A screening method for a panel for detecting residual micro-lesions in solid tumors

Abstract

The present invention discloses a screening method for a panel for detecting micro-residual lesions of solid tumors, including detecting ctDNA mutation information of solid tumor patients through a 142-gene detection panel (MRD Panel); and evaluating the genomic instability of solid tumor patients through low-depth WGS sequencing; combining ctDNA mutation information and genomic instability results to obtain an estimate of ctDNA content and give a corresponding risk classification. The present invention provides a method for detecting micro-residual lesions of solid tumors, and the present invention proposes a detection process that can simultaneously detect ctDNA mutation information and genomic instability. By simultaneously evaluating two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of tumor gene detection, and specifically to a screening method for a panel for detecting micro-residual lesions of solid tumors, and more particularly to an evaluation of tumor micro-residual lesions (MRD) based on mutation information (SNV, Indel, Fusion, CNV) and genomic instability information, which is used to determine the tumor burden in patients, evaluate the risk of tumor recurrence after surgery, predict the efficacy, etc. Background Art

[0002] The World Health Organization's International Agency for Research on Cancer (IARC) released the latest global cancer burden data for 2020. The data showed that there were 19.29 million new cancer cases and 9.96 million cancer deaths in 2020, and the global cancer burden remains severe;

[0003] In the early stage, surgical resection is the best treatment for tumors, but a considerable number of patients will still relapse after radical resection. Minimal residual disease (MRD) is considered to be the main cause of recurrence. Currently, imaging methods are mainly used in clinical practice to evaluate the presence of minimal residual lesions, but due to the limitation of detection sensitivity, residual lesions detected by imaging may indicate that the tumor has recurred. High sensitivity and real-time detection of minimal residual lesions can provide an ideal guidance plan for the postoperative management of tumor patients. Detecting tumor-derived signals at the molecular level, including methylation, mutation, copy number changes, etc., to capture information on minimal residual lesions that are invisible in imaging, thereby assessing the patient's tumor burden and risk of recurrence, is beneficial for the implementation of precise treatment for patients.

[0004] The current methods for evaluating microresidual lesions of solid tumors through ctDNA at the molecular level are as follows:

[0005] The application number is CN202110469995; 6, named as a circulating tumor DNA detection system and application for screening tiny residual lesions after colorectal cancer surgery and predicting the risk of recurrence, which discloses the detection of tumor-derived signals at the circulating tumor DNA (ctDNA) level. First, an open source database or a self-built database is used to screen tumor-specific gene information, and a detection panel is constructed. The panel is used to detect the mutation information (including SNV, indel, fusion, CNV, etc.) of the sample, and sequencing is performed by the second-generation sequencing method (NGS). Finally, the variation results at the ctDNA level are analyzed by bioinformatics algorithms. If the variation is positive, it means that MRD is positive and there are tiny residual lesions; if the variation is negative, it means that MRD is negative, and the tumor load is very low or does not exist at the current detection stage; the application number is CN201780007871; 8, named as variant-based disease diagnosis and tracking, a method for tracking the health of patients by longitudinally tracking genetic variants in patients, so that tumor or mutation classification markers can be provided. Longitudinal tracking improves the ability to detect minimal residual disease (MRD; a small number of cells that remain in the patient's body after treatment and / or during remission) and / or treatment response at an early stage, both of which can help guide treatment decisions and prevent missing different intra-tumor / inter-tumor responses in patients. It involves identifying and tracking the genetic diversity of individual tumors and / or patients in order to predict and understand treatment resistance and generate new antigens that can serve as targets for host immune responses. These changes represent the differences and basic hallmarks of tumors, which can ultimately be used to classify tumors and predict progression and treatment efficacy; the application number is CN201910074640X, which discloses a second-generation sequencing-based detection panel, detection kit and its application for pan-cancer targeting, chemotherapy and immunotherapy. Among them, the detection panel includes pan-cancer typing, treatment, and prognosis-related gene mutations, exon regions and microsatellite instability sites related to tumor mutation load calculation.The detection panel includes pan-cancer classification, treatment, prognosis-related gene mutations, tumor mutation load calculation-related exon regions and microsatellite instability sites. The gene information it includes is comprehensive and can directly perform joint detection of multiple tumor mutations. It can be used in the companion diagnosis of targeted drugs, chemotherapy drugs or immunotherapy drugs to obtain accurate results. In addition to mutations, the molecular characteristics of tumors in the above scheme also include structural variations and methylation information at the chromosome level. If only mutation information is used to evaluate microresidual lesions, there will be missed detections, resulting in false negative results. In other words, the large panel (425 genes) + NGS + bioinformatics analysis is used to detect microresidual lesions, which can only detect SNV, indel, fusion, and CNV at the gene level, but cannot detect chromosomal changes at the genome level. If the tumor sample only undergoes changes at the chromosome level, the MRD judgment of the above method will be negative, and there will be false negatives. Therefore, for the detection of microresidual lesions of tumors, it is necessary to detect markers that cover the molecular characteristics of tumors more comprehensively. Summary of the invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a screening method for a panel for detecting residual micro-lesions of solid tumors. The present invention proposes a detection process that can simultaneously detect ctDNA mutation information and genomic instability. By simultaneously evaluating the two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A screening method for a panel for detecting residual micro-lesions in solid tumors, comprising detecting ctDNA mutation information in patients with solid tumors through a 142-gene detection panel; and evaluating genomic instability in patients with solid tumors through low-depth WGS sequencing; combining ctDNA mutation information and genomic instability results to obtain an estimate of ctDNA content and provide a corresponding risk grading.

