Cholangiocarcinoma (CCA) constitutes a diverse group of malignancies emerging in the biliary tree. CCAs are divided into three subtypes depending on their anatomical site of origin: intrahepatic (iCCA), perihilar (pCCA) and distal (dCCA) CCA 1,2 (Fig. 1). Of note, considered as an independent entity, mixed HCC-CCA tumours are a rare type of liver malignancy sharing features of both iCCA and HCC and presenting an aggressive disease course and poor prognosis 3,4. iCCAs arise above the second-order bile ducts, whereas the point of anatomical distinction between pCCA and dCCA is the insertion of the cystic duct. pCCA and dCCA can also be collectively referred to as 'extrahepatic' (eCCA) 5. In the USA, pCCA is the single largest group, accounting for approximately 50-60% of all CCAs, followed by dCCA (20-30%) and iCCA (10-20%) 1,6,7. CCA is the second most common primary hepatic malignancy after hepatocellular carcinoma (HCC), comprising approximately 15% of all primary liver tumours and 3% of gastrointestinal cancers 1,6,7. CCAs are usually asymptomatic in early stages and, therefore, often diagnosed when the disease is already in advanced stages, which highly compromises therapeutic options, resulting in a dismal prognosis 1,8. CCA is a rare cancer, but its incidence (0.3-6 per 100,000 inhabitants per year) 1 and mortality (1-6 per 100,000 inhabitants per year, globally 9 , not taking into account specific regions with incidence >6 per 100,000 habitants such as South Korea, China and Thailand) have been increasing in the past few decades worldwide, representing a global health problem. Despite advances in

Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs.

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The human genome is replete with long non-coding RNAs (lncRNA), many of which are transcribed and likely to have a functional role. Microarray analysis of > 23 000 lncRNAs revealed downregulation of 712 (~3%) lncRNA in malignant hepatocytes, among which maternally expressed gene 3 (MEG3) was downregulated by 210-fold relative to expression in non-malignant hepatocytes. MEG3 expression was markedly reduced in four human hepatocellular cancer (HCC) cell lines compared with normal hepatocytes by real-time PCR. RNA in situ hybridization showed intense cytoplasmic expression of MEG3 in non-neoplastic liver with absent or very weak expression in HCC tissues. Enforced expression of MEG3 in HCC cells significantly decreased both anchorage-dependent and -independent cell growth, and induced apoptosis. MEG3 promoter hypermethylation was identified by methylation-specific PCR and MEG3 expression was increased with inhibition of methylation with either 5-Aza-2-Deoxycytidine, or siRNA to DNA Methyltransferase (DNMT) 1 and 3b in HCC cells. MiRNA-dependent regulation of MEG3 expression was studied by evaluating the involvement of miR-29, which can modulate DNMT 1 and 3. Overexpression of mir-29a increased expression of MEG3. GTL2, the murine homolog of MEG3, was reduced in liver tissues from hepatocyte-specific miR-29a/b1 knock-out mice compared with wild-type controls. These data show that methylation-dependent tissue-specific regulation of the lncRNA MEG3 by miR-29a may contribute to HCC growth and highlight the inter-relationship between two classes of non-coding RNA, miRNAs and lncRNAs, and epigenetic regulation of gene expression.

Hepatocellular carcinoma (HCC) is characterized by a propensity for multifocality, growth by local spread, and dysregulation of multiple signaling pathways. These features may be determined by the tumoral microenvironment. The potential of tumor cells to modulate HCC growth and behavior by secreted proteins has been extensively studied. In contrast the potential for genetic modulation is poorly understood. We investigated the role and involvement of tumor derived nanovesicles capable of altering gene expression, and characterized their ability to modulate cell signaling and biological effects in other cells. We show that HCC cells can produce nanovesicles, exosomes, that differ in both RNA and protein content from their cells of origin. These can be taken up and internalized by other cells, and can transmit a functional transgene. The microRNA content of these exosomes was examined, and a subset that is highly enriched within exosomes was identified. A combinatorial approach to identify potential targets identified transforming growth factor β activated kinase-1 (TAK1) as the most likely candidate pathway that could be modulated by these miRNA. Loss of TAK1 has been implicated in hepatocarcinogenesis and is a biologically plausible target for inter-cellular modulation. We showed that HCC cell derived exosomes can modulate TAK1 expression and associated signaling and enhance transformed cell growth in recipient cells. Conclusion: Exosome mediated miRNA transfer is an important mechanism of inter-cellular communication in HCC cells. These observations identify a unique inter-cellular mechanism that could potentially contribute to local spread, intrahepatic metastases or multifocal growth in HCC.

