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Background: Homologous recombination deficiency (HRD) is recognized as a pan-cancer predictive biomarker that potentially indicates who could benefit from treatment with PARP inhibitors (PARPi). Despite its clinical significance, HRD testing is highly complex. Here, we investigated in a proof-of-concept study whether Deep Learning (DL) can predict HRD status solely based on routine hematoxylin & eosin (H&E) histology images across nine different cancer types., Methods: We developed a deep learning pipeline with attention-weighted multiple instance learning (attMIL) to predict HRD status from histology images. As part of our approach, we calculated a genomic scar HRD score by combining loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST) from whole genome sequencing (WGS) data of n = 5209 patients across two independent cohorts. The model's effectiveness was evaluated using the area under the receiver operating characteristic curve (AUROC), focusing on its accuracy in predicting genomic HRD against a clinically recognized cutoff value., Results: Our study demonstrated the predictability of genomic HRD status in endometrial, pancreatic, and lung cancers reaching cross-validated AUROCs of 0.79, 0.58, and 0.66, respectively. These predictions generalized well to an external cohort, with AUROCs of 0.93, 0.81, and 0.73. Moreover, a breast cancer-trained image-based HRD classifier yielded an AUROC of 0.78 in the internal validation cohort and was able to predict HRD in endometrial, prostate, and pancreatic cancer with AUROCs of 0.87, 0.84, and 0.67, indicating that a shared HRD-like phenotype occurs across these tumor entities., Conclusions: This study establishes that HRD can be directly predicted from H&E slides using attMIL, demonstrating its applicability across nine different tumor types., (© 2024. The Author(s).)

Introduction: Whole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis., Methods: To assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics. Somatic and germline variants, copy-number alterations (CNAs), and complex biomarkers were investigated. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. The raw data were collected and re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability., Results: The mean positive percentage agreement (PPA) of somatic variant calling was 76 % while the positive predictive value (PPV) was 89 % in relation to a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88 % for all and 97 % for the clinically relevant variants. CNA calls were concordant for 82 % of genomic regions. Homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94 %, 93 %, and 93 % of calls, respectively. Variability of CNAs and complex biomarkers did not decrease considerably after harmonization of the bioinformatic processing and was hence attributed mainly to wet-lab differences., Conclusion: Continuous optimization of bioinformatic workflows and participating in round robin tests are recommended., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: MMT reports speaker and travel Expenses from Twist. JS reports speaker honoraria from DLS, Molecular Health, AstraZeneca and Biocartis, outside the submitted work. UL reports speaker fees from AstraZeneca, GSK, Novartis, Menarini, advisory board from AstraZeneca and Novartis. DH reports speaker honorary AstraZeneca, adboard BMS, WD speaker honoraries BMS & Novartis. SMB reports speaker honoraria, advisory board fees and research grants from AstraZeneca, Daiichi, Menarini, Novartis, Roche, BMS, Pfizer, Bayer, MSD, Merck, Amgen, Molecular Health, Targos, DLS, Janssen, GSK, QuIP, outside the submitted work. SL reports research grant from BMS, advisory board/speaker invitation from AstraZeneca, Eli Lilly, Roche and Takeda outside of this work. NTG reports research support from Janssen-Cilag and Advisory Boards from Janssen-Cilag, AstraZeneca, Daiichi-Sankyo and BMS outside the submitted work. WW reports research grants from Roche, MSD, BMS and AstraZeneca. Advisory board, lectures and speaker bureau fees from Roche, MSD, BMS, AstraZeneca, Pfizer, Merck, Lilly, Boehringer, Novartis, Takeda, Bayer, Janssen, Amgen, Astellas, Illumina, Eisai, Siemens, Agilent, ADC, GSK und Molecular Health. SO received reimbursement for travel expenses and payment for conference presentations from Illumina Inc. and Oxford Nanopore Technologies. CS reports research funding from BMS Stiftung Immunonkologie and institutional grants from Illumina outside the submitted work. CPS reports an investigator-initiated grant from Illumnia outside of the submitted work. PS reports grants from Inctye, BMS, Gilead, Falk, speakers bureau/advisory board from MSD, BMS, AstraZeneca, Incyte, Astellas, Janssen, Eisai, Amgen, Boehringer Ingelheim. DK reports personal fees for speaker honoraria from AstraZeneca, and Pfizer, personal fees for Advisory Board from Bristol-Myers Squibb, outside the submitted work. NP reports speaker fees from Novartis, Bayer, Roche, AstraZeneca, Illumina, BMS, MSD, PGDX/Labcorp, advisory board from Novartis, Lilly, Roche, Janssen, travel expenses from Novartis, AstraZeneca, Illumina, BMS, MSD, PGDX/Labcorp, Research grants from Illumina. JB reports grants from German Cancer Aid and consulting from MSD, outside the submitted work. AS reports participation in Advisory Board/Speaker’s Bureau for Astra Zeneca, AGCT, Bayer, Bristol-Myers Squibb, Eli Lilly, Illumina, Janssen, MSD, Novartis, Pfizer, Roche, Seattle Genetics, Takeda, and Thermo Fisher, grants from Bayer, Bristol-Myers Squibb, and Chugai, outside the submitted work. All other authors report no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The effect of grainyhead-like transcription factor 3 (GRHL3) on cancer development depends on the cancer subtypes as shown in tumor entities such as colorectal or oral squamous cell carcinomas. Here, we analyzed the subtype-specific role of GRHL3 in bladder carcinogenesis, comparing common urothelial carcinoma (UC) with squamous bladder cancer (sq-BLCA). We examined GRHL3 mRNA and protein expression in cohorts of patient samples, its prognostic role and its functional impact on tumorigeneses in different molecular and histopathological subtypes of bladder cancer. We showed for GRHL3 a reverse expression in squamous and urothelial bladder cancer subtypes. Stably GRHL3-overexpressing EJ28, J82, and SCaBER in vitro models revealed a tumor-suppressive function in squamous and an oncogenic role in the urothelial cancer cells affecting cell and colony growth, and migratory and invasive capacities. Transcriptomic profiling demonstrated highly subtype-specific GRHL3-regulated expression networks coined by the enrichment of genes involved in integrin-mediated pathways. In SCaBER, loss of ras homolog family member A (RHOA) GTPase activity was demonstrated to be associated with co-regulation of eukaryotic translation initiation factor 4E family member 3 (EIF4E3), a potential tumor suppressor gene. Thus, our data provide for the first time a detailed insight into the role of the transcription factor GRHL3 in different histopathological subtypes of bladder cancer., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Urothelial carcinoma (UC) is by far the most common malignant neoplasm of the urinary bladder; however, there are both benign and malignant changes of the urothelium which morphologically resemble urothelial carcinomas or other carcinomas of the urinary bladder. Thus, these mimickers can cause problems in the histomorphological diagnosis. This article provides an overview of possible mimickers and pitfalls of bladder cancer as well as practical notes on the diagnostic procedure, partly using case studies., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Due to the proliferation-induced high demand of cancer cells for folic acid (FA), significant overexpression of folate receptors 1 (FR1) is detected in most cancers. To our knowledge, a detailed characterization of FR1 expression and regulation regarding therapeutic and diagnostic feasibilities in prostate cancer (PCa) has not been described. In the present study, cell cultures, as well as tissue sections, were analyzed using Western blot, qRT-PCR and immunofluorescence. In addition, we utilized FA-functionalized lipoplexes to characterize the potential of FR1-targeted delivery into PCa cells. Interestingly, we detected a high level of FR1-mRNA in healthy prostate epithelial cells and healthy prostate tissue. However, we were able to show that PCa cells in vitro and PCa tissue showed a massively enhanced FR1 membrane localization where the receptor can finally gain its function. We were able to link these changes to the overexpression of GPI-transamidase (GPI-T) by image analysis. PCa cells in vitro and PCa tissue show the strongest overexpression of GPI-T and thereby induce FR1 membrane localization. Finally, we utilized FA-functionalized lipoplexes to selectively transfer pDNA into PCa cells and demonstrate the therapeutic potential of FR1. Thus, FR1 represents a very promising candidate for targeted therapeutic transfer pathways in PCa and in combination with GPI-T, may provide predictive imaging in addition to established diagnostics.

