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Abstract Urothelial bladder cancer (UC) has a wide tumor biological spectrum with challenging prognostic stratification and relevant therapy-associated morbidity. Most molecular classifications relate only indirectly to the therapeutically relevant protein level. We improve the pre-analytics of clinical samples for proteome analyses and characterize a cohort of 434 samples with 242 tumors and 192 paired normal mucosae covering the full range of UC. We evaluate sample-wise tumor specificity and rank biomarkers by target relevance. We identify robust proteomic subtypes with prognostic information independent from histopathological groups. In silico drug prediction suggests efficacy of several compounds hitherto not in clinical use. Both in silico and in vitro data indicate predictive value of the proteomic clusters for these drugs. We underline that proteomics is relevant for personalized oncology and provide abundance and tumor specificity data for a large part of the UC proteome ( www.cancerproteins.org ).

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide with lung adenocarcinoma (LUAD) being the most common type. Genomic studies of LUAD have advanced our understanding of its tumor biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD are still insufficiently explored. The prognosis for lung cancer patients is still mostly determined by the stage of disease at the time of diagnosis. Focusing on late-stage metastatic LUAD with poor prognosis, we compared the proteomic profiles of primary tumors and matched distant metastases to identify relevant and potentially druggable differences. We performed high-performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry (ESI–MS/MS) on a total of 38 FFPE (formalin‐fixed and paraffin‐embedded) samples. Using differential expression analysis and unsupervised clustering we identified several proteins that were differentially regulated in metastases compared to matched primary tumors. Selected proteins (HK1, ATP5A, SRI and ARHGDIB) were subjected to validation by immunoblotting. Thereby, significant differential expression could be confirmed for HK1 and ATP5A, both upregulated in metastases compared to matched primary tumors. Our findings give a better understanding of tumor progression and metastatic spreads in LUAD but also demonstrate considerable inter-individual heterogeneity on the proteomic level.

Metastatic prostate cancer (mPCa) is a widespread disease with high mortality. Unraveling molecular mechanisms of disease progression is of utmost importance. The microenvironment in visceral organs and the skeletal system is of particular interest as a harbinger of metastatic spread. Therefore, we performed a comprehensive transcriptomic analysis of prostate cancer lung metastases with a special focus on differentially expressed genes attributable to the microenvironment. Digital gene expression analysis using the NanoString nCounter analysis system was performed on formalin-fixed, paraffin-embedded (FFPE) tissue from prostate cancer (PCa) lung metastases (n = 24). Data were compared to gene expression data from primary PCa and PCa bone metastases. Bioinformatic analysis was performed using several publicly available tools. In comparison to prostate cancer bone metastases, 209 genes were significantly upregulated, and 100 genes were significantly downregulated in prostate cancer lung metastases. Among the up-regulated genes, the top 10 genes with the most significant P-value were HLA-DPB1, PTPRC, ITGB7, C3, CCL21, CCL5, ITGAM, SERPINA1, MFAP4, ARAP2 and among the down-regulated genes, the top 10 genes with the most significant P-value were FOXC2, TWIST1, CDK14, CHAD, IBSP, EPN3, VIT, HAPLN1, SLC44A4, TBX1. In PCa lung metastases genes associated with immunogenic responses were upregulated while genes associated with epithelial-mesenchymal transition were down-regulated. We also showed that CXCR3/CXCL10 axis plays a significant role in prostate cancer lung metastases in comparison to bone metastases. In this study, we comprehensively explored transcriptomic alterations in PCa lung metastases in comparison to primary PCa and PCa bone metastases. In PCa lung metastases genes associated with immunogenic responses are upregulated while genes associated with epithelial-mesenchymal transition are down-regulated. This points to a more immunogenic phenotype of PCa lung metastases thus potentially making patients more susceptible to immunotherapeutic approaches.

Alternative sources of tumour information need to be explored in patients with non‐small cell lung cancer (NSCLC). Here, we compared programmed cell death ligand 1 (PD‐L1) expression on cytology imprints and circulating tumour cells (CTCs) with PD‐L1 tumour proportion score (TPS) from immunohistochemistry staining of tumour tissue from patients with NSCLC. We evaluated PD‐L1 expression using a PD‐L1 antibody (28‐8) in representative cytology imprints, and tissue samples from the same tumour. We report good agreement rates on PD‐L1 positivity (TPS ≥ 1%) and high PD‐L1 expression (TPS ≥ 50%). Considering high PD‐L1 expression, cytology imprints showed a PPV of 64% and a NPV of 85%. CTCs were detected in 40% of the patients and 80% of them were PD‐L1+. Seven patients with PD‐L1 expression of

Abstract Background The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin β4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. Methods We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. Results We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. Conclusions These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies. Graphical Abstract

Sinonasal tumour diagnosis can be complicated by the heterogeneity of disease and classification systems. Here, the authors use machine learning to classify sinonasal undifferentiated carcinomas into 4 molecular classe with differences in differentiation state and clinical outcome.

Abstract Background Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. Methods TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell–cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. Results Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. Conclusions Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa. Graphical Abstract

Abstract Prostate cancer is a hormone-driven disease and its tumor cell growth highly relies on increased androgen receptor (AR) signaling. Therefore, targeted therapy directed against androgen synthesis or AR activation is broadly used and continually improved. However, a subset of patients eventually progresses to castration-resistant disease. To date, various mechanisms of resistance have been identified including the development of AR-independent aggressive variant prostate cancer based on neuroendocrine transdifferentiation (NED). Here, we review the highly complex processes contributing to NED. Genetic, epigenetic, transcriptional aberrations and posttranscriptional modifications are highlighted and the potential interplay of the different factors is discussed. Background Aggressive variant prostate cancer (AVPC) with traits of neuroendocrine differentiation emerges in a rising number of patients in recent years. Among others, advanced therapies targeting the androgen receptor axis have been considered causative for this development. Cell growth of AVPC often occurs completely independent of the androgen receptor signal transduction pathway and cells have mostly lost the typical cellular features of prostate adenocarcinoma. This complicates both diagnosis and treatment of this very aggressive disease. We believe that a deeper understanding of the complex molecular pathological mechanisms contributing to transdifferentiation will help to improve diagnostic procedures and develop effective treatment strategies. Indeed, in recent years, many scientists have made important contributions to unravel possible causes and mechanisms in the context of neuroendocrine transdifferentiation. However, the complexity of the diverse molecular pathways has not been captured completely, yet. This narrative review comprehensively highlights the individual steps of neuroendocrine transdifferentiation and makes an important contribution in bringing together the results found so far.

