Detection method and system for high-sensitivity detection of solid tumor ctDNA

Abstract

The invention relates to a detection method and system for high-sensitivity detection of solid tumor ctDNA. The method aims at 20 high-incidence solid tumors, and covers high-frequency main clone mutant genes, key genes related to common targeted therapy and genes related to prognosis. The design of the probe library and the kit is used for efficiently and accurately detecting the genes in a human sample through a high-throughput sequencing technology, so that comprehensive gene variation information is obtained. According to the method, the sensitivity and accuracy of solid tumor ctDNA detection can be remarkably improved, and a powerful tool is provided for early diagnosis, curative effect monitoring and prognosis evaluation of tumors.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis technology, and in particular to a detection method and system for high-sensitivity detection of ctDNA in solid tumors. Background Art

[0002] Circulating tumor DNA (ctDNA) is a DNA fragment released into the blood by tumor cells, which carries the genetic characteristics of tumor cells, including gene mutation, methylation, amplification or rearrangement, etc. These characteristics make ctDNA an important indicator for tumor screening, companion diagnosis, treatment efficacy evaluation and prognostic risk stratification.

[0003] Although traditional tumor detection methods, such as tissue biopsy, can provide direct information from tumor tissue, these methods are usually invasive and may not fully reflect the genetic heterogeneity of the tumor. In contrast, ctDNA, as a liquid biopsy method, has significant advantages such as non-invasive or minimally invasive, repeatable sampling, and short detection time. ctDNA detection can monitor the heterogeneity and dynamic changes of tumors in real time, and has important clinical value for early diagnosis, treatment monitoring, and prognosis evaluation.

[0004] However, since the concentration of ctDNA in the blood is usually low, especially in subjects with early and middle stage tumors, it is very difficult to detect ctDNA, which is like looking for a needle in a haystack. Therefore, how to keenly capture trace ctDNA signals has become the main technical challenge for effectively detecting minimal residual disease (MRD). In order to accurately detect MRD, the required sensitivity needs to reach the order of one in ten thousand, but the current detection methods are still limited in sensitivity and specificity.

[0005] For subjects who have undergone surgery for early or mid-stage solid tumors or are undergoing treatment for advanced solid tumors, the amount of ctDNA extracted is relatively small with the same amount of blood drawn, so multiple PCR cycles are often required before sequencing. This process may introduce DNA errors and lead to the amplification of DNA error signals through multiple PCR cycles. In addition, DNA errors may also occur during the sequencing process due to factors such as oxidative damage. Clonal hematopoiesis is another important factor that affects the specificity of plasma ctDNA and may interfere with the test results. Therefore, improving the specificity of the test to reduce false-positive results is crucial to ensure that subjects receive accurate diagnosis and appropriate treatment.

[0006] For subjects who receive early or mid-term postoperative treatment, existing imaging examinations and tumor marker detection methods are limited by clinical experience, limited biomarkers, and screening sensitivity, which may lead to delayed or inaccurate evaluation of treatment efficacy. When recurrence is clinically discovered, the subject may have lost the opportunity for a second surgery, thus affecting the prognosis.

[0007] At present, MRD detection strategies can be divided into two categories according to whether they are based on the genomic information of the subject's specific tumor: tumor-informed strategy and tumor-naïve strategy. In the tumor-informed strategy, the detection panel can be customized according to the genomic variation map of the subject's tumor tissue sample. This method is called personalized panel. The representative technology of personalized panel is Natera's Signatera technology, which analyzes the subject's tumor tissue sample through whole exome sequencing and screens out 16 sites for subsequent MRD monitoring. The advantage of this method is strong specificity, but because it can only provide 16 fixed mutation conditions, it cannot detect new mutations and other gene mutations caused by tumor heterogeneity. Another tumor-informed strategy is to use a population customized panel. This method does not rely on the specific variation of the subject, but selects a set of fixed monitoring panels for specific types of tumors (such as lung cancer, colorectal cancer, etc.). This fixed panel can detect existing mutations in the tissue and detect new mutations, overcoming tumor heterogeneity. Since conventional fixed panels often detect fewer mutations, their sample detection sensitivity is often limited.

[0008] In summary, the present invention aims to overcome the deficiencies in the prior art and construct a highly sensitive solid tumor ctDNA detection system to improve the sensitivity and accuracy of ctDNA detection and provide support for the development of precision tumor medicine. Summary of the invention

[0009] In response to the above problems, the present invention provides a high-sensitivity method for detecting circulating tumor DNA (ctDNA) in solid tumors. The method is particularly suitable for 20 solid tumors that are highly prevalent in the Chinese population, and can significantly improve the sensitivity and accuracy of detection, providing a powerful tool for early diagnosis, efficacy monitoring and prognosis evaluation of tumors.

[0010] To achieve the above object, the present invention provides the following technical solutions: A detection method for high-sensitivity detection of ctDNA in solid tumors, wherein the method is used for non-therapeutic and diagnostic purposes, comprising the following steps: Step 1, obtaining subject samples and extracting ctDNA, and setting the target cancer type; the subject samples include healthy control samples and tumor-positive samples; Step 2: Perform WES sequencing on ctDNA and compare it with the reference genome to obtain mutations in the gene coding region and screen for insertions and deletions (InDels) or single-base mutations (SNVs). The selected mutations need to have appeared in a certain number of samples. Step 3, analyzing the positions of the mutations obtained by screening, and merging the two mutations if the distance between them is less than a certain distance; if the length of the merged region is less than a certain distance, expanding the region to both sides; Step 4: After the region obtained in step 3 is merged with the COSMIC database, the microsatellite loci and fusion gene regions are included. For the obtained region, if the interval is less than or equal to a certain length, it is expanded to both sides. If it is greater than a certain length, it is split by sliding window to obtain the probe capture design interval; Step 5, according to the probe capture design interval, after removing the area with low specificity, the probe is designed; Step 6: Perform high-throughput sequencing of the sample to be tested by liquid phase capture method according to the designed probe, compare the sequencing results with the reference genome, and determine whether there is a mutation in the region captured by the probe.

[0011] The certain number refers to more than 10-40 samples.

[0012] The certain length mentioned refers to 5-15 bp.

[0013] Less than a certain distance means less than 5-15 bp, less than a certain length means less than 70-90 bp, and the extended region means making the full length 70-90 bp.

