CN118207322A - Application of HDR signaling pathway mutations in bile cfDNA in the prognostic evaluation of cholangiocarcinoma - Google Patents

Abstract

The invention discloses an application of HDR signal path mutation in bile cfDNA in bile duct cancer prognosis evaluation. The marker of HDR signal path mutation in the bile cfDNA is selected from one or more of SMC5, RAD51D, BRCA, TP53BP1 and RTEL 1. The reagent for detecting HDR signal channel mutation in the bile cfDNA comprises a bile cfDNA extraction reagent, a bile cfDNA library construction reagent and a targeted sequencing reagent. Based on bile of malignant bile duct tumor patients as a research sample, the invention adopts WGS sequencing technology to analyze the correlation between bile cfDNA mutation characteristics and clinical prognosis, and discovers that HDR signal path mutation is a liquid biopsy detection means which can be used for CCA prognosis evaluation, and 5 HDR signal path mutation genes of SMC5, RAD51D, BRCA2, TP53BP1 and RTEL1 can be used as biomarkers for CCA prognosis evaluation.

Description

Application of HDR signaling pathway mutations in bile cfDNA in the prognostic evaluation of cholangiocarcinoma

Technical Field

The present invention belongs to the field of biomedical detection technology, and specifically relates to the application of HDR signaling pathway mutations in bile cfDNA in the prognosis assessment of cholangiocarcinoma.

Background technique

Cholangiocarcinoma (CCA) is the second most common primary liver malignancy, accounting for 3% of all gastrointestinal cancers, especially in China, Thailand, and South Korea, where the incidence of CCA is greater than 6 cases per 100,000 residents. Due to its unclear early symptoms or non-specific clinical manifestations, patients are often diagnosed in the late stage of CCA. Late-stage patients are no longer suitable for surgery, resulting in a poor prognosis with a 5-year survival rate of 7%-20%. Even if some patients can be treated with surgical intervention, the tumor is likely to relapse after resection. High TNM stage of the tumor, positive margin, and lymph node metastasis are high-risk factors affecting the survival of cholangiocarcinoma after surgery, but these indicators require imaging or tissue specimens to confirm. Serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) are also used for postoperative monitoring of cholangiocarcinoma, but the sensitivity and specificity are low. In particular, there is a lack of biomarkers and detection methods for the prognosis prediction of cholangiocarcinoma in clinical practice. Therefore, finding reliable prognostic biomarkers has important clinical application value for the diagnosis and treatment of CCA.

At present, imaging, biopsy, puncture biopsy and serum tumor marker detection are the main methods for diagnosing cholangiocarcinoma. Among them, imaging methods mainly include ultrasound, CT (computed tomography), MRI (magnetic resonance imaging), PCT (percutaneous transhepatic cholangiography), ERCP (endoscopic retrograde pancreaticocholangiography), etc. Ultrasound is the preferred detection method for cholangiocarcinoma, but it cannot distinguish the stage of malignant tumors. CT and MRI are the main methods for diagnosing and staging cholangiocarcinoma. MRI can better show the anatomy of the liver and bile duct and the range of tumor involvement, but it is not effective in detecting tumors with a diameter of less than 5mm. ERCP brushing exfoliated cell examination is the preferred pathological diagnosis method for cholangiocarcinoma, but the sensitivity of brushing is low. When the brushing result is negative or uncertain, ERCP or ultrasound endoscopic guided fine needle aspiration biopsy can be considered, but it is easy to cause abdominal infection and tumor spread and metastasis. The current clinical guidelines do not recommend this technology for the first diagnosis of cholangiocarcinoma. CEA and CA19-9 are of certain significance in the diagnosis, efficacy, and monitoring of metastasis and recurrence of cholangiocarcinoma. Combined with ultrasound examination, they can be used as a preliminary examination method for high-risk groups, but their sensitivity and specificity are relatively low.

