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Renal cell carcinoma (RCC) is the deadliest primary genitourinary malignancy typically associated with asymptomatic initial presentation and poorly predictable survival. Next to established risk factors, tumor microenvironment may alter metastatic capacity and immune landscape. Due to their high concentrations, sulfoglycolipids (sulfatides) were among the first well-described antigens in RCC that are associated with worse prognosis. As sulfatide detection in routine diagnostics is not possible, we aimed to test the prognostic value of its protein counterpart, sulfatide-producing enzyme Gal3ST1. We performed retrospective long-term follow up analysis of Gal3ST1 expression as prognostic risk factor in a representative RCC patient cohort. We observed differentially regulated Gal3ST1 expression in all RCC types, being significantly more associated with clear cell RCC than to chromophobe RCC (p = 0.001). Surprisingly, in contrast to published observations from in vitro models, we could not confirm an association between Gal3ST1 expression and a malignant clinical behaviour of the RCC. In our cohort, Gal3ST1 did not significantly influence progression-free survival (Hazard Ratio (HR): 1.7 95% CI (0.6–4.9), p = 0.327). Particularly after adjusting for histology, T-stage, N-status and M-status at baseline, we observed no independent prognostic effect (HR = 1.0 95% CI (0.3–3.3), p = 0.96). The analysis of Gal3ST1 mRNA expression in a TCGA dataset supported the results of our cohort. Thus, Gal3ST1 might help to differentiate between chromophobe RCC and other frequent RCC entities but—despite previously published data from cell culture models—does not qualify as a prognostic marker for RCC. Further investigation of regulatory mechanisms of sulfatide metabolism in human RCC microenvironment is necessary to understand the role of this quantitatively prominent glycosphingolipid in RCC progression.

The thymus prevents autoimmune diseases through mechanisms that operate in the cortex and medulla, comprising positive and negative selection and the generation of regulatory T-cells (Tregs). Egress from the thymus through the perivascular space (PVS) to the blood is another possible checkpoint, as shown by some autoimmune/immunodeficiency syndromes. In polygenic autoimmune diseases, subtle thymic dysfunctions may compound genetic, hormonal and environmental cues. Here, we cover (a) tolerance-inducing cell types, whether thymic epithelial or tuft cells, or dendritic, B- or thymic myoid cells; (b) tolerance-inducing mechanisms and their failure in relation to thymic anatomic compartments, and with special emphasis on human monogenic and polygenic autoimmune diseases and the related thymic pathologies, if known; (c) polymorphisms and mutations of tolerance-related genes with an impact on positive selection (e.g. the gene encoding the thymoproteasome-specific subunit, PSMB11), promiscuous gene expression (e.g. AIRE, PRKDC, FEZF2, CHD4), Treg development (e.g. SATB1, FOXP3), T-cell migration (e.g. TAGAP) and egress from the thymus (e.g. MTS1, CORO1A); (d) myasthenia gravis as the prototypic outcome of an inflamed or disordered neoplastic ‘sick thymus’.

Thymomas exhibit a unique genomic landscape, comprising the lowest on average total mutational burden among adult human cancers; a unique point mutation in the GTF2I gene in WHO type A and AB thymomas (and rarely others); almost unique KMT2A-MAML2 translocations in rare WHO type B2 and B3 thymomas; a unique YAP1-MAML2 translocation in almost all metaplastic thymomas; and unique miRNA profiles in relation to GTF2I mutational status and WHO histotypes. While most thymomas can be diagnosed solely on the basis of morphological features, mutational analyses can solve challenging differential diagnostic problems. No molecular biomarkers have been identified that predict the response of unresectable thymomas to chemotherapy or agents with known molecular targets. Despite the common and strong expression of PDL1 in thymomas, immune checkpoint inhibitors are rarely applicable due to the poor predictability of common, life-threatening autoimmune side effects that are related to the unrivaled propensity of thymomas towards autoimmunity.

ZusammenfassungEine der primären Aufgaben der Rechtsmedizin ist die Aufklärung unerwarteter Todesfälle. Speziell das Erkennen seltener Todesursachen setzt ein fundiertes Wissen um die typischen makromorphologischen Befunde sowie das Einleiten und die Interpretation entsprechend geeigneter Ergänzungsuntersuchungen, etwa histologischer Verfahren, voraus. Wichtige Differenzialdiagnosen müssen bekannt sein, damit die Todesursache mit vertretbarem diagnostischem Aufwand festgestellt werden kann.In der vorliegenden Arbeit werden charakteristische makroskopische und mikroskopische Befunde der kardialen Sarkoidose (oder granulomatösen Myokarditis) und deren Abgrenzung zur Riesenzellmyokarditis anhand von 2 Fällen demonstriert. In beiden Fällen gab der plötzliche Tod einer erwachsenen Person mittleren Alters Anlass zu einer gerichtlichen Obduktion, die jeweils zur Diagnose einer kardialen Sarkoidose führte. Die unterschiedlichen Krankheitsverläufe und Befunde werden unter Berücksichtigung einschlägiger wissenschaftlicher Literatur diskutiert. Durch eine sorgfältige Erhebung der Vorgeschichte und der makromorphologischen Befunde kann die Verdachtsdiagnose einer kardialen Sarkoidose unmittelbar nach der Obduktion geäußert und durch histopathologische Untersuchungen bestätigt werden. Es wird deutlich, dass eine interdisziplinäre Fallarbeit bei seltenen Erkrankungen wie der kardialen Sarkoidose eine schnelle Diagnose ermöglicht.

