Prostate-specific Antigen Reverse Transcriptase-polymerase Chain Reaction Research Articles

Molecular staging of prostatic cancer with RT-PCR assay for prostate-specific antigen in peripheral blood and lymph nodes: 5 Year follow-up

Thirty percent of patients with localized prostate cancer undergoing radical prostatectomy experience biochemical recurrence with rising serum prostate-specific antigen (PSA). More than 50% of these develop distant metastases. Presence of PSA mRNA in pathologically normal pelvic lymph nodes from 154 patients undergoing radical prostatectomy was investigated with non-quantitative PSA reverse transcriptase polymerase chain reaction (RT-PCR). In 135 of these patients preoperative serum PSA RT-PCR was also assessed. RT-PCR positivity was correlated with biochemical recurrence and compared with other clinical risk factors. At a median follow-up of 58 months the biochemical failure-free survival of patients with positive versus negative lymph node RT-PCR was 68.4% and 76.7% respectively (p=0.2). Biochemical failure-free survival was not influenced by the serum PSA RT-PCR result either (72.3% versus 72.6%). Surgical margin status, preoperative serum PSA, pT category and Gleason score were independent prognostic risk factors for biochemical recurrence with a hazard ratio of 5.48, 2.56, 2.56 and 2.13 respectively. At 5 year follow-up after radical prostatectomy, both serum and lymph node RT-PCR are not correlated with biochemical failure-free survival. Established clinical risk factors have a much stronger impact on biochemical recurrence.

The detection and isolation of viable prostate-specific antigen positive epithelial cells by enrichment: A comparison to standard prostate-specific antigen reverse transcriptase polymerase chain reaction and its clinical relevance in prostate cancer

Purpose To isolate prostate epithelial cells from the peripheral blood and bone marrow, and compare prostate-specific antigen (PSA) reverse transcriptase polymerase chain reaction (RT-PCR) performed on unenriched or epithelial enriched peripheral blood and bone marrow samples. Patients and methods Peripheral blood samples from 371 patients with prostate cancer and 141 controls, and bone marrow samples from 292 patients with prostate cancer and 43 controls were obtained. One aliquot was assessed with PSA RT-PCR. Another was enriched for epithelial cells with paramagnetic immune microbeads and assessed for: (1) PSA immunohistochemistry, (2) PSA RT-PCR, and (3) immunofluorescent detection of epithelial cells. Results In the bone marrow ( P < 0.01), but not the peripheral blood ( P = 0.62), we observed significantly higher detection rates of disseminated PSA expressing epithelial cells after enrichment. The presence of epithelial cells with or without evidence of PSA production was uncommon among controls both in peripheral blood (1% and 0%) and bone marrow (11% and 0%). In patients with active prostate cancer, 46% to 74% had epithelial cells in peripheral blood, and 20% to 64% had PSA expressing epithelial cells. In bone marrow, 55% to 92% had epithelial cells, and 43% to 83% had PSA expressing epithelial cells. Particularly in bone marrow, circulating cells were frequently detected in men without evidence of disease after prostatectomy. With limited follow-up, the detection of epithelial cells or PSA expressing epithelial cells in peripheral blood or bone marrow before radical prostatectomy does not define a population of patients that will have biochemical failure. Conclusions Immunomagnetic enrichment frequently detects epithelial, presumably malignant, cells in the peripheral blood and, especially, the bone marrow of patients with prostate cancer. Viable cells can be acquired for gene expression and phenotyping studies.

