WO2006037994A2 - Egfr mutations altering the response to erbb receptor drugs - Google Patents

Abstract

The invention relates to a method for predicting the likelihood that a patient who is a candidate for treatment with an erbB receptor drug will respond to said treatment, comprising determining the sequence of EGFR in a tumour sample from the patient, whereby to predict an increased likelihood of response to the erbB receptor drug. In particular, determining the sequence of EGFR at any of the following positions as defined in SEQ ID NO: 1: position 2308 is A; position 2348 is T; position 2588 is A; or position 2689 is A or at any of the following positions as defined in SEQ ID NO: 2: position 770 is asparagine; 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine.

Description

METHOD

The phosphorylation of proteins on tyrosine residues is a key element of signal transduction within cells. Enzymes capable of catalysing such reactions are termed tyrosine kinases. A number of transmembrane receptors contain domains with tyrosine kinase activity and are classified as receptor tyrosine kinases (RTKs). There are several members of this family of RTKs, class I of which includes the erbB family, e.g. epidermal growth factor receptor (EGFR), erbB2, erbB3 and erbB4. The EGFR tyrosine kinase domain is activated by binding of a variety of ligands to the external domain. Activation causes EGFR itself and a number of cellular substrates to become phosphorylated on tyrosine residues. These phosphorylation reactions are a major component of growth factor induced proliferation of cells.

The erbB family of receptor tyrosine kinases are known to be frequently involved in driving the proliferation and survival of tumour cells (reviewed in Olayioye et al.). One mechanism by which this can occur is over expression of the receptor at the protein level, for example as a result of gene amplification. This has been observed in many common human cancers (reviewed in Klapper et al.) such as, non-small cell lung cancers (NSCLCs) including adenocarcinomas (Cerny et al.; Reubi et al.; Rusch et al.; Brabender et al.) as well as other cancers of the lung (Hendler et al.). It is now several decades since the study of retroviral mediated cellular transformation began to revolutionize our understanding of malignant transformation. Transformation was shown to be dependent on oncogenes carried by viruses and these were shown to have mammalian cellular counterparts, proto-oncogenes. In 1984, EGFR was described as the mammalian counterpart of the retroviral oncogene, v-erbB (Downward et al). This, coupled to earlier observations describing a two component autocrine growth promoting mechanism in cancer cells consisting of EGF ligand and its receptor EGFR (Sporn & Todaro), strengthened the hypothesis that EGFR signalling is an important contributor to tumourigenesis. Subsequent reports continued to provide evidence that EGFR is an attractive target for therapeutic intervention in Cancer (see Yarden & Sliwkowski for review). EGFR is markedly overexpressed across a large variety of epithelial cancers (see Salomon et al.) and some immunohistochemical studies have demonstrated EGFR expression is associated with poor prognosis. In addition to overexpression, it is recognised that there is potential for deregulated EGFR signalling in tumours via a number of alternative mechanisms including i) EGFR mutations ii) increased ligand expression and enhanced autocrine loop and iii) heterodimerisation and cross talk with other erbB receptor family members.

In addition, a wealth of pre-clinical information suggests that the erbB family of receptor tyrosine kinases are involved in cellular transformation. In addition to this, a number of pre-clinical studies have demonstrated that antiproliferative effects can be induced by knocking out one or more erbB activities by small molecule inhibitors, dominant negatives or inhibitory antibodies (reviewed in Mendelsohn et al).

Thus it has been recognised that inhibitors of these receptor tyrosine kinases should be of value as a selective inhibitor of the proliferation of mammalian cancer cells (Yaish et al.; Kolibaba et al.; Al-Obeidi et al.; Mendelsohn et al.).

A number of small molecule inhibitors of erbB family of receptor tyrosine kinases are known, particularly inhibitors of EGF and erbB2 receptor tyrosine kinases. For example European Patent Application No. 0566226 and International Patent Applications WO 96/33980 and WO 97/30034 disclose that certain quinazoline derivatives which possess an anilino substituent at the 4-position possess EGFR tyrosine kinase inhibitory activity and are inhibitors of the proliferation of cancer tissue including prostate cancer.

It has been disclosed (Woodbura et al.) that the compound N-(3-chloro-4- fluorophenyl)-7-methoxy-6-(3-moφholinopropoxy)quinazolin-4-amine is a potent EGFR tyrosine kinase inhibitor. This compound is also known as Iressa™ (registered trade mark), gefitinib (United States Adopted Name), by way of the code number ZD1839 and Chemical Abstracts Registry Number 184475-35-2. The compound is identified hereinafter as Iressa™.

