Cell-free DNA methylation markers for differential diagnosis of hepatocellular carcinoma
Biyuan LuoFang MaHao LiuJixiong HuLe RaoChunying LiuYongfang JiangShuyu Kuang-ZengXuan LinChenyang WangYiyu LeiZhongzhou SiGuangshun ChenNing ZhouChengbai LiangFang-Qing JiangFeng'e LiuWeidong DaiWei LiuYawen GaoZhihong LiXi LiGuangyu ZhouBingsi LiZhihong ZhangWeiqi NianLihua LuoXianling Liu
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Abstract Background Aberrant DNA methylation may offer opportunities in revolutionizing cancer screening and diagnosis. We sought to identify a non-invasive DNA methylation-based screening approach using cell-free DNA (cfDNA) for early detection of hepatocellular carcinoma (HCC). Methods Differentially, DNA methylation blocks were determined by comparing methylation profiles of biopsy-proven HCC, liver cirrhosis, and normal tissue samples with high throughput DNA bisulfite sequencing. A multi-layer HCC screening model was subsequently constructed based on tissue-derived differentially methylated blocks (DMBs). This model was tested in a cohort consisting of 120 HCC, 92 liver cirrhotic, and 290 healthy plasma samples including 65 hepatitis B surface antigen-seropositive (HBsAg+) samples, independently validated in a cohort consisting of 67 HCC, 111 liver cirrhotic, and 242 healthy plasma samples including 56 HBsAg+ samples. Results Based on methylation profiling of tissue samples, 2321 DMBs were identified, which were subsequently used to construct a cfDNA-based HCC screening model, achieved a sensitivity of 86% and specificity of 98% in the training cohort and a sensitivity of 84% and specificity of 96% in the independent validation cohort. This model obtained a sensitivity of 76% in 37 early-stage HCC (Barcelona clinical liver cancer [BCLC] stage 0-A) patients. The screening model can effectively discriminate HCC patients from non-HCC controls, including liver cirrhotic patients, asymptomatic HBsAg+ and healthy individuals, achieving an AUC of 0.957(95% CI 0.939–0.975), whereas serum α-fetoprotein (AFP) only achieved an AUC of 0.803 (95% CI 0.758–0.847). Besides detecting patients with early-stage HCC from non-HCC controls, this model showed high capacity for distinguishing early-stage HCC from a high risk population (AUC=0.934; 95% CI 0.905–0.963), also significantly outperforming AFP. Furthermore, our model also showed superior performance in distinguishing HCC with normal AFP (< 20ng ml −1 ) from high risk population (AUC=0.93; 95% CI 0.892–0.969). Conclusions We have developed a sensitive blood-based non-invasive HCC screening model which can effectively distinguish early-stage HCC patients from high risk population and demonstrated its performance through an independent validation cohort. Trial registration The study was approved by the ethic committee of The Second Xiangya Hospital of Central South University (KYLL2018072) and Chongqing University Cancer Hospital (2019167). The study is registered at ClinicalTrials.gov(# NCT04383353 ).Keywords:
Cell-free fetal DNA
A new noninvasive screening tool for colorectal neoplasia detects epigenetic alterations exhibited by gastrointestinal tumor cells shed into stool. There is insufficient existing data to determine temporal associations between colorectal cancer (CRC) progression and aberrant DNA methylation. To evaluate the feasibility of using fecal DNA methylation status to determine CRC progression, we collected stool samples from 14 male SD rats aged six weeks, and administered subcutaneous injections of either 1,2-dimethylhydrazine or saline weekly. p16 DNA methylation statuses in tumorous and normal colon tissue, and from stool samples were determined using methylation-specific PCR. Additionally, p16 methylation was detected in stool DNA from 85.7% of the CRC rats. The earliest change in p16 methylation status in the DMH-treated group stool samples occurred during week nine; repeatabilities were 57.1% in week 19 (p = 0.070) and 85.7% in week 34 (p = 0.005). A temporal correlation was evidenced between progression of CRC and p16 methylation status, as evidenced by DMH-induced rat feces. Using fecal DNA methylation status to determine colorectal tissue methylation status can reveal CRC progression. Our data suggests that p16 promoter methylation is a feasible epigenetic marker for the detection and may be useful for CRC screening.
