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Abstract Background Biliary tract cancer (BTC) is one of the most aggressive malignancies and surgery represents the only curative treatment approach. However, even in patients with complete tumor resection 5-year survival rates are below 30%. So far, prognostic markers to assess the outcome of these patients are lacking. We therefore evaluated bone mineral density (BMD) as a prognostic tool in patients receiving surgery for BTC. methods 76 BTC patients undergoing tumor resection in our clinic (Duesseldorf cohort) as well as an external validation cohort of 34 BTC patients (Cologne cohort) were included. BMD was analyzed at the first lumbar vertebra, using routine CT scans which has been proven comparable to DXA. Results Median overall survival (OS) of the Duesseldorf cohort after surgery was 527 days, one- and five-year survival probabilities were 62 and 18%. Patients with BMD above 156.5 HU had significantly improved OS (1435 days vs. 459 days; p = 0.002). The prognostic value for BMD was confirmed using Cox-regression analysis, as well as an external validation cohort. In subgroup analysis the prognostic effect of BMD was only present in female patients, suggesting sex specific differences. Conclusion BMD is a valuable, easily accessible and independent prognostic marker in patients receiving liver surgery for BTC.

Abstract In esophageal adenocarcinoma, the presence of lymph node metastases predicts patients' survival even after curative resection. Currently, there is no highly accurate marker for detecting the presence of lymph node metastasis. The SEMA3F/NRP2 axis was initially characterized in axon guidance and recent evidence has revealed its significant involvement in lymphangiogenesis, angiogenesis, and carcinogenesis. Hence, the objective of this study was to elucidate the roles of SEMA3F and its receptor NRP2 in esophageal adenocarcinoma. We conducted an immunohistochemical evaluation of SEMA3F and NRP2 protein expression in 776 patients with esophageal adenocarcinoma who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. Total and positive cancer cell counts were digitally analyzed using QuPath and verified by experienced pathologists to ensure accuracy. Positive expression was determined as a cell percentage exceeding the 50th percentile threshold. In our cohort, patients exhibiting SEMA3F positive expression experience significantly lower pT- and pN-stages. In contrast, positive NRP2 expression is associated with the presence of lymph node metastases. Survival analyses showed that the expression status of NRP2 had no impact on patient survival. However, SEMA3F positivity was associated with a favorable patient survival outcome (median OS: 38.9 vs. 26.5 months). Furthermore, SEMA3F could be confirmed as an independent factor for better patient survival in patients with early tumor stage (pT1N0-3: HR = 0.505, p = 0.014, pT1-4N0: HR = 0.664, p = 0.024, pT1N0: HR = 0.483, p = 0.040). In summary, SEMA3F emerges as an independent predictor for a favorable prognosis in patients with early-stage esophageal adenocarcinoma. Additionally, NRP2 expression is linked to a higher risk of lymph node metastases occurrence. We hypothesize that low SEMA3F expression could identify patients with early-stage tumors who might benefit from more aggressive treatment options or intensified follow-up. Furthermore, SEMA3F and its associated pathways should be explored as potential tumor-suppressing agents.

Abstract Esophageal adenocarcinoma (EAC) is one of the deadliest tumor entities worldwide, with a 5-year survival rate of less than 25%. Unlike other tumor entities, personalized therapy options are rare, partly due to the lack of knowledge about specific subgroups. In this publication, we demonstrate a subgroup of patients with EAC in a large screening cohort of 826 patients, characterized by specific morphological and immunohistochemical features. This subgroup represents approximately 0.7% (6/826) of the total cohort. Morphological features of this subgroup show a striking clear cytoplasm of the tumour cells and the parallel existence of rare growth patterns like yolk sac-like differentiation and enteroblastic differentiation. Immunohistochemistry reveals expression of the fetal gut cell-like proteins Sal-like protein 4 (SALL4), claudin-6, and glypican 3. Interestingly, we find a correlation with alterations of SWI/SNF-complex associated genes, which are supposed to serve as tumor suppressor genes in various tumour entities. Our results suggest a possible implication of rare tumour subtypes in the WHO classification for EACs according to the classification for gastric cancer. Furthermore, claudin-6 positive tumors have shown promising efficacy of CAR T cell therapy in the recently published BNT-211-01 trial (NCT04503278). This represents a personalized therapeutic option for this tumor subtype.

Abstract Pleomorphic dermal sarcomas are infrequent neoplastic skin tumors, manifesting in regions of the skin exposed to ultraviolet radiation. Diagnosing the entity can be challenging and therapeutic options are limited. We analyzed 20 samples of normal healthy skin tissue (SNT), 27 malignant melanomas (MM), 20 cutaneous squamous cell carcinomas (cSCC), and 24 pleomorphic dermal sarcomas (PDS) using mass spectrometry. We explored a potential cell of origin in PDS and validated our findings using publicly available single-cell sequencing data. By correlating tumor purity (TP), inferred by both RNA- and DNA-sequencing, to protein abundance, we found that fibroblasts shared most of the proteins correlating to TP. This observation could also be made using publicly available SNT single cell sequencing data. Moreover, we studied relevant pathways of receptor/ligand (R/L) interactions. Analysis of R/L interactions revealed distinct pathways in cSCC, MM and PDS, with a prominent role of PDGFRB-PDGFD R/L interactions and upregulation of PI3K/AKT signaling pathway. By studying differentially expressed proteins between cSCC and PDS, markers such as MAP1B could differentiate between these two entities. To this end, we studied proteins associated with immunosuppression in PDS, uncovering that immunologically cold PDS cases shared a “negative regulation of interferon-gamma signaling” according to overrepresentation analysis.

Summary: Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors. In this study, we develop a clinically useful computational pathology platform for NSCLC that can be a foundation for multiple downstream applications and provide immediate value for patient care optimization and individualization. We train the primary multi-class tissue segmentation algorithm on a substantial, high-quality, manually annotated dataset of whole-slide images with lung adenocarcinoma and squamous cell carcinomas. We investigate two downstream applications. NSCLC subtyping algorithm is trained and validated using a large, multi-institutional (n = 6), multi-scanner (n = 5), international cohort of NSCLC cases (slides/patients 4,097/1,527). Moreover, we develop four AI-derived, fully explainable, quantitative, prognostic parameters (based on tertiary lymphoid structure and necrosis assessment) and validate them for different clinical endpoints. The computational platform enables the high-precision, quantitative analysis of H&E-stained slides. The developed prognostic parameters facilitate robust and independent risk stratification of patients with NSCLC.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis, wherefore targeted therapies have experienced increasing interest. Zolbetuximab is a novel targeted therapy under investigation in patients with PDAC and targets Claudin 18.2 (CLDN18.2), which is a component of tight junctions and is of significance in various solid tumors. As its role in PDAC is not definitively elucidated, this study aims to clarify the significance of CLDN18.2 expression in PDAC in a real-world setting. Methods: All patients (n = 309) were recruited at one of the PANCALYZE study centers and received pancreatic resection with curative intention. Paraffin samples were analyzed using an antibody against CLDN18.2, which is known to be comparable to the antibody used by the SPOTLIGHT and GLOW studies. Results: 94 PDACs are positive for CLDN18.2 (30.4 %). Positive CLDN 18.2 expression was associated with significantly better cancer differentiation (p < 0.001). Patients with positive CLDN18.2 expression showed significantly better overall survival when compared to patients with negative expression (median OS: 30 versus 18 months, p = 0.003). Additionally, in multivariable analyses, CLDN18.2 expression was identified as an independent factor for better survival in patients with PDAC (HR = 0.686, 95 %CI = 0.492–0.956, p = 0.026). Conclusion: Significant improvement in survival could be demonstrated by adding Zolbetuximab to known chemotherapy regimes in patients with gastro-esophageal junction adenocarcinoma with at least 75 % CLDN18.2 positive cancer cells. Our findings demonstrate, that 30.4 % of the included patients with PDAC would potentially be eligible for therapy with Zolbetuximab in a real-world patient cohort. Results of trials targeting Claudin 18.2 are pending in patients with PDAC.

