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Pancreatic cancer features elaborate mechanisms of immune evasion. The potential of new immune molecules was explored to restore the antitumor immune response. If these immune molecules are associated with poor survival, specific drugs could take effect. Here, we analyze the expression of VISTA, LAG3, IDO, and TIM3 on tumor-infiltrating lymphocytes (TILs) and its impact on patient survival. We analyzed 153 pancreatic cancer patients from the prospectively managed database of the multicentered PANCALYZE study. Immunohistochemistry on a tissue microarray assessed VISTA, LAG3, IDO, and TIM3 expression of TILs from the patients undergoing primary resection. Complementarily, we analyzed publicly available transcriptomic data ( n = 903). Successful completion of chemotherapy, and lymph node status were independent predictors of survival in the multivariate analysis of the clinicopathologic parameters. Fifteen tumors were exclusively VISTA-positive, thirteen tumors expressed VISTA together with TIM3, and ten tumors expressed VISTA together with IDO. Patients featuring tumors with high numbers of IDO-positive TILs had better patient survival ( p = 0.037). VISTA, LAG3, and TIM3 expression did not correlate with survival. The analysis of publicly available data did not show survival differences. Tumors rarely co-express more than two immune molecules at the same time, and VISTA is most frequently co-expressed. Although IDO generally inhibits T-cell proliferation, a high expression of IDO was associated with improved survival. We expect immune checkpoint inhibitors against VISTA, LAG3, and TIM3 to be inefficient in a clinical application.

Based on gene expression profiles, diffuse large B cell lymphoma (DLBCL) is sub-divided into germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Two of the most common genomic aberrations in ABC-DLBCL are mutations in MYD88 , as well as BCL2 copy number gains. Here, we employ immune phenotyping, RNA-Seq and whole exome sequencing to characterize a Myd88 and Bcl2 -driven mouse model of ABC-DLBCL. We show that this model resembles features of human ABC-DLBCL. We further demonstrate an actionable dependence of our murine ABC-DLBCL model on BCL2. This BCL2 dependence was also detectable in human ABC-DLBCL cell lines. Moreover, human ABC-DLBCLs displayed increased PD-L1 expression, compared to GCB-DLBCL. In vivo experiments in our ABC-DLBCL model showed that combined venetoclax and RMP1-14 significantly increased the overall survival of lymphoma bearing animals, indicating that this combination may be a viable option for selected human ABC-DLBCL cases harboring MYD88 and BCL2 aberrations.

Objectives: The interferon-induced protein with multiple tetratricopeptide repeats 3 (IFIT3) seems to be associated with the prognosis in pancreatic cancer. Here we clarify whether the heterogeneity of IFIT3 expression affects previous IFIT3 analysis., Methods: This retrospective study analyzes pancreatic cancer tissue samples retrieved by surgery from 2 independent patient cohorts. Patients underwent either primary surgery (n = 72) or received neoadjuvant chemotherapy (n = 12). Immunohistochemistry assessed IFIT3 expression and its heterogeneity. Complementarily, we analyzed publicly available transcriptomic data (n = 903)., Results: Of the primarily resected tumors, 16.4% were heterogeneous. Patients with IFIT3-negative tumors did not survive longer compared with patients with IFIT3-positive tumors. An analysis of publicly available data confirmed this result. Patients developing lung metastases had the best prognosis (4.8 years) with significantly lower IFIT3 expression compared with liver metastasis (P = 0.0117). Patients receiving neoadjuvant therapy who are IFIT3 negative had a longer disease-free survival (1.2 vs 0.3 years, P = 0.0081)., Conclusions: Low IFIT3 expression is not associated with longer survival. Divergent results from tissue microarray analyses could be explained with tumor heterogeneity. As a single biomarker, IFIT3 is not suitable for predicting disease prognosis. Recurrence of lung metastases and response to neoadjuvant chemotherapy are associated with low IFIT3 expression.

