Giulio Milanese

Prognostic role of tumor necrosis, microvessel density, vascular endothelial growth factor and hypoxia inducible factor-1alpha in patients with clear cell renal carcinoma after radical nephrectomy in a long term follow-up.

Angiogenesis is a critical step in the growth, invasive progression and metastatic spread of solid tumors. We investigated the importance of tumor necrosis, and microvessel density (MVD), vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) immunohistochemical expression in a large series of clear cell renal carcinomas treated with radical nephrectomy and assessed the prognostic value of their expression in terms of patient survival at long-term follow up. Fifty patients with clear cell RCC were examined. The features considered when evaluating the patients were age, tumor size and grade, intratumoral vascular and renal capsula invasion, histological necrosis, and MVD, vascular and tumoral cell VEGF, and vascular, tumoral cytoplasmic and nuclear HIF-1α expression on the histologic specimens. All considered parameters were correlated with patient specific survival. Mean age was 62.06 ± 6.8 years. Median follow-up was 191.66 months; median survival was 120.86 months. Twenty-one patients developed metastases in the follow-up. Tumor necrosis, microvascular invasion and renal capsula infiltration are more likely to occur in high stage and grade RCC; cytoplasmic HIF-1α is highly expressed in high grade RCC. Survival is dependent upon tumor stage and grade, the presence of intratumoral vascular invasion and capsular infiltration, and tumor necrosis; MVD also resulted as being an important prognostic factor. VEGF and HIF-1α correlate with prognosis in high stage tumors where VEGF is the most important independent prognostic factor for cancer specific death. The histological and immunohistochemical parameters considered in our study can influence disease recurrence and survival in RCC.

Prognostic role of global DNA‐methylation and histone acetylation in pT1a clear cell renal carcinoma in partial nephrectomy specimens

Surgery is the main treatment for renal cell carcinoma (RCC); nephron sparing surgery can be performed as a treatment of choice for small peripheral lesions. Epigenetics configures a new entity that regulates gene expression throughout methylation, acetylation and chromatin remodelling. In addition to silencing as a result of mutations, loss of heterozygosity, or classic genetic events, epigenetic modification symbolizes essential events during carcinogenesis and tumour development. We investigated global methylation and histone acetylation expression in a series of small conventional clear cell renal carcinomas (i.e. less than 5 cm) (pT1a) treated with partial nephrectomy, to assess their possible role as diagnostic biomarkers. A total of 54 patients with conventional single RCC were selected and treated with partial nephrectomy; they were followed up to 186 months. Immunohistochemistry was performed on paraffin‐embedded sections, using anti‐5‐methylcytosine (5mc) and anti‐Acetyl‐Histone H3 (Lys 9). Our results confirm that the mean percentage of global cellular methylation in tumoural tissue was significantly higher compared to healthy peritumoural tissue, whereas the mean percentage of histone cellular acetylation in tumoural tissue was significantly lower. The percentage of methylation was significantly higher in grades 3 and 4 (P= 0.033), whereas the percentage of histone acetylation was significantly lower (P= 0.023), suggesting therefore that these markers could correlate with tumour aggressiveness in pT1a RCC. On univariate analysis of patient survival in relation to the different considered factors, Fuhrman grade was the most important survival factor. These epigenetic markers can give us interesting information about chromatin remodelling in RCCs; the percentage of global methylation increases with increasing Fuhrman grade, whereas histone acetylation decreases with increasing grade in small RCC; our results suggest that global hypermethylation and histone hypoacetylation can be assumed to be an early event in RCC and to correlate with tumour aggressiveness.

Upregulation of Tissue and Urinary Nicotinamide N-Methyltransferase in Bladder Cancer: Potential for the Development of a Urine-Based Diagnostic Test

