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Abstract Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research.

Abstract Personalized treatment for patients with advanced solid tumors critically depends on the deep characterization of tumor cells from patient biopsies. Here, we comprehensively characterize a pan-cancer cohort of 150 malignant serous effusion (MSE) samples at the cellular, molecular, and functional level. We find that MSE-derived cancer cells retain the genomic and transcriptomic profiles of their corresponding primary tumors, validating their use as a patient-relevant model system for solid tumor biology. Integrative analyses reveal that baseline gene expression patterns relate to global ex vivo drug sensitivity, while high-throughput drug-induced transcriptional changes in MSE samples are indicative of drug mode of action and acquired treatment resistance. A case study exemplifies the added value of multi-modal MSE profiling for patients who lack genetically stratified treatment options. In summary, our study provides a functional multi-omics view on a pan-cancer solid tumor cohort and underlines the feasibility and utility of MSE-based precision oncology.

Abstract Background Tertiary lymphoid structures (TLSs) are dense accumulations of lymphocytes in inflamed peripheral tissues, including cancer, and are associated with improved survival and response to immunotherapy in various solid tumors. Histological TLS quantification has been proposed as a novel predictive and prognostic biomarker, but lack of standardized methods of TLS characterization hampers assessment of TLS densities across different patients, diseases, and clinical centers. Methods We introduce an approach based on HookNet-TLS, a multi-resolution deep learning model, for automated and unbiased TLS quantification and identification of germinal centers in routine hematoxylin and eosin stained digital pathology slides. We developed HookNet-TLS using n = 1019 manually annotated TCGA slides from clear cell renal cell carcinoma, muscle-invasive bladder cancer, and lung squamous cell carcinoma. Results Here we show that HookNet-TLS automates TLS quantification across multiple cancer types achieving human-level performance and demonstrates prognostic associations similar to visual assessment. Conclusions HookNet-TLS has the potential to be used as a tool for objective quantification of TLS in routine H&E digital pathology slides. We make HookNet-TLS publicly available to promote its use in research.

Abstract Introduction Extended pelvic lymph node dissection (ePLND) in men undergoing robot-assisted laparoscopic radical prostatectomy (RARP) is a widely used procedure. However, little is known about anatomical site-specific yields and subsequent metastatic patterns in these patients. Patients and methods Data on a consecutive series of 1107 patients undergoing RARP at our centre between 2004 and 2018 were analysed. In men undergoing LN dissection, the internal, external and obturator nodes were removed and sent in separately. We performed an analysis of LN yields in total and for each anatomical zone, patterns of LN metastases and complications. Oncological outcome in pN+ disease was assessed including postoperative PSA persistence and survival. Results A total of 823 ePLNDs were performed in the investigated cohort resulting in 98 men being diagnosed as pN+ (8.9%). The median (IQR) LN yield was 19 (14–25), 10 (7–13) on the right and 9 (6–12) on the left side (P

Abstract Most invasive lobular breast carcinomas (ILBCs) are luminal‐type carcinomas with an HER2‐negative phenotype (ERBB2 or HER2 un‐amplified) and CDH1 mutations. Rare variants include ERBB2‐amplified subtypes associated with an unfavorable prognosis and less response to anti‐HER2 targeted therapies. We analyzed the clinicopathological and molecular features of ERBB2‐amplified ILBC and compared these characteristics with ERBB2‐unamplified ILBC. A total of 253 patients with ILBC were analyzed. Paraffin‐embedded formalin‐fixed tumor samples from 250 of these patients were added to a tissue microarray. Protein expression of prognostic, stem cell and breast‐specific markers was tested by immunohistochemistry (IHC). Hybrid capture‐based comprehensive genomic profiling (CGP) was performed for 10 ILBCs that were either fluorescent in situ hybridization (FISH) or IHC positive for HER2 amplification/overexpression and 10 ILBCs that were either FISH or IHC negative. Results were compared with a CGP database of 44,293 invasive breast carcinomas. The CGP definition of ERBB2 amplification was five copies or greater. A total of 17 of 255 ILBC (5%) were ERBB2 amplified. ERBB2‐amplified ILBC had higher tumor stage (p

Metabolic reprogramming and mitochondrial dynamics are pivotal in prostate cancer (PCa) progression and treatment resistance, making them essential targets for therapeutic intervention. In this study, we investigated the effects of the androgen receptor antagonist apalutamide (ARN) and the mitochondrial electron transport chain complex I inhibitor IACS-010759 (IACS) on the mitochondrial network architecture and dynamics in PCa cells. Treatment with ARN and/or IACS induced significant changes in mitochondrial morphology, particularly elongation, in androgen-sensitive PCa cells. Additionally, ARN and IACS modulated the mitochondrial fission and fusion processes, indicating a convergence of metabolic and androgen-signaling pathways in shaping mitochondrial function. Notably, the combination treatment with ARN and IACS resulted in increased apoptotic cell death and mitochondrial oxidative stress selectively in the androgen-sensitive PCa cells. Our findings highlight the therapeutic potential of targeting mitochondrial metabolism in prostate cancer and emphasize the need for further mechanistic understanding to optimize treatment strategies and improve patient outcomes.

Abstract ADP‐ribosylation (ADPR) of proteins is catalyzed by ADP‐ribosyltransferases, which are targeted by inhibitors (i.e. poly(ADP‐ribose) polymerase inhibitors [PARPi]). Although renal cell carcinoma (RCC) cells are sensitive in vitro to PARPi, studies on the association between ADPR levels and somatic loss of function mutations in DNA damage repair genes are currently missing. Here we observed, in two clear cell RCC (ccRCC) patient cohorts (n = 257 and n = 241) stained with an engineered ADP‐ribose binding macrodomain (eAf1521), that decreased cytoplasmic ADPR (cyADPR) levels significantly correlated with late tumor stage, high‐ISUP (the International Society of Urological Pathology) grade, presence of necrosis, dense lymphocyte infiltration, and worse patient survival (p

Abstract Defects in homologous recombination repair (HRR) in tumors correlate with poor prognosis and metastases development. Determining HRR deficiency (HRD) is of major clinical relevance as it is associated with therapeutic vulnerabilities and remains poorly investigated in sarcoma. Here, we show that specific sarcoma entities exhibit high levels of genomic instability signatures and molecular alterations in HRR genes, while harboring a complex pattern of chromosomal instability. Furthermore, sarcomas carrying HRDness traits exhibit a distinct SARC‐HRD transcriptional signature that predicts PARP inhibitor sensitivity in patient‐derived sarcoma cells. Concomitantly, HRDhigh sarcoma cells lack RAD51 nuclear foci formation upon DNA damage, further evidencing defects in HRR. We further identify the WEE1 kinase as a therapeutic vulnerability for sarcomas with HRDness and demonstrate the clinical benefit of combining DNA damaging agents and inhibitors of DNA repair pathways ex vivo and in the clinic. In summary, we provide a personalized oncological approach to treat sarcoma patients successfully.

