Non-invasive diagnosis of esophageal cancer by a simplified circulating cell-free DNA methylation assay targeting OTOP2 and KCNA3: a double-blinded, multicenter, prospective study.

BACKGROUND: Esophageal cancer (EC) is a highly lethal disease lacking early detection approaches. We previously identified that OTOP2 and KCNA3 were specifically hypermethylated in circulating cell-free DNA from patients with EC. We then developed a blood-based methylation assay targeting OTOP2 and KCNA3 (named "IEsohunter") for esophageal cancer noninvasive detection. This double-blinded, multicenter, prospective study aimed to comprehensively evaluate its clinical diagnostic performance. METHODS: Participants with EC, high-grade intraepithelial neoplasia (HGIN), other malignancies, benign gastrointestinal lesions, or no abnormalities were prospectively enrolled from 5 tertiary referral centers across China. Peripheral blood samples were collected, followed by plasma cell-free DNA methylation analysis using the IEsohunter test based on multiplex quantitative polymerase chain reaction adopting an algorithm-free interpretation strategy. The primary outcome was the diagnostic accuracy of IEsohunter test for EC. RESULTS: We prospectively enrolled 1116 participants, including 334 patients with EC, 71 with HGIN, and 711 controls. The areas under the receiver operating characteristic curves of the IEsohunter test for detecting EC and HGIN were 0.903 (95% CI 0.880-0.927) and 0.727 (95% CI 0.653-0.801), respectively. IEsohunter test showed sensitivities of 78.5% (95% CI 69.1-85.6), 87.3% (95% CI 79.4-92.4), 92.5% (95% CI 85.9-96.2), and 96.9% (95% CI 84.3-99.8) for stage I-IV EC, respectively, with an overall sensitivity of 87.4% (95% CI 83.4-90.6) and specificity of 93.3% (95% CI 91.2-94.9) for EC detection. The IEsohunter test status turned negative (100.0%, 47/47) after surgical resection of EC. CONCLUSIONS: The IEsohunter test showed high diagnostic accuracy for EC detection, indicating that it could potentially serve as a tool for noninvasive early detection and surveillance of EC.

Plasma methylated GNB4 and Riplet as a novel dual-marker panel for the detection of hepatocellular carcinoma.

Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.

Genome-wide methylation profiling identified methylated KCNA3 and OTOP2 as promising diagnostic markers for esophageal squamous cell carcinoma.

BACKGROUND: Early detection of esophageal squamous cell carcinoma (ESCC) can considerably improve the prognosis of patients. Aberrant cell-free DNA (cfDNA) methylation signatures are a promising tool for detecting ESCC. However, available markers based on cell-free DNA methylation are still inadequate. This study aimed to identify ESCC-specific cfDNA methylation markers and evaluate the diagnostic performance in the early detection of ESCC. METHODS: We performed whole-genome bisulfite sequencing (WGBS) for 24 ESCC tissues and their normal adjacent tissues. Based on the WGBS data, we identified 21,469,837 eligible CpG sites (CpGs). By integrating several methylation datasets, we identified several promising ESCC-specific cell-free DNA methylation markers. Finally, we developed a dual-marker panel based on methylated KCNA3 and OTOP2, and then, we evaluated its performance in our training and validation cohorts. RESULTS: The ESCC diagnostic model constructed based on KCNA3 and OTOP2 had an AUC of 0.91 [95% CI: 0.85-0.95], and an optimal sensitivity and specificity of 84.91% and 94.32%, respectively, in the training cohort. In the independent validation cohort, the AUC was 0.88 [95% CI: 0.83-0.92], along with an optimal sensitivity of 81.5% and specificity of 92.9%. The model sensitivity for stage I-II ESCC was 78.4%, which was slightly lower than the sensitivity of the model (85.7%) for stage III-IV ESCC. CONCLUSIONS: The dual-target panel based on cfDNA showed excellent performance for detecting ESCC and might be an alternative strategy for screening ESCC.