Abstract Background Recently, antibodies against the alpha isoform of the glial-fibrillary-acidic-protein (GFAPα) were identified in a small series of patients with encephalomyelitis. Coexisting autoantibodies (NMDA receptor, GAD65 antibodies) have been described in a few of these patients. We describe a patient with rapidly progressive encephalomyeloradiculitis and a combination of anti-ITPR1, anti-GFAP and anti-MOG antibodies. Case presentation and literature review A 44-year old caucasian woman with a flu-like prodrome presented with meningism, progressive cerebellar signs and autonomic symptoms, areflexia, quadriplegia and respiratory insufficiency. MRI showed diffuse bilateral T2w-hyperintense brain lesions in the cortex, white matter, the corpus callosum as well as a longitudinal lesion of the medulla oblongata and the entire spinal cord. Anti-ITPR1, anti-GFAP and anti-MOG antibodies were detected in cerebrospinal fluid along with lymphocytic pleocytosis. Borderline tumor of the ovary was diagnosed. Thus, the disease of the patient was deemed to be paraneoplastic. The patient was treated by surgical removal of tumor, steroids, immunoglobulins, plasma exchange and rituximab. Four months after presentation, the patient was still tetraplegic, reacted with mimic expressions to pain or touch and could phonate solitary vowels. An extensive literature research was performed. Conclusion Our case and the literature review illustrate that multiple glial and neuronal autoantibodies can co-occur, that points to a paraneoplastic etiology, above all ovarian teratoma or thymoma. Clinical manifestation can be a mixture of typically associated syndromes, e.g. ataxia associated with anti-ITPR1 antibodies, encephalomyelitis with anti-GFAPα antibodies and longitudinal extensive myelitis with anti-MOG antibodies.

AimNon-small cell lung cancer (NSCLC) remains the leading cause of cancer-related death globally despite promising progress of personalized therapy approaches. Cyclin-dependent kinase 7 (CDK7) is a kinase involved in transcription, overexpressed in a broad spectrum of cancer types and found to be associated with an unfavourable prognosis. In this study, we aimed to investigate the protein expression of CDK7 in a large cohort of NSCLC incorporating adenocarcinomas (adNSCLC) and squamous cell carcinomas (sqNSCLC) and to correlate its expression with clinicopathological data.MethodsWe performed immunohistochemical staining of CDK7 on our cohort of NSCLC including 258 adNSCLC and 101 sqNSCLC and measured protein expression via a semi-automated read out. According to the median value of CDK7 the cohort was stratified in a CDK7 high and low expressing group, respectively, and results were correlated with clinico-pathological data.ResultsCDK7 was significantly higher expressed in sqNSCLC than in adNSCLC. In the group of sqNSCLC, CDK7 expression was significantly higher in sqNSCLC with lymph node metastases than in sqNSCLC with N0 stage. We found a significantly worse overall survival and disease-free survival for patients with CDK7 high expressing NSCLC.ConclusionSince a high CDK7 expression seems to be linked with a poor prognosis it might serve as a promising novel prognostic biomarker and its assessment could be implied in future routine diagnostic workup of NSCLC samples. Considering that CDK7 inhibitors are currently tested in several trials for advanced solid malignancies, it may also be a new target for future anti-cancer therapy.

Abstract The only potentially curative treatment for lung adenocarcinoma patients remains complete resection of early-stage tumors. However, many patients develop recurrence and die of their disease despite curative surgery. Underlying mechanisms leading to establishment of systemic disease after complete resection are mostly unknown. We therefore aimed at identifying molecular signatures of resected lung adenocarcinomas associated with the risk of an early relapse. The study comprised 89 patients with totally resected stage IA–IIIA lung adenocarcinomas. Patients suffering from an early relapse within two years after surgery were compared to patients without a relapse in two years. Patients were clinically and molecular pathologically characterized. Tumor tissues were immunohistochemically analyzed for the expression of Ki67, CD45, CD4, CD8, PD1, PD-L1, PD-L2 and CD34, by Nanostring nCounter PanCancer Immune Profiling Panel as well as a comprehensive methylome profiling using the Infinium MethylationEPIC BeadChip. We detected differential DNA methylation patterns as well as significantly differentially expressed genes associated with an early relapse after complete resection. Especially, CD1A was identified as a potential biomarker, whose reduced expression is associated with an early relapse. These findings might help to develop biomarkers improving risk assessment and patient selection for adjuvant therapy as well as establish novel targeted therapeutic strategies.

Patients with head and neck squamous cell carcinoma (HNSCC) continue to have a rather poor prognosis. Treatment-related comorbidities have negative impacts on their quality of life. TRIM21 is a cytosolic E3 ubiquitin ligase that was initially described as an autoantigen in autoimmune diseases and later associated with the intracellular antiviral response. Here, we investigated the role of TRIM21 as a biomarker candidate for HNSCC in predicting tumor progression and patient survival. We analyzed TRIM21 expression and its association with clinical-pathological parameters in our HNSCC cohort using immunohistochemistry. Our HNSCC cohort included samples from 419 patients consisting of primary tumors (n = 337), lymph node metastases (n = 156), recurrent tumors (n = 54) and distant metastases (n = 16). We found that cytoplasmic TRIM21 expression was associated with the infiltration of immune cells into primary tumors. In addition, TRIM21 expression was significantly higher in primary tumors than in lymph node metastases, and increased TRIM21 expression was correlated with shorter progression-free survival in HNSCC patients. These results suggest that TRIM21 could be a new biomarker for progression-free survival.

Background & Aims: Cell differentiation in the colonic crypt is driven by a metabolic switch from glycolysis to mitochondrial oxidation. Mitochondrial and goblet cell dysfunction have been attributed to the pathology of ulcerative colitis (UC). We hypothesized that p32/gC1qR/HABP1, which critically maintains oxidative phosphorylation, is involved in goblet cell differentiation and hence in the pathogenesis of UC. Methods: Ex vivo, goblet cell differentiation in relation to p32 expression and mitochondrial function was studied in tissue biopsies from UC patients versus controls. Functional studies were performed in goblet cell-like HT29-MTX cells in vitro. Mitochondrial respiratory chain complex V-deficient, ATP8 mutant mice were utilized as a confirmatory model. Nutritional intervention studies were performed in C57BL/6 mice. Results: In UC patients in remission, colonic goblet cell differentiation was significantly decreased compared to controls in a p32-dependent manner. Plasma/serum L-lactate and colonic pAMPK level were increased, pointing at high glycolytic activity and energy deficiency. Consistently, p32 silencing in mucus-secreting HT29-MTX cells abolished butyrate-induced differentiation and induced a shift towards glycolysis. In ATP8 mutant mice, colonic p32 expression correlated with loss of differentiated goblet cells, resulting in a thinner mucus layer. Conversely, feeding mice an isocaloric glucose-free, high-protein diet increased mucosal energy supply that promoted colonic p32 level, goblet cell differentiation and mucus production. Conclusion: We here describe a new molecular mechanism linking mucosal energy deficiency in UC to impaired, p32-dependent goblet cell differentiation that may be therapeutically prevented by nutritional intervention.

We present the case of a patient with granulomatous endocarditis of the mitral valve leading to severe valve stenosis caused by granulomatosis with polyangiitis. Endocarditis is a rare complication of granulomatosis with polyangiitis that may be misdiagnosed as infectious endocarditis or, as in our case, thrombotic lesions. (Level of Difficulty: Intermediate.)