Abstract Background Colorectal cancer (CRC) frequently involves mutations in the KRAS gene, impacting therapeutic strategies and prognosis. The occurrence of KRAS mutations typically precludes the presence of RET fusions, with current medical literature suggesting a mutual exclusivity between these two genetic alterations. We present a unique case that challenges this notion. Case Presentation An 85-year-old female with metastatic CRC was found to have a combination of genetic anomalies that is to the best of our knowledge not yet described in the medical literature: a KRAS p.G12C mutation, associated with oncogenesis and treatment resistance, and an ANK3::RET fusion, an infrequent but targetable mutation in CRC. This molecular profile was uncovered through comprehensive genomic sequencing after the patient experienced metachronous tumor dissemination. The presence of both genetic events complicates the treatment approach. Conclusions The identification of both a KRAS p.G12C mutation and an ANK3::RET fusion in the same CRC patient adds a new layer to the oncogenic landscape and treatment considerations for CRC. It highlights the intricate decision-making required in the era of precision medicine, where targeted therapies must be carefully chosen and potentially combined to combat complex genetic profiles. The case emphasizes the urgency of investigating the clinical effects of concurrent or sequential use of KRAS p.G12C and RET inhibitors to inform future therapeutic guidelines and improve patient outcomes in similar cases.

AIM: In this study, we use Artificial Intelligence (AI), including Machine (ML) and Deep Learning (DL), to predict the long-term survival of resectable esophageal cancer (EC) patients in a high-volume surgical center. Our objective is to evaluate the predictive efficacy of AI methods for survival prognosis across different time points of oncological treatment. This involves comparing models trained with clinical data, integrating either Tumor, Node, Metastasis (TNM) classification or tumor biomarker analysis, for long-term survival predictions. METHODS: In this retrospective study, 1002 patients diagnosed with EC between 1996 and 2021 were analyzed. The original dataset comprised 55 pre- and postoperative patient characteristics and 55 immunohistochemically evaluated biomarkers following surgical intervention. To predict the five-year survival status, four AI methods (Random Forest RF, XG Boost XG, Artificial Neural Network ANN, TabNet TN) and Logistic Regression (LR) were employed. The models were trained using three predefined subsets of the training dataset as follows: (I) the baseline dataset (BL) consisting of pre-, intra-, and postoperative data, including the TNM but excluding tumor biomarkers, (II) clinical data accessible at the time of the initial diagnostic workup (primary staging dataset, PS), and (III) the PS dataset including tumor biomarkers from tissue microarrays (PS + biomarkers), excluding TNM status. We used permutation feature importance for feature selection to identify only important variables for AI-driven reduced datasets and subsequent model retraining. RESULTS: Model training on the BL dataset demonstrated similar predictive performances for all models (Accuracy, ACC: 0.73/0.74/0.76/0.75/0.73; AUC: 0.78/0.82/0.83/0.80/0.79 RF/XG/ANN/TN/LR, respectively). The predictive performance and generalizability declined when the models were trained with the PS dataset. Surprisingly, the inclusion of biomarkers in the PS dataset for model training led to improved predictions (PS dataset vs. PS dataset + biomarkers; ACC: 0.70 vs. 0.77/0.73 vs. 0.79/0.71 vs. 0.75/0.69 vs. 0.72/0.63 vs. 0.66; AUC: 0.77 vs. 0.83/0.80 vs. 0.85/0.76 vs. 0.86/0.70 vs. 0.76/0.70 vs. 0.69 RF/XG/ANN/TN/LR, respectively). The AI models outperformed LR when trained with the PS datasets. The important features shared after AI-driven feature selection in all models trained with the BL dataset included histopathological lymph node status (pN), histopathological tumor size (pT), clinical tumor size (cT), age at the time of surgery, and postoperative tracheostomy. Following training with the PS dataset with biomarkers, the important predictive features included patient age at the time of surgery, TP-53 gene mutation, Mesothelin expression, thymidine phosphorylase (TYMP) expression, NANOG homebox protein expression, and indoleamine 2,3-dioxygenase (IDO) expressed on tumor-infiltrating lymphocytes, as well as tumor-infiltrating Mast- and Natural killer cells. CONCLUSION: Different AI methods similarly predict the long-term survival status of patients with EC and outperform LR, the state-of-the-art classification model. Survival status can be predicted with similar predictive performance with patient data at an early stage of treatment when utilizing additional biomarker analysis. This suggests that individual survival predictions can be made early in cancer treatment by utilizing biomarkers, reducing the necessity for the pathological TNM status post-surgery. This study identifies important features for survival predictions that vary depending on the timing of oncological treatment.

Abstract Background Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease. Methods We describe an integrated whole‐exome sequencing and transcriptomic study of 37 iCCAs patients in Germany. Results We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke‐associated Asian iCCAs. Conclusions By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

Background Oropharyngeal squamous cell carcinoma (OPSCC) is the only subgroup of head neck cancer that presents with an increased incidence. Gender-specific studies in other cancer entities have revealed differences in treatment response and prognosis. However, only limited data in OPSCC according to gender and human papillomavirus (HPV) status exist. Therefore, we aimed to investigate sex-specific differences in OPSCC and how these may be distributed in relation to HPV and other risk factors. Methods This retrospective, bicentric study included 1629 patients with OPSCC diagnosed between 1992 and 2020. We formed subgroups based on TNM status, American Joint Cancer Committee 8th edition (AJCC8), HPV status, treatment modality (surgery (± radio(chemo)therapy (RCT) vs. definitive RCT) and patient-related risk factors and investigated gender differences and their impact on patients survival via descriptive-,uni- and multivariate analysis. Results With the exception of alcohol abuse, no significant differences were found in risk factors between men and women. Females presented with better OS than males in the subgroup T1-2, N + , independent of risk factors (p = 0.008). Males demonstrated significant stratification through all AJCC8 stages (all p

Background The individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides.Methods HLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses.Results We show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele.Conclusion The high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

Abstract Background Despite multiple therapeutic modalities, the overall survival of patients with gastric adenocarcinoma remains poor, especially for advanced tumor stages. Although the tyrosine kinase MerTK has shown therapeutic relevance in several tumor entities, its potential effects in gastric adenocarcinoma have not yet been sufficiently characterized. Methods MerTK expression and its influence on patient survival were evaluated by immunohistochemistry in a cohort of 140 patients with gastric adenocarcinoma. CRISPR/Cas9 knockout and siRNA knockdown of MerTK in the gastric cancer cell lines SNU1, SNU5, and MKN45 was used to analyze protein expression, growth, migration, and invasion properties in vitro and in a murine xenograft model. MerTK was pharmacologically targeted with the small molecule inhibitor UNC2025 in vitro and in vivo. Results In patients, high MerTK expression was associated with decreased overall survival (OS) and lymph node metastasis especially in patients without neoadjuvant therapy (p

Abstract Guidelines regulate how many (tumour-bearing) tissue particles should be sampled during gastric cancer biopsy to obtain representative results in predictive biomarker testing. Little is known about how well these guidelines are applied, how the number of tissue particles correlates with the actual tumour-infiltrated area and how many absolute tumour cells are captured. The study included endoscopic biopsies of untreated carcinomas of the upper gastrointestinal (GI)-tract during the 2016–2020 review period. Archival (H&E)-stained histological sections were digitised and the tumour areas were manually annotated. The tumour-bearing tissue area and absolute carcinoma cell count per case were determined by image analysis and compared with a reference primary surgical specimen. Biopsies from 253 patients were analysed. The following mean values were determined: (a) tumour tissue particle number: 6.5 (range: 1–25, standard deviation (SD) = 3.33), (b) number of tumour-bearing tissue particles: 4.7 (range: 1–20, SD = 2.80), (c) tumour-infiltrated area: 7.5 mm2 (range: 0.18–59.46 mm2, SD = 6.67 mm2), (d) absolute tumour cell count: 13,492 (range: 193–92,834, SD = 14,185) and (e) tumour cell count in a primary surgical specimen (tumour size: 6.7 cm): 105,200,176. The guideline-recommended tissue particle count of 10 was not achieved in 208 patients (82.2%) and the required tumour-bearing tissue particle count of 5 was not achieved in 133 patients (52.6%). Tissue particle count, tumour-infiltrated area and tumour cell count were only weakly correlated. Most cases featured an infiltrated area ≥ 4.5 mm2 (156, 61.7%). Cases with more tissue particles showed only a moderate increase in infiltrated area and tumour cells compared to cases with fewer particles. Biopsies are often used to determine predictive biomarkers, particularly Her2/neu and PD-L1. Diagnostic standards to ensure representative material have been suggested in guidelines to reduce false-negative predictions. However, the real-world practice seems to substantially deviate from recommended standards. To the best of our knowledge, this is the first systematic study describing the relationships between endoscopic tissue fragment number, actual infiltrated tumour area and carcinoma cell number. The data question the tissue particle number as a quality assessment parameter. We advocate histopathological reports indicating on which basis statements on therapy-relevant biomarkers were made. Digital pathology has the potential to objectively quantify the tissue for documentation, quality assessment and future clinical studies.

