WO2023145754A1 - Amorces et sonde pour détecter la présence d'un cancer de la vessie - Google Patents

Amorces et sonde pour détecter la présence d'un cancer de la vessie Download PDF

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WO2023145754A1
WO2023145754A1 PCT/JP2023/002178 JP2023002178W WO2023145754A1 WO 2023145754 A1 WO2023145754 A1 WO 2023145754A1 JP 2023002178 W JP2023002178 W JP 2023002178W WO 2023145754 A1 WO2023145754 A1 WO 2023145754A1
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probe
promoter
plekhs1
primers
mutations
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PCT/JP2023/002178
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Japanese (ja)
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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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    • 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
    • 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 primers, probes, etc. for detecting the presence of bladder cancer.
  • Liquid biopsy which detects tumor-derived genetic mutations in blood-circulating DNA (cell-free DNA: cfDNA), is a minimally invasive test, and has been reported in recent years to be effective in postoperative follow-up and assessment of therapeutic effects.
  • cfDNA analysis methods include the use of next-generation sequencing (NGS) and digital PCR.
  • NGS next-generation sequencing
  • Digital PCR has the advantage that it can be analyzed at a lower cost than NGS.
  • TERT telomerase reverse transcriptase
  • a primer set for detecting the presence of bladder cancer in a specimen comprising: a pair of primers, which are oligonucleotides with a length of 16 bases or more and 21 bases or less, targeting mutations in the PLEKHS1 promoter; one or more pairs of primers that target mutations in the TERT promoter; A primer set, including (2)
  • the PLEKHS1 promoter mutation is a G to A substitution at chromosome 10, position 115,511,590, a C to T substitution at chromosome 10, position 115,511,593, or both (1) Primer set described in .
  • a kit comprising the primer set according to any one of (1) to (4).
  • the kit according to (7) further comprising the probe according to (5) or (6).
  • a method for detecting the presence of bladder cancer in a specimen wherein the primer set according to any one of (1) to (4) or the probe according to (5) or (6) is used to detect the PLEKHS1 promoter.
  • the method according to (9), wherein the nucleic acid amplification method used in the detection step is digital PCR.
  • (11) The method according to (9) or (10), wherein the specimen is a liquid specimen.
  • Mutations in the PLEKHS1 promoter include G to A substitution at position 115,511,590 of chromosome 10 and C to T substitution at position 115,511,593 of chromosome 10.
  • the present inventors set the positions corresponding to these mutations near the center of the probe so as to maximize the Tm value.
  • the probe is usually designed as a 20-mer, 18-mer at the shortest, and 25-mer at the longest. bottom.
  • the primer was also designed with a different concept than usual.
  • the length of primers is designed to be about 20-25 mers. If the length of the primer is less than 20-mer, there is a possibility that the sequence that non-specifically binds to the base sequence other than the target increases, but the forward primer that detects the mutation of the PLEKHS1 promoter is a 16-mer. designed. This is in consideration of the need to lower the Tm value compared to wild-type/mutant probes.
  • the region containing the hotspot of the PLEKHS1 promoter region is an AT-rich sequence, but the primer design region is a sequence that cannot be said to be AT-rich, so the Tm value becomes high, and it is necessary to shorten the sequence. There is Furthermore, trying to avoid GC, for example, designing an A or T at the 3' end of the primer may reduce specificity for the template DNA. However, by conducting verification experiments, it was possible to design a shorter length with a lower Tm value than general designs.
  • the Tm value of the mutant probe is lower than that of the forward primer.
  • the annealing order is wild-type probe (53.92), forward primer (52.61), mutant probe (52.28), reverse primer (50.66).
  • the size of the amplicon to be amplified was designed to be less than 200 bp.
  • primers that target mutations in the PLEKHS1 promoter that are designed under a different concept as described above can be used to detect bladder cancer. It will be possible to cover patients who did not have it.
  • FIG. 2 shows the results of quantification of positive/negative droplets in mutation-positive patients.
  • Ch1 indicates Mut droplets
  • Ch2 indicates WT droplets
  • the lower left frame is WT and Mut negative droplets
  • the lower right frame is WT positive droplets
  • the upper left frame is Mut positive droplets
  • the upper right frame is WT. and Mut-positive droplets.
