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WO2009150256A1 - Marqueurs permettant de prévoir la réponse et la survie chez des patients subissant un traitement anti-egfr - Google Patents

Marqueurs permettant de prévoir la réponse et la survie chez des patients subissant un traitement anti-egfr Download PDF

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Publication number
WO2009150256A1
WO2009150256A1 PCT/EP2009/057394 EP2009057394W WO2009150256A1 WO 2009150256 A1 WO2009150256 A1 WO 2009150256A1 EP 2009057394 W EP2009057394 W EP 2009057394W WO 2009150256 A1 WO2009150256 A1 WO 2009150256A1
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Prior art keywords
patient
status
pten
braf
kras
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PCT/EP2009/057394
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English (en)
Inventor
Pierre Laurent-Puig
Original Assignee
Institut National De La Sante Et De La Recherche Medicale (Inserm)
Stone, Steve
Reid, Julia E.
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Application filed by Institut National De La Sante Et De La Recherche Medicale (Inserm), Stone, Steve, Reid, Julia E. filed Critical Institut National De La Sante Et De La Recherche Medicale (Inserm)
Publication of WO2009150256A1 publication Critical patent/WO2009150256A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • the invention generally relates to molecular diagnostics, and particularly to molecular markers for response and survival in patients treated with anti-EGFR therapeutics, as well as methods of use thereof.
  • Anti-EGFR antibody therapeutics headline the recent rise of targeted cancer therapies and have shown marked efficacy in many patients.
  • Such anti-EGFR antibody therapeutic include Cetuximab (marketed as Erbitux®) and Panitumumab (marketed as Vectibix®).
  • Cetuximab is widely used to treat metastatis colorectal cancer (mCRC) patients, either alone or in combination with 5 fluoruracil, irinotecan or oxaliplatin.
  • mCRC metastatis colorectal cancer
  • cetuximab only about 20% of mCRC patients respond well to cetuximab and its use is very costly.
  • cetuximab there is a great need to predict which patients will respond well to treatment.
  • the present invention is based on the identification of certain molecular markers for drug response and survival in mCRC patients.
  • the expression, activity, and/or sequence variation status of a few genes and/or their protein products have been determined as predictive of response and/or survival in cetuximab-treated patients.
  • an activating mutation in either of the following genes is independently predictive of poor response, shorter progression-free survival (PFS), and shorter overall survival
  • OS in cetuximab-treated patients: KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; Entrez GenelD No. 3845), BRAF (v-raf murine sarcoma viral oncogene homolog B 1 ; Entrez Genel D No. 673).
  • PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1 ); Entrez GenelD No. 5728) has been found to be independently predictive of better OS in cetuximab-treated patients. Tumours from 173 patients with mCRC were collected. All but one patient received cetuximab-based regimen as a second line or greater.
  • KRAS and BRAF status were assessed by allelic discrimination.
  • EGFR amplification was assessed by CISH and FISH, the expression of PTEN by immunochemistry.
  • PFS progression free survival
  • OS overall survival
  • BRAF mutation and PTEN expression status were associated with OS.
  • the invention provides a method of classifying a cancer, said method comprising determining for a patient the status of KRAS, BRAF, and/or PTEN, wherein a particular status indicates a particular classification.
  • Classifying a cancer can include many different determinations and/or predictions regarding a patient, includi ng but not lim ited to predicting a patient's response to, or likelihood of survival following, a particular therapy.
  • the particular therapy is an anti-EGFR therapy.
  • anti-EGFR therapy is an anti-EGFR antibody, e.g. , cetuximab.
  • determining expression level comprises at least one of: (a) determining the amount of polypeptide encoded by KRAS, BRAF, or PTEN;
  • determining activity level may comprise, e.g., determining the level of enzymatic activity of a polypeptide encoded by KRAS, BRAF, or PTEN; determining the level of interaction between said polypeptide and a binding partner; etc.
  • the invention provides compositions for use in the above methods.
  • compositions include, but are not limited to, nucleic acid probes hybridizing to at least one of KRAS, BRAF, or PTEN (or to any nucleic acids encoded by or complementary to any of said genes); nucleic acid primers and primer pairs suitable for amplifying all or a portion of any of said genes or any nucleic acids encoded thereby; antibodies binding immunologically to a polypeptide encoded by any of said genes; probe sets comprising a plurality of said nucleic acid probes, nucleic acid primers, antibodies, and/or polypeptides; microarrays comprising any of these; kits comprising any of these; etc.
  • the invention provides computer software and/or modules for use in the above methods.
  • Figure 1 shows the progression free survival (A) and overall survival (B) in wild-type KRAS patients according to BRAF status.
  • the lines correspond to BRAF mutants (V600E) or to non mutated tumours.
  • Figure 2 shows the overall survival in wild-type KRAS patients according to PTEN status.
  • the lines correspond to PTEN non-null or to PTEN null tumours.
  • Figure 3 shows a comparison of progression free survival of mutated KRAS patients and BRAF mutated patients status.
  • the lines correspond to BRAF mutants (BRAFM) or to KRAS mutants (KRASM).
  • the present invention is based on the identification of certain molecular markers for drug response and survival in mCRC patients.
  • the mutation and/or I HC status of a few genes and/or their protein products have been discovered to be associated with response and survival in cetuximab-treated patients.
  • an activating mutation in either of the following genes is independently predictive of poor response, progression-free survival (PFS), and overall survival (OS) in cetuximab- treated patients: KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; Entrez GenelD No.
  • BRAF v-raf murine sarcoma viral oncogene homolog B1 ; Entrez GenelD No. 673.