Abstract Esophageal adenocarcinoma exhibits one of the highest mortality rates among all cancer entities. Multimodal therapy strategies have improved patients’ survival significantly. However, patients in early stages are currently limited to receiving only local therapies, even though some patients within this group showcase short survival periods. Until now, there has been no widely established clinically used biomarker to detect these high-risk patients. Telomerase reverse transcriptase (TERT), a gene encoding a crucial subunit of the telomerase enzyme, plays a significant role in establishing cancer cell immortality and is under suspicion for its potential contribution to tumor progression. Therefore, we aimed to evaluate the clinical relevance of the TERT amplification status. We included 643 patients with esophageal adenocarcinoma, who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. The TERT amplification status was characterized using fluorescence in situ hybridization. Clinicopathological values and patients’ overall survival were compared between patients with and without TERT amplification. Further sub-cohort analyses were conducted for patients with pT1N0-3 tumor stage. Eighty-One patients (12.6%) exhibited TERT amplification. Patients with amplified TERT showed significantly worse overall survival (median OS: 22.6 vs. 36.8 months, p = 0.009). Interestingly, TERT amplification could be characterized as an independent risk factor for worse overall survival in multivariate analysis in patients with pT1N0-3 tumor stage (HR = 2.440, 95% CI 1.095–5.440, p = 0.029). In this study, we describe the TERT amplification status as an independent risk factor for worse survival in patients diagnosed with esophageal adenocarcinoma at pT1N0-3 tumor stage, encompassing cases involving tumor infiltration of the lamina propria, muscularis mucosae, and/or submucosa. Based on our findings, we put forth the proposition that evaluating the TERT amplification status may serve as a valuable tool in identifying a specific subgroup of patients, namely those with TERT amplification and pT1N0-3 tumor-stage esophageal adenocarcinoma. The patients of this subgroup could potentially benefit from enhanced follow-up protocols, more aggressive treatment approaches, or possible targeted TERT inhibition therapies, all aimed at improving their overall clinical outcomes.

Abstract Pathologic examination of prostate biopsies is time consuming due to the large number of slides per case. In this retrospective study, we validate a deep learning-based classifier for prostate cancer (PCA) detection and Gleason grading (AI tool) in biopsy samples. Five external cohorts of patients with multifocal prostate biopsy were analyzed from high-volume pathology institutes. A total of 5922 H&E sections representing 7473 biopsy cores from 423 patient cases (digitized using three scanners) were assessed concerning tumor detection. Two tumor-bearing datasets (core n = 227 and 159) were graded by an international group of pathologists including expert urologic pathologists (n = 11) to validate the Gleason grading classifier. The sensitivity, specificity, and NPV for the detection of tumor-bearing biopsies was in a range of 0.971–1.000, 0.875–0.976, and 0.988–1.000, respectively, across the different test cohorts. In several biopsy slides tumor tissue was correctly detected by the AI tool that was initially missed by pathologists. Most false positive misclassifications represented lesions suspicious for carcinoma or cancer mimickers. The quadratically weighted kappa levels for Gleason grading agreement for single pathologists was 0.62–0.80 (0.77 for AI tool) and 0.64–0.76 (0.72 for AI tool) for the two grading datasets, respectively. In cases where consensus for grading was reached among pathologists, kappa levels for AI tool were 0.903 and 0.855. The PCA detection classifier showed high accuracy for PCA detection in biopsy cases during external validation, independent of the institute and scanner used. High levels of agreement for Gleason grading were indistinguishable between experienced genitourinary pathologists and the AI tool.

Abstract Background The prognosis of esophageal adenocarcinoma (EAC) and gastric adenocarcinoma (GAC) remains poor, and new therapeutic approaches are urgently needed. Claudin 6 (CLDN6) is an oncofetal antigen that is largely absent in healthy tissues and upregulated in several cancers, making it a promising therapeutical target. In this study, the expression of CLDN6 was assessed in an large Caucasian EAC and GAC cohort. Methods RNA-Seq data from 89 EACs and 371 GACs were obtained from The Cancer Genome Atlas project and EAC/GAC cases were stratified by CLDN6 mRNA expression based on a survival-associated cutoff. For groups with CLDN6 expression above or below this cutoff, differential gene expression analyses were performed using DESeq, and dysregulated biological pathways were identified using the Enrichr tool. Additionally, CLDN6 protein expression was assessed in more than 800 EACs and almost 600 GACs using a CLDN6-specific immunohistochemical antibody (clone 58-4B-2) that is currently used in Phase I/II trials to identify patients with CLDN6-positive tumors (NCT05262530; NCT04503278). The expression of CLDN6 was also correlated with histopathological parameters and overall survival (OS). Results EACs and GACs with high CLDN6 mRNA levels displayed an overexpression of pathways regulating the cell cycle, DNA replication, and receptor / extracellular matrix interactions. CLDN6 protein expression was associated with shorter OS in EAC and GAC, both in treatment-naïve subgroups and cohorts receiving neoadjuvant therapy. In multivariate analysis, CLDN6 protein expression was an independent adverse prognostic factor in EAC associated with a shorter OS (HR: 1.75; p = 0.01) and GAC (HR: 2.74; p = 0.028). Conclusions High expression of CLDN6 mRNA is associated with the dysregulation of distinct biological pathways regulating cell growth, proliferation, and cell–matrix interactions. Clinically, the expression of CLDN6 protein is a valuable adverse prognostic marker in EAC and GAC.

Abstract Human Papilloma Virus (HPV)-associated oropharyngeal squamous cell cancer (OPSCC) represents an OPSCC subgroup with an overall good prognosis with a rising incidence in Western countries. Multiple lines of evidence suggest that HPV-associated tumors are not a homogeneous tumor entity, underlining the need for accurate prognostic biomarkers. In this retrospective, multi-institutional study involving 906 patients from four centers and one database, we developed a deep learning algorithm (OPSCCnet), to analyze standard H&E stains for the calculation of a patient-level score associated with prognosis, comparing it to combined HPV-DNA and p16-status. When comparing OPSCCnet to HPV-status, the algorithm showed a good overall performance with a mean area under the receiver operator curve (AUROC) = 0.83 (95% CI = 0.77-0.9) for the test cohort (n = 639), which could be increased to AUROC = 0.88 by filtering cases using a fixed threshold on the variance of the probability of the HPV-positive class - a potential surrogate marker of HPV-heterogeneity. OPSCCnet could be used as a screening tool, outperforming gold standard HPV testing (OPSCCnet: five-year survival rate: 96% [95% CI = 90–100%]; HPV testing: five-year survival rate: 80% [95% CI = 71–90%]). This could be confirmed using a multivariate analysis of a three-tier threshold (OPSCCnet: high HR = 0.15 [95% CI = 0.05–0.44], intermediate HR = 0.58 [95% CI = 0.34–0.98] p = 0.043, Cox proportional hazards model, n = 211; HPV testing: HR = 0.29 [95% CI = 0.15–0.54] p

Abstract Background Patients diagnosed with esophageal cancer demonstrate a low overall survival even despite the established multimodal therapy as the current standard of care. Therefore, further biomarkers for patients with high-risk and additional therapy options are needed. NANOG is a transcription factor, which can be found in stem cells and is known to support tumorigenesis. Methods Six hundred sixty patients with esophageal adenocarcinoma, who were operated at the University of Cologne with a curative intent, were included. Immunohistochemical stainings for NANOG were performed. The study population was divided into NANOG-positive and -negative subgroups. Results Positive NANOG expression correlates significantly with worse overall survival (p = 0.002) and could be confirmed as an independent risk factor for worse patient survival in multivariate analysis (HR = 1.40, 95%CI = 1.09–1.80, p = 0.006). This effect could be detected in the subgroup of primarily operated patients, but not in patients after neoadjuvant therapy. Conclusions We describe a NANOG-positive subgroup of patients with esophageal cancer, who exhibit worse overall survival in a large patient cohort. This discovery suggests the potential use of NANOG as a biomarker for both intensified therapy and stricter follow-up regimes. Additionally, NANOG-positive stem cell-like cancer cells could be used as a new antitumoral treatment target if validated in mechanistic and clinical studies.

