WO2013092960A1 - Procédés et kits destinés au diagnostic du cancer colorectal - Google Patents

Procédés et kits destinés au diagnostic du cancer colorectal Download PDF

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WO2013092960A1
WO2013092960A1 PCT/EP2012/076562 EP2012076562W WO2013092960A1 WO 2013092960 A1 WO2013092960 A1 WO 2013092960A1 EP 2012076562 W EP2012076562 W EP 2012076562W WO 2013092960 A1 WO2013092960 A1 WO 2013092960A1
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Prior art keywords
ddx55
cmtm7
fam60a
madcam1
encl
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PCT/EP2012/076562
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English (en)
Inventor
Amaia GARCÍA BILBAO
Guillermo M. López Vivanco
Iñaki Inza Cano
Mónica Betanzos Avellaneda
Paloma Aldamiz-Echebarria Zulueta
Rubén Armañanzas Arnedillo
Pedro Larrañaga Múgica
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Fundacion Gaiker
Administración General De La Comunidad Autónoma De Euskadi - Osakidetza
Universidad Del País Vasco
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Publication of WO2013092960A1 publication Critical patent/WO2013092960A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention is within the field of diagnosis, more specifically, in the field of diagnosis of colorectal cancer by means of using panels of genes the expression of which is altered in CRC samples compared to samples from a healthy subject.
  • Colorectal cancer is one of the most frequent neoplasias in the western world, it is the third cause of death in men, after lung cancer and prostate cancer and it is the second in frequency among women, after breast cancer.
  • CRC Colorectal cancer
  • Spain approximately 25,000 new cases are diagnosed every year, which constitutes an incidence of 50 new cases for every 100,000 inhabitants and year (www.aecc.org).
  • FFNI Mediterranean diet
  • L., et al., 2007 the Mediterranean diet
  • WO07032631 describes a method for the diagnosis of a colorectal tumor based on the determination of alterations in the expression levels of genes CTHRC1, CANP and KIAA0101.
  • WO06015742 describes a method for the diagnosis of a colorectal carcinoma based on the determination of the expression levels of a panel of 120 genes.
  • WO05044990 describes a method for the diagnosis of a colorectal carcinoma based on the determination of the expression levels of a panel of 93 genes.
  • EP1439393 describes a method for the diagnosis of colorectal carcinoma based on the determination of the expression levels of TIMP1.
  • WO03097872 describes a series of genes the expression levels of which are altered in colorectal carcinoma samples as well as methods for the diagnosis of colorectal carcinoma based on the determination of the expression levels of said genes.
  • WO04001072 describes methods for the diagnosis of colorectal cancer based on the determination of the expression levels of 52 genes the expression of which is high in colorectal carcinomas and in 376 genes the expression of which is repressed in colorectal carcinomas with respect to normal colorectal epithelium.
  • EP1355151 describes a method for the diagnosis of colorectal cancer based on the determination of the expression levels of a set of 39 genes.
  • the invention relates to a method for diagnosing colorectal cancer comprising determining the level of the expression of one or more genes selected from the group consisting of ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55 in a biofluid of a subject, wherein a change in the level of expression of said genes compared to the level in a reference value is indicative of colorectal cancer.
  • the invention in a second aspect, relates to a method for monitoring the effect of a therapy in a patient suffering from colorectal cancer and being treated with said therapy, comprising determining the levels of the expression of one or more genes selected from the group consisting of ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55 in a biofluid of said patient, wherein a change of the level of expression of said genes compared to the level in a reference value indicates a positive effect of said therapy.
  • invention in a third aspect, relates to a kit comprising a set of reagents which allow determining the expression levels of genes one or more genes selected from the group consisting of ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55.
  • the invention relates to a method for diagnosing colorectal cancer comprising determining the level of the expression of one or more genes selected from the group consisting ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55 in a biofluid of a subject, wherein a change in the level of expression of said genes compared to the level in a reference value is indicative of colorectal cancer.
  • the expression "method for the diagnosis” relates to a method that may essentially consist of the previously mentioned steps or may include additional steps.
  • the method in a preferred embodiment, is a method that is carried out in vitro, i.e., it is not carried out in the human or animal body.
  • Diagnosing as used herein relates to evaluating the probability according to which a subject suffers from a disease. As will be understood by persons skilled in the art, such evaluation, although it is preferred that it is, normally may not be correct for 100% of the subjects to be diagnosed. The term, however, requires that a statistically significant part of the subjects can be identified as suffering from the disease or having a predisposition for it.
  • the person skilled in the art can determine if a part is statistically significant without further delay by using several well known statistic evaluation tools, for example, determination of confidence intervals, determination of the p value, Student's t-test, Mann- Whitney test, etc. The details are in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • the preferred confidence intervals are of at least 50%, at least 60%, at least 70%>, at least 80%>, at least 90%>, at least 95%.
  • the p values are preferably p ⁇ 0.05, 0.01, etc.
  • colonal cancer includes any type of colon, rectal and appendix neoplasia and refers both to early and late adenomas and to carcinomas as well as to the hereditary, familial or sporadic cancer.
  • Hereditary CRC includes those syndromes which include the presence of polyps, such as the hamartomatous polyposis syndromes and the most known, familial adenomatous polyposis (FAP) as well as nonpolyposis syndromes such as hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome I.
  • said colorectal cancer is colon cancer, rectal cancer and/or vermiform appendix cancer.
  • said colorectal cancer is stage 0, stage I [please let us know whether you can provide any evidence for stages 0 and I], stage II, stage III and/or stage IV colorectal cancer.
  • the stages of CRC referred to herein correspond to the American Joint Committee on Cancer (AJCC) CRC staging, although other staging methods, such as Dukes and Astler-Coller staging, can equally be used.