Colorectal cancer is a notorious disease, with almost half of the patients succumbing to the disease. The prevalence and incidence rates of colorectal cancer are increasing in many parts of the world, highlighting the need to discover new biomarkers for diagnosis and therapy. Caldesmon (CaD), an actin-binding protein that plays a significant role in controlling cell motility, has emerged as a promising biomarker. The CALD1 gene encodes CaD as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight (h-CaD), expressed in smooth muscle, and low-molecular-weight (l-CaD), expressed in nonsmooth muscle cells. Most studies have suggested an oncogenic role of CaD in colorectal cancer, but the exact subcellular localization of the two CaD isoforms in tumor cells and stroma have not been clarified yet. Here, we analyzed tissue samples from 262 colorectal cancer patients by immunohistochemistry analysis using specific antibodies for l-CaD and h-CaD. The results showed elevated cytoplasmic expression levels of l-Cad in 187/262 (71.4%) cases. l-Cad was expressed at low levels in the normal colon mucosa and was also consistently expressed in the cancer-associated stroma of all cases, suggesting that it could play a role in modulating the tumor microenvironment. l-CaD expression in cancer cells was associated with preinvasive stages of cancer. Survival analysis indicated that patients with high l-CaD expression in tumor cells could respond poorly to selective chemotherapeutic 5FU, but not combination chemotherapy. h-CaD was expressed in colonic and vascular smooth muscle cells as expected and to a lesser extent in the tumor-associated stroma, but it was not expressed in the cancer cells or normal colon mucosal epithelial cells. Collectively, these data clarify how the expression patterns of CaD isoforms in colorectal cancer can have applications in the management of colorectal cancer patients.

Rovalpituzumab tesirine (Rova-T), an antibody-drug conjugate directed against Delta-like protein 3 (DLL3), is under development for patients with small cell lung cancer (SCLC). DLL3 is expressed on the majority of SCLC samples. Because SCLC is rarely biopsied in the course of disease, data regarding DLL3 expression in relapses is not available. The aim of this study was to investigate the expression of DLL3 in chemorelapsed (but untreated with Rova-T) SCLC samples and compare the results with chemonaive counterparts. Two evaluation methods to assess DLL3 expression were explored. Additionally, we assessed if DLL3 expression of chemorelapsed and/or chemonaive samples has prognostic impact and if it correlates with other clinicopathological data. The study included 30 paired SCLC samples, which were stained with an anti DLL3 antibody. DLL3 expression was assessed using tumor proportion score (TPS) and H-score and was categorized as DLL3 low (TPS < 50%, H-score ≤ 150) and DLL3 high (TPS ≥ 50%, H-score > 150). Expression data were correlated with clinicopathological characteristics. Kaplan–Meier curves were used to illustrate overall survival (OS) depending on DLL3 expression in chemonaive and chemorelapsed samples, respectively, and depending on dynamics of expression during course of therapy. DLL3 was expressed in 86.6% chemonaive and 80% chemorelapsed SCLC samples without significant differences between the two groups. However, the extent of expression varied in a substantial proportion of pairs (36.6% with TPS, 43.3% with H-score), defined as a shift from low to high or high to low expression. TPS and H-score provided comparable results. There were no profound correlations with clinicopathological data. Survival analysis revealed a trend toward a more favorable OS in DLL low-expressing chemonaive SCLC (p = 0.57) and, in turn, in DLL3 high-expressing chemorelapsed SCLC (p = 0.42) as well as in SCLC demonstrating a shift from low to high expression (p = 0.56) without being statistically significant. This is the first study to investigate DLL3 expression in a large cohort of rare paired chemonaive-chemorelapsed SCLC specimens. Comparative analysis revealed that DLL3 expression was not stable during the course of therapy, suggesting therapy-based alterations. Unlike in chemonaive samples, a high DLL3 expression in chemorelapsed samples indicated a trend for a more favorable prognosis. Our results highlight the importance to investigate DLL3 in latest chemorelapsed SCLC tumor tissue.

BackgroundHead and neck squamous cell carcinomas (HNSCCs) have a very moderate response rate to immune checkpoint inhibitor (ICI) treatment compared to other cancer types. Lacking predictive markers for treatment response, we analyzed the immune status of HNSCC and assessed the spatial distribution of immune cells.Materials and MethodsVia assessing hematoxylin–eosin (H&E) stains, we divided HNSCCs by the immune cell distribution in hot, cold, and excluded tumors. For each group, each with 10 tumors, we performed serial immunohistochemical (IHC) staining of the immune cell markers, checkpoint molecules, and immune regulators.ResultsThe spatial distributions were different for each immune cell type, allocating regulatory T cells (Tregs) and CD11b cells predominantly in the stroma. CD4 and CD8 cells were present either in the tumor stroma or between cancer cells. Interestingly, the expressions of PD-1 (programmed cell death 1 receptor) and PD-L1 (programmed death-ligand 1) were higher in hot tumors in comparison to cold and excluded tumors. The expression of pSMAD [indicating active transforming growth factor beta (TGF-β)] was higher in excluded tumors.ConclusionDifferent immune cell distribution patterns within tumors might be crucial for ICI treatment response since hot tumors have the highest expressions of PD-1 and PD-L1. TGF-β might be a key regulator for immune cell distribution and a promising therapeutic target that determines the formation of hot or excluded immune patterns.

Significant progress has been achieved in the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, results in patients with aggressive variant prostate cancer (AVPC) have been disappointing. Here, we report retrospectively collected data from intensively pretreated AVPC patients (n = 17; 88.2% visceral metastases; 82% elevation of neuroendocrine markers) treated with salvage chemotherapy consisting of cisplatin, ifosfamide, and paclitaxel (TIP). At the interim analysis, 60% of patients showed radiographic response or stable disease (PFS = 2.5 months; OS = 6 months). In men who responded to chemotherapy, an OS > 15 months was observed. Preclinical analyses confirmed the high activity of the TIP regimen, especially in docetaxel-resistant prostate cancer cells. This effect was primarily mediated by increased cisplatin sensitivity in the emergence of taxane resistance. Proteomic and functional analyses identified a lower DNA repair capacity and cell cycle machinery deficiency to be causative. In contrast, paclitaxel showed inconsistent effects, partially antagonizing cisplatin and ifosfamide in some AVPC models. Consequently, paclitaxel has been excluded from the TIP combination for future patients. In summary, we report for the first time the promising efficacy of TIP as salvage therapy in AVPC. Our preclinical data indicate a pivotal role for cisplatin in overcoming docetaxel resistance.

