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BACKGROUND AND AIMS Gallbladder cancer (GBC) is a highly aggressive malignancy of the biliary tract. Most cases of GBC are diagnosed in low-income 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 AND RESULTS To perform epigenome-wide methylation profiling, genomic DNA extracted from sections of formalin-fixed, paraffin-embedded gallbladder tissue was analyzed using Illumina Infinium MethylationEPIC BeadChips. Preprocessed, 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). The number and magnitude of epigenetic changes increased with disease development and predominantly involved the hypermethylation of cytosine-guanine dinucleotide 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 cyclin-dependent kinase inhibitor 2A, MDM2 proto-oncogene, tumor protein P53, and cyclin D1 genes. Gains in the targetable Erb-B2 receptor tyrosine kinase 2 gene were detected in 14% of 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.

Introduction: Nonalcoholic fatty liver disease covers a broad spectrum. Simple steatosis has usually a benign course while nonalcoholic steatohepatitis (NASH) can progress into hepatocellular carcinoma, and cirrhosis. Therefore, differentiating patients with benign steatosis and NASH is crucial. Liver biopsy, the usual gold standard for NASH diagnosis, cannot be used as a screening method due to its associated risks. This is especially problematic for obese patients with a prevalence of nonalcoholic fatty liver disease (NAFLD) in >80% of patients. The aim of this study was therefore to develop and validate a noninvasive NASH screening test in a cohort of high-risk, morbidly obese patients. Methods: This prospective study examined diagnostic accuracy in accordance with STARD guidelines. 112 liver biopsies were consecutively assigned to either a training or validation cohort. Using the Bedossa histological scoring system, the cohorts were subdivided into NASH versus NAFLD/No NAFLD. Predictors of NASH were evaluated with receiver operating characteristic (ROC) curves. A model was then constructed using a backward stepwise logistic regression and evaluated in an independent validation cohort. Results: 53.5% of the patients had NASH and 4 patients had cirrhosis. Mean body mass index (BMI) was 49.8 ± 7.5 kg/m2. Backward stepwise logistic regression identified 4 parameters associated with the presence of NASH: alanin-aminotransferase, albumin, BMI, and triglycerides. The noninvasive NASH detection score (NI-NASH-DS) had an ROC of 0.851 and 0.727 in the training and validation cohorts, respectively. Sensitivity and specificity were 77.1% and 88% in the training cohort and 88% and 48% in the validation cohort which was much better than the established noninvasive scores. Discussion/Conclusion: The NI-NASH-DS is easy-to-use, inexpensive, and noninvasive and can reliably detect NASH in patients with morbid obesity. Due to its simplicity, it can be used frequently and repeatedly.

Objective Evaluation of the outcome after resection for distal bile duct cancer (DBC) with focus on the impact of microscopic histopathological resection status R0 (>1 mm) vs R1 (≤1 mm) vs R1 (direct). Summary background data DBC is a rare disease for which oncologic resection offers the only chance of cure. Methods Prospectively collected data of consecutive patients undergoing pancreaticoduodenectomy for DBC were analyzed. Histopathological resection status was classified according to the Leeds protocol for pancreatic cancer (PDAC; R0 >1 mm margin clearance vs. R1 ≤1 mm vs. R1 direct margin involvement). Results A total of 196 patients underwent pancreaticoduodenectomy for DBC. Microscopic complete tumor clearance (R0>1 mm) was achieved in 113 patients (58%). Median overall survival (OS) of the entire cohort was 37 months (5- and 10-year OS rate: 40% and 31%, respectively). After R0 resection, median OS increased to 78 months with a 5-year OS rate of 52%. Negative prognostic factors were age >70 years (p 1000 ml (p=0.0009, HR 1.99), pN1 and pN2 status (p=0.0052 and p=0.0006, HR2.14 and 2.62, respectively) and American Society of Anesthesiologists score >II (p=0.0259, HR 1.61). Conclusions This is the largest European single-center study of surgical treatment for DBC and the first to investigate the prognostic impact of the revised PDAC resection status definition in DBC. The results show that this definition is valid in DBC and that "true" R0 resection (>1 mm) is a key factor for excellent survival. In contrast to PDAC, there was no survival difference between R1 (≤1 mm) and R1 (direct).

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.