INTRODUCTION: The WHO classification of pulmonary neuroendocrine tumors (PNETs) is also used to classify thymic NETs (TNETs) into typical and atypical carcinoid (TC and AC), large cell neuroendocrine carcinoma (LCNEC), and small cell carcinoma (SCC), but little is known about the usability of alternative classification systems. METHODS: One hundred seven TNET (22 TC, 51 AC, 28 LCNEC, and 6 SCC) from 103 patients were classified according to the WHO, the European Neuroendocrine Tumor Society, and a grading-related PNET classification. Low coverage whole-genome sequencing and immunohistochemical studies were performed in 63 cases. A copy number instability (CNI) score was applied to compare tumors. Eleven LCNEC were further analyzed using targeted next-generation sequencing. Morphologic classifications were tested against molecular features. RESULTS: Whole-genome sequencing data fell into three clusters: CNIlow, CNIint, and CNIhigh. CNIlow and CNIint comprised not only TC and AC, but also six LCNECs. CNIhigh contained all SCC and nine LCNEC, but also three AC. No morphologic classification was able to predict the CNI cluster. Cases where primary tumors and metastases were available showed progression from low-grade to higher-grade histologies. Analysis of LCNEC revealed a subgroup of intermediate NET G3 tumors that differed from LCNEC by carcinoid morphology, expression of chromogranin, and negativity for enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). CONCLUSIONS: TNETs fall into three molecular subgroups that are not reflected by the current WHO classification. Given the large overlap between TC and AC on the one hand, and AC and LCNEC on the other, we propose a morphomolecular grading system, Thy-NET G1-G3, instead of histologic classification for patient stratification and prognostication. peerReviewed

Rhabdomyosarcomas (RMS) are rare and often lethal diseases. It is assumed that the tumor microenvironment (TME) of RMS exerts an immunosuppressive function, but there is currently no systematic analysis of the immune cells infiltrating sarcoma tissue. Focusing on two common types of RMS (alveolar [RMA] and embryonal [RME]), we performed a comprehensive immunohistochemical analysis of tumor-infiltrating immune cells in the TME. We performed a qualitative estimation of infiltrating immune cells in the tumor microenvironment by an experienced pathologist as well as a quantitative digital pathology analysis. We found that (1) manual and automatic quantification of tumor-infiltrating immune cells were consistent; (2) RME tumors showed a higher degree of immune cell infiltration than RMA tumors but (3) the number of tumor infiltrating lymphocytes was low compared to other solid tumor types; (4) microvascular density correlated with immune cell infiltration and (5) CD163 positive macrophages as well as CD54 positive microvessels were more often detected in RME than in RMA and correlated with patient overall and event free survival. Our systematic analysis provides a comprehensive view of the immune landscape of RMS which needs to be taken into account for developing immunotherapies for this rare type of cancer.

Fatty acid-binding protein 5 (FABP5), a transport protein for lipophilic molecules, has been proposed as protein marker in prostate cancer (PCa). The role of FABP5 gene expression is merely unknown. In two cohorts of PCa patients who underwent radical prostatectomy (n = 40 and n = 57) and one cohort of patients treated with palliative transurethral resection of the prostate (pTUR-P; n = 50) FABP5 mRNA expression was analyzed with qRT-PCR. Expression was correlated with clinical parameters. BPH tissue samples served as control. To independently validate findings on FABP5 expression, three microarray and sequencing datasets were reanalyzed (MSKCC 2010 n = 216; TCGA 2015 n = 333; mCRPC, Nature Medicine 2016 n = 114). FABP5 expression was correlated with ERG-fusion status, TCGA subtypes, cancer driver mutations and the expression of druggable downstream pathway components. FABP5 was overexpressed in PCa compared to BPH in the cohorts analyzed by qRT-PCR (radical prostatectomy p = 0.003, p = 0.010; pTUR-P p = 0.002). FABP5 expression was independent of T stage, Gleason Score, nodal status and PSA level. FABP5 overexpression was associated with the absence of TMPRSS2:ERG fusion (p

Background: Perioperative cisplatin-based chemotherapy (CBC) can improve the outcome of patients with muscle-invasive bladder cancer (MIBC), but it is still to be defined which patients benefit. Mutations in DNA damage response genes (DDRG) can predict the response to CBC. The value of DDRG expression as a marker of CBC treatment effect remains unclear. Material and methods: RNA expression of the nine key DDRG (BCL2, BRCA1, BRCA2, ERCC2, ERCC6, FOXM1, RAD50, RAD51, and RAD52) was assessed by qRT-PCR in a cohort of 61 MICB patients (median age 66 y, 48 males, 13 females) who underwent radical cystectomy in a tertiary care center. The results were validated in the The Cancer Genome Atlas (TCGA) cohort of MIBC (n = 383). Gene expression was correlated with disease-free survival (DFS) and overall survival (OS). Subgroup analyses were performed in patients who received adjuvant cisplatin-based chemotherapy (ACBC) (Mannheim n = 20 and TCGA n = 75). Results: Low expression of RAD52 was associated with low DFS in both the Mannheim and the TCGA cohorts (Mannheim: p = 0.039, TCGA: p = 0.017). This was especially apparent in subgroups treated with ACBC (Mannheim: p = 0.0059, TCGA: p = 0.012). Several other genes showed an influence on DFS in the Mannheim cohort (BRCA2, ERCC2, FOXM1) where low expression was associated with poor DFS (p &lt, 0.05 for all). This finding was not fully supported by the data in the TCGA cohort, where high expression of FOXM1 and BRCA2 correlated with poor DFS. Conclusion: Low expression of RAD52 correlated with decreased DFS in the Mannheim and the TCGA cohort. This effect was especially pronounced in the subset of patients who received ACBC, making it a promising indicator for response to ACBC on the level of gene expression.

