Delphine Amsellem-Ouazana

microRNA expression profile in a large series of bladder tumors: Identification of a 3-miRNA signature associated with aggressiveness of muscle-invasive bladder cancer†

The aim of this study was to evaluate the expression levels of microRNAs (miRNAs) in bladder tumors in order to identify miRNAs involved in bladder carcinogenesis with potential prognostic implications. Expression levels of miRNAs were assessed by quantitative real‐time RT‐PCR in 11 human normal bladder and 166 bladder tumor samples (86 non‐muscle‐invasive bladder cancer (NMIBC) and 80 muscle‐invasive bladder cancer (MIBC)). The expression level of 804 miRNAs was initially measured in a well‐defined series of seven NMIBC, MIBC and normal bladder samples (screening set). The most strongly deregulated miRNAs in tumor samples compared to normal bladder tissue were then selected for RT‐PCR validation in a well‐characterized independent series of 152 bladder tumors (validation set), and in six bladder cancer cell lines. Expression levels of these miRNAs were tested for their association with clinical outcome. A robust group of 15 miRNAs was found to be significantly deregulated in bladder cancer. Except for two miRNAs, miR‐146b and miR‐9, which were specifically upregulated in MIBC, the majority of miRNAs (n = 13) were deregulated in the same way in the two types of bladder tumors, irrespective of pathological stage : three miRNAs were upregulated (miR‐200b, miR‐182 and miR‐138) and the other 10 miRNAs were downregulated (miR‐1, miR‐133a, miR‐133b, miR‐145, miR‐143, miR‐204, miR‐921, miR‐1281, miR‐199a and miR‐199b). A 3‐miRNA signature (miR‐9, miR‐182 and miR‐200b) was found to be related to MIBC tumor aggressiveness and was associated with both recurrence‐free and overall survival in univariate analysis with a trend to significance in the multivariate analysis (p = 0.05). Our results suggested a promising individual prognostic value of these new markers.

Surgery for bladder endometriosis: long-term results and concomitant management of associated posterior deep lesions

BACKGROUND Deep infiltrating endometriosis (DIE) is presented as a disease with high recurrence risk. Bladder DIE is the most frequent location in cases of urinary endometriosis. Surgical removal has been recommended for bladder DIE but long-term outcomes remains unevaluated. The objectives of this study are to evaluate the rate of recurrence after partial cystectomy for patients presenting with bladder DIE and to outline the surgical modalities for handling associated posterior DIE nodules. METHODS Seventy-five consecutive patients with histologically proved bladder DIE were enrolled at a single tertiary academic center between June 1992 and December 2007. A partial cystectomy was performed for each patient. Complete surgical exeresis of all associated symptomatic DIE lesions was carried out during the same surgical procedure. Bladder DIE patients were classified into three groups: patients with isolated bladder DIE (Group A); patients with associated symptomatic posterior DIE (Group B); patients with associated asymptomatic posterior DIE (Group C). Bladder DIE recurrence was defined as a clinical reappearance of the disease or radiological evidence that mandated a new surgical procedure. We assessed pelvic pain symptoms pre- and post-operatively using a 10-cm visual analogue scale. RESULTS In a series of 627 patients with DIE, we observed 75 patients (12%) with bladder DIE. With a 50.9 +/- 44.6 months mean follow-up after partial cystectomy no patient presented evidence of bladder DIE recurrence. Post-operatively, we observed a significant improvement with respect to pain symptoms, with only two patients (2.7%) developing major complications during follow-up. Among patients with non-operated associated asymptomatic posterior DIE lesions (n = 15), a second surgical procedure indicated for pain symptoms was necessary in only one patient (6.7%). CONCLUSIONS For patients presenting with bladder DIE, no patients required further surgery for bladder recurrence after radical surgery consisting in partial cystectomy. Exeresis of associated posterior DIE nodules is indicated only when they are symptomatic.

Intermittent androgen suppression in patients with prostate cancer

To evaluate intermittent androgen suppression (IAS) in patients with prostate cancer and to try to define predictive factors for biochemical progression.

