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Background & Aims Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. Methods We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. Results We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. Conclusions Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. Lay summary We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

Identifying target genetic mutations in hepatocellular carcinoma (HCC) for therapy is made challenging by intratumoral heterogeneity. Circulating cell-free DNAs (cfDNA) may contain a more complete mutational spectrum compared to a single tumor sample. This study aimed to identify the most efficient strategy to identify all the mutations within heterogeneous HCCs.Whole exome sequencing (WES) and targeted deep sequencing (TDS) were carried out in 32 multi-regional tumor samples from five patients. Matched preoperative cfDNAs were sequenced accordingly. Intratumoral heterogeneity was measured using the average percentage of non-ubiquitous mutations (present in parts of tumor regions). Profiling efficiencies of single tumor specimen and cfDNA were compared. The strategy with the highest performance was used to screen for actionable mutations.Variable levels of heterogeneity with branched and parallel evolution patterns were observed. The heterogeneity decreased at higher sequencing depth of TDS compared to measurements by WES (28.1% vs. 34.9%, p0.01) but remained unchanged when additional samples were analyzed. TDS of single tumor specimen identified an average of 70% of the total mutations from multi-regional tissues. Although genome profiling efficiency of cfDNA increased with sequencing depth, an average of 47.2% total mutations were identified using TDS, suggesting that tissue samples outperformed it. TDS of single tumor specimen in 66 patients and cfDNAs in four unresectable HCCs showed that 38.6% (26/66 and 1/4) of patients carried mutations that were potential therapeutic targets.TDS of single tumor specimen could identify actionable mutations targets for therapy in HCC. cfDNA may serve as secondary alternative in profiling HCC genome.Targeted deep sequencing of single tumor specimen is a more efficient method to identify mutations in hepatocellular carcinoma made from mixed subtypes compared to circulating cell-free DNA in blood. cfDNA may serve as secondary alternative in profiling HCC genome. Identifying mutations may help clinicians choose targeted therapy for better individual treatments.

Background/Aims: To assess the value of Dickkopf-1 (DKK1) for predicting clinical outcome of human hepatocellular carcinoma (HCC) in patients with HCC. Methods: Expression of DKK1 and b-catenin was investigated in HCC cell lines using qRT-PCR, Western blotting and immunofluorescence. Tissue microarrays representing 314 HCC patients were used to determine the expression patterns of DKK1 and b-catenin by immunohistochemistry, and prognostic significance was assessed by using Kaplan–Meier survival estimates and log-rank tests. Results: The expression level of DKK1 was associated with the staining pattern of b-catenin in HCC cell lines, and DKK1 overexpression correlated with b-catenin cytoplasmic/nuclear accumulation in clinical HCC samples (P = 0.011, correlation coefficient = 0.144). High DKK1 expression predicted unfavorable prognosis in HCC patients, especially in early stage patients and those with normal AFP levels. In multivariate analyses, DKK1 was an independent predictor for overall survival (OS) (P = 0.002) and disease-free survival (DFS) (P = 0.002) of HCC patients. Furthermore, the HCC patients with high DKK1 expression and cytoplasmic/nuclear b-catenin accumulation had very poor prognosis. Conclusions: Elevated expression of DKK1 is a critical event in patients with HCC that indicates poor clinical outcome. DKK1, alone or combined with b-catenin, is a novel prognostic predictor for HCC patients. 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.