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Background/Aims New prognostic markers are needed to identify patients with hepatocellular carcinoma (HCC) who carry a worse prognosis. Ultra-low-pass whole-genome sequencing (ULP-WGS) (≤0.5× coverage) of cell-free DNA (cfDNA) has emerged as a low-cost promising tool to assess both circulating tumor DNA (ctDNA) fraction and large structural genomic alterations. Here, we studied the performance of ULP-WGS of plasma cfDNA to infer prognosis in patients with HCC. Methods Plasma samples were obtained from patients with HCC prior to surgery, locoregional or systemic therapy, and were analyzed by ULP-WGS of cfDNA to an average genome-wide fold coverage of 0.3x. ctDNA and copy number alterations (CNA) were estimated using the software package ichorCNA. Results Samples were obtained from 73 HCC patients at different BCLC stages (BCLC 0/A: n=37, 50.7%; BCLC B/C: n=36, 49.3%). ctDNA was detected in 18 out of 31 patients who received systemic treatment. Patients with detectable ctDNA showed significantly worse overall survival (median, 13.96 months vs not reached). ctDNA remained an independent predictor of prognosis after adjustment by clinical-pathologic features and type of systemic treatment (hazard ratio 7.69; 95%, CI 2.09–28.27). Among ctDNA-positive patients under systemic treatments, the loss of large genomic regions in 5q and 16q arms was associated with worse prognosis after multivariate analysis. Conclusions ULP-WGS of cfDNA provides clinically relevant information about the tumor biology. The presence of ctDNA and the loss of 5q and 16q arms in ctDNA-positive patients are independent predictors of worse prognosis in patients with advanced HCC receiving systemic therapy.

Abstract The analysis of circulating tumor cells (CTCs) in blood is a powerful noninvasive alternative to conventional tumor biopsy. Inertial‐based separation is a promising high‐throughput, marker‐free sorting strategy for the enrichment and isolation of CTCs. Here, we present and validate a double spiral microfluidic device that efficiently isolates CTCs with a fine‐tunable cut‐off value of 9 μm and a separation range of 2 μm. We designed the device based on computer simulations that introduce a novel, customized inertial force term, and provide practical fabrication guidelines. We validated the device using calibration beads, which allowed us to refine the simulations and redesign the device. Then we validated the redesigned device using blood samples and a murine model of metastatic breast cancer. Finally, as a proof of principle, we tested the device using peripheral blood from a patient with hepatocellular carcinoma, isolating more than 17 CTCs/ml, with purity/removal values of 96.03% and 99.99% of white blood cell and red blood cells, respectively. These results confirm highly efficient CTC isolation with a stringent cut‐off value and better separation results than the state of the art.

Liquid biopsy, the molecular analysis of tumor components released into the bloodstream, has emerged as a noninvasive and resourceful means to access genomic information from cancers. Most data derived from translational studies showcase its numerous potential clinical applications. However, data from experimental models are scarce, and little is known about the underlying mechanisms and factors controlling the release of circulating tumor DNA (ctDNA) and cells (CTCs). This study aimed to model liquid biopsy in hepatocellular carcinoma xenografts and to study the dynamics of release of ctDNA and CTCs; this included models of intratumoral heterogeneity (ITH) and metastatic disease. We quantified ctDNA by quantitative polymerase chain reaction (PCR) targeting human long interspersed nuclear element group 1; targeted mutation analysis was performed with digital droplet PCR. CTCs were traced by flow cytometry. Results demonstrated the feasibility of detecting ctDNA, including clone‐specific mutations, as well as CTCs in blood samples of mice. In addition, the concentration of ctDNA and presence of tumor‐specific mutations reflected tumor progression, and detection of CTCs was associated with metastases. Our ITH model suggested differences in the release of DNA fragments impacted by the cell‐clone origin and the treatment. Conclusion: These data present new models to study liquid biopsy and its underlying mechanisms and highlighted a clone‐dependent release of ctDNA into the bloodstream.

