WO2008088885A2 - Mutations de récepteur de facteur de croissance épidermique et procédés d'utilisation - Google Patents

Mutations de récepteur de facteur de croissance épidermique et procédés d'utilisation Download PDF

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WO2008088885A2
WO2008088885A2 PCT/US2008/000701 US2008000701W WO2008088885A2 WO 2008088885 A2 WO2008088885 A2 WO 2008088885A2 US 2008000701 W US2008000701 W US 2008000701W WO 2008088885 A2 WO2008088885 A2 WO 2008088885A2
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egfr
mutation
residue
patient
combination
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Brian Loggie
Jason Foster
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Creighton University
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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
    • 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
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • a key driver for cell growth is the epidermal growth factor (EGF) and the receptor for EGF ("EGFR") (Goustin et al, Cancer Res., 1986, 46:1015-1029; Aaronson, 1991, 254:1146-1153).
  • the EGFR is a transmembrane receptor with an extracellular ligand-binding domain, a helical transmembrane domain, and an intracellular tyrosine kinase domain (Wells A, Int'l. J. Biochem. Cell Biol.
  • EGF and other ligands bind the EGFR extracellular domain to activate cellular signaling pathways that lead to cell proliferation.
  • EGFR plays an important role in cellular proliferation as well as apoptosis, angiogenesis and metastatic spread, processes that are crucial to tumor progression (Salomon et al., Crit. Rev. Oncology/Haematology, 1995, 19:183-232; Wu et al., J. Clin. Invest., 1995, 95:1897-1905; Karnes et al., Gastroenterology, 1998, 1 14:930-939; Woodburn et al., Pharmacol.
  • Cancer Res., 2005, 11 :2300-2304) evaluated the response of patients with malignant mesothelioma to gefitinib. Although 97% of the patients had EGFR overexpression, gefitinib was not active in the treatment of malignant mesothelioma.
  • the present invention represents an advance in the ability to characterize cancers, including malignant mesotheliomas, such as, for example, malignant peritoneal mesothelioma.
  • malignant mesotheliomas such as, for example, malignant peritoneal mesothelioma.
  • the inventors have discovered that the presence of mutations in epidermal growth factor receptor (EGFR) expressed in a cancer cell can be used to formulate an accurate prognosis of cancer patients, and aid in determining what treatments may be beneficial a patient.
  • EGFR epidermal growth factor receptor
  • the present invention includes methods for characterizing cancer cells in a patient.
  • the methods typically include providing polynucleotides from a cancer cell from a patient, and detecting the presence or absence of a mutation of an EGFR coding region.
  • the mutation may be, and preferably is, present in exon 18, 19, 20, 21, 22, 23, or 24.
  • An EGFR coding region may include more than one mutation, and the mutations may be present in the same exon or different exons.
  • the mutation is one that results in an EGFR polypeptide having a different amino acid at residue 731, 734, 785, 797, 801, 831, 868, or a combination thereof when compared to SEQ ID NO:2.
  • the mutation may also be one that results in an EGFR polypeptide having a different amino acid at residue 858, as well as 731, 734, 785, 797, 801, 831, 868, or a combination thereof when compared to SEQ ID NO:2.
  • the present invention also includes methods for determining a prognosis in a subject having or suspected of having a cancer.
  • the prognosis may be for, for instance, resectability of a cancer, survival of a patient having a cancer, or progression free survival of a cancer.
  • the cancer may be a malignant mesothelioma, such as a malignant peritoneal mesothelioma.
  • the methods typically include providing polynucleotides from a cancer cell from the patient, and detecting the presence or absence of a mutation of an EGFR coding region.
  • the mutation may be, and preferably is, present in exon 18, 19, 20, 21 , 22, 23, or 24.
  • An EGFR coding region may include more than one mutation, and the mutations may be present in the same exon or different exons.
  • the mutation is one that results in an EGFR polypeptide having a different amino acid at residue 731, 734, 785, 797, 801, 831, 858, 868, or a combination thereof when compared to SEQ ID NO:2.
  • the presence of a mutation indicates a prognosis of increased resectability, and absence of a mutation indicates a prognosis of decreased resectability.
  • the presence of a mutation indicates a prognosis of increased survival of the patient, and absence of a mutation indicates a prognosis of decreased survival of the patient.
  • the presence of a mutation indicates a prognosis of increased progression free survival, and absence of a mutation indicates a prognosis of decreased progression free survival.
  • the present invention further provides methods for evaluating treatment options for a patient having a cancer.
  • the methods typically include providing polynucleotides from a cancer cell from a patient, and detecting the presence or absence of a mutation of an EGFR coding region.
  • the mutation may be, and preferably is, present in exon 18, 19, 20, 21, 22, 23, or 24.
  • An EGFR coding region may include more than one mutation, and the mutations may be present in the same exon or different exons.
  • the mutation is one that results in an EGFR polypeptide having a different amino acid at residue 731, 734, 785, 797, 801, 831, 868, or a combination thereof when compared to SEQ ID NO:2.
  • the mutation may also be one that results in an EGFR polypeptide having a different amino acid at residue 858, as well as 731, 734, 785, 797, 801, 831, 868, or a combination thereof when compared to SEQ ID NO:2.
  • treatment options include surgery, administration of an EGFR kinase inhibitor, or a combination thereof.
  • the methods typically include providing polynucleotides from a cancer cell from the patient, and detecting the presence or absence of a mutation of an EGFR coding region.
  • the mutation may be, and preferably is, present in exon 18, 19, 20, 21, 22, 23, or 24.
  • An EGFR coding region may include more than one mutation, and the mutations may be present in the same exon or different exons.
  • the mutation is one that results in an EGFR polypeptide having a different amino acid at residue 731, 734, 785, 797, 801 , 831, 858, 868, or a combination thereof when compared to SEQ ID NO:2.
  • a patient having the mutation is treated by resection of the malignant mesothelioma.
  • the methods may also include treating the patient by administration of an EGFR kinase inihibitor.
  • the malignant mesothelioma may be a malignant peritoneal mesothelioma or a malignant pleural mesothelioma.
  • the polynucleotides provided may be ribonucleotide or deoxynucleotide, such as genomic DNA or cDNA.