Bone metastatic (BM) prostate cancer (PCa) belongs to the most lethal form of PCa, and therapeutic options are limited. Molecular profiling of metastases contributes to the understanding of mechanisms defining the bone metastatic niche. Our aim was to explore the transcriptional profile of PCa BM and to identify genes that drive progression. Paraffin-embedded tissues of 28 primary PCa and 30 BM were submitted to RNA extraction and analyzed by RNA sequencing using the Nanostring nCounter gene expression platform. A total of 770 cancer-related genes were measured using the Nanostring™ PanCancer progression panel. Gene Ontology (GO), KEGG, Reactome, STRING, Metascape, PANTHER, and Pubmed were used for data integration and gene annotation. We identified 116 differentially expressed genes (DEG) in BM compared to primaries. The most significant DEGs include CD36, FOXC2, CHAD, SPP1, MMPs, IBSP, and PTX3, which are more highly expressed in BM, and ACTG2, MYH11, CNN1, FGF2, SPOCK3, and CHRDL1, which have a lower expression. DEGs functionally relate to extracellular matrix (ECM) proteoglycans, ECM-receptors, cell-substrate adhesion, cell motility as well as receptor tyrosine kinase signaling and response to growth factors. Data integration and gene annotation of 116 DEGs were used to build a gene platform which we termed “Manually Annotated and Curated Nanostring-data Platform”. In summary, our results highlight the significance of certain genes in PCa BM to which essential pro-metastatic functions could be ascribed. Data from this study provide a comprehensive platform of genes that are related to PCa BM and provide evidence for further investigations.

Background: Patients with human papilloma virus (HPV)-related oropharyngeal cancer have a better prognosis than nonvirally associated patients, most likely because of better immune responses. Increased infiltration of T lymphocytes into the oropharyngeal tumor tissue has been observed, but the dynamics of circulating lymphocytes and monocytes are not fully understood. The aim of this study was to understand the population dynamics of circulating monocyte subsets in oropharyngeal cancer (OPC) patients with regard to the clinicopathological parameters and accompanying immunological consequences in view of the CD4/CD8 T cell subset composition, and the expression of checkpoint pathway proteins programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1). Materials and Methods: The abundance of circulating monocyte subsets and peripheral blood CD4/CD8 T cells of oropharyngeal cancer patients and their PD-L1 and PD-1 expression levels were analyzed by flow cytometry. Results: The studied oropharyngeal cancer patients revealed heterogeneous individual redistributions of CD14++CD16− (classical), CD14++CD16+ (intermediate), and CD14dim+CD16+ (nonclassical) monocyte subsets compared with healthy donors. These differences in monocyte subset alterations were independent in patients with TNM or HPV status but entailed further immunological consequences. Increased percentages of nonclassical monocytes significantly correlated with increased levels of monocytic PD-L1 expression. We observed significantly decreased levels of CD4+ effector T cells, which were accompanied by increased CD4+ effector memory T cells in OPC patients compared with healthy donors, each having a stronger effect in patients with decreased levels of classical monocytes. Conclusion: We conclude that oropharyngeal cancer, as a malignancy from a lymphoid-tissue-rich anatomical region, has a strong systemic impact on the differentiation and regulation of circulating innate and adaptive immune cells. Further comprehensive investigations are required for the possible future usability of the described immunological alterations as bioliquid parameters for prognosis or therapy response prediction.

Background: The approval of immune checkpoint inhibitors in combination with specific diagnostic biomarkers presents new challenges to pathologists as tumor tissue needs to be tested for expression of programmed death-ligand 1 (PD-L1) for a variety of indications. As there is currently no requirement to use companion diagnostic assays for PD-L1 testing in Germany different clones are used in daily routine. While the correlation of staining results has been tested in various entities, there is no data for head and neck squamous cell carcinomas (HNSCC) so far.Methods: We tested five different PD-L1 clones (SP263, SP142, E1L3N, 22-8, 22C3) on primary HNSCC tumor tissue of 75 patients in the form of tissue microarrays. Stainings of both immune and tumor cells were then assessed and quantified by pathologists to simulate real-world routine diagnostics. The results were analyzed descriptively and the resulting staining pattern across patients was further investigated by principal component analysis and non-negative matrix factorization clustering.Results: Percentages of positive immune and tumor cells varied greatly. Both the resulting combined positive score as well as the eligibility for certain checkpoint inhibitor regimens was therefore strongly dependent on the choice of the antibody. No relevant co-clustering and low similarity of relative staining patterns across patients was found for the different antibodies.Conclusions: Performance of different diagnostic anti PD-L1 antibody clones in HNSCC is less robust and interchangeable compared to reported data from other tumor entities. Determination of PD-L1 expression is critical for therapeutic decision making and may be aided by back-to-back testing of different PD-L1 clones.

Background The interplay of immune and cancer cells takes place in the tumor microenvironment where multiple signals are exchanged. The transforming growth factor beta (TGFB) pathway is known to be dysregulated in lung cancer and can impede an effective immune response. However, the exact mechanisms are yet to be determined. Especially which cells respond and where does this signaling take place with respect to the local microenvironment.Methods Human non-small cell lung cancer samples were retrospectively analyzed by multiplexed immunohistochemistry for SMAD3 phosphorylation and programmed death ligand 1 expression in different immune cells with respect to their localization within the tumor tissue. Spatial relationships were studied to examine possible cell-cell interactions and analyzed in conjunction with clinical data.Results TGFB pathway activation in CD3, CD8, Foxp3 and CD68 cells, as indicated by SMAD3 phosphorylation, negatively impacts overall and partially disease-free survival of patients with lung cancerindependent of histological subtype. A high frequency of Foxp3 regulatory T cells positive for SMAD3 phosphorylation in close vicinity of CD8 T cells within the tumor discriminate a rapidly progressing group of patients with lung cancer.Conclusions TGFB pathway activation of local immune cells within the tumor microenvironment impacts survival of early stage lung cancer. This might benefit patients not eligible for targeted therapies or immune checkpoint therapy as a therapeutic option to re-activate the local immune response.

Background: Head and neck squamous cell carcinoma (HNSCC) represents a common cancer worldwide. Past therapeutic advances have not significantly improved HNSCC prognosis. Therefore, it is necessary to further stratify HNSCC, especially with recent advances in tumor immunology.Methods: Tissue microarrays were assembled from tumor tissue samples and were complemented with comprehensive clinicopathological data of n = 419 patients. H&E whole slides from resection specimen (n = 289) were categorized according to their immune cell infiltrate as “hot,” “cold,” or “excluded.”Results: Investigating tumor immune cell patterns, we found significant differences in survival rates. Immunologic “hot” and “excluded” HNSCCs are associated with better overall survival than “cold” HNSCC patients (p < 0.05). Interestingly, the percentage of all three patterns is nearly identical in p16 positive and negative HNSCCs.Conclusions: Using a plain histological H&E approach to categorize HNSCC as being immunologic “hot,” “cold,” or “excluded” can offer a forecast of patients' prognosis and may thus aid as a potential prognostic tool in routine pathology reports. This “hot-cold-excluded” scheme needs to be applied to more HNSCC cohorts and possibly to other cancer types to determine prognostic meaning, e.g., regarding OS or DFS. Furthermore, our cohort reflects epidemiological data in the national, European, and international context. It may, therefore, be of use for future HNSCC characterization.

