Far-ultraviolet irradiation at 222 nm destroys and sterilizes the biofilms formed by periodontitis pathogens.

BACKGROUND: Periodontal disease is the leading cause of tooth loss, and an association between periodontal disease and non-oral systemic diseases has been shown. Formation of biofilm by periodontal pathogens such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans and their resistance to antimicrobial agents are at the root of persistent and chronic bacterial infections. METHODS: The bactericidal effect of far-ultraviolet (F-UV) light irradiation at 222 nm on periodontal bacteria was assessed qualitatively and quantitatively. The effect of biofilm disruption by F-UV light on periodontal bacteria was examined by crystal violet staining, and the morphologic changes of the biofilm after F-UV irradiation were explored by confocal laser microscopy and scanning electron microscopy. We developed a thin fiber-type 222 nm F-UV irradiator and studied its safety and effect of reducing bacteria in rodent models. RESULTS: F-UV light at 222 nm had a bactericidal effect on F. nucleatum, P. gingivalis, and S. mutans. Irradiation with F-UV light reduced the biofilm formed by the bacteria and sterilized them from within. Confocal laser microscopy showed a clear reduction in biofilm thickness, and scanning electron microscopy confirmed disintegration of the biofilm architecture. F-UV irradiation was less damaging to DNA and less cytotoxic than deep-ultraviolet light, and it reduced bacterial counts on the tooth surface. CONCLUSION: F-UV irradiation has the potential to destroy biofilm and act as a bactericide against pathogenic bacteria in the biofilm.

Determination of the Optimal Wavelength of the Hemolysis Index Measurement.

Many biochemical auto-analyzers have methods that measure the hemolysis index (HI) to quantitatively assess the degree of hemolysis. Past reports on HI are mostly in vitro studies. Therefore, we evaluated the optimal wavelength of HI measurement ex vivo using clinical samples. Four different wavelengths (410/451 nm: HI-1, 451/478 nm: HI-2, 545/596 nm: HI-3 and 571/596 nm: HI-4) were selected for HI measurement, and correlations were examined from the measurement results of 3890 clinical samples. Another set of 9446 clinical samples was used to examine the correlation of HI with lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and potassium (K). Strong correlations were found between HI-4 and HI-1 and between HI-4 and HI-3. HI-1 and HI-2 cannot correctly assess hemolysis for high bilirubin samples, and HI-3 cannot correctly assess hemolysis for high triglyceride samples. LDH, AST and K correlated positively with HI-4 in clinical samples. For every 1-unit increase in HI-4, LDH increased by 19.51 U/L, AST by 1.03 U/L and K by 0.061 mmol/L, comparable to reports of other studies. In clinical samples, HI-4 was less susceptible to bilirubin and chyle and reflected well the changes in LDH, AST and K caused by hemolysis. This suggested that the optimal wavelength for HI measurement is 571 nm.

Methylated SEPT9 assay-based liquid biopsy as a biomarker in molecular targeted agent-treated hepatocellular carcinoma.

BACKGROUND: The development of molecular targeted agents (MTAs) has changed the treatment strategy for hepatocellular carcinoma (HCC). However, currently, there are no established predictive biomarkers for the treatment efficacy of MTAs. Previously, we developed a novel liquid biopsy test for HCC screening using sensitive methylated DNA testing of septin 9 gene (SEPT9). Here, we hypothesized that SEPT9 could be used as a biomarker for MTA treatment efficacy. METHODS: We enrolled 157 patients receiving sorafenib or lenvatinib as a first-line therapy and allocated 85 and 72 patients to the training and validation cohorts, respectively. For the methylation assay, DNA was treated with methylation-sensitive restriction enzymes, followed by multiplex droplet digital PCR. Various clinical parameters were compared with clinical outcomes. RESULTS: The multivariate analysis revealed Eastern Cooperative Oncology Group performance status (≥ 1; p = 0.048), alpha-fetoprotein (AFP) (≥ 400 ng/mL; p < 0.001), and methylated-septin-9 (m-SEPT9) (≥ 205 copies/mL; p = 0.018) as significant predictors of poor overall survival (OS) in the training cohort. m-SEPT9 was identified as a predictor of poor OS in the validation cohort. We developed a predictive score, called the MTA score, consisting of these three significant OS parameters (two points were added for AFP and one point for each of the other predictors). Patients with MTA scores ≥ 2 showed a significantly poor prognosis compared to those with MTA scores ≤ 1 in both the training and validation cohorts. CONCLUSIONS: m-SEPT9 could be a potential predictive biomarker for survival in patients with HCC treated with MTAs.

