Method and prediction model for detecting tumour mutation burden

Abstract

The invention relates to the technical field of biomedicine, and in particular relates to a method and prediction model for detecting the tumour mutation burden. The method comprises the following steps of: S1, performing DNA extraction on tumour tissue and blood samples to be detected; S2, for tumour-related genes, constructing a target region targeted capture sequencing library; S3, obtaining original offline data by utilizing a high-throughput sequencing platform; and S4, obtaining the tumour mutation burden of the sample through a check-out process. The invention aims to provide the methodand prediction model for detecting the tumour mutation burden. Gene mutation is detected and calculated through the method and the prediction method; the method can achieve the same result as TMB detection by whole exon sequencing; and thus, the evaluation accuracy of high tumour mutation burden can be improved.

Description

technical field

[0001] The invention relates to the field of biomedical technology, in particular to a method for detecting tumor mutation load and a prediction model. Background technique

[0002] In recent years, immunotherapy has received extensive attention from the medical community, and it has achieved remarkable clinical efficacy in a variety of tumor treatments. Approved by the Drug Administration (FDA) and recommended by the National Comprehensive Cancer Network (NCCN) guidelines, it has become the first-line treatment option for advanced non-small cell lung cancer (NSCLC). Tumor immunotherapy mainly achieves the efficacy of identifying, controlling and eliminating tumors by activating the human immune system, and the most studied ones are monoclonal antibody immune checkpoint inhibitors such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4 ) monoclonal antibody, programmed death inhibitor protein and its ligand (PD-1 / PD-L1) monoclonal antibody, so far, seven...

Examples

Embodiment 1

[0052] In an embodiment, the samples to be tested are paraffin-embedded tissue samples known to have a high tumor mutation burden and corresponding blood control samples.

[0053] The test steps are as follows:

[0054] 1. Sample extraction and fragmentation: Genomic DNA was extracted from blood samples and paraffin-embedded tissue samples using a nucleic acid extraction kit (Magen or Promega), and quantified using Qubit; the sample DNA was fragmented using a Covaris M220 instrument to make The size of the DNA fragment is between 100 and 500 bp, and the QIAxcel (QIAGEN) instrument is used to detect whether the fragment size meets the requirements.

[0055] 2. Double UMI library construction:

[0056] 1) End repair and "A" addition: The fragmented sample DNA is subjected to end repair and "A" addition. The reaction system is shown in Table 1 below. Vortex and mix well, then centrifuge, and place on a thermal cycler. ℃ for 30 minutes, then 65 ℃ for 30 minutes.

[0057] Table ...

Embodiment 2

[0094] Whole exome sequencing and ChosenOne599 were performed on 1453 lung cancer samples Ⓡ Targeted sequencing, analyzed according to the above process, and counted ChosenOne599 Ⓡ The number of SNV synonymous mutations, the number of SNV non-synonymous mutations, the number of frameshift insertion mutations, the number of non-frameshift insertion mutations, the number of frameshift deletion mutations, and the number of non-frameshift deletion mutations in the The TMB value of the panel is used as the gold standard, and the TMB of the panel is calculated according to the multiple linear model.

[0095] Obtain the mutation data of the sample to be tested through the detection process, and calculate ChosenOne599 Ⓡ The number of SNV synonymous mutations Nsys, the number of SNV non-synonymous mutations Nnon, the number of frameshift insertion mutations Nis, the number of non-frameshift insertion mutations Nns, the number of frameshift deletion mutations Nds and the number of non-...

Embodiment 3

[0098] This embodiment carries out ChosenOne599 to 6 routine TMB standard items Ⓡ For targeted region sequencing, predict the results according to the TMB model, and compare the results with the gold standard as shown in Table 9 and Figure 6 shown.

[0099] Table 9 Results prediction and comparison results with gold standard

[0100]

technical field

[0001] The invention relates to the field of biomedical technology, in particular to a method for detecting tumor mutation load and a prediction model. Background technique

[0002] In recent years, immunotherapy has received extensive attention from the medical community, and it has achieved remarkable clinical efficacy in a variety of tumor treatments. Approved by the Drug Administration (FDA) and recommended by the National Comprehensive Cancer Network (NCCN) guidelines, it has become the first-line treatment option for advanced non-small cell lung cancer (NSCLC). Tumor immunotherapy mainly achieves the efficacy of identifying, controlling and eliminating tumors by activating the human immune system, and the most studied ones are monoclonal antibody immune checkpoint inhibitors such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4 ) monoclonal antibody, programmed death inhibitor protein and its ligand (PD-1 / PD-L1) monoclonal antibody, so far, seven...

Examples

Embodiment 1

[0052] In an embodiment, the samples to be tested are paraffin-embedded tissue samples known to have a high tumor mutation burden and corresponding blood control samples.

[0053] The test steps are as follows:

[0054] 1. Sample extraction and fragmentation: Genomic DNA was extracted from blood samples and paraffin-embedded tissue samples using a nucleic acid extraction kit (Magen or Promega), and quantified using Qubit; the sample DNA was fragmented using a Covaris M220 instrument to make The size of the DNA fragment is between 100 and 500 bp, and the QIAxcel (QIAGEN) instrument is used to detect whether the fragment size meets the requirements.

[0055] 2. Double UMI library construction:

[0056] 1) End repair and "A" addition: The fragmented sample DNA is subjected to end repair and "A" addition. The reaction system is shown in Table 1 below. Vortex and mix well, then centrifuge, and place on a thermal cycler. ℃ for 30 minutes, then 65 ℃ for 30 minutes.

[0057] Table ...

Embodiment 2

[0094] Whole exome sequencing and ChosenOne599 were performed on 1453 lung cancer samples Ⓡ Targeted sequencing, analyzed according to the above process, and counted ChosenOne599 Ⓡ The number of SNV synonymous mutations, the number of SNV non-synonymous mutations, the number of frameshift insertion mutations, the number of non-frameshift insertion mutations, the number of frameshift deletion mutations, and the number of non-frameshift deletion mutations in the The TMB value of the panel is used as the gold standard, and the TMB of the panel is calculated according to the multiple linear model.

[0095] Obtain the mutation data of the sample to be tested through the detection process, and calculate ChosenOne599 Ⓡ The number of SNV synonymous mutations Nsys, the number of SNV non-synonymous mutations Nnon, the number of frameshift insertion mutations Nis, the number of non-frameshift insertion mutations Nns, the number of frameshift deletion mutations Nds and the number of non-...

Embodiment 3

[0098] This embodiment carries out ChosenOne599 to 6 routine TMB standard items Ⓡ For targeted region sequencing, predict the results according to the TMB model, and compare the results with the gold standard as shown in Table 9 and Figure 6 shown.

[0099] Table 9 Results prediction and comparison results with gold standard

[0100]

Images