CN116516013A

CN116516013A - Method for monitoring urinary system tumor tiny residual lesion level based on urine ctDNA

Info

Publication number: CN116516013A
Application number: CN202310693903.1A
Authority: CN
Inventors: 李锋; 赵越; 李振东; 徐赞美
Original assignee: Shanghai Zhiben Medical Laboratory Co ltd; Origimed Technology Shanghai Co ltd
Current assignee: Shanghai Zhiben Medical Laboratory Co ltd; Origimed Technology Shanghai Co ltd
Priority date: 2023-06-12
Filing date: 2023-06-12
Publication date: 2023-08-01

Abstract

The invention relates to a method and a system for monitoring the level of tiny residual lesions of a urinary system tumor based on urine ctDNA. Wherein the method comprises the steps of: s1: treating a tumor tissue sample and a control blood sample, and extracting gDNA from the tumor tissue sample and the control blood sample; s2: fragmenting gDNA in the S1, respectively constructing a tumor tissue DNA library and a blood cell DNA library, performing Whole Exome Sequencing (WES) hybrid capture to obtain a captured DNA library, sequencing the captured DNA library, obtaining personalized monitoring site combination data based on WES results, and preparing a primer pool aiming at the personalized monitoring sites; s3: treating a urine sample, and extracting cfDNA from the urine; s4: constructing a urine sample ctDNA library, performing hybridization capture to obtain a captured ctDNA library, and sequencing the captured ctDNA library; s5: and (3) performing a belief generation analysis, combining the detection results of the personalized monitoring sites to obtain the MRD detection results of the samples, and if the number of positive sites of the MRD is greater than or equal to the recommended value of the MRD positive threshold value, determining that the MRD is positive, otherwise, determining that the MRD is negative.

Description

Method for monitoring urinary system tumor tiny residual lesion level based on urine ctDNA

Technical Field

The application belongs to the technical field of gene detection, and particularly relates to a method and a system for monitoring the level of micro residual lesions of urinary system tumors based on urine ctDNA.

Background

The urinary system cancer refers to cancers occurring in any part of the urinary system, such as kidney, ureter, bladder, urethra, etc. Among them, urothelial cancer (Urothelial Carcinoma, UC) is an invasive malignant tumor with hidden onset, high recurrence rate and high progression rate, and can be classified into renal pelvis cancer, ureter cancer, bladder cancer and urethra cancer according to the occurrence parts, and is one of the most common malignant tumors of urinary system in China. About 70-80% of the diagnosed cases of UC are non-myogenic invasive bladder urothelial carcinoma (Nonmuscle Invasive Urothelial Carcinoma, NMIBC), and conventional treatment regimens are typically transurethral bladder tumor resection (Transurethral Resection of Bladder Tumor, turbo) and are considered to receive bcg, chemotherapy, etc. based on recurrence risk. However, NMIBC patients, even if subjected to complete tumor resection, are at higher risk of recurrence or progression to more advanced disease due to the presence of minimal residual lesions (Minimal Residual Disease, MRD).

The concept of MRD is derived from hematological disorders, meaning that the small number of cancer cells remaining in the body after treatment may be small, and the number of remaining cancer cells may not cause any signs or symptoms, which are generally difficult to detect by conventional methods. Cystoscopy and urine-based abscission cytology are two widely used diagnostic means for UC. However, invasive cystoscopy and urine-based abscission cytology have limitations in identifying the detection of recurrence of UC, and such invasive detection means are not available as a long-term monitoring method. Imaging is considered to be the gold standard for current evaluation of cancer treatment efficacy, and recurrence detection and treatment response detection of metastatic tumors is typically performed by computed tomography (Computed Tomography, CT). However, CT is applied to MRD with low detection sensitivity and high false positive rate, so that MRD cannot be detected accurately and timely in practice, and the effect of treatment is evaluated by morphological change and has hysteresis, so that the effect of tumor treatment cannot be reflected timely.

ctDNA is a cell-free DNA (cfDNA) derived from a tumor, potentially carrying the same oncogenic mutations and genetic alterations as the primary tumor. Clinical researches show that the concentration of ctDNA is in direct proportion to the tumor load, and the detection of ctDNA mutation can effectively monitor tumor progress and auxiliary prognosis, so as to guide clinical personalized treatment. ctDNA detection has a hint on whether MRD exists or not, and whether tumor recurs or metastasizes can be monitored in real time. Compared with imaging and protein tumor marker detection, ctDNA has the greatest advantage of being capable of detecting MRD in an ultra-early stage, and the research proves that ctDNA detection can detect the progress of tumor several months in advance compared with CT. Thus, liquid biopsies using ctDNA-based are suitable for early warning of MRD and real-time detection of recurrence risk. The method has exploration in the aspects of new adjuvant therapy effect prediction, immunotherapy effect monitoring, systemic therapy effect monitoring of late patients, drug holiday prompting and the like, and has good application prospect.

Advanced sequencing-based urinary system cancer personalized analysis (urine Cancer Personalized Profiling by Deep Sequencing, uCAPP-Seq) is a highly sensitive high throughput sequencing method for detecting circulating tumor DNA (ctDNA) as reported by Washington university medical institute Aadel A. Chaudhur et al in PLOS Medicine using advanced sequencing (CAPP-Seq) and Integrated Digital Error Suppression (iDES). uCAPP-Seq is a method of detection of Tumor independent analysis (Tumor-diagnostic Assays), not by performing whole exome sequencing (Whole Exome Sequencing, WES) on Tumor tissue in advance, but by performing hybrid capture sequencing using a fixed panel containing 49 bladder cancers frequently mutated driver genes. uCAPP-Seq also attempted to tailor based on the data of 412 BLCA cases in TCGA, with the final panel covering a genomic space of about 311kb, including a region of 460 genes, showing only 6 median identifiable mutations per BLCA patient.

