Aaron E. Katz

Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay

OBJECTIVE Because up to 40 percent of surgically treated patients with prostate cancer are subsequently found to be clinically understaged, a more sensitive staging modality to identify extraprostatic disease prior to surgery is required. METHODS We describe an enhanced reverse transcriptase [RT] polymerase chain reaction (PCR) assay utilizing oligonucleotide primers specific for the human prostate-specific antigen (PSA). This assay identifies PSA-synthesizing cells from reverse transcribed mRNA. This assay was applied to RNAs extracted from the peripheral blood lymphocytes of 65 patients with clinically localized prostate cancer. In addition, blood from 20 women, 20 young men, 25 age-matched control men under treatment for benign prostatic hyperplasia (BPH), and 18 men with established, untreated metastatic prostate cancer was tested. RESULTS An RT-PCR assay for PSA can recognize one PSA-expressing cell diluted into one hundred thousand lymphocytes. The sensitivity of this assay can be enhanced by the addition of digoxigenin-modified nucleotides to the PCR reaction and this assay was applied to RNAs extracted from the peripheral lymphocyte fraction of 148 prostate cancer patients and controls at this institution. Although no specimen from women or men without cancer was positive in this assay, 14 of 18 metastatic prostate cancer patients were positive (77.8%). Additionally, 25 of 65 (38.5%) patients with clinically localized disease (T1-2b) were positive from blood specimens obtained prior to surgery. Final pathologic results from this group of patients identified a correlation between positivity on this assay and the presence of capsular tumor penetration (sensitivity, 68%; specificity, 84%) as well as strong correlation with the finding of carcinoma at the surgical margin (sensitivity, 87%; specificity, 76%). Logarithmic regression analysis of the results of the RT-PCR assay indicates its remarkable superiority to digital rectal examination, computed tomography scan, endorectal coil magnetic resonance imaging, PSA, prostate-specific antigen density, or Gleason score for predicting the true pathologic stage of prostate cancer in these surgically treated patients. CONCLUSIONS An RT-PCR assay using PSA primers to detect prostate cells in the peripheral circulation of surgical-candidate patients is significantly correlated with capsular penetration and tumor-positive surgical margins. This molecular assay provides a sensitive and specific means to stage correctly apparent localized prostate cancer prior to radical prostatectomy.

Best Practice Statement on Cryosurgery for the Treatment of Localized Prostate Cancer

Introduction The protracted natural history of clinically localized prostate cancer has confounded the development of a national consensus regarding the optimal treatment for this disease. In the AUA 2007 Guideline for the Management of Clinically Localized Prostate Cancer: 2007 Update, multiple treatment modalities are considered as options. This conundrum is further complicated by stage migration and lead time bias, both associated with PSA-based early detection strategies and the resultant increase in the detection of small volume clinically localized cancers. Since the majority of men currently diagnosed with prostate cancer are likely to have the disease eradicated by one of several treatment modalities, the clinical focus on HRQL associated with treatment has intensified. There are no published long-term data on the efficacy of cryosurgery on metastasis-free, prostate cancerspecific, or overall survival as there are with other more established forms of therapy; however, several large, single institution experiences, a pooled analysis, and several prospective evaluation studies report the efficacy and morbidity of cryosurgery of the prostate. Additionally, prostate cryosurgery has been found to result in acceptable HRQLbased outcomes with a reduced cost when compared to other local therapeutic options. Short-term PSA relapse-free survival outcomes following cryoablation of the entire prostate comparable to radiation therapy in men with intermediateand high-risk disease have been reported. Bio-

Salvage Prostate Cryoablation: Initial Results From the Cryo On-Line Data Registry

PURPOSE We report contemporary outcomes of salvage cryoablation at a large number of centers which have participated in the COLD (Cryo On-Line Data) Registry. MATERIALS AND METHODS A secure online database was developed to collect data for patients undergoing prostate cryoablation. Kaplan-Meier analysis was performed with biochemical failure defined using the American Society of Therapeutic Radiology and Oncology, and the Phoenix definitions. RESULTS Data from 279 patients who had undergone salvage cryoablation were entered. Average patient age was 70.0 +/- 7.1 years. Pretreatment prostate specific antigen was 7.6 +/- 8.2 ng/ml and Gleason score was 7.5 +/- 1.1 (median 7). Patients were followed for 21.6 +/- 24.9 months and 47 were followed longer than 5 years. The 5-year actuarial biochemical disease-free rates were 58.9% +/- 5.7% (American Society of Therapeutic Radiology and Oncology) and 54.5% +/- 4.9% (Phoenix). As predicted based on the preservation of some prostatic tissue, 83% +/- 3.5% of patients had a detectable prostate specific antigen 0.2 ng/ml or greater at 5 years. Positive biopsies were observed in 15 of the 46 patients (32.6%) who underwent prostate biopsy after salvage cryotherapy. The incontinence rate (requiring pad use) was 4.4%. The rectal fistula rate was 1.2% and 3.2% of patients underwent transurethral prostate resection to remove sloughed tissue. CONCLUSIONS Biochemical and local control rates support the use of salvage cryoablation for localized recurrence following failed radiation therapy. Efforts to continue to minimize these complications and to improve disease control in patients with persistent cancer following definitive radiotherapy should continue.