[0014] In step 4, the size of the sliding window is 100-140 bp, less than or equal to a certain length means less than or equal to 100-140 bp, and greater than a certain length means greater than 100-140 bp.

[0015] The COSMIC database is version V97.

[0016] The fusion genes are ALK, ROS1, RET, NTRK, NRG, FGFR, MET, EGFR, HER2 and BRAF.

[0017] The microsatellite loci are screened in the following manner: Obtain healthy subjects and colorectal cancer MSI-positive samples for WGS sequencing, find single nucleotide repeat sequences with a length of 10-20bp, and screen out microsatellite loci with a coverage depth greater than 40-60% of the average depth of the samples themselves in 70-90% of the samples; The obtained microsatellite loci were used as feature values ​​for binary classification of samples, and microsatellite loci with AUC values ​​greater than 0.75-0.95 were selected.

[0018] In step 5, the probe length is 110-130 bp.

[0019] The target cancer types include lung cancer, colorectal cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, bone and soft tissue sarcoma, head and neck tumors, urothelial carcinoma, kidney cancer, prostate cancer, melanoma, gastrointestinal stromal tumors, biliary tract tumors, lymphoma and central nervous system tumors.

[0020] The sample to be tested is derived from plasma free DNA.

[0021] A detection system for high-sensitivity detection of ctDNA in solid tumors, comprising: An extraction module, used to extract ctDNA from a subject's sample; The sequencing module is used to perform WES sequencing on ctDNA and compare it with the reference genome to obtain mutations in the gene coding region; The mutation type screening module is used to screen out insertion and deletion (InDel) or single base mutation (SNV) from the mutations obtained from the sequencing module, and the screened mutations need to have appeared in a certain number of samples; The first region merging module is used to analyze the positions of the mutations screened by the mutation type screening module. If the distance between the positions of two mutations is less than a certain distance, they are merged; if the length of the region after merging is less than a certain distance, the region is expanded to both sides; The second region merging module is used to merge the region obtained by the first region merging module with the COSMIC database, and then incorporate the microsatellite loci and fusion gene regions. For the obtained region, if the interval is less than or equal to a certain length, it is expanded to both sides. If it is greater than a certain length, it is split by sliding windows to obtain the probe capture design interval; The probe design module is used to design probes according to the probe capture design interval and remove the area with low specificity; The comparison module is used to obtain the sequencing results obtained by high-throughput sequencing of the sample to be tested through the sequencing module using the liquid phase capture method according to the designed probe, and compare the sequencing results with the reference genome to determine whether there is a mutation in the area captured by the probe.

[0022] Compared with the prior art, the present invention has significant advantages. First, as a non-invasive liquid biopsy method, the present invention avoids the invasiveness of traditional tissue biopsy, reduces the pain and risk of the subject, and can perform repeated sampling, which is convenient for real-time monitoring of the dynamic changes of the tumor. Secondly, the present invention significantly increases the number of gene mutations detected in the sample through a specific gene combination, improves the detection sensitivity, and thus enhances the ability to detect early diagnosis and minimal residual lesions (MRD). Through optimized test procedures and bioinformatics analysis algorithms, false positive and false negative results are reduced, and the accuracy of detection is improved. In addition, the present invention covers 20 major clonal mutation genes related to solid tumors that are highly prevalent in the Chinese population, important targeted drug-related mutations, and prognosis-related genes, providing comprehensive gene variation information.

[0023] By applying high-throughput sequencing technology, the present invention can process a large number of samples in a short period of time, improve detection efficiency, and can timely monitor ctDNA changes after surgery or other treatment plans are implemented to evaluate the treatment effect, provide real-time information for clinical treatment decisions, and reduce the risk of subjects being unable to undergo secondary surgery due to delayed diagnosis. In summary, the present invention can provide more accurate diagnostic information for tumor subjects, help guide clinical treatment decisions, and has important clinical application value and social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the coverage of the probe of the present invention on the genome.

[0025] Figure 2 When the probe of the present invention detects MRD quality control products EGFR -p.L858R& EGFR- p.T790M& EGFR -p.E746_A750del& IDH1 -p.R132G& TP53 -p.R273H& KRAS -IGV visualization of mutations such as p.G12C.

[0026] Figure 3 This is a distribution diagram of the proportion of the population in which the probe of the present invention detects specific mutations in highly prevalent cancers in China.

[0027] Figure 4 When the probe of the present invention detects MRD quality control products with different mutation abundances PIK3CA - IGV visualization of the p.H1047R mutation.

[0028] Figure 5 The probe of the present invention detects the MRD status of plasma samples of patients with early and middle stage lung cancer 3-7 days after surgery, and the survival curves of MRD-negative and -positive patients. DETAILED DESCRIPTION

[0029] In order to further explain the present invention, a specific embodiment is provided below for detailed explanation. However, it should be noted that the embodiments provided are only exemplary in nature, intended to illustrate the core concept of the present invention, and are not intended to limit the scope of the present invention. In the embodiments, for technical details or conditions not specifically described, reference can be made to the existing literature in the relevant field, or the relevant product instructions can be followed for operation. In addition, the reagents or instruments mentioned in the text are standard products that can be generally purchased on the market unless a brand or manufacturer is specifically specified.

[0030] The present invention relates to a detection method comprising a specific gene combination, a probe library for high-throughput sequencing, and a kit for implementing the detection method. The technical field includes, but is not limited to, biotechnology, medical research, disease diagnosis, gene detection, molecular biology, and personalized medicine. The present invention aims to provide a tool and method that can accurately and efficiently detect high-frequency major clone mutations in high-incidence solid tumors in the Chinese population, so as to play an important role in early diagnosis of tumors, efficacy monitoring, and prognosis evaluation.

[0031] In the description of the present invention, the term "high-throughput sequencing technology" is a broad concept, which covers the second-generation high-throughput sequencing technology and all the sequencing technologies developed thereafter with higher throughput characteristics. The application platforms of these sequencing technologies include but are not limited to the Illumina platform, the Ion Torrent platform, the BGI sequencing platform and other high-throughput sequencing platforms. When implementing the present invention, a suitable high-throughput sequencing technology platform can be selected according to actual needs to achieve the best experimental effect.

