Ioannis Katafigiotis

Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis

High resolution proteomics approaches have been successfully utilized for the comprehensive characterization of the cell proteome. However, in the case of quantitative proteomics an open question still remains, which quantification strategy is best suited for identification of biologically relevant changes, especially in clinical specimens. In this study, a thorough comparison of a label-free approach (intensity-based) and 8-plex iTRAQ was conducted as applied to the analysis of tumor tissue samples from non-muscle invasive and muscle-invasive bladder cancer. For the latter, two acquisition strategies were tested including analysis of unfractionated and fractioned iTRAQ-labeled peptides. To reduce variability, aliquots of the same protein extract were used as starting material, whereas to obtain representative results per method further sample processing and MS analysis were conducted according to routinely applied protocols. Considering only multiple-peptide identifications, LC-MS/MS analysis resulted in the identification of 910, 1092 and 332 proteins by label-free, fractionated and unfractionated iTRAQ, respectively. The label-free strategy provided higher protein sequence coverage compared to both iTRAQ experiments. Even though pre-fraction of the iTRAQ labeled peptides allowed for a higher number of identifications, this was not accompanied by a respective increase in the number of differentially expressed changes detected. Validity of the proteomics output related to protein identification and differential expression was determined by comparison to existing data in the field (Protein Atlas and published data on the disease). All methods predicted changes which to a large extent agreed with published data, with label-free providing a higher number of significant changes than iTRAQ. Conclusively, both label-free and iTRAQ (when combined to peptide fractionation) provide high proteome coverage and apparently valid predictions in terms of differential expression, nevertheless label-free provides higher sequence coverage and ultimately detects a higher number of differentially expressed proteins. The risk for receiving false associations still exists, particularly when analyzing highly heterogeneous biological samples, raising the need for the analysis of higher sample numbers and/or application of adjustment for multiple testing.

All You Need to Know About Urethrovesical Anastomotic Urinary Leakage Following Radical Prostatectomy

PURPOSE Radical prostatectomy is a challenging operation demanding a high level of surgical expertise and experience. Urinary leakage at the urethrovesical anastomosis is one of the most common short-term complications of radical prostatectomy, reaching an incidence of 0.3% to 15.4%. In this review we investigate and discuss all matters directly related to urethrovesical anastomotic leak, specifically how to diagnose it properly, how to determine when it is clinically significant and when intervention is required, how to prevent or predict it and, finally, the possible long-term sequelae. MATERIALS AND METHODS We conducted a systematic analysis of the literature searching for English and nonEnglish language publications from a preidentified time frame (1985 to 2011) using primary search databases (PubMed®, Web of Science®). Manual selection was performed by 2 authors and the third reviewed the final common selection. We also created an algorithm for the diagnosis and management of urethrovesical anastomotic leak. RESULTS A total of 72 studies were finally selected, including 48 (67%) observational case series, 16 (22.2%) prospective trials, 1 letter to the editor, 1 review and 1 systematic review which was focused only on laparoscopic radical prostatectomy. We also found 2 experimental studies performed in animal models and 3 case reports. Of these studies 7 reported results from fewer than 20 patients. No consensus was recorded on a strict definition of urethrovesical anastomotic leak. The factors determining possible definitions included postoperative day of urethrovesical anastomotic leak, amount of extravasation on cystography and the need for intervention. Urethrovesical anastomotic leak should be classified according to the Clavien classification system, depending on severity and the need for intervention. To our knowledge the role of the open, laparoscopic or robotic approach in the incidence of urethrovesical anastomotic leak has not been systematically investigated. Risk factors for urethrovesical anastomotic leak include obesity, prostate size, previous prostatic surgery, type of anastomosis technique, suture number and type, eversion of the mucosa, a difficult anastomosis or an anastomosis under tension, reconstruction of the musculofascial plate, blood loss, intraoperative flush test result and postoperative urinary tract infection. Diagnosis can be determined primarily by establishing the nature of the drain output. Retrograde cystography, computerized tomography cystography, transrectal ultrasound, contrast enhanced ultrasound and excretory urography are the indicated imaging modalities, and are not always necessary. Finally, the development of anastomotic stricture and incontinence due to urethrovesical anastomotic leak are additional complications. CONCLUSIONS We gathered all relevant critical information concerning urethrovesical anastomotic leak to encourage standardization in the diagnosis and management of this common complication. Systematic meta-analysis of each debatable issue is required to provide definite answers.

Implementation and external validation of Preoperative Aspects and Dimensions Used for an Anatomical (PADUA) score for predicting complications in 74 consecutive partial nephrectomies.

Study Type – Prognosis (case series)

Development and validation of urine-based peptide biomarker panels for detecting bladder cancer in a multi-center study

