Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias

Suehiro, Yutaka; Hashimoto, Shinichi; Higaki, Shingo; Fujii, Ikuei; Suzuki, Chieko; Hoshida, Tomomi; Matsumoto, Toshihiko; Yamaoka, Y.; Takami, Taro; Sakaida, Isao; Yamasaki, Takahiro

doi:10.18632/oncotarget.24768

Cited by 26 publications

(26 citation statements)

References 54 publications

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“…Thus, an increase in serum methylated SEPT9 may suggest the presence of other types of cancer. We have reported that serum methylated SEPT9 is significantly increased in patients with advanced colorectal adenoma and cancer . In the current study, colonoscopy was performed in all of the controls but not in all patients with CLD and with HCC, because it is difficult to perform a colonoscopy in patients who have no symptoms in relation to bowel movement.…”

Section: Discussionmentioning

confidence: 80%

“…We have reported that serum methylated SEPT9 is significantly increased in patients with advanced colorectal adenoma and cancer. (17) In the current study, colonoscopy was performed in all of the controls but not in all patients with CLD and with HCC, because it is difficult to perform a colonoscopy in patients who have no symptoms in relation to bowel movement. We suggest that colonoscopy should be indicated for patients with CLD who have elevated methylated SEPT9 levels, to rule out colorectal neoplasms.…”

Section: Discussionmentioning

confidence: 98%

“…The most unique feature of this novel assay is that it uses three methylation‐sensitive restriction enzymes in combination with droplet digital PCR (ddPCR). We have reported the performance of the serum CORD assay for methylated SEPT9 in patients with colorectal neoplasia, including cancer . In this study, we evaluated the potential application of this assay for methylated SEPT9 detection in HCC patients.…”

mentioning

confidence: 99%

“…We have reported the performance of the serum CORD assay for methylated SEPT9 in patients with colorectal neoplasia, including cancer. (17) In this study, we evaluated the potential application of this assay for methylated SEPT9 detection in HCC patients.…”

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confidence: 99%

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Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma

et al. 2020

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Liquid biopsies are not used in practice for hepatocellular carcinoma (HCC). Epi proColon is the first commercial blood‐based test for colorectal cancer screening based on methylated DNA testing of the septin 9 gene (SEPT9). However, Epi proColon has some disadvantages, including the requirement of a large amount of blood and lack of quantitative performance. Therefore, we previously developed a novel liquid biopsy test that can quantitatively detect even a single copy of methylated SEPT9 in a small amount of DNA. In the current study, we evaluated the application potential of this assay for diagnosing HCC. Study subjects included 80 healthy volunteers, 45 patients with chronic liver disease (CLD) without HCC, and 136 patients with HCC (stage 0, 12; stage A, 50; stage B, 31; stage C, 41; and stage D, 2), according to the Barcelona Clinic Liver Cancer staging system. For the assay, DNA was treated with methylation‐sensitive restriction enzymes in two steps, followed by multiplex droplet digital polymerase chain reaction. The median copy number of methylated SEPT9 was 0.0, 2.0, and 6.4 in the healthy control, CLD, and HCC groups, respectively, with significant differences among the groups (HCC vs. healthy control, P < 0.001; HCC vs. CLD, P = 0.002; CLD vs. healthy control, P = 0.008). Assay sensitivity and specificity were 63.2% and 90.0%, respectively (cutoff value, 4.6 copies), in detecting HCC when compared with healthy subjects. The positive rate of methylated SEPT9 increased with HCC progression (stage 0, 41.7%; stage A, 58.0%; stage B, 61.3%; stage C, 75.6%; and stage D, 100%). Conclusion: We developed a sensitive methylated SEPT9 assay that might serve as a liquid biopsy test for diagnosing HCC.

