WO2014191559A1 - Méthodes et trousses pour le pronostic du cancer colorectal - Google Patents

Méthodes et trousses pour le pronostic du cancer colorectal Download PDF

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WO2014191559A1
WO2014191559A1 PCT/EP2014/061278 EP2014061278W WO2014191559A1 WO 2014191559 A1 WO2014191559 A1 WO 2014191559A1 EP 2014061278 W EP2014061278 W EP 2014061278W WO 2014191559 A1 WO2014191559 A1 WO 2014191559A1
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genes
patient
expression
therapy
expression levels
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PCT/EP2014/061278
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Eduard BATLLE GÓMEZ
Elena Sancho Suils
David ROSSELL RIBERA
Alexandre CALON
Elisa ESPINET HERNÁNDEZ
Sergio PALOMO PONCE
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Institució Catalana De Recerca I Estudis Avançats
Fundació Institut De Recerca Biomèdica (Irb Barcelona)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to the field of diagnosis and, more in particular, to methods for predicting the risk of relapse of cancer patients as well as methods for providing personalized medicine to said patients.
  • the invention relates as well to kits for carrying out the diagnostic and predictive medicine methods.
  • 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. Colorectal cancer is the third most common cancer in men (663 000 cases, 10.0% of the total) and the second in women (571 000 cases, 9.4% of the total) worldwide. About 608 000 deaths from colorectal cancer are estimated worldwide, accounting for 8% of all cancer deaths, making it the fourth most common cause of death from cancer. (GLOBOCAN.iarc.fr)
  • the main treatment option for colorectal cancer is surgery, with or without adjuvant chemotherapy and/or radiotherapy, depending on the individual patient's staging and other medical factors.
  • TNM Tumor/Node/Metastasis
  • AJCC American Joint Committee on Cancer
  • histopathological variables such as high-risk features in stage 11 disease, are only directive when stratifying therapy.
  • lymph nodes When lymph nodes are invaded by tumor cells, the TNM test scores as bad prognosis and the patient is usually subjected to surgery followed by heavy chemotherapy.
  • Clinical studies show that for every 25 patients identified as high-risk stage 11 CRC, 20 will cure regardless of whether they receive treatment o not (Quasar collaborative group et al.. Lancet 2007; 370:2020-2029).
  • a subset of patients with stage III colon cancer treated only by surgery did not recur in 5 years even without adjuvant treatment (Ranghammar et al, Acta Oncologica 2001 ; 40: 282-308).
  • Adjuvant chemotherapy is standard recommendation for stage II I CRC, yet prospective identification of this subgroup of patients with stage III colon cancer could spare therapy.
  • an accurate and reliable method that identifies patients at greatest and least risk eg, "high-risk” stage II and "low-risk” stage III colon cancer) could improve the selection of individualized therapy within these groups.
  • Jorissen et al. have described a classifier formed by 128 genes which show reproducible variations between patients suffering stage A CRC (corresponding to stage I) and stage D (corresponding to stage IV). Moreover, at least two genes of the classifier (NPR3/C5orf23 y FLT1) are up- regulated in patients which suffered recurrence of the disease.
  • WO2010042228 describe the identification of a signature formed by 176 genes, the expression levels of which correlate with the prognosis of CRC.
  • WO2010124222 describes that colon cancer patients wherein the expression levels of FLT-1 (also known as VEGFR-1) are higher than a reference value show a higher probability of showing recurrence of the tumor after surgical resection.
  • FLT-1 also known as VEGFR-1
  • US7695913 describes a method for predicting the prognosis of a patient suffering CRC which comprises the determination of the normalized expression levels of the INHBA, MYBL2, FAP and Ki67 genes wherein an increased expression of INHBA and FAP negatively correlates with an increased probability of a positive prognosis and wherein the expression of the MYBL2 and Ki67 genes positively correlates with an increased possibility of positive prognosis.
  • the method described in this document forms the basis of the Oncotype DX kit although the kit includes the determination of 12 genes, including the INHBA, MYBL2, FAP and Ki67 genes.
  • WO02057787 reports the results of a study designed to determine whether
  • Survivin mR A can be used to predict death from recurrent colorectal carcinoma.
  • the study was based on data obtained from frozen tumour biopsies from 144 patients.
  • the study reportedly shows that Survivin expression is associated with a significantly greater risk of death due to recurrent cancer in patients with stage II colorectal cancer.
  • a test capable of quantifying likelihood of patient benefit from chemotherapy to identify more accurately Stage III patients for treatment would be extremely useful.
  • a patient having a low recurrence risk resembling that of a Stage II patient and a low likelihood of benefit from chemotherapy might elect to forego chemotherapy.
  • a patient with a high recurrence risk and a low likelihood of benefit from 5-FU based chemotherapy might elect an alternative treatment.
  • the invention relates to a method for determining whether a patient is likely to benefit from adjuvant therapy comprising a TGFbeta inhibitor after surgical resection of colorectal cancer wherein the TGFbeta inhibitor is selected from the group consisting of any inhibitor identified in Table 1, said method comprising the determination of the expression levels of the NPR3/C5orf23, CDK 2B and FLTl genes in a sample from said patient,
  • an increased expression level of said genes with respect to a reference value for said genes is indicative that the patient is likely to benefit from therapy after surgical treatment or
  • the invention relates to a TGF-beta inhibitor for use in the treatment of colorectal cancer in a patient after surgical treatment of the cancer, wherein the TGFbeta inhibitor is selected from the group consisting of any inhibitor identified in
  • the invention relates to the use of a kit comprising reagents adequate for determining the expression levels of the NPR3/C5orf23, CDKN2B and FLTl genes and, optionally, reagents for the determination of the expression levels of one or more housekeeping genes for selecting a suitable treatment in a patient suffering colorectal cancer or for selecting a patient which is likely to benefit from adjuvant therapy after surgical resection of colorectal cancer.
  • FIGURES Figure 1 TGF-beta signalling increases during adenoma-carcinoma transition during CRC progression.
  • the figure shows the levels of the TGFB1, 2 and 3 mRNA levels in CRC samples ( ⁇ ) and in adenomas (o).
  • FIG. 2 TGF-beta signalling is contributed by CRC associated fibroblasts (CAFs).
  • CAFs CRC associated fibroblasts
  • TGF-beta signalling acts preferentially over the stromal component of CRC.
  • FIG. 4 shows how the F-TBRS was derived.
