Group of biomarkers for evaluating alternative end ligation and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a group of biomarkers for evaluating alternative end ligation and application thereof, and the biomarkers comprise POLQ, DNA2, GEN1, MSH6, RRM2, PARP1, UNG, POLA1, LIG1 and LIG3. Compared with a conventional marker, the biomarker provided by the invention has a wider indication range. The conventional marker is limited to BRCA mutation or high HRD tumors, but the biomarker disclosed by the invention shows remarkable indication in all solid tumors. The biomarker can be used for clinical auxiliary screening, diagnosis, prognosis and the like of tumors, or can be used for designing diagnostic reagents, kits and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a set of biomarkers for evaluating alternative end joining and application thereof. BACKGROUND

[0002] Radiotherapy and targeted therapy are currently the main treatment methods for cancer in addition to surgical treatment. However, due to the heterogeneity of tumors, even if different cancer patients use the same treatment regimen, there will be great differences in treatment response and prognosis. How to solve the problem of tumor heterogeneity? The development of PARP inhibitors based on the genomic characteristics of cancer provides an important solution. For example, in ovarian cancer patients, the advent of targeted drugs PARP inhibitors greatly improves the survival of patients.

[0003] Although PARP inhibitors have excellent efficacy, their 5-year survival rate has long hovered around 40%, which is much lower than the requirement of the “Health China 2030” plan. In head and neck cancer patients, radiotherapy can be used as a major treatment method to destroy the genome of cancer cells, but its 5-year overall survival rate is still less than 50%. One of the main reasons hindering the further improvement of the 5-year survival rate of patients is that the treatment regimen lacks precision, leading to serious adverse reactions, radiotherapy resistance or drug resistance in patients. Improving the 5-year survival rate of patients by further guiding treatment methods or drug regimens is an important strategy, so developing new genetic markers for different patient genomic characteristics has important value for improving precise treatment regimens and guiding radiotherapy and PARP inhibitor use.

[0004] Currently, there are many technical solutions for developing precise cancer treatment based on genomic characteristics, but these technical solutions have different defects. In traditional radiotherapy regimens, the biological differences between tumors are often ignored, and this difference is a key factor affecting the prognosis of tumor radiotherapy. If the radiotherapy regimen does not take this factor into account, it will cause incomplete treatment of radioresistant tumors and excessive treatment of sensitive tumors.

[0005] In traditional targeted therapy regimens, the main markers developed for homologous recombination deficiency are BRCA1 / 2 and scarHRD (HRD). These markers can effectively distinguish between PARP inhibitor-sensitive and resistant populations, and the principle is that BRCA1 / 2 gene mutations will cause HRD, and HRD tumors will be particularly sensitive to PARP inhibitors due to “synthetic lethality”. However, due to the small number of BRCA1 / 2 mutation populations and the influence of factors other than homologous recombination deficiency on PARP inhibitor sensitivity, markers based on BRCA1 / 2 and HRD will miss many patients, and the benefit population will still be limited.

[0006] Therefore, the prior art still needs to be improved and developed. SUMMARY

[0007] In view of the deficiencies of the prior art described above, the purpose of the present application is to provide a set of biomarkers for evaluating alternative end joining and applications thereof, aiming to solve the problem of poor prognosis judgment for PARP inhibitor treatment.

[0008] The technical solutions of the present application are as follows:

[0009] In a first aspect of the present application, a set of biomarkers for evaluating alternative end joining is provided, and the biomarkers include POLQ, DNA2, GEN1, MSH6, RRM2, PARP1, UNG, POLA1, LIG1, and LIG3.

[0010] In a second aspect of the present application, the use of the above-mentioned biomarkers in the preparation of a product for evaluating the sensitivity of tumor patients to PARP inhibitors is provided.

[0011] In a third aspect of the present application, the use of reagents for detecting the above-mentioned biomarkers in the preparation of a product for evaluating the sensitivity of tumor patients to PARP inhibitors is provided.

[0012] In a fourth aspect of the present application, a product is provided, and the product includes reagents for detecting the above-mentioned biomarkers.

[0013] Optionally, the product is a kit, a reagent, or a test paper.

[0014] The present application has the following beneficial effects:

[0015] The present application provides a set of biomarkers for evaluating alternative end joining and applications thereof, and the present application provides a set of biomarkers for evaluating alternative end joining, which has a wider indication range than the previous biomarkers. The previous biomarkers are limited to BRCA mutations or high HRD tumors, while the biomarkers of the present application show significant indication in all solid tumors. The biomarkers can be used for clinical auxiliary screening, diagnosis, prognosis, and the like of tumors, or can be used for designing diagnostic reagents and kits, and the like. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Survival curve diagram of gene markers of TCGA solid tumor radiotherapy data set is shown;

[0017] Figure 2 ROC curve diagram of gene markers of TCGA solid tumor radiotherapy data set is shown;

[0018] Figure 3 Drug sensitivity analysis diagram of gene markers of GDSC2 solid tumor data set is shown; sensitivity analysis of Alt-EJ high and low score groups to PARP inhibitors Figure 3A-E); Alt-EJ high-score group and HRD high-score group respectively Figure 3 F), BRCA mutation group Figure 3 G-K) for comparison, analysis of sensitivity to PARP inhibitors;

[0019] Figure 4 The ROC curve of the gene marker of the GDSC2 solid tumor data set is shown.