  • AJCC American Joint Committee on Cancer
  • Stage 0 refers to the cancer in the earliest stage. It has not grown beyond the inner layer (mucosa) of the colon or rectum. This stage is also known as carcinoma in situ or intramucosal carcinoma.
  • Stress I refers to the cancer having grown through the muscularis mucosa into the submucosa or also having grown into the muscularis basement. It has not spread to nearby lymph nodes or distant sites.
  • “Stage ⁇ " including sub-stages A, B and C refers to the cancer (A) grown into the outermost layers of the colon or rectum but has not gone through them; it has not reached nearby organs and it has not yet spread to the nearby lymph nodes or distant sites; (B) grown through the wall of the colon or rectum but has not grown into other nearby tissues or organs; it has not yet spread to the nearby lymph nodes or distant sites; and/or (C) grown through the wall of the colon or rectum and is attached to or has grown into other nearby tissues or organs; it has not yet spread to the nearby lymph nodes or distant sites.
  • “Stage III” including sub-stages A, B and C refers to the following: (A) At least one of the following applies: (i) The cancer has grown through the mucosa into the submucosa and it may also have grown into the muscularis propria. It has spread to 1 to 3 nearby lymph nodes or into areas of fat near the lymph nodes but not the nodes themselves. It has not spread to distant sites, (ii) The cancer has grown through the mucosa into the submucosa. It has spread to 4 to 6 nearby lymph nodes. It has not spread to distant sites. (B) At least one of the following applies: (i) The cancer has grown into the outermost layers of the colon or rectum or through the visceral peritoneum but has not reached nearby organs.
  • the cancer has spread to 1 to 3 nearby lymph nodes or into areas of fat near the lymph nodes but not the nodes themselves. It has not spread to distant sites, (ii) The cancer has grown into the muscularis intestinal or into the outermost layers of the colon or rectum. It has spread to 4 to 6 nearby lymph nodes. It has not spread to distant sites, (iii) The cancer has grown through the mucosa into the submucosa or it may also have grown into the muscularis basement. It has spread to 7 or more nearby lymph nodes. It has not spread to distant sites. (C) At least one of the following applies: (i) The cancer has grown through the wall of the colon or rectum (including the visceral peritoneum) but has not reached nearby organs.
  • the cancer has spread to 4 to 6 nearby lymph nodes. It has not spread to distant sites, (ii) The cancer has grown into the outermost layers of the colon or rectum or through the visceral peritoneum but has not reached nearby organs. It has spread to 7 or more nearby lymph nodes. It has not spread to distant sites, (iii) The cancer has grown through the wall of the colon or rectum and is attached to or has grown into other nearby tissues or organs. It has spread to at least one nearby lymph node or into areas of fat near the lymph nodes. It has not spread to distant sites.
  • “Stage IV” including sub-stages A and B refers to the following: (A) The cancer may or may not have grown through the wall of the colon or rectum, and it may or may not have spread to nearby lymph nodes. It has spread to 1 distant organ (such as the liver or lung) or set of lymph nodes. (B) The cancer may or may not have grown through the wall of the colon or rectum, and it may or may not have spread to nearby lymph nodes. It has spread to more than 1 distant organ (such as the liver or lung) or set of lymph nodes, or it has spread to distant parts of the peritoneum (the lining of the abdominal cavity).
  • bio fluid is a biological fluid and refers to an aqueous fluid of biological origin. It may be obtained from any location (such as blood, plasma, serum, urine, bile, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion).
  • the biofluid is blood, plasma or serum. In a more preferred embodiment, it is blood.
  • subject refers to all animals classified as mammals and includes, but is not restricted to, domestic and farm animals, primates and humans, e.g., human beings, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.
  • the patient is a male or female human of any age or race.
  • a subject may or may not have or suffer from CRC. In other words, it is not known whether the subject has or suffers from CRC.
  • Marker genes in the diagnostic method of the invention include gene CMTM7 (CKLF-like MARVEL transmembrane domain containing 7 NM 138410)), FAM60A (family with sequence similarity 60, member A, NM_021238), ENC1 (ectodermal neural cortex- 1, NM_003633), AC ATI (acetyl-coenzyme A acetyltransferase 1, NM_000019), MADCAM1 (mucosal vascular addressing cell adhesion molecule 1, NM 130760) and gene DDX55 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 55, NM 020936).
  • CMTM7 CKLF-like MARVEL transmembrane domain containing 7 NM 138410
  • FAM60A family with sequence similarity 60, member A, NM_021238
  • ENC1 ectodermal neural cortex- 1, NM_003633
  • AC ATI acetyl
  • the methods of the invention preferably comprise the simultaneous determination of the expression levels of genes ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55.
  • the invention contemplates the use of a limited number of said genes, including cancer diagnosis methods in which the expression of genes ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55 is individually determined as well as diagnosis methods based on the determination of each of said genes individually as well as of subsets of markers formed by a combination of any two genes, any three genes, any four genes, any five genes or the six genes.
  • the methods of the invention consist of the determination of the expression level of ENC1.
  • the gene or genes used in the method of the invention are those which can diagnose colorectal cancer with an accuracy rate of at least 90%, at least 91%, at least at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%.
  • accuracy rate is defined as the condition of being true, exact or correct, that is the degree of agreement between the classification of a subject as healthy or illness and the real condition of said subject.