  • the detecting may be accomplished by using, for instance, a polymerase chain reaction (PCR) method, nucleotide sequence analysis, or a combination thereof.
  • the PCR method may be, but is not limited to, denaturing high-performance liquid chromatography.
  • the detecting may include detecting of portion of exon 18, exon 19, exon 20, exon 21, exon 22, exon 23, exon 24, or a combination thereof.
  • the EGFR polypeptide having the different amino acid may have increased tyrosine kinase activity compared to an EGFR polypeptide not having the mutation, and may have increased sensitivity to an EGFR kinase inhibitor compared to an EGFR polypeptide not having the mutation.
  • Preferred examples of different amino acids include a leucine at residue 731 , a glutamine at residue 734, an alanine at residue 785, a tyrosine at residue 797, a histidine at residue 801, a histidine or a leucine at residue 831, an arginine at residue 858, a glycine at residue 868, or a combination thereof.
  • the methods may also include obtaining a biological sample from a patient, where the biological sample includes the cancer cell.
  • Figure 1 Nucleotides sequence of an EGFR coding region.
  • the coding region is nucleotides 247-3879 and is SEQ ID NO:1.
  • the entire 5616 nucleotide sequence is SEQ ID NO:38.
  • Figure 2. Amino acid sequence of an EGFR polypeptide (SEQ ID NO:2) encoded by SEQ ID NO:1.
  • Figure 3 Alignment of the human wild- type EGFR sequence (bottom sequence, SEQ ID NO:3) with the Pfam model (top sequence, SEQ ID NO:4) of protein kinase domain.
  • Figure 4 Alignment of EGFR sequences of various organisms including cow, chimpanzee, chicken, human, mouse, rat, pig, zebrafish, dog, the CGl 0079-P A polypeptide of fruit fly, and the CG 10079-PB polypeptide of fruit fly using ClustalW Cow, SEQ ID NO:5; chimpanzee, SEQ ID NO:6; chicken , SEQ ID NO:7; human, SEQ ID NO:8; mouse, SEQ ID NO:9; rat, SEQ ID NO: 10; pig, SEQ ID NO:11 ; zebrafish, SEQ ID NO: 12; dog, SEQ ID NO: 13; CG10079-PA polypeptide, SEQ ID NO: 14; CGl 0079-PB polypeptide, SEQ ID NO:15.
  • the present invention includes methods for characterizing cells, preferably cancer cells.
  • the methods typically include providing polynucleotides from a cancer cell, and detecting the presence or absence of a mutation of an epidermal growth factor receptor (EGFR) coding region.
  • EGFR epidermal growth factor receptor
  • cancer cell used either in the singular or plural form, refers to a cell that has undergone a malignant transformation that makes it pathological to the host organism.
  • a feature of cancer cells is the tendency to grow in a manner that is uncontrollable by the host, but the pathology associated with a particular cancer cell may take any form.
  • Primary cancer cells for instance, cells obtained from near the site of malignant transformation
  • pathology techniques particularly histological examination.
  • a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • primary cells include, but are not limited to, cancer cells obtained from a subject having a cancer.
  • the subject can be a human patient, or an animal, such as a mouse or a rat, being used as an animal model to study a cancer.
  • the cancer can be those where a cancer cell has altered EGFR activity when compared to a normal cell. Examples of such cancers include those associated with mutations in an EGFR coding region, for example mutations that result in greater EGFR tyrosine kinase activity.
  • cancers include, but are not limited to, lung cancer (e.g., non-small cell lung cancer), head and neck cancer, and malignant mesothelioma (e.g., malignant peritoneal mesothelioma, malignant plural mesothelioma) (Rosell et al., Clin. Cancer Res., 2006, 12:7222-7231).
  • malignant mesothelioma e.g., malignant peritoneal mesothelioma, malignant plural mesothelioma
  • Other examples of cancers having altered EGFR activity include those associated with increased expression of EGFR.
  • examples of such cancers include, but are not limited to, breast cancer, bladder cancer, lung cancer, head and neck cancer, stomach cancer, and glioblastoma.
  • cancers include, but are not limited to, those classified as epithelial cancers, including, but not limited to, lung cancer (e.g., non-small cell lung cancer), ovarian cancer, breast cancer, brain cancer, colon cancer, and prostrate cancer. Further cancers include, but are not limited to, hepatocellular cancer, renal cancer, glioma, retina cancer, skin cancer, melanoma, pancreatic cancer, and genital-urinary cancer. In some aspects, the cancer is not a cancer of the upper aerodigestive tract, preferably a cancer of the tonsil.
  • Cancer cells may be obtained from a biological sample from a patient.
  • a biological sample refers to a sample of tissue or fluid isolated from a subject, including, but not limited to, saliva, blood, urine, stool, bone marrow, bile, spinal fluid, lymph fluid, ascites fluid, and tissue biopsies. Methods for obtaining a biological sample can depend on the type of cancer, and are known to a person skilled in the art and are routine.
  • obtaining a biological sample containing cancer cells from a patient having malignant peritoneal mesothelioma can be accomplished by, for example, radiographically guided biopsy via ultrasound or CT scan, laparoscopy, aspiration of ascites fluid, or by biopsy of a tumor during abdominal surgery.
  • Polynucleotides can be obtained from cells using routine methods known to a person skilled in the art (see, for example, Molecular Cloning: A Laboratory Manual (3-Volume Set) eds. Sambrook et al., Cold Spring Harbor Laboratory; 3rd edition (2001)).
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and single-stranded DNA and RNA, such as genomic DNA, cDNA, total RNA, and mRNA.
  • a polynucleotide may include nucleotide sequences having different functions, including for instance coding sequences such as exons, and non-coding sequences such as introns, regulatory sequences, and the like.
  • a polynucleotide can be obtained directly from a natural source, such as a cancer cell.
  • a polynucleotide can be linear or circular in topology.
  • Cells used as the source of the polynucleotides can be cells that have been recently removed from a patient or cells that have been subjected to conditions for storage, such as freezing or formalin fixing and paraffin embedding.
  • the polynucleotides obtained from a cell may be, for instance, genomic DNA, total RNA, or mRNA.
  • Genomic DNA may include all the DNA present in a cell (e.g., at least one copy of each coding region present in a cell), or may be a portion of the DNA present in a cell.
