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Cholangiocarcinoma constitutes a heterogeneous group of malignancies that can emerge at any point of the biliary tree. Cholangiocarcinoma is classified into intrahepatic, perihilar and distal based on its anatomical location. Histologically, conventional perihilar/distal cholangiocarcinomas are mucin-producing adenocarcinomas or papillary tumours; intrahepatic cholangiocarcinomas are more heterogeneous and can be sub-classified according to the level or size of the displayed bile duct. Cholangiocarcinoma develops through multistep carcinogenesis and is preceded by dysplastic and in situ lesions. Definition and clinical significance of precursor lesions, including biliary intraepithelial neoplasia, intraductal papillary neoplasms of the bile duct, intraductal tubulopapillary neoplasms and mucinous cystic neoplasm, are discussed in this review. A main challenge in diagnosing cholangiocarcinoma is the fact that tumour tissue for histological examination is difficult to obtain. Thus, a major clinical obstacle is the establishment of the correct diagnosis at a tumour stage that is amenable to surgery which still represents the only curable therapeutic option. Current standards, methodology and criteria for diagnosis are discussed. Cholangiocarcinoma represents a heterogeneous tumour with regard to molecular alterations. In intrahepatic subtype, mainly two distinctive morpho-molecular groups can currently be discriminated. Large-duct type intrahepatic cholangiocarcinoma shows a high mutation frequency of oncogenes and tumour suppressor genes, such as KRAS and TP53 while Isocitrate Dehydrogenase 1/2 mutations and Fibroblast Growth Factor Receptor 2-fusions are typically seen in small-duct type tumours. It is most important to ensure the separation of the given anatomical subtypes and to search for distinct subgroups within the subtypes on a molecular and morphological basis.

Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer. It is defined by cholangiocytic differentiation and has poor prognosis. Recently, epigenetic processes have been shown to play an important role in cholangiocarcinogenesis. We performed an integrative analysis on 52 iCCAs using both genetic and epigenetic data with a specific focus on DNA methylation components. We found recurrent isocitrate dehydrogenase 1 (IDH1) and IDH2 (28%) gene mutations, recurrent arm-length copy number alterations (CNAs), and focal alterations such as deletion of 3p21 or amplification of 12q15, which affect BRCA1 Associated Protein 1, polybromo 1, and mouse double minute 2 homolog. DNA methylome analysis revealed excessive hypermethylation of iCCA, affecting primarily the bivalent genomic regions marked with both active and repressive histone modifications. Integrative clustering of genetic and epigenetic data identified four iCCA subgroups with prognostic relevance further designated as IDH, high (H), medium (M), and low (L) alteration groups. The IDH group consisted of all samples with IDH1 or IDH2 mutations and showed, together with the H group, a highly disrupted genome, characterized by frequent deletions of chromosome arms 3p and 6q. Both groups showed excessive hypermethylation with distinct patterns. The M group showed intermediate characteristics regarding both genetic and epigenetic marks, whereas the L group exhibited few methylation changes and mutations and a lack of CNAs. Methylation-based latent component analysis of cell-type composition identified differences among these four groups. Prognosis of the H and M groups was significantly worse than that of the L group. Conclusion: Using an integrative genomic and epigenomic analysis approach, we identified four major iCCA subgroups with widespread genomic and epigenomic differences and prognostic implications. Furthermore, our data suggest differences in the cell-of-origin of the iCCA subtypes.

NUT carcinoma is a rarely diagnosed, poorly differentiated subtype of squamous cell carcinoma, defined by chromosomal rearrangements of the gene encoding nuclear protein of the testis (NUT). It is characterized by extremely aggressive clinical behavior resulting in a dismal prognosis, with a median survival of 6.7 months. Though most frequently detected along the body midline, NUT carcinoma can arise in any organ. Fewer than 100 cases have been reported in medical literature with the majority of patients being children or young adults. Here we present a case of sinonasal NUT in a 48-year-old male who came to our hospital due to progressive cephalalgia. Radiographically, an irregular mass in the left sphenoidal sinus suspicious for a malignant process was detected, and biopsies were taken. Histopathologically, a tumor of highly mitotic, predominantly small to middle-sized cells with a focal abrupt transition to mature-appearing, squamous epithelium was noted. Of critical importance for the diagnosis, the undifferentiated tumor cell population robustly expressed NUT. The diagnosis of NUT carcinoma was confirmed by the identification of BRD4-NUT fusion. This case integrates typical morphological, immunohistochemical and molecular characteristics of NUT carcinoma and highlights the need to consider this entity in cases of poorly differentiated squamous carcinoma.

UNLABELLED Several chronic inflammatory liver diseases, e.g., chronic hepatitis B or C viral infection and steatohepatitis, have been shown to predispose to the development of hepatocellular carcinoma (HCC). In patients with chronic liver disease, interleukin-6 (IL-6) serum levels are elevated and increase even more when HCC develops. However, the impact and regulatory mechanisms of IL-6 signaling during hepatocarcinogenesis are still poorly defined. Here, we show that gene expression profiles of patients with chromosome 8p loss correlate with increased IL-6 signaling. In addition, the chromosome 8p tumor suppressor genes Src homology 2 domain containing 4A (SH2D4A) and Sorbin and Src homology 3 domain containing 3 (SORBS3) together exerted greater inhibition of cell growth and clonogenicity compared to a single gene. Overexpression of SH2D4A and SORBS3 in HCC cells led to decreased IL-6 target gene expression and reduced signal transducer and activator of transcription 3 (STAT3) signaling. In situ and in vitro coimmunoprecipitation assays revealed that SH2D4A directly interacts with STAT3, thereby retaining STAT3 in the cytoplasm and inhibiting STAT3 transcriptional activity. On the other hand, SORBS3 coactivated estrogen receptor α signaling, leading indirectly to repression of STAT3 signaling. In human HCC tissues, SH2D4A was positively associated with infiltrating regulatory and cytotoxic T-cell populations, suggesting distinct immunophenotypes in HCC subgroups with chromosome 8p loss. Thus, the genetically linked tumor suppressors SH2D4A and SORBS3 functionally cooperate to inhibit STAT3 signaling in HCC. CONCLUSION The chromosome 8p tumor suppressor genes SORBS3 and SH2D4A are physically and functionally linked and provide a molecular mechanism of inhibiting STAT3-mediated IL-6 signaling in HCC cells. (Hepatology 2016;64:828-842).

BACKGROUND A major hallmark of malignant progression in human astrocytomas is the formation of new blood vessels. Antiangiogenic therapy using the anti-vascular endothelial growth factor (VEGF)-antibody bevacizumab leads to increased progression-free survival in glioblastoma patients but does not influence their overall survival. To date, it is unclear why antiangiogenic therapy fails in many glioblastoma patients, while a small subpopulation profits considerably from this treatment. METHODS The aim of our study was to determine the expression of VEGF-A and its (co-) receptors by immunohistochemistry and to test the association with patient survival in 350 glioma patients. Additionally, VEGF-A expression was analyzed by in-situ hybridization. In 18 patients, the protein expression was compared with the bevacizumab response according to extended and modified RANO criteria. RESULTS We found a heterogeneous expression pattern of VEGF and its receptors in glioblastoma patients with significantly lower levels in WHO grade II and III tumors and normal-appearing brain tissue (P < .001). Pilocytic astrocytomas (WHO grade I) showed significantly higher VEGFR-1, -2 and neuropilin-1 levels as compared to WHO grade II and III astrocytomas (P < .01) but at lower levels than glioblastomas. The expression of neuropilin-2 was low in all tumors. There was neither a significant correlation between protein expression and patient survival nor between protein levels and bevacizumab response after modified RANO criteria. CONCLUSION Since our data indicate that beneficial response to bevacizumab treatment is independent of the expression of VEGF-A and its (co-) receptors, further investigation is needed to decipher the underlying mechanisms of antiangiogenic treatment response.