Background Growing evidence supports the pivotal role of long non-coding RNAs (lncRNAs) in the regulation of cancer development and progression. Their expression patterns and biological function in muscle invasive bladder cancer (MIBC) remain elusive. Methods Transcript levels of lncRNA miR-31 host gene (MIR31HG) and its splice variants were measured in our MIBC cohort (n = 102) by qRT-PCR, and validated in silico by the TCGA cohort (n = 370). Kaplan-Meier and multiple Cox regression analysis were conducted to evaluate the survival significance of MIR31HG and its splice variants. Functional experiments were performed to examine the proliferation and migration abilities of MIR31HG and its splice variants by knockdown approaches. Results In this study, a decreased expression of MIR31HG was found in bladder cancer cells and tissues, except in the basal subtype. Survival analysis showed that high expression of MIR31HG was associated with poor overall survival (OS) and disease-free survival (DFS) in patients with MIBC of basal subtype. Two splice variants of MIR31HG lacking exon 1 (MIR31HGΔE1) and exon 3 (MIR31HGΔE3) were identified to have specific expression patterns in different molecular subtypes of our MIBC cohort. MIR31HGΔE3 was highly expressed in basal subtype tumors. A high expression of MIR31HGΔE1 and MIR31HGΔE3 was associated with worse OS and DFS in our cohort. In vitro experiments revealed that knockdown of MIR31HG inhibits cell proliferation, colony formation, and migration in bladder cancer. Cell proliferation and migration assays after knockdown of splice variants of MIR31HG showed corresponding roles for the full-length transcript. Conclusions Our study demonstrates that MIR31HG and its splice variants could serve as biomarkers for the classification and prognosis prediction of patients with MIBC.

Background The cell surface interleukin 22 (IL-22) receptor complex is mainly expressed in epithelial and tissue cells like pancreatitis cells. Recent studies described that IL-22R was overexpressed in malignant diseases and was associated with a poor overall survival (OS). The role of IL-22RA1 gene expression in muscle invasive bladder cancer (MIBC) has not been investigated, yet. Objectives The aim of this study was to analyze the role of IL-22RA1 gene expression in patients with MIBC. Methods In a cohort of 114 patients with MIBC who underwent radical cystectomy, IL-22RA1 gene expression was analyzed with qRT-PCR and correlated with clinical parameters. Furthermore, Kaplan-Meier and Cox regression analysis were performed. For validation, an in silico dataset (TCGA 2017, n=407) was reanalyzed. Results IL-22RA1 gene expression was independent of clinicopathological parameters like age (P=0.2681), T stage (P=0.2130), nodal status (P=0.3238) and lymph vascular invasion (LVI, P=0.5860) in patients with MIBC. A high expression of IL-22RA1 was associated with a shorter OS (P=0.0040) and disease-specific survival (P=0.0385). Furthermore, a shorter disease-free survival (DFS) was also associated with a high expression of IL-22RA1 (P=0.0102). In the multivariable analysis, IL-22RA1 expression was an independent prognostic predictors regarding OS (P=0.0096, HR=0.48). In the TCGA cohort, IL-22RA1 expression was independent regarding to OS and DFS. Conclusion A high IL-22RA1 gene expression was associated with worse outcome. Furthermore, IL-22RA1 represented an independent predictor regarding OS in our cohort and therefore might be used for risk stratification in patients with MIBC.

Paracrine and long-range signaling via extracellular vesicles, such as exosomes and microvesicles, is deemed crucial for tumorigenesis, invasion and spread of solid tumors. The ESCRT machinery (endosomal sorting complexes required for transport) and Rab-proteins act as key players in vesicular trafficking and secretion. Yet, their role in prostate cancer (PCa) is unknown. Therefore, this study aimed to elucidate the relevance of these components in PCa. In silico reanalysis of genes with known involvement in vesicular trafficking and secretion in an existing microarray dataset revealed low expression of RAB27A, RAB27B and VPS36 to be predictive for reduced BCR-free survival in patients with localized PCa (p=0.033, 0.025 and 0.005). In the same microarray dataset underexpression of RAB27A, RAB27B and VPS36 was seen in distant metastases (p

Summary Background The Thymectomy Trial in Non-Thymomatous Myasthenia Gravis Patients Receiving Prednisone (MGTX) showed that thymectomy combined with prednisone was superior to prednisone alone in improving clinical status as measured by the Quantitative Myasthenia Gravis (QMG) score in patients with generalised non-thymomatous myasthenia gravis at 3 years. We investigated the long-term effects of thymectomy up to 5 years on clinical status, medication requirements, and adverse events. Methods We did a rater-blinded 2-year extension study at 36 centres in 15 countries for all patients who completed the randomised controlled MGTX and were willing to participate. MGTX patients were aged 18 to 65 years at enrolment, had generalised non-thymomatous myasthenia gravis of less than 5 years' duration, had acetylcholine receptor antibody titres of 1·00 nmol/L or higher (or concentrations of 0·50–0·99 nmol/L if diagnosis was confirmed by positive edrophonium or abnormal repetitive nerve stimulation, or abnormal single fibre electromyography), had Myasthenia Gravis Foundation of America Clinical Classification Class II–IV disease, and were on optimal anticholinesterase therapy with or without oral corticosteroids. In MGTX, patients were randomly assigned (1:1) to either thymectomy plus prednisone or prednisone alone. All patients in both groups received oral prednisone at doses titrated up to 100 mg on alternate days until they achieved minimal manifestation status. The primary endpoints of the extension phase were the time-weighted means of the QMG score and alternate-day prednisone dose from month 0 to month 60. Analyses were by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00294658. It is closed to new participants, with follow-up completed. Findings Of the 111 patients who completed the 3-year MGTX, 68 (61%) entered the extension study between Sept 1, 2009, and Aug 26, 2015 (33 in the prednisone alone group and 35 in the prednisone plus thymectomy group). 50 (74%) patients completed the 60-month assessment, 24 in the prednisone alone group and 26 in the prednisone plus thymectomy group. At 5 years, patients in the thymectomy plus prednisone group had significantly lower time-weighted mean QMG scores (5·47 [SD 3·87] vs 9·34 [5·08]; p=0·0007) and mean alternate-day prednisone doses (24 mg [SD 21] vs 48 mg [29]; p=0·0002) than did those in the prednisone alone group. 14 (42%) of 33 patients in the prednisone group, and 12 (34%) of 35 in the thymectomy plus prednisone group, had at least one adverse event by month 60. No treatment-related deaths were reported during the extension phase. Interpretation At 5 years, thymectomy plus prednisone continues to confer benefits in patients with generalised non-thymomatous myasthenia gravis compared with prednisone alone. Although caution is appropriate when generalising our findings because of the small sample size of our study, they nevertheless provide further support for the benefits of thymectomy in patients with generalised non-thymomatous myasthenia gravis. Funding National Institutes of Health, National Institute of Neurological Disorders and Stroke.