  • total RNA may include all the RNA present in a cell (e.g., at least one copy of each ribonucleotide sequence present in a cell, including, for instance, mRNA and precursor RN A), or may be a portion of the RN A present in a cell.
  • the polynucleotides used in the methods of the present invention may be, for instance, genomic DNA, total RNA, or mRNA.
  • the mRNA may be processed to yield cDNA, which may be used in the methods of the present invention.
  • coding region is a nucleotide sequence that encodes a polypeptide and, when placed under the control of appropriate regulatory sequences expresses the encoded polypeptide.
  • the boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end.
  • a coding region may include introns. For instance, a coding region present in a sample of genomic DNA will typically include introns, while a coding region present in a sample of mRNA or cDNA will not include introns.
  • an "EGFR coding region” refers to a coding region that encodes an EGFR polypeptide.
  • An example of a normal genomic EGFR coding region is disclosed at Genbank Accession number NT 033968.5, and an example of a normal mRNA EGFR coding region is depicted as a cDNA at Genbank Accession number NM_005228.3 ( Figure 1, nucleotides 247-3869 SEQ ID NO:1).
  • a “mutation” refers to a difference present in a nucleotide of an exon of the analyzed polynucleotide when compared to the nucleotide sequence of a normal human EGFR coding region, and the mutation of the coding region results in a change of the amino acid sequence of the EGFR polypeptide encoded by the coding region.
  • An EGFR coding region having such a mutation is referred to herein as a "mutant EGFR coding region.”
  • the difference between the two nucleotide sequences can be the result of a transition or transversion of one or more nucleotides, or the result of an insertion or deletion of one or more nucleotides.
  • the change in amino acid sequence of an EGFR polypeptide encoded by the mutated EGFR coding region can be a single amino acid, multiple amino acids, or the addition or deletion of amino acids when compared to a normal EGFR polypeptide.
  • polypeptide refers broadly to a polymer of two or more amino acids joined together by peptide bonds.
  • polypeptide also includes molecules which contain more than one polypeptide joined by a disulfide bond, or complexes of polypeptides that are joined together, covalently or noncovalently, as multimers (e.g., dimers, tetramers).
  • peptide, oligopeptide, and protein are all included within the definition of polypeptide and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the polypeptide is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
  • An example of a normal EGFR polypeptide is disclosed at Genbank accession number NP_005219.2 ( Figure 2, SEQ ID NO:2).
  • An EGFR polypeptide having an amino acid sequence that is different than a normal EGFR polypeptide is referred to herein as a "mutant EGFR polypeptide.”
  • a mutation of an EGFR coding region may result in the change of at least one amino acid of an EGFR polypeptide present in the cancer cell when compared to a normal EGFR polypeptide. More preferably, the mutation results in a change of an amino acid present in the tyrosine kinase domain of an EGFR polypeptide. Most preferably, the mutation results in a change of an amino acid present in the tyrosine kinase domain of an EGFR polypeptide that increases the tyrosine kinase activity of the EGFR polypeptide.
  • An EGFR coding region in a cancer cell may include more than one mutation.
  • the tyrosine kinase domain of an EGFR polypeptide is encoded by exons 18 through 24.
  • An example of a nucleotide sequence of these exons in a normal EGFR coding region is disclosed at nucleotides 2308-3192 of SEQ ID NO: 1
  • the amino acid sequence of the tyrosine kinase domain of a normal EGFR polypeptide is amino acids 704-1019 of SEQ ID NO:2, preferably, amino acids 712-968.
  • amino acid residues that can be different in a mutant EGFR polypeptide include, but are not limited to, 731 (typically a tryptophan in a normal EGFR polypeptide), 734 (typically a glutamic acid in a normal EGFR polypeptide), 785 (typically a threonine in a normal EGFR polypeptide), 797 (typically a cysteine in a normal EGFR polypeptide), 801 (typically a tyrosine in a normal EGFR polypeptide), 831 (typically an arginine in a normal EGFR polypeptide), 858 (typically a leucine in a normal EGFR polypeptide), and/or 868 (typically a glutamic acid in a normal EGFR polypeptide).
  • 731 typically a tryptophan in a normal EGFR polypeptide
  • 734 typically a glutamic acid in a normal EGFR polypeptide
  • 785 typically a
  • Preferred examples of changes at these residues include a leucine at residue 731, a glutamine at residue 734, an alanine at residue 785, a tyrosine at residue 797, a histidine at residue 801 , a histidine or a leucine at residue 831 , an arginine at residue 858, and a glycine at residue 868.
  • residue 731 in a mutant EGFR polypeptide is not an arginine
  • residue 734 in a mutant EGFR polypeptide is not a lysine
  • residue 785 in a mutant EGFR polypeptide is not an isoleucine
  • residue 831 in a mutant EGFR polypeptide is not a cysteine
  • residue 858 in a mutant EGFR polypeptide is not an alanine, a glycine, a lysine, a methionine, a glutamine, and arginine, or not a tryptophan.
  • deletions include, but are not limited to, deletions of amino acids encoded by exon 19, such as a deletion of amino acids 746 through 753, or any range within 746 through 753, such as 746 - 750, 747 - 750, 747 - 751 , and 747 - 753 (Pugh et al., BMC Cancer, 2007, 7:128).
  • a cancer cell may include one or more of of these mutations in any combination.
  • Amino acid changes in an EGFR polypeptide are known to increase tyrosine kinase activity. Examples include the substitution of arginine at residue 858 for the leucine (L858R) present in a normal EGFR poylpeptide.
  • EGFR tyrosine kinase activity may result in greater EGFR tyrosine kinase activity.
  • Whether an amino acid change increases the tyrosine kinase activity of a mutant EGFR polypeptide can be accomplished by detecting EGFR-mediated phosphorylation of at least one EGFR substrate.
  • the EGFR tyrosine kinase activity of cancer cells removed from a patient can be assayed and, optionally, compared to the EGFR tyrosine kinase activity of a normal EGFR polypeptide.
  • Methods for assaying EGFR tyrosine kinase activity are known to the person skilled in the art and are routine.
  • RNA sequencing Various methods for analyzing the polynucleotides can be used, including, but not limited to, polymerase chain reaction (PCR), hybridization with allele-specific probes, enzymatic mutation detection, chemical cleavage of mismatches, mass spectrometry, or DNA sequencing.
