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WO2025063294A1 - Procédé de diagnostic du cancer colorectal et procédé d'évaluation de l'effet d'une chimiothérapie adjuvante appliquée au patient atteint d'un cancer colorectal - Google Patents

Procédé de diagnostic du cancer colorectal et procédé d'évaluation de l'effet d'une chimiothérapie adjuvante appliquée au patient atteint d'un cancer colorectal Download PDF

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Publication number
WO2025063294A1
WO2025063294A1 PCT/JP2024/033732 JP2024033732W WO2025063294A1 WO 2025063294 A1 WO2025063294 A1 WO 2025063294A1 JP 2024033732 W JP2024033732 W JP 2024033732W WO 2025063294 A1 WO2025063294 A1 WO 2025063294A1
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WO
WIPO (PCT)
Prior art keywords
gene
subject
colorectal cancer
base sequence
gpc6
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2024/033732
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English (en)
Japanese (ja)
Inventor
功士 三森
厚司 新井田
聡 長山
直輝 宇野
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Japanese Foundation for Cancer Research
Kyushu University NUC
Nagasaki University NUC
University of Tokyo NUC
Original Assignee
Japanese Foundation for Cancer Research
Kyushu University NUC
Nagasaki University NUC
University of Tokyo NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japanese Foundation for Cancer Research, Kyushu University NUC, Nagasaki University NUC, University of Tokyo NUC filed Critical Japanese Foundation for Cancer Research
Publication of WO2025063294A1 publication Critical patent/WO2025063294A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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 relates to a method for diagnosing colorectal cancer and a method for evaluating the effectiveness of adjuvant chemotherapy administered to colorectal cancer patients.
  • NSCLC non-small cell lung cancer
  • EGFR epidermal growth factor receptor
  • Liquid biopsy is the most effective approach for MRD monitoring, diagnosing recurrence earlier than conventional diagnostic tools such as high-resolution computed tomography (CT) and magnetic resonance imaging (MRI).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • customized blood CGP tests are expected to play a central role in MRD monitoring in the future and facilitate the rapid selection of appropriate drugs.
  • NGS next-generation sequencing
  • Ci rculating exosomal microRNA-203 is associated with metastasis possibly via inducing tumor-associated macrophages in colorectal cancer. Oncotarget 2017;8(45): 78598-78613.
  • the objective of the present invention is to provide a new technology that can predict the onset and recurrence of colorectal cancer more easily than conventional methods.
  • the present invention is as follows.
  • a method for detecting colon cancer in a subject comprising determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject.
  • the method according to any one of [1] to [3], wherein the determination of the methylation status is carried out based on a liquid sample derived from the subject.
  • the liquid sample is a blood sample.
  • [6] The method according to any one of [1] to [5], wherein the methylation status is determined by a PCR method.
  • a method for evaluating the effect of adjuvant chemotherapy being received by a subject comprising determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject receiving adjuvant chemotherapy for colorectal cancer.
  • the method described in [7] further comprising a step of determining the methylation status of the MSC gene in the subject.
  • a diagnostic kit for colon cancer including: (1) a PCR primer pair capable of amplifying a part or the whole of the nucleotide sequence of the FGD5 gene; and/or (2) A PCR primer pair capable of amplifying a part or the whole of the nucleotide sequence of the GPC6 gene.
  • a PCR primer pair capable of amplifying a part or the whole of the base sequence of the FGD5 gene is a nucleic acid having a base sequence shown in SEQ ID NO: 4 and a nucleic acid having a base sequence shown in SEQ ID NO: 5;
  • a PCR primer pair capable of amplifying a part or the whole of the base sequence of the GPC6 gene is a nucleic acid comprising the base sequence shown in SEQ ID NO: 6 and a nucleic acid comprising the base sequence shown in SEQ ID NO: 7.
  • PCR primer pair capable of amplifying a part or all of the base sequence of the MSC gene is a nucleic acid having a base sequence shown in SEQ ID NO: 8 and a nucleic acid having a base sequence shown in SEQ ID NO: 9.
  • the present invention makes it possible to determine whether a subject is suffering from colorectal cancer and whether colorectal cancer has recurred in a subject after radical surgery for colorectal cancer more quickly than with conventional methods. Furthermore, the present invention also makes it possible to evaluate whether adjuvant chemotherapy for colorectal cancer is effective in a subject receiving the chemotherapy.
  • Figure 1 shows an example of the calculation of methylated ctDNA levels by the AMUSE (amplicon of methylated sites using a specific enzyme) assay based on digital PCR findings (hereinafter, the methylated ctDNA level quantified using the AMUSE assay is referred to as the AMUSE score).
  • HapII is a methylation site-specific enzyme. Because RNase P contains genomic DNA sequences that cannot be methylated, RNase P cfDNA was used as a control group for HapII treatment. The cfDNA of the target gene and the cfDNA of the RNase P gene that was not treated with HapII were amplified, and the cfDNA (10)/RNase P (5) cfDNA ratio (2) was calculated.
  • FIG. 2 is a diagram outlining the flow of the search for methylation biomarkers related to the prediction of colorectal cancer recurrence employed in the present invention.
  • CRC colorectal cancer Verification of beta values of methylation sites of FDG5, GPC6, and MSC genes.
  • Figure 4 shows the basic characteristics of the three biomarkers selected in the present invention.
  • A) The methylation beta values of CpG islands of FGD5, GPC6, and MSC were investigated from 40 cases of colorectal cancer (stage I to IV) tumor/normal tissue pairs.