Disclosure of Invention

The existing detection means for urinary system tumor such as cystoscopy, urine liquid-based abscission cytology detection and the like cannot play a role in prompting whether MRD exists or not after a urinary system tumor patient is treated, and the technical problems that puncture bias, wound and the like cannot exist in a timely prediction or detection method for tumor recurrence, and cannot be used as a long-term monitoring means. Imaging is considered to be the gold standard for evaluating the efficacy of cancer treatment at present, and recurrence detection and therapeutic response detection of metastatic tumors are usually performed by CT. However, CT is not high in sensitivity and has high false positive rate when applied to MRD detection, and MRD cannot be detected accurately and timely.

ctDNA is released into the circulatory system by cancer cells that remain in the body after the patient has undergone treatment but cannot be detected by imaging means, confirming that the ctDNA content of the post-operative urine sample and its SNV site present are direct evidence of the presence or absence of MRD. The invention provides a noninvasive biopsy method for monitoring the micro residual lesion level of a urinary system based on urine ctDNA, which comprises the steps of performing WES detection on tumor tissues of a patient, matching control blood detection to accurately exclude non-tumor source variation, individually selecting monitoring sites according to WES detection results, generating a primer pool, and performing multiplex PCR library construction and sequencing on ctDNA obtained by urine extraction of the patient. The method aims at detecting whether MRD exists after operation of a patient and carrying out long-term recurrence detection and curative effect assessment to assist doctors in prognosis prediction.

Specifically, a first aspect of the present invention provides a method for monitoring urinary system micro residual lesions (MRD) levels based on urine ctDNA, comprising the steps of:

s1: treating a tumor tissue sample and a control blood sample, and extracting gDNA from the tumor tissue sample and the control blood sample;

s2: fragmenting gDNA in the S1, respectively constructing a tumor tissue DNA library and a blood cell DNA library, performing Whole Exome Sequencing (WES) hybrid capture to obtain a captured DNA library, sequencing the captured DNA library, obtaining personalized monitoring site combination data based on WES results, and preparing a primer pool aiming at the personalized monitoring sites;

s3: treating a urine sample, and extracting cfDNA from the urine sample;

s4: constructing a urine sample ctDNA library, performing hybridization capture to obtain a captured ctDNA library, and sequencing the captured ctDNA library;

s5: and (3) performing a belief generation analysis, combining the detection results of the personalized monitoring sites to obtain the MRD detection results of the samples, and if the number of positive sites of the MRD is greater than or equal to the recommended value of the MRD positive threshold value, determining that the MRD is positive, otherwise, determining that the MRD is negative. In one embodiment, wherein the personalized monitoring sites in S2 are SNV and Indel sites, and driver gene sites.

In a preferred embodiment, wherein the rules for selecting personalized monitoring sites in S2 are as follows:

1) Carrying out pyclone clustering on SNV variation points with the WES detection result of true, and selecting a main clone;

2) Preferably selecting clinically significant variant (report) and clinically insignificant variant (reportVUS) sites;

3) If condition 2) has fewer than 16 sites selected, then consider the other IGVs as true dominant gram Long Bianyi sites; primers were designed for the major cloning variation sites and driver gene sites that were frequently mutated in urinary system cancer.

In one embodiment, wherein said S5 comprises the steps of:

s51: reads pretreatment: the method comprises the steps of converting from a bcl2 file to a fastq file, removing a connector sequence, extracting and storing parameters related to sequencing quality, such as insert size, sequencing error rate and the like;

s52: alignment of Reads: the method comprises the steps of comparing reads and calibrating related indels, counting and recording the distribution and targeted sequencing areas of the reads, and reserving the obtained quality parameters in a bam file for subsequent mutation identification;

s53: data QC: the method comprises quality control of sequencing and reads comparison, wherein parameters such as target rate, pollution factor, average sequencing depth, median sequencing depth and the like are presented in the results so as to evaluate whether sequencing data are enough for mutation recognition, and SNV loci with the sequencing depth of less than 20000 multiplied by are filtered (the depth of 100000 multiplied by more is optimal);

S54: SNV recognition: it involves identifying the SNV of the target region and calculating the site coverage depth and the SNV frequency.

In one embodiment, the method for determining an MRD in S5 is as follows:

in one embodiment, wherein the tumor is a urinary system-related tumor.

In a preferred embodiment, wherein the tumor is renal cancer, urothelial cancer, prostate cancer.

In a preferred embodiment, wherein the urothelial cancer is ureteral cancer, bladder cancer, and urethra cancer.

In a second aspect, the present invention provides a method for detecting post-operative MRD and monitoring urinary system cancer recurrence based on ctDNA, comprising the steps of:

s3: treating a urine sample, and extracting cfDNA from the urine sample;

s5: performing raw message analysis, combining the detection results of the personalized monitoring sites to obtain the MRD detection results of the samples, wherein if the number of positive sites of the MRD is greater than or equal to the recommended value of the MRD positive threshold value, the MRD is positive, otherwise, the MRD is negative; if the MRD is positive, the patient is prompted to have a higher risk of cancer recurrence.

In one embodiment, wherein the personalized monitoring sites in S2 are SNV and Indel sites, and driver gene sites.

In one embodiment, wherein said S5 comprises the steps of:

S51: reads pretreatment: it includes conversion from bcl2 file to fastq file and elimination of linker sequence;

s52: alignment of Reads: it includes alignment of reads and indel-related calibration;

s53: data QC: the method comprises quality control of sequencing and reads comparison, wherein SNV loci with average sequencing depth lower than 20000 x are filtered, and the depth is more than 100000 x optimal;

In one embodiment, wherein the method of determining recurrence of urinary system cancer in S5 is as follows:

in one embodiment, wherein the urinary system cancer is renal cancer, urothelial cancer, prostate cancer.

