Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time sequencing.

The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure.

A simplified method to quantitatively predict the effect of lenvatinib on hepatocellular carcinoma using contrast-enhanced ultrasound with perfluorobutane microbubbles.

Contrast-enhanced computed tomography (CECT) is generally used to evaluate the response to treatment of hepatocellular carcinoma (HCC); however, CECT is unsuitable for the early prediction of therapeutic effects and frequent monitoring. We aimed to investigate the usefulness of our simplified method for the quantification of tumor vascularity using contrast-enhanced ultrasound (CEUS) with perfluorobutane microbubbles [Sonazoid® (GE Healthcare, Oslo, Norway)] to predict the therapeutic effect of lenvatinib. Among the 13 patients studied, nine who had more than a 20% reduction in tumor vascularity within 2 weeks of starting treatment experienced complete response or partial response at 8-12 weeks as assessed by CECT. In contrast, three patients without reductions and one patient with only a slight decrease in tumor vascularity had a poor response to lenvatinib. Quantitative assessment of tumor vascularity by our simplified CEUS-based method could be a useful predictor of therapeutic responses to lenvatinib in patients with HCC.

Mac-2 binding protein glycosylation isomer predicts tolerability and clinical outcome of lenvatinib therapy for hepatocellular carcinoma.

BACKGROUND: Many patients with hepatocellular carcinoma present with impaired hepatic function, which often requires interruption or withdrawal of lenvatinib due to associated adverse events. We aimed to identify pre-treatment predictors of tolerability and clinical outcome of lenvatinib therapy. METHODS: Eighty patients who received lenvatinib at our institution between 2018 and 2020 were included in this study. We assessed essential factors associated with prolonged progression-free survival (PFS), using Cox proportional hazards model. We also investigated the correlation between the factor identified as contributing most to PFS and the relative dose intensity (RDI), response rate, and duration of treatment with lenvatinib. RESULTS: Pre-treatment level of Mac-2-binding protein glycosylation isomer (M2BPGi) showed significant association with PFS (hazard ratio = 0.52, P = .0358). Low M2BPGi levels (<1.5) correlated significantly with longer PFS than higher levels (P = .0003). Patients with M2BPGi <1.5 achieved significantly higher RDI, objective response rate, and disease control rate, and maintained lenvatinib treatment for longer than those with baseline values ≥1.5. Patients with M2BPGi ≥1.5 had a higher incidence of adverse events such as fatigue and anorexia. CONCLUSIONS: Baseline M2BPGi levels may predict the tolerability and treatment response to lenvatinib. Patients with high M2BPGi levels may less likely to benefit from lenvatinib therapy.

Oncogenic transcriptomic profile is sustained in the liver after the eradication of the hepatitis C virus.

Hepatocellular carcinoma (HCC) developing after hepatitis C virus (HCV) eradication is a serious clinical concern. However, molecular basis for the hepatocarcinogenesis after sustained virologic response (SVR) remains unclear. In this study, we aimed to unveil the transcriptomic profile of post-SVR liver tissues and explore the molecules associated with post-SVR carcinogenesis. We analysed 90 RNA sequencing datasets, consisting of non-cancerous liver tissues including 20 post-SVR, 40 HCV-positive and 7 normal livers, along with Huh7 cell line specimens before and after HCV infection and eradication. Comparative analysis demonstrated that cell cycle- and mitochondrial function-associated pathways were altered only in HCV-positive non-cancerous liver tissues, whereas some cancer-related pathways were up-regulated in the non-cancerous liver tissues of both post-SVR and HCV-positive cases. The persistent up-regulation of carcinogenesis-associated gene clusters after viral clearance was reconfirmed through in vitro experiments, of which, CYR61, associated with liver fibrosis and carcinogenesis in several cancer types, was the top enriched gene and co-expressed with cell proliferation-associated gene modules. To evaluate whether this molecule could be a predictor of hepatocarcinogenesis after cure of HCV infection, we also examined 127 sera from independent HCV-positive cohorts treated with direct-acting antivirals (DAAs), including 60 post-SVR-HCC patients, and found that the elevated serum Cyr61 was significantly associated with early carcinogenesis after receiving DAA therapy. In conclusion, some oncogenic transcriptomic profiles are sustained in liver tissues after HCV eradication, which might be a molecular basis for the liver cancer development even after viral clearance. Among them, up-regulated CYR61 could be a possible biomarker for post-SVR-HCC.

