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Supplementary Figure 1 from Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy

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Major interest lies in the evaluation of immune infiltrate in bladder cancer. CD8+ cytotoxic lymphocytes are key effectors of adaptive immune response.The aims of the study were to set up a standardized methodology for CD8+ lymphocytes estimation in NMIBC and investigate how intra-tumoral heterogeneity influences CD8+ immune infiltrate.We considered 995 NMIBC included in the Spanish Bladder Cancer (SBC)/EPICURO Study. Duplicate 0.6mm TMA spots and paired full sections (FS) for 50 selected cases were double stained with anti-pan cytokeratin antibody and anti-CD8 antibody. Slides were digitalized and CD8+ cells were automatically counted after tissue recognition (tumor vs stroma). Spatial heterogeneity was assessed and a resampling strategy was applied to estimate the proper number of 0.6mm TMA spots providing an adequate CD8+ cell estimate. Association between CD8+ count and expression of urothelial differentiation markers was estimated. Cox regression models were performed to assess association between CD8+ cell count and risk of recurrence and progression.Microscopic examination of full sections showed spatial heterogeneity for CD8+ infiltrates. Simulation analyses demonstrated that 5 TMA regions provided a correct sampling of tumor and stromal compartments in Ta while 2 and 6 TMA regions were necessary in T1, respectively. CD8+ cells infiltration was associated with stage, regardless of the histological compartment analyzed (median CD8+ /mmDifferences identified between Ta and T1 tumours supported the hypothesis that rigorous efforts should be placed in proper study design. These results provide a new framework to investigate microenvironment complexity in bladder cancer.

INTRODUCTION/OBJECTIVE: DNA, RNA, and proteins are unavoidable human biomarkers. Today, blood remains the commonly used source of biomarkers despite numerous limitations. Therefore, other sources of biomarkers such as urine could be more appropriate for research in the field of bladder cancer. The aim of this study was to set up a new automated procedure for urinary DNA, RNA, and protein extraction and to evaluate their quality and quantity. MATERIALS AND METHODS: This study was conducted in the setting of the COBLAnCE cohort. Urinary DNA and RNA were extracted using the Maxwell 16 system, and urinary proteins were isolated by precipitation from the supernatant and the cell pellet. The concentration and purity of nucleic acids were determined by spectrophotometry. RNA integrity was determined by the Agilent Bioanalyzer. PCR assays were also used to ensure the quality of DNA and RNA samples. The quality of protein samples obtained was determined by Western blot analysis. RESULTS: PCR experiments performed highlighted that it is possible to use the DNA and RNA samples for amplification, gene expression, or genotyping. However, DNA and RNA recovery from urine was highly variable among patients, with a significant impact of the patient’s gender. The samples were highly degraded. Finally, our protocol of protein isolation was effective in extracting urinary supernatant proteins as well as pellet proteins. DISCUSSION: Therefore, urine samples could constitute valuable resources for subsequent investigations in bladder cancer. These samples will allow identifying new easy-access biomarkers for the early detection of cancer, monitoring cancer progression, and assessing response to therapy.

Basal/squamous (Ba/Sq) subtype represents an intrinsic and robust group in the consensus molecular classification of muscle-invasive bladder cancer (MIBC), with poor outcome and controversial chemosensitivity. We aimed to investigate the spectrum of intratumor heterogeneity (ITH) in the Ba/Sq subtype. First, we validated a 29-gene NanoString CodeSet to predict the Ba/Sq subtype for FFPE samples. We identified heterogeneous Ba/Sq tumors in a series of 331 MIBC FFPE samples using dual GATA3/KRT5/6 immunohistochemistry (IHC). Heterogeneous regions with distinct immunostaining patterns were studied separately for gene expression using the 29-gene CodeSet, for mutations by targeted next-generation sequencing, and for copy number alteration (CNA) by microarray hybridization. Among 83 Ba/Sq tumors identified by GATA3/KRT5/6 dual staining, 19 tumors showed heterogeneity at the IHC level. In one third of the 19 cases, regions from the same tumor were classified in different distinct molecular subtypes. The mutational and CNA profiles confirmed the same clonal origin for IHC heterogeneous regions with possible subclonal evolution. Overall, two patterns of intratumoral heterogeneity (ITH) were observed in Ba/Sq tumors: low ITH (regions with distinct immunostaining, but common molecular subtype and shared CNA) or high ITH (regions with distinct immunostaining, molecular subtype, and CNA). These results showed multilayer heterogeneity in Ba/Sq MIBC. In view of personalized medicine, this heterogeneity adds complexity and should be taken into account for sampling procedures used for diagnosis and treatment choice. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background & Aims: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs.Methods: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling.Results: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA (“Nestin High”, >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies.Conclusion: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. Lay summary: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Purpose: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare malignancy associated with an overall poor prognosis. We aimed to investigate the immune profile of cHCC-CCA and determine its impact on disease outcome. Experimental Design: We performed a multicenter study of 96 patients with cHCC-CCA. Gene expression profile was analyzed using nCounter PanCancer IO 360 Panel. Densities of main immune cells subsets were quantified from digital slides of IHC stainings. Genetic alterations were investigated using targeted next-generation sequencing. Results: Two main immune subtypes of cHCC-CCA were identified by clustering analysis: an “immune-high” (IH) subtype (57% of the cases) and an “immune-low” (IL) subtype (43% of the cases). Tumors classified as IH showed overexpression of genes related to immune cells recruitment, adaptive and innate immunity, antigen presentation, cytotoxicity, immune suppression, and inflammation (P < 0.0001). IH cHCC-CCAs also displayed activation of gene signatures recently shown to be associated with response to immunotherapy in patients with HCC. Quantification of immunostainings confirmed that IH tumors were also characterized by higher densities of immune cells. Immune subtypes were not associated with any genetic alterations. Finally, multivariate analysis showed that the IH subtype was an independent predictor of improved overall survival. Conclusions: We have identified a subgroup of cHCC-CCA that displays features of an ongoing intratumor immune response, along with an activation of gene signatures predictive of response to immunotherapy in HCC. This tumor subclass is associated with an improved clinical outcome. These findings suggest that a subset of patients with cHCC-CCA may benefit from immunomodulating therapeutic approaches.

Deep learning methods for digital pathology analysis are an effective way to address multiple clinical questions, from diagnosis to prediction of treatment outcomes. These methods have also been used to predict gene mutations from pathology images, but no comprehensive evaluation of their potential for extracting molecular features from histology slides has yet been performed. We show that HE2RNA, a model based on the integration of multiple data modes, can be trained to systematically predict RNA-Seq profiles from whole-slide images alone, without expert annotation. Through its interpretable design, HE2RNA provides virtual spatialization of gene expression, as validated by CD3- and CD20-staining on an independent dataset. The transcriptomic representation learned by HE2RNA can also be transferred on other datasets, even of small size, to increase prediction performance for specific molecular phenotypes. We illustrate the use of this approach in clinical diagnosis purposes such as the identification of tumors with microsatellite instability., RNA-sequencing of tumour tissue can provide important diagnostic and prognostic information but this is costly and not routinely performed in all clinical settings. Here, the authors show that whole slide histology slides—part of routine care—can be used to predict RNA-sequencing data and thus reduce the need for additional analyses.