In one embodiment, the methods of the invention may also be used in the setting of new aids for urinary system cancers, in the treatment of adjuvant therapy, in drug cues, in the treatment of disease progression, etc.

A third aspect of the present invention provides a urine biopsy detection system for monitoring the level of micro residual lesions (MRD) of the urinary system based on ctDNA, comprising the following modules:

A) Sample processing module: treating a tumor tissue sample and a control blood sample, and extracting gDNA from the tumor tissue sample and the control blood sample; treating a urine sample, and extracting cfDNA from the urine sample;

b) And the personalized monitoring module: respectively constructing a tumor tissue DNA library and a blood cell DNA library, performing full exome sequencing (WES) hybrid capture to obtain a captured DNA library, sequencing the captured DNA library, obtaining personalized monitoring site combination data based on WES results, and preparing a primer pool aiming at the personalized monitoring sites;

c) Urine detection module: constructing a urine sample ctDNA library, performing hybridization capture to obtain a captured ctDNA library, and sequencing the captured ctDNA library;

d) And an analysis module: and (3) performing a belief generation analysis, combining the detection results of the personalized monitoring sites to obtain the MRD detection results of the samples, and if the number of positive sites of the MRD is greater than or equal to the recommended value of the MRD positive threshold value, determining that the MRD is positive, otherwise, determining that the MRD is negative.

In one embodiment, wherein the personalized monitoring sites in B4) are SNV and Indel sites, and driver gene sites.

In a preferred embodiment, wherein the rules for selecting personalized monitoring sites in B4) are as follows:

In one embodiment, wherein the D) analysis module comprises the steps of:

d1 Reads pretreatment: it includes conversion from bcl2 file to fastq file and elimination of linker sequence;

d2 Alignment of Reads): it includes alignment of reads and indel-related calibration;

d3 Data QC: the method comprises quality control of sequencing and reads comparison, wherein SNV loci with average sequencing depth lower than 20000 x are filtered, and the depth is more than 100000 x optimal;

d4 SNV recognition: it involves identifying the SNV of the target region and calculating the site coverage depth and the SNV frequency.

In one embodiment, wherein the tumor is a urinary system-related tumor.

In one embodiment, wherein the tumor is renal cancer, urothelial cancer, prostate cancer.

In a fourth aspect, the invention provides the use of the above system in the monitoring of recurrence of cancer in the urinary system, neoadjuvant, adjuvant therapy efficacy, medication cues, and disease progression cues.

In one embodiment, wherein the cancer is renal cancer, urothelial cancer, prostate cancer.

Wherein, the specific scheme related to the recommended value of the MRD positive threshold value in all the above embodiments is as follows: CN202111349960.5, which is incorporated by reference in its entirety.

Including but not limited to, for example:

sensitivity x=positive detected/positive site=positive detected/(positive detected + false negative site)

Specificity y=negative site detected/negative site=negative site detected/(negative site detected+false positive site)

After single site sensitivity x (100%. Gtoreq.x.gtoreq.0) and specificity y (100%. Gtoreq.y.gtoreq.0) are calculated, the MRD is positive (a.gtoreq.b >0, a and b are integers) if the number of selected monitoring sites is a and at least b sites are positive:

1. Assuming that all a sites are positive, detecting that more than or equal to b sites are positive, and calculating sample sensitivity p according to binomial distribution, wherein the detected sites are positive;

2. assuming that all a sites are negative, detecting that the sites (a-b) are negative, namely MRD negative, and calculating sample specificity q according to binomial distribution;

the values of a and b are given according to the permutation and combination, a matrix is set and p and q of each pair of a and b are calculated, and the optimal a and b are selected according to p and q.

The method, the system and the excellent technical effects applied to the recurrence monitoring, the new assistance, the adjuvant therapy curative effect, the medication prompt and the disease progress prompt of the urinary system cancer are mainly characterized in that:

1) For the detection of urinary system tumor MRD, since ctDNA of tumor cells is directly released into urine and accumulated in bladder, the urine ctDNA-based detection method of the present invention has higher sensitivity and lower false negative compared to blood ctDNA-based MRD detection method.

2) According to the invention, ctDNA with the abundance of more than or equal to 0.02% can be stably detected, which is far higher than 0.1% of most hybridization capture sequencing methods, and the variation of non-Tumor sources is accurately eliminated through the design of contrast blood, and the design of selecting about 16-40 (preferably 20-30) personalized monitoring sites based on a Tumor-information assay strategy is adopted, so that the method has the advantages of high sensitivity, good compatibility, multiple effective sites and the like, and is beneficial to the subsequent MRD monitoring and other applications.

3) The invention carries out tissue prior based on WES 2 ten thousand genes, personalized and customized MRD monitoring panel, subsequent ultra-high depth sequencing, fully considers individual differences and can track mutation of primary tumor. Patient-personalized baseline design was performed by WES sequencing of 2 ten thousand genes, and with the support of an own algorithm, a good balance between noise reduction and sensitivity was achieved, providing more MRD tracking monitoring sites than the fixed panel.

Drawings

Fig. 1: a flow chart of a urine biopsy method for monitoring a tiny residual focus of the urinary system;

fig. 2: experimental flow chart of urine biopsy method for monitoring tiny residual focus of urinary system

Detailed Description

While this invention may be embodied in many different forms, there are disclosed herein specific illustrative embodiments thereof which embody the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, terms in the singular shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms (such as "include" and "contain") is not limiting. Furthermore, the scope provided in the specification and the appended claims includes all values between the endpoints and between the endpoints.

