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Background: To identify molecular biomarkers for tumor diagnosis and monitoring of disease progression, several noninvasive tests on liquid biopsy have been proposed for different cancers including those of urogenital origin. Among biomarkers, carbonic anhydrase IX (CAIX) has gained attention as it regulates extracellular pH and induces cytoplasmic alkalization contributing to malignant progression and poor treatment outcome. Works on tissues suggested the potential use of CAIX as a tumor biomarker for urogenital malignancies, but only few studies have been performed on its detection in urine., Scope: The aim of the present study is the measurement of CAIX messenger RNA (mRNA) in urine sediments of patients affected by kidney, prostate, and bladder cancers to evaluate the clinical sensitivity and specificity of the test., Procedures: The quantification of the total CAIX mRNA concentration and of its full-length isoform (CAIX FL) have been performed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) on RNA extracted from urine sediments of patients affected by urogenital cancers., Results: Urinary total CAIX mRNA expression resulted to be lower in patients with kidney and prostate cancer in comparison with the control group, but no statistically significant difference could be evidenced for bladder cancer. The evaluation of the relative percentage of FL isoform mRNA (FL%) showed a significant increase of FL% in urine from patients with cancer (median = 70.8%) in comparison with the healthy subjects (median = 2.6%) and this finding was confirmed for each cancer type separately. The comparison among receiver operating characteristic curves for total CAIX mRNA, CAIX FL mRNA, and FL% indicated that FL% shows the best diagnostic performance with 90% sensitivity and 72% specificity. Comparison of the results obtained in urine with those found in the corresponding tissues indicated 80% concordance., Conclusions: The CAIX mRNA expression in urine sediments can be considered a surrogate marker of CAIX expression in tumor tissues of urogenital origin. In particular, the analysis of FL% possesses the best characteristics to be a suitable noninvasive biomarker for urogenital cancer diagnosis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Introduction: The measurement of intracellular Ca(2+), cytosolic or stored in organelles, i.e., mitochondria, gave valuable data for numerous areas of research. In case of tumor cells, mitochondrial Ca(2+) levels play essential roles in apoptosis along with endoplasmic reticulum (ER) Ca(2+). In this study, we describe a Ca(2+) monitoring system that allows studying both adherent cells and tissues and discuss data obtained from hepatocellular carcinoma cells and rat thoracic aorta by using this system., Methods: For this purpose, two apparatus, one for adherent cells and the other for intact rat aorta, were designed and produced. With this system, changes in cytosolic Ca(2+) levels following store-operated calcium (SOC) entry induced by sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) blockers were recorded in different hepatocellular carcinoma cells. Furthermore, cytosolic and mitochondrial Ca(2+) levels were simultaneously measured in TRPC1-silenced Huh7 hepatocellular carcinoma cells. In addition, the effects of trifluoromethylphenylimidazole (TRIM) on cyclopiazonic acid (CPA)-, serotonin (5-HT)-, and phenylephrine (PE)-induced changes in isometric force and cytosolic Ca(2+) levels were determined simultaneously in rat thoracic aorta. The effects of aging on PE-induced responses were also investigated., Results: After SOC entry activation, cytosolic Ca(2+) levels were increased, as expected in all hepatocellular carcinoma cells. Mitochondrial Ca(2+) levels following CPA-induced ER depletion were significantly (p<.05) diminished in TRPC1-silenced Huh7 cells. In addition, TRIM partially inhibited both 5-HT-induced contractions and cytosolic Ca(2+) levels without affecting CPA and PE responses. PE-induced contractions and cytosolic Ca(2+) levels were similar in aorta from young and old (3 and 22 months, respectively) rats., Discussion: We confirmed that the system provides valuable data about intracellular Ca(2+) dynamics by allowing simultaneous measurements and sequential addition of compounds in adherent cells. The decrease in mitochondrial Ca(2+) loading following CPA-induced ER depletion in TRPC1-silenced Huh7 cells suggests a possible role of TRPC1 in hepatocellular carcinoma cell apoptosis. The system also enables the simultaneous measurement of isometric force and cytosolic Ca(2+) levels and promotes understanding vascular physiology and disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Embryonic rat aortic smooth muscle cells, A7r5, have been used extensively as an in vitro vascular smooth muscle cell model. They are usually provided at 11th passage by supplier and generally used before 25th passage. However, the exact passage number (P#) used is not reported in general., Methods: In this study, A7r5 cells (P#<18 and P#>23) were used in quantitative gene (5-HT2A and SERCA2b) expression as well as in functional analyses including measurements of real-time changes in cell proliferation and in 5-HT- and CPA-induced intracellular Ca(2+) levels., Results: CPA-induced SR Ca(2+) release and store-operated Ca(2+) entry significantly increased (p<.05) while cell proliferation decreased in P#>23 cells (p<.01). Furthermore, 5-HT-induced Ca(2+) elevations and 5-HT2A mRNA levels did not change whereas SERCA2b mRNA levels and CPA-induced [Ca(2+)]i levels were significantly elevated in P#>23 cells (p<.05, p<.01, respectively)., Discussion: Changes in SERCA2b expression and SOCE may contribute to suppression of cell proliferation during A7r5 subculturing. Therefore, passage numbers and subculturing procedure should be reported and taken into account during expressional and functional analyses for an accurate comparison of published data., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: In human