Background & Aims: α1-Antitrypsin (AAT) is a major protease inhibitor produced by hepatocytes. The most relevant AAT mutation giving rise to AAT deficiency (AATD), the 'Pi∗Z' variant, causes harmful AAT protein accumulation in the liver, shortage of AAT in the systemic circulation, and thereby predisposes to liver and lung injury. Although intravenous AAT augmentation constitutes an established treatment of AATD-associated lung disease, its impact on the liver is unknown., Methods: Liver-related parameters were assessed in a multinational cohort of 760 adults with severe AATD (Pi∗ZZ genotype) and available liver phenotyping, of whom 344 received augmentation therapy and 416 did not. Liver fibrosis was evaluated noninvasively via the serum test AST-to-platelet ratio index and via transient elastography-based liver stiffness measurement. Histologic parameters were compared in 15 Pi∗ZZ adults with and 35 without augmentation., Results: Compared with nonaugmented subjects, augmented Pi∗ZZ individuals displayed lower serum liver enzyme levels (AST 71% vs 75% upper limit of normal, P < .001; bilirubin 49% vs 58% upper limit of normal, P = .019) and lower surrogate markers of fibrosis (AST-to-platelet ratio index 0.34 vs 0.38, P < .001; liver stiffness measurement 6.5 vs 7.2 kPa, P = .005). Among biopsied participants, augmented individuals had less pronounced liver fibrosis and less inflammatory foci but no differences in AAT accumulation were noted., Conclusions: The first evaluation of AAT augmentation on the Pi∗ZZ-related liver disease indicates liver safety of a widely used treatment for AATD-associated lung disease. Prospective studies are needed to confirm the beneficial effects and to demonstrate the potential efficacy of exogenous AAT in patients with Pi∗ZZ-associated liver disease., (Copyright © 2024 AGA Institute. Published by Elsevier Inc. All rights reserved.)

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., (© 2023. Nature Publishing Group UK.)

Fibrosis, or tissue scarring, develops as a pathological deviation from the physiological wound healing response and can occur in various organs such as the heart, lung, liver, kidney, skin, and bone marrow. Organ fibrosis significantly contributes to global morbidity and mortality. A broad spectrum of etiologies can cause fibrosis, including acute and chronic ischemia, hypertension, chronic viral infection (e.g., viral hepatitis), environmental exposure (e.g., pneumoconiosis, alcohol, nutrition, smoking) and genetic diseases (e.g., cystic fibrosis, alpha-1-antitrypsin deficiency). Common mechanisms across organs and disease etiologies involve a sustained injury to parenchymal cells that triggers a wound healing response, which becomes deregulated in the disease process. A transformation of resting fibroblasts into myofibroblasts with excessive extracellular matrix production constitutes the hallmark of disease, however, multiple other cell types such as immune cells, predominantly monocytes/macrophages, endothelial cells, and parenchymal cells form a complex network of profibrotic cellular crosstalk. Across organs, leading mediators include growth factors like transforming growth factor-β and platelet-derived growth factor, cytokines like interleukin-10, interleukin-13, interleukin-17, and danger-associated molecular patterns. More recently, insights into fibrosis regression and resolution of chronic conditions have deepened our understanding of beneficial, protective effects of immune cells, soluble mediators and intracellular signaling. Further in-depth insights into the mechanisms of fibrogenesis can provide the rationale for therapeutic interventions and the development of targeted antifibrotic agents. This review gives insight into shared responses and cellular mechanisms across organs and etiologies, aiming to paint a comprehensive picture of fibrotic diseases in both experimental settings and in human pathology., Competing Interests: Declaration of competing interest IL has no conflicts of interest to declare. NTG has nothing to declare. RW has nothing to declare. FT's lab has received research funding from Allergan, Bristol-Myers Squibb, Gilead and Inventiva. FT has received honoraria for consulting or lectures from Astra Zeneca, Gilead, AbbVie, BMS, Boehringer, Madrigal, Intercept, Falk, Ionis, Inventiva, Merz, Pfizer, Alnylam, NGM, CSL Behring, Novo Nordisk, Novartis., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Surgical resection with complete tumor excision (R0) provides the best chance of long-term survival for patients with intrahepatic cholangiocarcinoma (iCCA). A non-invasive imaging technology, which could provide quick intraoperative assessment of resection margins, as an adjunct to histological examination, is optical coherence tomography (OCT). In this study, we investigated the ability of OCT combined with convolutional neural networks (CNN), to differentiate iCCA from normal liver parenchyma ex vivo., Methods: Consecutive adult patients undergoing elective liver resections for iCCA between June 2020 and April 2021 (n = 11) were included in this study. Areas of interest from resection specimens were scanned ex vivo, before formalin fixation, using a table-top OCT device at 1310 nm wavelength. Scanned areas were marked and histologically examined, providing a diagnosis for each scan. An Xception CNN was trained, validated, and tested in matching OCT scans to their corresponding histological diagnoses, through a 5 × 5 stratified cross-validation process., Results: Twenty-four three-dimensional scans (corresponding to approx. 85,603 individual) from ten patients were included in the analysis. In 5 × 5 cross-validation, the model achieved a mean F1-score, sensitivity, and specificity of 0.94, 0.94, and 0.93, respectively., Conclusion: Optical coherence tomography combined with CNN can differentiate iCCA from liver parenchyma ex vivo. Further studies are necessary to expand on these results and lead to innovative in vivo OCT applications, such as intraoperative or endoscopic scanning., (© 2023. The Author(s).)

