Methods for detecting mutations in microsatellite sequences

Abstract

To provide digital PCR diagnostic methods to detect microsatellite instability.SOLUTION: Provided herein is a method for detecting a mutation present in a microsatellite sequence locus of a target fragment derived from a DNA sample, the method comprising a step of subjecting the DNA sample to a digital polymerase chain reaction (dPCR) in the presence of a PCR solution, where the PCR solution comprises: a pair of primers for amplifying the target fragment of the DNA sample comprising the microsatellite sequence; a first MS oligonucleotide (MS) hydrolysis probe labeled with a first fluorophore and complementary to a wild type sequence comprising the microsatellite sequence; a second oligonucleotide reference (REF) hydrolysis probe positioned outside the microsatellite sequence and labeled with a second fluorophore, where the second oligonucleotide REF probe is complementary to the wild type sequence of the DNA fragment.SELECTED DRAWING: None

Description

[Background technology]

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

[0002] The MSI phenotype is primarily used as a molecular diagnostic tool for gastrointestinal, endometrial, and colorectal tumors, and its phenotype has significant 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 features, including a better prognosis associated with specific locations, low differentiation, high lymphocyte 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 gliablastoma, suggesting potentially broader applications of MSI screening in clinical practice (Hause et al., Nat Med. 2016 Nov.;22(11):1342-1350; Cortes-Ciriano et al., Nat Commun. 2017 June 6;8:15180).

[0004] In fact, MSI has recently emerged as the first pantumor biomarker likely to predict the clinical benefit of immune checkpoint blockade therapy (Le et al., N Engl J Med. June 25, 2015; 372(26): 2509-20; Le et al., Science June 8, 2017. pii:eaan6733). It is noteworthy that 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 through its accelerated approval process.

[0005] Molecular diagnostics of MSI are currently performed by examining PCR products of several useful 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 necessitates a minimum tumor cell density 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) has been used for greater sensitivity and accuracy in MSI detection (Salipante et al., Clin Chem June 30, 2014, 60(9), pp. 1192-1199; Hause et al., Nat Med. November 2016; 22(11): pp. 1342-1350; Cortes-Ciriano et al., Nat Commun. June 6, 2017; 8: 15180). While improvements over currently used methods in clinics are clear, the 1% sensitivity achieved by NGS still surpasses that of PCR-based assays.

[0006] Therefore, the development of highly sensitive MSI diagnostic methods applicable to circulating tumor DNA obtained from liquid biopsies remains of great clinical and therapeutic importance. [Overview of the project] [Problems that the invention aims to solve]

[0007] The authors designed a digital PCR diagnostic method for detecting 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 at which detection is likely to be reliably distinguishable from the blank limit) is 250 times lower than the minimum cell richness threshold (i.e., at least 20% cell richness) required to determine the MSI status by the quintuple assay currently used in clinical practice (see Bacher et al., 2004, Disease Markers 20:237-250, and Shi and Washington, Am J Clin Pathol 2012, 137:847-859). The results presented herein indicate that this novel MSI detection assay achieves high specificity and sensitivity, with sensitivity approaching 0.1%, at least theoretically. This innovative method also offers several other advantages, including the simplicity of the blood test and reduced analysis time. In summary, the MSI diagnostic method of the present invention offers the potential for improved diagnostic accuracy and unprecedented use of MSI biomarkers in liquid biopsies for disease treatment and diagnosis and monitoring of disease progression.

[0009] Similar techniques have been used to detect BRAF status in colorectal cancer (see Bidshahri et al., The Journal of Molecular Diagnostics 2016, 18(2):190-204). However, the use of such techniques has not been considered for the detection of mutant microsatellite sequences. In fact, due to the size of the microsatellite sequences, and more specifically their extreme repeatability, it is expected that probes that cover so many microsatellites (MS probes, see below) will slide over the repeating sequences, making effective or reliable hybridization of the probe impossible.

[0010] Dietmaier et al. (Laboratory Investigation, 2001) describe a technique for detecting microsatellite sequences by RT-PCR and by analyzing the melting point using hybridization probes of specific sequences of targeted markers. The Light Cycler HybProbes hybridization probes used in this document lack the ability to distinguish between wild-type (WT) and mutant microsatellite sequences. Therefore, additional melting point analysis is required after real-time PCR amplification to identify mutant microsatellites. Furthermore, Dietmaier et al.'s probes 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 is 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 fluorescence signal derived 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 is A pair of primers suitable for amplifying target fragments of DNA samples containing microsatellite sequences; A first oligonucleotide microsatellite (MS) hydrolysis probe labeled with a first fluorophore, wherein the first MS oligonucleotide probe is complementary to a wild-type sequence containing a microsatellite sequence; A second oligonucleotide reference (REF) hydrolysis probe labeled with a second fluorophore, wherein the second oligonucleotide REF probe does not contain the microsatellite sequence and is complementary to the wild-type sequence of the target DNA fragment. The present invention relates to a method for detecting mutations at microsatellite loci of a target fragment derived from a DNA sample, comprising the step of subjecting the DNA sample to a digital polymerase chain reaction (dPCR) in the presence of a PCR solution containing a certain substance.

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

[0014] Microsatellite loci may be selected from a 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. In addition, sensitivity may be increased by selecting microsatellite sequences located in genomic regions that are frequently amplified in cancer (e.g., the chr8q region of the human genome).

[0015] Generally, DNA samples are selected from a 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 related to REF and MS probes, wherein the maximum fluorescence intensity signal related to both REF and MS probes indicates the presence of a wild-type microsatellite sequence in a target DNA fragment, and a shift in the fluorescence intensity signal related to 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 a mutation in a mismatch repair (MMR) gene or a familial tumor predisposition in a subject, comprising the step of detecting a mutation at a microsatellite sequence locus of a target DNA derived from the above DNA sample, wherein the target fragment is derived from a tumor.

[0018] The present invention also relates to a method for predicting the prognosis of cancer, comprising the step of detecting a mutation at a microsatellite sequence locus of a target fragment derived from the above DNA sample, wherein the target fragment is derived from a tumor.

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

[0020] The present invention also relates to the step of detecting a mutation at a microsatellite sequence locus of a target fragment derived from the above DNA sample and the step of administering an immunotherapy to a subject when a mutation is identified in the microsatellite sequence locus of the target fragment, and also relates to a method for treating cancer in a subject in need thereof, comprising the above steps, wherein the target fragment of the DNA sample is derived from a tumor.

[0021] The present invention also relates to a method for monitoring patients diagnosed with or suffering from a tumor associated with DNA mismatch repair (MMR) impairment, comprising the step of detecting mutations at microsatellite loci of a target fragment derived from the above-mentioned DNA sample. The target fragments of the DNA sample originate from the tumor.