Abstract Rapid advances in high-throughput DNA sequencing technologies have enabled the conduct of whole genome sequencing (WGS) studies, and several bioinformatics pipelines have become available. The aim of this study was the comparison of 6 WGS data pre-processing pipelines, involving two mapping and alignment approaches (GATK utilizing BWA-MEM2 2.2.1, and DRAGEN 3.8.4) and three variant calling pipelines (GATK 4.2.4.1, DRAGEN 3.8.4 and DeepVariant 1.1.0). We sequenced one genome in a bottle (GIAB) sample 70 times in different runs, and one GIAB trio in triplicate. The truth set of the GIABs was used for comparison, and performance was assessed by computation time, F1 score, precision, and recall. In the mapping and alignment step, the DRAGEN pipeline was faster than the GATK with BWA-MEM2 pipeline. DRAGEN showed systematically higher F1 score, precision, and recall values than GATK for single nucleotide variations (SNVs) and Indels in simple-to-map, complex-to-map, coding and non-coding regions. In the variant calling step, DRAGEN was fastest. In terms of accuracy, DRAGEN and DeepVariant performed similarly and both superior to GATK, with slight advantages for DRAGEN for Indels and for DeepVariant for SNVs. The DRAGEN pipeline showed the lowest Mendelian inheritance error fraction for the GIAB trios. Mapping and alignment played a key role in variant calling of WGS, with the DRAGEN outperforming GATK.

Abstract Background Renal cell carcinoma (RCC) is a heterogeneous disease comprising histologically defined subtypes. For therapy selection, precise subtype identification and individualized prognosis are mandatory, but currently limited. Our aim was to refine subtyping and outcome prediction across main subtypes, assuming that a tumor is composed of molecular features present in distinct pathological subtypes. Methods Individual RCC samples were modeled as linear combination of the main subtypes (clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC)) using computational gene expression deconvolution. The new molecular subtyping was compared with histological classification of RCC using the Cancer Genome Atlas (TCGA) cohort (n = 864; ccRCC: 512; pRCC: 287; chRCC: 65) as well as 92 independent histopathologically well-characterized RCC. Predicted continuous subtypes were correlated to cancer-specific survival (CSS) in the TCGA cohort and validated in 242 independent RCC. Association with treatment-related progression-free survival (PFS) was studied in the JAVELIN Renal 101 (n = 726) and IMmotion151 trials (n = 823). CSS and PFS were analyzed using the Kaplan–Meier and Cox regression analysis. Results One hundred seventy-four signature genes enabled reference-free molecular classification of individual RCC. We unambiguously assign tumors to either ccRCC, pRCC, or chRCC and uncover molecularly heterogeneous tumors (e.g., with ccRCC and pRCC features), which are at risk of worse outcome. Assigned proportions of molecular subtype-features significantly correlated with CSS (ccRCC (P = 4.1E − 10), pRCC (P = 6.5E − 10), chRCC (P = 8.6E − 06)) in TCGA. Translation into a numerical RCC-R(isk) score enabled prognosis in TCGA (P = 9.5E − 11). Survival modeling based on the RCC-R score compared to pathological categories was significantly improved (P = 3.6E − 11). The RCC-R score was validated in univariate (P = 3.2E − 05; HR = 3.02, 95% CI: 1.8–5.08) and multivariate analyses including clinicopathological factors (P = 0.018; HR = 2.14, 95% CI: 1.14–4.04). Heterogeneous PD-L1-positive RCC determined by molecular subtyping showed increased PFS with checkpoint inhibition versus sunitinib in the JAVELIN Renal 101 (P = 3.3E − 04; HR = 0.52, 95% CI: 0.36 − 0.75) and IMmotion151 trials (P = 0.047; HR = 0.69, 95% CI: 0.48 − 1). The prediction of PFS significantly benefits from classification into heterogeneous and unambiguous subtypes in both cohorts (P = 0.013 and P = 0.032). Conclusion Switching from categorical to continuous subtype classification across most frequent RCC subtypes enables outcome prediction and fosters personalized treatment strategies.

The diagnosis of prostate cancer brain metastasis (PCBM) has increased. Here, the authors investigate the landscape of somatic genetic alterations in brain metastases in a PCBM cohort of 51 patients with non-synchronous matched primary samples available for 20 patients.

Transcriptional changes during prostate cancer progression are not yet fully understood. Here, the authors integrate a transcriptomics atlas of prostate cancer and validate it with preclinical models and single-cell RNA-seq, revealing the role of EZH2 and macrophage polarisation in tumour progression.

IntroductionCurrent evidence shows that serum miR-371a-3p can identify disease recurrence in testicular germ cell tumour (TGCT) patients and correlates with tumour load. Despite convincing evidence showing the advantages of including miR-371a-3p testing to complement and overcome the classical serum tumour markers limitations, the successful introduction of a serum miRNA based test into clinical practice has been impeded by a lack of consensus regarding optimal methodologies and lack of a universal protocol and thresholds. Herein, we investigate two quantitative real-time PCR (qRT-PCR) based pipelines in detecting disease recurrence in stage I TGCT patients under active surveillance, and compare the sensitivity and specificity for each method.MethodsSequential serum samples collected from 33 stage I TGCT patients undergoing active surveillance were analysed for miR-371a-3p via qRT-PCR with and without an amplification step included.ResultsUsing a pre-amplified protocol, all known recurrences were detected via elevated miR-371a-3p expression, while without pre-amplification, we failed to detect recurrence in 3/10 known recurrence patients. For pre-amplified analysis, sensitivity and specificity was 90% and 94.4% respectively. Without amplification, sensitivity dropped to 60%, but exhibited 100% specificity.DiscussionWe conclude that incorporating pre-amplification increases sensitivity of miR-371a-3p detection, but produces more false positive results. The ideal protocol for quantification of miR-371a-3p still needs to be determined. TGCT patients undergoing active surveillance may benefit from serum miR-371a-3p quantification with earlier detection of recurrences compared to current standard methods. However, larger cross-institutional studies where samples are processed and data is analysed in a standardised manner are required prior to its routine clinical implementation.

