Kit for detecting genes related to molecular typing, medication and genetic susceptibility of endometrial cancer and application of kit

Abstract

The invention relates to an endometrial cancer molecular typing, medication and genetic susceptibility related gene detection kit and application thereof. The detection kit comprises a probe group for targeted medication related genes, genetic susceptibility related genes, immunotherapy related genes and molecular typing related genes. On the basis of a next-generation sequencing technology, more than 100 genes highly related to development of endometrial cancer can be detected at a time by utilizing a hybrid capture method, and the genes comprise genes related to targeted medication, genes related to genetic susceptibility, genes related to immunotherapy and genes related to molecular typing. The probe is wide in coverage, high in sequencing depth and capable of detecting germline variation of all exon regions of related genes. The invention can report endometrial cancer targeted medication related variation and immunotherapy related variation, also can report all related diseases of genetic susceptibility related genes, and provides genetic modes corresponding to the diseases.

Description

Technical Field

[0001] This invention relates to a molecular subtyping, medication, and genetic susceptibility-related gene detection kit for endometrial cancer and its application, belonging to the field of multi-gene detection technology. Background Technology

[0002] Endometrial cancer (EC) is a type of epithelial malignant tumor that occurs in the uterine lining. It is one of the three most common malignant tumors of the female reproductive tract, accounting for approximately 20%-30% of gynecological malignancies, second only to cervical cancer in incidence. With economic development and changes in lifestyle, the incidence of endometrial cancer is showing an increasing trend year by year.

[0003] The occurrence and development of endometrial cancer are closely related to gene mutations, which not only affect molecular subtyping but also have significant implications for prognosis. The POLE gene encodes the catalytic subunit of DNA polymerase ε; endometrial cancers with pathogenic mutations in the POLE gene are classified as POLE mutants. These mutations are primarily concentrated in the exonuclease domain (EDM) of the POLE gene, impairing the proofreading function of the POLE protein and leading to genomic hypermutation. These tumors typically have a good prognosis. Mutations in mismatch repair genes (such as MLH1, MSH2, MSH6, and PMS2) result in DNA mismatch repair deficiency (dMMR), leading to microsatellite instability (MSI-H). dMMR / MSI-H endometrial cancer is also an important molecular subtype, accounting for approximately 20% of endometrial cancers. The prognosis of dMMR / MSI-H endometrial cancer is better than that of microsatellite stable (MSS) endometrial cancer, and these tumors respond well to immunotherapy. TP53 gene mutations are mainly found in serous carcinomas and high-grade endometrioid carcinomas, and are associated with a highly aggressive tumor phenotype. TP53 mutations often indicate a poor prognosis. Endometrial cancers lacking the above three characteristics are classified as nonspecific molecular markers (NSMP), and these tumors have an intermediate prognosis. Approximately 5%-10% of endometrial cancers are related to genetics. The most common is Lynch syndrome, an autosomal dominant genetic disorder in which patients carry germline mutations in mismatch repair genes (such as MLH1, MSH2, MSH6, PMS2, etc.), significantly increasing the risk of developing various cancers, including endometrial cancer and colorectal cancer. Therefore, gene testing plays a crucial role in the molecular subtyping, prognostic assessment, and genetic screening of endometrial cancer.

[0004] Precise genetic testing can screen patients who are more likely to respond to specific targeted therapies. Genetic testing can determine whether endometrial cancer patients have mismatch repair gene defects (dMMR) or microsatellite instability-high (MSI-H). For example, patients with dMMR / MSI-H endometrial cancer often respond well to immunotherapy. For endometrial cancer patients carrying HER-2 (ERBB2) gene amplification or overexpression, anti-HER-2 targeted drugs (such as trastuzumab) may have better treatment outcomes. For patients carrying NTRK gene fusions, tyrosine kinase inhibitors (entrectinib, larotrectinib, etc.) can provide significant remission.

[0005] Next-generation sequencing (NGS), also known as high-throughput sequencing, is a technology that can simultaneously detect mutations in multiple genes, offering broad coverage and high throughput. While it can comprehensively analyze the mutation types and sites of genes related to endometrial cancer, providing detailed and accurate genetic information for clinical diagnosis, treatment, molecular subtyping, prognosis, and genetic screening, the key challenge lies in the design of probes to capture mutation sites. Probes targeting each gene must not only be non-complementary but also free from overlap. Therefore, no probes have yet been published that can comprehensively cover gene mutations related to endometrial cancer. Summary of the Invention

[0006] The purpose of this invention is to provide a kit and application for detecting genes related to molecular subtyping, medication, and genetic susceptibility of endometrial cancer. Among them, a probe set is provided to detect genes related to targeted medication for endometrial cancer, which can be used to detect gene variations of endometrial cancer that have clear significance for medication, molecular subtyping, and genetic susceptibility.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a set of probes for detecting molecular subtyping, medication, and genes related to genetic susceptibility in endometrial cancer, characterized in that it comprises:

[0009] A probe set targeting genes related to molecular subtyping, medication, and genetic susceptibility of endometrial cancer, wherein the genes related to molecular subtyping, medication, and genetic susceptibility of endometrial cancer include genes related to targeted medication, genes related to genetic susceptibility, genes related to immunotherapy, and genes related to molecular subtyping;

