WO2014135655A1 - Compositions et méthodes de traitement du cancer de la vessie invasif pour le muscle - Google Patents

Compositions et méthodes de traitement du cancer de la vessie invasif pour le muscle Download PDF

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WO2014135655A1
WO2014135655A1 PCT/EP2014/054384 EP2014054384W WO2014135655A1 WO 2014135655 A1 WO2014135655 A1 WO 2014135655A1 EP 2014054384 W EP2014054384 W EP 2014054384W WO 2014135655 A1 WO2014135655 A1 WO 2014135655A1
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basal
muscle
bladder cancer
seq
expression level
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François RADVANYI
Sandra REBOUISSOU
Aurélie KAMOUN
Yves Allory
Aurélien De Reynies
Isabelle Bernard-Pierrot
Thierry LEBRET
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Centre National de la Recherche Scientifique CNRS
Institut Curie
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • 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, in particular of oncology. It relates to a new method to classify patients suffering from a muscle-invasive bladder cancer for therapeutic intervention.
  • Bladder carcinoma is one of the most common cancers in North America and Europe, accounting for approximately 200,000 new cases and 65,000 deaths in these regions in 2008. Bladder carcinoma may present as a non-muscle-invasive (70-80% of cases) or muscle-invasive (20-30%) of cases) disease, with highly divergent outcomes. Most patients with non-muscle- invasive bladder cancers (NMIBC) suffer multiple recurrences of the disease without developing a muscle-invasive neoplasm. In contrast, muscle-invasive bladder cancer (MIBC) is a major clinical issue, with cancer-related deaths of 40-50% at five years for patients with organ-confined tumors and more than 80% for those with lymph node involvement or distant metastasis.
  • NMIBC non-muscle-invasive bladder cancer
  • Radical cystectomy is the standard treatment for MIBC.
  • the addition of neoadjuvant and/or adjuvant chemotherapy has very modest benefits for overall survival (Sternberg et al, 2012).
  • Iterative bladder resection and radiotherapy alone are generally considered as palliative treatment options for patient unfit for cystectomy or as part of a multimodal bladder-preserving approach (Bellmunt et al., 2010).
  • MIBC is a highly heterogeneous disease in both molecular and clinical terms
  • tumor stratification is a key issue in the identification of appropriate targeted treatments.
  • a stratified approach to anti-EGFR therapy for MIBC may improve treatment efficacy.
  • the inventors searched for clinically-relevant molecularly-homogeneous subgroups of MIBC. They identified a subgroup of particularly aggressive MIBC tumors wherein the EGFR pathway was deregulated and they further provided evidence of a relationship between this subgroup of MIBC and sensitivity to anti-EGFR drugs.
  • the present invention concerns a method for determining whether a muscle-invasive bladder cancer has a basal-like phenotype, wherein the method comprises
  • the method comprises determining the expression level of K T5, K T6A and/or K T6B and the expression level of nuclear FOXAl in a cancer sample, the expression of K T5, K T6A and/or K T6B and the absence of nuclear FOXAl being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the expression level of K T5, K T6A and/or K T6B and the expression level of nuclear FOXAl may be assessed by immunohistochemistry.
  • the method comprises determining the expression level of PKP1 , IPPK, MAML3 and TGFBR3 genes in a cancer sample, high expression level of PKP 1 and IPPK genes and low expression level of MAML3 and TGFBR3 genes, being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the method comprises determining the expression level of the exon of SEQ ID NO: 24 in a cancer sample, low expression level of said exon being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the method comprises determining the expression level of the exon of SEQ ID NO: 37 in a cancer sample, high expression level of said exon is indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the method comprises determining the expression level of TGMl gene, high expression level is indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • this method further comprises determining the expression level of the exon of SEQ ID NO: 37.
  • the method comprises determining the expression level of TGM1 gene and determining the expression level of HDAC9 short isoform corresponding to the transcript ENST00000456174 and HDAC9 long iso forms corresponding to transcripts ENST00000406451 , ENST00000405010 and ENST00000428307.
  • the method further comprises calculating the ratio of HDAC9 short isoform to HDAC9 long iso forms, high expression level of TGM1 gene and high ratio are indicative that the muscle- invasive bladder cancer has a basal-like phenotype.
  • the method comprises determining the DNA methylation status of CpG islands of SEQ ID NO: 43, 45, 47 and 51 , hypermethylation of the CpG island of SEQ ID NO: 43 and hypomethylation of CpG islands of SEQ ID NO: 45, 47 and 51, being indicative that the muscle-invasive bladder cancer has a basal- like phenotype.
  • the method comprises determining the DNA methylation status of GpC sites of SEQ ID NO: 85, 86, 91 and 96, hypomethylation of said CpG islands being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the present invention concerns a method for predicting clinical outcome of a patient afflicted with a muscle-invasive bladder cancer, wherein the method comprises determining in a cancer sample from said patient whether the muscle-invasive bladder cancer has a basal-like phenotype with the method according to the invention, the presence of the basal- like phenotype being indicative of a poor prognosis.
  • the present invention concerns a method for selecting a patient afflicted with a muscle-invasive bladder cancer for a treatment comprising an EGFR kinase inhibitor and/or capecitabine, wherein the method comprises determining in a cancer sample from said patient whether the muscle-invasive bladder cancer has a basal-like phenotype with the method according to the invention, and optionally determining whether the muscle-invasive bladder cancer has a RAS-activating mutation, the presence of the basal-like phenotype being indicative that said patient is susceptible to benefit from a treatment comprising capecitabine, and the presence of the basal-like phenotype and the absence of a RAS-activating mutation being indicative that said patient is susceptible to benefit from a treatment comprising an EGFR kinase inhibitor.
  • the EGFR kinase inhibitor is selected from the group consisting of erlotinib, cetuximab, gefitinib, lapatinib, panitumumab, zalutumumab, nimotuzumab and matuzumab, and any combination thereof.
  • the EGFR kinase inhibitor is selected from the group consisting of erlotinib and cetuximab, and a combination thereof.
  • the present invention also concerns a method of predicting the sensitivity of a muscle- invasive bladder cancer to a treatment comprising an EGFR kinase inhibitor and/or capecitabine, wherein the method comprises determining whether the muscle-invasive bladder cancer has a basal-like phenotype with the method according to the invention, and optionally determining whether the muscle-invasive bladder cancer has a RAS-activating mutation, the presence of the basal-like phenotype in said cancer being indicative that said cancer is sensitive to a treatment comprising capecitabine, and the presence of the basal-like phenotype and the absence of RAS-activating mutation being indicative that said cancer is sensitive to a treatment comprising an EGFR kinase inhibitor.
  • the present invention concerns an EGFR kinase inhibitor for use in the treatment of muscle-invasive bladder cancer having a basal-like phenotype as determined with the method according to the invention and without RAS-activating mutation.
  • the present invention also concerns capecitabine for use in the treatment of muscle- invasive bladder cancer having a basal- like phenotype as determined with the method according to the invention.
  • the present invention concerns a kit and its use (i) for predicting clinical outcome of a patient afflicted with a muscle-invasive bladder cancer, (ii) for selecting a patient afflicted with a muscle-invasive bladder cancer for a treatment comprising an EGFR kinase inhibitor and/or capecitabine, and/or (iii) for predicting the sensitivity of a muscle- invasive bladder cancer to a treatment comprising an EGFR kinase inhibitor and/or capecitabine,
  • kit comprises detection means selected from the group consisting of a pair of primers, a probe and an antibody specific to
  • MT1X MT1X, RAB38, SFN, SAMD9, EGFR, CD44, IL1RAP, DSP, PKP1, SERPINB7, CELSR2, DUSP7, TBC1D2, ARL4D, IPPK, MTSSl and RGS20 genes, preferably PKPland IPPK, and at least 2 genes selected from the group consisting of PHC1, THYN1, TACC1, PPAP2B, NRXN3, GNA14, ZFHX3, TLE2, MAML3, EPS8, CACNA1D, RAB15, MAN1C1, SORL1, CHN2, TGFBR3, CAB39L, LIMCHl and BAMBI genes, preferably MAML3 and TGFBR3; and/or
  • the kit may further comprise detection means selected from the group consisting of a pair of primers, a probe and an antibody specific to the HDAC9 short iso form corresponding to the transcript ENST00000456174 and HDAC9 long iso forms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • the present invention also concerns a DNA chip and its use (i) for predicting clinical outcome of a patient afflicted with a muscle-invasive bladder cancer, (ii) for selecting a patient afflicted with a muscle-invasive bladder cancer for a treatment comprising an EGFR kinase inhibitor and/or capecitabine, and/or (iii) for predicting the sensitivity of a muscle-invasive bladder cancer to a treatment comprising an EGFR kinase inhibitor and/or capecitabine,
  • the DNA chip comprises a solid support which carries nucleic acids that are specific to
  • the DNA chip may further comprise nucleic acids that are specific to the HDAC9 short iso form corresponding to the transcript ENST00000456174 and HDAC9 long iso forms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • the present invention concerns a combined preparation, product or kit containing (a) capecitabine and (b) an alkylating agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a muscle-invasive bladder cancer having a basal-like phenotype as determined with the method of the invention.
  • the alkylating agent is cisplatin.
  • FIG. 1 Gene expression profiling identifies a basal-like molecular subtype of MIBC with specific genetic and immunohistochemical features.
  • A Hierarchical consensus clustering and
  • B principal component analysis of gene expression profiles of 85 MIBC (CIT series), showing two very distinct subgroups of tumors, the first (at left) comprising 64 tumors partitioned into several subclusters and the second (at right) comprising 21 tumors on which the inventors focused.
  • C Heatmap of the top 52 differentially-expressed genes between the two subgroups of MIBC, using row mean centered data.
  • D Immunohistochemical markers associated with the basal- like bladder cancer phenotype.
  • the graph below shows the distribution of the various CK5/6 and FOXA1 phenotypes in basal- like and non-basal- like MIBC (the P- value given was obtained in a chi-squared test for trend).
  • FIG. 2A Kaplan-Meier curves of overall survival in basal-like (BL) and non-basal-like (non-BL) muscle-invasive bladder carcinomas (MIBC) are shown for all patients (Fig. 2A), node negative, non-metastatic patients (NO, M0) (Fig. 2B), node positive, non-metastatic patients (Nl, M0) (Fig. 2C) and patients with metastases (Ml) (Fig. 2D). -values of log-rank tests for comparisons of overall survival between the BL and non-BL groups are shown for time points one, two and five years after diagnosis. Forest plots (Fig.
  • FIG. 3 Activation of the EGFR pathway in basal-like MIBC.
  • A qRT-PCR validation of gene expression array data comparing mRNA levels for EGFR and its ligands (AREG, EREG, HBEGF and TGFA) between basal- like and non-basal- like MIBC.
  • B Western-blotting analysis of EGFR and phospho-EGFR Tyrl068 (p-EGFR) protein levels in basal-like and non-basal-like MIBC; the tumor sample indicated by # displayed EGFR gene amplification. Down panel: representative immunoblots. Upper panel: immunoblot quantification (arbitrary units).
  • C Immunohistochemical analysis of phospho-EGFR Tyrl068.
  • FIG. 4 EGFR signaling is essential for the growth of basal- like bladder cancer cells in vitro and in vivo.
  • A Heatmap comparing levels of basal cell cytokeratins and EGFR pathway-related components between basal-like and non-basal-like bladder cancer cell lines. mRNA levels were quantified by qRT-PCR and protein levels were quantified by western blotting. Fold-change ratios are the mean value for basal-like tumors with respect to that for non-basal-like bladder cancer cell lines. -values indicate the significance of differences between the two groups of cell lines. Cell lines indicated by # displayed EGFR gene amplification and were excluded from fold-change ratio calculations for EGFR (mRNA and protein) and p-EGFR.
  • the cell lines indicated by * carried a RAS-activating mutation.
  • B Effect of erlotinib on the growth of basal- like and non-basal- like bladder cancer cell lines after 72 h of treatment. The GI50 for erlotinib is plotted for each cell line.
  • C Comparison of the GI50 for erlotinib between (BL) and non-basal-like (Non-BL) bladder cancer cell lines (box plot representation); (+) indicates the mean.
  • D Western-blot analysis of EGFR phosphorylation and downstream signaling proteins, following erlotinib treatment, in two basal-like (SCaBER and UMUC6) and two non-basal-like (JMSU1 and K 47) bladder cancer cell lines.
  • FIG. 5 An EGFR-driven autocrine mitogenic loop is activated in human basal-like bladder cancer cell lines.
