WO2022207760A1 - Prédicteur génomique de l'issue d'un cancer - Google Patents

Prédicteur génomique de l'issue d'un cancer Download PDF

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WO2022207760A1
WO2022207760A1 PCT/EP2022/058503 EP2022058503W WO2022207760A1 WO 2022207760 A1 WO2022207760 A1 WO 2022207760A1 EP 2022058503 W EP2022058503 W EP 2022058503W WO 2022207760 A1 WO2022207760 A1 WO 2022207760A1
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expression
genes
level
timp2
myb
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PCT/EP2022/058503
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Anna BIGAS SALVANS
Lluis ESPINOSA BLAY
Teresa LOBO JARNE
Laura SOLÉ FONT
Beatriz Bellosillo Paricio
Mar IGLESIAS COMA
Marta GUIX ARNAU
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Consorcio Centro De Investigación Biomédica En Red
Fundació Institut Mar D'investigacions Mèdiques (Imim)
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Priority to EP22713432.7A priority Critical patent/EP4314344A1/fr
Publication of WO2022207760A1 publication Critical patent/WO2022207760A1/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
    • C12Q2600/112—Disease subtyping, staging or classification
    • 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 present invention relates to the field of Medicine, particularly to cancer and more particularly to colorectal cancer, specifically to a new method for predicting the outcome of cancer.
  • the method has potential applications in the clinical management of cancer patients in terms of medical treatment.
  • CT Chemotherapy
  • SASP senescence-associated-secretory-phenotype
  • senescent cells may contribute to cancer progression and disease recurrence (e.g., after senescent cell reactivation) (Saleh et al., 2019). Therefore, even using chemotherapy in combination with other approaches, such as surgical removal of the tumor, not all cancer patients are cured.
  • CRC colorectal cancer
  • 5-FU 5-fluorouracil
  • Iri irinotecan
  • CRC remains the second leading cause of cancer-related death.
  • CT metastasis and chemotherapy
  • the inventors have found valuable informative markers about the prediction of the outcome of cancer.
  • the inventors have found that when a subject has a signature comprising differential expression of genes form table 1 , in particular, when the subject presents upregulation of the expression of certain genes of table 2 in combination with the downregulation of certain genes of table 3, then the subject has a bad prediction for the outcome (bad prognosis).
  • the inventors have identified that the genetic signature that was acquired by the TQL cells in vitro when using low doses of chemotherapy also predicted cancer outcome in cancer patients including patients which did not received yet chemotherapy (exemplified for CRC and demonstrated in three cohort of patients, see examples 3-5).
  • the inventors have demonstrated that ratio between the level of expression of genes of table 2 and genes of table 3 provide valuable information that predicted poor disease outcome of cancer patients (see example 5).
  • the inventors have found a signature useful for the prediction of the outcome of cancer, e.g., CRC, for the determination of the efficacy of an anti-cancer chemotherapy and for deciding or recommending a medical regime to a subject with cancer (e.g., with CRC).
  • CRC cancer-specific hormone
  • the first aspect of the invention refers to an in vitro method for the prediction of the outcome in a subject suffering cancer, the method comprising the step of: a. determining in an isolated sample of a subject the level of expression of
  • a second aspect of the invention refers to an in vitro method to determine the efficacy of an anti-cancer chemotherapy in a subject suffering cancer comprising the steps of:
  • step (a1) at least one gene selected from table 2 or any combination thereof, and (a2) at least one gene selected from table 3 or any combination thereof; and b) determining a ratio between the level of expression of the gene(s) determined in step (a1) and the gene(s) determined in step (a2) before and after starting the anti-cancer chemotherapy, wherein, if the ratio of gene expression after starting the anti-cancer chemotherapy is higher than or equal to the ratio obtained before starting the anti-cancer chemotherapy, this is indicative of the inefficiency of the anti cancer chemotherapy.
  • a third aspect of the invention refers to an in vitro method for deciding or recommending a medical regime to a subject suffering cancer the method comprising:
  • a fourth aspect of the invention refers to a use of a kit for predicting the outcome in a subject suffering cancer as defined in the method of the first aspect of the invention, or for determining the efficacy of an anti-cancer chemotherapy in a subject suffering cancer as defined in the second aspect of the invention, or for deciding or recommending a medical regime to a subject suffering cancer as defined in the method of the third aspect of the invention, wherein the kit comprises means for determining the level of expression of at least one of the genes selected from table 2 and at least one of the genes selected from table 3 or any combinations thereof.
  • a fifth aspect of the invention refers to a combined use of an expression product of at least one gene selected from table 2 and at least one gene selected form table 3 or any combination thereof, as a marker of prediction of progression of cancer, or of determining the efficacy of an anti-cancer chemotherapy in a subject suffering cancer, or of deciding or recommending a medical regime in a subject suffering cancer.
  • Fig. 1 Shows that CT-induced quiescent cells acquired a fetal intestinal stem cells signature: (A) western blotting (WB) analysis of control (untreated) and treated PD05 collected at the indicated time points after 5FU+lri treatment. (B) RT-qPCR analysis of selected p53 target genes from control (untreated) and IC20- treated PD05 cells.
  • WB western blotting
  • B RT-qPCR analysis of selected p53 target genes from control (untreated) and IC20- treated PD05 cells.
  • Fig.2. Shows that CT-induced quiescent cells displayed a fetal intestinal stem cell signature that was TP53 dependent:
  • the 88 remaining genes are indicated as white dots (within the upper right quadrant (genes upregulated) and the lower left quadrant (genes downregulated)).
  • B RT-qPCR analysis of normalized expression of selected 28up+8down-felSC signature genes in control (untreated) and treated PD05 as indicated.
  • C WB analysis of several feISC genes of control and treated PD05 collected at the indicated time points (in hours) after 5FU+lri treatment.
  • D, E RT-qPCR analysis of normalized relative expression of selected 28up+8down-felSC signature genes in control (untreated) and treated
  • E the TP53 mutant PD04.
  • Fig. 3 Identification of a fetal ISC signature with prognostic value in CRC: Kaplan-Meier representation of disease-free survival probability over time for patients with high or low expression of the 28up/8down-felSC signature selected according to the "Marisa”, “Jorissen” and “TCGA” colorectal cancer databases.
  • Fig. 4 Kaplan-Meier curves representing the disease-free survival of patient groups from (A) stage II and (B) stage II and III from Marisa colorectal cancer database, selected according to their cluster analysis of the 28up/8down-felSC signature. Hazard ratio (HR).
  • Fig. 5 Kaplan-Meier curves representing the disease-free survival of patient groups classified according to their cluster analysis of the 28up+8down-felSC signature for patient groups from TP53 wild type ("P53 WT”) and TP53 mutant (P53 MUT) in (A) Marisa and (B) TCGA colorectal cancer databases.
  • Fig. 6 Kaplan-Meier representation of disease-free survival probability over time of Marisa patient's tumors previously categorized as CMS4 and classified according to their cluster analysis of the 28up+8down-felSC signature.
  • Fig. 7 Kaplan-Meier representation of disease-free survival probability over time for patients with high or low expression of the 8up/8down-felSC signature according for the "Marisa”, “Jorissen” and “TCGA” colorectal cancer databases.
  • Fig. 8 Kaplan-Meier representation of disease-free survival probability over time for patients with high or low expression of the 8up/8down-felSC signature of (A) stage III and (B) stage II and III "Marisa” colorectal database, selected according to their unsupervised hierarchical cluster analysis.
  • Fig. 9. Shows the optimized 5up/3down-felSC signature: (A) RT-qPCR analysis of selected fetal genes from control (untreated) and IC20-treated PD05 cells. Kaplan-Meier representation of disease-free survival probability over time for patients with high or low expression of the 5up/3down-felSC signature (B) and 5up/4down signature (C) according for the "Marisa”, “Jorissen” and "TCGA” colorectal cancer databases.
  • Fig. 10 Kaplan-Meier representation of disease-free survival probability over time for patients with high or low expression of the 5up/3down-felSC signature of (A) stage II and (B) stage II and III "Marisa” colorectal database, selected according to their unsupervised hierarchical cluster analysis.
  • Kaplan-Meier representation of disease-free survival probability over time for patients with high or low expression of the 5up/4down-felSC signature of (C) stage II and (D) stage II and III "Marisa” colorectal database selected according to their unsupervised hierarchical cluster analysis.
  • Fig. 11 Shows that the acquisition of feISC by CT treatments was dependent of YAP1 activation:
  • A WB analysis of p53 and YAP1 protein levels in control and TP53-depleted PD05 KO# 3 cells collected after 72 hours of 5-FU+lri. treatment.
  • B WB analysis of TP53 wild type (HCT116 and Ls174T) and TP53 mutant (SW480 and HT29-M6) colorectal cancer cell lines untreated ("-") and collected after 72 hours of 5-FU+iri. Treatment ("+”).
  • C RT-qPCR analysis of normalized relative expression of selected 28up-felSC signature genes in control (untreated) and treated PD05 with 5-FU+lri.
  • Fig. 12 Kaplan-Meier representation of disease-free survival probability over time for patients from TCGA Stage I with high or low expression of the reduced signatures
  • A 5up/4down TIMP2, TSPAN4, TUBB6, MRAS and ARL4C up and MYB, AGMAT, CDX and HOOK1 down.
  • B rA 5up+3down.
  • C rG 28up+8down.
  • D 8up/8down.
