JP2024093463A - Methods for analyzing bladder cancer-associated mutant dna, and primers and probes for digital pcr therefor - Google Patents

Abstract

To provide less invasive and highly sensitive biomarkers to increase the opportunity of recurrence diagnosis in bladder cancer treatment.SOLUTION: Provided is an assay method for gene mutation(s) associated with bladder cancer in a subject, the method comprising the steps of: preparing a probe and/or primers for detecting mutation; and analyzing nucleic acid in a urine sediment obtained from the subject by digital PCR using the prepared probe and/or primers.SELECTED DRAWING: Figure 1

Description

Application for application of Article 30, Paragraph 2 of the Patent Act submitted. August 5, 2022: Public presentation at the 2nd Health Tech Device Forum Student Research Results Presentation Contest. [Publications, etc.] September 21, 2022: Public presentation via https://conference-apps-online.net/web/jca2022/ October 1, 2022: Public presentation at the 81st Annual Meeting of the Japanese Cancer Association.

The present invention relates to a method for analyzing mutant DNA associated with bladder cancer, and to digital PCR primers and probes for the same.

For non-metastatic bladder cancer, transurethral resection of the bladder tumor (TUR-BT) is performed as initial treatment, but in high-risk cancers (T1, high grade, carcinoma in situ (CIS), recurrent tumors, etc.), recurrence occurs in the bladder in approximately 75% of cases, most of which occur within two years after treatment.

As a follow-up method after bladder cancer treatment, cystoscopy 3 months after surgery, and then cystoscopy and urine cytology at intervals according to risk classification are recommended (Bladder Cancer Clinical Practice Guidelines 2019). However, although urine cytology has high specificity, its sensitivity is low, and for low-risk lesions, its sensitivity is only about 20% (Non-Patent Document 1). It is said that there is a 10-20% chance of missing a lesion with cystoscopy, and there are various issues, such as the fact that it is difficult to distinguish high-risk lesions such as CIS (carcinoma in situ) from inflammatory findings, that it is difficult to detect upper urinary tract recurrence and overlapping cancers with cystoscopy, and that evaluation varies depending on the subjective opinion of the surgeon. The usefulness of NMP22 (Nuclear Matrix Protein) and BTA (Bladder tumor antigen) has also been reported, but it has been reported that each has high sensitivity but low specificity. In addition, Urovision (fluorescence in situ hybridization (FISH) for bladder cancer) is known to be useful as an auxiliary test for diagnosing recurrence, but it is limited to twice every two years under insurance (Non-Patent Document 2). Prospective studies and expert opinions have also concluded that there are currently no sufficient urinary molecular markers to replace cystoscopy and urinary cytology (Bladder Cancer Clinical Practice Guidelines 2019).

In recent years, it has become known that circulating tumor DNA (ctDNA) can be detected in the blood of cancer patients. In gastrointestinal cancer, ctDNA was detected an average of five months earlier than recurrence could be diagnosed by imaging, demonstrating its validity as a biomarker for early recurrence diagnosis (Non-Patent Document 3).

Even if the specific mutations to be detected are identified through tumor sequence analysis, there are almost no commercially available dPCR probes, and it takes approximately one month to design, synthesize, and validate primers and probes corresponding to each mutation. To address this time lag, dPCR probe libraries have been created by selecting mutations that are common to a variety of cancers and are frequently detected based on publicly available databases such as COSMIC (Catalogue Of Somatic Mutations In Cancer, https://cancer.sanger.ac.uk/cosmic) (Patent Document 1).

Patent No. 6544783 (JP Patent Publication No. 2020-019738, International Publication WO2020/026776)

Bolenz, C., et al., Urinary cytology for the detection of urothelial carcinoma of the bladder--a flawed adjunct to cystoscopy? Urol Oncol, 2013. 31(3): p. 366-71. Sarosdy, M.F., et al., Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer. J Urol, 2002. 168(5): p. 1950-4. Iwaya, T., et al., Frequent Tumor Burden Monitoring of Esophageal Squamous Cell Carcinoma With Circulating Tumor DNA Using Individually Designed Digital Polymerase Chain Reaction. Gastroenterology, 2021. 160(1): p. 463-465.e4. Hayashi, Y. and K. Fujita, A new era in the detection of urothelial carcinoma by sequencing cell-free DNA. Transl Androl Urol, 2019. 8(Suppl 5): p. S497-S501. Ward, D.G., et al., Targeted deep sequencing of urothelial bladder cancers and associated urinary DNA: a 23-gene panel with utility for non-invasive diagnosis and risk stratification. BJU Int, 2019. 124(3): p. 532-544. Markus, H., et al., Analysis of recurrently protected genomic regions in cell-free DNA found in urine. Sci Transl Med, 2021. 13(581). Umansky, S.R. and L.D. Tomei, Transrenal DNA testing: progress and perspectives. Expert Rev Mol Diagn, 2006. 6(2): p. 153-63.