Tumors of the follicular dendritic cells (FDC-Sarcoma) represent a rare entity with only about 200 cases reported worldwide. The majority (60%) of cases arise primarily in cervical, abdominal or axillar lymph nodes, but extra nodal origin from secondary lymphatic tissue like the tonsils, Waldeyer's ring or MALT is also common (40%). The current report presents a characteristic course of a cervical FDC-Sarcoma, with its challenges in establishing the initial diagnosis and the struggle for therapeutic options. The FDC-Sarcoma presented recurrently for four times. Three different university hospitals in Germany were involved in the patients' treatment. Due to the patients' refusal, no adjuvant therapy was applied. In the end, a neck dissection was performed. The patient was closely followed up and has been recurrence-free for 10 years. This case suggests operative resection in combination with a neck dissection as a curative therapy for FDC-Sarcoma of the head and neck., (Copyright: © Schorn et al.)

Background: Indoleamine 2,3-dioxygenase (IDO) is an interferon-inducible immune checkpoint expressed on tumor-infiltrating lymphocytes (TILs). IDO is known as a poor prognostic marker in esophageal squamous cell cancer, while a positive effect was shown for breast cancer. A comprehensive analysis of IDO expression in a well-defined cohort of esophageal adenocarcinoma (EAC) is missing., Methods: We analyzed 551 patients with EAC using single-protein and multiplex immunohistochemistry as well as mRNA in situ technology for the expression and distribution of IDO on subtypes of TILs (INF- γ mRNA and CD4- and CD8-positive T lymphocytes)., Results: IDO expression on TILs was seen in up to 59.6% of tumors, and expression on tumor cells was seen in 9.2%. We found a strong positive correlation of IDO-positive TILs, CD3-positive T lymphocytes, and INF- γ mRNA-producing TILs in the tumor microenvironment of EACs showing significantly better overall survival (47.7 vs. 22.7 months, p < 0.001) with emphasis on early tumor stages (pT1/2: 142.1 vs. 37.1 months, p < 0.001). In multivariate analysis, IDO is identified as an independent prognostic marker., Conclusions: Our study emphasizes the importance of immunomodulation in EAC marking IDO as a potential biomarker. Beyond this, IDO might indicate a subgroup of EAC with an explicit survival benefit., Competing Interests: All authors declare that they have no conflict of interest., (Copyright © 2020 Heike Loeser et al.)

Purpose: We performed next-generation sequencing (NGS) in the CONKO-001 phase III trial to identify clinically relevant prognostic and predictive mutations and conducted a functional validation in The Cancer Genome Atlas (TCGA) sequencing data., Experimental Design: Patients of the CONKO-001 trial received curatively intended surgery for pancreatic adenocarcinoma (PDAC) followed by adjuvant chemotherapy with gemcitabine (Gem) or observation only (Obs). Tissue samples of 101 patients were evaluated by NGS of 37 genes. Cox proportional hazard models were applied for survival analysis. In addition, functional genomic analyses were performed in an NGS and RNA-sequencing dataset of 146 pancreatic tumors from TCGA., Results: The most common mutations in the CONKO cohort were KRAS (75%), TP53 (60%), SMAD4 (10%), CDKNA2 (9%), as well as SWI/SNF (12%) complex alterations. In untreated patients, TP53 mutations were a negative prognostic factor for disease-free survival (DFS; HR mut vs. WT 2.434, P = 0.005). With respect to gemcitabine treatment, TP53 mutations were a positive predictive factor for gemcitabine efficacy [ TP53 mut: HR for DFS Gem vs. Obs, 0.235 (0.130 - 0.423; P < 0.001); TP53 wt: HR for DFS Gem vs. Obs, 0.794 (0.417 - 1.513; P = 0.483)] with a significant test for interaction ( P = 0.003). In the TCGA dataset, TP53 mutations were associated with shortened DFS., Conclusions: In CONKO-001, the benefit from adjuvant gemcitabine was confined to the TP53 mut patient group. This potentially clinical relevant observation needs to be confirmed in independent prospective studies. The sensitivity of TP53mut PDAC to gemcitabine in CONKO-001 provides a lead for further mechanistic investigations., (©2020 American Association for Cancer Research.)