Background and objectives Malaria, a potentially life-threatening disease, is the most prevalent endemic infectious disease worldwide. In the modern era, the spectrum of glomerular involvement observed in patients after malarial infections remains poorly described. Design, setting, participants, & measurements We therefore performed a retrospective multicenter study to assess the clinical, biologic, pathologic, and therapeutic characteristics of patients with glomerular disease demonstrated by kidney biopsy in France within 3 months of an acute malaria episode. Results We identified 23 patients (12 men), all but 1 of African ancestry and including 10 patients with concomitant HIV infection. All of the imported cases were in French citizens living in France who had recently traveled back to France from an endemic area and developed malaria after their return to France. Eleven patients had to be admitted to an intensive care unit at presentation. Plasmodium falciparum was detected in 22 patients, and Plasmodium malariae was detected in 1 patient. Kidney biopsy was performed after the successful treatment of malaria, a mean of 24 days after initial presentation. At this time, all patients displayed AKI, requiring KRT in 12 patients. Nephrotic syndrome was diagnosed in 17 patients. Pathologic findings included FSGS in 21 patients and minimal change nephrotic syndrome in 2 patients. Among patients with FSGS, 18 had collapsing glomerulopathy (including 9 patients with HIV-associated nephropathy). In four patients, immunohistochemistry with an antibody targeting P. falciparum histidine-rich protein-2 demonstrated the presence of the malaria antigen in tubular cells but not in podocytes or parietal epithelial cells. An analysis of the apoL1 risk genotype showed that high-risk variants were present in all seven patients tested. After a mean follow-up of 23 months, eight patients required KRT (kidney transplantation in two patients), and mean eGFR for the other patients was 51 ml/min per 1.73 m2. Conclusions In patients of African ancestry, imported Plasmodium infection may be a new causal factor for secondary FSGS, particularly for collapsing glomerulopathy variants in an APOL1 high-risk variant background.

Extracellular vesicles released from cancer cells may play an important role in cancer progression by shuttling oncogenic information into recipient cells. However, our knowledge is still fragmentary and there remain numerous questions regarding the mechanisms at play and the functional consequences of these interactions. We have recently established a mesenchymal-like prostate cancer cell line (22Rv1/CR-1; Mes-PCa). In this study, we assessed the effects of the extracellular vesicles released by these cells on recipient androgen-dependent epithelial VCaP prostate cancer cells. Mes-PCa derived vesicles were found to promote mesenchymal features in the recipient epithelial-like prostate cancer cells. This transformation was accompanied by a modulation of androgen receptor signaling and activation of TGFβ signaling pathway. Moreover, recipient cells acquiring mesenchymal traits displayed enhanced migratory and invasive features as well as increased resistance to the androgen receptor antagonist, enzalutamide. Our results suggest a previously unappreciated role for Mes-PCa secreted vesicles in cancer promotion by transferring cell-mediated signals and promoting phenotypic changes in recipient prostate cancer cells.

Deep learning methods for digital pathology analysis have proved an effective way to address multiple clinical questions, from diagnosis to prognosis and even to prediction of treatment outcomes. They have also recently been used to predict gene mutations from pathology images, but no comprehensive evaluation of their potential for extracting molecular features from histology slides has yet been performed. We propose a novel approach based on the integration of multiple data modes, and show that our deep learning model, HE2RNA, can be trained to systematically predict RNA-Seq profiles from whole-slide images alone, without the need for expert annotation. HE2RNA is interpretable by design, opening up new opportunities for virtual staining. In fact, it provides virtual spatialization of gene expression, as validated by double-staining on an independent dataset. Moreover, the transcriptomic representation learned by HE2RNA can be transferred to improve predictive performance for other tasks, particularly for small datasets. As an example of a task with direct clinical impact, we studied the prediction of microsatellite instability from hematoxylin & eosin stained images and our results show that better performance can be achieved in this setting.

Background & aims The recent approval of direct acting anti-virals (DAA) has dramatically changed the landscape of hepatitis C virus (HCV) therapy. Whether viral clearance could promote liver carcinogenesis is debated. It has been hypothesized that changes in intrahepatic immune surveillance following viral cure could favour tumour growth. This study aimed at characterizing the intrahepatic immune changes induced by HCV cure following DAA therapy. Methods Patients with compensated cirrhosis who underwent surgical resection for hepatocellular carcinoma (HCC) after sustained virological response (SVR) to DAA therapy were included. A control group of untreated HCV-infected patients with compensated cirrhosis was selected. RNA was extracted from tumoral and non-tumoral tissues and analysed using the Nanostring Immuno-Oncology-360 panel. Immune cells were quantified by immunohistochemistry. Results Twenty patients were included: 10 patients with a DAA-induced SVR and 10 untreated controls. All of them had a de novo BCLC 0/A HCC. Non-tumoral tissue profiling showed down-regulation of interferon-related genes (including MX1, ISG15 and IFIT1) after DAA therapy. No other differences in immune profiles/immune cell densities were identified between the two groups. The intra-tumoral immune profiles of HCCs that occurred after DAA therapy were not qualitatively or quantitatively different from those of tumours occurring in untreated patients. Conclusion In conclusion, removal of HCV infection after DAA-based therapy results only in a down-regulation of interferon-stimulated genes in non-tumoral tissues from patients with cirrhosis who develop HCC. These minor changes in the liver immune microenvironment are unlikely to favour HCC occurrence or recurrence after DAA-induced SVR.

Despite the advent of several new treatment options over the past years, advanced/metastatic prostate carcinoma (PCa) still remains incurable, which justifies the search for novel targets and therapeutic molecules. Nucleophosmin (NPM1) is a shuttling nucleoprotein involved in tumor growth and its targeting could be a potential approach for cancer therapy. We previously demonstrated that the multivalent pseudopeptide N6L binds to NPM1 potently affecting in vitro and in vivo tumor cell growth of various tumor types as well as angiogenesis. Furthermore, NPM1 binds to androgen receptor (AR) and modulate its activity. In this study, we first investigated the implication of the NPM1 and its Thr199 and Thr234/237 phosphorylated forms in PCa. We showed that phosphorylated forms of NPM1 interact with androgen receptor (AR) in nucleoplasm. N6L treatment of prostate tumor cells led to inhibition of NPM1 phosphorylation in conjunction with inhibition of AR activity. We also found that total and phosphorylated NPM1 were overexpressed in castration-resistant PCa. Assessment of the potential therapeutic role of N6L in PCa indicated that N6L inhibited tumor growth both in vitro and in vivo when used either alone or in combination with the standard-of-care first- (hormonotherapy) and second-line (docetaxel) treatments for advanced PCa. Our findings reveal the role of Thr199 and Thr234/237 phosphorylated NPM1 in PCa progression and define N6L as a new drug candidate for PCa therapy.