[0009] As a further solution of the present invention, the steps of constructing the 142-gene detection panel include: confirming the covered cancer types; confirming the covered gene information; and retrieving the NGS test results of solid tumor patients from an existing database for effective area screening.

[0010] As a further solution of the present invention, the confirmed coverage of gene information includes: drug-sensitive and drug-resistant genes, MRD detection and dynamic monitoring areas, tumor driver genes, class I / II gene mutations, MSI sites, chemotherapy sites, covering CFDA, FDA, NCCN guideline recommendations and clinical research stage information sources.

[0011] As a further scheme of the present invention, the method of retrieving the NGS test results of solid tumor patients from an existing database for effective area screening specifically includes: classifying the data according to the type of cancer to obtain the number of samples of each cancer type, and for each cancer type data, evaluating the exons as units within the range of all coding exons of 142 genes; if the current exon contains somatic mutations and the mutation frequency in the population in the current cancer type is greater than or equal to 5%, retaining the exon; performing the same iterative evaluation in other cancer types and exons, retaining the exon regions that meet the conditions, and removing the exon regions that do not meet the conditions, to obtain the screened panel area.

[0012] As a further solution of the present invention, the algorithm for evaluating genomic instability includes: global copy number correction estimation and specific chromosome correction.

[0013] As a further solution of the present invention, the global copy number correction estimation includes whole genome sequencing data based on low coverage, the data is aligned to the human reference genome GRch37, the genome is divided into segments i of 1Mb, and the average coverage within the segment is calculated; coverage depth correction: the regional coverage depth is corrected, and the correction factors include GC content G and alignment degree M, and the GC content and alignment degree M in each segment are calculated.

[0014] Use loess regression fitting: Estimate the parameter coefficients of loess regression, obtain the parameters of the fitted model, calculate the corrected coverage d, and estimate the copy number: calculate the average ratio r for each segment i after correction: , the average ratio is related to the copy number Ci of segment i and the tumor purity p:

[0015] The estimated copy number C of each segment can be calculated by estimating the copy number C that best fits ri and the tumor purity p using the EM algorithm.

[0016] As a further embodiment of the present invention, the special chromosome correction includes: the correction factors for chromosome 19 include the PCR round number pn and the starting DNA amount m, The corrected copy number Cnorm was merged into the final result. The criteria for determining the presence of genomic instability were p>0 and at least >10M fragment copy number C!=2.

[0017] The present invention has the following beneficial effects: The present invention proposes a dual-indicator MRD detection method that can simultaneously detect ctDNA mutation information and genomic instability. By simultaneously evaluating the two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

[0018] In order to more clearly illustrate the structural features and effects of the present invention, the present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 , Figure 2 It is a flow chart of a detection system for a screening method of a panel for detecting residual micro-lesions of solid tumors provided by the present invention.