Relationships between reverse transcriptase-polymerase chain reaction for prostate specific antigen, survival, and various prognostic laboratory factors in patients with hormone refractory prostate cancer

The detection of prostate-specific antigen (PSA) mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) in the bloodstream of prostate cancer patients has been hypothesized as a prognostic marker, however little data are available concerning the association between this molecular marker and other laboratory values of potential importance. In this study, in patients with hormone-refractory prostate cancer (HRPC), relationships were determined between PSA RT-PCR positivity, survival, and various relevant markers including serum PSA, LDH, albumin, alkaline phosphatase and hemoglobin. A total of 19/30 HRPC patients were positive for PSA by RT-PCR. Positivity was significantly linked to serum PSA ( P = 0.004) and serum alkaline phosphatase ( P = 0.026) but not to the other laboratory variables. Median survival time for RT-PCR-positive patients was 9 months, compared to 19 months for RT-PCR-negative patients ( P = 0.035). Median survival time for patients with a hemoglobin ≥11 g/dL was 12 months, compared to 9 months for patients with <11 g/dL ( P = 0.005). Dichotomized (≥ or < median) serum PSA, LDH, alkaline phosphatase, and albumin were not significantly associated with survival in univariate analyses. In multivariate analysis, only dichotomized hemoglobin (<11g/dL vs. ≥11g/dL) remained statistically significant ( P = 0.019), indicating that RT-PCR had no independent association with survival after controlling for hemoglobin status in this study.

Detection and isolation of prostate cancer cells from peripheral blood and bone marrow

Objectives To detect and isolate disseminated prostate cancer cells because significant effort has been directed toward defining the characteristics of the primary tumor that predict progression, but little progress has been made on evaluating the disseminated prostate cancer cell. Prostate-specific antigen (PSA) reverse transcriptase-polymerase chain reaction results in the bone marrow (BM) and peripheral blood (PB) of men with prostate cancer suggest many have disseminated cancer cells. Methods Disseminated epithelial cells were isolated from the BM and PB using Miltenyi antibody-coated paramagnetic microparticle technology. In the two-step selection process, anti-CD45 and anti-CD61 were used for negative selection, and anti-human epithelial antigen was used for positive selection. Cells were then stained for Ber-EP4 (a distinct epitope of the human epithelial antigen) and PSA. PSA reverse transcriptase-polymerase chain reaction was performed on an enriched aliquot. Results The normal controls were negative. Before prostatectomy, PSA-expressing epithelial cells were detected in 54% of BM and 24% of PB samples. At a median of 4 months after prostatectomy, PSA-expressing cells were detected in 33% of BM and 9% of PB specimens from men without evidence of disease. In men more than 5 years after prostatectomy, PSA-expressing cells were detected in the BM of 4 (29%) of 14, 2 of whom subsequently developed evidence of disease recurrence. Conclusions The findings suggest that dissemination of cells is an early event in prostate cancer that is insufficient for the development of metastases. Isolation will allow interrogation of the phenotype and genotype of the cells.

DETECTION OF MICROMETASTATIC PROSTATE CANCER CELLS IN THE LYMPH NODES BY REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION IS PREDICTIVE OF BIOCHEMICAL RECURRENCE IN PATHOLOGICAL STAGE T2 PROSTATE CANCER.

We evaluated whether detecting prostate cancer cells by the nested reverse transcriptase-polymerase chain reaction (RT-PCR) in lymph nodes has predictive value for serum prostate specific antigen (PSA) recurrence in patients undergoing radical prostatectomy. We assessed the presence of prostate cancer cells by RT-PCR for prostate specific membrane antigen and PSA assay in lymph nodes dissected from 38 patients with localized prostate cancer treated with radical prostatectomy. The results of nested RT-PCR assay were compared with biochemical recurrence. Nested RT-PCR was positive in the lymph nodes of 2 of 18 patients (11%) with stage pT2a and 5 of 20 (25%) with stage pT2b disease. All 7 patients had biochemical recurrence. We noted a significant difference in the Kaplan-Meier recurrence-free actuarial probability curve in those with positive and negative nested RT-PCR results for prostate specific membrane antigen, PSA and prostate specific membrane antigen-PSA in the lymph nodes (p = 3.02x10(-7), 2.23x10(-7) and 3.02x10(-7), respectively). Multivariate analysis of serum PSA, Gleason score and preoperative RT-PCR assay in peripheral blood showed that nested RT-PCR for prostate specific membrane antigen, PSA and prostate specific membrane antigen-PSA in the lymph nodes were independent predictors of recurrence (p = 0.0089, 0.0075 and 0.0089, respectively). Nested RT-PCR of the lymph nodes may be a useful pretreatment prognostic test for patients undergoing radical prostatectomy. Further research is necessary using a much larger number of patients with a longer followup.

