Biomarker identification with ligand-targeted nucleoprotein assemblies.

AIMS: Since many biomarkers of both the tumor and its microenvironment are expected to involve differential expression of divalent proteins capable of protein or peptide ligand interaction, we are developing multivalent nanodevices for the identification of biomarkers in prostate cancer. PATIENTS & METHODS: We compared a multivalent thioredoxin-targeted nanodevice with monovalent thioredoxin in binding to human prostate cell line(s) and freshly frozen tissue specimens obtained after resection from patients with biopsy-proven prostate cancer. CONCLUSION: The nanodevice binds specifically with enhanced avidity to tumor microenvironment-associated stromal cells in prostate cancer tissue specimens. Cells that bind the nanodevice also reacted with antibodies to dimeric thioredoxin reductases 1 and 2, suggesting the utility of the nanodevice as a potentially specific and functional marker of tumor stromal cells.

Performance of a single assay for both type III and type VI TMPRSS2:ERG fusions in noninvasive prediction of prostate biopsy outcome.

BACKGROUND: TMPRSS2:ERG fusions are promising prostate cancer biomarkers. Because they can occur in multiple forms in a single cancer specimen, we developed a quantitative PCR test that detects both type III and type VI TMPRSS2:ERG fusions. The assay is quantified from a standard curve determined with a plasmid-cloned type III TMPRSS2:ERG fusion target. METHODS: We collected expressed prostatic secretion (EPS) under an institutional review board-approved, blinded, prospective study from 74 patients undergoing transrectal ultrasound-guided biopsy for prostate cancer. We compared the characteristic performance of the test for type III and type VI TMPRSS2:ERG fusions in predicting biopsy outcome and distinguishing between high and low Gleason scores with similar tests for the expression of PCA3 and DNA methylation levels of the APC, RARB, RASSF1, and GSTP1 genes. We used logistic regression to analyze the effects of multiple biomarkers in linear combinations. RESULTS: Each test provided a significant improvement in characteristic performance over baseline digital rectal examination (DRE) plus serum prostate-specific antigen (PSA); however, the test for type III and type VI TMPRSS2:ERG fusions yielded the best performance in predicting biopsy outcome [area under the curve (AUC) 0.823, 95% CI 0.728-0.919, P < 0.001] and Gleason grade >7 (AUC 0.844, 95% CI 0.740-0.948, P < 0.001). CONCLUSIONS: Although each test appears to have diagnostic value, PSA plus DRE plus type III and type VI TMPRSS2:ERG provided the best diagnostic performance in EPS specimens.

The impact of prostate gland weight in robot assisted laparoscopic radical prostatectomy.

PURPOSE: We determined whether prostate weight has an impact on the pathological and operative outcomes of robot assisted laparoscopic radical prostatectomy. MATERIALS AND METHODS: We reviewed the records of 1,847 consecutive patients who underwent robot assisted laparoscopic radical prostatectomy at our institution. Variables were compared across quartile distributions of prostate size as defined by weight, including group 1-less than 30 gm, group 2-30 to 49.9, group 3-50 to 69.9 and group 4-70 or greater. Factors assessed in this analysis were patient age, body mass index, prostate specific antigen, Gleason score, pathological stage, margin status, operative time, blood loss, transfusion rate, length of stay and rehospitalization rate. RESULTS: Patients with a larger prostate (group 4) were older (mean age 66.2 years), had higher pretreatment prostate specific antigen (median 6.5 ng/ml), lower Gleason score (mean 6.3), longer operative time (mean 3.2 hours), higher estimated blood loss (median 250 cc) and longer hospital stay (p = 0.0002). There was a trend toward higher risk disease based on D'Amico risk stratification and positive margin status in group 1, although evidence of extracapsular extension was more common in groups 2 and 3. There was no association between prostate size and body mass index, lymph node status, blood transfusion rate, seminal vesicle involvement and rehospitalization rate. CONCLUSIONS: Robot assisted laparoscopic radical prostatectomy in patients with an enlarged prostate is feasible with slightly longer operative time, urinary leakage rates and hospital stay. Pathologically larger prostates are generally associated with lower Gleason score and risk group stratification. One-year continence rates and biochemical recurrence rates are similar across all groups.

Multi-institutional study of symptomatic deep venous thrombosis and pulmonary embolism in prostate cancer patients undergoing laparoscopic or robot-assisted laparoscopic radical prostatectomy.