[0020] Figure 3 , Figure 4 They are respectively schematic diagrams of 2022-01-09 (sample ID: S2200039654): positive result of chromosome instability and 2022-04-11 (sample ID: S2200047668): negative result of chromosome instability in Example 3.

[0021] Figure 5 , Figure 6 They are schematic diagrams of 2021-11-05 (sample ID: PB5968): positive result of chromosome instability and 2022-06-07 (sample ID: PC3677): negative result of chromosome instability in Example 4.

[0022] Figure 7 , Figure 8 , Fig. 9 They are respectively 2022-03-14 (sample ID: S2200045588): negative result of chromosome instability, 2022-04-14 (sample ID: S2200047780): negative result of chromosome instability, and 2022-05-09 (sample ID: S2200050210): schematic diagram of negative result of chromosome instability in Example 5. DETAILED DESCRIPTION

[0023] The present invention will be further explained below in conjunction with the accompanying drawings and related knowledge, and described clearly and completely. Obviously, the described application is only a part of the embodiments of the present invention, rather than all the embodiments.

[0024] Reference Figure 1-Figure 2 As shown, a screening method for a panel for detecting residual micro-lesions in solid tumors includes detecting MRD in patients with solid tumors through a 142-gene detection panel; estimating the ctDNA content by evaluating whether patients with solid tumors have genomic instability; and providing risk grading based on the above test results.

[0025] Among them, the construction steps of the 142-gene detection panel include: confirming the covered cancer types; confirming the covered gene information; retrieving the NGS test results of solid tumor patients in the own database for effective area screening, and further optimizing and confirming the covered gene information including: medication guidance, MRD detection and dynamic monitoring, coverage of drug sensitive and resistant genes, tumor driver genes, class I / II gene mutations, MSI sites, chemotherapy sites, covering CFDA, FDA, NCCN guideline recommendations and clinical research stage information sources;

[0026] The 142 gene list of the present invention is shown in Table 1:

[0027] Table 1: List of 142 genes

[0028]

[0029] Note: After removing duplicates, there are 142 genes in total.

[0030] And retrieve the NGS test results of solid tumor patients in the existing database for effective area screening, specifically including: classify the data according to the type of cancer, obtain the number of samples of each cancer type, and evaluate the data of each cancer type within the range of all coding exons of 142 genes in exons. If the current exon contains somatic mutations and the mutation frequency in the population in the current cancer type is greater than or equal to 5%, retain the exon; perform the same iterative evaluation in other cancer types and exons, retain the exon regions that meet the conditions, remove the exon regions that do not meet the conditions, and obtain the screened panel area.

[0031] The present invention proposes a dual-indicator MRD detection method that can simultaneously detect ctDNA mutation information and genomic instability. By simultaneously evaluating the two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

[0032] In the present invention, the algorithm for assessing genomic instability includes: global copy number correction estimation and specific chromosome correction;

[0033] Further preferably, the global copy number correction estimation includes whole genome sequencing data based on low coverage, the data is aligned to the human reference genome GRch37, the genome is divided into 1Mb segments i, and the average coverage within the segment is calculated; coverage depth correction: the regional coverage depth is corrected, and the correction factors include GC content G and alignment degree M, and the GC content and alignment degree M in each segment are calculated.

[0034] Use loess regression fitting: Estimate the parameter coefficients of loess regression, obtain the parameters of the fitted model, calculate the corrected coverage d, and estimate the copy number:

[0035] After correction, the average ratio r is calculated for each segment i: , the average ratio is related to the copy number Ci of segment i and the tumor purity p:

[0036] The EM algorithm is used to estimate the copy number C of the maximum fit ri and the tumor purity p, and the estimated copy number C of each segment can be calculated; and the special chromosome correction includes: the correction factors for chromosome 19 include the PCR round number pn and the starting DNA amount m, The corrected copy number Cnorm was merged into the final result. The criteria for determining the presence of genomic instability were p>0 and at least >10M fragment copy number C!=2.

