Autophagy activity in cholangiocarcinoma is associated with anatomical localization of the tumor.

The presence of autophagy has been indicated in cholangiocarcinoma (CC), which disease has poor prognosis and limited treatment options. Recently, CC has been classified by anatomical localization as intrahepatic (iCC), perihilar (pCC) and distal (dCC), showing different clinical and molecular characteristics. Thus, our aim was to compare autophagy activity in CC samples resected from different anatomical locations. Further, we investigated whether autophagy could be modulated in cell lines originated from iCC and extrahepatic CC (eCC) following the treatments with autophagy inhibitory and inducing agents. Tissue microarrays were prepared from 70 CC (28 iCC, 19 pCC and 23 dCC), 31 adjacent non-tumorous and 9 hepatocellular carcinoma (HCC) samples. Autophagy markers LC3, p62 and Beclin1 as well as proliferation marker Ki-67 were monitored by immunohistochemistry and were associated with patients' survival. Modulation of autophagy was investigated in cell lines originated from iCC (HuH-28), eCC (TFK-1) and HCC (HepG2) by treating the cells with chloroquine (CQ) for inhibition and with Rapamycin, 5-Fluorouracil (5-FU) and Sorafenib for induction of autophagy. Our results indicated an inhibited autophagy in iCC and pCC tumor tissues, whereas active autophagy seemed to occur in dCC, especially in samples displaying low Ki-67 index. Additionally, low level of Beclin1 and high level of Ki-67 were associated with poor overall survival in dCC, suggesting the prognostic role of these proteins in dCC. Beside a baseline autophagy detected in each cell line, Rapamycin and 5-FU induced autophagy in iCC and HepG2 cell lines, Sorafenib in iCC cells. A chemotherapy agent in combination with CQ decreased IC50 effectively in the cell lines where basal and/or induced autophagy were present. In conclusion, we revealed differences in the autophagy activities of CC tissues and cell lines originated from different anatomical locations, which might influence patients' treatment. Our results also suggest a prognostic role of Beclin1 and Ki-67 in dCC.

Increased miR-20b Level in High Grade Cervical Intraepithelial Neoplasia.

Cervical cancer is a common malignant tumor worldwide ranking fourth in incidence and mortality among females, which was reduced significantly by cytology screening and human papilloma virus (HPV) DNA testing. The specificity of cytology is high; however, the sensitivity is low, in contrast to the HPV DNA testing. Despite the success of these measures, new biomarkers are still considered to aim increasing sensitivity and specificity of screening and diagnosis. Significant alterations in microRNA (miRNA) expression have been detected in several cancers with variable consistency. To investigate the stratification role of miRNAs between normal epithelium and cervical intraepithelial neoplasia (CIN2-3), we screened the expression of 667 miRNAs to identify significant markers (n = 10), out of them 9 miRNAs were applied in the study (miR-20b, -24, -26a, -29b, -99a, -100, -147, -212, -515-3p) along with RNU48 and U6 as the references. To benchmark the miRNAs, 22 paired (tumor-free and tumor tissue pairs) laser microdissection-obtained cervical formalin fixed, paraffin embedded tissue samples were assayed. The expression of miR-20b was 2.4 times higher in CIN2-3 samples as compared to normal tissues (p < 0.0001). In the HPV16-positive subsets of the samples (n = 13), miR-20b showed 2.9-times elevation (p < 0.001), whereas miR-515 was 1.15-times downregulated (p < 0.05) in CIN2-3 as compared to normal tissue. These results suggest the potential value of miR-20b as a statification biomarker in order to differentiate neoplastic and non-tumorous cases.

MicroRNA Expression in Focal Nodular Hyperplasia in Comparison with Cirrhosis and Hepatocellular Carcinoma.