OBJECTIVES: The true incidence of symptomatic deep venous thrombosis (DVT) and pulmonary embolism (PE) in patients undergoing laparoscopic radical prostatectomy is unknown. Our aim was to determine the incidence of symptomatic DVT and PE and the risk factors for these complications. METHODS: Fourteen surgeons from 13 referral institutions from both Europe and the United States provided retrospective data for all 5951 patients treated with laparoscopic radical prostatectomy (LRP), with or without robotic assistance, since the start of their institution's experience. Symptomatic DVT and PE within 90 d of surgery were regarded as venous thromboembolism (VTE). DVT was diagnosed mostly by Doppler ultrasound or contrast venography and PE by lung ventilation/perfusion scan or chest computed tomography or both. Statistical analysis included evaluation of incidence of symptomatic DVT and PE and risk factors as determined by exact methods and logistic regression. RESULTS: Of 5951 patients in the study, 31 developed symptomatic VTE (0.5%; 95% confidence interval [CI], 0.4%, 0.7%). Among patients with an event, 22 (71%) had DVT only, 4 had PE without identified DVT, and 5 had both. Two patients died of PE. Prior DVT (odds ratio [OR]=13.5; 95%CI, 1.4, 61.3), current tobacco smoking (OR=2.8; 95%CI, 1.0, 7.3), larger prostate volume (OR=1.18; 95%CI, 1.09, 1.28), patient re-exploration (OR=20.6; 95%CI, 6.6, 54.0), longer operative time (OR=1.05; 95%CI, 1.02, 1.09), and longer hospital stay (OR=1.05; 95%CI, 1.01, 1.09) were associated with VTE in univariate analysis. Neoadjuvant therapy, body mass index, surgical experience, surgical approach, pathologic stage, perioperative transfusion, and heparin administration were not significant predictors. CONCLUSIONS: The incidence of symptomatic VTE after LRP is low. These data do not support the administration of prophylactic heparin to all patients undergoing LRP, especially those without risk factors for VTE.

Can robotic assisted laparoscopic prostatectomy be recommended to obese patients?

Experienced robotic surgeons report lower estimated blood loss (EBL) and transfusion rates with similar clinical outcomes for robotic assisted laparoscopic radical prostatectomy (RALP) compared to open radical retropubic prostatectomy (RRP). We reviewed our experience with RALP to investigate how to counsel overweight and obese patients being considered for RALP regarding risk of major perioperative complications, oncological outcomes, and short-term recovery. We reviewed the data on 1,513 patients who underwent RALP by one of four experienced robotic surgeons between June 2003 and November 2006. These patients were categorized as normal, overweight, obese, and severely obese based on body mass index (BMI). Of these patients, 361 (23.9%) had a BMI under 25, 794 (52.5%) had a BMI between 25 and 30 (overweight), 290 (19.2%) had a BMI between 30 and 35 (obese), and 68 (4.5%) had a BMI over 35 (severely obese). Records were examined and analyzed with respect to BMI for major intra- and perioperative complications, operative time, margin status, EBL, transfusion rates, and length of hospital stay. Increasing BMI was associated with longer operative time, with mean operative times of 2.7, 2.8, 3.1, and 3.3 h for normal, overweight, obese and severely obese patients, respectively (P < 0.0001). Higher blood loss, though not clinically significant, was also associated with increased BMI, with obese and severely obese patients losing a median of 250 mL of blood compared to 200 mL in overweight and non-overweight patients (P < 0.0001). Transfusion rates, intra- and perioperative complications, margin status, and length of stay were not found to be associated with BMI group. Robotic assisted laparoscopic radical prostatectomy in experienced hands can be safely recommended to eligible patients regardless of BMI status.

Perioperative complications of laparoscopic and robotic assisted laparoscopic radical prostatectomy.

PURPOSE: While it remains controversial whether LRP or da Vinci RAP offers any advantages over radical retropubic prostatectomy, LRP and RAP are being used more frequently. We reviewed our experience with these minimally invasive techniques. MATERIALS AND METHODS: We reviewed intraoperative and early postoperative complications of 358 LRPs performed from October 2000 to January 2003 with those of 322 RAPs performed from June 2003 to June 2004. The transperitoneal approach with bilateral pelvic lymph node dissection was performed using each technique. Data acquisition was done independently of the 3 surgeons. RESULTS: The LRP and RAP groups had similar clinical characteristics in terms of patient race, body mass index, prostate specific antigen, risk group, and pathological tumor grade and stage. Median operative time and estimated blood loss for LRP and RAP were 4.1 and 3.1 hours, and 200 and 250 ml, respectively. No blood transfusions were given intraoperatively, although 8 patients with LRP (2.2%) and 5 with RAP (1.6%) were transfused postoperatively. Of the LRP and RAP patients 21 (5.9%) and 3 (0.3%), respectively, experienced intraoperative complications. Postoperatively 48 patients with LRP (13.4%) and 24 with RAP (6.8%) experienced urine leakage, while 19 with LRP (5.3%) and 9 with RAP (2.8%) had ileus. There were no deaths, myocardial infarctions, pulmonary emboli or cerebrovascular accidents. CONCLUSIONS: In our series surgeon experience derived from LRP may contribute to the lower complication rate and operative time of RAP. Dissemination of surgical technique and management of complications may lead to improved perioperative LRP and RAP morbidity. However, the morbidity of these 2 approaches compares favorably with that of radical retropubic prostatectomy.