Portal vein thrombosis due to essential thrombocythemia with limited cutaneous systemic sclerosis.

Portal vein thrombosis is caused by various diseases, including liver cirrhosis, cancer, abdominal infection, and myeloproliferative disorders. Essential thrombocythemia is one of the myeloproliferative disorders in which the bone marrow produces excessive amount of platelets and can be accompanied by various thrombotic diseases; however, essential thrombocythemia with limited cutaneous systemic sclerosis has not been reported yet. We herein report a case of extensive portal vein thrombosis due to essential thrombocythemia with limited cutaneous systemic sclerosis. A 49-year-old woman was referred to our hospital due to liver dysfunction. Extended portal vein thrombosis, splenomegaly, and thrombocytosis were founded. The examination of Janus kinase 2 V617F mutation in the bone marrow was positive. These findings resulted in the diagnosis of portal vein thrombosis due to essential thrombocythemia. Furthermore, Raynaud's phenomenon, finger's sclerosis, and positive anti-centromere antibody led to limited cutaneous systemic sclerosis. To further analyze the causal relationship between essential thrombocythemia and limited cutaneous systemic sclerosis, platelet-derived growth factor was examined. High level of serum platelet-derived growth factor, possibly caused by high platelet count due to essential thrombocythemia, was observed. As platelet-derived growth factor has been reportedly associated with the occurrence of systemic sclerosis, the present case indicates the possible causal link between essential thrombocythemia and limited cutaneous systemic sclerosis through high platelet-derived growth factor.

Branched-chain amino acid to tyrosine ratio is an essential pre-treatment factor for maintaining sufficient treatment intensity of lenvatinib in patients with hepatocellular carcinoma.

BACKGROUND/PURPOSE: Lenvatinib was recently approved as a novel agent for hepatocellular carcinoma. To maximize the therapeutic effect of anticancer drugs, it is essential to maintain treatment intensity by avoiding dose reduction or discontinuation. We aimed to identify essential factors contributing to achieve sufficient treatment intensity of lenvatinib. METHODS: Seventy-one patients who received treatment with lenvatinib were included in this study. We used the delivered dose intensity/body surface area ratio (DBR) to measure treatment intensity of lenvatinib. RESULTS: 2M-DBR (DBR for the first 60 days) of lenvatinib (≥206.7) was strongly correlated with objective response and was the significant factor contributing to prolonged progression-free survival (PFS). Patients with high 2M-DBR had significantly prolonged PFS compared with those who had low 2M-DBR (P < .001). Multivariate analysis revealed that pre-treatment α-fetoprotein and branched-chain amino acid to tyrosine ratio (BTR) were significant factors in maintaining high 2M-DBR of lenvatinib. Furthermore, patients with high pre-treatment BTR (≥4.50) showed significantly longer PFS than those with low BTR (P = .032). CONCLUSIONS: Maintaining high 2M-DBR of lenvatinib is essential to increase response rate and PFS. To achieve high 2M-DBR levels, preservation of pre-treatment BTR is essential, suggesting the importance of nutritional management in the treatment for hepatocellular carcinoma with lenvatinib.

Dose Intensity/Body Surface Area Ratio is a Novel Marker Useful for Predicting Response to Lenvatinib against Hepatocellular Carcinoma.