Today, pathology imaging is one of the most common and inexpensive diagnostic/prognostic tools used in oncology, while more sophisticated methods such as next generation sequencing (NGS) remain relatively expensive and not routinely used in a clinical setting. Deep convolutional neural networks (CNNs) have emerged as an important image analysis technology enhancing the workflow of pathologists and improving the prediction of patient prognosis and response to treatment​​. Recently, a few attempts have been made to predict molecular features from tissue imaging using CNNs. While these preliminary results are encouraging, there have been no systematic attempts to link Whole Slide Images (WSIs) to transcriptomic profiles. In this study, we developed a cutting-edge deep learning model named HE2RNA, specifically customized for the direct prediction of gene expression from H&E-stained WSIs without need for annotation from pathologists. Our model was trained and tested on 8,725 patients from 28 different cancer types available at The Cancer Genome Atlas (TCGA). HE2RNA accurately predicted the expression of six gene signatures related to well known cancer hallmarks (angiogenesis, hypoxia, DNA repair, cell cycle and immunity) and performed particularly well for signalling pathways involved in immune cell activation. This indicates that suitably designed deep learning models can recognize subtle structures in tissue imaging and relate them to molecular portraits. Moreover, HE2RNA is designed to generate a spatial representation (virtual map) of any well-predicted gene expression overlaying the H&E slide. Such a virtual map was validated on a double-stained H&E/CD3 slide obtained from an independent hepatocellular carcinoma sample. This spatialization could be useful in augmenting the pathologists' workflow by providing a virtual multiplexed staining for each H&E slide while overcoming the technical issues associated with immunohistochemistry. Various important prognostic factors, such as microsatellite instability (MSI), are derived from molecular features. Microsatellite instability refers to the hypermutability of short repetitive genomic sequences caused by impaired DNA mismatch repair. These mutations frequently observed in gastric and colorectal cancer are associated with better response to immunotherapy. We show that the transcriptomic representation learned by our model can be used to improve the performance of MSI status prediction for small datasets of WSI. This type of setting is common since large databases of matched RNA-Seq profiles and WSI are widely available, while databases of matched MSI status and WSI are more scarce. In the future, such technologies could therefore facilitate universal screening of molecular biomarkers and improved identification of patients that could benefit from new therapeutic strategies. Citation Format: Elodie Pronier, Benoît Schmauch, Alberto Romagnoni, Charlie Saillard, Pascale Maillé, Julien Calderaro, Meriem Sefta, Sylvain Toldo, Mikhail Zaslavskiy, Thomas Clozel, Matahi Moarii, Pierre Courtiol, Gilles Wainrib. HE2RNA: A deep learning model for transcriptomic learning from digital pathology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2105.

Members of the EGF-CFC (Cripto, FRL-1, Cryptic) protein family are increasingly recognized as key mediators of cell movement and cell differentiation during vertebrate embryogenesis. The founding member of this protein family, CRIPTO, is overexpressed in various human carcinomas. Yet, the biological role of CRIPTO in this setting remains unclear. Here, we find CRIPTO expression as especially high in a subgroup of primary prostate carcinomas with poorer outcome, wherein resides cancer cell clones with mesenchymal traits. Experimental studies in PCa models showed that one notable function of CRIPTO expression in prostate carcinoma cells may be to augment PI3K/AKT and FGFR1 signaling, which promotes epithelial-mesenchymal transition and sustains a mesenchymal state. In the observed signaling events, FGFR1 appears to function parallel to AKT, and the two pathways act cooperatively to enhance migratory, invasive and transformation properties specifically in the CRIPTO overexpressing cells. Collectively, these findings suggest a novel molecular network, involving CRIPTO, AKT, and FGFR signaling, in favor of the emergence of mesenchymal-like cancer cells during the development of aggressive prostate tumors.

BACKGROUND In view of the marked molecular heterogeneity of prostate cancer (PCa), clinical and pathologic parameters alone may be unreliable for predicting disease outcomes after surgical intervention. The development of biomarkers may be helpful to estimate tumor heterogeneity and stratify patients in terms of their risk of progression. Levels of v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), trefoil factor 3 (TFF3), and serine peptidase inhibitor, Kazal type 1 (SPINK1) are commonly elevated in PCa, but it is unclear whether the evaluation of these 3 markers can help to discriminate patients who will have different clinical outcomes. The authors investigated whether assessment of ERG, TFF3, and SPINK1 expression could help to define clinically relevant, distinct subsets of patients with PCa. METHODS The cohort consisted of 279 men with PCa who underwent radical prostatectomy at Henri Mondor Hospital. Expression levels of ERG, TFF3, and SPINK1 were evaluated immunohistochemically in the prostatectomy specimens. Potential associations of ERG, TFF3, and SPINK1 with age, prostate-specific antigen (PSA), tumor stage, Gleason score, and biochemical recurrence, defined by PSA failure, were investigated. RESULTS Although prognostic significance was not observed for ERG or TFF3, an exclusive pattern of expression was demonstrated for TFF3 and ERG. SPINK1 expression was observed exclusively in a subgroup of cancers that expressed TFF3 (41 of 175 tumors). Moreover, SPINK1 positivity was identified as predictive of biochemical recurrence in univariate (P = .0009) and multivariate (P = .0003) analyses. CONCLUSIONS The current results suggest that ERG and TFF3 characterize 2 distinct subsets of PCa, with a more aggressive subgroup of TFF3-expressing tumors that express SPINK1. Together, these findings support a rationale of screening for these biomarkers for prognostic purposes and molecular subtyping of the disease. Cancer 2015;121:1422–1430. © 2015 American Cancer Society.