One of the challenges in treating bladder cancer is the lack of biomarkers for diagnosing recurrence. To increase the chances of diagnosing recurrence, there is an urgent need to develop minimally invasive, highly sensitive biomarkers.

In bladder cancer, ctDNA can be detected in plasma in cases of bladder cancer that show muscle invasion or metastasis, but in the case of localized bladder tumors, especially non-muscle invasive bladder cancer, it is known that gene mutations can be detected only from "urinary DNA" (Non-Patent Document 4). Regarding fractionation of urinary DNA, it has been reported that the diagnostic sensitivity of detecting mutant DNA for bladder cancer is roughly equivalent in urinary supernatant and urinary sediment (Non-Patent Document 5). On the other hand, urinary supernatant DNA contains not only DNA derived from the urothelium, including the bladder, but also double-stranded DNA fragments of 130 bp or less derived from plasma that are filtered by the renal glomerulus (Non-Patent Documents 6 and 7). Therefore, unless DNA derived from urinary supernatant and sediment is distinguished, it is difficult to determine whether the detected mutation is derived from the urinary tract or blood, and if the information is specific to the urothelium, a test method specialized in urinary sediment-derived DNA is expected to be superior.

The inventors then used the dPCR method to monitor the amount of DNA in urinary sediment in line with the progress of treatment for gene mutations detected in the patient's bladder tumor tissue with high sensitivity, and discovered that this provided a variety of information and enabled highly sensitive diagnosis of bladder cancer recurrence, thus completing the present invention.

The present application provides the following:
[1] A method for analyzing a genetic mutation in a subject, comprising:
the mutation is associated with bladder cancer,
Providing probes and/or primers for detection of mutations;
The method comprises a step of analyzing nucleic acids in urinary sediment obtained from a subject by digital PCR using the prepared probe and/or primer.
[2] The method according to 1, wherein the mutation is any one selected from the following mutations in genes or promoter regions:
[3] The method according to 1, wherein the mutation is any one selected from the following mutations in genes or promoter regions:
[4] The method according to 1, performed in combination with analysis of ctDNA in blood.
[5] The method according to 1, for analyzing changes in variant allele frequency (VAF) in urinary sediment.
[6] The method according to any one of 1 to 5, which is periodically repeated.
[7] The method according to any one of 1 to 5, which is carried out for early detection of recurrence of bladder cancer.
[8] The method according to any one of 1 to 5, which is performed as a follow-up examination after surgery for bladder cancer.
[9] A polynucleotide which is a primer or probe for digital PCR and which consists of any one of SEQ ID NOs: 1 to 134, for use in the method according to any one of 1 to 5.
[10] The polynucleotide according to 9, wherein the primer or probe for digital PCR is a primer or probe consisting of any one of the following sequences (wherein lowercase a is 2-amino-deoxyadenine and lowercase c is 5-methyl-deoxycytosine):
[11] The polynucleotide according to 9, wherein the primer or probe for digital PCR is a primer or probe consisting of any one of the following sequences (wherein lowercase a is 2-amino-deoxyadenine and lowercase c is 5-methyl-deoxycytosine):
[12] The method according to claim 1,
A method in which the step of preparing probes and/or primers is a library composed of a plurality of probes and/or primers, the plurality of probes and/or primers including any of the polynucleotides described in 10.
[13] Use of DNA contained in urinary sediment as a biomarker for bladder cancer.

The method of the present invention can be used as a postoperative monitoring test for bladder cancer to complement cystoscopy, which is highly invasive and has low sensitivity.

The method of the present invention is expected to provide clinically significant recurrence diagnosis, such as improving prognosis through early therapeutic intervention based on early prediction of recurrence, and reducing the burden on patients by omitting testing for cases with a low risk of recurrence. Furthermore, it may become possible to use biomarkers for this purpose in daily medical care.

Gene mutation monitoring in non-recurrence cases. The photo shows cystoscopic findings, the inverted triangle shows the urine cytology results, and the bottom shows the VAF transitions in plasma and urinary sediment. The results of the urine cytology are shown in white for negative results, light gray for false positive results, and dark gray for positive results. Gene mutation monitoring in recurrent cases. The photo shows cystoscopic findings, the inverted triangle shows the urine cytology results, and the bottom shows the VAF transitions in plasma and urinary sediment. The results of the urine cytology are shown in white for negative results, light gray for false positive results, and dark gray for positive results.

