Molecular signatures associated with HCV-induced hepatocellular carcinoma and liver metastasis

Abstract

Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. In Italy, particularly Southern Italy, chronic hepatitis C virus (HCV) infection represents the main cause of HCC. Using high-density oligoarrays, we identified consistent differences in gene-expression between HCC and normal liver tissue. Expression patterns in HCC were also readily distinguishable from those associated with liver metastases. To characterize molecular events relevant to hepatocarcinogenesis and identify biomarkers for early HCC detection, gene expression profiling of 71 liver biopsies from HCV-related primary HCC and corresponding HCV-positive non-HCC hepatic tissue, as well as gastrointestinal liver metastases paired with the apparently normal peri-tumoral liver tissue, were compared to 6 liver biopsies from healthy individuals. Characteristic gene signatures were identified when normal tissue was compared with HCV-related primary HCC, corresponding HCV-positive non-HCC as well as gastrointestinal liver metastases. Pathway analysis classified the cellular and biological functions of the genes differentially expressed as related to regulation of gene expression and post-translational modification in HCV-related primary HCC; cellular Growth and Proliferation, and Cell-To-Cell Signaling and Interaction in HCV-related non HCC samples; Cellular Growth and Proliferation and Cell Cycle in metastasis. Also characteristic gene signatures were identified of HCV-HCC progression for early HCC diagnosis.

Conclusions: A diagnostic molecular signature complementing conventional pathologic assessment was identified.

Figure 1. Study design.

Gene signature distinguishing the different pathological stage of liver disease and potential molecular progression markers was defined in the first dataset (left panel) and defined/evaluated in the independent second dataset (right panel).

Figure 2. Unsupervised hierarchical clustering: Panel A.

Heat map based on 8,210 genes of 71 liver samples (HCV-related HCC, non-HCC counterpart, metastases, non cancerous counterpart and controls from healthy donors). Genes were filtered according to the following criteria: presence in 80% of all experiments, a >3 fold change ratio in at least one experiment. Red indicates over-expression; green indicates under-expression; black indicates unchanged expression; gray indicates no detection of expression (intensity of both Cy3 and Cy5 below the cutoff value). Each row represents a single gene; each column represents a single sample. The dendrogram at the top of the matrix indicates the degree of similarity between samples. Panel B Principal Component Analysis (PCA) 3D view for gene expression profiles from 71 liver samples. The PCA is based on log2 ratios and the expression profiles are across all the 36,000 genes in the microarrays. The green, blue, red, purple, and orange dots indicate metastasis, HCV-related HCC, HCV related non HCC, non metastatic counterpart, HCV-negative normal control and samples, respectively.

Figure 3. Heat map of the genes differentially expressed: identified by Class Comparison Analysis.

Analysis including HCV-related HCC and normal liver samples from control subjects (Panel A); analysis including HCV-related non-HCC liver tissues and liver samples from control subjects (Panel B); analysis including liver metastasis and liver samples from control subjects (Panel C); analysis including HCV-related HCC and their HCV-positive/cirrhotic counterpart (Panel D); analysis including all HCV-related HCC, their HCV-positive/cirrhotic counterpart and normal liver samples, each column represents the average of all samples belonging to the same group as it was a single array (Panel E). The expression pattern of the genes is shown, each row representing a single gene.

Figure 4. Ingenuity Pathways analysis: significant pathways at the nominal 0.01 level of the unpaired Student's t-test.

The 3 top-scoring pathways of genes up regulated in HCV-related HCC (Panel A), in HCV related non HCC (Panel B) and in Metastatic liver samples (Panel C).

Figure 5. Molecular signatures in Human Liver Cancer: Hierarchical clustering of the potentially gene markers up-regulated in: Panel A, HCV-related HCC liver samples (left panel first dataset, right panel second dataset); Panel B, HCV-related non HCC liver samples (left panel first dataset, right panel second dataset); Panel C, HCV-related HCC; Panel D, metastatic liver samples; Panel E, genes up-regulated considered possible markers of tumor progression.