[0020] Figure 5 The difference in gene marker score of the patient's epithelioid cancer cell before Niraparib "treatment" of the GEO data set is shown. DETAILED DESCRIPTION

[0021] The present application provides a set of biomarkers for evaluating alternative end joining and its application. In order to make the purpose, technical scheme and effect of the present application more clear and explicit, the present application is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0023] Among the DNA damage caused by radiotherapy, DNA double-strand breaks (DSB) are the most harmful and the main cause of cancer cell death. In addition to the classic non-homologous end joining (non-homologous end joining) and homologous recombination (homologous recombination), in recent years, more and more evidence shows that alternative non-homologous end joining (alternative non-homologous end joining, Alt-EJ) is also an important way for cancer cells to repair DSB. Unfortunately, as a potential factor leading to tumor radioresistance, there is currently no systematic investigation of the role of alternative non-homologous end joining in solid tumor radiotherapy. As a breakthrough in the study of alternative non-homologous end joining mechanism, the activity of Alt-EJ in radio-sensitive or radio-resistant solid tumors is compared, the PARP inhibitor and radio-sensitivity of alternative non-homologous end joining active solid tumors are investigated, and the value of Alt-EJ in precision medicine for solid tumors is determined.

[0024] Based on this, the embodiments of the present application provide a set of biomarkers for evaluating alternative end joining, which includes POLQ, DNA2, GEN1, MSH6, RRM2, PARP1, UNG, POLA1, LIG1, and LIG3.

[0025] The following detailed description uses specific examples.

[0026] Example 1 Data Download

[0027] Search for public gene scoring data and complete clinical annotations in the Genetic Scoring Integrated Database (GEO) and the Cancer Genome Atlas Database (TCGA) (https: / / www.cbioportal.org / );

[0028] https: / / gdc.cancer.gov / about-data / publications / pancanatlas;

[0029] https: / / gdc.cancer.gov / about-data / publications / PanCan-DDR-2018. Patients without survival information were removed from further evaluation. This example collected data from two eligible large cohorts of epithelioid-derived solid tumors (TCGA, GDSC) and one eligible single-cell cohort of ovarian cancer (GSE222557) for further analysis. For the TCGA dataset, pan-cancer sample batch-effect corrected RNA sequencing data for gene scoring were downloaded from the NIH GDC (https: / / gdc.cancer.gov / about-data / publications / pancanatlas) and further log-normalized. For GDSC drug sensitivity data, the data were downloaded from the Sanger Institute's Genomics of Drug Sensitivity in Cancer website (https: / / www.cancerrxgene.org / downloads / bulk_download), with the corresponding cell line scoring data GDSC1000 being... Microarray data were downloaded from GDSC1000 resource library (https: / / www.cancerrxgene.org / gdsc1000 / GDSC1000_WebResources / Home.html) after RMA algorithm normalization. The corresponding somatic gene mutation data were downloaded from COSMIC website of Sanger Institute (https: / / cancer.sanger.ac.uk / cosmic / download / cosmic). For the single-cell omics dataset GSE222557 of GEO, the original files were directly downloaded from the GEO website (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE222557), and the cell data were dimensionally clustered using the Seurat software package, and the cell markers were annotated using the CellMarjer2.0 website. All qualified solid tumor dataset information is summarized in Table 1 below, 10 genes are determined by functional experiments, and are verified in TCGA, GDSC, and GEO datasets.

[0030] Table 1

[0031]

[0032] Example 2

[0033] Based on the 10 genes of Example 1 functional experiment, a set of biomarkers for evaluating alternative end joining was constructed, which successively contained: POLQ, DNA2, GEN1, MSH6, RRM2, PARP1, UNG, POLA1, LIG1, and LIG3.

[0034] Example 3 Single-sample gene set enrichment analysis (ssGSEA)

[0035] The 10 genes in Example 2 were calculated by ssGSEA score, which reflects the enrichment direction and abundance of the 10 genes in each sample (patient tumor sample or tumor cell line), and the higher the score means the higher the total mRNA enrichment degree of the Alt-EJ gene set in each sample. Under this standard, the Alt-EJ score of each sample, cell line or epithelial-like cancer cell was calculated in TCGA, GDSC1000, and GSE222557 datasets, respectively.

