WO2024204599A1

WO2024204599A1 - Biomarker for bladder cancer

Info

Publication number: WO2024204599A1
Application number: PCT/JP2024/012784
Authority: WO
Inventors: 茂大塚原; 崇松元; 真己塩田; 正俊江藤; 東天康; 啓輔兒玉
Original assignee: デンカ株式会社; 国立大学法人九州大学
Priority date: 2023-03-29
Filing date: 2024-03-28
Publication date: 2024-10-03

Abstract

The present invention provides a biomarker for non-muscle invasive bladder cancer, wherein the biomarker is one or multiple mutations in a protein encoded by the ADGRG6 enhancer.

Description

Bladder Cancer Biomarkers

The present invention broadly relates to biomarkers for bladder cancer, etc.

In many cancers, including bladder cancer, point mutations occur in the promoter region of TERT (telomerase reverse transcriptase). In particular, the C to T substitution at position 1,295,228 of chromosome 5 and the C to T substitution at position 1,295,250 of chromosome 5 are known as so-called hotspot mutations commonly seen in multiple patients (Non-Patent Document 1).

In addition to the TERT promoter, hotspots are known in bladder cancer, such as ADGRG6, PLEKHS1, and WDR74, and it has been suggested that these could serve as biomarkers for bladder cancer (Non-Patent Document 2).

The present invention aims to provide new biomarkers for bladder cancer.

The inventors discovered that base changes in the ADGRG6 enhancer are found in non-muscle invasive bladder cancer, leading to the completion of the present invention.

That is, the present application includes the following inventions.
[1]
A biomarker for non-muscle invasive bladder cancer, comprising:
A biomarker that is one or more mutations in the ADGRG6 enhancer.
[2]
The biomarker according to [1], wherein the mutation is a single nucleotide polymorphism.
[3]
The biomarker according to [2], wherein the single nucleotide polymorphism is a mutation of guanine at position 142,706,206 and/or cytosine at position 142,706,209 of chromosome 6.
[4]
A biomarker according to any one of [1] to [3] for predicting the prognosis of non-muscle invasive bladder cancer.
[5]
The biomarker according to any one of [1] to [4], wherein the non-muscle invasive bladder cancer is high-grade non-muscle invasive bladder cancer.
[6]
The biomarker according to [4] or [5], wherein the prognosis is the length of recurrence-free survival or the level of risk of recurrence.
[7]
The biomarker according to any one of [4] to [6], which is a poor prognosis marker.
[8]
The biomarker according to any one of [1] to [7], wherein the mutation is present in circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), genomic DNA or complementary DNA (cDNA).
[9]
1. A method for detecting one or more mutations in the ADGRG6 enhancer in a sample from a subject having or suspected of having non-muscle invasive bladder cancer, comprising:
A method comprising detecting the presence of one or more mutations in the ADGRG6 enhancer in a sample obtained from the subject, compared to a wild-type ADGRG6 enhancer.
[10]
The method according to [9], wherein if a mutation is detected, the subject's prognosis is poor.
[11]
The method according to [9] or [10], wherein the nucleic acid amplification method used in the detection step is digital PCR.
[12]
The method according to any one of [9] to [11], wherein the sample is a liquid sample.
[13]
The method according to any one of [9] to [12], wherein the sample comprises circulating DNA (cfDNA).
[14]
1. A primer for detecting the presence of non-muscle invasive bladder cancer in a specimen, comprising:
A primer that is an oligonucleotide of at least 16 bases and no more than 21 bases in length that targets one or more mutations in the ADGRG6 enhancer.
[15]
A probe for detecting the presence of non-muscle invasive bladder cancer in a specimen, the probe targeting one or more mutations in the ADGRG6 enhancer.
[16]
A kit comprising the primer according to [14] and/or the probe according to [15].

Non-muscle invasive bladder cancer (NMIBC) is the most common type of untreated bladder cancer, and in particular high-grade non-muscle invasive bladder cancer, which has a high chance of recurrence, cases with poor prognosis require early detection of recurrence and metastasis, and prevention of further malignant progression. Therefore, mutations in the ADGRG6 enhancer can be a useful marker for non-muscle invasive bladder cancer.