  • A The effect of cetuximab on cell growth was evaluated at the clinically relevant concentration of 10 ⁇ g/ml for 11 basal- like and 11 non-basal- like bladder cancer cell lines, after 72 h of treatment. Results are expressed as the percentage of viable cells relative to untreated control cells.
  • B Comparison of cell growth following cetuximab treatment between (BL) and non-basal-like (Non-BL) bladder cancer cell lines (box plot representation); (+) indicates the mean.
  • Results are expressed as the percentage of viable cells with respect to untreated control. Data are represented as means ⁇ SEM and P- values, indicating the significance of differences between cells treated with the IgG mouse control and cells treated with the anti-AR, as assessed in a two-tailed Mann- Whitney test ( ** P ⁇ 0.001; *** P ⁇ 0.0001; ns: non significant)
  • E Effect of erlotinib and cetuximab on the transcription of EGFR ligand genes, as assessed by qRT-PCR after 8 h or 24 h of treatment of two basal-like bladder cancer cell lines, SCaBER and L1207. The L1207 cell line displayed EGFR gene amplification.
  • FIG. 6 Mouse BBN-induced bladder tumors have a basal-like molecular profile and are sensitive to anti-EGFR therapy (erlotinib).
  • A Expression levels of genes associated with the basal-like phenotype in human basal-like MIBC (BL MIBC) and in mouse basal-like BBN- induced bladder tumors (BL BBN-T) compared to normal urothelium (N), human or mouse.
  • BL MIBC human basal-like MIBC
  • BL BBN-T mouse basal-like BBN- induced bladder tumors
  • N normal urothelium
  • the heatmap on the left shows the level of expression (quantified by microarray) of each gene (rows) in each sample (columns).
  • the bar charts on the right show the mean expression value in basal-like tumors (T) for each gene relative to the mean expression value in the normal urothelium samples (N) (normalized to 1). Data are represented as means ⁇ s.e.m. -values for differences between basal-like tumors and normal urothelium were obtained in two-tailed Mann- Whitney tests (P ⁇ 0.01; ** P ⁇ 0.001).
  • B, C Effect of erlotinib treatment (100 mg/kg , 6 days per week) on the progression of BBN-induced mouse bladder tumors.
  • FIG. 7 Summary of the molecular and pathological features associated with the basal- like phenotype in 82 human MIBC from the CIT series.
  • a 40-gene predictor discriminating basal-like from non-basal-like bladder tumors was constructed from the CIT series. This predictor, validated in 6 independent transcriptome datasets, correctly classified MIBC into the basal-like and non-basal-like molecular subtypes, with the exception of one sample (CIT36), as shown by comparison with the clustering consensus approach used as the reference method for classification.
  • An immunohistochemical signature predictive of the basal-like subtype was established in a series of 62 MIBC. The CK5/6+FOXA1- phenotype was found in most of the basal-like MIBC analyzed.
  • mR A levels for seven genes were analyzed by qRT-PCR (EGFR, AREG, EREG, HBEGF, TGFA and ⁇ 63) or with gene expression arrays (KRT14).
  • the optimal cutoff point defining high and low levels of expression was obtained for each gene by regression tree analysis (RPart).
  • High levels of EGFR, AREG, EREG, HBEGF and TGFA expression were significantly associated with the basal- like subgroup in our study (indicated by *).
  • Overexpression of KRT14 and ⁇ 63 have been identified as single markers, associated with the expression of basal cell differentiation markers in previous studies by Kami-Schmidt et al, 2011 (#) and Volkmer et al, 2012 ( ⁇ ). Two-tailed Fisher's exact tests were used for statistical comparisons between basal-like and non-basal-like MIBC.
  • B TYMP protein expression measured by reverse phase protein array in human normal urothelial samples and in human MIBC presenting or not a basal-like signature.
  • C Correlation between mRNA level and protein level of TYMP in urothelial samples and in basal-like and non-basal- like MIBC. Statistical significance of correlation was assessed by Spearman's rank correlation test. Data are presented as mean ⁇ s.e.m. A two tailed Mann- Whitney test was used in A and B. *P ⁇ 0.05, ** PO.001; *** PO.0001, ns: not significant.
  • Figure 10 Class centroids for basal tumours and non basal tumours. Horizontal bars on the right (resp. left) sides quantify the inclusion (resp. exclusion) level of the exon undergoing a splicing change in each of the 19 genes.
  • FIG. 11 Expression of FOXA1 and CK5/6 assessed by immunohistochemistry in basal- like and non basal- like MIBC.
  • BL-MIBC are represented by plain black dots
  • NBL-MIBC are represented by open black dots.
  • the number beside each dot represents the number of tumors presenting the same expression of FOXAl and CK5/6 in case there are several tumors of the same type (BL-MIBC or NBL-MIBC) with the same expression of FOXAl and CK5/6.
  • the black lines represent the thresholds which separate the BL-MIBC from the NBL-MIBC: BL-MIBC present a high expression of CK5/6 and a low expression of FOXAl nuclear staining.
  • Figure 12 Alternatively spliced isoform of gene TGM1 lacking exon 9 is a specific marker for basal-like tumors.
  • Figure 14 Quantification of short (grey) and long (black) HDAC9 isoforms in basal- like and non basal-like bladder tumors.
  • Figure 17 Number of tumors exhibiting a high expression level of TGM1 gene and a high HDAC9 short/long isoform ratio according to exon array dataset. This representation includes all tumors analyzed by exon array including 30 basal- like and 177 non basal- like tumors. As shown in this figure, 15 basal- like and none non-basal- like tumors have a high expression level of TGM1 and a high HDAC9 short/long isoform ratio.
  • Figure 18 Number of tumors positive for overexpression of TGM1 and HDAC9 short/long isoform ratio according to exon array dataset. This representation includes tumors analyzed by RT-qPCR. As shown in this figure, 13 basal- like and none non-basal- like tumors have a high expression level of TGM1 and a high HDAC9 short/long isoform ratio.
  • FIG 19. Number of tumors positive for overexpression of TGM1 and HDAC9 short/long isoform ratio by RT-qPCR. All basal-like tumors are identified with this marker combination. As shown in this figure, 14 basal-like and none non-basal-like tumors have a high expression level of TGM1 and a high HDAC9 short/long isoform ratio.
  • Figure 20 Number of tumors positive for overexpression of TGM1 and HDAC9 short isoform by RT-qPCR. As shown in this figure, 13 basal- like and none non-basal- like tumors have a high expression level of TGM1 and a high HDAC9 short/long isoform ratio.
  • MIBC muscle-invasive bladder cancer
  • the levels of EGFR protein and its phosphorylated form were significantly higher in basal-like tumors than in non-basal-like tumors. However, these levels are not sufficient to distinguish these two subgroups. Accordingly, the inventors developed four methods to identify basal- like MIBC. This subgroup may be identified by immunohistochemical markers or transcriptomic, alternative splicing and DNA methylation signatures. They also demonstrated, with in vitro and in vivo preclinical models, that therapy targeting EGFR and/or comprising capecitabine, was particularly effective for basal-like tumors. Definitions
  • the methods of the invention as disclosed herein may be in vivo, ex vivo or in vitro methods.
  • the methods of the invention are in vitro methods.
  • the term “subject” or “patient” refers to an animal, preferably to a mammal, even more preferably to a human, including adult, child and human at the prenatal stage.
  • the term “subject” can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheeps and non-human primates, among others, that are in need of treatment.
  • sample means any sample containing cells derived from a subject, preferably a sample which contains nucleic acids.
  • samples include fluids such as blood, plasma, saliva, urine and seminal fluid samples as well as biopsies, organs, tissues, cell samples or cancer associated ascite fluids. The sample may be treated prior to its use.
  • sample may also refer to any sample containing free circulating nucleic acids.
  • cancer sample refers to any sample comprising tumor cells derived from a patient, preferably a sample which comprises nucleic acids. Preferably, the sample contains only tumor cells (i.e., no normal or healthy cell).
  • cancer sample may also refer to any sample comprising free circulating nucleic acids from tumor cells. Preferably, the sample contains only nucleic acids from tumor cells.
  • cancer refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features.
  • bladder cancer or “bladder tumor” is intended herein urinary bladder tumor, bladder cancer or urinary bladder cancer, and bladder neoplasm or urinary bladder neoplasm.
  • a bladder tumor can be a bladder carcinoma or a bladder adenoma, preferably a bladder carcinoma.
  • the most common staging system for bladder tumors is the TNM (tumor, node, metastasis) system. This staging system takes into account how deep the tumor has grown into the bladder, whether there is cancer in the lymph nodes and whether the cancer has spread to any other part of the body.
  • T part of TNM gives indication on how far into the bladder the cancer cells have grown: CIS/Ta: cancer cells are detected only in the innermost layer of the bladder lining; Tl : the cancer has started to grow into the connective tissue beneath the bladder lining; T2: the cancer has grown through the connective tissue into the muscle; T3: the cancer has grown through the muscle into the fat layer; and T4: the cancer has spread outside the bladder.
  • MIBC muscle-invasive bladder cancer
  • the term “poor prognosis” refers to a decreased patient survival and/or an early disease progression and/or an increased disease recurrence and/or an increased metastasis formation, preferably a decreased patient survival.
  • patient survival refers to the time interval between the date of diagnosis or the surgery and the date of death, preferably refers to the time interval between the date of cystectomy and the date of death.
  • treatment refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis and retardation of the disease.
  • such term refers to the amelioration or eradication of a disease or symptoms associated with a disease.
  • this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.
  • This term refers to the treatment at any stage of the disease. In particular, it can be an adjuvant therapy (chemo- or radiotherapy after surgery) or a neo-adjuvant therapy (chemo- or radiotherapy before surgery).
  • to treat a cancer or “treating a cancer” means reversing, alleviating, inhibiting the progress of, or preventing, either partially or completely, the growth of tumors, tumor metastases, or other cancer-causing or neoplastic cells in a patient.
  • the present invention relates to a method for determining whether a MIBC has a basal-like phenotype, i.e. belongs to the "basal-like" subgroup identified by the inventors.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining the expression level of K T5, K T6A and/or K T6B and the expression level of nuclear FOXA1 in a cancer sample.
  • the expression of K T5, K T6A and/or K T6B and the absence of nuclear FOXA1 are indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • Cytokeratin 6A (K T6A, also named cytokeratin 6C or 6D) is encoded by the gene
  • KRT6A (also named K6A, K6C, K6D, CK6A, CK6C, CK6D, KRT6C or KRT6D; Entrez Gene ID: 3853) located at 12ql3.13.
  • Cytokeratin 6B (KRT6B) is encoded by the gene KRT6B (also named K6B, PC2, CK6B, CK-6B, KRTL1; Entrez Gene ID: 3854) located at 12ql3.13.
  • Cytokeratin 5 (KRT5) is encoded by the gene KRT5 (also named K5, CK5, DDD, EBS2, KRT5A; Entrez Gene ID: 3852) located at 12ql3.13.
  • FOXA1 Forkhead box Al
  • FOXA1 also named HNF3A or TCF3A; Entrez Gene ID: 3169 located at 14ql2-ql3.
  • KRT5, KRT6A and/or KRT6B may be determined by any method known by the skilled person.
  • expression level may be determined (i) by measuring the quantity of mR A and/or (ii) by measuring the quantity of encoded protein.
  • the expression level of nuclear FOXA1 may be determined by any method known by the skilled person and that is suitable to distinguish cytoplasmic and nuclear expression of FOXA1.
  • Methods for determining the quantity of mRNA are well known in the art and include, but are not limited to, quantitative or semi-quantitative RT-PCR, real time quantitative or semiquantitative RT-PCR, Nanostring technology, sequencing based approaches or transcriptome approaches.
  • the nucleic acid contained in the sample may be first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions.
  • the extracted mRNA may be then detected by hybridization (e.g., Northern blot analysis) and/or amplification (e.g., RT-PCR).
  • Quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous.
  • primer pairs were designed in order to overlap an intron, so as to distinguish cDNA amplification from putative genomic contamination. Such primers may be easily designed by the skilled person.
  • ligase chain reaction LCR
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • the quantity of mRNA may also be measured using the Nanostring's NCOUNTERTM Digital Gene Expression System (Geiss et al, 2008) which captures and counts individual mRNA transcripts by a molecular bar-coding technology and is commercialized by Nanostring Technologies, or the QuantiGene ® Plex 2.0 Assay (Affymetrix).
  • the quantity of mRNA may further be determined using approaches based on high-throughput sequencing technology such as RNA-Seq (Wang et al, 2009).
