Methods for detecting mutations in microsatellite sequences

Abstract

The present invention relates to a method for detecting mutations at microsatellite sequence loci in a target fragment from a DNA sample, the method comprising subjecting the DNA sample to digital polymerase chain reaction (PCR) in the presence of a PCR solution comprising: a pair of primers suitable for amplifying a target fragment from the DNA sample, the target fragment containing a microsatellite sequence; a first MS oligonucleotide (MS) hydrolysis probe labeled with a first fluorophore, the first MS oligonucleotide probe being complementary to a wild-type sequence containing the microsatellite sequence; and a second oligonucleotide reference (REF) hydrolysis probe labeled with a second fluorophore, the second oligonucleotide REF probe being complementary to a wild-type sequence of the target DNA fragment that does not contain the microsatellite sequence. The present invention also encompasses methods for diagnosing and prognosing cancer and methods for determining the efficacy of cancer treatments. The present invention also includes kits for detecting mutations in microsatellite sequences. [Selection diagram] Figure 4

Description

[Background technology]

[0001] 〔preface〕 Microsatellites (MS) are tandem repeats of short DNA sequences abundant throughout the human genome. Due to their high mutation rate, microsatellite sequences have been widely used as polymorphic markers in population genetics and forensic science. Microsatellite instability (MSI) is a hypermutation phenotype that occurs in tumors with DNA mismatch repair deficiency (MMR) and is characterized by extensive length polymorphisms and frequent single nucleotide variants (SNVs) in microsatellite repeats due to DNA polymerase slippage. MSI results from the inactivation of MMR genes (e.g., MLH1, MSH2, MSH3, MSH6, and PMS2) by somatic mutations and increases the risk of cancer in individuals with inherited germline mutations (i.e., Lynch syndrome). MSI can also occur through epigenetic inactivation of MMR genes (e.g., hypermethylation of the MLH1 and MSH2 promoters associated with somatic BRAF V600E mutations or deletions at the 3' end of Ep-CAM) or downregulation of MMR genes by microRNAs. MSI events within coding regions can alter the reading frame, resulting in truncated, functionally impaired proteins (see also Cortes-Ciriano et al., Nat Commun. 2017 Jun 6;8:15180 and Copija et al., Int J Mol Sci. 2017 Jan 6;18(1).pii:E107).

[0002] MSI phenotyping is primarily used as a molecular diagnostic tool for gastrointestinal, endometrial, and colorectal tumors, and the phenotype has important implications for disease prognosis and rational treatment planning (Boland and Goel, Gastroenterology 2010 June;138(6):2073-2087.e3; Copija et al., Int J Mol Sci. 2017 January 6;18(1).pii:E107). MSI-positive tumors are known to exhibit unique histopathological and clinical characteristics, including a better prognosis associated with specific location, poor differentiation, high lymphocytic infiltration, and low frequency of distant metastasis (Boland and Goel, Gastroenterology 2010;138(6):2073-2087.e3).

[0003] Recent analyses have also identified MSI across several additional cancer types, including urinary tract, ovarian, prostate, lung, head and neck, liver, and glioblastoma, suggesting potentially broader application of MSI screening in clinical practice (Hause et al., Nat Med. 2016 Nov;22(11):1342-1350; Cortes-Ciriano et al., Nat Commun. 2017 Jun 6;8:15180).

[0004] Indeed, MSI has recently emerged as the first pan-tumor biomarker likely to predict clinical benefit from immune checkpoint blockade therapy (Le et al., N Engl J Med. 2015 Jun 25;372(26):2509-20; Le et al., Science 2017 Jun 8.pii:eaan6733). Of note, the FDA recently approved the use of anti-PD-1 blockade therapy for the treatment of adult and pediatric patients with unresectable or metastatic MSI-positive or MMR-deficient solid tumors under accelerated approval.

[0005] Molecular diagnosis of MSI is currently performed by examining PCR products at several informative microsatellite loci in DNA extracted from tumor samples (Bacher et al., Disease Markers 2004, pp. 237-250). Disadvantages of this method include the need for capillary electrophoresis to detect allele size shifts and the limited sensitivity of this technique, which requires a minimum tumor cellularity of 20% to achieve reliable and robust results (Shi and Washington, Am J Clin Pathol 2012, 137:847-859). In recent years, next-generation sequencing (NGS) methods have been used due to their higher sensitivity and greater accuracy in MSI detection (Salipante et al., Clin Chem 2014 Jun 30; 60(9):1192-1199; Hause et al., Nat Med. 2016 Nov;22(11):1342-1350; Cortes-Ciriano et al., Nat Commun. 2017 Jun 6;8:15180). While a clear improvement over methods currently used in the clinic, the 1% sensitivity achieved by NGS still exceeds that of PCR-based assays.

[0006] Therefore, the development of a highly sensitive MSI diagnostic method that can be used on circulating tumor DNA obtained from liquid biopsies remains of great clinical and therapeutic importance. Summary of the Invention [Problem to be solved by the invention]

[0007] The authors designed a digital PCR diagnostic method to detect microsatellite instability, which can be performed on DNA samples containing extremely low concentrations of target DNA. [Means for solving the problem]

[0008] The authors demonstrated that the achieved detection limit (i.e., the lowest concentration likely to be reliably distinguishable from the blank limit and at which detection is feasible) was 250-fold lower than the minimum cellularity threshold (i.e., at least 20% cellularity) required to determine MSI status by the 5-plex assay currently applied in clinical practice (see Bacher et al., 2004, Disease Markers 20:237-250; see also Shi and Washington, Am J Clin Pathol 2012, 137:847-859). According to the results presented herein, the new MSI detection assay is both highly specific and sensitive, with sensitivity approaching 0.1%, at least theoretically. This innovative approach also offers several other advantages, including the simplicity of blood testing and the reduction in analysis time. Taken together, the MSI diagnostic methods of the present invention promise greater diagnostic accuracy and unprecedented use of MSI biomarkers in liquid biopsies for diagnosing and monitoring disease treatment and disease progression.

[0009] Similar techniques have previously been used to detect BRAF status in colorectal cancer (see Bidshahri et al., The Journal of Molecular Diagnostic 2016, 18(2):190-204). However, the use of such techniques has not previously been envisioned for the detection of mutant microsatellite sequences. Indeed, due to the size, and more specifically, the highly repetitive nature, of microsatellite sequences, it would be expected that probes covering the microsatellite (MS probes, see below) would slip over the repetitive sequences to the extent that efficient or reliable hybridization of the probe could not be obtained.

[0010] Dietmaier et al. (Laboratory Investigation, 2001) describe a technique for detecting microsatellite sequences by RT-PCR and melting point analysis using hybridization probes of specific sequences of targeted markers. The Light Cycler HybProbes hybridization probes used in this paper are not capable of distinguishing between WT and mutant microsatellite sequences. Therefore, to identify mutant microsatellites, additional melting point analysis is required after real-time PCR amplification. Furthermore, the probes of Dietmaier et al. are not considered hydrolysis probes and are not appropriate in the context of digital PCR reactions.

[0011] The method of the present invention is based on a single reaction using two hydrolysis probes located within the same amplicon. The first probe covers the entire WT microsatellite sequence (MS probe). The second probe, a reference probe (REF) located in a non-variable region, does not contain the microsatellite sequence (MS) locus and is used to quantify droplets containing amplified DNA. Thus, wild-type (WT) sequences will exhibit a double-positive fluorescent signal resulting from hybridization of both the REF and MS probes, while droplets containing mutant microsatellite alleles will exhibit a signal shift resulting from hybridization of only the REF probe.

[0012] Therefore, the present invention provides a pair of primers suitable for amplifying a target fragment of a DNA sample, including a microsatellite sequence; a first oligonucleotide microsatellite (MS) hydrolysis probe labeled with a first fluorophore, the first MS oligonucleotide probe being complementary to a wild-type sequence comprising a microsatellite sequence; a second oligonucleotide reference (REF) hydrolysis probe labeled with a second fluorophore, the second oligonucleotide REF probe being complementary to the wild-type sequence of the target DNA fragment, the second oligonucleotide REF probe not including the microsatellite sequence; The present invention relates to a method for detecting a mutation in a microsatellite sequence locus of a target fragment derived from a DNA sample, the method comprising the step of subjecting the DNA sample to digital polymerase chain reaction (dPCR) in the presence of a PCR solution comprising:

[0013] The digital PCR (dPCR) is preferably digital droplet PCR (ddPCR). The target fragment of the DNA sample can be constitutive genomic DNA or genomic tumor DNA or circulating DNA.

[0014] The microsatellite loci may be selected from the group including 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. Additionally, microsatellite sequences located in regions of the genome frequently amplified in cancer (e.g., the chr8q region of the human genome) may be selected to increase sensitivity.

[0015] Generally, the DNA sample is selected from the group consisting of tumor tissue, disseminated cells, feces, blood cells, plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, cerebrospinal fluid, and serous fluid.

[0016] The present invention also relates to a method according to any one of the preceding claims, further comprising the step of measuring fluorescence signals associated with the REF and MS probes, wherein a maximum 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, and 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.