  • FIG. 1 shows detection accuracy (upper) and mutation rate (lower) of validation plasmids (wild-type (WT) and mutant-type (Mut)).
  • FIG. 2 shows the results of quantification of positive/
  • FIG. 3 shows changes over time in MAF: Mutation Allele Frequency after initial treatment (transurethral bladder tumor resection) of mutation-positive patients.
  • the arrows in the figure indicate mutations (or indicates the specific stage of the sample containing it).
  • the left figure shows the results of patient number 03 (Pt03), and the right figure shows the results of patient number 21 (Pt21).
  • Pt03 the results of patient number 03
  • Pt21 patient number 21
  • FIG. 4 illustrates the coverage of bladder cancer patients by the primers/probes of the present invention.
  • a primer set for detecting the presence of bladder cancer in a specimen comprising: a pair of primers, which are oligonucleotides with a length of 16 bases or more and 21 bases or less, targeting mutations in the PLEKHS1 promoter; one or more pairs of primers that target mutations in the TERT promoter;
  • a primer set comprising:
  • the specimen is not particularly limited as long as it can detect PLEKHS1 promoter mutation and TERT promoter mutation suggesting the presence of bladder cancer.
  • Fluid specimens containing cfDNA are preferred, such as blood or other bodily fluids. More specifically, the specimen is preferably blood, serum, plasma, urine, stool, saliva, sputum, interstitial fluid, cerebrospinal fluid, bodily fluids such as swabs, or dilutions thereof, particularly plasma.
  • Specimens are collected from subjects suspected of having bladder cancer.
  • Bladder cancer may be non-muscle invasive or muscle invasive.
  • the sample may be collected once or multiple times for purposes such as prognosis observation after bladder cancer treatment.
  • ctDNA bladder cancer-derived DNA
  • the PLEKHS1 promoter mutation is a G to A substitution (hereinafter also referred to as “590G>A”) at position 115,511,590 on chromosome 10, 115,511,593 on chromosome 10. It may be a C to T substitution at the position (hereinafter also referred to as “593C>T”) or both.
  • a primer is an oligonucleotide with a length of 16 bases or more and no more than 21 bases. Among them, those having a low Tm value are preferable. Specifically, by setting the Tm value of the reverse primer to be 1 to 3° C. higher than that of the probe, the probe is annealed to the template DNA first, and then the reverse primer is annealed.
  • the primers can be designed so that the amplicon size to be amplified is less than 200 bp.
  • SEQ ID NO: 1 having a base length of 16 bases
  • examples include the forward primer (gacctcttggcttcca) described above and the reverse primer (ctgcaaattttccatttcca) described in SEQ ID NO: 2, which has a base length of 21 bases. It is preferable to use a primer set consisting of the sequences set forth in SEQ ID NOS: 1 and 2. These primers may be combined with known primer sets that amplify the PLEKHS1 promoter region.
  • the TERT promoter mutation is a C to T substitution (hereinafter also referred to as “228C>T”) at position 1,295,228 on chromosome 5, 1,295,250 on chromosome 5 It may be a C to T substitution at the position (hereinafter also referred to as “250C>T”) or both.
  • 228C>T is known to be highly detectable in bladder cancer and 250C>T in non-muscle invasive bladder cancer.
  • Primers that amplify sequences containing probes that detect both the 228C>T and 250C>T mutations should be within 113 bp upstream or downstream of each mutation, i.e., primers targeting the former mutation. and within the region of positions 1,295,115 to 1,295,341 for the probe and within the region of positions 1,295,237 to 1,295,363 for primers and probes targeting the latter mutation. can be designed.
  • sequence shown in SEQ ID NO: 7 is obtained by converting the sequence into the sequence on the sense strand side and reflecting both the 228C>T and 250C>T mutations.
  • primers and probes that detect 228C>T the 227 bp sequence from positions 1,295,341 to 1,295,115 corresponding to positions 1,295,115 to 1,295,341 in SEQ ID NO: 7
  • Desired primers and probes can be appropriately designed from (SEQ ID NO: 8).
  • primers and probes for detecting 250C>T which can be designed from the 127 bp sequence (SEQ ID NO: 9) from positions 1,295,363 to 1,295,237.