(1) Background: IL-17A accelerates pancreatic intraepithelial neoplasia (PanIN) progression. In this study, we examined whether IL-17A/IL-17RA promotes pancreatic ductal adenocarcinoma (PDAC) aggressiveness in terms of survival and cancer stem cell modulation. (2) Methods: In vitro, the wound-healing assay, the sphere formation assay, and flow cytometry were applied to assess cancer stem cell features. In vivo, pancreatic tumors were induced in C57BL/6 mice using electroporation with oncogenic plasmids (P53-/- R172H; KrasG12V). Anti-IL-17 antibodies were administered as immunotherapy. We analyzed IL-17A/IL-17RA related survival using publicly available transcriptomic data ( n = 903). (3) Results: IL-17A/IL-17RA expression was not related to survival in PDAC patients. IL-17A neither induces stem cell markers nor increases sphere formation and cell motility in vitro. Blocking the IL-17A/IL-17RA axis in a murine pancreatic cancer model did not improve the survival of mice, but reduced the tumor burden slightly. (4) Conclusions: IL-17A does not promote stem cell expansion in PDAC cell lines. Blocking IL-17A/IL-17RA signaling does not interfere with pancreatic cancer development and progression and may not be considered as a promising monotherapy for PDAC.

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Background: We studied the prognostic effect of CD3-, CD8- and CD103-positive T lymphocytes in a cohort of 165 patients with resected pancreatic ductal adenocarcinomas (PDACs) of the treatment group (adjuvant gemcitabine) and the untreated control group of the CONKO-001 study., Methods: Immunohistochemical stainings on tissue microarrays (TMAs) against CD3, CD8 and CD103 were performed according to standard procedures., Results: A high number of CD8-positive lymphocytes were significantly and independently associated with longer disease-free survival (DFS) and overall survival (OS) in the overall study population. Median DFS/OS were 7.4/18.1 months for patients with a low number of CD8-positive intratumoural lymphocytes (≤42 per 1 mm tissue core) and 12.7/25.2 months for patients with high numbers (>42 per 1-mm tissue core; p = 0.008/0.020; HR 0.62/0.65). The ratio of intraepithelial to total CD103-positive lymphocytes, but not total numbers of CD103-positive lymphocytes or CD103-positive intraepithelial lymphocytes, was associated with significantly improved DFS and OS in the overall study population (p = 0.022/0.009). Median DFS/OS was 5.9/15.7 for patients with a ratio of intraepithelial to total CD103-positive intratumoural lymphocytes higher than 0.3 and 11.6/24.7 for patients with a lower ratio., Conclusion: T-lymphocyte subpopulations might be prognostic in resectable PDAC but need standardization and verification by further studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

There is evidence from studies on fibrotic diseases that a TH2 polarized immune response, characterized by the production of cytokines like IL-4, IL-5, and IL-13 is important for fibrosis development. Data concerning the role of TH2 polarized immune response for fibrogenesis in lymphomas are scarce. Using immunohistochemistry, we investigated paraffin embedded tissue specimens of 58 follicular lymphomas (FL) grade 1/2 with and without fibrosis. In FL with fibrosis, we detected more interfollicular GATA-3 positive lymphocytes and a shift towards a TH2 immune response. In areas of fibrosis, which were enriched with myofibroblasts, we observed a polarization of macrophages towards a M2 phenotype with expression of CD163. Our results hold some evidence that a polarization of the adaptive immune system towards a TH2 immune response in the tumor microenvironment is associated with increased fibrosis, which may indicate its functional relevance for either development or maintenance of fibrosis in FL.

During the preoperative diagnostics of an 80-year-old male patient prior to a planned endarterectomy, an unclear space-occupying lesion was detected in the right nasopharyngeal cavity. It proved to be a dense soft tissue space-occupying lesion of the right maxillary sinus. The histological investigations revealed a partially necrotically decomposed malignant tumor below normal respiratory mucosa free from dysplasia. This case demonstrates the difficulties in differential diagnostics, particularly involving (aberrant) expression of cytokeratin.

