Donata Villari

Quantitative methylation analysis of BCL2, hTERT, and DAPK promoters in urine sediment for the detection of non-muscle-invasive urothelial carcinoma of the bladder: A prospective, two-center validation study

OBJECTIVES Urinary hypermethylation of BCL2, hTERT, and DAPK promoters has been previously demonstrated as an accurate biomarker for the detection of urothelial carcinoma of the bladder (UCB) in patients undergoing radical cystectomy. In the present study, we investigated with a validation intent the frequency and levels of methylation of the same 3 genes in tumor tissue and urine sediment of patients undergoing transurethral resection (TUR) for non-muscle-invasive (NMI) UCB. MATERIALS AND METHODS A total of 108 consecutive patients with NMI UCB and 105 controls with no genitourinary malignancies were enrolled in this prospective study conducted in 2 tertiary referral academic urological departments with an advanced molecular laboratory. The frequency and levels of methylated BCL2, hTERT, and DAPK promoters were evaluated with quantitative methylation-specific real-time polymerase chain reaction in DNA extracted from tumor tissue and paired normal bladder mucosa retrieved at the time of TUR in patients, and from urine in patients and controls. RESULTS In tumor tissue, at least 1 gene was hypermethylated in 91% patients (BCL2 in 62%, hTERT in 53%, DAPK in 48%). Methylation of hTERT was significantly correlated with tumor grade (P = 0.026). In urine sediment sensitivity and specificity were 76% and 98%, respectively, using BCL2 and hTERT. The number of methylated genes was highly correlated with tumor grade (P = 0.005). Methylated BCL2 and hTERT in urine sediment were highly correlated with those of the corresponding bladder tumor qualitatively (P < 0.001), and only BCL2 also quantitatively (P = 0.005). Methylation levels of BCL2 and hTERT were variably associated with tumor grade and stage, but were significantly correlated with patient age (P = 0.004 and P = 0.027, respectively). CONCLUSIONS These findings suggest that quantitative methylation analysis of BCL2 and hTERT, but not DAPK, in urine sediment may be a useful tool in the diagnosis of NMI UCB, deserving future applicability studies.

ENDOLUMINAL STENT PLACEMENT AFTER PERCUTANEOUS TRANSLUMINAL ANGIOPLASTY IN THE TREATMENT OF POST-TRANSPLANT RENAL ARTERY STENOSIS

PURPOSE We report our experience with endoluminal stent placement after percutaneous transluminal angioplasty for the treatment of post-transplant renal artery stenosis. MATERIALS AND METHODS From October 1992 to September 1996, 8 stents were successfully implanted in 7 patients affected by resistant transplant renal artery stenosis. All transplanted kidneys were procured from cadaver donors. The patients were routinely evaluated with duplex sonography and the median interval between transplantation and stenosis detection was 7.4 months (range 0.5 to 17). When serious renal stenosis was diagnosed (greater than 50%), selected angiography and percutaneous transluminal angioplasty were performed. In 8 cases (7 patients) an endoluminal metallic Palmaz stent was placed in the site of the restenosis. One patient received 2 stents repeatedly positioned in different stenosis sites. RESULTS No major complications occurred. Clinical outcome was positive in 5 patients (71.4%) and Stenosis recurred in 2 (28.5%) (less than 50% and less than 35%, respectively). Median followup after stent placement was 14.8 months (range 1 to 37). CONCLUSIONS Percutaneous endoluminal stent procedures after resistant transplant renal artery stenosis or for ex novo treatment for severe anastomotic stenoses appears to be promising. Longer followup periods are necessary for true evaluation of this procedure.

