Application of maximum allelic mutation abundance in predicting mismatch repair protein-intact immunotherapy efficacy in metastatic colorectal cancer

Abstract

The application relates to application of maximum allelic mutation abundance in predicting immunotherapy efficacy of mismatch repair protein-intact metastatic colorectal cancer, in particular to application of maximum allelic mutation abundance and a reagent or system for detecting maximum allelic mutation abundance in preparation of a system for predicting immunotherapy efficacy of mismatch repair protein-intact metastatic colorectal cancer. The application is the first to use baseline ctDNA maxVAF as an immunotherapy efficacy prediction marker, fills the blank in the field, better guides individualized clinical treatment, uses a peripheral blood sample, calculates peripheral blood circulation maximum allelic mutation abundance, the sample is convenient to obtain, does not need invasive tissue biopsy, and is highly accepted by patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to the application of maximum allele mutation abundance (maxVAF) in predicting the efficacy of immunotherapy for metastatic colorectal cancer (mCRC) with mismatch repair protein integrity (pMMR). Background Technology

[0002] Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer death worldwide, with mismatch repair protein intact / microsatellite stable (pMMR / MSS) mCRC accounting for 95% of all mCRC cases. While systemic chemotherapy combined with targeted therapy has significantly prolonged survival for these patients, the efficacy of third-line therapy remains limited. The combination of anti-angiogenic tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) is currently the most studied treatment strategy for advanced mCRC; however, effective biomarkers are lacking for evaluating efficacy.

[0003] Currently, the development of biomarkers for evaluating efficacy mainly focuses on immunotherapy, including the expression of programmed cell death protein ligand 1 (PD-L1), tumor mutation burden (TMB) levels, microsatellite stability, and the presence or absence of mismatch repair proteins.

[0004] The above biomarkers have limitations in predicting the efficacy of immunotherapy in pMMR mCRC patients and require tissue samples for testing. Therefore, there is an urgent need to develop new biomarkers to guide clinical applications. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this application provides the application of maximum allele mutation abundance (maxVAF) or substances for detecting maximum allele mutation abundance (maxVAF) in a system for assessing the efficacy of immunotherapy for metastatic colorectal cancer (mCRC).

[0006] This application aims to provide effective biomarkers for predicting the efficacy of targeted immunotherapy for pMMR / MSS type mCRC, making up for the current lack of biomarkers for predicting the efficacy of targeted immunotherapy for pMMR / MSS type mCRC, and overcoming the limitations of relying mainly on the expression level of programmed cell death protein ligand 1 (PD-L1), tumor mutation burden (TMB) size, microsatellite stability, and the absence of mismatch repair proteins in predicting the efficacy of immunotherapy for pMMR / MSS type mCRC.

[0007] VAF, short for Variant Allele Frequency, is a key parameter in bioinformatics for measuring the frequency of genomic variations. It is defined as the proportion of reads supporting a mutation or substitution at a given locus relative to the total coverage depth. In VCF files, VAF is calculated using DP (Total Depth) and AD (Allele Depth), with the formula: VAF = Allele Depth / Total Depth. In diploid germline genotyping, the VAF at heterozygous sites is typically close to 50% at high depths (e.g., DP > 80), while a ratio close to 0.25 / 0.75 may suggest the presence of another copy of the genome. In cancer genomic analysis, VAF is crucial for assessing tumor heterogeneity, tumor purity, and prognosis.

[0008] Colorectal cancer can be divided into mismatch repair deficient type (dMMR) and mismatch repair intact type (pMMR). dMMR patients have excellent performance in PD-1 monoclonal antibody therapy, while pMMR patients have limited efficacy due to their lower immunogenicity and other reasons.

[0009] This application also provides the use of substances for detecting the abundance of maximum allele mutations in the preparation of kits for evaluating the efficacy of immunotherapy for metastatic colorectal cancer and / or the prognosis of immunotherapy for metastatic colorectal cancer.

[0010] As some embodiments of this application, the metastatic colorectal cancer includes metastatic colorectal cancer with intact mismatch repair proteins.

[0011] As some embodiments of this application, the maximum allele mutation abundance is one or more of the maximum allele mutation abundance in peripheral blood circulation (maxVAF) and the maximum allele mutation abundance in tissue samples.