Background: Biliary tract cancers (BTC) are rare malignant tumours with a poor prognosis. Previously, we have presented a detailed characterisation of the inflammatory infiltrate in BTC. Here, we analysed the impact of the expression of major histocompatibility complex class I (MHC I) on patient survival and the quantity, as well as the quality of tumour-infiltrating immune cell types in BTC. Methods: MHC I expression was assessed semi-quantitatively in 334 BTC, including extrahepatic (n=129) and intrahepatic cholangiocarcinomas (n=146), as well as adenocarcinomas of the gallbladder (n=59). In addition, 71 high-grade biliary intraepithelial lesions (BilIN 3) were included. Results were correlated with data on antitumour inflammation and investigated with respect to their association with clinicopathological variables and patient survival. Results: BTC showed a wide spectrum of different MHC I expression patterns ranging from complete negativity in some tumours to strong homogenous expression in others. In BilIN 3, significantly higher MHC I expression levels were seen compared to invasive tumours (P=0.004). Patients with strong tumoural MHC I expression had a significantly higher overall survival probability (median survival benefit: 8 months; P=0.006). MHC I expression strongly correlated with the number of tumour-infiltrating T-lymphocytes (CD4+ and CD8+) and macrophages. Conclusions: Differences of MHC I expression predict patient outcome and show correlations with specific components of the inflammatory infiltrate in BTC. These findings contribute to a better understanding of immune response and immune escape phenomena in cholangiocarcinogenesis.

Background Pancreas graft quality directly affects morbidity and mortality rates after pancreas transplantation (PTx). The criteria for pancreas graft allocation are restricted, which has decreased the number of available organs. Suitable pancreatic allografts are selected based on donor demographics, medical history, and the transplant surgeon's assessment of organ quality during procurement. Quality is assessed based on macroscopic appearance, which is biased by individual experience and personal skills. Therefore, we aim to assess the histopathological quality of unallocated pancreas organs to determine how many unallocated organs are potentially of suitable quality for PTx. Methods and analysis This is a multicenter cross-sectional explorative study. The demographic data and medical history of donor and cause of rejection of the allocation of graft will be recorded. Organs of included donors will be explanted and macroscopic features such as weight, color, size, and stiffness will be recorded by 2 independent transplant surgeons. A tissue sample of the organ will be fixed for further microscopic assessments. Histopathologic assessments will be performed as soon as a biopsy can be obtained. We will evaluate up to 100 pancreata in this study. Result This study will evaluate the histopathological quality of unallocated pancreas organs from brain-dead donors to determine how many of these unallocated organs were potentially suitable for transplantation based on a histopathologic evaluation of organ quality. Conclusion The comprehensive findings of this study could help to increase the pancreas graft pool, overcome organ shortage, reduce the waiting time, and also increase the number of PTx in the future. Registration number: ClinicalTrials.gov: NCT04127266.

Purpose Differential diagnosis of ulcerative colitis (UC) and Crohn's disease (CD) is of utmost importance for the decision making of respective therapeutic treatment strategies but in about 10-15% of cases, a clinical and histopathological assessment does not lead to a definite diagnosis. The aim of the study is to characterize proteomic differences between UC and CD. Experimental design Microproteomics is performed on formalin-fixed paraffin-embedded colonic tissue specimens from 9 UC and 9 CD patients. Protein validation is performed using immunohistochemistry (IHC) (nUC =51, nCD =62, nCTRL =10) followed by digital analysis. Results Microproteomic analyses reveal eight proteins with higher abundance in CD compared to UC including proteins related to neutrophil activity and damage-associated molecular patterns. Moreover, one protein, Aldo-keto reductase family 1 member C3 (AKR1C3), is present in eight out of nine CD and absent in all UC samples. Digital IHC analysis reveal a higher percentage and an increased expression intensity of AKR1C3-positive epithelial cells in CD compared to UC and in controls compared to inflammatory bowel disease (IBD). Conclusion and clinical relevance Overall, the results suggest that microproteomics is an adequate tool to highlight protein patterns in IBD. IHC and digital pathology might support future differential diagnosis of UC and CD.

Tyrosine kinase inhibitors currently confer the greatest survival gain for nonsmall cell lung cancer (NSCLC) patients with actionable genetic alterations. Simultaneously, the increasing number of targets and compounds poses the challenge of reliable, broad and timely molecular assays for the identification of patients likely to benefit from novel treatments. Here, we demonstrate the feasibility and clinical utility of comprehensive, NGS-based genetic profiling for routine workup of advanced NSCLC based on the first 3,000 patients analyzed in our department. Following automated extraction of DNA and RNA from formalin-fixed, paraffin-embedded tissue samples, parallel sequencing of DNA and RNA for detection of mutations and gene fusions, respectively, was performed using PCR-based enrichment with an ion semiconductor sequencing platform. Overall, 807 patients (27%) were eligible for currently approved, EGFR-/BRAF-/ALK- and ROS1-directed therapies, while 218 additional cases (7%) with MET, ERBB2 (HER2) and RET alterations could potentially benefit from experimental targeted compounds. In addition, routine capturing of comutations, e.g. TP53 (55%), KEAP1 (11%) and STK11 (11%), as well as the precise typing of fusion partners and involved exons in case of actionable translocations including ALK and ROS1, are prognostic and predictive tools currently gaining importance for further refinement of therapeutic and surveillance strategies. The reliability, low dropout rates (5%), minimal tissue requirements, fast turnaround times (6 days on average) and lower costs of the diagnostic approach presented here compared to sequential single-gene testing, highlight its practicability in order to support individualized decisions in routine patient care, enrollment in molecularly stratified clinical trials, as well as translational research.

Background A major molecular pathway of genetic instability in cancer is DNA mismatch repair deficiency. High-level microsatellite instability (MSI-H) is currently the best predictor of responsiveness towards immune checkpoint blockade. Data about the prevalence of high-level microsatellite instability in cholangiocarcinoma (CCA) has been conflicting. Methods We employed a cohort comprising 308 Western-world, non-liver fluke-associated CCAs (159 intrahepatic, 106 perihilar, and 43 distal). We analysed the mononucleotide microsatellite instability marker panel consisting of BAT25, BAT26, and CAT25 and detected MSI-H in 4/308 CCAs (1.3%). Results Patients affected by MSI-H CCA had mostly an atypical histomorphology (p = 0.004), showed a longer overall survival, although having a high tumour stage, and were of younger age. Correlation analysis of microsatellite instability status with tumour-infiltrating immune cells, MHC I, and PD-L1 expression in the same cholangiocarcinoma cohort showed higher numbers of CD8 + T cells, FOXP3 + regulatory T cells, CD20 + B cells and high or at least moderate MHC I expression levels in MSI-H CCAs. Conclusions Even though the overall number of MSI-H CCAs is low, the dismal prognosis of the disease and the therapeutic option of immune checkpoint blockade in the respective patients justify MSI testing of cholangiocarcinoma, particularly in younger patients showing an atypical histomorphology.

