Method for determining homologous recombination repair deficiency and kit therefor

Abstract

The present disclosure provides methods, systems, and kits for assessing a subject's homologous recombination repair deficiency (HRD) status. The present disclosure further provides methods, systems, and kits for identifying treatments based on a human subject's HRD status.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority of U.S. Provisional Application No. 63 / 135,622, filed on January 10, 2021, the content of which is hereby incorporated by reference in its entirety.

[0002] This disclosure relates to methods and kits for assessing homologous recombination repair deficiency (HRD) status.

Background Art

[0003] The poly(ADP - ribose) polymerase (PARP) pathway and the homologous recombination repair (HRR) pathway are involved in DNA damage repair. Inhibition of PARP can lead to the accumulation of unrepaired DNA single - strand breaks (SSBs) and stalled replication forks, resulting in replication fork collapse and the generation of double - strand DNA breaks (DSBs), which are repaired by the HRR pathway in normal cells. When HRR is deficient, synthetic lethality occurs in the presence of PARP inhibition. In recent years, PARP inhibitors have been developed as anti - cancer drugs for treating patients with homologous recombination repair deficiency (HRD).

[0004] For PARP inhibitor treatment, biomarker testing (i.e., BRCA1 / 2 mutation status) is mainly required before the start of PARP inhibitor treatment to identify the patients who will benefit most from the treatment. So far, only two companion diagnostic tests for PARP inhibitor treatment, Myriad myChoice and FoundationFocus, have been approved by the FDA. There is still a need to develop more companion diagnostic assays to determine the HRD status of patients.

Summary of the Invention

Means for Solving the Problems

[0005] In a general aspect, the disclosure is (1) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of a sample obtained from a subject, wherein more than 50% of the intervals between each two adjacent SNP loci have a length of 0.01 to 1 Mb, (2) Identifying the number of loss of heterozygosity (LOH) SNP loci and the number of non-homozygous SNP loci based on the sequencing results, (3) Calculating an LOH score, where the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and (4) Identifying the HRD status based on the LOH score A method for evaluating the homologous recombination repair deficiency (HRD) status in a subject, comprising the above steps.

[0006] In some embodiments, the number of SNP loci is 1,000 loci or more, 1,500 loci or more, 2,000 loci or more, 2,500 loci or more, 3,000 loci or more, 3,500 loci or more, 4,000 loci or more, 4,500 loci or more, 5,000 loci or more, 5,500 loci or more, 6,000 loci or more, 6,500 loci or more, 7,000 loci or more, 7,500 loci or more, 8,000 loci or more, 8,500 loci or more, 9,000 loci or more, 9,500 loci or more, 10,000 loci or more, 20,000 loci or more, 30,000 loci or more, 40,000 loci or more, 50,000 loci or more, 60,000 loci or more, 70,000 loci or more, 80,000 loci or more, 90,000 loci or more, 100,000 loci or more, 110,000 loci or more, 120,000 loci or more, 130,000 loci or more, 140,000 loci or more, 150,000 loci or more, 160,000 loci or more, 170,000 loci or more, 180,000 loci or more, 190,000 loci or more, 200,000 loci or more, 210,000 loci or more, 220,000 loci or more, 230,000 loci or more, 240,000 loci or more, 250,000 loci or more, 260,000 loci or more, 270,000 loci or more, 280,000 loci or more, 290,000 loci or more, or 300,000 loci or more. In some embodiments, the number of SNP loci is 1,000 - 260,000, 2,000 - 200,000, 3,000 - 100,000, 3,000 - 60,000, 6,000 - 11,000, 7,000 - 10,000, or 7,500 - 9,500. In some embodiments, the SNP loci are present on 1 pair or more, 2 pairs or more, 3 pairs or more, 4 pairs or more, 5 pairs or more, 6 pairs or more, 7 pairs or more, 8 pairs or more, 9 pairs or more, 10 pairs or more, 11 pairs or more, 12 pairs or more, 13 pairs or more, 14 pairs or more, 15 pairs or more, 16 pairs or more, 17 pairs or more, 18 pairs or more, 19 pairs or more, 20 pairs or more, 21 pairs or more, or 22 pairs of human chromosomes. In some embodiments, the SNP loci are present on autosomes.In some embodiments, the SNP loci are present on human chromosome arms 1p, 2p, 3p, 4p, 5p, 6p, 7p, 8p, 9p, 10p, 11p, 12p, 16p, 17p, 18p, 19p, 20p, 21p, 22p, 1q, 2q, 3q, 4q, 5q, 6q, 7q, 8q, 9q, 10q, 11q, 12q, 13q, 14q, 15q, 16q, 17q, 18q, 19q, 20q, 21q, and / or 22q. In some embodiments, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of the intervals between the SNP loci are 0.01 to 3 Mb, 0.02 to 2 Mb, 0.03 to 1 Mb, 0.06 to 1 Mb, 0.1 to 1 Mb, 0.1 to 0.5 Mb, or 0.06 to 0.6 Mb in length. In some embodiments, the average length of the intervals between the SNP loci is 0.01 to 3 Mb, 0.02 to 2 Mb, 0.03 to 1 Mb, 0.06 to 1 Mb, 0.1 to 1 Mb, 0.06 to 0.6 Mb, 0.1 to 0.5 Mb, or 0.2 to 0.4 Mb.

[0007] In some embodiments, the chromosomal aberration is loss of heterozygosity (LOH). In some embodiments, the HRD score is the LOH score. In some embodiments, the LOH score is the ratio of the number of non-homozygous SNP loci having a chromosomal aberration to the number of non-homozygous SNP loci. In some embodiments, the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci. In some embodiments, the non-homozygous SNP loci include heterozygous SNP loci and bi L OH SNP loci. In some embodiments, the heterozygous SNP loci are identified from the SNP loci.

[0008] In some embodiments, the LOH score is adjusted by excluding unbalanced chromosomal arms. In some embodiments, the LOH score is the ratio of the number of LOH SNP loci on non-unbalanced chromosomal arms to the number of heterozygous SNP loci on non-unbalanced chromosomal arms. In some embodiments, unbalanced chromosomal arms are characterized by a predetermined ratio of the number of LOH SNP loci to the number of heterozygous SNP loci on the chromosomal arm, where the predetermined ratio is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.