Carboxylesterase 2 (CES-2) is instrumental for conversion of ester-containing prodrugs in cancer treatment. CES-2 expression was analyzed by immunohistochemistry in colorectal cancer (CRC) compared to colonic inflammation as well as in liver and peripheral blood. In CRC, tumor grades showed no correlation with levels of CES-2 expression, which was heterogeneous within these tumors. Cellular infiltrates in the immediate tumor vicinity expressed high levels of CES-2. Thus, tissue adjacent to the tumor was a substantial source of CES-2 with high expression in plasma cells. CES-2(high) plasma cells were abundantly found in the colon of patients with inflammatory bowel disease. CES-2 expression is strong in hepatocytes of normal livers, while CES-2 expression in peripheral blood mononuclear cells of healthy donors was overall low at protein and mRNA levels. In summary, the conversion of ester-containing prodrugs by CES-2 is mainly to occur in the periphery, during liver passage and in the colon after enterohepatic recirculation. We here demonstrated plasma cells as strong producers of CES-2. Further studies should elucidate the role of CES-2(+) plasma cells in intestinal inflammation and cancer., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In pancreatic cancer there is a need for prognostic risk stratification and subsequent therapy strategies. Molecular analysis has shown in different cancers that variation in clinical behavior can be associated with specific alterations. The cell cycle regulators p16 and p53 belong to the most often alterated genes in pancreatic ductal adenocarcinoma (PDAC). We analyzed protein expression of p16, p53 and Ki67 by immunohistochemistry in 162 tumours of the CONKO-001 trial that investigated the role of adjuvant gemcitabine in pancreatic cancer patients. We could show that high proliferation of tumours and strong and consistent nuclear p53 expression by tumour cells is associated with a worse disease-free survival and overall survival in the overall study population. However, stratified analysis according to treatment arm revealed that the effect of deregulated p53 expression and high Ki67 expression was restricted to the disease free survival of patients treated with adjuvant gemcitabine. In multivariable survival analysis, p53 did not retain its prognostic status. Our study supports the important role of p53 and Ki67 expression in PDAC. They provide prognostic information in patients with adjuvant gemcitabine treatment and may contribute to treatment decision. However, these results should be validated in further studies., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Introduction: In a patient, it is usually not macroscopically possible to estimate the non-viable volume induced by radiofrequency ablation (RFA) after the procedure. The purpose of this study was to use an ex vivo bovine liver model to perform magnetic resonance (MR) volumetry of the visible tissue signal change induced by RFA and to correlate the MR measurement with the actual macroscopic volume measured in the dissected specimens., Materials and Methods: Sixty-four liver specimens cut from 16 bovine livers were ablated under constant simulated, close physiological conditions with target volumes set to 14.14 ml (3-cm lesion) and 65.45 ml (5-cm lesion). Four commercially available radiofrequency (RF) systems were tested (n=16 for each system; n=8 for 3 cm and n=8 for 5 cm). A T1-weighted turbo spin echo (TSE) sequence with inversion recovery and a proton-density (PD)-weighted TSE sequence were acquired in a 1.0-T open magnetic resonance imaging (MRI) system. After manual dissection, actual macroscopic ablation diameters were measured and volumes calculated. MR volumetry was performed using a semiautomatic software tool. To validate the correctness and feasibility of the volume formula in macroscopic measurements, MR multiplanar reformation diameter measurements with subsequent volume calculation and semiautomatic MR volumes were correlated., Results: Semiautomatic MR volumetry yielded smaller volumes than manual measurement after dissection, irrespective of RF system used, target lesion size, and MR sequence. For the 3-cm lesion, only 43.3% (T1) and 41.5% (PD) of the entire necrosis are detectable. For the 5-cm lesion, only 40.8% (T1) and 37.2% (PD) are visualized in MRI directly after intervention. The correlation between semiautomatic MR volumes and calculated MR volumes was 0.888 for the T1-weighted sequence and 0.875 for the PD sequence., Conclusion: After correlation of semiautomatic MR volumes and calculated MR volumes, it seems reasonable to use the respective volume formula for macroscopic volume calculation. Hyperacute MRI after ex vivo intervention may result in the underestimation of the real expansion of the produced necrosis zone. This must be kept in mind when using MRI for validating ablation success directly after RFA. One reason for the discrepancy between macroscopic and MRI appearance immediately after RFA may be that the transitional zone shows no or only partially visible MR signal change.