  • the expression level of a gene may also be determined by measuring the quantity of mRNA by transcriptome approaches, in particular by using DNA microarrays.
  • the sample optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface.
  • the labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art.
  • DNA biochips suitable to measure the expression level of the genes of interest include, but are not limited to, Human Genome U133 Plus 2.0 array (Affymetrix), or any other whole human genome microarray, such as those from Agilent or Illumina. Next Generation Sequencing methods (NGS) may also be used.
  • NGS Next Generation Sequencing methods
  • the quantity of mR A is measured by quantitative or semi-quantitative RT- PCR, by real-time quantitative or semi-quantitative RT-PCR, by Nanostring technology or sequencing based approaches, or by transcriptome approaches.
  • Methods for measuring the quantity or the activity of the encoded protein are also well- known by the skilled person and the choice of the method depends on the encoded protein.
  • these methods comprise contacting the sample with a binding partner capable of selectively interacting with the protein present in the sample.
  • the binding partner is generally a polyclonal or monoclonal antibody, preferably monoclonal.
  • the quantity of protein is measured by semi-quantitative Western blots, immunochemistry (enzyme-labeled and mediated immunoassays, such as ELISAs, biotin/avidin type assays, radioimmunoassay, Immunoelectrophoresis or immunoprecipitation) or by protein or antibody arrays.
  • the protein expression level may also be assessed by immunohistochemistry on a tissue section of the cancer sample (e.g. frozen or formalin- fixed paraffin embedded material).
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • Specific activity assays may also be used, in particular when the encoded protein is an enzyme.
  • the expression level of nuclear FOXA1 is determined by immunohistochemistry. More preferably, the expression level of KRT5, KRT6A and/or KRT6B and the expression level of nuclear FOXA1 are assessed by immunohistochemistry.
  • Antibodies specifically recognizing human KRT5, KRT6A, KRT6B and FOXA1 proteins are commercially available (e.g. FOXA1 : Ref:23738, Abeam, Cambridge, United Kingdom; CK5/6: clone D5/16 B4, DakoCytomation, Glostrup, Denmark).
  • Antibodies recognizing cytokeratins 5, 6A and 6B may react with only one of these cytokeratins or with two or three, preferably with all of cytokeratins 5, 6A and 6B.
  • the method comprises determining the expression level of KRT5, KRT6A and KRT6B. In another embodiment, the method comprises determining the expression level of one or two cytokeratins selected from the group consisting of KRT5, KRT6A and KRT6B. In a preferred embodiment, the method comprises determining the expression level of KRT6B.
  • the inventors analyzed transcriptomic data of 85 MIBC by two different unsupervised methods and identified a subgroup of 21 "basal- like" tumors wherein 761 genes were differentially expressed (P ⁇ 0.01 and fold change >2) by comparison with the others tumors. Using supervised analyses, the inventors then selected a set of predictive genes for distinguishing basal-like tumors from non basal-like tumors.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining, in a cancer sample, the expression levels of
  • KRT6B (Entrez Gene ID:3854), CSTA (Entrez Gene ID: 1475), DSC2 (Entrez Gene ID: 1824), MT1X (Entrez Gene ID:4501), RAB38 (Entrez Gene ID:23682), SFN (Entrez Gene ID:2810), SAMD9 (Entrez Gene ID:54809), EGFR (Entrez Gene ID:1956), CD44 (Entrez Gene ID:960), ILIRAP (Entrez Gene ID:3556), DSP (Entrez Gene ID: 1832), PKP1 (Entrez Gene ID:5317), SERPINB7 (Entrez Gene ID:8710), CELSR2 (Entrez Gene ID: 1952), DUSP7 (Entrez Gene ID: 1849), TBC1D2 (Entrez Gene ID:55357), ARL4D (Entrez Gene ID:379), IPPK (Entrez Gene ID:64768), MTSSI (Entrez Gene ID:9788), RGS20 (Entrez Gene ID:8601), PHC1 (
  • MT1X MT1X, RAB38, SFN, SAMD9, EGFR, CD44, ILIRAP, DSP, PKP1, SERPINB7, CELSR2, DUSP7, TBC1D2, ARL4D, IPPK, MTSSI and RGS20 genes, and at least one gene selected from the group consisting of PHC1, THYN1, TACC1, PPAP2B, NRXN3, GNA14, ZFHX3, TLE2, MAML3, EPS8, CACNA1D, RAB15, MAN1C1, SORLl, CHN2, TGFBR3, CAB39L, LIMCH1 and BAMBI genes; and/or
  • the method comprises determining the expression levels of at least 3, 4, 5, 10, 15, 20, 25, 30, 35 genes selected from the group consisting of PI3, KRT6B, CSTA, DSC2, MT1X, RAB38, SFN, SAMD9, EGFR, CD44, IL1RAP, DSP, PKP1, SERPINB7, CELSR2, DUSP7, TBC1D2, ARL4D, IPPK, MTSS1, RGS20, PHC1, THYNl, TACCl, PPAP2B, NRXN3, GNA14, ZFHX3, TLE2, MAML3, EPS8, CACNAID, RAB15, MANICI, SORLl, CHN2, TGFBR3, CAB39L, LIMCH1 and BAMBI genes. More preferably, the expression levels of all these genes are determined.
  • the method comprises determining the expression levels of at least two genes selected from the group consisting of PI3, KRT6B, CSTA, DSC2, MT1X RAB38, SFN, SAMD9, EGFR, CD44, IL1RAP, DSP, PKP1, SERPINB7, CELSR2, DUSP7, TBC1D2, ARL4D, IPPK, MTSS1 and RGS20 genes, and the expression level of at least two genes selected from the group consisting of PHCl, THYNl, TACCl, PPAP2B, NRXN3, GNA14, ZFHX3, TLE2, MAML3, EPS8, CACNAID, RAB15, MANICI, SORLl, CHN2, TGFBR3, CAB39L, LIMCH1 and BAMBI genes in a cancer sample.
  • the expression levels of at least 3, 4, 5, 6, 7, 8, 9, 10 or 15 genes of each group are determined.
  • the skilled person may easily select a combination comprising at least one gene selected from the group consisting of PI3, KRT6B, CSTA, DSC2, MT1X, RAB38, SFN, SAMD9, EGFR, CD44, IL1RAP, DSP, PKPl, SERPINB7, CELSR2, DUSP7, TBC1D2, ARL4D, IPPK, MTSS1 and RGS20 genes, and one gene selected from the group consisting of PHC1, THYN1, TACC1, PPAP2B, NRXN3, GNA14, ZFHX3, TLE2, MAML3, EPS8, CACNA1D, RAB15, MANICI, SORLl, CHN2, TGFBR3, CAB39L, LIMCHl and BAMBI genes, for determining whether a MIBC has a basal- like phenotype.
  • the method may comprise determining the expression levels of at least one of the following 2-gene combinations allowing to determine whether a MIBC has a basal-like phenotype with at least 98% specificity on the training CIT dataset: BAMBI, IPPK; TLE2, IPPK; RAB15, RAB38; EPS8, IPPK; SORLl, MTSS1; SORLl, ARL4D; SORLl, PKPl; SORLl, RAB38; TGFBR3, PI3; TGFBR3, RAB38; ZFHX3, PKPl; CAB39L, IPPK; CAB39L, RAB38; LIMCHl, DSC2; LIMCHl, IL1RAP; MAML3, IL1RAP; MAML3, IPPK; THYN1, IPPK; GNA14, IPPK; TACC1, IPPK; CACNA1D, IPPK; PPAP2B, IPPK; TGFBR3, IPPK; NRXN3, IPPK; CHN2, PK
  • the method may comprise determining the expression levels of at least one of the following 3 -gene combinations allowing to determine whether a MIBC has a basal- like phenotype with 100% sensibility and specificity on the training CIT dataset: BAMBI, SORLl, PKPl; BAMBI, TGFBR3, IPPK; TLE2, TGFBR3, IPPK; PPAP2B, TGFBR3, PI3; PPAP2B, MAML3, SFN; SORLl, ZFHX3, PKPl; SORLl, CAB39L, PKPl; TGFBR3, NRXN3, KRT6B; TGFBR3, NRXN3, PI3; TGFBR3, ZFHX3, DSC2; TGFBR3, ZFHX3, CSTA; TGFBR3, ZFHX3, IPPK; TGFBR3, LIMCHl, IPPK; TGFBR3, MANICI, PI3; NRXN3, LIMCHl, IL1RAP; NRX
  • the method may comprise determining the expression levels of at least one of the following 4-gene combinations allowing to determine whether a MIBC has a basal-like phenotype with 100% sensibility and specificity on the training CIT dataset: PPAP2B, SORLl, PI3, PKPl; PPAP2B, SORLl, PI3, CELSR2; PPAP2B, SORLl, SFN, CELSR2; PPAP2B, TGFBR3, PI3, DSC2; PPAP2B, TGFBR3, PI3, RAB38; PPAP2B, TGFBR3, PI3, SFN; PPAP2B, TGFBR3, PI3, CELSR2; PPAP2B, TGFBR3, IPPK, RAB38; PPAP2B, NRXN3, PI3, DSC2; PPAP2B, NRXN3, PI3, RAB38; PPAP2B, NRXN3, RAB38, CELSR2; PPAP2B
  • the inventors also calculated the sensibility and positive predictive value (true positives).
  • the method may comprise, in a particular embodiment, determining the expression levels of at least one of the following 4-gene combinations allowing to determine whether a MIBC has a basal-like phenotype with at least 95% sensibility, at least 95% specificity and a positive predictive value (true positives / (true positives + false positives)) of at least 85% on the above-identified cohorts : TGFBR3, ZFHX3, CSTA, SFN; NRXN3, MANICI, KRT6B, TBC1D2; PPAP2B, ZFHX3, KRT6B, TBC1D2; PPAP2B, ZFHX3, KRT6B, IPPK; CHN2, ZFHX3, KRT6B, MTSSl; PPAP2B, ZFHX3, KRT6B, DSC2; TGFBR3, ZFHX3, KRT6B, MTSSl; TGFBR3, ZFHX3, PI3, PKPl
  • the method comprises determining the expression levels of at least two genes selected from the group consisting of KRT6B, CSTA, EGFR, PKPl, TBC1D2 and MTSSl genes, preferably selected from the group consisting of KRT6B, PKPl, TBC1D2 and MTSSl genes, and the expression level of at least two genes selected from the group consisting of PPAP2B, NRXN3, ZFHX3, CHN2 and TGFBR3 genes, preferably selected from the group consisting of PPAP2B, ZFHX3 and TGFBR3 genes, in a cancer sample.
  • the method may comprise determining the expression levels of at least one of the following gene combinations allowing to determine whether a MIBC has a basal- like phenotype with 100% sensibility, at least 82% specificity and a positive predictive value (true positives / (true positives + false positives)) of at least 59% on the above- identified cohorts: CACNAID, ZFHX3, DSP, PKPl; CACNAID, ZFHX3, CD44, PKPl; CACNAID, ZFHX3, TBC1D2, PKPl; BAMBI, MANICI, CD44, SFN; MANICI, THYN1, TBC1D2, DSC2; CACNAID, ZFHX3, PKPl, SAMD9; EPS8, MANICI, CD44, CELSR2; CACNAID, ZFHX3, DSP, SAMD9; EPS8, MANICI, CD44, TBC1D2; MANICI, PHCl, DSP, TBC1D
  • the method comprises determining the expression levels of at least CD44 and TBC1D2 genes, and the expression level of at least two genes selected from the group consisting of CACNA1D, ZFHX3 andMANlCl genes, in a cancer sample.
  • the method comprises determining the expression level of at least PKPl, IPPK, MAML3 and TGFBR3 genes in a cancer sample, high expression level of PKPl and IPPK genes and low expression level of MAML 3 and TGFBR3 genes, being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the expression level of each gene may be determined from a cancer sample by a variety of techniques.
  • the expression level of each gene may be determined, as disclosed above, by measuring the quantity of mRNA and/or by measuring the quantity or the activity of encoded protein.
  • the expression level of a gene is determined by measuring the quantity of mRNA.
  • the phenotype of the MIBC may be determined using any commonly used suitable algorithm such as, for example, the nearest shrunken centroid (NSC) algorithm, the support vector machine (SVM) algorithm, or the k-nearest neighbour algorithm.
  • NSC nearest shrunken centroid
  • SVM support vector machine
  • the phenotype of the MIBC is obtained using the Nearest Shrunken Centroid method developed by Tibshirani et al, 2002.