Tumour-infiltrating lymphocytes (TIL), known to be of prognostic value in various solid tumours, have been in the focus of research in the last years. TIL are often quantified via IMMUNOSCORE ® (IS), a scoring system based on TIL cell densities. Recent studies were able to replicate these findings for muscle-invasive bladder cancer (MIBC), however data regarding non-muscle-invasive bladder cancer (NMIBC) are scarce. This study aimed to evaluate the value of a modified Immunoscore (mIS) as a predictive marker for NMIBC prognosis using tissue-micro-arrays (TMAs). We analysed two TMAs containing 316 samples from 158 patients with NMIBC, stained for CD3, CD8, CD45RO and FOXP3. Stained TIL were captured by digital pathology, cumulated, averaged, and reported as density (stained cells per mm²). The mIS was then constructed based on density of all four immune-cell types. Clinical, pathological and follow-up data were collected retrospectively. Univariable and multivariable cox regression analysis was performed to assess the potential value of mIS as a predictor for progression free survival (PFS) and recurrence-free-survival (RFS). Patients within “European Organisation for Research and Treatment of Cancer” (EORTC) risk groups were further substratified in high mIS and low mIS subgroups. Finally log-rank test was used to compare the different survival curves. The median age in our cohort was 68 years (Interquartile Range (IQR): 60 - 76), and 117 (74%) patients were male. A total of 26 patients (16.5%) were classified as EORTC low risk, 45 (28.5%) as intermediate risk and 87 (55.1%) as high risk. Patients in the EORTC high risk group with low mIS showed a shorter PFS in comparison to high mIS (HR 2.9, CI 0.79 – 11.0, p=0.082). In contrast, no predictive potential regarding PFS was observed in intermediate or low risk groups. Furthermore, mIS was not able to predict RFS in any EORTC risk group. mIS could be utilized to predict prognosis more accurately in high-risk patients with NMIBC by identifying those with higher or lower risk of progression. Therefore, mIS could be used to allocate these highrisk patients to more streamlined follow-up or more aggressive treatment strategies.

Abstract Similar to the influenza A pandemic in 1918/1919, the new Coronavirus disease 2019 (COVID‐19) has spread globally. The causes of death in COVID‐19 are frequently compared to a seasonal influenza outbreak. Complete COVID‐19 autopsy studies were almost non‐existent in the first months of the outbreak and are still rare with respect to the number of deaths. It has been recently reported that capillary microthrombi are significantly more prevalent in patients with COVID‐19 than in patients with influenza A. To date, the contribution of macrothrombi, i.e. visible thrombi in pulmonary arteries, to the death of patients with influenza A in comparison to COVID‐19 remains unaddressed. Here, we report autopsy findings in 411 patients who died from the ‘Spanish’ influenza A pandemic between May 1918 and April 1919 at the University Hospital Zurich, Switzerland. We compare these results with influenza A autopsies from 2009 to 2020, other influenza A autopsy series and all COVID‐19 autopsies published to date. No descriptions of any macroscopic thromboembolic events were mentioned in influenza A autopsy reports. In 75 published COVID‐19 autopsies, pulmonary artery thrombosis/embolism was reported in 36%. The direct comparison of macroscopic autopsy findings suggests a significantly greater degree of grossly visible pulmonary macrothrombi in patients with COVID‐19 in comparison to influenza A autopsies even though most patients received empiric thromboprophylaxis. This is consistent with the concept of a SARS‐related de novo coagulopathy with generalised in situ clot formation, which could explain the high incidence of pulmonary thrombosis/embolism with or without underlying deep vein thrombosis and in the absence of a history of venous thromboembolic events.

In May 2020, larotrectinib (Vitrakvi®) was approved as the first tumor-agnostic agent in Switzerland for the treatment of adult and pediatric patients with solid tumors that have a neurotrophic tyrosine receptor kinase (NTRK) gene fusion. While in certain types of tumors an NTRK gene fusion is often detectable, it is rare in some other types. As the detection of NTRK gene fusion opens up new treatment options for patients, it is vital to ensure that the detection methods and diagnostic algorithms in pathology institutes are optimized and of high quality. Detection can be performed by immunohistochemistry, next-generation sequencing or by fluorescence in situ hybridization. This review will discuss diagnostic algorithms based on underlying pathology that can be recommended for the detection of NTRK gene fusions in Switzerland.

Gene fusions involving the ERG transcription factor and point mutations in the ubiquitin ligase adaptor SPOP are two truncal mutations that are mutually exclusively present in prostate cancer. Here, the authors show that mutations in SPOP render prostate tumor cells sensitive to antiandrogen therapy and that the presence of ERG promotes sensitivity to high dose of androgen and SPOP inhibition.

Dormancy of disseminated cancer cells has been described in patients with breast cancer and associated with late metastatic relapses. Here the authors show that CD39+PD-1+CD8+ T cells correlate with increased disease free survival post-resection in breast cancer patients, and promote dormancy in a preclinical model.

Abstract Background A dual blockade against the novel immune checkpoint inhibitor lymphocyte activation gene-3 (LAG-3) and programmed cell death protein-1 (PD-1) is currently considered in advanced breast cancer. Nevertheless, PD-1 or LAG-3 expression within distant metastatic breast cancer tissue remains understudied. Methods To address this knowledge gap, we investigated the PD-1 and LAG-3 expression in combination with the CD8-based immune phenotype in intrapatient matched primary tumor distant metastases, representing 95 breast cancer patients with metastases occurring at four different anatomical locations. The immune phenotype was categorized into 2 categories: inflamed corresponding to the clinical category “hot” and exhausted or desert consistent with clinically “cold” tumors. Results Metastases of “cold” primary tumors always remained “cold” at their matched metastatic site. Expression of PD-1/LAG-3 was associated with a “hot” immune phenotype in both the primary tumors and metastases. We could not observe any association between the immune phenotype and the breast cancer molecular subtype. Brain and soft tissue metastases were more commonly inflamed with signs of exhaustion than other anatomical sites of metastases. Taken together, (i) the immune phenotype varied between sites of distant metastases, and (ii) PD-1+/LAG-3+ was strongly associated with a “hot” immune phenotype and (iii) was most prevalent in brain and soft tissue metastases among distant metastases. Conclusions Our data strongly support an integrated analysis of the immune phenotype together with the PD-1/LAG-3 expression in distant metastases to identify patients with inflamed but exhausted tumors. This may eventually improve the stratification and likelihood for advanced breast cancer patients to profit from immunotherapy.