Background: The incidence of oropharyngeal squamous cell carcinoma (OPSCC) is rapidly increasing in high income countries due to its association with persistent high-risk human papilloma virus (HPV) infection. Recent scientific advances have highlighted the importance of the tumor microenvironment in OPSCC. In this study, including 216 OPSCC patients, we analyze the composition of four established markers of cancer associated fibroblasts (CAFs) in the context of intratumoral CD8 T-cell infiltration.Methods: Immunohistochemical staining for fibroblast activation protein (FAP), platelet-derived growth factor receptor beta (PDGFRb), periostin, alpha smooth muscle actin (α-SMA) and CD8 were analyzed digitally and their association with survival, tumor- and patient characteristics was assessed.Results: Co-expression of CAF markers was frequent but not associated with HPV status. FAPhigh and PDGFRbhigh expression were associated with increased CD8 T-cell infiltration. Low expression of PDGFRb improved patient survival in female patients but not in male patients. We identified PDGFRblow periostinlow α-SMAlow status as an independent predictor of improved survival (hazard ratio 0.377, p = 0.006).Conclusion: These findings elucidate the co-expression of four established CAF markers in OPSCC and underscore their association with T-cell infiltration and patient survival. Future analyses of CAF subgroups in OPSCC may enable the development of individualized therapies.

BackgroundStaging, especially clinical lymph node staging in esophageal adenocarcinoma has only moderate sensitivity and specificity. Therefore, we evaluated combined molecular markers to predict prognosis.Patients and methods890 tumor tissue samples were obtained from patients who underwent surgery for esophageal adenocarcinoma with curative intent. These were stained by tissue micro array for 48 markers which are associated with tumorigenesis and correlated with clinical data (TNM-staging, overall survival) by multivariate Cox regression.ResultsTwo markers (preserved Y chromosome and high grade of (CD3+) T-cell infiltration) were found to be significantly and independently associated with better overall survival. We formed a score (called CY score) from the two markers. The more markers are positive and thus the higher the score (ranging from 0 to 2), the better the overall survival, independently of UICC. Moreover, we developed a combination score of the UICC and CY score based on cluster analysis. Patients with a UICC stage of III with the presence of both traits (CY=2) can be assigned to a better prognosis group (group II), whereas patients with a UICC stage of I without both traits (CY=0) must be assigned to a worse prognosis group (group II). Therefore, patients in stage I with adverse molecular signature might benefit of multimodal therapy.ConclusionIn summary, the CY score adds prognostic information to the UICC stage based on tumor biology in esophageal adenocarcinoma and warrants further evaluations in independent clinical cohorts.

Abstract The multitude of obesogenic diets used in rodent studies can hardly be overviewed. Since standardization is missing and assuming that individual compositions provoke individual effects, the choice of quality, quantity and combination of diet ingredients seems to be crucial for the outcome and interpretation of obesity studies. Therefore, the present study was conducted to compare the individual effects of three commonly used obesogenic diets, mainly differing in sugar and fat content. Besides basic phenotypic and metabolic characterization, one main aspect was a comparative liver proteome analysis. As expected, the obtained results picture differentiated consequences mainly depending on fat source and/or fat- and sugar quantity. By confirming the general presumption that the choice of nutritional composition is a pivotal factor, the present findings demonstrate that a conscious selection is indispensable for obtaining reliable and sound results in obesity research. In conclusion, we strongly recommend a careful selection of the appropriate diet in advance of a new experiment, taking into account the specific research question.

Loss of the Y chromosome (LoY) is frequently observed in somatic cells of elderly men. However, LoY is highly increased in tumor tissue and correlates with an overall worse prognosis. The underlying causes and downstream effects of LoY are widely unknown. Therefore, we analyzed genomic and transcriptomic data of 13 cancer types (2375 patients) and classified tumors of male patients according to loss or retain of the Y chromosome (LoY or RoY, average LoY fraction: 0.46). The frequencies of LoY ranged from almost absence (glioblastoma, glioma, thyroid carcinoma) to 77% (kidney renal papillary cell carcinoma). Genomic instability, aneuploidy, and mutation burden were enriched in LoY tumors. In addition, we found more frequently in LoY tumors the gate keeping tumor suppressor gene TP53 mutated in three cancer types (colon adenocarcinoma, head and neck squamous carcinoma, lung adenocarcinoma) and oncogenes MET, CDK6, KRAS, and EGFR amplified in multiple cancer types. On the transcriptomic level, we observed MMP13, known to be involved in invasion, to be up-regulated in LoY of three adenocarcinomas and down-regulation of the tumor suppressor gene GPC5 in LoY of three cancer types. Furthermore, we found enrichment of a smoking-related mutation signature in LoY tumors of head and neck and lung cancer. Strikingly, we observed a correlation between cancer type-specific sex bias in incidence rates and frequencies of LoY, in line with the hypothesis that LoY increases cancer risk in males. Overall, LoY is a frequent phenomenon in cancer that is enriched in genomically unstable tumors. It correlates with genomic features beyond the Y chromosome and might contribute to higher incidence rates in males.

Abstract Today, individual prognosis in patients with adenocarcinoma of the esophagus (EAC) is based on post-surgical TNM staging and valid biomarkers are still not implemented. Integrin beta1 (ITGB1) is widely expressed in epithelial cells and promotes cell adhesion and growth. Its impact on tumor progression was described for different tumor entities before, data on its function as a potential biomarker in EAC is not available. Aim of the study is to evaluate the expression level of ITGB1 in a large collective of EAC and its impact on patients´ prognosis. 640 patients with esophageal adenocarcinoma were analyzed immunohistochemically for ITGB1. The data was correlated with long term outcome, clinical, pathological and molecular data (TP53, HER2/neu, c-myc, GATA6, PIK3CA and KRAS). Of 640 patients to be analyzed, 127 (19.8%) showed expression of ITGB1. ITGB1 expression was associated with lymph node metastasis, expression of integrin alphaV and KRAS mutation status. Patients with high ITGB1 expression showed impaired overall survival (22.5 months (95% CI 15.3–29.7 months), vs. 34.1 months (95% CI 25.3–42.4 months), P = 0.024). This effect was particularly evident in the group of patients undergoing primary surgery without prior neoadjuvant therapy (10.2 months (95% CI 1.9–41.7 months) vs. 31.4 months (95% CI 21.1–144.2 months, P = 0.008). ITGB1 was also an independent prognostic marker in multivariable analysis (HR 1.696 (95% CI 1.084–2.653, P = 0.021) in patients that underwent primary surgery. We demonstrate for the first time the prognostic significance of ITGB1 expression in a large EAC patient population.

Introduction: Imaging of human clinical formalin-fixed paraffin-embedded (FFPE) tissue sections provides insights into healthy and diseased states and therefore represents a valuable resource for basic research, as well as for diagnostic and clinical purposes. However, conventional light microscopy does not allow to observe the molecular details of tissue and cell architecture due to the diffraction limit of light. Super-resolution microscopy overcomes this limitation and provides access to the nanoscale details of tissue and cell organization.Methods: Here, we used quantitative multicolor stimulated emission depletion (STED) nanoscopy to study the nanoscale distribution of the nuclear phosphatidylinositol 4,5-bisphosphate (nPI(4,5)P2) with respect to the nuclear speckles (NS) marker SON.Results: Increased nPI(4,5)P2 signals were previously linked to human papillomavirus (HPV)-mediated carcinogenesis, while NS-associated PI(4,5)P2 represents the largest pool of nPI(4,5)P2 visualized by staining and microscopy. The implementation of multicolor STED nanoscopy in human clinical FFPE skin and wart sections allowed us to provide here the quantitative evidence for higher levels of NS-associated PI(4,5)P2 in HPV-induced warts compared to control skin.Discussion: These data expand the previous reports of HPV-induced increase of nPI(4,5)P2 levels and reveal for the first time the functional, tissue-specific localization of nPI(4,5)P2 within NS in clinically relevant samples. Moreover, our approach is widely applicable to other human clinical FFPE tissues as an informative addition to the classical histochemistry.