[0012] This application can use peripheral blood to detect the abundance of the largest allele mutation, without relying on tissue samples, which greatly improves the convenience and operability of the test.

[0013] As some embodiments of this application, the maximum allele mutation abundance of the tissue sample is the maximum allele mutation abundance of the cancer tissue sample or the maximum allele mutation abundance of the adjacent normal tissue sample.

[0014] As some embodiments of this application, the maximum allele mutation abundance of the tissue sample is the maximum allele mutation abundance of the biopsy tissue sample.

[0015] As some embodiments of this application, the therapeutic agents for the immunotherapy efficacy of the mismatch repair protein intact metastatic colorectal cancer include one or more of PD-L1 antagonists, PD-1 antagonists, and CTLA-4 antagonists.

[0016] Immunotherapy agents, namely immune checkpoint inhibitors (ICIs), are a class of protein molecules developed to block inhibitory immune regulatory sites in the body's immune system and enhance the body's anti-tumor effects. They include PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors.

[0017] PD-1 / PD-L1 inhibitors restore T cell activity by blocking the interaction between PD-1 and PD-L1, enabling them to attack cancer cells. They have shown promising results in the treatment of various cancers, including melanoma, non-small cell lung cancer, and renal cell carcinoma.

[0018] CTLA-4 inhibitors enhance the immune system's ability to attack tumors by inhibiting the CTLA-4 protein, thereby increasing T cell activation and proliferation. CTLA-4 inhibitors have shown significant efficacy in the treatment of melanoma.

[0019] As some embodiments of this application, the immunotherapy efficacy of the intact mismatch repair protein in metastatic colorectal cancer is a targeted immunotherapy efficacy.

[0020] As some embodiments of this application, the reference for gene mutations is the hg19 human reference genome or the hg38 human reference genome.

[0021] The hg19 human reference genome (GRCh37) is a version of the human genome released in 2009, also known as GRCh37 (Genome Reference Consortium human genome build37).

[0022] The hg38 human reference genome (GRCh38) is a version of the human genome released in 2013, also known as GRCh38 (Genome Reference Consortium human genome build38). This version has some improvements over hg19, including better resolution of some difficult-to-map regions of the genome and more comprehensive annotation.

[0023] As some embodiments of this application, the gene mutation includes frameshift, non-frameshift, nonsense, and missense mutations.

[0024] This application also provides a method for generating the abundance of the largest allele mutation in peripheral blood circulation, the method comprising: sequencing a tissue sample or a peripheral blood ctDNA sample to obtain sequencing data; comparing the sequencing data obtained by the sequencing module with a reference genome to determine whether the tissue sample or the peripheral blood ctDNA has undergone mutation; calculating the abundance of the largest allele mutation in the tissue sample or the peripheral blood ctDNA sample; and determining whether the abundance of the largest allele mutation is greater than a threshold, wherein the threshold is 6.9%, based on the calculated result.

[0025] As some embodiments of this application, a cfDNA library generated from a peripheral blood sample is sequenced to obtain sequencing data, which includes sequencing data of 1249 cancer-related genes listed in Tables 1-4.

[0026] As some embodiments of this application, the obtained sequencing data is compared with a reference genome, and the obtained sequencing data is processed, including filtering variants, optimizing variant identification, variant annotation, excluding known germline SNPs, and retaining frameshift, non-frameshift, nonsense, and missense mutations.

[0027] In some embodiments of this application, the sequencing is next-generation sequencing. In some embodiments of this application, the sequencing mode is PE100 mode. In some embodiments of this application, variants are filtered using support counts, strand bias states, base quality, and the quality of variant mapping values. In some embodiments of this application, optimized variant identification is used to look for variants in short tandem repeat regions. In some embodiments of this application, variant annotation includes labeling single nucleotide polymorphisms (SNPs) and insertion / deletion mutations (indels). In some embodiments of this application, ANNOVAR is used to label single nucleotide polymorphisms (SNPs) and insertion / deletion mutations (indels) in the dbSNP(v138), 1000 Genomes, and ESP6500 databases. In some embodiments of this application, exclusion of known germline SNPs includes excluding SNPs with a population frequency ≥0.015 in the 1000 Genomes, dbSNP, and ESP6500 databases.

