Publications by authors named "Benjamin Goeppert"

73 Publications

Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis.

Gut 2021 Jan 19. Epub 2021 Jan 19.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany

Objective: A detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options.

Design: We analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling.

Results: Patients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but mutations occurred exclusively in invasive CCA and mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin.

Conclusion: Integrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1136/gutjnl-2020-322983DOI Listing
January 2021

Prohibitin, STAT3 and SH2D4A physically and functionally interact in tumor cell mitochondria.

Cell Death Dis 2020 11 30;11(11):1023. Epub 2020 Nov 30.

Department of General Pathology, Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Chromosome 8p is frequently deleted in various cancer entities and has been shown to correlate with poor patient survival. SH2D4A is located on chromosome 8p and prevents the nuclear translocation of the pro-tumorigenic transcription factor STAT3. Here, we investigated the interaction of SH2D4A and STAT3 to shed light on the non-canonical functions of STAT3 in cooperation with the tumor suppressor SH2D4A. Using an immunoprecipitation-mass spectrometry (IP-MS) approach, we identified the mitochondrial scaffold proteins prohibitin 1 (PHB1) and prohibitin 2 (PHB2) among other proteins to potentially bind to SH2D4A. Co-immunoprecipitation and proximity ligation assays confirmed direct interactions of STAT3, PHB1, and SH2D4A in situ and in vitro. In addition, cell fractionation and immunofluorescence staining revealed co-localization of these proteins with mitochondria. These interactions were selectively interrupted by the small molecule and PHB ligand FL3. Furthermore, FL3 led to a reduction of STAT3 protein levels, STAT3 transcriptional activity, and HIF1α protein stabilization upon dimethyloxalylglycine (DMOG) treatment. Besides, mitochondrial fusion and fission markers, L-OPA1, Mfn1, and FIS1, were dysregulated upon FL3 treatment. This dysregulated morphology was accompanied by significant reduction of mitochondrial respiration, thus, FL3 significantly diminished mitochondrial respirational capacity. In contrast, SH2D4A knockout increased mitochondrial respiration, whereas FL3 reversed the effect of SH2D4A knockout. The here described results indicate that the interaction of SH2D4A and PHB1 is involved in the mitochondrial function and integrity. The demonstrated interaction with STAT3, accompanied by its reduction of transcriptional activity, further suggests that SH2D4A is linking STAT3 to its mitochondrial functions, and inhibition of PHB-interaction may have therapeutic effects in tumor cells with STAT3 activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-03220-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705682PMC
November 2020

Identification of BCL-XL as highly active survival factor and promising therapeutic target in colorectal cancer.

Cell Death Dis 2020 10 17;11(10):875. Epub 2020 Oct 17.

Department of Medical Oncology, National Center for Tumor Diseases (NCT), University Hospital Heidelberg, 69120, Heidelberg, Germany.

Since metastatic colorectal cancer (CRC) is a leading cause of cancer-related death, therapeutic approaches overcoming primary and acquired therapy resistance are an urgent medical need. In this study, the efficacy and toxicity of high-affinity inhibitors targeting antiapoptotic BCL-2 proteins (BCL-2, BCL-XL, and MCL-1) were evaluated. By RNA sequencing analysis of a pan-cancer cohort comprising >1500 patients and subsequent prediction of protein activity, BCL-XL was identified as the only antiapoptotic BCL-2 protein that is overactivated in CRC. Consistently, pharmacologic and genetic inhibition of BCL-XL induced apoptosis in human CRC cell lines. In a combined treatment approach, targeting BCL-XL augmented the efficacy of chemotherapy in vitro, in a murine CRC model, and in human ex vivo derived CRC tissue cultures. Collectively, these data show that targeting of BCL-XL is efficient and safe in preclinical CRC models, observations that pave the way for clinical translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-03092-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568722PMC
October 2020

Epigenome-wide analysis of methylation changes in the sequence of gallstone disease, dysplasia, and gallbladder cancer.

Hepatology 2020 Oct 5. Epub 2020 Oct 5.

Institute of Medical Biometry and Informatics, University of Heidelberg, Heidelberg, Germany.

Background & Aims: Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low- and middle-income countries and research into this disease has long been limited. In this study we therefore investigate the epigenetic changes along the model of GBC carcinogenesis represented by the sequence gallstone disease → dysplasia → GBC in Chile, the country with the highest incidence of GBC worldwide.

Approach: To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of FFPE gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Pre-processed, quality-controlled data from 82 samples (gallstones n=32, low-grade dysplasia n=13, high-grade dysplasia n=9, GBC n=28) were available to identify differentially methylated markers, regions, and pathways as well as changes in copy number variations (CNVs).

Main Results: The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of CpG islands and gene promoter regions. The methylation of genes implicated in Wnt signaling, Hedgehog signaling, and tumor suppression increased with tumor grade. CNVs also increased with GBC development and affected CDKN2A, MDM2, TP53, and CCND1. Gains in the targetable ERBB2 were detected in 14% of the GBC samples.