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Mutants of hepatitis B virus surface antigen (HBsAg) are known as a cause of false-negative results in diagnostic tests for HBsAg; particularly when a diagnostic kit utilizes monoclonal antibodies to detect HBsAg. We compared seven HBsAg kits with regard to sensitivity for HBsAg subtypes (ad, ay) and their ability to detect nine different HBsAg mutants. Among them, the sensitivities of five kits were high and comparable to each other (0.2 - 0.3ng/ml). However, two kits were of lower sensitivity (0.8-1.3ng/ml, and 2.4-2.5ng/ml, respectively). Two kits, produced by the same company, reacted with all of the nine HBsAg mutants, but five kits showed false-negative results with one or more of the HBsAg mutants. These data indicate that there are differences in the detection sensitivities for HBsAg and abilities to detect HBsAg mutants among commercially available HBsAg kits, which may explain false-negative clinical results.
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DNA methylation measured in white blood cell DNA is increasingly being used as in studies of cancer susceptibility. However, little is known about the correlation between different assays to measure global methylation and whether the source of DNA matters when examining methylation profiles in different blood cell types. Using information from 620 women, 217 and 403 women with DNA available from granulocytes (Gran), and total white blood cells (WBC), respectively, and 48 women with DNA available from four different sources (WBC, Gran, mononuclear (MN), and lymphoblastoid cell lines (LCL)), we compared DNA methylation for three repetitive elements (LINE1, Sat2, Alu) by MethyLight, luminometric methylation assay (LUMA), and [3H]-methyl acceptance assay. For four of the five assays, DNA methylation levels measured in Gran were not correlated with methylation in LBC, MN, or WBC; the exception was Sat2. DNA methylation in LCL was correlated with methylation in MN and WBC for the [3H]-methyl acceptance, LINE1, and Alu assays. Methylation in MN was correlated with methylation in WBC for the [3H]-methyl acceptance and LUMA assays. When we compared the five assays to each other by source of DNA, we observed statistically significant positive correlations ranging from 0.3-0.7 for each cell type with one exception (Sat2 and Alu in MN). . Among the 620 women stratified by DNA source, correlations among assays were highest for the three repetitive elements (range 0.39-0.64). Results from the LUMA assay were modestly correlated with LINE1 (0.18-0.20). These results suggest that both assay and source of DNA are critical components in the interpretation of global DNA methylation patterns from WBC.
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In patients with chronic hepatitis B (CHB), loss of hepatitis B surface antigen (HBsAg) is considered a functional cure. However, HBsAg loss is uncommon with existing therapies, and predictive factors associated with HBsAg seroreversion are unknown. Using pooled data from three phase 3 clinical trials of patients with CHB treated with nucleos(t)ide analogue (NUC) monotherapy or peginterferon (Peg‐IFN) ± NUC combination therapy, we conducted a retrospective analysis to characterize patients who achieved sustained HBsAg loss, the predictors of HBsAg seroreversion, and the impact of hepatitis B surface antibody (anti‐HBs) seroconversion on durability of HBsAg loss. In these three international trials, 1,381 adults with CHB received either NUC monotherapy for up to 10 years or Peg‐IFN‐containing regimens for up to 1 year. A total of 55 patients had confirmed HBsAg loss, defined as two or more consecutive negative‐qualitative HBsAg results, with a minimum of one repeat result after the end of treatment. Throughout a median of 96 (quartile [Q]1, Q3, 46, 135) weeks follow‐up after HBsAg loss, HBsAg loss was durable in 82% (n = 45) of patients, with 10 patients experiencing HBsAg seroreversion. Anti‐HBs seroconversion was observed during follow‐up in 78% of patients who lost HBsAg and in 60% of those who subsequently seroreverted. In analyzing predictors of HBsAg seroreversion, study treatment was significant, yet anti‐HBs seroconversion and treatment duration after initial HBsAg loss were not. Risk of HBsAg seroreversion was observed to be lower if HBsAg loss was sustained through the off‐treatment week 24 visit (8/10 seroreversions occurred by posttreatment week 24). Conclusion: HBsAg loss after NUC or Peg‐IFN‐containing regimens was durable in 82% of patients with CHB. Anti‐HBs seroconversion and treatment duration after initial HBsAg loss were not significantly associated with durability of HBsAg loss.