[0017] The present invention also relates to a method for diagnosing cancer, a disease associated with mutations in mismatch repair (MMR) genes or familial tumor predisposition in a subject, comprising detecting a mutation in a microsatellite sequence locus in target DNA from the above-mentioned DNA sample, wherein the target fragment is derived from the tumor.

[0018] The present invention also relates to a method for predicting cancer prognosis, comprising the step of detecting mutations at microsatellite sequence loci in target fragments derived from a DNA sample as described above, wherein the target fragments are derived from a tumor.

[0019] The present invention also relates to a method for predicting the efficacy of a treatment in a subject suffering from cancer, comprising detecting a mutation in a microsatellite sequence locus of a target fragment derived from a DNA sample as described above, wherein the target fragment is derived from a tumor and the treatment is preferably an immune therapy such as an immune checkpoint therapy.

[0020] The present invention provides detecting mutations at microsatellite sequence loci of target fragments from said DNA sample; administering an immunotherapy to the subject if a mutation is identified in the microsatellite sequence locus of the target fragment; The present invention also relates to a method of treating cancer in a subject in need thereof, comprising:

[0021] The present invention also relates to a method for monitoring a patient diagnosed with or suffering from a tumor associated with a DNA mismatch repair (MMR) disorder, comprising the step of detecting a mutation in a microsatellite locus of a target fragment derived from the DNA sample described above, The target fragment of the DNA sample is derived from the tumor.

[0022] Finally, the present invention provides a pair of primers suitable for amplifying a target fragment from a DNA sample containing a microsatellite sequence; a first oligonucleotide hydrolysis probe (MS) labeled with a first fluorophore, the first oligonucleotide hydrolysis probe being complementary to a wild-type sequence comprising a microsatellite sequence; a second oligonucleotide hydrolysis probe (REF) labeled with a second fluorophore, the second oligonucleotide hydrolysis probe being complementary to the wild-type sequence of the amplified DNA fragment that does not contain the microsatellite sequence; Thermostable polymerase The present invention also includes a kit for identifying mutations in the microsatellite sequence region of a target fragment derived from a DNA sample comprising: [Brief explanation of the drawings]

[0023] [Figure 1A]Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1B] Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1C]Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1D] Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1E]Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1F] Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1G]Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1H] Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 1I]Figures A-C show two-dimensional fluorescence amplitude scatter plots of BAT-26 ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures D-F show two-dimensional fluorescence amplitude scatter plots of DEFB105A / B ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Figures G-I show two-dimensional fluorescence amplitude scatter plots of ACVR2A ddPCR MSI assays using HCT-116 cell line DNA (MSI-H), PBMC (WT), or a 10% dilution of HCT-116 in WT DNA. Droplets containing the WT allele are positive for both FAM and VIC signals, while droplets containing the MSI allele are positive for VIC signal only. [Figure 2A] Correlation curves obtained with the BAT-26 (A), DEFB105A / B (B), and ACVR2A (C) assays for observed versus expected MAFs at serial dilutions of reconstituted mutants (10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08%, 0.04%, 0.02%, 0.01%). Dotted lines: estimated upper 95% CI of false-positive calls in at least 53 independent ddPCR reactions containing LOB, WT DNA. [Figure 2B] Correlation curves obtained with the BAT-26 (A), DEFB105A / B (B), and ACVR2A (C) assays for observed versus expected MAFs at serial dilutions of reconstituted mutants (10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08%, 0.04%, 0.02%, 0.01%). Dotted lines: estimated upper 95% CI of false-positive calls in at least 53 independent ddPCR reactions containing LOB, WT DNA. [Figure 2C]Correlation curves obtained with the BAT-26 (A), DEFB105A / B (B), and ACVR2A (C) assays for observed versus expected MAFs at serial dilutions of reconstituted mutants (10%, 5%, 2.5%, 1.25%, 0.63%, 0.31%, 0.16%, 0.08%, 0.04%, 0.02%, 0.01%). Dotted lines: estimated upper 95% CI of false-positive calls in at least 53 independent ddPCR reactions containing LOB, WT DNA. [Figure 3A] Figure 1 shows the correlation between the percentage of ctDNA estimated by the BAT-26 (A), ACVR2A (B) or DEFB105A / B (C) ddPCR assays and the percentage of ctDNA estimated by a ddPCR assay specifically targeting the BRAFV600E mutation. [Figure 3B] Figure 1 shows the correlation between the percentage of ctDNA estimated by the BAT-26 (A), ACVR2A (B) or DEFB105A / B (C) ddPCR assays and the percentage of ctDNA estimated by a ddPCR assay specifically targeting the BRAFV600E mutation. [Figure 3C] Figure 1 shows the correlation between the percentage of ctDNA estimated by the BAT-26 (A), ACVR2A (B) or DEFB105A / B (C) ddPCR assays and the percentage of ctDNA estimated by a ddPCR assay specifically targeting the BRAFV600E mutation. [Figure 4] Figure 1 shows two-dimensional fluorescence amplitude scatter plots illustrating the fluorescent signals obtained in a triplex assay simultaneously targeting the BAT-26, ACVR2A, and DEFB105A / B microsatellite markers using a 10% dilution of the HCT-116 cell line in WT DNA. Results were obtained with an annealing temperature and extension time of 63°C for 3 minutes. Primer and probe concentrations were: BAT-26: 0.2x; ACVR2A: 0.6x; DEFB105A / B: 1x. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description A-definition The following definitions are intended to aid in providing a clear and consistent understanding of the scope and detail of the following terms used to describe and define the present invention: As used herein, the verb "comprise" and its conjugations in this description and claims are used in an open-ended sense to mean including items that follow the word, but not excluding items not specifically listed. In addition, referring to an element with the indefinite article "a" or "an" does not exclude the possibility that there is a plurality of elements, unless the context clearly requires that there is one or only one element. Thus, the indefinite article "a" or "an" typically means "at least one."

[0025] As used herein, a "tumor" or "neoplasm" (both terms may be used interchangeably) is an abnormal new growth of cells. Cells in a neoplasm usually grow more rapidly than normal cells and will continue to grow if untreated. As a neoplasm grows, it may affect and damage neighboring structures. The term neoplasm can refer to benign (usually treatable) or malignant (cancerous) growths.

[0026] Benign tumors, or neoplasms, are usually localized and do not spread to other parts of the body. Most benign tumors respond well to treatment. However, if left untreated, some benign tumors can grow large and, due to their size, can lead to serious illness. Benign tumors can also resemble malignant tumors and are therefore sometimes treated. Malignant tumors are cancerous growths. Malignant tumors are often resistant to treatment, can spread to other parts of the body (i.e., metastasize), and sometimes recur after removal.

[0027] The term "cancer" is used herein in reference to malignant tumors.

[0028] As used herein, "allele" refers to one of several alternative forms of a gene or DNA sequence at a particular chromosomal location (locus). At each autosomal locus, an individual carries two alleles, one inherited from the father and one from the mother.

[0029] As used herein, a "DNA polymorphism" refers to the presence of two or more alleles for a given locus within a population. As used herein, a "locus" or "genetic locus" refers to a unique location on a chromosome that defines the location of an individual gene or DNA sequence. As used herein, a "locus-specific primer" refers to a primer that specifically hybridizes to a portion of the described locus or its complementary strand, for at least one allele of that locus, and does not hybridize efficiently to other DNA sequences under the conditions used in the amplification method.

[0030] "Microsatellite sequence locus" or "microsatellite sequence," used interchangeably, refer to a region of genomic DNA that contains short repetitive sequence elements of one (1) to seven (7), usually one (1) to five (5), particularly one (1) to four (4) base pairs in length. Each sequence that is repeated at least once within a microsatellite locus is referred to herein as a "repeat unit." Each microsatellite locus typically contains at least seven repeat units, particularly at least 10 repeat units, and preferably at least 20 repeat units.

[0031] As used herein, "microsatellite instability" (hereinafter "MSI") refers to a form of genetic instability in which alleles of genomic DNA obtained from specific tissues, cells, or body fluids of a given subject are mutated at microsatellite loci.

[0032] Mutations at microsatellite loci typically involve deletion(s), addition(s), or substitution(s) of at least one repeat unit at the microsatellite locus. MSI generally results in a length change at the microsatellite locus, either by addition(s) or, most frequently, by deletion(s).

[0033] As used herein, the term "primer / probe set" refers to a group of a pair of oligonucleotide primers and two oligonucleotide probes, each hybridizing to a specific target nucleotide sequence. The oligonucleotide set consists of (a) a forward discrimination primer that hybridizes to a first position in a nucleic acid sequence; (b) a reverse discrimination primer that hybridizes to a second position in the nucleic acid sequence downstream of the first position; and (c) two probes that hybridize to the target sequence between the primers. In other words, a primer / probe set consists of a pair of specific oligonucleotides that anneal to opposite strands of a nucleic acid sequence (usually including a microsatellite sequence locus) to form an amplicon specific to the nucleic acid sequence during a PCR reaction, and two probes, preferably fluorescent, that hybridize (i.e., are complementary to) the specific target sequence of the amplicon.