  • Fig. 13 Kaplan-Meier representations of disease-free survival probability over time for patients with high or low expression of the reduced signatures selected according to the "Marisa”, “Jorissen” and “TCGA” colorectal cancer databases.
  • A rA: TIMP2, TSPAN4, TUBB6, MRAS, ARL4C up and MYB, AGMAT, CDX down.
  • B rB: TIMP2, TSPAN4 up and MYB, AGMAT down.
  • C rC: TIMP2, TUBB6 up and MYB, HOOK1 down
  • D rD: TIMP2, TSPAN4, TUBB6 up and MYB, HOOK down.
  • rE TIMP2, TUBB6 up and MYB, AGMAT, HOOK1 down.
  • F rF: TIMP2, TSPAN4, TUBB6 up and MYB, HOOK, AGMAT down.
  • G rG 28up+8down.
  • H rH: TIMP2, TSPAN4 up and MYB down.
  • I rl: TIMP2 up and MYB, AGMAT down.
  • J rJ: TIMP2, TUBB6 up and MYB down.
  • K rK: TIMP2 up and MYB, HOOK down.
  • L rL: TIMP2, TUBB6, TSPAN4 up and CDX1 down.
  • (M) rM TIMP2, TSPAN4 up and CDX1 down;
  • Fig. 14 Kaplan-Meier representations of disease-free survival probability over time for patients classified according to a gene set enrichment score, of the reduced signatures, selected by combining the "Marisa”, “Jorissen” and “TCGA” colorectal cancer databases, d are subset of up genes and c2 are subset of down genes from the reduced signatures. Quartiles were used to define high and low groups.
  • B rB TIMP2, TSPAN4 and MYB, AGMAT.
  • C rC TIMP2, TUBB6 and MYB, HOOK1.
  • the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering cancer, the method comprising the step of determining in an isolated sample from a subject the level of expression of at least one gene of Table 1, for example, the method comprising the step of determining in an isolated sample from a subject the level of expression of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • determining the level of expression of at least one gene e.g., 1, 2, 3, 4, 5,
  • prognosis is the natural evolution of the disease in terms of survival if there is no treatment in between.
  • bad outcome refers to recurrence, relapse, or progression of a cancer in a subject previously identified with the cancer.
  • good outcome refers to no recurrence, or no relapse of the cancer after at least 4 years of follow-up in a subject previously identified with cancer. Bad outcome is, therefore, bad prognosis and good outcome is good prognosis.
  • genes cited herein in tables 1, 2 and 3 are described by their number identifier in the public data base Ensembl (European Molecular Biology Laboratory-European Bioinformatics Institute, EMBL-EBI) and Entrez ID from the National Center for Biotechnology Information (NCBI) at day 25 November 2020. Moreover, for the genes indicated in tables 4 and 5 GenBank® (NCBI) and UniProt (EMBL-EBI) references are provided for expression products of the genes (see the material and method section below) (reference number at day 25 November 2020).
  • the "feISC signature” as described herein is characterized by the detection of at least one gene of Table 1; e.g., the upregulation of at least one gene of table 2 and the down regulation of at least one gene of table 3; e.g., the upregulation of at least one gene of table 4 and the down regulation of at least one gene of table 5; e.g., the "28up/8down” signature, or the “8up/8down” signature, or the "5up/3down” signature explained in the present invention.
  • the first aspect of the invention refers to an in vitro method for the prediction of the outcome in a subject suffering cancer, the method comprising the step of: a. determining in an isolated sample of a subject the level of expression of
  • the method for the prediction of the outcome further comprises the step: b) determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2) (ratio level of expression of the genes determined in step (a1):level of expression of the genes determined in step (a2)).
  • the method for the prediction of the outcome further comprises the step: b. determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2) (ratio level of expression of the genes determined in step (a1):level of expression of the genes determined in step (a2)); wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is, for example, higher than 1, for example is higher or equal to 1.01, and the subject is considered as having good outcome if the ratio obtained in step (b) is for example, lower than 1, for example, is lower or equal to 0.99.
  • step (b) It has been found that the risk of a bad outcome is directly proportional to the increase of the ratio obtained in step (b). This ratio can be considered as a hazard ratio, where patients scoring higher have a worst prognosis.
  • the method for the prediction of the outcome in a subject suffering cancer comprises the step: b. comparing the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of expression of the gene(s) of table 2 is higher, than the corresponding reference value, and wherein the level of expression of the gene(s) of table 3 is lower than the corresponding reference value.
  • the method comprising the determination of the level of expression product of at least 1, 2, 3,
  • the gene(s) form table 2 is(are) selected from the group consisting of: TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN, CXCL16, CD99L2, RHOD, and any combinations thereof.
  • the gene(s) form table 3 is(are) selected from the group consisting of: MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5, SLC27A2 genes, and any combinations thereof.
  • the in vitro method for the prediction of the outcome in a subject suffering cancer comprising determining in an isolated sample of a subject the level of expression of at least one gene (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 genes) selected from the group consisting of: TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN, CXCL16, CD99L2, RHOD (genes of table 4) and any combination thereof; and the level of expression of at least one gene (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 genes) selected from the group
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), wherein in step (a) it is determined the level of expression of:
  • (a1) at least one gene (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 gene(s)) selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR,
  • gene e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 gene(s) selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR,
  • gene e.g., 1, 2, 3, 4, 5, 6, 7, 8 gene(s)
  • the method for the prediction of the outcome in a subject suffering colorectal cancer comprises the steps: a. determining in an isolated sample of a subject the level of expression of:
  • (a1) at least one gene (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 gene(s)) selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN,
  • gene e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 gene(s) selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1,
  • the method comprises an alternative step (b) which comprises comparing the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression of any of the gene(s)of step (a1) is higher (e.g., at least 2-fold higher), than the corresponding reference value, and wherein the level of the expression for each one of the genes of step (a2) is lower (e.g., at least 2-fold lower) than the corresponding reference value.
  • step (b) which comprises comparing the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression of any of the gene(s)of step (a1) is higher (e.g., at least 2-fold higher), than the corresponding reference value, and wherein the level of the expression for each one of the genes of step (a2) is lower (e.g., at least 2-fold lower) than the corresponding reference value.
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC) wherein in step (a) it is determined the level of expression of:
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC) comprising the following steps: a. determining in an isolated sample of a subject the level of expression of (a1) at least each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes (e.g. in combination with at least one of the other genes of table 4, e.g., in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 gene(s) of table 4); and
  • step (a2) at least each one of MYB, AGMAT and CDX1 genes (e.g. in combination with at least one of the other genes of table 5, e.g. with 1, 2, 3, 4 or 5 gene(s) of table 5); and b) determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2); wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01, 1.05, 1.10, 1.2 or 1.22; and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99, 0.95 or 0.90.
  • This embodiment can comprise an alternative step (b) which compares the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes (e.g., also in combination with any other gene of table 4) is higher (e.g., at least 2-fold higher) than the corresponding reference value, and wherein the level of the expression of each one of MYB, AGMAT and CDX1 genes (e.g. also in combination with any other gene of table 5, is lower (e.g., at least 2-fold lower) with respect to the corresponding reference value.
  • step (b) which compares the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes (e.g., also in combination with any other gene of table 4) is higher
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), wherein in step (a) it is determined the level of expression of: (a1) at least each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, COL18A1, SERPINH1 and TPM2 genes (e.g., in combination with at least one of the other genes of table 4, e.g., in combination with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 gene(s) of table 4); and (a2) each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes.
  • CRC colorectal cancer
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), the method comprising the steps of: a. determining in an isolated sample of a subject the level of expression of
  • step (a2) each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes; and b. determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2); wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01, 1.05, 1.10, 1.2 or 1.22; and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99, 0.95 or 0.90.
  • This embodiment can comprise an alternative step (b) which compares the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, COL18A1, SERPINH1, and TPM2 genes (e.g., also in combination with any other gene of table 4) is higher (e.g., at least 2-fold higher) than the corresponding reference value, and wherein the level of the expression of each one of MYB,
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), the method comprising the step of: a. determining in an isolated sample of a subject the level of expression of
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), the method comprising the steps of: a. determining in an isolated sample of a subject the level of expression of
  • step (a1) determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2); wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01, 1.05, 1.10, 1.2 or 1.22; and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99, 0.95 or 0.90.
  • This embodiment can comprise an alternative step (b) which compares the level of the expression of each gene with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1,
  • SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN, CXCL16, CD99L2 and RHOD genes is higher (e.g., at least 2-fold higher), than the corresponding reference value, and wherein the level of the expression for each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes is lower (e.g., at least 2-fold lower) than the corresponding reference value.
  • step (a) contemplate determining in step (a) the level of expression of the following combinations of genes (reduced signatures): (a1) at least TIMP2 and TUBB6, and (a2) at least one gene selected from MYB,CDX1, HOOK1, and any combination thereof
  • the method comprises determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2) of the reduced signatures, wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01, 1.05, 1.10, 1.2 or 1.22; and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99, 0.95 or 0.90.
  • step (b) may comprise comparing the level of the expression of each gene disclosed for the reduced signatures with a corresponding reference value, wherein the subject is considered as having bad outcome if the level of the expression for each one of the genes in (a1) is higher (e.g., at least 2-fold higher), than the corresponding reference value, and the level of the expression for each one of the genes in (a2) is lower (e.g., at least 2-fold lower) than the corresponding reference value.