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 asTP53(35.5%),KRAS(28.0%),CDKN2A(14.5%), andSMAD4(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.

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.

Background Synaptophysin, chromogranin and CD56 are recommended markers to identify pulmonary tumors with neuroendocrine differentiation. Whether the expression of these markers in pulmonary adenocarcinoma and pulmonary squamous cell carcinoma is a prognostic factor has been a matter of debate. Therefore, we investigated retrospectively a large cohort to expand the data on the role of synaptophysin, chromogranin and CD56 in non-small cell lung cancer lacking morphological features of neuroendocrine differentiation. Methods A cohort of 627 pulmonary adenocarcinomas (ADC) and 543 squamous cell carcinomas (SqCC) lacking morphological features of neuroendocrine differentiation was assembled and a tissue microarray was constructed. All cases were stained with synaptophysin, chromogranin and CD56. Positivity was defined as > 1% positive tumor cells. Data was correlated with clinico-pathological features including overall and disease free survival. Results 110 (18%) ADC and 80 (15%) SqCC were positive for either synaptophysin, chromogranin, CD56 or a combination. The most commonly positive single marker was synaptophysin. The least common positive marker was chromogranin. A combination of ≤2 neuroendocrine markers was positive in 2–3% of ADC and 0–1% of SqCC. There was no significant difference in overall survival in tumors with positivity for neuroendocrine markers neither in ADC (univariate: P = 0.4; hazard ratio [HR] = 0.867; multivariate: P = 0.5; HR = 0.876) nor in SqCC (univariate: P = 0.1; HR = 0.694; multivariate: P = 0.1, HR = 0.697). Likewise, there was no significant difference in disease free survival. Conclusions We report on a cohort of 1170 cases that synaptophysin, chromogranin and CD56 are commonly expressed in ADC and SqCC and that their expression has no impact on survival, supporting the current best practice guidelines.

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 (

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.

Tumor heterogeneity encompasses both the malignant cells and their microenvironment. While heterogeneity between individual patients is well-known to affect the efficacy of anti-cancer drugs, most personalized treatment approaches do not account for intratumor heterogeneity. We addressed this issue by studying the heterogeneity of lymph node-derived B cell non-Hodgkin lymphoma (B-NHL) by single cell RNA-sequencing (scRNA-seq) and transcriptome-informed flow cytometry. We identified transcriptionally distinct malignant subclones and compared their drug response and genomic profiles. Malignant subclones of the same patient responded strikingly different to anti-cancer drugs ex vivo, which recapitulated subclone-specific drug sensitivity during in vivo treatment. Tumor 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 new insights into the heterogeneity of B-NHL and highlight the relevance of intratumor heterogeneity for personalized cancer therapies.

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.

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 BAP1 was revealed in both samples, with coverage data indicating a germline event. The patient was subsequently counseled by a human geneticist and underwent genetic testing, which confirmed the germline nature of this mutation. Collectively, these data led to the diagnosis of BAP1 (BRCA1-associated protein-1) tumor predisposition syndrome (TPDS). With the knowledge of an underlying BAP1 mutation and its known frequent association with epithelioid mesothelioma, the histology was reassessed and the diagnosis was revised to epithelioid mesothelioma. At this point, peritoneal involvement of mesothelioma could be diagnosed and histologically confirmed. This case illustrates the potential of integrated histopathologic and molecular diagnostics in helping to decipher CUP syndromes and establish the correct diagnosis. Additionally, this case highlights typical features of BAP1 TPDS with its general susceptibility to cancers, with pleural and peritoneal mesotheliomas as most prevalent clinical entities and the typically more benign course of these epithelioid mesotheliomas compared with BAP1-unrelated cases of mesotheliomas.