When approaching thyroid gland tumor classification, the differentiation between samples with and without "papillary thyroid carcinoma-like" nuclei is a daunting task with high inter-observer variability among pathologists. Thus, there is increasing interest in the use of machine learning approaches to provide pathologists real-time decision support. In this paper, we optimize and quantitatively compare two automated machine learning methods for thyroid gland tumor classification on two datasets to assist pathologists in decision-making regarding these methods and their parameters. The first method is a feature-based classification originating from common image processing and consists of cell nucleus segmentation, feature extraction, and subsequent thyroid gland tumor classification utilizing different classifiers. The second method is a deep learning-based classification which directly classifies the input images with a convolutional neural network without the need for cell nucleus segmentation. On the Tharun and Thompson dataset, the feature-based classification achieves an accuracy of 89.7% (Cohen's Kappa 0.79), compared to the deep learning-based classification of 89.1% (Cohen's Kappa 0.78). On the Nikiforov dataset, the feature-based classification achieves an accuracy of 83.5% (Cohen's Kappa 0.46) compared to the deep learning-based classification 77.4% (Cohen's Kappa 0.35). Thus, both automated thyroid tumor classification methods can reach the classification level of an expert pathologist. To our knowledge, this is the first study comparing feature-based and deep learning-based classification regarding their ability to classify samples with and without papillary thyroid carcinoma-like nuclei on two large-scale datasets.

With the advance of precision medicine, the availability of tumor tissue for molecular analysis has become a limiting factor. This is particularly the case for bone metastases which are frequently occurring in cancer types such as prostate cancer. Due to the necessary decalcification process it was long thought that transcriptome analysis will not be feasible from decalcified formalin-fixed, paraffin-embedded (DFFPE) in a large manner. Here we demonstrate that mRNA extraction from DFFPE is feasible, quick, robust and reproducible and that decalcification does not hamper subsequent gene expression analysis. This might assist in implementing transcriptome analysis from DFFPE into every day practice.

Head and neck squamous cell carcinomas (HNSCC) are among the most common cancers worldwide and are associated with a poor prognosis for patients. Among HNSCC, those originating in the hypopharynx have the worst prognosis. The histone demethylase LSD1 has been shown to promote cancer initiation, progression, and relapse through various mechanisms and is upregulated in many cancer tissues. LSD1 physically interacts with SNAIL and is required for SNAIL mediated transcriptional repression. Previous studies of the prognostic value of LSD1 in HNSCC have been limited in their analysis of sub-sites, and a correlation between LSD1 and SNAIL has not been shown in HNSCC patient samples. Here we used a large, representative, and clinically well-characterized cohort of 339 HNSCC patients to investigate the co-expression of LSD1 and SNAIL and their prognostic value in all HNSCC using immunohistochemical staining. Elevated LSD1 expression correlated with advanced tumor stage and poor progression-free survival (PFS) in HNSCC originating in the hypopharynx. Overexpression of the transcription factor SNAIL independently correlated with worse overall survival (OS) and PFS in HNSCC in general and prominently in tumors of the hypopharynx. Furthermore, increased LSD1 expression significantly correlated with elevated SNAIL expression in patient samples. Therefore, the presented data implicates LSD1 and SNAIL as independent prognostic biomarkers.

Self-sustained cell proliferation constitutes one hallmark of cancer enabled by aerobic glycolysis which is characterized by imbalanced glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) activity, named the Warburg effect. The C1q binding protein (C1QBP; gC1qR) is pivotal for mitochondrial protein translation and thus OXPHOS activity. Due to its fundamental role in balancing OXPHOS and glycolysis, c1qbp−/− mice display embryonic lethality, while gC1qR is excessively up-regulated in cancer. Although gC1qR encompasses an N-terminal mitochondrial leader it is also located in other cellular compartments. Hence, we aimed to investigate mechanisms regulating gC1qR cellular localization and its impact on tumor cell metabolism. We identified two caspase-1 cleavage sites in human gC1qR. GC1qR cleavage by active caspase-1 was unraveled as a cellular mechanism that prevents mitochondrial gC1qR import, thereby enabling aerobic glycolysis and enhanced cell proliferation. Ex vivo, tumor grading correlated with non-mitochondrial-located gC1qR as well as with caspase-1 activation in colorectal carcinoma patients. Together, active caspase-1 cleaves gC1qR and boosts aerobic glycolysis in tumor cells.

Abstract Oesophageal cancer (OC) has high mortality. This study aims at determining the feasibility of liquid biopsies for genomic profiling in early stage OC, comparing two different technologies for mutational analysis in circulating cell -free DNA (ccfDNA) and evaluating the clinical impact of these somatic alterations during primary staging. In 25 patients with locally advanced OC, endoscopic tumour biopsies and simultaneous blood samples were taken during primary staging. Genomic DNA from biopsies and ccfDNA were analysed for mutations using a 12 gene panel next-generation sequencing (NGS) assay as well as digital droplet PCR (ddPCR). Genetic data was correlated with patients’ outcome. In 21 of the tested biopsies (84%) at least one somatic mutation was detected by NGS. Mutations detected by NGS were detectable by ddPCR with similar allele frequencies. In three out of the 21 patients with proven mutations, the same mutations were also detectable in ccfDNA using NGS (14%). In contrast, ddPCR detected mutations in ccfDNA of five additional patients (8/21, 38%). Post-surgical outcome analysis was performed for those patients who had received complete tumour resection (n = 16). Five of them suffered from an early relapse within the first year after surgery, including four with detectable somatic mutations in ccfDNA during primary staging. Taken together, we showed a higher sensitivity for ddPCR compared to NGS in detecting mutated ccfDNA in OC. Detection of somatically altered ccfDNA during primary staging seems to be indicative for post-surgical tumour recurrence.

Abstract A venous tumor thrombus (VTT) is a potentially lethal complication of renal cell carcinoma (RCC) but virtually nothing is known about the underlying natural history. Based on our observation that venous thrombi contain significant numbers of viable tumor cells, we applied multiregion whole exome sequencing to a total of 37 primary tumor and VTT samples including normal tissue specimens from five consecutive patients. Our findings demonstrate mutational heterogeneity between primary tumor and VTT with 106 of 483 genes (22%) harboring functional SNVs and/or indels altered in either primary tumor or thrombus. Reconstruction of the clonal phylogeny showed clustering of tumor samples and VTT samples, respectively, in the majority of tumors. However, no new subclones were detected suggesting that pre-existing subclones of the primary tumor drive VTT formation. Importantly, we found several lines of evidence for “BRCAness” in a subset of tumors. These included mutations in genes that confer “BRCAness”, a mutational signature and an increase of small indels. Re-analysis of SNV calls from the TCGA KIRC-US cohort confirmed a high frequency of the “BRCAness” mutational signature AC3 in clear cell RCC. Our findings warrant further pre-clinical experiments and may lead to novel personalized therapies for RCC patients.