[Method of analyzing gene mutations]
The present invention provides a method for analyzing genetic mutations in a subject, comprising the steps of:
- A step of preparing a probe and/or primer for detecting a mutation. - A step of analyzing nucleic acids in a urine sediment obtained from a subject by digital PCR using the prepared probe and/or primer.

Analysis includes detection, quantification, etc. Subjects include cancer patients, people who have had cancer in the past, people at risk of cancer recurrence, people suspected of having cancer, etc. Analysis methods include methods that assist doctors in their actions, such as cancer-related diagnoses and quantitative evaluation of disease conditions. Assisting methods refer to methods performed by people other than doctors, such as clinical laboratory technicians, nurses, public health nurses, the subjects themselves, etc.

In the method of the present invention, the gene mutation is associated with bladder cancer. Bladder cancer is a general term for cancer that occurs in the bladder. Bladder cancer includes urothelial carcinoma, which occurs in the urothelium that covers the inside of the bladder, and also includes squamous cell carcinoma, adenocarcinoma, small cell carcinoma, and the like. The method of the present invention is particularly suitable for application to urothelial carcinoma, a type of bladder cancer. Urothelial carcinoma is classified into non-muscle invasive carcinoma and muscle invasive carcinoma depending on the depth of invasion of the cancer. The method of the present invention can be applied to either type.

One of the features of the method of the present invention is that nucleic acid (usually DNA) that serves as a template for digital PCR is obtained from the urinary sediment of a subject. Urinary sediment refers to the solid fraction obtained when urine is subjected to a solid-liquid separation operation. The liquid fraction is called the supernatant. In general, a test for urinary sediment is performed by centrifuging urine and examining the amount and type of solid components that have precipitated, such as red blood cells, white blood cells, uric acid crystals, cells, and bacteria, and methods for obtaining urinary sediment are well known to those skilled in the art.

Regarding urine fractionation to obtain urinary DNA, it is thought that the information expected will differ depending on whether the urine supernatant, urinary sediment, or both are included. The urine supernatant has the advantage of being less affected by proteins in the urine, but it contains a small amount of DNA and may contain plasma-circulating DNA (which may contain not only DNA derived from the urothelium, including the bladder, but also double-stranded DNA fragments of 130 bp or less derived from plasma filtered by the renal glomerulus), making it difficult to distinguish whether the DNA is derived from urine or blood. In contrast, a sufficient amount of DNA is obtained from urinary sediment, and it does not contain plasma-circulating DNA. Therefore, if the information is specific to the urothelium, a test method specialized in DNA derived from urinary sediment is expected to be superior. Note that there is no prior art that distinguishes between supernatant and sediment in the analysis of urinary tract cancer.

In the method of the present invention, digital PCR (dPCR) is used. In cancer diagnosis, the VAF of the target DNA is often 1% or less, and it is desirable to perform a highly sensitive analysis. dPCR (Vogelstein and Kinzler, Proc. Natl. Acad. USA, 96, 9236-9241, 1999) is an extension of conventional PCR and allows the amount of target nucleic acid to be directly quantified. dPCR is suitable for use in combination with an enrichment procedure to detect extremely low levels of mutations. In dPCR, the DNA sample is diluted to a single reaction unit, so that the starting material in each PCR reaction is either wild type or mutant. Enrichment is performed from a single molecule, and thousands of parallel enrichment reactions are performed simultaneously. After performing multiple PCR reactions from the same starting material, mutant molecules are isolated and detected. After PCR amplification, the absolute amount of nucleic acid can be calculated by counting the chambers containing the final PCR product. Various dPCR-based systems that can be used in the present invention are commercially available. The basic methodology of dPCR is described, for example, in Sykes et al., Biotechniques 13 (3): 444-449, 1992.

(Preferred embodiment)
The methods of the present invention can be performed in combination with analysis of ctDNA in blood.

The method of the present invention can be repeatedly performed on a subject on a regular basis. Regularly means, for example, every few months, every six months, or once a year. The period of regular continuation is not particularly limited, and can be, for example, one year, three years, five years, or ten years.

The method of the present invention is particularly suitable for early detection of recurrence of bladder cancer. In addition, the method of the present invention is particularly suitable for use as a follow-up test after surgery for bladder cancer. When subjects with a small amount of tumor cells, such as Stage I subjects, are used as subjects, tumor-derived DNA may not be detected in samples obtained from the subjects before treatment, after surgery, or during the follow-up period. For subjects with a certain amount of tumor cells, such as Stage II subjects, it can be confirmed that the tumor-derived DNA detected before treatment decreases to 0% after surgery. In addition, recurrence can be predicted by analyzing the presence or absence or amount of tumor-derived DNA in samples obtained from the subjects during follow-up, including postoperative adjuvant chemotherapy. With the method of the present invention, depending on the subject, a decrease in tumor-derived DNA associated with tumor shrinkage due to chemotherapy and radiation therapy can be confirmed. With the method of the present invention, recurrence may be diagnosed earlier than with conventional CT diagnosis.