[0009] In some embodiments, the ratio of heterozygous SNP loci with LOH to characterize unbalanced chromosomal arms is adjusted based on the value of the tumor purity of the sample. In some embodiments, the ratio of heterozygous SNP loci with LOH to identify unbalanced chromosomal arms is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100%. In some embodiments, the value of the tumor purity is 30% - 95% or 30% - 70%. In some embodiments, the value of the tumor purity is 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0010] In some embodiments, the HRD status is identified as positive or negative. In some embodiments, the cut-off value of the LOH score for identifying the HRD status is 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.6.

[0011] In a general aspect, the present disclosure (1) sequencing SNP loci of a sample obtained from a subject, wherein there is an interval between each two adjacent SNP loci, and 50% or more of the intervals have a length of 0.01 - 1 Mb, (2) calculating the ratio of the number of LOH SNP loci to the number of heterozygous SNP loci, and (3)Identifying the HRD state Relates to a method for evaluating the HRD state of a subject, including this.

[0012] In a general aspect, the present invention (1)Sequencing at least one HRR-related gene of a sample obtained from a subject (2)Determining whether any of the HRR-related genes have changes (3)Identifying the HRD state of the subject Relates to a method for evaluating the HRD state of a subject, including this.

[0013] In some embodiments, the HRD state is identified as positive when at least one of the genes has a change. In some embodiments, when none of the genes have a change, the HRD state is identified as negative.

[0014] In some embodiments, the change is selected from the group consisting of single nucleotide variants (SNVs), insertions, deletions, amplifications, gene fusions, and rearrangements. In some embodiments, the change is selected from the group consisting of SNVs, small insertions and deletions (INDELs), large-scale genomic rearrangements (LGRs), and copy number polymorphisms (CNVs). In some embodiments, the change is a germline change or a somatic change.

[0015] In a general aspect, the present invention (1)Sequencing a gene including BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof determining whether any of the BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, and RAD54L genes have a change, (3)identifying the HRD status relates to a method for assessing the HRD status of a subject, comprising the above.

[0016] In some embodiments, the method further comprises identifying treatment based on the HRD status of the subject and / or administering a therapeutically effective amount of treatment to the subject.

[0017] In some embodiments, the treatment comprises administering a drug, including but not limited to DNA damaging agents, anthracyclines, topoisomerase I inhibitors, radiation, and / or PARP inhibitors, or any combination thereof. In some embodiments, the PARP inhibitors include but are not limited to olaparib, niraparib, rucaparib, and talazoparib.

[0018] In some embodiments, the method for assessing the HRD status of a sample is performed on a next-generation sequencing (NGS) computing platform. In some embodiments, the sample is sequenced by an NGS assay. In some embodiments, NGS systems used in the NGS assay include, but are not limited to, the MiSeq sequencer, HiSeq sequencer, MiniSeq sequencer, iSeq sequencer, NextSeq sequencer, and NovaSeq sequencer manufactured by Illumina, Inc., the Ion Personal Genome Machine (PGM), Ion Proton, Ion S5 series, and Ion GeneStudio S5 series manufactured by Life Technologies, Inc., the BGlseq series, DNBseq series, and MGIseq series manufactured by BGI, and the MinlON / PromethlON sequencer manufactured by Oxford Nanopore Technologies.

[0019] In some embodiments, the sequencing reads are produced from nucleic acids amplified from the original sample or nucleic acids captured by baits. In some embodiments, the sequencing reads are produced from sequencers that require the addition of adapter sequences. In some embodiments, the sequencing reads are produced from methods including, but not limited to, hybrid capture, primer extension target enrichment, molecular inversion probe-based methods, or multiplex target-specific PCR.

[0020] In some embodiments, the sample is derived from a cell line, biopsy, primary tissue, frozen tissue, formalin-fixed paraffin-embedded (FFPE), liquid biopsy, blood, serum, plasma, buffy coat, body fluid, visceral fluid, ascites, puncture, cerebrospinal fluid, saliva, urine, tears, semen, vaginal fluid, aspirate, wash fluid, buccal swab, peripheral blood mononuclear cells (PBMC), circulating tumor cells (CTC), cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), DNA, nucleic acid, purified nucleic acid, or purified DNA.

[0021] In some embodiments, the sample is derived from a human subject. In some embodiments, the sample is a clinical sample. In some embodiments, the sample is derived from a diseased patient. In some embodiments, the sample is derived from a patient having cancer, a solid tumor, or a hematological malignancy. In some embodiments, the sample is derived from a patient having ovarian cancer, prostate cancer, breast cancer, or pancreatic cancer. In some embodiments, the sample is derived from a patient having brain cancer, breast cancer, colon cancer, endocrine gland cancer, esophageal cancer, female genital cancer, head and neck cancer, hepatobiliary cancer, kidney cancer, lung cancer, mesenchymal cell neoplasm, prostate cancer, skin cancer, stomach cancer, pancreatic exocrine tumor, or urinary system cancer. In some embodiments, the sample is derived from a pregnant woman, a child, a young adult, an elderly person, or an adult. In some embodiments, the sample is a research sample.

[0022] In some embodiments, the method further includes outputting the HRD status to an electronic storage medium or a display.

[0023] In a general aspect, the present disclosure discloses a method for evaluating the HRD status of a subject implemented on an NGS computing platform, including the following (1) to (3). (1) Assaying genetic changes in a sample obtained from a subject, including the following (1a) and (1b), (1a) Sequencing a gene including the BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, RAD54L gene, or any combination thereof; Determining whether any of the following genes have a change: BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, and RAD54L gene. (2) Calculating the HRD score of a sample, including the following (2a) and (2b). (2a) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of the sample; (2b) Calculating the HRD score of chromosomal abnormalities. (3) Identifying the HRD status.

[0024] In a general aspect, the present invention relates to a method for evaluating the HRD status of a subject implemented on an NGS computing platform, including the following (1) to (3). (1) Assaying changes in a plurality of genes in a sample obtained from a subject, including the following (1a) and (1b). (1a) Sequencing at least one HRR-related gene; (1b) Determining whether any of the HRR-related genes have a change. (2) Calculating the LOH score in a sample, including the following (2a) and (2b). (2a) Sequencing a plurality of SNP loci of the sample, where there is an interval between each two adjacent SNP loci, and more than 50% of the intervals have a length of 0.01 to 1 Mb; (2b) Calculating the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci. (3) Identifying the HRD status.

[0025] In some embodiments, the HRD state is identified as positive if at least one of the genes has a change or if the score (i.e., the LOH score or the HRD score) exceeds a cut-off value.