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Section: Discussionmentioning

confidence: 80%

Section: Discussionmentioning

confidence: 98%

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confidence: 99%

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confidence: 99%

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Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma

et al. 2020

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“…We have created an analytically sensitive technique, the combined restriction digital PCR (CORD) assay, which is a very sensitive methylation assay allowing for the counting of as little as one copy of a methylated gene in a small sample of DNA without necessitating DNA bisulfite treatment. Its sensitivity for minute quantities of the target methylated gene is more than 100 times higher than that of conventional bisulfite-based methylation assays and thus overcomes the issue of limited sample input [12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Blood Free-Circulating DNA Testing of Methylated RUNX3 Is Useful for Diagnosing Early Gastric Cancer

Hideura

Suehiro

Nishikawa

et al. 2020

Cancers

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The main modalities for gastric cancer screening are limited to upper gastrointestinal endoscopy and contrast radiography. The former is invasive, and the latter has high false-negative rates. Thus, alternative diagnostic strategies are required. One solution may be a liquid biopsy. Methylated RUNX3 is a well-known biomarker of gastric cancer but it is very difficult to detect with conventional bisulfite-based methylation assays when only a small amount of serum is available. We developed the combined restriction digital PCR (CORD) assay, a new methylation assay allowing for the counting of as little as one copy of a methylated gene in a small sample of DNA without necessitating DNA bisulfite treatment. We evaluated the sensitivity and specificity of the serum DNA testing of methylated RUNX3 by the CORD assay for the detection of early gastric cancer using 50 patients with early gastric cancer and 61 control individuals. The CORD assay had a sensitivity of 50.0% and a specificity of 80.3% for early gastric cancer. Methylated RUNX3 copies were significantly associated with tumor size, massive submucosal invasion, and lymph-vascular invasion. After the treatment, the median number of methylated RUNX3 copies was significantly decreased. The CORD assay may provide an alternative screening strategy to detect even early-stage gastric cancer.

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Methylation status and long‐fragment cell‐free DNA are prognostic biomarkers for gastric cancer

Kanazawa

Yamada

et al. 2021

Cancer Medicine

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Background Circulating tumor DNA (ctDNA) detected before surgery disappears after complete surgical resection of the cancer. Residual ctDNA indicates minimal residual disease (MRD), which is a cause of recurrence. The presence of long‐fragment circulating cell‐free DNA (cfDNA) or methylated cfDNA also implies the presence of cancer. In this study, we evaluated the prognostic value of cfDNA methylation and long‐fragment cfDNA concentration in gastric cancer patients undergoing curative surgery Methods Ninety‐nine gastric cancer patients were included. Peripheral blood samples were collected before and 1 month after surgery. In patients administered chemotherapy, samples were collected before starting chemotherapy. qPCR was performed to detect long‐ and short‐fragment LINE‐1. A plasma HELP (HpaII tiny fragment Enrichment by Ligation‐mediated PCR) assay to determine the concentration of HpaII small fragments was performed using ligation‐mediated PCR and HpaII was quantified as the HpaII:MspI ratio to detect methylation levels of cfDNA. Results Overall survival (OS) of patients with low methylation levels before starting treatment was significantly worse than that of patients with high methylation levels ( P = 0.006). In the 90 patients who underwent curative surgery, recurrence‐free survival (RFS) and OS of patients with low methylation levels before surgery were worse than those with high methylation levels (P=0.08 and P = 0.11, respectively). RFS and OS of patients with high concentrations of long‐fragment LINE‐1 after surgery were significantly worse than those with low concentrations of long‐fragment LINE‐1 ( P = 0.009, P = 0.04). Conclusions Pre‐surgical low methylation levels of LINE‐1 are a negative prognostic factor. Post‐surgical high concentrations of long‐fragment LINE‐1 indicate MRD and a high risk of recurrence.

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Blood free-circulating DNA testing by highly sensitive methylation assay to diagnose colorectal neoplasias

Cited by 26 publications

References 54 publications

Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma

Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma

Blood Free-Circulating DNA Testing of Methylated RUNX3 Is Useful for Diagnosing Early Gastric Cancer

Methylation status and long‐fragment cell‐free DNA are prognostic biomarkers for gastric cancer

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