By using the method of the present invention for postoperative monitoring tests for bladder cancer, it can complement conventional cystoscopy, which is highly invasive and has low sensitivity. In the future, application of this technology may enable the use of clinically significant recurrence diagnostic biomarkers in daily medical care, such as improving prognosis through early therapeutic intervention based on early prediction of recurrence, and reducing the burden on patients by omitting tests for cases with a low risk of recurrence.

(mutation)
The genetic mutation detected by the present invention is not particularly limited as long as it is related to bladder cancer, and is any one selected from the following mutations in the genes or promoter regions.

Preferably, the mutation is any one selected from the following mutations in genes or promoter regions. According to studies by the present inventors, it has been found that these mutations can cover multiple cases.

In the explanation of this invention, the term "mutation" refers to a mutation of a base (sometimes referred to as a "nucleotide"), unless otherwise specified. A mutation of a base includes replacement with another base, deletion (del), duplication (dup), etc.

In the present invention, the standard rules known to those skilled in the art are followed when describing gene mutations (https://varnomen.hgvs.org/recommendations/DNA/variant/duplication/, Hum Mutat 2016; 37: 564-569). In addition, in the present invention, the positions of mutations are based on the information published in the COSMIC database and the information published in the sequence listing, unless otherwise specified.

(Probes and/or primers)
The present invention provides a probe and/or primer for digital PDR to detect mutations associated with bladder cancer, suitable for use in the above-mentioned method. In the present invention, the term "probe and/or primer" refers to at least one of the probe and the primer, unless otherwise specified. In other words, the term "probe and/or primer" includes the case of only the probe, the case of only the primer, and the case of both the probe and the primer.

The probe may be for detecting a sequence that does not have the desired mutation (wild type), or for detecting a sequence that has the desired mutation (mutant). The device may be provided with both types of probes.

Primers are used to amplify the portion of the nucleic acid in the sample that contains the site of the target mutation, and are usually used in pairs: a forward primer that corresponds to the positive strand and a reverse primer that corresponds to the negative strand.

From the viewpoint of analyzing DNA in urinary sediments, it is preferable that the probe and/or primer are designed to amplify a relatively short nucleic acid fragment as a target sequence and detect the target mutation. The size of the amplified product in normal PCR is 100-150 bp, and the decrease in sensitivity and accuracy may be a problem. In order to improve analytical performance, it is considered important to shorten the size of the amplified product as much as possible (Antonov J,et.al. Lab Invest. 2005 Aug;85(8):1040-50, Kong H,et.al. Sci Rep. 2014 Nov 28;4:7246, Florent Mouliere et.al. PLOS ONE September 2011 Volume 6 Issue 9 e23418).

For example, a method has been developed that allows analysis using relatively short amplification products, for example, amplification products of 70 bp or less, by means of including modified nucleotides in the oligonucleotides of primers and probes. Primers and probes for such methods are known as Hypercool Primer & Probe ^TM (Nihon Gene Research Institute Co., Ltd., 2-5-2 Nakano 1-chome, Miyagino-ku, Sendai-shi), and a design method therefor has been published (http://ngrl.co.jp/wp/wp-content/uploads/2015/01/eb3607d6c1eb4174f10bb3bde67844fe.pdf). For detection of a low proportion of mutant sequences in the presence of a large excess of non-mutated sequences, see, for example, JP2010-535031A. This technology is based on a protocol in which the reaction mixture is treated at a temperature lower than the critical denaturation temperature or the melting temperature Tm of the non-mutated sequences.

In a preferred embodiment, the probe and/or primer contain modified bases (sometimes referred to as modified nucleotides). Modified bases include diamino-purine analogs (e.g., 2'-O-methyl-2,6-diaminopurine), uracil, peptide nucleic acid analogs, biotin-modified analogs, fluorophore-modified analogs, inosine, 7-deazaguanine, 2'-deoxy-2'-fluoro-β-D-arabinonucleic acid (2'F-ANA) nucleotides, locked nucleic acids (LNAs), ENAs: 2'-O,4'-C-ethylene bridged nucleic acids, and the like. These modifications can increase the difference in Tm between matched and mismatched bases, allowing for highly accurate analysis.

Particularly preferred examples of modified bases are 2-amino-deoxyadenine (2aA) and 5-methyl-deoxycytidine (5mC).

The probe and/or primer may also contain mismatched or unmatched bases. One of skill in the art of nucleic acid technology can determine duplex stability by considering a number of variables, such as the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, the ionic strength, and the percentage of mismatched bases. The stability of a nucleic acid duplex can be expressed by the melting temperature (sometimes called the denaturation temperature), Tm.