Detection of Prostate-Specific Antigen- or Prostate-Specific Membrane Antigen-Positive Circulating Cells in Prostatic Cancer Patients: Clinical Implications

Objectives: To evaluate the clinical benefit from using circulating prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSM) mRNA detection in prostate cancer staging and in follow-up. Methods: Nested reverse transcriptase-polymerase chain reaction (RT-PCR) assays were performed on RNA extracted from blood drawn from 56 patients with prostate cancer before any treatment. Additionally, assays were done on posttreatment samples from 50 patients who were followed up by serum PSA level, to determine whether any relationship exists between RT-PCR results and tumor recurrence. The prostate cell specificity of assays was evaluated by analysis of 21 blood samples from women or cystoprostatectomized men. Results: With PSM RT-PCR assay, good sensitivity and prostate cell specificity could not be attained together, since high PSM mRNA illegitimate expression has been shown in some healthy donor bloods. For this reason, only PSA RT-PCR assay was used as a clinical marker. PSA mRNA was detected in peripheral blood of 4 out of 31 patients with clinically localized prostate cancer. It showed no relationship to the pathologic stage, but significant relationship to metastatic status, lymph node involvement and Gleason score. During follow-up, circulating PSA mRNA was detected in 8 out of 17 (47%) patients in treatment failure and in only 1 out of 33 (3%) successfully treated patients, with significant relationship between RT-PCR results and concomitant serum PSA levels. Conclusion: Our study reveals no significant advantage to PSA RT-PCR assay (1) in improving the staging of clinically localized prostate cancer or (2) in follow-up treatment failure, as compared to the usual recurrence marker (serum PSA). Additional investigations are needed to determine the ultimate significance and the management of patients with positive PSA RT-PCR assays.

MOLECULAR DETECTION OF PROSTATE CELLS IN EJACULATE AND URETHRAL WASHINGS IN MEN WITH SUSPECTED PROSTATE CANCER

Purpose To determine whether prostatic cells were normally present in ejaculate and if the sensitivity and specificity of the detection of malignant prostate cells in ejaculate and urethral washings from men with suspected prostate cancer could be improved using the more sensitive molecular technique of reverse transcriptase-polymerase chain reaction (RT-PCR). Materials and Methods RT-PCR for prostate-specific antigen (PSA), prostate-specific membrane antigen (PSM) and Apoliprotein D (3 putative prostate-specific and/or cancer-specific markers) was performed on RNA extracts of ejaculate (80) and urethral washings (52) from 77 men with suspected prostate cancer and 12 young controls (<30 years of age) and urines from 5 men who had radical prostatectomies and 10 women. Results PSA, PSM and Apolipoprotein D expression was detected in ejaculates and urethral washings from both patient and control groups. No differences were observed in the results obtained for 58 men with suspected or 19 men with confirmed prostate cancer or the 18 vasectomized men within the patient group. Urines from the 5 men who had radical prostatectomies and 10 women were all negative for PSA, but PSM was detected in 2 female urines and in 3 radical prostatectomy samples. As few as 10 LNCaP prostate tumor cells could be detected by PSA RT-PCR when added to female urine. Conclusion We have established a sensitive method of detecting prostatic cells in ejaculate and urethral washings and shown that PSA RT-PCR is a reliable indicator of prostate cells in these samples. However, RT-PCR for PSA, PSM and Apoliprotein D were not useful for discriminating malignant from non-malignant prostate cells.