Conclusions: Our results indicate that GBC carcinogenesis comprises three main methylation stages: early (gallstone disease and low-grade dysplasia), intermediate (high-grade dysplasia), and late (GBC). The identified gradual changes in methylation and CNVs may help to enhance our understanding of the mechanisms underlying this aggressive disease and eventually lead to improved treatment and early diagnosis of GBC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.31585DOI Listing
October 2020

RNA Sequencing of Hepatobiliary Cancer Cell Lines: Data and Applications to Mutational and Transcriptomic Profiling.

Cancers (Basel) 2020 Sep 3;12(9). Epub 2020 Sep 3.

Institute of Medical Biometry and Informatics, University of Heidelberg, 69120 Heidelberg, Germany.

Cancer cell lines allow the identification of clinically relevant alterations and the prediction of drug response. However, sequencing data for hepatobiliary cancer cell lines in general, and particularly gallbladder cancer (GBC), are sparse. Here, we apply RNA sequencing to characterize 10 GBC, eight hepatocellular carcinoma, and five cholangiocarcinoma (CCA) cell lines. RNA extraction, quality control, library preparation, sequencing, and pre-processing of sequencing data were implemented using state-of-the-art techniques. Public data from the MSK-IMPACT database and a large cohort of Japanese biliary tract cancer patients were used to illustrate the usage of the released data. The total number of exonic mutations varied from 7207 for the cell line NOZ to 9760 for HuCCT1. Researchers planning experiments that require TP53 mutations could use the cell lines NOZ, OCUG-1, SNU308, or YoMi. Mz-Cha-1 showed mutations in ATM, SNU308 presented SMAD4 mutations, and the only investigated cell line that showed ARID1A mutations was GB-d1. SNU478 was the cell line with the global gene expression pattern most similar to GBC, intrahepatic CCA, and extrahepatic CCA. EGFR, KMT2D, and KMT2C generally presented a higher expression in the investigated cell lines than in Japanese primary GBC tumors. We provide the scientific community with detailed mutation and gene expression data, together with three showcase applications, with the aim of facilitating the design of future in vitro cell culture assays for research on hepatobiliary cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12092510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565451PMC
September 2020

[Cholangiocarcinoma-diagnosis, classification, and molecular alterations].

Pathologe 2020 Sep;41(5):488-494

Pathologisches Institut, Universitätsklinikum Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Deutschland.

Background: The entity cholangiocarcinoma comprises various malignant epithelial neoplasms of different morphology, etiology, and pathogenesis.

Aim: In this review, the etiology, pathogenesis, diagnostic criteria, classification, and molecular alterations of intra- and extrahepatic cholangiocarcinomas are summarized.

Material And Methods: This review is based on the currently available literature and personal knowledge.

Results And Discussion: Cholangiocarcinomas are morphologically and molecularly diverse neoplasms that can develop ubiquitously in the biliary tract. Since there is a significant histological and immunohistochemical overlap with frequent liver metastases, a definite diagnosis can often only be rendered in the clinical context. Cholangiocarcinomas are subclassified into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) tumors according to macro-anatomical criteria. Recent studies show that there are distinctive molecular differences in particular between iCCA and extrahepatic CCA (eCCA, including pCCA and dCCA). In addition, morphologically well-characterized precursor lesions have been identified, which in various frequencies can be assigned not only to the anatomical classification, but also to certain etiologies. An exact classification is therefore essential, especially with regard to the development of innovative, targeted therapeutic approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00292-020-00808-6DOI Listing
September 2020

miRNA profiling of biliary intraepithelial neoplasia reveals stepwise tumorigenesis in distal cholangiocarcinoma via the miR-451a/ATF2 axis.

J Pathol 2020 11 15;252(3):239-251. Epub 2020 Sep 15.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Distal cholangiocarcinoma (dCCA) is a biliary tract cancer with a dismal prognosis and is often preceded by biliary intraepithelial neoplasia (BilIN), representing the most common biliary non-invasive precursor lesion. BilIN are histologically well defined but have not so far been characterised systematically at the molecular level. The aim of this study was to determine miRNA-regulated genes in cholangiocarcinogenesis via BilIN. We used a clinicopathologically well-characterised cohort of 12 dCCA patients. Matched samples of non-neoplastic biliary epithelia, BilIN and invasive tumour epithelia of each patient were isolated from formalin-fixed paraffin-embedded tissue sections by laser microdissection. The resulting 36 samples were subjected to total RNA extraction and the expression of 798 miRNAs was assessed using the Nanostring® technology. Candidate miRNAs were validated by RT-qPCR and functionally investigated following lentiviral overexpression in dCCA-derived cell lines. Potential direct miRNA target genes were identified by microarray and prediction algorithms and were confirmed by luciferase assay. We identified 49 deregulated miRNAs comparing non-neoplastic and tumour tissue. Clustering of these miRNAs corresponded to the three stages of cholangiocarcinogenesis, supporting the concept of BilIN as a tumour precursor. Two downregulated miRNAs, i.e. miR-451a (-10.9-fold down) and miR-144-3p (-6.3-fold down), stood out by relative decrease. Functional analyses of these candidates revealed a migration inhibitory effect in dCCA cell lines. Activating transcription factor 2 (ATF2) and A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) were identified as direct miR-451a target genes. Specific ATF2 inhibition by pooled siRNAs reproduced the inhibitory impact of miR-451a on cancer cell migration. Thus, our data support the concept of BilIN as a direct precursor of invasive dCCA at the molecular level. In addition, we identified miR-451a and miR-144-3p as putative tumour suppressors attenuating cell migration by inhibiting ATF2 in the process of dCCA tumorigenesis. © The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.5514DOI Listing
November 2020