  • TGF-beta induced genes were obtained by microarray analysis of CCD-I8C0 normal colon fibroblasts in culture treated or not with TGFB. We further refined our classifier by analyzing their differential expression in FACS-purified CRC cell populations from patients (Venn diagram).
  • F-TBRS is composed of those 175 probes specifically upregulated in the CAF-enriched cell population compared with the other two fractions (>2 fold, p ⁇ 0.05).
  • 65 probes induced by TGF-beta in CCD-I8C0 were not significantly enriched in any of the three cell populations.
  • FIG. 6 Kaplan-Meier curves show the estimated probability of remaining disease- free upon therapy depending on the average expression level of F-TBRS.
  • Figure 7 Kaplan-Meier curves show the estimated probability of remaining disease- free upon therapy depending on the average expression level of F-TBRS in patient samples previously grouped according to their AJCC stage. P-values refer to overall differences between the three groups.
  • Figure 8 Kaplan Meier curves show survival depending on the average expression of the 3 predictors, CDKN2B, NPR3/C5orf23, FLT-1 for all patients (A), or for stage II (B) or stage III (C) patients.
  • Figure 9 shows incremental and approximately linear correlation between the expression of the 3 predictors, CDKN2B, NPR3/C5orf23, FLT-1 and the risk of recurrence.
  • Figure 10 A. Kaplan Meier curves show survival depending on the average expression of the 6 predictors FRMD6, ESMl, IGFBP3, FLTl, NPR3/C5orf23 and CDKN2B for Stage II patients. B. Incremental and approximately linear correlation between expression of the 6 predictors FRMD6, ESMl, IGFBP3, FLTl, NPR3/C5orf23 and CDKN2B and the risk of recurrence in all patients.
  • Figure 11 A. Kaplan Meier curves show survival depending on the average expression of the 6 predictors CDKN2B, NPR3/C5orf23, FLTl, GEM, FGF1 and MEX3B in Stage III patients.
  • Figure 12 In silico Validation. Colostage II predictor performance in a completely independent set of stage II CRC patients (GSE331 13).
  • A.- Kaplan Meier curves show probability of remaining disease-free upon therapy depending on the average expression of the Colostage II predictor.
  • Figure 13 In silico Validation. Colostage III signature performance in a completely independent dataset of stage II and stage III CRC patients (GSE37892).
  • A.- Kaplan Meier curves show probability of remaining disease-free upon therapy depending on the average expression of the Colostage III predictor.
  • Figure 14 Shows that TGFB2 and TGFB3 mR A levels in tumors predict CRC relapse.
  • B Hazard Ratios (HR) and p-values for recurrence- free survival probability over time comparing patients bearing low vs. medium, low vs. high and medium vs. high expression of TGFB1, TGFB2 and TGFB3.
  • TGFB2 and TGFB3 expression levels have statistically significant predictive power for disease free survival.
  • FIG 15 Distribution of CRC patients according to SCAD coefficient according to their TGF-beta 2 and -beta 3 expression levels for all patients.
  • Figure 16 Pharmacological inhibition of stromal TGF-beta signalling blocks metastasis initiation.
  • B Bio luminescence over time after intrasplenic inoculation of 5 x 10 5 colon cancer stem cells derived from a stage IV patient in NSG mice treated as in A. Intensities were normalized to day 0 and arbitrarily set to 100. Values are mean ⁇ SEM (*: p ⁇ 0.05).
  • the authors of the present invention have identified a set of genes which provide a reliable method for the identification of CRC patients at greatest and least risk (eg, "high-risk” stage II and "low-risk” stage III colon cancer) of suffering relapse.
  • a set of 127 genes induced by TGF- beta signaling in normal colon fibroblasts (CCD-co-18) is differentially expressed in cancer associated fibroblasts in response to TGF-beta signaling with respect to epithelial cells and leukocytes purified from colorectal tumours.
  • This set of genes allows predicting relapse of patients with a sensitivity that outperforms AJCC staging.
  • the authors of the present invention have selected a small subset of genes from the 127 gene signature that allows predicting the risk of recurrence.
  • the invention relates to a method (hereinafter personalized therapeutic method of the invention) for determining whether a patient which is likely to benefit from adjuvant therapy comprising a TGFbeta inhibitor after surgical resection of colorectal cancer wherein the TGFbeta inhibitor is selected from the group consisting of any inhibitor identified in Table 1 or any combination thereof, said method comprising the determination of the expression levels of the NPR3/C5orf23, CDKN2B and FLT1 in a sample from said patient, wherein an increased expression level of said genes with respect to a reference value for said genes is indicative that the patient is likely to benefit from therapy after surgical treatment or wherein a decreased expression level of said genes with respect to a reference value for said genes is indicative that the patient is unlikely to benefit from therapy after surgical treatment.
  • the term "determining”, is used herein to refer to the likelihood that a patient will have a particular response, whether positive or negative.
  • the predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient.
  • the predictive methods of the present invention are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as chemotherapy.
  • the prediction may include prognostic factors.
  • the prediction although preferred to be, need not be correct for 100% of the subjects to be diagnosed or evaluated.
  • the term requires that a statistically significant portion of subjects can be identified as having an increased probability of having a given outcome. Whether a subject is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, cross-validated classification rates and the like etc. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.
  • the p- values are, preferably, 0.01, 0,005 or lower.
  • treatment refers to clinical intervention in an attempt to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • patient 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.
  • colonal cancer is used in the broadest sense and refers to (1) all stages and all forms of cancer arising from epithelial cells of the large intestine and/or rectum and/or (2) all stages and all forms of cancer affecting the lining of the large intestine and/or rectum.
  • the colon and rectum are treated as one organ.
  • the patient has a stage I, a stage II, a stage III or a stage IV tumor, wherein Stage I is defined as either Tl NO M0 or T2 NO M0; Stage II is defined as T3 NO M0 or T4 NO M0; Stage III is defined as any T, Nl-2; M0 and Stage IV correspond to any T, any N, Ml .
  • TBM node, metastasis
  • Tl tumor invades submucosa
  • T2 tumor invades muscularis extern
  • T3 tumor invades through the muscularis basement into the subserose, or into the horric or perirectal tissues; T4: tumor directly invades other organs or structures, and/or perforates.
  • Node NO: no regional lymph node metastasis; Nl : metastasis in 1 to 3 regional lymph nodes; N2: metastasis in 4 or more regional lymph nodes.
  • Metastasis M0: mp distant metastasis; Ml : distant metastasis present.