Liquid biopsy is used for auxiliary diagnosis, efficacy evaluation, postoperative monitoring, and clinical medication guidance of tumor diseases based on its advantages such as minimally invasive or non-invasive sampling, small sample volume, and repeatable collection. Blood is the most widely used type of body fluid in liquid biopsy, but for cholangiocarcinoma, bile is secreted by hepatocytes and transferred from the bile duct to the gallbladder for concentrated storage and participation in the entire enterohepatic circulation. It can directly contact the liver, gallbladder, and digestive system, and is a better type of body fluid than blood. Studies have shown that the sensitivity of bile cfDNA in detecting somatic gene mutations in patients with biliary tract cancer is significantly better than that of blood cfDNA (PMID: 34653800). Targeted deep sequencing of bile cfDNA was performed, and the results were compared with those of tumor tissue, and it was found that the sensitivity and specificity of bile cfDNA detection were both greater than 90% (PMID: 31173267). However, research on whole genome sequencing of bile cfDNA is extremely lacking, which limits its application in revealing CCA disease progression, early diagnosis, and postoperative monitoring from the perspective of genomic mapping. Therefore, research data from whole-genome sequencing of bile cfDNA are urgently needed to assist in the clinical transformation of biomarkers such as early diagnosis, efficacy evaluation, and postoperative testing of CCA.

Summary of the invention

The purpose of the present invention is to provide an application of HDR signaling pathway mutations in bile cfDNA in the prognosis assessment of cholangiocarcinoma, to provide a liquid biopsy biomarker for the prognosis of CCA, to assist the postoperative treatment of CCA, and to prolong the survival time and quality of life of CCA patients.

The technical solution adopted by the present invention to achieve the above-mentioned purpose is as follows:

Application of reagents for detecting HDR signaling pathway mutations in bile cfDNA in the preparation of cholangiocarcinoma prognosis assessment products.

As a preferred embodiment, the markers of HDR signaling pathway mutations in bile cfDNA are selected from one or more of SMC5, RAD51D, BRCA2, TP53BP1, and RTEL1.

As a preferred embodiment, the reagents for detecting HDR signaling pathway mutations in bile cfDNA include bile cfDNA extraction reagents, bile cfDNA library construction reagents, and targeted sequencing reagents. Regarding bile cfDNA extraction reagents, the present invention adopts 3D-BCF, an extraction method suitable for bile cfDNA established by Shanghai Siludi Biomedical Technology Co., Ltd., and other commercialized body fluid cfDNA extraction kits can also be used. Regarding bile cfDNA library construction reagents, the TruSeq Nano DNA Library Prep Kit is used in the embodiment of the present invention to construct a bile cfDNA whole genome library. In addition, the following can be used: a. Scale ssDNA-seq lib prer kit for illumina (RK20222, ABclonal); b. Ultra ^TM II DNA Library Prep Kit for Illumina (#E7645S/#E7103S, NEB); c.KAPA DNA HyperPrep kit (KK8504, Roche, Switzerland). Targeted sequencing reagents are targeted sequencing of genes in the HDR signaling pathway, and then mutant genes are screened by comparing with reference sequences. Commonly used targeted sequencing platforms and reagents are: Illumina MiSeqDx instrument with MiSeq Reagent Kit v2 (MS-102-2003); ThermoFisher scientific Ion GeneStudio S5System sequencing instrument with Ion 540Chip Kit (A27766); BGI MGISEQ-2000 sequencing instrument with MGISEQ-2000RS high-throughput sequencing reagent set (1000013857).

The present invention also provides a cholangiocarcinoma prognosis assessment kit, which comprises a reagent for detecting HDR signaling pathway mutations in bile cfDNA.

As a preferred embodiment, the kit includes cfDNA extraction reagents, bile cfDNA library construction reagents, and targeted sequencing reagents.

As a preferred embodiment, the kit further comprises a bile sample collection reagent for collecting and preserving the bile of the sample to be tested.

As a preferred embodiment, the cfDNA extraction reagent includes a bile sample pretreatment reagent and a bile cfDNA extraction reagent. The method for pretreating the bile sample with the bile sample pretreatment reagent is as follows: the bile is centrifuged at 1,600g for 10 minutes at 4°C to remove cell debris, and after centrifugation, the bile supernatant is transferred to a new 1.5mL centrifuge tube. The bile supernatant is centrifuged again at 16,000g for 15 minutes at 4°C, and finally the supernatant is transferred and divided into 1.5mL centrifuge tubes at 1mL per tube and stored in a -80°C refrigerator.