Lenvatinib was recently approved as a novel first-line molecular targeted agent (MTA) for treating hepatocellular carcinoma (HCC). The importance of relative dose intensity (RDI) has been shown in the treatment of various types of cancers. However, RDI may not accurately reflect the treatment intensity of lenvatinib, as it is the first oral MTA where the dose is based on the patient's weight. We aimed to evaluate the utility of 2M-DBR (the delivered dose intensity/body surface area ratio at 60 days) by comparing the relationship between 2M-DBR, 2M-RDI (RDI at 60 days), and the therapeutic response. The therapeutic response to lenvatinib was evaluated in 45 patients who underwent computed tomography 8-12 weeks after treatment initiation. We also investigated the clinical factors associated with high 2M-DBR. The area under the receiver operating characteristic of 2M-DBR that predicts the response to lenvatinib was higher than that of 2M-RDI (0.8004 vs. 0.7778). Patients with high 2M-DBR achieved significantly better objective responses and disease control rates than those with low 2M-DBR (p < 0.0001 and 0.0008). Patients with high 2M-DBR experienced significantly longer progression-free survival (PFS) than those with low 2M-DBR (p = 0.0001), while there was no significant correlation between 2M-RDI levels and PFS (p = 0.2198). Patients who achieved higher levels of 2M-DBR had a significantly better modified ALBI grade (p = 0.0437), better CONUT score (p = 0.0222), and higher BTR (p = 0.0281). Multivariate analysis revealed that high 2M-DBR was the only significant factor associated with longer PFS. In conclusion, 2M-DBR could be an important factor that reflects treatment intensity and useful for predicting the response to lenvatinib against HCC, instead of 2M-RDI.

Utility of ultrasound-guided liver tumor biopsy for next-generation sequencing-based clinical sequencing.

AIM: Recent advances in next-generation sequencing (NGS) technologies allow for evaluation of genetic alterations in various cancer-related genes in daily clinical practice. Archival formalin-fixed paraffin-embedded (FFPE) tumor tissue is often used for NGS-based clinical sequencing assays; however, the success rate of NGS assays using archival FFPE tumor tissue is reported to be lower than that using fresh tumor tissue. We aimed to evaluate the feasibility and safety of ultrasound (US)-guided liver tumor biopsy for NGS-based multiplex gene assays. METHODS: We compared the success rate of NGS assays between archival FFPE tumor tissues and US-guided liver tumor biopsy tissues, and summarized the treatment progress of the patients. RESULTS: Next-generation sequencing assays using US-guided liver biopsy samples were successful in all patients (22/22), whereas the success rate with archival FFPE tumor tissue was 84.8% (151/178, P < 0.05). At least one potentially actionable genetic alteration was identified from the US-guided liver biopsy samples in 20 of 22 patients. Among the 18 patients with actionable genetic alterations targetable with drugs approved by the US Food and Drug Administration, eight initiated mutation-driven targeted therapies. Of these eight patients, four achieved partial response or stable disease for at least 4 months, and three were not assessable for response due to short exposure. There were no biopsy-related complications requiring additional treatment. CONCLUSION: Our findings suggest that US-guided liver tumor biopsy is a useful and safe method for obtaining high-quality samples for NGS-based clinical sequencing. In cases with metastatic liver tumors, US-guided biopsy should be considered to provide accurate and optimal sequencing results for patients.

Accelerated Progression of Hepatocellular Carcinoma during Immunosuppressive Therapy with Abatacept for Rheumatoid Arthritis.

Abatacept, a cytotoxic T lymphocyte antigen-4 immunoglobulin recombinant fusion protein, is an immunosuppressive agent indicated for rheumatoid arthritis. Although no significant increase in malignancy has been reported in abatacept-treated patients, whether or not abatacept accelerates tumor progression in specific cancer types remains unclear. We herein report a 66-year-old woman who showed unusually rapid progression of hepatocellular carcinoma following abatacept therapy for rheumatoid arthritis. Abatacept was speculated to have accelerated her hepatocellular carcinoma progression in the setting of her preexisting risk factors: autoimmune hepatitis and long-term methotrexate use. We propose close tumor surveillance be performed during abatacept therapy, especially for high-risk patients.