  • the patient the outcome of which is to be predicted is a patient which has been diagnosed with colorectal cancer and which has had surgical resection of the cancer.
  • the patient has had a surgical resection of a stage I tumor, of a stage II tumor, of a stage III tumor or of a stage IV tumor.
  • sample or "biological sample” means biological material isolated from a subject.
  • the biological sample can contain any biological material suitable for detecting the desired biomarker and can comprise cell and/or non-cell material of the subject.
  • the sample can be isolated from any suitable tissue or biological fluid such as for example, prostate tissue, blood, blood plasma, serum, urine, cerebrospinal liquid (CSF) or feces.
  • the samples used for the determination of the marker genes are preferably colorectal tissue samples obtained by biopsy.
  • the samples are bio fluid samples.
  • biological fluid and “biofluid” are used interchangeably herein and refer to aqueous fluids of biological origin.
  • the biofluid may be obtained from any location (such as blood, plasma, serum, urine, bile, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion), an exudate (such as fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (such as a normal joint or a joint affected by disease such as rheumatoid arthritis).
  • a location such as blood, plasma, serum, urine, bile, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion
  • an exudate such as fluid obtained from an abscess or any other site of infection or inflammation
  • a joint such as a normal joint or a joint affected by disease such as rheumatoid arthritis.
  • TGF inhibitor is understood as any compound capable of preventing signal transmission caused by the interaction between TGF and its receptor.
  • TGF i inhibitors that can be used according to the present invention include compounds preventing the competitive or allosteric binding of TGF to its receptor, compounds binding to TGF and compounds inhibiting the intracellular signalling of TGFp.
  • Proper assays to determine the inhibitory capacity of a TGF inhibitor include the in vitro inhibition of TGF biological activity by using the inhibitor in Mv-l-Lu cell proliferation assays as well as the in vivo inhibition of TGF biological activity by the inhibitor using a model of acute liver damage induced by CC14 (disclosed in WO200519244).
  • TGF-beta antagonists see also Wojtowicz-Praga (2003).
  • Suitable TGF inhibitors for use in the present invention are, without limitation, those defined in Table 2:
  • Meteiimumab as described by Denton, C. P. Arthritis Rheum. 56, 323-333 (2007) or any antigen-binding fragment thereof.
  • Pirfenidonc as described by Sheppard, D. Proc. Am. Thorac. Soc. 3, 4 13 41 7 (2006) and polymorphs, solvates, and hydrates thereof.
  • IMC-TR1 as described by Zhong, Z. et al. Clin. Cancer Res. 16, 1 191-1205 (2010) or any antigen-binding fragment thereof.
  • Fusion proteins comprising the ⁇ 2 and Fc
  • GB 1 201 (50, 102, 103) as described by Yao, E. 1 1. et al. Cardiov asc. Res. 81 , 797-804 (2009) and polymorphs, solvates, and hydrates thereof
  • GB 1 203 as described by Yao, E. H. et al. Cardiovasc. Res. 81 , 797 804 ( 2009) and polymorphs, solvates, and hydrates thereof.
  • n 1-2
  • Rl is hydrogen or C 1-C4 alkyl
  • R2 is selected from the group consisting of l-H-pyrrolo[2,3-b], l-H- pyrrolo[2,3c] pyridine, l-H-pyrazolol[3,4-b]pyridine and 7-H-pyrrolo[2,3- d]pyrimidine all of which may be optionally substituted with C1-C4 alkyl or phenyl
  • R is H, NHCOCH 2 N(CH 3 )2 or NH 2 and polymorphs, solvates, and hydrates thereof.
  • Y is 2-Cl and R is H or
  • Y is 6,8-(OMe) 2 and R is Me or
  • Y is 8-F and R is Me or
  • Y is 6-Br and R is Me or
  • TGFbeta inhibitors refers to a particular crystalline state of a substance, having particular physical properties described by X-ray diffraction patterns, IR spectra, phase transition point, and the like. The different polymorphs may result from differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism).
  • the TGFP inhibitor is a crystalline LY2157299 monohydrate further characterized by the solid state 13C nuclear magnetic resonance having a chemical shift (ppm) of 108.8, 115.6, 122.6, and 171.0 (+/- 0.2) ppm.
  • solvate means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloro methane, 2-propanol, or the like, bound by non-covalent intermolecular forces.
  • solvent such as water, acetone, ethanol, methanol, dichloro methane, 2-propanol, or the like, bound by non-covalent intermolecular forces.
  • solvent is water
  • hydrate is used instead of solvate.
  • the present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides.
  • exemplary antibodies include neutralizing antibodies, polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • Illustrative antibody fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and minimal recognition units.
  • the personalized therapeutic method according to the invention comprises the determination of the expression level of the NPR3/C5orf23, CDKN2B and FLT1 genes in a sample from said patient.
  • NPR3/C5orf23 refers to open reading frame 23 found in chromosome 5, corresponding to NPR3 natriuretic peptide receptor C/guanylate cyclase C (atrionatriuretic peptide receptor C), also known as FLJ14054 or hypothetical protein LOC79614.
  • the human NPR3/C5orf23, gene is depicted under accession number NG 028162.1 in the GenBank database.
  • CDK 2B refers to cyclin-dependent kinase inhibitor 2B, also known as pl5, MTS-22, MTS21, pl5 CDK inhibitor, INK4B1, P15, pl4_CDK inhibitor, TP15, pl5INK4b, CDK inhibitory protein, CDK4I1, pl4_INK4B2, multiple tumor suppressor 2, cyclin-dependent kinases 4 and 6 binding protein, pl4-INK4b, pl5_INK4B2, pl5-INK4b or pl5INK4B3.
  • the different iso forms of the human CDKN2B mR A are depicted in the GenBank database under accession numbers NM_078487.2 and NM_004936.3.
  • FLT1 refers to fms-related tyrosine kinase 1 and also known as vascular endothelial growth factor receptor, vascular permeability factor receptor or VEGFR-1.
  • the human gene encoding FLT1 is depicted under accession number NG O 12003.1 in the GenBank database.
  • the method according to the invention may further comprise the determination of one or more markers selected from the group consisting of FRMD6, IGFBP3, ESM1, FGF1, GEM, MEX3B, WNT2, NGF, MSC, SETBP1, FLJ10357, DACT, MURC and CollOAl , wherein increased expression levels of one or more of said genes with respect to a reference value is indicative that the patient shows increased risk of recurrence or wherein decreased expression levels of one or more of said genes with respect to a reference value for each gene is indicative that the patient shows low risk of recurrence.