CA19-9 in Combination with Methylated HOXA1 and SST Is Useful to Diagnose Stage I Pancreatic Cancer.

INTRODUCTION: We previously developed a novel methylation assay, the combined restriction digital PCR (CORD) assay, consisting of treatment of DNA with methylation-sensitive restriction enzymes and droplet digital PCR. METHODS: In this study, we assessed the diagnostic performance of serum methylated Homeobox A1 (mHOXA1) and methylated somatostatin (mSST) using the CORD assay in combination with CA19-9 for pancreatic cancer using serum samples from 82 healthy individuals, 13 patients with benign pancreatic disease, 3 patients with branched-duct intraductal papillary mucinous neoplasm, and 91 patients with pancreatic cancer. RESULTS: For the single marker tests, sensitivity for all stages of pancreatic cancer, stage I cancer, and specificity were, respectively, 71.4%, 50.0%, and 94.9% for CA19-9; 51.6%, 68.8%, and 90.8% for mHOXA1; and 50.1%, 68.8%, and 94.9% for mSST. Those for the combined marker tests were, respectively, 86.8%, 81.3%, and 85.7% for combined mHOXA1 and CA19-9; 86.8%, 87.5%, and 89.8% for combined mSST and CA19-9; and 89.0%, 87.5%, and 85.7% for all three markers combined. CONCLUSION: The combination of mHOXA1 and mSST with CA19-9 appears to be useful to detect pancreatic cancer even at an early stage.

MC180295 Inhibited Epstein-Barr Virus-Associated Gastric Carcinoma Cell Growth by Suppressing DNA Repair and the Cell Cycle.

DNA methylation of both viral and host DNA is one of the major mechanisms involved in the development of Epstein-Barr virus-associated gastric carcinoma (EBVaGC); thus, epigenetic treatment using demethylating agents would seem to be promising. We have verified the effect of MC180295, which was discovered by screening for demethylating agents. MC180295 inhibited cell growth of the EBVaGC cell lines YCCEL1 and SNU719 in a dose-dependent manner. In a cell cycle analysis, growth arrest and apoptosis were observed in both YCCEL1 and SNU719 cells treated with MC180295. MKN28 cells infected with EBV were sensitive to MC180295 and showed more significant inhibition of cell growth compared to controls without EBV infection. Serial analysis of gene expression analysis showed the expression of genes belonging to the role of BRCA1 in DNA damage response and cell cycle control chromosomal replication to be significantly reduced after MC180295 treatment. We confirmed with quantitative PCR that the expression levels of BRCA2, FANCM, RAD51, TOP2A, and CDC45 were significantly decreased by MC180295. LMP1 and BZLF1 are EBV genes with expression that is epigenetically regulated, and MC180295 could up-regulate their expression. In conclusion, MC180295 inhibited the growth of EBVaGC cells by suppressing DNA repair and the cell cycle.

Management of Systemic Therapies and Hepatic Arterial Infusion Chemotherapy in Patients with Advanced Hepatocellular Carcinoma Based on Sarcopenia Assessment.

Background: Sarcopenia, defined as the loss of skeletal muscle mass (MM), physical performance, and strength, has been associated with poor clinical outcomes in hepatocellular carcinoma (HCC) patients treated with several therapies. As systemic therapies, including molecular targeted agents, have a strong impact on sarcopenia, we aimed to review the impact of sarcopenia in patients receiving systemic therapies, especially sorafenib and hepatic arterial infusion chemotherapy (HAIC). Summary: Several studies have demonstrated that sarcopenia is associated with poor clinical outcomes in patients receiving sorafenib or lenvatinib, while HAIC has no association with overall survival (OS) and sarcopenia. Furthermore, based on our previous study, we developed the management of sorafenib score (MS score) to stratify patients' survival according to the positivity of three parameters (skeletal MM, disease control of sorafenib, and post-sorafenib therapy), ranging from 0 to 3. Patients with an MS score ≥2 (median survival time [MST], 16.4 months) showed significantly longer survival than those with an MS score ≤1 (MST, 8.4 months) (p < 0.001). This result indicates that patients need at least two positive parameters to prolong OS. Although performance status (PS) has been used in the Barcelona Clinic Liver Cancer staging system, we consider that the assessment of sarcopenia has the potential to replace PS. Key Messages: Sarcopenia is associated with poor clinical outcomes in patients of HCC receiving sorafenib or lenvatinib. The MS score, based on the positivity of three prognostic factors, including skeletal MM, in patients receiving sorafenib, can be a reliable indicator of prolonged survival.