Intrahepatic cholangiocarcinoma (iCCA) is a rare, understudied primary hepatic malignancy with dismal outcomes. Aiming to identify prognostically relevant single-nucleotide polymorphisms, we analyzed 11 genetic variants with a role in tumor-promoting inflammation (VEGF, EGF, EGFR, IL-1B, IL-6, CXCL8 (IL-8), IL-10, CXCR1, HIF1A and PTGS2 (COX-2) genes) and their association with disease-free (DFS) and overall survival (OS) in patients undergoing curative-intent surgery for iCCA. Genomic DNA was isolated from 112 patients (64 female, 48 male) with iCCA. Germline polymorphisms were analyzed with polymerase chain reaction-restriction fragment length polymorphism protocols. The IL-1B +3954 C/C (73/112, hazard ratio (HR) = 1.735, p = 0.012) and the IL-8 -251 T/A or A/A (53/112 and 16/112, HR = 2.001 and 1.1777, p = 0.026) genotypes were associated with shorter OS in univariable and multivariable analysis. The IL-1B +3954 polymorphism was also associated with shorter DFS (HR = 1.983, p = 0.012), but this effect was not sustained in the multivariable model. A genetic risk model of 0, 1 and 2 unfavorable alleles was established and confirmed in multivariable analysis. This study supports the prognostic role of the IL-1B C+3954T and the IL-8 T-251A variant as outcome markers in iCCA patients, identifying patient subgroups at higher risk for dismal clinical outcomes., (© 2023. The Author(s).)

Urothelial carcinoma of the upper urinary tract (upper tract urothelial carcinoma, UTUC) is less common than bladder carcinoma with nearly identical risk factors and has a poorer prognosis. The standard diagnostic procedure is imaging of the upper urinary tract by computed tomography urography. In cases of diagnostic uncertainty, a diagnostic ureterorenoscopy with biopsy sampling can be performed in addition to urine cytology. Treatment depends primarily on the stage and grading of the tumor. Depending on the extent and localization, organ-preserving treatment or radical nephroureterectomy is indicated. Perioperative systemic treatment in high-risk UTUC can be performed in both neoadjuvant and adjuvant settings, although the current data on neoadjuvant chemo- and immunotherapy do not yet allow standard application. For metastatic disease, a multimodal treatment approach consisting of cisplatin-based or carboplatin-based chemotherapy, immunotherapy, and treatment with enfortumab vedotin can be considered. Salvage surgery, radiotherapy and metastasectomy are available for rare individual cases., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Objective: Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC., Methods: Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions., Results: After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients., Conclusions: In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

The new World Health Organization (WHO) classification of urogenital tumors is still primarily based on anatomic location, but is also a hierarchical taxonomic classification without separate chapters for tumors of the upper urinary tract and the urethra. It clarifies aspects regarding grading and noninvasive entities. It consolidates the use of the Paris system for urinary cytology as well as various subtypes/special types of neoplasms, and incorporates general concepts of the 5th edition of the WHO blue book. In addition to mesenchymal tumors, well-differentiated neuroendocrine tumors and neuroendocrine carcinomas are addressed in separate chapters. Papillary non-invasive low- and high-grade carcinomas and carcinoma in situ remain, while dysplasia and urothelial proliferation of unknown malignant potential (UPUMP) are no longer treated as separate entities. Former variants of urothelial carcinoma are now called subtypes and aberrant differentiation and special types are more precisely defined., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Aims: Immune checkpoint inhibitor (ICI) therapy has become a viable treatment strategy in bladder cancer. However, treatment responses vary, and improved biomarkers are needed. Crucially, the characteristics of immune cells remain understudied especially in squamous differentiated bladder cancer (sq-BLCA). Here, we quantitatively analysed the tumour-immune phenotypes of sq-BLCA and correlated them with PD-L1 expression and FGFR3 mutation status., Methods: Tissue microarrays (TMA) of n = 68 non-schistosomiasis associated pure squamous cell carcinoma (SCC) and n = 46 mixed urothelial carcinoma with squamous differentiation (MIX) were subjected to immunohistochemistry for CD3, CD4, CD8, CD56, CD68, CD79A, CD163, Ki67, perforin and chloroacetate esterase staining. Quantitative image evaluation was performed via digital image analysis., Results: Immune infiltration was generally higher in stroma than in tumour regions. B-cells (CD79A) were almost exclusively found in stromal areas (sTILs), T-lymphocytes and macrophages were also present in tumour cell areas (iTILs), while natural killer cells (CD56) were nearly missing in any area. Tumour-immune phenotype distribution differed depending on the immune cell subset, however, hot tumour-immune phenotypes (high density of immune cells in tumour areas) were frequently found for CD8 + T-cells (33%), especially perforin + lymphocytes (52.2%), and CD68 + macrophages (37.6%). Perforin + CD8 lymphocytes predicted improved overall survival in sq-BLCA while high PD-L1 expression (CPS ≥ 10) was significantly associated with higher CD3 + , CD8 + and CD163 + immune cell density and high Ki67 (density) of tumour cells. Furthermore, PD-L1 expression was positively associated with CD3 + /CD4 + , CD3 + /CD8 + and CD68 + /CD163 + hot tumour-immune phenotypes. FGFR3 mutation status was inversely associated with CD8 + , perforin + and CD79A + lymphocyte density., Conclusions: Computer-based image analysis is an efficient tool to analyse immune topographies in squamous bladder cancer. Hot tumour-immune phenotypes with strong PD-L1 expression might pose a promising subgroup for clinically successful ICI therapy in squamous bladder cancer and warrant further investigation., (© 2023. The Author(s).)

Immunohistochemistry is widely used in diagnostic and scientific analysis of urothelial carcinoma. Objective interpretation of staining results is mandatory for accuracy and comparability in diagnostic and therapeutic patient care as well as research.Herein we summarize and explain standardized microscopic evaluation and scoring approaches for immunohistochemical stainings. We focus on commonly used and generally feasible approaches for different cellular compartments and comment on their utility in diagnostics and research practice., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Artificial intelligence (AI) can extract visual information from histopathological slides and yield biological insight and clinical biomarkers. Whole slide images are cut into thousands of tiles and classification problems are often weakly-supervised: the ground truth is only known for the slide, not for every single tile. In classical weakly-supervised analysis pipelines, all tiles inherit the slide label while in multiple-instance learning (MIL), only bags of tiles inherit the label. However, it is still unclear how these widely used but markedly different approaches perform relative to each other. We implemented and systematically compared six methods in six clinically relevant end-to-end prediction tasks using data from N=2980 patients for training with rigorous external validation. We tested three classical weakly-supervised approaches with convolutional neural networks and vision transformers (ViT) and three MIL-based approaches with and without an additional attention module. Our results empirically demonstrate that histological tumor subtyping of renal cell carcinoma is an easy task in which all approaches achieve an area under the receiver operating curve (AUROC) of above 0.9. In contrast, we report significant performance differences for clinically relevant tasks of mutation prediction in colorectal, gastric, and bladder cancer. In these mutation prediction tasks, classical weakly-supervised workflows outperformed MIL-based weakly-supervised methods for mutation prediction, which is surprising given their simplicity. This shows that new end-to-end image analysis pipelines in computational pathology should be compared to classical weakly-supervised methods. Also, these findings motivate the development of new methods which combine the elegant assumptions of MIL with the empirically observed higher performance of classical weakly-supervised approaches. We make all source codes publicly available at https://github.com/KatherLab/HIA, allowing easy application of all methods to any similar task., Competing Interests: Declaration of competing interest JNK declares consulting services for Owkin, France and Panakeia, UK. TJB reports owning a company that develops mobile apps, outside the scope of the submitted work (Smart Health Heidelberg GmbH, Handschuhsheimer Landstr. 9/1, 69120 Heidelberg). No other potential conflicts of interest are reported by any of the authors., (Copyright © 2022. Published by Elsevier B.V.)