Introducing locked nucleic acids into probes and/or primers improves the affinity for complementary sequences and increases the melting temperature by several steps (Braasch, D.A. and D.R. Corey, Chem. Biol. (2001), 8:1-7). Locked nucleic acids refer to a class of conformationally restricted nucleotide analogs (see, e.g., WO99/14226; Koshkin, A.A., et al., Tetrahedron (1998), 54: 3607-3630; and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404).

The probe may be modified near its terminus to facilitate analysis, and the modification may include a fluorescent substance. Examples of fluorescent substances include 6FAM (sometimes simply referred to as FAM), HEX, Tide Fluor ^™ 1, ATTO 390, ATTO 425, LC (registered trademark) 480Cyan500, ATTO 465, ATTO 488, Tide Fluor ^™ 2, ATTO 495, ATTO 514, ATTO 520, TET, JOE, CAL Fluor 540, Yakima Yellow, ATTO 532, ATTO Rho6G, LC (registered trademark) Yellow555, CAL Fluor 560, ATTO 542, Quasar (registered trademark) 570, Cy3, ATTO 550, TAMRA, Tide Fluor ^™ 3, ATTO 565, ATTO Rho3B, ROX, ATTO Rho11, ATTO Rho12, Cy3.5, ATTO Thio12, ATTO Rho101, LC® Red610, CAL Fluor 610, Tide Fluor ^™ 4, ATTO 590, TexasRed(SR101), ATTO 594, ATTO Rho13, ATTO 610, LC® Red670, ATTO 620, LC® Red640, ATTO Rho14, ATTO 633, ATTO 647, ATTO 647N, Quasar® 670, Tide Fluor ^™ 5, Cy5, ATTO 655, ATTO Oxa12, ATTO 665, Tide Fluor ^™ 6, Cy5.5, ATTO 680, LC® Red705, Quasar® 705, ATTO 700, ATTO 725, ATTO 740, Tide Fluor ^™ 7, Tide Fluor An example of such a probe is ^TM 8. By using different fluorescent substances for the wild type probe and the mutant probe, it becomes possible to simultaneously analyze each probe.

Probes may also be modified near their termini with quenchers, such as Dabcyl, BHQ-0®, BHQ-1®, BHQ-2®, BHQ-3®, TQ ^™ 1, TQ ^™ 2, TQ ^™ 3, TQ ^™ 4, TQ™ 5, TQ ^™ 6, ^{TQ™ 7} , TAMRA, ATTO540Q, ATTO 575Q, ATTO 580Q, ATTO 612Q, and BBQ-650®.

Examples of probes and/or primers provided by the present invention are shown in the table below. In the table, a lowercase a represents a modified adenine, a lowercase c represents a modified cytosine, and an example of a modified adenine is 2-amino-deoxyadenine, and an example of a modified cytosine is 5-methyl-deoxycytosine (as in other sequences shown in relation to the present invention).

Preferably, the probe and/or primer is any of the probes and/or primers having the following sequences. According to the studies of the present inventors, it has been found that these probes and/or primers can cover multiple cases.

(Implementation of dPCR)
When the above-mentioned primers and probes are used for dPCR, the amounts of the primers and probes can be appropriately determined by those skilled in the art. For example, the nucleic acid can be used in the range of 0.5 to 50 ng, the primers in the range of 0.02 to 2.0 μM, and the probe in the range of 0.01 to 1.0 μM per dPCR chip. When the probes and/or primers for detecting each mutation are configured to be stored in individual containers, for example, tubes, the typical concentrations in each tube are 450 to 3600 nM for the primers, preferably 500 to 2000 nM, more preferably 600 to 1200 nM, for example 900 nM, and 10 to 1000 nM for the probes, preferably 50 to 500 nM, more preferably 100 to 300 nM, for example 250 nM. In addition, when referring to the concentration of the primers or probes in the present invention, it is stated as the concentration of one type of primer and one type of probe, unless otherwise specified.

In dPCR, the system composition may include the amounts of nucleic acid, primers, and probes, as well as four types of dNTPs, a buffer, salts, surfactants, and an enzyme that catalyzes the chain extension reaction, as will be apparent to those skilled in the art.

According to the inventors' study, the primers designed based on the present invention, embodiments, and examples are preferably used in dPCR at a final concentration of 450 to 3600 nM. For many primers, an example of a suitable concentration is 900 nM. However, in some cases, the PCR reaction does not proceed efficiently at that concentration. In such cases, the PCR reaction can be allowed to proceed appropriately by using primers at a higher concentration. In such cases, specifically, the primers are used at 1000 to 3600 nM, preferably 1200 to 3000 nM, more preferably 1500 to 2100 nM, for example 1800 nM.