Generally, terms related to cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein, and techniques thereof, are well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook J. & Russell d.molecular Cloning: A Laboratory Manual, 3 rd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (2000); abbas et al, cellular and Molecular Immunology, 6 th edition, w.b. samaders Company (2010); harlow and Lane Using anti-cams A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1998); ausubel et al Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, wiley, john & Sons, inc. (2002); and Coligan et al, short Protocols in Protein Science, wiley, john & Sons, inc. (2003). The terms relating to analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are well known and commonly used terms in the art. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Definition of the definition

For a better understanding of the present invention, definitions and explanations of related terms are provided below. The following terms used in the present invention have the following meanings unless otherwise indicated. A particular term, unless otherwise defined, shall not be construed as being ambiguous or otherwise unclear, but shall be construed in accordance with the ordinary meaning in the art. When trade names are present in the present invention, it is intended to refer to their corresponding commercial products, compositions or active ingredients thereof.

As used herein, the term "free nucleic acid" refers to cell-free nucleic acid, cfNA, e.g., cfDNA, that can be released into the circulation by various forms of cell death (e.g., apoptosis, necrosis, autophagy, and necrotic apoptosis). cfDNA is fragmented and the fragments are distributed in a size range of 150-350bp to >10000bp (see Kalnina et al, world JGastroenterol, 201517; 21 (41): 11636-11653). For example, the size distribution of plasma DNA fragments in hepatocellular carcinoma (HCC) patients is in the range of 100-220bp in length, with peaks at a count frequency at about 166bp and highest tumor DNA concentration in fragments 150-180bp in length (see Jiang et al, proc Natl Acad Sci USA,112: E1317-E1325).

As used herein, the term "ctDNA" refers to circulating tumor DNA (circulating tumor DNA, ctDNA), DNA fragments derived from apoptosis, necrosis or secretion of tumor cells, containing the same genetic variations and apparent modifications as tumor tissue DNA, such as point mutations, gene rearrangements, fusions, copy number variations, methylation modifications, and the like. The detection of ctDNA can be applied to early cancer screening, diagnosis and stage division, targeted drug administration guidance, efficacy evaluation, recurrence monitoring and other aspects. Combining the information of mutation and methylation of tumor specific genes carried by ctDNA, the method is helpful for improving the sensitivity and specificity of detection, and can find cancer trails earlier, thus having important significance for early screening of tumors.

Wherein the ctDNA amplification/sequencing workflow may comprise generating a collection of amplicons by performing a multiplex amplification reaction on nucleic acids isolated from a urine sample from an individual, such as an individual suspected of having a cancer (e.g., renal cancer, urothelial cancer, or prostate cancer), or a portion thereof, wherein each amplicon in the collection of amplicons spans at least one single nucleotide variant locus in a collection of single nucleotide variant loci, such as SNV loci known to be associated with a cancer (e.g., renal cancer, urothelial cancer, or prostate cancer); and determining the sequence of at least one segment of each amplicon in the collection of amplicons, wherein the segment comprises a single nucleotide variant locus. In this way, this exemplary method determines whether a single nucleotide variant is present in the sample.

As used herein, the term "clinically significant variation (report)" refers to both primary and secondary variations in the hierarchical reporting of mutation sites: mutations of clear clinical or potential clinical significance, including: the administration therapeutic targets approved by the authorities such as the national food and drug administration (CFDA) and the American Food and Drug Administration (FDA); the blood tumor diagnosis and treatment guideline or expert consensus has the mutation with definite diagnosis, treatment and prognosis significance, and has not entered the diagnosis and treatment guideline or expert consensus, but has authoritative literature or reports the mutation with diagnosis and treatment significance in the blood tumor on a large scale; mutations that have diagnostic, therapeutic or prognostic significance in hematological tumors are reported based on a number of small-scale studies, but have not yet been agreed upon; somatic mutation of important structural domain of newly discovered disease related gene; "clinically insignificant mutation (reportVUS)" refers to a three-level mutation in the hierarchical report of mutation sites: clinically insignificant variants (Variants of Uncertain Significance, VUS), for mutation sites that are still in issue, the laboratory must formulate relevant rules to find mutations, i.e. report, and attach instructions and significance; it is also possible to report no or only small parts of the mutation results, with accompanying instructions, references and databases.

As used herein, the term "IGV" refers to comprehensive genomics visualization tool Itegrative Genomics Viewer, which can visualize genomic variations and is widely used in genomics research.

As used herein, the term "major cloning variation site" refers to a mutation in a gene that is present in most tumor cells.

As used herein, the term "cancer" refers to a broad group of various diseases characterized by uncontrolled growth of abnormal cells in the body. Refers to solid and non-solid tumors mediated by growth, proliferation or metastasis of any tumor or malignant cell that is responsible for a medical disorder.

Successful treatment of a disease (e.g., cancer) typically relies on early diagnosis, proper staging of the disease, selection of an effective treatment regimen, and close monitoring to prevent or detect recurrence. For cancer diagnosis, histological evaluation of tumor material obtained from tissue biopsies is generally considered the most reliable method. However, the invasive nature of biopsy-based sampling makes it unavailable for population screening and routine follow-up. Thus, the method of the present invention has the following advantages: which has a relatively low cost and a fast turnaround time. Multiplex PCR and bioinformatics methods that can be used increase throughput and reduce cost.

As used herein, the term "risk of cancer recurrence" may generally be determined by including, but not limited to:

renal carcinoma: after radical nephrectomy, recurrence is determined based primarily on imaging, either abdominal CT or MRI (at least abdominal B-mode ultrasound), or chest CT for routine follow-up monitoring. In the clinical suspicion of recurrence or metastasis, PET-CT can confirm recurrence and evaluate distant tumor metastasis of renal cancer.