[0032] When designing the probe for the MRD detection kit, the cancer types are preliminarily determined based on the mortality rates of cancer types in the literature: a) cancer types with a high incidence rate in the Chinese population (such as lung cancer, gastric cancer, intestinal cancer, breast cancer, cervical cancer, etc.) are selected; b) cancer types with a longer survival period, whose subject population will benefit more; c) cancer types with high clinical attention; d) the number of WES test samples for the selected cancer types is ≥80 cases.

[0033] Finally, 20 types of cancer were identified (lung cancer, colorectal cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, bone and soft tissue sarcoma, head and neck tumors, urothelial carcinoma, kidney cancer, prostate cancer, melanoma, gastrointestinal stromal tumors, biliary tract tumors, lymphoma, and central nervous system tumors). The samples included nearly 10,000 patent applications and the internal Chinese tumor WES sample library of the Gene. Probes were designed based on the enriched regions of the mutation sites of these samples, as well as coverage of the COSMIC database mutation hotspots and clinical driver mutation library.

[0034] The specific sample information is shown in Table 1: Table 1 Sample information

[0035] Specifically, the probe design process includes the following steps: 1. Filter common false mutations through the healthy population big data baseline database; 2. Perform statistics from the tumor WES sample library and screen mutations that occur in the gene coding region. The limited types include insertion deletion (InDel) or single nucleotide variation (SNV) mutations. More specific types can be missense mutations, frameshift mutations, nonsense mutations, non-frameshift mutations, splicing mutations, transcription start site mutations, stop codon mutations, synonymous mutations, etc. 3. The retained variant sites occurred in at least 20 samples; 4. Further filtering: 1) Filter multiple homologous genes on the genome, such as ( KRTAP2-2, KRTAP2-3, KRTAP4-4 etc.); for mutations that meet the conditions and whose hotspot coordinates are within 10 bp, the regions are merged. After merging, if the merged region is less than 80 bp, it can be extended to the same length at both ends to further expand to a region of 80 bp (or 81 bp). If the length of the merged region is greater than 80 bp, the region is not extended; 5. Statistical step: Obtain the performance of each interval of the site interval set in the pan-cancer data; Perform pan-cancer and cancer type count statistics. Pan-cancer types are mutations carried in any tumor sample, and are counted by sample (e.g., sample A has 2 mutations in interval α, but the sample count is recorded as 1).

[0036] 6. Adaptive weights are used to select pan-cancer and single-cancer contributions to intervals; 7. After tracing back the coordinates of the main contributing variant sites in each interval in the previous step and merging them with the COSMIC database (V97), further add microsatellite site regions and fusion regions. For the obtained intervals, if the interval is <=120bp, expand it to a 120bp interval; if the interval is already greater than 120bp, use 120bp as a sliding window and split it into 120bp regions in sequence. If the last region is less than 120, design from the end to the front; The microsatellite loci included here were screened in the following way: Screening of satellite sites: a. Whole exome sequencing (WES) data of healthy subjects and colorectal cancer MSI-positive samples (PCR method, obtained by testing 5 loci (NR-27, NR-24, NR-21, BAT-25 and BAT-26)) with qualified quality control (sequencing depth>150X), 100 samples each.

[0037] b. From the WES genome coverage area, single nucleotide repeat sequences with a length of 10 to 20 bp were screened as candidate satellite sites.

[0038] c. Preliminary screening of satellite loci: more than 80% of the samples must have a microsatellite locus coverage depth greater than 50% of the average depth of the samples themselves.

[0039] d. For the qualified satellite loci screened in the previous step, the coverage depth and the number of alleles of different lengths of each sample at each microsatellite locus were counted, and the frequency of each allele was calculated. The SVM algorithm was used as a feature for binary classification training, and the loci with an AUC value greater than 0.85 were selected as satellite loci with high MSI resolution.

[0040] The selection of fusion genes here is based on the recommendations of the Chinese Society of Clinical Oncology (CSCO) Guidelines for the Diagnosis and Treatment of Non-Small Cell Lung Cancer (2024 Edition), the NCCN Oncology Clinical Practice Guidelines for Non-Small Cell Lung Cancer (2024 Edition 11), the Chinese Clinical Diagnosis and Treatment Guidelines for Rare Target Targeted Therapy of Primary Lung Cancer (2024 Edition), and the Chinese Expert Consensus on Clinical Practice of Fusion Gene Detection in Non-Small Cell Lung Cancer (2023 Edition), and is selected as ALK, ROS1, RET, NTRK, NRG, FGFR, MET, EGFR, HER2, and BRAF.

[0041] 8. Use bam data of healthy human WES samples to test sequence region specificity. The test principle is to calculate the total number of sequences (N) in the specified region sequence, and the number of sequences with sequence mapQ values ​​less than 60 (L), and calculate the low sequence specificity ratio r=L / N. If r is lower than 80, it is considered that the region has low specificity, and this region is filtered out, and a 120bp length probe is designed; 9. Add microsatellite stability detection probes and gene fusion probes.

[0042] 10. Identify and eliminate probes with low-complexity base sequences; 11. Use BLAST to verify the uniqueness of the probe sequence on the genome, and further confirm the probe performance by reading the sequence alignment quality value of the bam data at the probe position for the multiple alignment probe.

[0043] 12. The above probes are combined into a probe library for high-sensitivity detection of solid tumor ctDNA (named MRD Ultrapanel) for MRD detection of solid tumors.

[0044] Based on the above methods, the design of high-sensitivity gene probes for solid tumor ctDNA detection was completed, and about 6,000 probes were developed, which were evenly distributed on the human genome. Each probe was synthesized separately and quality controlled to ensure uniform coverage and improve detection quality and accuracy. The design and sequence optimization of the detection gene probes were completed, and probes with poor specificity and coverage were removed by comparison with the database. The probe length ranged from 120 bases, and the GC ratio was between 40% and 60%. The conditions for the probe to bind to the target sequence were established.

[0045] The present invention also provides a method for using a high-sensitivity detection kit for solid tumor ctDNA under the DNBSEQ-T7 platform. The bioinformatics analysis results after the machine can be used for efficacy monitoring or prognosis evaluation of solid tumor subjects. The method of use is as follows: 1. ctDNA extraction: To extract circulating tumor DNA (ctDNA) from plasma and other body fluid samples, it is recommended to use a commercial nucleic acid extraction kit and strictly follow the instructions provided by the manufacturer.