Carcinoma of the bladder is one of the most common urologic malignancies occurring worldwide. Diagnosis and monitoring of bladder urothelial carcinoma (UC) are based on cystoscopy and urinary cytology. However, these diagnostic methods still have some limitations, mainly related to invasive nature and lack of sensitivity. New reliable and non-invasive biomarkers for bladder cancer detection are therefore required. To explore the involvement of enzymes of drug metabolism in bladder cancer, in the present study, we analyzed the gene expression profiles of tumor and normal looking tissues obtained from the same patient by cDNA macroarray. The enzyme nicotinamide N-methyltransferase (NNMT) was identified as a highly expressed gene in bladder cancer. RT-PCR, Real-Time PCR, Western blot analysis, and catalytic activity assay, performed on a large cohort of patients with bladder UC, confirmed NNMT upregulation. NNMT mRNA and protein levels were also determined in urine specimens obtained from patients with bladder UC and healthy subjects. We found that NNMT expression levels were significantly higher in patients with bladder tumor compared to controls that showed very low or undetectable amounts of NNMT transcript and protein. Our results indicate that a marked NNMT increase is a peculiar feature of bladder UC and suggest the potential suitability of urine NNMT expression levels determination for early and non-invasive diagnosis of bladder cancer.

Correlation Between Ultrasound Alterations of the Preprostatic Sphincter and Symptoms in Patients With Chronic Prostatitis-Chronic Pelvic Pain Syndrome

PURPOSE We investigated ultrasound alterations of the preprostatic sphincter in patients with chronic prostatitis-chronic pelvic pain syndrome. We evaluated the frequency of these alterations, standardized their ultrasound measurement and correlated them with symptoms in patients with chronic prostatitis-chronic pelvic pain syndrome. MATERIALS AND METHODS In 37 patients with chronic prostatitis-chronic pelvic pain syndrome and 23 healthy volunteers certain parameters were measured by transrectal ultrasound, including prostate volume, hypoechoic periurethral zone volume, posterior prostate lip thickness, bladder neck thickness, detrusor thickness and the degree of echogenicity of the anterior fibromuscular stroma. All patients were evaluated with the International Prostate Symptom Score and National Institutes of Health Chronic Prostatitis Symptom Index. Urinary flow rate and post-void residual urine volume were also considered in each patient. All assessments were done independently and consecutively by 3 operators. RESULTS A hypoechoic periurethral zone volume was found in 36 of 37 patients with chronic prostatitis-chronic pelvic pain syndrome. No significant intra-observer and interobserver differences were found in ultrasound parameter measurements. In the chronic prostatitis-chronic pelvic pain syndrome group ultrasound findings showed greater post-void residual urine volume, detrusor thickness and hypoechoic periurethral zone volume, increased posterior prostate lip thickness and bladder neck thickness, and greater anterior fibromuscular stroma hyperechogenicity. On multivariate analysis hypoechoic periurethral zone volume was an independent predictive factor for worse National Institutes of Health Chronic Prostatitis Symptom Index pain, urinary and total scores. Posterior prostate lip thickness was the only factor predictive of a worse International Prostate Symptom Score in patients with chronic prostatitis-chronic pelvic pain syndrome. A hypoechoic periurethral zone volume, posterior prostate lip thickness and bladder neck thickness with calculated threshold values revealed fair to excellent accuracy for identifying a patient with chronic prostatitis-chronic pelvic pain syndrome. CONCLUSIONS Ultrasound evaluation of the bladder neck-posterior urethra in patients with chronic prostatitis-chronic pelvic pain syndrome led us to identify a set of lesions that cannot be found in healthy subjects. The measurement of hypoechoic periurethral zone volume, posterior prostate lip thickness and bladder neck thickness could be useful for following patients with chronic prostatitis-chronic pelvic pain syndrome and maybe for better understanding the complicated pathophysiological mechanisms of chronic nonbacterial prostatitis.

Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma.

INTRODUCTION BRCA1-associated protein 1 (BAP1) is a gene situated on chromosome 3p in a region that is deleted in more than 90% of renal cell carcinomas (RCCs). In the present study, we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy; we assessed the prognostic value of their expression in terms of patients׳ survival at long-term follow-up. MATERIALS AND METHODS A total of 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis, and patient-specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BAP1 in tumoral tissue. RESULTS Median follow-up was 196.18 months and median survival was 125.34 months. Nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1-negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. In multivariate analysis, pathological stage and nuclear BAP1 expression resulted independent prognostic factors. CONCLUSION We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an influence on cancer-specific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant setting or in the metastatic setting or in both the settings.

Do DNA-methylation and histone acetylation play a role in clear cell renal carcinoma? Analysis of radical nephrectomy specimens in a long-term follow-up.

