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WO2024144237A1 - Procédé de détection du cancer pulmonaire par l'utilisation d'un gène marqueur de méthylation spécifique du cancer pulmonaire - Google Patents

Procédé de détection du cancer pulmonaire par l'utilisation d'un gène marqueur de méthylation spécifique du cancer pulmonaire Download PDF

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WO2024144237A1
WO2024144237A1 PCT/KR2023/021647 KR2023021647W WO2024144237A1 WO 2024144237 A1 WO2024144237 A1 WO 2024144237A1 KR 2023021647 W KR2023021647 W KR 2023021647W WO 2024144237 A1 WO2024144237 A1 WO 2024144237A1
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gene
helt
cpg island
lung cancer
onecut1
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Korean (ko)
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오태정
안성환
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Genomictree Inc
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    • 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
    • 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
    • C12Q2523/00—Reactions characterised by treatment of reaction samples
    • C12Q2523/10—Characterised by chemical treatment
    • C12Q2523/125—Bisulfite(s)
    • 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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—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
    • C12Q2535/00—Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
    • C12Q2535/122—Massive parallel sequencing
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a novel use as a lung cancer-specific methylation marker of one or more genes selected from the group consisting of AATF (Apoptosis antagonizing transcription factor), HELT (helt bHLH transcription factor), and ONECUT1 (one cut homeobox 1), specifically.
  • AATF Apoptosis antagonizing transcription factor
  • HELT helt bHLH transcription factor
  • ONECUT1 one cut homeobox 1
  • the present invention relates to a composition for diagnosing lung cancer by detecting methylation of one or more genes selected from the group consisting of AATF, HELT, and ONECUT1 as a biomarker, a kit containing the same, and a method of providing information for lung cancer diagnosis.
  • CpGs there are those that appear exceptionally densely, and these are called CpG regions (CpG islands).
  • the CpG site is 0.2 to 3 kb in length, has a distribution percentage of C and G bases exceeding 50%, and refers to a highly concentrated region with a distribution percentage of CpG of over 3.75%.
  • Approximately 45,000 CpG sites appear in the entire human genome, and are particularly concentrated in promoter regions that regulate gene expression.
  • CpG sites appear in the promoters of housekeeping genes, which account for about half of human genes (Cross, S. et al., Curr. Opin. Gene Develop., 5:309, 1995). It is known that abnormal DNA methylation mainly occurs in the 5' regulatory region of the gene and reduces the expression of the gene.
  • Methylation of the expression control region of tumor-related genes is an important indicator of cancer, and therefore, it can be used in various fields such as diagnosis and early diagnosis of cancer, prediction of cancer risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to anticancer therapy. It is possible. Recently, there have been active attempts to investigate the promoter methylation of tumor-related genes in blood, sputum, saliva, feces, urine, etc. and use it to treat various cancers (Ahlquist, D.A. et al., Gastroenterol., 119:1219, 2000 ).
  • the purpose of the present invention is to provide a use for diagnosing lung cancer using a lung cancer-specific methylation biomarker.
  • the purpose of the present invention is to provide a method of providing information for lung cancer diagnosis by using methylated lung cancer marker genes as lung cancer-specific methylation biomarkers.
  • the present invention is capable of detecting methylation of one or more gene CpG islands selected from the group consisting of AATF (Apoptosis antagonizing transcription factor), HELT (helt bHLH transcription factor), and ONECUT1 (one cut homeobox 1).
  • AATF Apoptosis antagonizing transcription factor
  • HELT helt bHLH transcription factor
  • ONECUT1 one cut homeobox 1
  • the present invention provides a substance that can detect CpG island methylation of one or more genes selected from the group consisting of AATF (Apoptosis antagonizing transcription factor), HELT (helt bHLH transcription factor), and ONECUT1 (one cut homeobox 1) in patients. , or detecting CpG island methylation of one or more genes selected from the group consisting of AATF (Apoptosis antagonizing transcription factor), HELT (helt bHLH transcription factor), and ONECUT1 (one cut homeobox 1) in a patient's sample.
  • a lung cancer diagnosis method including the step of processing a material is provided.
  • Figure 1 is a description of the CpG microarray analysis design for discovering new methylation biomarker candidates in lung cancer tissue.