Comparison of two primer-probe sets of Fusobacterium nucleatum using droplet digital polymerase chain reaction for the detection of colorectal neoplasia from faecal samples.

BACKGROUND: Although faecal DNA testing of Fusobacterium nucleatum (Fn) is expected to be useful for colorectal neoplasia detection, there is no standardized quantification method of Fn. We performed this study to establish a possible standardized method. METHODS: In this study, 322 participants including 71 subjects without colorectal neoplasia (control group), 31 patients with non-advanced colorectal adenoma, 93 patients with advanced colorectal adenoma, and 127 patients with colorectal cancer were enrolled. Faecal Fn were quantified by droplet digital PCR (ddPCR) using two PCR primer-probe sets reported previously that are tentatively named Fn1 and Fn2. Fn1 has been used in ddPCR by us and Fn2 has been widely used in quantitative real-time PCR. RESULTS: The Fn copy number using Fn1 was five times higher than that using Fn2, with a linear relationship shown between them. Receiver operating characteristic curve analysis showed the area under the curve (AUC) to be almost the same between Fn1 and Fn2 in discriminating between the control group and the colorectal cancer group (AUC = 0.81 and 0.81, respectively), and between the control/non-advanced colorectal adenoma group and the advanced colorectal adenoma/colorectal cancer group (AUC = 0.74 and 0.74, respectively). CONCLUSIONS: As the diagnostic performance was quite similar between Fn1 and Fn2, ddPCR-based Fn testing using Fn1 and Fn2 could be a possible standardized method for a colorectal neoplasia screening test, considering that Fn levels quantified by Fn1 are about five times higher than those by Fn2.

Successful Mitral Valve Replacement in an Infant with Neonatal Marfan Syndrome due to a Novel Missense Mutation of the FBN1 Gene.

Marfan syndrome is an autosomal dominant genetic disorder of the fibrous connective tissue caused by pathogenic mutations in the fibrillin-1 gene. Neonatal Marfan syndrome is a rare type of Marfan syndrome that is genotypically and phenotypically different from classical Marfan syndrome and has a poor prognosis. Most patients with neonatal Marfan syndrome die during infancy due to severe and rapidly progressive cardiovascular disorders. Here, we present a case of an 11-year-old girl with neonatal Marfan syndrome due to a novel missense mutation in exon 27 of the fibrillin-1 gene. Her condition was critical due to progressive mitral and tricuspid regurgitation. Mitral valve replacement, performed at the age of 6 months, improved her critical condition. Our case suggests that early mitral valve replacement may lead to better outcomes in patients with neonatal Marfan syndrome.

The bactericidal effect of far-UVC on ESBL-producing Escherichia coli.

Because extended-spectrum beta-lactamase (ESBL) infections can cause life-threatening disease and effective treatments need to be developed, we examined the bactericidal effect of far-ultraviolet C (far-UVC) light therapy on ESBL-producing Escherichia coli (E. coli). The bactericidal effect on 2 types of ESBL-producing E. coli was the same as that on the wild strain although the results of drug resistance tests varied among these strains. We believe that irradiation with far-UVC is effective in preventing infection by ESBL-producing E. coli in health care settings.

Vaccine-induced anaphylaxis in a child with cow's milk allergy: Lactose hydrate was the culprit.

Lactose hydrate was the cause of vaccine-induced anaphylaxis in a child with severe milk allergy. Although the amount of milk protein in lactose-containing vaccines is extremely small, physicians administering such a vaccine must be prepared for the potential risk of severe milk allergy.

Analysis of Relationships between Immune Checkpoint and Methylase Gene Polymorphisms and Outcomes after Unrelated Bone Marrow Transplantation.

Unrelated bone marrow transplantation (uBMT) is performed to treat blood disorders, and it uses bone marrow from an unrelated donor as the transplant source. Although the importance of HLA matching in uBMT has been established, that of other genetic factors, such as single-nucleotide polymorphisms (SNPs), remains unclear. The application of immunoinhibitory receptors as anticancer drugs has recently been attracting attention. This prompted us to examine the importance of immunoinhibitory receptor SNPs in uBMT. We retrospectively genotyped five single-nucleotide polymorphisms (SNPs) in the immune checkpoint genes, BTLA, PD-1, LAG3, and CTLA4, and two SNPs in the methylase genes, DNMT1 and EZH2, in 999 uBMT donor-recipient pairs coordinated through the Japan Marrow Donor Program matched at least at HLA-A, -B, and -DRB1. No correlations were observed between these SNPs and post-uBMT outcomes (p > 0.005). This result questions the usefulness of these immune checkpoint gene polymorphisms for predicting post-BMT outcomes. However, the recipient EZH2 histone methyltransferase gene SNP, which encodes the D185H substitution, exhibited a low p-value in regression analysis of grade 2-4 acute graft-versus-host disease (p = 0.010). Due to a low minor allele frequency, this SNP warrants further investigation in a larger-scale study.