Genome-wide methylation profiling identify hypermethylated HOXL subclass genes as potential markers for esophageal squamous cell carcinoma detection.

BACKGROUND: Numerous studies have revealed aberrant DNA methylation in esophageal squamous cell carcinoma (ESCC). However, they often focused on the partial genome, which resulted in an inadequate understanding of the shaped methylation features and the lack of available methylation markers for this disease. METHODS: The current study investigated the methylation profiles between ESCC and paired normal samples using whole-genome bisulfite sequencing (WGBS) data and obtained a group of differentially methylated CpGs (DMC), differentially methylated regions (DMR), and differentially methylated genes (DMG). The DMGs were then verified in independent datasets and Sanger sequencing in our custom samples. Finally, we attempted to evaluate the performance of these genes as methylation markers for the classification of ESCC. RESULTS: We obtained 438,558 DMCs, 15,462 DMRs, and 1568 DMGs. The four significantly enriched gene families of DMGs were CD molecules, NKL subclass, HOXL subclass, and Zinc finger C2H2-type. The HOXL subclass homeobox genes were observed extensively hypermethylated in ESCC. The HOXL-score estimated by HOXC10 and HOXD1 methylation, whose methylation status were then confirmed by sanger sequencing in our custom ESCC samples, showed good ability in discriminating ESCC from normal samples. CONCLUSIONS: We observed widespread hypomethylation events in ESCC, and the hypermethylated HOXL subclass homeobox genes presented promising applications for the early detection of esophageal squamous cell carcinoma.

Hypermethylated CDO1 and ZNF454 in Cytological Specimens as Screening Biomarkers for Endometrial Cancer.

We aimed to estimate the diagnostic value of DNA methylation levels in cytological samples of endometrial cancer (EC) and atypical hyperplasia (AH). Two hypermethylated genes, namely, cysteine dioxygenase type 1 (CDO1) and zinc finger protein 454 (ZNF454), in patients with EC were identified from The Cancer Genome Atlas database. In 103 endometrial histological specimens (the training set), the methylation levels of candidate genes were verified by quantitative methylation-specific polymerase chain reaction (qMSP). The methylation levels of another 120 cytological specimens (the testing set) were evaluated. Sensitivity (Se), specificity (Sp), accuracy, and area under the curve (AUC) were determined, with diagnosis verified by histopathological results. CDO1 and ZNF454 verified hypermethylation in histological specimens of patients with EC and AH compared with those with benign and normal endometrium (P < 0.001). In cytological specimens, hypermethylated CDO1 showed 86.36% Se and 90.79% Sp with the cutoff value of 6.0 to distinguish between malignant and benign groups; ZNF454 showed 79.55% Se and 93.42% Sp with the cutoff value of 7.1. When the two genes were combined, Se increased to 90.91% and Sp was 86.84%. AUC reached 0.931 (95% CI: 0.885-0.976). The diagnostic accuracy with cytology had no significant difference with endometrial tissue (P = 0.847 for CDO1, P = 0.108 for ZNF454, and P = 0.665 for their combination). Hypermethylated CDO1 and ZNF454 in endometrial cytology showed high Se, Sp, and AUC to detect EC and AH. Methylation analysis of endometrial cytology is promising biomarker for the screening of EC and AH.

The methylation of SDC2 and TFPI2 defined three methylator phenotypes of colorectal cancer.