[0026] In another general aspect, the present invention relates to a system for evaluating an HRD state, including a data storage device storing instructions for determining characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (3). (1) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of a sample, wherein there is an interval between each two adjacent SNP loci, and more than 50% of the intervals have a length of 0.01 to 1 Mb. (2) Calculating a loss of heterozygosity (LOH) score, wherein the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and (3) Identifying the HRD state.

[0027] In another general aspect, the present invention relates to a system for evaluating an HRD state, including a data storage device storing instructions for determining characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (3). (1) Sequencing a gene including the BRCA1 gene, the BRCA2 gene, the ARID1A gene, the ATM gene, the ATR gene, the ATRX gene, the BARD1 gene, the BRIP1 gene, the CDK12 gene, the CHEK1 gene, the CHEK2 gene, the FANCA gene, the FANCL gene, the FANCM gene, the HDAC2 gene, the NBN gene, the PALB2 gene, the PPP2R2A gene, the PTEN gene, the RAD51 gene, the RAD51B gene, the RAD51C gene, the RAD51D gene, the RAD54L gene, or any combination thereof. Determining whether any of the following genes has a change: BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, and RAD54L gene. (3) Identifying the HRD status.

[0028] In another general aspect, the present invention relates to a system for evaluating the HRD status, including a data storage device storing instructions for determining the characteristics of the HRD status, and a processor configured to execute the instructions to perform a method including the following (1) to (3). (1) Assaying changes in a plurality of genes in a sample, including the following (1a) and (1b). (1a) Sequencing a gene including the BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, RAD54L gene, or any combination thereof; Determining whether any of the following genes have changes: BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, and RAD54L gene. (2) Calculating the HRD score in a sample, including the following (2a) and (2b). (2a) Sequencing single nucleotide polymorphism (SNP) loci in the sample; (2b) Calculating the HRD score for chromosomal abnormalities. (3) Identifying the HRD status.

[0029] In another general aspect, the present invention relates to a system for evaluating the HRD status, including a data storage device storing instructions for determining the characteristics of the HRD status, and a processor configured to execute the instructions to perform a method including the following (1) and (2). (1) Assaying changes in a plurality of genes in a sample, including the following (1a) and (1b). (1a) Sequencing at least one HRR-related gene; and (1b) Determining whether any of the HRR-related genes have changes. (2) Calculating the LOH score of the sample, including the following (2a) and (2b). (2a) Sequencing a plurality of SNP loci of the sample, where there is an interval between each two adjacent SNP loci, and more than 50% of the intervals have a length of 0.01 - 1 Mb; (2b) Calculating the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci. (3) Identifying the HRD status.

[0030] In another general aspect, the present invention relates to a kit for evaluating the HRD state of a sample, comprising the following (1) to (3). (1) A set of oligonucleotides targeting a plurality of SNP loci (2) A set of oligonucleotides targeting a plurality of HRR-related genes, and (3) A computer program comprising instructions for performing a method for determining the HRD state.

[0031] In another general aspect, the present invention relates to a kit for evaluating the HRD state of a sample, comprising the following (1) and (2). (1) A reagent, comprising a set of oligonucleotides targeting a plurality of SNP loci, wherein an interval exists between each two adjacent SNP loci, and more than 50% of the intervals have a length of 0.01 to 1 Mb, the reagent (2) A computer program, instructions for calculating an LOH score, wherein the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci; and instructions for specifying the HRD state The computer program comprising.

[0032] In another general aspect, the present invention relates to a kit for evaluating the HRD state of a sample, comprising the following (1) and (2). (1) A reagent, A set of oligonucleotides targeting genes including the BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, RAD54L gene, or any combination thereof, reagent, (2) A computer program comprising: Instructions for determining whether any of the BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, and RAD54L gene has a change; and Instructions for identifying the HRD status A computer program comprising the above.

[0033] In another general aspect, the present invention relates to a kit for evaluating the HRD status of a sample, comprising the following (1) and (2). (1) A reagent comprising: A set of oligonucleotides targeting a plurality of SNP loci, wherein there is an interval between each two adjacent SNP loci, and more than 50% of the intervals have a length of 0.01 - 1 Mb; and, A set of oligonucleotides targeting at least one HRR-related gene A reagent comprising the above. (2) A computer program comprising: Instructions for calculating the LOH score, where the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci; and Identifying the HRD status; Instructions for determining whether any of the HRR-related genes have changes; and Instructions for identifying the HRD status A computer program comprising the above.

[0034] In another general aspect, the present invention relates to a kit for evaluating the HRD status of a sample, comprising the following (1) and (2). (1) A reagent comprising A set of oligonucleotides targeting a plurality of SNP loci of the sample; and A set of oligonucleotides targeting a gene comprising the BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, RAD54L gene, or any combination thereof A reagent, (2) A computer program comprising Instructions for calculating the HRD score of chromosomal abnormalities; Instructions for determining whether any of the BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, and RAD54L gene have changes; and Instruction for specifying the HRD state A computer program including

[0035] In some embodiments, the computer program further includes instructions for specifying treatment based on the HRD state of the subject.

Brief Description of the Drawings

[0036]

Figure 1A

Figure 1B

Figure 1C

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Modes for Carrying Out the Invention

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0038] As used herein, the term "HRR-related gene" refers to an HRR gene, or a regulator or modifier thereof. Changes in HRR-related genes can cause the presence of HRD. In some embodiments, the HRR-related gene is selected from the group consisting of BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, ABL1 gene, BAP1 gene, BARD1 gene, BLM gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, ERCC1 gene, ERCC3 gene, ERCC4 gene, FANCA gene, FANCC gene, FANCD2 gene, FANCE gene, FANCF gene, FANCG gene, FANCL gene, LIG3 gene, MRE11 gene, MSH2 gene, MSH6 gene, MLH1 gene, NBN gene, PALB2 gene, PTEN gene, PARP1 gene, POLB gene, RAD50 gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, RAD52 gene, RAD54L gene, UBE2A gene, XRCC2 gene, DNMT3A gene, IDH1 gene, IDH2 gene, STAG2 gene, and TP53 gene. In some embodiments, the HRR-related gene is selected from the group consisting of BRCA1 gene, BRCA2 gene, ARID1A gene, ATM gene, ATR gene, ATRX gene, BARD1 gene, BRIP1 gene, CDK12 gene, CHEK1 gene, CHEK2 gene, FANCA gene, FANCL gene, FANCM gene, HDAC2 gene, NBN gene, PALB2 gene, PPP2R2A gene, PTEN gene, RAD51 gene, RAD51B gene, RAD51C gene, RAD51D gene, and RAD54L gene.