Immunohistological expression of oestrogen receptor, progesterone receptor, mammaglobin, human epidermal growth factor receptor 2 and GATA-binding protein 3 in non-small-cell lung cancer.

Histopathology 2020 Dec 11;77(6):900-914. Epub 2020 Oct 11.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Aims: Non-small-cell lung cancer (NSCLC) and breast cancer are common entities. Staining for oestrogen receptor (ER), progesterone receptor (PgR), mammaglobin (MAMG) and GATA-binding protein 3 (GATA3) is frequently performed to confirm a mammary origin in the appropriate diagnostic setting. However, comprehensive data on the immunohistological expression of these markers in NSCLC are limited. Therefore, the aim of this study was to analyse a large cohort of NSCLCs and correlate the staining results with clinicopathological variables.

Methods And Results: A tissue microarray was stained for ER, PgR, MAMG, human epidermal growth factor receptor 2 (HER2), and GATA3, and included 636 adenocarcinomas (ADCs), 536 squamous cell carcinomas (SqCCs), 65 large-cell-carcinomas, 34 pleomorphic carcinomas, and 20 large-cell neuroendocrine carcinomas. HER2 status was determined for immunohistochemically positive cases with chromogenic in-situ hybridisation. Markers with a proportion of ≥5% positive cases in ADC and SqCC were considered for survival analysis. Among ADCs, 62 (10%), 17 (3%), one (<1%), seven (1%), and 49 (8%) cases were positive for ER, PgR, MAMG, HER2, and GATA3, respectively. Among SqCCs, 10 (2%), 14 (3%), two (<1%) and 109 (20%) cases were positive for ER, PgR, HER2, and GATA3, but none of the samples showed positivity for MAMG. ER positivity was associated with ADC, female sex, smaller tumour size, and lower clinical stage. None of the markers had an impact on survival.

Conclusion: We report on ER, PgR, MAMG, HER2 and GATA3 expression in a large cohort of NSCLCs. Interpretation of these markers in the differential diagnostic setting should be based on a multimarker panel.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/his.14203DOI Listing
December 2020

Dissecting intratumour heterogeneity of nodal B-cell lymphomas at the transcriptional, genetic and drug-response levels.

Nat Cell Biol 2020 07 15;22(7):896-906. Epub 2020 Jun 15.

Department of Medicine V, Hematology, Oncology and Rheumatology, University of Heidelberg, Heidelberg, Germany.

Tumour heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is known to affect the efficacy of cancer therapy, most personalized treatment approaches do not account for intratumour heterogeneity. We addressed this issue by studying the heterogeneity of nodal B-cell lymphomas by single-cell RNA-sequencing and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subpopulations and compared their drug-response and genomic profiles. Malignant subpopulations from the same patient responded strikingly differently to anti-cancer drugs ex vivo, which recapitulated subpopulation-specific drug sensitivity during in vivo treatment. Infiltrating T cells represented the majority of non-malignant cells, whose gene-expression signatures were similar across all donors, whereas the frequencies of T-cell subsets varied significantly between the donors. Our data provide insights into the heterogeneity of nodal B-cell lymphomas and highlight the relevance of intratumour heterogeneity for personalized cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-020-0532-xDOI Listing
July 2020

Expanding pancreas donor pool by evaluation of unallocated organs after brain death: Study protocol clinical trial (SPIRIT Compliant).

Medicine (Baltimore) 2020 Mar;99(10):e19335

Department of General, Visceral, and Transplantation Surgery, University of Heidelberg, Heidelberg.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MD.0000000000019335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7478640PMC
March 2020

NOTCH target gene HES5 mediates oncogenic and tumor suppressive functions in hepatocarcinogenesis.