[0037] Example 1

[0038] Reference Figure 1 As shown, a screening method for a panel for detecting residual micro-lesions of solid tumors, the present invention uses a dual-index MRD detection method that can simultaneously detect ctDNA mutation information and genomic instability, and can significantly improve the MRD detection rate and reduce false negatives by simultaneously evaluating two indicators;

[0039] The problem that the detection of tiny residual lesions using a large panel (425 genes) + NGS + bioinformatics analysis can only detect SNV, Indel, Fusion, and CNV at the gene level, but cannot detect chromosomal changes at the genome level has been solved. If the tumor sample only undergoes changes at the chromosome level, the MRD judgment of the above method will be negative, resulting in a false negative phenomenon.

[0040] Specifically include the following methods

[0041] Step 1: Provide a detection panel (MRD Panel) containing 142 genes, which can be used for MRD detection and medication guidance for patients with solid tumors. It can provide ctDNA content estimation and medication guidance information, etc. The MRD panel construction method is as follows; Step 1; Confirmation of cancer types covered: According to the latest global cancer burden data in 2020 released by the World Health Organization's International Agency for Research on Cancer (IARC), confirm the top 10 cancer types in China, and combine with the information in its own knowledge base to add 9 cancer types. Finally, the MRD Panel covers a total of 19 cancer types;

[0042] Step 2: Confirmation of covered gene information: This panel has functions such as medication guidance, MRD detection and dynamic monitoring, covering drug-sensitive and drug-resistant genes, tumor driver genes, class I / II gene mutations, MSI sites, chemotherapy sites, etc., covering information sources such as CFDA, FDA, NCCN guideline recommendations and clinical research stages, and the final number of genes is 142 (after deduplication);

[0043] Step 3: retrieve the NGS test results of about 110,000 solid tumor patients from the existing database for effective area screening. First, follow step 1; classify the data by cancer type to obtain the number of samples of each cancer type (N). For the data of each cancer type, evaluate the exons as units within the range of all coding exons of the 142 genes. If the current exon contains somatic mutations and the mutation frequency in the population in the current cancer type is >= 5% (based on N), retain the exon; perform the same iterative evaluation in other cancer types and exons. Keep the exon regions that meet the conditions, remove the exon regions that do not meet the conditions, and finally obtain the screened panel area;

[0044] Step 4: Result comparison: comparison of coverage area and sequencing depth, as shown in Table 2 below;

[0045] Table 2

[0046] Region size (Kb) Sequencing amount (G) Sequencing depth (X) All coding exons of 142 genes (b) 345 8.4 10513 142 gene screening region (c) 101 8.4 41942

[0047] In this embodiment, the detection panel of 142 genes, the incidence ranking: according to the IARC 2020 Global Cancer Report, the top 10 rankings are given, and other cancer types are not ranked and marked NA;

[0048] Refer to Table 3; Cancer type: cancer type; Number of genes: number of genes in the MRD Panel related to the current cancer type; Number of samples (N): number of samples of related cancer types in the own database; Ratio of covered samples (N): compare the position of somatic mutation information detected in each sample in the MRD Panel region with that in the own data. If the MRD Panel contains at least one known somatic mutation, the MRD Panel is determined to cover the sample, and the ratio of covered samples is obtained;

[0049] Table 3

[0050]

[0051] In this embodiment, the specific steps of evaluating whether a solid tumor patient has genomic instability to obtain an estimate of the ctDNA content include:

[0052] There are two steps: global copy number correction estimation and specific chromosome correction:

[0053] Global copy number correction estimate; based on low coverage (~1~2X) whole genome sequencing data. Data is aligned to the human reference genome GRch37. The genome is divided into 1Mb segments (i) and the average coverage within the segment is calculated. Coverage depth correction: Correction for regional coverage depth, correction factors include GC content (G) and alignment (M).

[0054] Calculate the GC content and alignment degree M in each segment, using loess regression fitting: Estimate the parameter coefficients of loess regression, obtain the parameters of the fitted model and calculate the corrected coverage (D).

[0055] Copy number estimation: After correction, the average ratio r is calculated for each segment i:

[0056] The average ratio is related to the copy number of segment i (Ci) and tumor purity (p):

[0057]

[0058] The estimated copy number (C) of each segment can be calculated by estimating the copy number C and purity p of the maximum fit ri through the EM algorithm.

