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Simple summary: Intraductal tubulopapillary neoplasms (ITPN) have recently been described as rare precursor lesions of pancreatic ductal adenocarcinoma and cholangiocarcinoma. Despite a high number of associated invasive adenocarcinomas at the time of diagnosis, patients with ITPN tend to have a much better clinical outcome than those with classical pancreato-biliary adenocarcinoma. Furthermore, rare molecular studies of ITPN show an unexpected lack of hotspot mutations in common driver genes of pancreato-biliary adenocarcinoma, including KRAS. This article reports the first large comprehensive and comparative molecular study of pancreato-biliary ITPN. In the absence of KRAS mutations, we found a high genetic heterogeneity with enrichment in core signaling pathways, including putative actionable genomic targets in one-third of the cases. Whereas, pancreatic ITPN demonstrates a highly distinct genetic profile, differing from classical pancreatic carcinogenesis, biliary ITPN and classical cholangiocarcinoma share common alterations in key genes of the chromatin remodeling pathway, and therefore, appear more closely related than pancreatic ITPN and classical pancreatic ductal adenocarcinoma PDAC. The molecular carcinogenesis of intraductal tubulopapillary neoplasms (ITPN), recently described as rare neoplasms in the pancreato-biliary tract with a favorable prognosis despite a high incidence of associated pancreato-biliary adenocarcinoma, is still poorly understood. To identify driver genes, chromosomal gains and losses, mutational signatures, key signaling pathways, and potential therapeutic targets, the molecular profile of 11 biliary and 6 pancreatic ITPNs, associated with invasive adenocarcinoma in 14/17 cases, are studied by whole exome sequencing (WES). The WES of 17 ITPNs reveals common copy number variants (CNVs) broadly distributed across the genome, with recurrent chromosomal deletions primarily in 1p36 and 9p21 affecting the tumor suppressors CHD5 and CDKN2A, respectively, and gains in 1q affecting the prominent oncogene AKT3. The identified somatic nucleotide variants (SNVs) involve few core signaling pathways despite high genetic heterogeneity with diverse mutational spectra: Chromatin remodeling, the cell cycle, and DNA damage/repair. An OncoKB search identifies putative actionable genomic targets in 35% of the cases (6/17), including recurrent missense mutations of the FGFR2 gene in biliary ITPNs (2/11, 18%). Our results show that somatic SNV in classical cancer genes, typically associated with pancreato-biliary carcinogenesis, were absent (KRAS, IDH1/2, GNAS, and others) to rare (TP53 and SMAD4, 6%, respectively) in ITPNs. Mutational signature pattern analysis reveals a predominance of an age-related pattern. Our findings highlight that biliary ITPN and classical cholangiocarcinoma display commonalities, in particular mutations in genes of the chromatin remodeling pathway, and appear, therefore, more closely related than pancreatic ITPN and classical pancreatic ductal adenocarcinoma., German Research Foundation (DFG)

Carcinomas of the pancreatobiliary system confer an especially unfavorable prognosis. The differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and its subtypes versus liver metastasis of ductal adenocarcinoma of the pancreas (PDAC) is clinically important to allow the best possible therapy. We could previously show that E-cadherin and N-cadherin, transmembrane glycoproteins of adherens junctions, are characteristic features of hepatocytes and cholangiocytes. We therefore analyzed E-cadherin and N-cadherin in the embryonally related epithelia of the bile duct and pancreas, as well as in 312 iCCAs, 513 carcinomas of the extrahepatic bile ducts, 228 gallbladder carcinomas, 131 PDACs, and precursor lesions, with immunohistochemistry combined with image analysis, fluorescence microscopy, and immunoblots. In the physiological liver, N-cadherin colocalizes with E-cadherin in small intrahepatic bile ducts, whereas larger bile ducts and pancreatic ducts are positive for E-cadherin but contain decreasing amounts of N-cadherin. N-cadherin was highly expressed in most iCCAs, whereas in PDACs, N-cadherin was negative or only faintly expressed. E- and N-cadherin expression in tumors of the pancreaticobiliary tract recapitulate their expression in their normal tissue counterparts. N-cadherin is a helpful marker for the differential diagnosis between iCCA and PDAC, with a specificity of 96% and a sensitivity of 67% for small duct iCCAs and 50% for large duct iCCAs.

