Immunovascular classification of HCC reflects reciprocal interaction between immune and angiogenic tumor microenvironments.

BACKGROUND AND AIMS: Immune cells and tumor vessels constitute important elements in tumor tissue; however, their detailed relationship in human tumors, including HCC, is still largely unknown. Consequently, we expanded our previous study on the immune microenvironment of HCC and analyzed the relationship among the immune microenvironment, inflammatory/angiostatic factor expression, angiogenic factor expression, and tumor vessel findings, including vessels encapsulating tumor clusters (VETC) and macrotrabecular-massive (MTM) patterns. APPROACH AND RESULTS: We classified HCC into four distinct immunovascular subtypes (immune-high/angiostatic [IH/AS], immune-mid/angio-mid [IM/AM], immune-low/angiogenic [IL/AG], and immune-low/angio-low [IL/AL]). IH/AS, IM/AM, and IL/AG subtypes were associated with decreasing lymphocytic infiltration and increasing angiogenic factor expression and VETC/MTM positivity, reflecting their reciprocal interaction in the tumor microenvironment of HCC. IL/AG subtype was further characterized by CTNNB1 mutation and activation of Wnt/ß-catenin pathway. IL/AL subtype was not associated with increased lymphocyte infiltration or angiogenic factor expression. Prognostically, IH/AS subtype and VETC/MTM positivity were independently significant in two independent cohorts. Increased angiogenic factor expression was not necessarily associated with VETC/MTM positivity and poor prognosis, especially when inflammatory/angiostatic milieu coexisted around tumor vessels. These results may provide insights on the therapeutic effects of immunotherapy, antiangiogenic therapies, and their combinations. The potential of evaluating the immunovascular microenvironment in predicting the clinical effect of these therapies in nonresectable HCC needs to be analyzed in the future study. CONCLUSIONS: HCC can be classified into four distinct immunovascular subtypes (IH/AS, IM/AM, IL/AG, and IL/AL) that reflect the reciprocal interaction between the antitumor immune microenvironment and tumor angiogenesis. In addition to its clinicopathological significance, immunovascular classification may also provide pathological insights on the therapeutic effect of immunotherapy, antiangiogenic therapy, and their combination.

Telomerase reverse transcriptase (TERT) promoter mutation correlated with intratumoral heterogeneity in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promoter mutations are frequently observed in hepatocellular carcinoma (HCC); however, the impact of TERT promoter mutations (TPMs) on clinical features and morphological patterns in HCC remains unresolved. Using DNA extracted from 97 HCCs, correlations between TPM status and both the clinical features of HCC and the immunohistochemically-based subgroups were evaluated. Morphological tumor patterns were semi-quantitatively analyzed using hematoxylin and eosin-stained slides of the whole tumor cross-sectional area. The percentages of tumor area occupied by early, well, moderate and poor histological patterns were calculated as a homogeneity index. TPMs were observed in 53 of 97 (55%) HCCs and were significantly associated with older age (P = 0.018) and HCV-related background (P = 0.048). The biliary/stem cell marker-positive subgroup was less likely to have TPMs (29%) compared to the Wnt/ß-catenin signaling marker-positive subgroup (60%). In contrast to TPM-negative HCCs, TPM-positive HCCs clearly exhibited intratumoral morphological heterogeneity (0.800 ± 0.117 vs 0.927 ± 0.096, P < 0.0001), characterized by two or more heterogeneous histological patterns (P < 0.0001) and had more well or early differentiated histological patterns (P = 0.024). Our findings showed that intratumoral heterogeneity was strongly related to TPM-positive HCCs, which established novel roles of TPMs, and may improve our understanding particularly about HCC development and diagnosis.

Ex vivo metabolism kinetics of primary to secondary bile acids via a physiologically relevant human faecal microbiota model.

Bile acids (BA) are synthesized in the human liver and undergo metabolism by host gut bacteria. In diseased states, gut microbial dysbiosis may lead to high primary unconjugated BA concentrations and significant perturbations to secondary BA. Hence, it is important to understand the microbial-mediated formation kinetics of secondary bile acids using physiologically relevant ex vivo human faecal microbiota models. Here, we optimized an ex vivo human faecal microbiota model to recapitulate the metabolic kinetics of primary unconjugated BA and applied it to investigate the formation kinetics of novel secondary BA metabolites and their sequential pathways. We demonstrated (1) first-order depletion of primary BA, cholic acid (CA) and chenodeoxycholic acid (CDCA), under non-saturable conditions and (2) saturable Michaelis-Menten kinetics for secondary BA metabolite formation with increasing substrate concentration. Notably, relatively lower Michaelis constants (Km) were associated with the formation of deoxycholic acid (DCA, 14.3 µM) and lithocholic acid (LCA, 140 µM) versus 3-oxo CA (>1000 µM), 7-keto DCA (443 µM) and 7-keto LCA (>1000 µM), thereby recapitulating clinically observed saturation of 7α-dehydroxylation relative to oxidation of primary BA. Congruently, metagenomics revealed higher relative abundance of functional genes related to the oxidation pathway as compared to the 7α-dehydroxylation pathway. In addition, we demonstrated gut microbial-mediated hyocholic acid (HCA) and hyodeoxycholic acid (HDCA) formation from CDCA. In conclusion, we optimized a physiologically relevant ex vivo human faecal microbiota model to investigate gut microbial-mediated metabolism of primary BA and present a novel gut microbial-catalysed two-step pathway from CDCA to HCA and, subsequently, HDCA.

Application of emerging technologies for gut microbiome research.

Microbiome is associated with a wide range of diseases. The gut microbiome is also a dynamic reflection of health status, which can be modified, thus representing great potential to exploit the mechanisms that influence human physiology. Recent years have seen a dramatic rise in gut microbiome studies, which has been enabled by the rapidly evolving high-throughput sequencing methods (i.e. 16S rRNA sequencing and shotgun sequencing). As the emerging technologies for microbiome research continue to evolve (i.e. metatranscriptomics, metabolomics, culturomics, synthetic biology), microbiome research has moved beyond phylogenetic descriptions and towards mechanistic analyses. In this review, we highlight different approaches to study the microbiome, in particular, the current limitations and future promise of these techniques. This review aims to provide clinicians with a framework for studying the microbiome, as well as to accelerate the adoption of these techniques in clinical practice.