High-grade non-muscle-invasive bladder cancer (HG NMIBC) patients are at high risk (HR) of progression to muscle-invasion. Bladder-preserving therapies for this patient subgroup are limited, and additional treatments are desirable. Recently, enfortumab vedotin, targeting cancer-associated NECTIN4, has been approved for the treatment of advanced urothelial carcinoma. However, data on the expression of NECTIN4 and its therapeutic potential for HR NMIBC are scarce. Here, NECTIN4 was immunohistochemically analyzed in urothelial HG NMIBC by studying cohorts of carcinoma in situ (CIS)/T1HG (N = 182 samples), HG papillary tumors from mixed-grade lesions (mixed TaHG) (N = 87) and papillary HG tumors without a history of low-grade disease (pure TaHG/T1HG) (N = 98) from overall 225 patients. Moreover, inter-lesional NECTIN4 heterogeneity in multifocal HG NMIBC tumors was determined. A high prevalence of NECTIN4 positivity was noted across HG NMIBC subgroups (91%, N = 367 samples), with 77% of samples showing moderate/strong expression. Heterogenous NECTIN4 levels were observed between HG NMIBC subgroups: non-invasive areas of CIS/T1HG and pure TaHG/T1HG samples showed NECTIN4 positivity in 96% and 99%, with 88% and 83% moderate/strong expressing specimens, respectively, whereas significantly lower NECTIN4 levels were detected in mixed TaHG lesions (72% positivity, 48% of samples with moderate/strong NECTIN4 expression). Moreover, higher NECTIN4 heterogeneity was observed in patients with multifocal mixed TaHG tumors (22% of patients) compared to patients with multifocal CIS/T1HG and pure TaHG/T1HG tumors (9% and 5%). Taken together, NECTIN4-directed antibody-drug conjugates might be promising for the treatment of HR NMIBC patients, especially for those exhibiting CIS/T1HG and pure TaHG/T1HG tumors without a history of low-grade disease., (© 2022. The Author(s).)

This study aims at analyzing the impact of the pharmacological inhibition of DNA damage response (DDR) targets (DNA-PK and ATR) on radiosensitization of bladder cancer cell lines of different molecular/histological subtypes. Applying DNA-PK (AZD7648) and ATR (Ceralasertib) inhibitors on SCaBER, J82 and VMCUB-1 bladder cancer cell lines, we revealed sensitization upon ionizing radiation (IR), i.e., the IC 50 for each drug shifted to a lower drug concentration with increased IR doses. In line with this, drug exposure retarded DNA repair after IR-induced DNA damage visualized by a neutral comet assay. Western blot analyses confirmed specific inhibition of targeted DDR pathways in the analyzed bladder cancer cell lines, i.e., drugs blocked DNA-PK phosphorylation at Ser2056 and the ATR downstream mediator CHK1 at Ser317. Interestingly, clonogenic survival assays indicated a cell-line-dependent synergism of combined DDR inhibition upon IR. Calculating combined index (CI) values, with and without IR, according to the Chou-Talalay method, confirmed drug- and IR-dose-specific synergistic CI values. Thus, we provide functional evidence that DNA-PK and ATR inhibitors specifically target corresponding DDR pathways retarding the DNA repair process at nano-molar concentrations. This, in turn, leads to a strong radiosensitizing effect and impairs the survival of bladder cancer cells.

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 ( LINC00152 ) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152 -driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2 . KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152 -miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.

Background and Aims: Perihilar cholangiocarcinoma (pCCA) is a hepatobiliary malignancy, with a dismal prognosis. Nerve fiber density (NFD)-a novel prognostic biomarker-describes the density of small nerve fibers without cancer invasion and is categorized into high numbers and low numbers of small nerve fibers (high vs low NFD). NFD is different than perineural invasion (PNI), defined as nerve fiber trunks invaded by cancer cells. Here, we aim to explore differences in immune cell populations and survival between high and low NFD patients., Approach and Results: We applied multiplex immunofluorescence (mIF) on 47 pCCA patients and investigated immune cell composition in the tumor microenvironment (TME) of high and low NFD. Group comparison and oncological outcome analysis was performed. CD8+PD-1 expression was higher in the high NFD than in the low NFD group (12.24 × 10 -6 vs. 1.38 × 10 -6 positive cells by overall cell count, p = 0.017). High CD8+PD-1 expression was further identified as an independent predictor of overall (OS; Hazard ratio (HR) = 0.41; p = 0.031) and recurrence-free survival (RFS; HR = 0.40; p = 0.039). Correspondingly, the median OS was 83 months (95% confidence interval (CI): 18-48) in patients with high CD8+PD-1+ expression compared to 19 months (95% CI: 5-93) in patients with low CD8+PD-1+ expression ( p = 0.018 log rank). Furthermore, RFS was significantly lower in patients with low CD8+PD-1+ expression (14 months (95% CI: 6-22)) compared to patients with high CD8+PD-1+ expression (83 months (95% CI: 17-149), p = 0.018 log rank)., Conclusions: PD-1+ T-cells correlate with high NFD as a prognostic biomarker and predict good survival; the biological pathway needs to be investigated.

Background: Microsatellite instability (MSI)/mismatch repair deficiency (dMMR) is a key genetic feature which should be tested in every patient with colorectal cancer (CRC) according to medical guidelines. Artificial intelligence (AI) methods can detect MSI/dMMR directly in routine pathology slides, but the test performance has not been systematically investigated with predefined test thresholds., Method: We trained and validated AI-based MSI/dMMR detectors and evaluated predefined performance metrics using nine patient cohorts of 8343 patients across different countries and ethnicities., Results: Classifiers achieved clinical-grade performance, yielding an area under the receiver operating curve (AUROC) of up to 0.96 without using any manual annotations. Subsequently, we show that the AI system can be applied as a rule-out test: by using cohort-specific thresholds, on average 52.73% of tumors in each surgical cohort [total number of MSI/dMMR = 1020, microsatellite stable (MSS)/ proficient mismatch repair (pMMR) = 7323 patients] could be identified as MSS/pMMR with a fixed sensitivity at 95%. In an additional cohort of N = 1530 (MSI/dMMR = 211, MSS/pMMR = 1319) endoscopy biopsy samples, the system achieved an AUROC of 0.89, and the cohort-specific threshold ruled out 44.12% of tumors with a fixed sensitivity at 95%. As a more robust alternative to cohort-specific thresholds, we showed that with a fixed threshold of 0.25 for all the cohorts, we can rule-out 25.51% in surgical specimens and 6.10% in biopsies., Interpretation: When applied in a clinical setting, this means that the AI system can rule out MSI/dMMR in a quarter (with global thresholds) or half of all CRC patients (with local fine-tuning), thereby reducing cost and turnaround time for molecular profiling., Competing Interests: Disclosure JNK declares consulting services for Owkin, France and Panakeia, UK. TJB reports owning a company that develops mobile apps, outside the scope of the submitted work (Smart Health Heidelberg GmbH, Handschuhsheimer Landstr. 9/1, 69120 Heidelberg). PQ has had paid roles in the English NHS bowel cancer screening programme over the course of this study. SBG is co-founder of Brogent International LLC with equity, outside the scope of the submitted work. All other authors have declared no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Fibroblast growth factor receptor (FGFR) inhibitor treatment has become the first clinically approved targeted therapy in bladder cancer. However, it requires previous molecular testing of each patient, which is costly and not ubiquitously available., Objective: To determine whether an artificial intelligence system is able to predict mutations of the FGFR3 gene directly from routine histology slides of bladder cancer., Design, Setting, and Participants: We trained a deep learning network to detect FGFR3 mutations on digitized slides of muscle-invasive bladder cancers stained with hematoxylin and eosin from the Cancer Genome Atlas (TCGA) cohort (n = 327) and validated the algorithm on the "Aachen" cohort (n = 182; n = 121 pT2-4, n = 34 stroma-invasive pT1, and n = 27 noninvasive pTa tumors)., Outcome Measurements and Statistical Analysis: The primary endpoint was the area under the receiver operating curve (AUROC) for mutation detection. Performance of the deep learning system was compared with visual scoring by an uropathologist., Results and Limitations: In the TCGA cohort, FGFR3 mutations were detected with an AUROC of 0.701 (p < 0.0001). In the Aachen cohort, FGFR3 mutants were found with an AUROC of 0.725 (p < 0.0001). When trained on TCGA, the network generalized to the Aachen cohort, and detected FGFR3 mutants with an AUROC of 0.625 (p = 0.0112). A subgroup analysis and histological evaluation found highest accuracy in papillary growth, luminal gene expression subtypes, females, and American Joint Committee on Cancer (AJCC) stage II tumors. In a head-to-head comparison, the deep learning system outperformed the uropathologist in detecting FGFR3 mutants., Conclusions: Our computer-based artificial intelligence system was able to detect genetic alterations of the FGFR3 gene of bladder cancer patients directly from histological slides. In the future, this system could be used to preselect patients for further molecular testing. However, analyses of larger, multicenter, muscle-invasive bladder cancer cohorts are now needed in order to validate and extend our findings., Patient Summary: In this report, a computer-based artificial intelligence (AI) system was applied to histological slides to predict genetic alterations of the FGFR3 gene in bladder cancer. We found that the AI system was able to find the alteration with high accuracy. In the future, this system could be used to preselect patients for further molecular testing., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