[0034] An "amplicon" refers to a nucleic acid fragment formed as the product of a natural or artificial amplification event or technique. Typically, an amplicon is produced by the polymerase chain reaction (PCR). As used herein, "amplifying" refers to the process by which multiple copies are made at a particular locus (i.e., the target sequence described above) of a nucleic acid, such as genomic DNA. Amplification is accomplished using PCR (Saiki et al., 1985, Science 230:1350-1354).

[0035] As used interchangeably herein, a "target (DNA) fragment" or a "target (DNA) region" refers to a fragment of a DNA sample that is amplified by a pair of primers of a primer / probe set. According to the present invention, such a target fragment comprises an MS locus. A "target sequence" or a "target DNA sequence," used interchangeably, refers to a DNA sequence that is complementary to a first or second oligonucleotide probe.

[0036] As used herein, "digital PCR" refers to an assay that allows for an endpoint measurement, providing the ability to quantify nucleic acids without the use of a standard curve as used in real-time PCR (see Sykes et al., 1992, "Quantitation of targets for PCR by use of limiting dilution," BioTechniques 13, 444-449; Vogelstein and Kinzler, 1999, "Digital PCR," Proc Natl Acad Sci USA, 96:9236-9241; and Pohl and Shihle, 2004, "Principle and applications of digital PCR," Expert Rev Mol Diagn, 4:41-47; see also Monya Baker, 2012, Nature Methods 9, 541-544).

[0037] In a typical digital PCR experiment, a PCR solution is prepared similarly to a classic TaqMan probe assay, typically containing a DNA sample, a fluorescence quenching probe (i.e., hydrolysis probe), primers, and a PCR master mix, which typically contains DNA polymerase, dNTPs, MgCl2, and a reaction buffer at optimal concentrations. The PCR solution is then randomly distributed into distinct (i.e., individual) sections or compartments, some of which contain no target DNA, while others contain one or more target DNA copies, most preferably one target DNA copy. Therefore, under these conditions, the reference signal associated with the presence of target DNA in a DNA sample within a given section or compartment should theoretically be 0 or 1. Obviously, due to biological variation within the population of sections or compartments, a cloud is observed corresponding to the theoretical values ​​of 0 or 1, respectively.

[0038] The sections are amplified separately until the terminal plateau (or end point) of the PCR and the fluorescence is read to determine the percentage of positive sections. If the sections are of equal volume, the number of target DNA molecules present is calculated by the following formula: λ=-ln(1-p) (1) The absolute concentration of target DNA can be calculated from the proportion of positive endpoint reactions using Poisson statistics according to the formula: where λ is the average number of target DNA molecules per replicate reaction and p is the proportion of positive endpoint reactions. From λ, an estimate of the absolute concentration of target DNA is calculated, along with the volume of each replicate PCR and the total number of replicates analyzed.

[0039] Microwell plates with nucleic acid binding surfaces, capillaries, oil emulsions, and arrays of miniaturized chambers can be used to compartmentalize samples into unique compartments or droplets. Thus, as used herein, digital PCR encompasses a variety of formats, including droplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification, and magnetic materials), and microfluidic chips.

[0040] "Droplet digital PCR" (ddPCR) refers to a digital PCR assay that measures absolute quantities by counting nucleic acid molecules encapsulated in separate, volumetrically defined, water-in-oil droplet compartments that support PCR amplification (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011). A single ddPCR reaction can consist of at least 20,000 compartmentalized droplets per well.

[0041] "Droplets" refer to discrete portions of a PCR solution in a droplet digital PCR assay. In the remainder of this application, digital PCR will be described in terms of droplet digital (or digital droplet PCR, used interchangeably), but as noted above, discrete portions of a PCR solution according to the principles of digital PCR can be obtained by a variety of techniques. Therefore, the methods of the present invention described below in terms of droplet digital PCR are not limited to this digital PCR technique, but may be applied in a similar manner to other digital PCR techniques.

[0042] Available technologies for digital PCR include PCR amplification on microfluidic chips (Warren et al., 2006, "Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR", Proc Natl Acad Sci USA 103, 17807-17812; Ottesen et al., 2006, "Microfluidic digital PCR enables multigene analysis of individual environmental bacteria", Science 314, 1464-1467; Fan and Quake, 2007, "Detection of aneuploidy with digital polymerase chain reaction", Anal Chem 79, 7576-7579). Other systems include separation on microarrays (Morrison et al., 2006, "Nanoliter high-throughput quantitative PCR," Nucleic Acids Res 34, e123) or spinning microfluidic disks (Sundberg et al., 2010, "Spinning disk platform for microfluidic digital polymerase chain reaction," Anal Chem 82, 1546-1550), and droplet technology based on oil-water emulsions (Hindson, Benjamin et al., 2011, "High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number," Analytical Chemistry 83(22):8604-8610). Generally, digital PCR is selected from droplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification, and magnetic material), and microfluidic chips. Preferably, the digital PCR is droplet digital PCR.

[0043] The droplets support PCR amplification of template molecule(s) using homogeneous assay chemistry and workflow similar to that widely used in real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83:8604-8610; Pinheiro et al., 2012, Anal. Chem. 84:1003-1011). Once generated, the droplets can be transferred to a PCR plate, and emulsion PCR reactions can be performed in a thermal cycler under a classical program, such as that described in BioRad's guidelines for ddPCR (http: / / www.bio-rad.com / webroot / web / pdf / lsr / literature / Bulletin_6407.pdf).

[0044] Droplet digital PCR may be performed using any platform that implements digital PCR assays that measure absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet compartments that support PCR amplification. The strategy of droplet digital PCR can be summarized as follows: a PCR solution containing a DNA sample is diluted and compartmentalized into thousands to millions of separate reaction chambers (water-in-oil droplets), each of which therefore contains one or zero copies of the nucleic acid molecule of interest.

[0045] The number of detected "positive" droplets (i.e., REF positive droplets according to the present invention) containing the target amplicon (i.e., the target DNA fragment) versus the number of "negative" droplets (i.e., REF negative droplets) not containing the target amplicon may be used to determine the copy number of the nucleic acid molecule of interest present in the original sample.

[0046] Examples of droplet digital PCR systems include the QX100™ Droplet Digital PCR System by Bio-Rad, which partitions a sample containing a nucleic acid template into 20,000 nanoliter-sized droplets; and the RainDrop™ Digital PCR System by RainDance, which partitions a sample containing a nucleic acid template into 1,000,000 to 10,000,000 picoliter-sized droplets.

[0047] Advantages of dPCR, and more specifically ddPCR technology, include: Absolute quantification, since ddPCR technology provides an absolute count of target DNA copies per input sample without the need for a standard curve.

[0048] Unparalleled precision, as the large-scale sample partitioning achieved by ddPCR allows small fold differences in target DNA sequence copy number between samples to be reliably measured.

[0049] Increased signal-to-noise ratio: High copy templates and background are diluted, effectively enriching the template concentration in the target-positive fraction, thereby allowing sensitive detection of dilute targets.

[0050] Elimination of PCR bias, as removing the amplification efficiency dependency of qPCR reduces the error rate, allowing detection of small differences (1.2-fold).

[0051] Simplified quantification, since no calibration standards or references are required for absolute quantification.

[0052] Reduced consumable costs due to reduced reagent usage and sample volume required for each data point, as reaction volumes are in the pico- to nanoliter range.

[0053] Lower equipment costs, as the emulsion-based reaction system means that PCR reactions can be carried out in standard thermal cyclers without the use of complex chips or microfluidics.

[0054] Superior partitioning: ddPCR technology generates 20,000 droplets per 20 μL of sample, or roughly 2,000,000 partitioned PCR reactions in a 96-well plate, while chip-based digital PCR systems only produce hundreds or thousands of partitions. A larger number of partitions also results in greater precision.

[0055] The term "melting temperature" or "Tm" refers to the temperature at which a polynucleotide dissociates from its complementary sequence. Generally, Tm can be defined as the temperature at which one-half of the Watson-Crick base pairs in a duplex nucleic acid molecule break or dissociate (i.e., "melt"), while the other half of the Watson-Crick base pairs remain intact in double-stranded form. In other words, Tm is defined as the temperature at which 50% of the nucleotides in two complementary sequences are annealed (double-stranded) and 50% of the nucleotides are denatured (single-stranded). Tm can be estimated by several methods, such as nearest-neighbor calculations according to Wetmur, 1991 (Wetmur, 1991, "DNA probes: applications of the principles of nucleic acid hybridization," Crit Rev Biochem Mol Biol 26:227-259, incorporated herein by reference), or by commercially available programs and programs available on the internet, including Oligo™ Primer Design. Alternatively, the Tm can be determined by actual experimentation, for example, using double-stranded DNA binding or intercalating dyes such as ethidium bromide or SYBR Green (Molecular Probes) in a melting curve assay to determine the actual Tm of a nucleic acid.

[0056] As used herein, the term "critical denaturation temperature" or "Tc" refers to the temperature below the Tm of the wild-type sequence at which a duplex of the wild-type sequence and a mutant sequence will melt. (In some instances, this temperature may also be the temperature at which a homoduplex of the mutant sequence will melt.)

