridization assays of EGFR gene (red) and chromosome 7(CEP 7; green) probes showed increased copy number; (B) relative amounts of EGFR gene copy number measured by quantitative PCR in patient 1 tumor, A431 cancer cell line (EGFR gene/nucleus 8.00; EGFR gene/CEP 72.57) and non-malignant RPE (EGFR gene/nucleus 1.60; EGFR gene/CEP 70.86) epithelial cell controls; (C) (D) measurement of liver metastasis with CT before (highest diameter, L line 4.4cm) and after treatment (highest diameter, M line 2.3cm) in patient 1 with moAb.

FIG. 5 inhibition of proliferation of colorectal cancer cell lines with cetuximab. (A) Proliferation of colorectal cancer cell lines in three independent experiments (mean ± SD) at increasing cetuximab concentrations. (B) EGFR protein levels measured in individual cell lines using Western blot. (C) EGFR gene copy number assessed with FISH in colorectal cancer cell lines. (D) Two-color fluorescence in situ hybridization assays with EGFR gene (red) and chromosome 7(CEP 7; green) probes showed increased copy number in the DiFi cell line.

Detailed Description

The term "copy number" is generally defined as the basis number of each genome. According to the present invention, the term "EGFR gene copy number" refers to the ratio of the number of EGFR genes per cell nucleus. According to the invention, this number varies between 1.0 and 8.2, or more preferably between 1.5 and 7.9.

According to the present invention, the term "increased or amplified EGFR gene copy number" means that, from a relative point of view, the ratio defined above in the cells of a specific tumor associated with a specific patient (which responds to an anti-EGFR antibody treatment) is higher or amplified than the specific ratio in the cells of a specific tumor associated with another specific patient. From a more absolute perspective, the term means that the ratio (EGFR gene factor/nucleus) is between 4.0 and 8.2, or 4.8 and 7.9, or 4.8 and 7.1, or 4.8 and 6.8, or 4.8 and 5.7. Preferably, said ratio is between 5.7 and 8.2, more preferably between 5.7 and 6.8, most preferably between 5.7 and 7.1.

According to these values applicable to "increased or amplified" EGFR gene copy number, the tumor cells exhibit a ratio of relatively decreased or reduced or non-amplified copy number in the range of 1.65-2.0 or 1.7-1.9 for patients who do not respond at all or do not respond effectively or do not respond positively to anti-EGFR antibody treatment.

EGFR gene copy number or ratio: the ratio of EGFR gene copy number/nucleus to EGFR gene copy/chromosome 7 centromeric probe (CEP7) was correlated. According to the invention, this EGFR gene/CEP 7 ratio is > 2 in patients who respond significantly to anti-EGFR antibody treatment, whereas in patients who do not respond, the ratio is usually close to 1.

According to the invention, "missense heterozygous mutation" refers to a mutation in one of the two alleles at which the codon for one amino acid becomes a codon for the other amino acid.

According to the invention, the term "in-frame deletion" refers to a mutation that alters the reading frame of an mRNA by deleting nucleotides.

According to the invention, "FISH (fluorescence in situ hybridization)" refers to the hybridization of cloned DNA to an intact chromosome, wherein the cloned DNA has been labeled with a fluorescent dye. This is a common method for determining chromosomal location, gene copy number (both increase and decrease), or chromosomal rearrangement.

Tumors from mCRC patients (31) that achieved the targeted response, i.e. stable disease or disease progression, after treatment with cetuximab or parlimumab were screened for genetic alterations in the EGFR gene or its direct intracellular effectors. Specifically, the EGFR gene copy number, mutation pattern of the EGFR catalytic domain, and exons of KRAS, BRAF, and PI3KCA genes that are frequently mutated in mCRC can be determined.

Mutational analysis of the EGFR tyrosine kinase Domain

To identify the molecular basis on which mCRC is based in response to matuzumab, parlimumab or cetuximab, the mutation status of the region corresponding to the catalytic domain of the EGFR gene was assessed in tumor samples of patients with different clinical effects after treatment with these mabs. Sequencing of EGFR exons 18, 19 and 21 did not reveal somatic mutations, except for one patient with 24 weeks of stable disease (tables 1 and 2). This patient showed a missense heterozygous mutation in exon 21(G857R) affecting residues in the activation loop located in the critical region of catalysis (FIG. 1). The G857R mutation is a single amino acid mutation in addition to the presently described L858R activating mutation found in lung cancer gefitinib and erlotinib responders (Lynch et al, 2004, N Engl J Med 350: 2129-.

Interestingly, mutations affecting similar residues in the BRAF gene (G595R) were previously detected in colorectal cancer (FIG. 1) (Rajagopalan et al, 2002, Nature 418: 934).

Based on current findings, the major molecular mechanism underlying response to mAb therapy is apparently not a mutation in the catalytic domain of EGFR. It is therefore believed that changes in EGFR gene copy number may be responsible for the observed antibody response.