Oncogene 2020 04 13;39(15):3128-3144. Epub 2020 Feb 13.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

NOTCH receptor signaling plays a pivotal role in liver homeostasis and hepatocarcinogenesis. However, the role of NOTCH pathway mutations and the NOTCH target gene HES5 in liver tumorigenesis are poorly understood. Here we performed whole-exome sequencing of 54 human HCC specimens and compared the prevalence of NOTCH pathway component mutations with the TCGA-LIHC cohort (N = 364). In addition, we functionally characterized the NOTCH target HES5 and the patient-derived HES5-R31G mutation in vitro and in an orthotopic mouse model applying different oncogenic backgrounds, to dissect the role of HES5 in different tumor subgroups in vivo. We identified nonsynonymous mutations in 14 immediate NOTCH pathway genes affecting 24.1% and 16.8% of HCC patients in the two independent cohorts, respectively. Among these, the HES5-R31G mutation was predicted in silico to have high biological relevance. Functional analyses in cell culture showed that HES5 reduced cell migration and clonogenicity. Further analyses revealed that the patient-derived HES5-R31G mutant protein was non-functional due to loss of DNA binding and greatly reduced nuclear localization. Furthermore, HES5 exhibited a negative feedback loop by directly inhibiting the NOTCH target HES1 and downregulated the pro-proliferative MYC targets ODC1 and LDHA. Interestingly, HES5 inhibited MYC-dependent hepatocarcinogenesis, whereas it promoted AKT-dependent liver tumor formation and stem cell features in a murine model. Thus, NOTCH pathway component mutations are commonly observed in HCC. Furthermore, the NOTCH target gene HES5 has both pro- and anti-tumorigenic functions in liver cancer proposing a driver gene dependency and it promotes tumorigenesis with its interaction partner AKT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41388-020-1198-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142020PMC
April 2020

Knockdown of Atg7 Induces Nuclear-LC3 Dependent Apoptosis and Augments Chemotherapy in Colorectal Cancer Cells.

Int J Mol Sci 2020 Feb 7;21(3). Epub 2020 Feb 7.

National Center for Tumor Diseases, University Hospital Heidelberg, 69120 Heidelberg, Germany.

Autophagy is a catabolic process that enables cells to degrade obsolete content and refuel energy depots. In colorectal cancer (CRC) autophagy has been shown to promote tumorigenesis through energy delivery in the condition of uncontrolled proliferation. With this study, we aimed at evaluating whether autophagy sustains CRC cell viability and if it impacts therapy resistance. Initially, a colorectal cancer tissue micro array, containing mucosa ( = 10), adenoma ( = 18) and adenocarcinoma ( = 49) spots, was stained for expression of essential autophagy proteins LC3b, Atg7, p62 and Beclin-1. Subsequently, central autophagy proteins were downregulated in CRC cells using siRNA technology. Viability assays, flow cytometry and immunoblotting were performed and three-dimensional cell culture was utilized to study autophagy in a tissue mimicking environment. In our study we found an upregulation of Atg7 in CRC. Furthermore, we identified Atg7 as crucial factor within the autophagy network for CRC cell viability. Its disruption induced cell death via triggering apoptosis and in combination with conventional chemotherapy it exerted synergistic effects in inducing CRC cell death. Cell death was strictly dependent on nuclear LC3b, since simultaneous knockdown of Atg7 and LC3b completely restored viability. This study unravels a novel cell death preventing function of Atg7 in interaction with LC3b, thereby unmasking a promising therapeutic target in CRC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21031099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038172PMC
February 2020

Microproteomics and Immunohistochemistry Reveal Differences in Aldo-Keto Reductase Family 1 Member C3 in Tissue Specimens of Ulcerative Colitis and Crohn's Disease.

Proteomics Clin Appl 2020 07 16;14(4):e1900110. Epub 2020 Feb 16.

Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, 69120, Heidelberg, Germany.

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) (n =51, n =62, n =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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prca.201900110DOI Listing
July 2020

Genomic Characterization of Cholangiocarcinoma in Primary Sclerosing Cholangitis Reveals Therapeutic Opportunities.

Hepatology 2020 Oct 19;72(4):1253-1266. Epub 2020 Aug 19.

Norwegian PSC Research Center Department of Transplantation Medicine, Division of Surgery, Inflammatory Medicine and Transplantation, Oslo University Hospital Rikshospitalet, Oslo, Norway.

Background And Aims: Lifetime risk of biliary tract cancer (BTC) in primary sclerosing cholangitis (PSC) may exceed 20%, and BTC is currently the leading cause of death in patients with PSC. To open new avenues for management, we aimed to delineate clinically relevant genomic and pathological features of a large panel of PSC-associated BTC (PSC-BTC).

Approach And Results: We analyzed formalin-fixed, paraffin-embedded tumor tissue from 186 patients with PSC-BTC from 11 centers in eight countries with all anatomical locations included. We performed tumor DNA sequencing at 42 clinically relevant genetic loci to detect mutations, translocations, and copy number variations, along with histomorphological and immunohistochemical characterization. Regardless of the anatomical localization, PSC-BTC exhibited a uniform molecular and histological characteristic similar to extrahepatic cholangiocarcinoma. We detected a high frequency of genomic alterations typical of extrahepatic cholangiocarcinoma, such as TP53 (35.5%), KRAS (28.0%), CDKN2A (14.5%), and SMAD4 (11.3%), as well as potentially druggable mutations (e.g., HER2/ERBB2). We found a high frequency of nontypical/nonductal histomorphological subtypes (55.2%) and of the usually rare BTC precursor lesion, intraductal papillary neoplasia (18.3%).

