No abstract

Background & Aims Hepatocellular carcinoma (HCC) is the second most lethal cancer due to lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. Methods HCC classification defined in our previous transcriptome meta-analysis (S1, S2, and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumors (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumors (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. Results HCC subclass-predictive indices were: steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-fetoprotein (>400 ng/mL) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184, and 53 to 30, 43, and 22 for S1, S2, and S3 subclass-targeting therapies, respectively. Conclusions HCC molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.

Background:Hepatocellular carcinoma (HCC) is one of the most important sanitary problems for its prevalence and poor prognosis. To date, no information is available on the prognostic value of the ov-serpin SERPINB3, detected in primary liver cancer but not in normal liver. The aim of the study was to analyse SERPINB3 expression in liver cancer in relation with molecular signatures of poor prognosis and with clinical outcome.Methods:Liver tumours of 97 patients were analysed in parallel for SERPINB3, TGF-β and β-catenin. In a subgroup of 67 patients with adequate clinical follow-up, the correlation of molecular findings with clinical outcome was also carried out.Results:High SERPINB3 levels were detectable in 22% of the patients. A significant correlation of this serpin with TGF-β at transcription and protein level was observed, whereas for β-catenin a strong correlation was found only at post-transcription level. These findings were in agreement with transcriptome data meta-analysis, showing accumulation of SERPINB3 in the poor-prognosis subclass (S1). High levels of this serpin were significantly associated with early tumour recurrence and high SERPINB3 was the only variable significantly associated with time to recurrence at multivariate analysis.Conclusions:SERPINB3 is overexpressed in the subset of the most aggressive HCCs.

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the rereplicating and arrested hepatocytes. Importantly, the DNA rereplication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis.

Objective The number of patients with hepatitis C virus (HCV)-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. Design We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1mg/dL), and platelet count (<100,000/mm3), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented utilizing a digital transcript counting (nCounter) assay specifically developed for clinical use, and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the U.S. (validation cohort, n=145, median follow-up 8 years). Results In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001), and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high (16%), intermediate (42%), or low (42%) risk group by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001), and all liver-related adverse events (HR=4.98, p<0.001). Conclusion A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction, and clinical trial stratification and enrichment for preventive interventions.

TAE averted the need for surgery in high-risk patients. Its role in low surgical risk patients or patients with small-bowel diverticular bleeding requires further study.

HCC patients transplanted beyond Milan criteria without gene signatures of progenitor markers (CK19 and S2) achieved survival rates similar as those within Milan criteria. Once prospectively validated, these markers may support a limited expansion of LT indications.

BackgroundArchived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis.MethodsGenome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks.ResultsRNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles.ConclusionsAS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.