Mutational analysis of EGFR intracellular effectors

In colorectal cancer, at least three intracellular molecules involved in EGFR signaling (KRAS, BRAF and PI3KCA) may be activated by point mutations. According to the present invention, it was analyzed whether the mutation status of the corresponding gene correlates with the clinical response to anti-EGFR antibodies (e.g., cetuximab, matuzumab or parlimumab). The exons (KRAS exon 2, BRAF exon 15, PI3KCA exons 9 and 20) of each of the three genes that have the highest frequency of mutation in colorectal cancer were analyzed. The nucleotide sequence corresponding to each exon can be amplified and directly sequenced from genomic DNA extracted from the tumor. Although activating mutations can be identified in KRAS genes (G12V, G12D, G12S and G13D), PI3KCA genes (E545K, H1047R) and BRAF (E599V), but are not associated with the clinical response of anti-EGFR mabs (RAS exon-2: p ═ 0.675; PI3K exon-9: p ═ 0.3; PI3K exon-20: p ═ 1; BRAF exon-15: p ═ 1; all these mutations: p ═ 0.44) (tables 1 and 2).

Analysis of the copy number of the EGFR Gene by FISH analysis

It was shown that there was no correlation between EGFR protein expression measured by Immunohistochemistry (IHC) and clinical response to anti-EGFR mAb in mCRC. These results, together with the results of the lack of correlation with the mutation status of EGFR and its downstream effectors, can lead to the hypothesis that the response to parlimumab, cetuximab, or matuzumab correlates with EGFR gene amplification.

According to the details in table 2 and figure 2, 9 of 10 patients with target response were FISH assessed, wherein 8/9 (88.8%) showed increased EGFR gene copy number (median EGFR gene/nucleus ratio of 6.80, range 1.65-35); of the 21 non-responsive patients, 20 were subjected to FISH assessment, of which 1/20 (5.0%) had increased EGFR gene copy number (median EGFR gene/nucleus ratio of 1.925), and the difference was found to be statistically significant.

Among the responders, 7 of the 9 patients evaluable with FISH had an increased EGFR gene copy number associated with an EGFR gene/CRP 7 ratio > 2, thus indicating the presence of amplification of the EGFR gene according to the criteria used for HER2 evaluation (Wiley, Diaz, 2004, Jama 291: 2019-2020). In patients 3 and 9, EGFR gene/nucleus ratios 7.10 and 3.38, correlated with EGFR gene/CEP 7 ratios 1.46 and 1.19, respectively, thus indicating the presence of an extra copy of the entire chromosome 7 (polysomy No. 7) (fig. 2C).

The tumor of patient 10 had a striking amplification of the EGFR gene, which was located in discrete foci, while the other malignant areas were indeed disfiguring. Notably, the region that showed EGFR gene amplification also showed strong EGFR protein expression by IHC evaluation; in contrast, the regions of the EGFR gene which show disodism do not express the corresponding protein (fig. 3).

Analysis of the copy number of the EGFR Gene by quantitative PCR (qPCR)

Increased EGFR gene copy number can be observed in patients responding to cetuximab, matuzumab, or parlimumab by FISH. To obtain independent measurements of EGFR locus status in tumor samples, qPCR analysis can be used. An increase in EGFR gene copy number was observed in patient 1 with responsive disease (fig. 4). The detection of increased EGFR gene copy number by qPCR was inconclusive for patient samples with a gene/chromosome ratio below 3. This is probably due to the limited number of EGFR genes that could not be consistently detected using previously reported methods (Layfield et al, 2003, J Surg Oncol 83: 227-. Furthermore, qPCR assays may be adversely affected by the contamination of DNA by ordinary somatic cells accompanying extraction, which can only be partially avoided during the cutting of paraffin-embedded samples. On the other hand, in situ analysis of gene copy number, e.g. obtained by FISH analysis, is not affected by the limitations of these techniques. This qPCR gene copy number measurement confirmed amplification.

Effect of cetuximab on cell lines with normal or increased copies of the EGFR gene

Previous studies using cellular cancer models have suggested that the response to cetuximab is related to the following factors: (i) overexpression of the EGFR receptor, (ii) constitutive phosphorylation of the receptor, (iii) amplification of the corresponding gene, and (iv) alteration of other members of the gene family.

Existing data indicate that responsiveness to parlimumab, matuzumab, or cetuximab in mCRC correlates with increased gene copy number at the EGFR locus. This prompted the inventors to evaluate the effect of cetuximab on a panel of colorectal cancer cell lines with normal or increased EGFR gene copy number measured with FISH (fig. 5). Cell proliferation measured by the BrdU incorporation assay was evaluated at increasing cetuximab concentrations. The cetuximab can obviously inhibit the proliferation of the DiFi cell line with the highest EGFR gene copy number, and can completely weaken the cetuximab concentration for the proliferation of the DiFi cells and cannot influence the cells with the non-amplified EGFR copy number. Interestingly, the SW620 cell line had 3 copies of the EGFR gene and Western blot showed that it did not express EGFR protein (fig. 5). SW620 cells therefore represent a functional knock-out of the EGFR gene, and therefore their proliferation is virtually unaffected by cetuximab.

