Clinical Use of PCA3 and TMPRSS2:ERG Urinary Biomarkers in African-American Men Undergoing Prostate Biopsy.

PURPOSE: Prostate specific antigen has decreased performance characteristics for the detection of prostate cancer in African-American men. We evaluated urinary PCA3 and TMPRSS2:ERG in a racially diverse group of men. MATERIALS AND METHODS: After institutional review board approval, post-examination urine was prospectively collected before prostate biopsy. PCA3 and TMPRSS2:ERG RNA copies were quantified using transcription mediated amplification assays (Hologic, San Diego, California). Prediction models were created using standard of care variables (age, race, family history, prior biopsy, abnormal digital rectal examination) plus prostate specific antigen. Decision curve analysis was performed to compare the net benefit of PCA3 and TMPRSS2:ERG. RESULTS: Of 304 patients 182 (60%) were African-American and 139 (46%) were diagnosed with prostate cancer (69% African-American). PCA3 and TMPRSS2:ERG scores were greater in men with prostate cancer, 3 or more cores, 33.3% or more cores, greater than 50% involvement of greatest biopsy core and Epstein significant prostate cancer (p <0.01). PCA3 added to the standard of care plus prostate specific antigen model for the detection of any prostate cancer in the overall cohort (0.747 vs 0.677, p <0.0001) in African-American men only (0.711 vs 0.638, p=0.0002) and nonAfrican-American men (0.781 vs 0.732, p=0.0016). PCA3 added to the model for the prediction of high grade prostate cancer for the overall cohort (0.804 vs 0.78, p=0.0002) and African-American men only (0.759 vs 0.717, p=0.0003) but not nonAfrican-American men. Decision curve analysis demonstrated improvement with the addition of PCA3. For African-American men TMPRSS2:ERG did not improve concordance statistics for the detection of prostate cancer. CONCLUSIONS: For African-American men urinary PCA3 improves the ability to predict the presence of any and high grade prostate cancer. However, the TMPRSS2:ERG urinary assay does not add significantly to standard tools.

Racial variation in prostate needle biopsy templates directed anterior to the peripheral zone.

OBJECTIVES: African Americans (AA) have been reported to have both increased incidence and increased aggressiveness of prostate cancer (PCa) located anterior to the peripheral zone (APZ). We sought to evaluate the utility of prostate biopsies directed toward the APZ in a predominantly AA cohort. METHODS AND MATERIALS: We reviewed all patients with PCa found on biopsy schema that included needle biopsies directed at both the peripheral zone (PZ) and APZ from 2010 to 2014. Self-identified race was recorded for all patients. To evaluate the reliability of APZ-directed prostate biopsies, we performed pathologic secondary review of 25 radical prostatectomy specimens. A series of the Mann-Whitney U and Chi-square tests were used to compare variables. RESULTS: We identified 398 men, of which 277 (70%) were AA. Compared with non-AA, AA had more National Comprehensive Cancer Network-defined intermediate or high-risk (50% vs. 39%, P = 0.25) PCa. Most patients had PCa limited to the PZ only (n = 190) or in both the PZ and APZ (n = 191). For 17 patients (4%), PCa was limited only to the APZ core(s), 14 (5%) AA vs. 3 (2%) non-AA (P = 0.24). Most of these 17 patients (n = 14, 82%) had Gleason 6 disease. Patients with PCa in both the PZ and APZ had higher serum prostate-specific antigen, prostate-specific antigen density, volume of disease, and increased grade and National Comprehensive Cancer Network category (all P<0.01). Of these patients, there were no differences in race (AA = 135, 71% vs. non-AA = 56, 29%; P = 0.48). In only 21 men (11%), without racial variation, APZ tumor grade was greater than PZ. Radical prostatectomy and APZ-directed biopsies demonstrated a concordance rate of 80% (20/25), false positive rate of 8% (2/25), and false negative rate of 12% (3/25). CONCLUSIONS: APZ-directed prostate biopsies are rarely the sole location of PCa and do not show a clear racial predilection. In those men with PCa identified in both regions, the APZ biopsy did not frequently change treatment recommendations. Biopsies directed at the APZ are not of greater benefit to AA than non-AA.