The immunopathological features of SARS-CoV-2 infection in the lungs remain unclear. Here, the authors provide a comprehensive characterization of post mortem lung tissues of COVID-19 patients and find two distinct patterns characterized by differential expression of interferon stimulated genes (ISGs), which correlate to viral loads, cytokines, lung damage and time of hospitalization, suggesting ISG profiles to mark disease progression

BackgroundLong-term sequelae of coronavirus disease 2019 (COVID-19), including the interaction between persisting viral-RNA and specific tissue involvement, pose a challenging issue. In this study, we addressed the chronological correlation (after first clinical diagnosis and postmortem) between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and organ involvement.MethodsThe presence of postmortem SARS-CoV-2 RNA from 35 complete COVID-19 autopsies was correlated with the time interval between the first diagnosis of COVID-19 and death and with its relationship to morphologic findings.ResultsSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be evident up to 40 days after the first diagnosis and can persist to 94 hours after death. Postmortem SARS-CoV-2 RNA was mostly positive in lungs (70%) and trachea (69%), but all investigated organs were positive with variable frequency. Late-stage tissue damage was evident up to 65 days after initial diagnosis in several organs. Positivity for SARS-CoV-2 RNA in pulmonary swabs correlated with diffuse alveolar damage (p = 0.0009). No correlation between positive swabs and other morphologic findings was present. Cerebral (p = 0.0003) and systemic hemorrhages (p = 0.009), cardiac thrombi (p = 0.04), and ischemic events (p = 0.03) were more frequent in the first wave, whereas bacterial pneumonia (p = 0.03) was more prevalent in the second wave. No differences in biometric data, clinical comorbidities, and other autopsy findings were found.ConclusionsOur data provide evidence not only of long-term postmortem persisting SARS-CoV-2 RNA but also of tissue damage several weeks after the first diagnosis of SARS-CoV-2 infection. Additional conditions, such as concomitant bacterial pulmonary superinfection, lung aspergillosis, thromboembolic phenomena, and hemorrhages can further worsen tissue damage.

Tumour progression is an evolutionary process in which different clones evolve over time, leading to intra-tumour heterogeneity. Interactions between clones can affect tumour evolution and hence disease progression and treatment outcome. Intra-tumoural pairs of mutations that are overrepresented in a co-occurring or clonally exclusive fashion over a cohort of patient samples may be suggestive of a synergistic effect between the different clones carrying these mutations. We therefore developed a novel statistical testing framework, called GeneAccord, to identify such gene pairs that are altered in distinct subclones of the same tumour. We analysed our framework for calibration and power. By comparing its performance to baseline methods, we demonstrate that to control type I errors, it is essential to account for the evolutionary dependencies among clones. In applying GeneAccord to the single-cell sequencing of a cohort of 123 acute myeloid leukaemia patients, we find 1 clonally co-occurring and 8 clonally exclusive gene pairs. The clonally exclusive pairs mostly involve genes of the key signalling pathways.

Alternative splicing is an essential regulatory mechanism for gene expression in mammalian cells contributing to protein, cellular, and species diversity. In cancer, alternative splicing is frequently disturbed, leading to changes in the expression of alternatively spliced protein isoforms. Advances in sequencing technologies and analysis methods led to new insights into the extent and functional impact of disturbed alternative splicing events. In this review, we give a brief overview of the molecular mechanisms driving alternative splicing, highlight the function of alternative splicing in healthy tissues and describe how alternative splicing is disrupted in cancer. We summarize current available computational tools for analyzing differential transcript usage, isoform switching events, and the pathogenic impact of cancer-specific splicing events. Finally, the strategies of three recent pan-cancer studies on isoform switching events are compared. Their methodological similarities and discrepancies are highlighted and lessons learned from the comparison are listed. We hope that our assessment will lead to new and more robust methods for cancer-specific transcript detection and help to produce more accurate functional impact predictions of isoform switching events.

Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) genes have been recently identified as the most enriched class of copy-number associated gene dependencies in human cancer. These genes are cell essential and render tumor cells highly sensitive to the expression of the remaining copy. Chromophobe renal cell carcinoma (chRCC) is characterized by frequent chromosomal deletions, but the relevance of CYCLOPS genes in this tumor subtype is unclear. We found 39 (31%) of 124 recently published candidate CYCLOPS genes (B. Paolella et al., eLife 2017;6:e23268) located on 7 autosomes that are frequently lost in chRCC. GISTIC and RNA-seq data obtained from the TCGA-KICH database showed that 62% of these CYCLOPS genes had significantly lower expression levels in samples with deletion of the respective gene. As copy number (CN) loss of the CYCLOPS gene SF3B1 (Splicing factor 3B subunit 1) has been recently reported in 71% chRCC, we explored the relevance of SF3B1 CN alteration and SF3B1 expression in a set of chRCC and additional oncocytic renal neoplasms. The frequency of SF3B1 CN loss (65%) was similar to that obtained from the TCGA-KICH database and correlated significantly with both lower SF3B1 mRNA (P

Abstract Background To validate the utility of the chemokine ligand 12 (CXCL12) as prognostic marker in patients with localized and metastatic germ cell tumors (GCT). Methods CXCL12 expression was analyzed on a tissue microarray consisting of 750 tissue cores of different histological tumor components, Germ cell neoplasia in situ (GCNIS) and adjacent normal tissue of 263 testicular cancer patients using a semi-quantitative score. The association between CXCL12 expression and recurrence-free survival (RFS) as well as overall survival (OS) was assessed using Kaplan-Meier curves with log-rank tests. Results CXCL12 expression was absent in all seminomas but was found in 52 of 99 (52.5%) non-seminomas. Follow-up was available for 260 patients of which 36 (13.8%) recurred. In patients with stage 1 non-seminoma GCT, CXCL12 expression was not associated with higher risk of disease recurrence (p = 0.270). In contrast, post chemotherapy RFS of patients with metastatic non-seminoma and positive CXCL12 expression was significantly shorter compared to CXCL12 negative patients (p = 0.003). OS differences were not statistically different between patients with CXCL12 positive or negative tumors for either localized or metastatic disease. Conclusions CXCL12 is almost exclusively expressed in non-seminoma. Pure seminoma, GCNIS and adjacent normal testicular tissue are CXCL12 negative. Our analysis suggests that patients with metastatic disease and a CXCL12-positive non-seminoma are at higher risk for disease recurrence after first-line chemotherapy and might thus be candidates for more intensive treatment and/or closer follow-up.