[0039] As used herein, "cutoff value" refers to a numerical value or other expression that is used to process between two or more classification states for a biological sample. In some embodiments of the present invention, the cutoff value is used to distinguish between a positive HRD state or a negative HRD state. If the HRD score exceeds the cutoff value, the HRD state is determined to be positive, or if the HRD score is less than the cutoff value, the HRD state is determined to be negative.

[0040] As used herein, "imbalanced chromosomal arm" means a loss or gain in the copy number of a chromosomal arm. In some embodiments, an imbalanced chromosomal arm refers to a chromosomal arm having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of the non-homozygous SNP loci with LOH.

[0041] As used herein, "tumor purity" is the proportion of cancer cells in a tumor sample. Tumor purity affects the accurate assessment of molecular and genomic features as assayed by NGS approaches. In some embodiments of the present disclosure, the sample has a tumor purity of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.

[0042] As used herein, "depth" refers to the number of sequencing reads per position. "Average depth" refers to the average number of reads across the entire sequencing region. Usually, the average depth affects the performance of the NGS assay. The higher the average depth, the lower the variability of the variant frequency. In some embodiments of the present disclosure, the average depth of the sample across the entire sequencing region is 200-fold or more, 300-fold or more, 400-fold or more, 500-fold or more, 600-fold or more, 700-fold or more, 800-fold or more, 900-fold or more, 1000-fold or more, 2000-fold or more, 3000-fold or more, 4000-fold or more, 5000-fold or more, 6000-fold or more, 8000-fold or more, 10000-fold or more, or 20000-fold or more.

[0043] As used herein, "coverage" refers to the depth at a given locus. "Coverage of target bases" refers to the proportion of the sequencing region that is sequenced at a depth exceeding a predetermined value. The coverage of target bases needs to specify the depth at which it is evaluated. In some embodiments, the coverage of target bases at 100-fold is 85%. That is, 85% of the target sequencing bases are covered by at least 100-fold sequencing read depth. In some embodiments, the coverage of target bases at 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 125-fold, 150-fold, 175-fold, 200-fold, 300-fold, 400-fold, 500-fold, 750-fold, 1000-fold exceeds 70%, 75%, 80%, 85%, 90%, or 95%.

[0044] As used herein, "subject" or "human subject" refers to a person with a formally diagnosed disease, a person without a formally recognized disease, a person receiving medical attention, a person at risk of developing a disease, and the like.

[0045] As used herein, "treating", "treatment", and "treatment thereof" include therapeutic treatments, prophylactic treatments, and applications that reduce the risk of a subject developing a disease or other risk factor. Treatment includes embodiments that do not require complete cure of the disease and that reduce symptoms or underlying risk factors.

[0046] As used herein, "therapeutically effective amount" means the amount of a therapeutically active molecule required to induce a desired biological or clinical effect. In a preferred embodiment of the present disclosure, "therapeutically effective amount" is the amount of a drug required to treat an HRD-positive cancer patient.

[0047] The present disclosure is further illustrated by the following examples, which are provided for purposes of demonstration and not limitation.

Examples

[0048] Example 1: Stability Test of Algorithms for LOH Scoring This test was designed to evaluate the stability of LOH scores derived from different algorithms.

[0049] In-silico downsampling was applied to 260K, 150K, 100K, 50K, 40K, 30K, 20K, 10K, 9K, 8K, 7K, 6K, 5K, 4K, 3K, 2K, and 1K SNP loci randomly selected from Affymetrix GeneChip Human Mapping 250K Nspl array data (Wang, Birkbak et al., 2012) published on GEO with GEO accession number: GSE39130. The inventors first assigned chromosomal arm information to each SNP locus in this array data and defined allele frequency ranges for homozygous, heterozygous, and loss of heterozygosity SNP loci. In-silico downsampling was performed by stratified sampling at the chromosomal arm level to obtain a specified number of SNPs. The inventors sampled the SNP loci of each chromosomal arm independently to ensure that the number of SNP loci of each chromosomal arm in the downsampled set was proportional to the original dataset. To evaluate the variation in LOH scores of different algorithms at different numbers of each SNP locus, 100 bootstrap sample sizes were generated. Equation 1 calculated the LOH score by considering the number of LOH SNPs and the ratio of the number of loss of heterozygosity SNPs to the number of non-homozygous SNPs. In contrast, Equation 2 determined the LOH score by considering the total length of LOH SNPs and the ratio of the total length of the loss of heterozygosity SNP region to the genome size. The analysis was performed using R (version 4.0.0).

[0050] [Number]

[0051] [Number]

[0052] Figures 1A to 1D show the LOH scoring results of two types of tumor samples (GSM956523 and GSM956527) and two types of normal samples (GSM956582 and GSM956597) using two different algorithms. The calculation results using Equation 1 show the median of the stable LOH scores at different numbers of SNP loci when the LOH score derived from Equation 2 is lower as the number of SNP loci is smaller.

[0053] Example 2: Verification of the LOH Scoring Algorithm In this test, an algorithm is selected that estimates the LOH score with a significant difference between the tumor group and the normal group at different numbers of SNP loci.

[0054] All samples used in the test were analyzed in this test (Wang, Birkbak et al., 2012). Tumor samples with BRCA2 LOH were clustered into the high genomic instability group (GI-H), and in contrast, the low instability group (GI-L) consisted of tumor samples without BRCA2 LOH. Since cells with BRCA2 LOH showed genomic instability and high sensitivity to DNA-damaging agents, the GI-H group in this test may potentially represent the drug-sensitive group, and the GI-L group may potentially represent the drug-resistant group. There were 12, 11, and 18 samples in the GI-H group, GI-L group, and normal group, respectively. The LOH scores of each sample were estimated by Equations 1 and 2 described in Example 1 with the numbers of SNP loci of 260K, 50K, 10K, 7K, 5K, 3K, 2K, and 1K. The Wilcoxon signed-rank test was applied to estimate the p-values of the LOH scores among the GI-H, GI-L, and normal samples.

[0055] The inventors found a significant difference in the LOH scores between the two tumor groups, GI-H and GI-L, using Equation 1 for all different numbers of SNP loci (p-value < 0.05). However, by using Equation 2, the LOH scores were significantly different between the two tumor groups only when the number of SNP loci was 7K or more.