  • markers selected from the group consisting of FRMD6, IGFBP3, ESM1, FGF1, GEM, MEX3B, WNT2, NGF, MSC, SETBP1, FLJ10357, DACT, MURC and CollOAl , wherein increased expression levels of one or more of said genes with respect to a reference value is indicative that the patient shows increased risk of recurrence or wherein decreased expression levels of one or more of said genes
  • FRMD6 refers to the FRMD6 domain containing 6, also known as EX1, Willin, C14orf31, MGC17921, cl4_5320.
  • the human FRMD6 gene is depicted in the GenBank database under accession number AL079307.7.
  • IGFBP3 refers to the Insulin-like growth factor binding protein 3, also known as IBP3 or BP-53.
  • the human IGFBP3 gene is depicted in the GenBank database under accession number NG 011508.1 (positions 5001 to 14028).
  • ESM1 refers to endothelial cell-specific molecule 1, also known as endocan.
  • the human ESM1 gene is depicted in the GenBank database under accession number NC 000005.9 (complement of positions 54273695 to 54281414.
  • FGF1 refers to fibroblast growth factor 1 (acidic), also known as AFGF, ECGF, FGFA, ECGFA, ECGFB, HBGF1, GLIO703, ECGF-beta or FGF-alpha.
  • the human FGF1 gene is depicted in the GenBank database under accession number NC 000005.9 (complement of positions 141971743 to 142077635).
  • GEM GTP binding protein overexpressed in skeletal muscle, also known as KIR or MGC26294.
  • the human GEM gene is depicted in the GenBank database under accession number NC 000008.10 (complement of positions 95261481 to 95274547).
  • MEX3B refers to a RNA-binding protein, also known as RKHD3, MEX-3B, RNF195, MGCl 17199 or DKFZp434J0617.
  • the human MEX3B gene is depicted in the GenBank database under accession number NC 000015.9 (complement of positions 82334128 to 82338361).
  • WNT2 refers to the wingless-type MMTV integration site family member 2, also known as IRP or INT ILL
  • the human WNT2 gene is depicted in the GenBank database under accession number NC 000007.13 (complement of positions 116916685 to 116963343).
  • NGF nerve growth factor
  • HSAN5 nerve growth factor
  • Beta-NGF nerve growth factor
  • MGC161426 MGC161428
  • MGC161428 MGC161428.
  • the human NGF gene is depicted in the GenBank database under accession number NG 007944.1 (positions 5001 to 57321).
  • MSC refers to the musculin gene, also known as ABFl, MYOR, ABF-1 or bHLHa22.
  • the human MSC gene is depicted in the GenBank database under accession number NC 000008.10 (complement of positions 72753777 to 72756731).
  • SETBP1 refers to SET binding protein 1, also known as SEB, KIAA0437 or DKFZp666J1210.
  • the human gene encoding SETBP1 is shown in the GenBank database under accession number G 027527.1 (positions 5001 to 393338).
  • FLJ10357 refers to Rho guanine nucleotide exchange factor (GEF) 40, also known as ARHGEF40, SOLO or Protein SOLO.
  • GEF Rho guanine nucleotide exchange factor
  • the human gene encoding FLJ10357 is shown in the GenBank database under accession number NC 000014.8 (positions 21538527 to 21558036).
  • the gene FLJ10357 is also known as ARHGEF40.
  • DACT1 refers to dapper, antagonist of beta-catenin, homologl, also known as DAPPER1, FRODO, DPR1, HDPR1, THYEX3, DAPPER, Hepatocellular carcinoma novel gene 3 protein, HNG3, or hDPRl .
  • the human gene encoding DACT1 is shown in the GenBank database under accession number NC 000014.8 (positions 59104757 to 59115039).
  • MURC muscle-related coiled-coil protein.
  • GenBank database accession number NC 000009.11 (positions 103340336 to 103350180).
  • CollOAl refers to collagen, type X, alpha 1.
  • the human gene encoding CollOAl is shown in the GenBank database under accession number NG 008032.1 (positions 5001 to 12212).
  • the method according to the present invention may comprise the determination of any naturally occurring polymorphic variant of one or more of the above genes.
  • the marker genes used in the first method of the invention are FRMD6, ESM1, IGFBP3, FLT1, NPR3/C5orf23 and CDKN2B and the patient is a stage II CRC patient.
  • the marker genes used in the first method of the invention are CDK 2B, NPR3/C5orf23, FLT1, GEM, FGF1 and MEX3B and the patient is a stage III CRC patient.
  • the method of the invention comprises the determination of the expression levels of the genes FRMD6, IGFBP3, ESM1, FGF1, GEM, MEX3B, WNT2, NGF, MSC, NPR3/C5orf23, CDK 2B, SETBP1, FLJ10357, DACT, MURC, FLT1 and CollOAl .
  • the method according to the present invention may further comprise the determination of the expression levels of one or more of the genes forming the F-TBRS, i.e. genes which are differentially expressed between the cell population enriched in cancer associated fibroblasts (enriched CAFs; EPCAM- CD45-) and the EPCAM+ and the EPCAM- cd45+ cell populations and having at least a 2-fold increase in the first cell population with respect to the second and third cell populations wherein increased expression levels of said genes with respect to reference values for said genes is indicative that the patient shows increased risk of recurrence or wherein decreased expression levels of said genes with respect to reference values for said gene is indicative that the patient shows low risk of recurrence.
  • genes forming the F-TBRS i.e. genes which are differentially expressed between the cell population enriched in cancer associated fibroblasts (enriched CAFs; EPCAM- CD45-) and the EPCAM+ and the EPCAM- cd45+ cell populations and having at least a 2-fold increase in the first cell population with respect to the second and
  • the method for predicting the outcome of a patient suffering colorectal cancer comprises the determination of the expression levels of the genes ANGPTL2, ANGPTL4, APBB2, BMPR2, BPGM, C13orf33, C5orfl3, NPR3/C5orf23, CACHD1, CALD1, CDH6, CDK 2B, CILP, CNTN1, COL10A1, COL12A1, COL27A1, DACT1, DIXDCl, DNAJB5, DNAJC18, ELTD1, EPHA4, ESM1, FAP, FGD6, FGF1, FGF2, FLJ10357, FLT-1, FN1 , FRMD4A, FRMD6, GAS1, GEM, GFPT2, GPR161, HAS2, HEY1, HICl, HS3ST3A1, IGFBP3, IGFBP7, IL11, INHBA, KALI, KIAA1755, KLF7, LARP6, LMCD1, LM04, LOC100128178
  • any conventional method can be used within the frame of the invention to detect and quantify the levels of said marker genes.
