Early-stage Hepatocellular Carcinoma Screening And Evolutionary Trajectories Of Ovarian Cancer Precursor Lesions Based On NGS

BACKGROUND&AIMS:Hepatitis B virus infection is one of the risk factors for hepatocellular carcinoma(HCC).This study aim to efficiently identify early stage HCCs from asymptomatic HBsAg-seropositive individuals in a community population by liquid biopsy base on cell free DNA(cfDNA)and proteins biomarkers.METHODS:Firstly,we developed a new cfDNA alterations detection technique named Mutation Capsule,which validated the performance of this method using genomic DNA standards containing known mutations and methylated DNA standards.Secondly,we designed a panel containing multiple primers covering the coding regions of TP53,CTNNB1,AXIN1,the promoter region of TERT,the HBV sequence.We collected 135 HCC or non-HCC individuals with clear diagnostic results,70 healthy individuals,and 331 HBsAg-positive but AFP/US-negative individuals without clinical symptoms.We determined the serum AFP and DCP levels of these samples,and detected the cfDNA genetic alterations simultaneously by Mutation Capsule.Finally,we developed a liquid biopsy assay,named HCCscreen,to identify HCC from HBsAg-seropositive,asymptomatic individuals in community population.These 135 cases who had liver nodules and/or elevated serum AFP levels were used as the training cohort.We further applied this assay to 331 individuals with normal liver US and serum AFP level,and early liver cancer screening was performed and followed up for 6-8 months.RESULTS:The validation results showed that the Mutation Capsule method can detect low-frequency mutations and methylation alterations accurately on low-quantity DNA samples.Our HCC panel can effectively detect a variety of HCC-related genetic variation and methylation changes in normal/tumor tissues,and these alterations can be used as biomarkers for distinguish between cancerous and non-cancerous conditions.The HCCScreen assay robustly separated those with HCC from those who were non-HCC with a sensitivity of 85%and a specificity of 93%in traning corhort.24 positive cases were identified and clinical follow-up for 6-8 months confirmed four had developed HCC.No HCC cases were diagnosed from the 307 test-negative individuals in the follow-up during the same time scale.Thus,the assay showed 100%sensitivity,94%specificity and 17%positive predictive value in the validation cohort.Notably,each of the four HCCs was at early stage(<3cm)when diagnosed.Conclusion:The Mutation Capsule can be used for mutation detection of cfDNA and supports simultaneous detection of genetic and methylation alterations.Our study indicates that the HCCScreen assay can effectively detect early-stage HCC from HBsAg-seropositive,asymptomatic individuals and is expected to be used in clinical practice.The clonal relationship between ovarian high-grade serous carcinoma(HGSC)and its presumed precursor lesion,serous tubal intraepithelial carcinoma(STIC),has been reported.However,when analyzing patients with concurrent ovarian carcinoma and tubal lesion,the extensive carcinoma tissues present at diagnosis may have effaced the natural habitat of precursor clones,obscuring tumor clonal evolutionary history,or may have disseminated to anatomically adjacent fimbriae ends,masquerading as precursor lesions.To circumvent these limitations,we analyzed the genomic landscape of incidental tubal precursor lesions including p53 signature,dormant STIC and proliferative STIC in women without ovarian carcinoma or any cancer diagnosis using whole-exome sequencing and target sequencing.In three of the four cancer-free women with multiple discrete tubal lesions we observed non-identical TP53 mutations between precursor lesions from the same individual.In one of the four women with co-existing ovarian HGSC and tubal precursor lesion we found non-identical TP53 mutations and a lack of common mutations shared between her precursor lesion and carcinoma.Analyzing the evolutionary history of multiple tubal lesions in the same four patients with concurrent ovarian carcinoma indicated distinct evolution trajectories.Collectively,the results support diverse clonal origins of tubal precursor lesions at the very early stages of tumorigenesis.Mathematical modeling based on lesion-specific proliferation rates indicated that p53 signature and dormant STIC may take a prolonged time(two decades or more)to develop into STIC,whereas STIC may progress to carcinoma in a much shorter time(6 years).The above findings may have implications for future research aimed at prevention and early detection of ovarian cancer.