We investigated global methylation and histone acetylation in 50 conventional clear cell renal carcinomas (RCC), treated with radical nephrectomy, to assess their possible role as diagnostic biomarkers. The features considered in this study were patient age, tumor size and grade, percentage and intensity of 5-methylcytosine (5mc) and Acetyl-Histone (Lys 9) expression in tumor tissue. All considered parameters were correlated with patient specific survival. The mean percentage of global cellular methylation in tumoral tissue was significantly higher compared to normal peritumoral tissue (p<0.0001), while the intensity of cellular methylation was significantly higher in normal tissue than in tumoral tissue (p=0.001). The mean percentage of histone cellular acetylation in tumoral tissue was significantly lower compared to normal peritumoral tissue (p=0.0005), while the intensity of mean acetylation in neoplastic tissue was similar to the normal tissue. The percentage of global DNA methylation was significantly higher in grades 3 and 4 tumors (p=0.033). Global DNA methylation and histone acetylation in tumoral tissue did not correlate with survival. Fuhrman grade was statistically significant for prognosis (p=0.031). In conclusion, global hypermethylation and histone hypoacetylation play an important role in RCC carcinogenesis; Fuhrman grade is still considered the most important factor for patient survival; 5mc can have a role as markers of aggressiveness.

Increased Urokinase-Type Plasminogen Activator Receptor and Epidermal Growth Factor Receptor in Serum of Patients With Prostate Cancer

PURPOSE Prostate cancer cell motility and invasion have been linked to the up-regulated signaling of epidermal growth factor receptor and urokinase-type plasminogen activator receptor. We analyzed the expression of serum urokinase-type plasminogen activator receptor and epidermal growth factor receptor in the serum of patients with clinical suspicion of prostate cancer to evaluate the possible role as prostate cancer markers. MATERIALS AND METHODS Serum was collected from 79 consecutive patients referred to our institution for transrectal ultrasound guided prostate biopsy. All blood samples were obtained before prostate biopsy. Total urokinase-type plasminogen activator receptor and epidermal growth factor receptor antigen in serum were measured by specific enzyme-linked immunosorbent assays. Gleason score, the number of positive cores, maximum percent of cancer and inflammation were considered on biopsy. Patients determined to have prostate adenocarcinoma underwent radical retropubic prostatectomy. Gleason score, pathological stage (extraprostatic extension), surgical margins, seminal vesicle involvement, perineural infiltration, lymphovascular invasion and cancer volume were evaluated in radical retropubic prostatectomy specimens. RESULTS The 30 patients with prostate cancer had significantly higher levels of serum urokinase-type plasminogen activator receptor and epidermal growth factor receptor in comparison to those without prostate cancer but not significantly higher levels of prostate specific antigen. Urokinase-type plasminogen activator receptor and epidermal growth factor receptor levels closely correlated in the serum of patients with prostate cancer. In a multivariate model high serum epidermal growth factor receptor increased the probability of positive biopsies by 1.9 times. ROC analysis revealed that serum epidermal growth factor receptor had 93.3% sensitivity and 98% specificity for detecting prostate cancer at a cutoff of 67.9 ng/ml. Urokinase-type plasminogen activator receptor and epidermal growth factor receptor were significantly higher in patients with extraprostatic extension, seminal vesicle involvement and perineural infiltration in the radical retropubic prostatectomy specimens. Serum urokinase-type plasminogen activator receptor was the only independent predictive serum marker of extraprostatic extension, seminal vesicle involvement and perineural infiltration. CONCLUSIONS The measurement of urokinase-type plasminogen activator receptor and epidermal growth factor receptor in the serum of patients with clinical suspicion of prostate cancer might provide clinically relevant information on the state of the prostate gland. Measuring serum epidermal growth factor receptor could help predict which patients have prostate cancer, while serum urokinase-type plasminogen activator receptor over expression seems to be related to tumor extraprostatic spread.