The Female Sexual Function Index (FSFI): Linguistic Validation of the Italian Version

INTRODUCTION Although several new measurements for female sexual dysfunction (FSD) have recently been developed, the Female Sexual Function Index (FSFI) remains the gold standard for screening and one of the most widely used questionnaires. The Italian translation of the FSFI has been used in several studies conducted in Italy, but a linguistic validation of the Italian version does not exist. AIM The aim of this study was to perform a linguistic validation of the Italian version of the FSFI. METHODS A multicenter cross-sectional study conducted in 14 urological and gynecological clinics, uniformly distributed over Italian territory. We performed all steps necessary to determine the reliability and the test-retest reliability of the Italian version of the FSFI. The study population was a convenience sample of 409 Italian women. MAIN OUTCOME MEASURES The reliability of the questionnaire was calculated using Cronbachs alpha, which was considered weak, moderate, or high if its value was found less than 0.6, between 0.6 and 0.8, or equal to or greater than 0.8, respectively. The test-retest reliability was assessed for all women in the sample by calculating Pearsons concordance correlation coefficient for each domain and for the total score, both at baseline and after 15 days (r range between -1.00 to +1.00, where +1.00 indicates the strongest positive association). RESULTS Cronbachs alpha coefficients for total and domain score were sufficiently high, ranging from 0.92 to 0.97 for the total sample. The test-retest procedure revealed that the concordance correlation coefficient was very high both for FSFI-I total score (Pearsons P = 0.93) and for each domain (Pearsons P always >0.92). CONCLUSION For the first time in the literature, our study has produced a validated and reliable Italian version of the FSFI questionnaire. Consequently, the Italian FSFI can be used as a reliable tool for preliminary screening for female sexual dysfunction for Italian women.

Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for the Measurement of Prostate-Specific Antigen mRNA in the Peripheral Blood of Patients with Prostate Carcinoma Using the TaqManTM Detection System

Abstract Circulating prostate cells can be detected in peripheral blood of patients with clinically localized or advanced prostate carcinoma. Traditionally, nested reverse transcriptase-polymerase chain reaction (RT-PCR) is used for this as a sensitive, but qualitative only, detection system. We developed a quantitative real-time RT-PCR method for measuring prostate-specific antigen (PSA) mRNA in peripheral blood of prostate cancer patients. A quantitative assay was developed using an external standard reference curve generated with RNA from the human prostate cell line LNCaP. Basal blood samples were collected from 44 patients without evidence of distant metastases and from 30 healthy controls. In 29 patients surgically treated with radical prostatectomy, the measurement of PSA mRNA was performed in blood samples collected before, at the end and 6 days after surgery. In 14 patients treated with radiotherapy, the measurements were repeated at 3-month intervals to evaluate time-related changes during therapy. The measurements were also performed for one year at 3-month intervals in one patient treated with anti-androgen therapy. We found detectable PSA mRNA in 14/44 (32%) basal blood samples. A wide range of values were observed in these patients, ranging from 0.5 to 1724 pg of total LNCaP RNA/ml blood. In patients undergoing radical prostatectomy, circulating PSA mRNA was detectable in eight patients in basal samples, and in seven of them also in blood specimens collected at the end of surgery, showing an increase in only two patients. In blood samples collected 6 days later, PSA mRNA was dramatically reduced in all patients, but still present in seven of them. In four patients, whose basal samples were negative, PSA mRNA was detectable in samples collected at the end of surgery and three of them were negative after 6 days. In patients who did not receive surgical treatment, a rapid decrease in PSA mRNA was demonstrated in five patients treated with radiotherapy and in one patient undergoing androgen deprivation. No detectable PSA mRNA was found in healthy controls. The levels of PSA mRNA in peripheral blood from patients with prostate carcinoma can be easily measured by this sensitive, quantitative and reliable procedure. This assay is a promising tool for the detection and follow-up of these patients.