Prostate cancer: prostate cancer recurrence includes biochemical recurrence and imaging recurrence

Biochemical recurrence (BCR) after Radical Prostatectomy (RP) refers to: prostate Specific Antigen (PSA) values can generally drop below 0.2ng/ml after RP, if PSA values rise back above 0.2ng/ml with an ascending trend, defined as PSA Recurrence (PSA Recurrence) or BCR after RP. If the PSA value fails to drop below 0.2ng/ml after RP, then this is called PSA Persistence (PSA Persistence), which is treated in accordance with the principles of PSA recurrence (BCR). Definition of biochemical recurrence after radical radiation therapy of prostate: the PSA value being 2ng/ml higher than the lowest point after radiotherapy is defined as biochemical recurrence after radiotherapy, no matter whether other treatment means are adopted at the same time or not, and no matter what the lowest value of PSA after radiotherapy is.

Imaging recurrence of prostate cancer:

local recurrence after radical prostatectomy: multiparameter magnetic resonance imaging (mpMRI) is currently the recommended method for detecting local recurrence of prostate cancer. Patients with PSA >0.5g/ml were examined with mpMRI using a transrectal coil for sensitivity up to 94% and with PSA <1ng/ml for sensitivity higher than 11C-choline PET/CT, as the first imaging examination to determine local recurrence. For patients with PSA <0.5ng/ml, the prostate specific membrane antigen PSMA PET/CT is a better option for detecting local recurrence.

Local recurrence after radical radiation therapy of prostate: prostate puncture biopsies over 18 months after radical radiotherapy of prostate cancer found cancer cells with concomitant elevation of PSA, whereas CT, MRI, bone scan or other imaging examinations did not find evidence of metastasis, defined as local recurrence after radiotherapy. Local recurrence is confirmed by needle biopsy. The detection rate of local recurrence by transrectal B-ultrasound is low, mpMRI is the first choice for evaluating the local recurrence condition, and a conditional person can conduct targeted puncture of a suspicious recurrence focus by using an mpMRI image on the basis of systematic puncture biopsy. PET/CT, PET/MRI, PSMA PET/CT are all optional imaging examinations, and there is no certainty about the superior or inferior contrast of such examinations with mpMRI in the diagnosis of local recurrence. Generalized recurrence also includes the occurrence of distant metastasis following treatment, i.e., the finding of distant metastasis lesions of tumors by imaging examination means such as mpMRI, bone ECT, PET/CT, PET/MRI, PSMA PET/CT, etc.

Bladder cancer:

the recurrence and metastasis risk after bladder cancer operation is related to the tissue pathology type and stage, the incidence rate after operation is highest in 24-36 months, and the later rate is relatively low. Conventional recommendation: patients in pT stage 1 are examined once a year, blood biochemically, chest X-ray, abdominal pelvic B-ultrasound, CT and/or MRI; 1 above examination was performed on patients in pT2 stage for 6 months; patients with stage pT3 tumor were performed 1 time every 3 months. For patients with pT 2-pT 3 stage tumor, 1 thoracoabdominal pelvic CT examination should be performed every half year.

Upper urinary tract urothelial cancer:

CT urography is a relatively new diagnostic imaging technique that produces high resolution images by rapidly acquiring thin slices during helical tomographic imaging. It is the most accurate imaging modality for diagnosing UTUC. The sensitivity range is between 67% and 100% and specificity is between 93% and 99% using MRI to detect UTUC has several drawbacks. Unlike non-enhanced CT scans, diagnosis of non-obstructive urinary tract stones can be difficult, and therefore, UTUC is difficult to diagnose based on the presence or absence of filling defects. Furthermore, the spatial resolution of MR images is significantly lower than CT urography and motion artifacts are more likely to occur.

As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include the values of any integer within the recited range, and fractions thereof (such as tenths and hundredths of integers) as appropriate, unless otherwise indicated.

The invention will be further described with reference to specific examples, which are, however, only intended to illustrate and not limit the scope of the invention. Also, the invention is not limited to any particular preferred embodiment described herein. It should be understood by those skilled in the art that equivalent substitutions and corresponding modifications to the technical features of the present invention are still within the scope of the present invention. The reagents used in the examples below are commercially available products, and the solutions may be formulated using techniques conventional in the art, unless otherwise specified.

Implementation of the embodimentsExample 1:

1.1 treatment of FFPE tumor tissue samples and blood samples

Treatment of 1.1.1FFPE tumor tissue samples

gDNA was extracted from FFPE tumor tissue samples using QIAamp DNAFFPE Tissue Kit (Qiagen) and the extracted gDNA was subjected to concentration determination using Qubit dsDNAHS Assay Kit (ThermoFisher). Taking 4ng of gDNA as a template, preparing an internal reference gene PCR reaction system, amplifying, and grading the DNA quality by agarose gel electrophoresis.

1.1.2 treatment of control blood samples

Human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMCs) were isolated from the centrifuged control blood samples. gDNA was extracted from PBMC using QIAamp DNAMini kit (Qiagen) and the extracted gDNA was subjected to concentration determination using Qubit dsDNAHS Assay Kit (ThermoFisher). When the viscosity of the sample is too high or macroscopic impurities (gDNA is not transparent clear liquid) are present after gDNA extraction, the method is used Nucleotide Removal Kit kit for purification.