Background/aim: 3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates the cellular effects of various growth factors. Increased PDK1 expression is present in various cancers, suggesting that PDK1 may be a critical oncogene in cancer progression. However, only limited data exist on PDK1 expression in ovarian serous cancer., Materials and Methods: We used tissue microarrays to analyze PDK1 expression in 253 primary ovarian serous carcinoma samples., Results: A statistically significant negative correlation between PDK1 expression and tumor grade was found. In the high-grade group of ovarian serous carcinomas (n=189), there was a statistically significant difference in overall survival between cases with positive and negative PDK1 expression (p=0.035); positive cases showed longer overall survival. Multivariate analysis confirmed that slight PDK1 expression was an independent indicator for prolonged overall survival (HR=0.51, 95% CI=0.28-0.92, p=0.025)., Conclusion: PDK1 appears to be a prognostic marker and a possible therapeutic target in ovarian serous carcinoma., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Primary high-grade serous peritoneal carcinoma (PPSC) is a rare malignancy with an ambiguous pathogenesis., Case Report: We report on a 51-year-old woman presenting with a routine smear test cytology suspicious of adenocarcinoma. She underwent hysteroscopy, laparsocopy with multiple biopsies and bilateral salpingoophorectomy. She was diagnosed with a serous tubal intraepithelial carcinoma in situ (STIC) in the right fallopian tube. Subsequently, she underwent radical surgery and was diagnosed with peritoneal high-grade serous carcinoma. Interestingly, both ovaries remained histologically tumour-free., Discussion: High-grade serous carcinomas that arise on the peritoneum with tumour-free ovaries are rare. The findings in this case, coupled with current evidence, strongly suggest a precursor lesion in the fallopian tube (STIC lesions). The clinical implications of this theory reside in the potential for improving early detection strategies. Nonetheless, more data on precursor lesions in the fallopian tubes and their transformation to serous carcinoma are required to plan for future screening methods.

The presence of tumor cells with monocytoid morphology in marginal zone lymphoma (MZL) has been described previously. Reactive monocytoid B cells bear a distinct immunophenotype and typically express T-bet, which clearly distinguishes them from nodal marginal zone B cells. The latter are positive for CD27 and negative for T-bet. We analyzed 74 MZLs for the expression of T-bet and correlated these results with the presence of monocytoid morphology. Expression of T-bet correlated with the presence of monocytoid morphology in MZLs. In analogy to reactive monocytoid B lymphocytes, we also found a significantly lower relative amount of intratumoral T lymphocytes in extranodal MZL with monocytoid morphology.

Formation of immune synapses (IS) between T cells and APC requires multiple rearrangements in the actin cytoskeleton and selective receptor accumulation in supramolecular activation clusters (SMAC). The inner cluster (central SMAC) contains the TCR/CD3 complex. The outer cluster (peripheral SMAC) contains the integrin LFA-1 and Talin. Molecular mechanisms selectively stabilizing receptors in the IS remained largely unknown. Here, we demonstrate that sustained LFA-1 clustering in the IS is a consequence of the combined activities of the actin-bundling protein L-plastin (LPL) and calmodulin. Thus, upon antigen-recognition of T cells, LPL accumulated predominantly in the peripheral SMAC. siRNA-mediated knock-down of LPL led to a failure of LFA-1 and Talin redistribution - however, not TCR/CD3 relocalization - into the IS. As a result of this LPL knock-down, the T-cell/APC interface became smaller over time and T-cell proliferation was inhibited. Importantly, binding of calmodulin to LPL was required for the maintenance of LPL in the IS and consequently inhibition of calmodulin also prevented stable accumulation of LFA-1 and Talin, but not CD3, in the IS.

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation-induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin-binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L-plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT). Mass spectrometry and site-directed mutagenesis revealed that Ser5 represents the only phospho-acceptor site of L-plastin in PBT. Wild-type L-plastin (wt-LPL) and a non-phosphorylatable 5A-L-plastin (5A-LPL) equally relocalized to the immunological synapse between PBT and APC. Yet importantly, cells expressing 5A-LPL showed a significantly lower expression of the T cell activation molecules CD25 and CD69 on the cell surface than cells expressing wt-LPL. This effect is due to a failure in the transport of CD25 and CD69 to the cell surface since the total amount of these proteins within the cells remained unchanged. In conclusion, phosphorylation of the actin bundling protein L-plastin represents a so-far-unknown mechanism by which costimulation controls the transport of activation receptors to the T cell surface.