[0059] Correction for special chromosomes: The above global correction and copy number estimation are applicable to chromosomes without overall GC content deviation. For chromosome 19, the GC content is higher than that of normal chromosomes, so further correction is required.

[0060] Correction factors include the number of PCR rounds (pn) and the amount of starting DNA (m) The corrected copy number (Cnorm) is incorporated into the final result, and the final criteria for determining the presence of genomic instability are p>0 and at least >10M fragment copy number C!=2. The present invention uses a dual-indicator MRD detection method that simultaneously detects ctDNA mutation information and genomic instability, and by simultaneously evaluating two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

[0061] Example 2

[0062] A screening method for a panel for detecting residual micro-lesions of solid tumors, wherein the overall detection process is implemented as follows:

[0063] Sample size: Two tubes of 10 ml peripheral blood, all used for plasma separation and cfDNA extraction, the total amount of extraction >= 20 ng can be used for subsequent NGS library construction, commercial library construction kit for WGS library construction, the original experimental conditions of 20 degrees 15 min (connector connection), changed to 4 degrees overnight, 100 ng library for WGS sequencing, sequencing data volume 6G (2x), the remaining library for hybridization capture with MRD panel; after capture, sequencing 4G (20000x); mutation analysis process for gene mutation analysis within the panel area (SNV, Indel, Fusion, CNV); genomic instability analysis process for WGS data analysis, assessment of genomic instability; comprehensive judgment of ctDNA content and MRD results.

[0064] Table 4: Results interpretation

[0065]

[0066] The present invention proposes a dual-indicator MRD detection method that can simultaneously detect ctDNA mutation information and genomic instability. By simultaneously evaluating the two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

[0067] Example 3

[0068] A screening method for a panel for detecting residual micro-lesions of solid tumors, detecting ctDNA mutation information of patients with sigmoid colon cancer through an MRD panel; and evaluating the genomic instability of the patient through low-depth WGS sequencing. This embodiment combines ctDNA mutation information and genomic instability results to obtain an estimate of ctDNA content and give a corresponding risk classification. The basic information of the sample is shown in Table 5;

[0069] Table 5: Basic information of samples

[0070] Sample ID S2200039654M; S2200047668M Basic Situation Sigmoid colon cancer, T3:N2b:M0, clinical stage: III MRD detection status Preoperative MRD: 2022-01-09; Postoperative MRD: 2022-04-11 in conclusion Preoperative MRD was positive (chromosomal instability was positive, mutation was negative), and postoperative MRD was negative (chromosomal instability was negative, mutation was negative). Increasing the chromosomal instability index has the function of reducing the false negative of the panel. Postoperative MRD negative indicates a low risk of recurrence. Non-shedding tumor: Tumor tissue exists, but does not release ctDNA, which is the main source of false negative results based on plasma cfDNA testing. This case proves that increasing the genomic instability characteristics can reduce false negatives.

[0071] The test results are summarized in Table 6.

[0072] Table 6: Summary of test results

[0073] Sampling time Sample Type MRD results ctDNA content Tumor burden Variant information summary 2022-01-09 blood Positive 4.56% □Extremely low load □Low load R High load Point mutation / fusion / insertion / deletion: --CNV: Positive 2022-04-11 blood Negative Not Detected R Very low load □ Low load □ High load Point mutation / fusion / insertion / deletion:--CNV:--

[0074] Reference Figure 3 :Positive result for chromosome instability (sample ID: S2200039654)

[0075] Each point represents a small chromosome segment, the horizontal axis represents the corresponding position on the chromosome, and the vertical axis represents the copy number abundance of the chromosome segment after standardization (taking the logarithm log value). Figure 3 The points corresponding to the segments with normal chromosome copy number are blue, the points corresponding to the segments with chromosome amplification are red, and the points corresponding to the segments with chromosome deletion are green. Figure 3-Figure 9 , please refer to this paragraph for annotation information)

[0076] Figure 4 : Negative result for chromosome instability (sample ID: S2200047668).