SALVAGE CRYOTHERAPY USING AN ARGON BASED SYSTEM FOR LOCALLY RECURRENT PROSTATE CANCER AFTER RADIATION THERAPY: THE COLUMBIA EXPERIENCE

PURPOSE Cryosurgical ablation of the prostate has been reported as potential treatment for radioresistant clinically localized prostate cancer. We report our experience with the safety and efficacy of salvage cryosurgery using the argon based CRYOCare system (Endocare, Inc, Irvine, California). MATERIALS AND METHODS Between October 1997 and September 2000, 38 men with a mean age of 71.9 years underwent salvage cryosurgery for recurrent prostate cancer after radiation therapy failed. All patients had biochemical disease recurrence, defined as an increase in prostate specific antigen (PSA) of greater than 0.3 ng./ml. above the post-radiation PSA nadir. Subsequently prostate biopsy was positive for cancer. Pre-cryosurgery bone scan demonstrated no evidence of metastatic disease. In addition, these patients received 3 months of neoadjuvant androgen deprivation therapy before cryotherapy. RESULTS The PSA nadir was 0.1 or less, 1 or less and greater than 1 ng./ml. in 31 (81.5%), 5 (13.2%) and 2 (5.3%) patients, respectively. Biochemical recurrence-free survival calculated from Kaplan-Meier curves was 86% at 1 year and 74% at 2 years. Reported complications included rectal pain in 39.5% of cases, urinary tract infection in 2.6%, incontinence in 7.9%, hematuria in 7.9% and scrotal edema in 10.5%. The rate of rectourethral fistula, urethral sloughing and urinary retention was 0%. CONCLUSIONS Our study supports cryosurgery of the prostate as safe and effective treatment in patients in whom radiation therapy fails. Using the CRYOCare machine resulted in a marked decrease in complications.

Whole Gland Primary Prostate Cryoablation: Initial Results From the Cryo On-Line Data Registry

PURPOSE We report the largest data set to date to our knowledge regarding outcomes for primary whole gland prostate cryoablation. MATERIALS AND METHODS The COLD (Cryo On-Line Data) Registry consists of case report forms obtaining pretreatment and posttreatment information for patients undergoing whole gland prostate cryoablation. A total of 1,198 patients were stratified into low, intermediate and high risk groups. Biochemical success was defined according to the traditional American Society for Therapeutic Radiology and Oncology definition (3 increases) and the newer (Phoenix) definition (nadir +2). Biopsy was performed at physician discretion but most commonly for cause if a patient had an increasing or suspicious prostate specific antigen. RESULTS Average patient age was 69.8 +/- 7.5 years. Pretreatment prostate specific antigen was 9.6 +/- 8.6 ng/ml and median Gleason sum was 7 (range 4 to 10). Patients were followed for 24.4 +/- 25.9 months with 136 having minimum 5-year data. The 5-year biochemical disease-free status for the entire population was 77.1% +/- 2.1% (American Society for Therapeutic Radiology and Oncology) and 72.9% +/- 2.1% (Phoenix). Five-year American Society for Therapeutic Radiology and Oncology biochemical disease-free status was 84.7% +/- 4.5%, 73.4% +/- 4.3% and 75.3% +/- 3.7% for the low, moderate and high risk groups, respectively. Using the Phoenix definition the biochemical disease-free status was 91.1% +/- 2.9%, 78.5% +/- 3.6% and 62.2% +/- 4.9%, respectively. As predicted based on intentional preservation of some prostatic tissue, 72.5 +/- 1.8% had a detectable prostate specific antigen 0.2 ng/ml or greater at 5 years. Biopsy after cryotherapy was positive during empiric without cause biopsy in 30 of 207 patients (14.5%), and the highly selected group biopsied based on suspicion of treatment failure due to abnormal or increasing prostate specific antigen had positive results in 38.0% (49 of 129). The rectal fistula rate was 0.4% and incontinence was 4.8% with 2.9% of patients using pads. Intercourse was reported by 25.2% but only 8.8% without pharmaceutical or device assistance. CONCLUSIONS Whole gland cryoablation, practiced in a spectrum of academic and community users, maintains efficacy and morbidity similar to that of single center reports.

Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia

OBJECTIVES Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cells ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH. METHODS A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker. RESULTS As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001). CONCLUSIONS Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.

Enhanced reverse transcriptase-polymerase chain reaction for prostate specific antigen as an indicator of true pathologic stage in patients with prostate cancer

Background. As up to 50% of all patients with prostate cancer who have undergone radical prostatectomy are found to be understaged subsequent to surgery, a more sensitive early staging modality currently is needed. A molecular assay that detects prostate specific antigen (PSA)‐synthesizing cells in the peripheral circulation of patients with prostate cancer is described.