Although human epidermal growth factor receptor 2 (HER2) may represent a therapeutic target, its evaluation in urothelial carcinoma of the bladder does not rely on a standardized scoring system by immunohistochemistry or fluorescent in situ hybridization (FISH), as reflected by various methodology in the literature and clinical trials. Our aim was to improve and standardize HER2 amplification detection in bladder cancer. We assessed immunohistochemical criteria derived from 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAPs) guidelines for breast cancer and investigated intratumoral heterogeneity in a retrospective multicentric cohort of 188 patients with locally advanced urothelial carcinoma of the bladder. Immunohistochemistry was performed on 178 primary tumors and 126 lymph node metastases, eligible cases (moderate/strong, complete/incomplete membrane staining) were assessed by FISH. HER2 overexpression was more frequent with 2013 ASCO/CAP than 2007 ASCO/CAP guidelines (p

The PI3K/AKT pathway is considered to play a major role in bladder carcinogenesis, but its relationships with other molecular alterations observed in bladder cancer remain unknown. We investigated PI3K/AKT pathway activation in a series of human bladder urothelial carcinomas (UC) according to PTEN expression, PTEN deletions and FGFR3, PIK3CA, KRAS, HRAS, NRAS and TP53 gene mutations. The series included 6 normal bladder urothelial samples and 129 UC (Ta n = 25, T1 n = 34, T2-T3-T4 n = 70). Expression of phospho-AKT (pAKT), phospho-S6-Ribosomal Protein (pS6) (one downstream effector of PI3K/AKT pathway) and PTEN was evaluated by reverse phase protein Array. Expression of miR-21, miR-19a and miR-222, known to regulate PTEN expression, was also evaluated. pAKT expression levels were higher in tumors than in normal urothelium (p < 0.01), regardless of stage and showed a weak and positive correlation with pS6 (Spearman coefficient RS = 0.26; p = 0.002). No association was observed between pAKT or pS6 expression and the gene mutations studied. PTEN expression was decreased in PTEN-deleted tumors, and in T1 (p = 0.0089) and T2-T3-T4 (p < 0.001) tumors compared to Ta tumors; it was also negatively correlated with miR-19a (RS = -0.50; p = 0.0088) and miR-222 (RS = -0.48; p = 0.0132), but not miR-21 (RS = -0.27; p = 0.18) expression. pAKT and PTEN expressions were not negatively correlated, and, on the opposite, a positive and moderate correlation was observed in Ta (RS = 0.54; p = 0.0056) and T1 (RS = 0.56; p = 0.0006) tumors. Our study suggests that PI3K/AKT pathway activation occurs in the entire spectrum of bladder UC regardless of stage or known most frequent molecular alterations, and independently of low PTEN expression.

International audience; BACKGROUND:Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer. Preclinical studies show that anti-FGFR3 treatment slows down tumor growth, suggesting that this tyrosine kinase receptor is a candidate for personalized bladder cancer treatment, particularly in patients with mutated FGFR3. We addressed tumor heterogeneity in a large multicenter, multi-laboratory study, as this may have significant impact on therapeutic response.PATIENTS AND METHODS:We evaluated possible FGFR3 heterogeneity by the PCR-SNaPshot method in the superficial and deep compartments of tumors obtained by transurethral resection (TUR, n = 61) and in radical cystectomy (RC, n = 614) specimens and corresponding cancer-positive lymph nodes (LN+, n = 201).RESULTS:We found FGFR3 mutations in 13/34 (38%) T1 and 8/27 (30%) ≥T2-TUR samples, with 100% concordance between superficial and deeper parts in T1-TUR samples. Of eight FGFR3 mutant ≥T2-TUR samples, only 4 (50%) displayed the mutation in the deeper part. We found 67/614 (11%) FGFR3 mutations in RC specimens. FGFR3 mutation was associated with pN0 (P < 0.001) at RC. In 10/201 (5%) LN+, an FGFR3 mutation was found, all concordant with the corresponding RC specimen. In the remaining 191 cases, RC and LN+ were both wild type.CONCLUSIONS:FGFR3 mutation status seems promising to guide decision-making on adjuvant anti-FGFR3 therapy as it appeared homogeneous in RC and LN+. Based on the results of TUR, the deep part of the tumor needs to be assessed if neoadjuvant anti-FGFR3 treatment is considered. We conclude that studies on the heterogeneity of actionable molecular targets should precede clinical trials with these drugs in the perioperative setting.

Calderaro J, Moroch J, Pierron G, Pedeutour F, Grison C, Maille P, Soyeux P, de la Taille A, Couturier J, Vieillefond A, Rousselet M C, Delattre O & Allory Y (2012) Histopathology 61, 428–435 SMARCB1/INI1 inactivation in renal medullary carcinoma Aims: Renal medullary carcinoma (RMC), a rare and highly aggressive tumour which occurs in patients with sickle-cell disease, shares many clinicopathological features with collecting duct carcinoma (CDC). The molecular mechanisms underlying RMC and CDC are mainly unknown, and there is ongoing debate about their status as distinct entities. Loss of expression of SMARCB1/INI1, a chromatin remodelling regulator and repressor of cyclin D1 transcription, has been reported recently in RMC. The aim of our study was to investigate if such loss of expression is specific for RMC. SMARCB1/INI1 genetic alterations and cyclin D1 expression were also studied. Methods and results: Using immunochemistry, neoplastic cells showed complete loss of SMARCB1/INI1 expression in all six cases of RMC but in only one of 22 cases of CDC. In two RMC cases investigated, comparative genomic hybridization demonstrated complete loss of one SMARCB1/INI1 allele, with no other genomic imbalances, and no mutations were found on the remaining allele. Cyclin D1 was expressed in all RMCs, suggesting that SMARCB1/INI1 inactivation may result in increased cyclin D1 transcription. Conclusions: The specific SMARCB1/INI1 inactivation observed in RMCs suggests that RMC and CDC are different entities.

The gene cyclin-dependent kinase inhibitor 2A (CDKN2A) is frequently inactivated by deletion in bladder carcinoma. However, its role in bladder tumourigenesis remains unclear. We investigated the role of CDKN2A deletion in urothelial carcinogenesis, as a function of FGFR3 mutation status, a marker for one of the two pathways of bladder tumour progression, the Ta pathway. We studied 288 bladder carcinomas: 177 non-muscle-invasive (123 Ta, 54 T1) and 111 muscle-invasive (T2–4) tumours. CDKN2A copy number was determined by multiplex ligation-dependent probe amplification, and FGFR3 mutations by SNaPshot analysis. FGFR3 mutation was detected in 124 tumours (43.1%) and CDKN2A homozygous deletion in 56 tumours (19.4%). CDKN2A homozygous deletion was significantly more frequent in FGFR3-mutated tumours than in wild-type FGFR3 tumours (p = 0.0015). This event was associated with muscle-invasive tumours within the FGFR3-mutated subgroup (p < 0.0001) but not in wild-type FGFR3 tumours. Similar findings were obtained for an independent series of 101 bladder carcinomas. The impact of CDKN2A deletions on recurrence-free and progression-free survival was then analysed in 89 patients with non-muscle-invasive FGFR3-mutated tumours. Kaplan–Meier survival analysis showed that CDKN2A losses (hemizygous and homozygous) were associated with progression (p = 0.0002), but not with recurrence, in these tumours. Multivariate Cox regression analysis identified CDKN2A loss as a predictor of progression independent of stage and grade. These findings highlight the crucial role of CDKN2A loss in the progression of non-muscle-invasive FGFR3-mutated bladder carcinomas and provide a potentially useful clinical marker for adapting the treatment of such tumours, which account for about 50% of cases at initial clinical presentation. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