Cancer testis antigens (CTAs) are an extensive gene family with a unique expression pattern restricted to germ cells, but aberrantly reactivated in cancer tissues. Studies indicate that the expression (or re-expression) of CTAs within the MAGE-A family is common in hepatocellular carcinoma (HCC). However, no systematic characterization has yet been reported. The aim of this study is to perform a comprehensive profile of CTA de-regulation in HCC and experimentally evaluate the role of MAGEA3 as a driver of HCC progression. The transcriptomic analysis of 44 multi-regionally sampled HCCs from 12 patients identified high intra-tumor heterogeneity of CTAs. In addition, a subset of CTAs was significantly overexpressed in histologically poorly differentiated regions. Further analysis of CTAs in larger patient cohorts revealed high CTA expression related to worse overall survival and several other markers of poor prognosis. Functional analysis of MAGEA3 was performed in human HCC cell lines by gene silencing and in a genetic mouse model by overexpression of MAGEA3 in the liver. Knockdown of MAGEA3 decreased cell proliferation, colony formation and increased apoptosis. MAGEA3 overexpression was associated with more aggressive tumors in vivo. In conclusion MAGEA3 enhances tumor progression and should be considered as a novel therapeutic target in HCC.

Without any treatment, the prognosis of hepatitis B in liver transplant recipients is very poor. So, antiviral prophylaxis is very important in patients with hepatitis B who undergo liver transplantation. Before liver transplantation, a suppression of viral replication has to be achieved by nucleos(t)ide analogs. Drugs used in the prophylaxis of post-transplant hepatitis B include immunoglobulin against HBV and nucleos(t)ide analogs. Prophylaxis against graft infection must be based on the individual risk of recurrence. When prophylactic measures have failed and graft infection has occurred, treatment of recurrent hepatitis B may be based on the resistance profile of the virus and previous antiviral exposure. Finally, lamivudine seems to be very effective in the prevention of de novo hepatitis B in patients transplanted with a graft from an anti-HBc positive donor.La infección por el virus de la hepatitis B en los receptores de trasplante hepático tiene un pronóstico malo en ausencia de tratamiento farmacológico. Por ello, en los pacientes trasplantados por una hepatopatía por virus B, la profilaxis de esta infección es imprescindible. Antes del trasplante, debe intentarse suprimir la replicación viral con un análogo de nucleót(s)idos. Tras el trasplante, la profilaxis de la recidiva de la hepatitis B se basa en el uso de los análogos de nucleót(s)idos y la inmunoglobulina frente al virus de la hepatitis B; la pauta de profilaxis debe basarse en el riesgo de recidiva de la hepatitis B que tenga cada paciente. En el caso de que se produzca una hepatitis B tras el trasplante, el tratamiento con análogos de nucleót(s)idos debe basarse en los estudios de resistencia. Por último, en los pacientes trasplantados que reciben un injerto de un donante antiHBc-positivo, la profilaxis con lamivudina parece ser muy eficaz.

The lack of antiviral cellular immune responses in patients with chronic hepatitis C virus (HCV) infection suggests that T-cell vaccines may provide therapeutic benefit. Due to the central role that dendritic cells (DC) play in the activation of T-cell responses, our aim was to carry out a therapeutic vaccination clinical trial in HCV patients using DC. Five patients with chronic HCV infection were vaccinated with three doses of 5 Ã 106 or 107 autologous DC transduced with a recombinant adenovirus encoding NS3 using the adapter protein CFh40L, which facilitates DC transduction and maturation. No significant adverse effects were recorded after vaccination. Treatment caused no changes in serum liver enzymes nor in viral load. Vaccination induced weak but consistent expansion of T-cell responses against NS3 and adenoviral antigens. Patientsâ DC, as opposed to murine DC or DC from healthy subjects, secreted high IL-10 levels after transduction, inducing the activation of IL-10âproducing T cells. IL-10 blockade during vaccine preparation restored its ability to stimulate anti-NS3 Th1 responses. Thus, vaccination with adenovirus-transduced DC is safe and induces weak antiviral immune responses. IL-10 associated with vaccine preparation may be partly responsible for these effects, suggesting that future vaccines should consider concomitant inhibition of this cytokine.