Method for detecting solid tumor molecule residual focus

Abstract

The invention belongs to the field of medical detection, and particularly provides a solid tumor molecule residual focus detection method which comprises tissue baseline detection, probe design, peripheral blood MRD detection and result analysis and interpretation. According to the present invention, the technical steps such as optimized pre-library construction and hybridization capture process, customized probe design, unique fusion gene probe design and the like are improved, such that the problems of low detection sensitivity, low specificity, low accuracy and the like in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the field of medical detection. Specifically, the present invention relates to a method for detecting molecular residual lesions in solid tumors. Background Art

[0002] Molecular residual disease, also known as minimal residual disease or measurable residual disease, abbreviated as MRD. In solid tumors, MRD usually refers to molecular residual disease, that is, through molecular abnormalities such as circulating tumor DNA (ctDNA) from tumors, potential lesions that cannot be detected by imaging or traditional laboratory methods are discovered, representing the persistence of tumors and the possibility of clinical progression. MRD can assist clinicians in identifying patients at risk of recurrence earlier and provide a basis for subsequent clinical decisions.

[0003] Circulating tumor DNA (ctDNA) is a free DNA fragment released by tumor cells into the plasma, carrying tumor-related specific gene characteristics and epigenetic changes. Whether there are tumor genome variant signals in the cfDNA of cancer patients after treatment is an important indicator for evaluating the treatment prognosis and recurrence and metastasis risks of patients. We can use minimally invasive blood samples to detect the ctDNA residue in the blood of tumor patients and dynamically monitor the treatment response and predict the recurrence risk.

[0004] Currently, the clinical application of circulating tumor gene monitoring has been recognized and recommended by many authoritative domestic and foreign guidelines and consensuses. The detection based on ctDNA MRD has been written into multiple tumor guidelines / expert consensuses such as "Expert Consensus on Molecular Residual Lesions in Non-Small Cell Lung Cancer", "Expert Consensus on Postoperative Recurrence Prediction of Non-Small Cell Lung Cancer Based on Molecular Markers", "NCCN Clinical Practice Guidelines for Colorectal Cancer", "Chinese Expert Consensus on High-Throughput Sequencing of Colorectal Cancer Molecules", and "Chinese Expert Consensus on Detection and Clinical Application of Molecular Residual Lesions in Gastric Cancer". Therefore, it is necessary to develop molecular residual detection technologies to meet clinical needs. Summary of the Invention

[0005] Aiming at the problems of low detection sensitivity, low specificity, and low accuracy in the prior art, the main purpose of the present invention is to provide a system for efficiently and accurately detecting molecular residual lesions in peripheral blood samples of solid tumor patients, which covers comprehensive baseline detection. Through multi-round optimized library construction strategies and unique probe design methods, the detection sensitivity, specificity, and accuracy can be effectively improved. To achieve the above technical effects, the present invention provides the following technical solutions:

[0006] First, in a first aspect, the present invention provides a method for detecting molecular residual lesions in solid tumors, and the method comprises the following steps:

[0007] S1. Perform whole exome sequencing on tissue DNA and whole exome sequencing on control peripheral blood leukocytes to obtain sequencing data;

[0008] S2. Screen for patient-specific somatic mutation sites based on the tissue baseline test results in S1, and design and screen personalized probes accordingly. Use the obtained personalized somatic mutation sites as monitoring sites for subsequent MRD evaluation;

[0009] S3. Perform MRD detection on peripheral blood plasma and control peripheral blood leukocytes;

[0010] S4. Calculate the combined p-value by combining the abundance of monitoring sites and the number of positive monitoring sites.