[0056] Example 3: LOH Scoring of Samples with Different Tumor Purity Levels, Considering or Not Considering Chromosome Arm Imbalance Factors This test is for evaluating the influence of chromosomal arm imbalance when calculating the LOH score.

[0057] Cancer cell line samples (NCL-H1395) with copy number changes were mixed with their matched normal samples to mimic different tumor purity levels. The experimental procedures included DNA extraction, library construction, and NGS sequencing, which followed Example 5. The LOH scores of the mixed samples were estimated by three different algorithms at different tumor purity levels. The first algorithm calculated the LOH score without considering the influence of chromosomal arm imbalance (Equation 1). The second and third algorithms considered the chromosomal arm imbalance factor by excluding SNPs located on imbalanced chromosomal arms (Equation 3). Imbalanced chromosomal arms were characterized by the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci on a chromosomal arm. The ratio in this example was 85%. The third algorithm further adjusted the ratio of non-homozygous SNP loci with LOH to characterize imbalanced chromosomal arms based on different tumor purity levels.

[0058]

Number

[0059] Figure 3 shows the LOH scoring results using three different algorithms at different tumor purity levels. The LOH score calculated by the first algorithm increased rapidly with the increase in tumor purity. In contrast, the LOH scores calculated by the second and third algorithms were stable when the tumor purity exceeded 30%.

[0060]

Table 1

[0061] Example 4: Determination of the HRD status of cancer samples An amplicon-based NGS panel was designed that targets the coding regions of Panel A, which includes ARID1A, ATM, ATR, ATRX, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, and RAD54L, and approximately 9000 SNP loci across the human genome. The average length of the intervals between SNP loci was approximately 0.3 Mb (Figure 4).

[0062] FFPE samples and PBMCs from cancer patients were collected and assayed using the NGS panel. Genomic DNA was extracted using the RecoverAll™ Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific). NGS libraries were constructed according to the user guide of the CleanPlex NGS panel (Paragon Genomics, USA). Briefly, 60 ng of DNA was amplified by multiplex PCR reaction using primers targeting the regions designed above. After purification using magnetic beads, CP digestion, and secondary purification, a second PCR reaction was performed using the i5 index primer and i7 index primer for Illumina according to the user guide. After additional purification, the samples were subjected to capillary electrophoresis (FragmentAnalyzer, AATI). Samples that passed library quality control (QC) were combined for sequencing on the NextSeq550 (Illumina, USA) according to the manufacturer's system guide and the Illumina NextSeq System Denature and Dilute Libraries Guide.

[0063] The raw reads generated by the sequencer were mapped to the hgl9 reference genome using BWA (version 0.7.17). SNVs and INDELs were identified using Pisces (version 5.2.5.20). All variants were annotated using VEP (Variant Effect Predictor) (version 88) with databases from Clinvar (version 20180729) and Genome Aggregation database r2.1.1. Coverage analysis was performed by Bedtools and Samtools to calculate the depth at each target base and target amplicon in the panel.

[0064] Sample QC was performed to confirm that the average sequencing depth of each sample reached 1000-fold.

[0065] To determine LGR and CNV, amplicons with read counts in the lowest 1 percentile and highest 0.5 percentile of all detectable amplicons, and amplicons with a coefficient of variation of 0.35 or greater were removed. The remaining amplicons were normalized to correct for pooling design bias. ONCOCNV (a method established to calculate copy number aberrations in sequencing data of amplicons by Boeva et al. (2014)) was applied to the normalization of the total number of amplicons, amplicon GC content, amplicon length, and technology-related bias, and subsequently, the samples were segmented with a gene recognition model. The observed copy number of each gene and exon was calculated using ONCOCNV. The tumour purity of each FFPE sample was calculated using Aberration Detection in Tumour Exome (ADTEx) software (Amarasinghe et al., 2014). The adjusted copy number of each gene was calculated by adjusting the tumour purity in the FFPE sample.

[0066] SNPs were determined as LOH or heterozygosity according to their variant allele frequencies. The LOH score of a sample was calculated by taking the proportion of SNPs with an LOH state according to Equation 3.

[0067] When an imbalance in the chromosomal arm is detected, all SNPs on the chromosomal arm are excluded from the analysis. Here, the chromosomal arm imbalance is detected as either an increase or a loss in the copy number relative to the entire chromosomal arm.

[0068] List the LOH scores of the tested samples in Table 2.

[0069]

Table 2

[0070] Example 5: LOH Distribution of Samples in Different Genotype Groups This test was designed to evaluate the LOH score distribution of samples with different genotypes in Panel A, including ARID1A, ATM, ATR, ATRX, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, and RAD54L.

[0071] By the assay of Example 4, a total of 92 ovarian cancer samples and 4 normal samples were sequenced, and the LOH score of each sample was calculated by Equation 3. Samples with pathogenic mutations or putative pathogenic mutations in the genes of Panel A were grouped into Panel A gene (harmful). In contrast, other samples without pathogenic mutations or putative pathogenic mutations in all genes of Panel A were regarded as Panel A gene (WT). The distribution of the sample LOH scores in each group is shown in Figure 5.