Prognostic markers such as base changes in the ADGRG6 enhancer, when combined with conventional prognostic markers such as TERT promoter mutations, which have been shown to be useful as poor prognostic markers in bladder cancer, will enable multifaceted clinical follow-up.

Furthermore, the newly designed primers and probes for detecting base changes in the ADGRG6 enhancer can be combined with conventional methods for detecting TERT promoter mutations to cover a larger number of bladder cancer patients.

FIG. 1 shows the coverage rate (proportion of positive detection) of biomarkers indicating TERT promoter mutations and TERT promoter and ADGRG6 enhancer mutations (43 cases).

The following describes an embodiment of the present invention (hereinafter referred to as the "present embodiment"); however, the scope of the present invention is not to be interpreted as being limited to the following embodiment.

(Biomarkers)
In a first aspect, a biomarker for non-muscle invasive bladder cancer is provided, the biomarker being one or more mutations in a protein encoded by the ADGRG6 enhancer.

Bladder cancer is a general term for cancer that occurs in the bladder, and originates from the urothelial mucosa of the bladder. The majority of bladder cancers are urothelial carcinomas. Bladder cancer is characterized by the fact that even if it is discovered early and the lesion is resected, it often recurs, yet the prognosis is difficult to predict. Depending on the depth of invasion, bladder cancer is divided into non-muscle invasive bladder cancer (NMIBC), in which the cancer remains in the mucosa and submucosa, and muscle invasive bladder cancer (MIBC), in which the cancer has spread to the muscle layer or connective tissue. The diagnosis of bladder cancer is known to those skilled in the art, and can be made, for example, in accordance with the Bladder Cancer Clinical Practice Guidelines (Japan Urological Association). According to the Bladder Cancer Clinical Practice Guidelines, the presence or absence of muscle invasion is determined by transurethral resection of bladder tumor (TURBT) or radical cystectomy specimens.

Bladder cancer is graded according to its grade, with two stages, low grade and high grade, or three stages, G1 to G3. High grade or G3 is the most malignant. The timing of recurrence of high-grade non-muscle-invasive bladder cancer varies from patient to patient, but in the case of Japanese patients, the recurrence-free survival (RFS) for each year from 1 to 5 years is 70% at 1 year, 55% at 3 years, and more than half at 5 years.

As used herein, "ADGRG6 enhancer" refers to the enhancer region of the ADGRG6 gene, which corresponds to positions 142,705,538 to 142,707,537 on chromosome 6.

As used herein, "a change in one or more bases" refers to a state in which one or more bases in the ADGRG6 enhancer are changed to one or more different bases compared to a control. Single nucleotide polymorphism changes such as SNPs and SNVs are preferred. For example, if the bases at positions 142,706,206 and/or 142,706,209 of chromosome 6 (the former is guanine and the latter is cytosine in the wild type), which are located in the enhancer region of the ADGRG6 gene, are replaced with other bases, the bases in the ADGRG6 enhancer are determined to be changed. Such a determination can be made by directly measuring the change in the bases in the ADGRG6 enhancer, or through the change in the corresponding bases in the antisense strand, etc. For example, such changes can be easily confirmed using a means for detecting circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), genomic DNA, or complementary DNA (cDNA). The nucleic acid for which the change is confirmed is not limited to DNA, but may be RNA such as mRNA or circulating tumor RNA. The base change may be evaluated as the amount of change in allele frequency. For example, if the mutant allele frequency increases over time, it may be evaluated as a sign of recurrence.

A control having a base before the change, which is used as a reference when determining the change in one or more bases of the ADGRG6 enhancer, may be a subject treated for bladder cancer or a healthy individual having a wild type for the corresponding base. The change in the base can be confirmed by repeatedly sequencing the ADGRG6 enhancer over time. Such sequencing can be performed at the timing of follow-up. The timing of follow-up is not particularly limited, and may be performed within several months to one year after treatment, for example, within 3 months to 10 months after treatment. The frequency is also expected to be several months, for example, about once every 3 months. Follow-up of bladder cancer is usually performed once every 3 months, but since non-muscle invasive bladder cancer is a type of cancer with a relatively good prognosis, if there is no recurrence for 2 years, the frequency may be once every 6 months.