  • the invention contemplates methods based on the determination of the level of a set of genes selected from the group consisting of ENCl and DDX55; ENCl and FAM60A; ENCl, FAM60A and DDX55; ENCl and CMTM7; ENC1,CMTM7 and DDX55; ENCl, CMTM7 and MADCAM1 ; ENCl and MADCAM1; ENCl, MADCAM1 and DDX55; ENCl, ACATl and MADCAM1; ENCl, ACATl, MADCAM1 and DDX55; ENCl, ACATl, CMTM7 and MADCAM1; ENCl, ACATl, CMTM7, MADCAM1 and DDX55; ENCl, ACATl, CMTM7, MADCAM1 and DDX55; ENCl, ACATl, CMTM7, FAM60A and MADCAM1; ENCl, CMTM7, MADCAM1 and DDX55
  • the invention contemplates methods based on the determination of the level of a set of genes selected from the group consisting of ACATl, MADCAM1 and DDX55; ACATl and MADCAM1; CMTM7 and MADCAM1; CMTM7, MAD CAM 1 and DDX55; CMTM7, FAM60A and MADCAM1; CMTM7, FAM60A, MADCAM1 and DDX55, ACATl, CMTM7, MADCAM1 and DDX55; ACATl, CMTM7 and MADCAM1; MADCAM and DDX55; FAM60A and MADCAM1; FAM60A, MADCAM 1 and DDX55.
  • a "change” as used herein, means an alteration of the expression level of the genes, for example an increase or a decrease.
  • the expression of a gene is considered increased in a sample of the subject under study when the levels increase with respect to the reference value by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, by at least 110%, by at least 120%, by at least 130%, by at least 140%, by at least 150%, or more.
  • the expression of a gene is considered decreased when its levels decrease with respect to the reference value by at least 5%, by at least 10%>, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100% (i.e., absent).
  • a “reference value”, as used herein, means a value determined in a sample obtained from a pool of healthy subjects which does not have a disease state or particular phenotype or from a bio fluid of said patient determined at the start of the therapy and/or determined previously during said therapy.
  • the suitable reference expression levels of genes can be determined by measuring the expression levels of said genes in several suitable subjects, and such reference levels can be adjusted to specific subject populations (for example, a reference level can be linked to the age so that comparisons can be made between expression levels in samples of subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group).
  • the reference value is obtained from several healthy subjects or from subjects without prior history of colorectal cancer.
  • the person skilled in the art will appreciate that the type of reference value can vary depending on the specific method to be performed
  • the expression profile of the genes in the reference value can preferably, be generated from a population of two or more individuals.
  • the population for example, can comprise 3, 4, 5, 10, 15, 20, 30, 40, 50 or more individuals.
  • the expression profile of the genes in the reference value and in the sample of the individual that is going to be diagnosed according to the methods of the present invention can be generated from the same individual, provided that the profiles to be assayed and the reference profile are generated from bio fluids taken at different times and are compared to one another. For example, a sample of an individual can be obtained at the beginning of a study period. A reference biomarker profile from this sample can then be compared with the biomarker profiles generated from subsequent samples of the same individual.
  • the reference value is from a pool of samples from several individuals.
  • the methods of the invention comprise the step of determining the expression levels of the marker genes.
  • any conventional method can be used within the frame of the invention to detect and quantify the levels of mRNA encoded by the marker genes.
  • the expression levels are determined by means of the quantification of the levels of mRNA encoded by said genes.
  • the latter can be quantified by means of using conventional methods, for example, methods comprising the amplification of mRNA and the quantification of the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Northern blot and the use of suitable probes, Northern blot and use of specific probes of the mRNA of the genes of interest or of their corresponding cDNA/cRNA, mapping with the SI nuclease, RT-PCR, hybridization, microarrays, etc.
  • conventional methods for example, methods comprising the amplification of mRNA and the quantification of the amplification product of said mRNA, such as electrophoresis and staining, or alternatively, by means of Northern blot and the use of suitable probes, Northern blot and use of specific probes of the mRNA of the genes of interest or of their corresponding cDNA/cRNA, mapping with the SI nuclease, RT-PCR, hybridization, microarrays, etc.
  • the levels of the cDNA/cRNA corresponding to said mRNA encoded by the marker genes can also be quantified by means of using conventional techniques; in this event, the method of the invention includes a step of synthesis of the corresponding cDNA by means of reverse transcription (RT) of the corresponding mRNA followed by the synthesis (RNA polymerase) and amplification of the cRNA complementary to said cDNA.
  • RT reverse transcription
  • RNA polymerase RNA polymerase
  • control RNA relates to RNA whose expression levels do not change or change only in limited amounts in tumour cells with respect to non-tumorigenic cells.
  • the control RNA is mRNA derived from housekeeping genes and which code for proteins which are constitutively expressed and carry out essential cellular functions.
  • housekeeping genes for use in the present invention include 18-S ribosomal protein, ⁇ -2- microglobulin, ubiquitin, cyclophilin, GAPDH, PSMB4, tubulin and ⁇ -actin.
  • the expression levels of the marker genes by means of the determination of the expression levels of the proteins encoded by said genes, since if the expression of genes is increased, an increase of the amount of corresponding protein should occur and if the expression of genes is decreased, a decrease of the amount of corresponding protein should occur.
  • the determination of the expression levels of the different proteins can be carried out using any conventional method.
  • said determination can be carried out using antibodies with the capacity for binding specifically to the protein to be determined (or to fragments thereof containing the antigenic determinants) and subsequent quantification of the resulting antigen-antibody complexes.
  • the antibodies that are going to be used in this type of assay can be, for example polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab' and F(ab')2, scFv, diabodies, triabodies, tetrabodies and humanized antibodies.
  • the antibodies may or may not be labeled.
  • markers include radioactive isotopes, enzymes, fluorophores, chemo luminescent reagents, enzyme cofactors or substrates, enzyme inhibitors, particles, dyes, etc.