  • FIG. 5-1 shows the confirmation of postoperative recurrence suspected on CT images using the AMUSE score.
  • the boxes (AC) under the graphs indicate the duration of ACT (similar to Figures 5B-F and 6).
  • the boxes in gray and black indicate the AMUSE score and serum CEA protein level, respectively.
  • the date of surgery (P), the suspected recurrence on the images (Q), and the recurrence diagnosis (R) are shown. These six cases have a higher Q than the 28 recurrent cases.
  • ACT adjuvant chemotherapy
  • AMUSE amplicon of methylated sites using a specific enzyme
  • CEA carcinoembryonic antigen
  • Figure 5-2 is a continuation of Figure 5-1.
  • Figure 6-1 shows the specificity of the AMUSE score in 19 cases without recurrence.
  • the box (AC) under the graph indicates the period of ACT.
  • Two patients (5G and 5I) showed false positive findings (arrows).
  • Figure 6-2 is a continuation of Figure 6-1. 7 shows the clinical benefit of the AMUSE assay.
  • D and E Consideration of recurrence cases (D) and all cases (E) by serum CEA.
  • AMUSE amplicon of methylated sites using a specific enzyme
  • CEA carcinoembryonic antigen
  • Figure 8 shows the stratification of postoperative changes in tumor burden affected by adjuvant chemotherapy.
  • ACT adjuvant chemotherapy
  • MRD minimal residual disease.
  • the present invention provides a method for detecting colorectal cancer in a subject (hereinafter, sometimes referred to as the "detection method of the present invention"), comprising a step of determining the methylation status of at least one of the FGD5 gene and the GPC6 gene in the subject.
  • the methylation status of at least one of the FGD5 gene and the GPC6 gene is determined.
  • the detection method of the present invention further determines the methylation status of the MSC gene.
  • the detection method of the present invention determines the methylation status of the FGD5 gene, the GPC6 gene, and the MSC gene.
  • the FGD5 gene (FYVE, RhoGEF and PH domain containing 5, Gene ID: 152273), also known as "ZFYVE23", is located at chromosomal position 8q13.3 in humans and is a gene with two exons.
  • the FGD5 gene is known to be highly expressed in the spleen and lungs in normal human individuals.
  • the base sequence of the human FGD5 gene is shown in SEQ ID NO:1.
  • the GPC6 gene (glypican 6, Gene ID: 10082), also known as "OMIMD1", is located at chromosomal location 13q31.3-q32.1 in humans and is a gene with 12 exons.
  • the GPC6 gene is known to be highly expressed in the gallbladder and urinary bladder in normal human individuals.
  • the base sequence of the human GPC6 gene is shown in SEQ ID NO: 2.
  • the MSC gene (musculin, Gene ID: 9242), also known as "ABF1,” “MYOR,” “ABF-1,” “bHLHa22,” etc., is located at chromosomal position 8q13.3 in humans, and the MSC gene, which is a gene with two exons, is known to be highly expressed in the placenta and gallbladder in normal humans.
  • the base sequence of the human MSC gene is shown in SEQ ID NO: 3.
  • the methylation state of the above-mentioned gene is determined, but the method of determining the methylation state of a gene is known to those skilled in the art, and any method may be used.
  • "determining the methylation state of gene A” may also mean “determining the amount of methylation in gene A”.
  • the methylation state of a gene can be detected, and the amount can be determined, for example, by methyl-DNA immunoprecipitation (MeDIP), methylation-sensitive restriction enzyme (MSRE) analysis, bisulfite sequencing, etc.
  • MeDIP methyl-DNA immunoprecipitation
  • MSRE methylation-sensitive restriction enzyme
  • the methylation state of a gene can be determined by combining methylation-sensitive restriction enzyme analysis with a PCR method.
  • the PCR method combined with methylation-sensitive restriction enzyme analysis is more preferably real-time PCR or digital PCR. Details of the determination of the methylation state by combining methylation-sensitive restriction enzyme analysis with a PCR method are described in the examples of this specification.
  • determining the methylation status of gene A means qualitatively analyzing the presence or absence of a methylated region in the entire length or a part of gene A.
  • the nucleotide length of the region for determining the methylation status is not particularly limited, but in one embodiment, it is preferable to set a length suitable for PCR amplification.
  • the nucleotide length of the region for determining the methylation status may be, but is not limited to, usually 30 to 1000, preferably 50 to 300, more preferably 75 to 200, and particularly preferably 75 to 150 nucleotides. Note that when the region is amplified using a primer, this nucleotide length includes the primer binding site.
  • the primer pair that can be used in the PCR reaction can be appropriately designed using a method known per se to amplify part or all of the regions that can be methylated in the FGD5 gene, the GPC6 gene, and the MSC gene.
  • the following primer pairs can be used, but are not limited to these.
  • Primer pair for FGD5 gene a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO:4 and a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO:5.
  • Primer pair for GPC6 gene a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO:6, and a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO:7.
  • Primer pair for MSC gene a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO:8, and a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO:9.
  • the present invention makes it possible to determine whether a subject is suffering from colorectal cancer, and whether colorectal cancer has recurred in a subject after radical surgery for colorectal cancer, more quickly than with conventional methods. Furthermore, the present invention also makes it possible to evaluate whether adjuvant chemotherapy for colorectal cancer is effective in a subject receiving the chemotherapy. Therefore, the present invention is extremely useful in the field of colorectal cancer treatment.