The liver disease focal nodular hyperplasia (FNH) has several histological features that resemble hepatic cirrhosis. Since cirrhosis may develop further into hepatocellular carcinoma (HCC) contrary to FNH, the aim of the present study was to identify microRNAs (miRNA), which, by their altered expression levels, may be associated with the benign, tumor-like nature of FNH. Altogether 106 surgically removed formalin-fixed paraffin-embedded liver samples were selected, including 22 FNH, 45 cirrhosis, 24 HCC and 15 normal liver tissues. Etiology of the cases of cirrhosis and HCC includes hepatitis C and alcoholism and the HCC cases developed in cirrhotic livers. Relative expression levels of 14 miRNAs were determined using TaqMan MicroRNA Assays. In comparison to normal liver, the levels of miR-34a and miR-224 were elevated not only in FNH but also in cirrhosis and HCC, while the expression of miR-17-5p, miR-18a and miR-210 was decreased in FNH. Further, the levels of miR-21 and miR-222 were increased in cirrhosis and HCC but were decreased in FNH and the expression of miR-17-5p, miR-18a, miR-195 and miR-210 was decreased in FNH as compared with cirrhosis and/or HCC. In conclusion, the elevation of miR-34a and miR-224 may be associated with both benign and malignant proliferative processes, nevertheless the increased expression of oncomiRs miR-21 and miR-222 in cirrhosis and HCC but not in FNH may be related to malignant processes of the liver. The decreased levels of miR-18a, miR-195 and miR-210 may further differentiate FNH from cirrhosis, reflecting the different pathogenesis of these two entities contrary to some histologically similar features.

[Treatment of metastatic progression following the synchronous occurrence of cutaneous and ocular primary melanomas]. / Metasztatikus progresszió kezelése primer cutan és ocularis melanoma szinkrón elofordulását követoen.

The incidence rates of cutaneous melanoma in non-Hispanic whites show an increasing tendency with age. While uveal melanoma in general is a rare disease, representing only 4% of all melanomas with an incidence rate of 0.6 per 100 000, it is still the most frequent malignancy of the eye. Synchronous occurrence of ocular and cutaneous melanoma is an exceptional rarity, due to the distinct genetic background of the diseases. We report the case of a 80-year-old man who underwent total excision of a cutaneous melanoma in 2008. In 2013, he was diagnosed with uveal melanoma as part of a routine work-up for reduced vision. The uveal melanoma was treated by brachytherapy. In 2015, liver metastases were suspected by routine ultrasonography. Core biopsy was carried out, and the histology confirmed melanoma metastases. The molecular analysis of the cutaneous lesion showed gain of function mutation of the BRAF V600 K gene, while we found a wild-type BRAF gene in the metastatic lesion. Based on the recommendation of the oncoteam, hepatic intra-arterial Epirubicin-Platidiam therapy was introduced. He received 11 doses of intra-arterial chemotherapy (IAC), in 21 cycles. IAC was well tolerated without any catheter-related complications or toxicities. Partial regression of the hepatic metastases were documented in February 2016. After completing the eleventh cycle of intrahepatic chemotherapy, the disease remained in complete remission for over a year. The parallel occurrence of cutaneous and ocular melanoma is rare, however, the metastatic progression in such cases make the selection of optimal medical therapy challenging. The distinct genetic background of two melanoma types may help the identification of the source of the metastatic lesions, in order to guide the treatment decisions. Orv Hetil. 2018; 159(16): 642-647.

Cytosolic localization of NADH cytochrome b5 oxidoreductase (Ncb5or).

Acyl-CoA desaturation in the endoplasmic reticulum (ER) membrane depends on cytosolic NADH or NADPH, whereas NADPH in the ER lumen is utilized by prereceptor glucocorticoid production. It was assumed that NADH cytochrome b5 oxidoreductase (Ncb5or) might connect Acyl-CoA desaturation to ER luminal redox. We aimed to clarify the ambiguous compartmentalization of Ncb5or and test the possible effect of stearoyl-CoA on microsomal NADPH level. Amino acid sequence analysis, fluorescence microscopy of GFP-tagged protein, immunocytochemistry, and western blot analysis of subcellular fractions unequivocally demonstrated that Ncb5or, either endogenous or exogenous, is localized in the cytoplasm and not in the ER lumen in cultured cells and liver tissue. Moreover, the involvement of ER-luminal reducing equivalents in stearoyl-CoA desaturation was excluded.

High tricellulin expression is associated with better survival in human hepatoblastoma.