Simple Summary Histopathological examination of lymph node (LN) specimens allows the detection of hematological diseases. The identification and the classification of lymphoma, a blood cancer with a manifestation in LNs, are difficult and require many years of training, as well as additional expensive investigations. Today, artificial intelligence (AI) can be used to support the pathologist in identifying abnormalities in LN specimens. In this article, we trained and optimized an AI algorithm to automatically detect two common lymphoma subtypes that require different therapies using normal LN parenchyma as a control. The balanced accuracy in an independent test cohort was above 95%, which means that the vast majority of cases were classified correctly and only a few cases were misclassified. We applied specific methods to explain which parts of the image were important for the AI algorithm and to ensure a reliable result. Our study shows that classifications of lymphoma subtypes is possible with high accuracy. We think that routine histopathological applications for AI should be pursued. Abstract The diagnosis and the subtyping of non-Hodgkin lymphoma (NHL) are challenging and require expert knowledge, great experience, thorough morphological analysis, and often additional expensive immunohistological and molecular methods. As these requirements are not always available, supplemental methods supporting morphological-based decision making and potentially entity subtyping are required. Deep learning methods have been shown to classify histopathological images with high accuracy, but data on NHL subtyping are limited. After annotation of histopathological whole-slide images and image patch extraction, we trained and optimized an EfficientNet convolutional neuronal network algorithm on 84,139 image patches from 629 patients and evaluated its potential to classify tumor-free reference lymph nodes, nodal small lymphocytic lymphoma/chronic lymphocytic leukemia, and nodal diffuse large B-cell lymphoma. The optimized algorithm achieved an accuracy of 95.56% on an independent test set including 16,960 image patches from 125 patients after the application of quality controls. Automatic classification of NHL is possible with high accuracy using deep learning on histopathological images and routine diagnostic applications should be pursued.

Simple Summary Gallbladder cancer (GBC) is an aggressive malignancy with poor prognosis. Currently, therapeutic options are mostly limited to palliative chemotherapy. Considering the advances of immunotherapy, we assessed the expression of the programmed cell death ligand-1 (PD-L1) as the most widely used predictive marker for immunotherapy response in a large Western-world GBC cohort. Additionally, we quantified the expression of the T-cell immunoreceptor with Ig and ITIM domains TIGIT/CD155 axis as an emerging immune checkpoint. Our results indicate that PD-L1 is heterogeneously expressed in Western-world GBC and associated with distinct histomorphological tumor subtypes and increased immune cell densities. We show that a high tumoral PD-L1 expression is a significant negative prognosticator. In a subset of patients, we identified expression of TIGIT in scattered immune cells, which correlated with tumoral expression of its ligand CD155. Our results suggest a subset of GBC patients to be candidates for immunotherapy via (combined) PD-L1 and TIGIT/CD155 inhibition. Abstract Inhibition of the programmed cell death protein-1/ligand-1 (PD-1/PD-L1) axis has opened a new era in the treatment of solid cancers. However, there is no data on the expression and relevance of PD-L1 in Western gallbladder cancer (GBC). We assessed PD-L1 immunohistochemically in 131 GBC patients as Tumor Proportion Score (TPS), Immune Cell Score (IC) and Combined Positivity Score (CPS). Tumor cells expressed PD-L1 in a subset of 14.7% GBC patients at a TPS cut-off of 1%. Higher PD-L1 levels above 10% and 25% TPS were reached in 4.7% and 3.1% of GBC cases, respectively. At a 10% cut-off, TPS was associated with distinct histomorphological subtypes and correlated with poor tumor differentiation. Survival analysis revealed a TPS above 10% to be a highly significant and independent negative prognosticator in GBC. PD-L1 expression was associated with increased CD4+, CD8+ and PD-1+ immune cell densities. In 14.8% of the cases, scattered immune cells expressed T-cell immunoreceptor with Ig and ITIM domains (TIGIT), which was correlated to tumoral expression of its ligand CD155. We here show that a high PD-L1 expression confers a negative prognostic value in Western-world GBC and highlight the TIGIT/CD155 immune checkpoint as a potential new target for GBC immunotherapy.

Simple Summary Research on gallbladder cancer (GBC) has been largely neglected and molecular GBC data is underrepresented in public databases. Cancer cell lines constitute a valuable tool to examine the mechanisms of malignant transformation and identify potential therapeutic targets. Here we use RNA sequencing to characterize 23 commercial hepatobiliary cancer cell lines, including ten GBC cell lines, and provide detailed mutation and gene expression data to the research community. We illustrate the practical utility of the released information by (1) assessing the presence of specific mutations in the investigated cancer cell lines, (2) comparing global gene expression patterns in cell lines and primary biliary tumours and (3) examining the expression levels of specific genes. The released data and showcase applications will ease the design of in vitro cell culture assays for future studies. Abstract 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.

Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.