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  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
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  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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Abstract

La présente invention concerne un procédé de détection du cancer colorectal chez un sujet, ledit procédé comprenant une étape de détermination de l'état de méthylation d'au moins l'un du gène FGD5 et du gène GPC6 chez le sujet.
PCT/JP2024/033732 2023-09-22 2024-09-20 Procédé de diagnostic du cancer colorectal et procédé d'évaluation de l'effet d'une chimiothérapie adjuvante appliquée au patient atteint d'un cancer colorectal Pending WO2025063294A1 (fr)

Applications Claiming Priority (2)

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JP2023-158487 2023-09-22
JP2023158487 2023-09-22

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WO2025063294A1 true WO2025063294A1 (fr) 2025-03-27

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022238560A1 (fr) * 2021-05-14 2022-11-17 Universal Diagnostics Sl Procédés pour la détection des maladies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022238560A1 (fr) * 2021-05-14 2022-11-17 Universal Diagnostics Sl Procédés pour la détection des maladies

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BARAULT LUDOVIC, AMATU ALESSIO, SIRAVEGNA GIULIA, PONZETTI AGOSTINO, MORAN SEBASTIAN, CASSINGENA ANDREA, MUSSOLIN BENEDETTA, FALCO: "Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer", GUT MICROBIOTA, BRITISH MEDICAL ASSOCIATION , LONDON, UK, vol. 67, no. 11, 1 November 2018 (2018-11-01), UK , pages 1995 - 2005, XP093024614, ISSN: 0017-5749, DOI: 10.1136/gutjnl-2016-313372 *
DMITRIEV ALEXEY A., RUDENKO EVGENIYA E., KUDRYAVTSEVA ANNA V., KRASNOV GEORGE S., GORDIYUK VASILY V., MELNIKOVA NATALIYA V., STAKH: "Epigenetic Alterations of Chromosome 3 Revealed by NotI-Microarrays in Clear Cell Renal Cell Carcinoma", BIOMED RESEARCH INTERNATIONAL, HINDAWI PUBLISHING CORPORATION, vol. 2014, 1 January 2014 (2014-01-01), pages 1 - 9, XP093293891, ISSN: 2314-6133, DOI: 10.1155/2014/735292 *

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