  • the training data set for the selected algorithm comprises MIBC with basal- like and non basal-like phenotypes.
  • the training data set may be selected, for example, from the group consisting of the CIT series disclosed in the experimental section, GSE13507 (Kim et al, 2010), GSE1827 (Blaveri et al, 2005, GSE19915 (Lindgren et al, 2010), GSE31684 (Riester et al, 2012), GSE5479 (Dyrskjot et al, 2007), E-TABM-147 (Stransky et al, 2006) and the cohort disclosed in Sanchez-Carbayo et al, 2006.
  • the method further comprises determining whether the expression levels of said genes are high or low compared to the reference expression level(s).
  • the reference expression level may be the expression level of a gene having a stable expression in different cancer samples, such as RPLPO, HPRT1, GAPDH, B2M, TBP and 18S genes.
  • the expression level of the gene of interest is considered as high if the level or quantity of mRNA is above a cut-off value easily adjusted by the skilled person depending on the gene of interest and the reference gene.
  • the cut-off value may be easily defined for each gene via a training data set by optimizing a criterion such as a chi-squared test.
  • the reference expression levels may also be the mean expression levels of said genes among a population of randomly selected MIBC samples.
  • the expression levels of the genes may be normalized using the expression level of an endogenous control gene having a stable expression in different cancer samples, such as RPLPO, HPRT1, GAPDH, B2M, TBP and 18S genes.
  • the inventors analyzed splicing changes in 191 MIBC samples and 7 samples of normal urothelium. They then selected alternative splicing events to distinguish basal-like and non basal-like tumors. In particular, the inventors showed that a single alternative splicing event identified in TGM1 gene was enough to correctly classify 70% of basal- like tumours based on numeric data from the exon arrays 3.17.
  • Alternative isoform including exon 9 appears to be ubiquitously expressed in basal-like, non basal- like and normal tissue. However, isoform lacking exon 9 is highly specific to basal- like tumours since the other samples only show a slightly detectable signal.
  • basal-like tumors can be correctly classified based on an alternative splicing event identified in HDAC9 gene.
  • Alternative isoform including exon 1 appears to be specific to basal-like tumors.
  • basal-like tumors can be classified based on the short/long HDAC9 isoform expression ratio wherein the HDAC9 short isoform corresponds to the transcript ENST00000456174 and comprises exon 1 (SEQ ID NO:37) and HDAC9 long iso forms correspond to transcripts ENST00000406451, ENST00000405010 and ENST00000428307 and do not include exon 1.
  • the inventors demonstrated that this ratio is higher in basal-like tumors than in non basal-like tumors or normal urothelium samples.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining the expression level of at least one exon selected from the group consisting of the exons of SEQ ID NO: 23 to 41 in a cancer sample.
  • the expression levels of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 exons selected from the group consisting of the exons of SEQ ID NO: 23 to 41 are determined. More preferably, the expression levels of all the exons of SEQ ID NO: 23 to 41 are determined.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining the expression level of the exon of SEQ ID NO:24 in a cancer sample.
  • the method may further comprise determining the expression level of at least one exon selected from the group consisting of the exons of SEQ ID NO: 23, and 25 to 41.
  • the method for determining whether a MIBC has a basal-like phenotype comprises determining the expression level of the exon of SEQ ID NO:37 in a cancer sample.
  • the method may further comprise determining the expression level of at least one exon selected from the group consisting of the exons of SEQ ID NO: 23 to 36 and 38 to 41, preferably of the exon of SEQ ID NO:24.
  • the expression level of each exon may be determined from a cancer sample by a variety of techniques, in particular by measuring the quantity of mRNA comprising said exon.
  • the method may further comprise determining whether the expression levels of said exons are high or low compared to the reference expression level.
  • the reference expression level may be the expression level of each exon in a normal sample, preferably a normal sample of urothelium.
  • the reference expression level may also be the expression level of other transcript iso forms from the same gene that do not comprise said exon.
  • the expression levels of the exons may be normalized using the expression level of an endogenous control gene having a stable expression in different cancer samples, such as RPLPO, HPRT1, GAPDH, B2M, TBP and 18S genes.
  • high expression levels of exons selected from the group consisting of the exons of SEQ ID NO:23, 25, 27, 28, 30, 31, 32 34, 37 and 39, and low expression levels of exons selected from the group consisting of the exons of SEQ ID NO:24, 26, 29, 33, 35, 36, 38, 40 and 41, are indicative that the muscle-invasive bladder cancer has a basal- like phenotype.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining the expression level of the exon of SEQ ID NO:24 in a cancer sample, a low expression level of said exon being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the expression level of the exon of SEQ ID NO:24 may be determined by in situ hybridization targeting the specific junction between exon 8 and exon 10.
  • the method may further comprise determining the expression level of at least one exon selected from the group consisting of the exons of SEQ ID NO: 23, and 25 to 41, high expression levels of exons selected from the group consisting of the exons of SEQ ID NO:23, 25, 27, 28, 30, 31, 32 34, 37 and 39, and low expression levels of exons selected from the group consisting of the exons of SEQ ID NO: 26, 29, 33, 35, 36, 38, 40 and 41, being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the method for determining whether a MIBC has a basal-like phenotype comprises determining the expression level of the exon of SEQ ID NO:37 in a cancer sample, a high expression level of said exon being indicative that the muscle- invasive bladder cancer has a basal-like phenotype.
  • the method may further comprise determining the expression level of at least one exon selected from the group consisting of the exons of SEQ ID NO: 23 to 36 and 38 to 41, high expression levels of exons selected from the group consisting of the exons of SEQ ID NO:23, 25, 27, 28, 30, 31, 32, 34 and 39, and low expression levels of exons selected from the group consisting of the exons of SEQ ID NO: 24, 26, 29, 33, 35, 36, 38, 40 and 41, being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the method for determining whether a MIBC has a basal-like phenotype comprises determining the expression level of HDAC9 short isoform corresponding to the transcript ENST00000456174 and HDAC9 long isoforms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • the transcript ENST00000456174 is 2,307 bp in length and the corresponding cDNA is set forth in SEQ ID NO: 109.
  • the transcript ENST00000406451 is 9,705 bp in length and the corresponding cDNA is set forth in SEQ ID NO: 110.
  • the transcript ENST00000405010 is 4,239 bp in length and the corresponding cDNA is set forth in SEQ ID NO: 111.
  • the transcript ENST00000428307 is 2,359 bp in length and the corresponding cDNA is set forth in SEQ ID NO: 112.
  • the method may further comprise calculating the ratio of HDAC9 short isoform expression level to HDAC9 long isoform expression levels, i.e. the ratio of the expression level of the transcript ENST00000456174 to the sum of the expression levels of the transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • this ratio can be calculated by determining the quantity of mRNA for HDAC9 short isoform corresponding to the transcript ENST00000456174 and the quantity of mRNA for HDAC9 long isoforms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • the quantity of mRNA for each transcript can be assessed by any method known by the skilled person, preferably by RT-qPCR.
  • the expression level of each transcript may be normalized, for example using the expression level of an endogenous control gene having a stable expression in different cancer samples, such as RPLPO, HPRT1, GAPDH, B2M, TBP and 18S genes.
  • the method may further comprise determining whether the calculated ratio is high or low compared to a reference level, a high ratio being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the reference level may be the ratio calculated in a normal sample, preferably a normal sample of urothelium. The ratio is considered as high if its value is above a cut-off value easily adjusted by the skilled person depending on the reference level.
  • the reference level is the ratio calculated in a normal urothelium sample and the ratio is considered as high if its value corresponds to at least 20 or 25 fold increase compared to the reference level, preferably at least 30 fold increase.
  • the method may further comprise determining the expression level of at least one exon selected from the group consisting of the exons of SEQ ID NO: 23 to 41, preferably selected from the group consisting of the exons of SEQ ID NO: 24 and 37, even more preferably of the exon of SEQ ID NO:24.
  • basal-like tumors can be correctly classified not only based on an alternative splicing event identified in TGM1 gene, but also on the global expression of TGM1 gene. Indeed, they observed that TGM1 gene is overexpressed in basal- like tumors by comparison with non-basal-like tumors.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining the expression level of TGM1 gene (Entrez Gene ID: 7051) in a cancer sample. High expression level of this gene is indicative that the MIBC has a basal-like phenotype.
  • Expression level of TGM1 gene may be determined from a cancer sample by a variety of techniques as disclosed above, in particular by measuring the quantity of mRNA.
  • the method may further comprises determining whether the expression level of TGM1 gene is high or low compared to the reference expression level(s).
  • the reference expression level may be the expression level of a gene having a stable expression in different cancer samples, such as RPLPO, HPRT1, GAPDH, B2M, TBP and 18S genes.
  • the expression level is considered as high if the level or quantity of mRNA is above a cut-off value easily adjusted by the skilled person.
  • the cut-off value may be easily defined via a training data set by optimizing a criterion such as a chi-squared test.
  • the reference expression level is the expression level of TGM1 gene in a normal urothelium sample.
  • the expression level of TGM1 gene is considered as high if its value corresponds to at least 3 fold increase compared to the reference level, preferably at least 4 fold increase.
  • the expression level of TGM1 gene may be normalized using the expression level of an endogenous control gene having a stable expression in different cancer samples, such as RPLPO, HPRT1, GAPDH, B2M, TBP and 18S genes.
  • the method for determining whether a MIBC has a basal- like phenotype comprises determining the expression level of TGM1 gene and determining (i) the expression level of the exon of SEQ ID NO:37 or (ii) the expression levels of HDAC9 short isoform corresponding to the transcript ENST00000456174 and HDAC9 long isoforms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • the method comprises determining the expression level of TGM1 gene, and determining the expression levels of HDAC9 short isoform corresponding to the transcript ENST00000456174 and HDAC9 long iso forms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • the method may further comprise calculating the ratio of HDAC9 short isoform to HDAC9 long iso forms as disclosed above.
  • the method comprises determining the expression level of TGM1 gene, determining the expression levels of HDAC9 short isoform corresponding to the transcript ENST00000456174 and HDAC9 long iso forms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307, calculating the ratio of HDAC9 short isoform expression level to HDAC9 long isoform expression levels, wherein a high expression level of TGM1 gene and a high ratio are indicative that the MIBC has a basal- like phenotype.
  • the inventors further identified DNA methylation signatures to distinguish basal- like and non basal-like MIBC.
  • the method for determining whether a MIBC has a basal- like phenotype comprises
  • the DNA methylation status may be determined using any method commonly known by the skilled person such as, for example, methods which used DNA methylation sensitive enzymes, or bisulfite treatment of DNA such as COBRA, methylation specific PCR (MSP), pyrosequencing, MethyLight, DNA arrays or highthroughput sequencing.
  • methods which used DNA methylation sensitive enzymes or bisulfite treatment of DNA such as COBRA, methylation specific PCR (MSP), pyrosequencing, MethyLight, DNA arrays or highthroughput sequencing.
  • the method may comprise determining the DNA methylation status of at least 5, 6, 7, 8, 9, 10, 11, 12 CpG islands selected from the group consisting of CpG islands listed on Table
  • the method may comprise determining the DNA methylation status of at least 5, 6, 7,
  • the method comprises determining
  • the method comprises determining the DNA methylation status of CpG islands of SEQ ID NO: 43, 45, 47 and 51, hypermethylation of the CpG island of SEQ ID NO: 43 and hypomethylation of CpG islands of SEQ ID NO: 45, 47 and 51, being indicative that the muscle-invasive bladder cancer has a basal-like phenotype.
  • the method comprises determining the DNA methylation status of GpC sites of SEQ ID NO: 85, 86, 91 and 96, hypomethylation of said CpG islands being indicative that the muscle- invasive bladder cancer has a basal-like phenotype.
  • the inventors demonstrated overall survival was significantly shorter for patients with basal-like MIBC. They further showed that the basal-like subtype was a prognosis factor independent of sex, node and metastasis status.
  • the present invention relates to a method for predicting clinical outcome of a patient afflicted with a muscle-invasive bladder cancer, wherein the method comprises determining in a cancer sample from said patient whether the muscle- invasive bladder cancer has a basal-like phenotype with the method according to the invention, the presence of the basal-like phenotype being indicative of a poor prognosis.
  • the method further comprises the step of providing a cancer sample from the patient.
  • the inventors Using in vitro and in vivo preclinical models, the inventors further demonstrate that, contrary to non-basal- like tumors, therapy targeting EGFR and/or comprising capecitabine was particularly effective for basal-like tumors.