Methodology of patient care for elevated PSA after prostate cancer treatment: A primary care perspective.

Prostate cancer (PCa) remains one of the most common cancers in men. Each year there are 230,000 new cases and it causes nearly 30,000 deaths. Elevations in prostate-specific antigen (PSA) after treatment with radiation or surgery can indicate the presence of PCa recurrence. About 15-18% of men treated for PCa will have an elevation of their PSA, which is a source of great concern and will lead to a management discussion with the patient. Approximately 25-33% of men with PCa will experience a recurrence of their cancer after surgery or radiation. This article will discuss the different options for men with a rising PSA after definitive therapy or an attempt at curative treatment for PCa. The purpose of this article is to help the primary care doctor, internist and geriatrician to become knowledgeable about the treatment options of recurrent PCa so that they can participate in the discussion with the patient and help the patient reach a decision regarding treatment and management of the elevated PSA levels, which signal recurrent disease.

A simplified baculovirus-AAV expression vector system coupled with one-step affinity purification yields high-titer rAAV stocks from insect cells.

Scalable methods of recombinant adeno-associated virus (rAAV) production have gained much recent interest as the field of rAAV-mediated gene therapy approaches the clinic. In particular, the production of rAAV vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of rAAV production. Here, we describe a method for the production of rAAV serotypes 1 and 2 in insect cells using a simplified baculovirus-AAV expression vector system coupled with particle purification via affinity chromatography. The number of separate baculovirus constructs required for rAAV production was reduced by genetically modifying the AAV rep gene to allow expression of the AAV-encoded replication enzymes, Rep78 and Rep52, from a single mRNA species and combining the modified rep gene with an AAV cap gene expression cassette in a single baculovirus construct. Additionally, we describe lysis, binding, and elution conditions compatible with a commercially available affinity medium (AVB Sepharose High Performance) used to purify rAAV particles to near homogeneity in a single chromatography step. Using the described method, we obtained an average yield of 7 x 10(4) purified rAAV particles per cell (range: 3.7 x 10(4) to 9.6 x 10(4)) from suspension cultures of recombinant baculovirus-infected insect cells.

Economized large-scale production of high yield of rAAV for gene therapy applications exploiting baculovirus expression system.

BACKGROUND: The versatility of recombinant adeno-associated vector (rAAV) as a gene delivery system is due to the vector's ability to transduce different cell types as well as dividing and non-dividing cells. Large-scale production of rAAV remains one of the major challenges for continued development of pre-clinical and clinical studies, and for its potential commercialization. The baculovirus expression vectors (BEVS) and insect cells represent a potential method to produce rAAV economically at large scale. This technology uses three different BEVs (Bac-Rep, Bac-GFP, and Bac-VP) each at a multiplicity of infection (MOI) of 3. We reported previously the production of rAAV at 40 L scale using a stirred-tank bioreactor (STB). However, production in larger volumes is limited by the stability of the BEVs and amount of BEVs needed to achieve the target MOI of 3 per BEV. Here, the production parameters were optimized and the baculovirus stability was determined. METHODS: The stability of the three types of baculovirus used to produce rAAV was determined for six expansion passages by protein expression analysis. To economize baculovirus, MOI and cell density at time of infection (TOI) were evaluated initially at small scale and then applied to the 10 L scale. RESULTS: An MOI = 0.03 and TOI cell density of 1 x 10(6) cells/mL produced high titer rAAV without comprising yield. To confirm the scalability of the process, rAAV was produced in a 10 L STB using the optimized parameters obtaining a 10x increase in yield ( approximately 1 x 10(14) rAAV DNAse-resistant particles per liter). CONCLUSION: These findings contribute to the process development for large-scale production of rAAV for gene therapy applications and its commercialization.