Iressa™ was developed as an inhibitor of epidermal growth factor receptor-tyrosine kinase (EGFR-TK), which blocks signalling pathways responsible for driving proliferation, invasion, and survival of cancer cells (Wakeling et al.). Iressa™ has provided clinical validation of small molecule inhibitors of EGFR. Potent anti-tumour effects as well as rapid improvements in NSCLC-related symptoms and quality of life have been observed in clinical studies that enrolled patients with advanced NSCLC who did not respond to platinum-based chemotherapy. The Phase II 'IDEAL' trials demonstrated that single agent Iressa™ resulted in objective anti-tumour activity, symptomatic improvement and limited toxicity in patients with advanced NSCLC and previously treated with cytotoxic chemotherapy (Fukuoka et al., Kris et al). Objective response rate (Complete Response + Partial Response) was 11.8 and 18.4% respectively in the Ideal 1 and Ideal 2 trials. Beyond objective responses, additional patients experienced stable disease and / or symptom improvement meaning that approximately 50% of patients overall benefit from Iressa™. The tumour response data has been the basis of initial regulatory approvals of Iressa™ in advanced NSCLC in several markets.

It is important to be able to understand the basis of response to anti-cancer therapeutic agents such as Iressa™ since this would allow clinicians to maximise the benefit/risk ratio for each patient, potentially via the development of diagnostic tests to identify patients most likely to benefit from Iressa™ treatment. An obvious candidate marker of response to Iressa™ has been EGFR expression level. However, Iressa™ inhibition of growth of some cancer-derived cell lines and tumour xenografts is not well correlated with the level of expression of EGFR. Furthermore, studies alongside the Ideal trials demonstrated that EGFR protein expression as measured by IHC was not an accurate predictor of response to Iressa™ (Bailey et al.). Although there are now several additional hypotheses based on genetics, genomics, proteomics, biochemical and other studies, there is still no pre-treatment predictive biomarker of Iressa™ response currentl\ available. Possibly the most significant recent breakthrough in understanding Iressa™ response has come from recent data (Lynch et al., Paez et al.) indicating that mutation in the EGFR kinase domain predicts Iressa™ hypersensitivity in NSCLC patients. Hypersensitivity is a vague term but in this field is generally understood to mean patients experiencing objective tumour responses (i.e. marked tumour regression, normally above 50%). As well as demonstrating that Iressa™ acts via the EGFR, this may provide a basis for venturing into other disease settings such as first line, adjuvant and possibly earlier cancer intervention with EGFR inhibitors in a targeted subpopulation in NSCLC patients and other types of cancers carrying the EGFR mutation.

Recent data supports the findings that mutations in the TK domain of EGFR are associated with sensitivity to Iressa™ or erlotinib (Tarceva, developed by Genentech, Inc., OSI Pharmaceuticals, Inc., and Roche) another ATP-competitive EGFR TBCI. It was reported that 81% of tumours from individuals who experienced partial responses or marked clinical improvement while taking Iressa™ or Tarceva, contain mutations in the EGFR TK domain. Conversely, none of the patients whom were unresponsive to treatment had such mutations (Pao et al.).

The differential response of patients to chemotherapy treatments indicates that there is a need to find methods of predicting which treatment regimes best suit a particular patient.

There is an increasing body of evidence that suggests that patients' responses to numerous drugs may be related to a patients' genetic, genomic, proteomic and/or biochemical profile and that determination of the genetic factors that influence, for example, response to a particular drug could be used to provide a patient with a personalised treatment regime. Such personalised treatment regimes offer the potential to maximise therapeutic benefit to the patient, whilst minimising, for example adverse events that may be associated with alternative and less effective treatment regimes.

Therefore there is a need for methods that can predict a patients' response to a drug based on the results of a test that indicates whether the patient is likely to respond to treatment or to be resistant to treatment.

It has been found that the sensitivity of certain cancers to chemotherapeutic agents can be predicted from a patients' genetic, genomic, proteomic and/or biochemical profile. Hence, determination of the genetic factors that influence, for example, response to a particular drug can be used to assess the suitability of a patient for treatment with such drugs.

The present invention permits the improved selection of a patient, who is a candidate for treatment with an erbB receptor drug, in order to predict an increased likelihood of response to the erbB receptor drug.

All the mutations in EGFR described herein have been identified in the tyrosine kinase domain. Without wishing to be bound by theoretical considerations it is considered likely that the mutations are likely to affect the activity of EGFR, e.g. ATP-binding and kinase activity. Mutations previously identified in EGFR include small deletions and point mutations, and are recognised to alter downstream signalling, constitutively activate the receptor, impair receptor downregulation, induce distinct patterns of phosphotyrosine proteins and/or abrogate antiapoptotic mechanisms. Putatively these effects are mediated by repositioning critical residues surrounding the ATP-binding cleft of the tyrosine kinase domain, thereby stabilising interactions with ATP and any competitive inhibitors of EGFR (Kobayashi et al., Pao and Miller). To illustrate, the mutation described herein at position 863 is immediately adjacent to the activation loop of EGFR and is likely to affect the regulation of activation.