Severe ureteral endometriosis: the intrinsic type is not so rare after complete surgical exeresis of deep endometriotic lesions

OBJECTIVE To evaluate the rate of intrinsic ureteral endometriosis in patients presenting with severe ureteral endometriosis. DESIGN Observational study between June 1992 and December 2007. SETTING University tertiary referral center. PATIENT(S) Twenty-nine patients presenting deeply infiltrating endometriosis (DIE) with severe ureteral endometriosis. Severe ureteral endometriosis was defined as DIE lesions causing significant obstruction to the urinary flow with ureteral stenosis. INTERVENTION(S) Complete surgical exeresis of DIE lesions. MAIN OUTCOME MEASURE(S) Pre- and peroperative evaluation associated with histologic analysis. Intrinsic ureteral endometriosis was defined as presence of DIE lesions infiltrating the ureteral muscularis. RESULT(S) In a series of 627 patients with histologic proved DIE, we observed 29 (4.6%) patients with severe ureteral endometriosis. Ureteral lesions (n = 34) were right sided in 7 (24.1%) patients, left sided in 17 (58.6%) patients, and bilateral in 5 (17.3%) patients. Eleven (37.9%) patients presented intrinsic lesions. Out of the 34 ureteral lesions 13 (38.2%) were intrinsic. In cases of radical ureteral surgery (n = 21 patients; n = 24 ureteral lesions) intrinsic ureteral DIE was observed in 52.4% (11 cases) of the patients and in 54.2% (13 cases) of the ureteral lesions. CONCLUSION(S) The prevalence of intrinsic ureteral endometriosis is underestimated. This result must be taken into account when specifying the surgical modalities for patients presenting with severe ureteral endometriosis.

Genomic expression and single-nucleotide polymorphism profiling discriminates chromophobe renal cell carcinoma and oncocytoma

BackgroundChromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC.MethodsGene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors.ResultsA novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities.ConclusionsGene expression profiles, high-throughput SNP genotyping, and pathway analysis effectively distinguish chRCC from oncocytoma. We have generated a novel transcript predictor that is able to discriminate between the two entities accurately, and which has been validated both in an internal and an independent data-set, implying generalizability. A cytogenetic alteration, loss of chromosome 1p, common to renal oncocytoma and chRCC has been identified, providing the opportunities for identifying novel tumor suppressor genes and we have identified a series of immunohistochemical markers that are clinically useful in discriminating chRCC and oncocytoma.

Large-scale Real-time Reverse Transcription-PCR Approach of Angiogenic Pathways in Human Transitional Cell Carcinoma of the Bladder: Identification of VEGFA as a Major Independent Prognostic Marker

BACKGROUND Actors of the angiogenesis pathways are targets for the new promising targeted therapies already used in several malignancies. In bladder cancer, antiangiogenic molecules could also add to already existing treatment options. OBJECTIVE To evaluate the involvement of angiogenesis pathways in bladder carcinogenesis and identify new molecular markers having a clinical implication. DESIGN, SETTING, AND PARTICIPANTS Expression levels of 40 genes involved in angiogenesis were assessed by quantitative real time RT-PCR in 157 urothelial tumour bladder samples obtained from patients who underwent transurethral bladder resection or radical cystectomy between 2001 and 2005. Pathologic tumour staging showed: 73 non-muscle-invasive bladder tumours (30 low-grade pTa, 14 high-grade pTa, and 29 high-grade pT1), and 84 muscle-invasive tumours (> or = pT2), all of high grade. RT-PCR results were associated with a survival analysis. RESULTS AND LIMITATIONS VEGFA, MET, CXCR4, and IL8 were significantly overexpressed in tumour samples as compared to normal bladder tissue. VEGFA overexpressions were found in 89% of non-muscle-invasive and 66% of muscle-invasive tumour samples. In univariate analysis, for invasive tumours, VEGFA overexpression was associated with a poorer outcome in both overall and disease-free survival (p=0.011 and 0.026 respectively) at a 13-mo median follow-up. Multivariate analysis retained T stage, N status, and VEGFA overexpression as independent prognostic factors in both overall and disease-free survival (p=0.02 and p=0.04, respectively, for VEGFA). CONCLUSIONS This study shows that, in bladder cancer, VEGFA status could be used as a prognostic factor at the individual level. VEGFA overexpression could guide a rationalized use of the costly antiangiogenic therapies which could therefore become part of the treatment options in bladder cancer.

Gene expression profiling of ERBB receptors and ligands in human transitional cell carcinoma of the bladder.