[0036] Example 4 Survival analysis and ROC curve analysis

[0037] All TCGA epithelioid solid tumor samples under radiation therapy (RT) were selected. Based on the median ssGSEA score, the samples were divided into a high-Alt-EJ score group and a low-Alt-EJ score group. Survival analysis was performed. The results showed that the survival time of patients in the high-Alt-EJ score group was significantly shorter than that in the low-Alt-EJ score group. A univariate Cox's Law analysis hazard ratio (HR) greater than 1 indicated a higher relative risk. Figure 1 This suggests that epithelioid solid tumors develop radioresistance through high Alt-EJ scores, and that Alt-EJ, as a novel biomarker, can effectively differentiate radioresistant patient populations. To evaluate the predictive performance of Alt-EJ as a novel biomarker for the prognosis of epithelioid solid tumors, a 5-year receiver operating characteristic (ROC) curve analysis was performed. The AUC value of Alt-EJ for predicting the prognosis of epithelioid solid tumors under radiotherapy was 0.577.

[0038] Example 5 Drug Sensitivity Analysis

[0039] Epithelioid solid tumor cell lines were selected and divided into high-scoring and low-scoring Alt-EJ cell lines based on the median ssGSEA score. Sensitivity analysis was then performed on PARP1 / PARP2 (PARP) inhibitors (Niraparib, Olaparib, Rucaparib, Talazoparib, Veliparib). CellTiter-Glo is a high-throughput cell viability assay based on ATP bioluminescence, which provides the half-maximal inhibitory concentration (IC50) of a cell line for a given drug. A lower IC50 value indicates greater sensitivity to the drug. Results showed that Alt-EJ effectively distinguished between PARP1 inhibitor-resistant and sensitive cell lines, and the high-scoring Alt-EJ group was more sensitive to PARP inhibitors. Figure 3 To further evaluate the performance of Alt-EJ as a novel biomarker in PARP inhibitor-targeted therapy compared to existing biomarkers, homologous recombination defect score scarHRD (HRD) and BRCA, this invention, based on the algorithms and annotation information from two publications (Dodson AE, et al., 2024, doi:10.1158 / 2767-9764.CRC-24-0316; Takamatsu S, et al., 2024, doi:10.1038 / s41597-024-03018-4), calculated and annotated the HRD of all GDSC cell lines. Based on the COMIC annotation information, the somatic mutation status of all GDSC cell lines was annotated. The results showed that, with an equal number of cell lines, the high Alt-EJ score group had significantly higher sensitivity to PARP than the high HRD score group (…). Figure 3 (F) and BRCA (Figure 3 The ssGSEA score model calculated based on the Alt-EJ marker of 10 genes can well predict the therapeutic effect of PARP inhibitors, and the best AUC value is 0.7428 (Olaparib) Figure 4 ), which indicates that Alt-EJ as a new marker is significantly better than the existing markers HRD and BRCA in the single-factor cancer cell environment.

[0040] Example 6 Single-cell omics difference analysis

[0041] Since the GDSC cell lines lack the in vivo microenvironment, this embodiment selects the GSE222557 dataset to perform Niraparib drug "pre-treatment" on non-BRCA mutant ovarian cancer patients, and the ssGSEA score of each cell obtained by clustering and identifying epithelial-like cancer cells by the R package Seurat is used for difference analysis. According to the prediction, the higher the Alt-EJ score of the tumor before treatment, the more the epithelial-like cancer cells of the patient will be eliminated after receiving Niraparib drug treatment, and therefore the treatment effect is better, that is, the patient with better treatment effect has a higher Alt-EJ score. The results show that the Alt-EJ_ssGSEA score of the epithelial-like cancer cells of the patient population before "treatment" is significantly higher than that of the patient population without response (No Response) Figure 5 ), indicating that Alt-EJ as a new marker not only can guide the precision use of PARP inhibitors in cell lines, but also has great potential as a guide for the precision use of PARP inhibitors in clinical practice.

[0042] In summary, the present application provides a set of biomarkers for evaluating alternative end joining and applications thereof, mainly by calculating the score of the biomarker through single-sample enrichment analysis (ssGSEA) technology, and based on the score, the marker enrichment degree of the tumor sample of the patient is distinguished. The Alt-EJ score is calculated in the TCGA epithelial-like solid tumor patient sample, the GDSC cell line solid tumor sample, and the tumor sample of the ovarian cancer patient before Niraparib treatment, respectively.

[0043] The Alt-EJ score indicates the radiosensitivity and PARP inhibitor sensitivity of the patient / cell line.

[0044] It should be understood that the application of the present application is not limited to the above examples, and those of ordinary skill in the art can make improvements or changes according to the above description, and all these improvements and changes shall belong to the protection scope of the appended claims of the present application.

Claims

1. A panel of biomarkers for assessing surrogate end connections, characterized in that, The biomarkers include POLQ, DNA2, GEN1, MSH6, RRM2, PARP1, UNG, POLA1, LIG1, LIG3.

2. Use of the biomarkers of claim 1 in the preparation of a product for evaluating the sensitivity of a tumor patient to a PARP inhibitor.

3. Use of a reagent for detecting the biomarkers of claim 1 in the preparation of a product for evaluating the sensitivity of a tumor patient to a PARP inhibitor.

4. A product characterized by, The product comprises a reagent for detecting the biomarkers of claim 1.

5. The product of claim 4, wherein, The product is a kit, a reagent, a test paper.

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