Immune checkpoints blockade (ICB) is a viable anti-cancer strategy. Here the authors show that nuclear receptor NR2F6 acts as an immune checkpoint in T cells and, using mouse models and human T cells, they show NR2F6 inhibition might improve current ICB therapy or work as an alternative therapeutic strategy.

The molecular nature of large-cell neuroendocrine lung carcinomas (LCNEC) has remained unclear. Here, the authors show LCNECs represent a distinct transcriptional subgroup among lung cancers and comprise two molecular subgroups, type I (TP53 and STK11/KEAP1 alterations) and type II (TP53 and RB1 inactivation).

Abstract Background MicroRNAs (miRNA) play a relevant role in carcinogenesis, cancer progression, invasion, and metastasis. Thus, they can serve as diagnostic/prognostic biomarkers. The knowledge on circulating miRNAs for clear cell renal cell carcinomas (ccRCC) is limited. Our study was designed to identify novel biomarkers for ccRCC patients. Results The serum small RNA expression profile was determined in 18 ccRCC and 8 patients with benign renal tumors (BRT) using small RNA sequencing. We detected 29 differentially expressed miRNAs (17 upregulated and 12 downregulated in ccRCC) in the expression profiling cohort. Based on the expression levels, we next validated serum miR-122-5p, miR-193a-5p, and miR-206 levels in an independent cohort (68 ccRCC, 47 BRT, and 28 healthy individuals) using quantitative real-time PCR. Serum expression levels of miR-122-5p and miR-206 were significantly decreased in ccRCC compared to healthy individuals. Both miRNAs were circulating at similar levels in ccRCC and BRT patients. miR-193a-5p expression levels were not different within the study cohort. High serum miR-122-5p and miR-206 levels were associated with adverse clinicopathological parameters: miR-122-5p levels were correlated with metastatic RCC and grade, and miR-206 with pT-stage and metastasis. Furthermore, high miR-122-5p and miR-206 serum levels were associated with a shorter period of progression-free, cancer-specific, and overall survival in patients with ccRCC. Conclusion We identified serum miR-122-5p and miR-206 as novel non-invasive prognostic biomarkers for patients with ccRCC.

Immune checkpoint inhibition has resulted in dramatic improvements in overall and relapse-free survival in patients with metastatic melanoma. The most commonly used immune checkpoint inhibitors are monoclonal antibodies targeting programmed cell death protein 1 and cytotoxic T-lymphocyte-associated protein 4. Unfortunately, a significant subset of patients fail to respond to these therapies, which has resulted in intense research efforts to identify the factors which are associated with treatment response. To this end, we investigated immune cell infiltration in primary melanomas and melanoma metastases, in addition to tumor cell PD-L1 expression, to determine whether these factors are associated with an improved outcome after immune checkpoint inhibition. Indeed, the extent of the immune cell infiltration in the primary melanoma, measured by the Immunoscore, was associated with a significantly improved response to immune checkpoint inhibition in terms of increased overall survival. However, the Immunoscore did not predict which patients would respond to treatment. The Immunoscore was significantly reduced in metastases when compared to primary melanomas. In contrast, PD-L1 expression, exhaustively tested using four commercially available anti-PD-L1 clones, did not differ significantly between primary tumors and melanoma metastases and was not associated treatment response. Whilst replication in larger, prospective studies is required, our data demonstrates the relevance of immune cell infiltration in the primary melanoma as a novel marker of improved overall survival in response to immune checkpoint inhibition.

Background: Stratifying prostate cancer (PCa) patients into risk groups at time of initial diagnosis enabling a risk-adapted disease management is still a major clinical challenge. Existing studies evaluating the prognostic potential of PSMA (prostate-specific membrane antigen) for PCa were performed on radical prostatectomy specimens (RPE), i.e., decision making for disease management was already completed at time of sample analysis. Aim of our study was to assess the prognostic value of PSMA expression for PCa patients on biopsies at time of initial diagnosis.Methods: PSMA expression was assessed by immunohistochemistry on 294 prostate biopsies with corresponding RPE, 621 primary tumor foci from 242 RPE, 43 locally advanced or recurrent tumors, 34 lymph node metastases, 78 distant metastases and 52 benign prostatic samples. PSMA expression was correlated with clinico-pathologic features. Primary endpoint was recurrence free survival. Other clinicopathologic features included WHO/ISUP grade groups, PSA serum level, TNM-stage, and R-status. Chi-square test, ANOVA-analyses, Cox-regression, and log-rank tests were performed for statistical analyses.Results: High PSMA expression on both biopsy and RPE significantly associates with a higher risk of disease recurrence following curative surgery. The 5-year-recurrence free survival rates were 88.2, 74.2, 67.7 and 26.8% for patients exhibiting no, low, medium, or high PSMA expression on biopsy, respectively. High PSMA expression on biopsy was significant in multivariate analysis predicting a 4-fold increased risk of disease recurrence independently from established prognostic markers. PSMA significantly increases during PCa progression.Conclusion: PSMA is an independent prognostic marker on biopsies at time of initial diagnosis and can predict disease recurrence following curative therapy for PCa. Our study proposes the application of the routinely used IHC marker PSMA for outcome prediction and decision making in risk-adapted PCa management on biopsies at time of initial diagnosis.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most common cancer worldwide [...]

It has been increasingly recognised that tumours are not made up of a homogeneous population of cells. Here, the authors show heterogeneous expression of five protein markers in renal cell cancer and demonstrate that the progression of the tumour does not influence the degree of heterogeneity in the tumour.

We have recently shown that centrosomal protein 57 (CEP57) is overexpressed in a subset of human prostate cancers. CEP57 is involved in intracellular transport processes, and its overexpression causes mitotic defects as well as abnormal microtubule nucleation and bundling. In the present study, we further characterized the prognostic and functional role of CEP57 in prostate cancer. Unexpectedly, we found that high CEP57 expression is an independent prognostic factor for a more favorable biochemical recurrence-free survival in two large patient cohorts. To reconcile this finding with the ability of CEP57 to cause cell division errors and thus potentially promote malignant progression, we hypothesized that alterations of microtubule-associated transport processes, in particular nuclear translocation of the androgen receptor (AR), may play a role in our finding. However, CEP57 overexpression and microtubule bundling had, surprisingly, no effect on the nuclear translocation of the AR. Instead, we found a significant increase of cells with disarranged microtubules and a cellular morphology suggestive of a cytokinesis defect. Because mitotic dysfunction leads to a reduced daughter cell formation, it can explain the survival benefit of patients with increased CEP57 expression. In contrast, we show that a reduced expression of CEP57 is associated with malignant growth and metastasis. Taken together, our findings underscore that high CEP57 expression is associated with mitotic impairment and less aggressive tumor behavior. Because the CEP57-induced microtubule stabilization had no detectable effect on AR nuclear translocation, our results furthermore suggest that microtubule-targeting therapeutics used in advanced prostate cancer such as docetaxel may have modes of action that are at least in part independent of AR transport inhibition.