[0022] Finally, the present invention is A pair of primers suitable for amplifying target fragments derived from DNA samples containing microsatellite sequences; A first oligonucleotide hydrolysis probe (MS) labeled with a first fluorophore, wherein the first oligonucleotide hydrolysis probe is complementary to a wild-type sequence containing a microsatellite sequence; A second oligonucleotide hydrolysis probe (REF) labeled with a second fluorophore, which does not contain the microsatellite sequence and is complementary to the wild-type sequence of the amplified DNA fragment; Thermally stable polymerase The kit also includes a method for identifying mutations in the microsatellite sequence regions of target fragments derived from DNA samples. [Brief explanation of the drawing]

[0023] [Figure 1A]Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1B] Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1C]Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1D] Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1E]Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1F] Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1G]Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1H] Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 1I]Figures A-C show two-dimensional fluorescence amplitude scatter plots of the BAT-26 ddPCR MSI assay 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 the DEFB105A / B ddPCR MSI assay 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 the ACVR2A ddPCR MSI assay 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 the VIC signal only. [Figure 2A] This figure shows correlation curves obtained by BAT-26(A), DEFB105A / B(B), and ACVR2A(C) assays for expected MAFs in 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%) for observed MAFs. The dotted line is estimated to be the upper 95% confidence interval (CI) for false positives in at least 53 independent ddPCR reactions, including LOB and WT DNA. [Figure 2B] This figure shows correlation curves obtained by BAT-26(A), DEFB105A / B(B), and ACVR2A(C) assays for expected MAFs in 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%) for observed MAFs. The dotted line is estimated to be the upper 95% confidence interval (CI) for false positives in at least 53 independent ddPCR reactions, including LOB and WT DNA. [Figure 2C]This figure shows correlation curves obtained by BAT-26(A), DEFB105A / B(B), and ACVR2A(C) assays for expected MAFs in 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%) for observed MAFs. The dotted line is estimated to be the upper 95% confidence interval (CI) for false positives in at least 53 independent ddPCR reactions, including LOB and WT DNA. [Figure 3] This figure shows the correlation between the proportion of ctDNA estimated by BAT-26(A), ACVR2A(B), or DEFB105A / B(C) ddPCR assays and the proportion of ctDNA estimated by ddPCR assays that specifically target the BRAFV600E mutation. [Figure 4] This figure shows a two-dimensional fluorescence amplitude scatter plot illustrating the fluorescence signals obtained in a triple assay simultaneously targeting BAT-26, ACVR2A, and DEFB105A / B microsatellite markers using a 10% dilution of HCT-116 cell line in WT DNA. Results were obtained at an annealing temperature and extension time of 63°C and 3 minutes. Primer and probe concentrations were: BAT-26: 0.2×; ACVR2A: 0.6×; DEFB105A / B: 1×. [Modes for carrying out the invention]

[0024] [Detailed explanation] A-definition The following definitions are intended to help provide a clear and consistent understanding of the scope and details of the terms used to describe and define the present invention: In this specification, the verb “comprise” and its conjugations as used in this description and claims are used in a non-restrictive sense to mean that the item following the word is included, but does not exclude any item not specifically mentioned. In addition, the reference of an element by the indefinite article “a” or “an” does not exclude the possibility of multiple elements being present unless the context clearly requires that there is one or only one element. Thus, the indefinite article “a” or “an” usually means “at least one.”

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

[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 sometimes 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] In this specification, "allele" refers to one of several alternative forms of a gene or DNA sequence located at a specific chromosomal locus. At each autosomal locus, an individual possesses two alleles, one inherited from the father and one from the mother.

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

[0030] The terms "microsatellite locus" or "microsatellite sequence" are used interchangeably and refer to a region of genomic DNA containing short repeat sequence elements of length 1(1) to 7(7), typically 1(1) to 5(5), and especially 1(1) to 4(4) base pairs. Each sequence 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 ten repeat units, and preferably at least twenty repeat units.

[0031] In this specification, "microsatellite instability" (hereinafter, "MSI") refers to a form of genetic instability in which alleles of genomic DNA obtained from a specific tissue, cell, or body fluid of a given subject are mutated at a microsatellite locus.

[0032] Mutations at microsatellite loci generally typically involve the deletion(s), addition(s), or substitution(s) of at least one repeat unit at the microsatellite locus. Generally, MSIs result in length changes at microsatellite loci through addition(s) or, most frequently, deletion(s).

[0033] In this specification, “primer / probe set” refers to a group of a pair of oligonucleotide primers and two oligonucleotide probes that hybridize to a specific target nucleotide sequence, respectively. The oligonucleotide set comprises (a) a forward-recognition primer that hybridizes to a first position in the nucleic acid sequence; (b) a reverse-recognition primer that hybridizes to a second position in the nucleic acid sequence downstream of the first position; and (c) two probes that hybridize to a target sequence between the primers. In other words, the primer / probe set comprises a pair of specific oligonucleotides that anneal to the opposite strand of the nucleic acid sequence (usually including a microsatellite locus) to form an amplicon specific to the nucleic acid sequence during the PCR reaction, and two probes that hybridize to a specific target sequence of the amplicon (i.e., complementary), preferably fluorescent probes.

[0034] An "amplicon" refers to a nucleic acid fragment formed as a product of a natural or artificial amplification event or technique. Generally, amplicons are produced by polymerase chain reaction (PCR). In this specification, "amplification" refers to the process of creating multiple copies at a specific locus (i.e., the target sequence mentioned above) of a nucleic acid, such as genomic DNA. Amplification is completed using PCR (Saiki et al., 1985, Science 230: pp. 1350-1354).

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

[0036] In this specification, “digital PCR” refers to an assay that enables endpoint measurement, providing the ability to quantify nucleic acids without using standard curves, as is done in real-time PCR (see Sykes et al., 1992, “Quantitation of targets for PCR by use of limiting dilution,” BioTechniques 13, pp. 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; and Monya Baker, 2012, Nature Methods 9, pp. 541-544).

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

[0038] Each segment is amplified individually until the terminal flat phase (or endpoint) of the PCR, and the proportion of positive segments is determined by reading the fluorescence. If the segments are of the same volume, the number of target DNA molecules present is given by the following formula: λ = -ln(1-p) (1) The percentage of positive endpoint reactions can be calculated using Poisson statistics, where λ is the average number of target DNA molecules per replication reaction and p is the percentage of positive endpoint reactions. From λ, an estimate of the absolute concentration of target DNA can be calculated, along with the volume of each replication PCR and the total number of replications analyzed.

[0039] Samples can be partitioned into specific compartments or droplets using arrays of microwell plates, capillaries, oil emulsions, and miniaturized chambers with nucleic acid binding surfaces. Therefore, digital PCR as used herein includes various forms, including droplet digital PCR (ddPCR), BEAMing (beads, emulsion, amplification, and magnetic material), and microfluidic chips.