In addition, in dPCR, when a region with a high GC content is targeted, the PCR reaction may not proceed smoothly. For example, the TERT promoter region has a high GC content, and amplification by primers may be difficult as is. In such cases, the annealing temperature in dPCR can be adjusted to the same temperature as in normal cases by adding 7-deaza-2'-deoxy-guanosine-5'-triphosphate (7-deaza dGTP) or dimethyl sulfoxide (DMSO) to the system. Specifically, when using 7-deaza dGTP, it is recommended to add it to the dPCR system at 20 to 500 μM, preferably 50 to 200 μM, more preferably 75 to 150 μM, for example 100 μM. Alternatively, when using DMSO, it is recommended to add it to the dPCR system at 0.15 to 3.8%, preferably 0.30 to 2.4%, more preferably 0.50 to 1.0%, for example 0.75%.

According to the inventors' investigations, in dPCR using primer and probe sets designed based on the present invention, embodiments, and examples, annealing is performed at 55 to 61°C. More specifically, annealing can be performed at the same temperature, for example 59 to 61°C, for 95% or more of the sets contained in the library.

Usually, in dPCR using sets of primers and probes with different amplification targets, the optimal conditions are often different. If the annealing temperature differs depending on the set, the PCR reaction will be divided into multiple runs unless a PCR device capable of simultaneously setting multiple conditions is used, which requires time and effort. However, according to the present invention, the embodiments, and the examples, by adjusting the primer concentration and adding reagents, the annealing temperature when using each set can be set to the same temperature in many cases (for example, 60°C). In exceptional sets, it may be preferable to set the annealing temperature to 58°C, 56°C, etc.
[Library]
The probes and/or primers used in the present invention may be a library. A library is a collection of probes and/or primers. A typical embodiment of the library is a collection of probes and/or primers in containers, in which the probes and/or primers for detecting each mutation are each stored in an individual container.

Although the library contains multiple oligonucleotides for detecting mutations at multiple sites, it can be used in various ways. Typically, it is used to analyze a target mutation (at one site) in a single subject. For example, it may be used to analyze whether a mutation detected in a patient's primary tumor before treatment is detected in the blood during the course of treatment, or to analyze whether the mutation is detected in the blood after treatment in order to diagnose the presence or absence of recurrence. The library may also be used to analyze multiple mutations at multiple sites in a single subject. The library may also be used to analyze a target mutation (at one site) in multiple subjects, or to analyze multiple mutations at multiple sites in each of multiple subjects.

The library may consist only of oligonucleotides capable of functioning as primers, may consist only of oligonucleotides capable of functioning as probes, or may contain both. Mutations are usually detected by amplifying a portion of nucleic acid that may contain the target mutation using a primer by polymerase chain reaction (PCR), and then detecting the presence of the target mutation in the amplified product using a probe. For this reason, it is preferable that the library contains a set of a probe and a primer pair for the target mutation. In PCR, the amplified product refers to the DNA amplified by PCR, and is sometimes called an amplicon.

The library is for detecting mutations associated with cancer, and in one embodiment, it can be capable of non-intermittently detecting mutations that may occur in a target gene. For example, for TP53, a library capable of non-intermittently detecting mutations in the coding region for 195 amino acids in which cancer-associated mutations are frequently observed specifically includes 1,755 primer-probe sets (see Patent Documents 1 and 2).

In another embodiment, the library includes primer-probe sets for mutations that may occur in a target gene or its promoter region and that occur with high frequency. According to the inventors' studies, the frequency of occurrence (reporting) varies greatly depending on the mutation, so by targeting frequent mutations in the library, more comprehensive detection of mutations becomes possible.

Therefore, one particularly preferred library is one designed to detect one or more types, for example, two or more types, preferably four or more types, more preferably six or more types, even more preferably ten or more types, and even more preferably twenty or more types, in order of frequency (in order of frequency).

More specifically, in one embodiment, the library is composed of multiple probes and/or primers for detecting cancer-associated mutations in the genes or their promoter regions shown in the table below.

Preferably, the library is composed of multiple probes and/or primers for detecting cancer-associated mutations in the genes or their promoter regions shown in the table below.

In one embodiment, the library contains at least one primer or probe having any one of the following sequences (wherein lowercase a is 2-amino-deoxyadenine and lowercase c is 5-methyl-deoxycytosine):

Preferably, the primers or probes contained in the library are primers or probes consisting of any one of the following sequences (wherein lower case a is 2-amino-deoxyadenine and lower case c is 5-methyl-deoxycytosine):

The library eliminates the need to design and synthesize primers and probes for each subject when analyzing mutations. By using a library containing highly versatile primers and probes, or primers and probes designed to detect mutations non-intermittently, recurrence of cancer after treatment can be diagnosed early. In addition, individualized follow-up observation after treatment can be easily performed for cancer patients.