[0072] The distribution of the LOH scores of the samples in different groups shows that the group of Panel A gene (harmful) has a higher LOH score than the other groups. Exemplary embodiments of the present invention are described below. <1> (1) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of a sample obtained from a subject, wherein there is an interval between each two adjacent SNP loci, and at least 50% of the intervals have a length of 0.01 to 1 Mb; (2) Identifying the number of loss of heterozygosity (LOH) SNP loci and the number of non-homozygous SNP loci based on the sequencing results; (3) Calculating an LOH score, where the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci; (4) Identifying a homologous recombination repair deficiency (HRD) state based on the LOH score A method for evaluating a homologous recombination repair deficiency (HRD) state in a subject, comprising: <2> The method according to <1>, wherein the number of the plurality of SNP loci is 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, 8000 or more, 8500 or more, 9000 or more, 9500 or more, 10000 or more, 20000 or more, 30000 or more, 40000 or more, 50000 or more, 60000 or more, 70000 or more, 80000 or more, 90000 or more, 100000 or more, 110000 or more, 120000 or more, 130000 or more, 140000 or more, 150000 or more, 160000 or more, 170000 or more, 180000 or more, 190000 or more, 200000 or more, 210000 or more, 220000 or more, 230000 or more, 240000 or more, 250000 or more, 260000 or more, 270000 or more, 280000 or more, 290000 or more, or 300000 or more. <3> The method according to <1>, wherein the number of the plurality of SNP loci is 2500 to 250000. <4> The method according to <1>, wherein the number of the plurality of SNP loci is 3000 to 60000. <5> The method according to <1>, wherein the number of the plurality of SNP loci is 6000 to 11000. <6> The method according to <1>, wherein the plurality of SNP loci are present on two or more pairs of chromosomes. <7> The method according to <1>, wherein the plurality of SNP loci are present on 22 pairs of chromosomes. <8> The method according to <1>, wherein the average length of the intervals is 0.01 to 3 Mb, 0.02 to 2 Mb, 0.03 to 1 Mb, 0.06 to 1 Mb, 0.1 to 1 Mb, 0.06 to 0.6 Mb, 0.1 to 0.5 Mb, or 0.2 to 0.4 Mb. <9> The method according to <1>, wherein step (3) further comprises adjusting the LOH score by excluding unbalanced chromosome arms. <10> The method according to <9>, wherein the LOH score is the ratio of the number of LOH SNP loci in non-unbalanced chromosome arms to the number of non-homozygous SNP loci in non-unbalanced chromosome arms. <11> The method according to <9>, wherein the unbalanced chromosome arms are characterized by a predetermined ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci in the chromosome arms, and the predetermined ratio is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100%. <12> The method according to <11>, wherein the predetermined ratio is further adjusted based on the tumor purity of the sample. <13> The method according to <12>, wherein the tumor purity is 30% to 95%. <14> The method according to <12>, wherein the tumor purity is 30% to 70%. <15> The method according to <1>, wherein the HRD status is identified as positive or negative. <16> The method according to <1>, wherein the cut-off value of the LOH score for identifying the HRD status is 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.6. <17> (1) Sequencing a gene including BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof in a sample obtained from a subject; (2) Determining whether the gene has a change; and (3) Identifying the HRD status based on the determination result A method for evaluating the HRD status of a subject, comprising: <18> The method according to <17>, wherein when the gene has a change, the HRD status is positive. <19> The method according to <17>, wherein when none of the genes have a change, the HRD status is negative. <20> The method according to <17>, wherein the change is a germline change or a somatic change. <21> The method according to <17>, wherein the change is selected from the group consisting of single nucleotide variants (SNVs), insertions, deletions, amplifications, gene fusions, and rearrangements. <22> The method according to <17>, wherein the change is selected from the group consisting of SNVs, small insertions and deletions (INDELs), large-scale genomic rearrangements (LGRs), and copy number variations (CNVs). <23> (1) Assaying for a change in a gene in a sample obtained from a subject, comprising the following (1a) and (1b): (1a) Sequencing a gene in the sample that comprises BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof; (1b) Determining whether the gene has a change (2) Calculating an HRD score for the sample, comprising the following (2a) and (2b): (2a) Sequencing a plurality of single nucleotide polymorphism (SNP) loci in the sample; (2b) Calculating an HRD score for chromosomal abnormalities (3) Identifying an HRD status based on the result of step (1b), step (2b), or both A method for evaluating the HRD status of a subject, comprising the above. <24> (1) Assaying for a change in a gene in a sample obtained from a subject, comprising the following (1a) and (1b): (1a) Sequencing an HRR-related gene; and (1b) Determining whether the HRR-related gene has a change (2) Calculating an LOH score for the sample, comprising the following (2a) and (2b): (2a) Sequencing a plurality of SNP loci in the sample, wherein there is an interval between each two adjacent SNP loci, and at least 50% of the intervals have a length of 0.01 to 1 Mb; and (2b) Calculating the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci (3) Identifying an HRD status based on the result of step (1b), step (2b), or both A method for evaluating the HRD status of a subject, comprising the above. <25> The method according to <23> or <24>, wherein the HRD status is positive when the gene in step (1) has a change or when the score in step (2) exceeds a cut-off value. <26> The method according to <1>, <17>, <23>, or <24>, further comprising the step of identifying a treatment based on the HRD state of the subject. <27> The method according to <26>, further comprising the step of administering to the subject a therapeutically effective amount of the treatment. <28> The method according to <26>, wherein the treatment is selected from the group consisting of DNA damaging agents, anthracyclines, topoisomerase I inhibitors, radiation, PARP inhibitors, and any combination thereof. <29> The method according to <28>, wherein the PARP inhibitor is selected from the group consisting of olaparib, niraparib, rucaparib, and talazoparib. <30> The method according to <1>, <17>, <23>, or <24>, wherein the method for assessing the HRD state of the sample is performed on a next-generation sequencing (NGS) computing platform. <31> The method according to <1>, <17>, <23>, or <24>, wherein the sample is sequenced by an NGS assay. <32> The method according to <1>, <17>, <23>, or <24>, wherein the sample is derived from a cell line, biopsy, primary tissue, frozen tissue, formalin-fixed paraffin-embedded (FFPE), liquid biopsy, blood, serum, plasma, buffy coat, body fluid, visceral fluid, ascites, puncture, cerebrospinal fluid, saliva, urine, tears, semen, vaginal fluid, aspirate, wash, buccal swab, circulating tumor cell (CTC), cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), DNA, RNA, nucleic acid, purified nucleic acid, purified DNA, or purified RNA. <33> The method according to <1>, <17>, <23>, or <24>, wherein the subject is a human. <34> The method according to <1>, <17>, <23>, or <24>, wherein the subject is a cancer patient. <35> The method according to <1>, <17>, <23>, or <24>, wherein the tumor purity of the sample is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%. <36> The method according to <1>, <17>, <23>, or <24>, further comprising the step of outputting the HRD state to an electronic storage medium or a display. <37> A system for evaluating the HRD state, including a data storage device storing instructions for determining the characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (4). (1) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of a sample obtained from a subject, wherein there is an interval between each two adjacent SNP loci, and more than 50% of the intervals have a length of 0.01 to 1 Mb. (2) Identifying the number of loss of heterozygosity (LOH) SNP loci and the number of non-homozygous SNP loci based on the result of the sequencing. (3) Calculating a loss of heterozygosity (LOH) score, wherein the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and (4) Identifying the HRD state based on the LOH score. <38> A system for evaluating the HRD state, including a data storage device storing instructions for determining the characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (3). (1) Sequencing a gene including BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof in a sample obtained from a subject. (2) Determining whether the gene has a change, and (3) Identifying the HRD state based on the determination result. <39> A system for evaluating the HRD state, including a data storage device storing instructions for determining the characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (3). (1) Assaying for changes in genes in a sample obtained from a subject, including the following (1a) and (1b). (1a) sequencing a gene of the sample, the gene comprising BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof; and, (1b) determining whether the gene has a change (2) calculating an HRD score of the sample, comprising the following (2a) and (2b), (2a) sequencing a plurality of single nucleotide polymorphism (SNP) loci of the human sample; and (2b) calculating the HRD score of chromosomal abnormalities, and (3) identifying an HRD status based on the result of step (1b), step (2b), or both. <40> A system for evaluating an HRD status, comprising a data storage device storing instructions for determining characteristics of an HRD status, and a processor configured to execute the instructions to perform a method comprising the following (1) to (3) (1) assaying for gene changes in a sample obtained from a subject, comprising the following (1a) and (1b), (1a) sequencing an HRR-related gene of the sample; and (1b) determining whether the HRR-related gene has a change, (2) calculating an LOH score of the sample, comprising the following (2a) and (2b), (2a) sequencing a plurality of SNP loci of the sample, wherein there is an interval between each adjacent two SNP loci, and more than 50% of the intervals have a length of 0.01 to 1 Mb; and (2b) calculating the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and (3) identifying the HRD status based on the result of step (1b), step (2b), or both. <41> The system according to <37> to <40>, further comprising the step of identifying a treatment based on the HRD status of the subject. <42> The system according to <41>, further comprising the step of administering a therapeutically effective amount of treatment to the subject. <43> A reagent, A reagent comprising a set of oligonucleotides targeting a plurality of SNP loci, wherein there is an interval between each adjacent two SNP loci, and 50% or more of the intervals have a length of 0.01 to 1 Mb, and A computer program, Instructions for calculating an LOH score, where the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and Instructions for identifying the HRD status A computer program comprising A kit for evaluating the HRD status of a subject, comprising <44> A reagent, A set of oligonucleotides targeting genes including BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof in a sample obtained from a subject, and A computer program, Instructions for determining whether the gene has a change, and Instructions for identifying the HRD status A computer program comprising A kit for evaluating the HRD status of a subject, comprising <45> A reagent, A set of oligonucleotides for targeting a plurality of SNP loci, wherein there is an interval between each adjacent two SNP loci, and 50% or more of the intervals have a length of 0.01 to 1 Mb; and A set of oligonucleotides for targeting HRR-related genes A reagent, comprising A computer program, Instructions for calculating an LOH score, where the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, Instructions for determining whether the HRR-related gene has a change, and Instructions for identifying the HRD status A computer program comprising A kit for evaluating the HRD status of a subject, comprising <46> A reagent, A set of oligonucleotides for targeting a plurality of SNP loci in a sample obtained from a subject; and A set of oligonucleotides for targeting genes comprising BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof A reagent comprising A computer program comprising Instructions for calculating the HRD score of chromosomal abnormalities; Instructions for determining whether the gene has a change; and Instructions for identifying the HRD status A computer program A kit for evaluating the HRD status of a subject comprising <47> The kit according to <43>, wherein when the LOH score exceeds a cut-off value, the HRD status is positive. <48> The kit according to <44>, wherein when the gene has a change, the HRD status is positive. <49> The kit according to <45>, wherein when the LOH score exceeds a cut-off value or the HRR-related gene has a change, the HRD status is positive. <50> The kit according to <46>, wherein when the HRD score exceeds a cut-off value or the gene has a change, the HRD status is positive. <51> The kit according to <43> to <46>, wherein the computer program further comprises instructions for identifying a treatment based on the HRD status of the subject.