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

[0041] A "droplet" refers to an individual portion of the PCR solution in a droplet digital PCR assay. In this application, digital PCR will be described in relation to droplet digital (or digital droplet PCR, used interchangeably), but as stated above, individual portions of the PCR solution according to the principle of digital PCR can be obtained by various techniques. Therefore, the method of the present invention described below with respect to droplet digital PCR is not limited to this digital PCR technique, but may be applied in a similar manner to other digital PCR techniques.

[0042] Technologies available 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, pp. 17807-17812; Ottesen et al., 2006, "Microfluidic digital PCR enables multigene analysis of individual environmental bacteria," Science 314, pp. 1464-1467; Fan and Quake, 2007, "Detection of aneuploidy with digital polymerase chain reaction," Anal Chem 79, pp. 7576-7579). Other systems include separation onto 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," Analytical Chem 82, pp. 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. Droplet digital PCR is preferred.

[0043] Droplets support PCR amplification of template molecules(s) using homogeneous assay chemistry and workflows similar to those widely used for real-time PCR applications (Hinson et al., 2011, Anal. Chem. 83: pp. 8604-8610; Pinheiro et al., 2012, Anal. Chem. 84: pp. 1003-1011). Once droplets are generated, they can be transferred to a PCR plate, and the emulsified PCR reaction can be performed in a thermal cycler under a classic program, such as those 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 performs a digital PCR assay that measures absolute amounts by counting nucleic acid molecules encapsulated in separate, volumetrically defined, water-in-oil droplet compartments supporting PCR amplification. The strategy for 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), so that each reaction chamber contains one copy or no copy of the nucleic acid molecule of interest.

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

[0046] Examples of droplet digital PCR systems include Bio-Rad's QX100® droplet digital PCR system, which partitions a sample containing nucleic acid templates into 20,000 nanoliter-sized droplets; and RainDance's RainDrop® digital PCR system, which partitions a sample containing nucleic acid templates into 1,000,000 to 1,000,000 picoliter-sized droplets.

[0047] The advantages of dPCR, or more specifically ddPCR technology, include the following: Absolute quantification is achieved by the ddPCR technique, which provides an absolute count of target DNA copies per sample without the need to perform a standard curve.

[0048] This unparalleled precision stems from the large-scale sample segregation achieved by ddPCR, which allows for the reliable measurement of small multiple differences in target DNA sequence copy number between samples.

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

[0050] Removing PCR bias reduces the error rate by eliminating the dependence on qPCR amplification efficiency, making it possible to detect small differences (1.2 times).

[0051] Simplified quantification is achieved by eliminating the need for calibration standards or references for absolute quantification.

[0052] The reduction in consumable costs is achieved by decreasing the amount of reagents and sample required for each data point, as the reaction volume is in the pico-nanolter range.

[0053] Lower equipment costs are due to the fact that emulsion-based reaction systems mean that PCR reactions can be performed in a standard thermocycle without the need for complex chips or microfluidics.

[0054] Superior compartmentalization is achieved through ddPCR technology, which produces 20,000 droplets per 20 μL of sample, resulting in nearly 2,000,000 compartmentalized PCR reactions in a 96-well plate, compared to chip-based digital PCR systems which produce only a few hundred or a few thousand compartments. A larger number of compartments also leads to higher accuracy.

[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 half of the Watson-Crick base pairs in a double-stranded nucleic acid molecule break or dissociate (i.e., “dissolve"), 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 the 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 the nearest nearest neighbor calculation by Wetmur 1991 (Wetmur, 1991, “DNA probes: applications of the principles of nucleic acid hybridization,” Crit Rev Biochem Mol Biol 26: pp. 227-259, incorporated herein by reference) or by commercial programs including Oligo™ Primer Design and programs available on the Internet. Alternatively, Tm can be determined by actual experiments. For example, the actual Tm of nucleic acids can be determined by using double-stranded DNA binding or intercalating dyes such as ethidium bromide or SYBR green (Molecular Probes) in a melting curve assay.

[0056] In this specification, the term “critical denaturation temperature” or “Tc” refers to a temperature lower than the Tm of the wild-type sequence, at which the double helix of the wild-type sequence and the mutant sequence dissolves. (In some examples, this temperature may also be the temperature at which the homodouble helix of the mutant sequence dissolves.)

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

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

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

[0060] This method uses the primer / probe set defined earlier.

[0061] It is generally preferable that the primer pair has a Tm lower than the reaction Tc. The primer pair can be designed using available computer programs. Generally, the probe according to the present invention is a hydrolysis probe (also named a TaqMan probe). The hydrolysis probe has a fluorophore and a quencher covalently bonded to the 5' end of the 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, AlexaFluor PET, Biosearch Blue (trademark), Marina Blue (registered trademark), Bothell Blue (registered trademark), and AlexaFluor (Alexa). Fluor) (registered trademark), 350FAM (trademark), SYBR (registered trademark) Green 1, Fluorescein, EvaGreen (trademark), Alexa Fluor (registered trademark), 488JOE (trademark), 25VIC (trademark), HEX (trademark), TET (trademark), CAL Fluor (registered trademark) Gold 540, Yakima Yellow (registered trademark), ROX (trademark), CAL Fluor (registered trademark) Red 610, Cy3.5 (trademark), Texas Red (registered trademark), Alexa Fluor (registered trademark) 568 Cry5 (trademark), Quasar (trademark) 670, LightCycler Red 640 (LightCycler The product is detected by a fluorescent label that can be selected from the group consisting of Red640 (registered trademark), AlexaFluor 633 Quasar (trademark) 705, Lightcycler Red 705 (registered trademark), AlexaFluor (registered trademark) 680, SYT0 (registered trademark) 9, LC Green (registered trademark), LC Green (registered trademark) Plus+, and Evergreen (trademark). The detectable label is preferably selected from 6-carboxyfluorescein, FAM or tetrachlorofluorescein (acronym: TET), Texas Red, cyanine 5, cyanine 3, or VIC (trademark).

[0063] The quencher may be an internal quencher or a quencher located at the 3' end of the probe. Typical quenchers are tetramethylrhodamine, TAMRA, black hole quenchers, or non-fluorescent quenchers. Hydrolysis probes usable under the present invention are well known in the art (see in particular http: / / www.sigmaaldrich.com / technical-documents / articles / biology / quantitative-pcr-and-digital-pcr-detection-mehods.html). Quenching molecules typically quench the fluorescence emitted by fluorophores when excited by a cycler light source, usually via FRET (Forster Resonance Energy Transfer). Quenching inhibits any fluorescence signal while the fluorophore and quencher are in proximity. Such probes are designed to anneal within a target region amplified by a specific set of primers. Taq polymerase extends the primer and synthesizes the nascent DNA chain. The 5'-3' exonuclease activity inherent in Taq DNA polymerase then cleaves the 3' quencher and 5' reporter, thereby generating a fluorescent signal in proportion 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 by practices established in the art to preferably minimize PCR artificial products and to specifically hybridize with the sequence defined below. The first and second probes are labeled with unique fluorophores to enable the separate detection of their respective signals.