[0057] The critical denaturation temperature (Tc) is the temperature below which PCR efficiency for a given nucleic acid sequence drops off sharply.

[0058] Methods for identifying mutations at microsatellite loci in B-DNA samples The present invention provides a pair of primers suitable for amplifying a target fragment of a DNA sample containing the MS locus; a first MS oligonucleotide probe labeled with a first fluorophore, the first MS oligonucleotide probe being complementary to a first wild-type target sequence comprising a microsatellite sequence; a second oligonucleotide reference (REF) probe labeled with a second fluorophore, the second oligonucleotide REF probe being complementary to a second wild-type target sequence of the amplified DNA fragment that does not contain the microsatellite sequence; The present invention relates to a method for detecting a mutation at a target microsatellite sequence (MS) locus in a target fragment from a DNA sample, the method comprising the step of subjecting the DNA sample to polymerase chain reaction (PCR) in the presence of

[0059] The DNA from the DNA sample, particularly the target DNA (or target DNA fragment), can be genomic DNA or DNA obtained by RNA reverse transcription. The genomic DNA can be constitutive DNA, DNA derived from a tumor (i.e., tumor genomic DNA), particularly a malignant tumor. Typically, the target DNA fragment is also cell-free DNA, such as circulating DNA. In particular, the target DNA fragment can be cell-free tumor DNA, particularly circulating tumor DNA, or cell-free fetal DNA (i.e., fetal DNA circulating in the maternal bloodstream).

[0060] The method uses the primer / probe sets defined above.

[0061] It is generally preferred that the primer pair be designed to have a Tm lower than the Tc of the reaction. Primer pairs can be designed using available computer programs. Generally, the probe according to the present invention is a hydrolysis probe (also called a TaqMan probe). Hydrolysis probes have a fluorophore and a quencher covalently attached to the 5' end of an oligonucleotide probe.

[0062] Oligonucleotide probes include, for example, FAM (5- or 6-carboxyfluorescein), VIC, NED, fluorescein, FITC, IRD-700 / 800, CY3, CY5, CY3.5, CY5.5, HEX, TET (5-tetrachlorofluorescein), TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor PET, Biosearch Blue™, Marina Blue®, Bothell Blue®, Alexa Fluor Fluor®, 350FAM™, SYBR® Green 1, Fluorescein, EvaGreen™, Alexa Fluor® 488 JOE™, 25VIC™, HEX™, TET™, CAL Fluor® Gold 540, Yakima Yellow®, ROX™, CAL Fluor® Red 610, Cy3.5™, Texas Red®, Alexa Fluor® 568 Cry5™, Quasar™ 670, LightCycler Red 640 The fluorescent label may be detectably labeled with a fluorescent label that may be selected from the group consisting of Alexa Fluor 633, Quasar™ 705, LightCycler Red 705™, Alexa Fluor™ 680, SYTO™ 9, LC Green™, LC Green™ Plus+, and Evergreen™. Preferably, the detectable label is selected from 6-carboxyfluorescein, FAM, or tetrachlorofluorescein (acronym: TET), Texas Red, Cyanine 5, Cyanine 3, or VIC™.

[0063] The quencher may be an internal quencher or a quencher located at the 3' end of the probe. Exemplary quenchers are tetramethylrhodamine, TAMRA, black hole quenchers, or non-fluorescent quenchers. Hydrolysis probes that can be used in accordance with the present invention are well known in the art (see, inter alia, http: / / www.sigmaaldrich.com / technical-documents / articles / biology / quantitative-pcr-and-digital-pcr-detection-methods.html). The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler's light source, usually via FRET (Förster Resonance Energy Transfer). While the fluorophore and quencher are in close proximity, quenching inhibits any fluorescent signal. Such probes are designed to anneal within the target region amplified by a specific set of primers. As Taq polymerase extends the primer and synthesizes the nascent strand, the 5'-3' exonuclease activity inherent in Taq DNA polymerase then separates the 3' quencher and 5' reporter, thereby generating a fluorescent signal proportional to the amplicon yield.

[0064] The first and second probes according to the present invention are located within the same amplicon. The probes are designed according to established practice in the art to preferably minimize PCR artifacts and specifically hybridize to the sequences defined below. The first and second probes are labeled with unique fluorophores, allowing their respective signals to be detected separately.

[0065] In some embodiments, hydrolysis probes according to the invention contain a minor groove binder (MGB) moiety at their 3' ends. Such MGBs generally increase the melting temperature (Tm) of the probe and stabilize the probe-target hybrid.

[0066] The oligonucleotide probe has a sequence length of about 10 to about 50 nucleotides. The oligonucleotide probe (particularly the MS probe) preferably has a sequence length of about 15 to 40, or 25 to 50, and particularly 15 to 35 nucleotides. The oligonucleotide probe (particularly the MS probe) preferably has a sequence length of about 20 to 40, or 30 to 50, and particularly 30 to 40 nucleotides.

[0067] The first probe (also referred to as an MS probe) of the present invention hybridizes to a first wild-type target sequence of an amplified target DNA fragment, which target sequence includes a microsatellite sequence locus. To ensure both proper binding and destabilization in the event of microsatellite instability, the probe preferably covers the entire wild-type microsatellite sequence and is extended by several additional nucleotides (generally 1-10, particularly 2-8, preferably 2-6, and most preferably 2-5 or 2-4 nucleotides) at each end. In other words, the probe size is designed to ensure proper binding to the wild-type microsatellite sequence but prevent hybridization of the MS probe when a mutation is present in the microsatellite sequence.

[0068] The second probe of the present invention (also called REF probe) hybridizes to a second wild-type target sequence of the amplified DNA fragment, which target sequence does not include the microsatellite sequence. In particular, the second probe may partially overlap the microsatellite sequence or may be located outside the microsatellite sequence. Preferably, the second probe of the present invention is located outside the microsatellite sequence.

[0069] Various microsatellite loci can be targeted in the first wild-type target sequence according to the present invention. Microsatellite loci or markers that can be targeted according to the present invention are particularly described in Bacher et al., 2004, Disease Markers 20, 237-250, and Hause et al., 2016, Nat Medicine Nov. 22, 11:1342-1350. The targeted microsatellite loci (or microsatellite markers) are preferably selected from microsatellites found to be highly associated with MSI-positive tumors based on the frequency of microsatellite instability in colon, endometrial, rectal, and gastric adenocarcinomas. The targeted microsatellite loci are preferably located within regions frequently amplified in tumors.

[0070] For example, the targeted microsatellite sequence locus can be selected from 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.

[0071] In one embodiment, the target microsatellite sequence locus may be selected from the Bethesda panel, which includes BAT-25, BAT-26, D2S123, D5S346, and D17S250.

[0072] Mononucleotide repeat loci have been shown to be highly susceptible to alteration in tumors containing dysfunctional DNA mismatch repair systems (Parsons, 1995 supra), making such loci particularly useful for detecting cancer and other diseases associated with dysfunctional DNA mismatch repair systems, such that mononucleotide MSI markers may be preferred.

[0073] In one embodiment of the present invention, the targeted microsatellite sequence loci are BAT-26 and / or ACVR2A and / or DEFB105A and DEFB105B.

[0074] More generally, suitable microsatellite loci that can be targeted by the present invention are short microsatellite loci (generally comprising 8 to 30, particularly 8 to 25, preferably 8 to 20, and most preferably 8 to 15 or 8 to 12 nucleotides), such as target microsatellite loci exemplified by the group consisting of D2S123, D5S346, D17S250, ACVR2A, DEFB105A, DEFB105B, RNF43, DOCK3, GTF2IP1, LOC100093631, PIP5K1A, MSH3, TRIM43B, PPFIA1, and TDRD1.

[0075] Depending on the microsatellite locus, probes of various sizes and G / C contents may also be used. For example, probes longer than 30 nucleotides and / or with a G / C content of less than 30% may be used. This is particularly the case when BAT-26 is involved. As an example, the MS probe of SEQ ID NO: 4, which hybridizes to a sequence containing the BAT-26 microsatellite sequence, may be used. The primers of SEQ ID NOs: 1-2 and the REF and MS probes of SEQ ID NOs: 3 and 4, respectively, represent an exemplary set of primers / probes that may be used in accordance with the present invention.

[0076] According to the present invention, the amplification of the target DNA fragment occurs by digital PCR technology. Generally, in such technology, a PCR solution is divided into multiple compartments or droplets, which are created for performing PCR individually. Also, generally, most of the compartments or droplets contain zero or one copy of the target DNA fragment to be amplified.

[0077] To circumvent the technical obstacles associated with amplifying low-complexity sequences such as microsatellite sequences, a series of modifications may be made to the Biorad guidelines for ddPCR described above to achieve proper hybridization of the MS probe to the WT allele. The reaction annealing temperature and / or extension time may be increased. A typical annealing temperature according to Biorad guidelines is 55°C. The annealing temperature may be advantageously increased by 3-15°C.