  • PCR polymerase chain reaction
  • enzymatic mutation detection e.g., a mutation in the polynucleotide
  • mass spectrometry e.g., a portion of one or more exons can be analyzed.
  • the invention provides a method of screening for mutations in an EGFR coding region, preferably exons 18-24, more preferably exons 18-21, by PCR or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science, 1988, 241 :1077-1080; and Nakazawa et al, Proc. Natl. Acad. Sci. USA, 1994, 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the EGFR-gene (see, Abravaya et al., Nucl. Acids Res., 1995, 23:675-682).
  • LCR ligation chain reaction
  • the method can include using primers for amplifying the target polynucleotide, the primers corresponding to one or more conserved regions of an EGFR coding region, amplifying with the primers using, as a template, a DNA or cDNA obtained from a biological sample and analyzing the PCR products. Comparison of the PCR products of the test biological sample to a control sample can indicate the presence of a mutation in the test biological sample. Likewise, determining the nucleotide sequence of a PCR product can indicate the presence of a mutation in the test biological sample.
  • Alternative amplification methods include, for example, self sustained sequence replication (see, Guatelli et al., Proc. Natl. Acad. Sci. USA, 1990, 87: 1874-1878), transcription-based amplification (see, Kwoh et al., Proc. Natl. Acad. Sci. USA, 1989, 86:1173-1177); Qb Replicase (see, Lizardi et al, BioTechnology, 1988, 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified polynucleotides using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of polynucleotides if such polynucleotides are present in very low numbers.
  • Primers useful in the present invention can be designed using amino acid sequences of the polynucleotide or polypeptide sequences disclosed at Figures 1 and 2, respectively, as a guide.
  • the primers are designed in the homologous regions wherein at least two regions of homology are separated by a divergent region having a mutation.
  • the homologous regions may be present in exons or, when genomic DNA is used, may be present in introns and/or exons.
  • Primers useful for the amplification of regions of an EGFR coding region, including exons 18-24, are known to the skilled person and are used routinely (Table 1). For instance, Pugh et al.
  • PCR primers that can be used to amplify nucleotides of exons 18-24
  • Chin et al. disclose primers that can be used to amplify nucleotides of exons 18-21
  • Bell et al. U.S. Patent Application Publication 2006/0147959 disclose PCR primers that can be used to amplify nucleotides of exons 18-24. Additional primers may be designed using a number of available computer programs, including, but not limited to, Primer Express® (Applied Biosystems, Foser City, CA), and IDT® Oligo Analyzer 3.0 (Integrated DNA Technologies, Coralville, IA). Primers may be labeled using labels known to one skilled in the art. Such labels include, but are not limited to radioactive, fluorescent, dye, and enzymatic labels.
  • Table 1 Examples of primers for amplification of nucleotides encoding an EGFR tyrosine kinase domain.
  • Analysis of amplification products can be performed using any method capable of separating the amplification products according to their size, including automated and manual gel electrophoresis, mass spectrometry, and the like.
  • the amplification products can be separated using sequence differences, using, for instance, single-stranded conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), two- dimensional gene scanning (TGGE), denaturing high-performance liquid chromatography (DHPLC), chemical cleavage, or restriction fragment polymorphisms as well as hybridization to, for example, a nucleic acid array.
  • SSCP single-stranded conformation polymorphism
  • DGGE denaturing gradient gel electrophoresis
  • TGGE two- dimensional gene scanning
  • DPLC denaturing high-performance liquid chromatography
  • chemical cleavage or restriction fragment polymorphisms as well as hybridization to, for example, a nucleic acid array.
  • Alterations in electrophoretic mobility can be used to identify mutations in an EGFR coding region.
  • SSCP may be used to detect differences in electrophoretic mobility between mutant and normal polynucleotides (see, for instance, Orita et al., Proc. Natl. Acad. Sci. USA, 1989, 86:2766; Cotton, Mutat. Res., 1993, 285:125-144; Keen et al., Trends Genet., 1991, 7:5; and Hayashi, Genet. Anal. Tech. Appl., 1992, 9:73-79). Single-stranded DNA fragments of sample and control EGFR polynucletides can be denatured and allowed to renature.
  • DDGE can be used to assay the movement of control and test polynucleotides in a polyacrylamide gel containing a gradient of denaturant (see, for instance, Myers et al., Nature 1985, 313:495) or a gradient of temperature (see, for instance, Rosenbaum and Reissner, Biophys. Chem., 1987, 265:12753).
  • DHPLC can be used to assay the movement of heteroduplexes that form in a PCR sample with sequence variations.
  • PCR products are resolved by chromatography, and heteroduplexes are identified by reduced column retention times relative to the homoduplex counterparts (see, for instance, Lilleberg, Curr. Opin. Drug Discov. Devel., 2003, 6:237-252; Chin et al., Clin. Chem., 2007, 53:62-70; and Janne et al., Clin. Cancer Res., 2006, 12:751-758).
  • Columns useful in DHPLC are known (see, for instance, Bonn et al., U.S. Patent 5,585,236) and are commercially available from, for instance, Transgenomic (San Jose, CA).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in an EGFR coding region include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (see, for instance, Myers et al., 1985, Science, 230:1242; Cotton et al., Proc. Natl. Acad. Sci. USA, 1988, 85:4397; and Saleeba et al., Methods Enzymol., 1992,
  • the mismatch cleavage reaction may employ one or more proteins that recognize mismatched base pairs in double-stranded DNA (often referred to as "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in EGFR cDNAs obtained from a cell (see, for example, Hsu et al., Carcinogenesis, 1994, 15:1657-1662; and Modrich et al., U.S. Patent 5,459,039).
  • DNA mismatch repair proteins that recognize mismatched base pairs in double-stranded DNA
  • primers may be prepared in which a known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found (see, for instance, Saiki et al., Nature, 1986, 324:163; and Saiki et al, Proc. Natl. Acad. Sci. USA, 1989, 86:6230).
  • Such allele specific polynucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the polynucleotides are attached to a hybridizing membrane and hybridized with labeled target DNA.
  • Allele specific amplification technology that depends on selective PCR amplification may be used in the present invention.