  • step (b) comprises comparing the level of the expression of each gene determined in (a1) and (a2) with a corresponding reference value and, subsequently, determining a ratio between the level of expression of the genes determined in step (a1) as compared to their reference values and the genes determined in step (a2) as compared to their reference values, wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01 and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99.
  • step (b) comprises: (b1) calculating the mean relative expression of all the genes determined in (a1), (b2) calculating the mean relative expression of all the genes determined in (a2), and (b3) determining the ration between (b1) and (b2), i.e. (b1)/(b2).
  • the relative expression is understood as the level of expression of the gene in the sample with respect to its corresponding reference value. More particularly, the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01, 1.05, 1.10, 1.2 or 1.22; and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99, 0.95 or 0.90.
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), wherein the method further comprises identifying the mutation status of TP53 gene (Gene ID: 7157) (ENSEMBL: ENSG0000014151 ), wherein the subject is considered as having bad outcome when the TP53 gene is the TP53 wild-type gene, or, alternatively, when the TP53 gene is a non-inactivating mutated TP53 (i.e., which does not lead to a loss of function of the p53).
  • This method can also comprise the detection of the p53 protein mutational status, wherein when functional p53 protein (i.e., a p53 WT or a p53 with non-inactivating mutation) is detected is indicative of the subject having bad outcome.
  • This method can also refer to the method wherein the subject is considered as having bad outcome when an inactivating TP53 mutation (i.e., which lead to a p53 with loss of function) it is present in a low percentage of tumoral cells.
  • An embodiment of the first aspect of the present invention optionally in combination with any of the embodiments provided above or below, refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), wherein the subject has received anti-cancer chemotherapy.
  • CRC colorectal cancer
  • reference value in the context of the present invention is to be understood as a predefined level of expression product of the genes in a sample or group of samples. This value is used as a threshold to discriminate subjects wherein the condition to be analysed is present from those wherein such condition is absent.
  • the samples are taken from a well-defined control subject or group of control subjects having no cancer, e.g., no CRC.
  • the skilled person in the art, making use of the general knowledge is able to choose the subject or group of subjects more adequate for obtaining the reference value. Methods for obtaining the reference value from the group of subjects selected are well known in the state of the art.
  • the reference value is determined from a subject or group of subjects that do not suffer from cancer.
  • the reference value is determined from a healthy subject or group of healthy subjects.
  • the reference value is normal tissue, for example, adjacent normal tissue, from the same subject suffering the cancer (e.g., CRC).
  • the reference value is from a reference tumor (e.g., a CRC) that does not present the signature of the present invention.
  • the reference value is obtained from a group of CRC tumoral tissue samples. More particularly, the CRC tumoral tissue samples are from patients suffering colorectal cancer at the same stage as the patient whose prognosis is being determined.
  • An embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), wherein the reference value is the level of expression of each gene in colorectal cancer tissue from a patient whose tumor does not relapse in the 4 first years of follow-up.
  • CRC colorectal cancer
  • the expression “higher than a reference value” is understood as any increase in the level of expression product, for example at least 1.2-fold, or 1.5-fold increase of expression product with respect to the reference value. In particular embodiments, “higher than a reference value” is understood as at least 2-fold increase of expression product with respect to the reference value.
  • the expression “lower than a reference value” is understood as any decrease in the level of expression product, for example at least 1.2-fold, or 1.5-fold decrease of expression product with respect to the reference value. In particular embodiments, “lower than a reference value” is understood as at least 2-fold decrease of expression product with respect to the reference value.
  • the subject is considered as having bad cancer outcome if the level of expression of the gene(s) product(s) selected form table 2 or table 4 is at least 1.2-fold higher, 1.5-fold higher, 2-fold higher, 3-fold higher, 4-fold higher, 5-fold higher, 6-fold higher, 7-fold higher, 8-fold higher, 9-fold higher, or 10-fold higher with respect to a corresponding reference value; and/or wherein the level of expression of the gene(s) product(s) selected from table 3 or table 5 is lower, e.g.
  • the subject is considered as having good outcome if the level of expression of the gene(s) of table 2 or table 4 is equal or lower, than the corresponding reference value, and wherein the level of expression of the gene(s) of table 3 or table 5 is equal or higher than the corresponding reference value.
  • Another embodiment of the first aspect of the present invention refers to an in vitro method for the prediction of the outcome in a subject suffering colorectal cancer (CRC), wherein it compares the level of expression with a tumor that it is known to contain the signature of the present invention.
  • CRC colorectal cancer
  • a second aspect of the invention refers to an in vitro method to determine the efficacy (i.e., efficiency or effectiveness) of an anti-cancer chemotherapy in a subject suffering cancer comprising the steps of:
  • (a1) at least one gene selected from the group consisting of at least one gene selected from Table 2 (e.g., least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
  • Table 2 e.g., least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • (a2) at least one gene selected from Table 3 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 gene(s)) in an isolated sample from the subject, and any combinations thereof;
  • Table 3 e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15, 17, 18, 17, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 gene(s)
  • step (b) ) optionally comprises determining a ratio between the level of expression of the gene(s) determined in step (a1) and the gene(s) determined in step (a2) before and after starting the anti-cancer chemotherapy, wherein, if the ratio of gene expression after starting the anti-cancer chemotherapy is higher than or equal to the ratio obtained before starting the anti-cancer chemotherapy this is indicative of the inefficiency of the anti cancer chemotherapy (thus, wherein, if the ratio of gene expression after starting the anti-cancer chemotherapy is lower than the ratio obtained before starting the anti-cancer chemotherapy this is indicative of the efficacy of the anti-cancer chemotherapy); alternatively, the efficacy of the anti-cancer chemotherapy can be performed by comparing the level of expression before and after starting the anti-cancer chemotherapy, wherein, if there is an increase between the level of the expression product of any gene of table 2 determined before starting the anticancer chemotherapy and there is a reduction between the level of the expression product of any gene of table 3 after starting the anticancer chemotherapy, this is indicative of the inefficiency of the
  • An anti-cancer chemotherapy is considered inefficient when the size of the tumor increases significantly (for example, a 20% or higher increase of its size in imaging tests) despite receiving the treatment.
  • An embodiment of the second aspect of the present invention refers to an in vitro method to determine the efficacy of an anti-cancer chemotherapy in a subject suffering colorectal cancer (CRC) comprising the steps of:
  • (a1) at least one gene (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 gene(s)) selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN, CXCL16, CD99L2, and RHOD (genes of table 4) and any combination thereof; and
  • (a2) at least one gene (e.g., 1, 2, 3, 4, 5, 6, 7, 8 gene(s)) selected from the group consisting of: MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 (genes of table 5) and any combination thereof; or, alternatively,
  • step (b) can refer to comparing the level of the expression before and after starting the anti-cancer chemotherapy, wherein, if there is an increase between the level of the expression of any gene of table 4 (e.g., of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 gene(s)) determined after the initiation of the therapy in comparison with before starting the anti-cancer chemotherapy and there is a reduction between the level of the expression of any gene of table 5 (e.g., of 1, 2, 3, 4, 5, 6, 7 or 8 gene(s)) determined after in comparison with before starting the anti-cancer chemotherapy; or, alternatively, when there is an increase between the level of the expression of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes (e.g., also of any other gene of table 4) determined after in comparison with before starting the anti-cancer chemotherapy and there is a reduction between the level of expression of each one of MYB, AGMAT and CDX1 genes (e.g.,
  • CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes determined after in comparison with before starting the anti-cancer chemotherapy, this is indicative of the inefficiency of the anti-cancer chemotherapy.
  • Other embodiments of the second aspect of the invention contemplate determining in step (a) the level of expression of the following combinations of genes (reduced signatures):
  • the method comprises determining in a step b) a ratio between the level of expression of the gene(s) determined in step (a1) and the gene(s) determined in step (a2) before and after starting the anti-cancer chemotherapy, wherein, if the ratio of gene expression after starting the anti-cancer chemotherapy is higher than the ratio obtained before starting the anti-cancer chemotherapy this is indicative of the inefficiency of the anti-cancer chemotherapy.
  • step (b) can refer to comparing the level of the expression before and after starting the anti-cancer chemotherapy, wherein, if there is an increase between the level of the expression of each of the genes in (a1) determined after the initiation of the therapy in comparison with before starting the anti-cancer chemotherapy and there is a reduction between the level of the expression of each of the genes in (a2) determined after in comparison with before starting the anti-cancer chemotherapy, this is indicative of the inefficiency of the anti-cancer chemotherapy.
  • step (b) comprises determining a ratio between the level of expression of the genes determined in step (a1) after starting the anti-cancer chemotherapy as compared to their level of expression before the anti-cancer therapy and the genes determined in step (a2) after starting the anti-cancer chemotherapy as compared to their level of expression before the anti-cancer therapy, wherein anti-cancer chemotherapy is considered to be ineffective if the ratio is higher than or equal to 1.01.
  • step (b) comprises: (b1) calculating the mean relative expression of all the genes determined in (a1), (b2) calculating the mean relative expression of all the genes determined in (a2), and (b3) determining the ratio between (b1) and (b2), i.e. (b1)/(b2).
  • the relative expression is understood, in this case, for the second aspect of the invention, as the level of expression of the gene in a sample obtained from the patient after the anti-cancer treatment with respect to the level of expression of the same gene in a sample obtained from the patient before the anti-cancer treatment. More particularly, the anti-cancer therapy is considered to be ineffective if the ratio obtained in step (b) is higher than or equal to 1.01, 1.05, 1.10, 1.2 or 1.22.