[0065] In some embodiments, the hydrolysis probe according to the present invention includes a secondary groove binder (MGB) moiety at its 3' end. Such an MGB generally increases the melting temperature (Tm) of the probe and stabilizes the probe-target hybrid.

[0066] Oligonucleotide probes have a sequence length of approximately 10 to approximately 50 nucleotides. Oligonucleotide probes (especially MS probes) preferably have a sequence length of approximately 15 to 40, or 25 to 50, particularly 15 to 35 nucleotides. Oligonucleotide probes (especially MS probes) preferably have a sequence length of approximately 20 to 40, or 30 to 50, particularly 30 to 40 nucleotides.

[0067] A first probe according to the present invention (also named an MS probe) hybridizes with a first wild-type target sequence of an amplified target DNA fragment, the target sequence containing a microsatellite locus. The probe preferably covers the entire wild-type microsatellite sequence and extends several additional nucleotides (generally 1-10, particularly 2-8, preferably 2-6, most preferably 2-5 or 2-4 nucleotides) at each end to confer both the ability to bind appropriately and the destabilization obtained in the case of microsatellite instability. In other words, the probe size is designed to confer the ability to bind appropriately to the wild-type microsatellite sequence while preventing hybridization of the MS probe in the presence of mutations in the microsatellite sequence.

[0068] The second probe of the present invention (also named the REF probe) hybridizes with a second wild-type target sequence of the amplified DNA fragment, the target sequence of which 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 within the first wild-type target sequence according to the present invention. Microsatellite loci or markers that can be targeted by the present invention are particularly described in Bacher et al., 2004, Disease Markers 20, pp. 237-250, and Hause et al., 2016, Nat Medicine, November 22 (11): pp. 1342-1350. The targeted microsatellite loci (or microsatellite marker) is preferably selected from microsatellites that have been 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 is preferably located within regions that are frequently amplified in tumors.

[0070] For example, the targeted microsatellite locus may 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 array locus may be selected from the Bethesda panel, including BAT-25, BAT-26, D2S123, D5S346, and D17S250.

[0072] Mononucleotide repeat loci have been shown to be highly susceptible to alteration in tumors involving dysfunctional DNA mismatch repair systems (Parsons, 1995, see above). Therefore, we created such loci that may be preferable for detecting cancer and other diseases associated with dysfunctional DNA mismatch repair systems, making them particularly useful.

[0073] In one embodiment of the present invention, the targeted microsatellite sequence locus is 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 sequences (generally containing 8 to 30, particularly 8 to 25, preferably 8 to 20, 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 content 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 especially true when BAT-26 is involved. As an example, the MS probe of SEQ ID NO: 4, which hybridizes with sequences 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 according to the present invention.

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

[0077] To avoid technical hurdles associated with the amplification of low-complexity sequences such as microsatellite sequences, a series of modifications may be made in addition to the Biorad guidelines for ddPCR described above, to achieve appropriate 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 increased favorably by 3–15°C.

[0078] Thermal cycling is performed to the endpoint. Therefore, after multiple PCR amplification cycles (i.e., after the PCR cycle is completed), raw PCR data is then collected by measuring the fluorescence signals associated with the REF and MS probes for each droplet. Droplets containing the WT target fragment exhibit a double-positive fluorescence signal (REF+ / MS+ droplet) derived from the hybridization of both the REF and MS probes. If the MS probe does not hybridize (or hybridization is inefficient) in droplets containing the mutant microsatellite allele, it results in a shift of the droplet cloud on the 2D graph toward a single REF-positive (REF+) population, which is proportional to the proportion of droplets containing the mutant microsatellite allele.

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

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

[0081] The total number of target DNA fragments in a sample can be quantified using the number of droplets that are positive for the reference probe (REF probe). Then, by fitting the proportion of positive droplets to a Poisson distribution, the absolute initial copy number of target DNA fragments in the input reaction mixture can be determined in copies / μL.

[0082] In droplets containing wild-type target DNA (no mutations in the targeted MS sequence), the maximum fluorescence signal is observed in both 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] Digital PCR reactions are most preferably designed to ensure that most droplets contain zero or one copy of the targeted DNA fragment (particularly depending on the amount of DNA loaded into the reaction). Under these conditions, optimal separation of WT (REF+ / MS+ signal) 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., non-significant) 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 as described above.

[0084] Generally, the frequency of mutant alleles can be determined from droplet counts by manually assigning WT and mutant microsatellite droplet clouds. As mentioned above, the identification of droplet populations with a single signal from the REF probe indicates the presence of mutant microsatellite sequences in the DNA sample.

[0085] As described above, the mutant allele frequencies that can be determined can be compared with the control mutant allele frequencies obtained from a control DNA sample. The control DNA sample may be a wild-type sample or a sample or cell line collected from a subject diagnosed with an MSI-positive tumor or a disease related to mutations in DNA mismatch repair at a time prior to the course of the disease and / or the course of treatment.

[0086] In this specification, the term “sample” means any that may contain DNA, in particular the DNA fragment to be amplified. In some embodiments, the “sample” contains RNA and is therefore subjected to the reverse transcription step. The sample may also be a biological sample such as a biological fluid or biological tissue. Examples of biological fluids include serous fluids such as urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, pancreatic juice, gastric juice, amniotic fluid, pericardial fluid, intrapleural fluid, or ascites.

[0087] Biological tissue is an aggregate of intercellular material and cells, usually special types of cells, that form one of the structural materials of human, animal, plant, bacterial, fungal, or viral structures, including connective, epithelial, muscle, and nerve tissue. Examples of biological tissue include organs, tumor tissue, lymph nodes, arteries, and disseminated cells. Tissue may be fresh, freshly frozen, or fixed, such as formalin-fixed paraffin-embedded (FFPE). Samples may be obtained by any means, such as surgical procedures including biopsy, or by minimally invasive methods including but not limited to dissection or fine-needle aspiration. DNA samples are preferably 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, intrapleural fluid, or ascites.

[0088] DNA, particularly target DNA fragments, may be genomic DNA or DNA obtained from reverse transcriptase. Genomic DNA may 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 have shown that tumor DNA is released into circulation and is present in particularly high concentrations in plasma and serum in several different types of cancer (Leon et al., 1977, Cancer Res 37: pp. 646-650; Stroun et al., 1989, Oncology 46: 318-322). Therefore, the DNA sample according to the present invention may 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 for 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, the DNA may be obtained from the reverse transcription of an RNA sample.

[0089] Generally, DNA samples according to the present invention are obtained from subjects. The subjects or patients (both terms may be used interchangeably) of the present invention are mammals, generally primates such as humans. In some embodiments, primates are monkeys or apes. Subjects may be male or female and may be of any appropriate age, including children, young adults, adolescents, adults, and elderly subjects. In some embodiments, subjects may be non-primate mammals such as rodents.