[0010] The probe set includes probes with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.220. The genes captured by the probe set cover endometrial-targeted drugs, immunotherapies, molecular typing, and genes related to genetic susceptibility; these include AKT1, AKT2, ALK, APC, AR, ARAF, ARID1A, ARID1B, ATM, ATR, ATRX, AXIN1, BARD1, BCOR, BRAF, BRCA1, BRCA2, BRIP1, BTK, CCND1, CCNE1, CDK12, CDKN2A, CHEK1, CHEK2, CREBBP, CTNNB1, DDR2, DICER1, DROSHA, MLH1, MLH3, and MRE. 11. MSH2, MSH3, MSH6, MTAP, MTOR, MUTYH, NBN, NF1, NRAS, NRG1, NTHL1, NTRK1, NTRK2, NTRK3, PALB2, PARP1, PBRM1, PDGFRA, PIK3CA, PIK3 R1, PMS1, PMS2, POLD1, POLE, EGFR, EPCAM, ERBB2, ERBB3, ERBB4, ESR1, FANCA, FANCB, FANCD2, FANCL, FBXW7, FGFR1, FGFR2, FGFR3, GATA3, IDH1, IDH2, JAK1, KDM6A, KIF5B, KIT, KMT2C, KMT2D, KRAS, MDM2, MDM4, MET, PP2R1A, PPP2R1A, PTEN, RAD51B, RAD51C, RAD51D, RAD54L, RAF1, RB1, RET, RIT1, RNF43, ROS1, SDHA, SDHB, SDHC, SDHD, SMARCA4, SMARCB1, SPOP, TERT, TP53, TSC1, TSC2, VEGFA, VHL; among which, important genes related to targeted therapy include: B The probe set includes RAF, ERBB2, NTRK1, NTRK2, NTRK3, and RET; important genes related to genetic susceptibility include AKT1, BRCA1, BRCA2, EPCAM, MLH1, MSH2, MSH6, PIK3CA, PMS2, POLD1, POLE, PTEN, SDHA, SDHB, SDHC, and SDHD; important genes related to immunotherapy include EPCAM, MLH1, MSH2, MSH6, PMS2, POLD1, and POLE; and genes related to molecular typing include EPCAM, MLH1, MSH2, MSH6, PMS2, POLE, and TP53. This probe set covers the full coding sequence (CDS) of most key genes and hotspot regions of other genes.

[0011] This invention also provides indications of the sensitivity of detected variant sites to different target drugs and drug levels. Based on the 2017 Chinese Expert Consensus on Next-Generation Sequencing Detection in Clinical Molecular Pathology Laboratories and the 2017 AMP / ASCO / CAP Standards and Guidelines for the Interpretation and Reporting of Tumor Cell Variations, the interpretation and annotation of the clinical significance of tumor somatic cell variations are divided into the following four levels of evidence:

[0012] Category A: Targets approved for drug use by the U.S. Food and Drug Administration (FDA) or the National Medical Products Administration (NMPA); variants with clear diagnostic / therapeutic / prognostic significance as included in domestic and international treatment guidelines.

[0013] Category B: Variants that have not yet been included in treatment guidelines but have been incorporated into expert consensus in the field.

[0014] Category C: Genetic variants approved by the FDA or NMPA for predictable treatment outcomes in other cancers, or variant sites in ongoing clinical trials.

[0015] Grade D: Preclinical studies or a small number of case reports, without expert consensus.

[0016] Secondly, the present invention also provides a molecular subtyping, medication and genetic susceptibility-related gene detection kit for endometrial cancer, including the probe set as described above.

[0017] More preferably, the 5' end of the probe is labeled with biotin, and the detection kit also includes streptomycin affinity magnetic beads that can specifically bind to biotin.

[0018] The detection kit described above preferably further includes genomic DNA extraction reagents, DNA library construction reagents, and next-generation sequencing library construction reagents.

[0019] Furthermore, the genomic DNA extraction reagent can be the Tiangen Blood Genome Extraction Kit, the DNA library construction reagent can be the Twist Library Preparation EF Kit 1, 2.0 from Twist, the next-generation sequencing library capture reagent can be the liquid phase hybridization capture kit from Twist, and the sequencing instruments or reagents can be the Illumina high-throughput sequencing platform and human genome sequences.

[0020] Thirdly, the present invention provides a method for constructing a library of genes related to molecular subtyping, medication, and genetic susceptibility of endometrial cancer for non-diagnostic / therapeutic purposes, comprising the following steps:

[0021] (1) Genomic DNA was extracted from ex vivo tumor tissue of endometrial cancer. The DNA was first fragmented, end-repaired and dA tailed. The dA-tailed product was ligated to Twist universal adapter. The ligation adapter product was purified and PCR amplified. The PCR reaction product was purified to obtain a DNA library.

[0022] (2) After the purified DNA library is concentrated, it is hybridized with probes with nucleotide sequences as shown in SEQ ID NO.1~220 to capture and purify the library, and then PCR amplification is performed again. The amplified product is purified again to obtain the next-generation sequencing library; the tag adapters used for different template DNAs are different.

[0023] (3) Perform second-generation sequencing and data analysis on the second-generation sequencing library to obtain the variation of genes related to targeted drug use for endometrial cancer and the molecular subtyping and genetic susceptibility genes related to endometrial cancer.

[0024] As described above, preferably, in step (1), the PCR amplification reaction system uses Twist's UDI primers; the amplification program is as follows: pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; extension: 72℃ for 1min, 4℃ for incubation.

[0025] Furthermore, the UDI primer is a 10bp universal UDI connector.

[0026] The method described above, preferably, in step (2), amplification is performed using Twist's Amplification Primers, and the amplification procedure is as follows:

[0027] Pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; Extension: 72℃ for 1min, followed by 4℃ heat preservation.

[0028] The library of genes related to targeted therapy for endometrial cancer, obtained using the library construction method described above, is used to assist in molecular subtyping of endometrial cancer, assess prognostic significance, guide medication use, and support genetic screening. Specifically, when a library of genes related to targeted therapy for endometrial cancer is obtained and relevant variants are detected, a local interpretation database related to targeted therapy, prognosis, molecular subtyping, and genetic susceptibility is established based on relevant guidelines, expert consensus, and literature reports. Inputting relevant variants will output corresponding interpretations.

[0029] The reference genome version used in this invention is GRCh37 / hg19.