Abstract Hwang et al. recently showed that VGF substantially contributes to the resistance of human lung cancer cells towards epidermal growth factor receptor kinase inhibitors. This was further linked to enhanced epithelial–mesenchymal transition. Here, we demonstrate that VGF is epigenetically modified in non-small cell lung cancer tissues compared to corresponding tumor-free lung tissues from the same donors by using methylome bead chip analyses. These epigenetic modifications trigger an increased transcription of the VGF gene within the tumors, which then leads to an increased expression of the protein, facilitating epithelial–mesenchymal transition, and the resistance to kinase inhibitors. These results should be taken into account in the design of novel therapeutic and diagnostic approaches.

Abstract Targeting checkpoint inhibitors using monoclonal antibodies results in significantly better outcome of cancer patients compared to conventional chemotherapy. However, the current companion diagnostics to predict response is so far suboptimal, since they base on more or less reliable immunohistochemical approaches. In order to overcome these limitations, we analyzed epigenetic modifications of PDCD1 (PD1), CD274 (PD-L1), and CTLA4 in NSCLC tissues from 39 patients. Results were correlated with transcriptome data. Significant differences in the CpG-methylation patterns between tumor tissues and matched controls were observed for CTLA4 and PDCD1 (PD1) showing a decreased methylation of these genes compared to matched tumor-free tissues from the same patients. Results were confirmed by bisulfide sequencing in an independent validation cohort. Hypomethylation also resulted in increased expression of these genes as shown by transcriptome data. These epigenetic pathways as a hallmark of NSCLC might be useful to generate more precise diagnostic approaches in the future.

Head and neck squamous cell carcinoma (HNSCC)is the 6th most common cancer in humans worldwide and is associated with a poor prognosis for patients. NR2F6 has been identified as an immune checkpoint molecule in tumor-infiltrating T lymphocytes and is associated with a poor prognostic outcome in various cancers. The prognostic value of NR2F6 in HNSCC has not been described yet. We used a large, representative and clinically well-characterized cohort of 383 HNSCC patients, of which 22.4% developed a local recurrence. The NR2F6 expression was analyzed by using immunohistochemistry and was afterward correlated with clinical characteristics and clinicopathological features of HNSCC patients. Primary tumors from patients who develop a local recurrence have a higher NR2F6 expression than primary tumors which do not develop a local recurrence. Furthermore, a high NR2F6 expression is associated with poorer recurrence-free survival, although there is no correlation with overall survival. NR2F6 expression is independent of the T stage and UICC stage. NR2F6 might be a new prognostic biomarker for the early detection of local recurrences in HNSCC patients. Therefore, it may help to improve the recognition of patients who would benefit from more frequent follow-up examinations.

The Mediator complex is a central integrator of transcription and a hub for the regulation of gene expression. Cyclin dependent kinase (CDK) 19 and its paralog CDK8 are part of its kinase domain and contribute to cancer progression in different cancer entities. STAT1 is an important immune modulator and a downstream substrate of CDK8/CDK19 mediated phosphorylation. So far, little is known about CDK19’s role in head and neck squamous cell carcinoma (HNSCC) progression, its link to STAT1 activity, and related immune modulation. Immunohistochemistry for CDK19, activated pSTAT1, and PD-L1, known to be affected by STAT1, was conducted on samples of 130 primary tumors, 71 local recurrences, 32 lymph node metastases, and 25 distant metastases of HNSCC. Compared to primary tumors, CDK19 is overexpressed in local recurrences and distant metastases as well as in primary tumors that developed local recurrence after initial therapy. Patients with high-CDK19-expressing primary tumors have a significantly shorter disease-free survival. CDK19 expression correlates with pSTAT1 expression in primary tumors associated with recurrent disease, local recurrent tumors, lymph node metastases, and distant metastases. pSTAT1 expression correlates with PD-L1 expression in recurrent tumors. Our findings identify CDK19 as a potential biomarker in HNSCC to predict recurrent disease and support recent developments to target CDK19 and its paralog CDK8 in advanced cancer.

Background: HNSCC is the sixth most common cancer in humans and has still a very poor prognosis. The treatment methods so far are very often associated with mutilation and impairment in the quality of life. Except for p16 expression, there are no reliable prognostic markers in HNSCC so far. Ecotropic Viral Integration Site 1 (EVI1) is a well-described prognostic marker in leukemia and different types of solid cancers. In these, a high EVI1 expression is associated with a poor prognosis. In HNSCC, it is not known so far if EVI1 has any prognostic relevance. Materials and Methods: We used our representative tissue cohort of 389 primary HNSCCs, of which 57.2% had one or more lymph node metastases. Here EVI1 expression was analyzed via immunohistochemistry and correlated with the clinical characteristics of these patients. Results: Although in HNSCC EVI1 expression does not predict poor survival, a high EVI1 expression in the primary tumor correlates with a lymph node metastatic disease. Conclusion: Consequently, EVI1 may serve as a biomarker to predict an occult lymph node metastasis in a clinical nodal negative (cN0) HNSCC.

Summary: Cdkn1a, which encodes p21, functions as a major route for p53-mediated cell-cycle arrest. However, the consequence of Cdkn1a gene dosage on tumor suppression has not been systematically investigated. Here, we employed BAC transgenesis to generate a Cdkn1aSUPER mouse, which harbors an additional Cdkn1a allele within its natural genomic context. We show that these mice display enhanced cell-cycle arrest and reduced apoptosis in response to genotoxic stress. Furthermore, using a chemically induced skin cancer model and an autochthonous Kras-driven lung adenocarcinoma model, we show that Cdkn1aSUPER mice display a cancer protection phenotype that is indistinguishable from that observed in Tp53SUPER animals. Moreover, we demonstrate that Tp53 and Cdkn1a cooperate in mediating cancer resistance, using a chemically induced fibrosarcoma model. Overall, our Cdkn1aSUPER allele enabled us to assess the contribution of Cdkn1a to Tp53-mediated tumor suppression. : Torgovnick et al. create a mouse model, carrying a third copy of Cdkn1a (p21), which shows enhanced cell-cycle arrest capacity and protection against DNA damage-induced apoptosis. The Cdkn1aSUPER animals display delayed epithelial regeneration and a robust cancer resistance phenotype, highlighting the importance of p21 in p53-dependent tumor suppression. Keywords: Cdkn1a, p21, p53, mouse model, cancer, tumor suppressor, cell cycle arrest, apoptosis, cancer protection

[This corrects the article DOI: 10.1371/journal.pone.0131702.].

Mammary analogue secretory carcinoma (MASC) is a newly defined entity among salivary gland malignancies which has just been established in the 4th edition of the WHO classification of head and neck tumors. MASC (synonym: secretory carcinoma) are characterized by a specific rearangement of the ETV6 gene locus. Here, we present a series of 3 MASC cases including clinical data with follow-up for up to 26 months. All tumours immunhistochemically displayed strong positivity for cytokeratin 7, and mammaglobin, focal positivity for S100, cytokeratin 5/6 and muc-4. In contrast, immunhistochemical stainings against cytokeratin 14, hormon receptors, Her2/neu, androgen receptor and prostate-specific antigen were consistently negative. FISH analysis showed translocation of the ETV6 gene locus in the majority of tumour cell nuclei. During clinical follow-up, no local relapse or metastasis was detected. As these carcinomas are clinically and radiologically indistinguishable from other salivary gland tumours and as therapeutic approaches and prognosis might differ, we need to be able to diagnose MASC correctly.