Tumor spread, in general, is the most important factor determining outcome in almost all malignant tumors. Lung tumors are unique with respect to potential routes for tumor dissemination, as apart from vascular, nodal, and distant spread of tumor cells, tumor spread through air spaces (STAS) might also occur. However, morphologic criteria for STAS and its prognostic impact have not been defined yet. We evaluated a series of 569 resected pulmonary adenocarcinomas (ADCs) for predefined morphologic criteria of limited and extensive STAS and correlated our findings with clinical, morphologic, molecular, and outcome data. Limited (21.6%) or extensive (29%) STAS was present in roughly half of all ADCs. The presence and type of STAS was tightly linked to specific growth patterns (P

Aims Pulmonary (ADC) and colorectal (CRC) adenocarcinomas are frequent entities in pathological routine diagnostics. Whereas the differential diagnosis is usually straightforward based on histomorphology, it can be challenging in small biopsies. In general, CDX-2, CK20, Napsin-A and TTF-1 are recommended immunohistological markers in this scenario. Hepatocyte nuclear factor 4 alpha (HNF4-α) and special AT-rich sequence-binding protein 2 (SATB2) were described recently as promising additional markers, but comprehensive large-scale data are lacking so far. Therefore, we analysed the expression of these six markers in 1021 non-small-cell lung cancers (NSCLC), including 472 ADC as well as in 80 pulmonary metastases of CRC. Methods and results Tissue microarrays of NSCLC and pulmonary metastases of CRC were stained for CDX-2, CK20, HNF4-α, Napsin-A, SATB2 and TTF-1 and staining results were correlated with clinicopathological variables. ADC exhibited expression of CDX-2, CK20, HNF4-α, Napsin-A, SATB2 and TTF-1 in nine (2%), 21 (4%), 17 (4%), 345 (73%), 35 (7%) and 408 (86%) samples, while 80 CRC were positive in 79 (99%), 74 (93%), 77 (96%), no (0%), 78 (98%) and five (6%) cases, respectively. Conclusions In addition to conventional immunomarkers, HNF4-α and particularly SATB2 may be helpful in the differential diagnosis of pulmonary ADC and metastases of CRC.

This case report presents a male patient with epithelioid mesothelioma that was initially misdiagnosed as cancer of unknown primary (CUP) and correctly identified using molecular panel sequencing. The patient had a prior history of colon and breast cancer. To assess the enlarged mediastinal lymph nodes, retrosternal lymphadenectomy was performed in 2016. The lymph nodes were histologically deemed unrelated to the known breast cancer by the reference pathologist, thus leading to the diagnosis of a CUP syndrome. When the patient presented to our center, targeted deep sequencing of both breast cancer and presumed CUP was performed to address the clonal relationship between both malignancies. A missense mutation in

The genetic landscape of rare benign tumours and their malignant counterparts is still largely unexplored. While recent work showed that mutant HRAS is present in subsets of poromas and porocarcinomas, a more comprehensive genetic view on these rare adnexal neoplasms is lacking. Using high-coverage next generation sequencing, we investigated the mutational profile of 50 cancer-related genes in 12 cases (six poromas and six porocarcinomas). Non-synonymous mutations were found in two-thirds of both poromas and porocarcinomas. Hotspot HRAS mutations were identified in two poromas (p.G13R and p.Q61R) and one porocarcinoma (p.G13C). While in poromas only few cases showed single mutated genes, porocarcinomas showed greater genetic heterogeneity with up to six mutated genes per case. Recurrent TP53 mutations were found in all porocarcinomas that harboured mutated genes. Non-recurrent mutations in porocarcinomas were found in several additional tumour suppressors (RB1, APC, CDKN2A, and PTEN), and genes implicated in PI3K-AKT and MAPK signalling pathways (ABL1, PDGFRA, PIK3CA, HRAS, and RET). UV-associated mutations were found in TP53, APC, CDKN2A, PTEN, and RET. In conclusion, our study confirms and extends the spectrum of genetic lesions in poromas and porocarcinomas. While poromas exhibited only few mutations, which did not involve TP53, the majority of porocarcinomas harboured UV-mediated mutations in TP53 with some of these cases showing considerable genetic heterogeneity that may be clinically exploitable.

AIMS: Deleted in malignant brain tumours 1 (DMBT1) exerts functions in the regulation of epithelial differentiation and inflammation and has been proposed as a tumour suppressor. Because chronic inflammation is a hallmark of cholangiocarcinogenesis, the aim of this study was to investigate the expression of DMBT1 in biliary tract cancer (BTC) and to correlate this expression with clinicopathological data.METHODS AND RESULTS: The expression of DMBT1 protein was examined immunohistochemically in 157 BTC patients [41 intrahepatic (ICC), 60 extrahepatic cholangiocarcinomas (ECC) and 56 adenocarcinomas of the gallbladder (GBAC)]. Additionally, 56 samples of high-grade biliary intraepithelial neoplasia (BilIN 3) and 92 corresponding samples of histological non-neoplastic biliary tract tissues were included. DMBT1 expression was increased significantly in BilIN 3 compared to normal tissue (P < 0.0001) and BTC (P < 0.0001). BTC showed no significant difference in DMBT1 expression compared to non-neoplastic biliary tissue (P = 0.315). Absent DMBT1 expression in non-neoplastic biliary tissue of BTC patients was associated with poorer survival (P = 0.027). DMBT1 expression was correlated significantly with patients' age (P < 0.001).CONCLUSION: DMBT1 is expressed differently in cholangiocarcinogenesis and poorer patients' survival rates are associated with absent DMBT1 expression in non-neoplastic biliary tissue, suggesting a tumour-suppressive role of DMBT1 in early cholangiocarcinogenesis.

Histologic scoring systems specific for primary sclerosing cholangitis (PSC) are not validated. We recently determined the applicability and prognostic value of three histological scoring systems in a single PSC cohort. The aim of this study was to validate their prognostic use and reproducibility across a multicenter PSC cohort. Liver biopsies from PSC patients were collected from seven European institutions. Histologic scoring was performed using the Nakanuma, Ishak, and Ludwig scoring systems. Biopsies were independently scored by six liver pathologists for interobserver agreement. The prognostic value of clinical, biochemical, and all three histologic scoring systems on predicting composite endpoints 1 (PSC-related death and liver transplantation), 2 (liver transplantation), and 3 (liver-related events), was assessed using univariable and multivariable Cox proportional hazards modeling. A total of 119 PSC patients were identified, and the median follow-up was 142 months. During follow-up, 31 patients died (20 PSC-related deaths), 31 patients underwent liver transplantation, and 35 patients experienced one or more liver-related events. All three staging systems were independent predictors of endpoints 2 and 3 (Nakanuma system: hazard ratio [HR], 3.16 [95% confidence interval (CI), 1.49-6.68] for endpoint 2 and HR, 2.05 [95% CI, 1.17-3.57] for endpoint 3; Ishak system: HR, 1.55 [95% CI, 1.10-2.18] for endpoint 2 and HR, 1.43 [95% CI, 1.10-1.85] for endpoint 3; Ludwig system: HR, 2.62 [95% CI, 1.19-5.80] for endpoint 2 and HR, 2.06 [95% CI, 1.09-3.89] for endpoint 3). Only the Nakanuma staging system was independently associated with endpoint 1: HR, 2.14 (95% CI, 1.22-3.77). Interobserver agreement was moderate for Nakanuma stage (κ = 0.56) and substantial for Nakanuma component fibrosis (κ = 0.67), Ishak stage (κ = 0.64), and Ludwig stage (κ = 0.62). Conclusion: We confirm the independent prognostic value and demonstrate for the first time the reproducibility of staging disease progression in PSC using the Nakanuma, Ishak, and Ludwig staging systems. The Nakanuma staging system—incorporating features of chronic biliary disease—again showed the strongest predictive value. (Hepatology 2017;65:907-919).