  • the expression levels are determined by means of the quantification of the levels of mRNA encoded by said genes or by means of the quantification of the protein levels.
  • the nucleic acid contained in the sample e.g., cell or tissue prepared from the patient
  • the sample e.g., cell or tissue prepared from the patient
  • the extracted mRNA is then detected by hybridization (e.g., Northern blot analysis or by oligonucleotide microarrays after converting the mRNA into a labeled cDNA) and/or amplification (e.g., RT-PCR).
  • hybridization e.g., Northern blot analysis or by oligonucleotide microarrays after converting the mRNA into a labeled cDNA
  • amplification e.g., RT-PCR
  • RT-PCR e.g., RT-PCR
  • quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi- quantitative RT-PCR is particularly advantageous.
  • primer pairs were designed in order to overlap an intron, so as to distinguish cDNA amplification from putative genomic contamination. Suitable primers may be easily designed by the skilled person.
  • Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).
  • LCR ligase chain reaction
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • the quantity of mRNA is measured by quantitative or semi-quantitative RT-PCR or by real-time quantitative or semi-quantitative RT-PCR.
  • 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.
  • 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.
  • reference values are the expression level of the genes being compared in a reference sample.
  • a “reference sample”, as used herein, means a sample obtained from a pool of healthy subjects which does not have a disease state or particular phenotype.
  • the reference sample may comprise samples from colon mucosa from patients which do not suffer colon cancer or which do not have a history of colon cancer.
  • the reference sample could be a sample or a pool of samples of colon cancer with a low risk of recurrence. This sample or pool of samples can be obtained from patients which have had surgical resection of the tumor and which have not suffered relapse, preferably in the absence of adjuvant chemotherapy.
  • the reference sample is a sample from a type I CRC or a pool of type I CRCs.
  • 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 sample is obtained from several healthy subjects or from subjects without prior history of colorectal cancer.
  • the reference sample is a sample or a pool of samples of colon cancer from patients which have had surgical resection of the tumor and which have not suffered relapse, preferably in the absence of adjuvant chemotherapy.
  • the references sample can vary depending on the specific method to be performed.
  • the references sample may be a pool of non-tumor colorectal tissue samples, either from individuals that do not have a history of colorectal cancer or from a pool of distal non-tumor tissues with respect to the respective tumor tissues, or a sample or a pool of samples of colon cancer from patients which have had surgical resection of the tumor and which have not suffered relapse, preferably in the absence of adjuvant chemotherapy.
  • the reference sample is preferably a sample obtained from said patient before starting the treatment.
  • the expression profile of the genes in the reference sample 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 sample 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 biological samples 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 sample is a pool of samples from several individuals and corresponds to portions of colorectal tissue that are far from the tumor area and which have preferably been obtained in the same biopsy but which do not have any anatomopathologic characteristic of tumor tissue.
  • 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 sample 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 1 10%, 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 sample 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).
  • the personalized therapeutic method according to the invention comprises the identification of those patients showing increased expression levels of said genes with respect to a reference value for said genes as patient which are likely to benefit from a therapy comprising a TGFbeta inhibitor after surgical treatment or patients showing a decreased expression level of said genes with respect to reference values for said genes as indicative that the patient is unlikely to benefit from a comprising a TGFbeta inhibitor after surgical treatment.
  • neoadjuvant therapy refers to any type of treatment of cancer given prior to surgical resection of the primary tumor, in a patient affected with a cancer.
  • the most common reason for neoadjuvant therapy is to reduce the size of the tumor so as to facilitate a more effective surgery.
  • adjuvant therapy refers to any type of treatment of cancer (e.g., chemotherapy or radiotherapy) given as additional treatment, usually after surgical resection of the primary tumor, in a patient affected with a cancer that is at risk of metastasizing and/or likely to recur.
  • the aim of such an adjuvant treatment is to improve the prognosis.
  • Personalized therapies of the invention also allow providing personalized therapies to patients suffering colorectal cancer.
  • patients which are considered as having a high risk of relapse will most likely benefit from an additional therapy after surgery.
  • patients showing low risk of relapse may do without additional therapeutic treatment following surgery.
  • the invention relates to a therapy comprising a TGF-beta inhibitor for use in the treatment of colorectal cancer in a patient after surgical removal of the cancer, wherein the patient has been selected by the first, second or third personalized therapeutic methods of the invention.
  • the invention relates to the use of a kit comprising reagents adequate for determining the expression levels of the NPR3/C5orf23, CDK 2B and FLT1 genes and, optionally, reagents for the determination of the expression levels of one or more housekeeping for selecting a patient which is likely to benefit from adjuvant therapy based on a TGFbeta inhibitor after surgical resection of colorectal cancer wherein the TGFbeta inhibitor is selected from the group consisting of any inhibitor identified in Table 2.
  • the kit for use according to the invention comprises reagents adequate for the determination of the expression levels of the NPR3/C5orf23, the CDK 2B, the FLT1 genes and one or more additional genes selected from the group consisting of FRMD6, IGFBP3, ESM1, FGF1, GEM, MEX3B, WNT2, NGF, MSC, SETBP1, FLJ10357, DACT, MURC and CollOAl genes.
  • the kit for use according to the invention comprises reagents adequate for the determination of the expression levels of the CDK 2B, NPR3/C5orf23, FLT1, FRMD6, IGFBP3 and ESM1 genes.
  • the kit for use according to the invention comprises reagents adequate for the determination of the expression levels of the CDK 2B, NPR3/C5orf23, FLT1, FGF1, GEM, and MEX3B genes.
  • the kit for use according to the present invention further comprises reagents adequate for the determination of the expression levels of one or more of the genes shown in Table 1 which are differentially expressed between the cell population enriched in cancer associated fibroblasts (enriched CAFs) and EPCAM+ and having at least a 2-fold increase in said first cell population.