　Follow-up methods vary depending on the type of bladder cancer. For example, in the case of non-muscle invasive bladder cancer (NMIBC), urinary cytology and cystoscopy are generally performed during follow-up. Because NMIBC is non-invasive and has a clinically low risk of metastasis to other organs, imaging diagnostics (CT) are often not performed. In the case of muscle invasive bladder cancer (MIBC), the bladder is completely removed during treatment, so cystoscopy is not indicated for follow-up. Instead, urinary cytology and imaging diagnostics using CT are performed during follow-up of MIBC.

In one embodiment, the ADGRG6 enhancer is sequenced over time in a subject after bladder cancer surgery to determine base changes compared to previous times. The base changes may be evaluated as the amount of change in allele frequency. For example, a patient who shows a relatively increased mutant allele frequency from the beginning of monitoring may be evaluated as having an indication of recurrence.

In one embodiment, the one or more base changes in the ADGRG6 enhancer are a guanine change at position 142,706,206 on chromosome 6 and/or a cytosine change at position 142,706,209 on chromosome 6.

A change in one or more bases in the ADGRG6 enhancer serves as an indicator (biomarker) for predicting the prognosis of bladder cancer. As used herein, "prognosis" refers to the medical outlook for the progress of a patient after some treatment for bladder cancer, such as transurethral bladder tumor resection, intravesical infusion therapy in which a drug is injected into the bladder, radical cystectomy, drug therapy, etc., and the patient's life expectancy. The treatment method is appropriately determined depending on the degree of progression of the cancer. Evaluation items related to the prognosis of bladder cancer include the presence or absence of recurrence, recurrence-free survival, survival rate, and survival time. These evaluation items include other primary and secondary evaluation items, such as disease-free survival (DFS), non-urothelial recurrence-free survival (NUTRFS), distant metastasis-free survival (DMFS), progression-free survival until second-line treatment (PFS2), etc. The use of the biomarker is preferably to predict prognosis, and more preferably to predict recurrence.

Prognosis is predicted during follow-up of non-muscle invasive bladder cancer (NMIBC). Follow-up of NMIBC includes urine cytology and cystoscopy. If a mutation is identified during follow-up, treatment can be initiated promptly. The timing of mutation monitoring is generally expected to be the same as that of urine cytology and cystoscopy.

Mutations in guanine at positions 142,706,206 and/or cytosine at positions 142,706,209 on chromosome 6 can be markers of poor prognosis. When two groups are examined, the group with significantly improved survival in terms of recurrence-free survival time and survival rate is judged to be the "good prognosis group," while the group with worsening survival is judged to be the "poor prognosis group."

(Detection Method)
In a second aspect, a method is provided for detecting one or more mutations in the ADGRG6 enhancer in a sample from a subject suffering from or suspected of suffering from non-muscle invasive bladder cancer, the method comprising detecting the presence of one or more mutations in the ADGRG6 enhancer in a sample obtained from the subject in comparison to a wild-type ADGRG6 enhancer.

　Samples are taken from subjects suspected of having bladder cancer. The bladder cancer may be either non-muscle invasive or muscle invasive. Samples may be taken once or multiple times for purposes such as prognosis monitoring after bladder cancer treatment.

The sample is not particularly limited as long as it can detect one or more base changes in the ADGRG6 enhancer, but is preferably a liquid sample containing cfDNA, such as blood or other body fluids. More specifically, the sample is preferably blood, serum, plasma, urine, stool, saliva, sputum, tissue fluid, cerebrospinal fluid, swab, or other body fluid or a dilution thereof, particularly preferably plasma.

Since the proportion of DNA (ctDNA) and RNA (ctRNA) derived from bladder cancer contained in the cfDNA and cfRNA obtained by liquid biopsy is very small, depending on the type of sample, a step of extracting ctDNA or ctRNA from the sample and a step of removing impurities may be carried out before nucleic acid amplification. Taking blood as an example, the recovery rate of ctDNA and ctRNA can be increased by performing known means such as plasmapheresis.

Nucleic acid amplification methods include PCR, which includes standard PCR, digital PCR, multiplex PCR, LAMP (Loop-mediated isothermal AMPlification), ICAN (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids), RCA (Rolling Circle Amplification), LCR (Ligase Chain Reaction), and SDA (Strand Displacement Amplification).

When targeting mutations in cfDNA or cfRNA, the nucleic acid amplification method is preferably digital PCR. Digital PCR may be droplet digital PCR.