  • a primer made by BioRad (TERT C228T_113: DHSAEXD72405942, TERT C250T_113: DHSAEXD466675715) (Ampuri Conto) (J Mol Diagn. 2019 MAR. 2019 MAR. See 21 (2) 274-285 ).
  • Such primers may be combined with known primer sets that amplify the TERT promoter region.
  • SEQ ID NO:6 GCCGGGGCCAGGGCTTCCCACGTGCAGCAGGACGCAGCGCTGCCTGAAACTCGCGCCGCGAGGAGAGGGCGGGGCCGCGGAAAGGAAGGGGAGGGGCTGGGAGGGCCCGGAGGGGGCTGGGCCGGGGACCCGGGAGGGGTCGGGACGGGGGGCGGGGTCCGCGCGGAGGAGGCGGAGCTGGAAGGTGAAGGGGCAGGACGGGTGCCCGGGTCCCCAGTCCCTCCGCCACGTGGGAAGCGCGGT CCTGGGMore SEQ ID NO: 7 CCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCCGTCCCGGGTCCCCCCTCCGGGCCCTCCCTCCTCCGGGCCCTCCCTCCTCCGGGCCCTCCCTCCCCTCCGGGCCCTCCCTCCCCTCCGGGCCCTCCCTCCCCTCCGGGCCCTCCCTCCCTCCTCCGGGCCCTCCCTCCCTCCTCCGGGCCCTCC
  • the nucleotides that make up the base sequences of primers and 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 solid-phase phosphoramidite method and triester method, according to the base sequence.
  • a nucleic acid amplification reaction using the above primers makes it possible to amplify sequences containing hotspot regions associated with bladder cancer.
  • Nucleic acid amplification methods include PCR methods, and in addition to normal PCR, digital PCR, multiplex PCR, LAMP (Loop-mediated isothermal amplification) method, ICAN (Isothermal and Chimeric primer-initiated amplification of nucleic acids) method, RCA ( Rolling Circle Amplification) method, LCR (Ligase Chain Reaction) method, SDA (Strand Displacement Amplification) method, and the like.
  • the nucleic acid amplification method is preferably digital PCR.
  • digital PCR it is possible to determine the ratio of mutant to normal types (MAF: Mutation Allele Frequency) for oncogenes.
  • Digital PCR may be droplet digital PCR.
  • a probe for detecting the presence of bladder cancer in a specimen is provided, the probe targeting mutations in the PLEKHS1 promoter.
  • the sequence that constitutes the probe is not limited as long as it can detect mutations in the PLEKHS1 promoter.
  • probes that can detect the 590G>A mutation or the 593C>T mutation are preferred.
  • the probe that detects the 590G>A mutation has the sequence set forth in SEQ ID NO: 4 (tgcaattgtttaattgcaaaaagc).
  • the probe consists of the sequence set forth in SEQ ID NO:4.
  • the probe that detects the 593C>T mutation has the sequence set forth in SEQ ID NO:5.
  • the probe preferably consists of the sequence (tgcaattattcaattgcaaaaagc) set forth in SEQ ID NO:5.
  • the probe may be partially modified, examples of which include those whose ends are aminated and those whose bases are partially modified with linker bases.
  • another base may be inserted into part of the base sequence, a part of the base sequence may be deleted, replaced with another base, or substituted with a substance other than a base. .
  • the probe may be modified with a labeling substance or the like, and may have, for example, a fluorescent dye and its quencher.
  • a fluorescent dye and its quencher Commercially available products can be used as such labeling substances.
  • the quencher is not particularly limited as long as it can quench the fluorescence from the fluorescent dye.
  • the nucleotide residues constituting the probe may themselves be modified, for example, substituted with artificially modified nucleotide residues.
  • kits in a second aspect, includes a primer set for detecting the presence of bladder cancer in a specimen.
  • the primer set described above can be used.
  • the kit may further contain probes targeting mutations in the PLEKHS1 promoter.
  • Probes for detecting the 590G>A and 593C>T mutations include those having the base sequences of SEQ ID NOS: 4 and 5, respectively.
  • the kit may further include one or more pairs of primers and probes targeting mutations in the TERT promoter.
