Probe group for evaluating whole-body tumor load of small cell lung cancer and application of probe group

Abstract

The invention discloses a probe set for evaluating whole-body tumor load of small cell lung cancer and application of the probe set, the probe set comprises a plurality of oligonucleotide probes for specifically capturing mutation regions of SCLC related genes in a targeted mode, and the genes are selected from TP53, RB1, CREBBP, EGFR, PTEN, PIK3CA, LRP1B and NOTCH1; and the probe set is capable of covering at least 94.9% of at least one mutation existing in an SCLC patient sample. The invention relates to the technical field of biotechnology and molecular diagnosis, and has the beneficial effects that based on a clinical queue, the probe group incorporates high-frequency mutant genes of SCLC, can be superposed with personalized probes for use, and is used for ctDNA detection of SCLC patients and evaluation of whole-body tumor load. The SCLC cancer species specific probe group provided by the invention plays a role in monitoring tumor evolution and new mutation, can overcome the space-time heterogeneity of tumors to a certain extent, and also can improve the capture efficiency at the same time.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and molecular diagnostics, and in particular to a probe set for assessing the systemic tumor burden of small cell lung cancer and its application. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly malignant and aggressive neuroendocrine tumor, accounting for approximately 15% of all lung cancers. It is characterized by rapid growth and early distant metastasis. Although sensitive to initial chemotherapy and radiotherapy, it is highly prone to recurrence and drug resistance, resulting in a very poor prognosis. Therefore, accurate and dynamic assessment of the patient's systemic tumor burden is crucial for developing precise individualized treatment plans, real-time monitoring of treatment efficacy, early warning of recurrence, and prognosis assessment.

[0003] Circulating tumor DNA (ctDNA) detection, as a non-invasive liquid biopsy technique, can reflect the genomic information of tumors in real time and comprehensively, overcoming the limitations of spatiotemporal heterogeneity in traditional tissue biopsies and providing a revolutionary tool for the quantitative assessment of tumor burden. By monitoring changes in the types and abundance of tumor-specific mutations in ctDNA before and after treatment, the dynamic changes in tumor burden can be accurately assessed.

[0004] However, applying ctDNA detection technology to SCLC clinical practice still faces significant challenges. Currently, there is a lack of targeted detection panels specifically optimized for the molecular characteristics of SCLC in clinical practice and on the market. While general-purpose large panels or whole-exome sequencing offer broad coverage, their capture efficiency for SCLC-specific, frequently mutated gene regions (such as TP53 and RB1) is uneven, and they are costly. While fully personalized probe designs offer high specificity, they cannot cover new clones generated during tumor evolution, and when faced with low-abundance ctDNA, they may miss some mutations due to insufficient tracking sites, resulting in insufficient detection sensitivity and failing to provide a stable and economical solution for the comprehensive management of SCLC.

[0005] Therefore, there is an urgent need in the field for a targeted probe solution that can take into account SCLC cancer type specificity, high capture efficiency, good stability and cost-effectiveness, in order to fill the gap in the current technology for accurately assessing the whole-body tumor burden of SCLC. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems by designing a probe set for assessing the systemic tumor burden of small cell lung cancer and its application.

[0007] To achieve the above objectives, the technical solution of the present invention is a probe set for assessing the systemic tumor burden of small cell lung cancer and its application. The probe set contains multiple oligonucleotide probes that specifically target and capture mutation regions of SCLC-related genes. The genes are selected from TP53, RB1, CREBBP, EGFR, PTEN, PIK3CA, LRP1B, and NOTCH1. The probe set can cover at least one mutation present in at least 94.9% of SCLC patient samples.

[0008] The probe set targets mutation regions including at least one of the following regions: exons 3-10 of the TP53 gene; exons 2, 3, 5, 7-17, 19-24 of the RB1 gene; exon 27 of the CREBBP gene; exons 18-21 of the EGFR gene; exon 3 of the PTEN gene; exon 10 of the PIK3CA gene; exons 3, 6, 65, 68, 75, 77 of the LRP1B gene; and exons 20 and 23 of the NOTCH1 gene.

[0009] The oligonucleotide probe is 100-150 bp in length, preferably 120 bp, and the 5' end of the probe is operably linked with a biotin label.

[0010] The probe set comprises the nucleotide sequences shown in SEQ ID NO: 1 to 97, or variants thereof that have at least 95% identity with the sequences and retain the same targeting and capture function.

[0011] On the other hand, the present invention also provides a combined probe system for assessing systemic tumor burden in small cell lung cancer (SCLC), comprising: (a) a set of SCLC-specific probes according to any one of claims 1-4;

[0012] (b) A personalized probe set based on the mutation profile of tumor tissue of an individual SCLC patient; wherein the SCLC-specific probe set and the personalized probe set can be used in combination for targeted capture of the same circulating tumor DNA sample.

[0013] On the other hand, the present invention also provides a kit for assessing the systemic tumor burden of small cell lung cancer (SCLC), comprising a probe set or a combination probe system, and at least one reagent for hybridization capture and high-throughput sequencing.

[0014] On the other hand, the present invention also provides a method for assessing the systemic tumor burden of small cell lung cancer (SCLC) for non-diagnostic purposes, comprising the following steps: Step S1, providing a liquid biopsy sample from a suspected or confirmed SCLC individual and obtaining circulating tumor DNA (ctDNA) therefrom; Step S2, using the probe set according to any one of claims 1-4 or the combined probe system according to claim 5 to target and capture the ctDNA; Step S3, performing high-throughput sequencing on the captured DNA; Step S4, analyzing the sequencing data and assessing the systemic tumor burden of the individual based on mutation information of the gene regions targeted by the probe set.

[0015] The liquid biopsy sample is plasma or serum; the method also includes using the assessment results to monitor treatment effectiveness, predict prognosis, or guide adjustments to the treatment plan.

[0016] On the other hand, the present invention also provides the use of the aforementioned probe sets, combined probe systems or kits in the preparation of products for assessing the systemic tumor burden of small cell lung cancer (SCLC).

[0017] On the other hand, the present invention also provides a non-transitory computer-readable storage medium storing instructions that, when executed by a processor, cause the processor to perform steps for analyzing sequencing data generated by the method of claim 7, the steps including: identifying and quantifying mutations from the gene of claim 1, and generating an assessment report reflecting the level of tumor burden.

[0018] This invention discloses a probe set for assessing systemic tumor burden in small cell lung cancer (SCLC) and its application. Based on a clinical cohort, this probe set incorporates high-frequency mutated genes in SCLC and can be used in conjunction with personalized probes for ctDNA detection in SCLC patients to assess systemic tumor burden. This SCLC-specific probe set of the invention monitors tumor evolution and new mutations, overcoming the spatiotemporal heterogeneity of tumors to some extent while improving capture efficiency.

[0019] Clinical samples have verified that the probe set of this invention has excellent capture efficiency and depth coefficient, good probe uniformity, and meets the requirements of clinical testing.

[0020] The probe set of this invention has excellent coverage of the SCLC population, with coverage rates of 94.9% (373 / 393) for clinical samples and 97.2% (280 / 288) for literature samples.

[0021] Clinical cohort validation has demonstrated that the probe set of this invention exhibits excellent clinical performance in assessing the systemic tumor burden of SCLC patients: the SCLC-specific probe set showed 100% sensitivity (30 / 30) in detecting baseline blood samples from SCLC patients, with a median detection of 2 tumor-derived mutations per sample. The combined use of the probe set and the personalized probe set further enhances the performance in assessing the systemic tumor burden of SCLC patients: while maintaining 100% sensitivity (30 / 30) for baseline blood samples, the median number of tumor-derived mutations monitored per sample significantly increased (from a median of 2 to a median of 20). Furthermore, the combined probe set detected a median of 20 tumor-derived mutations per plasma sample, allowing for more accurate assessment of tumor burden and further increasing detection sensitivity in samples with low tumor burden. Attached Figure Description

[0022] Figure 1 This is the capture efficiency test result of a probe set for evaluating the systemic tumor burden of small cell lung cancer and its application in the SCLC-specific probe set described in this invention.

[0023] Figure 2 This invention describes a probe set for assessing systemic tumor burden in small cell lung cancer and its application of a SCLC-specific probe set with a ≥0.2 times average depth percentage test result.

[0024] Figure 3 This invention describes a probe set for assessing systemic tumor burden in small cell lung cancer and its application of a SCLC-specific probe set with a ≥0.5 times average depth percentage test result.