Background For virtually every patient with colorectal cancer (CRC), hematoxylin–eosin (HE)–stained tissue slides are available. These images contain quantitative information, which is not routinely used to objectively extract prognostic biomarkers. In the present study, we investigated whether deep convolutional neural networks (CNNs) can extract prognosticators directly from these widely available images. Methods and findings We hand-delineated single-tissue regions in 86 CRC tissue slides, yielding more than 100,000 HE image patches, and used these to train a CNN by transfer learning, reaching a nine-class accuracy of >94% in an independent data set of 7,180 images from 25 CRC patients. With this tool, we performed automated tissue decomposition of representative multitissue HE images from 862 HE slides in 500 stage I–IV CRC patients in the The Cancer Genome Atlas (TCGA) cohort, a large international multicenter collection of CRC tissue. Based on the output neuron activations in the CNN, we calculated a “deep stroma score,” which was an independent prognostic factor for overall survival (OS) in a multivariable Cox proportional hazard model (hazard ratio [HR] with 95% confidence interval [CI]: 1.99 [1.27–3.12], p = 0.0028), while in the same cohort, manual quantification of stromal areas and a gene expression signature of cancer-associated fibroblasts (CAFs) were only prognostic in specific tumor stages. We validated these findings in an independent cohort of 409 stage I–IV CRC patients from the “Darmkrebs: Chancen der Verhütung durch Screening” (DACHS) study who were recruited between 2003 and 2007 in multiple institutions in Germany. Again, the score was an independent prognostic factor for OS (HR 1.63 [1.14–2.33], p = 0.008), CRC-specific OS (HR 2.29 [1.5–3.48], p = 0.0004), and relapse-free survival (RFS; HR 1.92 [1.34–2.76], p = 0.0004). A prospective validation is required before this biomarker can be implemented in clinical workflows. Conclusions In our retrospective study, we show that a CNN can assess the human tumor microenvironment and predict prognosis directly from histopathological images., Jakob Nikolas Kather and colleagues demonstrate the ability of deep convolutional neural networks to extract prognostically relevant information from routine histopathological images collected from colorectal cancer patients., Author summary Why was this study done? Colorectal cancer (CRC) is a common disease with a variable clinical course, and there is a high clinical need to more accurately predict the outcome of individual patients. For almost every CRC patient, histological slides of tumor tissue are routinely available. Deep learning can be used to extract information from very complex images, and we hypothesized that deep learning can predict clinical outcome directly from histological images of CRC. What did the researchers do and find? We trained a deep neural network to identify different tissue types that are abundant in histological images of CRC, especially nontumorous (“stromal”) tissue types. We showed that this deep neural network can decompose complex tissue into its constituent parts and that the abundance of each of these tissue parts can be aggregated in a prognostic score. In two independent, multicenter patient cohorts, we showed that this score improves survival prediction compared to the Union Internationale Contre le Cancer (UICC) staging system, which is the current state of the art. What do these findings mean? Deep learning is an inexpensive tool to predict the clinical course of CRC patients based on ubiquitously available histological images. Prospective validation studies are needed to firmly establish this biomarker for routine clinical use.

Muscle-invasive bladder cancer (MIBC) is characterized by high recurrence and rapid progression. Progression is linked to changes in glycan structures and altered levels of glycosyltransferases. The relationship of mRNA expression by glycosyltransferase genes B4GALT1, EXT1, MGAT5B, and POFUT1 to the probability of surviving MIBC after radical cystectomy has not yet been investigated. mRNA expression was analyzed using qRT-PCR in formalin-fixed and paraffin-embedded tumor samples (n = 105; 74% male patients and 26% female patients; median age = 72 years), correlated with histopathological variables, and evaluated by means of multivariable Cox regression analysis regarding to overall survival (OS), cancer-specific survival (CSS), and disease-free survival (DFS). Multivariable Cox regression analysis identified POFUT1 mRNA expression as superior prognostic marker, compared with currently used histological tumor stage methods, for CSS by MIBC patients following radical cystectomy. Thus, the patients with low POFUT1 mRNA were at a 4.9-fold greater risk for cancer-specific death according to the multivariable analysis (p = 0.0001). Low mRNA levels predicted poor survival according to the Kaplan-Meier analysis ((POFUT1:OS p = 0.0014; CSS p = 0.0007; DFS p = 0.0088); (EXT1:OS p = 0.0150; CSS p = 0.0130; DFS p = 0.0286); (B4GALT1:CSS p = 0.0134; DFS p = 0.0493)). A subgroup analysis of patients without lymph node metastasis (pN−; n = 73) indicated that low expression of POFUT1 predicted reduced OS (p = 0.0073), CSS (p = 0.0058,) and DSS (p = 0.0079). Low levels of POFUT1 mRNA are an independent prognostic indicator for OS and CSS in MIBC patients following radical cystectomy. This finding demonstrates the importance of altered glycosylation for the progress of MIBC., Highlights • Low POFUT1 mRNA expression is associated with a higher risk for overall and cancer-specific death in MIBC treated with RC. • MIBC patients with pN0 histology and, decreased POFUT1 mRNA levels showed poor outcome for OS, CSS and, DFS. • POFUT1 mRNA is an independent prognostic indicator for OS and CSS in multivariable analysis of MIBC patients following RC.