  • An embodiment of the second aspect of the present invention refers to the in vitro method to determine the efficacy of an anti cancer chemotherapy in a subject suffering colorectal cancer (CRC), wherein the method further comprises identifying the TP53 (Gene ID: 7157; ENSEMBL: ENSG0000014151; identifiers at day 25 November 2020) mutational status wherein when TP53 wild-type gene is determined, or, alternatively, when the TP53 gene is a non-inactivating mutated TP53 (i.e., which does not lead to a loss of function of the p53 protein) this is indicative of the inefficiency of the anti-cancer chemotherapy.
  • TP53 Gene ID: 7157; ENSEMBL: ENSG0000014151; identifiers at day 25 November 2020
  • This method can also comprise the detection of the p53 protein mutational status, wherein when functional p53 protein is detected is indicative of the inefficiency of the anti-cancer chemotherapy.
  • This method can also refer to the method wherein when an inactivating TP53 mutation it is present in a low percentage of tumoral cells, this is indicative of the inefficiency of the anti-cancer chemotherapy.
  • An embodiment of the second aspect of the present invention refers to the in vitro method to determine the efficacy of an anti cancer chemotherapy in a subject suffering cancer (e.g., CRC) wherein the time after starting the anti-cancer chemotherapy is at least after 4-6 cycles of chemotherapy treatment, for example, after 2-3 months after initiating the anti-cancer chemotherapy.
  • a subject suffering cancer e.g., CRC
  • the time after starting the anti-cancer chemotherapy is at least after 4-6 cycles of chemotherapy treatment, for example, after 2-3 months after initiating the anti-cancer chemotherapy.
  • An embodiment of the second aspect of the present invention refers to the in vitro method to determine the efficacy of an anti cancer chemotherapy in a subject suffering cancer (e.g., CRC) wherein the time after starting the anti-cancer chemotherapy is at any time after the completion of said chemotherapy treatment.
  • a subject suffering cancer e.g., CRC
  • the second aspect of the invention is also understood as an in vitro method to determine the efficacy of an anti-cancer chemotherapy in a subject suffering colorectal cancer (CRC) wherein in step (a) the determination of the level of expression in an isolated sample of a subject is performed comparing the level of expression in an intermediate time point during an anti-cancer chemotherapy and the level of expression in a posterior time point after starting said anti-cancer chemotherapy.
  • CRC colorectal cancer
  • the anti-cancer chemotherapy is selected from the group consisting of fluoropyrimidine (for example, 5-fluorouracile and/or capecitabine), oxaliplatin, irinotecan and any combination thereof, for example, 5-fluorouracile and irinotecan, for example, as commonly used to treat CRC patients.
  • fluoropyrimidine for example, 5-fluorouracile and/or capecitabine
  • oxaliplatin for example, 5-fluorouracile and irinotecan, for example, as commonly used to treat CRC patients.
  • the anticancer chemotherapy is combined with any chemotherapy of biological drug used to treat CRC patients, for example, is combined with an antiangiogenic drug (e.g., bevacizumab y aflibercept) and/or an EGFR inhibitor (e.g., cetuximab y panitumumab).
  • an antiangiogenic drug e.g., bevacizumab y aflibercept
  • an EGFR inhibitor e.g., cetuximab y panitumumab.
  • a third aspect of the invention refers to an in vitro method for deciding or recommending a medical regime to a subject suffering cancer the method comprising:
  • An embodiment of the third aspect of the present invention optionally in combination with any of the embodiments provided above or below, refers to an in vitro method for deciding or recommending a medical regime to a subject suffering colorectal cancer (CRC) the method comprising:
  • the medical regime decided or recommended is chemotherapy (for example, FOLFOX4, mFOLFOX6, FOLFIRI, CAPOX, FLOX, or de Gramont regime) combined with a YAP1 inhibitor, e.g., porphyrin compounds such as Verteporfin®, protoporphyrin ix or hematoporphyrin; e.g., Verteporfin®; e.g., is 5FU and irinotecan combined with Verteporfin®.
  • a YAP1 inhibitor e.g., porphyrin compounds such as Verteporfin®, protoporphyrin ix or hematoporphyrin; e.g., Verteporfin®; e.g., is 5FU and irinotecan combined with Verteporfin®.
  • the medical regime decided or recommended is surgery, a different anti-cancer chemotherapy, a different chemotherapy regime using the same anti-cancer chemotherapy, radiation therapy, immunotherapy, targeted therapy hormone therapy or any combination thereof.
  • the anti-cancer treatment is selected from the above-mentioned options based on type and stage of cancer, the results of clinical trials as well as histopathologic findings.
  • the medical regime decided or recommended is not a senolytic drug.
  • the subject is suffering colorectal cancer at stages II or III.
  • the level of expression of the gene is determined by measuring or determining the amount of corresponding mRNA or protein (e.g., full-length protein product or a proteolytic fragment thereof), depending on the detection technique to be used.
  • Determining the amount of mRNA can be performed by any method known to the skilled person, provided that said method permits the detection and quantification of mRNA in a biological sample. Included among the examples of these procedures are PCR, quantitative real-time PCR (QPCR), multiplex PCR, NASBA, LCR, RT-PCR, RNA sequencing, array hybridization or "Northern” transfer, or combinations of these.
  • the level of expression of the gene is determined (quantified) by measuring the amount of mRNA by a nucleic acid amplification-based technique (e.g., PCR, RT-PCR, quantitative PCR (qPCR), or quantitative RT-PCR (qRT-PCR or RT-qPCR)).
  • a nucleic acid amplification-based technique e.g., PCR, RT-PCR, quantitative PCR (qPCR), or quantitative RT-PCR (qRT-PCR or RT-qPCR).
  • the level of expression of the gene is determined (quantified) by measuring the amount of mRNA by qRT-PCR.
  • the amount of mRNA for each one of the gene markers provided by the invention via a hybridization technique, employing oligonucleotide probes.
  • the method may be carried out by combining isolated mRNA with reagents to convert to cDNA according to standard methods well known in the art, treating the converted cDNA with amplification reaction reagents (such as cDNA PCR reaction reagents) in a container along with an appropriate mixture of nucleic acid primers; reacting the contents of the container to produce amplification products; and analyzing the amplification products to detect the presence of one or more of the cancer markers in the sample.
  • the analysis step may be further accomplished by quantitatively detecting the presence of polynucleotide cancer markers in the amplification product and comparing the quantity of marker detected against a panel of expected values for the known presence or absence of such markers in normal and malignant tissue derived using similar primers.
  • the present invention optionally requires performing a ratio between the level of expression of any gene of table 2 and of table 3; or, alternatively, a ratio between the level of expression of any gene of table 4 and of table 5; or, alternatively, a ratio between the level of expression of the genes of any gen of step (a1) and of step (a2) of the above-mentioned methods of the invention.
  • the ratio is performed by performing the arithmetic mean of the level of expression of a subset of genes of table 2 divided by the arithmetic mean of the level of expression of a subset of genes of table 3, or alternatively, by performing the arithmetic mean of the level of expression of a subset of genes of table 4 divided by the arithmetic mean of the level of expression of a subset of genes of table 5.
  • the present invention optionally requires comparing the level of expression of the genes with a reference value.
  • the reference value is obtained from a control subject or group of control subjects or is a normal tissue, for example, adjacent normal tissue from the same subject suffering the cancer (e.g., CRC) or is a reference tumor (e.g., a CRC) that does not present the signature of the present invention.
  • the skilled person may use any available method to establish the described comparison. For instance, as method of relative quantification, the 2-MCt of Livak and Schmittgen may be employed (Methods, 2001 vol. 25, issue 4, p.402-8).
  • microarrays are used which include one or more probes corresponding to one or more of biomarkers identified in Tables 1, 2, 3, 4 or 5.
  • This method results in the production of hybridization patterns of labeled target nucleic acids on the array surface.
  • the resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid.
  • Representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement, light scattering, and the like.
  • the level of expression of the gene is determined by the detection and/or quantification of the protein, e.g., by a specific antibody or a fragment thereof able to bind to the target protein(s).
  • the expression product of the genes which is determined in the context of the present invention is the full-length protein encoded by the genes, or a fragment of said protein.
  • the level of the protein markers or fragments thereof is determined by a quantitative test selected from the group consisting of an immunological test, bioluminescence, fluorescence, chemiluminescence, electrochemistry and mass spectrometry.
  • the proteins and/or mRNAs to be determined are those shown in table 6 (identified with their GenBank® and/or UniProt reference number on day 25 November 2020).
  • the level of encoded protein or fragment thereof is detected by mass spectrometry, for example, by Shotgun Liquid Chromatography Mass Spectrometry (LC-MS/MS) or Multiple reaction monitoring (MRM) mass spectrometry, immunochemistry or by an immunoassay.
  • mass spectrometry for example, by Shotgun Liquid Chromatography Mass Spectrometry (LC-MS/MS) or Multiple reaction monitoring (MRM) mass spectrometry, immunochemistry or by an immunoassay.
  • immunochemistry refers to a variety of techniques for detecting antigens (in the present case any of the proteins encoded by the above genes or antigenic fragments thereof) in a sample by exploiting the principle of antibodies binding specifically to the target protein(s). Visualizing an antibody- antigen interaction can be then accomplished in a number of ways, usually by conjugating the antibody to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction, or to a fluorophore, such as fluorescein or rhodamine.