  • the present invention relates to a method for selecting a patient afflicted with a muscle-invasive bladder cancer for a treatment comprising an EGFR kinase inhibitor and/or capecitabine, wherein the method comprises determining in a cancer sample from said patient whether the muscle-invasive bladder cancer has a basal- like phenotype with the method according to the invention, and optionally determining whether the muscle-invasive bladder cancer has a RAS-activating mutation, the presence of the basal-like phenotype being indicative that said patient is susceptible to benefit from a treatment comprising capecitabine, and the presence of the basal- like phenotype and the absence of RAS-activating mutation being indicative that said patient is susceptible to benefit from a treatment comprising an EGFR kinase inhibitor.
  • the method further comprises the step of providing a cancer sample from the patient.
  • RAS-activating mutation refers to an activating mutation in a gene encoding a RAS protein.
  • RAS refers to a family of proteins (HRAS (SEQ ID NO: 100), KRAS (SEQ ID NO: 101), NRAS (SEQ ID NO: 102)) involved in signal transduction, in particular the signal transduction of tyrosine kinase receptors (Eswarakumar et al., 2005). They exist in two different states, an active state when bound to GTP and an inactive state when the GTP is converted to GDP because of the GTPase activity of RAS. In cancer, including bladder cancer, mutated form of all three RAS genes have been described.
  • RAS mutations have less GTPase activity so they remain in an active state.
  • An estimated rate of 17% of RAS mutations (HRAS, KRAS and NRAS mutations) in bladder cancer can be deduced from the Cosmic data base of the Sanger Institute (www.sanger.ac.uk/genetics/CGP/cosmic/).
  • the references for the different activating mutations of HRAS, KRAS and NRAS can also be found in the review of Schubbert et al. (2007).
  • the RAS-activating mutation can be selected among HRAS mutations G12S and G13V, KRAS mutations G12C and G12D and NRAS mutation M72I.
  • an overexpression of RAS is also contemplated.
  • the term "EGFR kinase inhibitor” refers to a molecule which inhibits or reduces the kinase activity of the epidermal growth factor receptor.
  • the activity of EGFR kinase can be easily assayed by any method known in the art for quantifying kinase activity or analyzing protein phosphorylation (see for example Olive, 2004).
  • the EGFR kinase inhibitor may be selected from the group consisting of a small molecule inhibiting the EGFR kinase activity, an antibody directed against the extracellular domain of the EGFR and a nucleic acid molecule interfering specifically with the expression of EGFR.
  • the EGFR kinase inhibitor is a small molecule inhibiting the EGFR kinase activity.
  • small molecule inhibiting the EGFR kinase activity refers to small molecule that can be an organic or inorganic compound, usually less than 1000 daltons, with the ability to inhibit or reduce the activity of the EGFR kinase activity.
  • This small molecule can be derived from any known organism (including, but not limited to, animals, plants, bacteria, fungi and viruses) or from a library of synthetic molecules.
  • the small molecule may be selected from the group consisting of erlotinib, gefitinib and lapatinib, and any combination thereof.
  • the EGFR kinase inhibitor is an antibody, preferably a monoclonal antibody, directed against the extracellular domain of the EGFR.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD and IgE, and humanized or chimeric antibody.
  • IgG and/or IgM are preferred because they are the most common antibodies in the physiological situation and they are most easily manufactured.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab', Fab, F(ab') 2, single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single chain Fv
  • scFv single chain Fv
  • a "humanized” antibody is an antibody in which the constant and variable framework region of one or more human immunoglobulins is fused with the binding region, e.g. the CDR, of an animal immunoglobulin.
  • Humanized antibodies contemplated in the present invention are chimeric antibodies from mouse, rat, or other species, bearing human constant and/or variable region domains, bispecific antibodies, recombinant and engineered antibodies and fragments thereof. Such humanized antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody.
  • a "chimeric" antibody is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • the term "antibody directed against the extracellular domain of EGFR” designates an antibody as described above which is able to bind to the extracellular domain of the EGF receptor and to block or reduce its activity. This inhibition can be due to steric hindrance or modification which prevents ligand binding.
  • the antibody directed against the extracellular domain of the EGFR may be selected from the group consisting of cetuximab, panitumumab, zalutumumab, nimotuzumab and matuzumab, and any combination thereof.
  • the EGFR kinase inhibitor a nucleic acid molecule interfering specifically with the expression of EGFR.
  • nucleic acid molecule includes, but is not limited to, RNAi, antisense and ribozyme molecules.
  • a "nucleic acid molecule specifically interfering with the expression of EGFR” is a nucleic acid molecule which is able to reduce or to suppress the expression of gene coding for EGFR, in a specific way.
  • RNAi or "interfering RNA” means any RNA which is capable of down-regulating the expression of the targeted protein. It encompasses small interfering RNA (siRNA), double-stranded RNA (dsRNA), single- stranded RNA (ssRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules.
  • siRNA molecules to inhibit gene expression, for example, WO 99/32619, US 20040053876, US 20040102408 and WO 2004/007718.
  • siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR (untranslated region) and 3'UTR are usually avoided.
  • the chosen siRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted.
  • Various products are commercially available to aid in the preparation and use of siRNA.
  • the RNAi molecule is a siRNA of at least about 15-50 nucleotides in length, preferably about 20-30 base nucleotides, preferably about 20-25 nucleotides in length.
  • RNAi can comprise naturally occuring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end of the molecule or to one or more internal nucleotides of the RNAi, including modifications that make the RNAi resistant to nuclease digestion.
  • RNAi may be administered in free (naked) form or by the use of delivery systems that enhance stability and/or targeting, e.g., liposomes, or incorporated into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors (WO 00/53722), or in combination with a cationic peptide (US 2007275923). They may also be administered in the form of their precursors or encoding DNAs. In a particular embodiment, RNAi are encapsulated within vesicles, preferably within liposomes.
  • Antisense nucleic acid can also be used to down-regulate the expression of EGFR.
  • the antisense nucleic acid can be complementary to all or part of a sense nucleic acid encoding a EGFR polypeptide e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence, and it thought to interfere with the translation of the target mRNA
  • the antisense nucleic acid is a RNA molecule complementary to a target mRNA encoding a EGFR polypeptide.
  • An antisense nucleic acid can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • antisense RNA molecules are usually 18-50 nucleotides in length.
  • An antisense nucleic acid for use in the method of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • antisense RNA can be chemically synthesized, produced by in vitro transcription from linear (e.g. PCR products) or circular templates (e.g., viral or non-viral vectors), or produced by in vivo transcription from viral or non-viral vectors.
  • Antisense nucleic acid may be modified to have enhanced stability, nuclease resistance, target specificity and improved pharmacological properties.
  • antisense nucleic acid may include modified nucleotides designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides.
  • Ribozyme molecules can also be used to decrease levels of EGFR.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single- stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA.
  • Ribozyme molecules can be designed, produced, and administered by methods commonly known to the art (see e.g., Fanning and Symonds, 2006, reviewing therapeutic use of hammerhead ribozymes and small hairpin RNA).
  • the EGFR kinase inhibitor is selected from the group consisting of erlotinib, gefitinib, lapatinib, cetuximab, panitumumab, zalutumumab, nimotuzumab and matuzumab, and any combination thereof.
  • the EGFR kinase inhibitor is selected from the group consisting of erlotinib and cetuximab, and combination thereof.
  • the present invention also relates to a method of predicting the sensitivity of a muscle- invasive bladder cancer to a treatment comprising an EGFR kinase inhibitor and/or capecitabine, wherein the method comprises determining whether the muscle-invasive bladder cancer has a basal- like phenotype with the method according to the invention, and optionally determining whether the muscle-invasive bladder cancer has a RAS-activating mutation, the presence of the basal-like phenotype in said cancer being indicative that said cancer is sensitive to a treatment comprising capecitabine, and the presence of the basal-like phenotype and the absence of RAS-activating mutation being indicative that said cancer is sensitive to a treatment comprising an EGFR kinase inhibitor.
  • the method further comprises the step of providing a cancer sample from the patient.
  • the present invention also relates to an EGFR kinase inhibitor for use in the treatment of muscle-invasive bladder cancer having a basal- like phenotype as determined with the method according to the invention and without RAS-activating mutation.
  • the present invention further relates to a method for treating a patient affected with a muscle-invasive bladder cancer having a basal-like phenotype as determined with the method according to the invention and without RAS-activating mutation, comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising an inhibitor of EGFR, and optionally a pharmaceutically acceptable carrier.
  • the present invention also relates to capecitabine for use in the treatment of muscle- invasive bladder cancer having a basal- like phenotype as determined with the method according the invention.
  • the present invention further relates to a method for treating a patient affected with a muscle-invasive bladder cancer having a basal-like phenotype as determined with the method according to the invention, comprising administering a therapeutically efficient amount of a pharmaceutical composition comprising capecitabine, and optionally a pharmaceutically acceptable carrier.
  • the muscle-invasive bladder cancer has a basal-like phenotype and RAS-activating mutation.
  • the EGFR inhibitor may be used in combination with capecitabine.
  • the EGFR inhibitor and/or capecitabine may also be used in combination with an alkylating agent.
  • alkylating agents include, but are not limited to, platinum-based chemotherapy drugs, cyclophosphamide, chlorambucil, uramustine, estramustine, ifosfamide, melphalan, bendamustine, carmustine, lomustine, semustine, streptozotocin, busulfan, dacarbazine, procarbazine, altretamine, adozelesin, thiotepa, mitozolomide and temozolomide.
  • Platinum-based chemotherapy drugs may include, but are not limited to, cisplatin, carboplatin, iproplatin, spiroplatin, nedaplatin, oxaliplatin, triplatin tetranitrate and satraplatin.
  • the alkylating agent is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, cyclophosphamide and ifosfamide, and any combination thereof. More preferably, the alkylating agent is cisplatin. Even more preferably, capecitabine is used in combination with an alkylating agent, preferably with cisplatin.
  • a “therapeutically efficient amount” is intended an amount of therapeutic agent administered to a patient that is sufficient to constitute a treatment of a bladder cancer.
  • the amount of EGFR inhibitor and/or capecitabine to be administered has to be determined by standard procedure well known by those of ordinary skill in the art.
  • Physiological data of the patient e.g. age, size, and weight
  • the routes of administration have to be taken into account to determine the appropriate dosage.
  • the appropriate dosage of each compound may also vary if it is used alone or in combination.
  • the present invention concerns a combined preparation, product or kit containing (a) capecitabine and (b) an alkylating agent, preferably cisplatin, as a combined preparation for simultaneous, separate or sequential use in the treatment of a muscle-invasive bladder cancer having a basal-like phenotype as determined with the method according to the invention, preferably a muscle-invasive bladder cancer having a basal- like phenotype and RAS- activating mutation.
  • the present invention also concerns a combined preparation, product or kit containing (a) an EGFR kinase inhibitor and (b) an alkylating agent, preferably cisplatin, as a combined preparation for simultaneous, separate or sequential use in the treatment of a muscle-invasive bladder cancer having a basal-like phenotype as determined with the method according to the invention, preferably a muscle-invasive bladder cancer having a basal-like phenotype and without RAS-activating mutation.
  • the present invention relates to a kit and its use (i) for predicting clinical outcome of a patient afflicted with a muscle-invasive bladder cancer, (ii) for selecting a patient afflicted with a muscle-invasive bladder cancer for a treatment comprising an EGFR kinase inhibitor and/or capecitabine, and/or (iii) for predicting the sensitivity of a muscle- invasive bladder cancer to a treatment comprising an EGFR kinase inhibitor and/or capecitabine,
  • kit comprises detection means selected from the group consisting of a pair of primers, a probe and an antibody specific to
  • DUSP7, TBC1D2, ARL4D, IPPK, MTSSl and RGS20 genes preferably PKPland IPPK, and at least 2 genes selected from the group consisting of PHC1, THYN1, TACC1, PPAP2B, NRXN3, GNA14, ZFHX3, TLE2, MAML3, EPS8, CACNA1D, RAB15, MANIC 1, SORL1, CHN2, TGFBR3, CAB39L, LIMCH1 and BAMBI genes, preferably MAML3 and TGFBR3; and/or
  • a leaflet providing guidelines to such use.
  • the kit may further comprise detection means selected from the group consisting of a pair of primers, a probe and an antibody specific to a RAS-activating mutation, in particular a mutation selected from the group consisting of HRAS mutations G12S and G13V, KRAS mutations G12C and G12D and NRAS mutation M72I.