  • Polynucleotides used as primers for specific amplification may carry a mutation of interest in the center of the molecule (so that amplification depends on differential hybridization; see, for instance, Gibbs et al., Nucl. Acids Res., 1989, 17:2437-2448) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (see, for instance, Prossner, Tibtech., 1993, 11 :238).
  • probes include hybridization probes and antibodies, such as monoclonal antibodies.
  • useful probes include hybridization probes and antibodies, such as monoclonal antibodies.
  • Those skilled in the art are familiar with the preparation of probes with particular specificities, and will recognize that a variety of variables can be adjusted to optimize the discrimination between two forms of a polynucleotide, including changes in salt concentration, temperature, pH, and addition of various compounds that affect the differential affinity of GC vs.
  • a probe may be designed to bind to, for example, at least three continuous nucleotides on both sides of the mutation. Such probes, when hybridized under the appropriate conditions, will bind to the mutant EGFR coding region, but will not bind to a normal EGFR coding region.
  • the detection of the presence or absence of a mutation can involve contacting a polynucleotide having the mutation with a probe, where the probe preferentially hybridizes with a form of the polynucleotide containing a complementary base at the mutation site as compared to hybridization to a form of the polynucleotide having a non-complementary base at the mutation site, where the hybridization is carried out under selective hybridization conditions.
  • a hybridization probe may span two or more mutation sites.
  • determining the presence or absence of a mutation can involve determining the sequence of a polynucleotide.
  • the nucleotide sequence of a portion or portions of an EGFR coding region, preferably, one or more of the exons encoding the tyrosine kinase domain is determined.
  • the portion is 500 nucleotides or less in length, more preferably 100 nucleotides or less, and most preferably 45 nucleotides or less in length.
  • sequencing can be carried out by various routine methods recognized by those skilled in the art, including the use of dideoxy termination methods, and the use of mass spectrometric methods. Sequencing may be used in conjunction with other methods, including PCR methods. For instance, a PCR product may be sequenced.
  • the present invention is further directed to methods for evaluating a human patient having or suspected of having a cancer.
  • a person suspected of having a cancer can be one that presents with symptoms and/or signs that they may have a cancer.
  • a person suspected of having a cancer can be one that has an increased likelihood of having a cancer due to, for instance, exposure to environmental conditions that may predispose a person to a cancer.
  • the methods generally include detecting the presence or absence of a mutation of an EGFR coding region as discussed above. Knowledge of the presence or absence permits one to determine a prognosis or determine what types of treatment may be appropriate for a patient.
  • prognosis generally refers to a forecast or prediction of a probable course or outcome.
  • Prognosis includes, but is not limited to, the forecast or prediction of any one or more of the following: likelihood resection of a tumor will be successful, likelihood of survival of a patient, duration of survival of a patient, and likelihood of progression free survival. Treatments may include, for instance, surgery, and the use of certain chemotherapeutic agents.
  • the methods of the present invention permit the identification of patients that have a greater likelihood of benefiting from surgery, and surviving a cancer for a longer duration. It is expected that the methods of the present invention permit the identification of patients that have cancers with greater sensitivity to tyrosine kinase inhibitors.
  • the cancers that can be evaluated include those with altered EGFR activity.
  • cancers include those associated with mutations in an EGFR coding region, for example mutations that result in greater EGFR tyrosine kinase activity.
  • cancers include, but are not limited to, lung cancer (e.g., non-small cell lung cancer), head and neck cancer, and malignant mesothelioma (e.g., malignant peritoneal mesothelioma, malignant plural mesothelioma) (Rosell et al., Clin. Cancer Res., 2006, 12:7222-7231).
  • Other examples of cancers having altered EGFR activity include those associated with increased expression of EGFR.
  • cancers examples include, but are not limited to, breast cancer, bladder cancer, lung cancer, head and neck cancer, stomach cancer, and glioblastoma.
  • Other cancers that can be evaluated include epithelial cancers, including, but not limited to, lung cancer (e.g., non- small cell lung cancer), ovarian cancer, breast cancer, brain cancer, colon cancer, and prostrate cancer.
  • Other cancers include, but are not limited to, hepatocellular cancer, renal cancer, glioma, retina cancer, skin cancer, melanoma, pancreatic cancer, and genital-urinary cancer.
  • the cancer is not a cancer of the upper aerodigestive tract, preferably a cancer of the tonsil.
  • telomere length is typically RO, Rl, or R2.
  • RO refers to negative gross and microscopic margins
  • Rl refers to negative gross margin and positive microscopic margin
  • R2 refers to positive gross and microscopic margin.
  • a post-operative scoring system is generally used that grades residual disease with a resection score of RO, Rl, R2a, R2b, or R2c.
  • the resection score is provided by the surgeon after the debulking surgery (also referred to as cytoreductive surgery) and is an estimate for completeness of resection.
  • Complete removal or destruction of visible disease is graded RO or Rl .
  • the 0/1 designation describes the clearance or presence, respectively, of microscopic margins.
  • Rl is the typical designation for complete clearance of malignant peritoneal mesothelioma.
  • complete surface clearance is often not possible, given the usually diffuse nature of disease.
  • a debulking surgery leaving visible surface disease or nodules estimated to be less than 5 mm in thickness is graded as an R2a resection.
  • R2b means leaving visible disease with nodules of maximum dimensions between 5 and 20 mm in thickness
  • R2c means that gross residual disease is present and greater than 20 mm in size.
  • the presence of one or more mutations in an EGFR coding region was found to correlate with increased likelihood of resectability (Example 2).
  • the presence of one or more mutations in an EGFR coding region as disclosed herein indicates a prognosis that a patient will have a resection score of at least R2a or greater
  • the presence of one or more mutations in an EGFR coding region as described herein, preferably in exons 18-24, more preferably in exons 18-21 , generally indicates whether a patient has an increased likelihood for survival while having a cancer and/or an increased duration of survival, such as a malignant mesothelioma, preferably malignant peritoneal mesothelioma or malignant plural mesothelioma, more preferably, malignant peritoneal mesothelioma.
  • a malignant mesothelioma preferably malignant peritoneal mesothelioma or malignant plural mesothelioma, more preferably, malignant peritoneal mesothelioma.
  • the presence of one or more of the mutations in a patient having malignant peritoneal mesothelioma is predictive of a greater likelihood of short term survival (e.g., increased likelihood of survival for at least 28 months after surgery).