1.2 WeS-based personalized monitoring site selection and primer pool preparation

1.2.1 library construction

The amount of gDNA extracted from FFPE tumor tissue samples and control blood samples was fragmented using LE220-plus Focused-ultrasonicator (Covaris) according to the corresponding procedure. Library construction was accomplished using KAPA Hyper Prep Kit (KAPA) and NEXTFLEX Unique Dual Index Barcodes (PerkinElmer) for end modification, a-tail addition, linker ligation and PCR amplification of the fragmented gDNA. The sequencing library was purified using Agencourt AMPure XP Beads (Beckman). UsingdsDNAHS Assay Kit (ThermoFisher) quantitated the library and the fragment distribution of the library was detected using LabChip GX Touch HT (Perkinelmer).

1.2.2WES hybrid Capture

The prepared sequencing library was subjected to WES hybridization capture using xGen Hybridization and Wash Kit (IDT) and zx6_ probes (WES panel), followed by PCR amplification using KAPA HiFi HotStart ReadyMix (KAPA) to prepare a hybridization capture library. The hybrid capture library was purified using Agencourt AMPure XP Beads (Beckman). The library was quantified using Qubit dsDNAHS Assay Kit (ThermoFisher) and the fragment distribution of the library was detected using LabChip GX Touch HT (PerkinElmer).

1.2.3 on-machine sequencing

The library was sequenced using a NovaSeq6000 sequencer or a NextSeq 550 sequencer.

1.2.4 selection of personalized monitoring sites and primer pool preparation

According to the WES detection result of the tissue sample, selecting clinically-defined SNV and Indel sites and adding driving gene sites which are frequently mutated in the urinary system cancer, wherein the selection rule is as follows:

3) If condition 2) has fewer than 16 sites selected, then consider the other IGVs as true dominant gram Long Bianyi sites;

primers are designed in batches by using Primer3 aiming at the selected main cloning mutation sites and the driving gene sites frequently mutated in urinary system cancers, the amplified fragment product is about 100bp, and SNV is positioned in the middle of the fragment as much as possible. Primers were synthesized by the biological engineering (Shanghai) Co., ltd according to the designed sequence and mixed into a primer pool.

1.3 preparation of urine ctDNA sequencing library based on multiplex PCR and on-machine

1.3.1 extraction of urine cfDNA

Free DNA urine sample sampling consumables (Jian Dan organisms) were selected to collect urine, which was filtered on cells using filters and syringes, and the filtered urine was stored in a urine cup with urine preservation solution. cfDNA was extracted from urine using the Quick-DNAUrine Kit (Zymo Research) and the extracted cfDNA was used for concentration determination using Qubit dsDNAHS Assay Kit (thermo fisher).

1.3.2 multiplex PCR

Multiplex PCR was performed using NEBNext Ultra II Q Master Mix with final primer pool concentration of 0.2 uM/strip in the amplification system and the amplified product was purified using Agencourt AMPure XP Regent (Beckman Coulter). Finally usedsDNAHS Assay Kit (ThermoFisher) quantitates the library.

1.3.3 library construction

UsingUniversal DNALibrary Prep Kit for Illumina V3 kit library construction was performed and the library was purified using an AMpure XP beams (Beckman Coulter). Use->dsDNAHS Assay Kit (ThermoFisher) quantitates the library.

1.3.4 library fragment distribution detection

The library obtained was diluted to 0.5-1 ng/. Mu.L and the library was tested using DNAHigh Sensitivity Reagent Kit (Perkinelmer) and the fragment distribution of the library after capture.

1.3.5 on-machine sequencing

1.4 Credit analysis, clinical annotation and report Generation

The effect of the belief algorithm is from the raw data generated by the sequencer to the clinical annotation stage, the algorithm software automatically compares short sequencing reads to the human genome, marks their positional information on the gene, and extracts the information of genetic variation. The process is divided into 4 steps, and is briefly described as follows:

Reads pretreatment: this module includes the conversion from bcl2 file to fastq file and the culling of the linker sequence. All parameters related to sequencing quality are extracted and saved, such as insert size and sequencing error rate.

Alignment of Reads: including alignment of reads and indel-related calibration. In this module, both the distribution of reads and the targeted sequencing region are counted and recorded. The aligned quality parameters are retained in the bam file for subsequent mutation identification.

Data QC: this module shows the quality of sequencing and reads alignment. The sequencing error rate and insert size of each sample are reported to the wet experimental link for quality control. The results are set forth in parameters such as target rate, contamination factor, average sequencing depth, and median sequencing depth to evaluate whether the sequencing data is sufficient for mutation identification. SNV sites with average sequencing depth below 20000 x are filtered, and the depth above 100000 x is optimal.

SNV recognition: SNVs of the target region are identified and site coverage depth and SNV frequency are calculated.

Example 2:

there have been documents that demonstrate a good prognosis and predictive effect on monitoring blood ctDNA for patients with basal invasive urothelial cancer (Powles, t., assaf, z.j., davarpah, n.et al, ctdnaguiniding adjuvant immunotherapy in urothelial carpinoma. Nature), the aim of this experiment was to confirm the monitoring effect of urine cfDNA and whether it is superior to plasma cfDNA, to consider the feasibility of urine cfDNA as a test sample for MRD of the urinary system and for dynamic monitoring of disease.

2.1 confirmation sample

Collecting urine and blood samples of 14 urothelial cancer patients and FFPE samples of tumor tissues, and comparing the detected variation in urine and blood cfDNA with the positive condition of MRD; repeatability tests were performed on samples with large amounts of urine cfDNA, and mutation detection and MRD results were compared.

2.2 acknowledgement scheme

2.2.1 Experimental design

Urine samples were collected by selecting free DNA urine sample sampling consumables (Jian Dan organisms), the urine was filtered on cells using filters and syringes, and the filtered urine was stored in a urine cup with urine preservation solution for a maximum of 1 month at room temperature. The cfDNA is extracted from urine by using Quick-DNAURINE Kit (Zymo Research), and the steps of subsequent multiplex PCR library establishment, machine starting and the like are the same as those of the solid tumor MRD.