  • non-labeled antibodies primary antibody
  • labeled antibodies secondary antibodies
  • these techniques include Western-blot or immunoblot, ELISA (enzyme- linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or protein microarrays including specific antibodies or assays based on the colloidal precipitation in formats such as reagent strips.
  • Other forms of detecting and quantifying the protein include affinity chromatography techniques, ligand-binding assays, etc.
  • the gene expression profiles of the invention can be used in combination with other genetic and non-genetic markers already known for the diagnosis of CRC.
  • the invention contemplates the use of the expression profiles previously mentioned together with the determination of the expression levels of one or several additional markers such as serum markers (for example, carcinoembrionic antigen) or analytes such as CA19-9, CA 125, CK-BB and the guanylyl cyclase C or together with methods based on the detection of blood in feces or the detection in feces of polymorphisms in genes K-ras, APC, p53 and BAT-26.
  • serum markers for example, carcinoembrionic antigen
  • analytes such as CA19-9, CA 125, CK-BB and the guanylyl cyclase C
  • the invention in a second aspect, relates to a method for monitoring the effect of a therapy in a patient suffering from colorectal cancer and being treated with said therapy, comprising determining the levels of the expression of ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55 in a bio fluid of said patient, wherein a change of the level of expression of said genes compared to the level in a reference value indicates a positive effect of said therapy
  • therapy refers to the attempted remediation of a health problem, usually following a diagnosis. As such, it is not necessarily a cure, i.e. a complete reversion of a disease. Said therapy may or may not be known to have a positive effect on CRC or, in other words, to be useful to treat CRC.
  • Therapies include, without limitation, surgery, radiotherapy, chemotherapy, or targeted therapies (e.g. using antibodies) and the choice of a therapy or a combination of therapies depends, for example, on the stage of the cancer, whether the cancer has recurred and the patient's general health.
  • surgery means any therapeutic procedure that involves methodical action of the hand or of the hand with an instrument, on the body of a human or other mammal, to produce a curative or remedial.
  • radiotherapy refers to multiple types of radiation therapy including internal and external radiation therapies or radio immunotherapy, and the use of various types of radiations including X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiations.
  • the therapy is neoadjuvant or adjuvant chemotherapy.
  • neoadjuvant therapy refers to any type of treatment of cancer given prior to surgical resection of the primary tumor, in a patient affected.
  • chemotherapy refers to the use of a chemical drug or a combination thereof for the treatment of cancer, tumors or malign neoplasia, including both cytotoxic or cytostatic drugs.
  • chemotherapy agents which may be in accordance to the present invention include: alkylating agents (for example mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, streptozocin, carmustine, lomustine, melphalan, busulfan, dacarbazine, temozolomide, thiotepa or altretamine); platinum drugs (for example cisplatin, carboplatin or oxaliplatin); antimetabolite drugs (for example 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine or pemetrexed); anti-tumor antibiotics (for example daunorubicin,
  • Suitable chemotherapy agents include but are not limited to alkylating agents such as for example, cyclophosphamide, carmustine, daunorubicin, mechlorethamine, chlorambucil, nimustine, melphalan and the like; anthracyclines, such as for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin and the like; taxane compounds, such as, for example, paclitaxel, docetaxel and the like; topoisomerase inhibitors such as for example, etoposide, teniposide, tuliposide, irinotecan and the like; nucleotide analogs such as for example, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate,
  • antitumor agent is understood as that chemical, physical or biological agent or compound with antiproliferative, antioncogenic and/or carcinostatic properties which can be used to inhibit tumor growth, proliferation and/or development.
  • antitumor agents which can be used in the present invention are (i) antimetabolites, such as antifolates and purine analogs; (ii) natural products, such as antitumor antibiotics and mitotic inhibitors; (iii) hormones and antagonist thereof, such as androgens and corticosteroids; and (iv) biological agents, such as viral vectors.
  • antimetabolites such as antifolates and purine analogs
  • natural products such as antitumor antibiotics and mitotic inhibitors
  • hormones and antagonist thereof such as androgens and corticosteroids
  • biological agents such as viral vectors.
  • the antitumor agent comprises antiangiogenic agents and signaling pathway inhibitors that, in another more particular embodiment, the antiangiogenic agent is Bevacizumab and the signaling pathway inhibitor is Cetuximab, Panitumumab or Erlotinib.
  • the reference value is from a biofluid of said patient determined at the start of the therapy and/or determined previously during said therapy.
  • Marker genes in the diagnostic method of the invention include gene CMTM7 (CKLF-like MARVEL transmembrane domain containing 7 NM 138410)), FAM60A (family with sequence similarity 60, member A, NM_021238), ENCl (ectodermal neural cortex- 1, NM_003633), ACATl (acetyl-coenzyme A acetyltransferase 1, NM_000019), MADCAMl (mucosal vascular addressing cell adhesion molecule 1, NM 130760) and gene DDX55 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 55, NM 020936).
  • the method is carried out by determining a set of genes wherein said set of genes is selected from the group consisting of ENCl and DDX55; ENCl and FAM60A; ENCl, FAM60A and DDX55;ENC1 and CMTM7; ENC1,CMTM7 and DDX55; ENC1,CMTM7 and MADCAMl; ENCl and MADCAMl; ENCl, MADCAMl and DDX55; ENCl, ACATl and MADCAMl; ENCl, ACATl, MADCAMl and DDX55; ENCl, ACATl, CMTM7 and MADCAMl; ENCl, ACATl, CMTM7, MADCAMl and DDX55; ENCl, ACATl, CMTM7, MADCAMl and DDX55; ENCl, ACATl, CMTM7, FAM60A and MADCAMl; ENCl, CMTM7, MADCAMl and DDX55; ENC
  • the method is carried out by determining a set of genes selected from the group consisting of ACATl, MADCAMl and DDX55;ACAT1 and MADCAMl; CMTM7 and MADCAMl; CMTM7, MADCAMl and DDX55; CMTM7, FAM60A and MADCAMl; CMTM7, FAM60A,MADCAM 1 and DDX55, ACAT1,CMTM7,MADCAM1 and DDX55; ACATl, CMTM7 and MADCAMl; MADCAM and DDX55; FAM60A and MADCAMl; FAM60A, MADCAMl and DDX55.