Bi-allelic inactivation of the VHL gene on chromosome 3p is the characteristic feature in most clear cell renal cell carcinomas (ccRCC). Frequent gene alterations were also identified in SETD2, BAP1 and PBRM1, all of which are situated on chromosome 3p and encode histone/chromatin regulators. The relationship between gene mutation, loss of protein expression and the correlations with clinicopathological parameters is important for the understanding of renal cancer progression. We analyzed PBRM1 and BAP1 protein expression as well as the tri-methylation state of H3K36 as a surrogate marker for SETD2 activity in more than 700 RCC samples. In ccRCC loss of nuclear PBRM1 (68%), BAP1 (40%) and H3K36me3 (47%) expression was significantly correlated with each other, advanced tumor stage, poor tumor differentiation (P

To evaluate the prognostic value of a modified Immunoscore (mIS) in a cohort of bladder cancer (BC) patients undergoing radical cystectomy (RC), two tissue microarrays of 159 BC patients were immunohistochemically stained for CD3/CD8/FOXP3 and CD45RO to detect Tumor-Infiltrating Lymphocytes (TIL). To predict progression free survival (PFS) and cancer specific survival (CSS), a predictive model cumulatively incorporating all four components was constructed and labeled as mIS. Patients were stratified into two risk groups; “high mIS/favorable risk” and “low mIS/unfavorable risk”. Kaplan–Meier analysis was used to test mIS within each American Joint Committee on Cancer (AJCC) stage group for BC. In a univariable cox regression analysis all single components used for mIS, showed a significant association with CSS. Patients with high mIS (all components) in the AJCC stage IIIa group additionally showed a significantly longer PFS (Hazard Ratio (HR): 2.7; p = 0.008) and CSS (HR: 3.5; p = 0.006) as compared to patients with low mIS. mIS is of prognostic value in BC patients undergoing RC and was able to stratify patients within AJCC stage IIIa and might thus serve as a prognostic marker to guide risk-adapted treatment or follow-up strategies after RC.

Background The human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level.Methods Human tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing.Results The initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference.Conclusion Given that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org.

Background: SARS-CoV-2 infection (COVID-19 disease) can induce systemic vascular involvement contributing to morbidity and mortality. SARS-CoV-2 targets epithelial and endothelial cells through the ACE2 receptor. The anatomical involvement of the coronary tree is not explored yet. Methods: Cardiac autopsy tissue of the entire coronary tree (main coronary arteries, epicardial arterioles/venules, epicardial capillaries) and epicardial nerves were analyzed in COVID-19 patients (n = 6). All anatomical regions were immunohistochemically tested for ACE2, TMPRSS2, CD147, CD45, CD3, CD4, CD8, CD68 and IL-6. COVID-19 negative patients with cardiovascular disease (n = 3) and influenza A (n = 6) served as controls. Findings: COVID-19 positive patients showed strong ACE2 / TMPRSS2 expression in capillaries and less in arterioles/venules. The main coronary arteries were virtually devoid of ACE2 receptor and had only mild intimal inflammation. Epicardial capillaries had a prominent lympho-monocytic endotheliitis, which was less pronounced in arterioles/venules. The lymphocytic-monocytic infiltrate strongly expressed CD4, CD45, CD68. Peri/epicardial nerves had strong ACE2 expression and lympho-monocytic inflammation. COVID-19 negative patients showed minimal vascular ACE2 expression and lacked endotheliitis or inflammatory reaction. Interpretation: ACE2 / TMPRSS2 expression and lymphomonocytic inflammation in COVID-19 disease increases crescentically towards the small vessels suggesting that COVID-19-induced endotheliitis is a small vessel vasculitis not involving the main coronaries. The inflammatory neuropathy of epicardial nerves in COVID-19 disease provides further evidence of an angio- and neurotrophic affinity of SARS-COV2 and might potentially contribute to the understanding of the high prevalence of cardiac complications such as myocardial injury and arrhythmias in COVID-19. Funding: No external funding was necessary for this study.

Abstract Objectives Critically ill coronavirus disease 2019 (COVID‐19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID‐19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils. Methods Regulated cell death phenotype of neutrophils isolated from critically ill COVID‐19 patients or healthy donors and stimulated with COVID‐19 or healthy plasma ex vivo was assessed by flow cytometry, time‐lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID‐19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID‐19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID‐19 patients. Results COVID‐19 plasma induced a necroptosis‐sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage‐associated molecular patterns (DAMPs), increased receptor‐interacting serine/threonine‐protein kinase (RIPK) 1 levels and mixed lineage kinase domain‐like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID‐19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF‐α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID‐19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF‐driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids. Conclusion Our results suggest a novel role for sFasL signalling in the TNF‐α‐induced RCD programme in neutrophils during COVID‐19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.

Clear cell renal cell carcinoma (ccRCC) displays a highly varying clinical progression, from slow growing localized tumors to very aggressive metastatic disease (mRCC). Almost a third of all patients with ccRCC show metastatic dissemination at presentation while another third develop metastasis during the course of the disease. Survival rates of mRCC patients remain low despite the development of novel targeted treatment regimens. Biomarkers indicating disease progression could help to define its aggressive potential and thus guide patient management. However, molecular markers that can reliably assess metastatic dissemination and disease recurrence in ccRCC have not been recommended for clinical practice to date. Liquid biopsies could provide an attractive and non-invasive method to determine the risk of recurrence or metastatic dissemination during follow-up and thus assist the search for surveillance biomarkers in ccRCC tumors. A wide spectrum of circulating molecules have already shown considerable potential for ccRCC diagnosis and prognostication. In this review, we outline state of the art of the key circulating analytes such as cfDNA, cfRNA, proteins, and exosomes that may serve as biomarkers for the longitudinal monitoring of ccRCC progression to metastasis. Moreover, we address some of the prevailing limitations in the past approaches and present promising adoptable technologies that could help to pursue the implementation of liquid biopsies as a prognostic tool for mRCC.