467 Background: Recent and independent muscle-invasive bladder cancer (MIBC) molecular classifications identified the basal / squamous-like (BASQ) tumours as an intrinsic and robust subtype, with a poor outcome and possible chemosensitivity to cisplatin based regimen, making mandatory the development of a diagnostic tool for their identification in routine samples. Our study aimed to evaluate the diagnostic accuracy of a Nanostring classifier for tumor subtype prediction on FPPE specimens. Methods: Two series of MIBC were used (CIT n = 51 & Stransky n = 22) for which BASQ tumours were identified previously using Affymetrix transcriptome data obtained from frozen samples (Rebouissou, Science Trans Med 2014). 29 genes were selected to predict the basal subtype, RNA expression of matched frozen and FFPE samples was studied using Nanostring technology. To define the classifiers for Affymetrix, frozen and FFPE Nanostring expression matrix on CIT samples the centroid of each cluster was calculated using the expression of 29 genes. Internal validation used leave-one-out cross-validation to train and test the prediction accuracy of the new classifier. For external validation, the CIT samples were used as training set and the Stransky samples as validation set. Predictive accuracy expressed as percentage of correctly classified samples is provided. Results: Correlations between Affymetrix, frozen and FFPE Nanostring data set were checked for gene expression and samples. Using CIT samples as train and test set, the predictive accuracy for BASQ tumour identification was for Affymetrix, frozen Nanostring and FFPE Nanostring classifiers, 90.20% [78.59%; 96.74%], 88.24% [76.13%; 95.56%] and 92.16% [81.12%; 97.82%], respectively. Using training on CIT samples and test on Stransky samples, this predictive accuracy was 90.91% [70.84%; 98.88%] both for Affymetrix, frozen Nanostring and FFPE Nanostring classifiers. Conclusions: The 29 gene expression Nanostring codeset was able to identify reliably basal / squamous like tumours on FFPE samples from MIBC in comparison with the gold standard approach based on transcriptomic profile, appearing as a promising diagnostic tool.

Occupational exposure to polycyclic aromatic hydrocarbons (PAH) is associated with an increased risk of urothelial carcinoma (UC). FGFR3 is found mutated in about 70% of Ta tumors, which represent the major group at diagnosis. The influence of PAH on FGFR3 mutations and whether it is related to the emergence or shaping of these mutations is not yet known. We investigated the influence of occupational PAH on the frequency and spectrum of FGFR3 mutations. We included on 170 primary urothelial tumors from five hospitals from France. Patients (median age, 64 yr) were interviewed to gather data on occupational exposure to PAH, revealing 104 non- and possibly PAH exposed patients, 66 probably and definitely exposed patients. Tumors were classified as follows: 75 pTa, 52 pT1, and 43 > or =pT2. Tumor grades were as follows: 6 low malignant potential neoplasms (LMPN) and 41 low-grade and 123 high-grade carcinomas. The SnaPshot method was used to screen for the following FGFR3 mutations: R248C, S249C, G372C, Y375C, A393E, K652E, K652Q, K652M, and K652T. Occupational PAH exposure was not associated with a particular stage or grade of tumors. Thirty-nine percent of the tumors harbored FGFR3 mutations. After adjustment for smoking, occupational exposure to PAH did not influence the frequency [OR, 1.10; 95% CI, 0.78-1.52], or spectrum of FGFR3 mutations. Occupational exposure to PAH influenced neither the frequency nor the spectrum of FGFR3 mutations and there was no direct relationship between these mutations and this occupational hazard.

Purpose: Abnormally high levels of epidermal growth factor receptor (EGFR) protein are associated with advanced tumor stage/grade. The objective of this study was to evaluate the effects of the specific EGFR tyrosine kinase inhibitor gefitinib on activation of the Akt and mitogen-activated protein kinase (MAPK) pathways in human urothelial cell carcinoma (UCC) cell lines and to identify potential markers of gefitinib responsiveness in biopsy samples of UCC. Experimental Design: Changes in markers of UCC growth and invasion after exposure to gefitinib were studied in six human UCC cell lines expressing various levels of EGFR. The findings were related to activation of Akt and MAPK. We studied the influence of gefitinib on intraepithelial expansion of the responsive 1207 cell line. EGFR, Akt, and MAPK activation was studied by Western blot analysis of a panel of 57 human UCC. Results: Gefitinib had a growth-inhibitory and anti-invasive effect in two of six UCC cell lines (i.e., 647V and 1207). Gefitinib was also able to block the expansion of 1207 at the expense of normal urothelial cells. These effects did not depend on the level of expression of EGFR but they were associated with the down-regulation of MAPK and Akt activity; in 1207 cells, gefitinib activity was associated with p27 up-regulation and p21 and matrix metalloproteinase-9 down-regulation. Similarly, the Akt and MAPK pathways were found to be strongly phosphorylated in association with EGFR activation in a subset of human UCC specimens. Conclusions: Activation of EGFR, Akt, and MAPK defines a subset of UCC which might provide information for the identification of gefitinib responders.

We evaluated the clinical significance of Th1(IL-2)/Th2(IL-10) urinary profiles during a weekly induction course lasting 6 weeks, followed by a weekly maintenance therapy schedule for 3 weeks. Urinary IL-2 and /IL-10 were measured by ELISA in 39 patients receiving BCG for superficial bladder cancer or carcinoma in situ. Measurements were made after each instillation of 81 mg of BCG Connaught (Immucyst) during the induction course and the 3-week maintenance therapy (given at 3, 6, 12, 18, 24, 30 and 36 months). Cytokine levels were correlated with the risk of recurrence, progression, leukocyturia and adverse events. Median follow-up was 35 months (range 7-72 months). Complete responses to BCG were obtained in 30 patients (77%); the remaining 9 patients relapsed (23%), and 4 of these patients progressed (10.2%). Failure to detect urinary IL-2 during BCG induction course and the first extended induction cycle (6+3 schedule) correlated with time to recurrence (p = 0.01) and progression (p = 0.01). During the extended induction cycle, the first instillation was associated with an IL-2 cytokine profile, whereas the second and third instillations were associated with a switch to an IL-10 cytokine profile. This switch was associated with leukocyturia (p = 0.0001) and adverse events (p = 0.03). The 6+3 schedule is associated with urinary IL-2 overproduction and improved recurrence- and progression-free survival. During the BCG extended induction cycle, the favorable IL-2 urinary cytokine pattern gradually switches to an IL-10 profile, suggesting that the schedule based on 3 weekly instillations may be unsuitable for some patients and that the dose and frequency of maintenance BCG instillations may be adapted to individual urinary cytokine levels.