[0078] Thermal cycling is carried out until the end point. Thus, after multiple PCR amplification cycles (i.e., after completing the PCR cycles), raw PCR data are then collected by measuring the fluorescent signals associated with the REF and MS probes for each droplet. Droplets containing WT target fragments exhibit double-positive fluorescent signals resulting from hybridization of both the REF and MS probes (REF+ / MS+ droplets). Non-hybridization (or inefficient hybridization) of the MS probe in droplets containing mutant microsatellite alleles results in a shift of the droplet cloud on the two-dimensional graph toward a single REF-positive (REF+) population, which is proportional to the proportion of droplets containing mutant microsatellite alleles.

[0079] Generally, raw dPCR (or ddPCR) data is collected after PCR cycling by reading or measuring the fluorescent signals associated with the REF and MS probes for each droplet.

[0080] The PCR data collection step is typically performed in an optical detector (e.g., a Bio-Rad QX-100 droplet reader can be used for ddPCR). At least a two-color detection system is preferably used (e.g., detecting FAM and either HEX or VIC fluorescent labels). A droplet cloud can typically be established on a two-dimensional graph by plotting the fluorescence level for each probe per droplet. In some embodiments, analysis can be accomplished with appropriate software (e.g., QuantaSoft v1.7.4 software for ddPCR or the ddPCR package in R [https: / / cran.r-project.org / web / packages / ddpcr / index.html]). QuantaSoft allows for manual assignment of droplets to single REF-positive or double REF / MS-positive populations (i.e., or clouds). The R package automatically defines thresholds to avoid bias that may be introduced by manual assignment.

[0081] The number of droplets that are positive for the reference probe (REF probe) can be used to quantify the total number of target DNA fragments in the sample. The proportion of positive droplets can then be fitted to a Poisson distribution to determine the absolute initial copy number of the target DNA fragment in the input reaction mixture in copies / µL.

[0082] In droplets containing wild-type target DNA (no mutations in the targeted MS sequence), maximum fluorescence signal is observed for both the REF and MS probes. Conversely, in droplets containing mutant sequences (i.e., mutations in the microsatellite sequence) in the amplified target DNA fragment, a shift in fluorescence intensity is observed in the signal associated with the MS probe.

[0083] It is most preferable to design the digital PCR reaction to ensure that most droplets contain zero or one copy of the targeted DNA fragment (especially depending on the amount of DNA loaded into the reaction). Under these conditions, optimal separation of WT (REF+ / MS+ signals) versus mutant microsatellite (or MSI) (single REF+ signal) clouds can be observed. It should be noted that due to biological variability, droplets classified as a single REF+ signal may contain residual (i.e., insignificant) MS signals. The threshold at which an MS signal is considered a "residual MS signal" can be determined by those skilled in the art using classical signal analysis techniques. The threshold can generally be set using the R package described above.

[0084] Generally, mutant allele frequencies can be determined from droplet counts by manually assigning WT and mutant microsatellite droplet clouds. As noted above, identification of a droplet population with a single signal from the REF probe indicates the presence of a mutant microsatellite sequence in the DNA sample.

[0085] The mutant allele frequency, which may be determined as described above, may be compared to a control mutant allele frequency obtained from a control DNA sample, which may be a wild-type sample or a sample or cell line collected in a subject diagnosed with an MSI-positive tumor or a disease associated with a mutation in DNA mismatch repair at an earlier time during the time course of the disease and / or treatment.

[0086] As used herein, the term "sample" refers to anything that may contain DNA, particularly DNA fragments to be amplified. In some embodiments, the "sample" contains RNA and is therefore subjected to a reverse transcription step. The sample may be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, semen ...

[0087] Biological tissues are aggregates of cells, usually of a specific type, and intercellular substances that form one of the structural materials of human, animal, plant, bacterial, fungal, or viral structures, including connective, epithelial, muscle, and nervous tissue. Examples of biological tissues include organs, tumor tissue, lymph nodes, arteries, and disseminated cell(s). Tissues can be fresh, freshly frozen, or fixed, such as formalin-fixed, paraffin-embedded (FFPE) tissue. Samples can be obtained by any means, including, but not limited to, surgical procedures such as biopsy, or minimally invasive methods, including, but not limited to, exfoliation or fine needle aspiration. Preferably, the DNA sample is selected from the group consisting of tumor tissue, disseminated cells, feces, blood cells, plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, cerebrospinal fluid, pericardial fluid, pleural fluid, or serous fluid, such as ascites.

[0088] The DNA, particularly the target DNA fragment, can be genomic DNA or DNA obtained from reverse transcriptase. Genomic DNA can be constitutive DNA, tumor DNA, or fetal DNA. In some embodiments, particularly when the sample is a biological fluid, the DNA sample may contain cell-free DNA (cfDNA) or circulating DNA. Early studies showed that tumor DNA is released into the circulation and is present at particularly high concentrations in plasma and serum in several different types of cancer (Leon et al., 1977, Cancer Res 37:646-650; Stroun et al., 1989, Oncology 46:318-322). Thus, the DNA sample according to the present invention can contain cell-free tumor DNA or circulating tumor DNA. In another embodiment, the DNA sample contains cell-free fetal DNA. Due to its high sensitivity, the method of the present invention can be used with plasma samples containing low concentrations of circulating or cell-free target DNA, such as cell-free or circulating tumor DNA or fetal DNA. In some embodiments of the present invention, DNA can be obtained by reverse transcription of an RNA sample.

[0089] Generally, a DNA sample according to the present invention is obtained from a subject. The subject or patient (both terms may be used interchangeably) of the present invention is a mammal, generally a primate such as a human. In some embodiments, the primate is a monkey or ape. The subject may be male or female and may be of any suitable age, including children, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.

[0090] In some embodiments of the invention, the subject has cancer, is in remission from cancer, or is at risk of developing cancer based particularly on family history, for example, in some embodiments the subject has a familial tumor predisposition.

[0091] In some embodiments, the subject has, is in remission, or has a familial predisposition to cancer, particularly, the subject has or is at risk of having a disease caused by a mutation in a mismatch repair (MMR) gene, such as constitutive mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome.

[0092] The cancer may be a solid tumor or a "liquid tumor" such as a cancer affecting the blood, bone marrow, and lymphatic system, also known as a tumor of the hematopoietic and lymphatic tissues, and particularly includes leukemia and lymphoma. Liquid tumors include, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), which includes various lymphomas such as mantle cell lymphoma or non-Hodgkin's lymphoma (NHL).

[0093] Solid tumors include, among others, cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, ameloblastoma, and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, neck, brain (such as meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma, and metastatic brain cancer), ovary, breast, head and neck region, testicle, prostate, and thyroid.

[0094] In some embodiments of the invention, the cancer is constitutive mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome.

[0095] In the context of the present invention, MSI-associated cancers (or tumors), also named MSI-positive cancers (or MSI-positive tumors), relate to cancers (or tumors) whose genomic tumor DNA exhibits at least one mutation in a microsatellite sequence. Thus, an MSI-positive cancer can be any of the cancers listed above, whose genomic tumor DNA exhibits at least one mutation in a microsatellite sequence.

[0096] Clinical applications: diagnostic and prognostic methods, therapeutic treatment and patient monitoring.

[0097] The method for identifying mutant microsatellite sequences in target DNA fragments described above has several major and immediate clinical applications.

[0098] First, as mentioned above, microsatellite instability is a hypermutator phenotype that occurs in tumors associated with DNA mismatch repair deficiency (MMR). Therefore, MSI has been associated with a wide variety of cancers, including, but not limited to, colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, urinary tract cancer, brain cancer, and breast cancer. MSI is most commonly a consequence of colorectal cancer. MSI is commonly seen in constitutive mismatch repair deficiency syndrome (CMMR syndrome) or Lynch syndrome.

[0099] Thus, detection of mutant microsatellite sequences by the above-described methods may be used to diagnose cancers as defined above, in particular cancers associated with DNA mismatch repair defects (as defined above), in particular MSI-positive cancers (or tumors).

[0100] In one embodiment of the present invention, the detection of mutated microsatellite sequences by the present method may also be used to diagnose diseases caused by mutations in mismatch repair (MMR) genes, in particular MSI-positive tumors such as constitutive mismatch repair deficiency syndrome (CMMR syndrome) or Lynch syndrome, or to diagnose familial tumor predisposition in a subject.

[0101] Thus, in one aspect, the present invention relates to a method for diagnosing cancer in a subject, particularly a disease associated with a mutation in a mismatch repair (MMR) gene, such as an MSI-positive tumor, and / or a familial tumor predisposition to cancer, comprising detecting a mutation at a microsatellite sequence locus in target DNA from a DNA sample according to the present invention. Generally, the target DNA is genomic DNA derived from a tumor. The sample may be obtained from the subject as described above. In one embodiment, detection of a mutant microsatellite sequence in a DNA sample from a subject indicates that the subject suffers from an MSI-positive tumor, particularly a disease caused by a mutation in an MMR gene, such as CMMRD or Lynch syndrome. Detection of a mutant microsatellite sequence in a DNA sample from a subject may also indicate that the subject has a familial tumor predisposition, such as CMMRD or Lynch syndrome.