  • the present invention also relates to a DNA chip and its use (i) for predicting clinical 5 outcome of a patient afflicted with a muscle-invasive bladder cancer, (ii) for selecting a patient afflicted with a muscle-invasive bladder cancer for a treatment comprising an EGFR kinase inhibitor and/or capecitabine, and/or (iii) for predicting the sensitivity of a muscle-invasive bladder cancer to a treatment comprising an EGFR kinase inhibitor and/or capecitabine,
  • the DNA chip comprises a solid support which carries nucleic acids that are 10 specific to
  • the DNA chip may further comprise nucleic acids that are specific to a RAS-activating 30 mutation, in particular a mutation selected from the group consisting of HRAS mutations G12S and G13V, KRAS mutations G12C and G12D and NRAS mutation M72I. Further aspects and advantages of the present invention will be described in the following examples, which should be regarded as illustrative and not limiting.
  • Table 1 Clinical data and identification of the basal-like molecular subtype in the CIT dataset
  • the Stransky series is composed of 39 MIBC samples.
  • RNA, DNA and protein extraction from tissues and cell lines RNA, DNA and protein extraction from tissues and cell lines.
  • RNA samples were frozen in liquid nitrogen and stored at -80°C until nucleic acid and protein extraction.
  • RNA, DNA and proteins were extracted from frozen human bladder samples and from human bladder cancer cell lines grown to 70% confluence, by centrifugation through a cesium chloride density gradient (Chirgwin et al, 1979; Coombs et al, 1990).
  • RNA was extracted from BBN-induced mouse bladder tumors or normal mouse urothelium, with Trizol.
  • the concentration, integrity and purity of each RNA sample were determined with the RNA 6000 LabChip Kit (Agilent Technologies) and an Agilent 2100 Bioanalyzer. DNA purity was assessed by determining the ratio of absorbances at 260 and 280 nm.
  • DNA concentration was determined with a Hoechst dye-based fluorescence assay (Labarca and Paigen, 1980). Proteins were resuspended in 1 x Laemmli buffer supplemented with protease and phosphatase inhibitors (Roche) and concentration was determined with a BCA Protein Assay-Reducing Agent Compatible kit (Pierce). Gene mutation analysis
  • FGFR3 mutations were studied with the SNaPshot method, as previously described (van Oers et al, 2005).
  • TP53 exons 2 to 11
  • KRAS exons 2-3
  • NRAS exons 2-3
  • HRAS exons 2-3
  • PIK3CA PIK3CA gene mutations were screened by direct sequencing with previously described primers and protocols (Boyault et al, 2007; Wallerand et al, 2005).
  • Basal-like bladder tumors and cell lines were analyzed for the presence of hotspot mutations in the EGFR gene (exons 18-21) by direct sequencing with previously described primers (Boyault et al, 2007).
  • the presence of the truncated EGFR variant III (EGFRvlll) was also assessed in these samples, by analysis of the cDNA with primers binding to sequences flanking the deletion of exons 2 to 7, after gel electrophoresis, as previously described (Sok et al, 2006). All mutations were confirmed by sequencing both strands of a second, independent PCR product.
  • Tissue samples were immunostained for CK5/6 and FoxAl on tissue microarrays (TMA) constructed with Beecher's Tissue arrayer®, according to the manufacturer's instructions (http://www.beecherinstruments.com), using three replicate cores (diameter: 0.6 mm) in each case.
  • TMA tissue microarrays
  • the sections were placed in citrate buffer pH 6, and microwaved for antigen retrieval, and then incubated with the primary rabbit polyclonal antibody against FoxAl (ref 23738, Abeam, Cambridge, United Kingdom) overnight (dilution 1 :200) at +4°C.
  • CK5/6 immunostaining mouse monoclonal antibody, clone D5/16 B4, dilution 1/100, DakoCytomation, Glostrup, Denmark
  • antigen retrieval was carried out in EDTA pH 9, and paraffin sections were processed on an automated instrument (Ventana Nexes; Ventana Medical Systems, Arlington AZ, USA) with an indirect biotin-avidin system, the Ventana Basic DAB Detection kit (Ventana Medical Systems), according to the manufacturer's instructions.
  • phospho-EGFR immunostaining nine whole tumor sections were used. The sections were placed in EDTA pH 9 and microwaved for antigen retrieval, then incubated with the rabbit monoclonal antibody (clone D7A5, ref 3777, Cell Signaling, Boston, MA) overnight (dilution l :50) at +4°C.
  • RNA levels were quantified on a LightCycler® 480 Instrument (Roche) in predesigned or custom gene expression assays, with gene-specific primers and a dye-labeled hydrolysis probe (TaqMan probes from Applied Biosystems or UPL (Universal ProbeLibrary) probes from Roche).
  • Predesigned assays were used to quantify human 18s rRNA (reference gene) and EREG gene expression (Gene: EREG, Assay ID: 110729, Supplier: Roche; Gene: Human 18s rRNA, Assay ID: 4319413E, Supplier: Applied Biosystems).
  • Custom assays were used for the other eight genes (Table 2).
  • primers and probes were designed with Probe Finder software via the Universal Probe Library Assay Design Center (Roche).
  • qRT-PCR was carried out with the LightCycler® 480 Instrument (Roche) in a 20 ⁇ reaction mixture containing lOng of reverse-transcribed RNA, 1 x LightCycler® 480 Probe Master, 25 ⁇ each of the forward and reverse primers and 10 ⁇ of the UPL probe (or 1 x the predesigned assay probe). All expression assays were run in the same thermal cycling conditions, including an initial step at 95°C (10 min), followed by 40 cycles at 95°C (10 s), 60°C (30 s) and 72°C (10 s). For each gene of interest, the amount of mRNA was normalized with respect to that of the ribosomal 18S (R18S) reference gene by the 2 ⁇ ACt method.
  • R18S ribosomal 18S
  • Affymetrix microarray profiling ofRNA extracted from human tumors CIT series
  • human cell lines CIT series
  • mouse samples CIT series
  • Expression profiles and associated clinical data for 326 MIBC samples were obtained from six public datasets (Blaveri et al, 2005; Dyrskjot et al, 2007; Kim et al, 2010; Lindgren et al, 2010; Riester et al, 2012; Stransky et al, 2006).
  • Expression profiles for bladder cancer cell lines were also obtained from two public series (Lee et al, 2007) (and an unpublished but publicly available dataset created by Wooster et. al. in collaboration with GlaxoSmithKline). A detailed description of these series is provided in the table below.
  • DNA copy number was analyzed for the 85 MIBC from the CIT series on the human genome-wide CIT-CGHarray (V6) designed by the CIT-CGH Consortium.
  • the overall survival of patients presenting with muscle-invasive bladder tumors was analyzed by combining seven independent datasets (CIT, Riester, Lindgren, Kim, Dyrskjot, Stransky, and Blaveri). Survival time was calculated from the date of cystectomy. Data were censored for patients lost to follow-up or alive at the time of last follow-up. Survival analysis was based on Kaplan-Meier curves (function surv), log rank tests (function survdiff) and Cox models (function coxph) using the survival package of R. Forest plots were generated with the R package rmeta.
  • the MGHU3, RT4 and SCaBER cell lines were a gift from Francisco Real (CNIO Madrid).
  • TCCSUP and KK47 were obtained from the laboratories of Miguel Chopin (Hopital Henri Mondor, Creteil, France) and Jennifer Southgate (previously of Cancer Research Unit St James's University Hospital Leeds, UK), respectively.
  • L1207 cell line was derived from tumor T1207, as previously described (De Boer et al., 1997). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. Erlotinib was obtained from LC laboratories. Cetuximab (5 mg/ml) was purchased from Merck KGaA (Darmstadt, Germany) and stored at 4°C. Assessment of cell viability
  • Cell viability was determined by colorimetric MTT assays.
  • cell lines were dispensed into 96-well plates at a density of 1500-8000 cells per well and incubated overnight. They were then treated for 72 h with various doses of erlotinib (0.01, 0.05, 5 0.1, 0.5, 1, 5 and 10 ⁇ , dissolved in 0.1% DMSO) or with 10 ⁇ g/ml cetuximab in 100 ⁇ of DMEM supplemented with 0.5% fetal bovine serum and 40 ⁇ g/ml transferrin.
  • Neutralizing antibody experiments were performed with a mouse monoclonal antibody 15 directed against human amphiregulin (MAB262) (purchased from R&D Systems). 24 h after the seeding of 96-well plates at a density of 4000-6500 cells per well, cells were treated for 6 days in DMEM (without serum) supplemented with 40 ⁇ g/ml transferrin and the neutralizing antibody directed against amphiregulin at a concentration of 5 ⁇ g/ml. Mouse IgG (5 ⁇ g/ml; R&D Systems) was included as a negative control. The culture medium was replaced after 3 0 days with medium containing fresh antibodies. All experiments were performed in triplicate and were carried out at least 3 times.
  • MAB262 human amphiregulin
  • cell viability was determined by adding MTT (1 mg/ml) to each well, after 1 hour of incubation at 37°C. The medium was then removed, and 100 ⁇ of DMSO was added, to lyse the cells and solubilize the 5 formazan.
  • mice Six-week-old female Swiss nu/nu mice (Charles River Laboratories) were raised in the 30 animal facilities of Institut Curie, in specified pathogen-free conditions. Animal care and housing conformed to the institutional guidelines of the French National Ethics Committee (Ministere de ⁇ Agriculture et de la Foret, Direction de la Sante et de la Protection Animale, Paris, France) and was supervised by accredited investigators. Mice received a subcutaneous injection, into each flank (dorsal region), of 2xl0 6 UMUC6 or 6x l0 6 K 47, JMSU1, BFTC905, VMCUB1 or L1207 bladder cancer cells in 100 ⁇ PBS.
  • mice were randomly separated into two groups of six mice when tumors reached a volume of 100 mm 3 ( ⁇ 20). The mice were treated six days per week by oral gavage with erlotinib (100 mg/kg) in one group and with vehicle (0.5% carboxymethylcellulose) in the other. Tumor size was measured twice weekly with calipers, and the volume in mm 3 was calculated with the formula: ⁇ /6 ⁇ (largest diameter) x (shortest diameter) 2 .
  • Bladder carcinogenesis was induced by supplying eight- week-old adult C57BL/6 male mice for 13 weeks with 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) (TCI Europe) in drinking water (el-Marjou et al, 2000). Mice were kept in the carcinogenesis room, with standard water, for a further two weeks. They were then randomly separated into two groups (20 mice/group), which were treated six days per week by oral gavage with erlotinib (100 mg/kg) or with vehicle (0.5% carboxymethylcellulose 0.5%) until the mice were killed.
  • BBN N-butyl-N-(4-hydroxybutyl) nitrosamine
  • Tumor formation was followed weekly, by ultrasound, in anesthetized mice, with a High-Resolution In Vivo Micro-Imaging System with a 707B probe (Vevo770, VisualSonics, Toronto, Canada). Tiny tumors accounting for 2 to 5% of bladder volume were detectable by this method. The time at which a tumor could first be unambiguously localized and its growth followed on a weekly basis was considered for tumor- free survival curve. Mice were killed when their tumors were 90-95%) the size of the bladder or when they showed signed of weakness (large weight loss), and this was the time considered for the overall survival curve. Tumors were removed. A part of each tumor was fixed in formalin and embedded in paraffin for histological analyses, and another part of the tumor was flash-frozen in liquid nitrogen for mRNA extraction in the Trizol protocol, for gene expression analysis.
  • Transcriptional profiling of muscle-invasive bladder cancers identifies a distinct molecular subtype, the basal-like subtype
  • genes (466 of 761) in the subgroup of interest was significantly enriched in genes associated with epithelial wound healing, including KRT6A, KRT16, KRT17 and PLAU, PLAUR (plasminogen activator/plasmin system) (Romer et al, 1994), keratinocyte differentiation, including several genes of the epidermal differentiation complex at lq21, and genes expressed in the basal layer of stratified epithelia, such as KRT5, KRT6A, KRT14 and EGFR.
  • the underexpressed genes included several bladder epithelial (urothelial) differentiation markers, such as uroplakin genes (UPK1A, UPK1B, UPK2, UPK3A) and FOXA1 (Varley et al, 2009).
  • uroplakin genes UPK1A, UPK1B, UPK2, UPK3A
  • FOXA1 FOXA1
  • the list of differentially-expressed genes was also significantly enriched in markers of a particular subgroup of breast cancer known as basal breast cancer (Perou et al, 2000). This, together with the enrichment in basal epithelial cell markers, led the inventors to call this subgroup "basal- like".