  • the kit for use comprises reagents adequate for the determination of the expression levels of the ANGPTL2, ANGPTL4, APBB2, BMPR2, BPGM, C13orf33, C5orfl3, NPR3/C5orf23, CACHD1, CALD1, CDH6, CDK 2B, CILP, CNTN1, COL10A1, COL12A1, COL27A1, DACT1, DIXDCl, DNAJB5, DNAJC18, ELTD1, EPHA4, ESM1, FAP, FGD6, FGF1, FGF2, FLJ10357, FLT-1, FN1, FRMD4A, FRMD6, GAS1, GEM, GFPT2, GPR161, HAS2, HEY1, HICl, HS3ST3A1, IGFBP3, IGFBP7, IL11, INHBA, KALI, KIAA1755, KLF7, LARP6, LMCD1, LM04, LOC100128178, LOC644242, LOC728264
  • the reagents adequate for the determination of the expression levels of one or more genes comprise at least 10%, at least 20%>, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the total amount of reagents adequate for the determination of the expression levels of genes forming the kit.
  • the reagents specific for said gene e.g.
  • probes which are capable of hybridizing under stringent conditions to the NPR3/C50RF23 gene, the CDKN2B and the FLTl genes comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% of the probes present in the kit.
  • the reagents adequate for the determination of the expression levels of one or more genes comprise at least 55%> at least 60%>, at least 65%>, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the total amount of reagents forming the kit.
  • 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 level of a gene means a compound or set of compounds that allows determining the expression level of a gene both by means of the determination of the level of mRNA or by means of the determination of the level 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 aptamers.
  • the reagents of the kit are nucleic acids which are capable of specifically detecting the mRNA level of the genes mentioned above and/or the level of proteins encoded by one or more of the genes mentioned above.
  • Nucleic acids capable of specifically hybridizing with the genes mentioned above can be one or more pairs of primer oligonucleotides for the specific amplification of fragments of the mRNAs (or of their corresponding cDNAs) of said genes.
  • the first component of the kit of the invention comprises a probe which can specifically hybridize to the genes mentioned above.
  • the term “specifically hybridizing”, as used herein, refers to conditions which allow hybridizing of two polynucleotide under high stringent conditions or moderately stringent conditions.
  • “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
  • “Stringent conditions” or “high stringency conditions”, as defined herein, typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Fico 11/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50%> formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1 % SDS, and 10% dextran sulfate at
  • Modely stringent conditions may be identified as described by Sambrook et al, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above.
  • washing solution and hybridization conditions e.g., temperature, ionic strength and % SDS
  • An example of moderately stringent conditions is overnight incubation at 37°C.
  • 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.
  • 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 of 5 to 50 nucleotides and, preferably, 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-stranded or double- stranded nucleic acids.
  • the array can contain all the specific probes of a certain mR A 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. Those probes in which a higher difference is observed between the signal of hybridization with the probe and its hybridization control are selected.
  • 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.
  • 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 mRNA. 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.
  • 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.
  • Probes suitable for use as expression controls correspond to genes 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, tyrosine 3- monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ) and beta- actin.
  • 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 mR A 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.
  • 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.
  • kits according to the present invention comprise reagents which are capable of specifically binding to said polypeptide or polypeptides.
  • the invention relates to the use of a kit comprising antibodies specific for the polypeptides encoded by the NPR3/C5orf23, the CDK 2B and the FLT1 genes.
  • the kit for use of the invention comprises antibodies specific for the NPR3/C5orf23, the CDK 2B, the FLT1 genes and for one or more additional polypeptides selected from the group consisting of the polypeptides encoded by the FRMD6, IGFBP3, ESM1, FGF1, GEM, MEX3B, WNT2, NGF, MSC, SETBP1, FLJ10357, DACT, MURC and CollOAl genes.
  • the kit for use according to the invention comprises antibodies specific for the CDK 2B, NPR3/C5orf23, FLT1, FRMD6, IGFBP3 and ESM1 genes.
  • the kit for use according to the invention comprises antibodies specific for the CDK 2B, NPR3/C5orf23, FLT1, FGF1, GEM, and MEX3B genes.
  • the kit for use according to the present invention further comprises antibodies specific for the polypeptides encoded by the genes shown in Table 1 which are differentially expressed between the cell population enriched in cancer associated fibroblasts (enriched CAFs) and EPCAM+ and having at least a 2-fold increase in said first cell population.
  • the kit for use according to the present invention comprises antibodies specific for the polypeptides encoded by the to the ANGPTL2, ANGPTL4, APBB2, BMPR2, BPGM, C13orf33, C5orfl3, NPR3/C5orf23, CACHD1, CALD1, CDH6, CDK 2B, CILP, CNTN1, COL10A1, COL12A1, COL27A1, DACT1, DIXDCl, DNAJB5, DNAJC18, ELTD1, EPHA4, ESM1, FAP, FGD6, FGF1, FGF2, FLJ10357, FLT-1, FN1, FRMD4A, FRMD6, GAS1, GEM, GFPT2, GPR161, HAS2, HEY1, HICl, HS3ST3A1, IGFBP3, IGFBP7, IL11, INHBA, KALI, KIAA1755, KLF7, LARP6, LMCD1, LM04, LOC100128178, LOC644242,
  • 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, 1- VII; MacBeath and Schreiber (2000) Science 289: 1760-1763; WO 01/40803 and WO 99/51773A1 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.
  • 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.
  • Human tissue samples were obtained from the Pathology Department of Hospital del Mar with the approval of the Bank Tumor Committee according to Spanish Ethical regulations. The study followed the guidelines of the Declaration of Helsinki and patient ' s identity of pathological specimens remained anonymous in the context of this study.
  • Freshly obtained tumors from CRC patients (n 8) treated at Hospital del Mar (Barcelona, Spain) were minced with sterile razor blade and incubated with rotation for 15-20 minutes at 37°C in 50% DMEM / 50% F12 (both from Gibco), containing 100X penicillin/streptomycin (Gibco); 0.1 % Hyaluronidase and 0.1 % CoUagenase 1A (both from Sigma). Pieces were then homogenized by pipetting and passed through consecutive 18G and 21G needles. Enzymatic reaction was stopped by adding 10%> FBS and single cells were collected by sequential filtering through cell strainers of 100 ⁇ ⁇ 70 ⁇ 40 ⁇ (BD Falcon).
  • CCD-I8C0 Fibroblasts were seeded at 60%> confluence and treated with TGFB1 (Peprotech; 5 ng/ml) for 8 hours.
  • Gene expression profiles were measured using HG- U133 plus 2.0 Affimetrix arrays and normalized via RMA.
  • a first signature was generated that contained genes up-regulated at least 2 fold in fibroblasts treated with TGF-beta in duplicate experiments (p ⁇ 0.05).