  • PTEN phosphatase and tensin homolog (mutated in multiple advanced cancers 1 ); Entrez Genel D No. 5728) has been found to be independently predictive of better OS in cetuximab- treated patients.
  • KRAS KRAS, BRAF or PTEN
  • PTEN protein products
  • the invention provides a method of classifying a cancer, said method comprising determining for a patient the status of KRAS, BRAF, and/or PTEN, wherein a particular status indicates a particular classification.
  • the "status" of a gene as used herein means whether said gene is normal, abnormal, high, low, present, or absent in some physical, chemical, or biological characteristic.
  • Particularly relevant characteristics for status according to the present invention are (a) whether and to what extent a gene is expressed; (b) whether and to what extent a gene is active; and (c) whether and to what extent a gene has sequence variation.
  • Each of these three characteristics can be either qualitattive (i.e. , binary, e.g., expressed vs. not expressed) or quantitative (e.g. , high expressed vs. normal or low expression).
  • sequence variation is meant any sequence variation compared to the wild-type sequence.
  • the wild-type sequence of KRAS is for example set forth in UniProtKB/Swiss-Prot Accession No. P01 1 16
  • the wild-type sequence of BRAF is for example set forth in UniProtKB/Swiss-Prot Accession No.
  • a patient sample can be assesed for
  • PTEN status In such a case, depending on the information sought, one can assign to a sample a status of "PTEN(+)" when some level of PTEN expression (e.g., PTEN protein as determined by IHC) is detected in said sample. Likewise, if no PTEN expression is detected the status assigned can be "PTEN(-)."
  • PTEN protein expression is determined by immunochemistry (IHC), for example on 3 ⁇ m paraffin sections.
  • the antibody may for example correspond to the PTEN antibody commercialized by R&D Systems, Lille, France (Reference No. AF847).
  • PTEN IHC control slides Reference No. 8106, Cell Signalling technology, Ozyme, Saint Quentin en Yvelines, France
  • normal endothelial cells may for example be used as positive controls.
  • the immunostaining and the detection can for example be carried out unsing a Benchmark XT immunostainer and a microscope.
  • the sample has a "PTEN(+)" status if PTEN expression is detected.
  • the sample has a "PTEN(-)” status if no PTEN expression can be detected.
  • the absence of PTEN expression (PTEN(-) status) can also be determined by comparison to a negative control indicative of loss of PTEN expression.
  • a sample can have a status designated "/CR/AS-activated” if the sample contains a KRAS gene harboring an activating mutation or "KRAS-wWd- type” if no such mutation is detected (same for BRAF).
  • the sample has a "/CR/AS-activated” status if the sample contains a KRAS gene that encodes a KRAS protein that comprises a mutation at codon 12 or 13 (codon numbering corresponding to the numbering given in UniProtKB/Swiss-Prot Accession No. P01 1 16). More specifically, the sample has a "/CR/AS-activated” status if the sample contains a KRAS gene harboring at least one of the following activating mutations:
  • the sample has a "KRAS- activated” status if the sample contains a KRAS gene that encodes a KRAS protein that comprises a mutation at codon 61 (codon numbering corresponding to the numbering given in UniProtKB/Swiss-Prot Accession No. P01 1 16). More specifically, the sample has a "/CR/AS-activated” status if the sample contains a KRAS gene harboring at least one of the following activating mutations:
  • the sample has a "BRAF- activated" status if the sample contains a BRAF gene that encodes a BRAF protein that comprises a mutation at codon 600, e.g. a Val600Glu activating mutation (codon numbering corresponding to the numbering given in UniProtKB/Swiss-Prot Accession No. P15056).
  • a BRAF gene that encodes a BRAF protein that comprises a mutation at codon 600, e.g. a Val600Glu activating mutation (codon numbering corresponding to the numbering given in UniProtKB/Swiss-Prot Accession No. P15056).
  • the invention provides a method of classifying a cancer comprising determining a patient's KRAS, BRAF, and/or PTEN status; wherein an activated KRAS status and/or and activated BRAF status indicates said patient has a poor prognosis for response, PFS, and/or OS; and wherein a PTEN(+) status indicates said patient has a good prognosis for OS.
  • the above method preferably comprises determining a patient's
  • BRAF and/or PTEN status may further comprise the step of determining a patient's KRAS status.
  • some aspects of the invention provide a method of classifying a cancer, said method comprising:
  • the cancer is a colon or a colorectal cancer, for example a metastatic colon or colorectal cancer.
  • the method of classifying a cancer comprises: a) determining the expression level of PTEN for a patient, for example by IHC; and, optionally, b) determining whether said patient has a sequence variation in KRAS and/or BRAF, e.g. an activating mutation in KRAS and/or BRAF.
  • Step (b) preferably comprises determining whether said patient has a sequence variation in KRAS (e.g. an activating mutation in KRAS).
  • the loss of PTEN expression is indicative of poor survival of the patient.
  • PTEN expression is indicative of a good prognosis for survival, more specifically for a good prognosis of OS.
  • the methods may further comprise the step of designing a treatment regimen for said patient.
  • An anti-EGFR antibody may be administered to patients exhibiting PTEN expression and having a wild-type KRAS genotype.
  • other drugs such as mTOR inhibitors should be administered to patients exhibiting loss of PTEN expression and having a wild-type KRAS genotype.
  • the methods in accordance with the invention are carried out in vitro, for examples on samples obtained from a patient.
  • the samples may correspond to a biopsy and/or a sample obtained from a biopsy (e.g. a DNA or protein preparation).
  • PTEN, BRAF and/or KRAS status is determined in tumor cells.
