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CN-122012719-A - Bladder cancer dynamic monitoring method based on circulating tumor DNA methylation marker

CN122012719ACN 122012719 ACN122012719 ACN 122012719ACN-122012719-A

Abstract

The invention discloses a dynamic bladder cancer monitoring method based on a circulating tumor DNA methylation marker, and belongs to the technical field of molecular diagnosis. The method comprises the steps of collecting urine of a patient at a plurality of continuous time points after operation, extracting circulating free DNA, constructing a targeted sequencing library aiming at a pre-selected two types of bladder cancer specific methylation difference regions, wherein a first type of region haplotypes represent tissue cloning stability, a second type of region haplotypes represent tumor malignancy progress, analyzing methylation haplotypes of each region through high-throughput sequencing, calculating passenger haplotype instability indexes reflecting cloning stability and driving haplotype loads reflecting malignancy potential, integrating the passenger haplotype instability indexes and the driving haplotype loads into dynamic methylation risk scores, and generating grading risk early warning according to continuous variation trend of the scores. The invention realizes high sensitivity, high specificity and noninvasive dynamic monitoring and early warning of bladder cancer recurrence and progression risk.

Inventors

  • ZHENG YICHUN
  • ZHAO JUNGANG
  • LIU DEKAI
  • JIANG YIWEI

Assignees

  • 浙江大学

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. A method for dynamically monitoring bladder cancer based on a circulating tumor DNA methylation marker, comprising the steps of: step 1, collecting urine samples at a plurality of continuous preset time points after operation of a bladder cancer patient and processing the urine samples to obtain urine supernatant for extracting circulating free DNA; Step 2, extracting circulating free DNA from urine supernatant, and quantifying and analyzing fragments of the extracted circulating free DNA; Step 3, constructing a targeted methylation haplotype sequencing library aiming at a pre-selected bladder cancer specific methylation difference region, wherein the methylation difference region comprises a first methylation difference region and a second methylation difference region, the first methylation difference region is a region in which the methylation haplotype is stable before and after cancer change, and the second methylation difference region is a region in which the methylation haplotype is rearranged in the tumor progress process; Step 4, carrying out high-throughput sequencing on the sequencing library, and analyzing methylation haplotypes of all continuous CpG sites in the two methylation difference regions; Step 5, calculating a passenger haplotype instability index and a driving haplotype load at each monitoring time point based on the analyzed methylation haplotype data, and integrating the passenger haplotype instability index and the driving haplotype load into a dynamic methylation risk score; the passenger haplotype instability index represents the degree of deviation of dominant haplotypes in the methylation difference region of the first type; And 6, generating a dynamic monitoring report and risk early warning according to the variation trend of the dynamic methylation risk score at the continuous time point.
  2. 2. The method for dynamic monitoring of bladder cancer based on a circulating tumor DNA methylation marker according to claim 1, wherein in step 1, the plurality of consecutive preset time points comprises 1 st month, 3 rd month, 6 th month, 12 th month after surgery, and is collected every 6 months; 100 milliliters of middle-section urine of a patient is collected each time, and is immediately injected into a urine preservation tube containing a DNase inhibitor, preserved at the temperature of 2-8 ℃ and treated within 4 hours after collection; the treatment comprises a two-step centrifugation process, wherein the first step centrifugation is carried out for 10 minutes at 4 ℃ and 1500 revolutions per minute, and the supernatant of the upper urine and the lower cell sediment are obtained by separation; And (3) transferring the urine supernatant obtained by the centrifugation in the first step to a new centrifuge tube by the centrifugation in the second step, centrifuging again for 10 minutes at the temperature of 4 ℃ and the speed of 13000 rpm, and removing cell fragments and impurities to obtain a final supernatant, namely the urine supernatant for extracting the circulating free DNA.
  3. 3. The method for dynamic bladder cancer monitoring based on circulating tumor DNA methylation markers according to claim 1, wherein in step 2, the circulating free DNA is extracted using a silica gel membrane adsorption column kit, and the pH and salt concentration of the binding buffer are adjusted during the extraction process to preferentially recover DNA fragments having a length of less than 200 base pairs; the quantitative and fragment analysis includes determining the total concentration of the extracted circulating free DNA using a fluorescent quantitative instrument; Meanwhile, the fragment size distribution of the circulating free DNA is analyzed by a microcapillary electrophoresis system, and the percentage of the circulating free DNA with the fragment size between 100 and 250 base pairs to the total circulating free DNA is calculated, wherein the percentage value is used as one of indexes for evaluating the enrichment degree of the circulating tumor DNA in the sample.
  4. 4. The method for dynamic bladder cancer monitoring based on a circulating tumor DNA methylation marker according to claim 1, wherein in step 3, the methylation difference regions are 5 methylation difference regions of a first type and 5 methylation difference regions of a second type; The methylation difference region of the first type is obtained by comparing and screening a large amount of genome-wide methylation sequencing data of normal urothelial tissues and bladder cancer tissues, wherein the screening standard is that the methylation difference region is in a hypermethylation state in normal and cancerous tissues, and haplotype patterns formed by continuous CpG sites in the methylation difference region are stable among different individuals and are consistent before and after the cancerous tissues of the same individual; The second methylation difference region is obtained by comparing and screening genome-wide methylation sequencing data of the non-myogenic layer invasive bladder cancer tissue and the myogenic layer invasive bladder cancer tissue, wherein the screening standard is that a specific hypermethylation state or haplotype mode is presented in the myogenic layer invasive bladder cancer tissue, and the frequency of occurrence of the haplotype mode is obviously increased in the conversion process of the non-myogenic layer invasive bladder cancer to the myogenic layer invasive bladder cancer.
