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Relationship between GPR126 mutation status and standard clinical pathological and biological parameters in bladder cancer

Survival curves of two patients groups according to GPR126 overexpression status in the series of 59 MIBC. x-axes represents the time (months). y-axes represents A : Recurrence-free-survival ; B: Overall survival. p=value is the result of log-rank test.

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

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

Supplementary Figure 5 from Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy

Supplementary Figure 3 from Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy

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

Supplementary Figure Legends 1-5 from Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy

Supplementary Figure 2 from Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy

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.

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. Patient summary: Oncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.

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.

The microenvironment of retinoblastoma, the solid malignancy of the developing retina, is immunosuppressive. To study the interactions between tumor-associated microglia/macrophages (TAMs) and tumor cells in retinoblastomas, we analyzed immunohistochemistry markers in 23 patient samples and characterized 105 secreted cytokines of 11 retinoblastoma cell models in culture. We detected profuse infiltration of CD163(+) protumoral M2-like polarized TAMs in eyes enucleated due to cancer progression. Previous treatment of patients increased the number of TAMs but did not affect M2-like polarization. M2-like microglia/macrophages were almost absent in five eyes obtained from children enucleated due to nontumoral causes. CD8(+) tumor-infiltrating lymphocytes (TILs) were moderately abundant in tumor eyes and very scarce in nontumoral ones. The expression of the immune checkpoint molecule PD-L1 was absent in 95% of the tumor samples, which is concordant with the finding of FOXP3(+) Tregs infiltrating tumors. We confirmed the pathology results using single-cell transcriptome analysis of one tumor. We identified the cytokines extracellular matrix metalloproteinase inducer (EMMPRIN) and macrophage migration inhibitory factor (MIF), both with reported immunosuppressive activity, secreted at high levels in retinoblastoma primary cell cultures. Gene expression analysis of a large retinoblastoma cohort and single-cell transcriptome analysis confirmed that MIF and EMMPRIN were significantly upregulated in retinoblastomas, which led us to quantify both proteins by immunoassays in liquid biopsies (aqueous humor obtained from more than 20 retinoblastoma patients). We found a significant increase in the concentration of MIF and EMMPRIN in cancer patients, compared to 12 noncancer ones. Finally, we showed that macrophages derived from peripheral blood mononuclear cells increased the expression of markers of M2-like polarization upon exposure to retinoblastoma-conditioned medium or recombinant MIF. Overall, our findings suggest that retinoblastoma cell secretions induce the protumoral phenotype of this tumor. Our results might have clinical impact in the fields of biomarkers and treatment. © 2022 The Pathological Society of Great Britain and Ireland.

Retinoblastoma is the most frequent intraocular malignancy in children, originating from a maturing cone precursor in the developing retina. Little is known on the molecular basis underlying the biological and clinical behavior of this cancer. Here, using multi-omics data, we demonstrate the existence of two retinoblastoma subtypes. Subtype 1, of earlier onset, includes most of the heritable forms. It harbors few genetic alterations other than the initiating RB1 inactivation and corresponds to differentiated tumors expressing mature cone markers. By contrast, subtype 2 tumors harbor frequent recurrent genetic alterations including MYCN-amplification. They express markers of less differentiated cone together with neuronal/ganglion cell markers with marked inter- and intra-tumor heterogeneity. The cone dedifferentiation in subtype 2 is associated with stemness features including low immune and interferon response, E2F and MYC/MYCN activation and a higher propensity for metastasis. The recognition of these two subtypes, one maintaining a cone-differentiated state, and the other, more aggressive, associated with cone dedifferentiation and expression of neuronal markers, opens up important biological and clinical perspectives for retinoblastomas., Retinoblastoma is the most frequent intraocular paediatric malignancy whose molecular basis remains poorly understood. Here, the authors perform multi-omic analysis and identify two subtypes; one in a cone differentiated state and one more aggressive showing cone dedifferentiation and expressing neuronal markers.