Preoperative Reverse Transcriptase Polymerase Chain Reaction for Prostate Specific Antigen Predicts Treatment Failure Following Radical Prostatectomy

PURPOSE We previously demonstrated than an enhanced reverse transcriptase-polymerase chain reaction assay for prostate specific antigen (PSA) can predict final pathological stage in radical prostatectomy patients. The potential role of the assay in predicting serum PSA recurrence after radical prostatectomy was explored. MATERIALS AND METHODS We evaluated 100 radical prostatectomy candidates by reverse transcriptase polymerase chain reaction preoperatively, and status was compared to serum PSA, Gleason score and final pathological results. Potential surgical failure was defined as tumor at the surgical margin or extending into the seminal vesicle. Patients were monitored postoperatively by serum PSA every 4 months. Kaplan-Meier analysis was used to evaluate the correlation between reverse transcriptase polymerase chain reaction and disease recurrence, defined as a PSA of 0.2 ng/ml. or greater. RESULTS Enhanced reverse transcriptase polymerase chain reaction for PSA had a stronger correlation with potential surgical failure than preoperative serum PSA or Gleason score (relative risks 15.2, 5.9 and 3.2, respectively). The correlation between these modalities and PSA recurrence was evaluated during a mean followup of 13.6 months (range 5 to 26). Of 36 patients with positive reverse transcriptase polymerase chain reactions 9 had failure by PSA compared to 3 of 64 (4.7%) with negative polymerase chain reactions (p<0.0286). The relative risk for failure by reverse transcriptase polymerase chain reaction was 3.6. Gleason score and serum PSA had higher correlations with postoperative PSA elevations (relative risk 13.2 and 7.6, respectively). A Cox regression analysis model demonstrated that reverse transcriptase polymerase chain reaction for PSA can be used in conjunction with Gleason score and provides statistically significant risk information. CONCLUSIONS Enhanced reverse transcriptase polymerase chain reaction for PSA is a statistically significant predictor of potential failure by pathological analysis and of disease recurrence by PSA. Longer followup data are required to define further the role of the assay in the management of patients with prostate cancer.

Salvage cryotherapy for recurrent prostate cancer after radiation therapy: the Columbia experience.

OBJECTIVES Cryotherapy of the prostate represents a potential treatment for localized recurrent prostate cancer after radiation therapy. We report our experience and evaluate the predictive factors for prostate-specific antigen (PSA) recurrence. METHODS Between October 1994 and April 1999, 43 patients underwent salvage cryoablation. All patients had biopsy-proven recurrent prostate cancer without seminal vesicle invasion, negative bone scans, and negative lymph node dissection. Patients had received 3 months of combined hormonal therapy before cryosurgery. Biochemical recurrence-free survival (bRFS) was defined as a PSA value less than 0.1 ng/mL. RESULTS Complications included incontinence (9%), obstruction (5%), urethral stricture (5%), rectal pain (26%), urinary infection (9%), scrotal edema (12%), and hematuria (5%). The mean follow-up was 21.9 months (range 1.2 to 54). Twenty-six patients (60%) reached a serum PSA nadir less than 0.1 ng/mL, 16 (37%) had a PSA less than 4 ng/mL, and 1 (3%) had a PSA less than 10 ng/mL. The bRFS rate was 79% at 6 months and 66% at 12 months. The bRFS rate was higher for patients who had an undetectable postcryotherapy PSA than for patients who did not reach a PSA less than 0. 1 ng/mL (73% versus 30%, P = 0.0076). Using multivariate analysis, a PSA nadir greater than 0.1 ng/mL was an independent predictor of PSA recurrence. CONCLUSIONS Current salvage cryotherapy of the prostate can result in undetectable serum PSA levels with low morbidity. Our data support the current safety and efficacy profile. We believe that cryotherapy is a viable option in the treatment of patients who have biopsy-proven local failure after radiation therapy for prostate cancer. Further refinements in technique and equipment may enhance cryosurgical results.

Microvessel density as a predictor of PSA recurrence after radical prostatectomy. A comparison of CD34 and CD31.

Whether prostate cancer recurrence can be predicted by microvessel density (MVD) measurements is controversial. One reason for the lack of agreement may be the differing antibodies used to determine MVD. We evaluated MVD using 2 different antibodies against endothelial cells, CD31 and CD34, on 102 patients who underwent radical prostatectomy without adjuvant hormonal therapy. The tumors from these cases were identified, and areas with the highest Gleason pattern were immunostained. Average MVD determined by CD31 (MVD/CD31) staining was significantly lower than that obtained by MVD/CD34 staining (60.1 vs 80.3). By using Kaplan-Meier analysis, prostate-specific antigen (PSA) recurrence was correlated with MVD/CD31 and MVD/CD34. MVD/CD34 and MVD/CD31 were associated strongly with PSA recurrence on a univariate level. However, only MVD/CD34 was an independent predictor of PSA failure. Therefore, some of the confusion about MVD value as a prognostic indicator may be due to the antibodies used.