Tankyrase-1 mRNA expression in bladder cancer and paired urine sediment: preliminary experience

Abstract Background: The enzyme tankyrase-1 (TNKS-1), a member of the growing family of poly(ADP-ribose) polymerases (PARPs), was identified as a component of the human telomeric complex. PARPs catalyze the formation of long chains of poly(ADP-ribose) onto protein acceptors using NAD+ as a substrate. TNKS-1 interacts with the telomeric DNA-binding protein TTAGGG repeat-binding factor 1 (TRF1), which is a negative regulator of telomere length. TNKS-1 is a positive regulator of telomere elongation and its activity appears to be upregulated in some human cancers. Methods: We evaluated for the first time TNKS-1 mRNA expression by real time RT-PCR in tumor tissue, paired normal mucosa and urine sediment in patients with transitional cell carcinoma (TCC) of the bladder. Samples were collected from 41 consecutive patients, 20 with non-muscle-invasive (pTa-pT1) and 21 with muscle-invasive (≥pT2) bladder TCC. Results obtained in urine sediment were compared with those from 40 healthy subjects matched for age and sex. Results: In pTa-pT1 tumor tissues, TNKS-1 mRNA levels were significantly higher than in ≥pT2 patients (p<0.0001). In urine sediment from TCC patients, independent of tumor stage, TNKS-1 mRNA levels were significantly higher than in healthy controls, with maximal levels in ≥pT2 patients. In particular, TNKS-1 mRNA levels in urine were elevated in 31/41 patients with a sensitivity of 81% in ≥pT2 tumors and 65% in pTa-pT1 TCC. Of patients with pTa-pT1 tumors, 11 had a recurrence within 18 months after initial transurethral resection. In these patients, urine levels of TNKS-1 mRNA were higher than in non-relapsing patients (p=0.038). Conclusions: In this preliminary study, TNKS-1 mRNA in urine sediment from patients with bladder TCC correlated with tumor stage, and higher preoperative levels were associated with increased risk of early recurrence. Clin Chem Lab Med 2007;45:862–6.

Renal Cell Carcinoma of Native Kidney After Renal Transplantation: Clinical Relevance of Early Detection

BACKGROUND Life expectancy after transplantation has improved, and cancer may soon be the leading cause of late death after transplantation. The guidelines of the American and European societies of nephrology and urology have not yet established the optimal frequency for screening for renal cell carcinoma (RCC) of native kidneys in patients who have undergone renal transplantation. OBJECTIVE To evaluate the prevalence, prognosis, and risk factors of RCC in a series of patients followed up for 16 years in our transplantation unit. MATERIALS AND METHODS Our study is a follow-up observational cohort study conducted in 694 consecutive renal transplant recipients admitted to our institution from July 1991 through July 2007. At our institution, ultrasound studies of the native kidneys were performed every 6 months after renal transplantation. RESULTS In the patient cohort studied, 10 patients developed a renal tumor (1.6% incidence). Three patients died of causes other than recurrence of RCC. Seven patients are alive with no evidence of RCC recurrence or metastatic disease after a mean (range) follow-up of 41 (12-96) months. Acquired cystic kidney disease and dialysis duration were positively associated with development of RCC. CONCLUSIONS The incidence of RCC in the literature varies between 0.3% and 4.8%. The variability depends on the timing of follow-up, with a higher incidence in prospective studies with strict follow-up. We advise ultrasound studies performed by specialized physicians every 6 months after transplantation. More detailed guidelines designed by the major international transplantation societies are necessary.

Des (1-3) IGF-I-stimulated growth of human stromal BPH cells is inhibited by a vitamin D3 analogue

Prostate growth and differentiation is under the control of androgens not only during fetal life and childhood but also in adulthood, and it has been proposed that increased prostatic concentration of androgens, or increased androgen responsiveness, causes benign prostatic hyperplasia (BPH). However, different androgen ablation strategies such as treatment with GnRH agonists and finasteride resulted in a modest decrease of the hyperplastic prostate volume. In the last few years it became evident that both androgen-dependent and androgen-independent growth factors promote prostate enlargement by inducing cell proliferation or reducing apoptosis. Therefore, new therapeutic strategies, aimed at reducing intraprostatic growth factor signaling, are under investigation. In this study, we report further evidence that a non hypercalcemic-analogue of vitamin D(3), analogue (V) decreases growth factor-induced human BPH cell proliferation and survival. We found that Des (1-3) insulin-like growth factor [Des (1-3) IGF-I], an IGF-I analogue, which does not bind to IGF-binding proteins, is a potent mitogen for BPH stromal cells via a dual mechanism: stimulation of cell growth and inhibition of apoptosis. Similar results were previously reported for another growth factor for BPH cells, keratinocyte growth factor (KGF). Accordingly, we speculate that both KGF and IGF might be involved in the pathogenesis of BPH. We also found analogue (V) not only inhibits the mitogenic activity of growth factors on BPH cells, but even decreased the basal expression of bcl-2, and induced apoptosis. Therefore, vitamin D(3) analogues might be considered for the medical treatment of BPH.