2.2.2 criteria for evaluation

The quality control standard of sample extraction is that the DNA yield is more than or equal to 10ng, and the sample extraction is judged to be qualified when the score is more than 0, otherwise, the sample extraction is disqualified, and the acceptable standard is that the qualification rate is more than or equal to 95%. The concentration of the constructed library is more than 0.5 ng/. Mu.L, and the acceptable standard is that the qualification rate is more than or equal to 95%. The remaining procedure QC criteria were the same as solid tumor MRD.

2.3 confirmation of results

2.3.1 qualification test for cfDNA extraction of urine samples

In order to verify the qualification rate of the cfDNA extraction of the urine sample (the preset value is more than or equal to 95%), 14 urine samples of patients with urinary system tumor are collected, less than or equal to 40mL of cfDNA of the sample is extracted by using the Quick-DNAURINE Kit (Zymo) and then dsDNAHS Assay Kit (ThermoFisher) quantitates the extracted DNA. Sample information and extraction results are shown in table 1 below.

Although there was a difference in yield between 14 samples, all achieved QC standards for this link, i.e. cfDNA total was ≡10ng, yield 14/14=100%. This result demonstrates that the method reagents used can ensure the qualification rate of cfDNA extraction.

TABLE 1 test results of cfDNA extraction yield for urine samples

2.3.2 stability test of construction of urine sample cfDNA library

To verify the stability of the urine sample library construction (the preset value is that the library concentration is not less than 0.5 ng/. Mu.L), the extracted cfDNA is selected by utilizing the tissue WES resultThe primers were spot designed for multiplex PCR. UsingUniversal DNALibrary Prep Kit for Illumina V3 (Vazyme) sample DNA library preparation was performed. Fragment sizes of the sample library were determined using LabChip GX Touch HT (Perkin Elmer). Sample information and library construction results are shown in Table 2 below. />

TABLE 2 urine sample library construction stability

Although there were differences in total library amounts between 14 samples, all achieved QC standards for this link, library concentrations were ≡0.5 ng/. Mu.l, yield 14/14=100%.

2.3.3 detection of urine and blood cfDNA variation and analysis of consistency of MRD results

2.3.3.1 detection of urine and blood cfDNA variation and MRD results

MRD positive is defined as: the number of positive sites of the MRD is greater than or equal to the MRD positive threshold recommendation, and table 3 below shows the mutation detection and MRD results for urine and blood samples from the same patient on the same date.

TABLE 3 urine and blood cfDNA variation detection and MRD result consistency statistics

2.3.3.2 analysis of results

From a comparison of the above results, the detection of variations in cfDNA in urine and blood is not completely consistent. Overall, the more sites are detected by urine, the more frequently. 13 samples of both urine and blood and 11 MRD results were consistent (85%). 1 example of LFG-T2 is positive for MRD in urine and negative for MRD in blood, and is likely to be more easily detected due to the fact that the urine contains more cfDNA released by urinary system tumors. 1 example WLM-T1 is positive for blood MRD, negative for urine MRD, low frequency of blood MRD detection sites, and 0.07% average VAF, possibly due to urine sample.

2.3.4 urine sample variation detection repeatability analysis

Since urine cfDNA sample yield was insufficient to complete the overall reproducibility verification, only samples with large extraction were selected for batch-to-batch reproducibility detection. Sample information and mutation detection results are shown in Table 4 below.

TABLE 4 urine sample cfDNA variation detection repeatability statistics

2.3.4.1 analysis of results

Together with 281, 266 was repeatedly detected (94.7%). Of the sites that failed to be repeatedly detected, 12/15 (80%) of the VAF was < 0.1%. The actual detection frequency of the sites around the LOD frequency is lower than the detection lower limit due to the floating up and down of the detected VAF, and is filtered as background noise. Of the sites with VAF > 0.1%, the 151/154 (98.1%) sites were repeatedly detected. Of the negative sites, the 110/110 (100%) site can be repeatedly detected.

2.4 confirmation of conclusion

The experiment verifies the feasibility of the cfDNA of the urine sample as the MRD monitoring of the tumor of the urinary system from the following aspects:

qualification rate of urine sample cfDNA extraction: 14 urine samples are subjected to extraction experiments, and the qualification rate is 14/14=100%, so that stable extraction can be realized.

Qualification rate of urine sample cfDNA library construction: 14 urine samples are subjected to library construction, and the qualification rate is 14/14=100%, so that the library can be stably built.

Blood and urine variant detection and MRD result consistency: urine and blood samples from 13 patients were tested for MRD, with 11 MRD results being consistent. Compared with a blood sample, the MRD in urine has more mutation sites and higher frequency, and is more suitable for being used as MRD monitoring of urinary system tumor.

Repeated analysis of urine sample variation detection: the urine samples of 7 patients are repeatedly detected, the total number of variation sites is 281, 94.7% (266/281) sites can be repeatedly detected; sites with poor 12/15 reproducibility VAF were <0.1% and sites that were not detected could be filtered for low frequency sites due to VAF fluctuations. Of the sites with VAF > 0.1%, the 151/154 (98.1%) sites were repeatedly detected. Of the negative sites, the 110/110 (100%) site can be repeatedly detected.

In conclusion, the urine sample meets verification requirements in terms of cfDNA extraction qualification rate, database establishment stability, coincidence rate with MRD detection in blood cfDNA, repeatability of detection results and the like, and can be used as a monitoring sample of urinary system tumor MRD.