Although the inflammation-associated cytokine Interleukin-6 (IL-6) has been implicated in cholangiocarcinoma growth, the relationship between IL-6 and oncogenic changes is unknown. IL-6 can increase expression of DNA methyltransferase 1 (DNMT-1) and epigenetically regulate the expression of several genes, including microRNAs (miRNAs). DNMT-1 up-regulation occurs in hepatobiliary cancers and is associated with a poor prognosis. To understand the potential regulation of DNMT-1 by IL-6 dependent miRNAs, we examined the expression of a group of miRNAs which have sequence complementarity to the 3′-UTR of DNMT-1, namely miR-148a, miR-152 and miR-301. The expression of these miRNAs was decreased in cholangiocarcinoma cells. Moreover, the expression of all three miRNAs was decreased in IL-6 over-expressing malignant cholangiocytes in vitro and in tumor cell xenografts. There was a concomitant decrease in expression of the methylation-sensitive tumor suppressor genes Rassf1a, and p16INK4a. Using luciferase reporter constructs, DNMT-1 was verified as a target for miR-148a and miR-152. Precursors to miR-148a and miR-152 decreased DNMT-1 protein expression, increased Rassf1a and p16INK4a expression and reduced cell proliferation. Conclusion These data indicate that IL-6 can regulate the activity of DNMT-1 and expression of methylation-dependent tumor suppressor genes by modulation of miR-148a and miR-152, and provide a link between this inflammation-associated cytokines and oncogenesis in cholangiocarcinoma.

The overexpression of microRNA-21 (miR-21) is linked to a number of human tumors including colorectal cancer, where it appears to regulate the expression of tumor suppressor genes including p21, phosphatase and tensin homolog, TGFβ receptor II, and B-cell leukemia/lymphoma 2 -associated X protein. Here we demonstrate that miR-21 targets and down-regulates the core mismatch repair (MMR) recognition protein complex, human mutS homolog 2 (hMSH2) and 6 (hMSH6). Colorectal tumors that express a high level of miR-21 display reduced hMSH2 protein expression. Cells that overproduce miR-21 exhibit significantly reduced 5-fluorouracil (5-FU)-induced G2/M damage arrest and apoptosis that is characteristic of defects in the core MMR component. Moreover, xenograft studies demonstrate that miR-21 overexpression dramatically reduces the therapeutic efficacy of 5-FU. These studies suggest that the down-regulation of the MMR mutator gene associated with miR-21 overexpression may be an important clinical indicator of therapeutic efficacy in colorectal cancer.

PurposeThe Medical Research Council Adjuvant Gastric Infusional Chemotherapy (MAGIC) trial established perioperative epirubicin, cisplatin, and fluorouracil chemotherapy as a standard of care for patients with resectable esophagogastric cancer. However, identification of patients at risk for relapse remains challenging. We evaluated whether pathologic response and lymph node status after neoadjuvant chemotherapy are prognostic in patients treated in the MAGIC trial.Materials and MethodsPathologic regression was assessed in resection specimens by two independent pathologists using the Mandard tumor regression grading system (TRG). Differences in overall survival (OS) according to TRG were assessed using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate analyses using the Cox proportional hazards method established the relationships among TRG, clinical-pathologic variables, and OS.ResultsThree hundred thirty resection specimens were analyzed. In chemotherapy-treated patients with a TRG of 1 or 2, median OS was not reached, whereas for patients with a TRG of 3, 4, or 5, median OS was 20.47 months. On univariate analysis, high TRG and lymph node metastases were negatively related to survival (Mandard TRG 3, 4, or 5: hazard ratio [HR], 1.94; 95% CI, 1.11 to 3.39; P = .0209; lymph node metastases: HR, 3.63; 95% CI, 1.88 to 7.0; P < .001). On multivariate analysis, only lymph node status was independently predictive of OS (HR, 3.36; 95% CI, 1.70 to 6.63; P < .001).ConclusionLymph node metastases and not pathologic response to chemotherapy was the only independent predictor of survival after chemotherapy plus resection in the MAGIC trial. Prospective evaluation of whether omitting postoperative chemotherapy and/or switching to a noncross-resistant regimen in patients with lymph node-positive disease whose tumor did not respond to preoperative epirubicin, cisplatin, and fluorouracil may be appropriate.