Construction of ordered protein arrays.

Artificially ordered protein arrays provide a facile approach to a variety of problems in biology and nanoscience. Current demonstration systems use either nucleic acid tethers or methyltransferase fusions in order to target proteins or peptides of interest to nucleic acid scaffolds. These demonstrations point to the large number of useful devices and assemblies that can be envisioned using this approach, including smart biological probes and drug delivery systems. In principle, these systems are now capable of imitating the earliest forms of prebiotic organisms and can be expected to reach the complexity of a small virus in the near future. Third-generation methyltransferase inhibitors provide an example of a smart chemotherapeutics that can be constructed with this approach. We describe the use of mechanistic enzymology, computer-aided design, and microfluidic chip-based capillary electrophoresis in assessing the final assembly and testing of designs of this type.

Prostate cancer molecular markers GSTP1 and hTERT in expressed prostatic secretions as predictors of biopsy results.

OBJECTIVES: To develop noninvasive diagnostic tools for the early detection of prostate cancer (PCa). Current screening for PCa lacks sensitivity and specificity. Two molecular markers, telomerase activity and aberrant methylation of the glutathione S-transferase P1 (GSTP1) promoter, are found in more than 90% of PCa specimens. Additionally, these markers can be detected in bodily fluids such as urine and postprostatic massage urethral washes. METHODS: Expressed prostatic secretions (EPS) from men being evaluated for PCa were analyzed for human telomerase reverse transcriptase (hTERT) expression (the critical factor for telomerase activity) and GSTP1 methylation status. The results were compared with the prostate needle biopsy findings. RESULTS: EPS could be obtained from 86% of all subjects, and 90% of these samples yielded sufficient RNA and/or DNA for assaying. hTERT expression from EPS (n = 49) had 36% sensitivity and 66% specificity, and GSTP1 methylation from EPS (n = 58) had 46% sensitivity and 56% specificity for the detection of PCa. The combined analysis (n = 32) of hTERT and GSTP1 had 73% sensitivity and 43% specificity, giving a positive predictive value of 40% and a negative predictive value of 75%. CONCLUSIONS: These results demonstrate that EPS can be successfully obtained and easily tested for hTERT expression and GSTP1 methylation. Tests with a high negative predictive value, such as our combination assay results, could be useful in augmenting current PCa diagnostic procedures. For example, the examination of EPS for hTERT and GSTP1 methylation in patients with an elevated prostate-specific antigen level might be used in predicting which patients will have negative biopsies. The use of this assay could potentially eliminate up to 30% of costly and invasive needle biopsies.

Mobility-shift analysis with microfluidics chips.

Electrophoretic mobility shift analysis (EMSA) is a well-characterized and widely used technique for the analysis of proten-DNA interaction and the analysis of transcription factor combinatorics. Currently implemented EMSA generally involves the time-consuming use of radiolabeled DNA and polyacrylamide gel electrophoresis. We are studying the bionanoscience of self-assembling supramolecular protein-nucleic nanostructures. We have undertaken these studies because they promise to enhance our understanding of assemblies formed during prebiotic evolution, provide tools for analysis of biological processes like DNA recombination, and may lead to the development of nanoscale biosensors designed for site-specific molecular targeting. During the course of that work, we noted that EMSA of these complex structures could be effectively implemented with microfluidics chips designed for the separation of DNA fragments. In this report we compare the two techniques and demonstrate that the microfluidics system is also capable of resolving complex mixtures produced by decorating DNA recombination intermediates with mixtures of DNA binding proteins. Moreover, the microfluidics chip system improves EMSA by permitting analysis with smaller samples, avoiding the use of radiolabeling, and reducing the time involved to a matter of minutes.