Fluorescence in situ hybridization analysis of CCND3 gene as marker of progression in bladder carcinoma.

The aim of this study was to assess patterns of CCND3 gene amplification in bladder cancer and correlate gene status with recurrence-free and progression-free survival. A sequential cohort series of 102 primary bladder tumor samples in which there was enough tissue material to assess CCND3 gene status by fluorescent in situ hybridization (FISH) was the study group. CCND3 gene FISH amplification present in 31.4 percent of bladder carcinomas, was related to tumor progression (p=0.021) and lower time to progression (mean+-SD; 25.75+-15.25 months) as compared to 33.29+-11.0 months in the CCND3 not amplified group (p=0.05). By immunohistochemistry, Cyclin D3 labeling index was higher in the CCND3 amplified group (mean+-SD, 76.69+-27.51) than in not amplified (mean+-SD, 21.57+-7.02) (p less than 0.0001). The univariate survival analysis showed CCND3 gene amplification to be associated to a shorter progression-free survival (p=0.020) together with WHO histological grade (p=0.001) and pT stage category (p less than 0.0001). Cox’s regression analysis selected CCND3 amplification as an independent predictor of progression-free survival (p= 0.030, RR3.561, 95 percent CI 1.128-11.236) together with pT category (p less than 0.0001, RR5.834, 95 percent CI 2.364-14.395). Our FISH analysis suggests that CCND3 gene amplification is a marker of aggressiveness and might be a predictor of tumor progression in bladder urothelial carcinoma.

WEE1 accumulation and deregulation of S-phase proteins mediate MLN4924 potent inhibitory effect on Ewing sarcoma cells.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of children and young adults in which finding effective new targeted therapies is imperative. Here, we report an in-depth preclinical study of the investigational cullin-RING ubiquitin ligase (CRL) inhibitor MLN4924 in ES, as we have recently demonstrated the implication of a CRL component in the ES pathogenesis. First, our results support a high sensitivity of ES cells to MLN4924 growth inhibition both in vitro (14 ES cell lines tested, median IC50=81 nM) and in tumor xenografts (tumor regression achieved with 60 mg/kg BID, subcutaneously, n=9). Second, we report a dual mechanism of action of MLN4924 in ES cells: while a wide range of MLN4924 concentrations (∼30-300 nM) trigger a G2 arrest that can only be rescued by WEE1 kinase inhibition or depletion, saturating doses of the drug (>300 nM) cause a delay in S-phase progression concomitant with unbalanced CDK2-Cyclin E and CDK2-Cyclin A relative levels (accumulation of the first and depletion of the latter). The aberrant presence of CDC6 in the nucleus at late S-phase cell cycle stage confirmed the loss of CDK2-Cyclin A-specific functions. Remarkably, other mechanisms explored (P27 accumulation and DNA damage signaling pathways) were found unable to explain MLN4924 effects, strengthening the specificity of our findings and suggesting the absence of functionality of some CRL substrates accumulated in response to MLN4924. This study renders a rationale for clinical trials and contributes molecular mechanisms for a better understanding of this promising antitumoral agent.

1q gain and CDT2 overexpression underlie an aggressive and highly proliferative form of Ewing sarcoma.

Despite extensive characterization of the role of the EWS-ETS fusions, little is known about secondary genetic alterations and their clinical contribution to Ewing sarcoma (ES). It has been demonstrated that the molecular structure of EWS-ETS lacks prognostic value. Moreover, CDKN2A deletion and TP53 mutation, despite carrying a poor prognosis, are infrequent. In this scenario identifying secondary genetic alterations with a significant prevalence could contribute to understand the molecular mechanisms underlying the most aggressive forms of ES.We screened a 67 ES tumor set for copy number alterations by array comparative genomic hybridization. 1q gain (1qG), detected in 31% of tumor samples, was found markedly associated with relapse and poor overall and disease-free survival and demonstrated a prognostic value independent of classical clinical parameters. Reanalysis of an expression dataset belonging to an independent tumor set (n=37) not only validated this finding but also led us to identify a transcriptomic profile of severe cell cycle deregulation in 1qG ES tumors. Consistently, a higher proliferation rate was detected in this tumor subset by Ki-67 immunohistochemistry. CDT2, a 1q-located candidate gene encoding a protein involved in ubiquitin ligase activity and significantly overexpressed in 1qG ES tumors, was validated in vitro and in vivo proving its major contribution to this molecular and clinical phenotype. This integrative genomic study of 105 ES tumors in overall renders the potential value of 1qG and CDT2 overexpression as prognostic biomarkers and also affords a rationale for the application of already available new therapeutic compounds selectively targeting the protein-ubiquitin machinery.

Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target.

BACKGROUND: Ewing sarcoma is a paradigm of solid tumour -bearing chromosomal translocations resulting in fusion proteins that act as deregulated transcription factors. Ewing sarcoma translocations fuse the EWS gene with an ETS transcription factor, mainly FLI1. Most of the EWS-FLI1 target genes still remain unknown and many have been identified in heterologous model systems. METHODS: We have developed a stable RNA interference model knocking down EWS-FLI1 in the Ewing sarcoma cell line TC71. Gene expression analyses were performed to study the effect of RNA interference on the genetic signature of EWS-FLI1 and to identify genes that could contribute to tumourigenesis. RESULTS: EWS-FLI1 inhibition induced apoptosis, reduced cell migratory and tumourigenic capacities, and caused reduction in tumour growth. IGF-1 was downregulated and the IGF-1/IGF-1R signalling pathway was impaired. PBK/TOPK (T-LAK cell-originated protein kinase) expression was decreased because of EWS-FLI1 inhibition. We showed that TOPK is a new target gene of EWS-FLI1. TOPK inhibition prompted a decrease in the proliferation rate and a dramatic change in the cell's ability to grow in coalescence. CONCLUSION: This is the first report of TOPK activity in Ewing sarcoma and suggests a significant role of this MAPKK-like protein kinase in the Ewing sarcoma biology.