Tissue‐resonance interaction method for the noninvasive diagnosis of prostate cancer: analysis of a multicentre clinical evaluation

To determine, in a multicentre prospective study, the accuracy of the tissue‐resonance interaction method (TRIMprob, new technology developed for the noninvasive analysis of electromagnetic anisotropy in biological tissues) in the diagnosis of prostate cancer.

Flow cytometric measurement of DNA content in human solid tumors: a comparison with cytogenetics.

The aims of this study were: (1) to test the accuracy of flow cytometery (FC) in the measurement of DNA content in human solid tumors, (2) to correlate the FC DNA-index (DI) with the chromosome modal number (CMN) provided by cytogenetic analysis (CG), and (3) to investigate the most frequent pitfalls in FC histograms classification. FC and CG analyses were performed in parallel on 113 samples of human solid tumors of different origin. FC provided an evaluable histogram in 110 out of 113 cases (97%), whereas a successful CG culture was obtained in 79 out of 113 samples (72%). In the 79 cases evaluable by both FC and CG, a concordant ploidy status was found in 66 cases (84%) (47 diploid and 19 aneuploid) (P < 0.001, chi-square test). In the 19 concordant aneuploid tumors a close correlation between the CMN and the DI was found (y = 0.019 x + 0.151; r = 0.860). Concerning the 13 discordant cases, 11 (85%) were classified as aneuploid by FC and as diploid by CG, while 2 cases (15%) were CG aneuploid (1 near-diploid and 1 tetraploid) and FC diploid. The current study suggests that FC is a reliable method for the measurement of tumor DNA content of the studied solid tumors. Special attention should be paid to the improvement of DNA histograms quality, in order to reduce the difficulties in the detection of near-diploid and near-tetraploid cell populations. Multiple sampling should be warranted whenever possible.

Uterus-Sparing Vaginal Surgery of Genitourinary Prolapse Employing Biocompatible Material

Objective: The study presents an original uterus sparing technique for transvaginal repair of total genitourinary prolapse. The technique employs a synthetic mesh of mixed polypropylene and 910 polyglactin fibers. Methods: The prosthesis creates a support for the cystocele, the cervix and the enterocele. It has four anchoring sites: two at the rear in the sacrospinous ligaments and two at the front in the arcus tendineous of the levator ani muscle. Between February 2001 and December 2004, 24 patients (mean age 66.9 years), presenting symptoms of uterine prolapse, cystocele and enterocele (POP-Q stage III-IV Aa associated to II-III-IV C), were treated with our procedure. Pre- and postoperative parameters were evaluated statistically. Results: No patient had any serious complications. The mean follow-up was 31.1 months (range 6–52). 19 patients (79.1%) have shown excellent results and have been completely cured. In 5 other cases (20.8%), the cystocele was completely cured and there was a significant improvement in the hysterocele and the enterocele. One patient required surgical treatment for postoperative stress incontinence. Statistical analysis of data regarding the pre- and postoperative prolapse stage demonstrated a high degree of objective cure rates (p < 0.0001). Conclusions: While hysterectomy remains the habitual treatment for severe uterine prolapse, our technique provides a promising alternative solution. It is also significant that there were no complications of erosion or infection associated with the prosthesis.