[0028] This application also provides an apparatus for predicting the efficacy of immunotherapy in metastatic colorectal cancer with intact mismatch repair protein (pMMR). The apparatus includes: a sequencing module for sequencing tissue samples or peripheral blood ctDNA samples to obtain sequencing data; an alignment module for aligning the sequencing data obtained by the sequencing module with a reference genome to determine whether the tissue sample or peripheral blood ctDNA has undergone mutation; a calculation module for calculating the maximum allele mutation abundance (maxVAF) in the tissue sample or peripheral blood ctDNA sample; and a judgment module for judging the efficacy of immunotherapy in metastatic colorectal cancer (mCRC) with intact mismatch repair protein (pMMR) based on the results obtained by the calculation module. When the maximum allele mutation abundance (maxVAF) is greater than a threshold, the patient is considered suitable for immunotherapy, and the threshold is 6.9%.

[0029] This application also provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the method described thereon.

[0030] This application also provides a computer processing device, which includes a processor and the computer-readable storage medium, wherein the processor operates the computer-readable storage medium to implement the method.

[0031] This application also provides an electronic terminal, which includes a communicator, a memory, and a processor. The communicator is used to communicate with an external identifier to receive sequencing data from detected tissue samples or peripheral blood ctDNA samples. The memory is used to store the computer program. The processor is used to execute the computer program stored in the memory so that after the electronic terminal analyzes the sequencing data, it analyzes the abundance of the largest allele mutation in the tissue sample or peripheral blood ctDNA sample. When the abundance of the largest allele mutation is greater than a threshold of 6.9%, it predicts that the patient with metastatic colorectal cancer with intact mismatch repair proteins will have a better response to immunotherapy.

[0032] This application also provides the use of any one of the aforementioned apparatus, computer-readable storage medium, computer processing device, and electronic terminal in preparing a system for predicting the efficacy of immunotherapy in metastatic colorectal cancer with intact mismatch repair proteins.

[0033] As described above, the application of the maximum allele mutation abundance in this application in predicting the efficacy of immunotherapy for metastatic colorectal cancer with intact mismatch repair proteins has the following beneficial effects:

[0034] (1) Currently, there is a lack of biomarkers for predicting the efficacy of targeted immunotherapy for pMMR / MSS mCRC. This application is the first to use baseline ctDNA maxVAF as a predictive marker for efficacy, filling the gap in this field and better guiding individualized clinical treatment.

[0035] (2) Peripheral blood samples are easy to obtain, do not require invasive tissue biopsy, and are highly accepted by patients. Attached Figure Description

[0036] Figure 1 The experimental procedure diagram of an embodiment of this application is shown;

[0037] Figure 2 A flowchart of the experimental processing according to an embodiment of this application is shown;

[0038] Figure 3 The differences in PR, SD and PD patients in 74 patient sample groups of this application are shown, including statistics of median maximum VAFs, comparison with values ​​calculated from the maximum Youden index, and comparison of ORR between different patients (patients in the low maxVAF group and the high maxVAF group).

[0039] Figure 4 The PFS and OS statistics for patients in the low maxVAF group and the high maxVAF group are shown. Detailed Implementation

[0040] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0042] Experimental Materials and Methods

[0043] 1. Implementation Approach

[0044] Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) in peripheral blood was performed on peripheral blood samples collected at baseline to predict the efficacy of targeted immunotherapy for pMMR / MSS type mCRC based on maxVAF values.

[0045] 2. Specific Implementation Process

[0046] (1) Blood sample processing and cell-free DNA (cfDNA) extraction

[0047] A venous blood sample of approximately 10 ml was collected prior to drug administration and centrifuged (1600×g, 4℃) for 10 min within 2 hours after collection. The plasma supernatant (5 ml) was transferred to a new EP tube and centrifuged (1600×g, 4℃) to remove any debris or residual cells. The supernatant was then transferred to a new EP tube, and cfDNA was extracted using the QIAamp Cyclic Nucleic Acid Kit (Qiagen). Genomic DNA was extracted from leukocytes using the QIAamp DNA Mini Kit (Qiagen), and DNA concentration was quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific).