  • absence of such a mutation in a patient having malignant peritoneal mesothelioma is predictive of a decreased likelihood of short term survival (e.g., decreased likelihood of survival for greater than 14 months after surgery) and increased likelihood of disease recurrence within 14 months after surgery.
  • the presence of one or more mutations in an EGFR coding region as described herein, preferably in exons 18-24, more preferably in exons 18-21, is expected to indicate whether a patient has an increased likelihood for progression free survival.
  • Progression free survival refers to patient survival without disease progression. In some patients it may not be possible to remove all diseased tissue, but the remaining diseased tissue may remain stable during progressive follow-up. This is expected to be particularly relevant in patients having one or more of the mutations described herein and treated with an EGFR kinase inhibitor.
  • the presence of one or more mutations in an EGFR coding region as described herein, preferably in exons 18-24, more preferably in exons 18-21, can also be used to aid in determining what types of treatment may be appropriate for a patient. Treatments may include, for instance, surgery and/or the use of certain therapeutic agents.
  • Surgical methods for resection of tumors depend upon the type and location of the tumor being removed, and such methods are known to the person skilled in the art and are routine. Typically, surgical resection is used with other therapies, including, for instance, intraperitoneal hyperthermic chemotherapy, systemic chemotherapy, and immunotherapy.
  • Intraperitoneal hyperthermic chemotherapy typically follows surgery, and may include one or more chemotherapeutic agents, such as, but not limited to, mitomycin C, carboplatin, cisplatin, fluorouracil, doxorubicin, and paclitaxel (see, for instance, Bridda et al., Med. Gen. Med., 2007, 9:32).
  • Systemic chemotherapy may be used, and may include, for instance, permetrexed and cisplatin.
  • Immunotherapy may be useful, and can include humanized anti-CD-3 antibodies, cytotoxic T lymphocytes, interferon alfa-2a, and autovaccine (see, for instance, Bridda et al., Med. Gen. Med., 2007, 9:32).
  • the presence of one or more mutations in an EGFR coding region as described herein, preferably in exons 18-24, more preferably in exons 18-21, is expected to indicate that a patient will have a greater benefit from the use of certain therapeutic agents than a patient that does not have one or more of the mutations.
  • the mutation(s) in an EGFR coding region results in an EGFR polypeptide having increased EGFR tyrosine kinase activity.
  • the therapeutic agents contemplated here include, but are not limited to, EGFR kinase inhibitors.
  • EGFR kinase inhibitor refers to any EGFR kinase inhibitor that is currently known in the art, and includes any chemical entity that, upon administration to a patient, results in inhibition of a biological activity associated with activation of the EGF receptor in the patient, including any of the downstream biological effects otherwise resulting from the binding to EGFR of its natural ligand.
  • Such EGFR kinase inhibitors include any agent that can block EGFR activation or any of the downstream biological effects of EGFR activation that are relevant to treating cancer in a patient.
  • Examples of how such an inhibitor can act include, but are not limited to, binding directly to the intracellular domain of the receptor and inhibiting its kinase activity, occupying the ligand binding site or a portion thereof of the EGFR, thereby making the receptor inaccessible to its natural ligand so that its normal biological activity is prevented or reduced, and modulating the dimerization of EGFR polypeptides, or interaction of EGFR polypeptide with other proteins, or enhance ubiquitination and endocytotic degradation of EGFR.
  • EGFR kinase inhibitors include, but are not limited to, low molecular weight inhibitors and antibodies or antibody fragments.
  • EGFR kinase inhibitors include, for example quinazoline EGFR kinase inhibitors, pyrido-pyrimidine EGFR kinase inhibitors, pyrimido-pyrimidine EGFR kinase inhibitors, pyrrolo-pyrimidine EGFR kinase inhibitors, pyrazolo- pyrimidine EGFR kinase inhibitors, phenylamino-pyrimidine EGFR kinase inhibitors, oxindole EGFR kinase inhibitors, indolocarbazole EGFR kinase inhibitors, phthalazine EGFR kinase inhibitors, isoflavone EGFR kinase inhibitors, quinalone EGFR kinase inhibitors, and tyrphostin EGFR kinase inhibitors, such as those described in the following patent publications, and all pharmaceutically acceptable salts and solvates of said EG
  • Patents 5,747,498 Schott al.
  • 5,789,427 Choen et al.
  • 5,650,415 Tang
  • 5,656,643 Spada
  • Additional non-limiting examples of low molecular weight EGFR kinase inhibitors include any of the EGFR kinase inhibitors described in Traxler, P., Exp. Opin. Ther. Patents, 1998, 8(12): 1599-1625.
  • low molecular weight EGFR kinase inhibitors that can be used according to the present invention include [6,7-bis(2- methoxyethoxy)-4-quinazolin-4-yl]-(3-ethynylphenyl)amine (also known as OSI-774, erlotinib, or TARCEVA (erlotinib HCl); OSI Pharmaceuticals/Genentech/Roche) (U.S. Patent 5,747,498 (Schnur);
  • EGFR kinase inhibitors also include, for example, multi-kinase inhibitors that have activity on EGFR kinase, i.e.
  • inhibitors that inhibit EGFR kinase and one or more additional kinases.
  • examples of such compounds include the EGFR and HER2 inhibitor CI- 1033 (formerly known as PD 183805; Pfizer); the EGFR and HER2 inhibitor GW-2016 (also known as GW-572016 or lapatinib ditosylate; GSK); the EGFR and JAK 2/3 inhibitor AG490 (a tyrphostin); the EGFR and HER2 inhibitor ARRY-334543 (Array BioPharma); BIBW-2992, an irreversible dual EGFR/HER2 kinase inhibitor (Boehringer Ingelheim Corp.); the EGFR and HER2 inhibitor EKB-569 (Wyeth); the VEGF- R2 and EGFR inhibitor ZD6474 (also known as Z ACTIMA.TM. ; AstraZeneca Pharmaceuticals), and the EGFR and HER2 inhibitor BMS-599626 (Bristol- My
  • Antibody-based EGFR kinase inhibitors include any anti-EGFR antibody or antibody fragment that can partially or completely block EGFR activation by its natural ligand.