  • an enzyme such as peroxidase
  • fluorophore such as fluorescein or rhodamine.
  • the immunochemistry technique can be direct or indirect.
  • Suitable immunoassay procedures include enzyme-linked immunosorbent assays (ELISA, such as multiplex ELISA), enzyme immunodot assay, agglutination assay, antibody-antigen-antibody sandwich assay, antigen- antibody-antigen sandwich assay, immunocromatography, or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmunoassay, as well as protein microarray formats.
  • ELISA enzyme-linked immunosorbent assays
  • enzyme immunodot assay enzyme immunodot assay
  • agglutination assay antibody-antigen-antibody sandwich assay
  • antigen- antibody-antigen sandwich assay antigen- antibody-antigen sandwich assay
  • immunocromatography or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmunoassay, as well as protein microarray formats.
  • the level of the protein is determined by an immunoassay.
  • the level of expression of protein is
  • antibody or a fragment thereof able to bind to the target protein(s) is to be understood as any immunoglobulin or fragment thereof able to selectively bind the target protein(s) referred in the aspects and embodiments of the present invention. It includes monoclonal and polyclonal antibodies.
  • fragment thereof encompasses any part of an antibody having the size and conformation suitable to bind an epitope of the target protein. Suitable fragments include F(ab), F(ab') and Fv.
  • An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies).
  • the mRNA and/or protein is at least one of the mRNA or protein described in table 6, or any combinations thereof.
  • the present invention also refers to a method of treatment of a subject suffering cancer (e.g., CRC) in need thereof, the method comprising:
  • the cancer is CRC and the CRC contains TP53 wild type gene, or a non inactivating TP53 mutation (i.e., which does not lead to a loss of function of the p53 protein) or a TP53 mutation that is present in a low percentage of tumoral cells.
  • This method can also comprise the detection of the p53 protein mutational status, wherein when functional p53 protein is detected is indicative of the inefficiency of the anti-cancer chemotherapy, or the anti-cancer chemotherapy is considered ineffective.
  • the treatment provided in step (b) is chemotherapy (for example, FOLFOX4, mFOLFOX6, FOLFIRI, CAPOX, FLOX, or de Gramont regime) combined with a YAP1 inhibitor, e.g., porphyrin compounds such as Verteporfin®, protoporphyrin ix or hematoporphyrin ; e.g., Verteporfin®; e.g., is 5FU and irinotecan combined with Verteporfin®.
  • chemotherapy for example, FOLFOX4, mFOLFOX6, FOLFIRI, CAPOX, FLOX, or de Gramont regime
  • a YAP1 inhibitor e.g., porphyrin compounds such as Verteporfin®, protoporphyrin ix or hematoporphyrin ; e.g., Verteporfin®; e.g., is 5FU and irinote
  • the treatment provided in step (b) is surgery, a different anti-cancer chemotherapy, a different chemotherapy regime using the same anti-cancer chemotherapy, radiation therapy, immunotherapy, targeted therapy hormone therapy or any combination thereof.
  • the anti-cancer treatment is selected from the above-mentioned options based on type and stage of cancer, the results of clinical trials as well as histopathologic findings.
  • the sample is an isolated tissue sample, or a or a biological fluid sample (e.g., blood, plasma, serum, ascitic fluid, broncoalveolar lavage, and urine).
  • a biological fluid sample e.g., blood, plasma, serum, ascitic fluid, broncoalveolar lavage, and urine.
  • the sample is an isolated colon tissue sample, a rectal tissue sample, a biological fluid sample (for example, blood, serum or plasma), or a stool sample.
  • a biological fluid sample for example, blood, serum or plasma
  • the sample is a tissue sample or a biological fluid sample suspected to contain tumoral cells, in particular a CRC tissue sample.
  • the sample is fresh, frozen, fixed or fixed and embedded in paraffin; in an example, the sample is a paraffin embedded cancer tissue.
  • the patient is a mammal, preferably is a human.
  • the patient can be of any age, gender or race.
  • the CRC is a colon or rectum adenocarcinoma.
  • the present invention also refers to a kit that comprises means for predicting the outcome of a subject suffering CRC by the method of the first aspect of the invention, or for determining the efficacy of an anti cancer chemotherapy in a subject suffering CRC by the method of the second aspect of the invention, or for deciding or recommending a medical regime to subject suffering CRC by the method of the third aspect of the invention, the kit comprising means for determining the level of expression of at least one (e.g., of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 gene(s)) of the genes in table 4 and at least one (e.g., of 1, 2, 3, 4, 5, 6, 7, 8 gene(s)) of the genes in table 5; or, alternatively, of each one of TIMP2, T
  • ABHD4, GSN, CXCL16, CD99L2, and RHOD genes and of each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes.
  • the kit can also comprise instructions (such as a leaflet) with the indication for performing the first, second, or third aspects or the fourth method of the present invention.
  • the kit can also comprise a reference sample, for example, a positive control (a sample from a tumor (e.g., CRC) with the signature of the present invention) and/or a negative control (for example from a normal tissue, or a reference tumor (e.g., a CRC) that does not present the signature of the present invention).
  • a positive control a sample from a tumor (e.g., CRC) with the signature of the present invention
  • a negative control for example from a normal tissue, or a reference tumor (e.g., a CRC) that does not present the signature of the present invention.
  • a fourth aspect of the invention refers to a use of a kit for predicting the outcome of a subject suffering CRC by the method of the first aspect of the invention, or for determining the efficacy of an anti-cancer chemotherapy in a subject suffering CRC by the method of the second aspect of the invention, or for deciding or recommending a medical regime to subject suffering CRC by the method of the third aspect of the invention
  • the kit comprising means for determining the level of expression of at least one (e.g., of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 gene(s)) of the genes in table 4 and at least one (e.g., of 1, 2, 3, 4, 5, 6, 7, 8 gene(s)) of the genes in table 5; or, alternatively, of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes (optionally in combination with at least any other gene of table 4), and of an expression product of each one of MYB, AGMAT and CDX1 genes (
  • the nature of the means depends on the technique selected to identify the gene. Details about their nature have been provided above (primers, probes, antibodies, and fluorescent dyes, among others).
  • the kit may additionally comprise further means (additives, solvents) to visualize the interactions (dipsticks, chemiluminescent reagents, turbidimetric reagents, etc.). Suitable additives, solvents and reagents to visualize the identification are disclosed in the examples.
  • the antibodies and/or primers as specific for the protein and/or mRNA, respectively, of any one of the products of expression included in table 6 of the present invention.
  • the antibodies and/or primers as specific for the protein and/or mRNA, respectively, of any one of the products of expression included in table 6 of the present invention can be at least one pair of primers disclosed in table 8 of the present invention.
  • the fifth aspect of the invention refers to a combined use of an expression product of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes, and of an expression product of each one of MYB, AGMAT and CDX1 genes; or, alternatively, of a combined use of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, COL18A1, SERPINH1 and TPM2 genes, and of an expression product of each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes; or, alternatively, a combined use of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, W
  • Another aspect of the invention refers to the use of the ratio of the level of expression of an expression product of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes, and the level of expression of an expression product of each one of MYB, AGMAT and CDX1 genes; or, alternatively, to the use of the ratio of the level of expression of an expression product of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, COL18A1, SERPINH1 and TPM2 genes, and the level of expression of an expression product of each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes; or, alternatively to the use of the ratio of the level of expression of an expression product of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A
  • the in vitro methods of the invention provide prognostic information, information for determining the efficacy of an anti-cancer chemotherapy or information for deciding or recommending a medical regime.
  • the methods of the invention further comprise the steps of (i) collecting said information, and (ii) saving the information in a data carrier.
  • a “data carrier” is to be understood as any means that contain meaningful information data for the prognosis of cancer, such as paper.
  • the carrier may also be any entity or device capable of carrying the prognosis data.
  • the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk.
  • the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means.
  • the carrier When the prognosis data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means.
  • Other carriers relate to USB devices and computer archives. Examples of suitable data carrier are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.
  • PDOs patient-derived organoids
  • Tumour spheres were collected and digested with an adequate amount of trypsin to single cells and re-plated in culture. Cultures were maintained at 37°C, 5% C0 2 and medium changed every week. PDOs were expanded by serial passaging and kept frozen in liquid Nitrogen for being used in subsequent experiments. Mutations for certain genes, including TP53, (see Table 7) in the PDOs were studied using lllumina® platform following manufacturer's instructions. In table 7 the corresponding chemotherapy concentrations that reduced a 20 and 30% of the cell growth (IC2oand IC30, respectively) are indicated for each PDO.
  • Patient-derived organoids (PDO) PD04, PD05, PD08, PDO10, PD011 and PD015 were deposited at MARBiobancfrom Hospital del Mar (Barcelona).
  • Patient- derived organoid 66 (PDO66) was kindly provided by Alberto Munoz Lab (Fernandez-Barral etal., 2020).
  • CRC cell lines HCT116 and Ls174T (KRAS mutated and TP53 WT), SW480 (KRAS and TP53 mutated) and HT29 (BRAF and TP53 mutated) were obtained from the American Type Culture Collection (ATCC, USA). All cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) plus 10% fetal bovine serum (Biological Industries) and were maintained in a 5% CO2 incubator at 37°C. 5-FU+lri.