  • This list was further refined by filtering through the expression profiles of cell populations purified from CRC patients: [CD45(+), Epcam(-): Leukocytic fraction], [CD45(-) Epcam(+); epithelial fraction] and [CD45(-) Epcam(-); CAF enriched fraction].
  • the F-TBRS corresponds to genes upregulated (>2 fold, p ⁇ 0.05) in the CAF enriched fraction compared to the other two populations. All p-values for fold change enrichment were obtained via moderated t- tests. Datasets
  • GSE 17537 and GSE 14333 are composed of 55 colon cancer patients treated at Vanderbilt University Medical Center (Vanderbilt, USA).
  • GSE1433330 contains a pool of 290 CRC patients treated at two different hospitals; Peter MacCallum Cancer Center (Australia) and H. Lee Moffitt Cancer Center (USA).
  • Available annotated clinical data for GSE17537 and GSE14333 datasets included AJCC staging, age, gender and disease free survival intervals.
  • the representation of tumor samples at different AJCC stages in these cohorts follows the natural distribution of CRC patients receiving standard treatment in the aforementioned hospitals.
  • expression levels for all genes were transformed to z-scores prior to pooling.
  • GSE331 13 contained a set of 90 AJCC stage II CRC patient material collected in the Academic Medical Center (AMC) in Amsterdam, The Netherlands. Extensive medical records were kept from these patients and long-term clinical follow-up was available for the large majority.
  • the cohort in GSE37892 contained a series of 130 colon cancer samples in which there were both stage II and stage III CRC patients.
  • GSEA gene set enrichment analysis
  • F-TBRS low expression ⁇ M - SD
  • F-TBRS medium expression > M - SD and ⁇ M
  • F- TBRS high expression > M
  • M is the average across all patients and SD the standard deviation.
  • the multivariate Cox model also included age, gender and AJCC stage. Non-significant variables were dropped from the model in a stepwise fashion until all variables were statistically significant. Statistical significance was defined at the 0.05 level. P-values in all Cox models were based on likelihood ratio tests.
  • a SCAD-based logistic regression model was fitted (Fan & Li, 1999, Journal of the American Statistical Association, 1999, 96, 1348-1360) to predict recurrence events based on patient age, gender, staging and gene expression, and selected the variables with non-zero coefficient estimates.
  • the SCAD penalization parameter was set via 10- fold cross-validation, as implemented in the R package ncvreg. We performed this analysis for stage II, III and also for all patients, which provided several short and highly predictive gene signatures.
  • the average signature expression was computed (i.e. across all genes in the signature) and fit multivariate Cox proportional-hazards models that included staging as an adjustment variable.
  • To visualize the results we stratified the patients according to their average signature expression and obtained Kaplan-Meier plots, and estimated the effect on the hazard ratio as a smooth function using quartic penalized splines (Eilers et al, 1996, Statistical Science, 11, 89-121) as implemented in the R package pspline.
  • TGF-beta signaling during CRC progression was explored.
  • Gene expression profiling of colon tumor samples confirmed elevated levels of TGFBI, TGFB2 and TGFB3 mRNAs in a subset of CRCs whereas all adenomas displayed low levels of the three TGF-beta iso forms (Fig. 1).
  • Characteristic features of the adenoma-CRC transition include increased desmoplastic reaction, inflammation and neovascularization, all of which involve several non-cancerous cell types that reside within the tumor stroma.
  • p-SMAD3 phosphorylation of SMAD3
  • Immunohistochemistry analysis on clinical material revealed prominent nuclear p-SMAD3 accumulation in epithelial cancer cells in 40% of the adenomas but only in 7% of the CRCs (Fig. 3). This finding may reflect the frequent acquisition of inactivating mutations in TGF-beta signaling pathway components during CRC progression.
  • epithelial p-SMAD3+ CRC cells may have impaired TGF- beta transcriptional response due to genetic alterations in SMAD4 (Liu et al, 1997, Genes dev., 11 : 3157-3167 and Alarcon, C, 2009, Cell, 139: 757-769).
  • tumor stromal cells displayed high levels of p-SMAD3 (Fig. 3). While the stroma of most adenomas contained few p- SMAD3 highly positive cells and stained weakly overall, a large proportion of CRCs (63%) were characterized by an abundance of stromal cells with strong nuclear p- SMAD3 staining.
  • F-TBRS TGF-beta response Signature
  • p-SMAD3 accumulated in the nucleus of different tumor stromal-associated cells including lymphocytes and endothelial cells. Yet, the most common stromal p-SMAD3+ cell-type in CRCs displayed a thin and elongated shape with numerous membrane extensions characteristic of fibroblasts. Cancer- Associated Fibroblasts (CAFs) are the most abundant population of the reactive cancer stroma and they participate in tumor growth, invasion and metastasis in several cancer types. To investigate the role of TGF-beta-stimulated fibroblasts in CRC, the set of genes regulated by TGF-beta in these cells was first identified.
  • TGF-beta normal colon fibroblasts (CCD-I8C0) were cultured in the presence or absence of TGF-beta and global gene expression profiles were assessed using microarrays. TGF-beta signalling up-regulated the expression levels of 280 genes in these cells (391 probes; >2 fold, p ⁇ 0.05). The cell- type specificity of this gene set was assessed using the purified tumor cell populations described in example 1. Out of the 391 probes induced by TGF-beta in CCD-I8C0 fibroblasts, 127 genes (detected by 175 probes) were differentially upregulated in the CAF-enriched cell population (Fig. 4; Table 1).
  • F-TBRS Fibroblast-specific TGF-beta Response Signature
  • Table 1 List of genes differentially regulated in TGF-beta stimulated colon fibroblasts wherein "CCD+ vs. CCD- fold change” refers to the fold change in gene expression level between TGF-beta stimulated CAF and normal colon fibroblasts and wherein "CD45-/Epcam- vs. CD45+ and Epcam+ Fold-Change” refers to the fold change in the gene expression between TGF-beta stimulated CAF and the average gene expression in Epcam+ (epithelial) cells and Epcam-Cd45+ cells (Leukocytes). NA: Not annotated.
  • stage I + II; FDR ⁇ 10 "6 ) and eventual cancer relapse (FDR ⁇ 10 ⁇ 6 ) (data not shown).