[0011] In one embodiment, in step S1, the whole exome sequencing of tissue DNA includes performing end repair and adding A to the fragmented tissue genomic DNA using the following PCR program:

[0012] The PCR reaction program is as follows:

[0013] Reaction temperature Reaction time 4℃ 1 min 30℃ 15 min 72℃ 20 min 4℃ Hold

[0014] In one embodiment, in step S1, the reaction system for ligation of adapters is as follows:

[0015] Component name Volume Reaction product 60 μL Adapter 5 μL Ligation buffer 30 μL Ligase 5 μL Total 100 μL

[0016] In one embodiment, in step S1, the following PCR reaction program is used for amplification:

[0017]

[0018]

[0019] In one embodiment, in step S1, the following hybridization step is included:

[0020] Take 30 μL of purified magnetic beads, 5 μL of blocking reagent, and 15 μL of pre-library amplification product. After vortexing and mixing, perform magnetic bead purification with reference to the above steps, and use the following eluent for elution:

[0021] Component Volume (μL) 2X Hybridi Buffer 8.5 Hybidization Buffer Enhancer 2.7 Whole exome capture probe 5.8 Total volume per reaction 17

[0022] After elution, perform overnight hybridization according to the following program:

[0023]

[0024] In one embodiment, in step S2, the length of the personalized probe is 120 bp, and it is required that the alignment position of the probe on the genome is unique.

[0025] In one embodiment, in step S4, the combined-pvalue calculation method includes three parts: (1) calculating the p-value based on abundance: vaf-pvalue; (2) calculating the p-value based on the number of positive monitoring sites: altsite-pvalue; (3) calculating the combined p-value based on Fisher's Method: combined-pvalue.

[0026] In a second aspect of the present invention, there is provided a computer detection device, which includes at least one processor capable of executing computer program instructions stored in a medium to implement the detection method for molecular residual lesions of solid tumors as described above.

[0027] Compared with the prior art, the present invention has the following remarkable improvements:

[0028] 1. Efficient library construction process: To achieve the detection of cfDNA with extremely low starting amounts, the present invention adopts a pre-library construction method optimized through multiple rounds. Key steps such as DNA end repair, A-tailing, and adapter ligation are optimized, adjusting the reaction system and reaction time, significantly reducing molecular loss, and improving library conversion efficiency; in addition, we have optimized the hybridization capture process, mainly including optimizing many parameters such as hybridization temperature, capture temperature, washing temperature, hybridization time, capture time, washing time, and number of washing times, improving the on-target rate, uniformity, and coverage of the hybridization capture process, thereby enhancing the sensitivity and specificity of the detection process; through the above optimization measures, efficient and accurate capture and analysis of free DNA fragments in plasma samples are achieved;

[0029] 2. Ingenious probe combination: The probe combination used in the present invention includes two parts, namely the core probe group and the customized probe group. Among them, the core probe group screens common drug use, drug resistance, and other related sites of common cancer types, a total of 21 genes, to be used for indicating new mutations, heterogeneous mutations, etc. The customized probe group is derived from patient-specific personalized mutation sites, a total of 150 sites, to monitor their mutation status. The combination of the two parts can effectively fix the probe target area to an appropriate size to prevent problems such as high data volume cost caused by an overly large probe target area and poor capture performance (low on-target rate, poor uniformity, low coverage) caused by an overly small probe target area. In addition, it can also take into account the patient's personalized sites to more accurately monitor the disease progression status;

[0030] 3. Unique fusion gene probe design and monitoring: Fusion is a common driver factor in tumors and is of great significance for tumor diagnosis, treatment selection, efficacy monitoring, and prognosis assessment. Especially in lung cancer, the overall incidence of fusion is relatively high, and there are also many targeted drugs. By monitoring the dynamic changes of fusion, clinical treatment decisions can be guided. However, there are two challenges in designing probes for fusion genes: 1) A clear fusion breakpoint at the DNA level is required, but most fusion breakpoints are in the intron region. Therefore, the tissue baseline detection panel needs to cover enough intron regions; 2) The capture efficiency of fusion probes is relatively poor compared to Snv / InDel probes. Kanghui tissue baseline uses a self-built WES probe to ensure coverage of the common intron region breakpoints of fusion genes. Customized fusion gene probes are designed with a probe at each end of the fusion breakpoint to improve the capture efficiency of fusion probes. When monitoring fusion genes, the number of junction reads is counted to determine whether the fusion gene is detected. At the same time, the fusion gene probe, as a component of the personalized probe set, assists in judging the MRD status by combining p-values at multiple points;

