Extracellular Vesicles in Hepatobiliary Cancer Management

Abstract

Cancer is a global health problem that will intensify in the upcoming years and decades. Hepatobiliary cancers, comprising hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA) and gallbladder carcinoma (GbCA), are among the most fatal types of cancers, owing to their commonly late-stage discovery and resulting bad prognoses with 5-year survival rates of 5-20%. Patient survival could considerably be improved if hepatobiliary cancers, especially biliary cancers (CCA and GbCA), were discovered and treated at earlier stages. Hepatobiliary cancers are a very heterogeneous group of tumors with considerably diverse treatment regimen. However, especially the distinction of HCC and iCCA remains an extremely difficult challenge that requires an invasive tissue biopsy for definitive diagnosis. Nonetheless, the distinction is crucial for following treatment decisions to ensure best patient care and survival. To date, no reliable serum biomarker exists for completely accurate detection and differentiation of these cancers. Extracellular vesicles (EVs), as a type of liquid biopsy, might offer the opportunity to not only discover hepatobiliary cancers at an earlier stage but also to differentiate between these cancer entities in an uncomplicated and easily applicable manner. EVs are phospholipid bilayer-enclosed vesicles that are released by every cell type including tumor cells. According to their size and mode of biogenesis, they can be classified into endosome-derived small EVs (40-100 nm) and plasma membrane-budding large EVs (100-1000 nm). They reflect the intracellular and surface composition of their parental cells and their potential as biomarkers for diseases has been demonstrated in a wide variety of studies.<br /> Here, the capability of EVs as biomarkers for differential diagnosis of hepatobiliary cancers, especially between HCC and iCCA, is assessed, in order to optimize patient care. In contrast to most biomarker studies on EVs that either focus on only one subtype or on the entirety of all EVs without specifying their molecular characteristics, this study comprehensively evaluates both large and small EVs separately in terms of their differential diagnostic capacity for hepatobiliary cancers.<br /> In this regard, the surface markers EpCAM, CD133, gp38 and CD44v6, that are all associated with carcinogenesis or cancer progression, were observed to be differentially expressed on human HCC and CCA tumor cell lines. Likewise, possible parental cell populations for EVs with combinations of the aforementioned markers on their surface were identified and showed a distinct expression pattern in several mouse organs, including liver and gallbladder. Large EVs (lEVs) featuring the corresponding surface marker combinations were isolated from peripheral patient blood, including HCC, CCA, GbCA, colorectal carcinoma (CRC), non-small cell lung carcinoma (NSCLC) and cirrhosis patients as well as healthy individuals. Flow cytometric analysis revealed that all investigated lEV populations served as biomarkers for biliary cancer with AnnV⁺CD44v6⁺ lEVs showing the best diagnostic performance (AUC: 0.80, sensitivity: 84.0%, specificity: 63.3%, PPV: 85.1%, NPV: 61.3%) for detecting biliary cancers out of a pool of healthy individuals. Furthermore, AnnV⁺CD44v6⁺ lEVs also displayed the best diagnostic capability for distinguishing between HCC and iCCA patients (AUC: 0.83, sensitivity: 93.3%, specificity: 71.0%, PPV: 60.9%, NPV: 95.7%). Importantly, when combining AnnV⁺CD44v6⁺ lEVs with the serum tumor marker AFP, a perfect separation of HCC and CCA was achieved with sensitivity, specificity, PPV and NPV all reaching 100%. Similarly, evaluation of several small EV (sEV) subtypes, characterized by CD9, CD63 or CD81 expression on their surface, by ExoView® scanning revealed a good diagnostic separation of HCC and iCCA patients with CD63⁺CD133⁺ sEVs showing the best diagnostic capability (AUC: 0.89, sensitivity: 87.5%, specificity: 100%, PPV: 100%, NPV: 88.9%). Even though small EVs achieved a similar diagnostic power for separation of HCC and iCCA than large EVs alone, the addition of APF in a combinational approach did not result in any diagnostically relevant improvement in this case.<br /> All in all, lEV profiling, especially AnnV⁺CD44v6⁺ lEVs, was proven to represent a diagnostically powerful marker for biliary cancers as well as for the clinically challenging differential diagnosis of iCCA and HCC that could be of great aid in complementing currently performed diagnostic procedures. As a minimal-invasive liquid biopsy, lEV profiling offers an easily accessible and applicable tool that is low in cost and risk and at the same time highly sensitive and specific. These advantages highlight the enormous potential of lEV profiling to improve informed decision-making during the challenging management of hepatobiliary cancers, thus maximizing best patient care and overall patient survival.