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WO2024241624A1 - Procédé d'aide au diagnostic du cancer hépatocellulaire - Google Patents

Procédé d'aide au diagnostic du cancer hépatocellulaire Download PDF

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WO2024241624A1
WO2024241624A1 PCT/JP2024/001080 JP2024001080W WO2024241624A1 WO 2024241624 A1 WO2024241624 A1 WO 2024241624A1 JP 2024001080 W JP2024001080 W JP 2024001080W WO 2024241624 A1 WO2024241624 A1 WO 2024241624A1
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hepatocellular carcinoma
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寛 末廣
勇希 國宗
隆弘 山▲崎▼
一成 佐伯
康貴 松井
朋美 星田
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Yamaguchi University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • 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 assisting in the diagnosis of hepatocellular carcinoma in a subject.
  • Liver cancer is the third leading cause of cancer deaths worldwide, with 830,000 deaths per year.
  • Hepatocellular carcinoma in particular accounts for 90.1% of primary liver cancers, and the relative 5-year survival rate is 62.9% for Stage I, 45.4% for Stage II, 15.9% for Stage III, and 4.5% for Stage IV according to the TNM Stage classification.
  • the prognosis worsens as the disease progresses, and it is still one of the cancer types with the poorest prognosis. Therefore, the best way to improve prognosis is to detect hepatocellular carcinoma early.
  • Tumor markers for hepatocellular carcinoma include ⁇ -fetoprotein (AFP) and protein induced by vitamin K absorption or antagonist-II (PIVKA-II).
  • AFP is a serum protein specific to the fetal stage that is produced in the fetal liver and yolk sac, and is used to diagnose hepatocellular carcinoma because its levels also increase in hepatocellular carcinoma.
  • AFP also increases in cancers other than hepatocellular carcinoma, such as chronic hepatitis, liver cirrhosis, neurodegenerative diseases, and nonseminomatous testicular cancer, so its use alone is considered undesirable.
  • PIVKA-II is an abnormal prothrombin with no clotting activity that is produced when there is a vitamin K deficiency.
  • PIVKA-II has higher sensitivity and specificity than AFP in diagnosing hepatocellular carcinoma.
  • PIVKA-II increases when warfarin is administered, when cephalosporin antibiotics are used, in patients with obstructive jaundice, in extremely malnutrition, and in cases of alcoholic liver damage, giving false positives.
  • caution is required as PIVKA-II normalizes when vitamin K preparations are administered even in the presence of hepatocellular carcinoma.
  • liver cancer treatment guidelines also recommend measuring two or more tumor markers when diagnosing small hepatocellular carcinoma.
  • AFP alone has a sensitivity of 53% and a specificity of 90%
  • PIVKA-II alone has a sensitivity of 61% and a specificity of 70%
  • the combined use of both improves sensitivity to 78%, but the specificity remains low at 62%, meaning that AFP and PIVKA-II alone have limitations in screening for early-stage hepatocellular carcinoma (see Non-Patent Document 2).
  • Non-Patent Document 3 there is a method for diagnosing hepatocellular carcinoma by performing sodium hydrogen sulfite treatment (bisulfite treatment) and detecting methylation of the SEPT9 gene (see Non-Patent Document 3), and it has been reported that the SEPT9 gene is an important epi-driver gene for carcinogenesis in the liver, as SEPT9 expression is suppressed by excessive methylation of the CpG sequence of SEPT9, and that the combination of AFP and the degree of methylation of the CpG sequence of the SEPT9 gene improves the detection sensitivity and specificity of hepatocellular carcinoma (see Non-Patent Documents 4 and 5).
  • the present inventors have focused on the relationship between methylation of CpG sequences and cancer, and have disclosed a method for predicting the presence or absence of a colon tumor by measuring the methylation level of one or more CpG sequences in or near the transcriptional regulatory region of Twist homolog 1 (Twist 1) (see Patent Document 1), a method for predicting the presence or absence of a colon tumor by measuring the methylation level of CpG in the TWIST1, NDRG4, BMP3, or SEPT9 genes in stool or serum (see Patent Document 2), a method for diagnosing cancer by measuring the methylation level of CpG in the RUNX3 gene (see Patent Document 3), and the fact that a combination of methylated SEPT9 and AFP in serum CpG sequences is useful for diagnosing hepatocellular carcinoma (see Non-Patent Documents 6 and 7).
  • methylation of the CpG sequence of Homeobox A1 (HOXA1), another tumor marker, may be useful in diagnosing cancer when combined with other cancer markers (see Non-Patent Documents 8 to 10).
  • Patent Document 1 sodium hydrogen sulfite treatment (bisulfite treatment) is used, which has the problem that quantitative analysis is not possible when the degree of methylation is low, and therefore a body fluid of 10 mL or more is required for whole blood and 3.5 mL or more for plasma.
  • Non-Patent Document 9 uses a combination of AFP and AFP-L3 as tumor markers, which are not covered by insurance in Japan, and analyzes the methylation of three genes, HOXA1, EMX1, and TSPYL5. For this reason, it is considered difficult to put this technology into practical use in terms of insurance coverage, test efficiency, and test costs.
  • Plasma mSEPT9 A Novel Circulating Cell-free DNA-Based Epigenetic Biomarker to Diagnose Hepatocellular Carcinoma. eBioMedicine Volume 30, April 2018, Pages 138-147 Villanueva A, Portela A, Sayols S, et al. DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma. Hepatology. 2015; 61: 1945-1956. Li B, Huang H, Huang R, et al. SEPT9 Gene methylation as a noninvasive marker for hepatocellular carcinoma. Dis Markers. 2020; 9:6289063. Kotoh Y, Suehiro Y, Saeki I, et al.
  • Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma. Hepatol Commun 2020; 4: 461-470.
  • Ayano Yamazaki et al. Screening for hepatocellular carcinoma by liquid biopsy using highly sensitive DNA methylation analysis technology: Comparison of diagnostic performance between methylated SST and methylated SEPT9, Yamaguchi Medical Journal, Vol. 70, No. 3, pp. 89-98, 2021 Paco A, de Bessa Garcia SA, Freitas R. Methylation in HOX Clusters and Its Applications in Cancer Therapy. Cells: 2020; 9: 1613.
  • the objective of the present invention is to provide a method for easily detecting hepatocellular carcinoma, without relying solely on AFP, by using bodily fluids such as serum collected during health checkups.
  • the inventors discovered a method for assisting in the diagnosis of hepatocellular carcinoma in a subject by measuring the CpG methylation level in DNA of the transcriptional regulatory region of the SEPT9 gene or the HOXA1 gene in addition to the AFP level or PIVKA-II level in the serum of the subject using a specified method, and evaluating these indicators in combination, thereby completing the present invention.