[0046] 2. Library construction: Use an end repair kit to perform DNA end repair operations according to the manufacturer's recommended conditions. At the same time, use T4 polynucleotide kinase to 5' phosphorylate the DNA fragments, and add an "A" tail to the 3' end under the action of Taq DNA polymerase and dATP; add universal short adapters to both ends of the fragment DNA (the universal adapter includes the amplification primer sequence and part of the sequencing primer sequence), and then introduce the Index sequence, the sequence complementary to the P5 / P7 oligo, and the remaining sequencing primer sequence through the Index Primer during the PCR process (using the molecular labeling technology described in the CN113373524A patent); magnetic bead purification will be performed after PCR to remove the reaction reagents and possible primer dimers and adapter dimers.

[0047] 5. Library enrichment: Utilizing the high affinity of biotin and streptavidin, the target region of genomic DNA bound to the probe (combination probe library for high-sensitivity detection of solid tumor ctDNA) is captured using streptavidin magnetic beads; a dedicated hybridization wash solution is used to remove non-specific DNA, and PCR amplification is performed to obtain the final captured library.

[0048] 6. Library quantification: Perform final qPCR amplification and quantification on the purified DNA product.

[0049] 7. Circularization and DNB preparation: The double-stranded DNA library is denatured into single-stranded DNA by high temperature, and then the two ends of the single-stranded DNA are connected to form a single-stranded circular DNA molecule through the complementary pairing of the splint oligo primer and the single-stranded DNA. The single-stranded circular DNA molecule is rolled circle amplified to form DNA nanoballs (DNBs).

[0050] 8. Sequencing and data analysis: DNBs are added to the array flow cell and high-throughput sequencing is performed using the DNBSEQ-T7 sequencer. After sequencing, the raw data is quality controlled and bioinformatics analyzed to obtain reliable biological information.

[0051] Example 1 Filtering false mutations before probe design / can improve detection specificity The present invention relates to a probe design method for improving the detection specificity of NGS kits. The method aims to reduce the detection rate of false mutations by introducing a multi-step screening process, thereby enhancing the accuracy of detection. Specifically, the screening process includes excluding common false mutations in the healthy population database, eliminating low-frequency mutations with mutation frequencies below a specific threshold, excluding mutations related to homologous genes, optimizing the detection contribution of mutation-related regions in pan-cancer and single cancer, using healthy population sample analysis to exclude low-specificity regions, and eliminating low-complexity probes. Tables 2-1 to 2-22 are some of the false mutations that have been filtered out after the above process analysis, and the mutation frequency of these mutations in the population is >98%. Through the above-mentioned comprehensive screening measures, the present invention can effectively improve the specificity of probe mutation detection, while reducing the cost of probe manufacturing and kit detection, and improving the efficiency of the probe.

[0052] Table 2-1 High-frequency false mutation table of healthy people database (Part I)

[0053] Table 2-2 High-frequency false mutation table of healthy people database (Part II)

[0054] Table 2-3 High-frequency false mutation table of healthy people database (Part 3)

[0055] Table 2-4 High-frequency false mutation table of healthy people database (Part 4)

[0056] Table 2-5 High-frequency false mutation table of healthy people database (Part 5)

[0057] Table 2-6 High-frequency false mutation table of healthy people database (Part 6)

[0058] Table 2-7 High-frequency false mutation table of healthy people database (Part VII)

[0059] Table 2-8 High-frequency false mutation table of healthy people database (Part 8)

[0060] Table 2-9 High-frequency false mutation table of healthy people database (Part 8)

[0061] Table 2-10 High-frequency false mutation table of healthy people database (Part 10)

[0062] Table 2-11 High-frequency false mutations in the healthy person database (Part 11)

[0063] Table 2-12 High-frequency false mutation table of healthy people database (Part 12)

[0064] Table 2-13 High-frequency false mutation table of healthy people database (Part 13)

[0065] Table 2-14 High-frequency false mutations in the healthy person database (Part 14)

[0066] Table 2-15 High-frequency false mutation table of healthy people database (Part 15)

[0067] Table 2-16 High-frequency false mutation table of healthy people database (Part 16)

[0068] Table 2-17 High-frequency false mutations in the healthy person database (Part 17)

[0069] Table 2-18 High-frequency false mutations in the healthy person database (Part 18)

[0070] Table 2-19 High-frequency false mutations in the healthy person database (Part 19)

[0071] Table 2-20 High-frequency false mutation table of healthy people database (Part 20)

[0072] Table 2-21 High-frequency false mutation table of healthy people database (Part 21)

[0073] Table 2-22 High-frequency false mutation table of healthy people database (Part 22)

[0074] Example 2 Testing the mutation type detection accuracy of the kit of the present invention We used the minimal residual lesion detection ctDNA quality control product (GW-OCTM800) to test the accuracy of mutation type detection. We selected 0% and 0.5% mutation frequency gradients in the quality control product for testing. The mutation results of these two samples were double-verified by ddPCR and high-throughput sequencing platforms. We used the detection results of these two platforms as the standard to test the mutation detection performance of the kit of the present invention. The 0.5% MRD quality control product contains 27 hotspot mutation sites covered by the kit of the present invention, involving AKT1, ALK, BRAF, EGFR, ERBB2, IDH1, IDH2, KIT, KRAS, NRAS, PDGFRA, PIK3CA, TP53 These genes are genes with relatively high mutation frequencies in solid tumors, and some of their mutations are targets for targeted therapeutic drugs. The test results can guide the subjects' targeted treatment.

