Methods for the surveillance, diagnosis and screening of bladder cancer

Abstract

The invention relates to a method of diagnosing or predicting a bladder cancer, or a risk of a developing a bladder cancer in a subject, which method comprises the detection of specific mutations in the FGFR3 gene in a first biological sample; and the measure of the degree of methylation of target genes in a biological sample obtained from said subject in a second biological sample,

1 . A method for the surveillance, diagnosis and screening of a bladder cancer, or a risk of a developing a bladder cancer in a subject, wherein said method comprises:
 a) a step of detecting a mutation in the FGFR3 gene in a first biological sample; and   b) a step of measuring the degree of methylation of at least two genes selected from the group consisting of SEPTIN 9, SLIT 2, TWIST 1, HS3ST2 and fragments or variants thereof in a second biological sample obtained from said subject,   wherein   said first and second biological samples are the same biological sample; and   said step a) is performed by:
 detecting a mutation selected from the group consisting of 742C→T, 746C→G, 1114G→T, and 1124A→G by reference to the nucleotide numbers of SEQ ID No 1; or 
 detecting a mutation in the group consisting of mutations Arg248Cys, Ser249Cys, Gly372Cys, and Tyr375Cys, by reference to the aminoacid numbers of SEQ ID NO. 2. 
   
     
     
         2 . The method according to  claim 1 , wherein step a) and step b) are performed in the same bisulfited treated DNA obtained from a single biological sample and wherein step a) is a step of determining the presence of:
 a guanine (G) in position 746 of the sequence depicted in SEQ ID NO. 1 which was bisulfite treated;   a thymine (T) in position 1114 of the sequence depicted in SEQ ID NO. 1 which was bisulfite treated; and/or   a guanine (G) in position 1124 of the sequence depicted in SEQ ID NO. 1 which was bisulfite treated.   
     
     
         3 . The method according to  claim 1 , wherein said method comprises a further step b′) after step b) of comparing the degree of methylation measured in step b) to a threshold value, wherein said threshold value distinguishes between a patient suffering or at risk of developing a bladder cancer and a patient who does not suffer from bladder cancer. 
     
     
         4 . The method according to  claim 1 , wherein:
 the presence of said mutation in the FGFR3 gene, and   the comparison of the methylation degree of at least one gene selected from the group consisting of SEPTIN 9, SLIT 2, TWIST 1, HS3ST2 and fragment or variant thereof to a threshold value   are indicative of a bladder cancer or of a risk of developing a bladder cancer.   
     
     
         5 . The method according to  claim 1 , wherein step a) of the method of the invention further comprises the detection of a mutation in TERT gene, said detection being performed by:
 detecting a mutation selected from the group consisting of mutations 77C→T and 99C→T by reference to the nucleotide numbers of SEQ ID No 32.   
     
     
         6 . The method according to  claim 1 , wherein step b) consists in:
 measuring the degree of methylation of the fragment of SEPTIN 9 as depicted in SEQ ID No 3;   measuring the degree of methylation of the fragment of SLIT 2 as depicted in SEQ ID No 4;   measuring the degree of methylation of the fragment of TWIST 1 as depicted in SEQ ID No 5; and   measuring the degree of methylation of the fragment of HS3ST2 as depicted in SEQ ID No 6.   
     
     
         7 . The method according to  claim 1 , wherein step b) further comprises the measure of the degree of methylation of DDR1 gene or one of its fragments or variants thereof. 
     
     
         8 . The method according to  claim 7 , wherein said fragment is depicted in SEQ ID NO: 7. 
     
     
         9 . The method according to  claim 1 , wherein said first and second biological samples are urine. 
     
     
         10 . The method according to  claim 1 , wherein step a) is performed by allele specific PCR (AS-PCR). 
     
     
         11 . The method according to  claim 10 , wherein step a) is performed using all the primers as depicted in SEQ ID NOS. 8 to 13. 
     
     
         12 . The method according to  claim 1 , wherein step b) is performed by quantitative real-time multiplex methylation specific polymerase chain reaction (Qm-PCR). 
     
     
         13 . The method according to  claim 12 , wherein said step b) is performed using the primers and probes as depicted in SEQ ID NOS. 16 to 30. 
     
     
         14 . The method according to  claim 12 , characterized in that it further comprises a step of measuring the degree of methylation of a housekeeping gene. 
     
     
         15 . The method according to  claim 14 , wherein said housekeeping gene is selected among albumin, β-Actin and β-Globin. 
     
     
         16 . The method according to  claim 3 , wherein said threshold is expressed in cumulative methylation index. 
     
     
         17 . The method according to  claim 1 , wherein said method further comprises a step c) of measuring the level of expression of a gene selected from the group consisting of BCLA-4 and BCAR-1. 
     
     
         18 . The method according to  claim 17 , wherein said step c) further comprises a step c′) of comparing said level of expression to the level of expression obtained in a healthy subject or a subject who overcame bladder cancer. 
     
     
         19 . A kit comprising:
 (a) at least one oligonucleotide selected from the group consisting of SEQ ID NOS. 8 to 13; and   (b) at least one oligonucleotide selected from the group consisting of SEQ ID NOS. 16 to 30.   
     
     
         20 . The method according to  claim 15 , wherein said housekeeping gene is albumin or a fragment or variant thereof.