  • a method for assisting in the diagnosis of hepatocellular carcinoma in a subject comprising: (A) measuring a CpG methylation level in a region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene, or a region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the HOXA1 gene in DNA extracted from a body fluid collected from the subject; and (B) measuring an ⁇ -fetoprotein (AFP) level or a protein induced by vitamin K absorption or antagonist-II (PIVKA-II) level in the body fluid collected from the subject; performing a statistical analysis based on at least the two indices (A) and (B); and based on a value obtained by the statistical analysis, The methylation level of CpG in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene, or the region of +1 to +1000 of the
  • step (c) amplifying a part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured using the DNA treated with the restriction enzyme in step (b) as a template; (d) measuring the methylation level of CpG in the DNA of the transcriptional regulatory region and/or the region of the transcription start site between +1 and +1000, which were amplified in step (c), based on the results of the amplification in step (c); a method for measuring a methylation level of CpG in a region of +1 to +1000 of a transcription regulatory region and/or a transcription start site of a gene to be measured, comprising steps (a) to (d) above, characterized in that the DNA in the region of +1 to +1000 of the transcription regulatory region and/or the transcription start site to be amplified in step (c) contains one or more sequences recognized by the restriction enzyme used in step (
  • [3] A method for assisting in the diagnosis of hepatocellular carcinoma in a subject according to [1] or [2] above, characterized in that a logarithmic transformation of the AFP level or PIVKA-II level is used in the statistical analysis.
  • [4] A method for assisting in the diagnosis of hepatocellular carcinoma according to [1] or [2] above, characterized in that in the statistical analysis, the statistical analysis is performed based on three indicators: the CpG methylation level, the AFP level, and the PIVKA-II level in DNA in the region +1 to +1000 of the transcriptional regulatory region and/or transcription start point of the SEPT9 gene, or the CpG methylation level, the AFP level, and the PIVKA-II level in DNA in the region +1 to +1000 of the transcriptional regulatory region and/or transcription start point of the HOXA1 gene.
  • [10] A method for assisting in the diagnosis of hepatocellular carcinoma in a subject according to [1] or [2] above, characterized in that, when two or more types of restriction enzymes are used in step (a), the DNA in the transcriptional regulatory region and/or the region from +1 to +1000 of the transcription start site that is the target of amplification in step (c) does not contain a sequence recognized by at least one of the restriction enzymes used in step (a).
  • [11] A method for assisting in the diagnosis of hepatocellular carcinoma in a subject described in [1] or [2] above, characterized in that in step (a), at least HhaI, HpaII and BstUI are added to the reaction vessel as restriction enzymes.
  • the present invention makes it possible to easily assist in the diagnosis of hepatocellular carcinoma in subjects using small amounts of serum, etc.
  • 1 is a graph showing the relationship between the number of methylated SEPT9 genes (copy numbers) obtained by a single treatment (condition 1) and the copy numbers obtained by a two-step treatment (condition 2).
  • 1 is a graph showing the relationship between the number of methylated HOXA1 genes (copy numbers) obtained by a single treatment (condition 1) and the copy numbers obtained by a two-step treatment (condition 2).
  • This figure shows the results of ROC analysis using indicators 1 to 3 out of four indicators, namely methylated SEPT9 (mSEPT9), methylated HOXA1 (mHOXA1), AFP, and PIVKA-II, in the control group and all viral hepatocellular carcinoma patient groups (Viral All Stage).
  • FIG. 13 shows the results of ROC analysis using indicators 1 to 3 out of four indicators, mSEPT9, mHOXA1, AFP, and PIVKA-II, in a group of patients with nonviral hepatocellular carcinoma (nonViral All Stage).
  • This figure shows the results of ROC analysis using indicators 1 to 3 out of four indicators, mSEPT9, mHOXA1, AFP, and PIVKA-II, in a group of patients with early viral hepatocellular carcinoma (Early Viral).
  • FIG. 1 shows the results of determining the AUC values for combinations of AFP, PIVKA-II, and mSEPT9 when they were normally distributed and when they were not normally distributed.
  • FIG. 13 shows the results of determining the AUC values for combinations of AFP, PIVKA-II, and mHOXA1 when they are normally distributed and when they are not normally distributed.
  • FIG. 1 shows the results of determining AUC values and statistical analysis results when AFP, PIVKA-II, and mSEPT9 were all normally distributed in the entire hepatocellular carcinoma.
  • FIG. 1 shows the results of determining the AUC value, sensitivity, and specificity in early stage hepatocellular carcinoma, early stage non-viral hepatocellular carcinoma, and early stage viral hepatocellular carcinoma when AFP, PIVKA-II, and mSEPT9 were all normally distributed.
  • FIG. 1 shows the results of determining AUC values and statistical analysis results when AFP, PIVKA-II, and mHOXA1 were all normally distributed in the entire hepatocellular carcinoma.
  • FIG. 1 shows the results of determining the AUC value, sensitivity, and specificity in early stage hepatocellular carcinoma, early stage non-viral hepatocellular carcinoma, and early stage viral hepatocellular carcinoma when AFP, PIVKA-II, and mHOXA1 were all normally distributed.
  • FIG. 13 shows the relationship between mSEPT9 and mHOXA1 measurement values when DNA was extracted from 1.0 mL or 0.4 mL of serum specimens.
  • the method for assisting in the diagnosis of hepatocellular carcinoma of the present invention comprises: A method for assisting in the diagnosis of hepatocellular carcinoma in a subject, comprising: (A) measuring a CpG methylation level in a region of +1 to +1000 of a transcriptional regulatory region and/or a transcriptional start site of a SEPT9 gene in DNA extracted from a body fluid collected from a subject, or a region of +1 to +1000 of a transcriptional regulatory region and/or a transcriptional start site of a HOXA1 gene; and (B) measuring an ⁇ -fetoprotein (AFP) level or a protein induced by vitamin K absorption or antagonist-II (PIVKA-II) level in the body fluid collected from the subject; and performing a statistical analysis based on at least the two indices (A) and (B); The methylation level of CpG in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene, or the region
  • step (c) amplifying a part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured using the DNA treated with the restriction enzyme in step (b) as a template; (d) measuring the methylation level of CpG in the DNA of the transcriptional regulatory region and/or the region of the transcription start site +1 to +1000 targeted for amplification in step (c) based on the results of the amplification in step (c); a method for measuring a methylation level of CpG in a region of +1 to +1000 of a transcription regulatory region and/or a transcription start site of a gene to be measured, comprising steps (a) to (d) above, characterized in that the DNA in the region of +1 to +1000 of the transcription regulatory region and/or the transcription start site to be amplified in step (c) contains one or more sequences recognized by the restriction enzyme used in step (a);
  • the subject in this specification is not particularly limited as long as it is a human, and can be, for example, a subject whose cancer is unknown. Such a subject also includes a subject who has had cancer in the past and has since been cured, but whose cancer is unknown at the time of examination.
  • the body fluid can be serum, plasma, blood, saliva, urine, etc.
  • serum is generally used in blood biochemistry tests.
  • plasma is limited to use in coagulation system tests and blood glucose tests.
