Method for Detecting a Mutation in a Microsatellite Sequence

Abstract

The invention relates to a method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising subjecting said DNA sample to a digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising:a pair of primers for amplifying said target fragment of the DNA sample including said microsatellite sequence;a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence; anda second oligonucleotide reference (REF) hydrolysis probe, labeled with a second fluorophore, wherein said second oligonucleotide REF probe is complementary to a wild-type sequence of said target DNA fragment which does not include said microsatellite sequence. The invention also encompasses methods for the diagnosis and prognosis of cancer and a method for determining the efficacy of a cancer treatment.

1 . A method for detecting a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, comprising a step of subjecting said DNA sample to a digital polymerase chain reaction (dPCR) in the presence of a PCR solution comprising:
 a pair of primers suitable for amplifying said target fragment of the DNA sample including said microsatellite sequence;   a first MS oligonucleotide (MS) hydrolysis probe, labeled with a first fluorophore, wherein said first MS oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence;   a second oligonucleotide reference (REF) hydrolysis probe, labeled with a second fluorophore, wherein said second oligonucleotide REF probe is complementary to a wild-type sequence of said target DNA fragment located outside of said microsatellite sequence.   
     
     
         2 . The method according to  claim 1 , wherein the target fragment of the DNA sample is constitutional genomic DNA. 
     
     
         3 . The method according to  claim 1 , wherein the target fragment of the DNA sample is genomic tumor DNA. 
     
     
         4 . The method according to  claim 1 , wherein the microsatellite sequence locus is selected from the group comprising BAT-25, BAT-26, BAT-34c4, BAT-40, NR21, NR24, MONO-27, D2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF2IP1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1 and TDRD1. 
     
     
         5 . The method according to  claim 1 , wherein the DNA sample is selected from the group consisting of tumor tissue, disseminated cells, feces, blood cells, blood plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, cerebrospinal fluid, serous fluids. 
     
     
         6 . The method according to  claim 1  further comprising a step of measuring the fluorescence signals associated with the REF and MS probes, wherein the maximal fluorescence intensity signal associated with both the REF and MS probes indicates the presence of a wild-type microsatellite sequence in the target DNA fragment, while a shift in the fluorescence intensity signal associated with the MS probe indicates the presence of a mutation in the microsatellite sequence of the target DNA fragment 
     
     
         7 . A method for the detecting cancers, diseases associated with mutations in mismatch repair (MMR) genes or familial tumor predisposition in a subject, comprising the detection of a mutation in a microsatellite sequence locus of a target DNA from a DNA sample according to  claim 1 , wherein the target fragment is originating from a tumor. 
     
     
         8 . A method for prognosis of cancers comprising the detection of a mutation in a microsatellite sequence locus of a target fragment from a DNA sample according to  claim 1 , wherein the target fragment is originating from a tumor. 
     
     
         9 . A method for predicting the efficacy of a treatment in a subject suffering from a cancer, comprising the detection of a mutation in a microsatellite sequence locus of a target fragment from a DNA sample according to  claim 1 , wherein the target fragment is originating from a tumor and wherein the treatment is preferably immune therapy such as immune checkpoint therapy. 
     
     
         10 . A method of treatment of a cancer in a subject in need thereof comprising:
 the detection of a mutation in a microsatellite sequence locus of a target fragment from a DNA sample according to  claim 1 , and   the administration to the subject of an immunotherapy if a mutation is identified in a microsatellite sequence locus of the target fragment,   
       wherein the target fragment of the DNA sample originates from a tumor. 
     
     
         11 . A method for the monitoring of a patient diagnosed with a tumor associated with impaired DNA mismatch repair (MMR), or having suffered from such tumor, comprising the detection of a mutation in a microsatellite sequence locus of a target fragment from a DNA sample, wherein the target fragment of the DNA sample originates from a tumor. 
     
     
         12 . A kit for identifying a mutation in a microsatellite sequence region of a target fragment from a DNA sample comprising:
 a pair of primers suitable for amplifying said target fragment from the DNA sample including said microsatellite sequence;   a first oligonucleotide hydrolysis probe (MS), labeled with a first fluorophore, wherein said first oligonucleotide probe is complementary to a wild-type sequence including the microsatellite sequence;   a second oligonucleotide hydrolysis probe (REF), labeled with a second fluorophore, wherein said second oligonucleotide probe is complementary to a wild-type sequence of said amplified DNA fragment located outside of said microsatellite sequence; and   a thermostable polymerase.