Gene expression in 1133 HCCs

Hepatocellular carcinomas (HCCs) exhibit a diversity of molecular phenotypes, raising major challenges in clinical management. HCCs detected by surveillance programs at an early stage are candidates for potentially curative therapies (local ablation, resection, or transplantation). In the long term, transplantation provides the lowest recurrence rates. Treatment allocation is based on tumor number, size, vascular invasion, performance status, functional liver reserve, and the prediction of early (<2 years) recurrence, which reflects the intrinsic aggressiveness of the tumor. Well-differentiated, potentially low-aggressiveness tumors form the heterogeneous molecular class of nonproliferative HCCs, characterized by an approximate 50% beta-catenin mutation rate. To define the clinical, pathological, and molecular features and the outcome of nonproliferative HCCs, we constructed a 1,133-HCC transcriptomic metadata set and validated findings in a publically available 210-HCC RNA sequencing set. We show that nonproliferative HCCs preserve the zonation program that distributes metabolic functions along the portocentral axis in normal liver. More precisely, we identified two well-differentiated, nonproliferation subclasses, namely periportal-type (wild-type beta-catenin) and perivenous-type (mutant beta-catenin), which expressed negatively correlated gene networks. The new periportal-type subclass represented 29% of all HCCs; expressed a hepatocyte nuclear factor 4A-driven gene network, which was down-regulated in mouse hepatocyte nuclear factor 4A knockout mice; were early-stage tumors by Barcelona Clinic Liver Cancer, Cancer of the Liver Italian Program, and tumor-node-metastasis staging systems; had no macrovascular invasion; and showed the lowest metastasis-specific gene expression levels and TP53 mutation rates. Also, we identified an eight-gene periportal-type HCC signature, which was independently associated with the highest 2-year recurrence-free survival by multivariate analyses in two independent cohorts of 247 and 210 patients. CONCLUSION: Well-differentiated HCCs display mutually exclusive periportal or perivenous zonation programs. Among all HCCs, periportal-type tumors have the lowest intrinsic potential for early recurrence after curative resection. (Hepatology 2017;66:1502-1518). Raw feature data from Gene Expression Omnibus (GSE16757, GSE20017, GSE14520, GSE25097, GSE17856, GSE54236, GSE20140 (subSeries GSE19977, subSeries GSE56140), GSE15765) were normalized and log 2 intensity expression summary values for each probe set were calculated using Robust Multiarray Average (RMA, packages Affy or Limma). Each dataset was assessed for the detection of outliers and inter-array, experiment-related batch effects by principal component analysis (PCA) and density plots. The outliers removed were: one sample from GSE17856, one sample from GSE15765 and two samples from GSE54236. When clear-cut inter-array experiment-related batch effects were observed within a dataset, this effect was corrected by the COMBAT method (package sva). GSE25097 was corrected for 2-batch effect and GSE14520 for 3-batch effect. Then, probes being detected over the background noise in at least one HCC were retained and quantile normalized (package preprocessCore). When raw data were not available, normalized data were directly used. The final metadata set include 1,133 HCCs and 9,542 genes.