  • plasma samples are generally used in liquid biopsies including methylation analysis. This is because when serum is used, white blood cells are destroyed during the coagulation process, and white blood cell-derived DNA increases in the serum, which can affect the measurement in liquid biopsy.
  • whole blood is used as a sample for liquid biopsy to extract DNA, the white blood cell-derived DNA accounts for the majority, and the tumor-derived DNA is relatively diluted, resulting in difficulty in detecting the tumor-derived DNA. For this reason, whole blood samples cannot be said to be optimal as samples for liquid biopsy.
  • blood biochemistry tests and methylation analysis, AFP, and PIVKA-II tests for the diagnosis of hepatocellular carcinoma can be performed with just one serum collection tube in a health checkup.
  • serum is generally used in blood biochemistry tests in health checkups.
  • plasma samples are generally used in liquid biopsies, including methylation analysis for cancer diagnosis. That is, when attempting to diagnose cancer in a health checkup using conventional methods, two blood collection tubes, one for serum and one for plasma, are required, and the large amount of blood collected places a great physical and mental burden on the subject.
  • both biochemical testing and methylation analysis can be performed using only one collection tube. Furthermore, because only a small amount of blood needs to be collected, the burden on the subject is reduced, and the workload of hospitals that collect blood from many subjects at once can also be reduced.
  • body fluid When analyzing all indicators, about 0.001 to 10 mL of body fluid can be used, and it may be 0.01 to 2 mL, 0.03 to 1 mL, 0.04 to 0.5 mL, 0.05 to 0.3 mL, 0.07 to 0.2 mL, or 0.08 to 0.1 mL. Since the method for assisting in the diagnosis of hepatocellular carcinoma does not require bisulfite treatment, it is possible to diagnose hepatocellular carcinoma with a small amount of body fluid.
  • CpG methylation levels and AFP levels in DNA in the region between +1 and +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene CpG methylation levels and AFP levels in DNA in the region between +1 and +1000 of the transcriptional regulatory region and/or transcription start site of the HOXA1 gene
  • CpG methylation levels and PIVKA-II levels in DNA in the transcriptional regulatory region and/or the region between +1 and +1000 of the transcriptional start site of the HOXA1 gene CpG methylation levels and PIVKA-II levels in DNA in the transcriptional regulatory region and/or the region between +1 and +1000 of the transcriptional start site of the HOXA1 gene
  • Three indexes may be used as at least the two indexes (A) and (B) above, and the following four combinations are exemplified.
  • indicators may be used as at least the two indicators (A) and (B) above, including a combination of the CpG methylation level in DNA in the region of the transcriptional regulatory region and/or the transcriptional start site of the SEPT9 gene from +1 to +1000, the CpG methylation level in DNA in the region of the transcriptional regulatory region and/or the transcriptional start site of the HOXA1 gene from +1 to +1000, the AFP level, and the PIVKA-II level.
  • the methylation level is measured using the CpG methylation level in the DNA of the transcriptional regulatory region and/or the region +1 to +1000 of the transcription start point of the SEPT9 gene or the CpG methylation level in the DNA of the transcriptional regulatory region and/or the region +1 to +1000 of the transcription start point of the HOXA1 gene as an indicator, it becomes possible to diagnose hepatocellular carcinoma without analyzing other methylation markers such as EMX1 and TSPYL5.
  • examples of multivariate analysis include logistic regression analysis, linear discriminant, and multiple regression analysis.
  • examples of continuous variables include the Mann-Whitney U test for two-group testing, the Kruskal-Wallis test and Dunn's test for multiple comparisons, and the chi-square test and Fisher's exact test for categorical variable testing.
  • indicators used in the statistical analysis indicators required for the FIB-4 index, such as sex, age, alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, and platelet count, may be added for analysis.
  • the AFP level, the PIVKA-II level, the CpG methylation level in DNA in the region of +1 to +1000 of the transcription regulatory region and/or the transcription start point of the SEPT9 gene, and/or the CpG methylation level in DNA in the region of +1 to +1000 of the transcription regulatory region and/or the transcription start point of the HOXA1 gene may be logarithmically or power-transformed, and a normally distributed value or a binarized value based on a predetermined cutoff value may be used.
  • the combination of values to be normally distributed is not particularly limited, and specific examples include the following: When only one indicator is normally distributed, only one of the following indicators can be normally distributed: AFP level, PIVKA-II level, CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start point of the SEPT9 gene, or CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start point of the HOXA1 gene.
  • the following two indicators can be normally distributed: AFP level and PIVKA-II level; AFP level and CpG methylation level in DNA of the +1 to +1000 region of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene; AFP level and CpG methylation level in DNA of the transcriptional regulatory region of the HOXA1 gene; PIVKA-II level and CpG methylation level in DNA of the +1 to +1000 region of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene; PIVKA-II level and CpG methylation level in DNA of the +1 to +1000 region of the transcriptional regulatory region and/or transcription start site of the HOXA1 gene.
  • the AFP level, the PIVKA-II level, and the CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene When the three indices are normally distributed, the AFP level, the PIVKA-II level, and the CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene; the AFP level, the PIVKA-II level, and the CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene;
  • the four indicators When the four indicators are normally distributed, the four indicators, AFP level, PIVKA-II level, CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the SEPT9 gene, and CpG methylation level in DNA in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site of the HOXA1 gene, can be normally distributed.
  • ⁇ 0 is the intercept (constant term)
  • ⁇ 1 to ⁇ 5 are regression coefficients
  • Sex is gender (substitute 1 for female and 0 for male)
  • Age is age
  • norm_APF is normally distributed AFP value
  • norm_PIVKA-II is normally distributed PIVKA-II value
  • norm_mSEPT9 is normally distributed methylated SEPT9 value.
  • norm means normally distributed.
  • the calculation can be based on the following formula (II).
  • ⁇ 0 is the intercept (constant term)
  • ⁇ 1 to ⁇ 5 are regression coefficients
  • Sex is gender (substitute 1 for female and 0 for male)
  • Age is age
  • norm_APF is normally distributed AFP value
  • norm_PIVKA-II is normally distributed PIVKA-II value
  • norm_mHOXA1 is normally distributed methylated HOXA1 value.
  • norm means normally distributed as above.
  • ⁇ Diagnosis of hepatocellular carcinoma> In the case of using the predicted value to assist in the diagnosis of hepatocellular carcinoma in a subject, a statistical analysis is performed based on at least two indices (A) and (B) in the body fluid collected from the subject, and the closer the value (predicted value) obtained by the statistical analysis is to 1, the higher the possibility of "having liver cancer", while the closer the predicted value is to 0, the higher the possibility of "not having liver cancer", so that hepatocellular carcinoma can be diagnosed based on the value (predicted value).