Purpose: Urothelial bladder cancer presents high recurrence rates, mandating continuous monitoring via invasive cystoscopy. The development of noninvasive tests for disease diagnosis and surveillance remains an unmet clinical need. In this study, validation of two urine-based biomarker panels for detecting primary and recurrent urothelial bladder cancer was conducted. Experimental Design: Two studies (total n = 1,357) were performed for detecting primary (n = 721) and relapsed urothelial bladder cancer (n = 636). Cystoscopy was applied for detecting urothelial bladder cancer, while patients negative for recurrence had follow-up for at least one year to exclude presence of an undetected tumor at the time of sampling. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was employed for the identification of urinary peptide biomarkers. The candidate urine–based peptide biomarker panels were derived from nested cross-sectional studies in primary (n = 451) and recurrent (n = 425) urothelial bladder cancer. Results: Two biomarker panels were developed on the basis of 116 and 106 peptide biomarkers using support vector machine algorithms. Validation of the urine-based biomarker panels in independent validation sets, resulted in AUC values of 0.87 and 0.75 for detecting primary (n = 270) and recurrent urothelial bladder cancer (n = 211), respectively. At the optimal threshold, the classifier for detecting primary urothelial bladder cancer exhibited 91% sensitivity and 68% specificity, while the classifier for recurrence demonstrated 87% sensitivity and 51% specificity. Particularly for patients undergoing surveillance, improved performance was achieved when combining the urine-based panel with cytology (AUC = 0.87). Conclusions: The developed urine-based peptide biomarker panel for detecting primary urothelial bladder cancer exhibits good performance. Combination of the urine-based panel and cytology resulted in improved performance for detecting disease recurrence. Clin Cancer Res; 22(16); 4077–86. ©2016 AACR.

Zinc α2-glycoprotein as a potential novel urine biomarker for the early diagnosis of prostate cancer.

Study Type – Diagnosis (exploratory cohort)

Role of white blood cell and neutrophil counts in predicting spontaneous stone passage in patients with renal colic

Study Type – Prognosis (case series)

IMAC fractionation in combination with LC–MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers

Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

Radical cystectomy over the age of 75 is safe and increases survival.

BackgroundRadical cystectomy (RC) is probably underused in elderly patients due to a potential increased postoperative complication risk, as reflected by their considerable comorbidities. Our objective was to estimate the overall complication rate and investigate a potential benefit to patients over the age of 75 subjected to RC in terms of disease-free survival.MethodsA total of 81 patients, 61 men and 20 women, from two urological departments, with a mean age of 79.2 ± 3.7 years, participated in the study. The mean follow-up period was 2.6 ± 1.6 years. All patients underwent RC with pelvic lymphadenectomy. An ileal conduit, an orthotopic ileal neobladder and cutaneous ureterostomies were formed in 48.1%, 6.2% and 45.7% of the patients, respectively. The perioperative and 90-day postoperative complications were recorded and classified according to the modified Clavien classification system. Survival plots were created based on the oncological outcome and several study parameters.ResultsThe perioperative morbidity rate was 43.2%; the 90-day morbidity rate was 37%, while the 30-day, 90-day and overall mortality rates were 3.7%, 3.7% and 21%, respectively. Overall mortality rates were recorded at the final year of data gathering (2009). Increased age, increased body mass index (BMI), longer hospitalization and age-adjusted Charlson comorbidity index (ACCI) more than six, were associated with greater hazard for 90-day morbidity. The cumulative mortality / metastasis-free rates for one, two, three and five years were 88.7%, 77.5%, 70.4%, and 62.3%, respectively. Tumour stage and positive nodes were prognostic predictors for oncological outcome.ConclusionsRC in patients over 75 is justified and feasible, due to acceptable complication rates and high 5-year cancer-specific survival, which support an aggressive approach. Prospective studies are needed for the verification of the above results.

The role of PSA density to predict a pathological tumour upgrade between needle biopsy and radical prostatectomy for low risk clinical prostate cancer in the modified Gleason system era

OBJECTIVES We evaluate the role of prostate-specific antigen (PSA) density to predict Gleason score upgrade between prostate biopsy material and radical prostatectomy specimen examination in patients with low-risk prostate cancer. METHODS Between January 2007 and November 2011, 133 low-risk patients underwent a radical prostatectomy. Using the modified Gleason criteria, tumour grade of the surgical specimens was examined and compared to the biopsy results. RESULTS A tumour upgrade was noticed in 57 (42.9%) patients. Organ-confined disease was found in 110 (82.7%) patients, while extracapsular disease and seminal vesicles invasion was found in 19 (14.3%) and 4 (3.0%) patients, respectively. Positive surgical margins were reported in 23 (17.3%) patients. A statistical significant correlation between the preoperative PSA density value and postoperative upgrade was found (p = 0.001) and this observation had a predictive value (p = 0.002); this is in contrast to the other studied parameters which failed to reach significance, including PSA, percentage of cancer in biopsy and number of biopsy cores. Tumour upgrade was also highly associated with extracapsular cancer extension (p = 0.017) and the presence of positive surgical margins (p = 0.017). CONCLUSIONS PSA density represents a strong predictor for Gleason score upgrade after radical prostatectomy in patients with clinical low-risk disease. Since tumour upgrade increases the potential for postoperative pathological adverse findings and prognosis, PSA density should be considered when treating and consulting patients with low-risk prostate cancer.

A male presenting with a primary mucinous bladder carcinoma: a case report

BackgroundThe primary mucinous adenocarcinoma of the bladder is an extremely rare urologic entity, which is found in less than 2% of all urinary bladder tumours and is often presented as metastatic.Case presentationA 69-year old male patient was diagnosed with a primary mucinous adenocarcinoma of the bladder after undergoing a transurethral resection of a bladder tumour and complete examination of the entire gastrointestinal tract to rule out other primary cites. Immunohistochemistry confirmed the nature of the tumour. The patient underwent a radical cystoprostatectomy with en block bilateral pelvic lymphadenectomy and urinary diversion with a Bricker ileostomy.ConclusionThe primary adenocarcinoma creates a diagnostic dilemma, since it cannot be easily differentiated by the adenocarcinoma that originates from the colon and the prostate. We advocate the radical surgical management, after exclusion of any primary malignant sites related to the gastrointestinal tract. The immunohistochemistry has a leading role, assisting with the differential diagnosis.