Thymome sind seltene Tumoren, zählen bei Erwachsenen aber zu den häufigsten Neoplasien des Mediastinums. Sie zeigen eine enorme histologische Variabilität selbst innerhalb einzelner Entitäten und einzigartige biologische und genetische Veränderungen. Außerdem sind Thymome mit verschiedenen histologischen Mustern teilweise häufiger als manche „reinen“ Histotypen. Da keine einzelnen immunhistologischen und molekularen Marker zur eindeutigen Klassifikation zur Verfügung stehen, sind für die Erkennung mancher Thymome oft komplexe diagnostische Algorithmen unerlässlich. Deren verfeinerte Darstellung steht im Zentrum der neuen WHO-Klassifikation der Thymome, die erstmals immunhistochemische Befundprofile zur besseren Reproduzierbarkeit der Klassifikation einführt. Ziel der vorliegenden Arbeit ist es, die konzeptionellen und diagnostischen Neuerungen der neuen WHO-Klassifikation der Thymome darzustellen und Antworten auf therapierelevante differenzialdiagnostische Fragen zu geben. peerReviewed

Reliable entity subtyping is paramount for therapy stratification in lung cancer. Morphological evaluation remains the basis for entity subtyping and directs the application of additional methods such as immunohistochemistry (IHC). The decision of whether to perform IHC for subtyping is subjective, and access to IHC is not available worldwide. Thus, the application of additional methods to support morphological entity subtyping is desirable. Therefore, the ability of convolutional neuronal networks (CNNs) to classify the most common lung cancer subtypes, pulmonary adenocarcinoma (ADC), pulmonary squamous cell carcinoma (SqCC), and small-cell lung cancer (SCLC), was evaluated. A cohort of 80 ADC, 80 SqCC, 80 SCLC, and 30 skeletal muscle specimens was assembled, slides were scanned, tumor areas were annotated, image patches were extracted, and cases were randomly assigned to a training, validation or test set. Multiple CNN architectures (VGG16, InceptionV3, and InceptionResNetV2) were trained and optimized to classify the four entities. A quality control (QC) metric was established. An optimized InceptionV3 CNN architecture yielded the highest classification accuracy and was used for the classification of the test set. Image patch and patient-based CNN classification results were 95% and 100% in the test set after the application of strict QC. Misclassified cases mainly included ADC and SqCC. The QC metric identified cases that needed further IHC for definite entity subtyping. The study highlights the potential and limitations of CNN image classification models for tumor differentiation.

Background Recently, the Food and Drug Administration (FDA)/European Medicines Agency (EMA) restricted first-line use of atezolizumab and pembrolizumab in patients with metastasised urothelial carcinoma by defining distinct programmed cell death ligand-1 cut-offs. We analysed the diagnostic performance of all FDA/EMA-approved programmed cell death ligand-1 assays with emphasis on new restrictions for first-line treatment with atezolizumab and pembrolizumab. Patients and methods Two hundred fifty-one urothelial carcinomas were analysed on tissue microarrays with four cores of each tumour. Stains were performed in certified laboratories on Ventana Benchmark Ultra and Dako Link 48 autostainers. Stains were read on an assay-by-assay basis by two trained pathologists. Overall percentage agreement (OPA) was calculated across the preset cut-offs. Positive percentage agreement (PPA) and negative percentage agreement (NPA) were calculated across different scoring algorithms. Venn diagrams were constructed to illustrate discordance according to the recent FDA/EMA guidelines. Results The Dako 28-8, 22c3 and the Ventana SP263 assays showed high interassay correlation (r-range 0.83–0.91). Interassay correlation between the Ventana SP142 and the three other assays was moderate (r-range 0.66–0.75). OPA of 93.3% was achieved between the Dako 28-8, 22c3 and Ventana SP263 assays. OPA including the SP142 was 84.1%. Pooled PPA and NPA of different scoring algorithms was 89.4% and 95.3% for the Dako 28-8, 22c3 and the SP263 assays, respectively. With the SP142 assay, pooled PPA was 59.1%. The SP142 assay identifies fewer eligible patients for first-line treatment with atezolizumab/pembrolizumab. Conclusion Dako 28-8, 22c3 and SP263 assays show interchangeable performance. The SP142 assay shows divergent staining results. Interassay variability leads to different detection rates of eligible patients for first-line treatment with atezolizumab and pembrolizumab.

Objective To evaluate the mRNA expression of lymphangiogenesis and proliferation markers and to examine its association with histopathological characteristics and clinical outcome in patients with urothelial carcinoma of the bladder (UCB) after radical cystectomy (RC). Patients and methods Gene expression analysis of the vascular endothelial growth factor-C and -D (VEGF-C/-D), its receptor VEGF receptor-3 (VEGFR-3), MKI67, and RACGAP1 was performed in 108 patients after radical cystectomy and their correlation with clinical-pathological parameters was investigated. Uni- and multivariate regression analyses were used to identify predictors for cancer-specific survival (CSS), recurrence-free survival (RFS) and overall survival (OS) after RC. Results The expression of RACGAP1 and VEGFR-3 showed an association with a higher pT stage ( P = 0.049; P = 0.009). MKI67 showed an association with a high-grade urothelial carcinoma of the bladder ( P = 0.021). VEGFR-3 expression was significantly associated with the presence of lymphovascular invasion (LVI) ( P = 0.016) and lymph node metastases (pN+) ( P = 0.028). With the univariate analysis, overexpression of VEGFR-3 ( P = 0.029) and the clinical-pathological parameters pT stage ( P P = 0.0004), LVI ( P P = 0.021) were significantly associated with a reduced CSS. Multivariate analysis identified a higher pT stage ( P = 0.017) and LVI ( P = 0.008) as independent predictors for reduced CSS. Independent predictors for reduced OS were a higher pT stage ( P = 0.0007) and LVI ( P = 0.0021), while overexpression of VEGF-D was associated with better OS ( P Conclusions The mRNA expression of the investigated markers showed associations with common histopathological parameters. Increased expression of VEGF-D is independently associated with better overall survival.

