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Th e capability of a tumor to grow and propagate seems to depend on a small sub-population of cells, called cancer stem cells (CSCs) [1]. We isolated and characterized putative bladder CSCs populations from primary Transitional Cell Carcinomas (TCCs). Th ese cells proliferated as urospheres and had abilities for extensive proliferation and self-renewal. Their positivity for stem cell markers and their potential to diff erentiate were assessed. Urospheres gradually showed loss of proliferation, adherence to the substrate, and morphological changes, which might refl ect their progressive loss of self-renewal ability. Conventional cytogenetic analysis was carried out on fresh chromosome spreads immediately aft er the isolation and aft er one week of culture. Th e data indicated important karyotype selection and the loss of complexity present in fresh tumors. Another purpose was to evaluate the performance of a targeted test (UroVysion assay) widely used for the detection of TCCs [2], on Formalin Fixed Paraffi n Embedded (FFPE) tissues and interphasic nuclei of urospheres: comparing tissue and aft er-culture data, we observed a great heterogeneity also among samples with the same histotype. The comparison between array-CGH and UroVysion assay evidenced the same heterogeneity on two different types of material derived from the same tumor: FFPE and Freshly Isolated interphasic Nuclei (FIN). Our results confi rmed the importance of use complementary techniques such array-CGH and FISH, as the former is able to detect alterations at the genome level, but the latter is able to maintain the individual data at the level of single cells, even if it focuses on few genomic regions.

The ERG gene belongs to the ETS family of transcription factors and has been found to be involved in atypical chromosomal rearrangements in several cancers. To gain insight into the oncogenic activity of ERG, we compared the gene expression profile of NIH-3T3 cells stably expressing the coding regions of the three main ERG oncogenic fusions: TMPRSS2/ERG (tERG), EWS/ERG and FUS/ERG. We found that all three ERG fusions significantly up-regulate PIM1 expression in the NIH-3T3 cell line. PIM1 is a serine/threonine kinase frequently over-expressed in cancers of haematological and epithelial origin. We show here that tERG expression induces PIM1 in the non-malignant prostate cell line RWPE-1, strengthening the relation between tERG and PIM1 up-regulation in the initial stages of prostate carcinogenesis. Silencing of tERG reversed PIM1 induction. A significant association between ERG and PIM1 expression in clinical prostate carcinoma specimens was found, suggesting that such a mechanism may be relevant in vivo. Chromatin Immunoprecipitation experiments showed that tERG directly binds to PIM1 promoter in the RWPE-1 prostate cell line, suggesting that tERG could be a direct regulator of PIM1 expression. The up-regulation of PIM1 induced by tERG over-expression significantly modified Cyclin B1 levels and increased the percentage of aneuploid cells in the RWPE-1 cell line after taxane-based treatment. Here we provide the first evidence for an ERG-mediated PIM1 up-regulation in prostate cells in vitro and in vivo, suggesting a direct effect of ERG transcriptional activity in the alteration of genetic stability.

Bladder cancer is the seventh most common cancer worldwide. Urothelial carcinoma (formerly known as transitional cell carcinoma, TCC) comprises the majority of bladder cancers, accounting for more than 90%. In general, TCC is grouped into high- or low-grade (HG- or LG) lesions, based on their genetic profile and clinical behavior. The majority of bladder primary TCCs are LG (grades I and II) noninvasive (NI, pathologic stage pTa) papillary lesions. TCC is associated with a high rate of recurrence (75% after 5 years), and a distinct rate (10%–30%) of progression to HG lesions (grade III), carcinoma in situ, or invasive (IN, pT1). The UroVysion Bladder Cancer Kit (UroVysion Kit) is a US Food and Drug Administration-approved fluorescence in situ hybridization (FISH) probe set for use in the detection of recurrent urothelial carcinoma and in patients with hematuria. It is a multi-target multi-color assay designed to detect aneuploidies for chromosomes 3, 7, 17, and loss of the 9p21 locus in urine specimens. In this study we applied the UroVysion test on freshly isolated interphase nuclei and paraffin-embedded tissue from 22 TCCs (9 HGIN, 3 HGNI, 1 LGIN, 9 LGNI), in order to compare numerical aberrations in the two cell samples from the same tumor. 100 morphologically abnormal nuclei were considered for each sample in both tests, recording hybridization signals corresponding to each probe. The signals were divided according to loss (number of signals/cell < 2), disomy (number of signals/cell = 2), and gain (number of signals/cell > 3). Although the number of samples is low, we observed a general heterogeneity in the copy number changes, also among samples with the same histotype and, surprisingly, within the same sample comparing the two tests carried out. In particular, in the HGIN and LGNI tumors we found differences in copy numbers of chromosomes 3, 7, and 17 (p < 0.01) in 67%–89% of samples; for the 9p21 locus the differences were less significant. Our data provide f