[0030] (III) Beneficial Effects

[0031] The beneficial effects of this invention are:

[0032] This invention provides a probe set for detecting genes related to targeted therapies in endometrial cancer. Based on next-generation sequencing and employing hybridization capture, it can simultaneously detect variations in 100 genes in a single sample, including single nucleotide variants (SNVs), insertions / deletions (InDels), and copy number variations (CNVs), comprehensively covering drug-related genes (such as BRAF, ERBB2, and RET). It also enables medication guidance, molecular subtyping (e.g., POLE hypermutant, mismatch repair deficient), screening for genetic susceptibility genes (e.g., Lynch syndrome-related genes), and assessment of MSI status.

[0033] The endometrial cancer targeted drug-related gene detection kit provided by this invention can detect genes with clearly defined drug targets in endometrial cancer in a single test, providing a one-stop solution with standardized and automated processes. Conventional methods using PCR or first-generation sequencing combined with immunohistochemistry are cumbersome, requiring multiple platforms for experimental operations, data analysis, and result interpretation. Result integration necessitates cross-platform data correlation, increasing the complexity of bioinformatics analysis and the risk of human error. This invention, however, completes all detection objectives through a single experimental procedure, reducing sample transfer and processing steps and lowering the risk of contamination. Bioinformatics analysis can achieve multi-dimensional result output (such as variant annotation, molecular typing, and MSI scoring) through a standardized process, simplifying subsequent interpretation.

[0034] The kit provided by this invention has high specificity and is designed for a single type of cancer. It can detect genes with clear drug significance, molecular subtype, and genetic susceptibility for endometrial cancer in a single test. It has strong specificity for endometrial cancer patients and can reduce costs and increase efficiency compared with multi-tumor gene testing.

[0035] The capture probe designed in this invention covers the full coding sequence (CDS) of most key genes and hotspot regions of other genes; it has high detection sensitivity, achieving 100% target region coverage and an average sequencing depth of up to 500x. The capture probe provided by this invention is used in next-generation sequencing (NGS), where the detection sensitivity for low-frequency mutations is far higher than that of first-generation sequencing, and it is particularly advantageous for samples with low tumor purity. It avoids sample exhaustion and prolonged detection time caused by sequential detection of single genes, providing a faster basis for patient treatment selection. Although the total cost of simultaneous multi-gene analysis by NGS is higher, the average detection cost per gene and single site is significantly reduced compared to traditional detection methods. Attached Figure Description

[0036] Figure 1 A schematic diagram of the NGS detection technology library construction process;

[0037] Figure 2To construct a good document library of fragment analysis results. Detailed Implementation

[0038] This invention addresses the core problem of inaccurate molecular typing in existing endometrial cancer gene detection technologies, such as PCR and first-generation sequencing combined with immunohistochemistry, which only cover some gene hotspot variants and easily miss rare or non-hotspot pathogenic mutations in important genes like POLE. It proposes a targeted drug-related gene detection kit based on second-generation sequencing and hybridization capture technology. The technical solution involves simultaneously detecting single nucleotide variants (SNVs), insertions / deletions (InDels), and copy number variations (CNVs) of 100 genes in a single system, comprehensively covering drug-related genes such as BRAF, ERBB2, and RET. This simultaneously enables drug guidance, molecular typing (e.g., POLE hypermutant, mismatch repair deficient), and genetic susceptibility gene screening (e.g., Lynch). This approach assesses the status of syndrome-related genes and microsatellite instability (MSI), and through standardized and automated experimental procedures and bioinformatics analysis, it addresses the problems of cumbersome traditional multi-platform testing processes, complex data integration, and high risk of human error. It boasts high specificity, high sensitivity, and high coverage (100% target region coverage, 500x sequencing depth), making it particularly suitable for samples with low tumor purity. Ultimately, it can significantly improve the accuracy of molecular subtyping and mutation detection rate, providing richer references for precision medicine in endometrial cancer and constructing a one-stop, efficient, and reliable precision diagnosis and treatment solution.

[0039] The molecular subtyping results and prognostic significance of endometrial cancer, referencing the 2024 NCCN Guidelines for the Diagnosis and Treatment of Uterine Tumors, the WHO Classification of Tumors of the Female Reproductive Organs (5th Edition), and other domestic and international guidelines and expert consensus, are classified as follows: POLE mutated, microsatellite instability or mismatch repair deficient (MSI-H / dMMR), nonspecific molecular profile (NSMP), and p53 aberrant (p53abn). According to the WHO Classification of Tumors of the Female Reproductive Organs (5th Edition), the POLE mutated type has a better prognosis, the MSI-H / dMMR type has a moderate prognosis, the NSMP type has a moderate to good prognosis, and the p53 abn type has a poor prognosis. Related genes for molecular subtyping include: EPCAM, MLH1, MSH2, MSH6, PMS2, POLE, and TP53. According to the guidelines and expert consensus, the interpretation order of molecular typing is as follows: if a hotspot mutation of POLE is present, it belongs to the POLE mut type; if there is no hotspot mutation of POLE, but there is a loss-of-function mutation of the MMR gene or microsatellite instability (MSI-H), it is interpreted as the MSI-H / dMMR type; if no loss-of-function mutation of the MMR gene is detected and the microsatellite is stable (MSS), and there is no hotspot mutation of POLE, and if no TP53 mutation is detected, it is the NSMP type; if a TP53 mutation is detected, it is interpreted as the p53abn type.

[0040] Inheritance patterns of OMIM diseases associated with genetic susceptibility genes: autosomal dominant (AD) and autosomal recessive (AR). Based on the 2015 ACMG guidelines, the clinical significance of germline variations is classified to accurately determine their pathogenicity. Germline variations are categorized as: pathogenic (P), suspected pathogenic (LP), clinically indeterminate (VUS), benign (B), and suspected benign (LB) variations. The corresponding genes are AKT1, BRCA1, BRCA2, EPCAM, MLH1, MSH2, MSH6, PIK3CA, PMS2, POLD1, POLE, PTEN, SDHA, SDHB, SDHC, and SDHD. Table 1 details the 16 genes associated with genetic susceptibility to endometrial cancer and related disease genes.

