Cell-free DNA testing for early hepatocellular carcinoma surveillance.

BACKGROUND: Liver cirrhosis (LC) is the highest risk factor for hepatocellular carcinoma (HCC) development worldwide. The efficacy of the guideline-recommended surveillance methods for patients with LC remains unpromising. METHODS: A total of 4367 LCs not previously known to have HCC and 510 HCCs from 16 hospitals across 11 provinces of China were recruited in this multi-center, large-scale, cross-sectional study. Participants were divided into Stage Ⅰ cohort (510 HCCs and 2074 LCs) and Stage Ⅱ cohort (2293 LCs) according to their enrollment time and underwent Tri-phasic CT/enhanced MRI, US, AFP, and cell-free DNA (cfDNA). A screening model called PreCar Score was established based on five features of cfDNA using Stage Ⅰ cohort. Surveillance performance of PreCar Score alone or in combination with US/AFP was evaluated in Stage Ⅱ cohort. FINDINGS: PreCar Score showed a significantly higher sensitivity for the detection of early/very early HCC (Barcelona stage A/0) in contrast to US (sensitivity of 51.32% [95% CI: 39.66%-62.84%] at 95.53% [95% CI: 94.62%-96.38%] specificity for PreCar Score; sensitivity of 23.68% [95% CI: 14.99%-35.07%] at 99.37% [95% CI: 98.91%-99.64%] specificity for US) (P < 0.01, Fisher's exact test). PreCar Score plus US further achieved a higher sensitivity of 60.53% at 95.08% specificity for early/very early HCC screening. INTERPRETATION: Our study developed and validated a cfDNA-based screening tool (PreCar Score) for HCC in cohorts at high risk. The combination of PreCar Score and US can serve as a promising and practical strategy for routine HCC care. FUNDING: A full list of funding bodies that contributed to this study can be found in Acknowledgments section.

The development of a cSMART-based integrated model for hepatocellular carcinoma diagnosis.

BACKGROUND: Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising. METHODS: Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes. RESULTS: An integrated diagnostic model called "Combined method" was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

The Landscape of Cell-Free HBV Integrations and Mutations in Cirrhosis and Hepatocellular Carcinoma Patients.

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.

[Adenovirus-mediated transduction of HBV/C genome into HepG2 cell line].

OBJECTIVE: To establish a method for efficient transfer of 1.3-fold HBV/C genome into HepG2 cell line using adenoviral vector system for studying the replication and antigen expression of HBV. METHODS: The 1.3-copy overlength genome of HBV genotype C was constructed and cloned into the shuttle vector pAdTrack. After confirmation of the constructed HBV genome by sequencing, the resultant plasmid linearized by digestion with Pme I was transformed into competent E.coli Adeasier-1 cells. The recombinants of pAdEasy-HBV/C were linearized by digestion with Pac I and transfected into the packaging cells (293 cells) via liposome. HepG2 cells were then infected with a proper quantity of the recombinant adenoviruses. The HBV DNA level and HBeAg and HBsAg titers were detected in the cell medium. RESULTS: The 1.3-fold overlength HBV/C genome was efficiently transferred into HepG2 cells via the adenoviral vector system, which resulted in initiation of the virus replication and protein expression in the cells using the viral replication mechanism. CONCLUSION: The adenovirus vector system (AdEasy) allows convenient and effective transfer of HBV genome into HepG2 cells, and provides a convenient means for screening therapeutic drugs against HBV.

[Characteristics of HBcAg(18-27) CTL epitopes of the main epidemic HBV strains in China].

OBJECTIVE: To study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China. METHOD: 30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites. RESULTS: We successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24). CONCLUSION: The last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.

[Distribution of hepatitis B virus genotype B subgenotype in China].

OBJECTIVE: To investigate the distribution of hepatitis B virus (HBV) genotype B subgenotype in China. METHODS: A cohort of 511 patients with chronic HBV genotype B infection, collected from 7 centers across China, were analyzed by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or nucleotide sequencing. RESULTS: For the 511 patients, only subgenotype Ba was identified and subgenotype Bj was not found. CONCLUSION: In China, subgenotype Ba was the most prevalent HBV strains, while subgenotype Bj was very rarely found.

Analysis of the CDR3 length of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B.

To clarify the characteristics of TCR alphabeta T cells in the peripheral blood of patients with chronic hepatitis B (CHB), we investigated the patterns of Complementarity Determining Region3 (CDR3) length distribution for all 24 TCR BV gene families and 32 TCR AV gene family in the peripheral blood lymphocytes of two patients with CHB and two healthy controls by immunoscope spectratyping technique. We found that the profiles of CDR3 length distribution for all TCR AV and TCR BV family showed Gaussian distribution (the polyclonal TCR alphabeta T cells) in healthy controls, however, the restricted usage of TCR BV and TCR AV family (the oligoclonal TCR alphabeta T cells) has been monitored in two CHB patients, furthermore, the oligoclonal TCR alphabeta T cells showed the different CDR3 sequences and length, it might be correlated to the different epitope of hepatitis B virus (HBV) or the different HLA type of the patients. Detailed analysis of the CDR3 length of TCR alphabeta T cells might be interesting to light on the further study of the individualized immunotherapy of CHB and the further research of the new T lymphocyte epitope vaccine of HBV.