[0031] 4. Ultra-high-depth white blood cell-assisted abnormal mutation filtering: By observing the mutation abundances of baseline mutations in multiple plasma monitoring samples, we found that the abundances of some mutations were significantly higher than those of all other mutations, and the mutation abundances remained basically unchanged during multiple monitorings. Therefore, we suspected that these mutations might be unrelated to the tumor and meaningless for monitoring tumor progression. Considering that the sequencing depth of the white blood cell control sample was relatively low (average sequencing depth of 150x) during WES tissue baseline detection, some low-abundance background or germline mosaic mutations might not be filtered out. Therefore, for samples with the above abnormal mutations, high-depth sequencing was also performed on the white blood cell control samples, and it was found that these mutations also existed in white blood cells and the abundances were similar to those in plasma, indicating that these mutations were indeed germline mosaics and needed to be removed from the subsequent monitoring sites to ensure the accuracy of the monitoring results. Testing with clinical samples with clear clinical endpoints and recurrence conclusions found that after filtering abnormal mutations with ultra-high-depth white blood cells, the accuracy of MRD detection results was significantly improved. Description of the Drawings

[0032] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0033] Figure 1 are the module and detection flowcharts of the system described in the present invention;

[0034] Figure 2To construct libraries using the library construction processes before and after optimization respectively, the library conversion rates of the two library construction methods were statistically analyzed based on the sequencing data. The library conversion rate before optimization was between 0.55 and 0.60, with an average of 0.58. The library conversion rate after optimization was between 0.67 and 0.70, with an average of 0.68. The library conversion rate increased by 0.1 after optimization compared to before optimization;

[0035] Figure 3 The figure shows the comparison of the optimized hybridization capture process. Libraries were constructed using the library construction processes before and after optimization respectively. According to the sequencing data, the target capture rate, uniformity (the proportion of 0.2X average depth), and coverage of the two library construction methods were statistically analyzed. The target capture rate before optimization was between 0.65 and 0.70, with an average of 0.67. The uniformity (the proportion of 0.2X average depth) was between 0.96 and 0.98, with an average of 0.975. The coverage was between 0.975 and 0.990, with an average of 0.985. The target capture rate after optimization was between 0.75 and 0.81, with an average of 0.78. The uniformity (the proportion of 0.2X average depth) was between 0.99 and 1, with an average of 0.994. The coverage was 1, with an average of 1. After optimization, the target capture rate increased by 0.11, the uniformity (the proportion of 0.2X average depth) increased by 0.019, and the coverage increased by 0.015 compared to before optimization. The capture performance was significantly improved after optimization compared to before optimization;

[0036] Figure 4 The figure shows the design of customized MRD fusion gene probes. Customized MRD fusion gene probes were designed upstream and downstream of the fusion gene junction site, covering multiple layers. The probe spans the junction site;

[0037] Figure 5 The figure shows the altsite ratio distribution. Altsite represents the number of positive sites, that is, the number of sites supported by mutant reads among the monitored sites. First, the altsite ratio distribution under different monitored sites was constructed. Through comparison, it was found that when the number of monitored sites ≥ 30, the altsite ratio approximately conformed to the normal distribution. Then, the mean and standard deviation of the altsite ratio when the number of monitored sites ≥ 30 were calculated;

[0038] Figure 6 The figure shows the sensitivity assessment results. Detailed implementation manners

[0039] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0040] Example 1 Module composition of the molecular residual disease detection system for peripheral blood samples of solid tumor patients

[0041] The modules and detection processes of the system are as Figure 1 shown.

[0042] 1. Tissue baseline detection module:

[0043] This module mainly includes the detection of the whole exome of tissue DNA and the detection of the whole exome of peripheral blood leukocyte controls. To achieve the detection of extremely low starting amounts of cfDNA, the present invention adopts a pre-library construction method optimized through multiple rounds. Key steps such as DNA end repair, A-tailing, and adapter ligation are optimized, the reaction system and reaction time are adjusted, significantly reducing molecular loss and increasing library conversion efficiency ( Figure 2 ). In addition, the hybridization capture process in this module is also optimized, mainly including many optimizations such as hybridization temperature, capture temperature, washing temperature, hybridization time, capture time, washing time, and number of washing times, improving the on-target rate, uniformity, and coverage of the hybridization capture process ( Figure 3 ), thereby improving the sensitivity and specificity of the detection process. The specific operations are as follows:

[0044] Detection of the whole exome of tissue DNA: First, extract tissue genomic DNA, then fragment the DNA, perform end repair and A-tailing, use ligase to ligate adapters with single-molecule barcode sequences (Barcodes) to both ends of the DNA molecules, and obtain a pre-library through amplification. The pre-library is hybridized in solution with biotin-labeled whole exome oligonucleotide probes, and the library bound to the probes is captured and enriched using streptavidin-coated magnetic beads. Finally, amplification is performed using primers with tag sequences (Indices) and polymerase to obtain a captured library. The captured library is sequenced by high-throughput sequencing to obtain sequencing data.

[0045] Detection of the whole exome of peripheral blood leukocyte controls: First, extract the genomic DNA of leukocytes from control blood, then construct a library for the DNA. The library construction process, capture process, and probes used are the same as above. The captured library is sequenced by high-throughput sequencing to obtain sequencing data.

[0046] The specific experimental steps for library construction are as follows:

[0047] 1.1 Fragmentation, end repair, and A-tailing:

[0048] The reaction system is as follows:

[0049]

[0050]

[0051] The PCR reaction program is as follows (hot lid at 105°C):

[0052] Reaction temperature Reaction time 4℃ 1 min 30℃ 15 min 72℃ 20 min 4℃ Hold

[0053] 1.2 Connection

[0054] The reaction system is as follows:

[0055] Component name Volume Product of previous reaction 60 μL Adapter (Yeasen 12960) 5 μL Ligation buffer (Yeasen 12805) 30 μL Ligase (Yeasen 12805) 5 μL Total 100 μL

[0056] The PCR reaction program is as follows (hot lid OFF):

[0057] Reaction temperature Reaction time 20℃ 15 min 4℃ Hold

[0058] 1.3 Magnetic bead purification

[0059] 1.3.1 Adding magnetic beads: Prepare a new 1.5 mL centrifuge tube, stick the sample number on the tube cap, and add 75 μL of CleanNGS magnetic beads.

[0060] 1.3.2 Adding the sample: After the PCR reaction is completed, take out the sample and place it on an ice box, and centrifuge briefly. Add all the sample to the 1.5 mL centrifuge tube, gently flick to mix, let stand for 8 min, adsorb with a magnetic stand for 5 min, and discard the waste liquid after the sample becomes clear (avoid sucking up the CleanNGS magnetic beads).

[0061] 1.3.3 Washing: Slowly add 200 μL of 80% ethanol along the side far from the CleanNGS magnetic beads, invert the tube gently to mix, and then let stand for 30 s, and discard the waste liquid. Then wash again with 80% ethanol.

[0062] 1.3.4 Drying: Centrifuge briefly, use a 10 μL pipette tip to suck out the waste liquid, open the lid and let dry for about 3 min until the magnetic beads are dry (the magnetic beads become matte).

[0063] 1.3.5 Elution: Add 25 μL of DNase / RNase-Free deionized water, gently flick to mix and let stand for 8 min, adsorb with a magnetic stand for 5 min, and pipette 23 μL of the supernatant into a new 0.2 mL PCR tube.

[0064] 1.4 Amplification

[0065] The reaction system is as follows:

[0066]

[0067] The PCR reaction program is as follows (hot lid at 105 °C):

[0068]

[0069] 1.5 Hybridization

[0070] Take 30 μL of purified magnetic beads, 5 μL of blocking reagent, and 15 μL of the pre-library amplification product. After vortexing and mixing, perform magnetic bead purification according to the above step 1.3, and use the following eluent for elution:

[0071]

[0072] After elution, perform overnight hybridization according to the following program:

[0073]

[0074] 1.6 Capture

[0075] Take 50 μL of capture magnetic beads into a PCR tube, wash the capture magnetic beads once with the magnetic bead washing solution, resuspend the magnetic beads with the following resuspension solution, and then quickly transfer the magnetic bead resuspension to the PCR hybridization tube for the capture reaction. After 20 min, vortex and mix, centrifuge, and then put it back into the PCR instrument to continue the capture reaction:

[0076] Component Reaction volume (μL) 2X Hybridi Buffer (IDT 1080584) 8.5 Hybidization Buffer Enhancer (IDT 1080584) 2.7 Enzyme-free water 5.8 Total volume per reaction 17

[0077] The capture program is as follows:

[0078]

[0079] 1.7 Washing and Hybridization

[0080] 1.7.1 Add 100 μL of preheated washing solution A at 65 °C to the PCR tube to wash the magnetic beads once;

[0081] 1.7.2 Add 140 μL of preheated washing solution S at 65 °C to the PCR tube to wash the magnetic beads twice, 5 min each time;

[0082] 1.7.3 Wash the magnetic beads once with 140 μL of washing solution A, 140 μL of washing solution B, and 140 μL of washing solution C in sequence, 5 min each time;

[0083] 1.7.4 Remove the supernatant to remove the residual liquid, then add 20 μL of enzyme-free water, vortex and mix briefly, and set aside for use.

[0084] 1.8 Amplification

[0085] The reaction system is as follows:

[0086] Component name Volume Magnetic bead resuspension of previous step 20 μL Tag primer CapIndex (Yeasen 12961) 5 μL KAPA Hifi Hotstart Ready Mix (KAPA KK2631) 25 μL Total volume per reaction 50 μL

[0087] The reaction program is as follows:

[0088]

[0089] 1.9 Purification

[0090] Add 75 μL of purified magnetic beads to the above reaction product, purify it according to the above magnetic bead purification steps in 1.3, and elute with 40 μL of eluent. The final library is obtained in this step.

[0091] 2. Probe Design Module:

[0092] This module is customized according to the screening of patient-specific mutation sites and personalized probe design. The specific operation is as follows:

[0093] Screen patient-specific somatic mutation sites based on the tissue baseline detection results. Preferentially screen non-synonymous mutations with high abundance and low background. If the number of mutations is insufficient, synonymous mutations can be used as a supplement. And accordingly design and screen personalized probes. The probe length is 120 bp, and it is required that the alignment position of the probe on the genome is unique. Then perform probe quality detection tests to evaluate the coverage, capture efficiency, and uniformity of the personalized probes. The on-target rate above 0.5 is qualified, the uniformity above 0.9 is qualified, and the coverage above 0.99 is qualified. The finally obtained personalized somatic mutation sites are used as monitoring sites for subsequent MRD evaluation. The specific rules for mutation site screening and probe design are as follows:

[0094] 1) Rules for screening somatic mutation sites

[0095] a. Filter out somatic mutations with high population frequencies and high background values;

[0096] b. Filter out somatic mutations located in repetitive regions and low-complexity regions;

[0097] c. Arrange non-synonymous mutations and synonymous mutations in descending order of abundance and ascending order of background value respectively. Preferentially screen the top 150 non-synonymous mutations. If there are not enough, supplement them with the top synonymous mutations;

[0098] d. Preferentially screen Tier Ⅰ / Ⅱ somatic mutations and fusion genes.

[0099] 2) Rules for personalized probe design

[0100] a. Extend 60 bp upstream and downstream of the mutation site to design a probe sequence with a length of 120 bp;

[0101] b. Require that the alignment position of the probe on the genome is unique;

[0102] c. Strictly require the probe alignment quality values: such as identity≥90, coverage≥100, score≥100, etc.

[0103] To ensure that the WES probe covers the common intron region breakpoints of fusion genes, we designed unique customized fusion gene probes by designing a probe at each end of the fusion breakpoint (Figure 4 (marked by red arrows) to improve the capture efficiency of the fusion probe, and count the number of junction reads during the monitoring of fusion genes. Figure 4 (marked by green) to determine whether the fusion gene is detected. At the same time, the fusion gene probe, as a component of the personalized probe set, assists in judging the MRD status by the combined p-value at multiple points.

[0104] 3. Peripheral blood MRD detection module

[0105] This module mainly includes the detection of cfDNA MRD in peripheral blood plasma and the detection of MRD in peripheral blood leukocyte controls.