  • targeted therapy refers to a type of medication that blocks the growth of or kills cancer cells by interfering with specific targeted molecules in or on tumour cells rather than by simply interfering with rapidly dividing cells (e.g. with traditional chemotherapy).
  • the main agent types used for targeted therapies are small molecules and antibodies.
  • patient refers to a subject as defined above which suffers and is known to suffer from CRC.
  • Start of said therapy means a time point just before the therapy is applied, preferably immediately before, e.g. before and on the same or the previous day.
  • priorly refers to any prior time point. Preferably though it means at least 1 day, at least 2 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 10 days, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 2 months or at least 3 months before.
  • step (i) can be compared to more than one level of step (ii), wherein the more than one levels of step (ii) are preferably determined at different time points, e.g. any of the time points recited above.
  • “Stagnation” preferably means a level within a 3%, a 5%, an 8%, a 10%, a 15%, or a 20%) range of a level determined before, e.g. previously as defined above. In a preferred embodiment, this "level determined before” is the value determined at the start of the therapy.
  • a “positive effect of a therapy” means that said therapy slows down disease progression, prevents disease progression, reduces or eliminates tumour burden (i.e. size and/or number of tumours), and/or cures the patient. It may also mean an increase in the duration of Recurrence-Free interval (RFI), an increase in the time of survival or Overall Survival (OS), an increase in the time of Disease-Free Survival (DFS), an increase in the duration of Distant Recurrence-Free Interval (DRFI), and/or the like.
  • RFI Recurrence-Free interval
  • OS overall Survival
  • DFS Disease-Free Survival
  • DRFI Distant Recurrence-Free Interval
  • the expression level of the genes is determined by determining the levels of the corresponding mRNAs. In a still more preferred embodiment, the determination of the levels of the mRNAs is determined by means of RT-PCR.
  • the biofluid is blood.
  • the reference value is preferably obtained from a sample of said patient before starting the treatment.
  • Other terms used to define the method of the second aspect have the meaning as defined with respect to the method of the first aspect. Equally, embodiments described to further characterize the method of the first aspect also further characterize the method of the second aspect, if applicable.
  • the invention in another aspect, relates to a kit for diagnosis of colorectal cancer comprising a set of reagents which allow determining the expression levels of genes ENC1, MADCAM1, CMTM7, FAM60A, AC ATI and DDX55.
  • kits are understood as a product containing the different reagents necessary for carrying out the methods of the invention packed so as to allow their transport and storage.
  • Materials suitable for packing the components of the kit include crystal, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, envelopes and the like.
  • the kits of the invention can contain instructions for the simultaneous, sequential or separate use of the different components which are in the kit.
  • Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like. Additionally or alternatively, the media can contain Internet addresses that provide said instructions.
  • Reagent which allows determining the expression levels of a gene means a compound or set of compounds that allows determining the expression levels of a gene both by means of the determination of the levels of mRNA and by means of the determination of the levels of protein.
  • reagents of the first type include probes capable of specifically hybridizing with the mRNAs encoded by said genes.
  • Reagents of the second type include compounds that bind specifically with the proteins encoded by the marker genes and preferably include antibodies, although they can be specific ap tamers.
  • the first component of the kit of the invention consists of a specific probe for each of genes ENC1, MADCAM1, CMTM7, FAM60A, ACAT1, and DDX55.
  • the reagents of the kit allow determining the expression levels of a set of genes and wherein said set is selected from the group consisting of ENCl and DDX55; ENCl and FAM60A; ENCl, FAM60A and DDX55;ENC1 and CMTM7; ENC1,CMTM7 and DDX55; ENC1,CMTM7 and MADCAM1 ; ENCl and MADCAM1 ; ENCl, M ADC AMI and DDX55; ENCl, ACATl and MADCAM1; ENCl, ACATl, MADCAM1 and DDX55; ENCl, ACATl, CMTM7 and MADCAM1; ENCl, ACATl, CMTM7, MADCAM1 and DDX55; ENCl, ACATl, CM
  • the reagents of the kit allow determining the expression levels of a set of genes and wherein said set is selected from the group consisting of ACATl, MADCAM1 and DDX55; ACATl and MADCAM1; CMTM7 and MADCAM1; CMTM7, MADCAM1 and DDX55; CMTM7, FAM60A and MADCAM1; CMTM7, FAM60A, MADCAM1 and DDX55, AC AT 1 ,CMTM7, M ADC AMI and DDX55; ACATl, CMTM7 and MADCAM1; MADCAM and DDX55; FAM60A and MADCAM 1; FAM60A, MADCAM 1 and DDX55.
  • the set of reagents in the kit of the invention which allow determining the expression levels of genes ENCl, MADCAM 1, CMTM7, FAM60A, ACATl, and DDX55 represents the 10%, 20%, 30%, 40%, 50%
  • the probes or antibodies forming the kit of the invention are coupled to an array.
  • the microarrays comprise a plurality of nucleic acids that are spatially distributed and stably associated to a support (for example, a biochip).