Clear-cell renal cell carcinomas (ccRCCs) are characterized by inactivation of the von Hippel-Lindau (VHL) gene and intracellular lipid accumulation by unknown pathomechanisms. The immunochemical analysis of 356 RCCs revealed high abundance of apoA-I and apoB, as well as scavenger receptor BI (SR-BI) in the ccRCC subtype. Given the characteristic loss of VHL function in ccRCC, we used VHL-defective and VHL-proficient cells to study the potential influence of VHL on lipoprotein uptake. VHL-defective patient-derived ccRCC cells and cell lines (786O and RCC4) showed enhanced uptake as well as less resecretion and degradation of radio-iodinated HDL and LDL (125I-HDL and 125I-LDL, respectively) compared with the VHL-proficient cells. The ccRCC cells showed enhanced vascular endothelial growth factor (VEGF) and SR-BI expression compared with normal kidney epithelial cells. Uptake of 125I-HDL and 125I-LDL by patient-derived normal kidney epithelial cells as well as the VHL-reexpressing ccRCC cell lines, 786-O-VHL and RCC4-O-VHL cells, was strongly enhanced by VEGF treatment. The knockdown of the VEGF coreceptor, neuropilin-1 (NRP1), as well as blocking of SR-BI significantly reduced the uptake of lipoproteins into ccRCC cells in vitro. LDL stimulated proliferation of 786-O cells more potently than 786-O-VHL cells in a NRP1- and SR-BI-dependent manner. In conclusion, enhanced lipoprotein uptake due to increased activities of VEGF/NRP1 and SR-BI promotes lipid accumulation and proliferation of VHL-defective ccRCC cells.

Abstract Background Cancer of unknown primary (CUP) is a distinct clinicopathological entity with poor prognosis, frequently resistant to chemotherapy. Comprehensive genomic profiling (CGP) by next‐generation sequencing potentially identifies novel treatment options for CUP patients. The objective of this study was to determine incidence and survival trends and to discuss the value of CGP in CUP patients. Methods Age‐standardized incidence rates (ASR) per 100 000 were calculated for 2935 CUP patients from 1981 to 2014 using cancer registry data of the canton of Zurich, Switzerland. Kaplan–Meier survival curves were estimated for sex, age, and histological groups. Cox proportional hazards regression models were used to estimate adjusted hazard ratios (HR). A literature review was conducted to assess the current use of CGP in CUP patients. Results ASR of CUP increased from 10.3 to 17.6 between 1981 and 1997 and decreased to 5.8/100 000 in 2014. Mean overall survival remained stable. Mortality was significantly lower for patients with squamous cell carcinoma (HR 0.48 [95% CI, 0.41‐0.57]) and neuroendocrine carcinoma (0.75 [0.63‐0.88]) and higher for unclassified neoplasms (1.25 [1.13‐1.66]) compared to adenocarcinomas. The literature review identified 10 studies using CGP of CUP tissue. Clinically relevant mutations were identified in up to 85% of CUP patients, of which 13%‐64% may benefit from currently available drugs. Conclusions CUP incidence decreased probably due to improved diagnostics, but mortality did not improve over the last 34 years. CGP testing may help to identify molecular signatures in CUP patients and enable targeted treatment.

Abstract Background Epithelioid angiomyolipoma is defined as potentially malignant mesenchymal neoplasm, characterized by proliferating epithelioid cells, whereas classic angiomyolipoma, composed of fat, smooth muscle cells and dysmorphic vessels, is defined as a potentially benign. The usual or classic angiomyolipoma is often found incidentally on imaging studies, relatively easily identified due to the presence of fat, in contrast to the epithelioid angiomyolipoma that can pose diagnostic challenges. Case presentation We report a 51-year-old female patient in which an ultrasonography examination showed a solid mass close to the right renal pelvis with hypoechoic and hyperechoic areas. A differential diagnosis of atypical sinus lipomatosis, lipoma and a transitional cell carcinoma was postulated whereas in a subsequent computed tomography a classic angiomyolipoma was postulated. A re-examination by contrast enhanced ultrasound revealed a striking perfusion difference of the hypoechoic and hyperechoic areas. The hypoechoic area showed homogenous and prolonged enhancement whereas the hypoechoic area displayed a marked slower contrast material flooding and a relatively rapid wash out. The histological analysis from the biopsy of the hyperechoic area showed a classic angiomyolipoma, whereas the sample of the hypoechoic central portion revealed an epithelioid angiomyolipoma. A nephrectomy was performed because of the malignant potential of the epithelioid variant of the angiomyolipoma. Conclusions A solid kidney mass with two sharply defined parts, one-part compatible with a classical angiomyolipoma and the other being suspected of carcinoma, is rare, but also illustrative and instructive. The combination of different imaging modalities in the work up of a solid renal mass facilitated to discriminate benign from malignant areas.

Renal cell carcinoma (RCC) represents a heterogeneous disease, encompassing an increasing number of tumor subtypes. Post-2016, the World Health Organization (WHO) classification recognized that the spectrum of papillary renal cell carcinoma is evolving and has long surpassed the dichotomic simplistic “type 1 versus type 2” classification. The differential diagnosis of pRCC includes several new provisional/emerging entities with papillary growth. Type 2 tumors have been cleared out of several confounding entities, now regarded as independent tumors with specific clinical and molecular backgrounds. In this work we describe the prevalence and characteristics of emerging papillary tumor entities in two renal tumor cohorts (one consisting of consecutive papillary tumors from a single institute, the other consisting of consultation cases from several centers). After a review of 154 consecutive pRCC cases, 58% remained type 1 pRCC, and 34% type 2 pRCC. Papillary renal neoplasm with reversed polarity (1.3%), biphasic hyalinizing psammomatous RCC (1.3%), and biphasic squamoid/alveolar RCC (4.5%) were rare. Among 281 consultation cases, 121 (43%) tumors had a dominant papillary growth (most frequently MiT family translocation RCCs, mucinous tubular and spindle cell carcinoma and clear cell papillary RCC). Our data confirm that the spectrum of RCCs with papillary growth represents a major diagnostical challenge, frequently requiring a second expert opinion. Papillary renal neoplasm with reversed polarity, biphasic hyalinizing psammomatous RCC, and biphasic squamoid/alveolar RCC are rarely sent out for a second opinion, but correct classification and knowledge of these variants will improve our understanding of the clinical behavior of renal tumors with papillary growth.

Biorespositories of formalin-fixed and paraffin-embedded (FFPE) or fresh frozen human tissues from malignant diseases generated as integral part of the diagnostic workup in many pathology departments have been pivotal resources for translational cancer studies. However, such tissue biobanks have traditionally contained only non-viable specimens and thus cannot enable functional assays for the discovery and validation of therapeutic targets or the assessment of drug responses and resistance to treatment. To overcome these limitations, we have developed a next-generation comprehensive biobanking platform that includes the generation of patient-derived in vitro cell models from colorectal, pancreatic and kidney cancers among others. As such patient-derived cell (PDC) models retain important features of the original human tumors, they have emerged as relevant tools for more dynamic clinical and experimental analyses of cancer. Here, we describe details of the complex processes of acquisition and processing of patient-derived samples, propagation, annotation, characterization and distribution of resulting cell models and emphasize the requirements of quality assurance, organizational considerations and investment into resources. Taken together, we show how clinical tissue collections can be taken to the next level thus promising major new opportunities for understanding and treating cancer in the context of precision medicine.