  • the inventors identified three regions significantly (Chi2 P ⁇ 0.01) more altered in the basal-like subgroup than in other MIBC (at 3pl4.2, 6p, 7pl 1.2).
  • the 3pl4.2 region which presented significantly more losses (both hemizygous and homozygous deletions), contained 52 genes, including the tumor suppressor gene FHIT.
  • the whole 6p arm showed significantly more gains.
  • the 7pl 1.2 region which presented a significant increase in copy number (both gains (up to four copies) and amplifications (more than four copies)) contained two genes, including the gene encoding the tyrosine kinase receptor EGFR.
  • the inventors determined from the KEGG and Bio carta databases those pathways most significantly altered in the basal-like subgroup with respect to other MIBC, using all seven available transcriptome datasets.
  • the pathway involving EGFR (“EGF signaling" in Biocarta and "ErbB signaling” in KEGG) was one of the most significantly altered pathways in the various datasets.
  • EGFR the genes encoding five of its six ligands (AREG, AREGB, EREG, HBEGF, TGFA), a downstream effector of EGFR (MYC), and genes known to be induced by EGFR activation in the urothelium (IL8, SOX9) (Ling et al, 2011 ; Perrotte et al, 1999) were overexpressed in basal- like tumors compared to non-basal- like MIBC. By contrast, ERBB3, another member of the ERBB family, was found to be underexpressed.
  • the inventors confirmed, by qRT-PCR, that EGFR and the genes encoding several of its ligands were significantly overexpressed in basal-like compared to non-basal-like tumors (Figure 3A). Moreover, the levels of EGFR protein and its phosphorylated form were significantly higher (2.4 times higher and 2 times higher, respectively) in basal-like tumor cells than in non-basal- like MIBC ( Figure 3B and 3C). Finally, they investigated the mechanisms underlying the upregulation of EGFR and its ligands, by analyzing mRNA levels as a function of gene copy number. They found a highly significant positive correlation between EGFR mRNA levels and EGFR copy number in basal- like tumors.
  • Table 4 Human basal-like bladder cancer gene expression predictor. A set of 40 predictive genes was selected to discriminate the basal-like samples from non basal-like samples using the CIT discovery cohort and the area under curve (AUC) criteria.
  • AUC area under curve
  • Table 5 Molecular subtype classification of 22 distinct human bladder cancer cell lines.
  • Bladder cancer cell line ID Dataset Chip basal- like subtype 0:
  • VMCUB1 Lee Affymetrix HG-U133A 1
  • VMCUB3 CIT Affymetrix EXON Hs 0
  • VMCUB3 Lee Affymetrix HG-U133A 0
  • the mRNA levels were analyzed for some of the genes typical of the basal-like phenotype (basal cytokeratins; EGFR and its ligands) by qRT-PCR and the levels of EGFR protein and its phosphorylated form by western blotting, in the 22 bladder cancer cell lines ( Figure 4A). The results obtained were consistent with the predicted basal-like/non-basal-like classification of the cell lines.
  • the two cell lines with the highest levels of EGFR and phospho- EGFR were basal- like and were the only cell lines displaying EGFR gene amplification (Black et al, 2008; Nicolle et al., 2006).
  • erlotinib a small-molecular inhibitor of EGFR
  • BBN N-butyl-N-(4-hydroxybutyl)nitrosamine
  • BBN induces muscle-invasive bladder tumors that typically have mixed histological features, displaying both urothelial and squamous cell differentiation (Becci et al, 1978; Tamano et al, 1991), as in the human basal- like subgroup.
  • the inventors previously showed that the growth of mouse cell lines derived from bladder tumors induced by chemical treatment with BBN is dependent on an autocrine loop involving EGFR and secreted growth factors (el-Marjou et al., 2000). They therefore hypothesized that BBN-induced mouse tumors could be used as a model of human basal-like tumors.
  • BBN-induced mouse bladder tumors closely resembled human bladder basal-like tumors, as nine out of the 11 mouse tumors showed a high correlation coefficient with the human basal-like centroid. Consistent with these results, analyses of the level of expression of genes typically associated with the basal-like phenotype showed the same profile of deregulation in mouse and human basal-like tumors, with respect to normal urothelium (mouse or human) ( Figure 6A).
  • the BBN mouse model of bladder cancer is a suitable model for the basal-like subtype of bladder tumors.
  • the inventors investigated the effect of erlotinib on bladder tumor-free survival and overall survival in this mouse model. Erlotinib treatment, initiated two weeks after the end of BBN treatment and administered six days per week, significantly delayed the detection of tumors assessed by echography (Figure 6B) and increased survival in mice ( Figure 6C).
  • the basal-like subgroup in MIBC was first identified from a non-supervised analysis of tumor transcriptomic data.
  • the inventors then developed, from their CIT series, a 40-gene predictor, which they validated in six independent transcriptomic datasets, with a sensitivity of 97% and a specificity of 88%.
  • This 40-gene predictor can therefore be used to identify basal- like MIBC.
  • Immunohistochemistry represents a simpler and more accessible assay in clinical practice. They showed that the expression of cytokeratins 5 and 6 and the lack of expression of FOXA1 can be used to identify basal- like MIBC in the CIT series ( Figure 1, Figure 7), with a specificity of 89%> and a sensitivity of 89%>.
  • a series of 85 muscle-invasive bladder carcinomas (31 pT2, 35 pT3 and 19 pT4) was collected from patients surgically treated from 1988 and 2006 at Henri Mondor Hospital, Institut Gustave Roussy (Villejuif, France), and Foch Hospital (Suresnes, France). Tumors were staged according to the 1997 TNM classification (Sobin and Fleming 1997) and graded according to the 1973 WHO classification (Mostofi, 1973). The clinical annotations for the patients and the pathologic features for each tumor were also recorded. Normal urothelial samples were obtained during organ procurement from cadaveric donors for transplantation, as previously described (Diez de Medina, 1998). All patients provided written informed consent and the study was approved by the ethics committees of the different hospitals.
  • RNA, DNA and proteins were extracted from bladder frozen samples and from bladder cancer cell lines grown to 70%> confluency, by cesium chloride density centrifugation(Chirgwin et al, 1979; Coombs et al, 1990).
  • concentration, integrity and purity of each RNA sample were determined with the RNA 6000 LabChip Kit (Agilent Technologies) and an Agilent 2100 Bioanalyzer. DNA purity was also assessed by determining the ratio of absorbances at 260 and 280 nm. DNA concentration was determined with a Hoechst dye-based fluorescence assay (Labarca et al, 1980). Proteins were resuspended in IX Laemmli buffer containing anti-protease and anti-phosphatase (Roche) and concentration was determined by a BCA Protein Assay-Reducing Agent Compatible kit (Pierce).
  • DSMZ (VMCUB1, JMSU1, BFTC-905) or ECACC (UMUC6).
  • K 47 were obtained from the laboratory of Jennifer Southgate (Cancer Resarch Unit St James'Hospital Leeds, UK). Cell line identity was confirmed by analyzing specific gene mutations previously described in each cell line.
  • the L1207 cell line was derived from tumor T1207, as previously described (De Boer et al, 1997). All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 U/mL streptomycin.
  • DMEM Dulbecco's modified Eagle's medium
  • Capecitabine (LC laboratories) was provided as a powder and suspended in vehicle (40 mM citrate buffer containing 0.5% of carboxymethylcellulose, pH 6.0) and was administrated by oral gavage.
  • Cisplatine was from Mylan Laboratory and was administrated by intraperitoneal injection.
  • mice Six-week-old female Swiss nu/nu mice (Charles River Laboratories) were raised in the animal facilities of the Curie Institute, in specified pathogen-free conditions. Their care and housing were in accordance with the institutional guidelines of the French National Ethics Committee (Ministere de I 'Agriculture et de la Foret, Direction de la Sante et de la Protection Animale, Paris, France), with supervision by authorized investigators. Mice receive a subcutaneous injection, into each flank (dorsal region), of 2xl0 6 UMUC6 or 6x 10 6 KK47, JMSU1, BFTC905 and L1207 bladder cancer cells in 100 ⁇ PBS.
  • mice were randomly separated into two groups of six mice. Mice were treated by oral gavage with vehicle control (40 mM citrate buffer containing 0.5% of carboxymethylcellulose, pH 6.0) or capecitabine (400 mg/kg) for 7 consecutive days followed by a one-week rest. Tumor size was measured twice a week with calipers and the volume in mm 3 was calculated using the formula: ⁇ /6 ⁇ (larger diameter) x (shorter diameter) 2 . Quantitative RT-PCR
  • RNA levels were quantified using pre- designed (for R18S) or custom gene expression (for TYMP and DPYD) assays containing gene specific primers and a dye-labeled hydrolysis probe (TaqMan probes from Applied Biosystems or UPL (Universal ProbeLibrary) probes from Roche).
  • primers and probes were designed with probeFinder software via the Universal ProbeLibrary Assay Design Center (Roche).
  • TYMP-sens 5 ' GC AC AC AGGAGGC ACCTT3 ' SEQ ID NO: 17
  • TYMP-anti-sens 5 ' CCTGGTCC AGC AGC ACTT3 ' SEQ ID NO: 18
  • TYMP-probe 5'CTCCAGCT3' SEQ ID NO: 19
  • DPYD-sens 5 ' C AAGAGCTGC AAAGGAAGGT3 ' SEQ ID NO: 20
  • DPYD-anti- sens 5 ' CCC ATC AGACCTGAG AC AGTG3 ' SEQ ID NO: 21
  • DPYD-probe 5'AGCTGGAG3' SEQ ID NO: 22
  • QRT-PCR were carried out with the LightCycler® 480 Instrument (Roche) in a 20 ⁇ reaction mixture containing, lOng of reverse transcribed RNA, l x LightCycler® 480 Probes Master, 25 ⁇ each of the forward and reverse primers and 10 ⁇ of the UPL probe (or IX of the pre-designed assay). All expression assays were run using the same thermal cycling conditions including initial step at 95°C (10 min), followed by 40 cycles at 95°C (10 s), 60°C (30 s) and 72 °C (10 s). For each gene of interest, the amounts of mRNA was normalized to that of the Ribosomal 18S (R18S) reference gene using the 2 "ACt method.
  • R18S Ribosomal 18S
  • Samples were deposited onto nitrocellulose covered slides (Schott Nexterion NC-C, Jena, Germany) using a dedicated arrayer (2470 Arrayer, Aushon Biosystems, Billerica, MA, USA). Four serial dilutions, ranging from 1.5 to 0.094 mg/ml, and four technical replicates per dilution were deposited for each sample. Arrays were revealed with an anti-TYMP antibody (HPA001072 from Sigma) using an Autostainer Plus robot (Dako, Trappes, France). Briefly, slides were incubated with avidin, biotin and peroxydase blocking reagents (Dako) before saturation with TBS containing 0.1% Tween-20 and 5% BSA (TBST-BSA).
  • Capecitabine is an orally administrated anticancer pro-drug of 5-fluorouracil (5-FU) that is selectively tumor-activated.
  • Capecitabine is metabolized in the liver to the intermediate 5'- deoxy-5-fluorouridine, which is subsequently converted into cytotoxic 5-FU by the thymidine phosphorylase (TYMP), an enzyme overexpressed in many tumors.
  • TYMP thymidine phosphorylase
  • Intracellular 5-FU is then catabolized to its inactive metabolites via dihydropyrimidine dehydrogenase (DP YD).
  • the ratio TYMP/DPYD mRNA is increased in basal-like MIBC and xenograft models of basal-like bladder tumors are sensitive to capecitabine treatment
  • TYMP/DPYD activity was found to be correlated with capecitabine susceptibility in various human cancer xenograft models (Ishikawa et al. 1998), we also investigated the expression level of DP YD by qRT-PCR in MIBC and normal urothelium samples. While, TYMP/DPYD mRNA ratio was significantly elevated both in basal-like (13- fold) and non basal-like MIBC (7 fold) compared to normal urothelium samples, this increase was much more important in basal-like tumors. These results led us to hypothesize that capecitabine may be particularly effective in the treatment of basal-like tumors.
  • capecitabine was further investigated in vivo on subcutaneous xenograft of four basal- like bladder cancer cell lines (LI 207, VMCUB1, UMUC6 and BFTC- 905) and two non-basal-like bladder cancer cell line (JMSU1 and RT112).
  • basal-like bladder cancer cell lines LI 207, VMCUB1, UMUC6 and BFTC- 905
  • JMSU1 and RT112 two non-basal-like bladder cancer cell line
  • the inventors further showed that the combination capecitabine + cisplatin provided a synergistic effect and greatly reduced tumor growth of the basal- like cell line BFTC-905 having a NRAS-activating mutation and that is resistant to a treatment using EGFR kinase inhibitor (cf. above) (Figure 13).