BACKGROUND: Methylated SDC2 and TFPI2 are widely used for colorectal cancer (CRC) detection. However, they often miss some CRCs, which directly diminishes the sensitivity. Further investigations of the underlying mechanisms leading to the missed samples will facilitate developing more eligible methylation markers. METHODS: CRC samples from TCGA and GEO datasets were divided into three groups, High-methylation/ High-methylation (HH), High-methylation/Low-methylation (HL), and Low-methylation/Low-methylation (LL) according to the methylation status of SDC2 and TFPI2 promoters. Variations in age, tumor location and microsatellite instable were then assessed between the three groups and verified in our custom cohort. RESULTS: Samples of HL group preferred to derive from left-sided CRCs (P < 0.05). HH samples showed the highest microsatellite instability and mutation load (mean nonsynonymous mutations for HH/HL/LL: 10.55/3.91/7.02, P = 0.0055). Almost all mutations of BRAF, one of the five typical CpG island methylator phenotype (CIMP) related genes, were observed in HH group (HH/HL/LL: 51/0/1, P = 0.018). Besides, older patients were frequently found in HH group. Expression analysis identified 37, 84, and 22 group-specific differentially expressed genes (DEGs) for HH, HL, and LL, respectively. Functional enrichment analysis revealed that HH-specific DEGs were mainly related to transcription regulation, while LL-specific DEGs were enriched in the biological processes of extracellular matrix interaction and cell migration. CONCLUSIONS: The current study revealed that the performance of methylation-based markers might be affected by tumor location, patient age, mutation load and MSI, and these respective sides should be considered when developing new methylation markers for CRC detection.

Identification of two methylated fragments of an SDC2 CpG island using a sliding window technique for early detection of colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancer types globally with a 5-year survival rate of < 50% in China. Aberrant DNA methylation is one of the hallmarks of tumor initiation, progression, and metastasis. Here, we investigated the clinical performance of two differentially methylated regions (DMRs) in SDC2 CpG islands for the detection of CRC. A sliding window technique was used to identify the DMRs, and methylation-specific PCR assay was used to assess the DMRs in 198 CRC samples and 54 normal controls. Two DMRs (DMR2 and DMR5) were identified using The Cancer Genome Atlas (TCGA) data, and the hypermethylation of DMR2 and DMR5 was detected in 90.91% (180/198) and 89.90% (178/198) of CRC samples, respectively. When combining DMR2 and DMR5, the sensitivity for CRC detection was 94.4% higher than that of DMR2 or DMR5 alone. Based on the above results, we propose using DMR2 and DMR5 as a sensitive biomarker to detect CRC.

Methylation of SDC2/TFPI2 and Its Diagnostic Value in Colorectal Tumorous Lesions.

Background: SDC2 methylation is a feasible biomarker for colorectal cancer detection. Its specificity for colorectal cancer is higher than 90%, but the sensitivity is normally lower than 90%. This study aims to improve the sensitivity of SDC2 detection through finding a high positive target from the false-negative samples of SDC2 detection based on analysis of the bowel subsite difference in methylation. Methods: Hypermethylated TFPI2 was identified in SDC2 hypomethylated colorectal cancer samples retrieved from TCGA database with the methylation level lower than 0.2. The methylation-specific PCR assay was developed and then evaluated using tissue samples (184 cancer and 54 healthy control samples) and stool samples (289 cancer, 190 adenoma, and 217 healthy control samples). Results: TFPI2 was hypermethylated in most SDC2 hypomethylated colorectal cancer samples. When the SDC2/TFPI2-combined PCR assay was performed in stool specimens, the AUC value of cancer vs. control was 0.98, with the specificity of 96.40% and sensitivity of 96.60%, and the AUC value of adenoma vs. control was 0.87, with the specificity of 95.70% and the sensitivity of 80.00%. The improvement in sensitivity was the most momentous in the left colon. As the detection index, the Ct value was better in improving the sensitivity of detection than the methylation level based on the 2-ΔΔCt value. Conclusion: TFPI2 can improve the sensitivity of SDC2 methylation-specific detection of colorectal tumorous lesions while maintaining high specificity, in particular reducing the missed detection of left colon cancer and adenoma.

When will the battle against novel coronavirus end in Wuhan: A SEIR modeling analysis.