In our study the Bladder Cancer Molecular Taxonomy Group collaborated to extend a first consensus report, addressing the need for a consensus molecular classification for muscle-invasive bladder cancer (MIBC) that would support basic research and clinical trials. We provide such a consensus classification and offer a single-sample classifier (http://cit.ligue-cancer.net:3838/apps/consensusMIBC_web).

Numerous pangenomic studies identified protein-coding genes and signaling pathways involved in bladder carcinogenesis. However, noncoding somatic alterations remain unexplored. A recent study revealed a mutational hotspot in intron 6 of GPR126 gene in 2.7% of a large breast cancer series. As GPR126 is highly expressed in bladder tissues, we investigated here the prevalence and the prognostic significance of these mutations in bladder cancer. We analyzed a cohort of 103 bladder cancers including 44 nonmuscle-invasive bladder cancers (NMIBC) and 59 muscle-invasive bladder cancers (MIBC). GPR126 mutations were analyzed by high-resolution melting and Sanger sequencing, and GPR126 expression levels were assessed using real-time quantitative RT-PCR. In NMIBC, somatic GPR126 noncoding mutations occurred in 47.7% of samples and were negatively associated with GPR126 mRNA levels. GPR126 mutations had higher frequencies in nonsmoker patients and were associated with a prior history of NMIBC. GPR126 overexpression was detected in 70.5% of samples. GPR126 mutation and overexpression status were not associated with outcome. In MIBC, somatic GPR126 mutations occurred in 44.1% of samples. Mutations were more frequent in females. GPR126 overexpression was detected in 27.1% of the sample. A trend toward significance was observed between GPR126 overexpression and better outcome. We identified the second most frequent mutational hotspot after TERT promoter (∼70%) in bladder cancer, with a mutation rate of approximately 50%. Implications: The GPR126 intronic mutational hotspot could be a promising clinical biomarker candidate to monitor tumor burden using circulating tumor DNA in bladder cancer.

Background: Muscle-invasive bladder cancer (MIBC) is a molecularly diverse disease with heterogeneous clinical outcomes. Several molecular classifications have been proposed, but the diversity of their subtype sets impedes their clinical application. Objective: To achieve an international consensus on MIBC molecular subtypes that reconciles the published classification schemes. Design, setting, and participants: We used 1750 MIBC transcriptomic profiles from 16 published datasets and two additional cohorts. Outcome measurements and statistical analysis: We performed a network-based analysis of six independent MIBC classification systems to identify a consensus set of molecular classes. Association with survival was assessed using multivariable Cox models. Results and limitations: We report the results of an international effort to reach a consensus on MIBC molecular subtypes. We identified a consensus set of six molecular classes: luminal papillary (24%), luminal nonspecified (8%), luminal unstable (15%), stroma-rich (15%), basal/squamous (35%), and neuroendocrine-like (3%). These consensus classes differ regarding underlying oncogenic mechanisms, infiltration by immune and stromal cells, and histological and clinical characteristics, including outcomes. We provide a single-sample classifier that assigns a consensus class label to a tumor sample's transcriptome. Limitations of the work are retrospective clinical data collection and a lack of complete information regarding patient treatment. Conclusions: This consensus system offers a robust framework that will enable testing and validation of predictive biomarkers in future prospective clinical trials. Patient summary: Bladder cancers are heterogeneous at the molecular level, and scientists have proposed several classifications into sets of molecular classes. While these classifications may be useful to stratify patients for prognosis or response to treatment, a consensus classification would facilitate the clinical use of molecular classes. Conducted by multidisciplinary expert teams in the field, this study proposes such a consensus and provides a tool for applying the consensus classification in the clinical setting. An international consortium of bladder cancer expert teams establishes a consensus reconciling the diverse molecular classifications of muscle-invasive bladder cancer. This work offers a robust framework that will enable testing and validating predictive biomarkers in future prospective clinical trials.