The term "ErbB receptor antagonist/inhibitor" refers to a biologically active molecule that can bind to and block or inhibit an ErbB receptor. Thus, by blocking the receptor, antagonists prevent binding of ErbB ligands (agonists), and activation of agonist/ligand receptor complexes. ErbB antagonists may be used for HER1(ErbB1, EGFR), HER2(ErbB2), ErbB3 and ErbB 4. Preferred antagonists of the invention are antagonists against the EGF receptor (EGFR, HER 1). The ErbB receptor antagonist may be an antibody or antibody fusion protein (immunoconjugate) or an immunotherapeutically effective fragment of an antibody or antibody fusion protein. According to the invention, preferred ErbB receptor antagonists are anti-EGFR antibodies, particularly and preferably the anti-EGFR antibodies mentioned above and below: murine, chimeric or humanized forms of cetuximab, parlimumab and matuzumab, including immunologically effective fragments (Fab, Fv) and immunoconjugates thereof, in particular immunocytokines.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homologous antibodies, i.e. each antibody comprised in the group is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, each monoclonal antibody is directed against a single determinant on the antigen, as compared to a polyclonal antibody preparation comprising different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that their synthesis avoids contamination with other antibodies. Methods for preparing monoclonal antibodies include The hybridoma method described by Kohler and Milstein (1975, Nature 256, 495) and in "monoclonal antibody Technology, The Production and Characterization of Rodent and human hybrids" (1985, Burdon et al, Laboratory technologies in biochemistry and Molecular Biology, Vol.13, Elsevier Science Publishers, Amsterdam), or by well-known recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Also useful are the methods described in, for example, Clackson et al, Nature, 352: 624-: 58, 1-597(1991), monoclonal antibodies are isolated from phage antibody libraries.

The term "chimeric antibody" refers to an antibody in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical to or homologous to corresponding sequences in an antibody derived from another species or belonging to another class or subclass; and fragments of such antibodies so long as they exhibit the desired biological activity (e.g., U.S. Pat. No. 4,816,567; Morrison et al, Proc. nat. Acad. Sci. U.S. 81: 6851-6855 (1984)). Methods for making chimeric and humanized antibodies are also known in the art. For example, methods for making chimeric antibodies include those described by Boss (Celltech) and Cabilly (Genentech) in patents (U.S. Pat. No. 4,816,397; U.S. Pat. No. 4,816,567).

A "humanized antibody" is a form of chimeric antibody that is non-human (e.g., rodent) and contains minimal sequences derived from non-human immunoglobulin. Humanized antibodies are mostly human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by a hypervariable region from a non-human species (donor antibody, e.g. mouse, rat, rabbit or non-human primate) which has the desired specificity, affinity and capacity. In some instances, residues of the Framework Region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not present in the recipient antibody or the donor antibody. These modifications were made to further improve the performance of the antibodies. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody may also optionally contain at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (US 5,225,539) and Boss (Celltech, US4,816,397).

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂Fv and Fc fragments, diabodies, linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. An "intact" antibody is one that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains CH1, CH2, and CH 3. Preferably, the intact antibody has one or more effector functions. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing a single antigen-binding site and CL, CH1 regions, and a residual "Fc" fragment, the name of which reflects the ability to crystallize readily. Typically, the "Fc" region of an antibody contains the CH2, CH3 and hinge regions of the broad class of IgG1 or IgG2 antibodies. The hinge region is a set of about 15 amino acid residues that connects the CH1 region and the CH2-CH3 region. The "Fab" fragment also contains the constant domain of the light chain and the first constant domain of the heavy chain (CH1) and has only one antigen binding site.

"Fab * fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the CH1 domain of the heavy chain, which CH1 domain comprises one or more residues from the hinge region of the antibodyAnd (c) cysteine. F (ab')₂Antibody fragments were originally prepared as Fab' fragment pairs with hinge cysteines between them. Other chemical couplings of antibody fragments are also known (see, e.g., Hermanson, Bioconjugate Techniques, Academic Press, 1996; U.S. Pat. No. 4,342,566). "Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present as a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the scFv to form the structure required for antigen binding. Single-chain FV Antibodies are known, for example, from Pluckthun (The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, Springer-Verlag, New York, p.269-315 (1994)), WO93/16185, US5,571,894, US5,587,458, Huston et al (1988, Proc. Natl.Acad.Sci.85, 5879) or Skerra and Plueuekthun (1988, Science 240, 1038).

Although the present invention preferably relates to colorectal or colorectal Cancer (CRC), it is in principle also applicable to other cancers and tumors that express or overexpress EGFR, that are present in patients with different EGFR gene copy numbers and that are treated with other ErbB antagonists (e.g., lung Cancer treated with IRESSA ®: e.g., Cancer Biology 2005, 4).

Thus, the terms "cancer" and "tumor" refer to or describe a physiological disease in mammals that is generally characterized by unregulated cell growth. Tumors, such as tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone marrow, blood, thymus, uterus, testis, cervix and liver, can be treated by the pharmaceutical composition of the present invention. The tumors that can be treated with the antibody molecules of the invention are preferably solid tumors or tumor metastases, e.g. breast, prostate, head and neck, SCLC, pancreatic cancers, expressing high amounts of ErbB receptors, in particular ErbB1(EGFR) receptor.

The term "biologically/functionally effective" or "therapeutically effective amount" refers to a drug/molecule that causes a biological function or a change in a biological function in vivo or in vitro, which is effective at a specified amount for treating a disease or condition in a mammal, preferably a human. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells, shrink tumor size, inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs, inhibit (i.e., slow to some extent and preferably stop) tumor metastasis, inhibit tumor growth to some extent, and/or alleviate one or more symptoms associated with cancer to some extent.

The term "immunotherapeutically effective" refers to a biomolecule that elicits an immune response in a mammal. More specifically, the term refers to molecules that can recognize and bind to antigens. In general, antibodies, antibody fragments, and antibody fusion proteins that contain their antigen binding sites (complementarity determining regions, CDRs) are effective for immunotherapy.