  • Non-limiting examples of antibody-based EGFR kinase inhibitors include those described in Modjtahedi, H., et al., Br. J. Cancer, 1993, 67:247-253; Teramoto, T., et al., Cancer, 1996, 77:639-645; Goldstein et al., Clin. Cancer Res. 1995, 1 :131 1-1318; Huang, S. M., et al., Cancer Res. 1999, 15:59(8):1935-40; and Yang, X., et al., Cancer Res.
  • the EGFR kinase inhibitor can be the monoclonal antibody Mab E7.6.3 (Yang, X. D. et al. Cancer Res. 1999, 59:1236-43), or Mab C225 (ATCC Accession No. HB-8508), or an antibody or antibody fragment having the binding specificity thereof.
  • Suitable monoclonal antibody EGFR kinase inhibitors include, but are not limited to, IMC-C225 (also known as cetuximab or ERBITUX; Imclone Systems), ABX-EGF (Abgenix), EMD 72000 (Merck KgaA, Darmstadt), RH3 (York Medical Bioscience Inc.), and MDX-447 (Medarex/Merck KgaA).
  • the present invention also provides methods for identifying an agent which decreases the tyrosine kinase activity of an EGFR polypeptide having a mutation, such as one or more of the mutations described herein.
  • the method may include contacting such an EGFR polypeptide with a test agent and determining the tyrosine kinase activity of the EGFR polypeptide in the presence of the test agent, wherein a change in the tyrosine kinase activity of the EGFR polypeptide in the presence of the test agent as compared to the tyrosine kinase activity of the EGFR polypeptide in the absence of the test agent is indicative of an agent which decreases the tyrosine kinase activity of the EGFR polypeptide.
  • test agents to be screened include, for instance, chemical compound libraries, fermentation media of bacteria and fungi, and cell extracts of plants and other vegetations. Methods for identifying compounds that decrease the tyrosine kinase activity of a receptor are generally known to those of skill in the art. An agent identified by this method is also provided.
  • the present invention further provides isolated EGFR polypeptides, or portions thereof, that contain one or more of the following mutations: W731L, E734Q, T785A, C797Y, Y801H, R831H, R831L, or E868G.
  • an "isolated" polypeptide or polynucleotide refers to a polypeptide or polynucleotide that has been either removed from its natural environment, produced using recombinant techniques, or chemically or enzymatically synthesized.
  • a portion of an isolated EGFR polypeptide may have between 10 and 1200amino acids, for instance, at least 10, at least 50, at least 100, at least 150, at least 200, or at least 250 amino acids and no greater than 1200, no greater than 1150, no greater than 1100, no greater than 1050, no greater than 1000, or no greater than 950 amino acids.
  • An isolated EGFR polypeptide of the present invention may have an amino acid sequence identical to the SEQ ID
  • an isolated portion of an EGFR polypeptide may have an amino acid identical to the corresponding portion of SEQ ID NO:2 except for the presence of one or more of the mutations described herein.
  • An isolated EGFR polypeptide, or portion thereof, of the present invention also includes those that are similar to SEQ ID NO:2 and include one of more of the mutations described herein.
  • the similarity is referred to as structural similarity and is generally determined by aligning the residues of the two amino acid sequences (i.e., a candidate amino acid sequence and the amino acid sequence of SEQ ID NO:2) to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • a candidate amino acid sequence is the amino acid sequence being compared to an amino acid sequence present in SEQ ID NO:2.
  • a candidate amino acid sequence can be isolated from an animal, preferably a human, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.
  • two amino acid sequences are compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison WI), or the Blastp program of the BLAST.2 search algorithm, as described by Tatusova, et al. (FEMS Microbiol Lett 1999, 174:247-250), and available through the World Wide Web, for instance at the internet site maintained by the National Center for Biotechnology Information, National Institutes of Health.
  • a polypeptide of the present invention also includes polypeptides with an amino acid sequence having at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, or at least 95% amino acid identity to SEQ ID NO:2.
  • a polypeptide having similarity to SEQ ID NO:2 and including one or more of the mutations described herein has a tyrosine kinase activity that is substantially identical to an EGFR polypeptide having a sequence that is identical to SEQ ID NO:2 with the same mutation(s).
  • a polypeptide having 80% identity to SEQ ID NO:2 and having the mutation R83 IH will have a tyrosine kinase activity that is substantially identical to the tyrosine kinase activity of a polypeptide having the sequence SEQ ID NO:2 and the same R831H mutation.
  • a candidate polypeptide having structural similarity to SEQ ID NO:2 may include conservative substitutions of amino acids present in SEQ ID NO:2.
  • a conservative substitution is typically the substitution of one amino acid for another that is a member of the same class.
  • an amino acid belonging to a grouping of amino acids having a particular size or characteristic such as charge, hydrophobicity, and/or hydrophilicity
  • conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: GIy, Ala, VaI, Leu, and He (representing aliphatic side chains); Class II: GIy, Ala, VaI, Leu, He, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulfur-containing side chains); Class V: GIu, Asp, Asn and GIn (carboxyl or amide group containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: GIy, Ala, Pro, Tip, Tyr, He, VaI,
  • the present invention also includes isolated polynucleotides that encode the isolated polypeptides of the present invention.
  • the population of nucleotide sequences encoding the polypeptides of the present invention is large but finite, and the nucleotide sequence of each member of the population can be readily determined by one skilled in the art by reference to the standard genetic code.
  • the present invention also provides a kit for practicing the methods described herein.
  • the kit includes two primers for use together to amplify an EGFR coding region, preferably one or more of exons 18-24, more preferably one or more of exons 18-21 , in a suitable packaging material in an amount sufficient for at least one amplification.
  • suitable packaging material preferably one or more of exons 18-24, more preferably one or more of exons 18-21 .
  • other reagents such as buffers and solutions needed to practice the invention are also included.
  • Instructions for use of the packaged primers are also typically included.
  • the phrase "packaging material” refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment.
  • the packaging material has a label which indicates that the primers can be used for the methods described herein.
  • the packaging material contains instructions indicating how the materials within the kit are employed to practice the methods.
  • the term "package” refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the primers.
  • a package can be a glass vial used to contain appropriate quantities of the primers.
  • "Instructions for use” typically include a tangible expression describing the conditions for use of the primers.