  • the invention pertains to a an in vitro method for determining whether a patient suffering from colon or colorectal cancer is a responder to a pharmaceutical treatment with an anti-EGFR antibody, and/or for determining the prognosis of said patient, said method comprising the steps of: a. obtaining a sample previously collected from said patient; b. determining PTEN expression level in said sample; and, optionally, at least one of (c) or (d): c. determining whether KRAS comprises a mutation in said sample; and/or d. determining whether BRAF comprises a mutation in said sample.
  • the invention pertains to a an in vitro method for determining whether a patient suffering from colon or colorectal cancer is a responder to a pharmaceutical treatment with an anti-EGFR antibody, and/or for determining the prognosis of said cancer, said method comprising the steps of: a. obtaining a sample previously collected from said patient; b. determining whether KRAS comprises a mutation in said sample; and c. determining at least one of: i. determining PTEN expression level in said sample; and/or ii. determining whether BRAF comprises a mutation in said sample.
  • a mutation in KRAS is indicative of a poor prognosis and of lack of response to an anti-EGFR therapy. If no mutation is found in KRAS: a mutation in BRAF is indicative of a poor prognosis (PFS and OS) and of lack of response to an anti-EGFR therapy; lack of PTEN expression is indicative of a poor prognosis (OS).
  • the patient should preferably be treated with other drugs than anti-EGFR antibodies, such as e.g. mTOR inhibitors; and
  • PTEN expression and the absence of a mutation in BRAF indicates that the patient is suited for an anti-EGFR therapy.
  • Determining PTEN expression may be done at the protein level
  • Determining whether BRAF and/or KRAS comprises a mutation may also be done at the protein level (e.g. using antibodies specifically binding to mutated proteins) or at the DNA or RNA level (e.g. by sequencing, by using microarrays or by using fluorescent probes allowing the specific detection of mutations).
  • classification of and “classifying” a patient's cancer refer to determining one or more clinically-relevant features of the cancer or determining the prognosis of the patient.
  • classifying a cancer includes, but is not limited to: (i) evaluating metastatic potential, potential to metastasize to specific organs, risk of recurrence, or course of the tumor; (ii) evaluating tumor stage; (iii) patient prognosis in the absence of therapy treatment of the cancer; (iv) prognosis of patient response to treatment (chemotherapy, radiation therapy, and/or surgery to excise tumor); (v) diagnosis of actual patient response to current and/or past treatment; (vi) determining a preferred course of treatment for the patient; (vii) prognosis for patient relapse after treatment; (viii) prognosis for patient survival (e.g., PFS and/or OS), etc.
  • the invention provides a method of classifying a cancer comprising: (1 ) identifying a patient with a particular type of cancer (e.g., mCRC); (2) making one or more of the determinations in (a), (b) or (c) above; and (3) classifying said cancer based on said determinations.
  • a particular type of cancer e.g., mCRC
  • a method according to the invention for determining a patient's prognosis comprises (a) determining for said patient the expression level of KRAS, BRAF, or PTEN; and/or (b) determining for said patient the activity level of a polypeptide encoded by KRAS, BRAF, or PTEN; (c) determining whether said patient has a sequence variation in KRAS, BRAF, or PTEN; wherein the presence of a difference from normal in expression level in (a), the presence of a difference from normal in activity level in (b), and/or the presence of a sequence variation in (c) indicates a particular cancer classification as indicated above.
  • any one of the determinations in (a)-(c) can be made in combination with any number of the others, or by using any combination of the compositions discussed herein, depending on the particular information desired. Indeed, one skilled in the art will recognize some overlap between the determinations in (a)-(c); e.g. , determining whether a patient has certain sequence variations can give information on whether the patient will also have differences in gene expression or activity levels (e.g., information about a mutation in codon 12 of KRAS can give information regarding the activity level of the KRAS protein).
  • determining whether a patient has certain sequence variations can give information on whether the patient will also have differences in gene expression or activity levels (e.g., information about a mutation in codon 12 of KRAS can give information regarding the activity level of the KRAS protein).
  • the invention provides a method of classifying a patient's cancer comprising making at least one of the determinations in (a)-(c) above for a plurality of KRAS, BRAF, or PTEN. In some embodiments determinations are made for at least 2 or 3 of KRAS, BRAF, or PTEN.
  • the invention provides methods of classifying a cancer by determining KRAS, BRAF, and/or PTEN status, wherein determining
  • KRAS, BRAF, and/or PTEN status comprises determining the expression level of KRAS, BRAF, and/or PTEN.
  • determining expression level comprises at least one of:
  • Some aspects of the i nvention provide methods com prisi ng determining expression level by determining whether a patient has copy number variation (CNV) in KRAS, BRAF, and/or PTEN.
  • CNV copy number variation
  • Various techniques for determining CNV are known in the art and suitable techniques include any technique that is able to provide detailed copy number information for KRAS, BRAF, or PTEN, including but not limited to, fluorescence in situ hybridization (FIS H), comparative genomic hybridization (CGH), array-based CG H (a-CGH), simple nucleotide polymorphism (SNP) microarray analysis, whole or partial genome sequencing, etc.
  • FIS H fluorescence in situ hybridization
  • CGH comparative genomic hybridization
  • a-CGH array-based CG H
  • SNP simple nucleotide polymorphism
  • CNV refers to a number of genomic copies of a particular nucleic acid that differs from the normal number. Thus CNV is determined as compared to some predefined "normal" copy number.
  • This normal copy number can be defined as, e.g. , the number of copies of any given nucleic acid expected in any normal diploid human sample, i.e., two copies.
  • the normal copy number can be defined as, e.g. , the actual number of copies of a given nucleic acid a patient has in his or her normal somatic tissue. Often, but not necessarily, this normal somatic tissue is tissue from the same organ or organ system where the suspected or known cancer resides.