[0041] Table 1. 16 genes and related diseases associated with genetic susceptibility to endometrial cancer.

[0042]

[0043]

[0044]

[0045] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0046] Example 1

[0047] This embodiment relates to the design and synthesis of a capture probe set for molecular subtyping of endometrial cancer, drug administration, and genes related to genetic susceptibility.

[0048] Endometrial cancer molecular subtyping, medication, and genetic susceptibility-related genes were screened using the following methods:

[0049] We manually reviewed the National Comprehensive Cancer Network (NCCN) guidelines, the Chinese Society of Clinical Oncology (CSCO) guidelines, and the World Health Organization (WHO) guidelines to organize, screen, and summarize information on targeted therapy, immunotherapy, molecular subtyping, and genes related to genetic susceptibility.

[0050] The relationship between genes related to genetic susceptibility and phenotype was obtained using the Online Mendelian Inheritance in Man (OMIM) database.

[0051] The pathogenic and potentially pathogenic variants of genes related to genetic susceptibility were screened using the ClinVar database. The probe design must include these variants. The obtained probe sequences are shown in SEQ ID NO.1-SEQ ID NO.220.

[0052] I. Probe Design and Synthesis

[0053] The probes provided by this invention cover endometrial targeted drug therapy, immunotherapy, molecular subtyping, and genes related to genetic susceptibility, including AKT1, AKT2, ALK, APC, AR, ARAF, ARID1A, ARID1B, ATM, ATR, ATRX, AXIN1, BARD1, BCOR, BRAF, BRCA1, BRCA2, BRIP1, BTK, CCND1, CCNE1, CDK12, CDKN2A, CHEK1, CHEK2, CREBBP, CTNNB1, DDR2, DICER1, DROSHA, MLH1, MLH3, and MRE11. MSH2, MSH3, MSH6, MTAP, MTOR, MUTYH, NBN, NF1, NRAS, NRG1, NTHL1, NTRK1, NTRK2, NTRK3, PALB2, PARP1, PBRM1, PDGFRA, PIK3CA, PIK3R1, PMS1, PMS2, POLD1, POLE, EGFR, EPCAM, ERBB2, ERBB3, ERBB4, ESR1, FANCA, FANCB, FANCD2, FANCL, FBXW7, FGFR1, FGFR2, FGFR3, GATA3, ID H1, IDH2, JAK1, KDM6A, KIF5B, KIT, KMT2C, KMT2D, KRAS, MDM2, MDM4, MET, PP2R1A, PPP2R1A, PTEN, RAD51B, RAD51C, RAD51D, RAD54L, RAF1, RB1, RET, RIT1, RNF43, ROS1, SDHA, SDHB, SDHC, SDHD, SMARCA4, SMARCB1, SPOP, TERT, TP53, TSC1, TSC2, VEGFA, VHL; among which, important genes related to targeted drug therapy include: BR AF, ERBB2, NTRK1, NTRK2, NTRK3, RET; Important genes related to genetic susceptibility include: AKT1, BRCA1, BRCA2, EPCAM, MLH1, MSH2, MSH6, PIK3CA, PMS2, POLD1, POLE, PTEN, SDHA, SDHB, SDHC, SDHD; Important genes related to immunotherapy include: EPCAM, MLH1, MSH2, MSH6, PMS2, POLD1, POLE; Genes related to molecular typing include: EPCAM, MLH1, MSH2, MSH6, PMS2, POLE, TP53.