  5. 5. The method for dynamically monitoring bladder cancer based on the methylation marker of circulating tumor DNA according to claim 4, wherein in the step 3, the specific steps of constructing the targeted methylation haplotype sequencing library are that firstly, the extracted circulating free DNA is subjected to bisulfite conversion treatment to convert unmethylated cytosine into uracil; Then, performing two-round amplification on the converted DNA by using a multiplex PCR primer pool designed for 10 methylation difference regions, wherein the first round of amplification introduces a sample specific index sequence, and the second round of amplification introduces a universal sequencing adapter; finally, purifying the amplified product and selecting fragments with specific size ranges to complete library construction; Sequencing libraries from all time points of the same patient were mixed in equimolar amounts after quantification for parallel sequencing in the same sequencing procedure.
  6. 6. The method for dynamic monitoring of bladder cancer based on circulating tumor DNA methylation markers according to claim 1, wherein in step 4, the depth of the high throughput sequencing is not less than 5000X per sample; the resolving methylation haplotypes includes aligning sequencing reads to a reference genome, mapping to a target methylation difference region; For the reading which is compared with each methylation difference region, sequentially judging the methylation state of each CpG site covered by the reading according to the sequence of the reading, wherein the methylation state is marked as 1, the non-methylation state is marked as 0, and a binary character string consisting of 0 and 1 is formed, and the character string is a methylation haplotype; all unique methylation haplotypes occurring in each methylation difference region and their corresponding sequencing read support numbers were counted.
  7. 7. The method for dynamically monitoring bladder cancer based on the methylation marker of circulating tumor DNA according to claim 6, wherein in step 5, the passenger haplotype instability index is calculated by determining a first postoperative monitoring time point as a base line time point for each methylation difference region of the first class, and identifying a dominant haplotype of the region at the base line time point; at each subsequent monitoring time point, determining whether the dominant haplotype for the region is the same as the dominant haplotype at the baseline time point; If the regions are different, judging that the haplotype offset of the regions occurs at the time point; The passenger haplotype instability index at a time point is equal to the ratio of the number of regions in which haplotype shifts occur in the 5 first type methylation difference regions at that time point divided by 5.
  8. 8. The method for dynamic monitoring of bladder cancer based on circulating tumor DNA methylation markers according to claim 6, wherein in step 5, the specific process of calculating the driving haplotype load is that for each second type methylation difference region, one or more specific methylation haplotypes which occur in the region and are strongly related to myometrium invasive bladder cancer are predefined as malignant haplotypes; At each monitoring time point, counting the number of reads for which the sequence matches exactly any predefined malignant haplotype in all sequencing reads aligned to that region; Calculating the percentage of the number to the total sequencing reads for the region; The driving haplotype load of one time point is equal to the weighted average value of the percentage values of 5 methylation difference regions of the second type, and the weight coefficient is preset according to the specificity of malignant haplotype of each region to the prediction of the myogenic invasive bladder cancer.
  9. 9. The method for dynamically monitoring bladder cancer based on the circulating tumor DNA methylation marker according to claim 7 or 8, wherein in step 5, the method for integrating the dynamic methylation risk score comprises the steps of inputting a pre-trained logistic regression model by taking the passenger haplotype instability index and the driving haplotype load at the same time point as two characteristic variables; the logistic regression model is trained based on historical patient cohort data including passenger haplotype instability index and driving haplotype load for bladder cancer patients known to eventually relapse or progress outcome at series monitoring time points; The model outputs a value between 0 and 1, which is the dynamic methylation risk score at that time point.
  10. 10. The method for dynamically monitoring bladder cancer based on the circulating tumor DNA methylation marker according to claim 1, wherein in the step 6, the specific method for generating the dynamic monitoring report and the risk early warning is that the dynamic methylation risk scores of the same patient at all continuous monitoring time points are connected in time sequence, and a risk trend graph is drawn; setting a first early warning threshold and a second early warning threshold, wherein the second early warning threshold is higher than the first early warning threshold; When the dynamic methylation risk score is continuously monitored and increased twice and the last score exceeds a first early warning threshold value, a recurrent low risk early warning is generated; And when the dynamic methylation risk score rapidly rises and the latest score exceeds a second early warning threshold value, and the driving haplotype load component at the time point is obviously higher than the historical monitoring value, generating a progress high risk early warning.