  • a statistical analysis is performed based on at least two indices (A) and (B) collected from the subject, and the value obtained by the statistical analysis is compared with the value obtained by the statistical analysis in a control subject not suffering from hepatocellular carcinoma, and when the value obtained by the statistical analysis based on at least two indices (A) and (B) of the subject is higher than the value obtained by the statistical analysis in a control subject not suffering from hepatocellular carcinoma, the subject can be evaluated as being highly likely to suffer from hepatocellular carcinoma, and when the compared value is lower, the subject can be evaluated as being low likely to suffer from hepatocellular carcinoma.
  • the value obtained by the statistical analysis of the control may be a value measured each time, or may be a value analyzed in advance.
  • the body fluids of the subject and the control to be compared are prepared by substantially the same method, or are obtained by substantially the same measurement method.
  • any cutoff value can be appropriately set to evaluate whether the value obtained by the statistical analysis based on at least the two indicators (A) and (B) in the body fluid collected from the subject is higher or lower than the value obtained by the statistical analysis in a control subject not suffering from hepatocellular carcinoma.
  • cutoff values include the mean value of "the value obtained by the statistical analysis in the control subject", the mean value ⁇ standard deviation (SD), the mean value ⁇ 2SD, the mean value ⁇ 3SD, the median, the median value ⁇ SD, the median value ⁇ 2SD, the median value ⁇ 3SD, etc.
  • the cutoff value can be calculated using the Youden Index, etc. by creating an ROC (Receiver Operating Characteristic) curve using statistical analysis software based on the data "of the value obtained by statistical analysis based on the two indicators (A) and (B) in the body fluid collected from the subject” and the data "of the value obtained by statistical analysis based on the two indicators (A) and (B) in the body fluid collected from the control.”
  • ROC Receiveiver Operating Characteristic
  • Hepatocellular carcinoma also includes early stage hepatocellular carcinoma.
  • the early stage hepatocellular carcinoma means hepatocellular carcinoma at stage 0 or A according to the BCLC staging system (Barcelona Clinical Liver Cancer Staging System) described in Non-Patent Document 4 mentioned above.
  • AFP levels and PIVKA-II levels can be measured by known techniques, specifically, enzyme immunoassay (EIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), chemiluminescent immunoassay (CLIA), chemiluminescent enzyme immunoassay (CLEIA), and immunochromatography.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • FIA fluorescent immunoassay
  • CLIA chemiluminescent immunoassay
  • CLIA chemiluminescent enzyme immunoassay
  • CLIA chemiluminescent enzyme immunoassay
  • CpG methylation refers to a state in which a methyl group is added to the carbon atom at the 5th position of the cytosine (C) residue in a CpG sequence, which is a two-base sequence (dinucleotide) in which a guanine (G) base appears next to a cytosine (C) base in DNA.
  • CpG sequence which is a two-base sequence (dinucleotide) in which a guanine (G) base appears next to a cytosine (C) base in DNA.
  • p represents the phosphodiester bond between cytosine and guanine.
  • the CpG methylation level in this specification can be evaluated by the number of DNA fragments in which the cytosines in the CpG are methylated (copy number) when a specific region of the DNA encoding the gene to be measured is amplified to obtain DNA fragments.
  • the level of methylation include the number of copies per unit amount of the solution treated with the restriction enzyme in step (c), for example, per 1 ⁇ L, the number of copies per total amount of the solution treated with the restriction enzyme in step (c), the number of copies per unit amount of the DNA source sample (e.g., per mL of serum), and the ratio of methylated DNA to the internal control.
  • the internal control may be any human DNA that does not have a cleavage site for a methylation-sensitive restriction enzyme within the PCR amplified region, and examples of the internal control include human TERT and RNase P.
  • the transcriptional regulatory region of a gene refers to a region that contains a nucleic acid sequence to which a transcriptional regulatory factor binds and that regulates the amount of transcription of the gene.
  • This transcriptional regulatory region includes promoter regions, enhancer regions, suppressor regions, etc.
  • the transcription start point refers to the site where transcription of mRNA begins, and corresponds to the position corresponding to the first base of mRNA.
  • the transcription start point is represented as "+1,” and the position one base upstream of the transcription start point is represented as "-1.” Therefore, +1000 of the transcription start point means the position 999 bases downstream when the transcription start point is +1.
  • the region of +1 to +1000 from the transcription start point in this specification includes exons and introns within the region of +1 to +1000 from the transcription start point, and examples of exons include exon 1, exon 2, and exon 3, and examples of introns include intron 1 and intron 2.
  • examples of the region of +1 to +1000 from the transcription start point include the region of +1 to +500 from the transcription start point, the region of +1 to +350 from the transcription start point, the region of +1 to +100 from the transcription start point, and the region of +1 to +60 from the transcription start point.
  • the transcriptional regulatory region and/or the region between +1 and +1000 from the transcription start point in this specification can include a region between +1 and +1000 from the transcription start point and 100 bases upstream to 100 bases downstream from the adenine (A) position of the start codon, preferably a region between 50 bases upstream to 50 bases downstream from the start codon.
  • the start codon refers to the codon that serves as the start point of protein synthesis when mRNA is translated into protein.
  • the reaction vessel in this specification is not particularly limited as long as it is a vessel capable of restriction enzyme treatment and is sealable.
  • sealed means a state in which the liquid in the reaction vessel cannot leak out to the outside, and “sealed” means, for example, putting a lid on a vessel for holding liquid, such as a microtube.
  • the gene to be measured in this specification is not particularly limited, so long as it contains one or more sequences recognized by the restriction enzyme used in step (a) in the transcriptional regulatory region and/or the region from +1 to +1000 of the transcription start point of the gene.
  • the recognition sequences of the four bases are HhaI (GCG/C), HpaII (C/CGG), and BstUI (CG/CG)
  • the recognition sequence of the five bases is Hpy99I (CGWCG/)
  • the recognition sequence of the six bases is SacII (CCGC/GG), SmaI (CCC/GGG), AatII (GACGT/C), AccI (GT/MKAC), Aor13HI (T/CCGGA), Aor51HI (AGC/GCT), BspT104I (TT/CGAA), BssHII (G/CGCGC), Cfr10I (R/CCGGY), ClaI (AT/CGAT), and Eco52I (C/G Examples of such a gene include
  • the transcriptional regulatory region and/or the region from +1 to +1000 of the transcriptional start site of the gene may contain one or more sequences recognized by the restriction enzyme described in step (a)(ii), preferably one or two sequences, and may contain one or more sequences recognized by BstUI, preferably one sequence.
  • W means A or T
  • M means A or C
  • K means G or T
  • R means A or G
  • Y means C or T.
  • genes to be measured include the SEPT9 gene, HOXA1 gene, SST gene, RUNX3 gene, ADAMTS2 gene, PCDH10 gene, SEMA5A gene, SPSB4 gene, BMP3 gene, NDRG4 gene, SDC2 gene, etc., in which the CpG methylation level in DNA in the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start point in a subject varies depending on the presence of cancer.
  • the DNA containing the gene to be measured can be obtained by extraction from the body fluid, and is preferably DNA extracted from serum.