  • Methods for determining DNA copy number include microarray- based technologies (see e.g. , U.S. Pat. No. 6,232,068; Pollack et al., Nat. Genet., 23(1 ):41 -6, (1999)), Southern blotting (see Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1995 supplement)), in situ hybridization such as fluorescence in situ hybridization (FISH) (see Angerer, METH . ENZYMOL. (1987) 152:649), comparative genomic hybridization (CGH), Amplification-based assays such as realtime quantitative PCR (see e.g.
  • a microarray with nucleic acid probes is employed to aid in determining whether a patient has CNV.
  • a microarray with nucleic acid probes is employed to aid in determining whether a patient has CNV.
  • a substrate or carrier e.g., a silicon chip or glass slide.
  • Target nucleic acid sequences to be analyzed can be contacted with the immobilized oligonucleotide probes on the microchip.
  • the multiple target nucleic acid sequences to be studied are fixed onto a substrate and an array of probes is contacted with the immobilized target sequences.
  • Drmanac et a/. NAT. BIOTECH NOL. (1998) 16:54-58.
  • Microchip technologies combined with computerized analysis tools allow fast screening in a large scale. The adaptation of the microchip technologies to the present invention will be apparent to a person of skill in the art apprised of the present disclosure. See, e.g. , U.S. Patent No. 5,925,525 to Fodor et a/; Wilgenbus et a/. , J. MOL. MED. (1999) 77:761 -786; Graber et a/.
  • An example of a microarray useful in the present invention is the Affymetrix ® 500K SNP microarray.
  • the high resolution of SNP microarrays (an average of about 5 Kb) and the huge number of probes (upwards of 1 million) makes them ideal for copy number analysis according to the present invention.
  • Nucleic acid arrays that are useful in the present invention include, but are not limited to, those that are commercially available from Affymetrix ® (Santa Clara, Calif.) under the brand name Genome-Wide Human SNP Array 5.0TM or Genome-Wide Human SNP Array 6.0TM.
  • Example arrays are shown on the Affymetrix website.
  • Another microarray supplier is illumina of San Diego, CA with example arrays shown on their website.
  • Some aspects of the invention provide methods comprising determining expression level by determining the amount of RNA encoded by KRAS, BRAF, and/or PTEN.
  • RNA encoded by KRAS, BRAF, and/or PTEN are well-adapted to such determinations.
  • microarrays in addition to providing CNV data, are easily adapted to use in determining how much RNA is being expressed in a sample.
  • One skilled in the art is aware of how to use microarrays, CGH, and a-CGH in this way (e.g., selecting different probes, etc.).
  • blotting e.g., Northern blotting
  • FISH can be used to quantitatively assess RNA expression levels.
  • Amplification techniques can also be used in measuring RNA expression levels according to the present invention by using isolated mRNA (or cDNA reverse transcribed therefrom) as the template rather than isolated genomic DNA.
  • Some aspects of the invention provide methods comprising determining expression level by determining whether a patient has a sequence variation in KRAS, BRAF, and/or PTEN that affects expression and/or activity.
  • KRAS a sequence variation in KRAS
  • BRAF cDNA reverse transcribed therefrom
  • PTEN e.g., PTEN that affects expression and/or activity.
  • One well-known, illustrative example of such a mutation is codon 12 of KRAS. When codon 12 is mutated, the resulting KRAS protein is constitutively activated.
  • Other mutations may, e.g., increase a transcript's stability and thus result in higher protein levels.
  • sequence variations in any gene of interest may provide information useful according to the invention.
  • a person's genome has been sequenced (somatic (e.g., tumor) and/or germline)
  • that sequence data may be reused in different applications, including looking for sequence variations relevant according to the present invention.
  • a particular gene of interest e.g., KRAS, BRAF, and/or PTEN
  • sequencing the whole genome especially, e.g., when looking for somatic sequence variations in tumor samples.
  • even full sequencing of an individual gene is not necessary.
  • Some aspects of the invention provide methods comprising determining expression level by determining the amount of polypeptide encoded by KRAS, BRAF, and/or PTEN.
  • the protein encoded by a target gene is a very useful gene product to be assayed in determining target gene expression levels.
  • Several useful techniques for identifying and quantifying such proteins are available to the skilled artisan including, but no limited to: electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, immunoelectrophoresis, IHC, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western Blot, etc.
  • Protein detection for tumor and cancer diagnostics and classification can be carried out by immunoassays — e.g., using antibodies directed against an expression product of a target gene.
  • an antibody that binds immunologically to PTEN protein would be particularly useful in determining the level of PTEN expression.
  • some aspects of the invention provide a method of classifying a cancer comprising (1 ) contacting a patient sample with an antibody that binds immunologically to PTEN; (2) determining the PTEN status for said patient sample; and (3) classifying said cancer based on the amount determined in (2).
  • the cancer is mCRC.
  • the antibodies (or fragments thereof) useful in the present invention can be employed histologically — e.g., IHC, immunofluorescence or immunoelectron microscopy — for in situ detection of peptides encoded by nucleic acids of interest.
  • In situ detection can be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or its fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • the biological sample that is subjected to protein detection can be brought in contact with and immobilized on a solid phase support or carrier, for example, nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles, or soluble proteins.
  • a solid phase support or carrier for example, nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles, or soluble proteins.
  • the support can then be washed with suitable buffers followed by treatment with the detectably-labeled, target gene-specific antibody.
  • the solid phase-support can then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on the solid support can then be detected by conventional means.