PURPOSE The ErbB driven growth pathway has been implicated in most human epithelial malignancies. Therefore, its blockade is a promising therapeutic strategy and several candidate drugs are currently undergoing clinical trials. Paradoxically little is known of the expression pattern or clinical significance of the 4 ErbB receptors and their 11 ligands in TCC of the bladder. MATERIALS AND METHODS To obtain further insight into the molecular pathogenesis of TCC we used quantitative real-time reverse transcriptase-polymerase chain reaction assay to quantify mRNA expression of the 4 ERBB and their 11 known ligand genes, including recently described EPGN/epigen, in 73 tumor samples. RESULTS The level of mRNA of 4 ligand genes (EGF, NRG1, NRG2 and NRG3) was extremely low, that is detectable but not quantifiable. Six genes were over expressed (ERBB2, TGFA, HB-EGF, AREG, EREG and EPGN), 3 were under expressed (ERBB1, ERBB4 and NRG4) and 2 were over or under expressed (ERBB3 and BTC). ERBB2 and AREG expression differed between early stage tumors (pTa grade 1) and normal samples. The most marked differences in expression were ERBB3, EREG and NRG4 between superficial and muscle invasive tumors (p = 0.0069, 0.00007 and 0.0000001, respectively), and TGFA and NRG4 between low and high grade superficial tumors, and between pT1 or greater and pTa tumors. CONCLUSIONS This study shows the involvement of the ERBB family and ligand genes in TCC. Most receptor and ligand genes are deregulated at different stages of carcinogenesis, implying that they should be studied simultaneously. Quantitative real-time reverse transcriptase-polymerase chain reaction could be used to determine ErbB signaling pathway status in individuals with a view to tailored therapy.

Confrontation of immunohistochemistry and fluorescent in situ hybridization for the assessment of HER-2/neu (c-erbb-2) status in urothelial carcinoma

Specific treatments targeted toward oncogenes expressed in cancer cells are currently under development. Patients with urothelial carcinomas showing HER-2/neu (human epidermal growth factor receptor 2) overexpression are candidates for such a specific treatment (trastuzumab). However, to be effective, this therapeutic approach requires an extremely reliable evaluation of HER-2/neu status in tumors. In order to assess the status of expression of this gene and to optimize its assessment, we analyzed a series of 64 primary urothelial carcinomas using immunohistochemistry (IHC) with the CB11 monoclonal antibody coupled with fluorescent in situ hybridization (FISH) in 21 cases. Strong HER-2/neu overexpression was detected using IHC in 15 of the 64 (23%) cases analyzed, and this rate rose to 33% for patients with metastases. HER-2/neu overexpression, as revealed using IHC, is strongly associated (95%) with gene amplification assessed using FISH. Patients with urothelial carcinomas overexpressing HER-2/neu using IHC are potential candidates for targeted chemotherapy.

Loss of chromosomes 9 and 11 may be recurrent chromosome imbalances in juxtaglomerular cell tumors

Juxtaglomerular cell tumor (JGCT), first described in 1967, is a rare tumor of the kidney that derived from specialized smooth muscle cells of the wall of the glomerular afferent arteriole. Less than 100 cases have been published, mainly as isolated case reports or small series. JGCTs are considered benign, but the clinical follow-up is short in most reported cases. Only 1 metastatic case has been reported to date, raising the question of tumors of uncertain malignant potential rather than clearly benign neoplasms. Genomic features have been studied in only 2 cases that showed gain of chromosome 10 as well as loss of chromosomes 9, 11q, and X. The present work studied the genomic characteristics of 2 additional cases of JGCT by comparative genomic hybridization. Similarly to the 2 previously reported cases, these 2 tumors showed loss of chromosomes 9 and 11, suggesting recurrent chromosomal imbalances. In addition, 1 case showed gain and loss of entire chromosomes, similar to a previous case studied by karyotyping. Such an aneuploid karyotype may reflect a potential for malignancy as previously reported. Thus, JGCT might be better considered as a tumor of uncertain malignant potential and then necessitates a prolonged follow-up. Future clinicopathologic and genomic studies of large retrospective and prospective series may help to better understand the biology of this fascinating entity.

Use of portfolios as a learning and assessment tool in a surgical practical session of urology during undergraduate medical training

We chose to introduce a portfolio as a learning and assessment tool in a practical training session of urological surgery for undergraduate medical students. Our primary objectives were to develop the students’ self reflexive ability in front of complex medical cases and to teach them how to identify their learning needs in a short period of time, on a specific topic. Students completed, during their training session, a portfolio on a urological topic under the constant supervision of a tutor. The students were evaluated on their portfolios presentation with a 20-point grade grid known in advance. Even in a surgical training session, a portfolio can be a useful learning and assessment tool. It clearly encourages self-reflection and pre-professional practice.