Purpose: Interleukin (IL)-2 and interferon-γ are released during T helper 1 lymphocyte responses, while IL-10 is released during T helper 2 responses. We evaluated the prognostic value of urinary IL-2, interferon-γ and IL-10 levels in patients with superficial bladder cancer treated with bacillus Calmette-Guerin (BCG) instillationMethods: Urinary IL-2, interferon-γ and IL-10 were measured by enzyme-linked immunosorbent assay in 37 patients receiving BCG for stages Ta/T1 superficial bladder cancer, and carcinoma in situ. Measurements were made after instillations 5 and 6 during a course of 6 weekly instillations of 150 mg. BCG, Pasteur strain. Correlations of cytokine levels with the clinical outcome were evaluated using the log rank testResults: Median followup was 29 months. Patients with urinary IL-2 less than 27 pg./μmol. creatinine were significantly more likely to have recurrences than those with higher values (log rank test p = 0.0009). Urinary IL-10 and interferon-γ levels had no apparent impact on...

In view of the marked molecular heterogeneity of prostate cancer (PCa), clinical and pathologic parameters alone may be unreliable for predicting disease outcomes after surgical intervention. The development of biomarkers may be helpful to estimate tumor heterogeneity and stratify patients in terms of their risk of progression. Levels of v-ets avian erythroblastosis virus E26 oncogene homolog (ERG), trefoil factor 3 (TFF3), and serine peptidase inhibitor, Kazal type 1 (SPINK1) are commonly elevated in PCa, but it is unclear whether the evaluation of these 3 markers can help to discriminate patients who will have different clinical outcomes. The authors investigated whether assessment of ERG, TFF3, and SPINK1 expression could help to define clinically relevant, distinct subsets of patients with PCa.The cohort consisted of 279 men with PCa who underwent radical prostatectomy at Henri Mondor Hospital. Expression levels of ERG, TFF3, and SPINK1 were evaluated immunohistochemically in the prostatectomy specimens. Potential associations of ERG, TFF3, and SPINK1 with age, prostate-specific antigen (PSA), tumor stage, Gleason score, and biochemical recurrence, defined by PSA failure, were investigated.Although prognostic significance was not observed for ERG or TFF3, an exclusive pattern of expression was demonstrated for TFF3 and ERG. SPINK1 expression was observed exclusively in a subgroup of cancers that expressed TFF3 (41 of 175 tumors). Moreover, SPINK1 positivity was identified as predictive of biochemical recurrence in univariate (P = .0009) and multivariate (P = .0003) analyses.The current results suggest that ERG and TFF3 characterize 2 distinct subsets of PCa, with a more aggressive subgroup of TFF3-expressing tumors that express SPINK1. Together, these findings support a rationale of screening for these biomarkers for prognostic purposes and molecular subtyping of the disease.

Muscle-invasive bladder carcinoma (MIBC) constitutes a heterogeneous group of tumors with a poor outcome. Molecular stratification of MIBC may identify clinically relevant tumor subgroups and help to provide effective targeted therapies. From seven series of large-scale transcriptomic data (383 tumors), we identified an MIBC subgroup accounting for 23.5% of MIBC, associated with shorter survival and displaying a basal-like phenotype, as shown by the expression of epithelial basal cell markers. Basal-like tumors presented an activation of the epidermal growth factor receptor (EGFR) pathway linked to frequent EGFR gains and activation of an EGFR autocrine loop. We used a 40-gene expression classifier derived from human tumors to identify human bladder cancer cell lines and a chemically induced mouse model of bladder cancer corresponding to human basal-like bladder cancer. We showed, in both models, that tumor cells were sensitive to anti-EGFR therapy. Our findings provide preclinical proof of concept that anti-EGFR therapy can be used to target a subset of particularly aggressive MIBC tumors expressing basal cell markers and provide diagnostic tools for identifying these tumors.

Previous studies indicated that transforming growth factor beta1 (TGFbeta1) is expressed by normal urothelial cells and exerts regulatory autocrine functions in urothelial maintenance and wound healing. However, little is known about the expression patterns of TGFbeta1 and its receptors in bladder tumors. Therefore, we studied the protein and mRNA localization of TGFbeta1 and TGFbeta receptor types I and II (TGFbetaRI and TGFbetaRII) in normal human urothelium and transitional cell carcinomas (TCCs) of different grades and stages. Expression of TGFbeta1 and its receptors was examined by immunocytochemistry and mRNA in situ hybridization in normal urothelium and TCCs using a semiquantitative method. By immunocytochemistry, the expression of TGFbeta1 and TGFbetaRII was higher in superficial and basal cell layers of normal urothelium than in the intermediate layer. A similar localization was seen in superficial TCCs. TGFbetaRI was mainly present in basal and intermediate cell layers of normal urothelium and superficial TCCs. In contrast, in muscle invasive TCCs, all tumor cells stained intensely for all three proteins. No correlation was found between immunostaining and TCC grade. In situ hybridization pointed out that all cell layers in normal urothelium exhibit similar TGFbeta1 mRNA levels. Elevated TGFbeta1 mRNA levels were noted in TCCs irrespective of grade or stage. In conclusion, these data indicate that in normal urothelium TGFbeta1, TGFbetaRI, and TGFbetaRII expression depend on maturation and differentiation. This pattern is particularly lost in muscle invasive TCCs, in which the expression of the three proteins is enhanced. These data suggest autocrine TGFbeta1 mechanisms in human TCC cells that may be more pronounced in muscle invasive TCC cells.

Renal medullary carcinoma (RMC) is a rare and highly aggressive neoplasm that most often occurs in young patients or children with sickle cell trait or disease (SCD). Although BAF47 staining is constantly negative in these tumors, the genetic mechanism underlying this phenotype remains unknown, since all cases reported so far show SMARCB1 hemizygous deletion only. By investigating a series of five RMC by gene expression profiling, array-CGH, and RNA and whole-exome sequencing, we demonstrated that SMARCB1 inactivation is related to an original mechanism of inter-chromosomal balanced translocations that disrupt SMARCB1 sequence and thus contribute to its inactivation. RMC were also characterised by a simple genome, with a low mutation rate and a lack of recurrent molecular alterations other than SMARCB1 deficiency, emphasizing its potency as single genetic event to drive oncogenesis. Finally, gene expression profiling revealed that RMC share common oncogenic pathways with pediatric malignant rhabdoid tumors, another lethal tumor subtype characterised by SMARCB1 deficiency Citation Format: Julien Calderaro, Julien Masliah-Planchon, Wilfrid Richer, Laetitia Planque, Pascale Maille, Alexandre de la Taille, Helene Boussion, Olivier Delattre, Yves Allory, Franck Bourdeaut. Balanced translocations disrupting SMARCB1 are hallmark recurrent genetic alterations in renal medullary carcinomas. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A29.