According to one aspect of the invention there is provided a method for predicting the likelihood that a patient who is a candidate for treatment with an erbB receptor drug will respond to said treatment, comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2142 is not G; position 2155 is not G; position 2308 is not G; position 2348 is not C; position 2364 is not C; position 2588 is not G; position 2689 is not G; or position 2877 is not A. Preferably the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2308 is not G; position 2348 is not C; position 2588 is not G; or position 2689 is not G.

According to another aspect of the invention there is provided a method for predicting the likelihood that a patient who is a candidate for treatment wrfr an erbB receptor drug will respond to said treatment, comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 719 is not glycine; position 770 is not aspartic acid; position 783 is not threonine; position 863 is not glycine; or position 897 is not valine. Preferably the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 770 is not aspartic acid; position 783 is not threonine; position 863 is not glycine; or position 897 is not valine.

In a preferred embodiment, the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2142 is A; position 2155 is A; position 2308 is A; position 2348 is T; position 2364 is T; position 2588 is A; position 2689 is A; or position 2877 is G. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 719 is serine; position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine. In a preferred embodiment, the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2308 is A; position 2348 is T; position 2588 is A; or position 2689 is A. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine.

According to another aspect, the method comprises predicting the likelihood that a patient who is a candidate for treatment with an erbB receptor drug will respond to said treatment, comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO:1: position 2308 is A; position 2348 is T; position 2588 is A; or position 2689 is A, or at any one of the following positions as defined in SEQ ID NO:2: position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine, whereby to predict an increased likelihood of response to the erbB receptor drug.

According to another aspect, the method comprises predicting the likelihood that a patient who is a candidate for treatment with an erbB receptor drug will respond to said treatment, comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO:1: position 2348 is T; position 2588 is A; or position 2689 is A, or at any one of the following positions as defined in SEQ ID NO:2: position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine.

In a preferred embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2348 is T; position 2588 is A; or position 2689 is A, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 783 is isoleucine; position 863 is aspartic acid; position 897 is isoleucine, whereby to predict an increased likelihood of response to the erbB receptor drug.

According to another aspect the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2308 is A; or position 2588 is A. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 770 is asparagine; or position 863 is aspartic acid. In a preferred embodiment the method comprises determining the sequence of

EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2308 is A; or position 2588 is A, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 770 is asparagine; or position 863 is aspartic acid, whereby to predict an increased likelihood of response to the erbB receptor drug.

According to another aspect the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2348 is T; or position 2588 is A. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 783 is isoleucine; or position 863 is aspartic acid.

According to another aspect the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2348 is T; or position 2588 is A, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 783 is isoleucine; or position 863 is aspartic acid, whereby to predict an increased likelihood of response to the erbB receptor drug.

According to another aspect of the invention there is provided a method as described herein comprising determining the sequence of EGFR in a tumour sample from the patient at any two of positions: i) 2308 as defined in SEQ ID NO: 1 or 770 as defined in SEQ ID NO:2; ii) 2348 as defined in SEQ ID NO: 1 or 783 as defined in SEQ ID NO:2; iii) 2588 as defined in SEQ ID NO: 1 or 863 as defined in SEQ ID NO:2; or iv) 2689 as defined in SEQ ID NO:1 or 897 as defined in SEQ ID NO:2.

According to another aspect of the invention there is provided a method as described herein comprising determining the sequence of EGFR in a tumour sample from the patient at any three of positions i) 2308 as defined in SEQ ID NO: 1 or 770 as defined in SEQ ID NO:2; ii) 2348 as defined in SEQ ID NO:1 or 783 as defined in SEQ ID NO:2; iii) 2588 as defined in SEQ ID NO: 1 or 863 as defined in SEQ ID NO:2; or iv) 2689 as defined in SEQ ID NO:1 or 897 as defined in SEQ ID NO:2.

According to another aspect of the invention there is provided a method as described herein comprising determining the sequence of EGFR in a tumour sample from the patient at any four of positions i) 2308 as defined in SEQ ID NO: 1 or 770 as defined in SEQ ID NO:2; v) 2348 as defined in SEQ ID NO:1 or 783 as defined in SEQ ID NO:2; vi) 2588 as defined in SEQ ID NO: 1 or 863 as defined in SEQ ID NO:2; or vii) 2689 as defined in SEQ ID NO:1 or 897 as defined in SEQ ID NO:2.

