Cristina Gazquez

Protamine 2 precursors (Pre-P2), protamine 1 to protamine 2 ratio (P1/P2), and assisted reproduction outcome

OBJECTIVE To determine whether the presence of protamine 2 precursors (pre-P2/P2 ratio) and the protamine 1 to protamine 2 ratio (P1/P2) are related to the assisted reproduction outcome. DESIGN Prospective study. SETTING Assisted Reproduction Unit and University laboratory. PATIENT(S) One hundred two infertile patients undergoing treatment at the Assisted Reproduction Unit of the Hospital Clinic of Barcelona. INTERVENTION(S) Intracytoplasmic sperm injection (ICSI) and/or IVF treatment of the infertile patients, sperm protamine analysis through electrophoresis and densitometry, and pre-P2 analysis through Western blot. MAIN OUTCOME MEASURE(S) The presence of protamine 2 precursors (pre-P2/P2 ratio), sperm P1/P2 ratio, fertilization rates by IVF and/or ICSI, and pregnancy outcome. RESULT(S) Pre-P2/P2 and P1/P2 ratios are positively associated with the pregnancy rate. In addition, the P1/P2 ratio is positively associated with the proportion of embryos obtained by IVF, but not by ICSI. The pre-P2/P2 ratio was not related to the fertilization rate. CONCLUSION(S) Decreased pre-P2/P2 and P1/P2 ratios are related to a poor pregnancy outcome, but not with the proportion of embryos obtained after ISCI.

A common protamine 1 promoter polymorphism (-190 C->A) correlates with abnormal sperm morphology and increased protamine P1/P2 ratio in infertile patients.

It is known that targeting the protamine 1 gene in mice leads to infertility, abnormal chromatin packaging, and abnormal sperm morphology. Because many infertile patients also have an abnormal sperm morphology and chromatin packaging, the human protamine 1 gene (PRM1) is an important candidate to screen for potential mutations. In this work, we have screened the PRM1 gene in search of potential mutations and determined the sperm morphology and the ratio between protamine 1 and protamine 2 (P1/P2 ratio). Direct sequencing of the PRM1 promoter led to the identification of a common single-nucleotide polymorphism (SNP; -190 C-->A). The -190 AA genotype was detected at a higher frequency (13.8%) in patients with markedly altered sperm morphology (<or=9% normal forms) compared with other patients (4.5%; P < .05) or compared with controls (2.97%; P < .005). The allelic frequency of the PRM1 -190 C-->A change was also consistently higher (.331) in infertile patients with a markedly altered morphology compared with population controls (.178; P < .01). Additionally, we have determined that the P1/P2 ratio is significantly increased in patients with the PRM1 -190 AA genotype compared with patients with the CA or CC genotypes (P = .006, Mann-Whitney). These findings indicate that the common PRM1 -190 C-->A polymorphism identified is associated with abnormal sperm head morphology and abnormal P1/P2 ratio in infertile patients.

Molecular characterization of upper urinary tract tumours.

To assess gene‐expression patterns of BIRC5, FGFR3, IGF2, KRT20, UPK2, EBF1, CDH1, FXYD3, HTERT, TP53, AGR2, HER2 and VEGF, widely known markers of bladder urothelial carcinoma (UC) in upper tract UC, and to determine their value as prognostic factors of tumour progression and cancer‐specific survival.

Utility of Urothelial mRNA Markers in Blood for Staging and Monitoring Bladder Cancer

OBJECTIVE To test the efficiency of 6 mRNA bladder markers in staging urothelial cell carcinoma (UCC) and monitoring UCC dissemination from blood samples. METHODS From 2002 to 2009, 347 blood samples were collected from 150 patients with UCC and 29 healthy controls. Sequential blood sampling was performed in patients undergoing cystectomy at surgery and 6, 12, 18, and 24 months postoperatively. The median follow-up was 33 months. The presence of KRT20, FXYD3, C10orf116, UPK2, AGR2, and KRT19 markers in blood was evaluated in all patients and controls by measuring the gene expression using preamplified cDNA and reverse transcriptase quantitative polymerase chain reaction. Gene expression data were correlated with the tumor risk, follow-up, and outcomes data. RESULTS Expression of C10orf116 and KRT19 genes differed between patients and controls (P<.001). KRT20, C10orf116, and AGR2 differentiated between low- and high-risk nonmuscle-invasive bladder cancer (P=.001, P=.011, and P=.001, respectively). FXYD3 differentiated between patients with high-risk nonmuscle-invasive bladder cancer and those with muscle-invasive bladder cancer (P=.009). In contrast, the 6 markers showed no differences in gene expression between metastatic and patients without metastases who had not undergone cystectomy (P=NS). None of the markers were significantly increased in the metastatic patients at 6, 12, 18, or 24 months after surgery. CONCLUSION The gene expression of bladder-specific mRNA markers in blood was different among the various tumor risk groups of patients with UCC. However, this gene expression analysis is not suitable for predicting metastases or monitoring UCC hematogenous dissemination in patients who have undergone cystectomy.