  • Methods for extracting DNA from body fluids include phenol extraction, phenol-chloroform extraction, alkaline dissolution, etc., and methods using commercially available DNA extraction reagents.
  • the restriction enzymes to be added to the reaction vessel include (ii) at least one, preferably two or more, more preferably two restriction enzymes selected from HhaI, HpaII, Hpy99I, SacII, SmaI, NotI, AatII, AccI, Aor13HI, Aor51HI, BspT104I, BssHII, Cfr10I, ClaI, CpoI, Eco52I, HaeII, MluI, NaeI, NruI, PmaCI, Psp1406I, PvuI, SalI, and SnaBI, and (iii) BstUI.
  • BstUI is also sold under the names AccII, Bsh1236I, BspFNI, BstFNI, FnuDII, or MvnI, depending on the manufacturer of the restriction enzyme.
  • the above restriction enzyme is a methylation-sensitive restriction enzyme.
  • a methylation-sensitive restriction enzyme is a restriction enzyme that can distinguish between methylated and unmethylated CpG in a sequence that the restriction enzyme recognizes.
  • methylation-sensitive restriction enzymes are used in a method for analyzing CpG methylation, and utilize the phenomenon that cytosine in a sequence that the restriction enzyme recognizes becomes methylated and the DNA cannot be cut.
  • Preferred combinations of the above restriction enzymes include two types, HhaI and BstUI, HpaII and BstUI, and three types, HhaI, HpaII and BstUI. Note that it is preferable to use heat-inactivated BstUI as BstUI, specifically BstUI that remains active even after treatment at 65°C for 20 minutes (or treatment at 60°C for 1 hour).
  • the restriction enzyme reaction buffer may be any buffer capable of cleaving DNA by the enzymatic reaction of the restriction enzyme used, and may be appropriately selected depending on the restriction enzyme used. In addition to commercially available restriction enzyme reaction buffers, PCR buffers may also be used.
  • Step (b) is a step of performing restriction enzyme treatment.
  • the treatment with the restriction enzyme has two temperature stages, and the treatment temperature in the first stage can be appropriately adjusted, but is, for example, within the range of 30 to 50° C., preferably within the range of 35 to 40° C., and more preferably within the range of 36 to 38° C.
  • the treatment time in the first stage can be appropriately adjusted, but is, for example, within the range of 0.5 to 16 hours, preferably 0.7 to 8 hours, more preferably 0.9 to 4 hours, and even more preferably 1 to 2 hours.
  • the processing temperature in the second stage can be adjusted as appropriate, but is, for example, within the range of 50 to 70°C, preferably within the range of 55 to 65°C, and more preferably within the range of 58 to 62°C.
  • the processing time in the second stage can be adjusted as appropriate, but is, for example, within the range of 0.5 to 20 hours, preferably within the range of 6 to 18 hours, more preferably within the range of 12 to 17 hours, and even more preferably within the range of 14 to 16 hours.
  • the first and second stages are performed consecutively while the reaction vessel is sealed.
  • the second stage is performed without releasing the sealed state, such as by opening the lid of the reaction vessel after the first stage.
  • the lid of the reaction vessel is opened after the first stage, a restriction enzyme is added, the lid is closed and sealed, and the second stage is performed. Therefore, the work of adding the restriction enzyme in the second stage is complicated and requires time and effort. There is also a problem that the liquid in the reaction vessel evaporates, changing the composition inside the reaction vessel.
  • all the restriction enzymes are added to the reaction vessel at once, so there is no need to open the lid of the reaction vessel after the first stage.
  • step (c) the DNA treated with the restriction enzyme in step (b) is used as a template to amplify a part or all of the region of +1 to +1000 of the transcription regulatory region and/or transcription start site of the gene to be measured (SEPT9 gene and/or HOXA1 gene).
  • the part or all of the region of +1 to +1000 of the transcription regulatory region and/or transcription start site of the gene to be measured to be amplified may be a region containing at least one sequence recognized by the restriction enzyme used in step (a), and may be a region containing one or more and four or less, preferably one or more and three or less.
  • step (a) there may be one, two, three, or four sequences recognized by one type of methylation-sensitive restriction enzyme used in step (a).
  • any one of the restriction enzymes may recognize only one sequence, or one type of restriction enzyme may recognize one sequence and another type of restriction enzyme may recognize two or more sequences, preferably two sequences.
  • the region to be amplified may be a part of the transcriptional regulatory region of the gene to be measured and a part of the region from +1 to +1000 of the transcription start site, only a part of the transcriptional regulatory region of the gene to be measured, only a part of the region from +1 to +1000 of the transcription start site of the gene to be measured, or only a part of the transcriptional regulatory region of the gene to be measured and a part of the region of exon 1.
  • the length of the transcriptional regulatory region of the gene to be measured and/or the part or whole of the region between +1 and +1000 of the transcription start site to be amplified can be 30 to 1000 bases, preferably 40 to 200 bases, and more preferably 50 to 150 bases.
  • the region may not contain a sequence recognized by at least one of the restriction enzymes used in step (a) in part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be amplified.
  • three types of restriction enzymes, X, Y, and Z may be used in step (a), and the region may contain one or more sequences recognized by the restriction enzymes X and Y independently in part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be amplified, but may not contain a sequence recognized by the restriction enzyme Z.
  • the restriction enzyme Z does not affect the measurement of methylation and is therefore unnecessary. However, by cutting the DNA even a little in the restriction enzyme treatment in step (b), the PCR amplification efficiency is improved, which is useful for measuring methylation.
  • the method for amplifying part or all of the transcriptional regulatory region and/or the region between +1 and +1000 of the transcription start site of the gene to be measured is not particularly limited, but examples include PCR methods such as digital PCR (polymerase chain reaction) and real-time PCR; LAMP (Loop-Mediated Isothermal Amplification); etc.
  • the "digital PCR” is a method for absolute quantitative determination of the amount of sample DNA.
  • the sample DNA is partitioned (diluted and distributed) into approximately 20,000 droplets or wells, and PCR is performed using a thermal cycler.
  • the principle is that the concentration of the gene to be measured in the sample can be obtained as an absolute value by counting the number of glowing droplets or wells and non-glowing droplets or wells.
  • the data is determined by the presence/absence of the target DNA in the droplets or wells, which is the same as a digital signal of 1/0, so it is called "digital PCR".
  • the "copy number count” in digital PCR refers to the process of measuring the fluorescence amount of the approximately 20,000 droplets and wells mentioned above one by one using a droplet reader, counting the number of droplets and wells that emit fluorescence and the number of droplets and wells with weak fluorescence intensity, and measuring the absolute value of the copy number of the gene by correction using a Poisson model.
  • the digital PCR is carried out as follows. (1) A PCR reaction solution containing template DNA is compartmentalized into approximately 20,000 droplets. (2) Approximately 20,000 droplets are collected in one tube. (3) Perform PCR reaction (PCR will be performed on approximately 20,000 droplets per tube). (4) The fluorescence intensity of approximately 20,000 droplets is measured one by one, the number of droplets with strong fluorescence intensity and those with weak fluorescence intensity are counted, and the copy number of the target gene in the sample is calculated by correction using a Poisson model.