[0048] (2) DNA library assembly and targeted capture

[0049] cfDNA libraries were generated using the KAPA Hyper Prep Kit (KAPA Biosystems), each library having a unique molecular barcode identifier. Index libraries used to capture targets underwent probe-based hybridization using a custom-designed NGS panel capable of detecting 1249 cancer-related genes. The names of the 1249 cancer-related genes are listed in Tables 1–4.

[0050] Table 1

[0051]

[0052] Table 2

[0053]

[0054] Table 3

[0055]

[0056] Table 4

[0057]

[0058] 5' biotinylated oligonucleotides (120 bp DNA) were synthesized as probe decoys (IDT, Coralville, IA). The concentration and distribution of fragment size were assessed using a Qubit™ 3.0 fluorescence spectrometer (Thermo Fisher Scientific) and a LabChip GX Touch HT analyzer (PerkinElmer).

[0059] (3) DNA sequencing, data processing and variant identification

[0060] The captured library was loaded onto NovaSeq 6000 (Illumina), and the paired ends of 100 bp were sequenced. The read sequences were mapped to the hg19 human reference genome using the Burrows-Wheeler Aligner. Variants were then filtered based on support count, strand bias state, base quality, and variant mapping value. Variant identification was also optimized to look for variants in short tandem repeat regions. Single nucleotide polymorphisms (SNPs) and insertion / deletion mutations (indels) from the dbSNP (v138), 1000 Genomes, and ESP6500 databases were tagged using ANNOVAR. Frameshift, non-frameshift, nonsense, and missense mutations were retained. Changes in copy number and gene rearrangement were then measured. All SNVs and indels in the target gene coding region were included in the analysis. SNPs with a population frequency ≥0.015 in the 1000 Genomes, dbSNP, and ESP6500 databases were excluded.

[0061] 3. Key conditions and parameters

[0062] (1) Extraction of cfDNA

[0063] (2) DNA library assembly and targeted capture

[0064] (3) DNA sequencing

[0065] See the experimental procedure. Figure 1 The experimental processing procedure is as follows: Figure 2 .

[0066] Example

[0067] The applicant tested the maxVAF of ctDNA in 74 patients (from patients with pMMR advanced colorectal cancer who had progressed after second-line standard treatment at the applicant's hospital) and used 6.9% as the cutoff value. The target immune response rate was higher in the low maxVAF group than in the high maxVAF group.

[0068] In the analysis, the baseline maxVAF of responders was lower than that of non-responders (median: 1.2% vs. 10.4%, P = 0.024). Figure 3 C). Among them, the median maximum VAFs for PR, SD, and PD patients were 1.2% (n=9), 5.7% (n=45), and 13.8% (n=17), respectively. Figure 3 A). The applicant further investigated the correlation between maxVAF and survival in 74 patients, selecting a median level of 6.9% as the cutoff value (high ≥6.9%, n=37; low <6.9%, n=37), consistent with the values ​​calculated using the maximum Youden index when grouping patients according to their response. Figure 3 B).

[0069] The results showed that the progression-free survival (PFS) of patients in the low maxVAF group was significantly longer than that of patients in the high maxVAF group (mPFS: 5.8 vs. 2.7 months, P<0.001). Figure 4 Similarly, the low maxVAF group had a longer OS (mOS: 19.3 vs. 9.0 months, P = 0.007). Figure 4 The ORR was significantly higher in patients with low maxVAF than in patients with high maxVAF (24% vs. 0, P = 0.001). Figure 3 C).

[0070] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. Application of substances for detecting the abundance of maximum allele mutations in the preparation of kits for evaluating the efficacy and / or prognosis of immunotherapy for metastatic colorectal cancer; The metastatic colorectal cancer is a metastatic colorectal cancer with intact mismatch repair proteins; The immunotherapy efficacy of the intact mismatch repair protein in metastatic colorectal cancer is the same as that of targeted immunotherapy. The maximum allele mutation abundance was calculated using data obtained from sequencing 1249 cancer-related genes listed in Tables 1-4. Based on the calculated results, it is determined whether the abundance of the largest allele mutation is greater than a threshold, which is 6.9%. The maximum allele mutation abundance refers to the maximum allele mutation abundance in peripheral blood circulation.