Effect of prostate biopsy on the results of the PSA RT-PCR test

Objectives. To evaluate the effect of prostate biopsy on the prostate-specific antigen (PSA) reverse transcriptase-polymerase chain reaction (RT-PCR) test. Methods. Ninety men who were scheduled to undergo prostate biopsy because of an elevated PSA or abnormal digital rectal examination, or both, were recruited to have PSA RT-PCR performed on peripheral blood samples drawn before and at 30 minutes, 1 week, and 1 month after undergoing prostate biopsy. PSA RT-PCR was performed and all PCR products were blotted and hybridized with phosphorus-32 ( 32-P)-PSA cDNA probe (exon 3 to 5). Results. Of 90 patients, 77 had a negative prebiopsy PSA RT-PCR result. Of these 77, 2 (2.6%) had a positive PSA RT-PCR result at some point after the biopsy procedure. Both of these patients had no evidence of malignancy on biopsy. The PSA RT-PCR test was positive at 30 minutes for 1 patient, but was negative at 1 week; the other was positive at 1 week but the patient did not return for the 1-month sample. Conclusions. Our study indicates that 2.6% of patients with an initially negative PSA RT-PCR will have a positive PSA RT-PCR test after biopsy has been performed. Although this is uncommon, it may have profound implications for those patients in whom it occurs. On the basis of our results, it appears that one should wait longer than 1 week after prostate biopsy before obtaining blood for PSA RT-PCR testing to decrease the likelihood of a spurious PSA RT-PCR result.

THE VALUE OF A REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION ASSAY IN PREOPERATIVE STAGING AND FOLLOWUP OF PATIENTS WITH PROSTATE CANCER

Purpose The reverse transcriptase polymerase chain reaction (RT-PCR) assay is an extremely sensitive technique of detecting cells expressing prostate specific antigen (PSA). Controversy exists regarding the ability of peripheral blood PSA RT-PCR testing to reflect pathological staging or treatment outcome. We examine the phenomenology of RT-PCR results in patients with prostate cancer, with particular emphasis on the RT-PCR test before and after radical prostatectomy, and correlations with pathological staging and treatment outcome. Materials and Methods Peripheral blood was obtained from a wide variety of patients with and without prostate cancer, including before and after radical prostatectomy. After ribonucleic acid isolation, complementary deoxyribonucleic acid was generated and amplified with a hot-start technique. RT-PCR results were compared with pathological stage, Gleason score, tumor volume and disease-free status. Correlations between preoperative and postoperative RT-PCR tests were also made. Results The RT-PCR test was positive in 1 of 56 controls (1.8%) without suspicion of prostate cancer. A positive test was obtained in 12 of 65 men (18.5%) with a suspicion of prostate cancer but a negative biopsy. Before radical prostatectomy a positive test was obtained in 13 of 75 men (17.3%) with pT2 disease versus 10 of 46 (21.7%) with pT3 disease. There was no significant difference in serum PSA, Gleason score or tumor volume between the men with positive or negative results. With repetitive testing an increasing percentage of men had at least 1 positive test preoperatively. With a median followup of 8 months 6 of the 7 patients in whom radical prostatectomy failed had had negative RT-PCR before treatment. Of patients with known metastatic disease or failed primary treatment a positive test was obtained in 32 to 75%. Radical prostatectomy and prostate needle biopsy appeared to have a negligible effect on RT-PCR tests immediately following these procedures. Following radical prostatectomy results were variable but many men who are RT-PCR positive preoperatively become RT-PCR negative postoperatively. Conclusions The PSA RT-PCR test in our laboratory cannot be used preoperatively to predict pathological stage of prostate cancer or treatment failure. Most cases that are positive preoperatively become negative postoperatively. While increasing tumor burden increases the likelihood of positive tests, there appears to be significant sampling error associated with the use of this test in the peripheral blood.