  • the strong fluorescence intensity of a certain droplet means that the target gene is contained in that droplet and the fluorescence intensity has been increased by PCR amplification.
  • PCR method examples include the TaqMan probe method, the intercalator method, the 5'-nuclease method, and the cycling probe method.
  • a part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the SEPT9 gene can specifically be the sequence of the region from the transcription start point (position 75,369,272 of human chromosome 17) to +295 to +356 of SEPT9 gene transcript variant 2 (positions 75,369,566 to 75,369,627 of human chromosome 17 in the position information in the GRCh37/hg19 database: SEQ ID NO: 1).
  • the sequence of the +295 to +356 region corresponds to the exon 1 region.
  • examples of the cytosine of the CpG for which the methylation level is to be measured in a part or all of the region between +1 and +1000 of the transcription regulatory region and/or transcription start site of the SEPT9 gene include the cytosine at positions 75,369,591, 75,369,593, 75,369,600, or 75,369,602 of human chromosome 17 (the cytosines at positions 26, 28, 35, and 37 in SEQ ID NO: 1); positions 75,369,591 and 75,369,593 of human chromosome 17; positions 75,369,591 and 75,369,600 on human chromosome 17; positions 75,369,591 and 75,369,602 of human chromosome 17; positions 75,369,593 and 75,369,600 on human chromosome 17; two cytosines at positions 75,369,593 and 75,369,602 of human chromosome 17;
  • the gene to be measured is the HOXA1 gene
  • a specific example of part or all of the region from +1 to +1000 of the transcription regulatory region and/or transcription start point of the HOXA1 gene is the sequence from the transcription start point of the HOXA1 gene (position 27,096,000 of human chromosome 7) to the region from +20 to +127 (positions 27,095,874 to 27,095,981 of human chromosome 7 in the location information in the GRCh38/hg38 database: SEQ ID NO: 2).
  • the sequence of the +20 to +127 region corresponds to the exon 1 region.
  • the cytosine of the CpG for which the methylation level is measured in part or all of the region between +1 and +1000 of the transcription regulatory region and/or transcription start point of the HOXA1 gene can be the cytosine at position 27,095,945 of human chromosome 7 in the exon 1 region (the 37th cytosine in SEQ ID NO: 2).
  • a primer set, probe, or a labeled product thereof for individually measuring the methylation of each cytosine of the CpG to be measured contained in a part or all of the region +1 to +1000 of the transcription regulatory region and/or transcription start point of the gene to be measured may be used, but it is preferable to use a primer set, probe, or a labeled product thereof for commonly measuring the methylation of each cytosine of the CpG to be measured in the DNA of a part or all of the region +1 to +1000 of the transcription regulatory region and/or transcription start point to be amplified.
  • the primers may be two pairs of primers for amplifying each CpG individually, or one pair of primers that can amplify two CpGs at once.
  • the probes may be two probes that hybridize to the base sequences of the respective PCR amplified regions, or one probe that hybridizes to the base sequences of both PCR amplified regions.
  • the CpG in steps (c) and (d) above is a CpG in a sequence that is recognized by at least one of the restriction enzymes used in step (a).
  • suitable examples include a forward primer consisting of the nucleic acid sequence shown in SEQ ID NO: 3, or a forward primer consisting of the nucleic acid sequence shown in SEQ ID NO: 3 in which one or several nucleic acids have been substituted, deleted, inserted, or added; a reverse primer consisting of the nucleic acid sequence shown in SEQ ID NO: 4, or a reverse primer consisting of the nucleic acid sequence shown in SEQ ID NO: 4 in which one or several nucleic acids have been substituted, deleted, inserted, or added; a probe consisting of the nucleic acid sequence shown in SEQ ID NO: 5, or a probe consisting of the nucleic acid sequence shown in SEQ ID NO: 5 in which one or several nucleic acids have been substituted, deleted, inserted, or added; and these labels.
  • suitable examples include a forward primer consisting of the nucleic acid sequence shown in SEQ ID NO: 6, or a forward primer consisting of the nucleic acid sequence shown in SEQ ID NO: 6 in which one or several nucleic acids have been substituted, deleted, inserted, or added; a reverse primer consisting of the nucleic acid sequence shown in SEQ ID NO: 7, or a reverse primer consisting of the nucleic acid sequence shown in SEQ ID NO: 7 in which one or several nucleic acids have been substituted, deleted, inserted, or added; a probe consisting of the nucleic acid sequence shown in SEQ ID NO: 8, or a probe consisting of the nucleic acid sequence shown in SEQ ID NO: 8 in which one or several nucleic acids have been substituted, deleted, inserted, or added; and these labels.
  • nucleic acid sequence in which one or several nucleic acids have been substituted, deleted, inserted, or added refers to a nucleic acid sequence in which, for example, 1 to 5 nucleic acids, preferably 1 to 3 nucleic acids, and more preferably 1 to 2 nucleic acids have been substituted, deleted, inserted, or added.
  • Labeling substances for the forward primer label, reverse primer label, and probe label include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apo-glucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; green fluorescent protein (GFP), cyan fluorescent protein (Cyan Fluorescein These include fluorescent proteins such as blue fluorescent protein (CFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and lucifer
  • step (d) the methylation level of CpG in DNA in the region of +1 to +1000 of the transcription regulatory region and/or transcription start site targeted for amplification in step (c) is measured based on the results of the amplification in step (c). Specifically, the methylation level of the CpG can be measured based on the degree of amplification of DNA that has escaped cleavage by a methylation-sensitive restriction enzyme due to methylation in step (c). The degree of DNA amplification can be evaluated by detecting the amplified DNA with a probe or the like.
  • step (d) "measuring the methylation level of CpGs” may involve not only measuring the methylation of CpGs in the region of +1 to +1000 of the transcriptional regulatory region and/or transcription start site to be amplified, but also measuring the methylation of the corresponding CpGs in the complementary strand.
  • the method described in the above Patent Document 2 performs restriction enzyme treatment by adding restriction enzymes in two stages. Specifically, in the first stage, two types of restriction enzymes are added to a DNA solution and reacted at 37° C. for 16 hours, and then in the second stage, an additional type of restriction enzyme is added and reacted at 60° C. for 16 hours.
  • a task of adding a restriction enzyme in the second stage occurs after the first stage of restriction enzyme treatment. For example, when 96 samples are handled, the task of adding a restriction enzyme in the second stage is as follows, and it takes about 60 minutes to perform all the steps.
  • the supernatant was removed with an aspirator so as not to suck up the white blood cells precipitated at the bottom of the tube, and this operation was repeated until the pellet (white blood cells) turned white.
  • the whitened pellet was transferred to a 1.5 mL tube, and nucleic acid extraction was performed using the nucleic acid extraction agent SepaGene (Sanko Junyaku Co., Ltd.). The extraction method followed the instructions for use.