  • An antibody specific to the expression product of a target gene can be detectably labeled, in one aspect, by linking the same to an enzyme, for example, horseradish peroxidase, alkaline phosphatase, or glucoamylase, and using it in an enzyme immunoassay (EIA) (see, e.g. , Voller, A., 1978, The Enzyme Linked Immunosorbent Assay (ELISA), Diagnostic Horizons, 2: 1 -7; Voller et al. , J. Clin. Pathol., 31 :507-520, 1978; Butler, J. E. , Meth. Enzymol., 73:482-523, 1981 ; Maggio, E.
  • EIA enzyme immunoassay
  • Enzyme Immunoassay CRC Press, Boca Raton, FIa. , 1980; and Ishikawa et al (eds), .Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981 ).
  • the enzyme bound to the antibody reacts with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety that can be detected, for example, by spectrophotometric or fluorimetric means, or by visual inspection.
  • the present invention provides the use of antibodies against an expression product of KRAS, BRAF, and/or PTEN in cancer classification.
  • Antibodies that specifically bind to the protein and polypeptides of interest can be produced by a variety of methods.
  • Such antibodies may include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') 2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, Nanobodies, epitope-binding fragments of any of the above, etc.
  • Such antibodies can be used, for example, in the detection of the target gene (i.e., its product), or its fingerprint or pathway genes involved in a particular biological pathway, which may be of physiological or pathological importance. These potential pathways or fingerprint genes, for example, may interact with the target gene's activity and be involved in tumorigenesis. Production of such antibodies to proteins is routine in the art.
  • Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen (e.g., the target gene product) as in the present invention, can be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to the hybridoma technique of Kohler and Milstein, (Nature, 256:495-497, 1975; and U.S. Pat. No. 4,376, 1 10), the human B-cell hybridoma technique (Kosbor et a/., Immunology Today, 4:72, 1983; Cole et a/. , Proc. Natl. Acad. Sci.
  • Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • the hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
  • chimeric antibodies can be made by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity (see Morrison et a/. , Proc. Natl. Acad. Sci. USA, 81 :6851 -6855, 1984; Neuberger et a/., Nature, 312:604-608, 1984; Takeda et a/., Nature, 314:452-454, 1985; and U.S. Pat. No. 4,816,567).
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, for example, those having a variable region derived from a murine mAb and a container region derived from human immunoglobulin.
  • Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') 2 fragments that can be produced by pepsin digestion of the antibody molecule, and the Fab fragments that can be generated by reducing the disulfide bridges of the F(ab') 2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., Science, 246: 1275-1281 , 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
  • Nanobodies are small antibodies consisting of only heavy chains
  • Nanobodies are described in, e.g., Hamers- Casterman et al. , Nature (1993) 363:446-448; Roovers et al. , Cancer Immunol. Immunother. (2007) 56:303-317; Harmsen et al. , Appl. Microbiol. Biotechnol. (2007) 77: 13-22.
  • the invention provides methods of classifying a cancer by determining KRAS, BRAF, and/or PTEN status, wherein determining
  • KRAS, BRAF, and/or PTEN status comprises determining the activity level of KRAS
  • BRAF BRAF
  • PTEN PTEN
  • PTEN for example, a technique for measuring phosphatase activity is discussed in Georgescu et al. , The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region, PROC NATL ACAD SCI USA (1999)
  • phosphatase reactions are performed in 50 ⁇ l of assay buffer containing 100 mM Tris-HCI pH 8, 10 mM DTT, and 200 ⁇ M water-soluble diC 8 -PIP3 (Echelon, Salt Lake City).
  • the reactions contain either 2.5 ⁇ g glutathione S-transferase-PTEN fusion proteins or PTEN proteins immunoprecipitated on protein
  • A/G-agarose beads (Santa Cruz Laboratories) from transfected cells. After immunoprecipitation the beads are washed twice in a low-stringency buffer containing 20 mM Hepes (pH 7.7), 50 mM NaCI, 0.1 mM EDTA and 2.5 mM MgCI 2 and once in phosphatase assay buffer lacking PIP 3 . The reactions are incubated for 40 min at 37°C and transferred to a 96-well plate. The release of phosphate from the substrate is measured in a colorimetric assay by using the Biomol Green Reagent (Biomol) in accordance with the instructions of the manufacturer. The absorbance at 650 nm is recorded in an ELISA plate reader.
  • Biomol Biomol Green Reagent
  • a standard curve is performed in each assay, and the amount of free phosphate is calculated from the standard curve line- fit data.
  • BRAF and KRAS for example, a method for measuring activity is discussed in Wan et al. , Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF, CELL (2004) 1 16:855-867.
  • Other techniques for measuring the enzymatic activities of KRAS, BRAF and/or PTEN are known to those skilled in the art.
  • a protein's activity lies in whole or in part in its protein- protein interactions.
  • Techniques for determining whether and to what extent a protein interacts with other proteins are known in the art. Examples include column chromatography and co-immunoprecipitation.
  • One skilled in the art is capable of adapting these and other known techniques to determining whether and/or to what extent KRAS, BRAF and/or PTEN interact with other proteins in a sample.
  • the invention provides methods of classifying a cancer by determining KRAS, BRAF, and/or PTEN status, wherein determining
  • KRAS, BRAF, and/or PTEN status comprises determining whether at least one of these genes has at least one sequence variation.
  • sequence variation information can in turn be useful in discovering information about expression and/or activity. Those skilled in the art are aware of various techniques for gathering sequence variation information.
  • whole-genome sequencing, whole gene sequencing, genotyping, etc. are all well-suited to use in the methods of the present invention that call for determining sequence variation.
  • One skilled in the art is familiar with techniques according to these methods for determining whether a patient (or patient sample) has a sequence variation including, but not limited to, TaqMan, microarray, Sanger sequencing, etc.