Conclusions: Genomic alterations in PSC-BTC include a significant number of putative actionable therapeutic targets. Notably, PSC-BTC shows a distinct extrahepatic morpho-molecular phenotype, independent of the anatomical location of the tumor. These findings advance our understanding of PSC-associated cholangiocarcinogenesis and provide strong incentives for clinical trials to test genome-based personalized treatment strategies in PSC-BTC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.31110DOI Listing
October 2020

Expression Analysis of ATP-Binding Cassette Transporters ABCB11 and ABCB4 in Primary Sclerosing Cholangitis and Variety of Pediatric and Adult Cholestatic and Noncholestatic Liver Diseases.

Can J Gastroenterol Hepatol 2019 10;2019:1085717. Epub 2019 Dec 10.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

ATP-binding cassette (ABC) transporters are the members of the efflux pumps that are responsible for the removal of cytotoxic substances by active transport. ABCB11, the bile salt efflux pump of hepatocytes, coordinates cellular excretion of numerous conjugated bile salts into the bile canaliculi, whereas ABCB4 acts as an ATP-dependent floppase translocating phosphatidylcholine from the inner to the outer leaflet of the bile canalicular membrane. Loss of functional ABCB11 and ABCB4 proteins causes early-onset refractory cholestasis or cholangiopathy. In this study, we investigated the expression and localization pattern of ABCB11 and ABCB4 using immunohistochemistry and RNA profiling in liver samples from patients with different types and stages of chronic cholestatic liver disease, with emphasis on primary sclerosing cholangitis (PSC), compared to a variety of cholestatic and noncholestatic hepatopathies. Therefore, ABCB11 and ABCB4 expressions were investigated on formalin-fixed and paraffin-embedded (FFPE) material in a patient cohort of total 43 patients with or without cholestatic liver diseases, on protein level using immunohistochemistry and on RNA level using nanoString technology. Intriguingly, our results demonstrated increased expression of ABCB11 and ABCB4 on protein as well as RNA level in PSC, and the expression pattern correlated with disease progression. We concluded from our study that patients with PSC demonstrate altered expression levels and pattern of ABCB11 and ABCB4 which correlated with disease progression; thereby, ABCB11 and ABCB4 analysis may be a useful tool for assessment of disease stages in PSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1155/2019/1085717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925824PMC
July 2020

HER2 gene (ERBB2) amplification is a low-frequency driver with potential predictive value in gallbladder carcinoma.

Virchows Arch 2020 Jun 14;476(6):871-880. Epub 2019 Dec 14.

Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.

Gallbladder carcinoma (GBC) is an aggressive type of cancer with a dismal prognosis. Recent case reports have highlighted the human epidermal growth factor receptor 2 (HER2) as a promising target for individualized therapy in biliary tract cancer; however, current data on HER2 positivity in GBC is contradictory. This study aimed to assess the proportion of HER2 positivity and its clinical implications in a large and well-characterized European GBC cohort. HER2 status was determined in 186 cases of surgically resected gallbladder adenocarcinoma and a subset of coexistent high-grade biliary intraepithelial neoplasia (BilIN, n = 74) in accordance with the up-to-date consensus for HER2 testing in gastric cancer by immunohistochemistry and dual-color chromogenic in situ hybridization. Positivity for HER2 was observed in 5.4% of all cases (n = 10). In those patients with concomitant high-grade BilIN, two of four positive samples also showed amplification in the precursor lesion, while in the two remaining cases, positivity was either confined to invasive tumor or high-grade BilIN, exclusively. Equivocal staining found in eleven cases was not accompanied by gene amplification. Staging of the HER2-positive group was significantly different from the HER2-negative group with most cases presenting at stage IV, paralleled by a trend towards decreased survival. One patient who received dual HER2 inhibition almost went into full clinical remission despite treatment initiation in a metastasized state. Our results reveal a low prevalence of HER2 positivity and highlight HER2 gene amplification as an early, potentially driving event in gallbladder carcinogenesis. Prospective standardized HER2 testing and randomized control studies are needed to prove clinical efficacy of targeted HER2 inhibition in GBC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00428-019-02706-6DOI Listing
June 2020

HER2 gene (ERBB2) amplification is a rare event in non-liver-fluke associated cholangiocarcinogenesis.

BMC Cancer 2019 Dec 5;19(1):1191. Epub 2019 Dec 5.

Institute of Pathology, University of Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.

Background: Cholangiocarcinoma is a rapidly fatal cancer entity with a median survival of less than one year. In contrast to many other malignancies, no substantial therapeutic breakthrough has been made in the past few decades, thereby limiting the treatment to cytotoxic chemotherapy with little beneficial effect for most patients. Targeted therapy tailored to the individual has shown substantial success in the recent past as a promising avenue for cancer therapy.

Methods: In this study, we determined the frequency of amplification of the HER2 gene in a comprehensive and well-characterized European cholangiocarcinoma cohort encompassing 436 patients including intrahepatic (n = 155), proximal (n = 155) and distal (n = 126) cholangiocarcinoma by strict application of a combined immunohistochemical and in situ hybridization algorithm following the current guidelines for HER2 assessment in gastric cancer.