Description

Bladder cancer dynamic monitoring method based on circulating tumor DNA methylation marker Technical Field The invention relates to the technical field of biology, in particular to a dynamic bladder cancer monitoring method based on a circulating tumor DNA methylation marker. Background Bladder cancer is a worldwide high-frequency malignancy of the urinary system, and a great challenge in clinical management is the extremely high postoperative recurrence rate, which can reach 60% -70% for non-myogenic invasive bladder cancer (NMIBC). Currently, the "gold standard" for post-operative monitoring of bladder cancer is cystoscopy in combination with pathological biopsy. However, this approach is invasive, can cause pain, hematuria, urinary tract infections, and other discomfort and risk to the patient, and is costly and has poor patient compliance. Traditional noninvasive monitoring means, such as urine abscission cytology examination, have high specificity, but have serious insufficient sensitivity (usually lower than 40%) to low-grade tumors, and cannot meet the requirements of early and accurate monitoring. Other methods based on urine protein markers (such as NMP 22) or Fluorescence In Situ Hybridization (FISH) also have the common limitations of insufficient sensitivity or specificity, many factors being affected, and the like. In recent years, liquid biopsy technology, particularly based on analysis of circulating tumor DNA (ctDNA), opens up a new path for noninvasive diagnosis and monitoring of cancer. In bladder cancer, urine is an ideal liquid biopsy sample enriched for ctDNA derived from a urinary system tumor. Among them, DNA methylation, a stable epigenetic modification, plays a key role in the development of tumorigenesis, and its abnormal pattern has tumor type specificity and stage specificity, and is considered as a very potential biomarker. Studies have demonstrated that assisted diagnosis of bladder cancer can be achieved by methylation analysis of urine DNA. However, the existing detection method based on DNA methylation still faces the following key problems when being applied to dynamic bladder cancer monitoring, and limits the clinical transformation and application effects of the detection method, namely, first, the impurity of marker functions leads to the impure monitoring signals. The existing detection mostly mixes and analyzes methylation difference regions (DMR) with different sources and different biological functions. Studies have shown that bladder cancer-related DMRs can be classified into different types, e.g. "passenger" DMRs (e.g. T1 DMR) reflect mainly the normal tissue origin of cells, whereas "driver" DMRs (e.g. T2 DMR) are directly involved in and drive the malignant progression of the tumor. In dynamic monitoring, the indiscriminate use of these markers can lead to background signal interference, making it difficult to capture key methylation changes that are truly indicative of tumor recurrence or malignant transformation. Second, there is a lack of efficient dynamic risk quantification models. The prior art focuses on qualitative or semi-quantitative diagnosis of "presence or absence" at a single time point, and continuous and dynamic quantitative assessment of the risk of relapse of a patient is not possible. The core value of post-operative monitoring is that early warning, a method that can integrate multidimensional methylation information and output time-dependent risk trends is needed to identify high risk patients in advance before imaging or cystoscopic recurrence. Third, the ability to predict tumor progression is inadequate. Distinguishing low-grade (LG)/non-myogenic invasive bladder cancer (NMIBC) from high-grade (HG)/Myogenic Invasive Bladder Cancer (MIBC) is critical to clinical treatment decisions. Existing methods have difficulty in effectively predicting whether a tumor is progressing from an inert to invasive phenotype by non-invasive means, which is the key information to decide whether more aggressive treatment is needed. Therefore, there is a need in the art for an innovative dynamic urine ctDNA methylation-based monitoring method that overcomes the above drawbacks by selecting and integrating methylation markers with specific biological functions to construct a dynamic risk assessment model, thereby achieving high-sensitivity and high-specificity bladder cancer recurrence early warning and progression risk prediction. Disclosure of Invention Based on the above purpose, the invention provides a dynamic bladder cancer monitoring method based on a circulating tumor DNA methylation marker, which comprises the following steps: step 1, collecting urine samples at a plurality of continuous preset time points after operation of a bladder cancer patient and processing the urine samples to obtain urine supernatant for extracting circulating free DNA; Step 2, extracting circulating free DNA from urine supernatant, and quantifying and analyzing fragments of the