  • Some aspects of the invention provide methods of treating a patient comprising the cancer classification methods discussed above.
  • the treatment methods of the invention often further comprise some action based on the determinations made in the classification steps (e.g. , prescribing or administering a particular course of treatment). For example, many courses of treatment are approved only for specific cancer types.
  • Such treatment may include administration of therapeutic compositions, surgery, etc.
  • Other actions (such as communicating, e.g., a particular diagnosis or prognosis to a patient) are also contemplated in the treatment methods of the invention.
  • a patient having a particular type of cancer may include administration of therapeutic compositions, surgery, etc.
  • Other actions such as communicating, e.g., a particular diagnosis or prognosis to a patient.
  • the invention provides a method of treatment comprising: (1 ) identifying a patient (e.g., an mCRC patient who lacks an activated
  • the invention provides a method of identifying a patient who will respond better to an M EK-inhibitor than to cetuximab, said method comprising (1 ) identifying a patient lacking /CR/AS-activated status and (2) determining the SR/AF-status of said patient, wherein an activated SR/AF-status indicates said patient is likely to respond better to MEK-inhibitors than to cetuximab.
  • PTEN(+) correlates with a good OS prognosis and PTEN(-) status correlates with a poor OS prognosis in cetuximab-treated mCRC patients.
  • the invention provides a method of determining whether to administer to a patient cetuximab or an mTOR inhibitor, said method comprising (1 ) identifying a patient lacking KRAS- activated status and (2) determining the PTEN status of said patient, wherein a
  • PTEN(+) status indicates said patient should be administered cetuximab and wherein a PTEN(-) status indicates said patient should be administered an mTOR inhibitor.
  • cetuximab is not administered if said patient has an activated BRAF status.
  • Some methods of the invention may also be practiced without direct use of a physical patient sample — i.e., some methods of the invention encompass analyzing expression, activity, or sequence data previously derived from a patient sample and contained in some medium (e.g., a computer-readable file).
  • some medium e.g., a computer-readable file.
  • a computer means is usually employed in collecting raw signal data from the arrays as well as in analyzing and converting these raw data into an intelligible CNV profile.
  • this CNV profile has been established for a patient (or a tumor sample from said patient), however, it can be analyzed and reanalyzed without the need for repeating the physical assay on the sample.
  • practicing the methods of the invention can be temporally and physically quite removed from any physical patient sample. This is equally true for expression, activity and sequence variation.
  • results of these and any other analyses according to the invention will often be communicated to physicians, genetic counselors and/or patients (or other interested parties such as researchers). Specifically the results can be cast in a transmittable form that can be communicated or transmitted to any of the above parties. Such a form can vary and can be tangible or intangible.
  • the results can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, graphs showing expression level, activity level, and/or sequence variation information for any of KRAS, BRAF and/or PTEN can be used in explaining the results. Diagrams showing, e.g., where a variant occurs in these or some additional target genes are also useful in indicating some testing results.
  • statements and visual forms can be recorded on a tangible medium such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible medium, e.g. , an electronic medium in the form of email or website on internet or intranet.
  • results can also be recorded in a sound form and transmitted through any suitable medium, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.
  • the information and data on a test result can be produced anywhere in the world and transmitted to a different location.
  • the information and data on a test result may be generated, cast in a transmittable form as described above, and then imported into the United States.
  • the present invention also encompasses a method for producing a transmittable form of information on at least one of the (a) expression level profile, (b) activity level profile, or (c) sequence variation profile of at least one patient sample.
  • the method comprises the steps of (1 ) making at least one of the determination (a), (b), or (c) above according to methods of the present invention; and (2) embodying the result of the determining step in a transmittable form.
  • the transmittable form is the product of the production method.
  • Some aspects of the invention comprise a kit useful in making one or more of the determinations (e.g., expression level, activity level, sequence variation) discussed above.
  • the kit may include a carrier for the various components of the kit in the form of, e.g. , bag, box, tube, rack, and is optionally compartmentalized. Often the kit will include instructions on its use (e.g. , how to run the particular reactions, care instructions for the various reagents, how to interpret the results, etc.).
  • the detection kit includes one or more oligonucleotides useful in detecting, quantitating, sequencing, genotyping, etc. KRAS, BRAF, and/or PTEN.
  • oligonucleotides can be used in any of the hybridization, amplification, and/or sequence variation-detecting techniques discussed above.
  • the oligonucleotides in this embodiment preferably have a nucleotide sequence that matches at least part of the nucleotide sequence (or complement thereof) of KRAS, BRAF, and/or PTEN. Under most conditions, a length for the oligonucleotide of 18 to 30 may be optimum.
  • the oligonucleotides should be designed such that they can be used in distinguishing one nucleotide sequence variant from another at a particular locus under predetermined stringent hybridization conditions.
  • the kit includes probes (e.g. fluorescent probes such as TaqMan probes) suitable for detecting a mutation in KRAS and/or in BRAF, preferably one of the activating mutations listed hereabove.
  • probes e.g. fluorescent probes such as TaqMan probes
  • the kit may further comprise primers for detecting a mutation in KRAS and/or in BRAF. Such primers allow amplifying the region comprising the mutation, which may then be detected by sequencing or by using fluorescent probes.
  • the kit includes one or more oligonucleotides suitable for use in detecting techniques such as ARMS, oligonucleotide ligation assay (OLA), and the like.
  • oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc.
  • the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.
  • the detection kit contains one or more antibodies that binds immunologically (or alternatively is specifically immunoreactive) with KRAS, BRAF, and/or PTEN proteins.
  • the kit may further comprise an antibody that binds immunologically (or alternatively is specifically immunoreactive) with proteins or polypeptides encoded by additional target genes.