Results: We identified a proportion of 1.4% (n = 6) patients that demonstrated HER2 gene amplification, with the highest rate among the distal cholangiocarcinoma patients (2.4%). None of the patients with equivocal (2+) immunohistochemical staining results exhibited gene amplification molecularly. In four of the five patients with HER2 positivity, gene amplification was already present in concomitantly tested high-grade biliary intraepithelial neoplasia (80%). HER2 gene amplification was not significantly associated with other clinical parameters, including survival.

Conclusions: This study identifies HER2 gene amplification as a rare event in cholangiocarcinoma of the Western population, occurring already in high-grade BilIN in a subset of patients. Furthermore, we provide a robust testing algorithm that may be used prior to therapy administration in future clinical trials evaluating the role of HER2 as a predictive marker in cholangiocarcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12885-019-6320-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896712PMC
December 2019

Karyopherin α2-dependent import of E2F1 and TFDP1 maintains protumorigenic stathmin expression in liver cancer.

Cell Commun Signal 2019 11 29;17(1):159. Epub 2019 Nov 29.

Institute of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld 224, 69120, Heidelberg, Germany.

Background: Members of the karyopherin superfamily serve as nuclear transport receptors/adaptor proteins and provide exchange of macromolecules between the nucleo- and cytoplasm. Emerging evidence suggests a subset of karyopherins to be dysregulated in hepatocarcinogenesis including karyopherin-α2 (KPNA2). However, the functional and regulatory role of KPNA2 in liver cancer remains incompletely understood.

Methods: Quantitative proteomics (LC-MS/MS, ~ 1750 proteins in total) was used to study changes in global protein abundance upon siRNA-mediated KPNA2 knockdown in HCC cells. Functional and mechanistic analyses included colony formation and 2D migration assays, co-immunoprecipitation (CoIP), chromatin immunoprecipitation (ChIP), qRT-PCR, immmunblotting, and subcellular fractionation. In vitro results were correlated with data derived from a murine HCC model and HCC patient samples (3 cohorts, n > 600 in total).

Results: The proteomic approach revealed the pro-tumorigenic, microtubule (MT) interacting protein stathmin (STMN1) among the most downregulated proteins upon KPNA2 depletion in HCC cells. We further observed that KPNA2 knockdown leads to reduced tumor cell migration and colony formation of HCC cells, which could be phenocopied by direct knockdown of stathmin. As the underlying regulatory mechanism, we uncovered E2F1 and TFDP1 as transport substrates of KPNA2 being retained in the cytoplasm upon KPNA2 ablation, thereby resulting in reduced STMN1 expression. Finally, murine and human HCC data indicate significant correlations of STMN1 expression with E2F1/TFPD1 and with KPNA2 expression and their association with poor prognosis in HCC patients.

Conclusion: Our data suggest that KPNA2 regulates STMN1 by import of E2F1/TFDP1 and thereby provide a novel link between nuclear transport and MT-interacting proteins in HCC with functional and prognostic significance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12964-019-0456-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883611PMC
November 2019

IgG4-related sclerosing mastitis in a 49-year-old patient with multiple, tumor-like nodules-Diagnostic accuracy of core needle biopsy.

Breast J 2019 11 6;25(6):1251-1253. Epub 2019 Jul 6.

Department of Pathology, University Hospital, Heidelberg, Germany.

Recently, it has been reported that IgG4-related disease may occur in the breast manifesting as nodular sclerosing interstitial mastitis. Here we report a case with multiple tumor-like nodules in one breast. The histologic diagnosis was established on core needle biopsies, and treatment was initiated without open biopsy. Diagnosis of IgG4-related sclerosing mastitis should be suspected in cases of tumor-like lesions on imaging with an interstitial plasma cell-rich sclerosing inflammation on histology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbj.13436DOI Listing
November 2019

Low frequency of mismatch repair deficiency in gallbladder cancer.

Diagn Pathol 2019 May 8;14(1):36. Epub 2019 May 8.

Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.

Background: DNA mismatch repair (MMR) deficiency is a major pathway of genomic instability in cancer. It leads to the accumulation of numerous mutations predominantly at microsatellite sequences, a phenotype known as microsatellite instability (MSI). MSI tumors have a distinct clinical behavior and commonly respond well to immune checkpoint blockade, irrespective of their origin. Data about the prevalence of MSI among gallbladder cancer (GBC) have been conflicting. We here analyzed a well-characterized cohort of 69 Western-world GBCs.

Methods: We analyzed the mononucleotide MSI marker panel consisting of BAT25, BAT26, and CAT25 to determine the prevalence of MMR deficiency-induced MSI.

Results: MSI was detected in 1/69 (1.4%) of analyzed GBCs. The detected MSI GBC had a classical histomorphology, i.e. of acinar/tubular/glandular pancreatobiliary phenotype, and showed nuclear expression of all four MMR proteins MLH1, MSH2, MSH6, and PMS2. The MSI GBC patient showed a prolonged overall survival, despite having a high tumor stage at diagnosis. The patient had no known background or family history indicative of Lynch syndrome.