// Bruno Christian Kohler 1, 2 , Benjamin Goeppert 2, 3 , Nina Waldburger 2, 3 , Kai Schlamp 4 , Peter Sauer 5 , Dirk Jager 1, 2 , Karl Heinz Weiss 2, 5 , Stephan Macher-Goppinger 6 , Henning Schulze-Bergkamen 7 , Peter Schirmacher 2, 3 and Christoph Springfeld 1, 2 1 Department of Medical Oncology, National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany 2 Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg, Germany 3 Department of Pathology, University of Heidelberg, Heidelberg, Germany 4 Department of Neuroradiology, University Hospital Heidelberg, Heidelberg, Germany 5 Department of Gastroenterology, University Hospital Heidelberg, Heidelberg, Germany 6 Institute of Pathology, University Medical Center Mainz, Mainz, Germany 7 Department of Internal Medicine II, Marien-Hospital, Wesel, Germany Correspondence to: Bruno Christian Kohler, email: bruno.koehler@nct-heidelberg.de Keywords: cholangiocarcinoma; Klatskin tumor; undifferentiated carcinoma; chemotherapy; endoscopic retrograde cholangiography Received: February 09, 2018 Accepted: March 22, 2018 Published: April 24, 2018 ABSTRACT Background: Neoplasms anatomically adjacent to the bile duct usually derive from malignantly transformed cholangiocytes forming cholangiocarcinoma (CCA). CCAs are divided in extrahepatic (eCCA) and intrahepatic (iCCA) tumors. Patients with irresectable CCAs are treated with systemic chemotherapy and have an unfavorable prognosis with a median survival of about one year. Here, we report a case of an undifferentiated carcinoma in Klatskin-position with long-term remission after systemic chemotherapy. Case Presentation: A 65-year-old Caucasian male presented with painless jaundice caused by an undifferentiated carcinoma in Klatskin-position (Type IIIb). Alpha fetoprotein (AFP; 3675 IU/mL) and carbohydrate antigen 19-9 (CA 19-9; 183 U/ml) were elevated. An exploratory laparotomy was carried out, but the patient was found to be irresectable due to severe fibrosis caused by biliary obstruction. Histology showed an undifferentiated carcinoma with high proliferation rate, and the patient was therefore subjected to poly-chemotherapy treatment according to the FOLFOX6-protocol. During therapy, AFP decreased to normal. Subsequent CT scans and ERC revealed a complete remission. Four years past initial diagnosis, a new suspicious lesion in the liver is visible on MRT; however, AFP and CA 19-9 are still in the normal range. Conclusions: Our case demonstrates that histopathological defined diagnosis may significantly inform therapeutic decision-making in irresectable cholangiocarcinoma even in regard to conventional systemic therapy. In case of an undifferentiated carcinoma poly-chemotherapy may provide significant success.

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.

Appendiceal intussusception is a rare, but relevant differential diagnosis of colorectal neoplasia on endoscopy. Misdiagnosis as polyp and endoscopic removal may result in severe iatrogenic complications.

Background: Brown tumor is a rare skeletal manifestation of secondary hyperparathyroidism. Although diagnosis of the disease is increasingly seen in early stages due to improved screening techniques, some patients still present in a progressed disease stage. The treatment depends on tumor mass and varies from a conservative approach with supportive parathyroidectomy to extensive surgical resection with subsequent reconstruction. Case presentation: We report a case of extensive mandibular brown tumor in a patient with a history of systemic lupus erythematosus, chronic kidney disease, and secondary hyperparathyroidism. Following radical resection of the affected bone, reconstruction could be successfully performed using a free flap. Conclusions: There were no signs of recurrence during five years of close follow-up. Increased awareness and multidisciplinary follow-ups could allow early diagnosis and prevent the need for radical therapeutical approaches.