In one embodiment, one or more bases of the ADGRG6 enhancer are guanine at position 142,706,206 and/or cytosine at position 142,706,209 of chromosome 6.

When detecting one or more base changes in the ADGRG6 enhancer, it is preferable to also detect mutations in hotspot regions in genes associated with bladder cancer, such as the TERT promoter region and the PLEKHS1 promoter region. Other steps and sample collection may be performed before or after the detection step. For example, a specimen may be collected from a subject suspected of having bladder cancer before the detection step. The detection step may be performed multiple times, for example, specimens from the same subject may be collected periodically and subjected to the detection step each time they are collected.

When a change in one or more bases in the ADGRG6 enhancer is detected, the prognosis of bladder cancer in the subject from whom the sample was derived is predicted. This can assist doctors in diagnosing bladder cancer and determining subsequent treatment methods. Such diagnoses and determinations include other known auxiliary diagnoses for bladder cancer, as well as definitive diagnoses such as urine cytology, bladder biopsy, and histopathological examination of biopsy tissue. Subjects diagnosed with recurrence can undergo surgery (surgical treatment) such as transurethral bladder tumor resection and radical cystectomy, chemotherapy (anticancer drug treatment) such as preoperative chemotherapy, postoperative chemotherapy, and recurrence chemotherapy, and radiation therapy.

In one embodiment, if a mutation in one or more bases in the ADGRG6 enhancer is detected, the subject's prognosis is determined to be poor.

(Primer)
In a third aspect, a primer for detecting the presence of non-muscle invasive bladder cancer in a sample is provided, the primer being an oligonucleotide of 16 bases or more and 21 bases or less in length that targets one or more mutations in the ADGRG6 enhancer.

In one embodiment, the one or more mutations in the ADGRG6 enhancer are a substitution of guanine at position 142,706,206 on chromosome 6 with adenine (hereinafter also referred to as "206G>A") and/or a substitution of cytosine at position 142,706,209 on chromosome 6 with thymine (hereinafter also referred to as "209C>T").

The primer is an oligonucleotide that is at least 16 bases long and at most 21 bases long. Among them, those with a low Tm value are preferable. Specifically, by setting the Tm value of the reverse primer 1 to 3°C higher than that of the probe, the probe anneals to the template DNA first, and then the reverse primer anneals.

Furthermore, the primers can be designed so that the size of the amplified amplicon is less than 200 bp.

Examples of primers based on the above design concept that amplify a sequence containing a probe for detecting the 206G>A and 209C>T mutations include the forward primer (TGCATATTTCACATGGAC) described in SEQ ID NO: 1, which has a base length of 20 bases, and the reverse primer (ATCCTGGAGGGAGAATAC) described in SEQ ID NO: 2, which has a base length of 20 bases. It is preferable to use primers consisting of the sequences described in SEQ ID NOs: 1 and 2. These primers may be combined with known primers that amplify hotspot regions in genes related to bladder cancer, such as the TERT promoter region and the PLEKHS1 promoter region.

In one embodiment, the TERT promoter mutation may be a C to T substitution at position 1,295,228 of chromosome 5 (hereinafter also referred to as "228C>T"), a C to T substitution at position 1,295,250 of chromosome 5 (hereinafter also referred to as "250C>T"), or both. It is known that 228C>T has a high detection rate in bladder cancer, and 250C>T has a high detection rate in non-muscle invasive bladder cancer.

Primers that amplify sequences containing probes that detect both the 228C>T and 250C>T mutations include BioRad primers (TERT C228T_113: dHsaEXD72405942, TERT C250T_113: dHsaEXD46675715) (amplicon size: 113 bp) (see also J Mol Diagn. 2019 Mar;21(2)274-285).

In one embodiment, the mutation in the PLEKHS1 promoter may be a G to A substitution at position 115,511,590 of chromosome 10 (hereinafter also referred to as "590G>A"), a C to T substitution at position 115,511,593 of chromosome 10 (hereinafter also referred to as "593C>T"), or both.

The nucleotides constituting the base sequence of a primer or the like may be either ribonucleotides or deoxyribonucleotides. These oligonucleotides can be synthesized by known methods, for example, by any nucleic acid synthesis method such as the solid-phase phosphoramidite method or the triester method, according to the base sequence.