[0075] Table 3 Mutation detection results of the kit of the present invention

[0076] The kit of the present invention was used to test two quality control products, and the test results are shown in Table 3. The quality control product with a mutation abundance of 0% was tested by ddPCR and a high-throughput sequencing platform, indicating that all 27 mutations were negative. Similarly, the test results of this kit were all negative. The quality control product with a mutation abundance of 0.5% was tested by ddPCR and a high-throughput sequencing platform, indicating that all 27 mutations were positive. The test results of this kit were also positive, and the base mutation form and amino acid mutation form of the detected mutations were 100% consistent with the standard results, as shown in Table 3. Figure 2 As shown ( EGFR -p.L858R& EGFR -p.T790M& EGFR -p.E746_A750del& IDH1 -p.R132G& TP53 -p.R273H& KRAS-p.G12C mutations as an example). The above results were statistically analyzed, and the results are shown in Table 4. When this kit was used for mutation detection, when this kit was used to detect MRD quality control products with a mutation abundance of 0.5%, the detection sensitivity was 100%; when the quality control products with a mutation abundance of 0% were detected, the detection specificity was 100%. Combining the two results, the detection accuracy can reach 100%.

[0077] Table 4 Mutation detection results of the kit of the present invention

[0078] In addition to the significant advantages of the kit of the present invention in detecting gene mutations, it can also identify gene fusions, thereby providing a wider range of mutation type detection. In order to verify the accuracy of our kit in detecting gene fusions, we screened plasma samples from the Shihe Gene Tumor Sample Database and tested them using the kit of the present invention. ALK, ROS1, RET, NTRK1, FGFR2, FGFR3 and EWSR1 Isogenic fusion.

[0079] Given that the above-mentioned gene detection kits for liquid biopsy have not yet been approved in China, and the DNA information in plasma samples usually comes from the patient's tumor samples, we used the tissue detection kit approved by the National Medical Products Administration NMPA on October 12, 2023-the Shihe No. 1 kit as a reference. The detection range of this kit covers the above-mentioned genes that we are concerned about. In order to further verify the accuracy of the kit of the present invention in detecting the results of plasma ctDNA fusions, we further selected from the screened samples those patients whose plasma samples were tested by the kit of the present invention and whose paired tumor tissue samples were sequenced by the Shihe No. 1 kit. Finally, we randomly screened out 40 patient samples for comparative analysis, and the test results are shown in Tables 5-1 to 5-2. There were a total of 43 fusions in the plasma of these patients, including 20 ALK Fusion, 8 ROS1 Fusion, 7 RET Fusion, 1 NTRK1 Fusion, 1 FGFR2 Fusion, 3 FGFR3 Fusion and 2 EWSR1 Fusion, the 43 fusion products detected by the kit of the present invention have all been verified in tumor tissue samples. The detection results of gene fusion products by the kit of the present invention are 100% consistent with the detection results of the kit approved by NMPA for marketing.

[0080] According to the classification of fusion products, there are 34 fusion products in total. The fusion breakpoints are diverse, but most of them occur in the intron region of the gene. This shows that for fusion detection, it is very necessary for the probe to cover the intron region of the gene. The patented kit of the present invention increases the probe coverage of the intron region for gene fusions related to clinical treatment / molecular typing / prognosis to prevent missed fusions. In addition to detecting reported gene fusion products, the patented kit of the present invention also detects 6 new fusions that have not been reported. CAPN13:exon23~ALK:exon20, CLASP1:exon15~ ALK:exon20, FGFR3:exon14~IGR (upstream PTPN13), IGR (upstream SEMA4A)~NTRK1: exon12, CCDC170:exon5~ROS1:exon33 and EWSR1:exon7~IGR (downstream BOD1L1) This indicates that this patented test kit also has outstanding detection advantages in the discovery of new fusions, increasing patients' opportunities for clinical targeted treatment, while also providing more information for the formulation of clinical plans after tumor recurrence in patients.

[0081] Table 5-1 Gene fusion types detected by the kit of the present invention and verification results (Part I)

[0082] Table 5-2 Gene fusion types detected by the kit of the present invention and verification results (Part II)

[0083] Example 3 Testing the average number of mutations detected by the kit of the present invention in highly prevalent cancers in China The kit of the present invention is a fixed panel for cancer types, and covers more genes than conventional liquid biopsy kits. Compared with personalized panels, the kit of the present invention can also detect new tumor mutations, overcome the influence of tumor heterogeneity, and provide convenience for subjects whose tissues are not accessible.

[0084] Since the number of sample mutations detected by the kit of the present invention is higher than that of the conventional kit, when the LOD (Limitation of Detection) of single-point mutations is the same, the more mutations detected, the lower the LOD of sample detection. In order to test the number of mutations detected by the kit of the present invention in high-incidence cancers in China, we used the kit to detect real-world clinical tumor samples from 490 subjects, including non-small cell lung cancer, colorectal cancer, gastric cancer, liver cancer and esophageal cancer. The number of mutations detected in each sample was detected and analyzed, and the average number of mutations detected in each cancer was counted according to the type of cancer. The analysis results showed that the average number of mutations in non-small cell lung cancer, colorectal cancer, gastric cancer, liver cancer and esophageal cancer samples was 14, 21, 16, 13 and 13, respectively. The number of mutations detected in colorectal cancer and gastric cancer samples was more, and the average number of mutations detected in all cancers was 17, which was higher than the average number of mutations detected in conventional liquid biopsy kits (about 3 mutations). In addition, we also analyzed the proportion of people with specific mutations detected in each cancer type, such as Figure 3 As shown, using the kit of the present invention for testing, 94% of the population is covered with ≥5 mutations, which is significantly better than conventional liquid biopsy kits.

[0085] Example 4 Testing the performance of the kit of the present invention in detecting MRD samples We used the ctDNA quality control product (GW-OCTM800) for minimal residual lesion detection to test the mutation detection performance. The quality control product contains 4 mutation frequency gradients, which are 0%, 0.005%, 0.05%, and 0.5%, respectively. The MRD quality control product contains 27 hotspot mutation sites covered by the kit of the present invention. We used the kit of the present invention to detect the 4 gradient MRD quality control products. The 4 gradient samples of 0%, 0.5%, 0.05%, and 0.005% were named M801~M804, respectively, and the sequencing depths were 28673X, 35889X, 37045X, and 23410X, respectively. The test results are shown in Table 6. The quality control product M801 with a mutation abundance of 0% and the quality control product M802 with a mutation abundance of 0.5% have been verified by ddPCR and high-throughput sequencing platforms. We used the detection results of these two platforms as standards to test the mutation detection performance of the kit of the present invention.

[0086] The M801 sample is a negative reference, which is a DNA sample with negative somatic mutation. The test results of the kit of the present invention for 27 mutation sites are also negative, with mutation abundances of 0% and a coincidence rate of 100% (27 / 27). The test results are correct, indicating that the specificity of mutation detection of the kit of the present invention is high.