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

[0091] In some embodiments, subjects are those who have, are in remission, or have a familial predisposition to cancer, and in particular, subjects have or are at risk of having a disease caused by mutations in mismatch repair (MMR) genes, such as constructive mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome.

[0092] Cancer can be a solid tumor or a "humoral tumor," such as a tumor of hematopoietic and lymphoid tissue, which affects the blood, bone marrow, and lymphatic system, and especially includes leukemia and lymphoma. Humoral tumors include chronic lymphocytic leukemia (CLL), which includes various lymphomas such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and mantle cell lymphoma or non-Hodgkins lymphoma (NHL).

[0093] Solid tumors include cancers that affect one of the following organs, selected from the group consisting of the 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, gliablastoma, low-grade astrocytoma, oligodendrocyte, pituitary tumor, schwannoma, and metastatic brain cancer), ovaries, chest, head and neck region, testes, prostate, and thyroid.

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

[0095] In the context of the present invention, cancer (or tumor) associated with MSI is also referred to as MSI-positive cancer (or tumor), and relates to cancer (or tumor) in which the genomic tumor DNA exhibits at least one mutation in the microsatellite sequence. Therefore, an MSI-positive cancer can be any of the cancers listed above, in which the genomic tumor DNA exhibits at least one mutation in the microsatellite sequence.

[0096] Clinical applications: Methods for diagnosis and prognosis prediction, therapeutic procedures, and patient monitoring.

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

[0098] Firstly, as mentioned above, microsatellite instability is a hypermutagenic phenotype that occurs in tumors associated with DNA mismatch repair deficiency (MMR). Therefore, MSI has been associated with a wide variety of cancers, though not limited to colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, urinary tract cancer, brain cancer, and breast cancer. MSI is most commonly an outcome of colorectal cancer. MSI is commonly seen in constructive mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome.

[0099] Therefore, detection of mutated microsatellite sequences by the method described above can be used in the diagnosis of cancers as defined earlier, particularly cancers associated with DNA mismatch repair disorders (as defined earlier), especially MSI-positive cancers (or tumors).

[0100] In one embodiment of the present invention, the detection of mutant microsatellite sequences by this method can be used for the diagnosis of diseases caused by mutations in mismatch repair (MMR) genes, particularly MSI-positive tumors such as constitutive mismatch repair deficiency syndrome (CMMRD syndrome) or Lynch syndrome, or for the diagnosis of familial tumor predisposition in subjects.

[0101] Accordingly, in one embodiment, the present invention relates to a method for diagnosing cancer in a subject, particularly diseases associated with mutations in mismatch repair (MMR) genes, such as MSI-positive tumors, and / or familial neoplastic predisposition to cancer, comprising the step of detecting mutations at microsatellite loci of target DNA derived 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 a subject as described above. In one embodiment, detection of a mutated microsatellite sequence in a subject-derived DNA sample indicates that the subject has an MSI-positive tumor, particularly a disease caused by a mutation in the MMR gene, such as CMMRD or Lynch syndrome. Detection of a mutated microsatellite sequence in a subject-derived DNA sample may also indicate that the subject has a familial neoplastic 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 is found at a lower prevalence in virtually all types of cancer (see Hause et al., 2016, Nature Medicine). As mentioned above, the MSI phenotype of cancer (i.e., positive or negative) has important implications for cancer prognosis and rational planning of treatment (Boland and Goel, Gastroenterology 2010). Therefore, even in cancers with low MSI-positive prevalence, it remains very important to identify whether a patient has an MSI-positive or MSI-negative tumor. Thus, the method of the present invention can be used to predict the prognosis of various cancers. Identification of positive MSI cancer is generally associated with a good prognosis.

[0104] Accordingly, the present invention also relates to a method for predicting the prognosis of cancer (as defined above), comprising the step of detecting mutations at microsatellite loci of 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 the tumor is MSI-positive.

[0105] In the therapeutic context described above, the method of the present invention is particularly useful because its outstanding 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 blood or plasma samples, and the target DNA is cell-free DNA such as circulating tumor DNA. This is particularly significant for diseases such as CMMRD, including brain tumors for which biopsy is not possible.

[0106] As reports have shown, for example, that patients with colorectal cancer who are MMR-deficient respond well to immunotherapy with PD-1 immune checkpoint blockade and show improved progression-free survival, the present invention also relates to methods for predicting the efficacy of treatment. Therefore, identifying patients with MSI-related cancers (i.e., MSI-positive cancers or tumors) is highly clinically relevant to selecting appropriate treatment strategies.

[0107] Accordingly, another aspect of the present invention relates to a method for predicting the efficacy of a treatment in a subject with cancer, the method comprising the step of detecting mutations at microsatellite loci of a target DNA fragment derived from the aforementioned subject DNA sample. The target DNA fragment is preferably derived from a tumor. Typically, the DNA sample is obtained from a subject with a tumor and / or a familial predisposition to cancer.

[0108] The present invention also proposes a method for treating cancer in subjects requiring it, comprising the step of detecting mutations at microsatellite loci of a target DNA fragment derived from a DNA sample by the method described herein. Typically, the target DNA fragment is derived from a tumor. Typically, DNA samples are also obtained from subjects who have a tumor and / or a familial predisposition to cancer.

[0109] Treatment is preferably immunotherapy. Immunotherapy includes, but is not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), monoclonal antibodies, and cancer vaccines.

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

[0111] If mutations are detected at microsatellite loci of target DNA derived from a DNA sample (particularly target tumor DNA), it is preferable to administer immunotherapy to the target.

[0112] Furthermore, the present invention's method for detecting microsatellite instability may be used to monitor subjects diagnosed with tumors associated with DNA mismatch repair deficiency. Such monitoring is preferably performed during the course of treatment. The method may also be used to monitor cancer recurrence in subjects with tumors associated with DNA mismatch repair deficiency. Therefore, in another embodiment, the present invention also provides a method for monitoring patients diagnosed with or suffering from tumors associated with DNA mismatch repair deficiency, comprising the step of detecting mutations in the microsatellite sequences of target tumor DNA derived from a DNA sample selected from a plasma or serum sample obtained from a subject diagnosed with or suffering from such a tumor. In patients with tumors associated with DNA mismatch repair deficiency, detection of microsatellite instability in circulating tumor DNA may serve as an indicator of recurrence.

[0113] A multiplexed assay to detect mutations at microsatellite loci of target DNA derived from DNA samples. The ability to detect the presence of MSI in tissues associated with specific diseases, such as cancerous tumors, can be greatly enhanced by multiplexed multi-marker assays. Therefore, in the context of the present invention, multiple sets of primers / probes defined above can be used in a multiplexed assay so that multiple microsatellite loci (i.e., panel microsatellite loci) can be targeted.

[0114] For example, the microsatellite loci of the panel of the multiplexed assay according to the present 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 previously defined groups.

[0115] As mentioned above, such multiplexed assays are particularly useful for clinical applications.