In the last four years, advances in Deep Learning technology have enabled the inference of selected mutational alterations directly from routine histopathology slides. In particular, recent studies have shown that genetic changes in clinically relevant driver genes are reflected in the histological phenotype of solid tumors and can be inferred by analysing routine Haematoxylin and Eosin (H&E) stained tissue sections with Deep Learning. However, these studies mostly focused on selected individual genes in selected tumor types. In addition, genetic changes in solid tumors primarily act by changing signaling pathways that regulate cell behaviour. In this study, we hypothesized that Deep Learning networks can be trained to directly predict alterations of genes and pathways across a spectrum of solid tumors. We manually outlined tumor tissue in H&E-stained tissue sections from 7,829 patients with 23 different tumor types from The Cancer Genome Atlas. We then trained convolutional neural networks in an end-to-end way to detect alterations in the most clinically relevant pathways or genes, directly from histology images. Using this automatic approach, we found that alterations in 12 out of 14 clinically relevant pathways and numerous single gene alterations appear to be detectable in tissue sections, many of which have not been reported before. Interestingly, we show that the prediction performance for single gene alterations is better than that for pathway alterations. Collectively, these data demonstrate the predictability of genetic alterations directly from routine cancer histology images and show that individual genes leave a stronger morphological signature than genetic pathways., Competing Interests: JK declares consulting services for Owkin, France and Panakeia, UK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Loeffler, Gaisa, Muti, van Treeck, Echle, Ghaffari Laleh, Trautwein, Heij, Grabsch, Ortiz Bruechle and Kather.)

Objective: Alpha-1 antitrypsin deficiency (AATD) is a common, potentially lethal inborn disorder caused by mutations in alpha-1 antitrypsin (AAT). Homozygosity for the 'Pi*Z' variant of AAT (Pi*ZZ genotype) causes lung and liver disease, whereas heterozygous 'Pi*Z' carriage (Pi*MZ genotype) predisposes to gallstones and liver fibrosis. The clinical significance of the more common 'Pi*S' variant remains largely undefined and no robust data exist on the prevalence of liver tumours in AATD., Design: Baseline phenotypes of AATD individuals and non-carriers were analysed in 482 380 participants in the UK Biobank. 1104 participants of a multinational cohort (586 Pi*ZZ, 239 Pi*SZ, 279 non-carriers) underwent a comprehensive clinical assessment. Associations were adjusted for age, sex, body mass index, diabetes and alcohol consumption., Results: Among UK Biobank participants, Pi*ZZ individuals displayed the highest liver enzyme values, the highest occurrence of liver fibrosis/cirrhosis (adjusted OR (aOR)=21.7 (8.8-53.7)) and primary liver cancer (aOR=44.5 (10.8-183.6)). Subjects with Pi*MZ genotype had slightly elevated liver enzymes and moderately increased odds for liver fibrosis/cirrhosis (aOR=1.7 (1.2-2.2)) and cholelithiasis (aOR=1.3 (1.2-1.4)). Individuals with homozygous Pi*S mutation (Pi*SS genotype) harboured minimally elevated alanine aminotransferase values, but no other hepatobiliary abnormalities. Pi*SZ participants displayed higher liver enzymes, more frequent liver fibrosis/cirrhosis (aOR=3.1 (1.1-8.2)) and primary liver cancer (aOR=6.6 (1.6-26.9)). The higher fibrosis burden was confirmed in a multinational cohort. Male sex, age ≥50 years, obesity and the presence of diabetes were associated with significant liver fibrosis., Conclusion: Our study defines the hepatobiliary phenotype of individuals with the most relevant AATD genotypes including their predisposition to liver tumours, thereby allowing evidence-based advice and individualised hepatological surveillance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Perihilar cholangiocarcinoma (pCCA) is a rare primary liver malignancy. Even in patients amenable to surgery, outcomes are often dismal. Here, we aimed to identify prognostic markers for patient outcomes by analyzing functionally relevant single-nucleotide polymorphisms (SNPs) in genes with a role in tumor inflammation and angiogenesis. We analyzed 11 polymorphisms in the inflammation-angiogenesis axis ( VEGF, EGF, EGFR, IL-1b, IL-6, CXCL8 (IL-8), IL-10, CXCR1, HIF1A , and COX2 genes) for their prediction of tumor recurrence and survival in pCCA patients undergoing surgery in a curative intent., Methods: Samples were obtained from 111 patients with pCCA undergoing liver resection in curative intent. DNA was extracted and analyzed using polymerase chain reaction-restriction fragment length polymorphism protocols and correlated with patients' outcomes., Results: Out of the assessed variants, only the CXCR1 (also: interleukin-8-receptor alpha - IL-8RA ) +860C>G heterozygous polymorphism (rs2234671) was associated with decreased disease-free survival (DFS), cancer-specific survival (CSS), and overall survival (OS) (18/111 (16.2%), median DFS 14 months, log-rank p = 0.007; median CSS 31 months, log-rank p = 0.007; and median OS 6 months, log-rank p = 0.002), compared to the GG genotype (92/111 (82.9%), median DFS 55 months, median CSS 63 months, and median OS 33 months). In the multivariate analysis, +860C>G remained an independent prognostic factor for DFS (adjusted p = 0.008), CSS (adjusted p = 0.001), and OS (adjusted p = 0.001)., Conclusion: Genetic variant of CXCR1 +860C>G may serve as a molecular marker for DFS, CSS, and OS in patients undergoing curative-intent surgery for pCCA, indicating that the analysis of SNPs in genes involved in immune-mediated angiogenesis may help to identify patient subgroups at high risk for dismal oncological and overall outcome., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2022 by S. Karger AG, Basel.)