Typically, a therapeutically effective amount of an anti-EGFR antibody or fragment thereof is an amount sufficient to achieve a plasma concentration of from about 0.01 micrograms (μ g)/milliliter (ml) to about 100 μ g/ml, preferably from about 1 μ g/ml to about 5 μ g/ml and typically about 5 μ g/ml, when administered in a physiologically tolerable composition. In other words, the dosage may vary from about 0.1mg/kg to about 300mg/kg, preferably from about 0.2mg/kg to about 200mg/kg, most preferably from about 0.5mg/kg to about 20mg/kg, in one or more administrations per day over one or more days. Preferred plasma concentrations, expressed as molarity, are from about 2 micromolar (. mu.M) to about 5 millimolar (mM), preferably from about 100. mu.M to 1mM, of the antibody antagonist.

The pharmaceutical compositions of the invention may comprise treating a subject with agents that reduce or avoid the side effects associated with the combination therapies of the invention ("adjuvant therapy"), including, but not limited to, those agents that, for example, reduce the toxic effects of anticancer drugs, e.g., bone resorption inhibitors, cardioprotective agents. The adjuncts prevent or reduce the incidence of nausea and vomiting associated with chemotherapy, radiation therapy or surgery, or reduce the incidence of infection associated with administration of spinal cord inhibitory anti-cancer drugs. Adjuncts are well known in the art. According to the present invention, the immunotherapeutic agent may be additionally administered together with an adjuvant such as BCG and an immune system stimulant. In addition, the compositions may contain immunotherapeutic or chemotherapeutic agents, including those with radiolabeled isotopes of cytotoxic effect, or other cytotoxic agents, such as cytotoxic peptides (e.g., cytokines) or cytotoxic drugs, and the like.

Other features and advantages of the present invention will be apparent from the following more detailed examples, which illustrate, by way of example, the principles of the invention. In particular, specific values or terms set forth above and below are not limiting of the invention and may be extrapolated as deemed necessary by those skilled in the art.

Examples

Example 1: patients and treatments using anti-EGFR monoclonal antibodies

Clinical trials of treatment of EGFR expressing mCRC with anti-EGFR moAb parlimumab or cetuximab were performed on ospedae Niguarda Ca' Granda enrolled patients, of which 31 patients were evaluated with radioactively demonstrated tumor sensitivity or tolerance to the therapy (table 1). Patient selection is based on the availability of sufficient tumor tissue for current studies. All patients had mCRC expressing EGFR, as ≧ 1% EGFR-stained malignant cells, assessed by IHC in the central laboratory using the DAKO EGFRPHARmDX kit per clinical protocol (Cunningham et al, 2004, N EngI J Med 351: 337-. Cetuximab (chimeric IgG1 moAb; Erbitux)^®Merck, Milan, Italy) and parquinan antibody (whole human IgG2 moAb; amgen, thunder Oaks, CA, usa) targets the ligand binding domain of EGFR. In addition to the lower incidence of fusion reactions observed with intact human parlimumab, the clinical activity of both was expected to be comparable, so this study analyzed patients treated with both moabs together. anti-EGFR moAb treatment consists of a monotherapy with cetuximab (n ═ 12), cetuximab + based on irinotecan (Campt oa)^®；Aventis，Milan, Italy) chemotherapy (n ═ 9), or paparma monotherapy (n ═ 10). In particular, cetuximab (400 mg/m) as a single drug²Intravenous dose and weekly 250 mg/m thereafter²Until advanced) as first line therapy in the EMR 202-600 phase II trial or as third line therapy in the monotherapy group of the BOND phase II trial for irinotecan-resistant patients. Cetuximab (same dose and procedure as monotherapy) + irinotecan (same dose and procedure as mCRC alone exhibits tolerability) was administered as a three-line therapy to irinotecan-resistant patients in the BOND trial combination group and in the MABEL phase II trial until progression. In the latter case, resistance to irinotecan is defined as the occurrence of the recorded progression of the disease during or within 3 months after irinotecan administration. A single dose of palimumab (6 mg/kg intravenously every 2 weeks until progression) was used as a three-or four-wire therapy in phase III ABX-EGF 20020408 test and cross ABX-EGF 20020194 test for patients who were resistant to oxaliplatin (oxaliplatin) and irinotecan-containing drugs. Institutional Ethics Committee approved the treatment protocol, and patients written consent for EGFR analysis and receiving investigational treatment. Tumor responses were evaluated by research institutes and independent radiologists according to clinical protocols using coherent imaging techniques (CT or MRI) according to RECIST (response assessment criteria in solid tumors).