Claims

1. (1) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of a sample obtained from a subject, wherein an interval exists between each two adjacent SNP loci, and at least 50% of the intervals have a length of 0.01 to 1 Mb; (2) Identifying the number of loss of heterozygosity (LOH) SNP loci and the number of non-homozygous SNP loci based on the sequencing results; (3) Calculating an LOH score, where the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and the non-homozygous SNP loci include heterozygous SNP loci and LOH SNP loci; (4) Identifying a homologous recombination repair deficiency (HRD) state based on the LOH score A method for evaluating a homologous recombination repair deficiency (HRD) state in a subject, comprising the above steps.

2. The method according to claim 1, wherein the number of the plurality of SNP loci is 1,000 or more, 1,500 or more, 2,000 or more, 2,500 or more, 3,000 or more, 3,500 or more, 4,000 or more, 4,500 or more, 5,000 or more, 5,500 or more, 6,000 or more, 6,500 or more, 7,000 or more, 7,500 or more, 8,000 or more, 8,500 or more, 9,000 or more, 9,500 or more, 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, 80,000 or more, 90,000 or more, 100,000 or more, 110,000 or more, 120,000 or more, 130,000 or more, 140,000 or more, 150,000 or more, 160,000 or more, 170,000 or more, 180,000 or more, 190,000 or more, 200,000 or more, 210,000 or more, 220,000 or more, 230,000 or more, 240,000 or more, 250,000 or more, 260,000 or more, 270,000 or more, 280,000 or more, 290,000 or more, or 300,000 or more.

3. The method according to claim 1, wherein the number of the plurality of SNP loci is 2,500 to 250,000.

4. The method according to claim 1, wherein the number of the plurality of SNP loci is 3,000 to 60,000.

5. The method according to claim 1, wherein the number of the plurality of SNP loci is 6,000 to 11,000.

6. The method according to claim 1, wherein the plurality of SNP loci are present on two or more pairs of chromosomes.

7. The method according to claim 1, wherein the plurality of SNP loci are present on 22 pairs of chromosomes.

8. The method according to claim 1, wherein the average length of the intervals is 0.01 to 3 Mb, 0.02 to 2 Mb, 0.03 to 1 Mb, 0.06 to 1 Mb, 0.1 to 1 Mb, 0.06 to 0.6 Mb, 0.1 to 0.5 Mb, or 0.2 to 0.4 Mb.

9. The method according to claim 1, wherein step (3) further comprises adjusting the LOH score by excluding unbalanced chromosomal arms.

10. The method according to claim 9, wherein the LOH score is the ratio of the number of LOH SNP loci on chromosomal arms that are not unbalanced to the number of non-homozygous SNP loci on chromosomal arms that are not unbalanced.

11. The unbalanced chromosomal arms are characterized by a predetermined ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci on the chromosomal arms, and the predetermined ratio is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100%. The method according to claim 9.