  • the present invention provides a substance that can detect CpG island methylation of one or more genes selected from the group consisting of AATF (Apoptosis antagonizing transcription factor), HELT (helt bHLH transcription factor), and ONECUT1 (one cut homeobox 1) in patients. , or detecting CpG island methylation of one or more genes selected from the group consisting of AATF (Apoptosis antagonizing transcription factor), HELT (helt bHLH transcription factor), and ONECUT1 (one cut homeobox 1) in a patient's sample.
  • a lung cancer diagnosis method including the step of processing a material is provided.
  • the HELT gene may include, for example, the sequence of SEQ ID NO: 2.
  • the CpG island of the HELT gene may be characterized as being present at positions -2,068 to -1,805 from the transcription start site (+1: C underlined in SEQ ID NO: 2) in the sequence of SEQ ID NO: 2.
  • the AATF target position is (based on the underlined C in the transcription start point sequence number 2) -2,068 ⁇ -2,024 GAGTGCGAAGTGTGTGCGCGCTCCTTTTTACCCATTCACGTTAGTTA , -1,849 ⁇ -1,805
  • Another method for detecting nucleic acids containing methylated CpG includes contacting a sample containing nucleic acids with an agent that modifies unmethylated cytosine and using a methylation-independent oligonucleotide primer. It includes the step of amplifying the CpG-containing nucleic acid of the sample using.
  • the oligonucleotide primer may be characterized in that it amplifies nucleic acids without distinguishing between modified methylated and unmethylated nucleic acids.
  • the amplified product is sequenced by the Sanger method using sequencing primers, or is described in connection with bisulfite sequencing for detection of methylated nucleic acids by next generation sequencing.
  • the markers included in the reverse primer are biotin, FAM, Cy5, Cy3, FITC, EDANS (5-(2'-aminoethyl)amino-1-naphthalene sulfate), tetramethylrhodamine (TMR), and tetramethylrhodamine isocyanate. It may be (TMRITC), x-rhodamine, DIG, or antibody, or a nanoparticle bound thereto, but is not limited thereto.
  • It may further include processing a probe capable of complementary hybridization to one or more genes selected from the group consisting of methylated AATF, HELT, and ONECUT1 specifically amplified by the primers.
  • the conditions used to achieve a particular level of stringency vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC/AT composition ratio), and the nucleic acid type (e.g., RNA, DNA) are considered in selecting hybridization conditions. A further consideration is whether the nucleic acid is immobilized, for example on a filter.
  • pruritic refers to the property of being susceptible to the above-mentioned cell growth abnormalities.
  • a pruritic specimen refers to a specimen that does not yet have a cell growth abnormality, but in which a cell growth abnormality exists or has an increased tendency to exist.
  • CpG-containing genes are typically DNA.
  • the method of the present invention can be applied to samples containing, for example, DNA or RNA including DNA and mRNA, where the DNA or RNA may be single-stranded or double-stranded, or may be a DNA-RNA hybrid. It can be characterized as a sample containing.
  • Nucleic acid mixtures may also be used. “Multiple” as used in the present invention includes both the case where there are a plurality of specific nucleic acid sequence regions to be detected in a kind of gene and the case where a plurality of target DNAs are included in one tube (single reactor).
  • the specific nucleic acid sequence to be detected may be a fraction of a larger molecule, or the specific sequence may initially exist in the form of a separate molecule that makes up the entire nucleic acid sequence.
  • the nucleic acid sequence need not be a nucleic acid that exists in pure form; the nucleic acid may be a minor fraction of a complex mixture, such as that containing the entire human DNA.
  • the present invention is intended to detect methylation of a plurality of target DNAs among samples in a single reactor.
  • the sample may include a plurality of multiple target DNAs, and the target DNA is abnormally methylated and expressed as well as the control gene. If this gene is suppressed or suppressed, any gene that affects the occurrence or progression of lung cancer can be used without limitation.
  • sample refers to a wide range of body fluids, including all biological body fluids obtained from individuals, body fluids, cell lines, tissue cultures, etc., depending on the type of analysis being performed. Methods for obtaining body fluid and tissue biopsies from mammals are commonly known.
  • the present invention relates to a kit for detecting methylation of target DNA comprising the composition.
  • the kit includes a compartmentalized carrier means for holding a sample, a container containing a reagent, a material capable of detecting methylation of the CpG island of the AATF gene, and a material capable of detecting methylation of the CpG island of the HELT gene. and a container containing one or more substances selected from the group consisting of substances capable of detecting methylation of the CpG island of the ONECUT1 gene. In some cases, it may further include a container containing a probe for detecting each of one or more methylated gene amplification products selected from the group consisting of AATF, HELT , and ONECUT1 .
