WO2006037994A2 - Methode - Google Patents

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Publication number
WO2006037994A2
WO2006037994A2 PCT/GB2005/003814 GB2005003814W WO2006037994A2 WO 2006037994 A2 WO2006037994 A2 WO 2006037994A2 GB 2005003814 W GB2005003814 W GB 2005003814W WO 2006037994 A2 WO2006037994 A2 WO 2006037994A2
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WO
WIPO (PCT)
Prior art keywords
seq
egfr
patient
following positions
erbb receptor
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PCT/GB2005/003814
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English (en)
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WO2006037994A3 (fr
Inventor
Gillian Ellison
Ruth Eleanor March
Alan Wookey
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Astrazeneca Ab
Astrazeneca Uk Limited
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Priority claimed from GB0422158A external-priority patent/GB0422158D0/en
Priority claimed from GB0508493A external-priority patent/GB0508493D0/en
Application filed by Astrazeneca Ab, Astrazeneca Uk Limited filed Critical Astrazeneca Ab
Publication of WO2006037994A2 publication Critical patent/WO2006037994A2/fr
Publication of WO2006037994A3 publication Critical patent/WO2006037994A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism

Definitions

  • tyrosine kinases A number of transmembrane receptors contain domains with tyrosine kinase activity and are classified as receptor tyrosine kinases (RTKs).
  • RTKs receptor tyrosine kinases
  • 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.).
  • 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.
  • 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.
  • the compound N-(3-chloro-4- fluorophenyl)-7-methoxy-6-(3-mo ⁇ holinopropoxy)quinazolin-4-amine is a potent EGFR tyrosine kinase inhibitor.
  • This compound is also known as IressaTM (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 IressaTM.
  • IressaTM 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.). IressaTM 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.
  • EGFR-TK epidermal growth factor receptor-tyrosine kinase
  • IressaTM anti-cancer therapeutic agents
  • An obvious candidate marker of response to IressaTM has been EGFR expression level.
  • IressaTM inhibition of growth of some cancer-derived cell lines and tumour xenografts is not well correlated with the level of expression of EGFR.
  • studies alongside the Ideal trials demonstrated that EGFR protein expression as measured by IHC was not an accurate predictor of response to IressaTM (Bailey et al.).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method comprises determining the sequence of
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method as described hereinabove may be used to assess the pharmacogenetics of an erbB receptor drug.
  • 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.
  • 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.
  • 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
  • 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
  • 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.
  • an erbB receptor drug is an EGFR drug, preferably an EGFR inhibitor, and most preferably an EGFR tyrosine kinase inhibitor.
  • 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.
  • the erbB receptor drug is selected from any one of: i) an EGFR drug; ii) an EGFR inhibitor; iii) an EGFR tyrosine kinase inhibitor;
  • 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 (ZactimaTM) or any of the compounds as disclosed hi WO2004/006846 or WO2003/082290.
  • 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.
  • cetuximab Erbitux, C225
  • matuzumab EMD-72000
  • panitumumab ABX-EGF/ rHuMAb-EGFR
  • MRl-I MRl-I
  • IMC-11F8 EGFRLl 1
  • 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.
  • 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.
  • 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.
  • non-solid tumours such as leukaemia, multiple myeloma or lymphoma
  • solid tumours for example bile duct, bone, bladder, brain/CNS,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a method for the detection of a mutation in mRNA encoded by a mutant EGFR gene in another aspect, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a tumour sample is a tumour tissue sample.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 IressaTM, or those drugs which are active against EGFR and other erbB receptors, for example ZD6474.
  • 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.
  • tumour sample could include for example, tumour section or slide, fresh or frozen tissue, fine needle aspirate, circulating tumour cells or bronchial lavage material.
  • 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 45 0 C before adding chloroform (lOO ⁇ L). 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 (50 ⁇ L).
  • PCR was performed on 5 ⁇ l of 1:5 and 1:10 dilutions of the extracted genomic DNA. A total reaction volume of 50 ⁇ l 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, 400 ⁇ M dNTPs and 0.3 ⁇ M of each primer. Cycle conditions were as follows: 95°C for 10 minutes followed by 13 cycles of 94 0 C for 20 second?.. 61 0 C for 1 minute (dropping 0.5°C/cycle), 72 0 C for 1 minute). Standard cycling conditions were then carried out for a further 29 cycles at an annealing temperature of 54 0 C.
  • PCR products (10 ⁇ l) were treated with ExoSAP-IT (1 ⁇ l, 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 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 IressaTM (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.
  • PR partial response Note on Table 1 : Counts 27 and 32 were treated with IressaTM 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 IressaTM.
  • Detection of a mutation in the EGFR gene in a tumour sample can be used to select patients for treatment with IressaTM or other inhibitors of the EGFR tyrosine kinase, either as monotherapy or in combination therapy.

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Abstract

L'invention concerne une méthode permettant de prédire le degré de probabilité selon lequel un patient candidat à un traitement par médicament à récepteur erbB répondra audit traitement. Cette méthode consiste à déterminer la séquence du facteur EGFR (facteur de croissance épidermique) dans un échantillon de tumeur prélevé sur le patient afin de prédire un accroissement de la probabilité d'une réponse au médicament récepteur de EGFR. En particulier, l'invention porte sur la détermination de la séquence d'EGFR à l'une quelconque des positions suivantes telles que définies dans SEQ ID NO: 1: position 2308 correspondant à A; position 2348 correspondant à T; position 2588 correspondant à A; ou position 2689 correspondant à A; ou à l'une quelconque des positions suivantes telles que définies dans SEQ ID NO: 2: position 770 correspondant à l'aspargine; 783 à l'isoleucine; position 863 à l'acide aspartique; ou position 897 à l'isoleucine.
PCT/GB2005/003814 2004-10-06 2005-10-04 Methode WO2006037994A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
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WO2006103421A2 (fr) * 2005-04-01 2006-10-05 Astrazeneca Ab Procede
CN106755297A (zh) * 2016-11-15 2017-05-31 上海派森诺医学检验所有限公司 一组基于arms荧光定量pcr检测egfr基因t790m突变的引物组及其制备方法

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WO2005094357A2 (fr) * 2004-03-31 2005-10-13 The General Hospital Corporation Procede permettant de determiner la reponse d'un cancer a des traitements cibles par le recepteur du facteur de croissance epidermique

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006103421A2 (fr) * 2005-04-01 2006-10-05 Astrazeneca Ab Procede
WO2006103421A3 (fr) * 2005-04-01 2007-03-22 Astrazeneca Ab Procede
CN106755297A (zh) * 2016-11-15 2017-05-31 上海派森诺医学检验所有限公司 一组基于arms荧光定量pcr检测egfr基因t790m突变的引物组及其制备方法

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