[0106] Detection of cfDNA MRD in peripheral blood plasma: First, extract cell-free DNA from plasma, then perform end repair and A-tailing on cfDNA, use ligase to ligate adapters with single-molecule barcode sequences (Barcodes) to both ends of the DNA molecule, and obtain a pre-library through amplification. The pre-library is hybridized in solution with a personalized probe set (personalized probe + core probe) labeled with biotin, and the library bound to the probe is captured and enriched using magnetic beads coated with streptavidin. Finally, amplification is performed using primers and polymerase with tag sequences (Indices) to obtain a captured library. The captured library is sequenced by high-throughput sequencing to obtain sequencing data. The sequencing depth meets or exceeds 100,000×.

[0107] The experimental steps refer to the baseline detection, and the differences are as follows:

[0108] (1) In step 1.1, the interrupted end-repair mixture is replaced with the end-repair mixture.

[0109] (2) In step 1.5, the whole-exome capture probe is replaced with a customized probe.

[0110] (3) In step 1.8, the number of amplification cycles is changed from 11 to 14.

[0111] Detection of MRD in peripheral blood leukocyte controls: First, extract genomic DNA from the leukocytes of the control blood, then perform library construction on the DNA. The library construction process, capture process, and probes used are the same as above. The captured library is sequenced by high-throughput sequencing to obtain sequencing data.

[0112] The experimental steps refer to the baseline detection, and the differences are as follows:

[0113] (1) In step 1.5, the whole-exome capture probe is replaced with a customized probe.

[0114] (2) In step 1.8, the number of amplification cycles is changed from 11 to 14.

[0115] Based on the fastq sequencing data of peripheral blood plasma cfDNA and peripheral blood leukocyte controls, adapter trimming and low-quality filtering are performed to obtain clean fastq. Then, the clean fastq is aligned to the hg19 reference genome. After further deduplication and correction, the final bam file is obtained. Finally, de novo mutation detection is carried out, the abundances of monitored sites in peripheral blood plasma cfDNA samples are calculated, and a personalized mutation background pool is constructed.

[0116] 4. MRD Result Analysis and Interpretation Module

[0117] This module is used for the result determination of MRD. Specifically:

[0118] Combining the abundance of monitored sites and the number of positive monitored sites (the number of sites with abundance greater than 0 among the monitored sites), the combined p-value can be calculated. When the combined p-value is less than the threshold, MRD is positive. Additionally, when a de novo somatic mutation is detected within the coverage area of the core probe set, MRD is also positive. Otherwise, MRD is negative.

[0119] The calculation method of the combined p-value includes three parts: (1) Calculate the p-value based on abundance: vaf-pvalue; (2) Calculate the p-value based on the number of positive monitored sites: altsite-pvalue; (3) Calculate the combined p-value based on Fisher's Method: combined-pvalue.

[0120] (1) Calculate vaf-pvalue

[0121] 1) Construct a mutation background pool

[0122] Statistically analyze the mutation abundances (i.e., vaf) of various mutation types at each site within 50 bp upstream and downstream of each baseline mutation site, and construct a background mutation database.

[0123] 2) Calculate vaf-pvalue by permutation test

[0124] Calculate the mean μ of the baseline mutations in the plasma sample, then randomly select mutations with the number of baseline mutations from the background pool and calculate the mean μi. Repeat the operation 10,000 times. Assume the number of times μi > μ is c, then vaf-pvalue = c / 10,000.

[0125] 3) MRD positive judgment

[0126] If vaf-pvalue < 0.01, it is considered that the significance of baseline mutations is higher than that of background mutations, and MRD is positive.

[0127] Otherwise, it is negative.

[0128] (2) Calculate altsite - pvalue

[0129] 1) Construct the altsite ratio distribution model for healthy populations (such as Figure 5 );

[0130] In Figure 2 , altsite represents the number of positive sites, that is, the number of sites supported by mutant reads among the monitored sites. First, construct the altsite ratio distribution under different monitored sites. Through comparison, it is found that when the number of monitored sites ≥ 30, the altsite ratio approximately conforms to the normal distribution. Then, calculate the mean and standard deviation of the altsite ratio when the number of monitored sites ≥ 30.