BACKGROUND: Recent outbreak of 2019-nCoV in Wuhan raised serious public health concerns. By February 15, 2020 in Wuhan, the total number of confirmed infection cases has reached 37 914, and the number of deaths has reached 1123, accounting for 56.9% of the total confirmed cases and 73.7% of the total deaths in China. People are eager to know when the epidemic will be completely controlled and when people's work and life will be on the right track. METHOD: In this study we analyzed the epidemic dynamics and trend of 2019-nCoV in Wuhan by using the data after the closure of Wuhan city till February 12, 2020 based on the SEIR modeling method. RESULTS: The optimal parameters were estimated as R0 = 1.44 (interquartile range: 1.40-1.47), TI = 14 (interquartile range = 14-14) and TE = 3.0 (interquartile range = 2.8-3.1). Based on these parameters, the number of infected individuals in Wuhan city may reach the peak around February 19 at about 47 000 people. Once entering March, the epidemic would gradually decline, and end around the late March. It is worth noting that the above prediction is based on the assumption that the number of susceptible population N = 200 000 will not increase. If the epidemic situation is not properly controlled, the peak of infected number can be further increased and the peak time will be a little postponed. It was expected that the epidemic would subside in early March, and disappear gradually towards the late March. CONCLUSIONS: The epidemic situation of 2019-nCoV in Wuhan was effectively controlled after the closure of the city, and the disease transmission index also decreased significantly. It is expected that the peak of epidemic situation would be reached in late February and end in March.

The Jujube Genome Provides Insights into Genome Evolution and the Domestication of Sweetness/Acidity Taste in Fruit Trees.

Jujube (Ziziphus jujuba Mill.) belongs to the Rhamnaceae family and is a popular fruit tree species with immense economic and nutritional value. Here, we report a draft genome of the dry jujube cultivar 'Junzao' and the genome resequencing of 31 geographically diverse accessions of cultivated and wild jujubes (Ziziphus jujuba var. spinosa). Comparative analysis revealed that the genome of 'Dongzao', a fresh jujube, was ~86.5 Mb larger than that of the 'Junzao', partially due to the recent insertions of transposable elements in the 'Dongzao' genome. We constructed eight proto-chromosomes of the common ancestor of Rhamnaceae and Rosaceae, two sister families in the order Rosales, and elucidated the evolutionary processes that have shaped the genome structures of modern jujubes. Population structure analysis revealed the complex genetic background of jujubes resulting from extensive hybridizations between jujube and its wild relatives. Notably, several key genes that control fruit organic acid metabolism and sugar content were identified in the selective sweep regions. We also identified S-locus genes controlling gametophytic self-incompatibility and investigated haplotype patterns of the S locus in the jujube genomes, which would provide a guideline for parent selection for jujube crossbreeding. This study provides valuable genomic resources for jujube improvement, and offers insights into jujube genome evolution and its population structure and domestication.

Removing PCR for the elimination of undesired DNA fragments cycle by cycle.

A novel removing polymerase chain reaction (R-PCR) technique was developed, which can eliminate undesired genes, cycle by cycle, with efficiencies of 60.9% (cDNAs), 73.6% (genomic DNAs), and ~ 100% (four DNA fragments were tested). Major components of the R-PCR include drivers, a thermostable restriction enzyme - ApeKI, and a poly(dA) adapter with mismatched restriction enzyme recognition sites. Drivers were generated from the undesired genes. In each cycle of R-PCR, drivers anneal to complementary sequences and allow extension by Taq DNA polymerase. Thus, ApeKI restriction sites in the undesired genes are recovered, and adapters of these undesired DNA fragments are removed. Using R-PCR, we isolated maize upregulated defense-responsive genes and Blumeria graminis specialized genes, including key pathogenesis-related effectors. Our results show that after the R-PCR reaction, most undesired genes, including very abundant genes, became undetectable. The R-PCR is an easy and cost-efficient method to eliminate undesired genes and clone desired genes.