Muscle-invasive urothelial carcinoma of the urinary bladder (UCB) often recurs following radical cystectomy (RC). An altered expression of sex-steroid hormone receptors has been associated with oncological outcomes of UCB and may represent therapeutic targets. Here the expression of different hormone receptors was measured on mRNA levels in patients treated by RC and associated with outcomes. Androgen receptor (AR), estrogen receptor 1 (ESR1), and progesterone receptor (PGR) mRNA expression was assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in RC samples of 87 patients with a median age of 66 (39–88) years. Univariate and multivariate analyses were performed to test associations with pathological and clinical characteristics as well as recurrence-free (RFS) and disease-specific survival (DSS). AR mRNA expression was lower in comparison with ESR1 and PGR expression (p

ObjectiveThe aim of the study was to analyze the effect of thymectomy on the proposed disease-specific microRNA (miRNA) biomarkers miR-150-5p and miR-21-5p in patients from the prospective randomized trial of thymectomy in myasthenia gravis (MGTX trial) and to evaluate the longitudinal changes in clinical patterns compared with these miRNA levels.MethodsSerum samples were obtained from 80 patients with MG who were included in the MGTX trial. Thirty-eight patients were randomized to thymectomy plus prednisone treatment, and 42 patients were randomized to prednisone treatment. Serum samples were analyzed for the expression of miR-150-5p and miR-21-5p, with quantitative reverse transcriptase PCR at baseline and at 12, 24, and 36 months after randomization. The inclusion criteria for participation in the MGTX trial were age 18–65 years, generalized myasthenia gravis (Myasthenia Gravis Foundation of America Class II–IV), disease duration of less than 5 years, and seropositivity for acetylcholine receptor antibodies (AChR+).ResultsPatients treated with thymectomy had lower levels of miR-150-5p at 24 months, both compared with baseline values (p = 0.0011) and the prednisone group (p = 0.04). No change in miRNA levels was found in the prednisone group. Levels of miR-21-5p displayed a negative correlation with the prednisone dose within the prednisone-only group (p ≤ 0.001).ConclusionsThymectomy lowers the levels of the proposed biomarker miR-150-5p, which strengthens its position as a potential disease-specific biomarker for AChR+ MG.

The thymectomy specimens from the "thymectomy trial in non-thymomatous myasthenia gravis patients receiving prednisone therapy" (MGTX) underwent rigid and comprehensive work-up, which permits analysis of the spatial distribution of histological and immunohistological features. This analysis revealed strong intra- and inter-case variability. While many histological features (e.g. median percent fat content among different specimens) can easily be correlated with clinical parameters, intra-case spatial variability of histological features has yet defied quantification and statistical evaluation. To overcome this gap in digital pathology, we here propose intra-case entropy of measured histological features in all available slides of a given thymectomy specimen as a quantitative marker of spatial histological heterogeneity. Calculation of entropy led to one value per specimen and histological feature. Through these 'entropy values' the so far neglected degree of spatial histological heterogeneity could be fed into statistical analyses, extending the scope of clinico-pathological correlations.

Background Tumor budding, meaning a detachment of tumor cells at the invasion front of colorectal carcinoma (CRC) into single cells or clusters (

INTRODUCTION: Gene expression analyses have identified similarities between bladder and breast cancer, where clinical risk stratification is based on Her2, ESR1, PGR and Ki67 expression. The aim of the study was to assess the respective marker gene expression in patients treated with radical cystectomy for muscle-invasive bladder cancer (MIBC) and to evaluate the applicability of breast cancer subtypes for MIBC risk stratification. MATERIALS & METHODS: 102 patients treated with radical cystectomy for MIBC were assessed. Using routine FFPE tissue and an IVD validated kit, mRNA expression was measured by single step RT-qPCR. Partition test were employed to define cut-off values for high or low marker gene expression. Association of expression with outcome was assessed using Kaplan-Meier analysis and multivariate cox regression analysis. Finally, we performed validation of our results in the MD-Anderson cohort (n=57). RESULTS: Cancer specific survival (CSS) was impaired in patients with high gene expression of Her2 ( P =0.0009) and ESR1 ( P =0.04). In the multivariate regression model Her2 expression remained significant for the prediction of CSS (HR=2.11, CI 1.11-4.21, P =0.024). Furthermore, molecular stratification by breast cancer subgroups was significant ( P =0.023) for CSS prediction. Especially the differentiation between Her2 -positive and Luminal A (HR=4.41, CI 1.53-18.71, P =0.004) and Luminal B (HR=1.96, CI 0.99-4.08, P =0.053) respectively was an independent prognostic parameter for CSS. External validation resulted in comparable risk stratification with differences in fractional subgroups distribution. CONCLUSION: Gene expression of Her2, ESR1, PGR, Ki67 and corresponding breast cancer subtypes allow a risk-stratification in MIBC, whereby Her2 overexpressing tumors reveal a particularly poor prognosis.