It should be understood that while the present invention has been described by way of example in terms of its preferred embodiments, it is not limited to the above embodiments, but is capable of numerous modifications and variations by those skilled in the art. The selection and application of particular reagents may be adapted and varied according to particular needs. It will thus be appreciated that those skilled in the art will be able to devise numerous alternative arrangements which, although not explicitly described herein, embody the principles of the invention and are included within its spirit and scope.

Claims

1. A method of monitoring urinary system tumor micro residual lesions (MRD) levels based on urine ctDNA, comprising the steps of:

s3: treating a urine sample, and extracting cfDNA from the urine sample;

s5: and (3) performing a belief generation analysis, combining the detection results of the personalized monitoring sites to obtain the MRD detection results of the samples, and if the number of positive sites of the MRD is greater than or equal to the recommended value of the MRD positive threshold value, determining that the MRD is positive, otherwise, determining that the MRD is negative.

2. The method of claim 1, wherein the personalized monitoring sites in S2 are SNV and Indel sites, and driver gene sites.

3. The method of claim 2, wherein the rules for selecting personalized monitoring sites in S2 are as follows:

1) Carrying out Pyclone clustering on SNV variation points with the WES detection result of true, and selecting a main gram Long Bianyi locus;

4. The method of claim 1, wherein the S5 comprises the steps of:

s51: reads pretreatment: including conversion from bcl2 file to fastq file and elimination of adaptor sequence, and extracting and saving sequencing quality related parameters, such as insert size, sequencing error rate, etc.;

s53: data QC: the quality control comprises sequencing and reads comparison, and parameters such as target rate, pollution factor, average sequencing depth, median sequencing depth and the like are presented in the results to evaluate whether sequencing data are enough for mutation recognition, and SNV loci with the sequencing depth of less than 20000 multiplied by are filtered, wherein the depth is preferably more than 100000 multiplied by;

5. The method of claim 1, wherein the tumor is a urinary system-related tumor.

6. The method of claim 5, wherein the tumor is renal cancer, urothelial cancer, and prostate cancer.

7. The method of claim 6, wherein the urothelial cancer is ureteral cancer, bladder cancer, and urethra cancer.

8. A method of monitoring urinary system cancer recurrence based on urine ctDNA comprising the steps of:

treating a tumor tissue sample and a control blood sample, and extracting gDNA from the tumor tissue sample and the control blood sample;

s3: treating a urine sample, and extracting cfDNA from the urine sample;

S5: performing raw message analysis, combining the detection results of the personalized monitoring sites to obtain the MRD detection results of the samples, wherein if the number of positive sites of the MRD is greater than or equal to the recommended value of the MRD positive threshold value, the MRD is positive, otherwise, the MRD is negative; if the MRD is positive, the patient is prompted to have a high risk of cancer recurrence or disease progression.

9. The method of claim 8, wherein the personalized monitoring sites in S2 are SNV and Indel sites, and driver gene sites.

10. The method of claim 9, wherein the rules for selecting personalized monitoring sites in S2 are as follows:

11. The method of claim 8, wherein the S5 comprises the steps of:

s53: data QC: the method comprises quality control of sequencing and reads comparison, wherein parameters such as target rate, pollution factor, average sequencing depth, median sequencing depth and the like are presented in the results to evaluate whether sequencing data are enough for mutation recognition, and SNV loci with the sequencing depth of less than 20000 multiplied by are filtered, and the depth is preferably more than 100000 multiplied by;

12. The method of claim 8, wherein the urinary system cancer is renal cancer, urothelial cancer, and prostate cancer.

13. The method of claim 12, wherein the urothelial cancer is ureteral cancer, bladder cancer, and urethra cancer.

14. A system for monitoring urinary system Micro Residual Disease (MRD) levels based on urine ctDNA, comprising the following modules:

B) And the personalized monitoring module: respectively constructing a tumor tissue DNA library and a blood cell DNA library, performing Whole Exome Sequencing (WES) hybrid capture to obtain a captured DNA library, sequencing the captured DNA library, obtaining personalized monitoring site combination data based on WES results, and preparing a primer pool aiming at the personalized monitoring sites;

15. The system of claim 14, wherein the personalized monitoring sites in B4) are SNV and Indel sites, and driver gene sites.

16. The system of claim 15, wherein the rules for selecting personalized monitoring sites in B4) are as follows:

17. The system of claim 14, wherein the D) analysis module comprises the steps of:

d1 Reads pretreatment: the method comprises the steps of converting from a bcl2 file to a fastq file, removing a connector sequence, extracting and storing parameters related to sequencing quality, such as insert size, sequencing error rate and the like;

d2 Alignment of Reads): the method comprises the steps of comparing reads and calibrating related indels, counting and recording the distribution and targeted sequencing areas of the reads, and reserving the obtained quality parameters in a bam file for subsequent mutation identification;

d3 Data QC: the method comprises quality control of sequencing and reads comparison, wherein parameters such as target rate, pollution factor, average sequencing depth, median sequencing depth and the like are presented in the results to evaluate whether sequencing data are enough for mutation recognition, and SNV loci with the sequencing depth of less than 20000 multiplied by are filtered, and the depth is preferably more than 100000 multiplied by;

18. The system of claim 14, wherein the tumor is a urinary system-related tumor.

19. The system of claim 18, wherein the tumor is renal cancer, urothelial cancer, prostate cancer.

20. Use of the system according to any one of claims 14-19 for the recurrence monitoring, neoadjuvant, adjuvant therapy efficacy, medication cues, disease progression cues of urinary system cancer.

21. The use of claim 20, wherein the cancer is renal cancer, urothelial cancer, and prostate cancer.

22. The use of claim 21, wherein the urothelial cancer is ureteral cancer, bladder cancer, and urethra cancer.