[0102] Mutations in MMR genes include additions, deletions or substitutions, particularly single nucleotide variations (SNVs) and epimutations (such as DNA hypermethylation).

[0103] The prevalence of MSI-positive tumors is higher in colorectal cancer, gastric cancer, and endometrial cancer. However, MSI has been found at a lower prevalence in virtually all types of cancer (see Hause et al., Nature Medicine, 2016). As mentioned above, the MSI phenotype (i.e., positive or negative) of a cancer has important implications for cancer prognosis and rational treatment planning (Boland and Goel, Gastroenterology, 2010). Therefore, even in the case of cancers with a low prevalence of MSI positivity, it is still very important to identify whether a patient has an MSI-positive or MSI-negative tumor. Therefore, the method of the present invention can be used to predict the prognosis of various cancers. Identification of MSI-positive cancers is generally associated with a favorable prognosis.

[0104] Thus, the present invention also relates to a method for prognosticating cancer (as defined above) comprising detecting mutations at microsatellite loci in a DNA sample according to the present invention. In some embodiments, the identification of mutated microsatellite sequences in a sample, preferably a tumor-derived DNA sample, indicates that said tumor is MSI-positive.

[0105] In the above-mentioned therapeutic context, the method of the present invention is particularly useful because its exceptional sensitivity allows for the detection of microsatellite instability in DNA samples containing extremely low concentrations of target DNA. Therefore, the method of the present invention can be routinely performed on biological samples such as blood samples, plasma samples, urine, or even feces. Generally, the method of the present invention is performed on a blood or plasma sample, and the target DNA is cell-free DNA, such as circulating tumor DNA. This is particularly relevant for diseases such as CMMRD, including brain tumors, where biopsy is not possible.

[0106] The present invention also relates to methods for predicting treatment efficacy, as reports have shown that, for example, MMR-deficient colorectal cancer patients have a superior response to immunotherapy with PD-1 immune checkpoint blockade and show improved progression-free survival. Therefore, identifying patients suffering from MSI-associated cancers (i.e., MSI-positive cancers or tumors) is of high clinical relevance for selecting appropriate therapeutic strategies.

[0107] Therefore, another aspect of the present invention relates to a method for predicting the efficacy of a treatment in a subject suffering from cancer, said method comprising detecting a mutation at a microsatellite locus in a target DNA fragment from a DNA sample of the subject as described above. Preferably, the target DNA fragment is derived from a tumor. Typically, the DNA sample is obtained from a subject suffering from a tumor and / or with a familial cancer predisposition.

[0108] The present invention also provides a method for treating cancer in a subject in need thereof, comprising detecting a mutation at a microsatellite locus in a target DNA fragment from a DNA sample by the method described herein. Typically, the target DNA fragment is derived from a tumor. Typically, the DNA sample is also obtained from a subject suffering from a tumor and / or with a familial cancer predisposition.

[0109] Preferably, the treatment is immunotherapy, including but not limited to immune checkpoint modulators (i.e., inhibitors and / or agonists), monoclonal antibodies, and cancer vaccines.

[0110] Most preferably, the treatment involves the administration of an immune checkpoint modulator, such as an anti-PD-1 and / or anti-PDL-1 inhibitor.

[0111] If a mutation is detected in a microsatellite sequence locus in target DNA from a DNA sample (particularly target tumor DNA), immunotherapy is preferably administered to the subject.

[0112] Furthermore, the method of the present invention for detecting microsatellite instability may be used to monitor subjects diagnosed with tumors associated with DNA mismatch repair disorders. Preferably, the monitoring is performed over a time course of treatment. The method may also be used to monitor cancer recurrence in subjects suffering from tumors associated with DNA mismatch repair disorders. Thus, in another aspect, the present invention also provides a method for monitoring patients diagnosed with or suffering from tumors associated with DNA mismatch repair disorders, comprising detecting mutations in microsatellite sequences in target tumor DNA from a DNA sample selected from a plasma or serum sample obtained from a subject diagnosed with or suffering from a tumor associated with DNA mismatch repair disorders. In patients suffering from tumors associated with DNA mismatch repair disorders, detecting microsatellite instability in circulating tumor DNA may be an indicator of recurrence.

[0113] A multiplexed assay for detecting mutations at microsatellite sequence loci in target DNA from DNA samples The power to detect the presence of MSI in tissues associated with a particular disease, such as cancerous tumors, can be greatly increased by multiplexing multiple markers. Thus, in the context of the present invention, multiple sets of primers / probes as defined above can be used in a multiplexed assay, such that multiple microsatellite loci as defined above (i.e., panel microsatellite loci) can be targeted.

[0114] By way of example, the microsatellite sequence loci of a panel of multiplexed assays according to the invention may be selected from the group consisting of 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, as well as from any of the groups defined above.

[0115] Such multiplexed assays are particularly useful for clinical applications, as discussed above.

[0116] In multiplexed assays, primer pairs are preferably designed using available computer programs so that the resulting amplicons that undergo amplification are predicted to have the same melting temperature.

[0117] In the digital range (when all compartments contain either zero or one target molecule), each target-containing reaction will proceed with the target specifically binding to the primers / probes, but no reaction will occur in the target-free compartments, allowing for multiplexed qPCR assays without concerns about competition or cross-reactivity. Having each molecule in a separate reaction compartment allows for counting both high- and low-abundance targets in the same experiment without concern about "swamping out" the low-abundance target (since each compartment contains at most one target, regardless of its concentration in the average sample volume). When multiple targets are counted (e.g., in a duplex assay format), an "absolute ratio" can be quantified from the ratio of counts of one target to another (e.g., mutant allele vs. wild-type allele) using one of the targets as an internal normalization reference (e.g., how many amplifiable genome equivalents were loaded) that was run in the same experiment as the other targets assayed.

[0118] Additionally, because dPCR is performed as an end-point reaction (PCR is run to completion before measuring fluorescence), having exactly one target molecule per isolate allows multiplexing based on probe intensity (Zhong, Bhattacharya, et al., 2011, "Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR," Lab Chip, 11:2167-2174). By adding a target-specific fluorescent assay at a limiting concentration, the compartment containing that target molecule is PCR-positive but has limited brightness at the PCR endpoint. To count a second target type, a different target-specific probe with the same "color" (i.e., the same fluorophore) is added at a different concentration. The compartment containing the second target will have a brighter signal at the PCR endpoint than the compartment containing the first target, resulting in a separate cloud and thus allowing separate counting for each target. Therefore, a combination of both different colored and different concentrations of probes can be used to achieve higher levels of multiplexing.

[0119] kit: The present invention provides a pair of primers suitable for amplifying a target DNA fragment in a DNA sample, including a microsatellite sequence; a first oligonucleotide probe labeled with a first fluorophore, the first oligonucleotide probe being complementary to a wild-type sequence comprising the microsatellite sequence; a second oligonucleotide probe labeled with a second fluorophore, the second oligonucleotide probe being complementary to a wild-type sequence of the amplified DNA fragment located outside the microsatellite sequence; Thermostable DNA polymerase Also included is a kit for identifying mutations in microsatellite sequence regions of a DNA sample, comprising a primer / probe set comprising:

[0120] Thermostable DNA polymerases are generally described in Newton and Graham 1994 In: PCR, BIOS Scientific Publishers, Ltd., Oxford, UK 13. Advantageously, the thermostable polymerase is Taq polymerase.

[0121] In one embodiment, the kit comprises a plurality of primer / probe sets that allow for the amplification and detection of target DNA fragments that contain unique microsatellite sequences.

[0122] The above-mentioned kits can be used in the aforementioned clinical applications. [Example]

[0123] result Materials and Methods Primer and probe design Primers and probes were designed with the assistance of Primer3Plus Software (Whitehead Institute for Biomedical Research). All primers were checked for nonspecific binding and the absence of secondary structure using Primer BLAST. Primers were designed to generate amplicons of less than 140 bp to optimally amplify cell-free DNA (cfDNA) and fragmented DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor samples. The oligonucleotide sequences used in this study are provided in Table 1. BAT-26 alone: ​​SEQ ID NOS: 1-4; ACVR2A alone: ​​SEQ ID NOS: 5-8; DEFB105A / B alone: ​​SEQ ID NOS: 9-12; BRAF V600E alone: ​​SEQ ID NOS: 13-16; BAT-26-ACVR2A-DEFB105A / B triplex: SEQ ID NOS: 1-5, 7, 9, 11, 17-20. Desalted primers and HPLC-purified probes were manufactured by Invitrogen and Applied Biosystems UK.