Genetic variation in the transmembrane channel-like (TMC)6/TMC8 region has been linked to β-type human papillomavirus (HPV) infection and squamous cell carcinoma (SCC) of the skin and the head and neck, α-type HPV persistence and progression to cervical cancer. The functional variant rs7208422 of the TMC8 gene was suggested to have a high impact on susceptibility to β-papillomaviruses and their oncogenic potential and to also have an influence on α-type HPV-related disease. The aim of the present study was to evaluate a possible influence of rs7208422 on penile cancer risk, a known α-type HPV-related malignancy. Therefore, the distribution of rs7208422 was determined by direct Sanger sequencing of 104 Caucasian penile SCC cases and compared to data of 3,810 controls taken from the literature. HPV detection was performed by usage of GP5+/6+ primers and subtype-specific PCR. It was observed that the distribution of rs7208422 followed the Hardy-Weinberg equilibrium in both cases and controls. HPV DNA was detected in 39% of the penile SCC cases. Overall, there was no significant difference in the distribution of rs7208422 neither between cases and controls (P=0.726) nor between HPV-positive and -negative penile SCC cases (P=0.747). There was also no association between rs7208422 genotypes and age of disease onset (P=0.740). In conclusion, the present data argue against a significant impact of rs7208422 on the risk for the development of penile SCC in Caucasians. Even in combination with the HPV status, the SNP appears not to influence the risk of penile SCC in HPV-positive cases., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020, Spandidos Publications.)

Histologically, bladder cancer is a heterogeneous group comprising urothelial carcinoma (UC), squamous cell carcinoma, adenocarcinomas (ACs), urachal carcinomas (UrCs), and small cell neuroendocrine carcinomas (SCCs). However, all bladder cancers have been treated so far uniformly, and targeted therapy options are still limited. Thus, we aimed to determine the protein expression/molecular status of commonly used cancer targets (programmed cell death 1 ligand 1 (PD-L1), mismatch repair (MMR), androgen and estrogen receptors (AR/ER), Nectin-4, tumor-associated calcium signal transducer 2 (Tacstd2, Trop-2), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and fibroblast growth factor receptor 3 (FGFR3)) to give first insights into whether patients with SCC, AC/UrCs, and squamous-differentiated carcinomas (Sq-BLCA) of the bladder could be eligible for targeted therapies. In addition, for MMR-deficient tumors, microsatellite instability was analyzed. We completed our own data with molecular data from The Cancer Genome Atlas (TCGA). We present ratios for each drug and cumulative ratios for multiple therapeutic options for each nonurothelial subtype. For example, 58.9% of SCC patients, 33.5% of AC/UrCs patients, and 79.3% of Sq-BLCA patients would be eligible for at least one of the analyzed targets. In conclusion, our findings hold promise for targeted therapeutic approaches in selected patients in the future, as various drugs could be applied according to the biomarker status.

Background: Response to immunotherapy in gastric cancer is associated with microsatellite instability (or mismatch repair deficiency) and Epstein-Barr virus (EBV) positivity. We therefore aimed to develop and validate deep learning-based classifiers to detect microsatellite instability and EBV status from routine histology slides., Methods: In this retrospective, multicentre study, we collected tissue samples from ten cohorts of patients with gastric cancer from seven countries (South Korea, Switzerland, Japan, Italy, Germany, the UK and the USA). We trained a deep learning-based classifier to detect microsatellite instability and EBV positivity from digitised, haematoxylin and eosin stained resection slides without annotating tumour containing regions. The performance of the classifier was assessed by within-cohort cross-validation in all ten cohorts and by external validation, for which we split the cohorts into a five-cohort training dataset and a five-cohort test dataset. We measured the area under the receiver operating curve (AUROC) for detection of microsatellite instability and EBV status. Microsatellite instability and EBV status were determined to be detectable if the lower bound of the 95% CI for the AUROC was above 0·5., Findings: Across the ten cohorts, our analysis included 2823 patients with known microsatellite instability status and 2685 patients with known EBV status. In the within-cohort cross-validation, the deep learning-based classifier could detect microsatellite instability status in nine of ten cohorts, with AUROCs ranging from 0·597 (95% CI 0·522-0·737) to 0·836 (0·795-0·880) and EBV status in five of eight cohorts, with AUROCs ranging from 0·819 (0·752-0·841) to 0·897 (0·513-0·966). Training a classifier on the pooled training dataset and testing it on the five remaining cohorts resulted in high classification performance with AUROCs ranging from 0·723 (95% CI 0·676-0·794) to 0·863 (0·747-0·969) for detection of microsatellite instability and from 0·672 (0·403-0·989) to 0·859 (0·823-0·919) for detection of EBV status., Interpretation: Classifiers became increasingly robust when trained on pooled cohorts. After prospective validation, this deep learning-based tissue classification system could be used as an inexpensive predictive biomarker for immunotherapy in gastric cancer., Funding: German Cancer Aid and German Federal Ministry of Health., Competing Interests: Declaration of interests JNK declares consulting roles for OWKIN France and Panakeia (UK) without any direct connection to this work; these roles started in April, 2021, after conducting the present study. JNK also declares honoraria from MSD and Eisai. DC declares grants from Medimmune/AstraZeneca, Clovis, Eli Lilly, 4SC, Bayer, Celgene, Leap, and Roche, and Scientific Board Membership for OVIBIO. DJ declares consulting services and advisory board participation for CureVac AG, Definiens, F Hoffmann-La Roche, Genmab A-S, Life Science Inkubator GmbH, VAXIMM AG, OncoOne Research & Development Research GmbH, and Oncolytics Biotech; payment or honoraria from SKK Kliniken Heilbronn, Georg Thieme Verlag, Terrapinn, Touch Medical Medica, BMS GmbH & Co KG, and MSD; reimbursements for expert opinion on medical questions from Wilhelm-Sander Foundation, Else-Kröner-Fresenius Foundation, Scherer Foundation, and NordForsk; meeting support (ie, for travel) from Amgen, Oryx GmbH, Roche Glycart AG, Parexel.com, IKTZ HD GmbH, and BMS; and leadership in the BMS Foundation Immunooncology. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

In the future, precision medicine with agents targeting specific genetic alterations will play an important role in bladder cancer. This includes both single genetic alterations (e.g. FGFR3) and gene panel analyses in patients with no further therapeutic options, rare cancer subtypes or unusual clinical courses. These molecular analyses can be carried out on formalin-fixed paraffin-embedded tumor samples and the results should be discussed in interdisciplinary molecular tumor boards in order to either recommend approved targeted therapies or suggest patients for molecular-based clinical trials, compassionate use programs or off-label use of drugs. The remuneration of molecular diagnostics is largely well-represented for the outpatient sector in Germany; however, the covering of treatment costs must currently be approved by the health insurances., (© 2021. Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