[0087] Table 6 Mutation detection results of the kit of the present invention for detecting four mutation abundance gradient MRD quality control products

[0088] The gene mutations in the samples M802~M804 were in three mutation abundance gradients, namely 0.5%, 0.05% and 0.005%. When the kit of the present invention detected the M802 sample, 27 mutations were detected 100%, and the average mutation abundance was 0.51%, which was consistent with the standard abundance of 0.5%, indicating that the kit of the present invention had high detection accuracy when detecting mutations with a mutation abundance of 0.5%. The mutation abundance was further lowered to 0.05% (M803 sample), and the kit of the present invention detected 22 mutations with an average mutation abundance of 0.05%, which was completely consistent with the standard abundance of 0.05%, indicating that the kit of the present invention still had good detection ability when detecting a mutation abundance of 0.05%. When the mutation abundance reached a very low level of 0.005% (M804 sample), the kit of the present invention was still able to detect 5 mutations, and the base mutation form and amino acid mutation form of the detected mutations were 100% consistent with the standard results, such as Figure 4 As shown (taking PIK3CAp.H1047R mutation as an example), it is shown that the kit of the present invention still has a certain detection ability under ultra-low mutation abundance.

[0089] In summary, if any mutation is detected in the sample according to the MRD evaluation criteria, it is MRD positive. For samples with MRD levels of 0%, 0.5%, 0.05% and 0.005%, the commercial MRD quality control product was tested by the kit of the present invention, and the accuracy of evaluating the MRD status was 100%.

[0090] Next, we used the gradient dilution method to increase the mutation abundance gradient to 7, which were 0.1%, 0.05%, 0.03%, 0.01%, 0.005%, 0.003%, and 0%. At the same time, the detection performance was tested by repeating the batches twice and repeating the batches twice. The test results are shown in Table 7. When detecting samples with a mutation abundance ≥ 0.005%, the MRD positivity rate was 100% (15 / 15). When detecting samples with a mutation abundance of 0.003%, the MRD positivity rate was 50% (2 / 4). When detecting MRD negative quality control products, the results of 4 batches were all negative, with a specificity of 100% (4 / 4).

[0091] Table 7 Mutation detection results of 7 mutation abundance gradient MRD quality control products detected by the kit of the present invention

[0092] Example 5 Testing the feasibility of using the kit of the present invention in high-sensitivity detection of solid tumor ctDNA In order to test the feasibility of the kit of the present invention in the high-sensitivity detection of ctDNA in solid tumors, the main application scenarios include the recurrence risk and prognosis assessment after radical treatment of early and middle-stage patients, the evaluation of the efficacy of neoadjuvant therapy, the guidance of auxiliary program formulation, and the monitoring of the therapeutic efficacy of patients with advanced tumors. Taking patients with early and middle-stage lung cancer who underwent radical surgery as an example, we used the kit of the present invention to detect 3-7 days of plasma ctDNA samples of 63 lung cancer patients who underwent radical surgery, analyzed the postoperative plasma MRD status, and associated the patients' disease-free survival (DFS) to explore the correlation between MRD status and DFS.

[0093] The basic clinical information of the 63 patients is shown in Table 8. The median age was 63 years old, and the majority of them were male, accounting for about 63.5%. In terms of staging, patients in stages I, II, and III each accounted for about one-third, and the overall staging was early. At this time, the patient's tumor was small and most of them had no metastasis. In terms of pathological type, half of the patients had lung adenocarcinoma, and about 40% had lung squamous cell carcinoma.

[0094] Table 8 Clinical information of lung cancer patients

[0095] The kit of the present invention was used to detect plasma ctDNA samples of 63 patients 3-7 days after surgery. Among them, mutations were detected in plasma ctDNA of 25 patients, which were defined as MRD positive, and the other 38 patients were MRD negative. Survival curves were drawn, as shown in Fig. Figure 5 The DFS of MRD-positive patients was significantly shorter than that of MRD-negative patients. P <0.0001, indicating that the kit of the present invention has good clinical application value in high-sensitivity detection of solid tumor ctDNA.

Claims

1. A method for high-sensitivity detection of ctDNA in solid tumors, wherein the method is used for non-therapeutic and diagnostic purposes, characterized in that: The following steps are involved: Step 1, obtaining a subject sample, extracting ctDNA and setting a target cancer type; the subject sample includes a healthy control sample and a tumor-positive sample; Step 2: Perform WES sequencing on ctDNA and compare it with the reference genome to obtain mutations in the gene coding region, screen out insertion and deletion mutations or single-base mutations, and the screened mutations need to have appeared in a certain number of samples; Step 3, analyzing the positions of the mutations obtained by screening, and merging the two mutations if the distance between them is less than a certain distance; if the length of the merged region is less than a certain distance, expanding the region to both sides; Step 4: After the region obtained in step 3 is merged with the COSMIC database, the microsatellite loci and fusion gene regions are included. For the obtained region, if the interval is less than or equal to a certain length, it is expanded to both sides. If it is greater than a certain length, it is split by sliding window to obtain the probe capture design interval; Step 5, according to the probe capture design interval, after removing the area with low specificity, the probe is designed; Step 6: Perform high-throughput sequencing of the sample to be tested by liquid phase capture method according to the designed probe, compare the sequencing results with the reference genome, and determine whether there is a mutation in the region captured by the probe.

2. The detection method for high-sensitivity detection of ctDNA in solid tumors according to claim 1, characterized in that: The certain number mentioned refers to more than 10-40 samples; the certain length mentioned refers to 5-15bp.

3. The detection method for high-sensitivity detection of ctDNA in solid tumors according to claim 1, characterized in that: Less than a certain distance means less than 5-15 bp, less than a certain length means less than 70-90 bp, and the extended region means making the full length 70-90 bp.

4. The detection method for high-sensitivity detection of solid tumor ctDNA according to claim 1, characterized in that: In step 4, the size of the sliding window is 100-140 bp, less than or equal to a certain length means less than or equal to 100-140 bp, and greater than a certain length means greater than 100-140 bp.