Background: Entropy belongs to the few basic measurable entities in nature. It measures the distance of a closed or open ‘statistical’ system from its present stage to its final stage, and analyzes the probability distribution of the included elements, independently from their meaning. The development can be predicted by use of an ‘ideal transformation’, i.e. additive formula (for example Shannon’s entropy) or by mathematical derivatives such as the more generalized q – entropy, for example Tsallis entropy. Herein, the internal structures of the system are described which include so – called macro – systems. They are created by individual elements or basic micro – systems, and transformed into essentials of tissue – based diagnosis. Entropy and neighborhood: The basic entropy approaches consider a spatially force – independent system, i.e., the calculation of the elements’ probability distribution does not take into account the formation of macro – systems, or the position of the individual elements within the system or between individual elements. The receiver of an informative signal cannot distinguish whether it has been generated in the center or at the boundary of the system. Only the signal’s probability within the information chain and the formation of the chain are informative. However, neighborhood plays an important role in development, maturation, degradation, and dissolution of biological systems. Most cells are generated by cellular division and neighboring cells are more similar in morphology and function than non-neighboring cells. This observation also holds true for ‘higher order’ biological systems such as animal colonies, forests, or even human societies. Thus, a potentially successful approach of estimating the development of a biological system should include neighborhood definitions and considerations. Neighborhood conditioned (MST) entropy and entropy flow: Any definition of a neighborhood condition is based upon the distances between different elements, called objects. The distances can be weighted by additional object features, might be ‘directed’, or might include certain ‘shadow’ conditions (hidden behind another object). The most frequently used algorithm has been introduced by Voronoi in 1902. It can be successfully formulated in graph theory and derived approaches. In microscopic morphology, the construction of weighted minimum spanning trees (MST) is a convincing approach. Living biological systems are open and not closed. They exchange energy, information, and directives for future development with their environment. They have to stabilize their own entropy level against that of their environment. The mandatory entropy exchange or entropy flow from the individual element into its environment or vice versa reflects to the system’s stability and impact on its environment. Tissue – based diagnosis and entropy: Tissue – based diagnosis includes all technical procedures to ascertain a ‘medical diagnosis’, such as microscopic, electron microscopic investigations, gene analysis, proteomics, syntactic structure analysis, liquid biopsies, etc. Herein, the transformation and applicability of the entropy approach are described and discussed., Diagnostic Pathology, Vol 3 No 1 (2017): 2017

Until recently, tissue fibrosis—ultimately leading to permanent scaring—has been considered an irreversible process. However, recent findings indicate that it may be reversible after all. Vesicourethral anastomotic stenosis (VUAS) as fibrous narrowing is a frequent complication after radical prostatectomy with high recurrence rates and requires invasive treatment. The pathophysiology is poorly understood. Therefore, a combined mRNA and miRNA transcription profiling in tissue from VUAS was performed using nCounter technology. To assess tissue morphology and fiber composition, histochemical staining was performed. RNA expression of healthy and fibrotic tissue of twelve patients was analyzed using the human miRNA panel v3 and mRNA PanCancer pathway panel on the nCounter gene1 system and qRT-PCR. Differential expression data analysis was performed using the nSolver software implementing the R-based advanced pathway analysis tool. miRWalk2.0 was used for miRNA target prediction. More linearized tissue architecture, increased collagens, and decreased elastic fibers were observed in VUAS samples. 23 miRNAs and 118 protein coding genes were differentially expressed (p

Blood vessels in cancer Intra-tumoral blood vessels are of supreme importance for tumor growth, metastasis and therapy. Yet, little is known about spatial distribution patterns of these vessels. Most experimental or theoretical tumor models implicitly assume that blood vessels are equally abundant in different parts of the tumor, which has far-reaching implications for chemotherapy and tumor metabolism. In contrast, based on histological observations, we hypothesized that blood vessels follow specific spatial distribution patterns in colorectal cancer tissue. We developed and applied a novel computational approach to identify spatial patterns of angiogenesis in histological whole-slide images of human colorectal cancer. A characteristic spatial pattern of blood vessels in colorectal cancer In 33 of 34 (97%) colorectal cancer primary tumors blood vessels were significantly aggregated in a sharply limited belt-like zone at the interface of tumor tissue to the intestinal lumen. In contrast, in 11 of 11 (100%) colorectal cancer liver metastases, a similar hypervascularized zone could be found at the boundary to surrounding liver tissue. Also, in an independent validation cohort, we found this vascular belt zone: 22 of 23 (96%) samples of primary tumors and 15 of 16 (94%) samples of liver metastases exhibited the above-mentioned spatial distribution. Summary and implications We report consistent spatial patterns of tumor vascularization that may have far-reaching implications for models of drug distribution, tumor metabolism and tumor growth: luminal hypervascularization in colorectal cancer primary tumors is a previously overlooked feature of cancer tissue. In colorectal cancer liver metastases, we describe a corresponding pattern at the invasive margin. These findings add another puzzle piece to the complex concept of tumor heterogeneity.

The initial clinical management of mediastinal masses essentially depends on the assumption whether surgery is indicated or not. Thymic masses that are thought to be operable based on imaging studies may not need to be biopsied, if surgery is deemed to be the treatment of choice. However, if nonsurgical therapies (as is the case in lymphomas) or neoadjuvant strategies are options, core needle biopsies may be an invaluable tool to guide therapeutic decisions. Like small biopsies elsewhere, sampling error is also a key problem in the prevascular and visceral mediastinum where it is, however, aggravated by (i) the high degree of microscopic similarity between normal thymus, thymic hyperplasias, T-cell-rich thymomas, and T-lymphoblastic lymphoma; (ii) the common histological heterogeneity across individual type B thymomas; (iii) the striking morphological overlap between spindle cell lesions with thymic epithelial, neuroendocrine, mesenchymal, and dendritic differentiation; (iv) the occurrence of acquired thymic cysts, the development of which is driven by different inflammatory processes and a broad spectrum of mediastinal tumors that may be dwarfed by the cystic process and escape sampling in small biopsies; and (v) reactive epithelial proliferations that can mimic neoplasias. These challenges encountered in small biopsies imply inevitable diagnostic limitations, suggest cautious reporting of diagnoses and argue not only for the thoughtful use of ancillary immunohistological and molecular studies but also for thorough clinicopathological correlation. The below sections on thymic epithelial tumors, various types of thymic hyperplasias and thymic cysts pinpoint diagnostic pitfalls and suggest strategies to avoid them.