Renal medullary carcinoma (RMC), a rare and highly aggressive tumour which occurs in patients with sickle-cell disease, shares many clinicopathological features with collecting duct carcinoma (CDC). The molecular mechanisms underlying RMC and CDC are mainly unknown, and there is ongoing debate about their status as distinct entities. Loss of expression of SMARCB1/INI1, a chromatin remodelling regulator and repressor of cyclin D1 transcription, has been reported recently in RMC. The aim of our study was to investigate if such loss of expression is specific for RMC. SMARCB1/INI1 genetic alterations and cyclin D1 expression were also studied.Using immunochemistry, neoplastic cells showed complete loss of SMARCB1/INI1 expression in all six cases of RMC but in only one of 22 cases of CDC. In two RMC cases investigated, comparative genomic hybridization demonstrated complete loss of one SMARCB1/INI1 allele, with no other genomic imbalances, and no mutations were found on the remaining allele. Cyclin D1 was expressed in all RMCs, suggesting that SMARCB1/INI1 inactivation may result in increased cyclin D1 transcription.The specific SMARCB1/INI1 inactivation observed in RMCs suggests that RMC and CDC are different entities.

The gene cyclin-dependent kinase inhibitor 2A (CDKN2A) is frequently inactivated by deletion in bladder carcinoma. However, its role in bladder tumourigenesis remains unclear. We investigated the role of CDKN2A deletion in urothelial carcinogenesis, as a function of FGFR3 mutation status, a marker for one of the two pathways of bladder tumour progression, the Ta pathway. We studied 288 bladder carcinomas: 177 non-muscle-invasive (123 Ta, 54 T1) and 111 muscle-invasive (T2-4) tumours. CDKN2A copy number was determined by multiplex ligation-dependent probe amplification, and FGFR3 mutations by SNaPshot analysis. FGFR3 mutation was detected in 124 tumours (43.1%) and CDKN2A homozygous deletion in 56 tumours (19.4%). CDKN2A homozygous deletion was significantly more frequent in FGFR3-mutated tumours than in wild-type FGFR3 tumours (p = 0.0015). This event was associated with muscle-invasive tumours within the FGFR3-mutated subgroup (p0.0001) but not in wild-type FGFR3 tumours. Similar findings were obtained for an independent series of 101 bladder carcinomas. The impact of CDKN2A deletions on recurrence-free and progression-free survival was then analysed in 89 patients with non-muscle-invasive FGFR3-mutated tumours. Kaplan-Meier survival analysis showed that CDKN2A losses (hemizygous and homozygous) were associated with progression (p = 0.0002), but not with recurrence, in these tumours. Multivariate Cox regression analysis identified CDKN2A loss as a predictor of progression independent of stage and grade. These findings highlight the crucial role of CDKN2A loss in the progression of non-muscle-invasive FGFR3-mutated bladder carcinomas and provide a potentially useful clinical marker for adapting the treatment of such tumours, which account for about 50% of cases at initial clinical presentation.

Objectifs L’impact pronostique de l’Immunoscore dans le cancer du colon conduit les chercheurs a s’interesser a l’infiltration lymphocytaire dans les carcinomes urotheliaux. L’absence de standardisation des techniques d’immunohistochimie (IHC) et de comptage rend les resultats des etudes peu reproductibles. L’objectif etait d’evaluer une methode de comptage automatique standardisee de l’infiltrat inflammatoire lymphocytaire de tumeurs non invasives du muscle (TVNIM) en comparant les resultats au comptage manuel. Methodes L’analyse a ete realisee sur lame de TMA comprenant 142 spots de 0,6 mm de TVNIM Ta G2 (WHO 1973). La lame a ete marquee sur un plateforme d’IHC automatisee (automate Ventana™) par un anticorps anti-pan cytokeratine AE1–AE3 (identification du tissus tumoral) puis un anticorps anti-CD8. Apres scannage des lames, le comptage des CD8 du compartiment tumoral a ete realise manuellement, puis automatiquement a l’aide du logiciel Calopix (societe Tribvn™). Le logiciel permet la segmentation des compartiments stromal et tumoral puis le comptage des elements marques ainsi que la mesure de la surface d’analyse et de marquage permettant la normalisation resultats en μm 2 . Resultats Au total, 142 spots ont ete analyses manuellement, puis a l’aide de Calopix. Pour chaque spot le logiciel a rapporte le nombre d’elements ainsi que la surface d’analyse ( Fig. 1 ). Le coefficient de correlation de Pearson entre comptage manuel et automatique etait de 0,72 ( Fig. 2 ). En cas de forte infiltration lymphocytaire le comptage automatique etait plus performant. Pour 10 % des spots une forte discordance a ete observee due a un defaut de segmentation tumeur/stroma conduisant au comptage des lymphocytes du stroma avec les lymphocytes tumoraux. Apres correction du procede de segmentation, le coefficient de correlation etait de 0,84. Conclusion La correlation entre comptage manuel et automatique etait elevee. En cas de forte infiltration lymphocytaire le comptage automatique est plus performant. Une fois validee, cette technique standardisee pourrait palier au probleme de reproductibilite des techniques d’IHC et de comptage et conduire a des etudes a large echelle pour evaluer l’impact pronostique de l’infiltrat lymphocytaire dans les TVNIM.

Expression of class III β-tubulin (βIII-tubulin) correlates with tumor progression and resistance to taxane-based therapies for several human malignancies, but its use as a biomarker of tumor behavior in prostate cancer (PCa) remains largely unexplored. Here, we describe βIII-tubulin immunohistochemical staining patterns of prostate tumors obtained from a broad spectrum of PCa patients, some of whom subsequently received docetaxel therapy for castration-resistant PCa (CRPC). Elevated βIII-tubulin expression was significantly associated with tumor aggressiveness in PCa patients with presumed localized disease, as it was found to be an independent marker of biochemical recurrence after treatment. Additionally, βIII-tubulin expression in tumor cells was an independent predictor of lower overall survival for patients receiving docetaxel-based chemotherapy for CRPC. Manipulation of βIII-tubulin expression in human PCa cell lines using a human βIII-tubulin expression vector or βIII-tubulin small interfering RNA altered cell survival in response to docetaxel treatment in a manner that supports a role for βIII-tubulin expression as a mediator of PCa cell resistance to docetaxel therapy. Our findings suggest a role for βIII-tubulin as candidate theranostic biomarker to predict the response to docetaxel-based chemotherapy as well as to target for treatment of docetaxel-resistant CRPC. Cancer Res; 70(22); 9253–64. ©2010 AACR.