5. The detection method for high-sensitivity detection of ctDNA in solid tumors according to claim 1, characterized in that: The COSMIC database is version V97; the fusion genes refer to ALK fusion, ROS1 fusion, RET fusion, NTRK fusion, NRG fusion, FGFR fusion, MET fusion, EGFR fusion, HER2 fusion and BRAF fusion.

6. The detection method for high-sensitivity detection of ctDNA in solid tumors according to claim 1, characterized in that: Healthy people and colorectal cancer MSI-positive samples were obtained for WGS sequencing, and single nucleotide repeat sequences with a length of 10-20bp were found in them, and microsatellite loci with a coverage depth greater than 40-60% of the average depth of their own samples in 70-90% of the samples were screened out; the obtained microsatellite loci were used as characteristic values ​​according to their allele frequencies, and the samples were binary classified, and microsatellite loci with AUC values ​​greater than 0.75-0.95 were selected.

7. The detection method for high-sensitivity detection of ctDNA in solid tumors according to claim 1, characterized in that: In step 5, the probe length is 110-130bp; the target cancer types are lung cancer, colorectal cancer, gastric cancer, liver cancer, esophageal cancer, breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, bone and soft tissue sarcoma, head and neck tumors, urothelial carcinoma, kidney cancer, prostate cancer, melanoma, gastrointestinal stromal tumors, biliary tract tumors, lymphomas and central nervous system tumors.