  • concentrations that reduced 30% of each cell growth were as follows: HCT116, 0.01 pg/mL 5-FU and 0.004 pg/mL Iri.; Ls174T, 0.025 pg/mL 5-FU and 0.01 pg/mL Iri.; SW480, 0.28 pg/mL 5-FU and 0.11 pg/mL Iri.; HT29, 0.33 pg/mL 5-FU and 0.13 pg/mL Iri.
  • Antibodies used Mouse monoclonal anti-p53 DO-1 (ab1101) (RRID:AB_297667), Rabbit monoclonal anti-p21 [EPR362] (ab 109520) (RRI D : AB_10860537), Rabbit monoclonal anti-CKN2A/p16INK4a [EPR1473] (ab108349) (RRI D : AB_10858268) , Rabbit polyclonal anti-CD99L2 (ab224164), Mouse monoclonal anti-
  • TIMP2 [3A4] (ab1828) (RRID:AB_2256129), Rabbit polyclonal anti-MRas (ab26303) (RRID:AB_470849), Anti- TUBB6 (PA5-P8948), Recombinant Anti-ICAMI antibody [EPR4776] (ab109361) (RRI D : AB_10958467) , Recombinant Anti-Hsp47 antibody [EPR4217] (ab109117) (RRI D : AB_10888995), Recombinant Anti-YAP1 antibody [EP1674Y] (ab52771) (RRID:AB_2219141), Anti-Histone H3 antibody-Nuclear Marker and ChIP Grade (ab791) (RRID:AB_302613) and Anti-Histone H4 antibody-ChIP Grade (ab1015) (RRID:AB_296888) from Abeam.
  • Mouse monoclonal anti-yH2AX (pS139) (564719) (RRID:AB_2738913) from BD, Biosciences; Mouse monoclonal anti-Ki67 (MM1) (NCL-Ki67-MM1) (RRI D : AB_442101) from Leica Biosystems; Rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) (9661) (RRID:AB_2341188) from Cell Signaling; Goat polyclonal anti-EphB2 (AF467) (RRID:AB_355375) from RD Systems; TSPAN4 Polyclonal Antibody (PA5-69344) (RRID:AB_2688603) from Thermo Fisher Scientific; Monoclonal Anti-S100A4 antibody produced in mouse
  • D-Luciferin (Cat#LUCK) from Goldbio. PhosSTOP phosphatase inhibitor cocktail (Cat#PHOSS-RO) and Complete Mini protease inhibitor cocktail (Cat#11836170001) from Roche. DAPI Fluoromount-G (Cat#0100-20) from Southern Biotech. Protein A- Sepharose CL-4B (Cat#17-0780-01) and Protein G-Sepharose 4 Fast Flow (Cat#17-0618-01) from GE Healthcare.
  • APC BrdU Flow Kit (Cat#552598), from BD Biosciences. Senescence b-Galactosidase Staining Kit (Cat#9860S) from Cell Signaling. Cell Event Senescence Green Flow Cytometry Assay KiT (Cat#C10840) and Annexin V Apoptosis Detection Kit APC (Cat#88-8007) from Invitrogen. CometAssay Kit (Cat#4250-050- K) from Trevigen. RNeasy Micro Kit (Cat#74004) from Qiagen. SYBR Green I Master Kit (Cat#04887352001) from Roche. Lenti-X Concentrator (Cat#631232) from Clontech.
  • RRID:SCR_015687 ChIP-seeker package from Bioconductor. Corrplot, survimer, survival, heatmaply and pheatma packages from CRAN. TopHat (RRID:SCR_013035), from Kim etal. 2013. HTSeq (RRID:SCR_05514) from Anders etal. 2015. The stats package from R Core Team. Oligonucleotides: Table 8 (see below) shows the oligonucleotides for RT-qPCR and ChIP-qPCR and sgRNA for CIRSPR/Cas9 knockout used (SEQ ID NO: 1-71).
  • tumoroids were plated in 96-well plates in Matrigel and after 6 days in culture were treated with combinations of 5-FU and Irinotecan. Following 72 hours of treatment, it was changed to fresh medium and treated with increasing concentrations of either 5-FU, Irinotecan, dasatinib or combinations for 72 hours at the indicated concentrations. Cell viability was determined as described. Tumor-initiating assays
  • PDOs patient-derived organoids
  • Paraffin blocks were obtained from tissues and PDOs, previous fixation in 4% formaldehyde overnight at room temperature. Paraffin-embedded sections of 4 pm, for tissues, and 2.5 pm, for PDOs, were de-paraffinized, rehydrated and endogenous peroxidase activity was quenched (20 min, 1.5% H 2 0 2 ). EDTA- or citrate-based antigen retrieval was used depending on the primary antibody used. All primary antibodies were diluted in PBS containing 0.05% BSA, incubated overnight at 4 °C and developed with the Envision+ System HRP Labelled Polymer anti-Rabbit or anti-Mouse and 3,3'-diaminobenzidine (DAB). Samples were mounted in DPX mountant and images were obtained with an Olympus BX61 microscope.
  • DAB Envision+ System HRP Labelled Polymer anti-Rabbit or anti-Mouse and 3,3'-diaminobenzidine
  • Fluorescent in-situ hybridization (FISH) FISH analyses from control and IC30-treated PDOs were performed using commercial probes (Abbott Molecular Inc, Des Plaines, IL, USA), one including the centromeric alfa-satellite region specific for chromosome 8 (probe "30-70008 CEP 8 SpOrange”), and a second one containing locus-specific probes from the long arm of chromosome 13 and 21 (probe "33-171076 Aneuvysion 13 Sgreen/21 SpOrange”).
  • a cytospin to concentrate nuclei in the FISH slide was performed. Slides were pre-treated with pepsin for 5 minutes at 37°C.
  • Samples and probe were co-denaturated at 80°C for five minutes and hybridized overnight at 37°C in a hot plate (Hybrite chamber, Abbot Molecular Inc.). Post-hybridization washes were performed at 73°C in 2xsodium salt ctrate buffer (SSC) and at room temperature in 2xSSC, 0.1% NP-40 solution. Samples were counterstained with 4,6-diamino-2-phenilindole (DAPI) (Abbott Molecular Inc, Des Plaines, IL, USA). Results were analyzed in a fluorescence microscope (Olympus, BX51) using the Cytovision software (Applied Imaging, Santa Clara, CA). A minimum of 50 nuclei per case was analysed.
  • SSC 2xsodium salt ctrate buffer
  • DAPI 4,6-diamino-2-phenilindole
  • Cell senescence assays Cell senescence was identified by the presence of SA- -galactosidase activity using two different approaches. On one hand, staining for SA- -galactosidase activity in cultured cells was carried out using the Senescence b-Galactosidase Staining Kit. Briefly, PDOs were seeded in 24-well plates (3000 cells per well). After 6 days, PDOs were treated with combinations of 5-FU and Irinotecan for 72 hours and were subsequent stained with the b-Galactosidase Staining Solution for 2 hours, according to the manufacturer's instructions. Sections embedded in paraffin were counterstained with Fast Red for nuclei visualization.
  • Annexin V binding was determined by flow cytometry using the standard Annexin V Apoptosis Detection Kit APC. Single cells of treated PDOs with indicated combinations of 5-FU+lri. were obtained and stained according to the manufacturer's instructions, with Propidium Iodide staining for the DNA content. The cells were analysed in the Fortessa analyser.
  • PDO Initiating Capacity assay 300 or 600 single PDO cells were plated in 96-well plates in 10 pL Matrigel. After 11 days in culture, the number of PDOs in each well was counted, photographs were taken for PDO diameter determination and cell viability was measured.
  • Tumoroid Initiating Capacity assay 300 or 600 single PDO cells were plated in 96-well plates in 10 pL Matrigel. After 11 days in culture, the number of PDOs in each well was counted, photographs were taken for PDO diameter determination and cell viability was measured.
  • Treated PDOs were lysed for 20 min on ice in 300 pL of PBS plus 0.5% Triton X-100, 1 mM EDTA, 100 mM NA-orthovanadate, 0.2 mM phenyl-methylsulfonyl fluoride, and complete protease and phosphatase inhibitor cocktails. Lysates were analysed by western blotting using standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. In brief, protein samples were boiled in Laemmli buffer, run in polyacrylamide gels, and transferred onto polyvinylidene difluoride (PVDF) membranes.
  • PVDF polyvinylidene difluoride
  • the membranes were incubated with the appropriate primary antibodies overnight at 4°C, washed and incubated with specific secondary horseradish peroxidase-linked antibodies. Peroxidase activity was visualized using the enhanced chemiluminescence reagent and autoradiography films.
  • RNA from treated PDOs was extracted with the RNeasy Micro Kit, and cDNA was produced with the RT-First Strand cDNA Synthesis Kit.
  • RT-qPCR was performed in LightCycler 480 system using SYBR Green I Master Kit. Samples were normalized to the mean of the housekeeping genes TBP, HPRT1 and ACTB. Primers used for qPCR are listed in Table 8 (SEQ ID NO: 1 to 60) (see below).
  • Chromatin-immunoprecipitation assay ChIP
  • Control and IC20-treated PDOs were subjected to ChIP following standard procedures. Briefly, PDO cells were extracted with formaldehyde crosslinked for 10 min at room temperature and lysed for 20 min on ice with 500 pL of H 2 0 plus 10 mM Tris-HCI pH 8.0, 0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 20 mM b- glycerol-phosphate, 100 mM NA-orthovanadate, 10 mM NaButyrate and complete protease inhibitor cocktail.
