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WO2024204599A1 - Biomarqueur pour le cancer de la vessie - Google Patents

Biomarqueur pour le cancer de la vessie Download PDF

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Publication number
WO2024204599A1
WO2024204599A1 PCT/JP2024/012784 JP2024012784W WO2024204599A1 WO 2024204599 A1 WO2024204599 A1 WO 2024204599A1 JP 2024012784 W JP2024012784 W JP 2024012784W WO 2024204599 A1 WO2024204599 A1 WO 2024204599A1
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Prior art keywords
bladder cancer
adgrg6
biomarker
enhancer
mutations
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PCT/JP2024/012784
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English (en)
Japanese (ja)
Inventor
茂大 塚原
崇 松元
真己 塩田
正俊 江藤
東天 康
啓輔 兒玉
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デンカ株式会社
国立大学法人九州大学
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Publication of WO2024204599A1 publication Critical patent/WO2024204599A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention broadly relates to biomarkers for bladder cancer, etc.
  • Non-Patent Document 1 In many cancers, including bladder cancer, point mutations occur in the promoter region of TERT (telomerase reverse transcriptase).
  • TERT telomerase reverse transcriptase
  • 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).
  • Non-Patent Document 2 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.
  • a biomarker for non-muscle invasive bladder cancer comprising: A biomarker that is one or more mutations in the ADGRG6 enhancer.
  • the biomarker according to [1], wherein the mutation is a single nucleotide polymorphism.
  • 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.
  • 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.
  • 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 [14] and/or the probe according to [15].
  • Non-muscle invasive bladder cancer 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.
  • 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).
  • 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.
  • NMIBC non-muscle invasive bladder cancer
  • MIBC muscle invasive bladder cancer
  • bladder cancer 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.
  • TURBT transurethral resection of bladder tumor
  • 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.
  • 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.
  • 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.
  • 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.
  • NMIBC non-muscle invasive bladder cancer
  • MIBC muscle invasive bladder cancer
  • 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.
  • 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.
  • 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.
  • 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.
  • DFS disease-free survival
  • NUTRFS non-urothelial recurrence-free survival
  • DMFS distant metastasis-free survival
  • PFS2 progression-free survival until second-line treatment
  • the use of the biomarker is preferably to predict prognosis, and more preferably to predict recurrence.
  • NMIBC non-muscle invasive bladder cancer
  • 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.
  • 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.”
  • a method 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 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.
  • a step of extracting ctDNA or ctRNA from the sample and a step of removing impurities may be carried out before nucleic acid amplification.
  • 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).
  • PCR 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).
  • the nucleic acid amplification method is preferably digital PCR.
  • Digital PCR may be droplet digital PCR.
  • 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.
  • telomeres may be detected 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.
  • 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.
  • 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.
  • 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.
  • the subject's prognosis is determined to be poor if a mutation in one or more bases in the ADGRG6 enhancer is detected.
  • 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.
  • 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.
  • the primers can be designed so that the size of the amplified amplicon is less than 200 bp.
  • 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.
  • 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).
  • 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.
  • 590G>A G to A substitution at position 115,511,590 of chromosome 10
  • 593C>T C to T substitution at position 115,511,593 of chromosome 10
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits 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.
  • 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.
  • a labeling substance capable of specifically recognizing the amplified products may be used.
  • labeling substances include fluorescent dyes, biotin, and digoxigenin.
  • 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.
  • the intercalator there are no particular limitations on the intercalator, so long as it is a substance that intercalates into double-stranded DNA and emits fluorescence.
  • detection may be performed by known methods, such as electrophoresis using polyacrylamide or agarose gels.
  • electrophoresis the presence of an amplification product can be identified by its mobility relative to that of a marker of known molecular weight.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TERT and ADGRG6 mutations were used to detect TERT and ADGRG6 mutations by dPCR.
  • 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.
  • 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)).
  • the amplicon size of both types is 113 bp.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un biomarqueur pour le cancer de la vessie invasif non musculaire, le biomarqueur étant une ou plusieurs mutations dans une protéine codée par l'activateur ADGRG6.
PCT/JP2024/012784 2023-03-29 2024-03-28 Biomarqueur pour le cancer de la vessie WO2024204599A1 (fr)

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WO2021066038A1 (fr) * 2019-10-02 2021-04-08 国立大学法人九州大学 Biomarqueur, procédé, kit et réseau pour prédire des effets thérapeutiques d'une thérapie par perfusion intravésicale de bcg dans le traitement du cancer de la vessie

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