Conclusions: Even though the overall number of MSI tumors is low in GBC, the potentially therapeutic benefit of checkpoint blockade in the respective patients may justify MSI analysis of GBC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13000-019-0813-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506936PMC
May 2019

Profiling of gallbladder carcinoma reveals distinct miRNA profiles and activation of STAT1 by the tumor suppressive miRNA-145-5p.

Sci Rep 2019 03 18;9(1):4796. Epub 2019 Mar 18.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

Gallbladder carcinoma (GBC) is a biliary tract cancer with few treatment options and poor prognosis. Radical surgery is the only potentially curative treatment option but most patients diagnosed with GBC are unresectable. Thus, there is a great need for the development of new treatment options including targeted therapy. Here, we aimed at identifying deregulated miRNAs and affected pathways involved in GBC development and progression. We performed global miRNA profiling of 40 GBC and 8 normal gallbladder tissues and identified large differences with 30% of miRNAs being differentially expressed (false discovery rate: FDR < 0.001). We found 24 miRNAs to be differentially regulated in GBC with poor outcome (p < 0.05) of which miR-145-5p was the most downregulated miRNA. Overexpression of miR-145-5p significantly reduced cell proliferation and colony formation. Gene expression analysis of cells expressing miR-145-5p mimics revealed activation of the Signal transducer and activator of transcription 1 (STAT1) signaling pathway which is mainly tumor suppressive. Furthermore, the activation of STAT1 by miR-145-5p was specifically observed in gallbladder carcinoma and cholangiocarcinoma but not in hepatocellular carcinoma cells. The Protein Tyrosine Phosphatase Receptor Type F (PTPRF) is downregulated upon miR-145 expression and may be involved in STAT1 regulation. In addition, we found that the STAT1-regulated protein IRF7 is downregulated in GBC compared to normal gallbladder tissue and low IRF7 expression is associated with significantly lower overall survival of GBC patients. Thus, this study identified GBC patient subgroups and provides new mechanistic insights in the tumor suppressive function of miR-145-5p leading to activation of STAT1 signaling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-40857-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423323PMC
March 2019

Anatomical, histomorphological and molecular classification of cholangiocarcinoma.

Liver Int 2019 05;39 Suppl 1:7-18

Department of Movement, Human and Health Sciences, Division of Health Sciences, University of Rome "Foro Italico", Rome, Italy.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/liv.14093DOI Listing
May 2019

Prognostic Impact of Carboxylesterase 2 in Cholangiocarcinoma.

Sci Rep 2019 03 13;9(1):4338. Epub 2019 Mar 13.

Institute for Medical Immunology, Campus Virchow, Berlin, Charité, Germany.

Carboxylesterase 2 (CES2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. Novel treatment strategies are exceedingly needed for cholangiocarcinoma (CCA) patients. Here, we assessed CES2 expression by immunohistochemistry in a CCA cohort comprising 171 non-liver fluke associated, intrahepatic (n = 72) and extrahepatic (perihilar: n = 56; distal: n = 43) CCAs. Additionally, 80 samples of high-grade biliary intraepithelial neoplastic tissues and 158 corresponding samples of histological normal, non-neoplastic biliary tract tissues were included. CES2 expression was highest in non-neoplastic biliary tissue and significantly decreased in CCA. Patients showing any CES2 expression in tumor cells had a significantly better overall survival compared to negative cases (p = 0.008). This survival benefit was also maintained after stratification of CES2-positive cases, by comparing low, medium and high CES2 expression levels (p-trend = 0.0006). Evaluation of CCA subtypes showed the survival difference to be restricted to extrahepatic tumors. Correlation of CES2 expression with data of tumor-infiltrating immune cells showed that particularly CD8+ T cells were more frequently detected in CES2-positive CCAs. Furthermore, treatment of CCA cell lines with the prodrug Irinotecan reduced cell viability, increased cytotoxicity and modulated inflammatory gene expression. In conclusion, reduced CES2 expression is associated with poor outcome and low CD8+ T cell infiltration in CCA patients. Further clinical studies could show, whether CES2 expression may serve as a predictive marker in patients treated with prodrugs converted by CES2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-40487-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416336PMC
March 2019

NUT carcinoma in a nutshell: A diagnosis to be considered more frequently.

Pathol Res Pract 2019 Jun 30;215(6):152347. Epub 2019 Jan 30.

Institute of Pathology, Heidelberg University Hospital, University of Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.prp.2019.01.043DOI Listing
June 2019

Combined targeted DNA and RNA sequencing of advanced NSCLC in routine molecular diagnostics: Analysis of the first 3,000 Heidelberg cases.

Int J Cancer 2019 08 19;145(3):649-661. Epub 2019 Feb 19.

Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ijc.32133DOI Listing
August 2019

Programmed cell death ligand 1 (PD-L1, CD274) in cholangiocarcinoma - correlation with clinicopathological data and comparison of antibodies.