[0025] Figure 4 This is the depth coefficient test result of a probe set for assessing the systemic tumor burden of small cell lung cancer and its application, as described in this invention.

[0026] Figure 5 This invention relates to a probe set for assessing systemic tumor burden in small cell lung cancer and the coverage of SCLC-specific probe sets for SCLC patients.

[0027] Figure 6 This invention describes a probe set for assessing the systemic tumor burden of small cell lung cancer and the coverage of mutations detected in 30 SCLC tumor tissues by the SCLC-specific probe set used in this invention.

[0028] Figure 7 This invention describes a probe set for assessing systemic tumor burden in small cell lung cancer and the number of tissue-derived mutations detected in the baseline blood of 30 SCLC patients using a SCLC-specific probe set.

[0029] Figure 8This invention relates to a probe set for assessing systemic tumor burden in small cell lung cancer and the statistical analysis of mutation counts monitored in 30 SCLC patients using a personalized probe set.

[0030] Figure 9 This invention describes a probe set for assessing systemic tumor burden in small cell lung cancer and the number of mutations detected in the baseline blood of 30 patients using a patient-specific probe set. Detailed Implementation

[0031] The present invention will now be described in detail with reference to the accompanying drawings, such as... Figure 1-9 As shown, a probe set for assessing systemic tumor burden in small cell lung cancer and its application;

[0032] Example 1: Target screening and probe sequencing for SCLC-specific probe sets

[0033] Using the test results of 393 clinical SCLC samples, the high-frequency mutated genes and regions of SCLC were screened according to the following rules.

[0034] The screening rules for high-frequency mutant genes and regions in SCLC are as follows:

[0035] (1) Obtain the mutation information of each sample from the SCLC dataset, and annotate the corresponding transcripts and exons based on the mutation information;

[0036] (2) Calculate the mutation density coefficient of the probe covering the target exon:

[0037] Target exon probe mutation density coefficient = (a / m) / length

[0038] a: The total number of mutations in the target exon in the population.

[0039] m: The total number of mutations in the population.

[0040] length: The length of the probe covering the target exon, estimated based on the length of the exon.

[0041] Exon length ≤ 40 bp, probe length: 120 bp, exon extension length ≥ 40 bp.

[0042] Exon > 40 bp, probe length: exon length + 80 bp, with each end of the exon extending outward by 40 bp;

[0043] (3) Calculation of the incidence rate of exons carrying mutations:

[0044] Prevalence of exons carrying mutations = b / n

[0045] b: Number of patients carrying the mutation in the target exon

[0046] n: Total number of patients;

[0047] First, sort the genes / regions in descending order based on the mutation density coefficient of the target exon probes, and then sort them in descending order based on the incidence of exons carrying the mutations in the population. Genes / regions that are ranked higher have higher priority.

[0048] (5) Select the high-priority SCLC high-frequency mutant genes and regions according to the sorting until the expected panel size (about 8kb) is reached.

[0049] Based on the test results of 393 clinical SCLC samples, the high-frequency mutated genes and regions of SCLC were screened as follows:

[0050] Gene area TP53 Exons 3-10 RB1 Exons 2, 3, 5, 7-17, 19-24 CREBBP Exon 27 EGFR Exons 18-21 PTEN Exon 3 PIK3CA Exon 10 LRP1B Exons 3, 6, 65, 68, 75, 77 NOTCH1 Exons 20 and 23

[0051] Based on the above high-frequency mutated genes and regions in SCLC, a tumor-specific probe set for SCLC was designed, and the probe sequences are as follows.