8. The detection method for high-sensitivity detection of ctDNA in solid tumors according to claim 1, characterized in that: In the step 1, among the mutations in the gene coding region, it is necessary to delete the false mutations; The pseudomutation is obtained through sequencing data of healthy people; The positions of the pseudomutations on the genome are chr11:108141955, chr8:145738768, chr2:215657183, chr18:60794268, chr20:43963724, chr19:36214871, chr6:152265282, chr11:69466115, chr18:48584855, chr17:56435161, chr9:8341281, chr3:10128985, chr16:9934671, chr10:89725230, chr2:141625541, chr13:489 53655, chr17:29508819, chr22:29687622, ​​chr22:41550984, chr20:43963730, chr4:55152284, chr21:42866994, chr6:157099427, chr9:13939770 7. chrX:44935925, chr3:52621586, chr4:55956320, chr18:60794267, chrX:44935925, chr17:7577679, chr6:117642992, chr6:157099313, chr13:4 8954283, chr16:9934671, chrX:44935878, chr3:30691871, chr12:58142004, chr4:187532982, chr2:43793963, chr2:202141738, chr11:10818826 7. chr7:116409675, chr10:123242780, chr6:117642993, chr5:112162713, chr1:26227631, chr21:42867553, chr19:10602180, chr4:55598211, chr 2:141625630, chr2:48032613, chr17:74733099, chr12:6705138, chr11:119144809, chr4:55161517, chr1:27107263, chr18:48584855, chr2:9584 9385, chr12:6705122, chr21:42867554, chr4:66468047, chr11:12549076 6. chr5:112111310, chr11:108141956, chr17:7579644, chr21:42873564,chr13:48953656、chr4:185340726、chr2:47635523、chr4:187532981、chr11:119144792、chr22:29685620、chr4:55598210、chr7:116381122、chr10:8111346、chr11:118353054、chr6:162683815、chr16:9934691、chr9:5073682、chr15:91303326、chr13:48954160、chr11:108121411、chr16:9934691、chr6:163899920、chr22:41545024、chr1:115256669、chr21:42871731、chr5:170837514、chr13:48955363、chr16:67646013、chr18:48584855、chr8:92998419、chr17:47696481、chr7:103202407、chr4:1806012、chr2:209113049、chr20:43962266、chr3:41268676、chr21:42867842、chrX:44935877、chr1:161332092、chr2:173435579、chr2:29445298、chr22:41551232、chr2:42499218、chr9:80343588、chr21:42866995、chr21:42877365、chr3:178927848、chr10:123240430、chr12:58142005、chrX:123204993、chr2:47635524、chr2:48033891、chr11:102056691、chr15:90631775、chr10:8115668、chr13:48953655、chr4:55598212、chr3:142274740、chr5:112111309、chr8:145738582、chr1:156844822、chr13:48939205、chr6:117645779、chr22:41550985、chr3:49406080、chr3:10128966、chr8:92983160、chr4:55139978、chr4:25678531、chr22:41565469、chr2:47641559、chr22:41550984、chr6:117725383、chr11:108141956、chr3:10128965、chr6:114292040、chr12:133250138、chr11:69465873、chr3:10128966、chr7:116409676、chr2:29446721、chr9:80343587、chr10:123241332、chr2:141202033、chr4:1809111、chr10:8111345、chr11:108117898、chr11:108117897、chrX:44935925、chr21:42866995、chr12:6705123、chr2:141283603、chr6:117725384、chr4:1809124、chr11:100922404、chr3:70008317、chr12:58142005、chr11:108141956、chr11:119144791、chr6:134494548、chr21:42877162、chr2:173435578、chr17:29528406、chr2:141267744、chr3:41265953、chr2:47635524、chr22:30051706、chr6:134494709、chr1:27023451、chr2:198266943、chr9:135773000、chr10:123240904、chr11:108114662、chr2:29447253、chr10:89720633、chr21:42871730、chr11:125490766、chr22:30051706、chr13:48922985、chr11:119144792、chr2:48032740、chr11:125490766、chr10:8115669、chr3:10128965、chr7:81381621、chr22:29683752、chr3:89448446、chr1:161332092、chr3:10128966、chr5:170837514、chr2:111886181、chr7:142460261、chr11:118353037、chr17:7577678、chr2:141625631、chr3:142274739、chr20:43964407、chr2:48032740、chr5:149784087、chr2:111886180、chr2:43793963、chr1:161332091、chr16:9934670、chr7:116381121、chr10:123279745、chrX:123204979、chr10:8115687、chr1:243736211、chr18:60794193、chr21:42866995、chr8:128750607、chr11:108188266、chr11:118369265、chr2:42499218、chr2:48032875、chr18:48584856、chr9:126217129、chr6:134494708、chr3:178938736、chr6:117725383、chr22:41565470、chr3:41268677、chr11:125490765、chr13:48922984、chr2:47702451、chr4:185340725、chr22:30051705、chr5:131931452、chr17:7579795、chr9:80343588、chr4:55598212、chr20:43963735、chr8:30969355、chr10:89692731、chr11:118369266、chr21:42867553、chr17:29508820、chr11:108119616、chr8:145738582、chr16:3828847、chr22:24135969、chr16:3828848、chr22:29687480、chr2:47702452、chr7:116409676、chr11:108114662、chr4:1806013、chr13:49051012、chr11:102056722、chr8:128749940、chr2:215657183、chr2:48032754、chr8:69104521、chr15:91303326、chr3:52621587、chr13:48955676、chr2:95849361、chr11:108119615、chr4:1809128、chr22:29685621、chr4:55956321、chr3:47129781、chr17:7578645、chr9:139399409、chr15:66727689、chr6:157099403、chr3:169988365、chr21:42867842、chr2:48033891、chr4:1809129、chr4:55598202、chr3:178927849、chr9:126217129、chr17:29508820、chr4:55139691、chr7:116409676、chr4:187532981、chr18:42533130、chr13:48954282、chr3:169988365、chr4:25679582、chr10:8115668、chr13:48955364、chr9:126217129、chr12:6705123、chr11:108121411、chr2:48033890、chr10:8115669、chr16:67670473、chr11:108141955、chr11:118369266、chr4:55161391、chr11:118369266、chr11:118353038、chr8:145738582、chr2:48032937、chr17:7577679、chr9:5073681、chr22:30051706、chr11:118353037、chrX:123184950、chr17:7578115、chr1:27107264、chr22:24135968、chr15:66727689、chr6:117642993、chr14:38061517、chr2:48032741、chr2:47702451、chr22:24135969、chr6:117645778、chr11:125490765、chr3:41274764、chr11:108114661、chr4:153268228、chr4:185340725、chr10:89720634、chrX:123204978、chrX:44935878、chr5:112111310、chr9:8341281、chr20:43964130、chr4:55161254、chr10:123240429、chr10:123240904、chr3:181431227、chr7:18688075、chr11:118369265、chr2:48033891、chr17:29508819、chr5:131931451、chr15:66727689、chr2:209113048、chr6:117725384、chr2:215657183、chr4:1806013、chr8:92983159、chr10:8115669、chr9:126217129、chr4:187532982、chrX:66765161、chr14:23652346、chr4:1803497、chr12:416952、chr15:91303325、chr10:8106232、chr21:42867843、chr4:25678659、chr18:60794323、chr1:156845493、chr2:47641746、chr13:48954094、chr2:141598473、chr2:48032741、chr6:163899794、chrX:66765243、chr3:181431226、chr2:47641747、chr21:42867554、chr1:243736211、chr3:41265954、chr17:29508820、chr4:1807894、chr3:47129782、chr18:60795032、chr1:161332092、chr2:111886181、chr6:157099333、chr1:243736210、chr2:141259447、chr10:123242724、chr11:102056723、chr22:41545025、chr11:119144792、chr10:123240287、chr8:30969356、chr22:30051706、chr17:47696482、chr6:117725384、chr4:153268228、chr5:1295257、chr7:103202426、chr11:118353038、chr17:29528407、chr4:1809127、chr22:30051722、chr2:47702451、chr5:170837513、chr17:29528325、chr14:23652347、chr22:24135968、chr3:52621607、chr11:118344186、chr22:41545024、chr11:102056722、chr11:108119616、chr7:6426941、chr1:27100182、chr20:43964124、chr4:1809128、chr22:29130813、chr1:243736211、chr9:37034095、chr12:58142004、chr2:42499218、chr2:95849362、chr7:116409676、chr4:55151958、chr2:173435579、chr12:58142004、chr20:43963047、chr2:111886181、chr6:114292039、chr8:145738583、chr3:52621587、chr5:112164561、chr21:36206932、chr8:11607662、chr2:141032173、chr3:169988364、chr18:60791876、chr6:114292110、chr4:153267968、chr18:60794324、chr9:8341280、chr3:52621587、chr3:37067099、chr22:41551231、chr7:103202407、chr2:141201763、chr2:141665484、chr12:6705122、chr1:243736211、chr2:47635523、chr5:149784088、chr6:163899795、chr8:30969356、chr2:42499217、chr16:9934671、chr5:112175770、chr8:128750605、chr8:145738581、chr11:102056723、chr6:117642992、chr2:42499219、chr2:16082314、chr11:108141956、chr8:128749934、chr2:141625835、chr12:416953、chr7:103202407、chr6:159191717、chr6:134494709、chr18:60791876、chr12:6705122、chr3:30691872、chr8:145738768、chr3:52621586、chr4:25678801、chr2:141625834、chrX:123204979、chr11:69465988、chr11:108188267、chr13:48953655、chr7:18688076、chr21:42867843、chr9:80343587、chr18:60794194、chr11:125490766、chr1:27107284、chr10:123242026、chr11:118353037、chr2:141625542。、 9. A detection system for high-sensitivity detection of ctDNA in solid tumors, characterized in that: include: An extraction module, used to extract ctDNA from a subject's sample; The sequencing module is used to perform WES sequencing on ctDNA and compare it with the reference genome to obtain mutations in the gene coding region; The mutation type screening module is used to screen out insertion / deletion mutations or single-base mutations from the mutations obtained from the sequencing module, and the screened mutations need to have appeared in a certain number of samples; The first region merging module is used to analyze the positions of the mutations screened by the mutation type screening module. If the distance between the positions of two mutations is less than a certain distance, they are merged; if the length of the region after merging is less than a certain distance, the region is expanded to both sides; The second region merging module is used to merge the region obtained by the first region merging module with the COSMIC database, and then incorporate the microsatellite loci and fusion gene regions. For the obtained region, if the interval is less than or equal to a certain length, it is expanded to both sides. If it is greater than a certain length, it is split by sliding windows to obtain the probe capture design interval; The probe design module is used to design probes according to the probe capture design interval and remove the area with low specificity; The comparison module is used to obtain the sequencing results obtained by high-throughput sequencing of the sample to be tested through the sequencing module using the liquid phase capture method according to the designed probe, and compare the sequencing results with the reference genome to determine whether there is a mutation in the area captured by the probe.

10. A computer readable medium, characterized in that The invention also contains computer program code for executing the method according to any one of claims 1 to 8.

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