  • Mutations in the TERT promoter include 228C>T and 250C>T. within the region of positions 1,295,115 to 1,295,341 for primers and probes targeting 228C>T; It can be designed within the region of 295,363 positions.
  • a reaction solution containing the above primer set, probe, sample, and DNA polymerase is prepared. After being distributed to wells or droplets, the reaction solution is subjected to an amplification reaction.
  • a labeling substance that can specifically recognize the amplification product may be used to detect the amplification product obtained in the nucleic acid amplification reaction.
  • labeling substances include fluorescent dyes, biotin, digoxigenin and the like.
  • fluorescence can be detected using a fluorescence microscope, a fluorescence plate reader, or the like.
  • a substance that intercalates with the amplification product can also be used as a labeling substance.
  • the intercalator is not particularly limited as long as it is a substance that intercalates with the double-stranded DNA and emits fluorescence.
  • detection may be performed by known methods such as electrophoresis using polyacrylamide or agarose gel.
  • electrophoresis the presence of an amplification product can be identified from the mobility of the amplification product relative to the mobility of a marker of known molecular weight.
  • Detection method In a third aspect, a method for detecting the presence of bladder cancer in a specimen is provided, comprising detecting mutations in the PLEKHS1 promoter using the primer set or kit described above.
  • the primer set includes primers that target mutations in the TERT promoter, and that mutations in the TERT promoter are also detected in the detection process.
  • Other steps may be included before or after the detection step, for example, a specimen may be obtained from a subject suspected of having bladder cancer. The detection step may be performed multiple times, for example, specimens derived from the same subject may be collected periodically and subjected to the detection step each time they are collected.
  • a mutation in the PLEKHS1 promoter is detected, it is determined that bladder cancer is present in the specimen. This can assist doctors in diagnosing bladder cancer and determining subsequent treatment methods.
  • surgery such as transurethral bladder tumor resection, radical cystectomy, preoperative chemotherapy, postoperative chemotherapy, chemotherapy such as recurrent chemotherapy (anti-cancer drug therapy) and radiotherapy can be performed.
  • transurethral bladder tumor resection it will be possible to diagnose muscle invasion from the results of transurethral bladder tumor resection. If the tumor does not invade the muscle layer, treatment is completed with only transurethral resection of the bladder tumor, followed by follow-up.
  • preoperative chemotherapy will be started, and if recurrence or metastasis is found after completion of treatment, recurrent chemotherapy will be introduced.
  • Preoperative chemotherapy can improve the prognosis, but it may not be given depending on the histology of the tumor and the patient's wishes.
  • Postoperative chemotherapy is often added when intraoperative findings suggest residual disease. If radical cystectomy is considered difficult due to age or general condition, radiation may be performed instead.
  • primer dimer evaluation is performed by PCR electrophoresis, PCR efficiency for each primer pair was confirmed by quantitative PCR. Since cfDNA was the target, the amplicon was within the range of 80 to 150 mers, and the Tm value calculated from the probe was taken into consideration for the sequence and number of bases of the primers. Taking into account PCR efficiency, primer dimers, and non-specific bands, the most efficient primer pair was determined, and correct amplification of the target region was confirmed by Sanger sequencing.
  • tDNA tumor tissue-derived DNA
  • gDNA tumor tissue-derived DNA
  • gDNA tumor tissue-derived DNA
  • gDNA tumor tissue-derived DNA
  • gDNA tumor tissue-derived DNA
  • plasmids for validation were prepared in the following order.
  • the vector is introduced into Escherichia coli, and about 10 to 20 colonies are isolated and cultured in large quantities.
  • Vectors were recovered by mini prep, and whether each vector was WT or Mutant was confirmed by Sanger sequencing.
  • the primer/probe detection accuracy was confirmed using the validation plasmid in the following order.
  • the forward primer for detecting wild-type PLEKHS1 and mutant PLEKHS1 consists of the sequence (gacctcttggcttcca) set forth in SEQ ID NO: 1, and the reverse primer consists of the sequence set forth in SEQ ID NO: 2 (ctgcaaaattttccatttcca). used something.
  • a probe consisting of the sequence (tgcaattgttcaattgcaaaaagc) set forth in SEQ ID NO: 3 was used as a probe for detecting wild-type PLEKHS1.