The English abbreviations involved in the present invention are annotated as follows:

cfDNA: partially degraded endogenous DNA that is free from cells

CCA: Cholangiocarcinoma

HDR: homologous dependent recombination (HDR) pathway

SMC5: Gene encoding SMC5 protein in the chromosome structure maintenance protein SMC5/6 complex

RAD51D: Gene encoding a member of the Rad51 protein family of DNA repair proteins

BRCA2: breast cancer 2, early onset

TP53BP1: Gene encoding tumor protein p53 binding protein 1

RTEL1: Gene encoding DNA helicase RTEL1

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention uses bile from patients with malignant bile duct tumors as research samples, extracts bile cfDNA, and uses whole-genome sequence second-generation sequencing technology to detect single nucleotide variations (SNVs) and insertion/deletion mutations (Indels) in bile cfDNA. By comparing somatic mutations with whole exome sequencing results of tumor tissues from an external cohort (n=239), the correlation between bile cfDNA mutation characteristics and clinical prognosis is analyzed, and it is found that HDR signaling pathway mutations are a liquid biopsy detection method that can be used for CCA prognosis assessment.

2. The present invention uses the Illumina NovaSeq 6000 sequencing platform to sequence the bile cfDNA library, and then compares it with the human reference genome sequence and then annotates it with the gene map of the own leukocytes, and finally screens the mutant genes. For the first time, it was found that the five HDR signaling pathway mutant genes SMC5, RAD51D, BRCA2, TP53BP1 and RTEL1 in the bile cfDNA of patients with cholangiocarcinoma can be used as liquid biopsy biomarkers for CCA prognosis assessment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the distribution characteristics of bile cfDNA fragments in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the whole genome mutation characteristic analysis of bile cfDNA in an embodiment of the present invention.

FIG3 is a schematic diagram of the comparative analysis results of bile cfDNA whole genome mutation characteristics and tumor tissue in an embodiment of the present invention.

FIG. 4 is a schematic diagram of the results of mutation analysis of 10 core pathways of DNA damage response in an embodiment of the present invention.

FIG5 is a schematic diagram of the results of mutation analysis of 10 carcinogenic signaling pathways in an embodiment of the present invention.

FIG6 is a schematic diagram of the analysis results of the Cox proportional hazard model in an embodiment of the present invention.

FIG. 7 is a schematic diagram of the Kaplan-Meier survival curve analysis results of patients with HDR pathway mutations in an embodiment of the present invention.

FIG8 is a schematic diagram of the analysis results of CA19-9 protein levels and HDR pathway mutations in an embodiment of the present invention.

Detailed ways

The technical solution of the present invention is described in detail below in conjunction with the embodiments. The reagents and biological materials used below are all commercial products unless otherwise specified.

Example 1

1) Sample source

Twenty-two patients with malignant CCA were selected, including 4 patients with intrahepatic cholangiocarcinoma, 13 patients with hilar cholangiocarcinoma, and 5 patients with distal cholangiocarcinoma. The average age of the patients at diagnosis was 60.5 years (age range, 34-75 years), of which 17 patients underwent radical resection, 4 patients underwent R1 resection, and 1 patient underwent R2 resection. Other clinical characteristics such as gender, AJCC tumor stage, lymph node metastasis, and serum tumor marker concentrations are shown in Table 1.

Table 1

2) Extraction of bile cfDNA

Bile and blood samples were collected from all 22 patients with malignant CCA before surgery. Each enrolled patient was given an accurate pathological diagnosis after surgery. Bile cfDNA was extracted from the bile samples of each CCA patient after pretreatment.

2.1) Pretreatment of bile and blood samples from patients with cholangiocarcinoma

Bile and blood samples of CCA patients were collected in 10 mL free nucleic acid storage tubes (BEAVERCell Free DNA Tubes, 43803, China) before surgery and shipped at room temperature. After receiving the samples, bile and blood were first centrifuged at 1,600 g for 10 min at 4 ° C to remove cell debris. After centrifugation, the bile supernatant was transferred to a new 1.5 mL centrifuge tube. After centrifugation, the blood sample was aspirated with white blood cells into a 1.5 mL centrifuge tube and stored in a -80 ° C refrigerator. The bile supernatant was centrifuged again at 16,000 g for 15 min at 4 ° C, and the supernatant was finally transferred and divided into 1.5 mL centrifuge tubes at 1 mL per tube and stored in a -80 ° C refrigerator.