Reaktive und neoplastische Veranderungen des Thymus stehen im Zentrum der Differenzialdiagnose bei tumorartigen Veranderungen des vorderen und mittleren Mediastinums, wahrend bei Tumoren des hinteren Mediastinums besonders an neurogene Neoplasien zu denken ist (die hier nicht behandelt werden). Bei Neugeborenen und Sauglingen stellen Keimzelltumoren, kongenitale Zysten und „echte Thymushyperplasien“, die haufigsten Veranderungen im vorderen Mediastinum dar. Bei Kleinkindern dominieren Teratome vor Dottersacktumoren und Zysten. Bei alteren Kindern und Jugendlichen sind Lymphome am pravalentesten, wahrend Thymuskarzinome und Thymome Raritaten sind. Zusatzlich sind dann auch entzundlich bedingt Thymushyperplasie bei Myasthenia gravis und „Uberschusshyperplasien“ des Thymus z. B. nach Chemotherapie zu bedenken. Sarkome sind ab der Geburt seltene aber wichtige Differenzialdiagnosen. Ausgehend von der Schilderung eines seltenen Falls einer rezidivierten „echten Thymushyperplasie“ wird die Differenzialdiagnose dieser nichtneoplastischen aber potenziell lebensbedrohlichen Erkrankung diskutiert.

Background Prostate cancer with neuroendocrine differentiation (NEPCA) shares similarities in tumor biology with small-cell lung cancer. While immunotherapies were successfully tested in small-cell lung cancer, and programmed death ligand 1 (PD-L1) expression arises as an essential predictive biomarker, the local immune status in NEPCA is still poorly described. Patients and Methods Paraffin-embedded tissue samples of 39 patients (7 adenocarcinomas with neuroendocrine differentiation [ACA NED], 20 small-cell neuroendocrine carcinomas, 2 well-differentiated neuroendocrine tumors of NEPCA, and 10 adenocarcinoma liver metastases) were examined retrospectively by immunohistochemistry of chromogranin A (CGA), CD56, synaptophysin (SYN), CD3, and PD-L1. Laser capture microdissection was used for neuroendocrine hot-spot evaluation for additional real-time reverse transcription–quantitative PCR analysis (PD-L1, CGA, CD56, SYN, GRP, ASCL1, and DLK1). Results PD-L1 immunohistochemistry expression in NEPCA was observed by assay E1L3N in 5 (20.8%) of 24 samples, but not by assay 22c3. Gene expression of PD-L1 could be evaluated in 18 (62%) of 29 samples. Nine (69%) of 13 prostate specimens and 2 (40%) of 5 liver metastases were positive for PD-L1. In ACA NED 4 (80%) of 5 and in small-cell neuroendocrine carcinomas 6 (50%) of 12 specimens were positive for PD-L1. Tumor-infiltrating lymphocytes ≥ 10% were observed in 9 (37.5%) of 24 specimens. Low ASCL1 expression was observed in liver metastases. Conclusion These data identify molecular PD-L1 features in NEPCA. The predictive role of PD-L1 status and tumor-infiltrating lymphocytes in NEPCA remains to be established.

Aim: To evaluate the prognostic role of the p53-upstream inhibitors MDM2, MDM4 and its splice variant MDM4-S in patients undergoing radical cystectomy (RC) for muscle-invasive bladder cancer (MIBC). Materials and Methods: mRNA Expression levels of MDM2, MDM4 and MDM4-S were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) in 75 RC samples. Logistic regression analyses identified predictors of recurrence-free (RFS) and cancer-specific survival (CSS). Results: High expression was found in 42% (MDM2), 27% (MDMD4) and 91% (MDM4-S) of tumor specimens. Increased MDM2 expression was significantly associated with higher tumor stage (p=0.05) and lymphovascular invasion (LVI) (p=0.041). In the univariate analysis, low MDM4 expression (hazard ratio (HR)=5.93; p=0.002; HR=3.00; p=0.047), but not MDM2 (HR=1.63; p=0.222; HR=1.59; p=0.27), were associated with RFS and CSS. In the multivariate analysis, the combination of low MDM4 and high MDM2 was significant for RFS and CSS (HR=14.9; p=0.001; HR=5.63; p=0.019). Conclusion: The combination of MDM2 and MDM4 expression is an independent predictor in patients undergoing RC for MIBC.

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer that responds to PD-1/PD-L1 immune checkpoint inhibitors. CD200 is another checkpoint modulator whose receptor is found on tumor-promoting myeloid cells, including M2 macrophages. We found high CD200 mRNA expression in MCC tumors, and CD200 immunostaining was demonstrated on 95.5% of MCC tumors. CD200R-expressing myeloid cells were present in the MCC tumor microenvironment. MCC-associated macrophages had a higher average CD163:CD68 staining ratio (2.67) than controls (1.13), indicating an immunosuppressive M2 phenotype. Accordingly, MCC tumors contained increased densities of FOXP3+ regulatory T-cells. Intravenous administration of blocking anti-CD200 antibody to MCC xenograft mice revealed specific targeting of drug to tumor. In conclusion, MCC are highly CD200 positive and associated with immunosuppressive M2 macrophages and regulatory T-cells. As anti-CD200 antibody effectively targets CD200 on MCC tumor cells in vivo, this treatment may provide a novel immunotherapy for MCC independent of PD-1/PD-L1 blockade.

4525Background: Transcriptome expression studies identified distinct bladder cancer subtypes of prognostic relevance and closely related with breast cancer molecular subclasses. Here we developed a...