In a specific embodiment, the method as described hereinabove may be used to assess the pharmacogenetics of an erbB receptor drug.

In another aspect of the invention an erbB receptor drug can be used in preparation of a medicament for treating a disease in a human determined as having a mutation in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: 2142, 2155, 2308, 2348, 2364, 2588, 2689 or 2877, or in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: 719, 770, 783, 863 or 897.

Preferably an erbB receptor drug can be used in preparation of a medicament for treating a disease in a human determined as having a mutation in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : 2308, 2348, 2588 or 2689 or at any one of the following positions as defined in SEQ ID NO: 2: 770, 783, 863 or 897.

According to another aspect of the invention there is provided a method of treating a human in need of treatment with an erbB receptor drug in which the method comprises detection of a mutation, comprising: (i) determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: positions 2142, 2155, 2308, 2348, 2364, 2588, 2689 or 2877; or

(ii) determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: positions 719, 770, 783, 863 or 897; and administering an effective amount of the drug.

Preferably the method of treating a human in need of treatment with an erbB receptor drug in which the method comprises detection of a mutation, comprises: (i) determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: positions 2308, 2348, 2588 or 2689; or

(ii) determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: positions 770, 783, 863 or 897; and administering an effective amount of the drug.

In one embodiment an erbB receptor drug or any anti-cancer drug, for example chemotherapy or cytotoxic therapy, e.g. taxol or platinum-based therapy can be used in preparation of a medicament for treating a disease in a human determined as having a mutation in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: 2142, 2155, 2308, 2348, 2364, 2588, 2689 or 2877, or in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO:2: 719, 770, 783, 863 or 897. In a preferred embodiment an erbB receptor drug is an EGFR drug, preferably an EGFR inhibitor, and most preferably an EGFR tyrosine kinase inhibitor. In one embodiment an erbB receptor drug or any anti-cancer drug, for example chemotherapy or cytotoxic therapy, e.g. taxol or platinum-based therapy can be used in preparation of a medicament for treating a disease in a human determined as having a mutation in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : 2308, 2348, 2588 or 2689 in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: 770, 783, 863 or 897.

In a preferred embodiment the erbB receptor drug is selected from any one of: i) an EGFR drug; ii) an EGFR inhibitor; iii) an EGFR tyrosine kinase inhibitor;

In a more preferred embodiment the EGFR tyrosine kinase inhibitor is selected from gefitinib, erlotinib (Tarceva, OSI-774, CP-358774), PKI-166, EKB-569, HKI-272 (WAY-177820), lapatinib (GW2016, GW-572016, GSK572016), canertinib (CI-1033, PDl 83805), AEE788, XL647, BMS 5599626, ZD6474 (Zactima™) or any of the compounds as disclosed hi WO2004/006846 or WO2003/082290. In an alternative embodiment the EGFR inhibitor is selected from an anti-EGFR antibody such as cetuximab (Erbitux, C225), matuzumab (EMD-72000), panitumumab (ABX-EGF/ rHuMAb-EGFR), MRl-I, IMC-11F8 or EGFRLl 1. We contemplate that erbB receptor drugs may be used as monotherapy or in combination with other drugs.

In a further preferred embodiment the EGFR tyrosine kinase inhibitor is selected from gefitinib, erlotinib or ZD6474. In a most preferred embodiment the EGFR tyrosine kinase inhibitor is gefitinib or ZD6474, especially gefitinib.

In a preferred embodiment the present invention is particularly suitable for use in predicting the response to the erbB receptor drug as described hereinbefore, in patients with a tumour which is dependent alone, or hi part, on an EGF tyrosine kinase receptor. Such tumours include, for example, non-solid tumours such as leukaemia, multiple myeloma or lymphoma, and also solid tumours, for example bile duct, bone, bladder, brain/CNS, glioblastoma, breast, colorectal, cervical, endometrial, gastric, head and neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural/peritoneal membranes, prostate, renal, skin, testicular, thyroid, uterine and vulval tumours. In a more preferred embodiment the present invention is particularly suitable for use in predicting the response to the erbB receptor drug as described hereinbefore in patients with head and neck, colorectal and breast tumours. In an especially preferred embodiment the present invention is particularly suitable in predicting the response to the erbB receptor drug in those patients with NSCLC, more particularly advanced NSCLC including advanced adenocarcinoma. The present invention offers considerable advantages in the treatment of tumours such as NSCLC, especially advanced NSCLC by identifying "individual cancer profiles" of NSCLC and so determining which tumours would respond to gefitinib.