Autophagy is a cellular “self-digestion” process known to be essential for various physiological and pathological pathways, including cancer, where its role appears to be context-dependent. In this work, we aimed to investigate the level of autophagy by evaluating the expression of key autophagy-related proteins (ATGs) in testicular germ cell tumors (TGCT) for which autophagy has been rarely investigated. We decided to use an immunohistochemical (IHC) staining approach employing a tissue microarray (TMA). Software-based evaluation of the integrated optical densities (IODs) of these proteins indicated a significant downregulation of ATG1, ATG5, and ATG16L1. Accordingly, reduced levels of microtubule-associated proteins 1A/1B light chain 3B (LC3B) were found to parallel increases in sequestosome-1 (SQSTM1 or p62), a protein normally degraded via autophagy, suggesting an in vivo reduction in autophagy with TGCT. Thus, our work provides evidence for a tumor suppressive function of autophagy in the development of TGCT and supports the concept of a context-dependent role of autophagy in tumorigenesis which is tumor type-dependent.

Tertiary lymphoid structures (TLS) are associated with favorable outcome in non-metastatic colorectal carcinoma (nmCRC), but the dynamics of TLS maturation and its association with effective anti-tumor immune surveillance in nmCRC are unclear. Here, we hypothesized that not only the number of TLS but also their composition harbors information on recurrence risk in nmCRC. In a comprehensive molecular, tissue, laboratory, and clinical analysis of 109 patients with stage II/III nmCRC, we assessed TLS numbers and degree of maturation in surgical specimens by multi-parameter immunofluorescence of follicular dendritic cell (FDC) and germinal center (GC) markers. TLS formed in most tumors and were significantly more prevalent in highly-microsatellite-instable (MSI-H) and/or BRAF-mutant nmCRC. We could distinguish three sequential TLS maturation stages which were characterized by increasing prevalence of FDCs and mature B-cells: [1] Early TLS, composed of dense lymphocytic aggregates without FDCs, [2] Primary follicle-like TLS, having FDCs but no GC reaction, and [3] Secondary follicle-like TLS, having an active GC reaction. A simple integrated TLS immunoscore reflecting these parameters identified a large subgroup of nmCRC patients with a very low risk of recurrence independently of clinical co-variables such as ECOG performance status, age, stage, and adjuvant chemotherapy. We conclude that (1) mismatch repair and BRAF mutation status are associated with the formation of TLS in nmCRC, (2) TLS formation in nmCRC follows sequential maturation steps, culminating in germinal center formation, and (3) this maturation process harbors important prognostic information on the risk of disease recurrence.

BACKGROUND: Recurrent mutations in the Speckle-Type POZ Protein (SPOP) gene occur in up to 15% of prostate cancers. However, the frequency and features of cancers with these mutations across different populations is unknown. OBJECTIVE: To investigate SPOP mutations across diverse cohorts and validate a series of assays employing high-resolution melting (HRM) analysis and Sanger sequencing for mutational analysis of formalin-fixed paraffin-embedded material. DESIGN, SETTING, AND PARTICIPANTS: 720 prostate cancer samples from six international cohorts spanning Caucasian, African American, and Asian patients, including both prostate-specific antigen-screened and unscreened populations, were screened for their SPOP mutation status. Status of SPOP was correlated to molecular features (ERG rearrangement, PTEN deletion, and CHD1 deletion) as well as clinical and pathologic features. RESULTS AND LIMITATIONS: Overall frequency of SPOP mutations was 8.1% (4.6% to 14.4%), SPOP mutation was inversely associated with ERG rearrangement (P < .01), and SPOP mutant (SPOPmut) cancers had higher rates of CHD1 deletions (P < .01). There were no significant differences in biochemical recurrence in SPOPmut cancers. Limitations of this study include missing mutational data due to sample quality and lack of power to identify a difference in clinical outcomes. CONCLUSION: SPOP is mutated in 4.6% to 14.4% of patients with prostate cancer across different ethnic and demographic backgrounds. There was no significant association between SPOP mutations with ethnicity, clinical, or pathologic parameters. Mutual exclusivity of SPOP mutation with ERG rearrangement as well as a high association with CHD1 deletion reinforces SPOP mutation as defining a distinct molecular subclass of prostate cancer.

TFE3 translocation renal cell carcinoma (tRCC) is defined by chromosomal translocations involving the TFE3 transcription factor at chromosome Xp11.2. Genetically proven TFE3 tRCCs have a broad histologic spectrum with overlapping features to other renal tumor subtypes. In this study,we aimed for characterizing RCC with TFE3 protein expression. Using next-generation whole transcriptome sequencing (RNA-Seq) as a discovery tool, we analyzed fusion transcripts, gene expression profile, and somatic mutations in frozen tissue of one TFE3 tRCC. By applying a computational analysis developed to call chimeric RNA molecules from paired-end RNA-Seq data, we confirmed the known TFE3 translocation. Its fusion partner SFPQ has already been described as fusion partner in tRCCs. In addition, an RNAread-through chimera between TMED6 and COG8 as well as MET and KDR (VEGFR2) point mutations were identified. An EGFR mutation, but no chromosomal rearrangements, was identified in a control group of five clear cell RCCs (ccRCCs). The TFE3 tRCC could be clearly distinguished from the ccRCCs by RNA-Seq gene expression measurements using a previously reported tRCC gene signature. In validation experiments using reverse transcription-PCR, TMED6-COG8 chimera expression was significantly higher in nine TFE3 translocated and six TFE3-expressing/non-translocated RCCs than in 24 ccRCCs (P

Abstract The combinations of genetic alterations that cooperate with von Hippel–Lindau (VHL) mutation to cause clear cell renal cell carcinoma (ccRCC) remain poorly understood. We show that the TP53 tumour suppressor gene is mutated in approximately 9% of human ccRCCs. Combined deletion of Vhl and Trp53 in primary mouse embryo fibroblasts causes proliferative dysregulation and high rates of aneuploidy. Deletion of these genes in the epithelium of the kidney induces the formation of simple cysts, atypical cysts and neoplasms, and deletion in the epithelia of the genital urinary tract leads to dysplasia and tumour formation. Kidney cysts display a reduced frequency of primary cilia and atypical cysts and neoplasms exhibit a pro‐proliferative signature including activation of mTORC1 and high expression of Myc, mimicking several cellular and molecular alterations seen in human ccRCC and its precursor lesions. As the majority of ccRCC is associated with functional inactivation of VHL, our findings suggest that for a subset of ccRCC, loss of p53 function represents a critical event in tumour development.