[0124] ddPCR conditions Droplet digital PCR (ddPCR) was performed using a Bio-Rad QX100 system as instructed by the manufacturer. PCR reactions were prepared in a 20 μL volume containing 10 μL of dUTP-free 2× Supermix for Probes (Bio-Rad, Reference 1863024), 900 nM of each primer, 250 nM of each TaqMan® probe, and up to 16.5 ng of DNA template equivalent to 5000 copies. The PCR reactions were then transferred to a disposable droplet generator cassette (Bio-Rad, Reference 864008). 70 μL of droplet generation oil (Bio-Rad Reference 1863005) was added, and the cassette was loaded into the droplet generator. Generated droplets (40 μL) were transferred to a 96-well PCR plate (Eppendorf Reference 0030 128.575). Emulsion PCR reactions were then performed using a C1000 thermal cycler (Bio-Rad) under the following cycling conditions: denaturation at 95°C for 10 min, followed by 40 amplification cycles at 94°C for 30 s, 61°C for 3 min (BAT-26), 59°C for 3 min (DEFB105A / B), 55°C for 3 min (ACVR2A), or 60°C for 1 min (BRAFV600E); a final hold at 98°C for 10 min. The ramp rate was set at 2.5°C / s. Each run included a no-DNA control and controls containing 100% WT or 100% mutant DNA. Cluster thresholding and quantification were performed using QuantaSoft v1.7.4 software (Bio-Rad). For ddPCR MSI assays, droplets were manually assigned as WT or MSI-positive based on fluorescence amplitude: WT, VIC. + / FAM + ; MSI positive (mutant), VIC + / FAM - / low Template-free droplets were then collected by VIC. - / FAM - Assay optimization was performed on genomic DNA (gDNA) from the HCT-116 cell line (an MSI-positive colon cancer cell line) diluted or not in WT DNA from peripheral blood mononuclear cells (PBMCs). Mutant allele frequencies (MAFs) were determined from droplet counts with manual assignment.

[0125] LOB and LOD calculation The background signal or false-positive rate for each assay was estimated using at least 53 replicates of WT DNA. The upper limit of blank space (LOB) was defined as the upper 95% confidence limit of the mean false-positive measurement. Analytical sensitivity was estimated using serial dilutions of HCT-116 cell line in WT DNA at mutant allele frequencies (MAF) ranging from 10% to 0.01% (1:2 serial dilutions). The total number of replicates per dilution point ranged from 3 to 8 (10% and 5%, 3x; 2.5% and 1.25%, 4x; 0.63% to 0.16%, 6x; 0.08% to 0.01%, 8x) to maximize detection of rare events. The limit of detection (LOD) was estimated as the lowest mutant concentration likely to be reliably distinguished from LOB.

[0126] Validation of the ddPCR MSI assay in patient samples The ddPCR MSI assay was validated using formalin-fixed, paraffin-embedded (FFPE) tumor tissue, plasma, or serum samples primarily from patients with colorectal cancer (CRC) or endometrial cancer (EC). All samples were obtained from patients enrolled in clinical trials treated at the Institut Curie (Paris, France) under approval of the Institution's Clinical Research Ethical Board. Samples were selected from pools of microsatellite-stable (MSS) or microsatellite-unstable (MSI-H) tumors and identified by a 5-plex PCR method (Bacher et al., 2004) with or without immunohistochemical staining (IHC) for mismatch repair (MMR) proteins (MLH1, MSH2, MHS6, and PMS2). Tumor tissue-derived gDNA was extracted using the Qiagen DNA FFPE Tissue Kit (Qiagen reference 56404) according to the manufacturer's instructions and stored at -20°C. cfDNA was extracted from 0.5–1.8 mL of plasma or serum using the QIAamp® Circulating Nucleic Acid Kit (Qiagen reference 55114) according to the manufacturer's recommendations and stored at −20°C. DNA was quantified using the Qubit dsDNA HS assay and LINE-1 amplification (Rago et al., 2007). ddPCR reactions were performed as described above. The total amount of DNA per reaction varied from 2.5 ng to 10 ng for FFPE samples and from 1 ng to 10 ng for plasma or serum samples.

[0127] result The BAT-26, ACVR2A, and DEFB105A / B MSI ddPCR assays reliably detect allele size variation within microsatellites located internal to the MSH2, ACVR2A, and DEFB105A and B genes, respectively. Three mononucleotide poly(A) microsatellite (MS) markers: BAT-26, a quasi-monomorphic long A located in the fifth intron of the MSH2 gene; 27We developed ddPCR assays capable of detecting allele size variations for the BAT-26 repeat, as well as two shorter A8 and A9 repeats located in exon 10 of ACVR2A and the second intron of the DEFB105A / B paralogous genes, respectively (Table 1). BAT-26 is one of five microsatellite markers widely used in clinical practice to determine the MSI status of colorectal and endometrial tumors (Suraweera et al., 2002). Microsatellites located within the ACVR2A and DEFB105A / B genes are novel discriminatory markers recently identified from analysis of TCGA exome sequencing data as being periodically unstable in MSI-H tumors compared with MSS tumors (Hause et al., 2016; Maruvka et al., 2017). Three assays are based on a drop-off ddPCR strategy, which identifies mutant alleles based on the absence of WT signal (Decraene et al., 2018). Two TaqMan hydrolysis probes were designed for each microsatellite marker within the same amplicon: a VIC-labeled reference probe (REF) that hybridizes to nonvariable sequences upstream or downstream of the microsatellite region, and a FAM-labeled drop-off probe (MS) that spans the entire polyA homopolymer and is flanked by +2–4 bases to confer the ability to properly bind and destabilize in the case of mutant alleles associated with microsatellite instability. The REF probe quantifies the total copy number of the amplicon (i.e., BAT-26, ACVR2A, or DEFB105A / B DNA fragment), while the MS probe distinguishes between WT and MSI alleles due to inefficient hybridization to mutant sequences. Thus, with this type of assay, a two-dimensional scatter plot of VIC and FAM fluorescence amplitudes reveals three possible clusters of droplets: template-free droplets (VICs); - / FAM - ), droplets containing the WT allele (VIC + / FAM + ) and droplets containing MSI-positive alleles (VIC + / FAM - / low ) can be shown (Figures 1A to 1I).

[0128] Given the low complexity of the MS probes, it was necessary to adjust the standard ddPCR conditions (BioRAD guidelines) to achieve specific hybridization to the WT allele. We observed that a thermal cycling protocol with increased annealing temperature and annealing / extension time significantly improved the specificity of the MS probes to the WT allele and, therefore, improved the separation of WT and MSI-positive samples. The optimized assays were able to specifically detect MSI alleles in DNA extracted from the HCT-116 MSI-H cell line, whereas no instability was observed in WT DNA obtained from peripheral blood mononuclear cells (PBMCs) (Figures 1A and 1B for BAT-26; Figures 1D and 1E for DEFB105A / B; Figures 1G and 1H for ACVR2A). Furthermore, the three assays were able to accurately quantify MSI alleles at 1 / 10 dilutions of the HCT-116 cell line in a WT background (Figures 1C, 1F, and 1I).

[0129] The BAT-26, ACVR2A, and DEFB105A / B ddPCR assays are highly specific, reaching detection limits of 0.1% or less. The analytical specificity of the BAT-26, ACVR2A, and DEFB105A / B ddPCR MSI assays was assessed by measuring false-positive MSI calls in at least 53 separate ddPCR reactions of wild-type DNA from PBMCs (mean copy numbers per reaction: 4520 for BAT-26, 3380 for ACVR2A, and 3740 for DEFB105A / B). The mean false-positive rates were as follows: 0.006908 ± 0.01366% for BAT-26 (MSI calls in 11 / 53 reactions), 0.006136 ± 0.01623% for ACVR2A (MSI calls in 7 / 55 reactions), and 0.005604 ± 0.01911% for DEFB105A / B (MSI calls in 5 / 55 reactions). The upper blank limit (LOB) for each assay was estimated at 0.01067% for BAT-26 (Figure 2A), 0.01077% for DEFB105A / B (Figure 2B), and 0.01052% for ACVR2A (Figure 2C). Analytical sensitivity was estimated using serial dilutions of HCT-116 cell line in WT PBMC DNA at mutant allele frequencies (MAFs) ranging from 10% to 0.01% (1:2 serial dilutions). The total number of replicates per dilution point ranged from 3 to 8 (10% and 5%, 3x; 2.5% and 1.25%, 4x; 0.63% to 0.16%, 6x; 0.08% to 0.01%, 8x) to maximize detection of rare events. For the three assays, excellent linear correlations were observed between the expected and observed MAFs, indicating that the three assays can accurately quantify MSI over a wide range of frequencies. 2 = 0.9984 p < 0.0001 (Figure 2A), R for DEFB105A / B 2 = 0.9964 p < 0.0001 (Figure 2B) and R for ACVR2A 2= 0.9955 p < 0.0001 (Figure 2C). The limits of detection (LOD), estimated as the lowest concentration of variant likely to accurately distinguish from LOB, were estimated to be 0.04% for BAT-26 (Figure 2A) and 0.08% for both the DEFB105A / B (Figure 2B) and ACVR2A (Figure 2C) markers. We conclude that the three MSI ddPCR assays are highly sensitive and specific, offering the promise of improved diagnostic accuracy and unprecedented use of MSI biomarkers in liquid biopsies to diagnose and monitor disease treatment and progression.