  • the kit may also comprise an antibody specific for specific amino acid variants of any of these proteins. Methods for producing and using such antibodies are known in the art and discussed above.
  • the detection kit of this invention may also be included in the detection kit of this invention.
  • examples of such components include, but are not limited to, Taq polymerase, deoxyribonucleotides, dideoxyribonucleotides other primers suitable for the amplification of a target DNA sequence, RNase A, mutS protein, and the like.
  • the detection kit preferably includes instructions on using the kit for detecting nucleotide variants in auxiliary gene sequences.
  • the kit may also include one or more microarrays.
  • microarrays include, but are not limited to, high-density SNP microarray chips - such as the Genome-Wide Human SNP Array 5.0TM or Genome-Wide Human SNP Array 6.0TM from Affymetrix ® of Santa Clara, CA or those available from illumina of San Diego, CA — useful in determining CNV profiles for tumor samples, expression microarray chips, etc.
  • the kit may further comprise a computer system and or a computer program product of the present invention for copy number profile comparison and analysis, which are described above.
  • the kit for classifying cancer comprises: (a) reagents for detecting mutations in KRAS (e.g. primers and/or fluorescent probes such as TaqMan probes);
  • reagents for detecting mutations in BRAF e.g. primers and/or fluorescent probes such as TaqMan probes
  • reagents for determining PTEN expression level e.g. an antibody suitable for detection by IHC or ELISA.
  • Such a kit preferably comprises (a) and (c), (a) and (b), or (a), (b) and (c).
  • Another aspect of the invention pertains to an anti-EGFR antibody for use in the treatment of individuals suffering from colon or colorectal cancer wherein the tumor cells have a PTEN(+) phenotype.
  • the tumor cells have a wild-type KRAS genotype and/or a wild-type BRAF genotype.
  • Still another aspect of the invention pertains to an anti-EGFR antibody for use in the treatment of individuals suffering from colon or colorectal cancer wherein the tumor cells have a wild-type KRAS genotype and a wild-type BRAF genotype.
  • tumor cells having a PTEN(+) phenotype is meant tumor cells expressing PTEN.
  • tumors cells having a wild-type KRAS genotype or “tumors cells having a wild-type BRAF genotype” is meant tumors cells, the genotype of which is devoid of activating mutations in the KRAS gene or the BRAF gene.
  • BRAF V600E mutation detection was assessed by allelic discrimination using Taqman probes following the same protocol as KRAS mutation.
  • ZytoDot SPEC EGFR Probe digoxigenylated (CE-Marked), the ZytoDot Pretraitment kit and the ZytoDot CISH Polymer detection kit according to the protocol given by the supplier (Zytovision, Clinisciences, Montrouge, France).
  • the DNA probe and sections were denatured at 95°C and hybridized at 37°C overnight using a HYBriteTM instrument.
  • An overlook was made with an X40 dry objective to assess the heterogeneity of the staining on all tumour cells and 60 nucleus were scored with a X60 oil objective in at least 4 areas by two observers independently (AC/FPL).
  • tumours with four or more copies of the EGFR gene were controlled by FISH. All other tumours were considered to be FISH negative.
  • FISH experiments were performed using EGFR specific sequence probe (LSI® EGFR) and control chromosome enumeration probe 7 (CEP7) according to the manufacturers' recommended protocol (Vysis-Abbott Molecular Diagnostics, Rungis, France), but with some minor modifications.
  • the DNA probes and tissue sections were denatured at 85°C for 5 min using a HYBrite TM instrument. An additional wash in distilled water was added before counterstaining and mounting with a solution of 4, 6-diamidino-2-phenylindole (DAPI).
  • DAPI 4, 6-diamidino-2-phenylindole
  • tumours with four or more copies of the EGFR gene i n ⁇ 40% of the cells (high polysomy) or tumours with EGFR gene amplification were considered to be FISH positive, whereas all other tumours were considered to be FISH negative.
  • PTEN expression As defined previously by Hirsch et al. (Increased EGFR gene copy number detected by fluorescent in situ hybridization predicts outcome in non-small-cell lung cancer patients treated with cetuximab and chemotherapy. J Clin Oncol 26:3351 -7, 2008), tumours with four or more copies of the EGFR gene i n ⁇ 40% of the cells (high polysomy) or tumours with EGFR gene amplification (gene-to-chromosome ratio ⁇ 2 or presence of gene cluster or ⁇ 10 gene copies in ⁇ 10% of the cells) were considered to be FISH positive, whereas all other tumours were considered to be FISH negative.
  • PTEN expression As defined previously by Hirsch et al. (Increased EGFR gene copy number detected by fluorescent in situ hybrid
  • KRAS mutations are reproducibly associated with outcome after cetuximab therapy, it is important to adjust for KRAS status when looking for other molecular determinants of drug efficacy. This adjustment should preclude the chance of identifying false associations that may result from the uneven distribution of KRAS mutations in subsequent partitions of the patient cohort. Therefore, when we considered whether BRAF mutations were associated with drug efficacy, we restricted our analysis to KRAS wild-type patients only.
  • the V600E mutation in BRAF gene was determined in the entire series. Five tumours (3%) out of 171 were mutated, as expected all were found in KRAS wild-type tumours. BRAF mutations were tested for association with treatment outcome.
  • EGFR amplification status was determined in 138 of 173 cases. The high dropout rate was due to a technical problem related to the fixation process, and therefore, is not expected to create patient ascertainment bias.
  • EGFR high polysomy or amplification FISH positive phenotype
  • FISH positive phenotype was found in 22 cases. Among them, 17 (77.3%) occurred in the 96 KRAS wild-type tumours (17.7%).