Tumor VEGF expression correlates with tumor stage and identifies prognostically different groups in patients with clear cell renal cell carcinoma

OBJECTIVES Vascular endothelial growth factor (VEGF) is a potent inducer of tumor angiogenesis and represents the key element in the pathogenesis of clear cell renal cell carcinoma (ccRCC). The aim of this study was to investigate the use of tumor VEGF expression as a parameter to identify tumor stage and prognostically different patient groups. METHODS AND MATERIALS We retrospectively collected clinical data of 137 patients treated with partial or radical nephrectomy at our institutions for organ-confined, locally advanced, and metastatic ccRCCs between 1984 and 2013. Tumor cell VEGF immunohistochemical expression was compared with pathological and clinical features including age, sex, tumor stage, and Fuhrman grade. Comparison of VEGF expression levels between tumor stages was performed via Kruskal-Wallis nonparametric test. Survival analysis was conducted via Kaplan-Meier product-limit method, and Mantel-Haenszel log-rank test was employed to compare survival among groups. RESULTS Median age at diagnosis was 61 years (range: 33-85 y). Tumor stage was pT1N0M0 in 67 patients (49%), pT2N0M0 in 5 (4%), and pT3N0M0 in 25 (18%), while 40 patients (29%) had metastatic tumors at diagnosis. Fuhrman nuclear grade was G1 in 22 patients (16%), G2 in 60 (44%), G3 in 33 (24%), G4 in 13 patients (9%), and unknown in 9 patients. Tumor VEGF was differentially expressed among different stages (P<0.001) and in low (G1-2) and high (G3-4) Fuhrman grade tumors (P<0.001). No significant differences were found when stratifying by sex (P = 0.06) or age (P = 0.29). Median overall survival (OS) from partial or radical nephrectomy was 161 months (range: 1-366). We observed a significantly longer OS in patients with low (<25%) vs. high (>25%) VEGF expression levels (median OS 206 vs. 65 mo, P<0.001). CONCLUSIONS Our data show that tumor cell VEGF expression is significantly associated with tumor stage and Fuhrman grade and is able to predict patient outcome, suggesting a potential use of this parameter in identifying prognostically different patients with ccRCC.

The role of the prostatic stroma in chronic prostatitis/chronic pelvic pain syndrome

ObjectiveTo confirm the hypothesis of prostatic stromal involvement in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS).Materials and methodsA literature review to analyze mechanisms commonly indicated as a cause of CP/CPPS that can interfere with the processes of cell growth of smooth muscle fibrocells and may cause smooth muscle cell hypertrophy, periurethral edema, and inflammation.ResultsOur review strongly suggests a prevalent stromal involvement, specifically of the smooth muscle cells, in CP/CPPS physiopathology. The involvement of the endocrine system, in particular the role of estrogens, the neurological pathway mediated by noradrenalin, and the presence of inflammation, support the hypothesis that CP/CPPS could be a disease with a prevalent role of smooth muscle stromal cells rather than glandular structures. Neurogenous inflammation, oxidative stress and psychological factors may be involved in the chronic nature of the disease.ConclusionsWe believe that new studies regarding chronic prostatitis should also be focused on prostatic stromal involvement in the inflammatory pathway.

Non-muscle invasive high grade urothelial carcinoma of the bladder. Which factors can influence understaging at the time of radical cystectomy?

OBJECTIVE To evaluate the main factors which influence understaging in patients with T1G3 non-muscle invasive bladder cancer (NMIBC). MATERIALS AND METHODS 109 patients with T1/G3 underwent transurethral resection of bladder tumor (TURBT) and then radical cystectomy (RC) with pelvic lymph nodes dissection. A number of variables were considered when evaluating the detection of understaging. We considered the patients age and gender, as well as the size, number, location and morphology of their tumor. We also considered coexistence of bladder carcinoma in situ (CIS), microscopic vascular invasion and deep lamina propria invasion. The level of experience of the surgeon was also analyzed. RESULTS in RC samples muscle invasion, that is understaging, was detected in 74 (67.9%) patients, while 35 (32.1%) patients were appropriately staged. In these cohort of patients with high grade tumors, understaging was associated with deep lamina propria and microscopic vascular invasion, multiple tumors, tumor size > 6 cm, tumor location (trigone and dome), presence of residual tumor; age, gender, tumor morphology, CIS associated, and experience of urological surgeon were not associated with clinical understaging. CONCLUSIONS in our study, evaluating patients with high grade NMIBC at first TURBT, we identified some risk factors that need to be considered and that are able to increase the risk of understaging: deep lamina propria and microscopic vascular invasion, multiple tumors, tumor size > 6 cm, tumor location (trigone and dome), presence of residual tumor. When these risk factors are present, performing an early cystectomy, and not a re-TURBT, could lower the risk of worse pathological finding due to rapid disease progression of the high grade tumors, and can prolong survival.