Summary: Background: Gastric cancer (GC) is clinically heterogenous according to location (cardia/non-cardia) and histopathology (diffuse/intestinal). We aimed to characterize the genetic risk architecture of GC according to its subtypes. Another aim was to examine whether cardia GC and oesophageal adenocarcinoma (OAC) and its precursor lesion Barrett’s oesophagus (BO), which are all located at the gastro-oesophageal junction (GOJ), share polygenic risk architecture. Methods: We did a meta-analysis of ten European genome-wide association studies (GWAS) of GC and its subtypes. All patients had a histopathologically confirmed diagnosis of gastric adenocarcinoma. For the identification of risk genes among GWAS loci we did a transcriptome-wide association study (TWAS) and expression quantitative trait locus (eQTL) study from gastric corpus and antrum mucosa. To test whether cardia GC and OAC/BO share genetic aetiology we also used a European GWAS sample with OAC/BO. Findings: Our GWAS consisting of 5816 patients and 10,999 controls highlights the genetic heterogeneity of GC according to its subtypes. We newly identified two and replicated five GC risk loci, all of them with subtype-specific association. The gastric transcriptome data consisting of 361 corpus and 342 antrum mucosa samples revealed that an upregulated expression of MUC1, ANKRD50, PTGER4, and PSCA are plausible GC-pathomechanisms at four GWAS loci. At another risk locus, we found that the blood-group 0 exerts protective effects for non-cardia and diffuse GC, while blood-group A increases risk for both GC subtypes. Furthermore, our GWAS on cardia GC and OAC/BO (10,279 patients, 16,527 controls) showed that both cancer entities share genetic aetiology at the polygenic level and identified two new risk loci on the single-marker level. Interpretation: Our findings show that the pathophysiology of GC is genetically heterogenous according to location and histopathology. Moreover, our findings point to common molecular mechanisms underlying cardia GC and OAC/BO. Funding: German Research Foundation (DFG).

Abstract Current recommendations suggest neoadjuvant treatment in node-positive esophageal cancer or tumors staged T3 and upwards but some T2 N0 patients might benefit from neoadjuvant therapy. It is of clinical relevance to identify this subgroup. Loss of epithelial apicobasal polarity is a key factor in the development of invasive capabilities of carcinoma. The oncofetal stem/progenitor cell marker NOPE is expressed in adult depolarized murine hepatocytes and in murine/human hepatocellular carcinoma. We analyzed NOPE expression in 363 patients with esophageal adenocarcinoma using an RNA Scope Assay on a tissue microarray and correlated results with clinical data. Median follow-up was 57.7 months with a 5-year survival rate of 26.6%. NOPE was detectable in 32 patients (8.8%). In pT1/2 stages, NOPE expression was associated with a significantly reduced median OS of 6.3 months (95% CI 1.2–19.4 months), the median OS is not reached in the NOPE-negative group (calculated mean OS 117.1 months) (P = 0.012). In advanced tumor stages, a NOPE dependent survival difference was not detected. This is the first report of NOPE expression demonstrating a prognostic value in esophageal cancer. Early stage, NOPE positive patients are at a high risk of tumor progression and may benefit from neoadjuvant treatment analogous to advanced stage cancer.

The tumor microenvironment comprises multiple cell types, like cancer cells, endothelial cells, fibroblasts, and immune cells. In recent years, there have been massive research efforts focusing not only on cancer cells, but also on other cell types of the tumor microenvironment, thereby aiming to expand and determine novel treatment options. Fibroblasts represent a heterogenous cell family consisting of numerous subtypes, which can alter immune cell fractions, facilitate or inhibit tumor growth, build pre-metastatic niches, or stabilize vessels. These effects can be achieved through cell–cell interactions, which form the extracellular matrix, or via the secretion of cytokines or chemokines. The pro- or antitumorigenic fibroblast phenotypes show variability not only among different cancer entities, but also among intraindividual sites, including primary tumors or metastatic lesions. Commonly prescribed for arterial hypertension, the inhibitors of the renin–angiotensin system have recently been described as having an inhibitory effect on fibroblasts. This inhibition leads to modified immune cell fractions and increased tissue stiffness, thereby contributing to overcoming therapy resistance and ultimately inhibiting tumor growth. However, it is important to note that the inhibition of fibroblasts can also have the opposite effect, potentially resulting in increased tumor growth. We aim to summarize the latest state of research regarding fibroblast heterogeneity and its intricate impact on the tumor microenvironment and extracellular matrix. Specifically, we focus on highlighting recent advancements in the comprehension of intraindividual heterogeneity and therapy options within this context.

Abstract Background According to current guidelines, treatment of patients with hepatic oligometastasis in pancreatic cancer is not reflected and systemic chemotherapy is recommended in those patients. Retrospective data suggest beneficial outcomes in patients with hepatic oligometastasis, though prospective data from clinical trials addressing this particular patient group is not available. Methods In this single arm, phase-2 trial, survival data from patients receiving neoadjuvant chemotherapy followed by R0/R1 resection will be compared to historic data from patients with oligometastatic adenocarcinoma of the pancreas. The clinical trial will focus on a well-defined patient collective with metastatic load limited to the liver as target organ with a maximum of five metastases. The combination of liposomal irinotecan (nal-IRI), oxaliplatin (OX) and 5-fluouracil (5-FU)/folinic acid (FA) (nal-IRI + OX+ 5-FU/FA, NAPOX) was chosen as neoadjuvant chemotherapy; the choice was based on an ongoing clinical study in which NAPOX appeared manageable, with promising anti-tumor activity in first-line treatment of patients with metastatic pancreatic adenocarcinoma. In total 150 patients will be enrolled for this trial with an aim of 55 patients receiving a complete macroscopic synchronous tumor and metastatic resection. Discussion This is the first clinical study to prospectively evaluate the value of multimodality therapy concepts in oligometastatic pancreatic cancer. Trial registration numbers EudraCT 2019–002734-37 ; NCT04617457 .

Background Specific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA).Methods RNA expression was assessed by NanoString in tumor samples of 41 treatment-naïve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing, in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion, clonality and cytotoxic activity of expanded T cells.Results 68.3% of patients expressed ≥5 TAAs simultaneously with coregulated clusters, which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ≥1 TAA were detectable in 75.0% and 53.7% of patients, respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal, could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare.Conclusions We identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA.

Kinase inhibitors are widely used to treat cancer, however patients frequently develop resistance. Here, the authors investigate adaption mechanisms during drug persistence and show that stimulation of the innate immunity sensor RIG-I enhances cancer cell death when combined with kinase inhibition.

Abstract The immune response against cancer is orchestrated by various parameters and site-dependent specificities have been poorly investigated. In our analyses of ten different cancer types, we describe elevated infiltration by regulatory T cells as the most common feature, while other lymphocyte subsets and also expression of immune-regulatory molecules on tumor-infiltrating lymphocytes showed site-specific variation. Multiparametric analyses of these data identified similarities of renal and liver or lung with head and neck cancer. Co-expression of immune-inhibitory ligands on tumor cells was most frequent in colorectal, lung and ovarian cancer. Genes related to antigen presentation were frequently dysregulated in liver and lung cancer. Expression of co-inhibitory molecules on tumor-infiltrating T cells accumulated in advanced stages while T-cell abundance was related to enhanced expression of genes related to antigen presentation. Our results promote evaluation of cancer-specific or even personalized immunotherapeutic combinations to overcome primary or secondary resistance as major limitation of immune-checkpoint inhibition.