  • the nucleic acids have a sequence complementary to particular subsequences of genes the expression of which is to be detected, therefore are capable of hybridizing with said nucleic acids.
  • a microarray comprising an array of nucleic acids is put into contact with a preparation of nucleic acids isolated from the patient object of the study.
  • the incubation of the microarray with the preparation of nucleic acids is carried out in conditions suitable for the hybridization. Subsequently, after the elimination of the nucleic acids which have not been retained in the support, the hybridization pattern is detected, which provides information on the genetic profile of the sample analyzed.
  • the microarrays are capable of providing both qualitative and quantitative information of the nucleic acids present in a sample, the invention requires the use of arrays and methodologies capable of providing quantitative information.
  • the invention contemplates a variety of arrays with regard to the type of probes and with regard to the type of support used.
  • the probes included in the arrays that are capable of hybridizing with the nucleic acids can be nucleic acids or analogs thereof which maintain the hybridization capacity such as for example, nucleic acids in which the phosphodiester bond has been substituted with a phosphorothioate, methylimine, methylphosphonate, phosphoramidate, guanidine bond and the like, nucleic acids in which the ribose of the nucleotides is substituted with another hexose, peptide nucleic acids (PNA).
  • PNA peptide nucleic acids
  • the length of the probes can be of 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100 nucleotides and vary in the range of 10 to 1000 nucleotides, preferably in the range of 15 to 150 nucleotides, more preferably in the range of 15 to 100 nucleotides and can be single-strand or double-strand nucleic acids.
  • probes for the different target genes are carried out such that they bind specifically to the target nucleic acid with a minimum hybridization to non-related genes.
  • probes of 20 nucleotides which are not unique for a certain mR A. Therefore, probes directed to said sequences will show a cross- hybridization with identical sequences that appear in mRNA of non-related genes.
  • probes that do not specifically hybridize with the target genes in the conditions used because of secondary structures or of interactions with the substrate of the array. This type of probe must not be included in the array. Therefore, the person skilled in the art will observe that the probes that are going to be incorporated in a certain array must be optimized before their incorporation to the array.
  • the optimization of the probes is generally carried out by generating an array containing a plurality of probes directed to the different regions of a certain target polynucleotide. This array is put into contact firstly with a sample containing the target nucleic acid in an isolated form and, secondly, with a complex mixture of nucleic acids. Probes which show a highly specific hybridization with the target nucleic acid but low or no hybridization with the complex sample are thus selected for their incorporation to the arrays of the invention. Additionally, it is possible to include in the array hybridization controls for each of the probes that is going to be studied. In a preferred embodiment, the hybridization controls contain an altered position in the central region of the probe. In the event that high levels of hybridization are observed between the studied probe and its hybridization control, the probe is not included in the array.
  • the array contains a plurality of probes complementary to subsequences of the target nucleic acid of a constant length or of variable length in a range of 5 to 50 nucleotides.
  • the array can contain all the specific probes of a certain mRNA of a certain length or can contain probes selected from different regions of an mRNA. Each probe is assayed in parallel with a probe with a changed base, preferably in a central position of the probe.
  • the array is put into contact with a sample containing nucleic acids with sequences complementary to the probes of the array and the signal of hybridization with each of the probes and with the corresponding hybridization controls is determined.
  • the optimization process can include a second round of optimization in which the hybridization array is hybridized with a sample that does not contain sequences complementary to the probes of the array. After the second round of selection, those probes having signals of hybridization lower than a threshold level will be selected. Thus, probes which pass both controls, i.e., which show a minimum level of unspecific hybridization and a maximum level of specific hybridization with the target nucleic acid are selected.
  • microarrays of the invention contain not only specific probes for the polynucleotides indicating a determined pathophysiological situation, but also containing a series of control probes, which can be of three types: normalization controls, expression level controls and hybridization controls.
  • Normalization controls are oligonucleotides that are perfectly complementary to labeled reference sequences which are added to the preparation of nucleic acids to be analyzed.
  • the signals derived from the normalization controls after the hybridization provide an indication of the variations in the hybridization conditions, intensity of the marker, efficiency of the detection and another series of factors that can result in a variation of the signal of hybridization between different microarrays.
  • the signals detected from the rest of probes of the array are preferably divided by the signal emitted by the control probes, thus normalizing the measurements. Virtually any probe can be used as normalization control. However, it is known that the efficiency of the hybridization varies according to the composition of nucleotides and the length of the probe.
  • preferred normalization probes are those which represent the mean length of the probes present in the array, although they can be selected such that they include a range of lengths that reflect the rest of probes present in the array.
  • the normalization probes can be designed such that they reflect the mean composition of nucleotides of the rest of probes present in the array.
  • a limited number of normalization probes is preferably selected such that they hybridize suitably, i.e., they do not have a secondary structure and do not show sequence similarity with any of the probes of the array is used.
  • the normalization probes can be located in any position in the array or in multiple positions in the array to efficiently control variations in hybridization efficiency related to the structure of the array.
  • the normalization controls are preferably located in the corners of the array and/or in the center thereof.
  • the expression controls levels are probes which hybridize specifically with genes which are expressed constitutively in the sample which is analyzed.
  • the expression level controls are designed to control the physiological state and the metabolic activity of the cell.
  • the examination of the covariance of the expression level control with the expression level of the target nucleic acid indicates if the variations in the expression levels are due to changes in the expression levels or are due to changes in the overall transcriptional rate in the cell or in its general metabolic activity.
  • the observation of a decrease both in the expression levels of the target gene as in the expression levels of the control is expected.