Example 2: mutation analysis

DNA was extracted from paraffin-embedded samples. 10 sections were prepared per patient. Additional representative sections were deparaffinized, stained with hematoxylin-eosin and subjected to detailed morphological analysis. The area showing tumor tissue was marked and the tissue was extracted with 0.2M NaOH/1mM EDTA and then neutralized with 100mM Tris-TE. After extraction, the DNA was purified using Qiagen PCR purification kit (catalog No. 28104) according to the manufacturer's instructions. Exon-specific primers and sequencing primers were designed using Primer3 software (http:// frodo. wi. mit. edu/cgi-bin/Primer3/Primer 3-www.cgi) and by Invitrogen^TMAnd (6) analyzing. The primer sequences are: forward, reverse, and sequencing of each exonThe primers are as follows:

EGFR-Ex18

GCTGAGGTGACCCTTGTCTC；ACAGCTTGCAAGGACTCTGG；TGGAGCCTCTTACACCCAGT；

EGFR-Ex19

CCCAGTGTCCCTCACCTTC；CCACACAGCAAAGCAGAAAC；GCTGGTAACATCCACCCAGA；

EGFR-Ex21

TGATCTGTCCCTCACAGCAG；TCAGGAAAATGCTGGCTGAC；TTCAGGGCATGAACTACTTGG；

PI3K CA-Ex9

GGGAAAAATATGACAAAGAAAGC；CTGAGATCAGCCAAATTCAGTT；

TAGCTAGAGACAATGAATTAAGGGAAA；

PI3K CA-Ex20

CTCAATGATGCTTGGCTCTG；TGGAATCCAGAGTGAGCTTTC； TTGATGACATTGCATACATTCG

Ras ex2

GGTGGAGTATTTGATAGTGTATTAACC；AGAATGGTCCTGCACCAGTAA；

TCATTATTTT TATTATAAG GCCTGCTG.

the conditions for amplifying exon-specific regions from tumor genomic DNA by PCR and identifying mutations have been described previously (Bardelli et al, 2003, Science 300: 949). PCR was performed in 20L volumes using the descending PCR procedure as described previously (Pao et al, 2004, Proc NatlAcad Sci USA 101: 13306-. The purified PCR products were sequenced using BigDye ® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) and analyzed using a 3730 ABI capillary electrophoresis system. The analysis of the mutations was performed as described previously. Due to the limited amount of tumor tissue from patient 13, mutation analysis of all exons was technically impossible.

Example 3: analysis of EGFR Gene by Fluorescence In Situ Hybridization (FISH)

Tissue sections were processed according to the procedure used in Her2 FISH detection kit (Dakocytomation, Glostrup, DK). The sample was placed in the pretreatment solution at 96 ℃ for 30 minutes and then digested with pancreatin solution at room temperature for 30 minutes. Two-color dual-target FISH assay was performed using LSI EGFR Spectrum Orange/CEP7 Spectrum Green Probe (Vysis, Downers Green, IL). Briefly, tissue sections were covered with 10 μ L of probe solution, incubated with the co-denatured EGFR and CEP7 probes for 5 minutes at 75 ℃ and hybridized overnight at 37 ℃. Both the co-denaturation and the hybridization were carried out sequentially in a microprocessor-controlled system (Hybridizer, Dakocytomation, Glostrup, DK). Post hybridization stringency washes were performed in a 65 ℃ water bath for 10 minutes. After washing twice and drying at room temperature for 15 minutes, the tissue sections were covered with 4', 6-diamidino-2-phenylindole (DAPI II, Vysis), chromatin counterstained and examined microscopically. The analysis was performed with a fluorescence microscope (Zeiss Axioskop, Gottingen, Germany) equipped with a Chromowin workstation (Amplimedical, Milan, Italy). The EGFR gene was visually observed as a red signal with a tetramethyl-rhodamine isothiocyanate (TRITC) filter, the chromosomal 7 α -centromere (CEP7) sequence was visually observed as a green signal with a Fluorescein Isothiocyanate (FITC) filter, and the nucleus was visually observed as a blue signal with a DAPI filter. Representative images of each sample were obtained in a single color layer using a Hamamatsu C5895 cooled CCD camera (Upstate Technical Equipment co., New York, usa) and subsequently fused using the Casti Imaging FISH Multicolor software (amplification). Two independent observers (SMV and RB) scored at least 200 non-overlapping interphase nuclei using a predefined scoring index. The observer is unaware of the clinical characteristics of the patient and the evaluation and scoring of the samples against each other. Within each nucleus, the copy number of EGFR and the number of probes for chromosome 7 were evaluated independently. The status of the EGFR gene was scored with the EGFR/nucleus and EGFR/CEP7 ratios. Normal controls consisted of a cultured Retinal Pigment Epithelial (RPE) cell line and normal colorectal mucosa in the vicinity of a single malignancy. The amplified EGFR gene control consisted of an a431 human squamous cell carcinoma cell line. The increased EGFR gene copy number is arbitrarily defined as EGFR gene copy number/nucleus ≧ 3. Samples from patients 4 and 15 were only taken as 10 μ sections, and despite various efforts, definitive FISH analysis could not be generated due to the excessive thickness of the tissue.

Example 4: analysis of EGFR Gene by quantitative polymerase chain reaction (qPCR)

Use of ABI PRISM^®7900HT apparatus (Applied biosystems) determines copy number of the corresponding EGFR locus using real-time PCR. DNA content was normalized to Line-1, which, as previously mentioned, is a repetitive element with similar copy number per diploid genome in all human cells (normal or malignant) (Wang et al, 2002, Proc Natl Acad Sci USA 99: 16156-. Change of copy number is shown in equation 2^{(Dt-Dline)-(Nt-Nline)}Calculations were performed where Dt is the average threshold cycle number observed in tumor cell extracted DNA with the experimental primers, Dline is the average threshold cycle number observed in tumor cell extracted DNA with the Line-1 primers, Nt is the threshold cycle number observed in normal reference DNA extracted from RPE cells, and Nline is the threshold cycle number observed in normal reference DNA extracted from RPE cells with the Line-1 primers. The amplification conditions were as follows: 1 cycle at 95 ℃ for 10 minutes followed by 45 cycles at 95 ℃ for 15 seconds and 60 ℃ for 1 minute. The threshold cycle number is determined by ABIPRISM^®7900HT sequence detection system software. PCR was performed in triplicate for each primer set and the mean threshold cycle number was calculated. Primers for the EGFR gene (designed to span the 100-200bp non-repeat region) were: forward direction: GAATTCGGATGCAGAGCTTC and the reverse: GACATGCTGCGGTGTTTTC are provided. The primers for the Line-1 repeat elements were: forward AAAGCCGCTCAACTACATGG and reverse: TGCTTTGAATGCGTCCCAGAG are provided.