cancers, carbonic anhydrase IX (CAIX) influences cell proliferation and tumor progression, maintaining intracellular and extracellular pH under hypoxic conditions. An alternative CAIX isoform, lacking of exons 8-9 (AS) and independent from the levels of hypoxia, was recently demonstrated in cancer cells. AS-CAIX competes with the full-length (FL) isoform in the regulation of the extracellular pH, mainly in a mild hypoxic status. In the present study, we evaluated mRNA expression of the 2 CAIX isoforms and their clinical relevance in bladder cancers and urine sediments., Materials and Methods: We measured mRNA expression of FL- and AS-CAIX isoforms in tumor tissues and benign mucosa from 45 patients with bladder transitional cell carcinoma. The expression of the 2 isoforms was also measured in urine sediment of 81 bladder cancer patients and 93 control subjects., Results: Expression of FL-CAIX mRNA was lower than AS-CAIX in benign mucosa (P = 0.006) whereas in paired bladder cancers FL-CAIX mRNA was higher (P = 0.007). Consequently, the percentage of FL-CAIX in bladder cancers [median: 62.6%] was significantly higher than in benign mucosa [15.0%] (P < 0.0001). In the urinary sediments of bladder cancer patients FL-CAIX mRNA was significantly higher in comparison with normal controls (P = 0.003). FL-CAIX percentage appeared dramatically higher in urine sediments of bladder cancer patients [64.5%] in comparison with controls [7.5%] (P < 0.0001). In addition, FL-CAIX% was significantly different in sediments from pTa-pT1 and ≥ pT2 patients [51.5% and 91.7%, respectively] (P = 0.016). Stratification according tumor grade indicated that FL-CAIX% was significantly lower in G1 bladder cancers [33.3%] in comparison with G2-G3 [88.6%] (P = 0.005) The clinical sensitivity for FL-CAIX% in urine sediments was 0.93, with a 0.76 specificity. Using the same cut-off positive predictive value (PPV) was 0.78, whereas negative predictive value (NPV) was 0.93., Conclusions: Our results seem to indicate that in bladder cancers and related urine sediments, FL-CAIX is the prevalent and is the most accurate clinically relevant variant surrogate of hypoxic stress., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives: We retrospectively evaluated the outcome of the surgical treatment of patients with renal cell carcinoma (RCC) and extensive inferior vena cava (IVC) involvement. Our aim was to investigate if a particular surgical technique could reduce morbidity and complications associated with this condition., Materials and Methods: From 1996 to 2007, 22 patients with RCC and extensive IVC involvement underwent radical surgical treatment with the intention to avoid, whenever possible, sternotomy and cardiopulmonary bypass. The level of the tumor thrombus was I (<2 cm above the renal vein) in 2 patients, II (below the intrahepatic vena cava) in 9 patients, III (intrahepatic vena cava below the diaphragm) in 7 patients, and IV (atrial) in 4 patients. Extracorporeal vascular bypass was used for 4 patients with level IV and for 2 patients with level III tumor thrombi, with hypothermic circulatory arrest in 2 patients. Extensive liver mobilization techniques were adopted in 16 patients. Overall and cancer-specific survival (CSS) were analyzed based on tumor extent (N0M0, N+M+), pathologic stage (pT3b, pT3c, pT4), thrombus level, and caval wall infiltration., Results: Two patients died within 1 month of surgery and the remaining 20 patients have a mean follow-up of 32.2 months (range 6-90): 8 are alive (overall survival 40%), but 2 with disease (CSS 30%). A total of 10 severe complications developed in 8 patients (36%). Both overall and CSS were significantly associated with tumor stage (Log-rank P = 0.0237 and 0.0465), presence of nodal or systemic metastases (Log-rank P = 0.0835 and 0.0669; Wilcoxon's test P = 0.0407 and 0.0411), and caval wall infiltration (Log-rank P = 0.0200 and 0.0418)., Conclusions: Despite the low overall survival, related to the high percentage of nodal and systemic metastases, aggressive surgical management with resection of synchronous metastatic disease for symptom palliation and cytoreduction, followed by immunotherapy is justified in this setting. A transabdominal approach to RCC and IVC involvement, even in patients with level III thrombus, can provide the surgeon with an exposure similar to thoracoabdominal incisions without the complications associated with thoracotomy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Purpose: To assess whether PSA density (PSAD) and PSA density of the transition zone (PSADTZ) are more accurate than PSA alone in predicting the pathological stage of prostate cancer., Materials and Methods: One hundred and nine consecutive patients with clinically localized prostate cancer and preoperative PSA values over the whole range, treated with radical retropubic prostatectomy and limited pelvic lymph node dissection were included in this prospective study. Total prostate and transition zone volumes were measured by transrectal ultrasound using the prolate ellipsoid method. PSA, PSAD, and PSADTZ were compared to percentage of positive biopsy cores (% PC), biopsy and surgical Gleason score, and pathological stage, using univariate and multivariate analysis., Results: Pathological stage was pT2a, pT2b, pT3a, and pT3b in 25.6%, 37.7%, 25.6%, and 11.1% of patients, respectively. Lymph node metastases were found in 4.6% of patients. PSA, PSAD, and PSADTZ were significantly related to % PC, biopsy, and surgical Gleason score and pathological stage (P < 0.001), and were equally able to predict higher pathological stage, i.e., seminal vesicle invasion and lymph node metastases. Only by adding % PC in multivariate analysis was it possible to discriminate intra- from extracapsular tumors., Conclusions: The results of the present study demonstrate that PSAD and PSADTZ failed to outperform PSA in preoperative stage prediction of prostate cancer, possibly because the formula used to calculate them does not eliminate the contribution to total PSA of the nonmalignant portion of the gland.