Cancers acquire multiple somatic mutations that can lead to the generation of immunogenic mutation-induced neoantigens. These neoantigens can be recognized by the host's immune system. However, continuous stimulation of immune cells against tumor antigens can lead to immune cell exhaustion, which allows uncontrolled outgrowth of tumor cells. Recently, immune checkpoint inhibitors have emerged as a novel approach to overcome immune cell exhaustion and reactivate antitumor immune responses. In particular, antibodies blocking the exhaustion-mediating programmed death receptor (PD-1)/programmed death receptor ligand (PD-L1) pathway have shown clinical efficacy. The effects were particularly pronounced in tumors with DNA mismatch repair (MMR) deficiency and a high mutational load, which typically occur in the colon and endometrium. Here, we report on a 24-yr-old woman diagnosed with extrahepatic cholangiocarcinoma who showed strong and durable response to the immune checkpoint inhibitor pembrolizumab, although treatment was initiated at an advanced stage of disease. The patient's tumor displayed DNA MMR deficiency and microsatellite instability (MSI) but lacked other features commonly discussed as predictors of response toward checkpoint blockade, such as PD-L1 expression or dense infiltration with cytotoxic T cells. Notably, high levels of HLA class I and II antigen expression were detected in the tumor, suggesting a potential causal relation between functionality of the tumor's antigen presentation machinery and the success of immune checkpoint blockade. We suggest determining MSI status in combination with HLA class I and II antigen expression in tumors potentially eligible for immune checkpoint blockade even in the absence of conventional markers predictive for anti-PD-1/PD-L1 therapy and in entities not commonly linked to the MSI phenotype. Further studies are required to determine the value of these markers for predicting the success of immune checkpoint blockade.

Background: Uncontrolled proliferation is a hallmark of malignant tumour growth. Its prognostic role in non-small cell lung cancer (NSCLC) has been investigated in numerous studies with controversial results. We aimed to resolve these controversies by assessing the Ki-67 proliferation index (PI) in three large, independent NSCLC cohorts. Methods: Proliferation index was retrospectively analysed by immunohistochemistry in a cohort of 1065 NSCLC and correlated with clinicopathological data including outcome and therapy. Results were validated in two independent cohorts of 233 squamous cell carcinomas (SQCC) and 184 adenocarcinomas (ADC). Results: Proliferation index (overall mean: 40.7%) differed significantly according to histologic subtypes with SQCC showing a mean PI (52.8%) twice as high as ADC (25.8%). In ADC PI was tightly linked to growth patterns. In SQCC and ADC opposing effects of PI on overall (OS), disease-specific and disease-free survival were evident, in ADC high PI (optimised validated cut-off: 25%) was a stage-independent negative prognosticator (hazard ratio, HR OS: 1.56, P=0.004). This prognostic effect was largely attenuated by adjuvant radio-/chemotherapy. In SQCC high PI (optimised validated cut-off: 50%) was associated with better survival (HR OS: 0.65, P=0.007). Conclusions: Our data demonstrate that PI is a clinically meaningful biomarker in NSCLC with entity-dependent cut-off values that allow reliable estimation of prognosis and may potentially stratify ADC patients for the need of adjuvant therapy.

Aims Molecular characterization of non-small-cell lung cancer (NSCLC) has revealed multiple druggable mutations for targeted therapies. Recently, chromosomal rearrangements involving c-ros oncogene 1, receptor tyrosine kinase (ROS1) were identified, and patients seem to benefit from crizotinib treatment. The aim of this study was to identify the clinicopathological characteristics of NSCLC with ROS1 expression and translocation. Methods and results We screened 1478 NSCLCs with a ROS1-specific antibody, and tested positive cases with FISH. All positive cases were analysed for associated clinicopathological characteristics, including survival and molecular tumour composition. Sixty-eight cases (4.6%) showed ROS1 immunoreactivity, and ROS1 translocations were confirmed in nine cases (0.6%). ROS1 expression was predominantly found in female adenocarcinoma patients, in patients with low T stages, and in association with TTF1 and napsin expression, and certain histomorphological adenocarcinoma patterns (lepidic, acinar, and solid). ROS1 translocations occurred in conjunction with other driver mutations (EGFR, KRAS, and BRAF). ROS1 expression was found to be a stage-independent predictor of favourable survival. Conclusions ROS1 translocations are rare events in resected NSCLCs from Caucasian patients. Immunohistochemical screening for ROS1 expression and clinicopathological parameters, including female sex, early tumour stages, adenocarcinomas with TTF1 and/or napsin expression, and a distinct histomorphological growth pattern, strongly facilitate case enrichment. Molecularly driven multistep concepts might not be optimal for case selection.

Cholangiocarcinoma (CC) is a rare malignancy of the extrahepatic or intrahepatic biliary tract with an outstanding poor prognosis. Non-surgical therapeutic regimens result in minimally improved survival of CC patients. Global genomic analyses identified a few recurrently mutated genes, some of them in genes involved in epigenetic patterning. In a previous study, we demonstrated global DNA methylation changes in CC, indicating major contribution of epigenetic alterations to cholangiocarcinogenesis. Here, we aimed at the identification and characterization of CC-related, differentially methylated regions (DMRs) in potential microRNA promoters and of genes targeted by identified microRNAs. Twenty-seven hypermethylated and 13 hypomethylated potential promoter regions of microRNAs, known to be associated with cancer-related pathways like Wnt, ErbB, and PI3K-Akt signaling, were identified. Selected DMRs were confirmed in 2 independent patient cohorts. Inverse correlation between promoter methylation and expression suggested miR-129-2 and members of the miR-200 family (miR-200a, miR-200b, and miR-429) as novel tumor suppressors and oncomiRs, respectively, in CC. Tumor suppressor genes deleted in liver cancer 1 (DLC1), F-box/WD-repeat-containing protein 7 (FBXW7), and cadherin-6 (CDH6) were identified as presumed targets in CC. Tissue microarrays of a representative and well-characterized cohort of biliary tract cancers (n=212) displayed stepwise downregulation of CDH6 and association with poor patient outcome. Ectopic expression of CDH6 on the other hand, delayed growth in the CC cell lines EGI-1 and TFK-1, together suggesting a tumor suppressive function of CDH6. Our work represents a valuable repository for the study of epigenetically altered miRNAs in cholangiocarcinogenesis and novel putative, CC-related tumor suppressive miRNAs and oncomiRs.