[0054] The hybridization capture probe sequences designed based on the aforementioned related genes are shown in the nucleotide sequences of SEQ ID NO.1-SEQ ID NO.220. The sequence numbers and their corresponding gene names are as follows: SEQ ID NO.1-2: MTOR; SEQ ID NO.3-4: SDHB; SEQ ID NO.5-6: ARID1A; SEQ ID NO.7-8: MUTYH; SEQ ID NO.9-10: RAD54L; SEQ ID NO.11-12: JAK1; SEQ ID NO.13-14: NRAS; SEQ ID NO.15: RIT1; SEQ ID NO.16-17: NTRK1; SEQ ID NO.18-19: SDHC; SEQ ID NO.20-21: DDR2; SEQ ID NO.22-23: MDM4; SEQ ID NO.24-25: PARP1; SEQ ID NO.26-27: ALK; SEQ ID NO.28-29: EPCAM; SEQ ID NO.22-23: MDM4; SEQ ID NO.24-25: PARP1; SEQ ID NO.26-27: ALK; SEQ ID NO.28-29: EPCAM; SEQ ID NO.22-23: MTOR; SEQ ID NO.24-25 ... MDM4; SEQ ID NO.26-27: ALK; SEQ ID NO.28-29: EPCAM; SEQ ID NO.22-23: MTOR; SEQ ID NO.24-25: MDM4; SEQ ID NO.24-2 ID NO.30-31: MSH2; SEQ ID NO.32-33: MSH6; SEQ ID NO.34-35: FANCL; SEQ ID NO.36-37: IDH1; SEQ ID NO.38-39: ERBB4; SEQ ID NO.40-41: BARD1; SEQ ID NO.42-43: PMS1; SEQ ID NO.44-45: FANCD2; SEQ ID NO.46-47: VHL; SEQ ID NO.48-49: RAF1; SEQ ID NO.50-51: MLH1; SEQ ID NO.52-53: CTNNB1; SEQ ID NO.54-55: PBRM1; SEQ ID NO.56-57: ATR; SEQ ID NO.58-59: PIK3CA; SEQ ID NO.60-61: FGFR3; SEQ ID NO.62-63: PDGFRA; SEQ ID NO.64-65: KIT; SEQ ID NO.66-67: FBXW7; SEQ ID NO.68-69: SDHA; SEQ ID NO.70-71: TERT; SEQ ID NO.72-73: DROSHA; SEQ ID NO.74-75: PIK3R1; SEQ ID NO.76-77: MSH3; SEQ ID NO.78-79: APC; SEQ ID NO.80-82: VEGFA; SEQ ID NO.83-84: ROS1; SEQ ID NO.85-86: ESR1; SEQ ID NO.87-88:ARID1B;SEQ ID NO.89-90:PMS2;SEQ ID NO.91-92:EGFR;SEQ ID NO.93-94:MET;SEQ ID NO.95-96:BRAF;SEQ ID NO.97-98:KMT2C;SEQ ID NO.99-100:NRG1;SEQ IDNO.101-102:FGFR1;SEQ ID NO.103-104:NBN;SEQ ID NO.105-106:MTAP;SEQ ID NO.107-108:CDKN2A;SEQ ID NO.109-110:NTRK2;SEQ ID NO.111-112:TSC1;SEQ ID NO.113-114:GATA3;SEQ ID NO.115-116:KIF5B;SEQ ID NO.117-118:RET;SEQ ID NO.119-120:PTEN;SEQ ID NO.121-122:FGFR2;SEQ ID NO.123-124:CCND1;SEQ ID NO.125-126:MRE11;SEQID NO.127-128:ATM;SEQ ID NO.129-130:SDHD;SEQ ID NO.131-132:CHEK1;SEQ IDNO.133-134:KRAS;SEQ ID NO.135-136:KMT2D;SEQ ID NO.137-138:ERBB3;SEQ IDNO.139-140:MDM2;SEQ ID NO.141-142:POLE;SEQ ID NO.143-144:BRCA2;SEQ ID NO.145-146:RB1;SEQ ID NO.147-148:RAD51B;SEQ ID NO.149-150:MLH3;SEQ ID NO.151-152:DICER1;SEQ ID NO.153-154:AKT1;SEQ ID NO.155-156:NTRK3;SEQ ID NO.157-158:IDH2;SEQ ID NO.159-160:AXIN1;SEQ ID NO.161-162:NTHL1;SEQ ID NO.163-164:TSC2;SEQ IDNO.165-166:CREBBP;SEQ ID NO.167-168:PALB2;SEQ ID NO.169-171:FANCA;SEQ IDNO.172-173:TP53;SEQ ID NO.174-175: NF1; SEQ ID NO. 176-177: RAD51D; SEQ ID NO. 178-179: CDK12; SEQ ID NO. 180-181: ERBB2; SEQ ID NO. 182-183: BRCA1; SEQ ID NO. 184-185: SPOP; SEQ ID NO. NO.186-187: RNF43; SEQ ID NO.188-189: RAD51C; SEQ ID NO.190-191: BRIP1; SEQ ID NO.192-193: SMARCA4; SEQ ID NO.194-195: CCNE1; SEQ ID NO.196-197: AKT2; SEQ ID NO.198-199: POLD1; SEQ ID NO.200-201: PPP2R1A; SEQ ID NO.202-203: SMARCB1; SEQ SEQ ID NO. 204-205: CHEK2; SEQ ID NO. 206-207: FANCB; SEQ ID NO. 208-209: BCOR; SEQ ID NO. 210-211: KDM6A; SEQ ID NO. 212-213: ARAF; SEQ ID NO. 214-215: AR; SEQ ID NO. 216-217: ATRX; SEQ ID NO. 218-219: BTK; SEQ ID NO. 220: MTOR. These probes cover the entire coding region of the aforementioned genes.

[0055] The probe was synthesized by Twist and biotin was labeled at the 5' end of the probe.

[0056] The capture probes described above are specific capture probes designed for known sequences of endometrial cancer susceptibility genes. These probes only pair complementary to the target gene sequences. For detection, genomic DNA samples are first extracted from endometrial cancer tissue to construct a genomic DNA library: DNA fragmentation, end repair, and dA tailing are performed. The dA-tailed product is then ligated with a Twist universal adapter. The adapter-ligated product is then subjected to PCR amplification, and the PCR reaction product is purified to obtain the DNA library.

[0057] (2) The purified DNA library is concentrated and hybridized with probes whose nucleotide sequences are shown in SEQ ID NO. 1~220. The probes act like molecular magnets, specifically binding to the corresponding target gene fragments in the sample, while non-target gene fragments do not bind. Subsequent washing and elution steps remove unbound non-target fragments, thus enriching the target gene fragments. Library capture amplification is then performed, followed by further purification to obtain a next-generation sequencing library. Sequencing and signal analysis: The enriched target gene fragments are subjected to PCR amplification and high-throughput sequencing. If the probe and the gene fragment in the sample are perfectly paired (including specific pairing at mutation sites), a stable signal (such as a fluorescence signal or an electrical signal) will be generated, and the fluorescence intensity of a perfectly paired sample is significantly higher than that of a mismatch.

[0058] Bioinformatics analysis to locate mutation sites: Sequencing data is compared with standard reference genome sequences, and bioinformatics tools are used to identify differences such as base substitutions, insertions, and deletions. False positive results are eliminated by combining signal intensity screening, and finally the mutation sites on the target gene are accurately located.

[0059] Based on the detected genes, the OMIM disease and its corresponding inheritance pattern are output through bioinformatics annotation, as shown in Table 12 (Results of Genetic Susceptibility Gene Detection).

[0060] Example 2

[0061] This embodiment describes a method based on the probe kit from Example 1 for detecting molecular subtyping, medication, and genes related to genetic susceptibility in endometrial cancer. The kit includes probes with nucleotide sequences as shown in SEQ ID NO. 1-220, and genomic DNA extraction reagents are available from Tiangen Blood Genomic Extraction Kit.