[0052] SEQ IDNO Gene nucleotide sequence 1 LRP1B GAAAAGGTATGGACTTTCAAATATACAATGGTATTTACTTACAATCAAGCCACTGTTTGCTTGCCTTTTTTCTCTGCCATGGATCCTTTTGTAGAAAATTCCGCCTGGATTAAACTGAGT 2 LRP1B ACTCCAAATAATCATATCTCTTTGATAATATACATCCATCCCTGTTATTCTTGAATTATGTTCAATATGAGAAATTTGTTGATGATCGCCACTGTAGTTGAATGGATATATAAAACCCAG 3 LRP1B GATATCAGTGTCATTAGCAATGTAGAGAACTTGATTCTTCAGAGCCTGGAGATTATTATAATAAAATACAAAAATAAAGTAAATTTCAACTAATGTTAAAAGCAATAATTGTACTTTTAAA 4 LRP1B GAAAAGTACTCTACCTTCACATTGTCTGTTTTCATGTTTCTCTGAAATCCAGGCTTACAGCGACAGAAAACAGATGTTTTTTATTTGATTACAATATGCATCATCTCCACATGGATTCAC 5 LRP1B TTTCTCTGAAATCCAGGCTTCAGCGACAGAAAACAGATGTTTTTATTTGATTACAATATGCATCATCTCCACATGGATTCACATTATCTTCACAGGTATATTCAGTAGGAGCTGGGATT 6 LRP1B GCTTTCTTTTTGATTTTACAAAGATGCCTACCTCATCAGAGCCATCTGCACAGTCAAAATCTCCATCACACCAAAACCTTGAAGAAACACAGTCCCCATTGGCACAGAGGAAATCTTTCA 7 LRP1B GCCATCTGCACAGTCAAAATCTCCATCACACCAAAACCTTGAAGAAACACAGTCCCCATTGGCACAGAGGAAATCTTTCAATGTACATGTCTGTGGCTCTGGGGATAAAAAAACAGCACA 8 LRP1B TTGAGGTGACTTACGACAGTCTCTTTCATCTTCCTCATCACCACAGTCATCTTGCCCATTACATCTTAAGTTTACTGGGATACATTTCTGGTTCTTGGTACATTTGAATTGACCTGACAG 9 LRP1B CTTCCTCATCACCACAGTCATCTTGCCCATTACATCTTAAGTTTACTGGGATACATTTCTGGTTCTTGGTACATTTGAATTGACCTGACAGGCAGACATGTGTGTCTAGAACATAGAAGG 10 LRP1B GAGAATCCAGCCTACGGGTCTGTAAATGAAATCAGTAAAGGGTGGCATATAATATTTAATTGAAACACTTACTGTGGAAGGATTGAAGAATATTGATTGTCCATTCATCTGTTAATCCTC 11 LRP1B CTGCTTTTGTTATCTGGATACATTTGAGTTGATTTGAAGATTCTCTTGATTCAATCCAACAAATCATATCTTCATTATAAATAAAATCCAGAGTATGAATTTCATTTCCATTGACTGAGC 12 LRP1B TTAGAGTTGCCATTTTACTTCCATTAAGATAGAAAACCTCAATTGTTTCAAAATTTGCAATTAATAGTATAGGTGGTCTATCTGTAGGTTCTGGAATAAAATAGAAAAAGAGAAGTAAAT 13 LRP1B GGACTCACCCTGACAATGTACTCCTTCGTCATACCCATCTGGGCAGTCCAAGACACCATTGCACAGCTGGGATAAATGAACACATTTGTTGGTACCAAGGCAAGCAATGTGATTCAAGGG 14 LRP1B CCATCTGGGCAGTCCAAGACACCATTGCACAGCTGGGATAAATGAACACATTTGTTGGTACCAAGGCAAGCAATGTGATTCAAGGGGCACTTGATTTCTACCTCCTCGGGACCTGAAAAG 15 PIK3CA ATTTTATTTTACAGAGTAACAGACTAGCTAGAGACAATGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGAGCAGGAGAAAG 16 PIK3CA TGAATTAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTGAAATCACTGAGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAATGGAG 17 EGFR CTTGTCCCCCCCAGCTTGTGGAGCCTCTTACACCCAGTGGAGAAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCCGGTGCGT 18 EGFR GGAGAAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCCGGTGCGTTCGGCACGGTGTATAAGGTAAGGTCCCTGGCACAGGCC 19 EGFR CAGTTAACGTCTTCCTTCTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGG 20 EGFR CTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTGGGG 21 EGFR ATGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCA 22 EGFR TCACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTATGTCCGGGAACACAAAGACAATATTGGCTCCCAGTACCTGCTCAACTGGTGTGTGCAGATCGCAAAGGTAATCAGGGAAGGGA 23 EGFR CAGCAGGGTCTTCTCTGTTTCAGGGCATGAACTACTTGGAGGACCGTCGCTTGGTGCACCGCGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATT 24 EGFR TTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTCAGCCAGCATTTTCCTGACACCAGGGACCAGGCTGCCTTCC 25 NOTCH1 GCTGCCCCACACGCCCCACCCGCCTGGGCGCGGCACCCACCGGTGTGACCAGCACGGCACTCGCAGTGGAAGTCATTGACGCGCTGCACGCAGTTCTGGGTGCCACGGGCGTCGCAGGGA 26 NOTCH1 TTGGACAGGCACTCGTTGACATCCCCCTCACAGCGCTCACCCACGAAGCCCGGCGGGCAGGTGCAGCTGTAGCCGCCCACCTGGTCCACGCAGGTGCCGTTGTTAAAGCACTTGGGGCTC 27 NOTCH1 CGGGACACGGGGTCAACGGGGGGATTGCAGTCGTCCACGTTGATCTCACAGTGCACACCTGCGGGGCCAGGTTTCGTCAGTGGCCCAAGCCCGCCACACCCCGGCCCTGCCGTGCCGCGT 28 NOTCH1 CTGGGTGGGCACAGCAGGTTACCTTGTCGCTGCGCAGCCACCTCACAGGACACGCTGGGCACGTCGCAGTAAAGGCCGGTCCAGCCGCTGGGGCACTCGCAGCGGTACTGGGTGTGGGTC 29 NOTCH1 TGCCAGCATTTGCCGCCGTTCTTGCAGGGCGAGGAGTCACACCAGTGCACAAGGTTCTGGGGACAGATTGGGGTCAGCTGGGTGCCCGCGCCCCGGCCATTTCCCCGGTAGCTCAGAACG 30 PTEN ATTTCAAATGTTAGCTCATTTTTGTTAATGGTGGCTTTTTGTTTGTTTGTTTTGTTTTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACAATCTGTAAGTATGTTTTC 31 PTEN TTAATGGTGGCTTTTTGTTTGTTTTTTGTTTTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACAATCTGTAAGTATGTTTTCTTATTTGTATGCTTGCAAATATCT 32 PTEN GTTGTTTTGTTTTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACAATCTGTAAGTATGTTTTCTTATTTGTATGCTTGCAAATATCTTCTAAAACAACTATTAAGTG 33 RB1 TTCATTTGGTAGGCTTGAGTTTGAAGAAACAGAAGAACCTGATTTTACTGCATTATGTCAGAAATTAAAGATACCAGATCATGTCAGAGAGAGAGCTTGGTTAACTTGGGAGAAAGTTTC 34 RB1 TGATTTTACTGCATTATGTCAGAAATTAAAGATACCAGATCATGTCAGAGAGAGAGCTTGGTTAACTTGGGAGAAAGTTTCATCTGTGGATGGAGTATTGGTAAGGATTTTCTTAAAACG 35 RB1 TGATCTTTATTTTTTGTTCCCAGGGAGGTTATATTCAAAAGAAAAAGGAACTGTGGGGAATCTGTATCTTTATTGCAGCAGTTGACCTAGATGAGATGTCGTTCACTTTTACTGAGCTAC 36 RB1 GGAACTGTGGGGAATCTGTATCTTTATTGCAGCAGTTGACCTAGATGAGATGTCGTTCACTTTTACTGAGCTACAGAAAAACATAGAAATCAGGTAAAGTTTCTTGTATAAATATAAGCC 37 RB1 AGAAAACTACTATGACTTCTAAATTACGAAAAAATGTTAAAAAGTCATAATGTTTTTCTTTTCAGGACATGTGAACTTATATATTTGACACAACCCAGCAGTTCGTAAGTAGTTCACAGA 38 RB1 ACGAAAAAATGTTAAAAAGTCATAATGTTTTTCTTTTCAGGACATGTGAACTTATATATTTGACACAACCCAGCAGTTCGTAAGTAGTTCACAGAATGTTATTTTTCACTTAAAAAAAAA 39 RB1 AAAGTCATAATGTTTTTCTTTTCAGGACATGTGAACTTATATATTTGACACAACCCAGCAGTTCGTAAGTAGTTCACAGAATGTTATTTTTCACTTAAAAAAAAAGATTTTTATGGAATA 40 RB1 AAAATGTACATTTTTTTTTCAGGGGAAGTATTACAAATGGAAGATGATCTGGTGATTTCATTTCAGTTAATGCTATGTGTCCTTGACTATTTTATTAAACTCTCACCTCCCATGTGCTC 41 RB1 AATGGAAGATGATCTGGTGATTTCATTTCAGTTAATGCTATGTGTCCTTGACTATTTTATTAAACTCTCACCTCCCATGTTGCTCAAAGAACCATATAGTAAGTATTTAATTTATGCCCC 42 RB1 CCACTTTTACAGAAACAGCTGTTATACCCATTAATGGTTCACCTCGAACACCACGGCGAGGTCAGAACAGGAGTGCACGGATAGCAAAACAACTAGAAAATGATACAAGAATTATTGAAG 43 RB1 CACCTCGAACACCCAGGCGAGGTCAGAACAGGAGTGCACGGATAGCAAAACAACTAGAAAATGATACAAGAATTATTGAAGTTCTCTGTAAAGAACATGAATGTAATATAGATGAGGTAA 44 RB1 ATTTTAATGATCATGTTGTAACTTCATCTTTTTCAGGTGAAAAATGTTTATTTCAAAAATTTTATACCTTTTATGAATTCTCTTGGACTTGTAACATCTAATGGACTTCCAGAGGTAATC 45 RB1 TGTAACTTCATCTTTTTCAGGTGAAAAATGTTTATTTCAAAAATTTTATACCTTTTATGAATTCTCTTGGACTTGTAACATCTAATGGACTTCCAGAGGTAATCTGAAAGGAAATTTAAT 46 RB1 CAGGTGAAAAATGTTTATTTCAAAAATTTTATACCTTTTATGAATTCTCTTGGACTTGTAACATCTAATGGACTTCCAGAGGTAATCTGAAAGGAAATTTAATAAAATATTAATGTTTTG 47 RB1 TTGTCAGTGACTTTTTTCTTTCAAGGTTGAAAATCTTTCTAAACGATACGAAGAAATTTATCTTAAAAATAAAGATCTAGATGCAAGATTATTTTTGGATCATGATAAAACTCTTCAGAC 48 RB1 CAAGGTTGAAAATCTTTCTAAACGATACGAAGAAATTTATCTTAAAAATAAAGATCTAGATGCAAGATTATTTTTGGATCATGATAAAACTCTTCAGACTGATTCTATAGACAGGTATTG 49 RB1 TTTCTAAACGATACGAAGAAATTTATCTTAAAAATAAAGATCTAGATGCAAGATTATTTTTGGATCATGATAAAACTCTTCAGACTGATTCTATAGACAGGTATTGCACATGGTATATTT 