  • Restricting our analysis to KRAS wild-type tumours only, association between EGFR high polysomy or amplification and treatment outcome was tested. In the KRAS wild-type sub-group, EGFR FISH positive phenotype was associated with higher response rate (71 %) than seen in tumours with normal EGFR copy number (37%). This difference was significant (p-value 0.015, Table 1A). In addition, there was a trend towards longer PFS and OS in patients with FISH positive phenotype, but the difference did not reach the threshold of significance (Table 1A).
  • PTEN status was determined by IHC in 162 samples.
  • the evaluation of PTEN cytoplasmic as a continuous trait did not demonstrate any significant association with response or PFS or OS.
  • the PTEN status for each patient was defined as a dichotomous trait of either expressing PTEN in the cytoplasmic compartment of the cell (i.e. any positive tumour cells) or lacking PTEN expression.
  • Thirty-one tumours (19%) were negative for PTEN expression in the tumour cell cytoplasmic compartment.
  • 22 (19.8%) occurred in the group of 1 1 1 wild-type KRAS patients.
  • PTEN(+) status was associated with a longer OS. This effect was not significant in the KRAS or BRAF mutant patient population. PTEN expression was not associated with response, or with length of PFS in either KRASIBRAF mutant or non-mutant populations. Although it was not significant, we found that responding patients with PTEN(-) tumors survived longer than non-responding patients with PTEN(-) tumors. PTEN status was not predictive of survival in patients with KRAS or BRAF mutant tumors. In this study, we have identified molecular determinants of response, PFS and/or OS in the context of cetuximab treatment.
  • a multivariate analysis of response and OS was performed to determine the respective roles of EGFR, BRAF and PTEN in determining treatment outcome for colon cancer patients with KRAS wild-type tumours.
  • the multivariate analysis also included adjustments for other variables such as gender, age, number of previous chemotherapy regimens before the use of anti-EGFR therapy, and type of tumour material used for the determination of biomarker status (i.e. primary or metastatic tumour).
  • EGFR and BRAF were significant predictors of response even after adjustment for all other covariates (Table 2A). This association was also significant for EGFR in the subset of patients who were wild- type for both KRAS and BRAF (Table 2B).
  • PTEN and BRAF were joint predictors of overall survival (Table 3A and 3B).
  • BRAF BRAF
  • BRAF was an independent predictor of response (as could be deduced from the fact that no patient carried both mutations).
  • BRAF was also an independent predictor of PFS.
  • predictive models that included BRAF were significantly better predictors than KRAS only models.
  • Skin toxicity was weakly associated with OS after including KRAS (p-value 0.02).
  • BRAF and PTEN were completely independent predictors of OS. In fact, the predictive power of PTEN expression was only evident in the KRAS WT population.
  • KRAS activating mutations in KRAS are strong predictors of resistance to cetuximab.
  • KRAS is not a perfect predictor since only 40% to 60% of patients with wild-type KRAS tumours respond to treatment.
  • KRAS wild-type patients 1 ) mutations in BRAF are associated with response, PFS, and OS; 2) EGFR amplification is associated with response; and 3) PTEN protein expression is associated with OS.
  • KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer, CANCER RES . (2006) 66:3992-3995; Di Fiore et al. , Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy, BR. J .
  • KRAS is not a perfect predictor since only 40-50% of patients with wild- type KRAS respond to treatment.
  • BRAF are /CR/AS-independent predictors of response, PFS, and OS.
  • PTEN protein expression is a /CR/AS-independent predictor of OS.
  • BRAF mutations predict benefit from cetuximab.
  • BRAF is an important regulatory molecule in the RAS signaling pathway, situated between RAS and MEK in the canonical kinase cascade.
  • BRAF mutations have been reported to occur in 10% of colon cancer cases (Barault et al. , Mutations in the RAS-MAPK, PI(3)K (phosphatidylinositol-3-OH kinase) signaling network correlate with poor survival in a population-based series of colon cancers, INT. J. CANCER (2008) 122:2255-2259). However, mutations in the present cohort were less frequent (-3%).
  • BRAF status should be considered before considering anti-EGF R therapies for mCRC patients.
  • I ndeed regardless of mutation frequency, the effect on the individual patient could be significant.
  • BRAF provides /CR/AS-independent information about response, it may ultimately be true that both genes should be screened for mutations before prescribing cetuximab.
  • BRAF mutations have been shown to be associated with sensitivity to MEK inhibitors (Solit et al. , BRAF mutation predicts sensitivity to MEK inhibition, NATURE (2006) 439:358-362). Perhaps this class of drug will prove highly effective in KRAS or BRAF mutant colon cancer.
  • the frequency of BRAF mutations is expected to be higher, and therefore more clinically relevant, i n patients with MS I tu mou rs or (assu m ing anti-EGFR therapies a re eventually approved for adjuvant care) in patients with stage I I I CRC, where up to 16% of tumours could carry somatic BRAF mutations.
  • PTEN expression is associated with OS in a cetuximab-treated cohort. This association does not appear to be related to response. In fact, some PTEN(-) tumors respond to treatment, but in the end, these patients have worse outcomes compared to patients whose tumors have detectable PTEN expression. These data strongly support the view that PTEN is prognostic (i.e.

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Abstract

L'invention porte de manière générale sur des diagnostics moléculaires et plus particulièrement sur des marqueurs moléculaires permettant de prévoir la réponse et la survie de patients qui suivent une thérapie anti-EGFR, ainsi que sur des procédés d'utilisation correspondants.
PCT/EP2009/057394 2008-06-13 2009-06-15 Marqueurs permettant de prévoir la réponse et la survie chez des patients subissant un traitement anti-egfr WO2009150256A1 (fr)

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