[0062] DNA library construction reagents are available from Twist's Twist Library Preparation EF Kit 1, 2.0; next-generation sequencing library construction reagents are available from Twist's Liquid Hybridization Capture Kit.

[0063] The instructions for using the kit include:

[0064] 1. Genomic DNA extraction

[0065] Endometrial cancer tumor tissue samples were collected from the subjects and genomic DNA was extracted using a commercially available genomic DNA extraction kit.

[0066] 2. Constructing a DNA library

[0067] A flowchart illustrating the process of constructing a DNA library is shown below. Figure 1 As shown, specifically:

[0068] The genomic DNA from the tissue samples obtained above was used to construct a DNA library using the Twist Library Preparation EF Kit 1, 2.0 (lot number 34000005688) from Twist Corporation. The specific library construction process is as follows:

[0069] (1) DNA fragmentation, end repair and dA tailing

[0070] A 50 μL reaction system was used: X μL (200 ng) of genomic DNA from tissue samples, 4 μL of Frag / AT Buffer, 6 μL of Frag / AT Enzymes, and 40-X μL of nuclease-free water. After preparation, the system was mixed thoroughly by pipetting and incubated in a PCR instrument for DNA fragmentation, end repair, and dA tailing. The specific reaction program was: 37℃ for 10 min, 65℃ for 30 min, and 4℃ Hold to obtain the DNA fragmentation, end repair, and dA tailing products.

[0071] (2) Connect the Twist universal connector

[0072] The obtained DNA fragmentation, end repair, and dA tailing products were prepared into a 75 μL reaction mixture, specifically including 50 μL of the DNA fragmentation, end repair, and dA tailing products, 5 μL of artificially synthesized sequencing adapters (Twist universal Adapters), and 20 μL of Ligation Master Mix. After mixing, the mixture was incubated in a PCR instrument with the lid closed and the reaction program set to 20°C for 15 min, followed by incubation at 4°C.

[0073] (3) Purification

[0074] a. Add 60 μL of DNA Purification Beads (Twist) (0.8 times the sample volume) equilibrated to room temperature and thoroughly mixed to the sample from the previous step, mix by pipetting 30 times, and incubate at room temperature for 5 min;

[0075] b. Instantly detach the sample, place it on a magnetic rack for 5 minutes, and discard the supernatant;

[0076] c. Wash with 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0077] d. Repeat step c;

[0078] e. Immediately collect the residual liquid at the bottom of the tube, place it on a magnetic rack, then discard the residual liquid and let it air dry at room temperature for 5 minutes until the alcohol has completely evaporated;

[0079] f. Add 18 μL of nuclease-free water, mix well by pipetting, and incubate at room temperature for 5 min to obtain adapter-ligated DNA;

[0080] g. Briefly separate the sample and place it on a magnetic rack for 2 minutes until the liquid is clear. Take 15 μL of the supernatant into a new PCR tube for PCR amplification. The PCR amplification reaction system is 50 μL, specifically including 15 μL of adapter-ligated DNA, 25 μL of Equinox Library Amp Mix (2X), and 10 μL of UDI primers (Twist bioscience, unique dual-index primers, sequencing adapters, compatible with Illumina's Novaseq sequencer).

[0081] After mixing the above-mentioned system thoroughly by pipetting, place it in a PCR instrument for incubation. The PCR reaction program is as follows: heat cover 98℃, specifically pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; extension: 72℃ for 1min, then incubate at 4℃.

[0082] (4) Purify again

[0083] a. Add 50 μL of DNA Purification Beads equilibrated to room temperature and thoroughly mixed to the PCR product from the previous step, mix by pipetting 30 times, and incubate at room temperature for 5 min;

[0084] b. Instantly detach the sample, place it on a magnetic rack for 5 minutes, and discard the supernatant;

[0085] c. Add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0086] d. Repeat step c;

[0087] e. Immediately collect the residual liquid at the bottom of the tube, place it on a magnetic rack, then discard the residual liquid and let it air dry at room temperature for 5 minutes until the alcohol has completely evaporated;

[0088] f. Add 24 μL of nuclease-free water, mix well by pipetting, and let stand at room temperature for 5 min;

[0089] g. Instantly centrifuge, place on a magnetic rack for 2 minutes until the liquid is clear, take 22 μL of supernatant into a new centrifuge tube to obtain the DNA library, and vortex to mix.

[0090] Take 2 μL of the above DNA library, dilute it 10-fold, and quantify it using the Equalbit 1×dsDNA HS Assay Kit, recording the library concentration. Take 1 μL of the DNA library diluted 10-fold in the previous step and determine the library fragment length using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit). The library length is approximately between 260-350 bp. 3. Library pooling, hybridization capture and PCR amplification, and library purification. (If possible, use 1-8 pre-libraries constructed as described in the above steps.)

[0091] a. The total amount of each library pool is 1.5-4.0 μg, and the pooling volume of each library is calculated based on the concentration of each library;

[0092] b. Mix each library in a 1.5 mL centrifuge tube, gently shake, and centrifuge. (1) Library hybridization and capture a. Library concentration, the concentration system is as follows: pooling library X (the volume of 1-8 pre-libraries added together), universal blockers (Twist) 8 μL, blocking solution (Twist) 5 μL, probe 4 μL (the nucleotide sequence of the probe is as shown in SEQ ID NO.1-220, the concentration and amount of the probe is 0.2 fmol / probe / person)

[0093] b. The reaction tube was dried completely at 45°C in a vacuum dryer.

[0094] (2) Library hybridization

[0095] a. After drying, add 20 μL of the preheated (65℃) Fast Hybridization Mix (Twist) to a centrifuge tube, gently tap to mix, let stand at room temperature for 5 min, and then transfer to a new PCR tube.

[0096] b. Add 30 μL of Hyb Enhancer (Twist) to the surface of the liquid in the PCR tube, briefly remove air bubbles, and place it in the PCR instrument. Heat the tube at 98°C. The amplification program for the hybridization reaction system is: 95°C for 5 min, 60°C for 2 h, and 60°C Hold.