The present invention is particularly useful in the treatment of patients with advanced NSCLC who have failed previous chemotherapy, such as platinum-based chemotherapy. The present invention is also particularly useful in the treatment of patients with locally advanced (stage IIIB) or metastasized (stage IV) NSCLC who have received previous chemotherapy, such as platinum-based chemotherapy.

The present invention is also useful in adjuvant, or as a first-line, therapy. In another aspect of the invention there is provided a method as described hereinabove wherein the method for detection of a nucleic acid mutation is selected from amplification refractory mutation system and restriction fragment length polymorphism. According to a further aspect of the invention there is provided a primer or an oligonucleotide probe capable of detecting a mutation in the EGFR gene in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : 2142, 2155, 2308, 2348, 2364, 2588, 2689 or 2877. Preferably the primer or oligonucleotide probe is capable of detecting a mutation in the EGFR gene in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : 2308, 2348, 2588 or 2689. Details of these and other general molecular biology techniques can be found in Current Protocols in Molecular Biology Volumes 1-3, edited by F M

Asubel, R Brent and R E Kingston; published by John Wiley, 1998 and Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

In another aspect of the invention there is provided a method as described hereinabove wherein the method for detection of a nucleic acid mutation comprises determining the sequence of cDNA generated by reverse transcription of EGFR mRNA extracted from archival tumour sections or other clinical material. Extraction of RNA from formalin fixed tissue has been described (O Bock et al.), procedures for extraction of RNA from non fixed tissues, and protocols for generation of cDNA by reverse transcription, PCR amplification and sequencing are described in Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. In another aspect of the invention there is provided a method as described hereinabove wherein the method for detection of a nucleic acid mutation comprises amplification of individual exons of the EGFR gene, annealing of individual exons followed by digestion with CeI I and analysis by dHPLC (denaturing High Peformance Liquid Chromatography). This technique is known as WAVE analysis. Details of these techniques may be found at www.transgenomic.com.

In another aspect, the invention provides a mutant human EGFR polynucleotide comprising any one of the following nucleic acid bases at the following positions as defined in SEQ ID NO: 1 : an A at position 2142; an A at position 2155; a T at position 2348; an A at position 2308; a T at position 2364; an A at position 2689; an A at position 2588; or a G at position 2877, or a fragment thereof comprising at least 20 nucleic acid bases provided that the fragment comprises the mutation at position 2142, 2155, 2308, 2348, 2364, 2588, 2689 or 2877. Preferably, the invention provides a mutant human EGFR polynucleotide comprising any one of the following nucleic acid bases at the following positions as defined in SEQ ID NO: 1 : an A at position 2308; a T at position 2348; an A at position 2588; or an A at position 2689, or a fragment thereof comprising at least 20 nucleic acid bases provided that the fragment comprises the mutation at at least one of positions 2308, 2348, 2588 or 2689.

In a further aspect the invention provides a mutant human EGFR polypeptide comprising any one of the following amino acid residues at the following positions as defined in SEQ ID NO: 2: a serine at position 719; an asparagine at position 770; an isoleucine at position 783; an aspartic acid at position 863; or an isoleucine at position 897, or a fragment thereof comprising at least 10 amino acid residues provided that the fragment comprises the allelic variant at position 719, 770, 783, 863 or 897. Preferably, the invention provides a mutant human EGFR polypeptide comprising any one of the following amino acid residues at the following positions as defined in SEQ ID NO: 2: an asparagine at position 770; an isoleucine at position 783; an aspartic acid at position 863; or an isoleucine at position 897, or a fragment thereof comprising at least 10 amino acid residues provided that the fragment comprises the mutation at at least one of positions 770, 783, 863 or 897.

In another aspect, there is provided a method for the detection of a mutation in mRNA encoded by a mutant EGFR gene. In another aspect of the invention there is provided a method as described herein wherein the method for detection of an amino acid mutation is selected from, for example, an immunohistochemistry-based assay or application of an alternative proteomics methodology. According to another aspect the invention comprises an antibody specific for a mutant human EGFR polypeptide as defined hereinabove.

A further aspect of the invention provides a diagnostic kit, comprising an antibody specific for a mutant human EGFR polypeptide as defined hereinabove, or a primer or oligonucleotide probe capable of detecting a mutation in the EGFR gene as defined hereinabove, for use in a method of predicting the responsiveness of a patient or patient population with a tumour, to treatment with chemotherapeutic agents, especially erbB receptor drugs.