Example 5: cell proliferation inhibition assay and Western blot

Colorectal cancer cell lines (HT-29, HCT-116, DLD-1, SW48, SW480, and LoVo cells) were from the ATCC deposit; the DiFi cells are a gift from Jose Baselaga (Vall d' Hebronuniversity, Barcelona, E). Except that the DiFi cells were grown in F-12 cultures supplemented with 10% Fetal Calf Serum (FCS) and antibioticsCells were grown in DMEM supplemented with 10% FCS and antibiotics, except for the nutrient medium. For cell proliferation inhibition assays, cells were grown in 96-well black plates (Culture plates)^TM96F Packard Bioscience) supplemented with 2% FBS, and incubated with 0.01-100nM cetuximab (purchased from Komtur Pharmaceuticals, Freiburg, D) for 5 days. Cell proliferation was measured by BrdU incorporation using the chemiluminescent ELISA method (Roche cat # 1669915). The cell density per well was as follows: DiFi, 4000; LoVo, 4000; DLD, 500; HCT116, 1000; HT29, 1000; SW480, 1000; SW387, 4000; SW48, 500; SW620, 500. The BrdU assay was performed according to the manufacturer's instructions and terminated 20 hours after the addition of the labeling solution. Three independent experiments were set up in triplicate for each cell line. The percentage of cell proliferation at each cetuximab concentration (experimental group) was calculated using the following formula: (experimental-blank)/(control-blank). times.100, where control refers to cells grown in media only (no drug) and blank refers to cells grown in DMEM with 0.02% Triton X. Western blots were performed as described previously (Lynch and Yang, 2002, Semin Oncol 29: 47-50).

Table 1: tumor-associated clinical features and EGFR gene molecular alterations in mCRC patients

Patient number and UPN	Treatment with anti-EGFR antibodies	Tumor response		Molecular analysis of EGFR
						Optimum response	Duration of response (week)	Number of copiesb	Sequencing＊
		1-MR120653	Cetuximab and CTa	PR	48					Increased of	WT
2-LM090846	Cetuximab and CT					PR	36	Increased of	WT
		3-RP180336	Cetuximab and CT	PR	36+					Increased of	WT
4-LS250848	Cetuximab					PR	30	Not evaluatedc	WT
		5-AC201146	Palmann single resistance	PR	33					Increased of	WT
6-GL240243	Palmann single resistance					PR	24	Increased of	WT
		7-FC151048	Palmann single resistance	PR	16					Increased of	WT
8-PA260526	Cetuximab					PR	16+	Is normal	WT
		9-AM180627	Palmann single resistance	PR	12+					Increased of	WT
10-GM281120	Cetuximab					PR	8+	Increased of	WT
		11-SM070445	Cetuximab	SD	30					Is normal	WT
12-LC280946	Cetuximab and CT					SD	24	Is normal	WT
		13-AG080530	Cetuximab and CT	SD	24					Is normal	Exon 21G857R
14-MM180625	Cetuximab					SD	36+	Is normal	WT
		15-GM160553	Palmann single resistance	SD	32					To evaluatec	WT
16-CC090234	Palmann single resistance					SD	16+	Is normal	WT
		17-GT030547	Cetuximab	PD	N.A.					Increased of	WT
18-SM140851	Cetuximab and CT					PD	N.A.	Is normal	WT
		19-AC230643	Cetuximab	PD	N.A.					Is normal	WT
20-DS010731	Cetuximab and CT					PD	N.A.	Is normal	WT
		21-RV110964	Cetuximab and CT	PD	N.A.					Is normal	WT
22-CC041133	Cetuximab					PD	N.A.	Is normal	WT
		23-GT050933	Cetuximab	PD	N.A.					Is normal	WT
24-RT161027	Cetuximab					PD	N.A.	Is normal	WT
		25-CB280630	Cetuximab	PD	N.A.					Is normal	WT
26-FL020230	Cetuximab					PD	N.A.	Is normal	WT
		27-PC020849	Palmann single resistance	PD	N.A.					Is normal	WT
28-CF141238	Palmann single resistance					PD	N.A.	Is normal	WT
		29-WB030428	Cetuximab	PD	N.A.					Is normal	WT
30-GA240151	Palmann single resistance					PD	N.A.	Is normal	WT
		31-IM100640	Palmann single resistance	PD	N.A.					Is normal	WT

^aChemotherapy (CT) consists of irinotecan-based therapy (details see herein);^bincreased EGFR gene copy number, consisting of balanced polysomy in examples 3 and 9, and gene amplification in other examples (see results);^cfor technical reasons, multiple FISH attempts are uncertain (see methods). FISH, fluorescence in situ hybridization; PR, partial response; SD, stable disease condition; PD, disease progression; UPN, unique patient number; WT, wild type; + indicates that the response was maintained at the time of filing the article (month 2 2005).