Deep Ultraviolet Light-Emitting Diode Light Therapy for Fusobacterium nucleatum.

BACKGROUND: Fusobacterium nucleatum, which is associated with periodontitis and gingivitis, has been detected in colorectal cancer (CRC). METHODS: We evaluated the bactericidal effect of deep ultraviolet (DUV) light-emitting diode (LED) light therapy on F. nucleatum both qualitatively and quantitatively. Two DUV-LEDs with peak wavelengths of 265 and 280-nm were used. DNA damage to F. nucleatum was evaluated by the production of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). RESULTS: DUV-LEDs showed a bactericidal effect on F. nucleatum. No colony growth was observed after 3 min of either 265 nm or 280 nm DUV-LED irradiation. The survival rates of F. nucleatum under 265 nm DUV-LED light irradiation dropped to 0.0014% for 10 s and to 0% for 20 s irradiation. Similarly, the survival rate of F. nucleatum under 280 nm DUV-LED light irradiation dropped to 0.00044% for 10 s and 0% for 20 s irradiation. The irradiance at the distance of 35 mm from the DUV-LED was 0.265 mW/cm2 for the 265 nm LED and 0.415 mW/cm2 for the 280 nm LED. Thus, the radiant energy for lethality was 5.3 mJ/cm2 for the 265 nm LED and 8.3 mJ/cm2 for the 280 nm LED. Amounts of CPD and 6-4PP in F. nucleatum irradiated with 265 nm DUV-LED light were 6.548 ng/µg and 1.333 ng/µg, respectively. CONCLUSIONS: DUV-LED light exerted a bactericidal effect on F. nucleatum by causing the formation of pyrimidine dimers indicative of DNA damage. Thus, DUV-LED light therapy may have the potential to prevent CRC.

Combination of CA19-9 and Blood Free-Circulating Methylated RUNX3 May Be Useful to Diagnose Stage I Pancreatic Cancer.

BACKGROUND: Although serum carbohydrate antigen 19-9 (CA19-9) is widely used as a useful biomarker of pancreatic cancer for monitoring the response to therapy, it is not recommended for screening of early pancreatic cancer because of its limited sensitivity for small tumors. Thus, it is critical to discover novel serum biomarkers to complement CA19-9 in order to improve sensitivity. Although methylated runt-related transcription factor 3 (RUNX3) is a biomarker of pancreatic cancer, its detection by conventional bisulfite-based methylation assays from a small serum sample amount is very difficult. Therefore, we developed a new methylation assay, the combined restriction digital PCR (CORD) assay, that enables counting of even one copy of a methylated gene in a small DNA sample amount without DNA bisulfite treatment. OBJECTIVES: We evaluated the sensitivity and specificity of serum DNA testing of methylated RUNX3 by the CORD assay in combination with and without CA19-9 for the detection of pancreatic cancer in 55 patients with pancreatic cancer, 12 patients with benign pancreatic disease, and 80 healthy individuals. RESULTS: The CORD assay of methylated RUNX3 had a sensitivity of 50.9% (28/55) and specificity of 93.5% (86/92). Combination of the CORD assay of methylated RUNX3 and CA19-9 resulted in a sensitivity of 85.5% (47/55) and specificity of 93.5% (86/92) for all stages of pancreatic cancer and a sensitivity of 77.8% (7/9) for stage I pancreatic cancer. CONCLUSIONS: ombination of the CORD assay and CA19-9 may provide an alternative screening strategy for detecting early-stage pancreatic cancer.

Mesenchymal Stem Cells Induce a Fibrolytic Phenotype By Regulating mmu-miR-6769b-5p Expression in Macrophages.