Numerous pangenomic studies identified protein-coding genes and signaling pathways involved in bladder carcinogenesis. However, noncoding somatic alterations remain unexplored. A recent study revealed a mutational hotspot in intron 6 of

High plasma exposure to estrogens is often associated with prostate cancer. Reducing this phenomenon may present therapeutic benefits. The involvement of estrone sulphate (E1S), the most abundant circulating estrogen in men, has been partially studied in this age-related pathology. To investigate the consequences of plasma E1S overload on blood and prostate sex steroid levels and inflammatory tissue responses, young and middle-aged male rats were treated with E1S with or without steroid sulfatase (STS) inhibitor STX64 for 21 consecutive days. A plasma and prostate tissue steroid profile was determined. STS activity, mRNA expression of E1S organic anion transporting polypeptides (slco1a2, slco2b1, slco4a1) and pro-inflammatory cytokines (Il1-beta, Il6, TNF-alpha) were evaluated in prostate tissue according to age and treatment group. A significant correlation between plasma and prostate steroid levels related to hormone treatment was observed in all rat age groups. However, while the E1S level in prostate tissue increased in middle-aged treated rats (p

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

Prostate cancer (PCa) is androgen sensitive in its development and progression to metastatic disease. Hedgehog (Hh) pathway activation is important in the initiation and growth of various carcinomas including PCa. We and others have observed aberrations of Hh pathway during the progression of PCa to the castration-resistant state. The involvement of androgen signalling in Hh pathway activation, however, remains largely elusive. Here we investigate the direct role of androgen signalling on Hh pathway. We examined the effect of Dihydrosterone (DHT), antiandrogen, bicalutamide, and Hh pathway inhibitor, KAAD-cyclopamine in four human prostate cell lines (two cancerous: LNCaP, VCaP, and two normal: PNT2 and PNT2-ARm which harbours a mutant version of androgen receptor (AR) that is commonly found in LNCaP). Cell proliferation as well as Hh pathway members (SHH, IHH, DHH, GLI, PTCH) mRNA expression levels were assessed. We showed that KAAD-cyclopamine decreased cell proliferation of DHT-stimulated LNCaP, VCaP and PNT2-ARm cells. SHH expression was found to be downregulated by DHT in all AR posititve cells. The negative effect of DHT on SHH expression was counteracted when cells were treated by bicalutamide. Importantly, KAAD-cyclopamine treatment seemed to inhibit AR activity. Moreover, bicalutamide as well as KAAD-cyclopamine treatments induced GLI and PTCH expression in VCaP and PNT2-ARm. Our results suggest that Hh pathway activity can be regulated by androgen signalling. Specifically, we show that the DHT-induced inhibition of Hh pathway is AR dependent. The mutual interaction between these two pathways might be important in the regulation of cell proliferation in PCa.

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.

Background: Microenvironmental conditions in normal or tumour tissues and cell lines may interfere on further biological analysis. To evaluate transcript variations carefully, it is common to use stable housekeeping genes (HKG) to normalise quantitative microarrays or real-time polymerase chain reaction results. However, recent studies argue that HKG fluctuate according to tissues and treatments. So, as an example of HKG variation under an array of conditions that are common in the cancer field, we evaluate whether hypoxia could have an impact on HKG expression. Methods: Expression of 10 commonly used HKG was measured on four cell lines treated with four oxygen concentrations (from 1 to 20%). Results: Large variations of HKG transcripts were observed in hypoxic conditions and differ along with the cell line and the oxygen concentration. To elect the most stable HKG, we compared the three statistical means based either on PCR cycle threshold coefficient of variation calculation or two specifically dedicated software. Nevertheless, the best HKG dramatically differs according to the statistical method used. Moreover, using, as a reference, absolute quantification of a target gene (here the proteinase activating receptor gene 1 (PAR1) gene), we show that the conclusions raised about PAR1 variation in hypoxia can totally diverge according to the selected HKG used for normalisation. Conclusion: The choice of a valid HKG will determine the relevance of the results that will be further interpreted, and so it should be seriously considered. The results of our study confirm unambiguously that HKG variations must be precisely and systematically determined before any experiment for each situation, to obtain reliable normalised results in the experimental setting that has been designed. Indeed, such assay design, functional for all in vitro systems, should be carefully evaluated before any extension to other experimental models including in vivo ones.