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17ß-Estradiol, an epigenetic modulator, is involved in the increased prevalence of migraine in women. Together with the prophylactic efficacy of valproate, which influences DNA methylation and histone modification, this points to the involvement of epigenetic mechanisms. Epigenetic studies are often performed on leukocytes, but it is unclear to what extent methylation is similar in other tissues. Therefore, we investigated methylation of migraine-related genes that might be epigenetically regulated (CGRP-ergic pathway, estrogen receptors, endothelial NOS, as well as MTHFR) in different migraine-related tissues and compared this to methylation in rat as well as human leukocytes. Further, we studied whether 17ß-estradiol has a prominent role in methylation of these genes. Female rats (n = 35) were ovariectomized or sham-operated and treated with 17β-estradiol or placebo. DNA was isolated and methylation was assessed through bisulphite treatment and mass spectrometry. Human methylation data were obtained using the Illumina 450k genome-wide methylation array in 395 female subjects from a population-based cohort study. We showed that methylation of the Crcp, Calcrl, Esr1 and Nos3 genes is tissue-specific and that methylation in leukocytes was not correlated to that in other tissues. Interestingly, the interindividual variation in methylation differed considerably between genes and tissues. Furthermore we showed that methylation in human leukocytes was similar to that in rat leukocytes in our genes of interest, suggesting that rat may be a good model to study human DNA methylation in tissues that are difficult to obtain. In none of the genes a significant effect of estradiol treatment was observed.
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DNA promoter methylation of tumor suppressor genes and global DNA hypomethylation are common features of head and neck cancers. Our goal was to identify early DNA methylation changes in oral premalignant lesions (OPL) that may serve as predictive markers of developing oral squamous cell carcinoma (OSCC). Using high-throughput DNA methylation profiles of 24 OPLs, we found that the top 86 genes differentially methylated between patients who did or did not develop OSCC were simultaneously hypermethylated, suggesting that a CpG island methylation phenotype may occur early during OSCC development. The vast majority of the 86 genes were nonmethylated in normal tissues and hypermethylated in OSCC versus normal mucosa. We used pyrosequencing in a validation cohort of 44 patients to evaluate the degree of methylation of AGTR1, FOXI2, and PENK promoters CpG sites that were included in the top 86 genes and of LINE1 repetitive element methylation, a surrogate of global DNA methylation. A methylation index was developed by averaging the percent methylation of AGTR1, FOXI2, and PENK promoters; patients with a high methylation index had a worse oral cancer-free survival (P = 0.0030). On the other hand, patients with low levels of LINE1 methylation had a significantly worse oral cancer-free survival (P = 0.0153). In conclusion, AGTR1, FOXI2, and PENK promoter methylation and LINE1 hypomethylation may be associated with an increased risk of OSCC development in patients with OPLs.
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DNA methylation is a well-characterized epigenetic modification that plays an important role in the regulation of gene expression. There is growing evidence on the involvement of epigenetic mechanisms in disease onset, including cancer. Environmental factors seem to induce changes in DNA methylation affecting human health. However, little is known about basal methylation levels in healthy people and about the correlation between environmental factors and different methylation profiles. We investigated the effect of seasonality on basal methylation by testing methylation levels in the long interspersed nucleotide element-1 (LINE-1) and in two cancer-related genes (RASSF1A and MGMT) of 88 healthy male heavy smokers involved in an Italian randomized study; at enrolment the subjects donated a blood sample collected in different months. Methylation analyses were performed by pyrosequencing. Mean methylation percentage was higher in spring and summer for the LINE1, RASSF1A and MGMT genes (68.26%, 2.35%, and 9.52% respectively) compared with autumn and winter (67.43%, 2.17%, and 8.60% respectively). In particular, LINE-1 was significantly hypomethylated (p = 0.04 or 0.05 depending on the CpG island involved) in autumn and winter compared with spring and summer. Seasonality seems to be a modifier of methylation levels and this observation should be taken into account in future analyses.
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