Introduction: The treatment of patients (pts) with rrMM remains challenging and response is often limited in depth and duration. In contrast to many other malignant diseases, targeted therapy in MM is hampered by the limited number of actionable targets. Activating mutations of BRAF have been found in 2-4% of newly diagnosed MM and in up to 8% of rrMM. Several case reports have reported clinical efficacy of downstream pathway inhibition in MM. This phase II trial is evaluating the safety and efficacy of combined BRAF/MEK inhibition in rrMM pts with BRAF V600E mutation. Aims: The primary objective of this study was to demonstrate the therapeutic efficacy of encorafenib in combination with binimetinib. The primary endpoint was the overall response rate (ORR), defined as best response within 1 year of treatment. Main secondary endpoints included progression-free survival (PFS) and duration of response. Safety analyses included all adverse events (AEs) with those of grade 1/2 only being assessed further if considered to be related to study medications by the investigator. Methods: A total of 15 pts with rrMM who have failed at least 2 prior lines of therapy, including a proteasome inhibitor (PI) and an immunomodulatory drug (IMID), were planned to be enrolled in this open-label phase II multicenter trial. Key inclusion criteria were presence of a BRAF V600E/K mutation confirmed by both mutation-specific immunohistochemistry (IHC) and next-generation sequencing in more than 50% of MM cells. Exclusion criteria included plasma cell leukemia, CNS involvement, cardiac dysfunction, or a history of retinal vein occlusion. Pts received encorafenib 450mg p.o. daily and binimetinib 45mg p.o. twice daily. Responses were assessed using IMWG 2016 criteria. The primary endpoint was analyzed by testing the one-sided null hypothesis of ORR Results: As of July 2020, 12 pts have been enrolled and were evaluable for safety and 11 pts were evaluable for response as 1 pt had just completed cycle 1 with confirmation of response pending at the time of data lock. Subtypes of MM were IgG in 6 pts, IgA in 2 pts, and Bence Jones in 4 pts. Pts had received a median of 5 prior lines of therapy (range 2-14). All pts had failed previous treatment with both a PI and an IMID and in addition carfilzomib, pomalidomid, and/or anti-CD38 antibodies in 8/4/6 pts, respectively. The study already reached its primary endpoint. The ORR was 82% (lower limit of the 95% CI 56.4%, one-sided exact binomial test, p Adverse events of all grades, assessed to be at least possibly related to a study drug, occurred in 9/12 pts and included blurred vision, macula edema, cramps, arthralgia, diarrhea, skin rash, and decreased left ventricular function. Grade 3 or 4 AEs irrespective of causality occurring in more than 1 pt were reported in 8 pts with anemia, hypertension and thrombocytopenia (3/ 3/ 2 pts, respectively). SAEs were reported in 2 pts, respiratory tract infection in both pts and tooth decay in 1 pt that was not related to the study medication. No deaths occurred while on treatment or within 30 days of the end of treatment. Preliminary results of biomarker assessments reveal new RAS mutations and amplification of the BRAF locus at the time of relapse while on study treatment as potential markers of resistance. Analysis of pharmacodynamic markers by phospho-IHC reveals suppression of BRAF/MEK signaling at cycle 1 / day 28 and restoration of expression at the time of relapse. Conclusion: Targeting activating BRAF mutations in rrMM by combining the BRAF inhibitor, encorafenib, and the MEK inhibitor, binimetinib, induces rapid and deep responses in the majority of pts. No new safety signal has been observed when compared to prior reports on these compounds. The study reached its primary endpoint with fewer pts than expected. The primary efficacy and safety analysis will be presented at the conference. Disclosures Raab: Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Scheid:BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Besemer:GSK: Honoraria; Janssen: Honoraria. Metzler:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; GSK: Consultancy; BMS: Consultancy. Khandanpour:Astra Zeneca: Research Funding; Sanofi: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Celgene: Consultancy. Trautmann-Grill:Janssen: Consultancy; Celgene: Consultancy; Sanofi: Consultancy; Takeda: Consultancy, Honoraria; GSK: Consultancy; BMS: Honoraria. Goldschmidt:Mundipharma GmbH: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck Sharp and Dohme (MSD): Research Funding; Chugai: Honoraria, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; University Hospital Heidelberg, Internal Medicine V and National Center for Tumor Diseases (NCT), Heidelberg, Germany: Current Employment; GlaxoSmithKline (GSK): Honoraria; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Grants and/or provision of Investigational Medicinal Product:, Research Funding; Novartis: Honoraria, Research Funding; Molecular Partners: Research Funding; Incyte: Research Funding; Johns Hopkins University: Other: Grants and/or provision of Investigational Medicinal Product; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; Dietmar-Hopp-Foundation: Other: Grants and/or provision of Investigational Medicinal Product:. Weisel:Abbvie: Consultancy, Honoraria; Adaptive: Consultancy, Honoraria; GlaxoSmithKline: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. OffLabel Disclosure: Encorafenib and Binimetinib in BRAF V600E mutant multiple myeloma

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.

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. Electronic supplementary material The online version of this article (10.1186/s13000-019-0813-5) contains supplementary material, which is available to authorized users.

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

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.

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. Electronic supplementary material The online version of this article (10.1186/s12885-018-5254-0) contains supplementary material, which is available to authorized users.

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.

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.

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.

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.

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: 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.

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

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.

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.

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)