Biomarkers vs conventional histological analysis to detect lymph node micrometastases in bladder cancer: a real improvement?

Study Type – Therapy (case series)

866 DIFFERENTIAL MIRNA EXPRESSION PROFILES IN URINES FROM BLADDER CANCER PATIENTS

duced with a luciferase gene (MBT-2 luc), was instilled in the bladder of 6 week old, female C3H/He mice (3x10 cells/50ul). Bioluminescent imaging was performed to determine tumor burden on day 5 and mice were randomized to treatment groups. Intravesical treatments (10 C.cresc. in 50ul of H2O or H2O as control) were performed on days 6, 8, 11 and 15 and tumor growth was examined by bioluminescence after i.p. injection of luciferin. Mice were euthanized on day 20 and mouse bladders processed for histology and immuno-histochemistry. RESULTS: In vivo, intravesical treatment of MBT-2 luc tumor bearing C3H/He mice showed a significant inhibition of tumor growth compared to the control group, while no side-effects due to the intravesical C.cresc. application were observed. Additionally performed in vitro experiments revealed that incubation of MBT-2 cells with C.cresc. resulted in a concentration dependent growth inhibition. CONCLUSIONS: These preliminary results support the safety and efficacy of Caulobacter crescentus as a novel intravesical treatment for bladder cancer. Further investigation is needed to reveal the exact mechanism of action and to distinguish, if a direct anti-tumor effect of C.cresc. or the host immune-response is the driving component of the anti-tumor effect.

358 PREDICTING THE RESPONSE TO NEOADJUVANT CHEMOTHERAPY IN PATIENTS WITH UROTHELIAL CELL CARCINOMA BY MIRNA EXPRESSION PROFILE

INTRODUCTION AND OBJECTIVES: To analyze the miRNA expression profile of bladder cancer patients according to the response to neoadjuvant chemotherapy (NA CT), in order to select the differentially expressed miRNA between both groups and to identify a predictive miRNA panel of response to chemotherapy. METHODS: A total of 81 patients with urothelial cell carcinoma of the bladder (UCC) whom received NA CT followed by radical surgery between 1991 and 2008 at the Hospital Clinic of Barcelona were identified. Of these patients, 10 responders and 10 nonresponders to NA CT were analyzed in this analysis. RNA was extracted from the FFPE tissue blocks obtained in the TUR prior to the cystectomy by a commercial kit (RecoverAll Total Nucleic Acid Isolation, Ambion). Real Time Quantitative PCR (qRT-PCR) was performed using TaqMan Arrays containing 738 miRNAs. RESULTS: Sixty three miRNAs were found differentially expressed between responders and non-responders to NA CT (p 0.05). A miRNA panel containing 9 miRNAs (hsa-miR-17 ; hsa-miR-193b; hsa-miR-200c ; hsa-miR-489; hsa-miR-548b-5p; hsa-miR-548c-5p; hsa-miR-622; hsa-miR-744 ; hsa-miR-886-5p) allows differentiating responders from non-responders to NA CT with sensibility and specificity of 100%. Furthermore, the study of the more differentially expressed miRNAs shows that some of them are related to other cancers. CONCLUSIONS: In this initial study we show that miRNA expression profile is able to differentiate between responders and non-responders to NA CT. With that knowledge, we built a miRNA set panel capable to identify those patients that will not respond to the treatment. However, a validation of this miRNA set panel in a larger series of patients, which is currently under investigation in our laboratory by using patient’s samples from 2 centres, is necessary before definitive conclusions can be drawn.