50 RB1 TTTCAGTATGTGAATGACTTCACTTATTGTTATTTAGTTTTGAAACACAGAGAACACCACGAAAAAGTAACCTTGATGAAGAGGTGAATGTAATTCCTCCACACACTCCAGTTAGGTATG 51 RB1 TTAGTTTTGAAACACAGAGAACACCACGAAAAAGTAACCTTGATGAAGAGGTGAATGTAATTCCTCCACACACTCCAGTTAGGTATGAATTTTCCTACTTTTAATTATATTATAATTTTG 52 RB1 TTCATTGCTTAACACATTTTCCTATTTTTATCCCCCTCTAGGACTGTTATGAACACTATCCAACAATTAATGATGATTTTAAATTCAGCAAGTGATCAACCTTCAGAAAATCTGATTTCCT 53 RB1 TTTTATCCCCTCTAGGACTGTTATGAACACTATCCAACAATTAATGATGATTTTAAATTCAGCAAGTGATCAACCTTCAGAAAATCTGATTTCCTATTTTAACGTAAGCCATATATGAAA 54 RB1 GAACACTATCCAACAATTAATGATGATTTTAAATTCAGCAAGTGATCAACCTTCAGAAAATCTGATTTCCTATTTTAACGTAAGCCATATATGAAACATTATTTATTGTAATATCTTGGC 55 RB1 TTTTTACCTCCTAAAGAACTGCACAGTGAATCCAAAAAAGTATACTGAAAAGAGTGAAGGATAGGATACATCTTTAAAGAAATTTGCTAAAGCTGTGGGACAGGTTGTGTCGA 56 RB1 AGTATACTGAAAAGAGTGAAGGATAGGATACATCTTTAAAGAGAAATTTGCTAAAGCTGTGGGACAGGGTTGTGTCGAAATTGGATCACAGGTAACTTGAATTCATTGTAATTCGTGG 57 RB1 GTGATTTTCTAAAATAGCAGGCTCTTATTTCTTTTTGTTTGTTTGTAGCGATACAAACTTGGAGTTCGCTTGTTACCGAGTAATCCATGCTTAAATCAGTAAGTTAAAAAC 58 RB1 GTTTGTTTGTAGCGATACAAACTTGGAGTTCGCTTGTATTACCGAGTAATGGAATCCATGCTTAAATCAGTAAGTAAAACAATAATAAAAAATTTCAGCCGGGCGCGGTGGCTCACGC 59 RB1 TCAATGCTGACACAAATAAGGTTTCAATTAAACAACTTCTTTTTTTTTTTTTAAATTATCTGTTTCAGGAAAGAACGATTATCCATTCAAATTTTAGTAAATTTTTTACTTTTAGT 60 RB1 AATTAACAACTTCTTTTTTTTTTTTTAATTTATCTGTTTCAGGAAGAAGAACGATTATCCAAATTTTAGGTAATTTTTACTTTTAGTAAAAAATTTTTTTCTTTTTATAGAA 61 RB1 TTTTTTTTTTTTTAAATTATCTGTTTCAGGAAGAAGAACGATTATCCATTCAAATTTTAGGTAATTTTTACTTTTAAAAAATTTTTCTTTTTTTATAGAAGTAAGTATTTTATA 62 RB1 GAAGTAAGTATTTTATAATCTTTTTTTTTTTCCTTTAGCAAACTTCTGAATGACAACATTTTCATATGTCTTTATTGGCGTGCGCTCTTGAGGTTGTAATGGCCACATATAGCAGTAAG 63 RB1 CTTTAGCAAACTTCTGAATGACAACATTTTTCATATGTCTTTTATTGGCGTGCGCTCTTGAGGTTGTAATGGCCACATATAGCAGTAAGTTAAATTTTCATAAATAAACACTTTTGTTCAA 64 RB1 TTCATTTTTAGGAAGTACATCTCAGAATCTTGATTCTGGAACAGATTTGTCTTTCCCATGGATTCTGAATGTGCTTAATTTAAAAGCCTTTGATTTTTACAAAGTGATCGAAAGTTTTAT 65 RB1 CAAAGCAGAAGGCAACTTGACAAGAGAAATGATAAAACATTTAGAACGAATGTGAACATCGAATCATGGAATCCCTTGCATGGCTCTCAGTAAGTAGCTAAATAATTGAAGAAATTCATTC 66 RB1 AATATATCTAGGTATCTTTCTCCTGTAAGATCTCCAAAGAAAAAAGGTTCAACTACGCGTGTAAATTCTACTGCAAATGCAGAGACACAAGCAACCTCAGCCTTCCAGACCCAGAAGCCA 67 RB1 AAAGGTTCAACTACGCGTGTAAATTCTACTGCAAATGCAGAGACACAAGCAACCTCAGCCTTCCAGACCCAGAAGCCATTGAAATCTACCTCTCTTTCACTGTTTTATAAAAGGTTAG 68 RB1 TTCCCACAGTGTATCGGCTAGCCTATCTCCGGCTAAATACACTTTGTGAACGCCTTCTGTCTGAGCACCCAGAATTAGAACATATCATCTGGACCCTTTTCCAGCACACCCTGCAGAATG 69 RB1 TACACTTTGTGAACGCCTTCTGTCTGAGCACCCAGAATTAGAACATATCATCTGGACCCTTTTCCAGCACACCCTGCAGAATGAGTATGAACTCATGAGAGACAGGCATTTGGACCAAGT 70 RB1 CTGACTACTTTTACATCAATTTATTTACTAGATTATGATGTGTTCCATGTATGGCATATGCAAAGTGAAGAATATAGACCTTAAATTCAAAATCATTGTAACAGCATACAAGGATCTTCC 71 RB1 GTGTTCCATGTATGGCATATGCAAAGTGAAGAATATAGACCTTAAATTCAAAATCATTGTAACAGCATACAAGGATCTTCCTCATGCTGTTCAGGAGGTAGGTAATTTTCCATAGTAAGT 72 RB1 TTTTTTACTGTTCTTCCTCAGACATTCAAACGTGTTTTGATCAAAGAAGAGGAGTATGATTCTATTATAGTATTCTATAACTCGGTCTTCATGCAGAGACTGAAAACAAATATTTTGCAG 73 RB1 CAAAGAAGAGGAGTATGATTCTATTATAGTATTCTATAACTCGGTCTTCATGCAGAGACTGAAAACAAATATTTTGCAGTATGCTTCCACCAGGGTAGGTCAAAAGTATCCTTTGATTGG 74 RB1 CATTAAATAAATAATCTACTTTTTTGTTTTTGCTCTAGCCCCCTACCTTGTCACCAATACCTCACATTCCTCGAAGCCCTTACAAGTTTCCTAGTTCACCCTTACGGATTCCTGGAGGGA 75 RB1 ACATCTATATTTCACCCCTGAAGAGTCCATATAAAATTTCAGAAGGTCTGCCAACACCAACAAAAATGACTCCAAGATCAAGGTGTGTGTTTTCTCTTTAGGGAAGTAGTAAAGAATGAG 76 RB1 TTATGCTCATCTCTGCAAAATTGTATATGGTTTTTTATTACTAATTGGTATTTCATCTTAACTTGACAGAATCTTAGTATCAATTGGTGAATCATTCGGGGTGAGTATTTTCTTTCTATG 77 RB1 TATGGTTTTTTATTACTAATTGGTATTTCATCTTAACTTGACAGAATCTTAGTATCAATTGGTGAATCATTCGGGGTGAGTATTTTCTTTCTATGAAATATAATAGTATGCATTGTAAGT 78 RB1 AATTGGTATTTCATCTTAACTTGACAGAATCTTAGTATCAATTGGTGAATCATTCGGGGTGAGTATTTTCTTTCTATGAAATATAATAGTATGCATTGTAAGTATAAAAGAAATTAAAGC 79 CREBBP TTCCTTCAGCGCCGGGTACCTTGTAGTCATGGATGATCCGCTCTGCAAACGCCTTGTCCAGCATCTTTTTGTACCACTCCTGCAGTCGTTTTGGCTTGGGTATTTTTTGATCAGGTGGGT 80 CREBBP GGCAATGGAAGATGTAATCATCTCCTTCACTTGGAGGACAGGCCCAGATGTGCCCTGTCACATACCTGCAGGACCCACGCACACACGTCAGATGAACGTGCCAGTGAAATCGGCCCTGCC 81 TP53 GAAGGGGCTGAGGTCACTCACCTGGAGTGAGCCCTGCTCCCCCCTGGCTCCTTCCCAGCCTGGGCATCCTTGAGTTCCAAGGCCTCATTCAGCTCTCGGAACATCTCGAAGCGCTCACGC 82 TP53 TGCTCCCCCCTGGCTCCTTCCCAGCCTGGGCATCCTTGAGTTCCAAGGCCTCATTCAGCTCTCGGAACATCTCGAAGCGCTCACGCCCACGGATCTGCAGCAACAGAGGAGGGGGAGAAG 83 TP53 CTGGAAACTTTCCACTTGATAAGAGGTCCCAAGACTTAGTACCTGAAGGGTGAAATATTCTCCATCCAGTGGTTTCTTCTTTGGCTGGGGAGAGGAGCTGGTGTTGTTGGGCAGTGCTAG 84 TP53 CTTAGTACCTGAAGGGTGAAATATTCTCCATCCAGTGGTTTCTTCTTTGGCTGGGGAGAGGAGCTGGTGTTGTTGGGCAGTGCTAGGAAAGAGGCAAGGAAAGGTGATAAAAGTGAATCT 85 TP53 TCCTGCTTGCTTACCTCGCTTAGTGCTCCCTGGGGGCAGCTCGTGGTGAGGCTCCCCTTTCTTGCGGAGATTCTCTTCCTCTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACACGCAC 86 TP53 TGAGGCTCCCCTTTCTTGCGGAGATTCTCTTCCTCTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACACGCACCTCAAAGCTGTTCCGTCCCAGTAGATTACCACTACTCAGGATAGGA 87 TP53 AGGGTGGCAAGTGGCTCCTGACCTGGAGTCTTCCAGTGTGATGATGGTGAGGATGGGCCTCCGGTTCATGCCGCCCATGCAGGAACTGTTACACATGTAGTTGTAGTGGATGGTGGTACA 88 TP53 GATGGTGAGGATGGGCCTCCGGTTCATGCCGCCCATGCAGGAACTGTTACACATGTAGTTGTAGTGGATGGTGGTACAGTCAGAGCCAACCTAGGAGATAACACAGGCCCAAGATGAGGC 89 TP53 CCCCAGTTGCAAACCAGACCTCAGGCGGCTCATAGGGCACCACCACACTATGTCGAAAAGTGTTTCTGTCATCCAAATACTCCACACGCAAATTTCCTTCCACTCGGATAAGATGCTGAG 90 TP53 ATAGGGCACCACCACACTATGTCGAAAAGTGTTTCTGTCATCCAAATACTCCACACGCAAATTTCCTTCCACTCGGATAAGATGCTGAGGAGGGGCCAGACCTAAGAGCAATCAGTGAGG 91 TP53 AGCCCCAGCTGCTCACCATCGCTATCTGAGCAGCGCTCATGGTGGGGGCAGCGCCTCACAACCTCCGTCATGTGCTGTGACTGCTTGTAGATGGCCATGGCGCGGACGCGGGTGCCGGGC 92 TP53 GGGGGTGTGGAATCAACCCACAGCTGCACAGGGCAGGTCTTGGCCAGTTGGCAAAACATCTTGTTGAGGGCAGGGGAGTACTGTAGGAAGAGGAAGGAGACAGAGTTGAAAGTCAGGGCA 93 TP53 GATACGGCCAGGCATTGAAGTCTCATGGAAGCCAGCCCCTCAGGGCAACTGACCGTGCAAGTCACAGACTTGGCTGTCCCAGAATGCAAGAAGCCCAGACGGAAACCGTAGCTGCCCTGG 94 TP53 TAGGTTTTCTGGGAAGGGACAGAAGATGACAGGGGCCAGGAGGGGGCTGGTGCAGGGGCCGCCGGTGTAGGAGCTGCTGGTGCAGGGGCCACGGGGGGAGCAGCCTCTGGCATTCTGGGA 95 TP53 GCTTCATCTGGACCTGGGTCTTCAGTGAACCATTGTTCAATATCGTCCGGGGACAGCATCAAATCATCCATTGCTTGGGACGGCAAGGGGGACTGTAGATGGGTGAAAAGAGCAGTCAGA 96 TP53 CCCAGCCCCCCAGCCCTCCAGGTCCCCAGCCCTCCAGGTCCCCAGCCCAACCCTTGTCCTTACCAGAACGTTGTTTTCAGGAAGTCTGAAAGACAAGAGCAGAAAGTCAGTCCCATGGAA 97 TP53 CTCCAGGTCCCCAGCCCAACCCTTGTCCTTACCAGAACGTTGTTTTCAGGAAGTCTGAAAGACAAGAGCAGAAAGTCAGTCCCATGGAATTTTCGCTTCCCACAGGTCTCTGCTAGGGGG