[0130] ddPCR MSI testing in clinical samples We next evaluated the performance of the BAT-26, ACVR2A, and DEFB105A / B ddPCR MSI assays in 177 FFPE tumor samples obtained primarily from patients with colorectal or endometrial cancer (Table 2). These samples had previously been characterized as MSI-positive (MSI-H, n=94) or MSI-negative (MSS, n=83) using a standard multiplex PCR-capillary electrophoresis method assessing microsatellite instability at five microsatellite markers: BAT-26, NR-21, BAT-25, MONO-27, and NR-24. Samples that showed instability for at least two of the five markers were considered MSI-positive (MSI-H), while samples showing no instability were classified as MSI-negative (MSS). Importantly, the ddPCR and subsequent analyses were performed blinded and without knowledge of the samples' MSI status. As shown in Table 2, MSI ddPCR identified unstable alleles for the BAT-26, ACVR2A, and DEFB105A / B markers in 92, 87, and 81 samples, respectively. It is noteworthy that concordant results between capillary electrophoresis and ddPCR were obtained for BAT-26 in 172 of the 177 samples tested. In three of the five discordant samples, the status of BAT-26 could not be determined by capillary electrophoresis and was defined as unstable by ddPCR. In the other two discordant samples, BAT-26 was classified as unstable by capillary electrophoresis but reported as stable by ddPCR and was not determined. Assuming that a sample is considered MSI-H if instability is found in at least two of the three ddPCR markers analyzed, MSI ddPCR correctly classified 100% (83 / 83) of MSS samples as MSS and 94% (88 / 94) of MSI-H samples as MSI-H. Notably, most of the discrepancies corresponded to endometrial tumor samples (4 / 6), which are more difficult to classify than colorectal cancer and are more prone to false-negative results (Suraweera et al., 2002; Wang et al., 2017).

[0131] Given the high sensitivity and specificity of the MSI ddPCR assay, we next evaluated its performance on 22 plasma or serum samples collected from 12 patients with stage IV MSI-H colorectal or endometrial tumors. Notably, the MSI ddPCR assay was able to detect microsatellite instability in all samples tested, including those with mutant allele frequencies as low as approximately 0.2% (Table 3). Furthermore, five of these 12 patients had BRAF-mutated tumors (BRAF V600E). Therefore, the mutant allele frequencies reported by the MSI ddPCR assay can be directly compared with those obtained with a ddPCR assay specifically targeting the BRAF V600E mutation. Excellent correlation (R for BAT-26) was observed. 2 =0.9852 p<0.0001, R for ACVR2A 2 =0.9603 p<0.0001 and R for DEFB105A / B 2 = 0.9275 p < 0.0001), a correlation further supporting the reliability of the ddPCR MSI assay for the detection and quantification of circulating tumor DNA (Figures 3A-C). Taken together, these results demonstrate that the MSI ddPCR assay can accurately detect MSI in patient samples and can therefore be used as an alternative method to MSI testing in tumor tissue and liquid biopsy in clinical practice.

[0132] Multiplex assay development We next aimed to develop a multiplex MSI ddPCR assay capable of simultaneously detecting the MSI status of the BAT-26, ACVR2A, and DEFB105A / B markers in a single reaction. The multiplexing strategy varied primer and probe concentrations to alter endpoint fluorescence, allowing for differentiation of WT and MSI-positive clusters of droplets for the three markers (see Bio-Rad droplet digital PCR multiplexing guidelines). Different primers and probes, as well as various combinations of primer and probe concentrations, annealing temperatures, and extension times, were tested, several of which produced satisfactory results. An example obtained with an annealing / extension temperature / time of 63°C for 3 minutes and the following primer / probe combinations: BAT-26, SEQ ID NOs: 1-4, 0.2x; ACVR2A, SEQ ID NOs: 5, 7, 17, and 18, 0.6x; and DEFB105A / B, SEQ ID NOs: 9, 11, 19, and 20, 1x, is shown in Figure 4. Although preliminary, these results demonstrate the feasibility of a multiplexed ddPCR assay targeting diverse microsatellite sequences in a single reaction.

[0133] [Table 1]

[0134] [Table 2] TIFF0007766398000003.tif207149 TIFF0007766398000004.tif202149 TIFF0007766398000005.tif207149 TIFF0007766398000006.tif101149

[0135] [Table 3]

[0136] [References] ·Hause RJ, Pritchard CC, Shendure J,Salipante SJ (2016) Classification and characterization of microsatelliteinstability across 18 cancer types. Nature Medicine 22(1):1342-1350 ·Rago C, Huso DL, Diehl F, Karim B, LiuG, Papadopoulos N, Samuels Y, Velculescu VE, Vogelstein B, Kinzler KW, Diaz LAJr (2007) Serial assessment of human tumor burdens in mice by the analysis ofcirculating DNA. Cancer Research 67(19:9364-9370 ·Suraweera N, Duval A, Reperant M, VaurtC, Furlan D, Leroy K, Seruca R, Lacopetta B, Hamelin R (2002) Evaluation oftumor microsatellite instability using five quasimonomorphic mononucleotiderepeats and pentaplex PCR. Gastroenterology 123:1804-1811 ·Maruvka Y, Mouw KW, Karlic R,Parasuraman P, Kamburov A, Polak P, Haradhvala NJ, Hess JM, Rheinbay E, BrodyY, Koren A, Braunstein LZ, D’Andrea A, Lawrence MS, Bass A, Bernards A, Michor F, Getz G (2017)Analysis of somatic microsatellite indels identifies driver events in humantumors. Nature Biotechnology 35:951-959 ·Decraene C, Silveira AB, Bidard FC,Vallee A, Michel M, Melaabi S, Vincent-Salomon A, Saliou A, Houy A, Milder M,Lantz O, Ychou M, Denis MG, Pierga JY, Stern MH, Proudhon C (2018) Multiplehotspot mutations scanning by single droplet digital PCR. Clinical Chemistry64:317-328 ·Wang Y, Shi C, Eisenberg R,Vnencak-Jones CL (2017) Differences in microsatellite instability profilesbetween endometrioid and colorectal cancers. The Journal of MolecularDiagnostics 19:57-64

Claims

1. 1. A method for detecting microsatellite instability in a target fragment from a DNA sample, comprising: a) a pair of primers suitable for amplifying a target fragment of a DNA sample, the target fragment comprising a microsatellite sequence; a first MS oligonucleotide (MS) hydrolysis probe labeled with a first fluorophore, the first MS oligonucleotide probe being complementary to a wild-type sequence comprising said microsatellite sequence; a second oligonucleotide reference (REF) hydrolysis probe labeled with a second fluorophore, the second oligonucleotide REF probe being complementary to a wild-type sequence of the target DNA fragment located outside the microsatellite sequence; subjecting the DNA sample to digital polymerase chain reaction (dPCR) in the presence of a PCR solution comprising: b) measuring fluorescence signals associated with the REF and MS probes, wherein a maximum 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, and a shift in the fluorescence intensity signal associated with the MS probe indicates the presence of the microsatellite instability in the target DNA fragment; c) comparing with a control DNA sample.

2. The method of claim 1 , wherein the target fragment of the DNA sample is constitutional genomic DNA.

3. 3. The method of claim 1 or 2, wherein the target fragment of the DNA sample is genomic tumor DNA.

4. 4. The method of any one of claims 1 to 3, wherein the method targets a microsatellite locus 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. 5. The method of any one of claims 1 to 4, wherein the DNA sample is selected from the group consisting of tumor tissue, disseminated cells, stool, blood cells, plasma, serum, lymph nodes, urine, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, and serous fluid.

6. 6. A method for aiding in the diagnosis of cancer, a disease associated with mutations in mismatch repair (MMR) genes or familial tumor predisposition in a subject, comprising a method for detecting microsatellite instability in target DNA from a DNA sample according to any one of claims 1 to 5, wherein the target fragment is derived from a tumor.

7. A method for assisting in cancer prognosis, comprising detecting microsatellite instability in a target fragment derived from a DNA sample according to any one of claims 1 to 5, wherein the target fragment is derived from a tumor.

8. 6. A method for aiding in the prediction of efficacy of a treatment in a subject suffering from cancer, comprising detecting microsatellite instability in a target fragment derived from a DNA sample according to any one of claims 1 to 5, wherein the target fragment is derived from a tumor.

9. The method described in claim 8, wherein the treatment is immunotherapy.

10. 6. A method to aid in the monitoring of a patient diagnosed with or suffering from a tumor associated with a DNA mismatch repair (MMR) disorder, comprising a method for detecting microsatellite instability in a target fragment from a DNA sample according to any one of claims 1 to 5, wherein the target fragment from the DNA sample is derived from the tumor.

11. a pair of primers suitable for amplifying a target fragment from a DNA sample containing a microsatellite sequence; a first oligonucleotide hydrolysis probe (MS) labeled with a first fluorophore, the first oligonucleotide probe being complementary to a wild-type sequence comprising said microsatellite sequence; a second oligonucleotide hydrolysis probe (REF) labeled with a second fluorophore, the second oligonucleotide probe being complementary to a wild-type sequence of the amplified DNA fragment located outside the microsatellite sequence; Thermostable polymerase A kit for identifying microsatellite instability in a target fragment derived from a DNA sample, comprising:

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