2.2) Extraction of bile cfDNA and leukocyte gDNA from patients with cholangiocarcinoma

The 3D-BCF method established by Shanghai Silidi Biomedical Technology Co., Ltd. was used to extract bile cfDNA. The specific process was as follows: after the bile sample was taken out from -80℃, it was placed in a 37℃ water bath until it was completely melted, and then centrifuged at 4℃, 16,000g for 5min, and 0.99mL of bile supernatant was aspirated into a new 1.5mL centrifuge tube. 5.55μL Carrier RNA (1017647, Qiagen, Shanghai, China) was added to 792μL of commercial lysis buffer BufferACL (939017, Qiagen, Shanghai, China), mixed, and 792μL of the mixture was added to the bile, followed by 99μL of proteinase K (19133, Qiagen, Shanghai, China), inverted to mix, and incubated in a metal bath at 60℃ for 30min. After incubation, 1.782 mL of precipitation reagent Buffer ACB (1069275, Qiagen, Shanghai, China) was added and placed in a -20 °C refrigerator for 5 min to promote the precipitation of cfDNA to obtain a bile mixture. 700 μL of bile mixture was added to the QIA-quick adsorption column (28115, Qiagen, Shanghai, China). After the first sample was loaded, it was first allowed to stand at room temperature for 5 min, and then the centrifugation operation was repeated until all samples passed through the adsorption column. 500 μL of Buffer PE (19065, Qiagen, Shanghai, China) was added to the adsorption column and centrifuged twice. The adsorption column was placed in a new 1.5 mL centrifuge tube and centrifuged at high speed to remove the remaining liquid in the adsorption column. 30 μL of elution buffer (19086, Qiagen, Shanghai, China) was added to the adsorption column. After incubation at room temperature for 3 min, the bile cfDNA was eluted and collected by centrifugation, and then placed in a -20 °C or -80 °C refrigerator for long-term storage.

Leukocyte gDNA was extracted using DNA Mini kit (51304, Qiagen, Shanghai, China). For specific operation procedures, please refer to the product manual.

3) Detection of bile cfDNA and leukocyte gDNA concentration and fragment distribution

The concentration of bile cfDNA and leukocyte gDNA was detected by Qubit 3.0 fluorescence quantification instrument and supporting reagents (Q32854, Thermo Fisher, USA). The fragment distribution of bile cfDNA and leukocyte gDNA extracted was analyzed using 2100 analyzer and supporting chips and reagents (5067-4626, Agilent, USA). The specific detection process refers to the instructions of the kit. See Figure 1 for the distribution characteristics of bile cfDNA fragments. The results show that bile cfDNA is mainly large fragments, which is consistent with the fragment distribution characteristics of bile cfDNA in the published article (PMID: 31173267).

4) Bile cfDNA and leukocyte gDNA WGS sequencing and bioinformatics analysis

Bile cfDNA and leukocyte gDNA were first sheared into a main peak of 350bp using an M220 ultrasonic shearer, and then the library was constructed using the TruSeq Nano DNA Library Prep Kit (20015964, Illumina, USA). The input amount of each bile cfDNA and leukocyte gDNA was 400ng. For specific operation procedures, please refer to the product manual. The constructed library was sequenced by Illumina NovaSeq 6000 for double-end sequencing, with a sequencing depth of 50X for bile cfDNA and 20X for leukocyte gDNA.

The raw sequencing data were aligned to the human reference genome (UCSC hg19) using the software BWA-MEM (Version: 0.7.17-r1188), and the Sentieon Dedup algorithm (Sentieon-genomics-201911) was used to mark and remove duplicate data. The sequencing data were analyzed for quality control using Samtools stats (version: 1.3). Leukocyte gDNA samples were used as a control group to remove false positive mutations. The software Strelka2 (version: 2.9.10) was used to detect somatic SNVs and INDELs in each tumor/leukocyte sample pair. To improve the detection performance of INDELs, Strelka2 was run with INDEL candidates generated by Manta (version: 1.6.0) and annotated using ANNOVAR (version date: 2018-04-16). The final screening criteria for somatic variants were as follows: (1) sites with allele frequencies (AFs) not less than 0.01 in leukocyte samples (1000Genome Project, ExAC, ESP6500, and gnomAD) were removed; (2) total coverage ≥ 8, alternative allele reads ≥ 3; (3) allele mutation frequency ≥ 0.05.