  • any probe corresponding to a gene expressed constitutively such as genes encoding proteins which exert essential cell functions such as ⁇ -2-microglobulin, ubiquitin, ribosomal protein 18S, cyclophilin A, transferrin receptor, actin, GAPDH and the like, can be used.
  • the expression levels controls are GAPDH, tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein (YWHAZ), ubiquitin, beta-actin and ⁇ -2- microglobulin.
  • Hybridization controls can be included both for the probes directed to target genes and for the probes directed to the expression level or to the normalization controls.
  • Error controls are probes of oligonucleotides identical to the probes directed to target genes but which contain mutations in one or several nucleotides, i.e., which contain nucleotides in certain positions which do not hybridize with the corresponding nucleotide in the target gene.
  • the hybridization controls are selected such that, applying the suitable hybridization conditions, the target gene should hybridize with the specific probe but not with the hybridization control or with a reduced efficiency.
  • the hybridization controls preferably contain one or several modified positions in the center of the probe. The hybridization controls therefore provide an indication of the degree of unspecific hybridization or of cross-hybridization to a nucleic acid in the sample to a probe different from that containing the exactly complementary sequence.
  • the arrays of the invention can also contain amplification and sample preparation controls which are probes complementary to subsequences of selected control genes because they normally do not appear in the biological sample object of the study, such as probes for bacterial genes.
  • the R A sample is supplemented with a known amount of a nucleic acid which hybridizes with the selected control probe. The determination of the hybridization to said probe indicates the degree of recovery of the nucleic acids during their preparation as well as an estimation of the alteration caused in the nucleic acids during the processing of the sample.
  • a set of probes showing the suitable specificity and a set of control probes are provided, the latter are arranged in the array in a known position such that, after the steps of hybridization and of detection, it is possible to establish a correlation between a positive signal of hybridization and the particular gene from the coordinates of the array in which the positive signal of hybridization is detected.
  • the microarrays can be high density arrays with thousands of oligonucleotides by means of photolithographic in situ synthesis methods (Fodor et al, 1991, Science, 767- 773). This type of probe is usually redundant, i.e., they include several probes for each mRNA which is to be detected.
  • the arrays are low density arrays or LDA containing less than 10000 probes per square centimeter.
  • the different probes are manually applied with the aid of a pipette in different locations of a solid support (for example, a crystal surface, a membrane).
  • the supports used to fix the probes can be obtained from a large variety of materials, including plastic, ceramics, metals, gels, membranes, crystals and the like.
  • the microarrays can be obtained using any methodology known for the person skilled in the art.
  • a step of washing is necessary to eliminate said non-hybridized nucleic acid.
  • the step of washing is carried out using methods and solutions known by the person skilled in the art.
  • the microarray comprising the target nucleic acids bound to the array with the other components of the system necessary to cause the reaction giving rise to a detectable signal.
  • the target nucleic acids are labeled with biotin
  • the array is put into contact with conjugated streptavidin with a fluorescent reagent in suitable conditions so that the binding between biotin and streptavidin occurs.
  • a step of washing to eliminate all the molecules which have bound non- specifically to the array.
  • the washing conditions will be determined by the person skilled in the art using suitable conditions according to the system generating the detectable signal and which are well known for the person skilled in the art.
  • the resulting hybridization pattern can be viewed or detected in several different ways, said detection being determined by the type of system used in the microarray.
  • the detection of the hybridization pattern can be carried out by means of scintillation counting, autoradiography, determination of a fluorescent signal, calorimetric determinations, detection of a light signal and the like.
  • endonucleases suitable for this treatment include the SI nuclease, mung bean nuclease and the like.
  • the treatment with endonuclease is carried out in an assay in which the target nucleic acid is not labeled with a directly detectable molecule (for example, in an assay in which the target nucleic acid is biotinylated)
  • the treatment with endonuclease will be carried out before putting the microarray into contact with the other members of the system producing the detectable signal.
  • the hybridization pattern is detected and quantified, for which the signal corresponding to each point of hybridization in the array is compared to a reference value corresponding to the signal emitted by a known number of terminally labeled nucleic acids in order to thus obtain an absolute value of the number of copies of each nucleic acid which is hybridized in a certain point of the microarray.
  • compositions containing at least one specific antibody for each of the genes are useful.
  • the arrays of antibodies such as those described by De Wildt et al. (2000) Nat. Biotechnol. 18:989- 994; Lueking et al. (1999) Anal. Biochem. 270: 103-111; Ge et al. (2000) Nucleic Acids Res. 28, e3, I- VII; MacBeath and Schreiber (2000) Science 289: 1760-1763; WO 01/40803 and WO 99/51773 Al are useful.
  • the antibodies of the array include any immunological agent capable of binding to a ligand with high affinity, including IgG, IgM, IgA, IgD and IgE, as well as molecules similar to antibodies which have an antigen binding site, such as Fab', Fab, F(ab')2, single domain antibodies or DABS, Fv, scFv and the like.
  • the techniques for preparing said antibodies are very well known for the person skilled in the art and include the methods described by Ausubel et al. ((Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons (1992)).
  • the antibodies of the array can be applied at high speed, for example, using commercially available robotic systems (for example, those produced by Genetic Microsystems or Biorobotics).
  • the substrate of the array can be nitrocellulose, plastic, crystal or can be of a porous material as for example, acrylamide, agarose or another polymer.
  • cells producing the specific antibodies for detecting the proteins of the invention by means of their culture in array filters. After the induction of the expression of the antibodies, the latter are immobilized in the filter in the position of the array where the producing cell was located.