^＊Mutation status of EGFR gene, exons 18, 19 and 21.

Table 1 b: additional clinical features of mCRC patients evaluated in this study

Patient number and UPN	Sex	Age (age)	PSa	No	Use of metastatic diseaseMedicineb
						1-MR120653	F	52	0	3	5-FU/FA, FOLFOX, irinotecan
2-LM090846	M	59	0	3	5-FU/FA，FOLFOX，FOLFIRI
						3-RP180336	M	69	0	2	FOLFOX，FOLFIRI
4-LS250848	M	57	1	3	5-FU/FA，FOLFOX，FOLFIRI
						5-AC201146	M	59	0	3	FOLFOX, capecitabine, FOLFIRI
6-GL240243	F	62	1	2	FOLFOX， FOLFIRI
						7-FC151048	M	57	1	2	FOLFIRI，FOLFOX
8-PA260526	M	79	1	0	Not applicable to
						9-AM180627	F	78	1	3	FOLFOX, capecitabine, FOLFIRI
10-GM281120	M	85	1	0	Not applicable to
						11-SM070445	M	60	0	1	Irinotecan
12-LC280946	M	59	0	2	FOLFOX，FOLFIRI
						13-AG080530	M	75	0	2	FOLFOX，FOLFIRI
14-MM180625	F	80	1	0	Not applicable to
						15-GM160553	F	52	0	4	FOLFOX, irinotecan, capecitabine, FOLFIRI
16-CC090234	M	71	1	2	FOLFOX，FOLFIRI
						17-GT030547	M	58	0	0	Not applicable to
18-SM140851	M	54	1	2	FOLFOX, irinotecan + weekly high dose of 5-FU/AF
						19-AC230643	M	62	1	0	Not applicable to
20-DS010731	M	74	0	3	Irinotecan, capecitabine, FOLFOX, FOLFIRI
						21-RV110964	M	41	0	3	FOLFOX, FOLFIRI, irinotecan
22-CC041133	F	72	1	0	Not applicable to
						23-GT050933	M	72	1	0	Not applicable to
24-RT161027	M	7B	1	0	Not applicable to
						25-CB280630	F	75	1	0	Not applicable to
26-FL020230	M	75	1	0	Not applicable to
						27-PC020849	M	56	1	1	oxaliplatin-irinotecan-5-FU/FA
28-CF141238	F	67	0	2	FOLFOX，FOLFIRI
						29-WB030428	M	77	1	0	Not applicable to
30-GA240151	M	54	0	2	FOLFOX，FOLFIRI
						31-IM100640	F	65	1	3	5-FU/FA，FOLFOX，FOLFIRI

No: number of previous chemotherapies for metastatic disease

Physical status at the start of anti-EGFR monoclonal antibody treatment (ECOG).

b chemotherapeutic agents consisting of 5-FU/FA: 5-fluorouracil + formyltetrahydrofolate (multiple protocol); FOLFOX: oxaliplatin + 5-fluorouracil + leucovorin; FOLFIRI: irinotecan + 5-fluorouracil + leucovorin.

Table 2: molecular alterations found in mCRC tumor patients

Patient UPN		Copy number of gene		Mutation analysis
											Tumor response	EGFRgene/CEP7	EGFRgene/nucleus	EGFR exon-18	EGFR exon-19	EGFR exon-21	Ras exon-2	Pl3K exon-9	Pl3K exon-20	BRAF exon-15
	1 -MR120653	PR	3.37	7.90	WT	WT	WT	WT	E545K	WT											WT
2 -LM090846											PR	2.28	5.70oo	WT	WT	WT	WT	WT	WT	WT
	3 -RP180336	PR	1.42	7.10	WT	WT	WT	WT	WT	WT											WT
4 -LS250848											PR	n.e.a	n.e.a	WT	WT	WT	WT	WT	WT	WT
	5 -AC201146	PR	2.50	4.80	WT	WT	WT	WT	WT	WT											WT
6 -GL240243											PR	2.13	6.80	WT	WT	WT	G13D	WT	WT	WT
	7 -FC151048	PR	3.27	8.20	WT	WT	WT	G12D	WT	WT											WT
8 -PA260526											PR	1.03	1.65	WT	WT	WT	WT	WT	WT	WT
	9 -AM180627	PR	1.19	3.38	WT	WT	WT	WT	WT	WT											WT
10 -GM281120											PR	8.75 clustered	35 clustered	WT	WT	WT	WT	WT	WT	WT
	11 -SM70445	SD	0.98	1.8	WT	WT	WT	WT	WT	WT											WT
12 -LC280946											SD	1.05	1.9	WT	WT	WT	WT	WT	WT	WT
	13 -AG080530	SD	0.95	1.75	WT	WT	G857R	WT	WT	WT											n.e.a
14 -MM180625											SD	1.06	1.80	WT	WT	WT	WT	WT	WT	WT
	15 -GM160553	SD	n.e.a	n.e.a	WT	WT	WT	G13D	WT	WT											WT
16 -CC090234											SD	1.04	1.88	WT	WT	WT	G12V	WT	WT	WT
	17 -GT030547	PD	4.68 clustered	20.2 clustered	WT	WT	WT	WT	WT	H1047R											WT
18 -SM140851											PD	1.04	2.00	WT	WT	WT	G13D	WT	WT	WT
	19 -AC230643	PD	0.70	1.72	WT	WT	WT	WT	WT	WT											WT
20 -DS010731											PD	0.99	1.95	WT	WT	WT	G12V	WT	WT	WT
	21 -RV110964	PD	0.95	2.00	WT	WT	WT	WT	WT	WT											WT
22 -CC041133											PD	1.00	1.90	WT	WT	WT	G12S	WT	WT	WT
	23 -GT050933	PD	1.20	2.10	WT	WT	WT	WT	WT	WT											WT
24 -RT161027											PD	1.16	1.98	WT	WT	WT	G12D	WT	WT	WT
	25 -CB280630	PD	0.90	1.75	WT	WT	WT	WT	WT	WT											WT
26 -FL020230											PD	0.96	1.85	WT	WT	WT	G12D	WT	WT	WT