  • IC20-treated PD05 was subjected to ChIP as previously described (Mulero etal., 2013). Briefly, formaldehyde crosslinked cell extracts were sonicated, and chromatin fractions were incubated for 16 h with anti-p53 [Abeam ab 1101] antibody in RIPA buffer and then precipitated with protein A/G-sepharose [GE Healthcare, Refs. 17- 0618-01 and 17-0780-01], Crosslinkage was reversed, and 6-10 ng of precipitated chromatin was directly sequenced in the genomics facility of Parc de Recerca Biomedica de Barcelona (PRBB) using lllumina® HiSeq platform.
  • PRBB Parc de Recerca Biomedica de Barcelona
  • Raw single-end 50-bp sequences were filtered by quality (Q > 30) and length (length > 20 bp) with Trim Galore (Krueger etal., 2012). Filtered sequences were aligned against the reference genome (hg38) with Bowtie2 (Langmead & Salzberg, 2012). MACS2 software (Zhang et al., 2008) was run first for each replicate using unique alignments (q-value ⁇ 0.1). Peak annotation was performed with ChIPseeker package and peak visualization was done with Integrative Genomics Viewer (Robinson etal., 2011).
  • lentiCRISPR v2 was used for knock-out experiments.
  • Three sgRNA against TP53 gene were designed using Benchling (table 8, SEQ ID NO: 69-71). Lentiviral production was performed transfecting in HEK293T cells the lentiviral vectors (pMD2.G and pCMVR8.2 plasmids) and the plasmid of interest (lentiCRISPR v2 as CRISPR control plasmid and the 3 sgRNA).
  • PDOs were infected by resuspending single cells in concentrated virus diluted in complete medium, centrifuged for 1 h at 650 ref, and incubated for 5 hours at 37°C. Cells were then washed in complete culture medium and seeded as described above.
  • RNA from untreatred and treated PDOs was extracted using RNeasy Micro Kit.
  • the RNA concentration and integrity were determined using Agilent Bioanalyzer (Agilent Technologies, following manufacturer's instructions). Libraries were prepared at the Genomics Unit of Barcelona Biomedical Research Park (PRBB) (Barcelona, Spain) using standard protocols, and cDNA was sequenced using lllumina® HiSeq platform (HiSeq® 2500 Sequencing System, following manufacturer's instructions), obtaining ⁇ 25-30million 50-bp single-end reads per sample. Adapter sequences were trimmed with Trim Galore. Sequences were filtered by quality (Q > 30) and length (> 20 bp).
  • the CRC cell line LS174T was treated with sublethal doses of 5FU+irinotecan for 72h, alone or in combination with the YAP1 inhibitor verterporfin®, at a concentration of 5mM.
  • CRC cell line LS 174T ( TP53 WT) was obtained from ATCC, was grown in Dulbecco's modified Eagle's medium (Invitrogen) plus 10% fetal bovine serum (Biological Industries) and was maintained in a 5% CO 2 incubator at 37°C.
  • the membranes were incubated with the appropriate primary antibodies overnight at 4°C, washed and incubated with specific secondary horseradish peroxidase-linked antibodies. Peroxidase activity was visualized using the enhanced chemiluminescence reagent and autoradiography films.
  • Example 1 Low-dose CT treatment of colorectal cancer PDOs induced a non-senescent quiescent phenotype in the absence of sustained DNA damage.
  • CRC PDOs were treated with serial dilutions of the first-line CT agents 5-FU+lri. to define doses that reduced cell numbers about 20- 30% after 72 hours of treatment (hereafter referred as IC20 and IC30, for Inhibitory Concentration 20 and 30), which were specific for each PDO (see table 7).
  • IC20 and IC30 were treated with serial dilutions of the first-line CT agents 5-FU+lri. to define doses that reduced cell numbers about 20- 30% after 72 hours of treatment (hereafter referred as IC20 and IC30, for Inhibitory Concentration 20 and 30), which were specific for each PDO (see table 7).
  • Microscopy analysis of PDOs indicated that IC20 and IC30 did not promote detectable cell death after 72 hours but imposed a dose-dependent growth arrest that persisted for at least 2 weeks after drug washout.
  • SA senescence-associated
  • IHC immunohystochemistry
  • Example 2 The TQL phenotype was associated with acquisition of a fetal intestinal stem cell (feISC) signature.
  • feISC fetal intestinal stem cell
  • RNA sequencing RNA sequencing (RNAseq) of control, IC20- and IC30- treated PD05 cells and differentially expressed genes (DEGs) between conditions were assessed.
  • RNAseq RNA sequencing of control, IC20- and IC30- treated PD05 cells and differentially expressed genes (DEGs) between conditions were assessed.
  • DEGs differentially expressed genes
  • GSEA Gene Set Enrichment Analysis
  • GSEA analysis also detected association with the NF-KB, apoptosis and interferon gamma (IFNy) pathways (p53 pathway, TNFA siganling via NFKB, coagulation, myogenesis, epithelial-mesenchymal transition, Kras signalling Down (DN), apoptosis, Kras signalling up and Interferon gamma response pathways in descending order of enrichment score (normalized enrichment score).
  • IFNy interferon gamma pathway
  • Example 3 The feISC signature shows a coordinate expression in human CRC and is dependent of functional p53.
  • the genes included in the 28up/8down signature were the following: "28up” were TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN, CXCL16, CD99L2 and RHOD genes (see table 4 below); and the "8down” were MYB, AGMAT, CDX1, H00K1,
  • PDSS1, HUNK, KCNK5 and SLC27A2 genes see table 5 below.
  • ChIP-sequencing ChIP-sequencing assay of 5FU+iri (IC20) treated-PD05 cells, it was only detected p53 binding at the promoter of PLK2, PHLDA3 and GSN genes, consistent with the fact that only 5 of the 28up-felSC genes had been previously identified as p53 targets.
  • RNAseq data from Lee and collaborators demonstrated that feISC genes were expressed in the epithelial component of untreated tumors, particularly in state 1, 5 and 6 that were associated with secretory and migratory pathways.
  • acquisition of the TQL phenotype was linked with the expression of specific fetal ISC signature that was differentially expressed in cancer cells and dependent on the presence of a functional p53 pathway.
  • This feISC signature was expressed in a coordinate manner in untreated human CRC tumors in the CMS4 subtype and tumors in the secretory and migratory epithelial states 1 , 5 and 6 from Guinney and collaborators.
  • TQL cancer cells displayed in vitro and in vivo tumor initiation capacity Whether TQL cells preserved the tumor initiation capacity of untreated PDOs was studied. There were seeded 300 single cells from untreated or PDO cells treated for 72 hours in 3D cultures as indicated in the material and method section. It was found that CT-treated and untreated cells displayed comparable tumor initiation capacity (TIC) as indicated by the equal number of PDOs formed, although CT-treatment resulted in dose- dependent reduction of PDO diameter consistent with their low proliferation rates. In contrast, IC20- 5Fu+iri pre-treatment of p53 mutant PD04 and PD08 cells resulted in TIC abrogation, which was in agreement with the massive accumulation of DNA damage detected in comer assays.
  • TIC tumor initiation capacity
  • a p53 WT PD05 carrying a doxycycline-inducible histone-GFP reporter that has been previously demonstrated to label the quiescent tumor population after doxycycline withdrawal (Puig et al., 2018).
  • PD05 cells were treated with 5-FU+lri. for 72h and, after 2 weeks of doxycycline washout, cells were analyzed by flow cytometry and GFP high and GFP
  • Sorted GPF high which represented the quiescent population of CT-treated cells, displayed identical capacity for organoid generation as GFP high plus GFP
  • IC20 and IC30-treated PDOs were inoculated comparable numbers of untreated, IC20 and IC30-treated PDOs in the cecum of nude mice (2, 4 and 5 mice, respectively). Tumor growth was evaluated by palpation of the mice cecum weekly.
  • IHC analysis of the proliferation marker ki67 indicated that tumors generated from untreated PDOs contained 60-80% of proliferating cells, whereas tumors derived from IC20 and IC30-treated PDOs showed a mixed pattern of quiescent (ki67 negative) and proliferative (ki67 positive) areas.
  • 40,000 single PD05 cells (untreated, IC20 or IC30) expressing a luciferase reporter were injected intracardiac to NSG mice. Mice were analysed weekly for metastatic growth using the IVIS animal imaging system. PD05 treated with 5-FU+lri displayed a slightly superior metastatic capacity than untreated cells.
  • mice transplanted with untreated PD05 cells contained metastatic lesions at week 15 after transplantation.
  • 4 of 6 mice transplanted with IC20-treated cells and 5 of 6 mice with IC30-treated cells showed visible implants 15 weeks after injection.
  • Example 5 The CT induced feISC signature was predictive of poor patient prognosis, with higher statistical power in p53 WT tumors. The possibility that the feISC signature present in TQL cells was associated with cancer patient's outcome was studied. It was analyzed the predictive capacity of 28up/8down-felSC gene signature in the Marisa (Marisa etal., 2013), Jorissen (Jorissen etal., 2009) and TCGA (TCGA Portal) CRC data sets (following the method described for the "Association of the signatures with clinical outcome” in the material and method section above).
  • the 8up/8down-felSC signature included TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, C0L18A1, SERPINH1, TPM2 genes as the 8up genes, and MYB, AGMAT, CDX1, H00K1, PDSS1, HUNK, KCNK5 and SLC27A2 as the 8down genes.