[0131] 2) Calculate altsite - pvalue

[0132] We use altsite - pvalue to evaluate the difference degree between the number of positive sites of the sample to be tested and that of healthy populations. When the number of monitored sites ≥ 30, the altsite ratio of the sample to be tested approximately follows the normal distribution, and its occurrence probability altsite - pvalue can be calculated using the z - test, with the mean and standard deviation referring to the results of the previous step.

[0133] aitsite - pvalue = F(X > altsite_ratio) = 1 - F(X ≤ altsite_ratio) (Formula 1)

[0134] (3) Calculate combined - pvalue

[0135] Integrate vaf - pvalue and altsite - pvalue into combined - pvalue by Fisher's Method. Combining the reference product and clinical sample data, when combined - pvalue < 0.05, then MRD is positive. If the number of monitored sites < 30 or altsite ratio < 0.1, then when combined - pvalue < 0.01, MRD is positive.

[0136] Example 2 Evaluation of System Sensitivity, Specificity, and Detection Limit

[0137] To explore the efficacy of the system described in Example 1, we used lymphocyte line standard products with known concentrations for pre - library construction, hybridization capture, on - machine sequencing, and bioinformatics analysis. The experimental process and bioinformatics analysis refer to Example 1. Then, conduct sensitivity, specificity, and detection limit studies respectively.

[0138] 1. Sensitivity assessment

[0139] Reference products with variant allele frequencies (VAFs) of 0.0012%, 0.0025%, 0.0063%, 0.0125%, and 0.03% were prepared by mixing lymphocyte line DNA. Libraries were constructed with starting DNA amounts of 10, 30, and 50 ng respectively, and the effects of different starting amounts and different numbers of monitored loci on the sensitivity of MRD detection were evaluated through these samples at a sequencing depth of 100,000x.

[0140] From the above results, it can be seen that as the number of monitored loci (Monitor Mutation Count) increases, the sensitivity of customized MRD detection also increases. When the number of monitored loci is equal to 150, the sensitivity at all VAFs reaches 95%. Continuing to increase the number of monitored loci, the change in the sensitivity of customized MRD detection is not significant. In addition, the greater the starting amount of DNA, the higher the sensitivity of customized MRD detection. Especially when the VAF is lower, increasing the starting amount of DNA can significantly improve the sensitivity of MRD detection.

[0141] 2. Specificity assessment

[0142] At a sequencing depth of 100,000x, 30 healthy human plasma samples were used to evaluate the specificity of the MRD detection system described in Example 1.

[0143] Known positive Known negative MRD+ 0 0 / MRD- 0 30 NPV = 100% / NPA = 100%

[0144] From the above results, it can be seen that both the NPA and NPV of the MRD detection method are 100%, and the specificity verification results meet the requirements.

[0145] 3. Limit of detection assessment

[0146] Reference products with VAFs of 0.0012%, 0.0025%, 0.0063%, 0.0125%, and 0.03% were prepared by mixing lymphocyte line DNA. Libraries were constructed with starting DNA amounts of 10, 30, and 50 ng respectively, and each starting amount was replicated 3 times for library construction. The VAF with a sample detection rate ≥ 95% was determined as the limit of detection of MRD. The results are as Figure 6 and the following table shows.

[0147]

[0148] From the above results, it can be seen that the limit of detection varies with different starting amounts and numbers of monitored loci. The higher the starting amount and / or the more the number of monitored loci, the lower the limit of detection. For example, when the starting amount of DNA is 50 ng and the number of monitored loci is 150, the limit of detection can reach 0.0012%. When the starting amount of DNA is 10 ng and the number of monitored loci is 100, the limit of detection can reach 0.0025%.

[0149] Example 3 Application of Clinical Samples

[0150] Three clinical samples were selected for customized MRD detection. The sample numbers were Sample 1, Sample 2, and Sample 3 respectively. The specific steps are as follows:

[0151] 1. Tissue Baseline Detection

[0152] After performing WES detection on the tumor baseline tissue samples, somatic mutation sites were screened according to the rules described in Example 1, and screening probes were designed to obtain the monitored mutation sites. The following table shows the monitored mutation sites corresponding to the three samples.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] #Vaf: Mutation abundance; Positive_Site: YES indicates positive site, NO indicates negative site.