International audience; INTRODUCTION AND OBJECTIVES: Prostatic inflammation could be a key component in prostate enlargement and benign prostatic hyperplasia (BPH) progression. Our aim was to characterize inflammatory cells infiltrate within BPH tissue and to correlate inflammation and clinical data. MATERIAL AND METHODS: Inflammation was profiled on three clinical outcome tissue microarrays (TMAs), including 282 patients treated by surgery for a complicated and/or symptomatic BPH. Inflammation score was defined by combining six cytological parameters and five markers on immunohistochemistry (IHC). Cytological parameters were lymphocytes, macrophages, and polynuclears leukocytes infiltrates, and three glandular aspect modifications: glandular atrophy, glandular destruction, and tissue fibrosis. IHC markers were CD3, CD4, and CD8 decorating T-lymphocytes, CD20 decorating B-lymphocytes, and CD163 decorating macrophages. RESULTS: The majority of patients had inflammatory cells infiltrating BPH tissues: 81% had T-lymphocytes markers (CD3), 52% had B-lymphocytes markers (CD20), and 82% had macrophages markers (CD163). IPSS score (21 vs. 12; P = 0.02) and prostate volume (77 cm(3) vs. 62 cm(3); P = 0.002) were significantly higher in patients with high-grade prostatic inflammation. CONCLUSION: We characterized inflammatory cells infiltrate in a large cohort of surgically treated BPH specimens. The role of inflammation in BPH development was highlighted by the strong correlation between histological inflammation, IPSS, and prostate volume. Prostate enlargement due to chronic inflammatory process may progressively conduce to BPH progression. Therefore, inflammation is a therapeutic target for BPH.

Background: Class III β-tubulin (βIII-tubulin) is expressed in tissues of neuronal lineage and also in several human malignancies, including non-small-cell lung carcinoma, breast and ovarian cancer. Overexpression of βIII-tubulin in these tumours is associated with an unfavourable outcome and resistance to taxane-based therapies. At present, βIII-tubulin expression remains largely uncharacterised in prostate cancer. Methods: In this report, we evaluated the expression of βIII-tubulin in 138 different human prostate tumour specimens by immunohistochemistry from patients with hormone-treated or hormone-untreated prostate cancer. βIII-tubulin expression was also examined in various prostatic cancer cell lines including in androgen-sensitive human prostate cancer cells, LNCaP, grown in androgen-depleted medium in 2D cultures or as tumour xenografts when the host mouse was castrated. Results: Whereas moderate-to-strong βIII-tubulin expression was detected in only 3 out of 74 (4%) hormone-naive tumour specimens obtained from patients who never received hormone therapy, 6 out of 24 tumour specimens (25%) from patients treated for 3 months with neoadjuvant hormone therapy and 24 out of 40 (60%) castration-resistant tumour specimens from chronic hormone-treated patients were found to express significant levels of βIII-tubulin. These findings were supported by in vitro and in vivo settings. Conclusion: Our data indicate that βIII-tubulin expression is augmented in prostate cancer by androgen ablation and that the expression of this β-tubulin isoform is associated with the progression of prostate cancer to the castration-resistant state, a stage largely responsible for mortality from prostate cancer.

Prostatic inflammation could be a key component in prostate enlargement and benign prostatic hyperplasia (BPH) progression. Our aim was to characterize inflammatory cells infiltrate within BPH tissue and to correlate inflammation and clinical data.Inflammation was profiled on three clinical outcome tissue microarrays (TMAs), including 282 patients treated by surgery for a complicated and/or symptomatic BPH. Inflammation score was defined by combining six cytological parameters and five markers on immunohistochemistry (IHC). Cytological parameters were lymphocytes, macrophages, and polynuclears leukocytes infiltrates, and three glandular aspect modifications: glandular atrophy, glandular destruction, and tissue fibrosis. IHC markers were CD3, CD4, and CD8 decorating T-lymphocytes, CD20 decorating B-lymphocytes, and CD163 decorating macrophages.The majority of patients had inflammatory cells infiltrating BPH tissues: 81% had T-lymphocytes markers (CD3), 52% had B-lymphocytes markers (CD20), and 82% had macrophages markers (CD163). IPSS score (21 vs. 12; P = 0.02) and prostate volume (77 cm(3) vs. 62 cm(3); P = 0.002) were significantly higher in patients with high-grade prostatic inflammation.We characterized inflammatory cells infiltrate in a large cohort of surgically treated BPH specimens. The role of inflammation in BPH development was highlighted by the strong correlation between histological inflammation, IPSS, and prostate volume. Prostate enlargement due to chronic inflammatory process may progressively conduce to BPH progression. Therefore, inflammation is a therapeutic target for BPH.

International audience; Recent studies have revealed the potential involvement of Hedgehog (Hh) signalling in proliferation and invasive behaviour of prostate carcinoma (PCa). The aim of this study was to specify the role of Sonic Hh (Shh), Desert Hh (Dhh) and Indian Hh (Ihh) in the natural history of PCa. Hh ligands expression was compared in primary hormone-naive PCa (HNPC), hormone-treated PCa (HTPC) and hormone-refractory PCa (HRPC), using immunohistochemistry. Shh and Dhh were expressed by both epithelial and stromal cells of prostate tissues. Ihh was only expressed by stromal cells. For the three ligands, mRNA and immunostaining were not correlated. In HNPC, Shh epithelial expression was significantly associated with high Gleason scores (p = 0.03), metastatic lymph nodes (p = 0.004) and Dhh epithelial staining was associated with high pT stages (p = 0.003), seminal vesicle invasion (p = 0.03) and bladder neck invasion (p = 0.0008). Negative Shh staining in stromal cells was associated with high Gleason scores (p = 0.015), high pT stages (p = 0.01) and bladder neck invasion (p = 0.04). Concomitant absence of Shh and Dhh expression in stromal cells was an independent prognostic parameter for biological recurrence on multivariate analysis (p = 0.01). Epithelial expression of Shh and Dhh was increased in HTPC compared to HNPC (p = 0.02 and p = 0.04). Interestingly, in vitro, transcript analysis also showed increased expression of these 2 Hh ligands when androgen-sensitive LNCaP cells were maintained in androgen-free medium mimicking hormonal therapy. Epithelial expression of Dhh was increased (p < 0.0001) in HRPC compared to HNPC, while stromal expression of Shh and Dhh was decreased (p < 0.0001). In conclusion, the Hh signalling pathway is associated with pejorative pathological parameters in HNPC and is up-regulated in epithelial cells of HTPC and HRPC. Moreover, the lack of Hh molecules in stromal cells seems to be associated with invasive and hormone-refractory behaviours and suggests specific changes in stromal-epithelial crosstalks during PCa progression.