[0053] Example 2: Evaluation of the capture efficiency and uniformity of SCLC-specific probe sets for clinical samples:

[0054] The performance of the SCLC-specific probe set was tested using clinical cfDNA samples to evaluate the probe capture efficiency and uniformity.

[0055] (1) Experimental procedure:

[0056] Step ① cfDNA extraction: After the whole blood sample was centrifuged at 1,600 g and 16,000 g to separate plasma and remove cell debris from the plasma, cfDNA was extracted from the plasma using the MagMAX™ Cell-Free DNA Separation Kit (Thermo Fisher) via magnetic beads.

[0057] Step ② Library Construction: The cfDNA was end-repaired and an "A" was added using a customized human molecular residual lesion (MRD) detection kit (GenePlus). Then, the DNA underwent adapter ligation, purification, pre-capture PCR (Non-C-PCR), and further purification to obtain a pre-capture intermediate library. Samples with acceptable intermediate library concentrations were then subjected to subsequent hybridization and elution.

[0058] Step ③ Hybridization Capture: Using a customized human molecular residual lesion (MRD) detection kit (GenePlus), the concentration-controlled library was subjected to pooling, evaporation, hybridization with mixed probes, elution, PCR of the elution products, and purification to obtain a hybridized general library. The general library was then sequenced after passing concentration and fragment distribution quality control.

[0059] Step 4 Sequencing and FASTQ data output: Paired-end (PE100) sequencing was performed using the Gene+Seq2000 sequencer, and the data was split into fastq files after being downloaded from the sequencer.

[0060] Step 5: Data alignment and BAM file generation: Before data alignment, Realseq2 software (version: 1.1.6) was used to: (1) remove the UMI from the ends of the reads and save it in the read name; (2) filter low-quality reads. The obtained fastq was aligned to the human reference genome (version: hs37d5) using BWA software (version: 0.7.15-r1140) to generate the initial alignment result BAM file. Then, Realseq2 software (version: 201808) was used to perform clustering and error correction on the PCR repeat reads in the initial alignment result file with the help of UMI. The indel regions within a 50bp range extending from both ends of the detection chip were re-aligned using common indel mutations from the 1000-person database and the dbSNP (version: 138) database. The base quality values ​​within a 50bp range extending from both ends of the detection chip were re-corrected using information from the 1000-person database, the dbSNP (version: 138) database, and the COSMIC database.

[0061] Step 6 Sample Quality Control: (1) Sample Pairing Errors: The bioinformatics workflow determines sample pairing by calculating the consistency between homozygous sites in the control sample extending 50 bp from both ends of the chip interval and those in the tumor sample. (2) Sample Contamination: The Calculate Contamination module in GATK (version: 4.1.4) software, combined with the BAM file information of the control and tumor samples, assesses the cross-contamination of samples by reading and statistically analyzing the reads of supporting reference bases in homozygous sites in the test samples.

[0062] Step 7 Mutation calling: This product detects single nucleotide variants (SNVs) and insertion / deletion mutations (Indels) within a 50 bp range extending from both ends of the chip capture region, as well as SVs. The mutations obtained in the above detection process are annotated using the following databases: (1) Gene Annotation Database (version: NCBI release 104); (2) dbSNP database (version: 147); (3) tgp database (version: phase3); (4) COSMIC database (version: v80); (5) ExAC database (version: 0.3.1); (6) clinvar database (version: 20200701). The mutations obtained in the above steps are filtered, and reliable mutations are retained.

[0063] In this embodiment, cfDNA samples from three clinical patients were used to test probe capture efficiency, uniformity, and probe depth coefficient. The probes used in step ③, hybridization capture, were either a personalized probe set for detecting SCLC (hereinafter referred to as the SCLC personalized probe set) used alone, or the SCLC-specific probe set provided in this application (hereinafter referred to as the SCLC-specific probe set) used alone, or a combination of both.

[0064] The sequences of the three SCLC-specific probe sets used in the test are as follows:

[0065] Probe Name Gene Probe Sequence T1 EPHA5 ACACACATGTATAATTTGCCATCATACTTACTTTTGGTCACCACACCTTCTAAATGGATGATGTTAGGATGATCAAACTGTCCCATGATACTTGCTTCACCTAGGAAATCTCTGCGTTGC T1 FAT1 TCACAGAGGGCCCCGTGCAGGCAAGGGTTTCCAGAGCACTCGTCGATATCACTCTGACACCTGCCAAGGAAGTCAGGAATGAGGAGAGACCTCTGTAGCATACGCCAGCGTATGTCAGGC T1 FAT1 CGATCTTATTCCCAATTTTAAATCCTGACTTGTGCCCAAAACTCATCAAACCGTATGCAGGTTCTCCTCAGTGTAACCCATACGGACAACGAAAATGAGGGGGAATGCTTACCAGTGATG T1 FAT1 CATCAAACCGTATGCAGGTTCTCCTCAGTGTAACCCATACGGACAACGAAAATGAGGGGGAATGCTTACCAGTGATGTCAGGGTATGCCAGGAGGCTCCACAGATATTACTCCAATCATG T1 FAT1 TGCCAGGAGGCTCCACAGATATTACTCCAATCATGTGAGCAACGGGGTCACTTTCCATCACAGTAAAGGTAAAAAATGATTCTTCAAATGAAATGGGCTCCAGGGACGGTTTGGGCTTGG T1 MAP3K1 GGCGGCAGCTGCGCAAAGTGCGGAGTGTGGAGCTGGACCAGCTGCCTGAGCAGCCGCTCTTCCTTGCCGCCTCACCGCCGGCCTCCTCGACTTCCCCGTCGCCGGAGCCCGCGGACGCAG T1 APC TTTCAGTGCCAGCTCCTGTTGAACATCAGATCTGTCCTGCTGTGTGTGTTCTAATGAAACTTTCATTTGATGAAGAGCATAGACATGCAATGAATGAACTAGGTAAGACAAAAATGTTTT T1 GABRA6 CCCCTTAACATCAGTGGTGATAATTGTTTCATCCCTCTGGGCTAATTTCAGGTGCTGTCACTGAAGTCAAAACAGACATTTATGTGACCAGTTTTGGGCCCGTGTCAGATGTGGAGATGG T1 CSMD3 AAACGTTAAATGGTGCTAATCTCACATTCATGTTTTTGTTCATTTTGGCATAATATACTTCACCTTGACAGGCAGGGACTGGCGCATCCCATGCATGATACCCATAGGAAGACTTTCTGC T1 PTCH1 CCAGCGCAGCATGGTTAGACAGGCATAGGCGAGCTGCAAGCAGAACAATGGGGGCACAGAACAAAAGCCGAACATTAGAATGTGTTGTGATTCTAATGTTTCCCTCCACCCATCACCCCA T1 CDH23 GCCTTCGAGCGCAACGCCCGCACAGAATCCGCCAAATCCACACCCCTGCACAAACTTCGCGACGTGATCATGGAGACCCCCCTGGAGATCACAGAGCTGTGACTAGACAGGGAAGCCTTG T1 ETV6 GCTTTGGGATTACGTCTATCAGTTGCTTTCTGACAGCCGGTACGAAAACTTCATCCGATGGGAGGACAAAGAATCCAAAATATTCCGGATAGTGGATCCCAACGGACTGGCTCGACTGTG T1 PTPRO ACGTGGCTCTACCTTTTTGCATGCTTAACTAGCAGAGTCCTGGGTCATCCTAACAGTGAACTGAAAAATTAATGCTTGTTTTTGGCTTTTTTCTCTAAAGGCTGACGAGATGCAGGATGT T1 CUL4A GTTTAACATTTGCTTATCAACTCTCTGATGTCTTGATTATTCTCGTCTGGTTATAAATAGGTAAAGATGTCTTTGAAGCATTTTATAAAAAAGATTTGGCAAAAAGACTCCTTGTTGGGA T1 CTCF GACCCAGACTATCAGCCACCAGCCAAAAAAACAAAGAAAACCAAAAAGAGCAAACTGCGTTATACAGAGGAGGGCAAAGATGTAGATGTGTCTGTCTACGATTTTGAGGAAGAACAGCAG T1 SMARCA4 TCTACCCCTGAGGTCACCCCGCTGACCCTGTTCTCCTCTGTGCCCGTCAGGAACCACGAAGGCGGAGGACCGGGGCATGCTGCTGAAAACCTTCAACGAGCCCGGCTCTGAGTACTTCAT T1 BCOR GGGTGGCCACCTTCTCTTTCTTTCATCTCCAACTCTGAGAAGCGCATCATTGCACGCTAGAAAGAGAACGGAGATGGAAAAAAAAAAAAACAACACCTTACCATAAAAGCTATTTCATTT T1 RARA-CUEDC1 GGCTCTGTGGATGTTTGTGCACATGCATGAACACGCATGCCGTGGTGTGCGGGCTCACGGTTGAGGATGGTTTGTGTGTAGCTGCAAGGACCTGTTTGCGAGTCTGGCTGGCTGTGTGTC T1 RARA-CUEDC1 AGGGCTCTGTGGATGTTTGTGCACATGCATGAACACGCATGCCGTGGTGTGCGGGCTCACGGCCAAAATATTACATAATGATTGTTAATTACTACCAAATACTTATTAAGCCCCTAGCAC T1 RARA-CUEDC1 TTGTGCTAGGGGCTTAATAAGTATTTGGTAGTAATTAACAATCATTATGTAATATTTTGGCCGGGCGTGGTGGCTCATACCTATAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATC T2 SPTA1 CACATCCCTGCAGTCATTAACAAGAGCTCCAACCAAAGAAGATAGCAAGCATTACAGGGCAAACGGTCCAACAGAACTCACAGCAAGGACCATCCTAGTTGACCCAGGAGCAATTATCCT T2 LRP1B TCTCTCTTACTCGGTTAAATATTTTAACCTCCTTCAGGTTGACTCCAAGGCCGGTTCTCATGGTTATCGTTTCTGTGGCATCATTCTTGTGGCCCCTTCTGACAGACCCGTTTGCATGTG T2 MST1R CCTTCCCTGAGGCGGCCTTGAGCACCGCACACCCTCATGCCCTGTCCTTTTGCTTCACCCCAGCTACTCTGGACTCTCACATGCAGTTCCGCGCAGCCAGGTCCCTGTGCACAAACTTCT T2 FAT1 GTAGCCTCGACTGTGAGGTTGTAGTTTGACTTCTGTTCTGCATCAAGAGGTTTGGCAACAATGATGGTTCCAGTTCCCTTGTCCACATCGAAGTGACTGTCGTAGTTGCCACCTAATTTG T2 SPINK1 TGTTGGATCAAACTGTTCCAGTCTGAGAAATAAGAAATTACAAATATCTCTTTACCTCTCTTCCCAGGGAGTCAGCTCCAGTGTTACCTAGAAATAAATCAGATATGGTAAGTTGGGTCC T2 NSD1 CATTGATAAGATGAGACATGATGTGGGAGAGTTCCCAGTCCTCTTTTTTGGATCTAATGACTATTTGTGGACTCACCAGGCCCGAGTCTTCCCTTACATGGAGGGTGACGTGAGCAGCAA T2 KEL TTTCTACTCTGTAAGCCTTACATTCCCTCCCCTCCAGTGGGGGCAGGACTTTTGCACACAGCCACCCTCACCCCTCCCCGCTAAGCCTCTGACTCCAAAAGGGGACTTACCATCTGTCTA T2 CSMD3 TGCCAAGAAAATAGTAGACCATTTTTAGAAATAATGAACTCTAGAAAAACAAATGACATTACTAACCACTTAGCTTTGGGCTGGGTGTTCCCAGTGCAGGTCTAGGATCTTTCACCAATA T2 CSMD3 TTCCCTACACAGGTCAAAACAGCAGGGAAGGATAGCTCATAGCCTGGAGAACAGATGTAGCTAATACTAAAGCCCCAGTCGAAATTTGTTCCTTCCAGCCTTCCATTAGAGATCTGGGGA T2 FAM135B TTTCAGACATTAGGAAGTCCAGTTTTCCTCCAGGGAGCCCCAGTTCTATGAAAGTCTTTACCAGCCGGAGGTCTGCACTGTTCCCTAAAAATGACAGATAACCCCCATCCCAGTCCCGTA T2 RET CTTCCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCCGCAACTCCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATCTCATT T2 CYP2C8 TGGGGAATTGCCTCTTCCAGAAAACTCCTCTCCATTATCAATCAGGGCTTCCTTCACTGCCTCATATCCATGAAACACCACTATGGGATTCATGCCAAAATACACGGTGAACACAGGACC