Despite the individually different molecular alterations in tumors, the malignancy associated biological traits are strikingly similar. Results of a previous study using renal cell carcinoma (RCC) as a model pointed towards cancer-related features, which could be visualized as three groups by microarray based gene expression analysis. In this study, we used a mathematic model to verify the presence of these groups in RCC as well as in other cancer types. We developed an algorithm for gene-expression deviation profiling for analyzing gene expression data of a total of 8397 patients with 13 different cancer types and normal tissues. We revealed three common Cancer Transcriptomic Profiles (CTPs) which recurred in all investigated tumors. Additionally, CTPs remained robust regardless of the functions or numbers of genes analyzed. CTPs may represent common genetic fingerprints, which potentially reflect the closely related biological traits of human cancers.

BACKGROUND:In vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE). AIM:To investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers. METHODS:Past and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician's perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed. RESULTS:IVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0-4%, p

BACKGROUND:Brain metastasis is a common endpoint in patients suffering from malignant melanoma. However, little is known about factors that predispose to brain metastases. OBJECTIVE:We performed a retrospective clinical and pathological investigation of melanoma patients with brain metastases in order to better characterise this patient population. METHODS:193 melanoma patients with brain metastasis histologically diagnosed between 1990 and 2015 at the University Hospital Zurich were retrospectively identified and further specified for sex, age at diagnosis and detection of brain metastasis, and localisation. In addition, data were extracted regarding the subtype of primary melanoma, Breslow tumour thickness, Clark Level, mutation status, extent of metastatic spread and history of a second melanoma. RESULTS:We found a significant male predominance (n = 126/193; 65%; p < 0.001). Breslow tumour thickness showed a wide range from 0.2 to 12.0 mm (n = 99; median 2.3 mm). 14 of 101 melanomas (14%) were classified as T1, thereof 11 (79%) were found in men. In 32 of 193 patients (17%), the primary melanoma was unknown. CONCLUSIONS:Of special interest in our series is the high incidence of male predominance (79%) in cases of thin metastasing melanoma (14%), implicating genetic or epigenetic (hormonal) gender differences underlying tumour progression. Additionally, the high percentage of unknown primary melanoma (17%), at least partly representing completely regressed melanomas, indicates the importance of immune surveillance in melanoma progression.

Abstract Type II endometrial carcinomas are a highly aggressive group of tumour subtypes that are frequently associated with inactivation of the TP53 tumour suppressor gene. We show that mice with endometrium‐specific deletion of Trp53 initially exhibited histological changes that are identical to known precursor lesions of type II endometrial carcinomas in humans and later developed carcinomas representing all type II subtypes. The mTORC1 signalling pathway was frequently activated in these precursor lesions and tumours, suggesting a genetic cooperation between this pathway and Trp53 deficiency in tumour initiation. Consistent with this idea, analyses of 521 human endometrial carcinomas identified frequent mTORC1 pathway activation in type I as well as type II endometrial carcinoma subtypes. mTORC1 pathway activation and p53 expression or mutation status each independently predicted poor patient survival. We suggest that molecular alterations in p53 and the mTORC1 pathway play different roles in the initiation of the different endometrial cancer subtypes, but that combined p53 inactivation and mTORC1 pathway activation are unifying pathogenic features among histologically diverse subtypes of late stage aggressive endometrial tumours.

Background: Melanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition. Patients and Methods: We used 2 methods to analyze the BRAF mutation status in 52 of 62 melanoma patients. Method 1 (mutation-specific real-time PCR) specifically detects the most frequent BRAF mutations, V600E and V600K. Method 2 (denaturing gel gradient electrophoresis and direct sequencing) identifies any mutations affecting exons 11 and 15. Results: Eighteen BRAF mutations and 15 wild-type mutations were identified with both methods. One tumor had a double mutation (GAA) in codon 600. Results of 3 samples were discrepant. Additional mutations (V600M, K601E) were detected using method 2. Sixteen DNA samples were analyzable with either method 1 or method 2. There was a significant association between BRAF V600E mutation and survival. Conclusion: Standardized tissue fixation protocols are needed to optimize BRAF mutation analysis in melanoma. For melanoma treatment decisions, the availability of a fast and reliable BRAF V600E screening method may be sufficient. If other BRAF mutations in exons 11 and 15 are found to be of predictive value, a combination of the 2 methods would be useful.

We have explored MICA/B expression and its relationship with innate inflammatory infiltrate in renal cell carcinoma (RCC). The expression of MICA/B, CD16, CD56, and CD68 in 140 RCC lesions contained in a tissue microarray (TMA) was investigated by immunohistochemistry. MICA/B gene and protein expressions in Caki-1 cells were analyzed by reverse transcription-polymerase chain reaction and flow cytometry, respectively. Natural killer (NK) cells were studied by flow cytometry. All the RCC lesions (n = 140) were MICA/B-positive. MICA/B was mainly expressed in the cytoplasm of tumor cells, whereas stromal cells were negative. Renal cell carcinoma lesions showed low NK cell infiltration, although they were rich in CD16+CD56- cells, strongly resembling macrophages. CD16+ macrophage infiltration was more frequently detectable in metastatic lesions compared with primary tumors (P = .0223) and was associated with poor RCC differentiation (P = .007). To investigate mechanisms potentially underlying the lack of NK cells infiltration into MICA/B-positive RCC lesions, we used Caki-1 RCC cells. Caki-1 expressed MICA and MICB genes. However, MICA protein was not detectable in Caki-1 cells, whereas MICB protein was detectable in their cytoplasm and on the cell membrane. Coculture of peripheral blood mononuclear cells with Caki-1, K562, HCT116, respectively, resulted in CD56+CD16+ NK cells deletion without affecting CD56+/CD16- NK subset and immature NK cells generated in vitro from CD34+ cells. Natural killer cell apoptosis seemed to be preferentially triggered by cancer cells because HLA-A0201+ NK cells were only marginally affected by allogeneic HLA-A0201- peripheral blood mononuclear cells. Caki-1 cell-mediated NK cell apoptosis was reduced by an anti-β2-integrin (CD18) monoclonal antibody but was NKG2D-, granule exocytosis-, and caspase-independent.