[0116] In multiplexed assays, it is preferable that primer pairs be designed using available computer programs so that the amplicons obtained after amplification are predicted to have the same melting temperature.

[0117] When the reaction is within the digital range (all compartments contain either 0 or 1 target molecule), each target-containing reaction proceeds with the target specifically binding to the primer / probe, but no reaction occurs in compartments without targets, allowing for multiplex qPCR assays without competition or cross-reactivity. By having each molecule in a separate reaction compartment, it becomes possible to count both high and low-abundance targets in the same experiment without considering "swamping out" low-abundance targets (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 dual assay format), one of the targets can be used as an internal standardization reference (e.g., how many amplified genomic equivalents were loaded) after completing the same experiment as the other targets assayed, allowing for the quantification of an "absolute ratio" from the ratio of one target's count to another target (e.g., mutant alleles versus wild-type alleles).

[0118] In addition, because dPCR is performed as an endpoint reaction (completed before PCR fluorescence is measured), having exactly one target molecule in the isolate allows for probe intensity-based multiplexing (Zhong, Bhattacharya et al., 2011, "Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR," Lab Chip, 11: pp. 2167-2174). By adding a target-specific fluorescence assay at a limiting concentration, the compartment containing the target molecule becomes PCR-positive but exhibits limited brightness at the PCR endpoint. To count a second target type, different target-specific probes with the same "color" (i.e., the same fluorophore) are added at different concentrations. The compartment containing the second target will have a brighter signal at the PCR endpoint than the compartment containing the first target, resulting in separate clouds and thus allowing for separate counting for each target. Therefore, multiplexing can be performed at a higher level using combinations of probes of different colors and probes of different concentrations.

[0119] kit: The present invention A pair of primers suitable for amplifying target DNA fragments in a DNA sample, including microsatellite sequences; A first oligonucleotide probe labeled with a first fluorophore, wherein the first oligonucleotide probe is complementary to a wild-type sequence containing a microsatellite sequence; A second oligonucleotide probe labeled with a second fluorophore, the second oligonucleotide probe located outside the microsatellite sequence and complementary to the wild-type sequence of the amplified DNA fragment; Thermally stable DNA polymerase It also includes a kit for identifying mutations in the microsatellite sequence regions of DNA samples, including a primer / probe set containing [specific components / technology].

[0120] Thermally stable DNA polymerases are generally described in Newton and Graham 1994 In:PCR, BIOS Scientific Publishers, Ltd., Oxford, UK 13. It is advantageous for thermally stable polymerases to be Taq polymerases.

[0121] In one embodiment, the kit comprises multiple primer / probe sets, which enable amplification and detection of target DNA fragments containing specific microsatellite sequences.

[0122] The kit mentioned above can be used for the aforementioned clinical applications. [Examples]

[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 verified for nonspecific binding and the absence of secondary structures using Primer BLAST. The 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 triple: 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. The PCR reaction was prepared in a 20 μL volume containing 10 μL of dUTP-free 2× Supermix for Probes (Bio-Rad, reference 1863024), 900 nM primers, 250 nM TaqMan® probes, and up to 16.5 ng of DNA template equivalent to 5000 copies. The PCR reaction was 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 placed in the droplet generator. The generated droplets (40 μL) were transferred to a 96-well PCR plate (Eppendorf, reference 0030 128.575). The emulsified PCR reaction was then performed using a C1000 thermal cycler (Bio-rad) under the following cycle conditions: denaturation at 95°C for 10 minutes, followed by 40 amplification cycles of 30 seconds at 94°C, 3 minutes at 61°C (BAT-26), 3 minutes at 59°C (DEFB105A / B), 3 minutes at 55°C (ACVR2A), or 1 minute at 60°C (BRAFV600E); and a final retention of 10 minutes at 98°C. The ramp rate was set to 2.5°C / second. Each run included a DNA-free control and a control 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 A droplet without a mold, VIC - / FAM - The assay was optimized using genomic DNA (gDNA) from HCT-116 cell lines (MSI-positive colorectal cancer cell lines) diluted or undiluted in peripheral blood mononuclear cell (PBMC)-derived WT DNA. Mutant allele frequencies (MAFs) were determined from droplet counts obtained by manual allocation.

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

[0126] Validation of 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 prepared at Institut Curie (Paris, France) under the approval of the Institution's Clinical Research Ethical Board and obtained from patients enrolled in clinical trials. Samples were selected from a pool of microsatellite-stable (MSS) or microsatellite-unstable (MSI-H) tumors and identified by quintuple PCR (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 DNA amount 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 allelic size variations within microsatellites located inside the MSH2, ACVR2A, and DEFB105A and B genes, respectively. Three mononucleotide poly(A) microsatellite (MS) markers: BAT-26, a quasi-mononucleotide of length A located in the 5th intron of the MSH2 gene. 27We developed a ddPCR assay that can detect allelic size variations for two shorter A8 and A9 repeats located in exon 10 of ACVR2A and intron 2 of the DEFB105A / B paralogous genes, respectively (Table 1). BAT-26 is one of five microsatellite markers widely used to determine the MSI status of colorectal and endometrial tumors in clinical practice (Suraweera et al., 2002). Microsatellites located within the ACVR2A and DEFB105A / B genes are novel discriminative markers recently identified from the analysis of TCGA exome sequencing data as being periodically unstable in MSI-H tumors compared to MSS tumors (Hause et al., 2016; Maruvka et al., 2017). The three assays are based on the drop-off ddPCR strategy, which identifies mutant alleles based on the absence of the WT signal (Decraene et al., 2018). For each microsatellite marker, two Taqman hydrolysis probes were designed within the same amplicon. A VIC-labeled reference probe (REF) that hybridizes to invariant sequences upstream or downstream of the microsatellite region and a FAM-labeled drop-off probe (MS) that encompasses the entire polyA homopolymer and has +2-4 bases on both sides to confer the destabilization obtained in the case of mutant alleles related to microsatellite instability and the ability to bind appropriately. The REF probe quantifies the total copy number of the amplicon (i.e., BAT-26, ACVR2A, or DEFB105A / B DNA fragment), and the MS probe discriminates between WT and MSI alleles by inefficient hybridization to the mutant sequence. Therefore, with this type of assay, the two-dimensional scatter plot of VIC and FAM fluorescence amplitudes can show three possible classes of droplets: droplets without template (VIC - / FAM - ), droplets containing the WT allele (VIC + / FAM + ), and droplets containing the MSI-positive allele (VIC + / FAM - / low ) (Figures 1A-1I).