In a further aspect a panel of cell lines expressing either the wild type or a mutant EGFR could be used in screening programmes to identify novel EGFR inhibitors with specificity for the mutant EGFR phenotype or novel inhibitors with activity against the phenotype associated with the wild type receptor. The availability of a panel of cell lines expressing mutant EGFRs will assist in the definition of the signaling pathways activated through the EGFR and may lead to the identification of additional targets for therapeutic intervention. In another aspect the invention provides a method of preparing a personalised genomics profile for a patient comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2142; position 2155; position 2308; position 2348; position 2364; position 2588; position 2689; or position 2877, and creating a report summarising the data obtained by said analysis. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 719 is serine; position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine, and creating a report summarising the data obtained by said analysis. Preferably the method comprises of preparing a personalised genomics profile for a patient comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2308; position 2348; position 2588; or position 2689, and creating a report summarising the data obtained by said analysis. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 2: position 770; position 783; position 863; or position 897, and creating a report summarizing the data obtained by said analysis.

In another aspect of the invention there is provided a method of selecting a patient with a tumour for treatment with an erbB receptor drug comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2142; position 2155; position 2308; position 2348; position 2364; position 2588; position 2689; or position 2877, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample, as defined by the positions in SEQ ID NO: 2 at any one of: position 719 is serine; position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine.

In another aspect the invention provides a method of selecting a patient with a tumour for treatment with an erbB receptor drug comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2308; position 2348; position 2588; or position 2689, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample, as defined by the positions in SEQ ID NO: 2 at any one of: position 770; position 783; position 863; or position 897, whereby to predict an increased likelihood of response to the erbB receptor drug.

In another aspect there is provided a method of predicting the responsiveness of a patient, or patient population, with cancer to treatment with an erbB receptor drug, or for selecting patients, or patient populations, that will respond to an erbB receptor drug comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : position 2142; position 2155; position 2308; position 2348; position 2364; position 2588; position 2689; or position 2877, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample, as defined by the positions in SEQ ID NO: 2 at any one of: position 719 is serine; position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine. In another aspect there is provided a method of predicting the responsiveness of a patient, or patient population, with cancer to treatment with an erbB receptor drug, or for selecting patients, or patient populations, to an erbB receptor drug comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2308; position 2348; position 2588; or position 2689, whereby to predict an increased likelihood of response to the erbB receptor drug. In an alternative embodiment the method comprises determining the sequence of EGFR in a tumour sample, as defined by the positions in SEQ ID NO: 2 at any one of: position 770; position 783; position 863; or position 897, whereby to predict an increased likelihood of response to the erbB receptor drug. In another aspect there is provided a method as described hereinabove comprising determining the sequence of EGFR in a tumour sample from the patient at any two of, any three of or any four of positions 2308, 2348, 2588 or 2689, as defined in SEQ ID NO:1 or positions 770, 783, 863 or 897 as defined in SEQ ID NO:2.

In another aspect the tumour sample is any tumour tissue or any biological sample that contains a sample which originated from the tumour, for example bronchial lavage material or a blood sample containing a shed antigen. Preferably a tumour sample is a tumour tissue sample. In a preferred embodiment the biological sample would have been obtained using a minimally invasive technique to obtain a small sample of tumour, or suspected tumour, from which to determine the EGFR sequence. Such techniques include, for example tumour biopsy, such as transbronchial biopsy. The sequence of EGFR in transbronchial biopsy specimens whose size is about 1 mm may be determined for example using a suitable amplification procedure. In a preferred embodiment the biological sample comprises either a single sample, which may be tested for any of the mutations as, described hereinabove, or multiple samples, which may be tested for any of the mutations as, described hereinabove.

In a preferred embodiment the present invention includes administration of an erbB receptor drug to a mammal selected according the methods described hereinabove. According to another aspect of the invention there is provided a method of using the results of the methods described above in determining an appropriate dosage of an erbB receptor drug.

In another aspect there is provided a method of treating a patient, or a patient population, having NSCLC identified according to the method as described herein comprising administering to said patients an erbB receptor drug.

The term "erbB receptor drug" includes drugs acting upon the erbB family of receptor tyrosine kinases, which include EGFR, erbB2 (HER), erbB3 and erbB4 as described in the background to the invention above, including those drugs which are specific for EGFR, for example Iressa™, or those drugs which are active against EGFR and other erbB receptors, for example ZD6474.

Examples The invention is illustrated by the following non-limiting examples. General molecular biology techniques are described in "Current Protocols in Molecular Biology Volumes 1-3, edited by F M Asubel, R Brent and R E Kingston; published by John Wiley, 1998 and Sambrook, J. and Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

Example 1 -Identification of mutations in NSCLC tumour sections

The mutations have been detected in tumour sections, taken from patients at time of diagnosis or surgery. The sections have varied in thickness from 5-20 microns. Regions of the section containing tumour were identified by histopathology of a master slide and tumour material was recovered from the relevant area of adjacent slides cut from the same tumour sample.