  • lung cancer can be diagnosed by examining the methylation of one or more genes selected from the group consisting of marker genes AATF, HELT , and ONECUT1 .
  • One or more marker genes selected from the group consisting of AATF, HELT , and ONECUT1 using a sample showing a normal phenotype.
  • Cellular transformation means a change in the characteristics of a cell from one form to another, such as from normal to abnormal, non-neoplastic to neoplastic, undifferentiated to differentiated, or stem cell to non-stem cell. Additionally, the transformation can be recognized by cell morphology, phenotype, biochemical properties, etc.
  • “early identification” of cancer refers to discovering the possibility of cancer before it metastasizes, preferably before morphological changes are observed in sample tissues or cells. Additionally, “early identification” of cell transformation refers to the likelihood that transformation will occur at an early stage before the cell becomes a transformed form.
  • the present invention can use the AATF, HELT , or ONECUT1 genes individually as diagnostic or predictive markers for lung cancer, or use one or more genes, two or more genes, or three genes selected from the group consisting of AATF, HELT , and ONECUT1 in combination. .
  • AATF, HELT , and ONECUT1 genes it can be used in the form of a panel display. At this time, the number of genes that are methylated together and their importance can be ranked, weighted, and the level of possibility of developing cancer can be selected.
  • genomic DNA of cancer tissue and adjacent normal tissue obtained from surgical tissue of 13 lung cancer patients (stage I-III, provided by Chungnam National University Hospital) was ultrasonically pulverized ( Vibra Cell, SONICS) to produce genomic DNA fragments of approximately 200 to 300 bp.
  • the methyl binding domain (MBD), which is known to bind to methylated DNA, was used as described by Oh et al. (J. Mol. Diag. 2013;15(4):498-507).
  • MBD2bt After amplifying the obtained methylated DNA bound to MBD2bt according to the method described in Oh et al ((J. Mol. Diag. 2013;15(4):498-507)), human 244K human Hybridization was performed on a CpG microarray (Agilent, USA) ( Figure 1). After the hybridization, a series of washing processes were performed and then scanning was performed using a Laser scanner (Agilent, USA). The signal value was calculated from the microarray image using the Feature Extraction program v.9.5.3.1 (Agilent, USA) to calculate the relative difference in signal intensity between the lung cancer tissue of a lung cancer patient and the adjacent normal tissue sample.
  • probes with reliable signals were selected using the GeneSpring 7.3.1 program (Agilent, USA). From this, in order to select probes that were specifically hypermethylated in lung cancer, an ANOVA test was performed by comparing normal tissue and lung cancer tissue adjacent to lung cancer, and 6,854 probes with a P value of 0.05 or less were selected. Selected. From this, 621 probes that were hypermethylated more than 4 times on average in lung cancer tissue compared to normal tissue were re-selected, and among the genes that simultaneously showed hypermethylation in two or more contiguous probes in the promoter region, methylation was not reported in lung cancer. Three new biomarker gene candidates ( AATF, HELT, ONECUT1 ) were discovered ( Figure 2).
  • Example 2 Confirmation of methylation of three biomarker candidate genes in lung cancer cell lines
  • PCR reaction solution (20 ng of bisulfite-converted genomic DNA, TOPsimpleTM DryMIX-HOT (Enzynomics, P581H, Korea), and 2 uL (10 pmole) of PCR primers were treated at 95 degrees for 5 minutes, then incubated at 95 degrees. The reaction was performed a total of 45 times for 30 seconds, 60 degrees for 30 seconds, and 72 degrees for 30 seconds, and then the amplification of the PCR product was confirmed by electrophoresis using a 2.0% agarose gel.
  • Example 3 Measurement of methylation of biomarker genes in lung cancer patient cancer tissue and adjacent normal tissue
  • PCR reaction solution (20 ng of bisulfite-converted genomic DNA, TOPsimple DryMIX-HOT (Enzynomics, P581H, Korea), and 2 uL (10 pmole) of PCR primers were treated at 95 degrees for 5 minutes, then incubated at 95 degrees for 30 minutes. The reaction was performed a total of 45 times at 60 degrees for 30 seconds and at 72 degrees for 30 seconds, and then the amplification of the PCR product was confirmed by electrophoresis using a 2.0% agarose gel.
  • Table 3 shows the average methylation values of AATF, HELT, ONECUT1 genes in lung cancer tissue and normal tissue adjacent to it. A chi-square test was performed to determine whether there was a statistically significant difference in the methylation levels between lung cancer tissue and normal tissue. As a result, it was confirmed that the P value was less than 0.0001, indicating a high level of significance (Table 3).
  • methylation was analyzed in bronchial lavage fluid from lung cancer patients and patients with benign lung diseases other than lung cancer.
  • qMSP quantitative methylation-specific real time PCR
  • Example 5 Evaluation of lung cancer diagnostic ability of ONECUT1 novel biomarker gene in blood