　By using the above primers in a nucleic acid amplification reaction, it becomes possible to amplify sequences containing hotspot regions associated with bladder cancer.

(probe)
In a fourth aspect, there is provided a probe for detecting the presence of bladder cancer in a specimen, the probe targeting one or more mutations in the ADGRG6 enhancer.

The sequence constituting the probe is not limited as long as it can detect a mutation in the ADGRG6 enhancer, and a probe that can detect, for example, the 206G>A or 209C>T mutation is preferred.

In one embodiment, the probe for detecting the 206G>A mutation has the sequence set forth in SEQ ID NO:3 (AGGCTCTTTGTATGTTTATACAAAG). The probe preferably consists of the sequence set forth in SEQ ID NO:3.

In one embodiment, the probe for detecting the 209C>T mutation has the sequence set forth in SEQ ID NO: 4 (AGGCTCTTTGTATATTCATACAAAG). The probe preferably consists of the sequence set forth in SEQ ID NO: 4.

The probe may be partially modified, for example, with aminated ends or with some bases modified with linker bases. In addition, a different base may be inserted into part of the base sequence, some bases in the base sequence may be deleted or replaced with another base, or replaced with a substance other than a base.

Furthermore, the probe may be modified with a labeling substance, etc., and may have, for example, a fluorescent dye and its quencher. Such labeling substances can be commercially available. There are no particular limitations on the quencher, so long as it can quench the fluorescence from the fluorescent dye. The nucleotide residues themselves that constitute the probe may be modified, and may be replaced with, for example, artificially modified nucleotide residues.

(kit)
In a fifth aspect, there is provided a kit comprising a primer and/or a probe for detecting the presence of bladder cancer in a specimen. The primer may be any of those described above.

The kit may further include a probe that targets a mutation in the ADGRG6 enhancer. An example of a probe that detects the 206G>A mutation is one having the base sequence set forth in SEQ ID NO:3. An example of a probe that detects the 209C>T mutation is one having the base sequence set forth in SEQ ID NO:4.

The kit may further include one or more pairs of primers and probes targeting hotspot regions in genes associated with bladder cancer, such as the TERT promoter region, the PLEKHS1 promoter region, etc.

When amplifying nucleic acids using digital PCR, a reaction solution is prepared containing the above primers, probe, sample, and DNA polymerase. The reaction solution is distributed into wells or droplets and then subjected to an amplification reaction.

To detect the amplified products obtained in a nucleic acid amplification reaction, a labeling substance capable of specifically recognizing the amplified products may be used. Examples of such labeling substances include fluorescent dyes, biotin, and digoxigenin. When fluorescent substances are used as the labeling substance, the fluorescence can be detected using a fluorescent microscope, a fluorescent plate reader, etc.

A substance that intercalates into the amplified product can also be used as the labeling substance. There are no particular limitations on the intercalator, so long as it is a substance that intercalates into double-stranded DNA and emits fluorescence.

Alternatively, detection may be performed by known methods, such as electrophoresis using polyacrylamide or agarose gels. For example, in electrophoresis, the presence of an amplification product can be identified by its mobility relative to that of a marker of known molecular weight.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these.

1. Primer/probe design 1) Determination of probe recognition site The base sequence around positions 142,706,206 and/or 142,706,209 of chromosome 6 was obtained from NCBI etc., and the recognition site of the detection probe was set at the site containing the mutation. The recognition position and base length of the probe were determined taking into consideration the GC content and Tm value.

2) Primer design Several primer pairs were designed to amplify sequences including the probe, and the presence or absence of nonspecific amplicon formation in gDNA and cfDNA, and primer dimer evaluation were confirmed by PCR electrophoresis, and the PCR efficiency for each primer pair was confirmed by quantitative PCR. In order to target cfDNA, the amplicon was in the range of 80-150 mer, and the sequence and number of bases of the primer were determined taking into account the Tm value calculated from the probe. The most efficient primer pair was determined taking into account PCR efficiency, primer dimers, and nonspecific bands, and it was confirmed by Sanger sequencing that the target region was correctly amplified.

3) Determination of Probe Sequence The Tm value of each primer was compared with that of the probe, and the base sequence of the probe was determined as to whether it should be bound to the sense strand or the antisense strand.

The sequences determined by the above procedure are shown in the table below.