The oncological role of the density of nerve fibers (NFs) in the tumor microenvironment (TME) in intrahepatic cholangiocarcinoma (iCCA) remains to be determined. Therefore, data of 95 iCCA patients who underwent hepatectomy between 2010 and 2019 was analyzed regarding NFs and long-term outcome. Extensive group comparisons were carried out and the association of cancer-specific survival (CSS) and recurrence-free survival (RFS) with NFs were assessed using Cox regression models. Patients with iCCA and NFs showed a median CSS of 51 months (5-year-CSS = 47%) compared to 27 months (5-year-CSS = 21%) in patients without NFs ( p = 0.043 log rank). Further, NFs (hazard ratio (HR) = 0.39, p = 0.002) and N-category (HR = 2.36, p = 0.010) were identified as independent predictors of CSS. Patients with NFs and without nodal metastases displayed a mean CSS of 89 months (5-year-CSS = 62%), while patients without NFs or with nodal metastases but not both showed a median CCS of 27 months (5-year-CSS = 25%) and patients with both positive lymph nodes and without NFs showed a median CCS of 10 months (5-year-CSS = 0%, p = 0.001 log rank). NFs in the TME are, therefore, a novel and important prognostic biomarker in iCCA patients. NFs alone and in combination with nodal status is suitable to identify iCCA patients at risk of poor oncological outcomes following curative-intent surgery.

In the 2016 WHO classification of genitourinary tumors Muellerian tumors of the urinary tract (MTUT) comprise clear cell adenocarcinomas and endometrioid carcinomas. Since these rare tumors remained understudied, we aimed to characterize their molecular background by performing DNA- and RNA-based targeted panel sequencing. All tumors ( n = 11) presented single nucleotide alterations (SNVs), with ARID1A mutations being the most prevalent (5/11, 45%). Besides frequent ARID1A mutations, loss of ARID1A protein is not a suitable marker since protein expression is (partly) preserved also in mutated cases. Copy number alterations (CNVs) were found in 64% of cases (7/11), exclusively gene amplifications. Interestingly, a functionally relevant RSPO2 gene fusion/microdeletion was discovered in the endometrioid adenocarcinoma case. Comparing our findings with mutational profiles of other tumor entities, absence of TERT promoter mutations argues for a non-urothelial origin. No similarities were also found between MTUT and kidney cancers while parallels were observed for specific SNVs with endometrial carcinomas. In conclusion, immunohistochemical PAX8-positivity and lack of TERT promoter mutations could serve as key diagnostic features in difficult cases. Thus, understanding the molecular background of these tumors helps to refine treatment options and offers the possibility of targeted therapies in cases where needed.

Background: The reduction of perioperative morbidity is a main surgical goal in patients undergoing partial hepatectomy for hepatocellular carcinoma (HCC). Here, we investigated clinical determinants of perioperative morbidity in a European cohort of patients undergoing surgical resection for HCC., Methods: A total 136 patients who underwent partial hepatectomy for HCC between 2011 and 2017 at our institution were included in this analysis. The associations between major surgical complications (Clavien-Dindo ≥ 3) and overall morbidity (Clavien-Dindo ≥ 1) with clinical variables were assessed using univariate and multivariable binary logistic regression analysis., Results: Multivariable analysis identified the Child-Pugh-Score (CPS, HR = 3.23; p = 0.040), operative time (HR = 5.63; p = 0.003), and intraoperatively administered fresh frozen plasma (FFP, HR = 5.62; p = 0.001) as independent prognostic markers of major surgical complications, while only FFP (HR = 6.52; p = 0.001) was associated with morbidity in the multivariable analysis. The transfusion of FFP was not associated with perioperative liver functions tests., Conclusions: The intraoperative administration of FFP is an important independent predictor of perioperative morbidity in patients undergoing partial hepatectomy for HCC.

This study aims at characterizing the role of the putative tumor suppressor ITIH5 in basal-type bladder cancers (BLCA). By sub-classifying TCGA BLCA data, we revealed predominant loss of ITIH5 expression in the basal/squamous-like (BASQ) subtype. ITIH5 expression inversely correlated with basal-type makers such as KRT6A and CD44. Interestingly, Kaplan-Meier analyses showed longer recurrence-free survival in combination with strong CD44 expression, which is thought to mediate ITIH-hyaluronan (HA) binding functions. In vitro, stable ITIH5 overexpression in two basal-type BLCA cell lines showing differential CD44 expression levels, i.e., with (SCaBER) and without squamous features (HT1376), demonstrated clear inhibition of cell and colony growth of BASQ-type SCaBER cells. ITIH5 further enhanced HA-associated cell-matrix attachment, indicated by altered size and number of focal adhesion sites resulting in reduced cell migration capacities. Transcriptomic analyses revealed enrichment of pathways and processes involved in ECM organization, differentiation and cell signaling. Finally, we provide evidence that ITIH5 increase sensitivity of SCaBER cells to chemotherapeutical agents (cisplatin and gemcitabine), whereas responsiveness of HT1376 cells was not affected by ITIH5 expression. Thus, we gain further insights into the putative role of ITIH5 as tumor suppressor highlighting an impact on drug response potentially via the HA-CD44 axis in BASQ-type BLCA.

The c-Jun N-terminal kinase (JNK) signaling pathway mediates adaptation to stress signals and has been associated with cell death, cell proliferation, and malignant transformation in the liver. However, up to now, its function was experimentally studied mainly in young mice. By generating mice with combined conditional ablation of Jnk1 and Jnk2 in liver parenchymal cells (LPCs) (JNK1/2 LPC-KO mice; KO, knockout), we unraveled a function of the JNK pathway in the regulation of liver homeostasis during aging. Aging JNK1/2 LPC-KO mice spontaneously developed large biliary cysts that originated from the biliary cell compartment. Mechanistically, we could show that cyst formation in livers of JNK1/2 LPC-KO mice was dependent on receptor-interacting protein kinase 1 (RIPK1), a known regulator of cell survival, apoptosis, and necroptosis. In line with this, we showed that RIPK1 was overexpressed in the human cyst epithelium of a subset of patients with polycystic liver disease. Collectively, these data reveal a functional interaction between JNK signaling and RIPK1 in age-related progressive cyst development. Thus, they provide a functional linkage between stress adaptation and programmed cell death (PCD) in the maintenance of liver homeostasis during aging., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Aims: Malignant germ cell tumours (GCTs) of the testis are rare neoplasms, but the most common solid malignancies in young men. World Health Organization guidelines divide GCTs into five types, for which numerous immunohistochemical markers allow exact histological subtyping in the majority of cases. In contrast, a germ cell origin is often hard to prove in metastatic GCTs that have developed so-called somatic malignant transformation. A high percentage, up to 89%, of GCTs are characterised by the appearance of isochromosome 12p [i(12p)]. Fluorescence in-situ hybridisation has been the most common diagnostic method for the detection of i(12p) so far, but has the disadvantages of being time-consuming, demanding, and not being a stand-alone method. The aim of the present study was to establish a quantitative real-time polymerase chain reaction assay as an independent method for detecting i(12p) and regional amplifications of the short arm of chromosome 12 by using DNA extracted from formalin-fixed paraffin-embedded tissue., Methods and Results: A cut-off value to distinguish between the presence and absence of i(12p) was established in a control set consisting of 36 tumour-free samples. In a training set of 149 GCT samples, i(12p) was detectable in 133 tumours (89%), but not in 16 tumours (11%). In a test set containing 27 primary and metastatic GCTs, all 16 tumours with metastatic spread and/or somatic malignant transformation were successfully identified by the detection of i(12p)., Conclusion: In summary, the qPCR assay presented here can help to identify, further characterise and assign a large proportion of histologically inconclusive malignancies to a GCT origin., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA) are both deadly cancers and they share many biological features besides their close anatomical location. One of the main histological features is neurotropism, which results in frequent perineural invasion. The underlying mechanism of cancer cells favoring growth by and through the nerve fibers is not fully understood. In this review, we provide knowledge of these cancers with frequent perineural invasion. We discuss nerve fiber crosstalk with the main different components of the tumor microenvironment (TME), the immune cells, and the fibroblasts. Also, we discuss the crosstalk between the nerve fibers and the cancer. We highlight the shared signaling pathways of the mechanisms behind perineural invasion in PDAC and CCA. Hereby we have focussed on signaling neurotransmitters and neuropeptides which may be a target for future therapies. Furthermore, we have summarized retrospective results of the previous literature about nerve fibers in PDAC and CCA patients. We provide our point of view in the potential for nerve fibers to be used as powerful biomarker for prognosis, as a tool to stratify patients for therapy or as a target in a (combination) therapy. Taking the presence of nerves into account can potentially change the field of personalized care in these neurotropic cancers.