Abstract Background Expression of CXCR4, a chemokine (C-X-C motif) receptor that plays a central role in tumor growth and metastasis of circulating tumor cells, has been described in a variety of solid tumors. A high expression of CXCR4 has a prognostic significance with regard to overall and progression-free survival and offers a starting point for targeted therapies. In this context, [68]Ga-Pentixafor-Positron Emission Tomography/Computer Tomography (PET/CT) offers promising possibility of imaging the CXCR4 expression profile. We set out to compare a [18F] fluorodeoxyglucose (FDG)-PET/CT and a [68Ga]Pentixafor-PET/CT in (re-)staging and radiation planning of patients with localized esophageal cancer. Materials and methods In this retrospective analysis, ten patients, with adeno- or squamous cell carcinoma of the esophagus (n = 3 and n = 7, respectively), which were scheduled for radio (chemo) therapy, were imaged using both Pentixafor and FDG PET/CT examinations. All lesions were visually rated as Pentixafor and FDG positive or negative. For both tracers, SUVmax was measured all lesions and compared to background. Additionally, immunohistochemistry of CXCR4 was obtained in patients undergoing surgery. Results FDG-positive tumor-suspicious lesions were detected in all patients and a total of 26 lesions were counted. The lesion-based analysis brought equal status in 14 lesions which were positive for both tracers while five lesions were FDG positive and Pentixafor negative and seven lesions were FDG negative, but Pentixafor positive. Histopathologic correlation was available in seven patients. The CXCR4 expression of four non-pretreated tumour lesion samples was confirmed immunohistochemically. Conclusion Our data shows that additional PET/CT imaging with Pentixafor for imaging the CXCR4 chemokine receptor is feasible but heterogeneous in both newly diagnosed and pretreated recurrent esophageal cancer. In addition, the Pentixafor PET/CT may serve as complementary tool for radiation field expansion in radiooncology.

Abstract Patients with metastasized malignant melanomas (MM) are regularly treated with immune checkpoint inhibitors (CPI). Within our study, we evaluated the predictive value of tumor infiltrating lymphocyte (TIL) clusters in primary MM and its association to molecular subtypes to predict response to CPI treatment. A cohort of 90 MM patients who received CPI treatment were collected from a single center, as well as a validation cohort of 351 patients from the TCGA database (SKCM) who received standard of care. A deep-convolutional-neural network (U-Net) was trained to detect viable tumor areas on H&E whole-slide-images, following a quantitative detection of TILs with help of a separate additional neural network. The number of TIL clusters was associated with response to CPI in 90 MM patients (AUC = 0.6), even more pronounced within the sub-cohort of BRAF V600 E/K -mutated MM patients (AUC = 0.7, n = 32). Interestingly, the TIL clusters in NRAS-mutated as well as wildtype MM (BRAF-wt, NRAS-wt) tumors, did not demonstrate a predictive value of CPI response (AUC = 0.5, n = 25). Moreover, PD-L1 expression had a limited predictive value within our cohort. In parallel, within an independent cohort of MM patients (TCGA, n = 351), the number of TIL clusters was associated with improved survival in BRAF V600 E/K mutated MM (p

Abstract Background Helicobacter pylori, a 2 × 1 μm spiral-shaped bacterium, is the most common risk factor for gastric cancer worldwide. Clinically, patients presenting with symptoms of gastritis, routinely undergo gastric biopsies. The following histo-morphological evaluation dictates therapeutic decisions, where antibiotics are used for H. pylori eradication. There is a strong rational to accelerate the detection process of H. pylori on histological specimens, using novel technologies, such as deep learning. Methods We designed a deep-learning-based decision support algorithm that can be applied on regular whole slide images of gastric biopsies. In detail, we can detect H. pylori both on Giemsa- and regular H&E stained whole slide images. Results With the help of our decision support algorithm, we show an increased sensitivity in a subset of 87 cases that underwent additional PCR- and immunohistochemical testing to define a sensitive ground truth of HP presence. For Giemsa stained sections, the decision support algorithm achieved a sensitivity of 100% compared to 68.4% (microscopic diagnosis), with a tolerable specificity of 66.2% for the decision support algorithm compared to 92.6 (microscopic diagnosis). Conclusion Together, we provide the first evidence of a decision support algorithm proving as a sensitive screening option for H. pylori that can potentially aid pathologists to accurately diagnose H. pylori presence on gastric biopsies.

Abstract Mucosal and skin cancers are associated with infections by human papillomaviruses (HPV). The manner how viral oncoproteins hijack the host cell metabolism to meet their own energy demands and how this may contribute to tumorigenesis is poorly understood. We now show that the HPV oncoprotein E7 of HPV8, HPV11 and HPV16 directly interact with the beta subunit of the mitochondrial ATP-synthase (ATP5B), which may therefore represent a conserved feature across different HPV genera. By measuring both glycolytic and mitochondrial activity we observed that the association of E7 with ATP5B was accompanied by reduction of glycolytic activity. Interestingly, there was a drastic increase in spare mitochondrial respiratory capacity in HPV8-E7 and an even more profound increase in HPV16-E7 expressing cells. In addition, we could show that ATP5B levels were unchanged in betaHPV positive skin cancers. However, comparing HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) we noticed that, while ATP5B expression levels did not correlate with patient overall survival in HPV-negative OPSCC, there was a strong correlation within the HPV16-positive OPSCC patient group. These novel findings provide evidence that HPV targets the host cell energy metabolism important for viral life cycle and HPV-mediated tumorigenesis.

Abstract Background Adenocarcinoma of the gastroesophageal junction (GEJ) Siewert type II can be resected by transthoracic esophagectomy or transhiatal extended gastrectomy. Both allow for a complete tumor resection, yet there is an ongoing controversy about which surgical approach is superior with regards to quality of life, oncological outcomes and survival. While some studies suggest a better oncological outcome after transthoracic esophagectomy, others favor transhiatal extended gastrectomy for a better postoperative quality of life. To date, only retrospective studies are available, showing ambiguous results. Methods This study is a multinational, multicenter, randomized, clinical superiority trial. Patients (n = 262) with a GEJ type II tumor resectable by both transthoracic esophagectomy and transhiatal extended gastrectomy will be enrolled in the trial. Type II tumors are defined as tumors with their midpoint between ≤1 cm proximal and ≤ 2 cm distal of the top of gastric folds on preoperative endoscopy. Patients will be included in one of the participating European sites and are randomized to either transthoracic esophagectomy or transhiatal extended gastrectomy. The trial is powered to show superiority for esophagectomy with regards to the primary efficacy endpoint overall survival. Key secondary endpoints are complete resection (R0), number and localization of tumor infiltrated lymph nodes at dissection, post-operative complications, disease-free survival, quality of life and cost-effectiveness. Postoperative survival and quality of life will be followed-up for 24 months after discharge. Further survival follow-up will be conducted as quarterly phone calls up to 60 months. Discussion To date, as level 1 evidence is lacking, there is no consensus on which surgery is superior and both surgeries are used to treat GEJ type II carcinoma worldwide. The CARDIA trial is the first randomized trial to compare transthoracic esophagectomy versus transhiatal extended gastrectomy in patients with GEJ type II tumors. Several quality control measures were implemented in the protocol to ensure data reliability and increase the trial’s significance. It is hypothesized that esophagectomy allows for a higher rate of radical resections and a more complete mediastinal lymph node dissection, resulting in a longer overall survival, while still providing an acceptable quality of life and cost-effectiveness. Trial registration The trial was registered on August 2nd 2019 at the German Clinical Trials Register under the trial-ID DRKS00016923 .

Abstract Background Gastric cancer is one of the deadliest cancer entities worldwide. While surgery is the only curative treatment option in early tumors, for locally advanced and metastatic patients further therapeutic targets are needed. Several studies not only reported mutations but also amplifications of the KRAS locus in different cancer entities. More recently, KRAS amplification was discussed as a new therapeutic target. Little is known about the (prognostic) relevance and (heterogenic) distribution of KRAS amplification in gastric adenocarcinomas, especially in Non-Asian patients. Methods Amplification of the KRAS locus and corresponding protein expression was analyzed in 582 gastric adenocarcinomas employing fluorescence in-situ hybridization (FISH) and immunohistochemistry. Amplification status was correlated with clinico-pathological features, clinical outcome and molecular tumor data including a correlation to the TCGA subtypes of gastric carcinoma. Results KRAS amplification was detected in 27 out of 470 analysable tumors (5.7%) and correlated with protein expression of KRAS in all amplified tumors. Within the KRAS amplified gastric tumors 14/27 (51.9%) showed a heterogeneous distribution with also KRAS non-amplified tumor parts. According to TCGA 24 tumors (88.8%) were related to chromosomal instable tumors (CIN). The survival analysis of the entire patient cohort did not show any difference in overall survival in dependence on the KRAS status. However, a significant survival difference with a worse outcome for patients with KRAS amplified tumors was identified when analysing patients without neoadjuvant pre-treatment. Conclusions We confirm the unfavorable prognosis of KRAS amplified tumors reported by other studies in (Asian) patient groups, at least in patients without neoadjuvant pre-treatment. Within KRAS amplified tumors we revealed intratumoral heterogeneity that may define a (more aggressive) tumor cell population which is more frequently observed in patients with lymph node metastases. Despite the heterogeneous distribution of KRAS amplified tumor clones, KRAS amplified locally advanced or metastasized gastric adenocarcinomas represent a therapeutically highly relevant tumor subgroup.