[0097] (3) Document capture

[0098] a. Take 100 μL of streptavidin binding beads (Twist) into a 1.5 mL tube (let it stand at room temperature for 30 min beforehand and shake to mix).

[0099] b. Add 200 μL of Fast Binding Buffer (Twist), mix well, place on a magnetic rack for 1 min, discard the supernatant, and repeat this washing step 2 times for a total of 3 times; then add 200 μL of Fast Binding Buffer, vortex to resuspend and mix thoroughly.

[0100] c. After library hybridization is complete, open the PCR tube cap on the PCR instrument, quickly transfer all the hybridization solution to the magnetic bead solution, and immediately mix by pipetting.

[0101] d. Incubate the centrifuge tubes on a centrifuge at room temperature and 1200 rpm for 30 min. Centrifuge the centrifuge tubes rapidly on a magnetic rack for 1 min, then discard the supernatant.

[0102] e. Add 200 μL of Fast Wash Buffer 1 (Twist) preheated to 67°C (do not remove from the metal bath), mix gently, and then quickly incubate at 67°C for 5 min.

[0103] f. Place on a magnetic rack for 1 min, remove the supernatant, add 200 μL of preheated Fast Wash Buffer 1 (Twist), mix lightly, and then quickly incubate at 67°C for 5 min.

[0104] g. Transfer all liquid in the tube to a new 1.5 mL tube, place on a magnetic rack for 1 min, and discard the supernatant.

[0105] h. Add 200 μL of preheated Wash Buffer 2 (Twist) at 48°C (do not remove from the metal bath), mix gently, and then quickly incubate at 48°C for 5 min, on a magnetic rack for 1 min. Remove the supernatant and wash twice more with Wash Buffer 2, for a total of three times.

[0106] I. After detaching, place the sample on a magnetic rack and use a 10μL pipette tip to aspirate any remaining liquid.

[0107] J. Add 45 μL H2O, mix well by pipetting, and incubate on ice (Note: If PCR is not performed immediately, store the magnetic bead mixture at -20°C).

[0108] (4) PCR amplification

[0109] The PCR amplification reaction system used was a 50 μL system, which specifically included 22.5 μL of magnetic bead mixture, 25 μL of high-fidelity hot-start PCR premix (KAPA HIFI HotStart ReadyMix, Twist), and 2.5 μL of amplification primers (Twist), for a total volume of 50 μL.

[0110] After preparing the reaction system, mix it thoroughly by pipetting and place it on the PCR instrument for reaction. Heat the instrument to 98°C. The specific PCR reaction program is as follows: pre-denaturation: 98°C for 45s; denaturation-annealing-extension: 98°C for 15s, 60°C for 30s, 72°C for 30s, with 8 cycles; final extension: 72°C for 1min; incubate at 4°C.

[0111] (5) Purification

[0112] a. Add 90 μL of DNA Purification Beads (Twist) equilibrated to room temperature and thoroughly mixed to the PCR product from the previous step, mix by pipetting 30 times, and incubate at room temperature for 5 min;

[0113] b. Instantly detach the sample, place it on a magnetic rack for 5 minutes, and discard the supernatant;

[0114] c. Add 200 μL of freshly prepared 80% ethanol, let stand at room temperature for 1 min, and discard the supernatant;

[0115] d. Repeat step c;

[0116] e. Immediately collect the residual liquid at the bottom of the tube, place it on a magnetic rack, then discard the residual liquid and let it air dry at room temperature for 3-5 minutes until the alcohol has completely evaporated;

[0117] f. Add 34 μL of nuclease-free water, mix well by pipetting, and let stand at room temperature for 5 min;

[0118] g. Instantly centrifuge, place on a magnetic rack for 2 minutes until the liquid is clear, take 32 μL of supernatant into a new centrifuge tube to obtain the hybridized and captured library, and shake to mix.

[0119] Take 2 μL of the amplified next-generation sequencing library and quantify it using the Equalbit 1×dsDNA HS Assay Kit (Vzayme). Record the library concentration, which is approximately 1-15 ng / μL. Take 1 μL of the amplified next-generation sequencing library and determine the library fragment length using the Agilent 2100 Bioanalyzer system (Agilent DNA 1000 Kit). The library length is approximately 300-450 bp.

[0120] 4. Sequencing

[0121] The amplified next-generation sequencing library obtained in step 3 above was sequenced using Illumina's Nextseq 2000. Bioinformatics analysis was then performed: the sequencing results were analyzed and annotated using bioinformatics software.

[0122] Example 3

[0123] This embodiment uses the probe set from Example 1 and performs clinical sample testing according to the detection method of Example 2. The detection process is as follows:

[0124] One tumor tissue sample was collected from each of two endometrial cancer patients. The samples were analyzed for medication use, molecular subtyping, and genetic susceptibility gene variations. Genomic DNA was first extracted from the tumor tissue samples of the two endometrial cancer patients. Then, library construction was performed according to the method described in Example 1, and the libraries were successfully constructed (see [link to example]). Figure 2 The library was sequenced on an Illumina high-throughput sequencer with sequencing parameters PE: 2 × 150. After quality control of the sequencing data, bioinformatics analysis was performed using software such as BWA / GATK, with the reference genome version being GRCh37 / hg19. Relevant databases such as gnomAD / Clinvar / OMIM were annotated. Somatic variants were classified according to clinical significance according to guidelines such as AMP / ASCO / CAP: Grade I (variables with clear clinical significance), Grade II (variables with potential clinical significance), Grade III (variables with unclear clinical significance), and Grade IV (benign or possibly benign variants). Germline variants were classified according to clinical significance according to ACMG guidelines: pathogenic (P), suspected pathogenic (LP), undetermined clinical significance (VUS), benign (B), and suspected benign (LB) variants. Finally, each locus was validated using first-generation sequencing, with a 100% concordance rate. Sequencing quality is shown in Table 2.