2. Definitive diagnosis of bladder cancer and classification of patients by pathological diagnosis According to the Bladder Cancer Treatment Guidelines (Japan Urological Association), patients suspected of having bladder cancer were definitively diagnosed as having bladder cancer or not, and then the nature of the cancer was evaluated by pathological diagnosis, and the patients were classified. The procedure is as follows.
- Urine cytology is performed on patients suspected of having bladder cancer, and a positive or negative result is determined to make a definitive diagnosis of bladder cancer.
- The study will target patients with a differential diagnosis of bladder cancer who have no history of treatment and are experiencing their first case of the disease.
- For patients who have been definitively diagnosed with bladder cancer, a pathological diagnosis is performed using tissue specimens obtained through transurethral resection of bladder tumor (TURBT) or radical cystectomy to determine whether or not the bladder cancer has invaded the muscularis propria.

As a result of differential diagnosis based on the presence or absence of invasion into the proper muscle layer, 43 cases that were diagnosed as having non-muscle invasive bladder cancer and 36 cases that were diagnosed as having muscle invasive bladder cancer were recruited for this study. Based on the findings of the surgically removed tissue specimens, the malignancy of the tumor was classified as low-grade or high-grade histological atypia.

3. Methods for collecting specimens after definitive diagnosis and methods for extracting nucleic acid from specimens From patients who were definitively diagnosed with bladder cancer and able to participate in this study, blood samples were taken during treatment or follow-up after consent to participate in the study was obtained. Tumor tissue was collected only when transurethral bladder tumor resection or radical cystectomy was performed. For cases undergoing treatment or follow-up, blood was collected every time. Blood was collected using standard blood collection methods (EDTA blood collection tubes) to collect peripheral white blood cells and plasma, and tumor tissue was removed during surgery and frozen. The tissue was stored frozen at -80°C until extraction and analysis.

From the obtained samples, DNA derived from tumor tissue was extracted as tDNA (tumor DNA), DNA derived from somatic cells (peripheral white blood cells) as gDNA (genomic DNA), and DNA circulating in the plasma as cfDNA (cell-free DNA). DNA was extracted by column purification using a kit (QIAGEN QIAamp DNA Blood Mini Kit or QIAamp Circulating Nucleic Acid Kit).

Tumor tissue and blood samples were collected from patients with bladder cancer (both non-muscle invasive and muscle invasive) as subjects. Blood samples were collected every three months from patients diagnosed with muscle invasive bladder cancer, even after treatment.

4. Gene Mutation Analysis Method by Digital PCR 4.1. Experimental Flow Using digital PCR, it was confirmed whether or not there was a mutation in ADGRG6 in the blood sample obtained as described above. First, the validity of the primers designed in the following order was checked.
(i) Amplification of the fragment by PCR.
(ii) DNA purification.
(iii) Confirmation of the target fragment and primer dimer by electrophoresis.
(iv) Sequence confirmation by Sanger sequencing of the fragments.

Next, validation plasmids were prepared in the following order:
(i) Extraction of genomic DNA from patient tumor samples.
(ii) Amplification by PCR of a DNA fragment containing the region at positions 142,706,206 or 142,706,209 of wild-type ADGRG6.
(iii) Insertion of the ADGRG6 fragment into the vector.
(iv) The vector is introduced into E. coli, and about 10 to 20 colonies are isolated and cultured in large quantities.
(v) The vectors were recovered by mini prep, and each vector was confirmed to be WT or mutant by Sanger sequencing.
(vi) If only WT was recovered, introduction of the 206G>A and 209C>T mutations by Mutagenesis Kit using the wild-type ADGRG6 introduction plasmid as a template.
(vii) Obtaining a mutant ADGRG6 introduction plasmid.