  • An array of antibodies can be put into contact with a labeled target and the binding level of the target to the immobilized antibodies can be determined. If the target is not labeled, a sandwich type assay can be used in which a second labeled antibody specific for the polypeptide which binds to the polypeptide which is immobilized in the support is used. The quantification of the amount of polypeptide present in the sample in each point of the array can be stored in a database as an expression profile. The array of antibodies can be produced in duplicate and can be used to compare the binding profiles of two different samples.
  • the invention relates to the use of a kit of the invention for the diagnosis of colorectal cancer or for determining the effect of a treatment in a colorectal cancer patient.
  • RNA samples from patients with colorectal cancer (6% stage I, 33% stage II, 44% stage III and 17%> stage IV) were processed at Hospital de Cruces and 10 blood samples from healthy patients were processed at Gaiker.
  • the RNA was obtained using Qiamp Blood RNA kit (Qiagen) according to the manufacturer's instructions.
  • the essential parameter in a real-time PCR is the threshold cycle (or Ct). It is defined as the number of PCR cycles from which the amplified product is detected (HU, N., et al, 2006). It is inversely proportional to the number of copies of the gene of interest.
  • Values of a gene above 1 indicate that that gene is expressed that number of time more (fold-change) in the condition tested with respect to the control. Values below 1 indicate that that gene is more expressed in the control.
  • Table 1 Expression levels ofENCl, ACT1, CMTM7, FAM60A, MADCAMl and DDX5 genes in blood samples from patients with colorectal cancer.
  • the main objective of the following bioinformatics and data mining analysis is to gain insight in the discrimination-diagnosis of both phenotypes (tumoral-T vs control- NT), making use of the exposed RT-PCR dataset.
  • each gene is measured by means of a well-known non-parametric (not assuming any specific data shape distribution) test called symmetrical uncertainty (SU): where X is the predictive variable (in our case, each gene), C is the class label to be predicted (it takes both phenotypic values: non-tumoral and tumoral), H(X) denotes the entropy of X (level of uncertainty in its values) and H(X
  • SU symmetrical uncertainty
  • genes' relevance can be ranked in the following way by means of SU (for each gene, in brackets, the value of the SU correlation degree), denoting a different degree of relevance among them: CMTM7 (0.487), ENC1 (0.431), DDX55 (0.423), FAM60A (0.395), AC ATI (0.39), MADCAM1 (0.286).
  • CMTM7 0.487
  • ENC1 0.431
  • DDX55 0.423
  • FAM60A 0.95
  • AC ATI 0.09
  • MADCAM1 MADCAM1
  • Multivariate diagnostic model Based on these results on the statistical significance of their expressions, all six genes are included in the following multivariate analysis stage. Multivariate diagnostic model.
  • Bayesian network structure known as naive Bayes
  • naive Bayes assumes that all predictive genes are independent when the phenotype is known. It obtains 83.92% accuracy rate.
  • the associated confusion matrix is shown below (rows correspond to real- observed phenotype values, whereas columns collect the predictions of the model):
  • the number of 'false negative' samples is notably reduced, while the number of 'false positive' cases is maintained.
  • the 'sensitivity' value of the model is 0.943, while 'specificity' is 0.9.
  • the results of the same type of k-dependence Bayesian classifier can be improved if the gene values are discretized using the supervised multi-interval discretization technique.
  • This discretization technique tends to fix the binning intervals of the variable in mid-points that induce notably different distributions of the class phenotype.
  • conditional probability tables of the Bayesian network structures which show the strength of modeled 'parent-child' relationships, are not displayed.
  • the following items can be highlighted:
  • CMTM7 and ACAT1 genes strongly condition the values of the FAM60A gene.
  • Table 2 shows the accuracy rate, sensitivity and specificity of different combinations of markers.
  • ENC1-MADCAM1 96.43% 1.000 0.200
  • ENC1-ACAT1-MADCAM1 96.43% 1.000 0.200
  • ENC1-ACAT1-CMTM7-MADCAM1 94.64% 0.978 0.200
  • ENC1-ACAT1-CMTM7-FAM60A-MADCAM1 94.64% 0.978 0.200
  • ACAT1-MADCAM1 94.64% 0.957 0.100
  • CMTM7-MADCAM1 94.64% 0.935 0,000
  • CMTM7-FAM60A-MADCAM1 94.64% 0.935 0,000 TPR FPR
  • ACAT1-CMTM7-MADCAM1 92.86% 0.935 0.100
  • MADCAM-DDX55 91.07% 0.890 0,000
  • FAM60A-MADCAM1 91.07% 0.891 0,000
  • FAM60A-MADCAM1-DDX55 91.07% 0.891 0,000
  • ENC1-CMTM7-MADCAM1 91.07% 0.935 0.200
  • ENC1-CMTM7-MADCAM1-DDX55 91.07% 0.935 0.200
  • ENC1-CMTM7-FAM60A-MADCAM1 91.07% 0.935 0.200
  • ENC1-CMTM7-FAM60A-MADCAM1-DDX55 91.07% 0.935 0.200
  • ENC1-ACAT1-FAM60 91.07% 0.935 0.200
  • ENC1-ACAT1-FAM60A-MADCAM1 91.07% 0.935 0.200
  • ENC-ACAT1-FAM60A-MADCAM1-DDX55 91.07% 0.935 0.200
  • Bayesian network models allow a more clear interpretation and comprehension of the underlying modeled relationships.

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Abstract

La présente invention concerne des procédés destinés au diagnostic du cancer colorectal basés sur la détermination des niveaux d'expression d'un ensemble de gènes, dont l'expression modifiée par rapport à une valeur de référence permet de diagnostiquer ledit cancer avec une haute fiabilité.
PCT/EP2012/076562 2011-12-22 2012-12-21 Procédés et kits destinés au diagnostic du cancer colorectal WO2013092960A1 (fr)

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