Single‐cell transcriptomics have revolutionized our understanding of the cell composition of tumors and allowed us to identify new subtypes of cells. Despite rapid technological advancements, single‐cell analysis remains resource‐intense hampering the scalability that is required to profile a sufficient number of samples for clinical associations. Therefore, more scalable approaches are needed to understand the contribution of individual cell types to the development and treatment response of solid tumors such as esophageal adenocarcinoma where comprehensive genomic studies have only led to a small number of targeted therapies. Due to the limited treatment options and late diagnosis, esophageal adenocarcinoma has a poor prognosis. Understanding the interaction between and dysfunction of individual cell populations provides an opportunity for the development of new interventions. In an attempt to address the technological and clinical needs, we developed a protocol for the separation of esophageal carcinoma tissue into leukocytes (CD45+), epithelial cells (EpCAM+), and fibroblasts (two out of PDGFRα, CD90, anti‐fibroblast) by fluorescence‐activated cell sorting and subsequent RNA sequencing. We confirm successful separation of the three cell populations by mapping their transcriptomic profiles to reference cell lineage expression data. Gene‐level analysis further supports the isolation of individual cell populations with high expression of CD3, CD4, CD8, CD19, and CD20 for leukocytes, CDH1 and MUC1 for epithelial cells, and FAP, SMA, COL1A1, and COL3A1 for fibroblasts. As a proof of concept, we profiled tumor samples of nine patients and explored expression differences in the three cell populations between tumor and normal tissue. Interestingly, we found that angiogenesis‐related genes were upregulated in fibroblasts isolated from tumors compared with normal tissue. Overall, we suggest our protocol as a complementary and more scalable approach compared with single‐cell RNA sequencing to investigate associations between clinical parameters and transcriptomic alterations of specific cell populations in esophageal adenocarcinoma.

Gene amplifications of PIK3CA or KRAS induce a downstream activation of the AKT-mTOR or RAF-ERK-pathways. Interactions of the active AKT pathway have been implicated in the inflammatory tumor microenvironment. Nothing is known about these interactions or prognostic power in esophageal adenocarcinoma (EAC). We retrospectively analyzed a large cohort of 685 EAC considering KRAS and PIK3CA gene amplification using fluorescence in situ hybridization (FISH) and immunohistochemistry. These results were correlated with clinical and molecular data as well as the inflammatory tumor microenvironment. Amplifications of KRAS were seen in 94 patients (17.1%), PIK3CA amplifications in 23 patients (5.0%). KRAS amplifications significantly correlated with nodal positive patients and poorer overall survival (OS) in the subgroup without neoadjuvant treatment (p = 0.004), coamplifications of Her2 (p = 0.027), and TP53 mutations (p = 0.016). PIK3CA amplifications significantly correlated with a high amount of tumor infiltrating T cells (p = 0.003) and showed a tendency to better OS (p = 0.068). A correlation with checkpoint makers (PD-L1, LAG3, VISTA, TIM3, IDO) could not be revealed. Our findings are the first to link the KRAS amplified genotype with lymphonodal positivity and poor prognosis and the PIK3CA-amplified genotype with a T cell–rich microenvironment in EAC. Future studies must show whether these two genotype subgroups can be therapeutically influenced. A dual inhibition of MEK and SHP2T could be effective in the subgroup of KRAS amplified EACs and an immune checkpoint blockade may prove to be particularly promising in the subgroup of PIK3CA-amplified EACs.

Abstract Background The SWI/SNF complex is an important chromatin remodeler, commonly dysregulated in cancer, with an estimated mutation frequency of 20%. ARID1A is the most frequently mutated subunit gene. Almost nothing is known about the other familiar members of the SWI/SNF complexes, SMARCA2 (BRM), SMARCA4 (BRG1) and SMARCB1 (INI1), in oesophageal adenocarcinoma (EAC). Methods We analysed a large cohort of 685 patients with EAC. We used four different antibodies to detect a loss-of-protein of ARID1A BRM, BRG1 and INI1 by immunohistochemistry and correlated these findings with molecular and clinical data. Results Loss of ARID1A, BRG1, BRM and INI1 was observed in 10.4, 3.4, 9.9 and 2% of EAC. We found a co-existing protein loss of ARID1A and BRM in 9.9% and of ARID1A and BRG1 in 2.2%. Patients with loss of ARID1A and TP53 wildtype EACs showed a shortened overall survival compared with AIRDA1A-positive tumours [median overall survival was 60.1 months (95%CI 1.2–139.9 months)] in patients with ARIDA-1A expression and 26.2 months (95%CI 3.7–19.1 months) in cases of ARIDA-1A loss (p = 0.044). Tumours with loss or expression of ARID1A and TP53 loss were not associated with a difference in survival. Only one tumour revealed high microsatellite instability (MSI-H) with concomitant ARID1A loss. All other ARID1A loss-EACs were microsatellite-stable (MSS). No predictive relevance was seen for SWI/SNF-complex alterations and simultaneous amplification of different genes (PIK3CA, KRAS, c-MYC, MET, GATA6, ERBB2). Conclusion Our work describes, for the first time, loss of one of the SWI/SNF ATPase subunit proteins in a large number of adenocarcinomas of the oesophagus. Several papers discuss possible therapeutic interventions for tumours showing a loss of function of the SWI/SNF complex, such as PARP inhibitors or PI3K and AKT inhibitors. Future studies will be needed to show whether SWI/SNF complex-deficient EACs may benefit from personalized therapy.

Abstract Background To date, patients with metastasized pancreatic ductal adenocarcinoma (PDAC M1) are regarded as a uniform collective. We hypothesize the existence of oligometastatic disease (OMD): a state of PDAC M1 disease with better tumor biology, limited metastasis, and increased survival. Methods Data of 128 PDAC M1 patients treated at the University of Cologne between 2008 and 2018 was reviewed. Interdependence between clinical parameter was calculated using the Mann-Whitney U-Test. Survival curves were generated using the Kaplan-Meier method and analyzed using the log-rank test. Results Eighty-one (63%) patients had metastases confined to one organ (single organ metastasis, SOG) whereas the remaining 47 (37%) showed multiple metastatic sites (multi-organ metastasis, MOG). Survival analysis revealed a median overall survival (OS) of 12.2 months for SOG vs 4.5 months for MOG (95% CI 5.7–9.8; p

Advanced salivary gland carcinomas (SGC) often lack therapeutic options. Agents targeting CD138 have recently shown promising results in clinical trials for multiple myeloma and a preclinical trial for triple-negative breast cancer. Immunohistochemistry for CD138 was performed for all patients who had undergone primary surgery for SGC with curative intent. Findings were validated using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging. Overall, 111 primary SGC and 13 lymph node metastases from salivary duct carcinomas (SaDu) were evaluated. CD138 expression was found in 60% of all SGC with differing expression across entities (p < 0.01). A mean of 25.2% of the tumor cells in mucoepidermoid carcinoma (MuEp) were positive, followed by epithelial-myoepithelial carcinoma (20.9%), acinic cell carcinoma (16.0%), and SaDu (15.2%). High-/intermediate-grade MuEp showed CD138 expression in a mean of 34.8% of tumor cells. For SaDu, lymph node metastases showed CD138 expression in a mean of 31.2% of tumor cells which correlated with CD138 expression in their primaries (p = 0.01; Spearman’s ρ = 0.71). MALDI-MS imaging confirmed the presence of the CD138 protein in SGC. No significant association was found between clinicopathological data, including progression-free survival (p = 0.50) and CD138 expression. CD138 is expressed in the cell membrane of different entities of SGC and SaDu lymph node metastases and therefore represents a potential target for CD138 targeting drugs.