The need for continuous immunosuppression with cyclosporin A to maintain an experimental model of uveal melanoma.

We investigated the need for continuous immunosuppression to maintain experimental tumours derived from human uveal melanoma cells implanted in the choroid of pigmented rabbits. Two groups of pigmented rabbits immunosuppressed with cyclosporin A (CsA) were implanted with human uveal melanoma cells in the suprachoroidal space. After 5 weeks, CsA was discontinued in group 2. Animals were treated with prophylactic antibiotics and examined weekly for tumour growth, weight and secondary effects; blood urea nitrogen levels were measured every two weeks. Autopsies and histopathological studies were performed after death or euthanasia at the end of week 12. The difference between the groups in the development of ophthalmoscopic tumours was not statistically significant 5 weeks after implantation. Tumours in group 1 grew progressively throughout the experiment, whereas group 2 tumours showed marked regression 3-4 weeks after discontinuing CsA. Tumours in group 1 were significantly larger and had greater mitotic activity and showed more ciliary body, optic nerve and extrascleral invasion than tumours in group 2, which showed massive fibrosis, minimal mitotic activity and marked inflammatory cell infiltration. Continuous immunosuppression with CsA seems to be necessary to maintain tumour growth in this experimental model of uveal melanoma.

The mouse as a model to study adhesion formation following endoscopic surgery: a preliminary report.

Our aim was to investigate the feasibility of a mouse model to study adhesion formation following endoscopic surgery. Following preliminary studies to establish anaesthesia and pneumoperitoneum pressure, a prospective randomized study was carried out to investigate the effect of CO2 pneumoperitoneum on postoperative adhesions. In group I (control group), the duration of pneumoperitoneum was shorter than 5 min. In groups II, III and IV, pneumoperitoneum was maintained for 60 min without flow, with a continuous low flow (1 ml/min) and a continuous high flow (10 ml/min) through the abdominal cavities of the mice using non-humidified CO2, respectively. Adhesions were scored after 7 days by laparotomy. The total adhesion scores were 0.9 +/- 0.8 (n = 15) in control group, 2.4 +/- 0.8 (n = 15) (P < 0.001 versus control group) in group II with no flow, 2.6 +/- 1.3 (n = 15) (P < 0.001 versus control group) in group III with a continuous low flow and 4.3 +/- 0.9 (n = 15) (P < 0.001 versus control group and P < 0.001 versus group II and III) in group IV with a continuous high flow. In conclusion, the mouse can be used as a model to study adhesion formation following endoscopic surgery. Duration of CO2 pneumoperitoneum is a co-factor in adhesion formation.

Fetoscopy in the pregnant rabbit at midgestation.

OBJECTIVE: To develop a small animal model for fetoscopy. METHODS: In 12 time-dated pregnant rabbits at 22 days' gestational age (term 32 days) one amniotic sac in each uterine horn (n = 24) was used for a fetoscopic procedure. After laparotomy, a 2- to 3-mm microsurgical myometrial incision was made to expose the chorionic and amniotic membrane. Under microscopic control, a 2-mm needle was inserted into the amniotic sac. Through this a 1.2-mm endoscope was passed to carry out fetoscopy during maximally 10 min, using 5-10 ml saline amnioinfusion. Mean outcome measurements were ability to visualize the placenta, umbilical cord and the different fetal elements during fetoscopy, as well as fetal survival and weight at second-look operation at 30 days. The untreated amniotic sacs served as negative controls. RESULTS: In all cases, fetoscopy could be carried out successfully, and all fetuses survived till delivery without significant influence on fetal birth weight. CONCLUSION: The midgestational rabbit can be used to perform fetoscopy.

The effect of training and duration of surgery on adhesion formation in the rabbit model.

In order to evaluate the effect of training upon postoperative adhesions, standard bipolar and mechanical, nonopposing injuries were performed in the uterine horns and side walls of 52 mature female rabbits using a conventional three-puncture laparoscopy, by an endoscopic surgeon with limited experience. An additional injury, either bipolar or mechanical or both, was performed in the retro-uterine space. With experience, the duration of surgery decreased progressively from 12 +/- 2 to 8 +/- 1 min in the first and last 10 animals respectively. The amount of perioperative bleeding was not affected by experience. With experience the postoperative adhesions decreased in extent (P = 0.0001), tenacity (P = 0.004), type (P = 0.002) and inflammation (P = 0.003) and for total score (P = 0.0002). These changes were correlated with the briefer duration of surgery but not with the amount of perioperative bleeding. The strong correlations of adhesion scores in the pouch of Douglas, and around both uterine horns confirmed the importance of the inter-animal variability in making adhesions. By logistic regression, the adhesions in the pouch of Douglas were explained simultaneously by the adhesions on the uterine horns (P = 0.0004, thus correcting for inter-animal variability) by the amount of bleeding (P = 0.01) and the duration of surgery (P = 0.05). No major differences were found in adhesions following a mechanical or a bipolar injury or following such a lesion in the pouch of Douglas or at the uterine horns. In conclusion, experience, expressed by the duration of surgery and to a lesser extent perioperative bleeding, is a major co-factor in postoperative adhesions, suggesting that duration of surgery should be strictly standardized in endoscopic adhesion studies. The important inter-animal variability can be circumvented by using a standard control lesion, making each animal its own control.