Liver transplantation is the only radical treatment for decompensated cirrhosis, but its use is limited owing to a shortage of donors; hence, there is an urgent need for new treatments. Previously, we developed a liver-regeneration therapy using autologous bone marrow-derived mesenchymal stem cells (BMSCs), which is under clinical investigation. Cell-cell interactions between BMSCs and macrophages (Mφs) participate in the improvement of liver function and alleviation of liver fibrosis, although the associated mechanisms have not been elucidated. Therefore, in this study, we investigated phenotypic changes in Mφs caused by interactions with BMSCs, as well as the underlying mechanisms. Co-culturing lipopolysaccharide (LPS)-stimulated murine bone marrow-derived Mφs (BMDMs) with BMSCs substantially upregulated matrix metalloproteinase 9 (Mmp9), Mmp12, and Mmp13 expression, and downregulated tumor necrosis factor alpha (Tnfα) expression. To identify humoral factors involved in phenotypic changes occurring in Mφs, microarray analysis was performed with microRNAs (miRNAs) derived from extracellular vesicles in the supernatant of co-cultured BMSCs and LPS-stimulated BMDMs. We found that miR-6769b-5p was highly expressed and that transfecting miR-6769b-5p mimic upregulated MMP9 in LPS-stimulated BMDMs and downregulated Tnfα and interleukin-1 beta (Il-1ß). MiR-6769b-5p expression in BMDMs was decreased by LPS stimulation but was increased by co-culture with BMSCs. Microarray and pathway analyses of gene expression in LPS-stimulated, miR-6769b-5p-transfected BMDMs revealed changes in the eukaryotic initiation factor 2-signaling pathway and decreased the expression of activating transcription factor 4 (Atf4). LPS-stimulated BMDMs exhibited increased MMP9 expression and decreased the expression of Tnfα and Il-1ß by ATF4 knockdown. These findings indicate that upregulating miR-6769b-5p in BMDMs induced a fibrolytic phenotype, where MMP9 was highly expressed and inflammatory cytokine expression was decreased by the suppression of ATF4 expression. These findings imply that regulating miR-6769b-5p or ATF4 expression in BMDMs may be helpful for treating chronic liver disease.

What is the best wavelength for the measurement of hemolysis index?

BACKGROUND: Hemolysis is a common problem in the handling of serum specimens. The hemolysis index (HI) provides a warning of hemolysis in auto-analyzers. However, HI has not been standardized, and each laboratory's original method is applied. Especially, the wavelength used for HI measurement is different in each laboratory. Thus, we investigated the warning ability of HI at various wavelengths. METHODS: We selected 4 wavelength types, and each HI was measured and calculated (410 nm/HI-1, 451 nm/HI-2, 545 nm/HI-3, and 571 nm/HI-4). To compare the 4 HI types, we investigated the influence of 3 interference components using artificially hemolyzed specimens (AHSs). We also investigated both the relationship between HI and hemoglobin concentration (Hb) and that between HI and 31 biochemical test values in AHSs. RESULTS: In the interference assessment, only HI-4 showed no influence on the 3 interference components. The correlation between Hb and HI-4 was very strong (rS = 0.9987). A 1-unit increase in HI-4 corresponded to a 14.8-mg/dL increase in Hb. CONCLUSION: We found the best wavelength for HI to be at or near 571 nm.

Fecal DNA Testing of TWIST1 Methylation Identifies Patients With Advanced Colorectal Adenoma Missed by Fecal Immunochemical Test for Hemoglobin.

INTRODUCTION: We have reported previously that fecal DNA testing of TWIST1 methylation in combination with the fecal immunochemical test for hemoglobin (FIT) (combination test) is useful for colorectal neoplasia screening. In this study, using larger sample sizes, we studied the clinical performance of the combination test for the detection of colorectal neoplasia and, especially, advanced colorectal adenoma. METHODS: We performed a prospective study in which FIT, fecal DNA testing of TWIST1 methylation, and colonoscopy were performed on 372 patients with colorectal neoplasia and 71 subjects without colorectal neoplasia. We assessed the individual clinical performance of each of FIT and fecal DNA testing of TWIST1 methylation and of the combination test for the detection of colorectal neoplasia including advanced adenoma based on morphologic subtypes. RESULTS: The FIT alone had a sensitivity of 7.5% (3/40) for nonadvanced adenoma, 32.3% (41/127) for advanced adenoma, and 93.7% (192/205) for colorectal cancer and a specificity of 87.3% (62/71). The combination test had a sensitivity of 35.0% (14/40) for nonadvanced adenoma, 68.5% (87/127) for advanced adenoma, and 95.6% (196/205) for colorectal cancer and a specificity of 80.3% (57/71). For morphological subtypes of advanced adenoma, the sensitivity of FIT was only 28.2% (20/71) for polypoid type and 16.1% (5/31) for nonpolypoid type, whereas the combination test increased the sensitivities to 64.8% (46/71) and 71.0% (22/31), respectively. DISCUSSION: The combination of the fecal DNA test with FIT seemed to be useful to detect colorectal neoplasia and, especially, advanced adenoma of the nonpolypoid type.

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

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.

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

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.