T2 ATM TTCTTCCCACTTTGCTTTATATTTGGACTCAACATAGGCTTAATGATTCTTTAAAAGAAGTCATTATTGAATTATTTCAACTGCAAATTTATATCCATCATCCGAAAGGAGCCAAAACCC T2 RB1 GCAACCTCAGCCTTCCAGACCCAGAAGCCATTGAAATCTACCTCTCTTTCACTGTTTTATAAAAAAGGTTAGTAGATGATTATTTTCAAGAGCATGGACTCTGAAACTAGGCTGACTGGG T2 MLH3 TGCATGCAGAGGTGTTTGATCACTGCTATGTTGAAGGGCTTACCATGGCAGGCTTGGGATGCCAACACCTTCTGGACAGTCAGTGGCAATGTCCCTTGGATGCCTCCGGTGGTCTGGAGT T2 TYRO3 TCCTGGGTTTTCCTTTAGGCAAGCCTTTGACAGTGTCATGGCCCGGGGAGAGCCAGCCGTTCACTTCCGGGCAGCCCGGTCCTTCAATCGAGAAAGGCCCGAGCGCATCGAGGCCACATG T2 PRKCB GTGTAAGGAAAACATCTGGGATGGGGTGACAACCAAGACATTCTGTGGCACTCCAGACTACATCGCCCCCGAGGTGAGAGCTGCTGGGCACACGTTCACATTGCGGTGATGTACCCTCTG T2 ZFHX3 AGTTACACACCTCGCAGCGGAAAGGCTTGTAACCACACGTGTAGCTCTCGCCTCGTGCCAGCCGGGGGTGGGGCTGCCCGCTTTTGCAGTAGACACAGGAGCCCCCCGGCTCCGGGTGCT T2 PKD1L2 CTCAGGTGTCAGAAGGCCGTCCTCCATGGGCTGTGGGGAGGGAGTCAGGTTGGGGGACCCCCGGTCCCATTTTCCAGTGTTCTGCTCCTTCGCGACCCGGGGACGGGTGTTCTGAAAGAT T2 RAD51C TATCTTTCAGCCTCAGGGATTTAGAGATACTGTTGTTACTTCTGCATGTTCATTGCAAACAGAAGGTTCCTTGAGCACCCGGAAACGGTCACGAGACCCAGAGGAAGAATTATAACCCAG T3 XPO1 AAAGAACACGTCTTGTTAAATCCCATGAAAACTTTTAAGAAAAGGTAGAAATACTTACTTTGGTTGCCTGCTGGATTATACTATCCCACACTTGATTAGGGAGTAACATGTACTTTTCTA T3 LRP1B CTAGCCTTGCCACATTAATTTGCGTTTCATCAAATTCTGAGCTAATCCAGTATAAATTACGTGACACCCAATCCACTGCTAGCCCTCTGATACTCTGAATATCTATAAAACAGGAAAGAG T3 ERBB4 CTTACCCTATTCGAGTCAATTCTTGCTCTGGAAGTATAGATGGGAGGTGGGATGTTGAAAGCCTGAGGGACCAAGTACTCCTCAGCATCCATCATATCTTCCAAATCCTCTTCATCCAAG T3 BARD1 CAGCTTCATTGCTGAGGGACTAGACATCACTCGCCTGTAACTTGAACTACTTAATGTAGAAGGTGGTGTACCTGGTGAAAGACTAATGAATTCATCGGACATGTTACTGTTTTTCCTCCC T3 CBLB ACCTTTGGTGAACCCGTTGGGCTGCCACAGGGAGATCTAACTATGCCTTTCTGAATTAGATCCAGGCGAGGAGGCACGGGTGGCAGGCTTAGATGTGGGATCTGGAGTGGGTCAGGCTGT T3 KDR TCTCACACGAAATGATGCTTTGCATTTATTTCCAGTAGTTACCATTTTATTTCTGGGGGTGGGTAACCAAGGTACTTCGCAGGGATTCTGACACGCTCCCCCACCGTGGCTTCCACCAGA T3 MYB GGTCCGAAACGTTGGTCTGTTATTGCCAAGCACTTAAAGGGGAGAATTGGAAAACAATGTAGGGAGAGGTGGCATAACCACTTGAATCCAGAAGTTAAGAAAACCTCCTGGACAGAAGAG T3 EPHB6 ACCCAGGGGAAAGGTCTGGAGCTTGGGGCTAGAGCCTGGGAAAGCCAGGGAGGGTAGATGCAAACCTAAAGAAAACTGAGGAGAGGGGCTAAGATGAAGAGGAGACCTTGACCCTGCTTG T3 MLL3 AAATTAGACAATATGAACATACTGCTTACCAGCAATAAGAAGACTATCTGTGTCAAGACTTTCTGAGGGATGACTCTTCTGTTGCTCTTCAGTGTGGACTTGAACCGCTGTGAGTAACAC T3 GATA3 ACCCGGGCCTCAGCCACTCCTACATGGACGCGGCGCAGTACCCGCTGCCGGAGGAGGTGGATGTGCTTTTTAACATCGACGGTCAAGGCAACCACGTCCCGCCCTACTACGGAAACTCGG T3 PIK3C2G TTAGTGCAACTTCTACTCCACCGCTCCTTGCAGAGCATCCAGGTTGCCCATCGTCTTTACTGGTAAGATTAACTAAATCAGGCAAGGATGCCTTTTTAATTGCCACCTTCTCTATGGGGC T3 CDK4 GGGGTGCCTTGTCCAGATATGTCCTTAGGTCCTGGTCTACATGCTCAAACACCAGGGTTACCTTGATCTCCCGGTCAGTTCGGGATGTGGCACAGACGTCCATCAGCCTGACCAGAGTAA T3 NAV3 CCTGGGATAGTCCTGAGGAACTGAAAAAACCAGAAGAAGATTTTGACAGCCATGGGGATGCTGGTGGCAAGTGGAAGACTGTGTCCTCTGGACTTCCTGAAGACCCCGAGAAGGCAGGGC T3 NAV3 AGTTCAGAGAACCATTTTTGTTGGATGTGTAATTTGGAAGTTTTGTTTACATTATGTCCTTAGGGGTTTTCTTTGTTTTAACAGCATGCAGCTTGACAGAAATACACTACCCAAAAAGGG T3 FLT1 CTCTGCCCTCCGGCCGCCCCATCGCAGCCCGCCTCAGGCCCCGGCCCCCAGCCGCGCCTCACCTGTGAGAAGCAGACAGCTGAGCAGCGCGCACAGCAGGACCCCGGTGTCCCAGTAGCT T3 RB1 AAAAGCCTTTGATTTTTACAAAGTGATCGAAAGTTTTATCAAAGCAGAAGGCAACTTGACAAGAGAAATGATAAAACATTTAGAACGATGTGAACATCGAATCATGGAATCCCTTGCATG T3 RB1 AGCAGAAGGCAACTTGACAAGAGAAATGATAAAACATTTAGAACGATGTGAACATCGAATCATGGAATCCCTTGCATGGCTAAATAATTGAAGAAATTCATTCATGTGCATATGGCTAAC T3 RB1 AAATAATTGAAGAAATTCATTCATGTGCATATGGCTAACAAATTATTGTTAGTGAGAGGTGTTTCTTAACATCTACCTCAAGAACATATAGGGAATTTAATGAATAATGTTATTTCAGTC T3 TYRO3 GACTGAGGTTTTCTGGCCCCAGGGGTGACCCAGAGCACCATGTTTTCCTGTGAAGCTCACAACCTAAAAGGCCTGGCCTCTTCTCGCACAGCCACTGTTCACCTTCAAGGTAGGAGGGCT T3 MC1R CATCGCCTACTACGACCACGTGGCCGTCCTGCTGTGCCTCGTGGTCTTCTTCCTGGCTATGCTGGTGCTCATGGCCGTGCTGTACGTCCACATGCTGGCCCGGGCCTGCCAGCACGCCCA T3 SMARCA4 ACCTGGGACGCACTGTTTTCTCTTTTGTTTCTCCCTACATGTAGGTATGGGAGGGCCCAACATGCCTCCCCCAGGACCCTCGGGCGTGCCCCCCGGGATGCCAGGCCAGCCTCCTGGAGG T3 SMARCA4 TGCAGCCAAGTACAAGCTCAACGTGGACCAGAAGGTGATCCAGGCCGGCATGTTCGACCAGAAGTCCTCCAGCCATGAGCGGCGCGCCTTCCTGCAGGCCATCCTGGAGCACGAGGAGCA T3 POLD1 CAGCCTGCCCATTGACACGCAGTACTACCTGGAGCAGCAGCTGGCCAAGCCCCTCCTGCGCATCTTCGAGCCCATCCTGGGCGAGGGCCGTGCCGAGGCTGTGCTACTGCGTACGGGGGC T3 KDM5C CAGCCTCGGCAAAGTTGTAGCCTTGGTTGAAGCCGCTGTGGTAAGCACGGGGGAAGGTGATGACAAACTCTCCTGCACACTGGTTTGTGCGGACAACCTGAAGAACACAAAAGGCCATGG