The NOTCH pathway is an evolutionary conserved signaling pathway that is known to play a pivotal role in physiological liver development and regeneration. Thereby, the NOTCH pathway controls cell fate decisions of bipotential liver progenitor cells promoting a biliary lineage rather than hepatic differentiation. Furthermore, aberrant NOTCH signaling is also a potential driver of liver inflammation, formation and progression of hepatocellular (HCC) and intrahepatic cholangiocarcinoma (iCCA). Previous studies mainly focused on the expression levels of the NOTCH receptors (NOTCH1-4) and their ligands (DLL1/3/4, JAG1/2). However, little is known regarding mutational effects within components of the Notch pathway and their relevance in liver cancer. In a whole exome sequencing approach of 54 human HCC samples, we discovered 19 amino acid-altering mutations in 15 different NOTCH pathway genes which were confirmed by Sanger sequencing to be somatic. In summary, these 15 mutations affected 25.9% of patients (14 out of 54 patients) and presented at least one mutation in one of the included NOTCH pathway components (NOTCH1/3/4, DLL1, DTX1/3/4, HES5, DVL2, LFNG, NUMB, NCSTN, DMXL2, GXYLT2). Consistently, 30% of HCC patients have been reported to contain a tumor-associated hyper-activated NOTCH pathway. Using multiple online tools to predict the effect of the mutations on protein function and tumor relevance, we observed that HES5-Arg31Gly mutation ranked highest. In order to test, whether HES5 is activated by NOTCH in HCC cells, we generated cell lines with inducible expression of NOTCH1 or NOTCH3 intracellular domain and found that the transcription factor HES5 but not HES1 is strongly activated in several HCC cell lines. Furthermore, induction of HES5 in an inducible Hep3B cell line resulted in increased expression of stem cell markers and epithelial-mesenchymal transition markers. In contrary, CCND1, HNF4alpha and HES1 were downregulated. Interestingly, HES5-Arg31Gly failed to modulate the expression of the here tested target genes. The loss of function of HES5-Arg31Gly may be partially explained by lower nuclear translocation of HES5-Arg31Gly protein. In vivo, mouse models using hydrodynamic transduction of transposons expressing MYC and HES5 indicated a tumor suppressive role of HES5, whereas, in AKT-driven liver tumorigenesis, HES5 exhibited oncogenic potential. Consistent with our in vitro experiments, AKT/HES5-driven murine tumors exhibited partial downregulation of HNF4alpha and upregulation of the cholangiocyte marker cytokeratin suggesting a transdifferentiation towards a cholangiocytic phenotype. Thus, we identified somatic mutations in NOTCH pathway genes in 25.9% of HCC patients. Our functional in vitro and in vivo analyses suggest that HES5 has oncogenic or tumor suppressive properties depending on the underlying oncogene or tumor subtype, respectively. Citation Format: Sarah Luiken, Matthias Bieg, Angelika Fraas, Raisatun Sugiyanto, Benjamin Goeppert, Stephan Singer, Stefan Pusch, Arianeb Mehrabi, Thomas Longerich, Peter Schirmacher, Stephanie K. Roessler. NOTCH/HES5 signaling exhibits distinct pro- and anti-tumorigenic roles in liver carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3487.

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2′deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients. Conclusion: We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis. (Hepatology 2014;59:544–554)

Biliary tract cancers (BTC) are a diverse group of tumours that arise from the biliary tract epithelium. Biliary tract cancers can be divided into three major clinical phenotypes: cholangiocarcinomas (CC) of intrahepatic (ICC) and extrahepatic (ECC) origin and adenocarcinomas of the gallbladder (GBAC). In the United States and Europe, risk factors for BTC include chronic biliary tract diseases, such as primary sclerosing cholangitis, hepatolithiasis, choledochal cysts, and other anatomical malformations of the bile ducts (Blechacz et al, 2011). Non-biliary diseases such as heavy alcohol abuse, obesity, non-alcoholic fatty liver disease, chronic hepatitis C, and cirrhosis are also more prevalent in BTC patients compared with the general population. For BTC patients with locally advanced or metastatic disease, the prognosis is poor with median survival less than 1 year (Hezel et al, 2010). Common therapeutic options include surgery and chemotherapy but as BTC patients often present in an unresectable state, there is an urgent need of novel chemotherapeutic options (Patel, 2011). As some BTC patients are in a general condition insufficient to undergo aggressive systemic treatment, finding better and reliable prognostic markers for BTC is of importance for further stratification of patients for therapeutic trials. Tumour cells are often surrounded by infiltrating inflammatory cells, particularly lymphocytes and macrophages (Smyth et al, 2006). Tumour antigens drive the development of tumour-specific adaptive immune responses (Boon et al, 1994). CD4+ and CD8+ T lymphocytes are crucial components of tumour-specific cellular adaptive immunity. CD8+ T lymphocytes attack tumour cells presenting tumour-associated antigen peptide with major histocompatibility complex class I (MHC I) on their surface by producing interferon-γ. Interferon-γ-dependent mechanisms of tumour cell cytostasis and killing comprise cell cycle inhibition, apoptosis, angiostasis, and induction of antitumourigenic activity of macrophages (Dunn et al, 2004). Immunohistochemical studies have found that tumour-infiltrating CD8+ T lymphocytes have a favourable effect on patient survival in several malignant tumours including colorectal (Galon et al, 2006), ovarian (Zhang et al, 2003), breast (Mahmoud et al, 2011), and pancreatic (Fukunaga et al, 2004) cancer. In colorectal cancer (CRC), density and location of CD8+ T lymphocytes have a prognostic value superior to and independent of the International Union Against Cancer (UICC) – TNM classification (Mlecnik et al, 2011). Further, density of immune cells at the invasive margin of metastatic CRC is predictive for response to chemotherapy (Halama et al, 2011). In BTC, sub-populations of immune cells have so far only been studied in parts, using comparably small patient cohorts and without specifying anatomical/histological subtypes (Oshikiri et al, 2003; Takagi et al, 2004). Therefore, our aim was to analyse the influence of density and distribution of tumour-infiltrating immune cells on patient prognosis in a large, well-characterized series of BTC patients with long-time follow-up.

Aims In adult humans, the follicle-stimulating hormone receptor (FSHR) is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. Recently, it has been shown that FSHR is expressed selectively on the surface of blood vessels in a wide range of tumours. So far, the expression of FSHR in mesenchymal tumours has not been studied. Methods and results We performed a semiquantitative evaluation of FSHR protein expression in a large cohort of soft tissue sarcomas (STS; n = 335), including 11 subtypes. FSHR-positive vessels were detected in all sarcoma subtypes analysed. Among liposarcomas, significantly more cases of dedifferentiated liposarcomas (28 of 44) showed FSHR expression compared to well-differentiated liposarcomas (WDLS; four of 21; P