27 -PC020849	PD	0.91	1.70	WT	WT	WT	WT	WT	WT	WT
											28 -CF141238	PD	1.02	2.00	WT	WT	WT	G13D	WT	WT	WT
29 -WB030428	PD	1.00	2.05	WT	WT	WT	WT	WT	WT	WT
											30 -GA240151	PD	1.03	2.00	WT	WT	WT	WT	WT	WT	WT
31 -IM100640	PD	1.18	2.10	WT	WT	WT	WT	WT	H1047R	E599V

^aFISH and mutation analysis measurements are uncertain for technical reasons (see methods); WT, wild type; PR, partial response; SD, stable disease condition; PD, advanced disease condition; UPN, unique patient number; n.e., not evaluated.^ooIncreased EGFR gene copy number was demonstrated to occur in both primary colorectal tumors before moAb treatment and in liver metastases at the time of disease progression after moAb treatment.

Claims (23)

1. A method for detecting and analyzing in vitro whether a patient has a cancer that overexpresses EGF receptor (EGFR) and that can respond positively to the administration of an anti-EGFR antibody or immunologically effective fragment thereof, comprising determining in vitro the EGFR gene copy number of a sample of tumor cells obtained from said patient, and selecting said patient for administration of said anti-EGFR antibody if said patient's tumor cells exhibit an amplified EGFR gene copy number.

2. The method of claim 1, wherein the copy number of the EGFR gene is measured as a ratio of the number of EGFR genes per cell nucleus.

3. The method of claim 2, wherein the ratio has a value between 4.0 and 8.2.

4. The method of claim 2 or claim 3, wherein the ratio has a value between 5.7 and 7.1.

5. The method of claim 1, wherein the copy number of the EGFR gene is measured as a ratio of EGFR gene basis factors per CEP 7.

6. The method of claim 5, wherein the ratio is > 2.

7. The method according to any of claims 1 to 6, wherein the EGFR gene copy number is measured by FISH analysis (fluorescence in situ hybridization).

8. The method according to any of claims 1-7, wherein the amplified EGFR gene copy number is specific for the tumor.

9. The method according to any of claims 1 to 7, wherein the amplified EGFR gene copy number is specific for the individual cancer tissue spectrum of the patient.

10. The method of claim 9, wherein the individual's cancer tissue profile further has a molecular alteration.

11. The method of claim 10, wherein the molecular alteration is a point mutation within the EGFR gene.

12. The method according to any one of claims 1 to 11, wherein the anti-EGFR antibody is selected from the group consisting of cetuximab (mAb c225), matuzumab (mAb h425) and parlimumab (mAb ABX) or specific murine, chimeric or humanized forms thereof.

13. The method according to any one of claims 1-12, wherein the cancer is colorectal cancer (CRC), lung cancer, head and neck cancer, and breast cancer.

14. Use of an anti-EGFR antibody, or immunologically effective fragment thereof, in the manufacture of a medicament for treating cancer in a patient, wherein the cancer overexpresses EGFR and exhibits amplified EGFR gene copy number.

15. The use of claim 14, wherein the EGFR gene copy number is measured as a ratio of EGFR genes per cell nucleus and the value of the ratio is between 4.0 and 8.2.

16. Use according to claim 15, wherein the value of the ratio is between 5.7 and 7.1.

17. The use according to any of claims 14-16, wherein the cancer treatment is more effective than treatment with the same antibody at the same dose in a cancer patient whose cancer cells do not exhibit an amplified copy number of EGFR.

18. The use according to any of claims 14-17, wherein the amplified EGFR gene copy number is specific for the tumor.

19. Use according to any of claims 14 to 18, wherein the amplified EGFR gene copy number is specific for the individual cancer tissue profile of the patient.

20. The use of claim 19, wherein the individual has a tissue spectrum of cancer with genetic mutations.

21. The use of any one of claims 14-20, wherein the EGFR-expressing tumor is colorectal cancer (CRC), lung cancer, breast cancer, or head and neck cancer.

22. Use according to any one of claims 14 to 21, wherein the anti-EGFR antibody is selected from the group consisting of cetuximab (mAb c225), matuzumab (mAb h425) and parlimumab (mAb ABX) or specific murine, chimeric or humanized forms thereof.

23. A method for in vitro detection and measurement of EGFR gene copy number in EGFR overexpressing tumor tissue by determining the response of cancer patients to administration of anti-EGFR antibodies using Fluorescence In Situ Hybridization (FISH) in an assay.