B cells and tertiary lymphoid structures (TLS) are reported to be important in survival in cancer. Pancreatic Cancer (PDAC) is one of the most lethal cancer types, and currently, it is the seventh leading cause of cancer-related death worldwide. A better understanding of tumor biology is pivotal to improve clinical outcome. The desmoplastic stroma is a complex system in which crosstalk takes place between cancer-associated fibroblasts, immune cells and cancer cells. Indirect and direct cellular interactions within the tumor microenvironment (TME) drive key processes such as tumor progression, metastasis formation and treatment resistance. In order to understand the aggressiveness of PDAC and its resistance to therapeutics, the TME needs to be further unraveled. There are some limited data about the influence of nerve fibers on cancer progression. Here we show that small nerve fibers are located at lymphoid aggregates in PDAC. This unravels future pathways and has potential to improve clinical outcome by a rational development of new therapeutic strategies.

Tumor angiogenesis plays a pivotal role in hepatocellular carcinoma (HCC) biology. Identifying molecular prognostic markers is critical to further improve treatment selection in these patients. The present study analyzed a subset of 10 germline polymorphisms involved in tumor angiogenesis pathways and their impact on prognosis in HCC patients undergoing partial hepatectomy in a curative intent. Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from 127 HCC patients at a German primary care hospital. Genomic DNA was extracted, and genotyping was carried out using polymerase chain reaction (PCR)-restriction fragment length polymorphism-based protocols. Polymorphisms in interleukin-8 (IL-8) (rs4073; p = 0.047, log-rank test) and vascular endothelial growth factor (VEGF C + 936T) (rs3025039; p = 0.045, log-rank test) were significantly associated with disease-free survival (DFS). After adjusting for covariates in the multivariable model, IL-8 T-251A (rs4073) (adjusted p = 0.010) and a combination of "high-expression" variants of rs4073 and rs3025039 (adjusted p = 0.034) remained significantly associated with DFS. High-expression variants of IL-8 T-251A may serve as an independent molecular marker of prognosis in patients undergoing surgical resection for HCC. Assessment of the patients' individual genetic risks may help to identify patient subgroups at high risk for recurrence following curative-intent surgery.

Aims: Pseudosarcomatous myofibroblastic proliferations (PSMPs) of the urinary bladder are diagnostically challenging. Diagnostic difficulties are mainly due to frequent cytokeratin expression, variable ALK expression and worrisome morphological features suggestive of malignancy. Conversely, sarcomatoid urothelial carcinoma (UC) may show bland inflammatory myofibroblastic tumour (IMT)-like morphology. TERT promoter mutations are characteristic events in urothelial cancers, but have not been studied in PSMPs., Methods and Results: We compared histomorphological and immunohistochemical features and TERT promoter status in 16 PSMPs and 18 sarcomatoid UC. In a subset of PSMPs, RNA sequencing was performed. At least focal IMT-like morphology was seen in nine of 17 sarcomatoid UC. Atypical mitoses, differentiated urothelial component and heterologous elements were the most reliable distinguishing histomorphological features of sarcomatoid UC, if present. A panel of immunohistochemistry (IHC) including ALK (clone D5F3), p53 pattern, p63 and GATA3 reliably distinguished PSMP from sarcomatoid UC. GATA3 (P = 0.001) and p53 patterns (mutant versus wild-type; P < 0.001) were differentially expressed between PSMPs and sarcomatoid UC. Diffuse pancytokeratin staining was significantly associated with PSMPs (10 of 13) compared to four of 14 sarcomatoid UCs (P = 0.012). TERT promoter mutations were found in 17 of 18 sarcomatoid UC versus none of 16 PSMPs (P < 0.001). RNA sequencing revealed ALK genetic rearrangements in one of two ALK-positive and one of 10 ALK-negative PSMPs, which revealed a novel FN1/RET gene fusion., Conclusion: Careful histomorphological analysis and differential IHC reliably distinguish the majority of PSMPs and sarcomatoid UC. In equivocal cases, TERT promoter mutation analysis and/or detection of ALK expression/rearrangements are valuable additional diagnostic adjuncts, strongly supporting sarcomatoid UC and PSMP, respectively., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Dysfunction of the SWI/SNF complex has been observed in various cancers including urothelial carcinomas. However, the clinical impact of the SWI/SNF complex in squamous-differentiated bladder cancers (sq-BLCA) remains unclear. Therefore, we aimed to analyze potential expression loss and genetic alterations of (putative) key components of the SWI/SNF complex considering the co-occurrence of genetic driver mutations and PD-L1 expression as indicators for therapeutic implications. Assessment of ARID1A , SMARCA2 , SMARCA4 , SMARCB1/INI1 , SMARCC1 , SMARCC2 and PBRM1 mutations in a TCGA data set of sq-BLCA ( n = 45) revealed that ARID1A was the most frequently altered SWI/SNF gene (15%) while being associated with protein downregulation. Genetic alterations and loss of ARID1A were confirmed by Targeted Next Generation Sequencing (NGS) (3/6) and immunohistochemistry (6/116). Correlation with further mutational data and PD-L1 expression revealed co-occurrence of ARID1A loss and TP53 mutations, while positive correlations with other driver mutations such as PIK3CA were not observed. Finally, a rare number of sq-BLCA samples were characterized by both ARID1A protein loss and strong PD-L1 expression suggesting a putative benefit upon immune checkpoint inhibitor therapy. Hence, for the first time, our data revealed expression loss of SWI/SNF subunits in sq-BLCA, highlighting ARID1A as a putative target of a small subgroup of patients eligible for novel therapeutic strategies.