  • Example 3 alternative splicing signature to identify basal-like MIBC
  • the set of 191 bladder carcinomas were obtained from tumour tissue banks at Henri Mondor Hospital, Institut Gustave Roussy (Villejuif, France), and Foch Hospital (Suresnes, France). These cancers were selected randomly from a consecutive set, to cover the different 15 stages of bladder cancer as follows: 51 Ta, 37 Tl, 27 T2, 46 T3, and 30 T4 tumours. 7 normal urothelial samples were also used for the analysis. They were obtained from fresh urothelial cells scraped from the normal bladder wall and dissected from the lamina intestinal during organ procurement from cadaveric donors for transplantation. All patients provided written informed consent and the study was approved by the ethics committees of the different hospitals.
  • RNA from the 198 samples was processed and labelled using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay as outlined in the manufacturer's instructions. Hybridization to Affymetrix Human Exon 1.0 ST arrays was carried out independently at the Institut Curie Affymetrix platform. Affymetrix Expression 25 Console Software was used to perform quality assessment.
  • the ARH method (Rasche et al., 2010) was used to identify exons differentially included in transcripts between tumours and normal samples. ARH scores by gene and ARH splicing deviations by exon were computed for each tumour using median values over normal samples as reference. ARH splicing deviations give a measure of inclusion level differences of each exon in the corresponding gene for each tumour compared to normal samples. The values are centered in 0, negative values suggest a loss of exon whereas positive values suggest a gain of exon in the tumour relative to normal samples. Fisher exact test was used to identify relationships between each splicing change and molecular or clinical data. The computations were performed using R platform (R development Core Team, 2011). Nearest shrunken centroid ASE classifier
  • the Nearest Shrunken Centroid method developed by Tibshirani (Tibshirani et al., 2002; Hastie et al, 2011) was used to build a classifier identifying basal tumours based on alternative splicing changes.
  • the classifier was trained using basal/non basal class prediction based on the established transcriptomic signature and the ARH splicing deviation data obtained for each tumour and each exon were used to train and classify the samples.
  • the method uses a training set to computes a standardized centroid for each class, which is derived from the average splicing deviation for each exon in each class of tumours divided by the within-class standard deviation for that exon.
  • the classifier takes the exon splicing deviation profile of a new sample, and compares it to each of these class centroids (see class centroids on figure 10).
  • the class whose centroid that it is closest to, in squared distance, is the predicted class for that new sample.
  • Affymetrix Human Exon arrays were used to analyse splicing changes in 191 tumour samples and 7 samples of normal urothelium. ARH scores and splicing deviations were used to assess significance to every putative splicing event in tumours compared to normal samples. A total of 26 038 alternative splicing changes were found to occur in at least 10% of tumours considering p-values ⁇ 0.05 and fold-changes > 2. For each of those events Fisher's exact tests were used to identify the relationships between splicing changes, molecular and clinical features of the disease, including basal-like phenotype. Only splicing changes linked to basal tumours with the smallest significant p-value were selected to identify a basal-like-specific alternative splicing signature (1 309 events).
  • the inventors trained a classifier based on a subset of 103 tumours: only muscle-invasive tumours were selected for the training phase, so that the classification would not depend on the invasiveness of tumours. According to the training phase results, the inventors chose to select 19 alternative splicing events that were sufficient to distinguish basal- like and non basal- like tumours with only 2 misclassification errors, which corresponds to the best ratio [error rate / number of markers] achieved. The same efficiency was observed when applying the classifier on the whole dataset of 191 tumours.
  • a simple hierarchical clustering based on ARH splicing deviations for the 19 markers performs as well for separating basal- like and non basal- like tumours (data not shown).
  • the 19 alternative splicing changes selected therefore define an alternative splicing signature for the basal-like tumours which can be used by itself to characterize this aggressive subgroup of tumours with a sensitivity of 96% and a specificity of 99%.
  • the genes and exons (ranked according to the longest coding transcript they belong to) included in the signature are listed in Table 6 below.
  • Table 6 Signature of alternative splicing changes specific to basal-like tumours. 9 exons are down-regulated and 10 exons are up-regulated in basal-like tumours compared to non-basal-like tumours and normal samples.
  • the ENSE and ENST identifiers refer to Ensembl database.
  • transcript ID transcript vs Non Basal
  • TGMl alternative splicing pattern enabled the characterization of two unknown transcript structures involving mutually exclusive exons ( Figure 12).
  • Alternative isoform including exon 9 appears to be ubiquitously expressed in basal-like, non basal-like and normal tissue.
  • isoform lacking exon 9 is highly specific to basal-like tumours since the other samples only show a slightly detectable signal.
  • in situ hybridization targeting the specific junction between exon 8 and exon 10 provides a good marker to detect basal- like tumours.
  • Example 4 methylation signature to identify basal- like MIBC Material and methods
  • a set of 68 bladder carcinomas were obtained from tumour tissue banks at Henri Mondor Hospital, Institut Gustave Roussy (Villejuif, France), and Foch Hospital (Suresnes, France). These cancers were selected randomly from a consecutive set, to cover the different stages of bladder cancer as follows: 20 Ta, 9 Tl, 11 T2, 17 T3, and 11 T4 tumours. 4 normal urothelial samples and 3 normal bladder muscle samples were also used for the analysis. Normal urothelium samples were obtained from fresh urothelial cells scraped from the normal bladder wall and dissected from the lamina intestinal during organ procurement from cadaveric donors for transplantation. All patients provided written informed consent and the study was approved by the ethics committees of the different hospitals.
  • the Nearest Shrunken Centroid method developed by Tibshirani (Tibshirani et al., 5 2002; Hastie et al, 2201 1) was used to build a classifier identifying basal- like tumours based on their DNA methylation profile.
  • the classifier was trained using basal-like/non basal-like class prediction based on the established transcriptomic signature and the ⁇ - values obtained for each tumour and each CpG site (or each CpG island) were used to train and classify the samples.
  • the method uses a training set to compute a standardized centroid for each class, which0 is derived from the average ⁇ -values of CpG sites (or CpG islands) in each class of tumours divided by the within-class standard deviation.
  • the classifier takes the profile of ⁇ -values of a new sample, and compares it to each of these class centroids (see class centroids on figures 1 and 2). The class whose centroid that it is closest to, in squared distance, is the predicted class for that new sample. 5 Thresholds computations
  • Table 7 Signature of CpG island methylation specific to basal-like tumours. 2 CpG islands are hypermethylated and 11 CpG islands are hypomethylated in basal-like tumours compared to non-basal-like tumours.
  • Threshold eg Threshold.
  • I Gene symbol CpG ID CpG position chrl : 155931629- hypo 0,37 0,22 ARHGEF2 eg 155931629 155931858 20847292
  • Table 8 Forward sequence of the area targeted with the array. CpG sites are in brackets.
  • CTTCCC[CG]GTGTGGTCTCGCTGTTCCCTTTGAGAGTGCGATGCTGCCGCTTCAG CCAGGACGTTCTCA (SEQ ID NO :68)
  • Table 9 Signature of CpG site methylation specific to basal-like tumours. All these CpG sites are hypomethylated in basal-like tumours compared to non-basal-like tumours. CpG sites are in brackets.
  • Threshold gene CpG ID Chr. CpG position FOR WARD SEQUENCE
  • Example 5 Identification of basal-like MIBC using basal-like specific mRNA isoform of HDAC9 gene and/or HDAC9 short/long isoform ratio and/or overexpression of TGM1 gene
  • HDAC9 short and long iso forms are the result of alternative promoter usage.
  • HDAC9 long iso forms correspond to transcripts ENST00000406451, ENST00000405010 and ENST00000428307 as described on the Ensembl database.
  • Short isoform corresponds to the transcript ENST00000456174.
  • bladder tumors were collected from patients surgically treated between 1988 and 2006 at Henri Mondor Hospital, Institut Gustave Roussy (Villejuif, France), and Foch Hospital (Suresnes, France). 5 normal bladder urothelium samples were also collected.
  • Probe to exons assignments were drawn from the custom CDF files of Dai et al (Dai et al., 2005) in version 12 for Ensembl exons. Only a subset of well- annotated core probe sets from the exon array was used for the study.
  • ARH alternative promoter usage from exon array data was conducted based on a published statistical method called ARH (Rasche et al., 2010). The method enables to compare two samples and measure significance of the possible differences in transcript structures for each gene between the samples.
  • ARH scores by gene and ARH splicing deviations by exon were computed for each tumor using median values over normal samples as reference (bladder urothelium samples).
  • ARH scores are linked to p-values referring to the hypothesis that the gene is alternatively spliced (has at least one exon differentially included in the gene) between tumor and normal samples.
  • ARH splicing deviations give a measure of inclusion level differences of each exon in the corresponding gene for each tumor compared to normal samples.
  • RNA 2 ⁇ g were reverse transcribed using the High capacity cDNA reverse transcription kit (Applied Biosystems, Courtaboeuf, France). For all samples, lOng of cDNA were used for amplification with a LightCycler 480 Instrument (Roche Diagnostics, Meylan, France). All samples were run in duplicate. Amplification was performed using SybrGreen master mix (Roche Diagnostics). Forward/reverse primers used for TGM1 gene expression are 5 '-AACTCCCTGGATGACAATGG-3 ' (SEQ ID NO : 103) / 5'- GCAGCACTGTGGTGGTC A-3 ' (SEQ ID NO : 104).
  • Forward reverse primers used for HDAC9 short and long isoforms are 5 '-TCACAGTGTAGCTTGAGAAAAATG-3 ' (SEQ ID NO : 105) / 5 '-TGCAACTTGATATGCTCCTGA-3 ' (SEQ ID NO : 106) and 5'- CAGATGGGGTGGCTGGAC-3 ' (SEQ ID NO : 107) / 5'-TGCTTCTGGATTTGTTGCTG-3' (SEQ ID NO : 108) respectively.
  • 18S housekeeping gene expression was quantitatively measured in each sample using a pre-designed gene expression assay containing gene specific primers and a dye-labeled hydrolysis probe Assay-on demand (Applied Biosystems).
  • Quantification was performed on 20 basal-like and 20 no basal-like tumors randomly chosen from samples analyzed on exon array.
  • HDAC9 short isoform corresponding to the transcript ENST00000456174 comprises exon 1 contrary to HDAC9 long isoforms corresponding to transcripts ENST00000406451, ENST00000405010 and ENST00000428307.
  • Figure 15 shows short/long HDAC9 isoform expression ratios in basal- like and non- basal-like bladder tumors. These results demonstrate that this ratio is overexpressed in basal- like and then discriminates basal-like and non-basal-like bladder tumors.
  • TGM1 gene expression in basal-like and non-basal-like bladder tumors was performed by RT-qPCR (Figure 16).
  • the inventors observed that TGM1 gene was overexpressed in basal-like tumors by comparison with non-basal-like tumors and showed that basal-like MIBC can be classified based on global expression of TGM1 gene.
  • combining TGMl gene expression and short/long HDAC9 isoform expression ratio resulted in highly efficient classification of basal-like and non basal like MIBC.
  • the qPCR cut-off values were chosen to optimize both specificity and sensibility.
  • the HDAC9 short/long isoform ratio was considered as "high” if its value corresponded to 30 fold increase compared to normal urothelium sample.
  • the expression level of TGMl gene was considered as "high” if its value corresponded to 4 fold increase compared to normal urothelium sample.
  • R A language and environment for statistical computing.

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Abstract

La présente invention concerne un procédé pour classifier des patients souffrants du cancer de la vessie invasif pour le muscle pour une intervention thérapeutique, en particulier pour la sélection d'un patient atteint du cancer de la vessie invasif pour le muscle pour un traitement comprenant un inhibiteur de kinase EGFR et/ou de capécitabine.
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WO2016105503A1 (fr) * 2014-12-24 2016-06-30 Genentech, Inc. Méthodes de traitement, de diagnostic et de pronostic du cancer de la vessie
CN113528659A (zh) * 2020-09-18 2021-10-22 深圳汇芯生物医疗科技有限公司 肾癌和膀胱癌的风险评估装置
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US11248054B2 (en) 2017-06-12 2022-02-15 Bluefin Biomedicine, Inc. Anti-IL1RAP antibodies and antibody drug conjugates
CN116144790A (zh) * 2022-11-30 2023-05-23 扬州大学 一种湖羊肌肉细胞增值相关的标志物及其检测引物和应用
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