[0125] Based on the mutation information obtained from bioinformatics analysis, the detection results of targeted drug-related gene mutations for Patient 1 are shown in Tables 3-7, and the detection results of targeted drug-related gene mutations for Patient 2 are shown in Tables 8-12. Furthermore, according to the 2017 Chinese Expert Consensus on Next-Generation Sequencing Detection in Clinical Molecular Pathology Laboratories and the 2017 AMP / ASCO / CAP Standards and Guidelines for the Interpretation and Reporting of Tumor Cell Mutations, the interpretation and annotation of the clinical significance of tumor somatic cell mutations are divided into the following four levels of evidence:

[0126] Category A: Targets approved for drug use by the U.S. Food and Drug Administration (FDA) or the National Medical Products Administration (NMPA); variants with clear diagnostic / therapeutic / prognostic significance as included in domestic and international treatment guidelines.

[0127] Category B: Variants that have not yet been included in treatment guidelines but have been incorporated into expert consensus in the field.

[0128] Category C: Genetic variants approved by the FDA or NMPA for predictable treatment outcomes in other cancers, or variant sites in ongoing clinical trials.

[0129] Grade D: Preclinical studies or a small number of case reports, without expert consensus.

[0130] A and B correspond to Level I; C and D correspond to Level II. The interpretation of the gene mutation results related to targeted drug use in Patient 1 is shown in Table 4, and the interpretation of the gene mutation results related to targeted drug use in Patient 2 is shown in Table 10.

[0131] Table 2: Sequencing quality:

[0132]

[0133] Table 3: Results of targeted therapy-related gene mutation detection in Patient 1

[0134]

[0135] Table 4: Interpretation of Targeted Therapy-Related Gene Variation Results in Patient 1

[0136]

[0137] Table 5: Molecular subtyping results of endometrial cancer in patient 1

[0138]

[0139] Table 6: Results of genetic susceptibility testing for Patient 1

[0140]

[0141] Table 7: Results of immune-related gene testing for Patient 1

[0142]

[0143] The test results for Patient 2 are shown in Tables 8-12 below.

[0144] Table 8: Results of targeted therapy-related gene mutation detection in patient 2

[0145]

[0146] Table 9: Interpretation of Targeted Therapy-Related Gene Variation Results in Patient 2

[0147]

[0148]

[0149] Table 10: Molecular subtyping results of endometrial cancer in patient 2

[0150]

[0151] Table 11: Results of genetic susceptibility testing for Patient 2

[0152]

[0153] Table 12: Results of immune-related gene testing in Patient 2

[0154]

[0155] The test results show that the probe coverage of the kit described in this invention can reach 100%, and the average depth of the target region is above 500x, with quality control meeting the requirements. Test results on routine samples indicate that the probes, methods, and kits described in this invention can accurately detect gene mutations in endometrial cancer, enabling medication guidance, molecular subtyping, and screening for genetic susceptibility genes.

[0156] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A set of probes for detecting molecular subtyping, medication, and genetic susceptibility-related genes in endometrial cancer, characterized in that, include: A probe set targeting genes related to molecular subtyping, medication, and genetic susceptibility of endometrial cancer, wherein the genes related to molecular subtyping, medication, and genetic susceptibility of endometrial cancer include genes related to targeted medication, genes related to genetic susceptibility, genes related to immunotherapy, and genes related to molecular subtyping; The probe set includes probes with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

220.

2. A kit for detecting molecular subtyping, medication, and genetic susceptibility-related genes in endometrial cancer, characterized in that, It includes probes with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.

220.

3. The detection kit as described in claim 2, characterized in that, Each probe is labeled with biotin at its 5' or 3' end, and the assay kit also includes streptomycin affinity magnetic beads that can specifically bind to biotin.

4. The detection kit as described in claim 2, characterized in that, It also includes genomic DNA extraction reagents, DNA library construction reagents, and next-generation sequencing library construction reagents.

5. The detection kit as described in claim 2, characterized in that, The genomic DNA extraction reagent is the Tiangen Blood Genomic Extraction Kit, the DNA library construction reagent is the Twist Library Preparation EF Kit 1, 2.0 from Twist, and the next-generation sequencing library capture reagent is the Twist Liquid Hybridization Capture Kit from Twist.

6. A method for detecting endometrial cancer molecular subtyping, medication, and genetic susceptibility-related genes for non-diagnostic / therapeutic purposes, characterized in that, Includes the following steps: (1) Genomic DNA was extracted from ex vivo tumor tissue of endometrial cancer. The DNA was first fragmented, end-repaired and dA tailed. The dA-tailed product was ligated to Twist universal adapter. The ligation adapter product was purified and PCR amplified. The PCR reaction product was purified to obtain a DNA library. (2) After the purified DNA library is concentrated, it is hybridized with the probes with nucleotide sequences as shown in SEQ ID NO.1~220 to capture and purify the library, and then PCR amplification is performed again. The amplified product is purified again to obtain the next-generation sequencing library. (3) Perform second-generation sequencing and data analysis on the second-generation sequencing library to obtain the variation of genes related to targeted drug use for endometrial cancer and the molecular subtyping and genetic susceptibility genes related to endometrial cancer.

7. The detection method according to claim 6, characterized in that, In step (1), the PCR amplification reaction system uses Twist's UDI primers; the amplification procedure is as follows: Pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; Extension: 72℃ for 1min, followed by 4℃ heat preservation.

8. The detection method according to claim 6, characterized in that, In step (2), amplification is performed using Twist's Amplification Primers, and the amplification procedure is as follows: Pre-denaturation: 98℃ for 45s; 98℃ for 15s, 60℃ for 30s, 72℃ for 30s cycled 8 times; Extension: 72℃ for 1min, followed by 4℃ heat preservation.

Images