[0128] Given the low complexity of the MS probe, it was necessary to adjust standard ddPCR conditions (BioRAD guidelines) to achieve specific hybridization for WT alleles. Thermal cycling protocols with increased annealing temperature and annealing / extension time were observed to significantly improve the specificity of the MS probe for WT alleles and, therefore, improve the separation of WT and MSI-positive clouds. The optimized assays were able to specifically detect MSI alleles in DNA extracted from the HCT-116 MSI-H cell line, but 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 in a 1 / 10 dilution 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 and reach a detection limit of less than 0.1%. The analytical specificity of the BAT-26, ACVR2A, and DEFB105A / B ddPCR MSI assays was evaluated by measuring false-positive MSI determinations in at least 53 individual ddPCR reactions of WT DNA obtained from PBMCs (average copy number per reaction: 4520 for BAT-26; 3380 for ACVR2A; and 3740 for DEFB105A / B). The average false-positive rates were as follows: 0.006908±0.01366% for BAT-26 (MSI determinations in 11 / 53 reactions), 0.006136±0.01623% for ACVR2A (MSI determinations in 7 / 55 reactions), and 0.005604±0.01911% for DEFB105A / B (MSI determinations in 5 / 55 reactions). The upper limit of blank (LOB) for each assay was estimated to be 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 lines in WT PBMC DNA at mutant allele frequencies (MAF) from 10% to 0.01% (1:2 serial dilutions). The total number of replicas per dilution point ranged from 3 to 8 (10% and 5%, 3×; 2.5% and 1.25%, 4×; 0.63% to 0.16%, 6×; 0.08% to 0.01%, 8×) to maximize the detection of rare events. For the three assays, excellent linear correlations were observed between expected MAF and observed MAF, indicating that the three assays can accurately quantify MSI across a wide range of frequencies. (For BAT-26, R) 2 =0.9984 p<0.0001 (Figure 2A), R in the case of DEFB105A / B 2 =0.9964 p<0.0001 (Figure 2B) and in the case of ACVR2A, R 2=0.9955 p<0.0001 (Figure 2C). The estimated limit of detection (LOD), which represents the lowest concentration of variants likely to be accurately distinguishable from LOBs, was estimated at 0.04% for BAT-26 (Figure 2A) and 0.08% for both the DEFB105A / B marker (Figure 2B) and ACVR2A (marker Figure 2C). We conclude that the three MSI ddPCR assays are highly sensitive and specific, and that they offer the potential for superior diagnostic accuracy and unprecedented use of MSI biomarkers in liquid biopsies for disease management and diagnosis and monitoring of disease progression.

[0130] ddPCR MSI testing in clinical samples The results of BAT-26, ACVR2A, and DEFB105A / B ddPCR MSI assays were evaluated in 177 FFPE tumor samples, primarily obtained from patients with colorectal or endometrial cancer (Table 2). These samples have been characterized as MSI-positive (MSI-H, n=94) or MSI-negative (MSS, n=83) using standard multiplex PCR capillary electrophoresis to assess microsatellite instability in five microsatellite markers: BAT-26, NR-21, BAT-25, MONO-27, and NR-24. Samples showing instability in at least two of the five markers were considered MSI-positive (MSI-H), and samples showing no instability were classified as MSI-negative (MSS). Importantly, ddPCR and the following analyses were performed blindly and without knowledge of the sample's MSI status. As shown in Table 2, unstable alleles for BAT-26, ACVR2A, and DEFB105A / B markers were identified in 92, 87, and 81 samples, respectively, by MSI ddPCR. It is noteworthy that for BAT-26, matching results between capillary electrophoresis and ddPCR were obtained in 172 out of 177 samples tested. In three of the five mismatched samples, the status of BAT-26 could not be determined by capillary electrophoresis and was defined as unstable by ddPCR. In the other two mismatched samples, BAT-26 was classified as unstable by capillary electrophoresis but reported as stable by ddPCR, and therefore could not be determined. If a sample is considered MSI-H if instability is detected in at least two of the three ddPCR markers analyzed, then 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 mismatches corresponded to endometrial tumor samples (4 / 6), which are more difficult to classify than colorectal cancer and have a higher tendency for false negative results (Suraweera et al., 2002; Wang et al., 2017).

[0131] Given the high sensitivity and specificity of the MSI ddPCR assay, its performance was evaluated on 22 plasma or serum samples collected from 12 patients with stage IV MSI-H colorectal or endometrial tumors. The remarkable MSI ddPCR assay was able to detect microsatellite instability in all samples tested, including those with a low mutant allele frequency of 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 to those obtained by ddPCR assays specifically targeting the BRAF V600E mutation. Excellent correlation (in the case of BAT-26, R 2 =0.9852 p<0.0001, R in the case of ACVR2A 2 =0.9603 p<0.0001 and R in the case of DEFB105A / B 2 A correlation of 0.9275 (p<0.0001) was obtained, and this correlation further supports the reliability of the ddPCR MSI assay for detection and the quantification of circulating tumor DNA (Figures 3A-3C). 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 to liquid biopsy in clinical practice.

[0132] Development of multiple assays The next goal was to develop a multiplexed MSI ddPCR assay capable of simultaneously detecting the MSI status of BAT-26, ACVR2A, and DEFB105A / B markers in a single reaction. The multiplexing strategy involved varying the primer and probe concentrations to alter endpoint fluorescence, allowing for differentiation between WT and MSI-positive clusters of droplets for the three markers (see Bio-Rad Droplet Digital PCR Multiplexing Guidelines). Various combinations of different primers and probes, as well as primer and probe concentrations, annealing temperature, and extension time, were tested, and several produced satisfactory results. An example obtained with an annealing / extension temperature / time of 63°C for 3 minutes and the following primer / probe combinations is shown in Figure 4. Although preliminary, these results demonstrate the feasibility of multiplexed ddPCR assays targeting diverse microsatellite sequences in a single reaction.

[0133] [Table 1]

[0134] [Table 2] TIFF2023106439000003.tif207149 TIFF2023106439000004.tif202149 TIFF2023106439000005.tif207149 TIFF2023106439000006.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 human tumors. 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 profiles between endometrioid and colorectal cancers. The Journal of MolecularDiagnostics 19:57-64

[0137] Preferred embodiments of the present invention are as follows. [1] A pair of primers suitable for amplifying target fragments of DNA samples containing microsatellite sequences; A first MS oligonucleotide (MS) hydrolysis probe labeled with a first fluorophore, wherein the first MS oligonucleotide probe is complementary to the wild-type sequence containing the microsatellite sequence; A second oligonucleotide reference (REF) hydrolysis probe labeled with a second fluorophore, the second oligonucleotide REF probe located outside the microsatellite sequence and complementary to the wild-type sequence of the target DNA fragment; A method for detecting mutations at microsatellite loci of a target fragment derived from a DNA sample, comprising the step of subjecting the DNA sample to a digital polymerase chain reaction (dPCR) in the presence of a PCR solution containing a [specific compound]. [2] The method according to [1], wherein the target fragment of the DNA sample is constitutive genomic DNA. [3] The method according to [1] or [2], wherein the target fragment of the DNA sample is genomic tumor DNA. [4] The method according to any one of [1] to [3], wherein the microsatellite array locus is 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. [5] The method according to any one of [1] to [4], wherein 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. [6] The method according to any one of [1] to [5], further comprising the step of measuring the fluorescence signals associated with the REF and MS probes, wherein the 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 the 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 diagnosing cancer, a disease associated with mutations in mismatch repair (MMR) genes, or a familial neoplastic predisposition in a subject, comprising the step of detecting a mutation at a microsatellite sequence locus of a target DNA derived from any of the DNA samples described in [1] to [6], wherein the target fragment is derived from a tumor. [8] A method for predicting the prognosis of cancer, comprising the step of detecting a mutation at a microsatellite sequence locus of a target fragment derived from a DNA sample described in any of [1] to [6], wherein the target fragment is derived from a tumor. [9] A method for predicting the efficacy of a treatment in a patient with cancer, comprising the step of detecting mutations at microsatellite loci of a target fragment derived from a DNA sample described in any of [1] to [6], wherein the target fragment is derived from a tumor and the treatment is preferably an immunotherapy such as immune checkpoint therapy.