IntroductionKnowledge of the high microsatellite-instability (MSI-H)/mismatch repair deficiency (MMRd) status is of increasing interest for personalized neoadjuvant or adjuvant therapy planning. Only a few studies are available on MSI-H distribution in the Northern European Caucasian patient population. In this study, we focused on a large cohort of tumors of the upper gastrointestinal tract.Materials and MethodsSurgical material from a total of 1,965 patients was analyzed for MSI-H/MMRd status (including 1,267 carcinomas of the esophagus or stomach). All tumors were analyzed with an internationally recommended immunohistochemical panel consisting of four antibodies (MLH1, MSH2, PMS2, and MSH6). The results were molecularly objectified.ResultsAdenocarcinomas with MSI-H/MMRd were detected with the following distribution: esophagus (1.4%), stomach (8.3%), small intestine (18.2%), large intestine (8.5%), intrahepatic bile ducts (1.9%), and pancreas (0%). In case of gastric tumors with MSI-H/MMRd, neoadjuvant therapy did not influence the prognosis of patients (p = 0.94). Within all tumor entities with MSI-H/MMRd, patients with a UICC stage 4 were also represented. In this advanced stage, 11.7% of patients with MSS tumors were diagnosed compared to 0.5% of patients with MSI-H tumors relative to the entire tumor collective.DiscussionIn this study, the proportion of MSI-H/MMRd tumors in the stomach is smaller than would have been expected in knowledge of the data published by TCGA or AGRC. Negative prognostic effects regarding MSI-H status and neoadjuvant therapy as described by the MAGIC study group were not seen in our cohort. The extent to which the MSI-H/MMRd status should be known for neoadjuvant therapy planning must be clarified in prospective studies in the future. At present, there is no convincing data to dispense the neoadjuvant therapy for gastric carcinoma. Due to the very convincing, positive data regarding the response rates of MSI-H tumors to treatment with PD1/PD-L1 inhibitors, every metastatic carcinoma of the gastrointestinal tract should be tested for its MSI-H status.

Abstract Background Molecular markers predicting survival in esophageal adenocarcinoma (EAC) are rare. Specifically, in favorable oncologic situations, e.g. nodal negativity or major neoadjuvant therapy response, there is a lack of additional risk factors that serve to predict patients’ outcome more precisely. This study evaluated X-linked inhibitor of apoptosis protein (XIAP) as a potential marker improving outcome prediction. Methods Tissue microarrays from 362 patients that were diagnosed with resectable EAC were included in the study. XIAP was stained by immunohistochemistry and correlated to clinical outcome, molecular markers and markers of the cellular tumor microenvironment. Results XIAP did not impact on overall survival (OS) in the whole study collective. Subgroup analyses stratifying for common genetic markers (TP53, ERBB2, ARID1A/SWI/SNF) did not disclose any impact of XIAP expression on survival. Detailed subgroup analyses of [1] nodal negative patients, [2] highly T-cell infiltrated tumors and [3] therapy responders to neoadjuvant treatment revealed a significant inverse role of high XIAP expression in these specific oncologic situations; elevated XIAP expression detrimentally affected patients’ outcome in these subgroups. [1]: OS XIAP low: 202 months (m) vs. XIAP high: 38 m; [2]: OS 116 m vs. 28.2 m; [3]: OS 31 m vs. 4 m). Conclusions Our data suggest XIAP expression in EAC as a worthy tool to improve outcome prediction in specific oncologic settings that might directly impact on clinical diagnosis and treatment of EAC in the future.

Abstract Background Carcinomas of the small bowel are rare tumors usually with dismal prognosis. Most recently, some potentially treatable molecular alterations were described. We emphasize the growing evidence of individualized treatment options in small bowel carcinoma. Methods We performed a DNA- based multi-gene panel using ultra-deep sequencing analysis (including 14 genes with up to 452 amplicons in total; KRAS, NRAS, HRAS, BRAF, DDR2, ERBB2, KEAP1, NFE2L2, PIK3CA, PTEN, RHOA, BRCA1, BRCA2 and TP53) as well as an RNA-based gene fusion panel including ALK, BRAF, FGFR1, FGFR2, FGFR3, MET, NRG1, NTRK1, NTRK2, NTRK3, RET and ROS1 on eleven formalin fixed and paraffin embedded small bowel carcinomas. Additionally, mismatch-repair-deficiency was analyzed by checking the microsatellite status using the five different mononucleotide markers BAT25, BAT26, NR-21, NR-22 and NR-27 and loss of mismatch repair proteins using four different markers (MLH1, MSH6, MSH2, PMS2). Results In five out of eleven small bowel carcinomas we found potentially treatable genetic alterations. Three patients demonstrated pathogenic (class 5) BRCA1 or BRCA2 mutations – one germline-related in a mixed neuroendocrine-non neuroendocrine neoplasm (MiNEN). Two additional patients revealed an activating ERBB2 mutation or PIK3CA mutation. Furthermore two tumors were highly microsatellite-instable (MSI-high), in one case associated to Lynch-syndrome. We did not find any gene fusions. Conclusion Our results underscore, in particular, the relevance of potentially treatable molecular alterations (like ERBB2, BRCA and MSI) in small bowel carcinomas. Further studies are needed to proof the efficacy of these targeted therapies in small bowel carcinomas.

Up to 40% of esophageal carcinomas have a biallelic intact TP53 gene. It is largely unclear how these carcinoma cells prevent apoptosis, what is the kind of pathway alterations, or whether therapeutically relevant alterations exist in this subgroup. We evaluated The Cancer Genome Atlas (TCGA) data to compare TP53-mutated with TP53–wild-type tumors regarding copy number variations, gene mutations, and expression patterns of protein-coding genes and miRNAs. Additionally, we analyzed up to 428 esophageal adenocarcinomas (EACs) in total using an ultra-deep parallel sequencing panel, immunohistochemistry, as well as fluorescence in situ hybridization. In the TCGA cohort, 17.3% has a biallelic intact TP53 gene. This group has a smaller average total size of somatic copy number variations. Some protein coding genes and miRNAs were differentially expressed between the TP53-wild-type and TP53-mutated group to emphasize mdm2, CCND2, TP73, or miRNA 150, 488, or 4662a. In addition, 50% of the TP53–wild-type tumors carry somatic mutations in at least one of the genes involved in the TP53 pathway. Our patient cohort revealed 41.3% TP53–wild-type tumors; 5.6% were MDM2 amplified. In accordance with the TCGA data, we did not find a prognostic relevance of TP53 in our tumor cohort as well. The mutation status of TP53 defines an important subtype in esophageal carcinoma. Our comprehensive molecular analysis revealed important and potentially therapeutically relevant genomic alterations in this subgroup.

Abstract Background HER2 (ERBB2 or HER2/neu) is a tyrosine-kinase increasing cell proliferation. Overexpression/amplification of HER2 is correlated with worse prognosis in solid malignancies. Consequently, HER2 targeting is established in breast and upper gastrointestinal tract cancer. There are conflicting data concerning the impact of HER2 overexpression on esophageal adenocarcinoma (EAC), as most studies do not differ between cancers of the esophagus/gastroesophageal junction and the stomach. The aim of this study was to analyze the expression/amplification of HER2 in EAC in correlation to clinicopathological data to verify its prognostic impact. Methods We analyzed 428 EAC patients that underwent transthoracic thoraco-abdominal esophagectomy between 1997 and 2014. We performed HER2 immunohistochemistry (IHC) according to the guidelines and fluorescence-in-situ-hybridization (FISH) for IHC score2+, using tissue micro arrays (TMA) with up to eight biopsies from the surface and infiltration area of a single tumor for evaluating HER2-heterogeneity and single-spot TMA. The HER2-status was correlated with clinicopathological data. Results HER2-positivity was found in up to 14.9% in our cohort (IHC score 3+ or IHC score 2+ with gene amplification) and demonstrated a significantly better overall survival (OS) in correlation to HER2-negative tumors (median OS 70.1 vs. 24.6 months, p = 0.006). HER2-overexpression was more frequently seen in lower tumor stages (pT1/pT2, p = 0.038), in the absence of lymphatic metastases (pN0/pN+, p = 0.020), and was significantly associated with better histological grading (G1/G2) (p = 0.041). Conclusion We demonstrated a positive prognostic impact of HER2 overexpression in a large cohort of EAC, contrary to other solid malignancies including gastric cancer and breast cancer, but consistent to the results of a large study on EAC from 2012.