[0066] Probe capture efficiency;

[0067] The probe capture efficiency test results for clinical samples are as follows: Figure 1 As shown in the figure, the capture efficiency using only the SCLC personalized probe set ranged from 21.9% to 38.8%, exhibiting significant fluctuations, with a median capture efficiency of 36.7%. The capture efficiency of the SCLC specific probe set ranged from 65.5% to 69.6%, with a median capture efficiency of 67.7%. The capture efficiency of using both the SCLC personalized and specific probe sets in combination ranged from 63.7% to 65.5%, with a median capture efficiency of 64.0%. These results indicate that compared to the SCLC personalized probe set, the SCLC specific probe set significantly increased the capture efficiency with less fluctuation. Furthermore, the combined use of the SCLC personalized and specific probe sets significantly improved the capture efficiency and increased its stability.

[0068] Probe uniformity;

[0069] The probe uniformity test results using the SCLC probe set alone are as follows: Figures 2-3 As shown, the percentage of probes at depths ≥0.2 times the average depth was 100% in all three cfDNA samples, while the percentage of probes at depths ≥0.5 times the average depth ranged from a minimum of 96.7% to a maximum of 98.6%, with a median of 97.6%. These results indicate that the SCLC-specific probe set exhibits excellent homogeneity, meeting the needs of clinical testing.

[0070] Probe depth coefficient;

[0071] The depth coefficient results of the SCLC specific probe group are as follows: Figure 4 As shown in the figure. The test results show that the median values ​​of the probe depth coefficients for the three samples are 0.99, 0.98 and 0.97, respectively, indicating that the SCLC specific probe group has a relatively consistent depth and the depth coefficients are stable in different samples.

[0072] Example 3: Coverage of SCLC-specific probe group in SCLC patients, such as... Figure 5 As shown;

[0073] The coverage of the SCLC-specific probe group for SCLC patients (the proportion of samples covering at least one mutation) was evaluated using clinical and literature samples, respectively. The evaluation results showed that the coverage of the SCLC-specific probe group for clinical and literature samples was 94.9% (373 / 393) and 97.2% (280 / 288), respectively. Therefore, the SCLC-specific probe group has excellent coverage of the SCLC population.

[0074] References:

[0075] George J, Lim JS, Jang SJ, et al. Comprehensive genomic profiles of small cell lung cancer[J]. Nature, 2015, 524(7563): 47-53.

[0076] Wang H, Wu S, Li Z, et al. Molecular subtyping of small-cell lungcancer based on mutational signatures with different genomic features and therapeutic strategies[J]. Cancer Science, 2023, 114(2): 665-679.

[0077] Example 4: The clinical effectiveness of SCLC-specific probe sets in assessing systemic tumor burden;

[0078] Thirty patients with extensive-stage SCLC were included to validate the clinical effectiveness of SCLC-specific probe groups in assessing systemic tumor burden. Comprehensive genomic analysis of tumor tissues from these 30 patients was performed to identify patient-specific tumor mutation profiles. First, the coverage of mutations detected in SCLC patient tumor tissues by the SCLC-specific probe group was evaluated (the proportion of samples covering at least one mutation). The results showed that the SCLC-specific probe group achieved 100% coverage in all 30 patients, with a median coverage of two mutations per sample, further demonstrating the excellent coverage of the SCLC-specific probe group in the SCLC population.

[0079] Then, the SCLC-specific probe set was used to perform targeted capture and ultra-high-depth sequencing on baseline blood samples from 30 patients before treatment. Evaluation results showed that all 30 baseline blood samples were identified as ctDNA positive, and the sensitivity of the SCLC-specific probe set for detecting baseline blood samples from SCLC patients was 100% (30 / 30). A total of 53 tumor-derived mutations were detected in the 30 baseline blood samples, including 3 samples with 3 mutations each, 17 samples with 2 mutations each, and 10 samples with 1 mutation each, with a median of 2 tumor-derived mutations detected per sample. These results demonstrate that the SCLC-specific probe set has excellent detection sensitivity for assessing the systemic tumor burden in SCLC patients.

[0080] Based on the mutation profiles of tumor tissues from 30 patients, 2-20 mutations were selected for each patient to create a customized probe set (the personalized probe selection algorithm is described in patent ZL 2023 1 0890251.0). The number of mutations monitored by the customized probes for each patient was statistically analyzed. 77% of patients (23 / 30) had 20 mutations monitored, and 97% of patients (29 / 30) had at least 10 mutations monitored, with a median of 20 mutations monitored per patient. Literature reports that increasing the number of mutations tracked can improve analytical sensitivity, thereby increasing the detection rate of samples with low tumor burden (refer to the consensus on molecular residual lesion detection in solid tumors).

[0081] A patient-specific probe set was created by combining a personalized probe set for SCLC with a SCLC-specific probe set. Baseline blood samples were then targeted for capture and subjected to ultra-high-depth sequencing to assess the systemic tumor burden in SCLC patients. Results showed that the sensitivity of the patient-specific probe set in detecting baseline blood samples from SCLC patients was 100% (30 / 30). A total of 550 tumor-derived mutations were detected in 30 baseline blood samples. Specifically, 18 samples each had 20 mutations, 4 samples each had 19 mutations, 3 samples each had 18 mutations, 2 samples each had 12 mutations, and one sample each had 16, 13, and 7 mutations, respectively. The median number of tumor-derived mutations detected per sample was 20. These results demonstrate that the combination of the SCLC-specific and personalized probe sets still exhibits excellent detection sensitivity for assessing the systemic tumor burden in SCLC patients. The combined use of the SCLC-specific and personalized probe sets can track more tissue-derived mutations and detect more tissue-derived mutations in the blood. Tracking more mutations can increase the detection sensitivity of low tumor burden samples and further improve the accuracy of tumor burden assessment.

[0082] The above technical solutions only embody the preferred technical solutions of the present invention. Any modifications that may be made by those skilled in the art to certain parts thereof embody the principles of the present invention and fall within the protection scope of the present invention.

Claims

1. A probe set for assessing systemic tumor burden in small cell lung cancer (SCLC), characterized in that, The probe set contains multiple oligonucleotide probes that specifically target and capture mutation regions of SCLC-related genes, which are selected from TP53, RB1, CREBBP, EGFR, PTEN, PIK3CA, LRP1B, and NOTCH1; and the probe set can cover at least one mutation present in at least 94.9% of SCLC patient samples.

2. The probe assembly according to claim 1, characterized in that, The probe set targets mutation regions including at least one of the following regions: exons 3-10 of the TP53 gene; exons 2, 3, 5, 7-17, 19-24 of the RB1 gene; exon 27 of the CREBBP gene; exons 18-21 of the EGFR gene; exon 3 of the PTEN gene; exon 10 of the PIK3CA gene; exons 3, 6, 65, 68, 75, 77 of the LRP1B gene; and exons 20 and 23 of the NOTCH1 gene.

3. The probe assembly according to claim 1 or 2, characterized in that, The oligonucleotide probe is 100-150 bp in length, preferably 120 bp, and the 5' end of the probe is operably linked with a biotin label.

4. The probe assembly according to any one of claims 1-3, characterized in that, The probe set comprises the nucleotide sequences shown in SEQ ID NO: 1 to 97, or variants thereof that have at least 95% identity with the sequences and retain the same targeting and capture function.

5. A combined probe system for assessing systemic tumor burden in small cell lung cancer (SCLC), characterized in that, include: (a) The SCLC-specific probe set according to any one of claims 1-4; (b) A personalized probe set based on the mutation profile of tumor tissue of an individual SCLC patient; wherein the SCLC-specific probe set and the personalized probe set can be used in combination for targeted capture of the same circulating tumor DNA sample.

6. A kit for assessing systemic tumor burden in small cell lung cancer (SCLC), characterized in that, It comprises a probe set as described in any one of claims 1-4 or a combined probe system as described in claim 5, and at least one reagent for hybridization capture and high-throughput sequencing.

7. A method for assessing the systemic tumor burden of small cell lung cancer (SCLC) for non-diagnostic purposes, characterized in that, Includes the following steps: Step S1: Provide a liquid biopsy sample from a suspected or confirmed SCLC individual and obtain circulating tumor DNA (ctDNA) from it; Step S2: Use the probe set according to any one of claims 1-4 or the combined probe system according to claim 5 to target and capture the ctDNA; Step S3: Perform high-throughput sequencing on the captured DNA; Step S4: Analyze the sequencing data and assess the individual's systemic tumor burden based on the mutation information of the gene regions targeted by the probe set.

8. The method according to claim 7, characterized in that, The liquid biopsy sample is plasma or serum; the method also includes using the assessment results to monitor treatment effectiveness, predict prognosis, or guide adjustments to the treatment plan.

9. The use of the probe set according to any one of claims 1-4, the combined probe system according to claim 5, or the kit according to claim 6 in the preparation of a product for assessing the systemic tumor burden of small cell lung cancer (SCLC).

10. A non-transitory computer-readable storage medium storing instructions that, when executed by a processor, cause the processor to perform steps for analyzing sequencing data generated by the method of claim 7, the steps comprising: Identify and quantify mutations from the gene described in claim 1, and generate an assessment report reflecting the level of tumor burden.

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