Hepatocellular nodules in cirrhosis include regenerative (large regenerative, LRN) and dysplastic (low and high grade, LGDN and HGDN) nodules, early and grade 1 HCC (eHCC-G1), and overt HCC. The differential diagnosis may be particularly difficult when lesions such as HGDN and eHCC-G1 are involved. We investigated the diagnostic yield of a panel of 3 putative markers of hepatocellular malignancy such as HSP70, glypican 3 (GPC3), and glutamine synthetase (GS). We selected 52 surgically removed nonmalignant nodules (15 LRNs, 15 LGDNs, 22 HGDNs) and 53 HCCs (10 early, 22 grade 1, and 21 grade 2-3) and immunostained them for HSP70, GPC3, and GS. The sensitivity and specificity of the individual markers for the detection of eHCC-G1 were 59% and 86% for GS, 69% and 91% for GPC3, and 78% and 95% for HSP70. We identified 2 main phenotypes: (1) all negative, seen in 100% LRN and LGDN, 73% HGDN and 3% eHCC-G1; (2) all positive, a feature detected in less than half the eHCC-G1. Using a 3-marker panel, when at least 2 of them, regardless which, were positive, the sensitivity and specificity for the detection of eHCC-G1 were respectively 72% and 100%; the most sensitive combination was HSP70+/GPC3+ (59%) when a 2-marker panel was used. Conclusion: The adopted panel of 3 markers is very helpful in distinguishing eHCC-G1 from dysplastic nodules arising in cirrhosis. (Hepatology 2007;45:725–734.)

Genetic determinants of the early steps of carcinogenesis on cirrhosis are still poorly understood. We aimed to evaluate the occurrence of telomerase reverse transcriptase (TERT) promoter mutations in the transformation of cirrhotic nodules into hepatocellular carcinoma (HCC). We analyzed a series of 268 liver samples, including 96 nodules developed in 58 patients with cirrhosis and 114 additional cirrhosis. All samples were screened for TERT promoter mutations, and in 31 nodules, for 10 genes recurrently mutated in HCC. Immunohistochemistry (IHC) analyses were performed for glypican 3, glutamine synthase, and heat shock protein 70. Six liver pathologists reviewed all the samples. Among The 96 nodules, 88 were firmly diagnosed as low‐grade dysplastic nodules (LGDNs; 32 cases), high‐grade dysplastic nodules (HGDNs; 16 cases), early HCC (eHCC; 23 cases), or small and progressed HCC in 17 cases. The agreement between the initial diagnosis from pathological report and the final expert consensus report was moderate for the diagnosis of benign versus malignant nodules (weighted kappa = 0.530). TERT promoter mutations were highly related to the step‐wise hepatocarcinogenesis because mutations were identified in 6% of LGDNs, 19% of HGDNs, 61% of eHCCs, and 42% of small and progressed HCC. TERT promoter mutation is the most frequent molecular alteration in eHCC given that the IHC criteria for diagnosis of malignancy were found in only 39% of the cases. TERT promoter mutation was also the earliest genetic alteration because mutations in 10 other genes were only identified in 28% of the small and progressed HCC. Conclusion: Frequency of TERT promoter mutations rapidly increases during the different steps of the transformation of premalignant lesions into HCC on cirrhosis. Consequently, somatic TERT promoter mutation is a new biomarker predictive of transformation of premalignant lesions into HCC. (Hepatology 2014;60:1982–1991)

We investigated the clinical significance of a vascular growth pattern of hepatocellular carcinoma (HCC), the vessels that encapsulate tumor clusters (VETC), previously linked to HCC metastatic dissemination. VETC was assessed in a large multi‐institutional cohort of 541 resected HCCs from Italy, Korea and Japan, and matched against a full spectrum of clinical and pathological variables. The VETC phenotype (defined as ≥ 55% tumor area by CD34 immunostaining) was easily reproducible and reliably detectable in whole sections and small‐sized tissues of tissue microarray. VETC HCCs represented 18.9% of the whole series, the lowest proportion occurring in the cohort with smallest tumors (8.7%, Japanese series). VETC was significantly associated with several clinical and pathological features such as high alfa‐fetoprotein (AFP) level, tumor size greater than 5 cm, poor differentiation, macrotrabecular pattern, less compact pattern, less inflammatory infiltrates, and frequent microvascular invasion. VETC was associated with early recurrence (hazard ratio [HR]: 1.52 [1.06‐2.19], P = 0.023), disease‐free survival (HR: 1.66 [1.21‐2.27], P = 0.002), and overall survival (HR: 2.26 [1.37‐3.72], P = 0.001) at multivariable analysis. VETC affected the survival in HCC patients stratified for etiology (hepatitis C virus/hepatitis B virus), vascular invasion, and specific molecular phenotypes (β‐catenin/GS+). This distinct vascular pattern was enriched in the recently reported macrotrabecular massive HCC subtype, which was seen in 7.8% (42 of 541) of patients and associated with high AFP levels and poor differentiation. Conclusion: The VETC pattern was found to be easily detectable in a consistent fraction of HCC and a powerful pathological finding affecting survival. This study suggests that the heterogeneous pattern of angiogenesis is involved in HCC behavior.

Although the growth suppressing Hippo pathway has been implicated in hepatocellular carcinoma (HCC) pathogenesis, it is unknown at which stage of hepatocarcinogenesis its dysregulation occurs. We investigated in rat and human preneoplastic lesions whether overexpression of the transcriptional co-activator Yes-associated protein (YAP) is an early event. The experimental model used is the resistant-hepatocyte (R-H) rat model. Gene expression was determined by qRT-PCR or immunohistochemistry. Forward genetic experiments were performed in human HCC cells and in murine oval cells. All foci of preneoplastic hepatocytes, generated in rats 4weeks after diethylnitrosamine (DENA) treatment, displayed YAP accumulation. This was associated with down-regulation of the β-TRCP ligase, known to mediate YAP degradation, and of microRNA-375, targeting YAP. YAP accumulation was paralleled by the up-regulation of its target genes. Increased YAP expression was also observed in human early dysplastic nodules and adenomas. Animal treatment with verteporfin (VP), which disrupts the formation of the YAP-TEAD complex, significantly reduced preneoplastic foci and oval cell proliferation. In vitro experiments confirmed that VP-mediated YAP inhibition impaired cell growth in HCC and oval cells; notably, oval cell transduction with wild type or active YAP conferred tumorigenic properties in vitro and in vivo. These results suggest that (i) YAP overexpression is an early event in rat and human liver tumourigenesis; (ii) it is critical for the clonal expansion of carcinogen-initiated hepatocytes and oval cells, and (iii) VP-induced disruption of the YAP-TEAD interaction may provide an important approach for the treatment of YAP-overexpressing cancers.