International audience; Despite the high prevalence of histological Benign Prostatic Hypeplasia (BPH) in elderly men, little is known regarding the molecular mechanisms and networks underlying the development and progression of the disease. Here, we explored the effects of a phytotherapeutic agent, Lipidosterolic extract of the dwarf palm plant Serenoa repens (LSESr), on the mRNA gene expression profiles of two representative models of BPH, BPH1 cell line and primary stromal cells derived from BPH. Treatment of these cells with LSESr significantly altered gene expression patterns as assessed by comparative gene expression profiling on gene chip arrays. The expression changes were manifested three hours following in vitro administration of LSESr, suggesting a rapid action for this compound. Among the genes most consistently affected by LSESr treatment, we found numerous genes that were categorized as part of proliferative, apoptotic, and inflammatory pathways. Validation studies using quantitative real-time PCR confirmed the deregulation of genes known to exhibit key roles in these biological processes including IL1B, IL1A, CXCL6, IL1R1, PTGS2, ALOX5, GAS1, PHLDA1, IL6, IL8, NFkBIZ, NFKB1, TFRC, JUN, CDKN1B, and ERBB3. Subsequent analyses also indicated that LSESr treatment can impede the stimulatory effects of certain proinflammatory cytokines such as IL6, IL17, and IL15 in these cells. These results suggest that LSESr may be useful to treat BPH that manifest inflammation characteristics. This also supports a role for inflammation in BPH presumably by mediating the balance between apoptosis and proliferation.

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.

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.

Sir, We have read with great interest the study by Kontos et al (2010) in British Journal of Cancer about the use of L-DOPA decarboxylase as a prognostic marker for colorectal adenocarcinoma. To assess the expression level of L-DOPA decarboxylase transcripts, the authors have performed qRT-PCR using GAPDH as the dedicated housekeeping gene (HKG) to normalise their results. However, no results concerning the stability of GAPDH that has lead to its use as the best housekeeper in their model system were available. We have recently shown on human cell lines that choosing an unstable internal control gene could generate dramatic misinterpretations (Caradec et al, 2010a). In our study, and in our conditions, GAPDH was one of the most variable HKG so far impairing its use as a relevant normaliser. As this gene is likely to be emblematical to normalise gene expression results, we have developed a specific qRT-PCR with calibration curve to specifically study GAPDH expression in different cell lines grown with various hypoxic conditions. Our results unambiguously showed that GAPDH expression varies according to oxygen tension. We have also analysed GAPDH variability comparing meta-analysis data from microarray experiments on human samples available online (https://www.oncomine.com/resource/login.html). Using Oncomine 4.3, a powerful tool allowing rapid gene expression comparison between healthy and/or tumour human samples, we report here that GAPDH variability differs largely from one study to another and more importantly may largely vary between patients in a given study (Figure 1). As, aside still unidentified factors, hypoxia can be considered as a major one to have a critical role in cancer development, especially in colorectal cancer (Baba et al, 2010), we would be interested in learning how Kontos et al have tested HKGs variability in their system and found GAPDH to be the most relevant. Figure 1 GAPDH fold change in different colon adenocarcinoma studies (Graudens et al, 2006; Clarke et al, 2003; Notterman et al, 2001 and Bittner, not published (International Genomics Consortium, Expression Project for Oncology - Colon Samples, http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= ... Another unclear point concerns qRT-PCR Ct intervals between GAPDH and L-DOPA target gene amplification. Indeed, the study results of Kontos et al. showed a ΔCt equal to 13 (Ct (GAPDH) 15, Ct (L-DOPA) 28), signifying that GAPDH transcripts are likely to be expressed 213 (8200) times higher than those encoding L-DOPA. As we stressed this particular point very recently, discussing about the use of r18S as a normaliser (Caradec et al, 2010b), we would be very interested to know Kontos et al. opinion about the accuracy of such disproportion. Definitely, the choice of a valid HKG set will determine the relevance of the results that will be further interpreted, and so it should be seriously considered.

Reply: Choosing a stable housekeeping gene and protein is essential in generating valid gene and protein expression results