In a preferred embodiment, the method of the present invention can be carried out as follows:
From the library of the present invention prepared in advance, a set deemed appropriate (e.g., a set of primers and probes for analyzing case-specific mutations detected by mutation analysis of the primary lesion) is obtained, and if necessary, its operation is confirmed, after which the mutant allele frequency (%) of mutant DNA in samples appropriately collected from the subject is monitored.

The following examples are provided for further explanation, but the present invention is not limited to these examples.

[Method]
1. Gene mutation analysis of tumor tissues Transurethral bladder tumor resection is performed for bladder cancer, and tumor tissue DNA is extracted from the formalin-fixed (FFPE) specimen of the tumor tissue resection obtained during the procedure. Comprehensive gene mutation analysis is performed on the tumor tissue DNA by targeted sequencing, and for hot spot mutations (C228T, C250T) in the TERT promoter region, which are detected in approximately 70% of bladder cancer cases, mutation analysis is performed on all cases by digital PCR (dPCR). This is because the TERT promoter region is not included in the analysis region depending on the type of sequencing panel.

2. Selection of gene mutations to be tracked From the gene mutation analysis data for each tumor obtained by these two types of gene analysis, the mutations to be monitored for gene mutations in bodily fluids are selected. Selection criteria, referring to the database, include whether the gene is a cancer-related gene, whether it is not polymorphic, and whether it is a mutation that has been frequently reported in the COSMIC (Catalogue Of Somatic Mutations In Cancer, https://cancer.sanger.ac.uk/cosmic) database. The dPCR primers and probes used for monitoring will be OTS probes owned by QuantDetect Co., Ltd., and if a gene mutation not included in the library is to be selected, the company will be requested to synthesize the primers and probes.

3. Time-course monitoring of gene mutations Urine samples will be collected before and after treatment and at regular examinations thereafter, and urinary sediment DNA will be extracted. For gene mutations selected as targets for tracking using the method described above, urinary sediment DNA will be analyzed by dPCR to monitor the dynamics of the variant allele frequency (VAF).

The sequences of the primers and probes used in dPCR are shown in the table below. Production was outsourced to Japan Gene Research Institute Co., Ltd. (1-5-28 Nakano, Miyagino-ku, Sendai, Miyagi Prefecture, Japan 983-0013).

In the table, the lowercase letters a and c in the primer and probe sequences represent the modified bases 2aA and 5mC used in the Hypercool primer and probe, respectively. The boxed areas are the mutation sites or the sites corresponding to the mutations.

4. Urinary Sediment DNA Extraction Procedure Urinary sediment DNA is extracted using the Quick-DNATM Urine Kit' (ZYMO RESEARCH, California, US). After collecting 40 ml of urine, immediately mix with 2.5 ml of urine conditioning buffer provided with the kit and store in a cool place. After storing in a cool place, centrifuge within 5 days and discard the supernatant, leaving the urinary sediment. Extract DNA using the kit and store at -20°C.

5. dPCR Analysis Procedure
dPCR analysis was performed using a QuantStudio 3D Digital PCR System (Thermo Fisher Scientific) and a QX200 (Biorad). DNA samples were used in amounts of 10-20 ng per analysis. The thermal cycler settings for the QuantStudio 3D Digital PCR System were (1) 96°C for 10 min, (2) 98°C for 30 s and 60°C for 1 min, repeated for 39 cycles, (3) 60°C for 2 min, and then stopped at 10°C. The thermal cycler settings for the QX200 were (1) 95°C for 10 min, (2) 94°C for 30 s and 60°C for 1 min, repeated for 39 cycles, (3) 98°C for 10 min, and then stopped at 4°C.

[result]
Figures 1 and 2 show the progression of VAF and clinical information data for two cases in which dPCR monitoring was actually performed. In cases in which TUR-Bt (transurethral resection of bladder tumor) was performed as an initial treatment for bladder cancer and thereafter there was no recurrence (Figure 1) and cases in which recurrence was detected (Figure 2), the VAF in plasma and urinary sediment showed different progressions. In cases without recurrence (Figure 1), the gene mutations detected in the urinary sediment before surgery decreased to below the detection sensitivity early after treatment, and the VAF then remained at low values of less than 1%. On the other hand, in cases in which recurrence was detected (Figure 2), gene mutations were continuously detected in the urinary sediment after treatment and remained at high values, and the mutant allele frequency remained at high values until the time when recurrence was actually detected.

[Discussion]
In preparation for this study, we attempted to collect DNA from both the supernatant and sediment of urine samples; however, the DNA yield in the supernatant was low, and we were concerned that it might also contain blood DNA filtered by the kidney.