  • L858R has been identified in non-small cell lung cancer patients associated with response to gefitinib, while the other mutations (W731L, E734Q, T785A, C797Y, Y801H, R831H, R831 L, and E868G) have not been reported.
  • the EGFR missense mutations identified in human cancers were analyzed using computational analysis tools to determine the effects of these mutations on EGFR function.
  • the effect of the EGFR missense mutations and non-synonymous polymorphisms on the protein domain structure of EGFR was analyzed using the Pfam computational analysis tool (Roberio et al., Genet MoI Res., 2005 4(3):590-8).
  • Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein families based on the Swissprot 44.5 and SP-TrEMBL 27.5 protein sequence databases.
  • the positions of the EGFR missense mutations appear in the Pfam model of protein kinase domain.
  • the EGFR is part of a subfamily of four closely related receptors EGFR (or ErbB-1), Her 2/neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).
  • Receptors exist as inactive single units or monomers that, on activation by ligand binding, pair to form an active dimer.
  • the two receptors that form a pair are not necessarily identical, for example an EGF receptor (EGFR) may pair with another EGF receptor, giving a so-called homodimer, or an EGFR may pair with another member of the receptor family, such as Her 2/neu, to give an asymmetrical heterodimer.
  • Known EGFR phosphorylation sites include Thr678, Thr693, Ser695, Tyr869, Serl070, SerlO71, TyrlO92, Tyrl 1 10, Tyrl 172, and Tyrl 197 which are summarized below in Table 3.
  • the known EGFR phosphorylation sites are underlined.
  • EGFR phosphorylation sites were identified by computational analysis using the NetPhos computational analysis tool.
  • NetPhos produces neural network predictions for serine, threonine and tyrosine phosphorylation sites in eukaryotic proteins (Blom et al, J. MoI. Biol., 1999, 294(5): 1351- 1362).
  • Potential EGFR phosphorylation sites predicted by NetPhos are summarized in Table 3.
  • the known EGFR phosphorylation sites are underlined.
  • W731 and E734 are close to T725, and E868 is directly adjacent to Y869.
  • mutations at the positions W731, E734, and E868 may influence the phosphorylation patterns of nearby sites.
  • PROSITE is a database of protein families and domains. It consists of biologically significant sites, patterns and profiles that help to reliably identify to which known protein family (if any) a new sequence belongs as well as to identify potential sites for protein modification (HuIo N et al., Nucl. Acids. Res., 2004, 32:D134-D137; Sigrist CJA et al., Brief Bioinform., 2002, 3:265-274; Gattiker A et al., Applied Bioinformatics, 2002, 1 :107-108).
  • a search using PROSITE showed that the other potential sites for protein modification are predicted at EGFR amino acid positions, 220-222 and 752-754 as PKC phosphorylation sites as summarized below in Table 4. Also, EGFR amino acids 718-745 matched to consensus of an ATP -binding site. Two missense mutations, W731L and E734Q, are located within the ATP -binding site. These mutations may potentially influence the ATP binding. EGFR amino acids 833-845 match with the consensus of a tyrosine kinase active site. The R83 IH and R83 IL mutations are adjacent to this region and may influence tyrosine kinase activity. The predicted N- myristoylation sites are also listed. A mutation, T785A, is a single amino acid away from the one of these sites at amino acids 779-784.
  • EGFR missense mutations on EGFR biological function was further analyzed by peptide sequence alignment.
  • Known EGFR sequences of various organisms including cow, chimpanzee, chicken, human, mouse, rat, pig, zebrafish, dog, the CG10079-PA polypeptide of fruit fly, and the CG 10079- PB polypeptide of fruit fly were obtained from GenBank and aligned using ClustalW (Chenna et al., Nucleic Acids Res., 2003, 31 :3497-500) (Fig. 4).
  • ClustalW is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of divergent sequences.
  • R831 H K831 is present in fruitfly
  • MMMs Malignant peritoneal mesotheliomas
  • MPMs Malignant peritoneal mesotheliomas
  • MM malignant mesothelioma
  • the median survival for these tumors is less than a year, and like other peritoneal surface malignancies this is due primarily to intra-abdominal recurrence/progression.
  • MM malignant mesothelioma
  • MM malignant mesothelioma
  • MM malignant mesothelioma
  • the median survival for these tumors is less than a year, and like other peritoneal surface malignancies this is due primarily to intra-abdominal recurrence/progression.
  • IPHC IPHC including Mitomycin C (30 mg + 10 mg) or Carboplatin (800 mg/M2) for 90 to 120 minutes at 40.5 0 C, and complete clinical data including age, sex, cytoreductive score, histology, mutation, and survival was available for comparison of the group containing a mutation and the group not containing a mutation.
  • the median age was 56, and 71% of the patients were male with a median follow-up time (MFT) of 14.5 months. Mutations were found in 31% (9/29) of the tumors. Seven of these mutations were novel (W731L, E734Q, T785A, C797Y, Y801H, R831H, R831L, and E868G), and the eighth one was the L858R mutation previously described in non-small cell lung cancer (NSCLC). Of the 25 patients managed surgically there were 7 patients with a mutation in the catalytic TK domain of EGF receptor (mut+) and 18 patients without a mutation in the catalytic TK domain of EGF receptor (mut-) (see Table 7).
  • the tyrosine kinase domain of the EGFR protein appears to be a common site for mutation in MPM, with the identification of novel and known activation mutation in 31% of tumors.
  • the presence of a mutation appears to be predictive of patients who can undergo optimal CRS/IPHC and benefit from the procedure).

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Abstract

La présente invention concerne des procédés pour caractériser des cellules cancéreuses chez un patient, pour déterminer un pronostic chez un sujet ayant un cancer, ou susceptible d'en avoir un, et pour évaluer des options de traitement pour un patient ayant un cancer, tel qu'un mésothéliome malin, y compris un mésothéliome péritonéal malin. Les procédés comprennent la détection de la présence ou de l'absence d'une mutation d'une région de codage d'un récepteur de facteur de croissance épidermique (EGFR), où la mutation a pour conséquence un polypeptide d'EGFR ayant un acide aminé différent au niveau d'un résidu (731, 734, 785, 797, 801, 831, 858, 868), ou leur combinaison par comparaison à un polypeptide d'EGFR normal.
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