[0077] Example 4

[0078] Method for detecting micro-residual lesions of breast cancer: detecting ctDNA mutation information of breast cancer patients through MRD panel; and evaluating genomic instability of solid tumor patients through low-depth WGS sequencing. This embodiment combines ctDNA mutation information and genomic instability results to obtain an estimate of ctDNA content and give a corresponding risk classification. The basic information of the samples is shown in Table 7.

[0079] Table 7: Basic information of samples

[0080] Sample ID PB5968; PC3677 Basic Situation Original breast cancer, surgical treatment, and normal life afterwards. MRD test on 2021-11-05 found ctDNA abnormalities (chromosomal instability and mutation double positive), indicating recurrence. PET-CT test showed no abnormalities in the breast, but abnormalities in the liver. After surgical treatment, MRD test was performed again, and ctDNA was negative (double negative) MRD detection status Preoperative MRD test: 2021-11-05; Postoperative MRD test: 2022-06-07 in conclusion The sample was from a patient with breast cancer. It was MRD-positive (chromosomal instability positive, mutation positive), indicating disease recurrence. PET-CT confirmed liver metastasis of breast cancer. After surgical treatment, MRD was negative (chromosomal instability negative, mutation negative), indicating a low risk of recurrence.

[0081] The test results are summarized in Table 8.

[0082] Table 8: Summary of test results

[0083] Sampling time Sample Type MRD results ctDNA content Tumor burden Variant information summary 2021-11-05 blood Positive 8.10% □Extremely low load □Low load R High load Point mutation / fusion / insertion / deletion: positive CNV: positive 2022-06-07 blood Negative Not Detected R Very low load □ Low load □ High load Point mutation / fusion / insertion / deletion:--CNV:--

[0084] The mutation positive results are shown in Table 9

[0085] Table 9: Mutation positive results

[0086] Mutation Level Gene Exon Nucleotide variation Amino acid variation Abundance Category I PIK3CA exon21 c.3140A>G p.H1047R 5.49% Category III SF3B1 exon15 c.2219G>A p.G740E 3.55%

[0087] Reference Figure 5 As shown, the result of chromosome instability is positive (sample ID: PB5968);

[0088] Reference Figure 6 As shown, negative result for chromosomal instability (sample ID: PC3677).

[0089] Example 5

[0090] A method for detecting micro-residual lesions of rectal cancer, detecting ctDNA mutation information of patients with solid tumors of rectal cancer by MRD panel; and evaluating genomic instability of patients with solid tumors by low-depth WGS sequencing. In this embodiment, by combining ctDNA mutation information and genomic instability results, an estimate of ctDNA content is obtained, and corresponding risk classification is given. The basic information of the samples is shown in Table 10.

[0091] Table 10: Basic information of samples

[0092] Sample ID S2200045588M; S2200047780M; S2200050210M Basic Situation Rectal cancer, T2:N1:M0, clinical stage: III; surgery time: 2021 / 12 / 24, first treatment time: 2022 / 02 / 15, XELOX4 cycle, last follow-up time: 2022 / 05 / 07, last follow-up survival status: alive MRD detection status Postoperative MRD first time: 2022-03-14; Postoperative MRD second time: 2022-04-14; Postoperative MRD third time: 2022-05-09 in conclusion Postoperative MRD (chromosomal instability, mutation) double negative, 3 times, if the MRD result continues to be negative, the risk of recurrence is low, which can indicate potential cure.

[0093] The test results are summarized in Table 11:

[0094] Table 11: Summary of test results

[0095] Sampling time Sample Type MRD results ctDNA content Tumor burden Variant information summary 2022-03-14 blood Negative Not Detected R Very low load □ Low load □ High load Point mutation / fusion / insertion / deletion:--CNV:-- 2022-04-14 blood Negative Not Detected R Very low load □ Low load □ High load Point mutation / fusion / insertion / deletion:--CNV:-- 2022-05-09 blood Negative Not Detected R Very low load □ Low load □ High load Point mutation / fusion / insertion / deletion:--CNV:--

[0096] Reference Figure 7 As shown, the result of chromosome instability was negative; (sample ID: S2200045588);

[0097] Reference Figure 8 As shown, the result of chromosome instability is negative; (sample ID: S2200047780);

[0098] Reference Fig. 9 As shown, the result of chromosome instability is negative; (Sample ID: S2200050210).

