Pathologic Biomarkers that Indicate the Aggressiveness and Prognosis of Hepatocellular Carcinoma

Pathological features related to prognosis of hepatocellular carcinoma (HCC) include tumor differentiation grade, tumor size, vascular invasion and intrahepatic metastasis. HCCs are classified into early HCCs and progressed HCCs. Early HCCs are suggested to be the earliest lesion of HCC, corresponding to carcinoma in situ of the other organs. Early HCCs are well differentiated without vascular invasion and its prognosis is very good. In contrast, progressed HCCs are usually moderate to poor differentiated, even in the small sized (< 2cm) HCCs, and vascular invasion and intrahepatic metastasis are found in 27 % and 10%, respectively. The changes in transcriptomes of early HCCs are modest and homogenous, whereas extensive genetic alterations and subsequent activation of prognostic adverse signaling pathways occur only late during hepatocarcinogenesis, and are centered on TGF-s, WNT, NOTCH, and epithelial-mesenchymal transition (EMT)-related genes highlighting the molecular diversity of progressed HCCs. Recently, new HCC subtypes have been reported based on pathological-molecular classification, and some of them have been reported to be related to poor prognosis. HCCs expressing stemness markers including K19 have been reported to have an aggressive behavior. HCCs with high expression of stemness markers (K19, EpCAM) show activation of YAP pathway, TP53 mutation, FGF19 amplification, and they correlate with aggressive gene expression signatures including S2 (Hoshida et al.) and G1 (Boyault et al.) subclasses. There are some characteristic clinicopathological features that have been more frequently seen in HCCs expressing stemness-related markers compared to those without these markers. HCCs with K19 expression, approximately 10-28% of HCCs show high serum alpha-fetoprotein (AFP) levels, chronic hepatitis B, and decreased overall and recurrence-free survival. HCCs with K19 expression are less frequently encapsulated compared to those without, imparting a more infiltrative growth pattern. Vascular invasion is more frequent in HCCs with K19 expression compared to those without. A fibrous stromal component is more frequently identified in HCCs with K19 expression compared to those without. There is a crosstalk between tumor epithelial cells and stromal cells, and K19 expression is regulated by fibroblast-derived HGF via a MET-ERK1/2-AP1 and SP1 Axis. HCCs with K19 expression are associated with increased expression of EMT-related genes and their proteins, and they are also associated with longer telomeres, increased hTERT expression and increased chromosomal instability, suggesting that HCCs with K19 expression have a survival advantage over those without by maintaining telomeres despite the increased chromosomal instability. Hypoxic microenvironment is known to be important in the generation and maintenance of stemness, and we found that HCCs with stemness (K19, EpCAM) and hypoxia (CAIX)-related markers showed more resistance to transarterial chemoembolization (TACE) and poorer outcome compared those without, and their tumor microenvironment was found to be altered under TACE-induced hypoxia, which might promote the aggressive biology of HCC. The expression of stemness and hypoxia-related markers were correlated each other, and evaluation for both markers of stemness and hypoxia may have an additional value in predicting HCC outcome, especially for TACE-treated HCCs. Scirrhous HCC subtype, which frequency is 4% of HCCs shows dense intratumoral fibrosis > 50% of tumor, and correlates with aggressive gene expression signature of G2 subclass (Boyault et al.). Its key molecular features are TSC1/2 mutations, and TGF-s signaling activation. The fibrous stroma of scirrhous HCC contains abundant cancer-associated fibroblasts and tumor-infiltrating macrophages, suggesting a possible role for this complex tumor microenvironment in the aggressive behavior of scirrhous HCC. In fact, the majority of scirrhous HCCs have been demonstrated to express K19 or other “stemness”-related markers, and therefore, K19-positive HCCs and scirrhous HCCs may lie on the same spectrum of HCCs, with the latter being defined as containing more intratumoral fibrous stroma. Mactrotrabecular massive (MTM) HCC subtype is defined as trabecular pattern with more than 6-10 cells thickness in 50% of tumor. Its frequency is 5% of HCCs and reported to have a poor prognosis. It correlates with aggressive gene expression signature of G3 subclass (Boyault et al.). Its key molecular features are TP53 mutations and FGF19 amplication. Vessels that encapsulate tumor clusters (VETC), which is defined as ≥ 55% tumor area by CD34 immunostaining is a new HCC subtype related to poor prognosis. VETC was significantly associated to high serum AFP level, tumor size >5 cm, poor differentiation, macrotrabecular pattern, and frequent microvascular invasion. Therefore, the identification of pathologic biomarkers related to aggressiveness and poor prognosis on HCC tissues obtained by biopsy, resection or transplantation could provide useful information for treatment of HCC patient. Moreover, the recent accumulation of molecular-pathological data will hopefully lead to the development of targeted therapy for this aggressive subset of HCCs.