  • the nucleic acid pellet obtained by extraction was air-dried and dissolved in 100 ⁇ L of TE buffer to prepare a SEPT9 unmethylated control DNA or HOXA1 unmethylated control DNA sample.
  • methylated control DNA (EpiScope Methylated HCT116 gDNA) was added to a fixed concentration (0.8 ng/ ⁇ L) of unmethylated control DNA to prepare a dilution series of methylated control DNA samples: 0%, 3.1%, 6.3%, 12.5%, 25%, 50%, and 100%.
  • Table 1 shows the relationship between the concentration and dilution series (%) of the methylation control DNA (SEPT9 methylation control or HOXA1 methylation control) added to each sample.
  • ⁇ Condition 1 Single treatment> One-step enzyme treatment of DNA: simultaneous treatment with HhaI, HpaII, ExoI, and BstUI 1. Place 10 ⁇ L of the DNA sample prepared according to Table 1 in a 1.5 mL tube. 2. Add 1 ⁇ L of AmpliTaq Gold buffer II (included in AmpliTaq Gold DNA Polymerase Kit N808-0241: Thermo Fisher Scientific). Add 1 ⁇ L of 3.25 mM MgCl2 solution (included in AmpliTaq Gold DNA Polymerase Kit N808-0241: Thermo Fisher Scientific). 4. Add 1 ⁇ L (10 U) of HhaI (Thermo Fisher Scientific). 5.
  • ⁇ Condition 2 Two-step treatment> Two-step enzyme treatment of DNA: treatment with HhaI, HpaII, and ExoI, followed by treatment with BstUI.
  • 1.1 Place 10 ⁇ L of the DNA sample prepared according to Table 1 in a 5 mL tube. 2.
  • 1 ⁇ L of AmpliTaq Gold buffer II (included in AmpliTaq Gold DNA Polymerase Kit N808-0241: Thermo Fisher Scientific).
  • Add 1 ⁇ L of 3.25 mM MgCl2 solution (included in AmpliTaq Gold DNA Polymerase Kit N808-0241: Thermo Fisher Scientific).
  • 4. Add 1 ⁇ L (10 U) of HhaI (Thermo Fisher Scientific). 5.
  • HhaI, HpaII, and BstUI are methylation-sensitive restriction enzymes that recognize 5'-GCGC-3', 5'-CCGG-3', and 5'-CGCG-3', respectively, and cleave the base sequence. If the cytosine of the CpG in this base sequence is methylated, this base sequence will remain uncleaved, making it possible to amplify it in the subsequent PCR.
  • Table 2 shows the base sequence of the region to be amplified by PCR.
  • the underlined bases in the base sequence column of the region to be amplified by PCR indicate sites that may be cut by the restriction enzymes HhaI, HpaII, or BstUI.
  • these restriction enzymes are methylation-sensitive restriction enzymes, if the cytosine (C) of the underlined CG in Table 2 is methylated, the recognition site of the above restriction enzymes containing the methylated cytosine will not be cut.
  • each amplified region is part of the exon 1 region.
  • the RefSeq of SEPT9 mRNA in the NCBI Reference Sequence Database is NM_001113493 (updated June 14, 2013), and the RefSeq of HOXA1 mRNA is NM_005522 (updated February 17, 2023) or NM_153620 (updated February 17, 2023), and the transcription start site of HOXA1 in this specification is represented based on NM_005522.
  • digital PCR is a method for absolutely quantifying the amount of DNA in a sample.
  • a digital PCR system QX200 Droplet Digital PCR System: Bio-Rad
  • This DNA-containing PCR reaction solution consisted of 1 ⁇ L each of 20 ⁇ M primer mix and 5 ⁇ M probe (total 4 ⁇ L) for amplifying specific regions of the SEPT9 gene and HOXA1 gene and measuring specific methylated cytosines, and 10 ⁇ L of 2x ddPCR master mix (Bio-Rad). Multiplex PCR was performed using the above DNA-containing PCR reaction solution.
  • the base sequences of the primer and probe for SEPT9 are as follows: Forward primer: 5'-GCCCACCAGCCATCATGT-3' (SEQ ID NO: 3) Reverse primer: 5'-GTCCGAAATGATCCCCATCCA-3' (SEQ ID NO: 4) Probe: 5'-FAM-CCGCGGTCAACGC-MGB-3' (SEQ ID NO:5)
  • the base sequences of the primers and probe for HOXA1 are as follows: Forward primer: 5'-CCCATGGAGGAAGTGAGAAA-3' (SEQ ID NO:6) Reverse primer: 5'-GGGGTATTCCAGGAAGGAGT-3' (SEQ ID NO: 7) Probe: 5'-FAM-GCACAGTCACGCCGG-MGB3' (SEQ ID NO: 8)
  • Droplets were made from this DNA-containing PCR reaction solution using an Automated Droplet Generator (Bio-Rad). Using a thermal cycler, the droplets were preheated at 95°C for 10 minutes, and then the DNA was amplified by repeating 40 cycles of thermal denaturation at 94°C for 30 seconds and annealing at 56°C for 60 seconds, and finally heated at 98°C for 10 minutes.
  • Bio-Rad Automated Droplet Generator
  • the fluorescent dye derived from the TaqMan probe in each droplet was detected using a QX200 Droplet Reader (Bio-Rad) and QuantaSoft software (Bio-Rad), and the number of DNA fragments (copy numbers) in which cytosines in CpGs in the amplified region of the target gene were methylated, which had escaped cleavage by methylation-sensitive restriction enzymes, was measured.
  • the base sequence (5'-GCGC-3', 5'-CCGG-3', 5'-CGCG-3') containing the underlined CpG in Table 2 of DNA in the amplification target region of SEPT9 or HOXA1 can be recognized by any of the three restriction enzymes used (HhaI, HpaII, BstUI).
  • HhaI, HpaII, BstUI restriction enzymes used
  • SEPT9 if the CpGs of 5'-GCGC-3' and/or 5'-CGCG-3' are not methylated, they are recognized and cleaved by the restriction enzymes HhaI and/or BstUI, and are not amplified by PCR, and therefore are not detected.
  • HOXA1 if the CpG at 5'-CCGG-3' is not methylated, it is recognized by HpaII, cleaved, and not amplified by PCR, and therefore not detected. On the other hand, if the CpG at 5'-CCGG-3' is methylated, it escapes cleavage by the restriction enzyme and is amplified by PCR, and therefore is detected.
  • cytosine of the CpG in the base sequence CCGG in the PCR amplification target region of HOXA1 in the template DNA shown in Table 2 is methylated, cleavage of CCGG does not occur even if restriction enzyme treatment is performed by single treatment (condition 1) or two-step treatment (condition 2), and the region can be amplified by subsequent PCR, so that it can be counted as the number of methylated HOXA1 copies by digital PCR.