If tumor cells in the bladder are released into the urine due to necrosis or mechanical stimulation, they are expected to be separated into the urinary sediment by centrifugation, and since sufficient DNA was obtained from the sediment in the preparation stage, it is considered that urinary sediment should be analyzed.

It is known that ctDNA is detected in plasma DNA when muscle invasion or metastasis occurs. Based on the above, DNA monitoring of urine sediments, or urine sediments and plasma, allows for highly sensitive diagnosis of bladder cancer recurrence.

[Other primers/probes]
The table below shows the genes, mutation information (nucleotide change, amino acid change) and synthesis (commissioned to Japan Gene Research Institute Co., Ltd. (1-5-28 Nakano, Miyagino-ku, Sendai, Miyagi Prefecture, Japan 983-0013) of the gene mutations used in monitoring of other target cases, as well as the sequence information of the primer/probe whose operation was confirmed. dPCR was similarly performed using these primers/probes, and their operation was confirmed. In the table, the lowercase letters a and c in the primer and probe sequences represent the modified bases 2aA and 5mC, respectively, used in the Hypercool method primers and probes. The boxed areas are the mutation sites or the sites corresponding to the mutations.

The following table shows the mutations that covered multiple cases among the synthesized primers/probes. These primers/probes are

[Sequences listed in the sequence listing]
SEQ ID NO:1 Forward primer, STAG2, c.646C>T
SEQ ID NO:2 Reverse primer, STAG2, c.646C>T
SEQ ID NO:3 Probe Wt, STAG2, c.646C>T
SEQ ID NO:4 Probe Mt, STAG2, c.646C>T
SEQ ID NO:5 Forward primer, TERT promoter 228C>T, Chr5:1295228 C>T on hg19, 124bp upstream of the translation start; Forward primer, TERT promoter 250C>T, Chr5:1295250 C>T on hg19, 146bp upstream of the translation start
SEQ ID NO:6 Reverse primer, TERT promoter 228C>T, Chr5:1295228 C>T on hg19, 124bp upstream of the translation start; Reverse primer, TERT promoter 250C>T, Chr5:1295250 C>T on hg19, 146bp upstream of the translation start
SEQ ID NO:7 Probe Wt, TERT promoter 228C>T, Chr5:1295228 C>T on hg19, 124bp upstream of the translation start
SEQ ID NO:8 Probe Mt, TERT promoter 228C>T, Chr5:1295228 C>T on hg19, 124bp upstream of the translation start
SEQ ID NO:9 Probe Wt, TERT promoter 250C>T, Chr5:1295250 C>T on hg19, 146bp upstream of the translation start
SEQ ID NO:10 Probe Mt, TERT promoter 250C>T, Chr5:1295250 C>T on hg19, 146bp upstream of the translation start
SEQ ID NOs:11-134 other primers and probes

Claims (13)

1. A method for analyzing genetic mutations in a subject, comprising:
the mutation is associated with bladder cancer,
Providing probes and/or primers for detection of mutations;
The method comprises a step of analyzing nucleic acids in urinary sediment obtained from a subject by digital PCR using the prepared probe and/or primer. The method according to claim 1 , wherein the mutation is any one selected from the following mutations in genes or promoter regions:
The method according to claim 1 , wherein the mutation is any one selected from the following mutations in the genes:
The method according to claim 1, which is performed in combination with analysis of ctDNA in blood. The method according to claim 1, wherein changes in variant allele frequency (VAF) in urine sediment are analyzed. The method according to any one of claims 1 to 5, which is periodically and repeatedly performed on a subject. The method according to any one of claims 1 to 5, which is performed for early detection of recurrence of bladder cancer. The method according to any one of claims 1 to 5, which is performed as a follow-up examination after surgery for bladder cancer. A polynucleotide that is a primer or probe for digital PCR and has any one of the sequences of SEQ ID NOs: 1 to 134 for use in the method according to any one of claims 1 to 5. The polynucleotide according to claim 9, wherein the primer or probe for digital PCR is a primer or probe consisting of any one of the following sequences (wherein lowercase a is 2-amino-deoxyadenine and lowercase c is 5-methyl-deoxycytosine):
The polynucleotide according to claim 9, wherein the primer or probe for digital PCR is a primer or probe consisting of any one of the following sequences (wherein lowercase a is 2-amino-deoxyadenine and lowercase c is 5-methyl-deoxycytosine):
2. The method of claim 1 ,
A method, wherein the step of preparing probes and/or primers is a library composed of a plurality of probes and/or primers, the plurality of probes and/or primers comprising any one of the polynucleotides described in claim 10. The use of DNA contained in urinary sediment as a biomarker for bladder cancer.