The detection accuracy of the primers/probes was confirmed using the validation plasmid in the following order.
(i) Confirmation of detection accuracy by dPCR using a wild-type primer/probe set and a mutant-type primer/probe set for each of the wild-type ADGRG6-introduced plasmids and the mutant-type ADGRG6-introduced plasmids.
The forward primer used to detect wild-type ADGRG6 and mutant ADGRG6 was the sequence (TGCATATTTCACATGGAC) set forth in SEQ ID NO: 1, and the reverse primer was the sequence (ATCCTGGAGGGAGAATAC) set forth in SEQ ID NO: 2. At this stage, the validation plasmid was confirmed by Sanger sequencing, and the exact mutation rate of less than 10% was not known. Therefore, at this stage, it was also verified whether the validation plasmid was almost 100% wild-type or almost 100% mutant type and therefore worthy of use in the next step (ii).
(ii) Wild-type and mutant plasmids were spiked in in multiple steps to prepare 100%, 10%, 1%, 0.5%, 0.25%, 0.125%, 0.0625%, and 0% mutant plasmids.
(iii) Confirmation of the detection accuracy of the mutant plasmid.

Patient samples were used to detect TERT and ADGRG6 mutations by dPCR.
(i) Mutation detection confirmation using tDNA and gDNA samples (27 samples each) using the validated wild-type ADGRG6 primers/probes and mutant-type ADGRG6 primers/probes.
(ii) Similarly, mutation detection confirmation was performed using tDNA and gDNA (27 samples each) using TERT primers/probes. The TERT primers/probes were commercially available products (BioRad primer/probe set (TERT C228T_113: dHsaEXD72405942, TERT C250T_113: dHsaEXD46675715) (amplicon size: 113 bp)). Although their sequences are unknown, the amplicon size of both types is 113 bp. In addition, since primers are generally designed around the mutation site and the probe is designed so that the mutation site is hit, it is considered that the primers and probes are located within a range of 113 bp upstream or downstream from C228 or C250 as the starting point. Design may be performed within a range of 50 to 200 bp upstream or downstream from the above mutation site.
(iii) In cases where tDNA was confirmed to be mutation-positive and gDNA was confirmed to be mutation-negative, mutation detection and MAF were calculated using cfDNA as a sample. If the mutation frequency in the tumor was low, it was expected that detection in the blood would be difficult, so cases with a mutation rate of 10% or more in tDNA were considered mutation-positive.

The materials and methods used for digital PCR are described below.
material:
Sample DNA (-20°C)
2x ddPCR Supermix for Probes (No dUTP) (4℃)
20x primers/probes:
Restriction enzyme: HaeIII

method:
1. Adjust the concentration of DNA solution to <50 ng/well (up to 7 μL) (15 ng can be added for 10-50 copies)
2. Vortex at room temperature, centrifuge and store protected from light.
3. Prepare the mix (restriction enzyme stock solution 10 U/μL)

4. Vortex the mix, centrifuge and leave at room temperature for 3 minutes. 5. Add 20 μL of the mix to the sample well and 70 μL of Droplet Generation Oil to the oil well of the DG8® cartridge.
6. Place in the Droplet Generator to create droplets in solution. 7. Place 40 μL of droplets in a PCR 96 well and seal with aluminum foil.

8. Analysis with Droplet Reader

4.2. Confirmation of detection accuracy Plasmids (wild type, mutant type) for validation were prepared and the detection accuracy was confirmed. For the detection of ADGRG6 206G>A, the detection accuracy of the wild type and mutant type was 99.98% and 100.00%, respectively. For the detection of ADGRG6 209C>T, the detection accuracy of the wild type and mutant type was 99.99% and 100.00%, respectively. In addition, when detecting a DNA solution in which the mutant type was diluted with the wild type, 0.063% of mutations could be significantly detected (206G>A: p=0.0017, 209C>T: p<0.001).

When dPCR was performed using nucleic acids (tDNA, cfDNA) extracted from the patient's blood sample as a template, the detection signals were good for both the wild type and the mutant type, and positive and negative droplets could be clearly separated in both samples.

When the Mutant Allele Frequency (MAF) of cfDNA from mutation-positive patients was analyzed over time, it did not exceed 0.3% over time in any patient. This is thought to reflect the fact that no patient has been diagnosed with recurrence by imaging to date.

