Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation.

BACKGROUND: Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. METHODS: Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. RESULTS: Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. CONCLUSIONS: EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.

The tumour microenvironment and immune milieu of cholangiocarcinoma.

Tumour microenvironment is a complex, multicellular functional compartment that, particularly when assembled as an abundant desmoplastic reaction, may profoundly affect the proliferative and invasive abilities of epithelial cancer cells. Tumour microenvironment comprises not only stromal cells, mainly cancer-associated fibroblasts, but also immune cells of both the innate and adaptive system (tumour-associated macrophages, neutrophils, natural killer cells, and T and B lymphocytes), and endothelial cells. This results in an intricate web of mutual communications regulated by an extensively remodelled extracellular matrix, where the tumour cells are centrally engaged. In this regard, cholangiocarcinoma, in particular the intrahepatic variant, has become the focus of mounting interest in the last years, largely because of the lack of effective therapies despite its rising incidence and high mortality rates worldwide. On the other hand, recent studies in pancreatic cancer, which similarly to cholangiocarcinoma, is highly desmoplastic, have argued against a tumour-promoting function of the tumour microenvironment. In this review, we will discuss recent developments concerning the role of each cellular population and their multifaceted interplay with the malignant biliary epithelial counterpart. We ultimately hope to provide the working knowledge on how their manipulation may lead to a therapeutic gain in cholangiocarcinoma.

Extracellular Vesicles in Liver Diseases: Meeting Report from the International Liver Congress 2018.

Extracellular vesicles (EVs) are small and heterogeneous membrane-bound structures released by cells and found in all biological fluids. They are effective intercellular communicators, acting on a number of close and/or distant target cells. EV cargo may reflect the cell of origin as well as the specific stress that induces their formation and release. They transport a variety of bioactive molecules, including messenger RNA, noncoding RNAs, proteins, lipids, and metabolites, that can be transferred among cells, regulating various cell responses. Alteration in the concentration and composition of EVs in biological fluids is a typical hallmark of pathologies in different liver diseases. Circulating EVs can serve as biomarkers or as messengers following uptake by other cells. This review is a meeting report from the International Liver Congress 2018 (European Association for the Study of the Liver) celebrated in Paris (Symposium: Extracellular vesicles and signal transmission) that discusses the role of EVs in several liver diseases, highlighting their potential value as disease biomarkers and therapeutic opportunities.

Extracellular Vesicles in Liver Diseases: Diagnostic, Prognostic, and Therapeutic Application.

Extracellular vesicles, comprising exosomes, microvesicles, and apoptotic bodies, represent an emerging field in disease diagnostics and prognosis. They can be isolated from peripheral blood of patients as well as from other body fluids and can therefore be considered a minimally invasive liquid biopsy screening tool. Especially their surface antigen composition can reveal information about disease backgrounds. For several liver diseases, including fatal hepatocellular and cholangiocellular carcinoma as well as other nonmalignant liver disorders such as nonalcoholic fatty liver disease, alcoholic hepatitis, or acute liver failure, it has been shown that extracellular vesicle (EV) surface profiling can be useful for disease diagnosis and prognosis. This review focuses on latest advances in these areas to improve liver disorder detection and management. Additionally, the authors will discuss possible therapeutic applications of EVs in liver diseases, which might be a potent treatment option in the future.

Cancer-associated circulating large extracellular vesicles in cholangiocarcinoma and hepatocellular carcinoma.

BACKGROUND & AIMS: Large extracellular vesicles, specifically AnnexinV+ EpCAM+ CD147+ tumour-associated microparticles (taMPs), facilitate the detection of colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) as well as pancreas carcinoma (PaCa). Here we assess the diagnostic value of taMPs for detection and monitoring of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Specifically, the aim of this study was to differentiate liver taMPs from other cancer taMPs, such as CRC and NSCLC. METHODS: Fluorescence-activated cell scanning (FACS) was applied to detect various taMP populations in patients' sera that were associated with the presence of a tumour (AnnexinV+ EpCAM+ CD147+ taMPs) or could discriminate between cirrhosis (due to HCV or HBV) and liver cancers (AnnexinV+ EpCAM+ ASGPR1+ taMPs). In total 172 patients with liver cancer (HCC or CCA), 54 with cirrhosis and no liver neoplasia, and 202 control subjects were enrolled. RESULTS: The results indicate that AnnexinV+ EpCAM+ CD147+ taMPs were elevated in HCC and CCA. Furthermore, AnnexinV+ EpCAM+ ASGPR1+ CD133+ taMPs allowed the distinction of liver malignancies (HCC or CCA) and cirrhosis from tumour-free individuals and, more importantly, from patients carrying other non-liver cancers. In addition, AnnexinV+ EpCAM+ ASGPR1+ taMPs were increased in liver cancer-bearing patients compared to patients with cirrhosis that lacked any detectable liver malignancy. The smallest sizes of successfully detected cancers were ranging between 11-15mm. AnnexinV+ EpCAM+ ASGPR1+ taMPs decreased at 7days after curative R0 tumour resection suggesting close correlations with tumour presence. ROC values, sensitivity/specificity scores and positive/negative predictive values (>78%) indicated a potent diagnostic accuracy of AnnexinV+ EpCAM+ ASGPR1+ taMPs. CONCLUSION: These data provide strong evidence that AnnexinV+ EpCAM+ ASGPR1+ taMPs are a novel biomarker of HCC and CCA liquid biopsy that permit a non-invasive assessment of the presence and possible extent of these cancers in patients with advanced liver diseases. LAY SUMMARY: Microparticles (MPs) are small vesicles that bleb from the membrane of every cell, including cancer cells, and are released to circulate in the bloodstream. Since their surface composition is similar to the surface of their underlying parental cell, MPs from the bloodstream can be isolated and by screening their surface components, the presence of their parental cells can be identified. This way, it was possible to detect and discriminate between patients bearing liver cancer and chronic liver cirrhosis.