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Table S5

Purpose:Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear.Experimental Design:A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study.Results:A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone.Conclusions:Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Purpose: Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. Experimental Design: A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. Results: A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. Conclusions: Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

Background: Patients with liver cirrhosis are at higher risk of developing hepatocellular carcinoma (HCC). Due to the low accuracy of current methods, new non-invasive strategies for early HCC diagnosis in cirrhotic patients are needed. Methods: A case-control study recruited a total of 2,250 patients with liver cirrhosis (LC), 508 with HCC, and 476 healthy controls (CTRL), from 13 hospitals in 11 provinces of China. Four genomic features of cell-free DNA (cfDNA), including nucleosome footprint, fragmentation, base mismatch and 5-hydroxymethylcytosines were profiled. Training and validation cohorts helped develop the integrated diagnostic model, and a test cohort was used to evaluate its performance. Findings: We established a classifier of each feature for HCC against non-HCC (cirrhosis & healthy control). By integrating these four genomic features, we developed a novel diagnostic model (HIFI, 5-Hydroxymethylcytosines/base mIsmatch/Fragmentation/nucleosome footprInt), and achieved a higher accuracy in differentiating HCCs from liver cirrhosis (AUC=0.997 [0.994-0.999], sensitivity 95.42%, specificity 97.67% in test set) than AFP or PIVKA-II, irrespective of the following demographics and clinical features, age, HBV status, Child-Pugh score, BCLC stage, tumor size, and AFP status. In AFP positive (AFP>20ug/L) liver cirrhosis patients and AFP/PIVKA-II negative (AFP 400μg/L: 100% in liver cirrhosis patients; AFP

To observe the therapeutic effects of Pegasys (pegylated interferon alpha-2a) in chronic hepatitis B (CHB) patients with low-level alanine transaminase (ALT)2 x upper limit of normal (ULN).One hundred and seven CHB patients were randomized enrolled including 52 with ALT2 x ULN and liver tissues inflammation activityor = G2 as observational group and 55 with ALT2 x ULN as control group. All the enrolled patients received pegasys treatment for 48 weeks and the responses between two groups were compared. Measurement data were analyzed using t test and numeration data were analyzed using chi square test.The reductions of HBV DNA in observational group at different time points were all less than control group (all P0.05). At the end of treatment, the HBV DNA negative rate, HBeAg seroconversion rate and HBsAg loss rate in the observational group were 51.9%, 48.8% and 1.9% , respectively, which were all lower than control group (67.3% , 66.7% and 7. 3% , respectivley) ( all P0. 05). The ALT normalization rates of two groups were 75% and 76.4% (P0. 05).Pegasys is efficient for CHB patients with ALT2 x ULN and liver tissues inflammation activityor = G2, while it is still inferior to those with ALT2 x ULN.