4.3. Gene Mutation Analysis by Digital PCR Among 104 patients who were definitively diagnosed as suffering from urothelial carcinoma, 25 cases of upper urinary tract cancer anatomically considered to be renal pelvis cancer and ureter cancer were excluded, leaving 79 cases of bladder cancer under follow-up, of which 43 cases were definitively diagnosed as non-muscle invasive bladder cancer (NMIBC) were further examined. The study began on the date of sampling of samples from 43 patients who had undergone treatment. Although the start of the study varied depending on when each patient joined the study, samples were obtained during follow-up 36 months after the start of the study for the first patient. As a result, the observation period ranged from 10 months to 36 months. Regarding the results obtained by subjecting the specimen to digital PCR, cases in which a mutation from G (guanine) at position 142,706,206 of ADGRG6 to C (cytosine) or a mutation from C (cytosine) at position 142,706,209 to T (thymine) could be detected were registered as mutation-positive cases. In addition, cases in which the 206th G>A and 209th C>T mutations were found simultaneously were also registered in the same way. In this case, cases in which mutations were found at two locations simultaneously were also counted as "1" in the number of cases.

36 months after the start of the study, the number of recurrent and non-recurrent cases in 43 cases of non-muscle invasive carcinoma (NMIBC) was counted, and then the presence or absence of TERT and ADGRG6 gene mutations was evaluated. Cases were considered to have recurred when local recurrence or metastasis to other organs was found in imaging tests (CT) of the chest to pelvis during follow-up. In addition, the presence or absence of mutations in the TERT and ADGRG6 genes was counted for both low grade and high grade histological atypia.

In the recurrent and non-recurrent groups of non-muscle invasive cancer (NMIBC), 1) TERT only and 2) both TERT and ADGRG6 were confirmed simultaneously, and the proportion of mutation-positive cases was calculated. In low-grade and high-grade NMIBC, 1) TERT only and 2) both TERT and ADGRG6 were confirmed simultaneously, and the proportion of mutation-positive cases was calculated. The results are shown in Figure 1.

In low-grade non-muscle invasive bladder cancer (NMIBC) cases, the coverage rate did not increase significantly when ADGRG6 enhancer mutations were evaluated in addition to conventional prognostic markers such as TERT promoter mutations, whereas in high-grade cases, the coverage rate increased significantly by combining both mutations. Non-muscle invasive bladder cancer, which accounts for the majority of bladder cancer cases, requires early detection of recurrence and metastasis and prevention of further malignant progression, so changes in the ADGRG6 enhancer can be a poor prognostic marker for non-muscle invasive bladder cancer (NMIBC), especially high-grade non-muscle invasive bladder cancer, which is highly malignant.

Claims

A biomarker for non-muscle invasive bladder cancer, comprising:
A biomarker that is one or more mutations in the protein encoded by the ADGRG6 enhancer.
The biomarker of claim 1, wherein the mutation is a single nucleotide polymorphism.
The biomarker according to claim 2, wherein the single nucleotide polymorphism is a mutation of guanine at position 142,706,206 and/or cytosine at position 142,706,209 on chromosome 6.
The biomarker described in claim 1 or 2 for predicting the prognosis of non-muscle invasive bladder cancer.
The biomarker according to claim 4, wherein the non-muscle invasive bladder cancer is high-grade non-muscle invasive bladder cancer.
The biomarker according to claim 4, wherein the prognosis is the length of recurrence-free survival or the risk of recurrence.
The biomarker according to claim 4, which is a poor prognosis marker.
The biomarker of claim 1 or 2, wherein the mutation is present in circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), genomic DNA or complementary DNA (cDNA).
1. A method for detecting one or more mutations in the ADGRG6 enhancer in a sample from a subject having or suspected of having non-muscle invasive bladder cancer, comprising:
A method comprising detecting the presence of one or more mutations in the ADGRG6 enhancer in a sample obtained from the subject, in comparison to a wild-type ADGRG6 enhancer.
The method according to claim 9, wherein if a mutation is detected, the subject has a poor prognosis.
The method according to claim 9 or 10, wherein the nucleic acid amplification method used in the detection step is digital PCR.
The method according to claim 9 or 10, wherein the sample is a liquid sample.
The method of claim 9 or 10, wherein the sample comprises circulating DNA (cfDNA).
1. A primer for detecting the presence of non-muscle invasive bladder cancer in a specimen, comprising:
A primer that is an oligonucleotide of at least 16 bases and no more than 21 bases in length that targets one or more mutations in the ADGRG6 enhancer.
A probe for detecting the presence of non-muscle invasive bladder cancer in a specimen, the probe targeting one or more mutations in the ADGRG6 enhancer.
A kit comprising the primer according to claim 14 and/or the probe according to claim 15.