[0099] The present invention adopts a dual-indicator MRD detection method that simultaneously detects ctDNA mutation information and genomic instability. By simultaneously evaluating the two indicators, the MRD detection rate can be significantly improved and false negatives can be reduced.

[0100] The technical principle of the present invention is described above in combination with the specific embodiments, which are only preferred implementations of the present invention. The protection scope of the present invention is not limited to the above embodiments, and all technical solutions under the idea of ​​the present invention belong to the protection scope of the present invention. Those skilled in the art can think of other specific implementations of the present invention without creative work, and these methods will fall within the protection scope of the present invention.

Claims

1. A method for screening a panel for detecting residual micro-lesions of solid tumors, characterized in that: The construction steps of the detection panel include: confirming the cancer types covered, where the cancer types are specifically lung cancer, intestinal cancer, gastric cancer, breast cancer, liver cancer, esophageal cancer, thyroid cancer, pancreatic cancer, prostate cancer, cervical cancer, ovarian cancer, endometrial cancer, melanoma, bladder cancer, kidney cancer, head and neck tumors, gliomas, biliary tumors, and gastrointestinal stromal tumors; confirming the covered gene information; retrieving the NGS test results of solid tumor patients in the existing database for effective area screening; the confirmed covered gene information includes: drug sensitive and resistant genes, MRD detection and dynamic monitoring areas, tumor driver genes, class I / II gene mutations, MSI sites, chemotherapy sites, covering CFDA, FDA, NC The CN guideline recommendation and clinical research stage information source, the final number of genes obtained after deduplication is 142; the said retrieval of the NGS test results of solid tumor patients in the existing database for effective area screening specifically includes: classifying the data according to the type of cancer, obtaining the number of samples of each cancer type, and evaluating the data of each cancer type within the range of all coding exons of 142 genes, with exons as units. If the current exon contains somatic mutations and the mutation frequency of the population in the current cancer type is greater than or equal to 5%, the exon is retained; the same iterative evaluation is performed in other cancer types and exons, the qualified exon regions are retained, and the unqualified exon regions are removed to obtain the screened panel area; The evaluation algorithm of genomic instability includes: global copy number correction estimation and special chromosome correction; the global copy number correction estimation includes whole genome sequencing data based on low coverage, the data is aligned to the human reference genome GRch37, the genome is divided into 1Mb segments i, and the average coverage within the segment is calculated; coverage depth correction: the regional coverage depth is corrected, the correction factors include GC content G and alignment degree M, the GC content and alignment degree M in each segment are calculated, and loess regression fitting is used: d~αG+βM to estimate the parameter coefficients of loess regression, α and β are the fitting coefficients of the regression model G and M respectively, and the parameters of the fitting model are obtained to calculate the corrected coverage d; copy number estimation: after correction, the average ratio r is calculated for each segment i: The average ratio is related to the copy number Ci of segment i and the tumor purity p: is the length of the jth segment on the entire genome, and the copy number and tumor purity p of the maximum fit ri are estimated by the EM algorithm to calculate the estimated copy number C of each segment; The 142 gene list specifically includes: Detect point mutation + insertion and deletion + copy number variation Detect point mutation + insertion / deletion mutation + fusion mutation + copy number variation Chemotherapy-related genes After deduplication, there are a total of 142 genes, including the detection of ctDNA mutation information of solid tumor patients through a 142-gene detection panel; and the assessment of genomic instability of solid tumor patients through low-depth WGS sequencing. Combining ctDNA mutation information and genomic instability results, an estimate of ctDNA content is obtained, and the corresponding risk classification is given.

2. A method for screening a panel for detecting residual micro-lesions of solid tumors according to claim 1, characterized in that: The special chromosome correction includes: the correction factors for chromosome 19 include the PCR round number pn and the starting DNA amount m, Cnorm=αpn+βm, and the corrected copy number Cnorm is merged into the final result. The standard for determining the instability of the genome is p>0 and at least >10M fragment copy number C≠2.

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