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Abstract

La présente invention concerne une nouvelle utilisation d'un ou de plusieurs gènes choisis dans le groupe constitué du facteur de transcription antagoniste de l'apoptose (AATF), du facteur de transcription bHLH helt (HELT) et de homeobox 1 onecut (ONECUT1) en tant que marqueur(s) de méthylation spécifique du cancer pulmonaire et, plus particulièrement, à : une composition pour diagnostiquer le cancer pulmonaire en utilisant un ou plusieurs gènes choisis dans le groupe constitué de AATF, HELT et ONECUT1 comme biomarqueur(s) de manière à détecter la méthylation ; un kit comprenant la composition ; et un procédé pour fournir des informations pour le diagnostic du cancer pulmonaire.
PCT/KR2023/021647 2022-12-27 2023-12-27 Procédé de détection du cancer pulmonaire par l'utilisation d'un gène marqueur de méthylation spécifique du cancer pulmonaire Ceased WO2024144237A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271455A1 (en) * 2013-03-14 2014-09-18 City Of Hope Dna methylation biomarkers for small cell lung cancer
WO2019144057A1 (fr) * 2018-01-19 2019-07-25 The University Of North Carolina At Chapel Hill Marqueurs de méthylation pour mélanome et utilisations associées
CN114651073A (zh) * 2019-11-11 2022-06-21 通用诊断公司 结肠直肠癌和/或进行性腺瘤的检测

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271455A1 (en) * 2013-03-14 2014-09-18 City Of Hope Dna methylation biomarkers for small cell lung cancer
WO2019144057A1 (fr) * 2018-01-19 2019-07-25 The University Of North Carolina At Chapel Hill Marqueurs de méthylation pour mélanome et utilisations associées
CN114651073A (zh) * 2019-11-11 2022-06-21 通用诊断公司 结肠直肠癌和/或进行性腺瘤的检测

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIANG RUNZHANG; LI XIAOSONG; LI WEIQUAN; ZHU XIAO; LI CHEN: "DNA methylation in lung cancer patients: Opening a "window of life" under precision medicine", BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, FR, vol. 144, 13 October 2021 (2021-10-13), FR , XP086861504, ISSN: 0753-3322, DOI: 10.1016/j.biopha.2021.112202 *
LU FENG, WU JUAN, ZOU HONGPENG, YANG XIN, WU YONGBING, XU JIANJUN: "The Oncogenic and Immunological Roles of Apoptosis Antagonistic Transcription Factors in Human Tumors: A Pan-Cancer Analysis", OXIDATIVE MEDICINE AND CELLULAR LONGEVITY, HINDAWI PUBLISHING CORPORATION, US, vol. 2022, 12 October 2022 (2022-10-12), US , pages 1 - 16, XP093187335, ISSN: 1942-0900, DOI: 10.1155/2022/3355365 *

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