Other types of tumour sample could include for example, tumour section or slide, fresh or frozen tissue, fine needle aspirate, circulating tumour cells or bronchial lavage material.

Example 2 - DNA extraction from slide section

Volumes are given for extraction of one section. Region of tumour identified by histopathology on one section and isolated from adjacent sections by scraping relevant area from slide into an eppendorf tube. The material from a 20 micron section was resuspended in 100 μL 0.5% Tween-20 (Sigma Aldrich), heated to 9O⁰C for 10 minutes then cooled to 55⁰C. Proteinase K (2 μL, 10mg/m L) was added to the suspension, the solution was mixed and incubated at 55⁰C for 3 hours with occasional mixing. Chelex-100 (100 μL, 5% in Tris EDTA) was added and the suspension was incubated at 99⁰C for 10 minutes. The extracted DNA was recovered by centrifugation at 10500 x g for 15 minutes, the solution below the wax layer which formed was transferred to a clean tube. The solution was heated to 45⁰C before adding chloroform (lOOμL). The suspension was mixed before centrifugation at 10500 x g, DNA was then recovered from the upper aqueous layer by ethanol precipitation. The DNA pellet was rinsed in 70% ethanol, recovered by centrifugation, air dried and dissolved in water (50μL).

Example 3 - PCR amplification ofexons in EGFR tyrosine kinase domain Primers

Tags

PCR was performed on 5μl of 1:5 and 1:10 dilutions of the extracted genomic DNA. A total reaction volume of 50μl was used for each PCR. 3.75 Units of Amplitaq gold DNA polymerase was used in each reaction with final concentrations of 2mM magnesium chloride, 400μM dNTPs and 0.3μM of each primer. Cycle conditions were as follows: 95°C for 10 minutes followed by 13 cycles of 94⁰C for 20 second?.. 61⁰C for 1 minute (dropping 0.5°C/cycle), 72⁰C for 1 minute). Standard cycling conditions were then carried out for a further 29 cycles at an annealing temperature of 54⁰C. PCR products (10 μl) were treated with ExoSAP-IT (1 μl, 1:2 dilution) to remove unincorporated oligonucleotides and nucleotides. Other groups have needed to perform PCR in two stages and have only been able to detect mutations by sequencing secondary amplification products (Lynch et al.). Example 4 — DNA sequencing

Sequencing reactions were performed using ABI Big Dye Terminator chemistry (at a 1:16 dilution) and then run on an ABI 3730 sequencer as described in the Applied Biosystems manual. Sequence chromatograms were analysed using Mutation Surveyor software.

Example 5 — Mutation detection

Mutations were detected using WAVE analysis. AU exons coding for the kinase domain of EGFR (exons 18-24) were amplified using the primers and PCR conditions described above. Prior to WAVE analysis, the products were subjected to a heteroduplex step (the sample was heated to 95⁰C for 5 minutes and allowed to slowly cool to 25⁰C on a

PCR block). Heteroduplexed samples were digested using Cell enzyme as described on the Transgenomic website

(<http://www.transgenomic.com/asp/ShowFile.asp?file=602084.pdf&sid=679298689&cyi d=l>). Samples were run on the WAVE under non-denaturing conditions using Navigator software and a fluorescent detector. Samples were analysed by eye. Any samples containing mutations were confirmed by fragment collection followed by sequencing.

Example 6 — Amplification Refractory Mutation System An Amplification Refractory Mutation System assay (ARMS) is used to detect the presence of a nucleotide base change in the EGFR gene compared to a background of normal DNA. Each ARMS assay is specific for a given mutation e.g. designed to detect a change from G to A at position 2308, or C to T at position 2348, or G to A at position 2588 or G to A at position 2689. The assay is multiplexed with a second PCR reaction that detects the presence of DNA in the reaction, thereby indicating successful PCR. TaqMan technology is used to detect the PCR products of both reactions using TaqMan probes labelled with different fluorescent tags.

Example 7 — Analysis of patient samples Paraffin blocks of tumour material were obtained from 41 patients with NSCLC prior to treatment with Iressa™ (ZDl 839, gefitinib). Genomic DNA was isolated from tumour material and quantified as described. Exons 18-24 of the EGFR gene were amplified by PCR and sequenced. All products were analysed in the forward and reverse direction and were analysed separately by two individuals.

Analysis of the clinical response data showed that 50% of evaluable patients had shown an objective (complete or partial) response to Iressa™ treatment. All patients possessing a mutation in the tyrosine kinase domain of the EGFR gene showed an objective response. Table 1

PR = partial response Note on Table 1 : Counts 27 and 32 were treated with Iressa™ and count 34 was given a placebo. Without wishing to be bound by theoretical considerations it is considered likely the patient who exhibited an objective response to placebo is likely to respond to any therapy, including treatment with Iressa™.