AIMS: The more differentiated fetal component of hepatoblastoma (HB) is characterized by increased expression of tight junction (TJ) proteins claudin-1 and -2 when compared with embryonal component. Expression patterns of the recently identified TJ protein tricellulin and the epigenetic regulator enzyme EZH2 were investigated in epithelial subtypes of HB and related to survival. METHODS AND RESULTS: Twenty-one cases of epithelial HBs subtyped as pure fetal (n = 12) and embryonal/fetal (n = 9), along with 16 non-tumorous samples from surrounding liver, were analysed by immunohistochemistry for tricellulin, ß-catenin and EZH2 expression. No significant differences were revealed in overall survival between fetal and embryonal/fetal types of HBs. The fetal component, however, showed considerably increased tricellulin expression while the embryonal component displayed significantly increased nuclear EZH2 positivity, in comparison to other epithelial subtypes and non-tumorous surrounding hepatocytes. Strong nuclear ß-catenin staining was notably more frequent in embryonal than in fetal types. High tricellulin expression was associated with significantly increased overall survival (P = 0.03), while elevated EZH2 expression was linked to the presence of distant metastases (P = 0.013). CONCLUSIONS: Our data indicate that patients with treated HBs showing high expression of tricellulin have significantly better overall survival, independent of histological subtype. Increased nuclear expression of EZH2 was associated with the presence of distant metastases.

MicroRNA expression might predict prognosis of epithelial hepatoblastoma.

Hepatoblastoma (HB) is the most common primary liver cancer in childhood. The fetal and mixed embryonal/fetal epithelial subtypes of HB differ not only in grade of differentiation but probably also in prognosis. We aimed to determine microRNA (miRNA) expression patterns of the main subtypes of epithelial HBs to reveal differences and relate them to survival. We studied 20 cases of epithelial HB, subtyped as pure fetal (n = 12) or embryonal/fetal (n = 8). Tissues were sampled according to subtype to arrive at 15 purely fetal and eight purely embryonal samples (n = 8) and 15 samples of non-tumorous surrounding liver (SL). Relative expression of miR-17-5p, miR-18a, miR-21, miR-34a, miR-96, miR-122, miR-181a, miR-195, miR-210, miR-214, miR-221, miR-222, miR-223, and miR-224 was determined by TaqMan MicroRNA Assays applying miR-140 as reference. A higher level of miR-18a (p < 0.01) was found in embryonal samples than in fetal samples. Lower miR-17-5p, miR-195, miR-210, miR-214, and higher miR-221 levels were detected in fetal samples (p < 0.02) in comparison with SL samples, whereas a lower miR-122 level was observed in embryonal samples (p < 0.003). Histological subtype did not correlate with survival; however, high miR-21, low miR-222, and low miR-224 levels proved to be independently prognostic for HB with significantly increased overall survival (p < 0.03). The fetal and embryonal components of epithelial HB, as well as SL, revealed different miRNA expression patterns. Furthermore, miR-21, miR-222, and miR-224 levels predict overall survival of HB patients regardless of epithelial subtype.

Enhancer of zeste homologue 2 (EZH2) is a reliable immunohistochemical marker to differentiate malignant and benign hepatic tumors.

BACKGROUND: The immunohistochemical demonstration of Enhancer of zeste homologue 2 (EZH2) proved to be a useful marker in several tumor types. It has been described to distinguish reliably hepatocellular carcinomas from liver adenomas and other benign hepatocellular lesions. However, no other types of malignant liver tumors were studied so far. METHODS: To evaluate the diagnostic value of this protein in hepatic tumors we have investigated the presence of EZH2 by immunohistochemistry in hepatocellular carcinomas and other common hepatic tumors.EZH2 expression was examined in 44 hepatocellular carcinomas, 23 cholangiocarcinomas, 31 hepatoblastomas, 16 other childhood tumor types (rhabdomyosarcoma, neuroblastoma, Wilms' tumor and rhabdoid tumor), 17 metastatic liver tumors 24 hepatocellular adenomas, 15 high grade dysplastic nodules, 3 biliary cystadenomas, 3 biliary hamartomas and 3 Caroli's diseases. RESULTS: Most of the malignant liver tumors were positive for EZH2, but neither of the adenomas, cirrhotic/dysplastic nodules, reactive and hamartomatous biliary ductules stained positively. CONCLUSIONS: Our immunostainings confirm that EZH2 is a sensitive marker of hepatocellular carcinoma, but its specificity is very low, since almost all the investigated malignant liver tumors were positive regardless of their histogenesis. Based on these results EZH2 is a sensitive marker of malignancy in hepatic tumors. In routine surgical pathology EZH2 could be most helpful to diagnose cholangiocarcinomas, because as far as we know this is the first marker to distinguish transformed and reactive biliary structures. Although hepatoblastomas also express EZH2, the diagnostic significance of this observation seems to be quite limited whereas, the structurally similar, other blastic childhood tumors are also positive. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1173195902735693.