12. The method according to claim 11, wherein the predetermined ratio is further adjusted based on the tumor purity of the sample.

13. The method according to claim 12, wherein the tumor purity is 30% to 95%.

14. The method according to claim 12, wherein the tumor purity is 30% to 70%.

15. The method according to claim 1, wherein the HRD status is specified as positive or negative.

16. The method according to claim 1, wherein the cut-off value of the LOH score for specifying the HRD status is 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, or 0.

6.

17. (1) Assaying genetic changes in a sample obtained from a subject, comprising the following (1a) and (1b): (1a) Sequencing HRR-related genes; and (1b) Determining whether the HRR-related genes have changes (2) Calculating the LOH score of the sample, comprising the following (2a) and (2b): (2a) Sequencing a plurality of SNP loci of the sample, wherein there is an interval between each two adjacent SNP loci, and 50% or more of the intervals have a length of 0.01 to 1 Mb; and (2b) Calculating the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, wherein the non-homozygous SNP loci include heterozygous SNP loci and LOH SNP loci (3) Specifying the HRD status based on the result of step (1b), step (2b), or both A method for evaluating the HRD status of a subject, comprising the above steps.

18. The method according to claim 17, wherein when the gene in step (1) has changes, or when the LOH score in step (2) exceeds the cut-off value, the HRD status is positive.

19. The method according to claim 1 or claim 17, further comprising the step of specifying treatment based on the HRD status of the subject.

20. The method according to claim 19, wherein the treatment is selected from the group consisting of DNA damaging agents, anthracyclines, topoisomerase I inhibitors, radiation, PARP inhibitors, and any combination thereof.

21. The method according to claim 20, wherein the PARP inhibitor is selected from the group consisting of olaparib, niraparib, rucaparib, and talazoparib.

22. The method according to claim 1 or claim 17, wherein the method for evaluating the HRD status of a sample is performed on a next-generation sequencing (NGS) computing platform.

23. The method according to claim 1 or claim 17, wherein the sample is sequenced by an NGS assay.

24. The method according to claim 1 or claim 17, wherein the sample is derived from a cell line, biopsy, primary tissue, frozen tissue, formalin-fixed paraffin-embedded (FFPE), liquid biopsy, blood, serum, plasma, buffy coat, body fluid, visceral fluid, ascites, puncture, cerebrospinal fluid, saliva, urine, tears, semen, vaginal fluid, aspirate, wash fluid, buccal swab, circulating tumor cell (CTC), cell-free DNA (cfD NA), circulating tumor DNA (ctDNA), DNA, RNA, nucleic acid, purified nucleic acid, purified DNA , or purified RNA.

25. The method according to claim 1 or claim 17, wherein the subject is a human.

26. The method according to claim 1 or claim 17, wherein the subject is a cancer patient.

27. The method according to claim 1 or claim 17, wherein the tumor purity of the sample is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%.

28. The method according to claim 1 or claim 17, further comprising the step of outputting the HRD state to an electronic storage medium or a display.

29. A system for evaluating an HRD state, comprising a data storage device storing instructions for determining characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (4). (1) Sequencing a plurality of single nucleotide polymorphism (SNP) loci of a sample obtained from a subject, wherein there is an interval between each two adjacent SNP loci, and 50% or more of the intervals have a length of 0.01 to 1 Mb. (2) Identifying the number of loss of heterozygosity (LOH) SNP loci and the number of non-homozygous SNP loci based on the result of the sequencing. (3) Calculating a loss of heterozygosity (LOH) score, wherein the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and the non-homozygous SNP loci include heterozygous SNP loci and LOH SNP loci, and (4) Identifying the HRD state based on the LOH score.

30. A system for evaluating an HRD state, comprising a data storage device storing instructions for determining characteristics of the HRD state, and a processor configured to execute the instructions to perform a method including the following (1) to (3). (1) Assaying genetic changes in a sample obtained from a subject, including the following (1a) and (1b). (1a) sequencing the HRR-related gene of the sample; and (1b) determining whether the HRR-related gene has a change, (2) calculating the LOH score of the sample, including the following (2a) and (2b), (2a) sequencing a plurality of SNP loci of the sample, wherein there is an interval between each adjacent two SNP loci, and at least 50% of the intervals have a length of 0.01 to 1 Mb; and (2b) calculating the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, wherein the non-homozygous SNP loci include heterozygous SNP loci and LOH SNP loci, and (3) identifying the HRD state based on the result of step (1b), step (2b), or both.

31. The system according to claim 29 or claim 30, further comprising the step of identifying a treatment based on the HRD state of the subject.

32. The system according to claim 31, further comprising the step of performing a treatment in a therapeutically effective amount on the subject.

33. A reagent, comprising a set of oligonucleotides targeting a plurality of SNP loci, wherein there is an interval between each adjacent two SNP loci, and at least 50% of the intervals have a length of 0.01 to 1 Mb, and A computer program, instructions for calculating an LOH score, wherein the LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and the non-homozygous SNP loci include heterozygous SNP loci and LOH SNP loci, and instructions for identifying an HRD state A computer program comprising A kit for evaluating the HRD status of a subject, comprising

34. A reagent comprising A set of oligonucleotides for targeting a plurality of SNP loci, wherein there is an interval between each adjacent two SNP loci, and at least 50% of said intervals have a length of 0.01 to 1 Mb; and A set of oligonucleotides for targeting HRR-related genes A reagent comprising A computer program comprising Instructions for calculating an LOH score, wherein said LOH score is the ratio of the number of LOH SNP loci to the number of non-homozygous SNP loci, and said non-homozygous SNP loci include heterozygous SNP loci and LOH SNP loci, Instructions for determining whether said HRR-related gene has a change, and Instructions for identifying an HRD status A computer program comprising A kit for evaluating the HRD status of a subject, comprising

35. The kit according to claim 34, wherein said HRR-related gene comprises BRCA1, BRCA2, ARID1A, ATM, ATR, ATRX, BARD1, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCL, FANCM, HDAC2, NBN, PALB2, PPP2R2A, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, or any combination thereof.

36. The kit according to claim 33, wherein when said LOH score exceeds a cut-off value, said HRD status is positive.

37. The kit according to claim 34, wherein when said LOH score exceeds a cut-off value, or when said HRR-related gene has a change, said HRD status is positive.

38. The kit according to claim 33 or claim 34, wherein the computer program further includes instructions for specifying a treatment based on the HRD state of the subject.

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