  • the amplified region of SEPT9 contains the BstUI and HhaI cleavage sites but not the HpaII cleavage site, and the amplified region of HOXA1 contains the HpaII cleavage site but not the BstUI and HhaI cleavage sites.
  • Example 2 From the above Example 1, it was confirmed that the methylation level can be easily measured by the method of Condition 1. Therefore, it was examined whether hepatocellular carcinoma can be diagnosed by combining the copy number of methylated SEPT9 or methylated HOXA1 with other hepatocellular carcinoma markers.
  • the subjects included 247 control subjects (154 healthy subjects [Health Control] and 93 chronic liver disorder patients [CLI]) and 269 hepatocellular carcinoma patients (HCC).
  • the chronic liver disorder patients consisted of 62 with chronic hepatitis (CH) and 31 with liver cirrhosis (LC).
  • CH chronic hepatitis
  • LC liver cirrhosis
  • HCC group hepatocellular carcinoma patient group
  • BCLC stage 0/A hepatocellular carcinoma
  • HBV hepatocellular carcinoma
  • NBNC non-viral hepatocellular carcinoma
  • hepatocellular carcinoma The progression of hepatocellular carcinoma was assessed using the criteria of the BCLC staging system, with Stage 0/A being considered early hepatocellular carcinoma, and of the 109 patients, 61 had viral hepatocellular carcinoma and 48 had non-viral hepatocellular carcinoma.
  • the AFP level was measured by EIA, using the AFP concentration (ng/mL) in 0.025 mL of serum.
  • the test reagent was ST E test "TOSOH” II (AFP) (Tosoh Corporation), and measurements were performed according to the attached protocol.
  • the measurement device used was AIA-2000 (Tosoh Corporation).
  • the PIVKA-II level was measured by EIA using the PIVKA-II concentration (mAU/mL AU: arbitrarily unit standard) in 0.025 mL of serum.
  • the test reagent used was E-test "TOSOH" II (PIVKA-II) (Sekisui Medical Co., Ltd.), and measurements were performed according to the attached protocol.
  • the measurement device used was AIA-2000 (Tosoh Corporation).
  • DNA Extraction The frozen serum specimen was thawed, and DNA was extracted from 1.0 mL of the serum specimen out of the approximately 3 mL of serum obtained above using Maxwell (registered trademark) RSC ccfDNA plasma Kit (Promega). The DNA was then eluted in 50 ⁇ L of elution buffer and frozen and stored at ⁇ 20° C. Note that since the amount of DNA required for the enzyme treatment in Example 1 above is 10 ⁇ L, a total of five enzyme treatments can be performed with the above 50 ⁇ L of DNA solution. Since the amount of DNA required for one enzyme treatment is 10 ⁇ L derived from 0.2 mL of serum, it can be said that methylation analysis is possible with a small amount of serum. Note that in the digital PCR reaction in the following methylation analysis, half of the enzyme-treated DNA sample (5 ⁇ L as DNA, 0.1 mL of serum-derived DNA) is used for one test.
  • the copy numbers of methylated SEPT9 (mSEPT9) and methylated HOXA1 (mHOXA1) were measured by first carrying out the same enzyme treatment as in Example 1 above, and then using the enzyme-reacted DNA (containing 0.1 mL of serum-derived DNA), a specific region of the SEPT9 gene and the HOXA1 gene was amplified by multiplex digital PCR to measure the copy numbers.
  • the DNA-containing PCR reaction solution was the same as in Example 1 above, except that it was prepared by mixing 8 ⁇ L of the enzyme-reacted DNA solution treated under Condition 1 or Condition 2 above and 14 ⁇ L of the PCR reaction solution.
  • ROC receiver operating characteristic
  • each index was logarithmically transformed to obtain a normal distribution and then analyzed.
  • ⁇ 0 is the intercept (constant term)
  • ⁇ 1 to ⁇ 5 are regression coefficients
  • Sex is gender (substitute 1 for female and 0 for male)
  • Age is age
  • norm_APF is the normally distributed AFP value
  • norm_PIVKA-II is the normally distributed PIVKA-II value
  • norm_mSEPT9 is the normally distributed methylated SEPT9 value.
  • norm means normally distributed.
  • ⁇ 0 to ⁇ 5 are as follows.
  • ⁇ 0 is the intercept (constant term)
  • ⁇ 1 to ⁇ 5 are regression coefficients
  • Sex is gender (substitute 1 for female and 0 for male)
  • Age is age
  • norm_APF is the normally distributed AFP value
  • norm_PIVKA-II is the normally distributed PIVKA-II value
  • norm_mHOXA1 is the normally distributed methylated HOXA1 value.
  • norm means normally distributed.
  • Figures 7 and 8 show that the AUC value is high when AFP, PIVKA-II, mSEPT9, and mHOXA1, especially PIVKA-II, are normally distributed. Even in the case of a combination of PIVKA-II and mSEPT9 or mHOXA1 without AFP, the AUC was high at 0.935 when PIVKA-II was normally distributed.
  • the overall hepatocellular carcinoma in mSEPT9 is shown in Figure 9A, early hepatocellular carcinoma, early non-viral hepatocellular carcinoma, and early viral hepatocellular carcinoma in Figure 9B, and the overall hepatocellular carcinoma in mHOXA1 is shown in Figure 10A, and early hepatocellular carcinoma, early non-viral hepatocellular carcinoma, and early viral hepatocellular carcinoma in Figure 10B.
  • the cutoff value for mSEPT9 was calculated using the Youden Index to be 35 (copy number/mL), and the cutoff value for mHOXA1 was calculated using the Youden Index to be 310 (copy number/mL).
  • the cutoff value was set to 0.60 when mSEPT9 was combined using the Youden Index, and 0.61 when mHOXA1 was combined, and the sensitivity and specificity were calculated at that time.

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Abstract

La présente invention a pour but de procurer un procédé permettant de détecter facilement le carcinome hépatocellulaire sans dépendre uniquement de l'AFP, par l'utilisation de sérum ou d'éléments similaires recueillis dans le cadre d'un diagnostic de santé ou similaire.　Dans un fluide corporel prélevé sur un sujet, (A) le niveau de méthylation de CpG dans l'ADN dans une région de +1 à +1000 d'une région de régulation transcriptionnelle et/ou d'un point d'initiation transcriptionnelle du gène SEPT9 ou dans une région de +1 à +1000 d'une région de régulation transcriptionnelle et/ou d'un point d'initiation transcriptionnelle du gène HOXA1 et (B) le niveau d'AFP ou le niveau de PIVKA-II sont mesurés par des procédés prédéterminés, une analyse statistique est effectuée à partir d'au moins deux indices (A) et (B), et le diagnostic de carcinome hépatocellulaire chez le sujet est assisté à partir de la valeur obtenue par l'analyse statistique.
PCT/JP2024/001080 2023-05-23 2024-01-17 Procédé d'aide au diagnostic du cancer hépatocellulaire Pending WO2024241624A1 (fr)

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