[0165] 2. Peripheral Blood MRD Detection

[0166] Peripheral blood plasma cfDNA MRD detection and peripheral blood leukocyte control MRD detection were performed on the peripheral blood plasma cfDNA of the three clinical samples to obtain the monitored mutation site abundances and the number of positive monitored sites, as shown in the 6th and 7th columns of the above table respectively.

[0167] 3. Comprehensive Judgment of MRD Status

[0168] According to the monitored mutant site abundance and the number of positive monitoring sites, calculate the combined p-value according to the method described in the MRD result analysis and interpretation module in Example 1, and determine the MRD status. The MRD status of 3 clinical samples is as follows in the table:

[0169] Sample MRD status Combined p-value Number of monitoring sites Number of positive sites Sample 1 Negative 0.929560704392956 143 1 Sample 2 Negative 0.21981272489163367 94 10 Sample 3 Positive 0 148 66

[0170] #Combined p-value, with thresholds of 0.05 or 0.01.

[0171] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these changes and modifications.

Claims

1. A method for detecting molecular residual lesions of solid tumors, characterized in that: The method comprises the following steps: S1. Perform whole exome testing on tissue DNA and whole exome testing on control peripheral blood leukocytes to obtain sequencing data; S2. Screen patient-specific somatic mutation sites based on the tissue baseline test results in S1, and design and screen personalized probes accordingly, using the obtained personalized somatic mutation sites as monitoring sites for subsequent MRD assessment; S3. Perform MRD detection on peripheral blood plasma and control peripheral blood leukocytes; S4. Calculate the joint p-value by combining the abundance of monitoring sites and the number of positive monitoring sites.

2. The method according to claim 1, characterized in that In step S1, the whole exome detection of tissue DNA includes performing end repair and A addition on the interrupted tissue genomic DNA using the following PCR procedure: The PCR reaction procedure is as follows: The reaction was carried out at 4°C for 1 min; at 30°C for 15 min; at 72°C for 20 min; and maintained at 4°C.

3. The method according to claim 1, characterized in that In step S1, the reaction system for connecting the linker is: Reaction product: 60 μL Adapter: 5 μL Ligation buffer: 30 μL Ligase: 5 μL 100 μL total.

4. The method according to claim 1, characterized in that In step S1, the following PCR reaction program is used for amplification: react at 98°C for 45s, 1 cycle; react at 98°C for 10s, react at 58°C for 30s, react at 72°C for 30s, 11 cycles; react at 72°C for 1min, 1 cycle; and maintain at 4°C.

5. The method according to claim 1, characterized in that The step S1 includes the following hybridization steps: Take 30 μL purified magnetic beads, 5 μL blocking reagent, and 15 μL pre-library amplification product, vortex and mix well, and then perform magnetic bead purification according to the above steps. The eluent components used are: 2X Hybrid Buffer 8.5μL, Hybidization Buffer Enhancer 2.7μL, all-exon capture probe 5.8μL, and the total volume is 17μL; After elution, overnight hybridization was performed according to the following hybridization program: 95°C for 2 min, 65°C for 10 h, and 65°C for maintenance.

6. The method according to claim 1, characterized in that In the step S1, after hybridization, capture and elution, the following PCR program is used for secondary amplification: react at 98°C for 45 sec, 1 cycle; react at 98°C for 15 sec, 58°C for 30 sec, 72°C for 30 sec, 11 cycles; react at 72°C for 1 min, 1 cycle; maintain at 4°C.

7. The method according to claim 1, characterized in that The length of the personalized probe is 120 bp, and the probe meets the requirement that the alignment position on the genome is unique.

8. The method according to claim 1, characterized in that In step S4, the combined p-value calculation method includes three parts: (1) calculating the p-value based on abundance; (2) calculating the p-value based on the number of positive monitoring sites; (3) calculating the combined p-value based on Fisher's Method.

9. A computer detection device, comprising at least one processor, wherein the processor is capable of executing computer program instructions stored in a medium to implement the method for detecting molecular residual lesions of solid tumors as described in any one of claims 1 to 8.

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