  • the CRC patient cohort was stratified into three groups according to low, medium or high average expression of F-TBRS genes (Fig. 6). Large differences in the relative risk of cancer relapse were observed between the three groups. During 10 years of follow-up, 55% CRC patients with F-TBRS highly positive primary tumors suffered tumor recurrence, whereas all patients with F-TBRS low tumors remained disease-free (Fig. 6).
  • Table 2 Multivariate analysis using Cox Proportional Hazards Model to assess dependency of F-TBRS and AJCC staging in the prediction of cancer relapse
  • the F-TBRS was further analyzed as described in Materials and Methods in order to identify the minimal subset of genes that provides good prediction of disease- free survival.
  • a subset of three genes (minisignature) formed by the CDK 2B, NPR3/C5orf23, and FLT-1 genes was identified which associated with time to recurrence in a statistically significant manner in all patients analysed as well as in patients from stage II and stage III subgroups.
  • Figure 8 shows Kaplan Meier curves wherein survival of patients is plotted depending on the average expression of the CDKN2B, NPR3/C5orf23 and FLT-1 genes in all patients (panel A), in stage II patients (panel B) or in stage III patients (panel C).
  • the slope of the three gene expression signature shows an approximate incremental linear relationship with the risk of recurrence.
  • stage II the expression signature formed by CDKN2B, NPR3/C5orf23, FLT-1, FRMD6, IGFBP3 and ESMl allowed prediction of time to recurrence with a p ⁇ 0.0039 (Figure 10).
  • stage III the expression signature formed by CDK 2B, NPR3/C5orf23, FLT-1, FGF1, GEM and MEX3B allowed prediction of time to recurrence with a p ⁇ 0.0001 (figure 11).
  • the signature expression displayed an incremental and approximately linear effect on the risk of recurrence (figures 10B and 11B).
  • FIG. 12 shows Kaplan Meier curves wherein survival of patients is plotted depending on the average expression of the CDKN2B, NPR3/C5orf23, FLT-1, FRMD6, IGFBP3 and ESMl genes in all patients (stage II) of GSE 33113 (Fig. 12A). For every increment (+1SD) in the average expression of the colostage II predictor there is a 1.47 increase in the risk to experience recurrence (Fig. 12B).
  • Figure 13 shows Kaplan Meier curves wherein survival of patients is plotted depending on the average expression of the CDKN2B, NPR3/C5orf23, FLT-1, FGF1, GEM and MEX3B genes in all patients of GSE 37892 (Fig. 13 A). For every increment (+1SD) in the average expression of the colostage III predictor there is a 1.52 increase in the risk to experience recurrence (Fig. 13B).
  • TGF-beta 2 and TGF-beta 3 can predict recurrence in colorectal cancer
  • TGFB2 and TGFB3 had similar predictive power over disease relapse as the expression levels of the genes forming the F-TBRS (Fig. 14).
  • TGFB2 and TGFB3 are independent predictors for recurrence in colorectal cancer. As shown in figure 15, there is a correlation between the SCAD coefficient and the percentage of recurrence.
  • tumour stage is an information available to the oncologist at the time of diagnosis, the prognostic value of TGFB2 and TGFB3 levels in combination with staging was evaluated. Again, this allows for the identification of a group of patients at very low risk of disease recurrence (local or distant) in both groups.
  • CoCSCs human colon cancer stem cells
  • TGF-beta inhibitor LY2157299 conferred resistance to the formation of subcutaneous tumours by primary CoCSC-derived cells (Fig. 16A). Remarkably, this TGF-beta inhibitor regime also reduced formation of liver metastasis by CoCSCs inoculated via the spleen (Fig. 16B). Kinetics of metastatic colonization showed that LY2157299 reduced the number of cells that engrafted the liver immediately after inoculation (Fig. 16B, - inset).

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Abstract

L'invention concerne des méthodes de prédiction du risque de récidive de patients cancéreux, ainsi que des procédés pour fournir un médicament personnalisé auxdits patients, sur la base des niveaux d'expression de différents gènes, dont l'expression est induite en réponse à une stimulation par TGF-bêta. L'invention concerne également des trousses pour la mise en œuvre des méthodes médicales de diagnostic et de prédiction.
PCT/EP2014/061278 2013-05-30 2014-05-30 Méthodes et trousses pour le pronostic du cancer colorectal WO2014191559A1 (fr)

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WO2019105217A1 (fr) * 2017-11-30 2019-06-06 苏州科睿思制药有限公司 Forme cristalline du galunisertib, son procédé de préparation et son utilisation
WO2019178283A1 (fr) * 2018-03-13 2019-09-19 Baylor Research Institute Méthodes et compositions pour traiter et diagnostiquer le cancer colorectal
WO2020003213A1 (fr) * 2018-06-27 2020-01-02 Medpacto, Inc. Méthodes de diagnostic et de traitement de patients atteints d'un cancer exprimant des niveaux élevés de signature de réponse au tgf-b
CN113189345A (zh) * 2018-07-09 2021-07-30 清紫生物科技（深圳）有限公司 Pdlim4用作胃癌标志物的应用
CN113234829A (zh) * 2021-06-23 2021-08-10 至本医疗科技（上海）有限公司 结肠癌预后评估基因集及其构建方法

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CN107106706A (zh) * 2015-01-20 2017-08-29 安徽医科大学 lmo4基因表达的抑制剂在制备银屑病外用型治疗药物中的用途
CN107106706B (zh) * 2015-01-20 2021-10-08 安徽医科大学 lmo4基因表达的抑制剂在制备银屑病外用型治疗药物中的用途
WO2019105217A1 (fr) * 2017-11-30 2019-06-06 苏州科睿思制药有限公司 Forme cristalline du galunisertib, son procédé de préparation et son utilisation
WO2019178283A1 (fr) * 2018-03-13 2019-09-19 Baylor Research Institute Méthodes et compositions pour traiter et diagnostiquer le cancer colorectal
WO2020003213A1 (fr) * 2018-06-27 2020-01-02 Medpacto, Inc. Méthodes de diagnostic et de traitement de patients atteints d'un cancer exprimant des niveaux élevés de signature de réponse au tgf-b
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CN113189345B (zh) * 2018-07-09 2022-08-19 广东格锐科技有限公司 Pdlim4用作胃癌标志物的应用
CN113234829A (zh) * 2021-06-23 2021-08-10 至本医疗科技（上海）有限公司 结肠癌预后评估基因集及其构建方法
CN113234829B (zh) * 2021-06-23 2022-02-01 至本医疗科技（上海）有限公司 结肠癌预后评估基因集及其构建方法

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