  • the 590G>A mutant probe consists of the sequence (tgcaattgtttaattgcaaaaagc) set forth in SEQ ID NO: 4, and the probe for detecting the 593C>T mutation consists of the sequence set forth in SEQ ID NO: 5 (tgcaattattcaattgcaaaaagc). It was used.
  • (ii) Generation of 0.1% mutant plasmids at a ratio of wild-type:mutant 999:1.
  • primers and probes exist within a range of 113 bp upstream or downstream from C228 or C250. It may be designed within the range of 50 to 200 bp upstream or downstream from the mutation site.
  • Method 1. Prepare ⁇ 50 ng/well (up to 7 ⁇ L) DNA solution (15 ng can be added for 10-50 copies) 2. Thaw at room temperature, vortex, centrifuge, and protect from light. 3. Prepare Mix (restriction enzyme undiluted solution 10 U/ ⁇ L) 4. Vortex/centrifuge and leave at room temperature for 3 minutes5. Add 20 ⁇ L of mix to the sample well of the DG8® cartridge and 70 ⁇ L of Droplet Generation Oil to the oil well. 6. 7. Place in Droplet Generator to create droplets in solution. Put 40 ⁇ L of Droplet into PCR 96well and seal (aluminum foil) 8. Analysis with Droplet Reader
  • dPCR was used to detect tumor DNA from 26 specimens, and the TERT promoter and PLEKHS1 promoter were positive in 50.0% (13/26) and 42.3% (11/26) of the patients, respectively. rice field.
  • one patient with multiple PLEKHS1 promoter mutations was counted. With the TERT promoter alone, 50.0% of target patients could be observed, whereas the addition of the PLEKHS1 promoter improved to 57.7% (15/26). Also, multiple mutations could not be observed in the same patient with the TERT promoter alone, i.e., 0.0% (0/26), compared to 34.6% (9/26) with the addition of the PLEKHS1 promoter. Patients can now be observed with multiple mutations.
  • the advantage of being able to analyze mutations by dPCR not only in the TERT promoter but also in the PLEKHS1 promoter is that the number of patients who can be analyzed with the same assay increases. Since tumor mutations in bladder cancer are diverse, the mainstream method is to analyze mutations that differ from patient to patient over time. Compared to this method, hotspot detection is advantageous in terms of reducing analysis costs. Second, by analyzing multiple MAFs, it is possible to more accurately assess the disease state of the patient. Multiple events in which MAF could not be detected were observed despite the diagnosis of systemic metastasis, suggesting the risk of being judged as false negative when follow-up is performed for a single mutation. be. It is believed that observing multiple MAFs can reduce the possibility of a false negative evaluation.

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Abstract

La présente invention propose un ensemble d'amorces pour détecter la présence d'un cancer de la vessie dans un échantillon, ledit ensemble d'amorces contenant une paire d'amorces étant des oligonucléotides ciblant une mutation du promoteur de PLEKHS1 et présentant une longueur de 16 à 21 bases incluses, et une ou plusieurs paires d'amorces ciblant une mutation du promoteur de TERT.
PCT/JP2023/002178 2022-01-26 2023-01-25 Amorces et sonde pour détecter la présence d'un cancer de la vessie WO2023145754A1 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAXTER L., GORDON N. S., OTT S., WANG J., PATEL P., GOEL A., PIECHOCKI K., SILCOCK L., SALE C., ZEEGERS M. P., CHENG K. K., JAMES : "Properties of non-coding mutation hotspots as urinary biomarkers for bladder cancer detection", SCIENTIFIC REPORTS, vol. 13, no. 1, XP093080486, DOI: 10.1038/s41598-023-27675-4 *
WU SONG, OU TONG, XING NIANZENG, LU JIANG, WAN SHENGQING, WANG CHANGXI, ZHANG XI, YANG FEIYA, HUANG YI, CAI ZHIMING: "Whole-genome sequencing identifies ADGRG6 enhancer mutations and FRS2 duplications as angiogenesis-related drivers in bladder cancer", NATURE COMMUNICATIONS, vol. 10, no. 1, XP093080485, DOI: 10.1038/s41467-019-08576-5 *

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