Gleason grading is the best independent predictor for prostate cancer (PCa) progression. Recently, a new PCa grading system has been introduced by the International Society of Urological Pathology (ISUP) and is recommended by the World Health Organization (WHO). Following studies observed more accurate and simplified grade stratification of the new system. Aim of this study was to compare the prognostic value of the new grade groups compared to the former Gleason Grading and to determine whether re-definition of Gleason Pattern 4 might reduce upgrading from prostate biopsy to radical prostatectomy (RP) specimen. A cohort of men undergoing RP from 2002 to 2015 at the Hospital of Goeppingen (Goeppingen, Germany) was used for this study. In total, 339 pre-operative prostatic biopsies and corresponding RP specimens, as well as additional 203 RP specimens were re-reviewed for Grade Groups according to the ISUP. Biochemical recurrence-free survival (BFS) after surgery was used as endpoint to analyze prognostic significance. Other clinicopathological data included TNM-stage and pre-operative PSA level. Kaplan–Meier analysis revealed risk stratification of patients based on both former Gleason Grading and ISUP Grade Groups, and was statistically significant using the log-rank test (p

IntroductionBladder cancer (BCa) is among the most frequent cancer entities and relevantly contributes to cancer-associated deaths worldwide. The multi-protein Mediator complex is a central regulator of the transcriptional machinery of protein-coding genes and has been described to be altered in several malignancies. MED1, a subunit of the tail module, was described to negatively modulate expression of metastasis-related genes and to be downregulated in melanoma and lung cancer. In contrast, MED1 hyperactivity was described in breast and prostate cancer, likely due its function as a hub for nuclear hormone receptors. So far, only little is known about the function of the Mediator complex in BCa. The aim of this study was therefore to investigate the role of MED1 in BCa as a prognostic biomarker and a biomarker of disease progression.MethodsThe protein expression of MED1 was assessed by immunohistochemistry (IHC) on tissue microarrays from 224 patients: benign urothelium n = 31, non-muscle invasive BCa (pTis, pT1) n = 72, and muscle invasive BCa (pT2–T4) n = 121. Comprehensive clinicopathological information including follow-up were available. Quantification of MED1 protein expression was evaluated by the semiquantitative image analysis program Definiens.ResultsMED1 expression significantly decreased during BCa progression from benign urothelium to advanced BCa. Muscle invasion, the crucial step in BCa progression, was associated with low MED1 protein expression. Accordingly, decreased MED1 expression was found in primary BCa samples with positive lymphonodal status and distant metastases. Furthermore, cancer-specific survival was significantly worse in the group of low MED1 expression.ConclusionOur findings show that the downregulation of MED1 is associated with muscle invasion, metastatic spread, and shorter overall survival in BCa.

Clear cell renal cell carcinoma (ccRCC) is a common human malignancy. Despite numerous efforts, there is still no reliable biomarker or combination of biomarkers available for daily practice. Our study was designed to explore the expression profile of messenger RNA (mRNA) and long non-coding RNA (lncRNA) transcripts in ccRCC in order to identify potential diagnostic biomarkers for patients with ccRCC. Total RNA from corresponding normal and malignant tissue of 15 patients with ccRCC was isolated. Expression profiling was performed using a custom Agilent gene expression microarray which allowed the analysis of 34,144 mRNA and 32,183 lncRNA transcripts. We observed that a subset of mRNA (n = 1064; 3.1%) and lncRNA (n = 1308; 4.1%) transcripts are dysregulated (fold change > 2) in ccRCC tissue. The relative higher number of differentially expressed lncRNAs indicates that lncRNA profiling may be better suited for diagnostic purposes; a number of so far unknown RNAs with potential diagnostic interest in ccRCC are identified by our gene expression profiling study. The data are deposited in the Gene Expression Omnibus (GSE61763).

Neuroendocrine prostate cancer (NEPC), also referred to as anaplastic prostate cancer, is a lethal tumor that most commonly arises in late stages of prostate adenocarcinoma (PCA) with predilection to metastasize to visceral organs. In the current study, we explore for evidence that Aurora kinase A (AURKA) and N-myc (MYCN) gene abnormalities are harbingers of treatment-related NEPC (t-NEPC). We studied primary prostate tissue from 15 hormone naïve PCAs, 51 castration-resistant prostate cancers, and 15 metastatic tumors from 72 patients at different stages of disease progression to t-NEPC, some with multiple specimens. Histologic evaluation, immunohistochemistry, and fluorescence in situ hybridization were performed and correlated with clinical variables. AURKA amplification was identified in overall 65% of PCAs (hormone naïve and treated) from patients that developed t-NEPC and in 86% of metastases. Concurrent amplification of MYCN was present in 70% of primary PCAs, 69% of treated PCAs, and 83% of metastases. In contrast, in an unselected PCA cohort, AURKA and MYCN amplifications were identified in only 5% of 169 cases. When metastatic t-NEPC was compared to primary PCA from the same patients, there was 100% concordance of ERG rearrangement, 100% concordance of AURKA amplification, and 60% concordance of MYCN amplification. In tumors with mixed features, there was also 100% concordance of ERG rearrangement and 94% concordance of AURKA and MYCN co-amplification between areas of NEPC and adenocarcinoma. AURKA and MYCN amplifications may be prognostic and predictive biomarkers, as they are harbingers of tumors at risk of progressing to t-NEPC after hormonal therapy.

Next generation sequencing (NGS) technologies have revolutionized cancer research allowing the comprehensive study of cancer using high throughput deep sequencing methodologies. These methods detect genomic alterations, nucleotide substitutions, insertions, deletions and copy number alterations. SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) is a promising technology generating billions of 50 bp sequencing reads. This robust technique, successfully applied in gene identification, might be helpful in detecting novel genes associated with cancer initiation and progression using formalin fixed paraffin embedded (FFPE) tissue. This study’s aim was to compare the validity of whole exome sequencing of fresh-frozen vs. FFPE tumor tissue by normalization to normal prostatic FFPE tissue, obtained from the same patient. One primary fresh-frozen sample, corresponding FFPE prostate cancer sample and matched adjacent normal prostatic tissue was subjected to exome sequencing. The sequenced reads were mapped and compared. Our study was the first to show comparable exome sequencing results between FFPE and corresponding fresh-frozen cancer tissues using SOLiD sequencing. A prior study has been conducted comparing the validity of sequencing of FFPE vs. fresh frozen samples using other NGS platforms. Our validation further proves that FFPE material is a reliable source of material for whole exome sequencing.