Bile cfDNA isolated from 22 patients with malignant CCA was used to detect single nucleotide variations (SNVs), insertion and deletion mutations (Indels) and other mutational features of bile cfDNA using WGS next-generation sequencing technology. A total of 46,528 mutations were detected in 22 patients with malignant CCA. Among all mutations, 2.19% (1019/46,528) occurred in the exon region, which is consistent with the exon percentage of the human genome. See Figure 2 for a schematic diagram of the analysis of the whole genome mutation features of bile cfDNA. As shown in Figure 2, TP53 is the gene with the highest mutation frequency, followed by KRAS. These mutated genes mainly occur in the TP53 signaling pathway (TP53), RTK/RAS signaling pathway (KRAS, ERBB3, SCRIB, RASGRP2, PDGFRA, RASGRF2, NRAS, EGFR and KSR2), HDR (SMC5, RAD51D, BRCA2, TP53BP1 and RTEL1), HIPPO (CRB1, FAT1, NF2, LLGL2 and WWTR1), WNT, BER and FA signaling pathways. See Figure 3 for a schematic diagram of the comparative analysis results of the whole genome mutation characteristics of bile cfDNA and tumor tissue. The data of tumor tissue samples are from the data of the published article (doi:10.1038/ng.3375), which is included in the EGA database (accession number: EGA00001000950). Comparison with whole-exome sequencing results of tumor tissue from an external cohort (n = 239) revealed that 10 non-silent mutations were found in both studies (TP53, KRAS, ARID1B, BRCA2, CACNA1A, ELF3, ERBB3, FLNA, NRAS, and SPTA1).

5) Correlation analysis between whole genome mutation characteristics of bile cfDNA and clinical prognosis

The mutation characteristics of bile cfDNA were analyzed by Kaplan-Meier curve, and the log-rank test was used to compare the correlation between different signaling pathway mutation groups of bile cfDNA and clinical prognosis.

In order to identify the relevant pathways that promote CCA tumorigenesis, mutant genes (SNVs and INDELs) in 10 core DNA damage response pathways and 10 oncogenic signaling pathways were studied. See Figures 4 and 5. Figure 4 is a schematic diagram of the analysis results of mutations in 10 core DNA damage response pathways, and Figure 5 is a schematic diagram of the analysis results of mutations in 10 oncogenic signaling pathways. The analysis results show that RTK/RAS is the most common mutation pathway (n=8), followed by TP53 signaling pathway (n=6, all TP53 mutations) and HDR signaling pathway (n=5). The Cox proportional hazard model was then used to determine clinical or genetic characteristics associated with prognosis. See Figure 6 for a schematic diagram of the analysis results of the Cox proportional hazard model. The results showed that HDR signaling pathway mutations had a higher hazard ratio (HR=15.77, 95%CI:1.571-158.4). Kaplan-Meier curves were further used for survival analysis. See Figure 7 for a schematic diagram of the Kaplan-Meier survival curve analysis results for patients with HDR pathway mutations. See Figure 8 for a schematic diagram of the analysis results of CA19-9 protein levels and HDR pathway mutations. The results showed that patients with HDR pathway mutations had poor overall survival (OS) and higher CA19-9 protein levels.

The above analysis results show that patients with HDR pathway mutations have poor overall survival, and HDR signaling pathway mutations in bile cfDNA can be used to evaluate the prognosis of cholangiocarcinoma. The prognosis of cholangiocarcinoma patients can be predicted by detecting five HDR signaling pathway mutation genes, SMC5, RAD51D, BRCA2, TP53BP1, and RTEL1, in bile cfDNA.

The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims (8)

1. Application of reagents for detecting HDR signaling pathway mutations in bile cfDNA in the preparation of cholangiocarcinoma prognosis assessment products. 2. The use according to claim 1, characterized in that the markers of HDR signaling pathway mutations in bile cfDNA are selected from one or more of SMC5, RAD51D, BRCA2, TP53BP1, and RTEL1. 3. The use according to claim 1 is characterized in that: the reagents for detecting HDR signaling pathway mutations in bile cfDNA include bile cfDNA extraction reagents, bile cfDNA library construction reagents, and targeted sequencing reagents. 4. A cholangiocarcinoma prognosis assessment kit, characterized in that: the kit includes a reagent for detecting HDR signaling pathway mutations in bile cfDNA. 5. The cholangiocarcinoma prognosis assessment kit according to claim 4, characterized in that the kit comprises a cfDNA extraction reagent, a bile cfDNA library construction reagent, and a targeted sequencing reagent. 6 . The cholangiocarcinoma prognosis assessment kit according to claim 5 , characterized in that the kit further comprises a bile sample collection reagent. 7. The cholangiocarcinoma prognosis assessment kit according to claim 5, characterized in that the cfDNA extraction reagent includes a bile sample pretreatment reagent and a bile cfDNA extraction reagent. 8. Application of one or more of the markers of HDR signaling pathway mutations in bile cfDNA, including SMC5, RAD51D, BRCA2, TP53BP1, and RTEL1, in the prognosis assessment of cholangiocarcinoma.