[10] A step of detecting mutations at microsatellite loci of a target fragment derived from a DNA sample described in any of [1] to [6], The steps include administering immunotherapy to a subject when a mutation is identified in the microsatellite sequence locus of the target fragment, and A method for treating cancer in a subject requiring the treatment thereof, wherein the target fragment of the DNA sample is derived from a tumor.

[11] A method for monitoring a patient diagnosed with or suffering from a tumor associated with DNA mismatch repair (MMR) impairment, comprising the step of detecting mutations at microsatellite loci of a target fragment derived from a DNA sample, wherein the target fragment of the DNA sample originates from the tumor.

[12] A pair of primers suitable for amplifying target fragments derived from DNA samples containing microsatellite sequences; A first oligonucleotide hydrolysis probe (MS) labeled with a first fluorophore, wherein the first oligonucleotide probe is complementary to the wild-type sequence containing the microsatellite sequence; A second oligonucleotide hydrolysis probe (REF) labeled with a second fluorophore, the second oligonucleotide probe located outside the microsatellite sequence and complementary to the wild-type sequence of the amplified DNA fragment; Thermally stable polymerase A kit for identifying mutations in the microsatellite sequence regions of target fragments derived from DNA samples, including [specific component / tool].

Claims

1. A method for detecting a mutation in a microsatellite sequence locus of a target fragment derived from a DNA sample, comprising: 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 detectable label, 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 detectable label, 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 droplet polymerase chain reaction (ddPCR) in the presence of a PCR solution comprising:

2. The hydrolysis probe, a quencher, or The method of claim 1 , having a detectable label and a quencher covalently attached to the 5′ end of the oligonucleotide probe.

3. The method described in claim 1 or 2, wherein the detectable label is a fluorescent label or a fluorophore.

4. The detectable label or the fluorescent label is selected from the group consisting of FAM (5- or 6-carboxyfluorescein), TET (5-tetrachlorofluorescein), cyanine 5, cyanine 3, VIC (trademark), NED, fluorescein, FITC, IRD-700 / 800, CY3.5, CY5.5, HEX, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor PET, Biosearch Blue (trademark), Marina Blue (registered trademark), Bothell Blue (registered trademark), Alexa Fluor Fluor®, 350 FAM™, SYBR® Green 1, Alexa Fluor® 488 JOE™, 25 VIC™, CAL Fluor® Gold 540, CAL Fluor® Red 610, Alexa Fluor® 568 Cry5™, Quasar™ 670, LightCycler Red 640®, Alexa Fluor 633, Quasar™ 705, LightCycler Red 705®, Alexa Fluor® 680, SYT0® 9, LC Green 4. The method of claim 3, wherein the active ingredient is selected from the group consisting of LC Green®, LC Green® Plus+, and Evergreen™.

5. The method of any one of claims 1 to 4, wherein the target fragment of the DNA sample is constitutional genomic DNA.

6. The method of any one of claims 1 to 4, wherein the target fragment of the DNA sample is genomic tumor DNA.

7. 7. The method of any one of claims 1 to 6, wherein the microsatellite sequence loci are 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.

8. 8. The method of any one of claims 1 to 7, 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.

9. 9. The method of claim 1, further comprising measuring the fluorescent signals associated with the REF and MS probes, wherein a maximum fluorescent 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 fluorescent intensity signal associated with the MS probe indicates the presence of a mutation in the microsatellite sequence of the target DNA fragment.

10. 10. 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, the method comprising detecting mutations in microsatellite loci of target DNA from a DNA sample according to any one of claims 1 to 9, wherein the target fragment is derived from a tumor.

11. 10. A method for assisting in cancer prognosis, comprising the method of detecting a mutation in a microsatellite locus of a target fragment derived from a DNA sample according to any one of claims 1 to 9, wherein the target fragment is derived from a tumor.

12. 10. A method for aiding in the prediction of efficacy of a treatment in a subject suffering from cancer, the method comprising the method of detecting mutations in microsatellite loci of a target fragment derived from a DNA sample according to any one of claims 1 to 9, wherein said target fragment is derived from a tumor and said treatment is preferably an immunotherapy such as an immune checkpoint therapy.

13. 10. A method to aid in the monitoring of a patient diagnosed with or who has suffered from a tumor associated with a DNA mismatch repair (MMR) disorder, comprising detecting a mutation in a microsatellite locus of a target fragment derived from a DNA sample according to any one of claims 1 to 9, wherein the target fragment of the DNA sample is derived from a tumor.

14. A kit for identifying mutations in a microsatellite sequence region of a target fragment derived from a DNA sample, comprising: 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 detectable label, 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 detectable label, the second oligonucleotide probe being complementary to a wild-type sequence of the amplified DNA fragment located outside the microsatellite sequence; Thermostable polymerase Including, The hydrolysis probe is a quencher or A kit having a detectable label and a quencher covalently attached to the 5' end of said oligonucleotide probe.

15. The kit of claim 14, wherein the detectable label is a fluorescent label or a fluorophore.

16. The method of claim 16, wherein the detectable label or the fluorescent label is selected from the group consisting of FAM (5- or 6-carboxyfluorescein), TET (5-tetrachlorofluorescein), cyanine 5, cyanine 3, VIC™, NED, fluorescein, FITC, IRD-700 / 800, CY3.5, CY5.5, HEX, 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 PET, and the like. Fluor®, 350 FAM™, SYBR® Green 1, Alexa Fluor® 488 JOE™, 25 VIC™, CAL Fluor® Gold 540, CAL Fluor® Red 610, Alexa Fluor® 568 Cry5™, Quasar™ 670, LightCycler Red 640®, Alexa Fluor 633, Quasar™ 705, LightCycler Red 705®, Alexa Fluor® 680, SYT0® 9, LC Green 16. The kit of claim 14 or 15, wherein the immunoglobulin is selected from the group consisting of LC Green®, LC Green® Plus+, and Evergreen™.

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