2. The application according to claim 1, characterized in that, The substances used to detect the abundance of the largest allele mutations include next-generation sequencing and data processing equipment.

3. The application according to claim 1, characterized in that, Therapeutic agents for enhancing the immunotherapy efficacy of the mismatch repair protein in metastatic colorectal cancer include one or more of PD-L1 antagonists, PD-1 antagonists, and CTLA-4 antagonists.

4. The application according to claim 1, characterized in that, The reference for gene mutations is the hg19 human reference genome or the hg38 human reference genome.

5. The application according to claim 1, characterized in that, The gene mutations include frameshift, nonframeshift, nonsense, and missense mutations; 6. The application according to claim 1, characterized in that, The methods for detecting the abundance of the largest allele mutation include: Peripheral blood ctDNA samples were sequenced to obtain sequencing data; The obtained sequencing data is compared with the reference genome to determine whether peripheral blood ctDNA has mutated. Calculate the abundance of the largest allelic mutation in peripheral blood ctDNA samples; The obtained sequencing data is compared with the reference genome, and the obtained sequencing data is processed, including filtering variants, optimizing variant identification, variant annotation, excluding known germline SNPs, and retaining frameshift, non-frameshift, nonsense, and missense mutations. Based on the calculated results, it is determined whether the abundance of the largest allele mutation is greater than a threshold, which is 6.9%.

7. The application according to claim 6, characterized in that, The method for calculating the maximum allele mutation abundance in peripheral blood ctDNA samples is as follows: Sequencing is performed on the cfDNA library generated from the peripheral blood samples to obtain sequencing data, which includes sequencing data of 1249 cancer-related genes listed in Tables 1-4; the obtained sequencing data is then compared with the reference genome, and the obtained sequencing data is processed, including filtering variants, optimizing variant identification, variant annotation, excluding known germline SNPs, and retaining frameshift, non-frameshift, nonsense, and missense mutations.

8. A device for predicting the efficacy of immunotherapy in metastatic colorectal cancer with intact mismatch repair proteins, characterized in that, The device includes: The sequencing module is used to sequence peripheral blood ctDNA samples and obtain sequencing data. The alignment and data processing module is used to align the sequencing data obtained by the sequencing module with the reference genome to determine whether peripheral blood ctDNA has mutated. The calculation module is used to calculate the abundance of the largest allelic mutation in peripheral blood ctDNA samples; The determination module is used to determine the efficacy of immunotherapy for patients with metastatic colorectal cancer whose mismatch repair proteins are intact, based on the results obtained by the calculation module. When the abundance of the largest allele mutation is greater than a threshold of 6.9%, the patient is considered suitable for immunotherapy. The maximum allele mutation abundance was calculated using data obtained from sequencing 1249 cancer-related genes listed in Tables 1-4.

9. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by the processor, it implements the application described in claim 1.

10. A computer processing device, characterized in that, The computer processing device includes a processor and a computer-readable storage medium as described in claim 9, wherein the processor operates the computer-readable storage medium to implement the application as described in claim 1.

11. An electronic terminal, characterized in that, The electronic terminal includes a communicator, a memory, and a processor; The communicator is used to establish a communication connection with an external identifier to receive sequencing data from peripheral blood ctDNA samples; The memory is used to store the computer program; The processor is used to execute the computer program stored in the memory so that after the electronic terminal analyzes the sequencing data, it analyzes the abundance of the largest allele mutation in the peripheral blood ctDNA sample. When the abundance of the largest allele mutation is greater than a threshold, which is 6.9%, it predicts that the immunotherapy efficacy of patients with metastatic colorectal cancer with intact mismatch repair proteins is good. The maximum allele mutation abundance was calculated using data obtained from sequencing 1249 cancer-related genes listed in Tables 1-4.

12. The use of any one of the apparatus of claim 8, the computer-readable storage medium of claim 9, the computer processing device of claim 10, or the electronic terminal of claim 11 in preparing a system for predicting the efficacy of immunotherapy in metastatic colorectal cancer with intact mismatch repair proteins.

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