BMC Cancer 2019 Jan 15;19(1):72. Epub 2019 Jan 15.

Institute of Pathology, University Heidelberg, Im Neuenheimer Feld 224, Heidelberg, Germany.

Background: Cholangiocarcinoma (CCA) may arise in the intra- or extrahepatic biliary tract and is associated with a poor prognosis. Despite recent advances, to date there is still no established targeted therapeutic approach available. Non-surgical therapeutic agents are urgently needed, as most patients are non-eligible to surgical resection. Anti-PD-L1 therapy prevents cancer cells from evading the immune system and has emerged as a new treatment option in several cancer entities. Recently, PD-L1 expression has been analyzed in comparably small CCA patient cohorts. However, a systematic validation of different PD-L1 antibodies has not been performed in CCA so far.

Methods: We stained a tissue microarray consisting of 170 patients, including 72 intrahepatic cholangiocarcinomas (iCCAs), 57 perihilar cholangiocarcinomas (pCCAs) and 41 distal cholangiocarcinomas (dCCAs) by immunohistochemistry and evaluated PD-L1 positivity in tumor and stromal cells. We analyzed three different PD-L1 antibodies (clones 28-8, SP142, and SP263) that are frequently used and recommended for predictive diagnostic testing in other cancer types.

Results: For PD-L1 antibody clone SP263, 5% of iCCAs, 4% of pCCAs and 3% of dCCAs exhibited PD-L1 expression on tumor cells, thereby showing the highest frequencies of PD-L1 positivity. Accordingly, highest PD-L1 positivity rates of stromal cells with 31% in iCCA, 40% in pCCA and 61% in dCCA were detected for clone SP263. Agreement of PD-L1 positivity in tumor cells was moderate for clone 28-8 and SP263 (κ = 0.44) and poor between 28-8 and SP142 (κ = 0.13), as well as  SP142 and SP263 (κ = 0.11), respectively. Statistical analyses of PD-L1 expression (clone SP263) on tumor cells with clinicopathological data revealed a positive correlation with shortened overall survival in CCA patients.

Conclusions: Selection of appropriate PD-L1 antibodies and careful evaluation of immunohistochemical staining patterns have a significant impact on PD-L1 testing in CCA. Clinical trials are necessary to investigate the putative beneficial effects of PD-L1 targeted immunotherapy in CCA patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12885-018-5254-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6332835PMC
January 2019

[Spindle and giant cell type undifferentiated carcinoma of the distal bile duct: a case report].

Z Gastroenterol 2019 Jan 14;57(1):52-56. Epub 2019 Jan 14.

Pathologisches Institut Heidelberg, Universitätsklinikum Heidelberg, Universität Heidelberg, Im Neuenheimer Feld 224, 69120 Heidelberg.

Undifferentiated carcinoma of the biliary tract is rare and more frequently occurs in the gall bladder than in the extrahepatic bile ducts. We report on an extremely rare case of a spindle and giant cell type undifferentiated carcinoma of the distal bile duct. In the presented tumor, atypical glands and single-cell clusters arising from the bile duct epithelium gradually transitioned into a predominantly sarcomatoid architecture, which constituted more than 98 % of the whole tumor volume. Focally, osteoclast-like giant cells were intermixed with the spindle cells. The tumor showed a high proliferation activity (Ki-67) and was demonstrated to harbor mutations in the genes for cyclin D3 , fibroblast growth factor receptor 4 , neurofibromin 1 and , as assessed by next-generation sequencing analysis. The presented case underscores the relevance of this tumor entity beyond the pancreas and gall bladder and emphasizes the indispensable combination of morphology and immunohistochemistry regarding the diagnostic process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1055/a-0784-8763DOI Listing
January 2019

Integrative Analysis Defines Distinct Prognostic Subgroups of Intrahepatic Cholangiocarcinoma.

Hepatology 2019 05 28;69(5):2091-2106. Epub 2019 Feb 28.

Institute of Pathology, University Clinic of Heidelberg, Heidelberg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/hep.30493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594081PMC
May 2019

Nuclear Translocation of RELB Is Increased in Diseased Human Liver and Promotes Ductular Reaction and Biliary Fibrosis in Mice.

Gastroenterology 2019 03 13;156(4):1190-1205.e14. Epub 2018 Nov 13.

Department of Medical Oncology, National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany; Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg, Germany. Electronic address:

Background & Aims: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction.

Methods: We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin β (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (Cyld mice and Cyld/Relb mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB.

Results: In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of Cyld mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/Relb mice. Compared with livers from control mice, livers from Cyld mice (but not Cyld/Relb mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl exposure. Cyld mice and Cyld/Relb mice had improved liver function on the DDC diet compared with control mice fed the DDC diet.

Conclusion: Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in Cyld mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2018.11.018DOI Listing
March 2019

Mismatch repair deficiency is a rare but putative therapeutically relevant finding in non-liver fluke associated cholangiocarcinoma.

Br J Cancer 2019 01 31;120(1):109-114. Epub 2018 Oct 31.

Department of Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41416-018-0199-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325153PMC
January 2019