BRCA1-associated protein 1 (BAP1) is a deubiquitinating enzyme that functions as a tumor suppressor gene. Double hit BAP1 inactivation has been reported in a range of tumor types, including intrahepatic cholangiocarcinoma (ICC), sometimes in association with germline mutation. We performed immunohistochemistry for BAP1 on a well-characterized cohort of 211 ICC patients undergoing surgical resection with curative intent at 3 institutions based in 3 different countries. The median age at diagnosis was 65 years (range, 36.5–86) and 108 (51%) were men. Negative staining for BAP1 (defined as completely absent nuclear staining in the presence of positive internal controls in nonneoplastic cells) occurred in 55 ICCs (26%). BAP1 loss predicted a strong trend toward improved median survival of 40.80 months (95% CI, 28.14–53.46) versus 24.87 months (95% CI, 18.73–31.01), P = 0.059). In a multivariate model including age, sex, BAP1 status, tumor stage, tumor grade, lymphovascular invasion, and tumor size, female sex was associated with improved survival (hazard ratio [HR] 0.54; 95% CI, 0.34–0.85), while advanced tumor stage and lymphovascular invasion (HR 1.89; 95% CI, 1.09–3.28) correlated with decreased survival. In a multivariate analysis, high grade tumors were associated with BAP1 loss (odds ratio [OR] 3.32; 95% CI, 1.29–8.55), while lymphatic invasion was inversely associated with BAP1 loss (OR 0.36; 95% CI, 0.13–0.99). In conclusion, we observed a trend toward improved prognosis in ICC associated with absent expression of BAP1 and an association of BAP1 loss with higher histological grade and absent lymphatic invasion. Female sex was associated with improved survival while advanced tumor stage and lymphatic invasion were associated with decreased survival.

As in most solid tumors, colorectal cancer prognosis strongly depends on the extent of local invasion and lymph node and distant metastases. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a transmembrane glycoprotein that activates matrix metalloproteinases, a group of enzymes that play an important role in tumor invasion and metastasis formation. This study investigates the EMMPRIN expression in a large cohort of patients with colorectal cancer. Immunohistochemical analysis of tissue microarrays from 285 patients shows that increased EMMPRIN protein expression does not correlate with clinicopathologic parameters and is an independent prognostic factor of poor survival, with mean survival times of 103 months in EMMPRIN negative/low versus 57 months in EMMPRIN intermediate/high patients (P < .001). This pronounced association of increased EMMPRIN levels and--on average--a 45% reduction in overall survival could help improve the risk stratification in patients with colorectal cancer; moreover, the lack of correlations with classical measures of cancer invasion/spreading may suggest the relevance of alternative EMMPRIN pathways beyond matrix metalloproteinase activation.

Objective This study retrospectively analyzes the characteristics and kinetics of pulmonary changes in Pneumocystis jiroveci pneumonia (PJP) before and after treatment as depicted by thin-section-CT in HIV-negative patients. Materials and methods Serial CT scans of 84 consecutive HIV-negative PJP patients were reviewed retrospectively encompassing a median follow-up of 76 (range, 37–506) days. Along with underlying disease and time span between the onset of symptoms and specific antimicrobial therapy, early and late pulmonary CT-findings were evaluated. Results Imaging findings at initial diagnosis differed from those in the posttherapeutic setting. In the acute (initial) PJP-phase, most frequent finding was symmetric, apically distributed ground glass opacities (GGO) with peripheral sparing 43% ( n = 36). These initial changes resolved up to 1st follow-up-examination in 57% ( n = 48), and finally in all except for two patients after a median period of 13 (mean 26, range 1–58) days following application of specific therapy. In 42% ( n = 35) architectural distortions occurred, but they resolved after a median period of 27 (mean 60, range 11–302) days. Only in 9 patients, complete resolution could not be documented. Significant correlations of the underlying disease or the time span between the onset of symptoms and specific antibiotic therapy and morphologic kinetic could not be found. Conclusion Thin-section CT-findings of PJP usually resolve soon after onset of specific therapy. Postinfectious fibrosis rarely occurs following PJP in HIV-negative patients.

The purpose of this work was to determine efficacy, toxicity, and patterns of recurrence after concurrent chemoradiation (CRT) in patients with extrahepatic bile duct cancer (EHBDC) and hilar cholangiocarcinoma (Klatskin tumours) in case of incomplete resection or unresectable disease. From 2003–2010, 25 patients with nonmetastasized EHBDC and hilar cholangiocarcinoma were treated with radiotherapy and CRT at our institution in an postoperative setting (10 patients, 9 patients with R1 resections) or in case of unresectable disease (15 patients). Median age was 63 years (range 38–80 years) and there were 20 men and 5 women. Median applied dose was 45 Gy in both patient groups. Patients at high risk (9 times R1 resection, 1 pathologically confirmed lymphangiosis) for tumour recurrence after curative surgery had a median time to disease progression of 8.7 months and an estimated mean overall survival of 23.2 months (6 of 10 patients are still under observation). Patients undergoing combined chemoradiation in case of unresectable primary tumours are still having a poor prognosis with a progression-free survival of 7.1 months and a median overall survival of 12.0 months. The main site of progression was systemic (liver, peritoneum) in both patient groups. Chemoradiation with gemcitabine is safe and can be applied safely in either patients with EHBDC or Klatskin tumours at high risk for tumour recurrence after resection and patients with unresectable tumours. Escalation of systemic and local treatment should be investigated in future clinical trials.

The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down-regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12β promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5-aza-2′deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA-mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR-183 and miR-186 are up-regulated in CL and DN and are able to target AKAP12. Conclusion: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis. (HEPATOLOGY 2010;.)

Invasive carcinomas arising within fibroepithelial tumors represent an uncommon manifestation of breast cancer. We report the case of a 70-year-old woman who underwent mastectomy for a malignant phyllodes tumor measuring 6 cm. Histological workup of the specimen revealed a high-grade invasive ductal carcinoma 2.5 cm in diameter within the phyllodes tumor. DNA was isolated from microdissected epithelial and stromal components of the phyllodes tumor as well as from the invasive ductal carcinoma cells. Using multiplex polymerase chain reaction, a comparative allelotyping was performed with a panel of 11 microsatellite markers. The malignant stroma of the phyllodes tumor showed loss of heterozygosity at chromosome 16q23, 17q12, 17q25, and 22q13; the epithelial tumor component shared the loss of 16q23; whereas the invasive carcinoma had lost divergent alleles at 16q23, 17q12, and 17q25, indicating a lack of clonality between phyllodes tumor and invasive ductal carcinoma. Although our data are compatible with a previously postulated common origin of epithelial and stromal components of phyllodes tumors, the coexisting invasive ductal carcinoma appears to represent a true collision tumor.

The inhibition of heat shock protein 90 (Hsp90) has emerged as a promising antineoplastic strategy in diverse human malignancies. Hsp90 has been predicted to be involved in hepatocellular carcinoma (HCC) development; however, its role in hepatocarcinogenesis remains elusive. Using chemically distinctive Hsp90 inhibitors, we show that Hsp90 capacitates the aberrant expression and activity of crucial hepatocarcinogenesis-driving factors (e.g., insulin-like growth factor receptor 1, hepatocyte growth factor receptor, protein kinase B, v-raf-1 murine leukemia viral oncogene homolog 1, and cyclin-dependent kinase 4). In vitro, Hsp90 inhibition with both geldanamycin analogs (17-allylamino-17-desmethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-desmethoxygeldanamycin (17-DMAG)) and the non-quinone compound 8-(6-iodobenzo[d][1,3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine (PU-H71) reduced the viability of various HCC cell lines, induced the simultaneous degradation of numerous hepatocarcinogenic factors, and caused substantial cell cycle arrest and apoptosis. In contrast, nontumorigenic hepatocytes were less susceptible to Hsp90 inhibition. Because conventional geldanamycin-derivate Hsp90 inhibitors induce dose-limiting liver toxicity, we tested whether novel Hsp90 inhibitors lacking the benzoquinone moiety, which has been deemed responsible for hepatotoxicity, can elicit antineoplastic activity without causing significant liver damage. In HCC xenograft mouse models, PU-H71 was retained in tumors at pharmacologically relevant concentrations while being rapidly cleared from nontumorous liver. PU-H71 showed potent and prolonged in vivo Hsp90 inhibitory activity and reduced tumor growth without causing toxicity. Conclusion: Hsp90 constitutes a promising therapeutic target in HCC. Non-quinone Hsp90 inhibitors exhibit tumor-specific accumulation and exert potent antineoplastic activity without causing significant hepatotoxicity. (HEPATOLOGY 2009.)