Example 8 - Selection of patients for treatment

Detection of a mutation in the EGFR gene in a tumour sample can be used to select patients for treatment with Iressa™ or other inhibitors of the EGFR tyrosine kinase, either as monotherapy or in combination therapy.

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Claims

Claims

1. A method for predicting the likelihood that a patient who is a candidate for treatment with an erbB receptor drug will respond to said treatment, comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO:1: position 2142 is not G; position 2155 is not G; position 2308 is not G; position 2348 is not C; position 2364 is not C; position 2588 is not G; position 2689 is not G; or position 2877 is not A, or at any one of the following positions as defined in SEQ ID NO:2: position 719 is not glycine; position 770 is not aspartic acid; position 783 is not threonine; position 863 is not glycine; or position 897 is not valine.

2. A method according to claim 1 comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2308 is not G; position 2348 is not C; position 2588 is not G; or position 2689 is not G, or at any one of the following positions as defined in SEQ ID NO:2: position 770 is not aspartic acid; position 783 is not threonine; position 863 is not glycine; or position 897 is not valine.

3. A method according to any of the preceding claims comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO:1: position 2308 is A; position 2348 is T; position 2588 is A; or position 2689 is A, or at any one of the following positions as defined in SEQ ID NO:2: position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine.

4. A method according to claim 3 comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2308 is A; position 2348 is T; position 2588 is A; or position 2689 is A, or at any one of the following positions as defined in SEQ ID NO:2: position 770 is asparagine; position 783 is isoleucine; position 863 is aspartic acid; or position 897 is isoleucine, whereby to predict an increased likelihood of response to the erbB receptor drug.

5. A method according to claim 4 comprising determining the sequence of EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: position 2348 is T; position 2588 is A; or position 2689 is A, or at any one of the following positions as defined in SEQ ID NO:2: position 783 is isoleucine; position 863 is aspartic acid; or position 897is isoleucine, whereby to predict an increased likelihood of response to the erbB receptor drug.

6. A method according to any of claims 1 to 4 comprising determining the sequence of EGFR in a tumour sample from the patient at positions: i. 2308 as defined in SEQ ID NO:1 or 770 as defined in SEQ ID

NO:2; ii. 2348 as defined in SEQ ID NO:1 or 783 as defined in SEQ ID

NO:2; iii. 2588 as defined in SEQ ID NO:1 or 863 as defined in SEQ ID NO:2; and iv. 2689 as defined in SEQ ID NO:1 or 897 as defined in SEQ ID

NO:2.

7. Use of a method according to any of the preceding claims to assess the pharmacogenetics of an erbB receptor drug.

8. Use of an erbB receptor drug in preparation of a medicament for treating a disease in a human determined as having a mutation in EGFR in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1: 2308, 2348, 2588 or 2689, or at any one of the following positions as defined in SEQ ID NO: 2: 770, 783, 863 or 897.

9. A method according to any one of claims 1-8 wherein the erbB receptor drug is selected from any one of: i. an EGFR inhibitor; ii. an EGFR tyrosine kinase inhibitor; iii. gefitinib, erlotinib or ZD6474.

10. A method according to claim 9 wherein the erbB receptor drug is gefitinib.

11. A method according to any one of the preceding claims wherein the method for detection of a nucleic acid mutation is selected from any one of amplification refractory mutation system, restriction fragment length polymorphism or WAVE analysis.

12. A primer or an oligonucleotide probe capable of detecting a mutation in the EGFR gene in a tumour sample from the patient at any one of the following positions as defined in SEQ ID NO: 1 : 2308, 2348, 2588, or 2689.

13. A mutant human EGFR polynucleotide comprising any one of the following at positions as defined in SEQ ID NO: 1:

2308 is A; 2348 is T;

2588 is A; or

2689 is A, or a fragment thereof, comprising at least 20 nucleic acid bases provided that the fragment comprises the mutation at at least one of positions 2308, 2348, 2588 or 2689.

14. A mutant human EGFR polypeptide comprising any one of the following at positions as defined in SEQ ID NO:2: an asparagine at position 770; an isoleucine at position 783; an aspartic acid at position 863; or an isoleucine at position 897, or a fragment thereof, comprising at least 10 amino acid residues provided that the fragment comprises the mutation at at least one of positions 770, 783, 863 or 897.

15. An antibody specific for a mutant human EGFR polypeptide as defined in claim 14.

16. A diagnostic kit comprising an antibody of claim 15 or a primer or oligonucleotide probe of claim 12.