  • genes were selected by two ways. First, genes were scored by their coordinate expression taking into account the 3 CRC datasets analysed. Then it was evaluated the prognosis value by adding a value of single genes to the simplest signature composed by the highest scored 28up plus the highest scored 8down-felSC. This method uncovered a more simplified signature (see Table 9), named as "5up+3down” signature, which included 5 upregulated genes TIMP2, TSPAN4, TUBB6, MRAS, and ARL4C, and 3 down regulated genes MYB, AGMAT and CDX1.
  • genes were selected by two conditions: first by a correlation score higher than 0.8 (or if not, higher than the maximum of its table) and second, statistically significant, this is a correlation lower than a significant level of 5% . Then it was evaluated the prognosis value by adding a value of single genes to the simplest signature composed by genes that appeared in at least two of the three cohorts analyzed or that comprised the "5up+3down” signature.
  • rC "TUBB6" and “TIMP2” as up-regulated genes and “MYB” and “HOOK” as down-regulated genes.
  • rD “TUBB6”, “TIMP2” and “TSPAN4" as up-regulated genes and “MYB” and “HOOK” as down-regulated genes.
  • rE “TUBB6” and “TIMP2” as up-regulated genes and “MYB”, “HOOK” and “AGMAT” as down-regulated genes.
  • rF “TUBB6", “TIMP2” and “TSPAN4" as up-regulated genes and "MYB”, “HOOK” and “AGMAT” as down-regulated genes.
  • rJ "TUBB6" and “TIMP2” as up-regulated genes and "MYB” as down-regulated gene.
  • rK “TIMP2” as up-regulated gene and “MYB” and “H00K1” as down-regulated genes.
  • rL “TIMP2, “TUBB6” and “TSPAN4" as up-regulated genes and "CDX1” as down-regulated gene.
  • rM “TIMP2” and “TSPAN4" as up-regulated genes and "CDX1” as down-regulated gene.
  • the arithmetic mean of the 28up genes divided by the arithmetic mean of the 8down genes was analysed, either for the group with highest 28up and lowest 8down- felSC levels (poorest outcome), as well as for the group with lowest 28up and highest 8down-felSC levels, in the Marisa dataset. This gave rise to a ratio of 1.22 within the patients with the poorest outcome. It was performed a series of supervised analysis for TCGA, Jorissen and Marisa dataset for the purpose of finding the optimized ratios.
  • Example 6 Acquisition of feISC by CT treatment was linked to and dependent on YAP1 activation and the feISC signature was reversed by using the YAP1 inhibitor Verteporfin®
  • FIG. 11 A An in vitro experiment using PD05 cells (Fig. 11 A) and a various CRC cell lines (Fig. 11 B) showed increased YAP1 expression after 5-FU+lri treatment, that was restricted to cells carrying WT P53.
  • Nuclear (active) YAP1 accumulated in the IC20- and IC30- derived PD05 tumors at 2 months after implantation in mice.
  • IHC analysis of ki67 in 62 paired human CRC samples collected at diagnosis (biopsy) and after DNA damaging-based neoadyuvant treatment (surgery) the proliferation status of the tumors was determined.
  • tumors exhibited similar proliferation rates after treatment, as determined by Ki67 staining (type 1), a large subset of tumors displayed reduced proliferation with no morphological evidences of senescence (type 2), such as enlarged nuclei or expression of the senescence marker p16 which were detected in scarce tumors at surgery (type 3). No differences were detected in patient prognosis when comparing type 1 and type 2 tumors that were readily observed in patient carrying type 3 tumors (no events of relapse in the follow up period).
  • nuclear YAP1 which was already detected in few epithelial cells of untreated tumors, was massively increased in neoadjuvant treated tumors independently of the proliferation status associated with expression of the feISC markers S100A4 and SERPINH1.
  • YAP1 activity was required for transcriptional induction of feISC genes by CT
  • PD05 cells were incubated with the YAP1 inhibitor verteporfin® (Fig. 11C), which precluded the induction of all tested feISC genes following IC205FU+irinotecan treatment.
  • treatment of the colorectal p53 WT cell line (cell line LS174T) with IC205FU+irinotecan for 72h led to an increase in YAP1, SERPINH1 and TSPAN4 protein levels, which was abrogated by the verterporfin® (Fig. 11D).
  • Low CT doses led to nuclear YAP1 accumulation that was abrogated by incubation with verteporfin®.
  • Verteporfin® also led to a reduction in protein levels of YAP1 and the feISC markers SERPINH1 and TSPAN4 (Fig. 11 D). Importantly, verteporfin® alone or in combination with low doses of CT promoted tumor cell death specifically in the P53 WT PD05. In contrast, PD08 carrying mutant P53 showed higher resistance to YAP1 inhibition.
  • Table 6 Expression products of the genes of tables 4 and 5: mRNA Ref SEQ and protein RefSeq were the reference sequence of GenBank; HGNC, HUGO Gene Nomenclature Committee at 25 November 2020.
  • Table 7 Patient-derived organoids used. The mutations and the corresponding chemotherapy concentrations that reduce a 20 and 30% of the cell growth (IC20 and IC30, respectively) are indicated for each PDO.
  • Table 8 List of oligonucleotides for RT-qPCR and ChIP-qPCR and sgRNA for CIRSPR/Cas9 knockout used.
  • Table 9 Pearson correlation punctuation score for the genes included in the 28up/8down firm.
  • (a1) at least one gene selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4,
  • GSN GSN, CXCL16, CD99L2, RHOD (genes of table 4) and any combination thereof; and (a2) at least one gene selected from the group consisting of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5, SLC27A2 (genes of table 5) and any combination thereof.
  • the in vitro method for the prediction of the outcome which further comprises the step: b) determining a ratio between the level of expression of the genes determined in step (a1) and the genes determined in step (a2); wherein the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01 and the subject is considered as having good outcome if the ratio obtained in step (b) is lower than or equal to 0.99.
  • step (b) 3.
  • the subject is considered as having bad outcome if the ratio obtained in step (b) is higher than or equal to 1.01 , 1.05, 1.10 or 1.2; and the subject is considered as having good outcome if the ratio obtained in step
  • (b) is lower than or equal to 0.99, 0.95 or 0.90.
  • step (a) comprises determining in an isolated sample of a subject the level of expression of (a1) at least each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes;
  • step (a) comprises determining in an isolated sample of a subject the level of expression of (a1) at least each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, COL18A1, SERPINH1 and
  • step (a) comprises determining in an isolated sample of a subject the level of expression of
  • the method further comprises identifying the mutation status of TP53 gene, wherein the subject is considered as having bad outcome when the TP53 gene is the TP53 wild-type gene or, alternatively, when the TP53 gene is a non-inactivating mutated TP53.
  • (a1) at least one gene selected from the group consisting of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, LAPTM5, COL18A1, SERPINH1, CRIP2, ICAM1, VAMP5, TPM2, PLAUR, S100A4, GPC1, ANXA1, PHLDA3, CLU, PLK2, KIFC3, IL1RN, GLIPR1, WTIP, ABHD4, GSN, CXCL16, CD99L2, and RHOD (genes of table 4) and any combination thereof; and
  • (a2) at least one gene selected from the group consisting of: MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 (genes of table 5) and any combination thereof; or, alternatively,
  • An in vitro method for deciding or recommending a medical regime to a subject suffering colorectal cancer (CRC) comprising:
  • kits for predicting the outcome of a subject suffering colorectal cancer as defined in any one of claims 1 to 8 or 11 or 13, or for determining the efficacy of an anti-cancer chemotherapy in a subject suffering colorectal cancer as defined in any one of claims 9 to 11 or 13, for deciding or recommending a medical regime to subject suffering colorectal cancer as defined in any one of claims 12-13 the kit comprising means for determining the level of expression of at least one of the genes in table 4 and at least one of the genes in table 5; or, alternatively, of each one of TIMP2, TSPAN4, TUBB6, MRAS and ARL4C genes, and of each one of MYB, AGMAT and CDX1 genes; or, alternatively, of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C, COL18A1, SERPINH1 and
  • TPM2 genes and of each one of MYB, AGMAT, CDX1, HOOK1, PDSS1, HUNK, KCNK5 and SLC27A2 genes; or, alternatively, of each one of TIMP2, TSPAN4, TUBB6, MRAS, ARL4C,
  • TopHat2 Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biology, doi.org/10.1186/gb-2013-14-4-r36.

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Abstract

La présente invention concerne un procédé in vitro pour la prédiction de l'issue du cancer chez un sujet en étant atteint, en particulier pour le cancer colorectal. La présente invention concerne également un procédé in vitro pour déterminer l'efficacité d'une chimiothérapie anticancéreuse chez un patient cancéreux et un procédé in vitro pour décider ou recommander un schéma médical à un sujet atteint de cancer, en particulier de cancer colorectal ; les procédés comprenant la détermination dans un échantillon isolé à partir d'un sujet du niveau d'expression de gènes sélectionnés.
PCT/EP2022/058503 2021-03-31 2022-03-30 Prédicteur génomique de l'issue d'un cancer WO2022207760A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115572769A (zh) * 2022-12-07 2023-01-06 中国人民解放军军事科学院军事医学研究院 用于肝癌患者良性预后预测的基因标志物组合
CN115572769B (zh) * 2022-12-07 2023-03-14 中国人民解放军军事科学院军事医学研究院 用于肝癌患者良性预后预测的基因标志物组合

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