Aquaporin-4 (AQP4), the most prominent CNS water channel, is restricted to the glia limitans and astrocytic endfeet. We previously showed the loss of spatial AQP4 expression in glioblastomas and a redistribution across the cell surface. However, opposing AQP4 functions have been described: protective in vasogenic but detrimental in cytotoxic brain edema. Thus, specific AQP4 induction to prevent or reduce vasogenic edema is suggested. To elucidate the AQP4 role in brain tumors, we investigated 189 WHO grade I-IV gliomas by immunohistochemistry and the prognostic significance for patients' survival. In gliomas, a remarkable de novo AQP4 redistribution was observed in comparison with normal CNS tissue. Surprisingly, the highest membraneous staining levels were seen in pilocytic astrocytomas WHO grade I and grade IV glioblastomas, both significantly higher than in WHO grade II astrocytomas. AQP4 up-regulation was associated with brain edema formation; however, no association between survival and WHO grade-dependent AQP4 expression was seen. Hence, AQP4 redistribution may go along with other tumor properties, such as vascular proliferation and resulting blood-brain barrier disturbance, features usually prominent in pilocytic astrocytomas WHO I and glioblastomas WHO grade IV. In summary, our findings question the protective role of AQP4 in vasogenic brain edema. Although AQP4 was associated with brain edema formation, one has to question the suitability of AQP4 induction as a promising approach in vasogenic brain edema prevention and treatment. In addition, our results provide unexpectedly high AQP4 levels in pilocytic astrocytomas and present AQP4 as tumor progression marker in WHO grade II-IV astrocytomas.

A multistep model of disease progression and genomic landscape has been firmly established for colorectal cancer (CRC) primaries, but the genetic makeup of related metastases and the dynamics of genetic changes during metastatic progression are scarcely known. To address these issues, we used multigene high-coverage next-generation sequencing of 24 microsatellite-stable CRC primaries, matched normal tissue, and related multiple metastases to nodes, liver, lung, and brain with a CRC-specific gene panel to infer the degree of clonal evolution during metastatic progression of the disease. Somatic mutations were detected in 40% of CRC-related genes, and we observed a striking 100% genetic concordance between primary and multiple secondary sites for APC, KRAS, FBXW7, PIK3CA, BRAF, SMAD4, and ACVR2A. Except for true de novo mutations in 4 cases (affecting SYNE1, CTNNB1, TP53, and PTEN), all remaining cases (84.4%) shared the genetic lesions of the primary tumors with all investigated metastases irrespective of the site of metastasis or time lapse between primary tumor resection and the occurrence of metastatic spread. Putative biomarkers and druggable targets were identified in 25% of the cases. Our data proves that genetic alterations occurring early in CRC carcinogenesis are remarkably stable during metastatic progression, indicating (i) a very low degree of genetic heterogeneity between primary and multiple secondary sites with respect to CRC driver mutations and (ii) that genetic interrogation of archived primary tumor samples appears to be sufficient for the application of cancer precision medicine in the metastatic setting.

Background Liver resection (LR) for hilar cholangiocarcinoma (HCA) remains challenging because of the occurrence of unanticipated vascular and longitudinal bile duct invasion. Operative strategies to achieve negative resection margins vary, and the benefit of routine portal vein resection (PVR) is discussed controversially. Methods The data of 60 consecutive patients who underwent LR for HCA were analyzed. Twenty-one patients (35.0%) underwent LR plus PVR and 39 (65.0%) LR only. Clinicopathologic data were evaluated by the use of uni- and multivariate analyses. Results The majority of resections was performed for Bismuth–Corlette type III/IV tumors (97.3%). Hepatectomy involved trisectionectomies in 41 patients (68.3%). R1 resection margin status was identified as adverse prognosis factor for survival (hazard ratio 3.61; P = .003). PVR increased the perioperative morbidity (P = .04). The 90-day mortality rate was comparable between both groups (P = .70). Negative resection margin status was similar between groups (P = .70). The lymph node clearance was equal (P = .86). PVR was not associated with a beneficial long-term outcome, the 5-year and disease-free survival were comparable (LR only 17.8% vs LR plus PVR 20.0% [P = .89] and LR only 10.6% vs LR plus PVR 21.4% [P = .63]). PVR was no prognostic factor for tumor-dependent or disease-free survival (hazard ratio 0.64; P = .26 and hazard ratio 0.76; P = .47). Conclusion The presented data indicate that simultaneous PVR has no beneficial impact on oncologic long-term outcome in patients undergoing LR for HCA. Because it increases the perioperative morbidity, a recommendation for routine application cannot be given.

Apoptosis is critical for maintaining tissue homeostasis, and apoptosis evasion is considered as a hallmark of cancer. However, increasing evidence also suggests that proapoptotic molecules can contribute to the development of cancer, including liver cancer. The aim of this study was to further clarify the role of the proapoptotic B-cell lymphoma 2 homology domain 3 (BH3)-only protein BH3 interacting-domain death agonist (BID) for chronic liver injury (CLI) and hepatocarcinogenesis (HCG). Loss of BID significantly delayed tumor development in two mouse models of Fah-mediated and HBsTg-driven HCG, suggesting a tumor-promoting effect of BID. Liver injury as well as basal and mitogen-stimulated hepatocyte proliferation were not modulated by BID. Moreover, there was no in vivo or in vitro evidence that BID was involved in DNA damage response in hepatocytes and hepatoma cells. Our data revealed that CLI was associated with strong activation of oxidative stress (OS) response and that BID impaired full activation of p38 after OS. Conclusion: We provide evidence that the tumor-promoting function of BID in CLI is not related to enhanced proliferation or an impaired DNA damage response. In contrast, BID suppresses p38 activity and facilitates malignant transformation of hepatocytes. (Hepatology 2015;62:816–828)

OBJECTIVES Recently, aberrations in the gene encoding for ataxia-telangiectasia-mutated (ATM) protein kinase have been reported for pancreatic ductal adenocarcinomas (PDAC). These findings argue that ATM deficiency may play a role during carcinogenesis. Therefore, in this study, we investigated the clinical relevance of ATM expression and ATM activation in PDAC. METHODS Both ATM expression and nuclear phosphoSer1981-ATM levels were assessed by immunohistochemistry in a cohort of 133 PDAC and correlated with clinicopathological parameters. RESULTS We found stratification in prognostic subgroups. Complete loss of Ser1981-ATM was indicative of the worst prognosis (median survival, 10.8 vs 14.3 months [low expression] vs 31.1 months [high expression], P < 0.001). Similarly, analysis of ATM expression demonstrated absent expression levels of ATM to be associated with dismal prognosis (median survival, 9.6 months), whereas expression of ATM in general was associated with increased survival (17.7 months, P = 0.001). CONCLUSIONS Our analysis shows that both ATM expression and activated ATM are prognostic markers in PDAC with respect to standard clinicopathological parameters. These results suggest that ATM should be further explored as prognostic as well as predictive factor with respect to conventional chemotherapies and for putative synthetic lethal approaches.

Background: Several lines of evidence indicate that Sirt1, a class III histone deacetylase (HDAC) is implicated in the initiation and progression of malignancies and thus gained attraction as druggable target. Since data on the role of Sirt1 in pancreatic ductal adenocarcinoma (PDAC) are sparse, we investigated the expression profile and prognostic significance of Sirt1 in vivo as well as cellular effects of Sirt1 inhibition in vitro. Methods: Sirt1 expression was analyzed by immunohistochemistry in a large cohort of PDACs and correlated with clinicopathological and survival data. Furthermore, we investigated the impact of overexpression and small molecule inhibition on Sirt1 in pancreatic cancer cell culture models including combinatorial treatment with chemotherapy and EGFR-inhibition. Cellular events were measured quantitatively in real time and corroborated by conventional readouts including FACS analysis and MTT assays. Results: We detected nuclear Sirt1 expression in 36 (27.9%) of 129 PDACs. SIRT1 expression was significantly higher in poorly differentiated carcinomas. Strong SIRT1 expression was a significant predictor of poor survival both in univariate (p = 0.002) and multivariate (HR 1.65, p = 0.045) analysis. Accordingly, overexpression of Sirt1 led to increased cell viability, while small molecule inhibition led to a growth arrest in pancreatic cancer cells and impaired cell survival. This effect was even more pronounced in combinatorial regimens with gefitinib, but not in combination with gemcitabine. Conclusions: Sirt1 is an independent prognosticator in PDACs and plays an important role in pancreatic cancer cell growth, which can be levered out by small molecule inhibition. Our data warrant further studies on SIRT1 as a novel chemotherapeutic target in PDAC.

Numerous studies have been published on single aspects of pulmonary adenocarcinoma (ADC). To comprehensively link clinically relevant ADC characteristics, we evaluated established morphological, diagnostic and predictive biomarkers in 425 resected ADCs. Morphology was reclassified. Cytokeratin-7, thyroid transcription factor (TTF)1, napsin A, thymidylate synthase and excision repair cross-complementing rodent repair deficiency complementation group-1 expression, anaplastic lymphoma kinase rearrangements as well as epidermal growth factor receptor ( EGFR ), V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue ( KRAS ) and v-Raf murine sarcoma viral oncogene homologue B1 ( BRAF ) mutations were analysed. All characteristics were correlated with clinical and survival parameters. Morphological ADC subtypes were significantly associated with smoking history and distinct patterns of diagnostic biomarkers. KRAS mutations were prevalent in male smokers, while EGFR mutations were associated with female sex, nonsmoking and lepidic as well as micropapillary growth patterns. TTF1 expression (hazard ratio (HR) for overall survival 0.61, p=0.021) and BRAF mutations (HR for disease-free survival 2.0, p=0.046) were found to be morphology- and stage-independent predictors of survival in multivariate analysis. Adjuvant radio-/chemotherapy, in some instances, strongly impacted on the prognostic effect of both diagnostic and predictive biomarkers. Our data draw a comprehensive picture of the prevalence and interplay of established histological and molecular ADC characteristics. These data will help to develop time- and cost-effective diagnostic and treatment algorithms for ADC.

BRAF mutations have emerged as an important predictive biomarker for metastasized melanoma. Other types of cancer may also benefit from BRAF mutation-targeted therapies. In biliary tract cancer, reported BRAF mutation rates are highly controversial, ranging from 0 to 33% in adenocarcinoma of the gallbladder and 0 to 22% in cholangiocarcinoma. We here analyzed tissue microarrays of a large cohort of biliary tract cancer (n=377) including 159 intrahepatic cholangiocarcinomas, 149 extrahepatic cholangiocarcinomas, and 69 adenocarcinomas of the gallbladder for BRAF V600E mutation using a highly sensitive immunohistochemical screening approach implementing the BRAF V600E protein-specific antibody VE1. All VE1-positive cases as well as 42 VE1-negative cases were additionally analyzed by Sanger sequencing. In total, only 5 VE1-positive cases were detected (5/377; 1%). BRAF V600E mutation was confirmed by direct sequencing in all cases. All 5 mutated cases were intrahepatic cholangiocarcinomas (5/159; 3%). None of the extrahepatic cholangiocarcinomas and adenocarcinomas of the gallbladder were VE1 positive. Apart from the subtype restriction of BRAF V600E mutation to intrahepatic cholangiocarcinoma and a female predominance (4 female, 1 male), no significant correlation with clinicopathological data and patient outcome was detected. In conclusion, we demonstrate that BRAF V600E mutation is a rare event in biliary tract cancer, accounting for only 1% of all subtypes, and is restricted to intrahepatic cholangiocarcinoma. In addition, we demonstrate that VE1 immunohistochemistry is a feasible approach to routinely screen for BRAF V600E mutation in biliary tract cancer patients, thereby facilitating the detection of rare patients who may benefit from BRAF mutation-targeted therapies.

The molecular mechanisms underlying the genesis of cholangiocarcinomas (CCs) are poorly understood. Epigenetic changes such as aberrant hypermethylation and subsequent atypical gene expression are characteristic features of most human cancers. In CC, data regarding global methylation changes are lacking so far. We performed a genome-wide analysis for aberrant promoter methylation in human CCs. We profiled 10 intrahepatic and 8 extrahepatic CCs in comparison to non-neoplastic biliary tissue specimens, using methyl-CpG immunoprecipitation (MCIp) combined with whole-genome CpG island arrays. DNA methylation was confirmed by quantitative mass spectrometric analysis and functional relevance of promoter hypermethylation was shown in demethylation experiments of two CC cell lines using 5-aza-2'deoxycytidine (DAC) treatment. Immunohistochemical staining of tissue microarrays (TMAs) from 223 biliary tract cancers (BTCs) was used to analyze candidate gene expression at the protein level. Differentially methylated, promoter-associated regions were nonrandomly distributed and enriched for genes involved in cancer-related pathways including Wnt, transforming growth factor beta (TGF-β), and PI3K signaling pathways. In CC cell lines, silencing of genes involved in Wnt signaling, such as SOX17, WNT3A, DKK2, SFRP1, SFRP2, and SFRP4 was reversed after DAC administration. Candidate protein SFRP2 was substantially down-regulated in neoplastic tissues of all BTC subtypes as compared to normal tissues. A significant inverse correlation of SFRP2 protein expression and pT status was found in BTC patients.We provide a comprehensive analysis to define the genome-wide methylation landscape of human CC. Several candidate genes of cancer-relevant signaling pathways were identified, and closer analysis of selected Wnt pathway genes confirmed the relevance of this pathway in CC. The presented global methylation data are the basis for future studies on epigenetic changes in cholangiocarcinogenesis.

The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.