Influence of Blood Storage and Sample Processing on Molecular Detection of Circulating Prostate Cells in Cancer

Although not in routine use, molecular techniques appear able to detect small numbers of solid tumor cells in blood, lymph nodes, and bone marrow (for review see 1 ). Provided that a given mRNA is expressed exclusively in tumor tissue and not by nucleated blood cells, lymph node cells, or bone marrow cells, its presence outside the organ identifies disseminated metastatic cells. In prostate cancer (CaP), early detection of metastases by molecular staging could help in selecting which patients would benefit most from surgical therapy (2). Several reverse transcriptase-polymerase chain reaction (RT-PCR) protocols are able to amplify the mRNAs encoding prostate-specific antigen (PSA) or prostate-specific membrane antigen (PSMA) in blood (1), lymph nodes (3), or bone marrow (4). Nevertheless, the results reported have varied so widely (e.g., positive PSA RT-PCR results ranging from 25% (5) to 80% (6) for metastatic CaP patients) that definitive conclusions about the clinical relevance of this new molecular tool have not yet been established. The heterogeneity of both preanalytical and analytical steps of PCR protocols may explain the discrepancies observed between studies. To our knowledge, no systematic studies of the successive steps of the RT-PCR method have yet been performed. To better standardize the assay, we have assessed the effects of the storage conditions of blood samples and delay before processing. To compare the effect of delayed vs immediate sample processing, we collected 250-mL blood samples in 25 10-mL EDTA-treated tubes (Becton Dickinson) from 4 …

Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow.

This study was designed to evaluate the potential of reverse-transcriptase polymerase chain reaction (RT-PCR) analyses for the detection of micrometastatic carcinoma cells in bone marrow (BM). The specificity of RT-PCR assays with primers specific for various tumor-associated and organ-specific mRNA species was examined by analysis of 53 BM aspirates from control patients with no epithelial malignancy. In addition, BM samples from 63 patients with prostate cancer (n = 53) or breast cancer (n = 10) were analyzed by RT-PCR with primers specific for prostate-specific antigen (PSA) mRNA. As a reference method, all samples were analyzed simultaneously by an established immunocytochemical assay, using monoclonal antibodies (mAbs) against cytokeratins (CK) for tumor-cell detection. Seven of eight marker species could be detected in a considerable number of BM samples from control patients: epithelial glycoprotein-40 (EGP-40; 53 of 53 samples), desmoplakin I (DPI I; five of five), carcinoembryonic antigen (CEA; five of 19), erb-B2 (five of seven), erb-B3 (six of seven), prostate-specific membrane antigen (PSM; four of nine), and CK18 (five of seven). Only PSA mRNA was not detected in any of the 53 control BM samples. In serial dilution experiments, the PSA RT-PCR assay was able to detect five LNCaP prostate carcinoma cells in 4 x 10(6) BM cells. CK-positive cells were found in 20 patients (37.7%) with prostate cancer, while PSA mRNA was found in only 15 (28.3%; P = .04). Moreover, despite the recent observation that PSA is also expressed in mammary carcinomas, none of the 10 CK-positive BM samples were PSA mRNA-positive. Limiting factors in the detection of micrometastatic tumor cells by RT-PCR are (1) the illegitimate transcription of tumor-associated or epithelial-specific genes in hematopoietic cells, and (2) the deficient expression of the marker gene in micrometastatic tumor cells.

Reverse transcriptase-polymerase chain reaction for prostate-specific antigen may be a prognostic indicator in breast cancer.

Among women with node-negative breast cancer and small tumours, it is important to identify those with tumours that will recur, so that they may receive adjuvant therapy, while sparing those with tumours that will not recur the hazards of adjuvant treatment. A reverse transcriptase-polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) may be used to identify circulating metastatic cells in patients with prostate cancer. Approximately 30% of breast cancer cells also produce PSA. Therefore, we tested the PSA RT-PCR assay on blood specimens from women with breast cancer. We evaluated 78 women at Mount Sinai Medical Center with histologically confirmed breast cancer. Venous blood (5 cm3) from the women was collected in ethylene diaminetetraacetic acid (EDTA)-treated collection tubes and approximately 400 ng of RNA from each sample was subjected to an RT-PCR. We were able to detect the amplified PSA fragment in 18 of 78 women with breast cancer; 7 of the 18 women with the PSA fragment had localised, small, node-negative tumours, both oestrogen receptor (ER) positive and ER negative. We could not detect the amplified PSA fragment in 20 normal women and 22 normal men. We conclude that PSA RT-PCR may be a useful method for determining the presence of circulating metastatic cells in some women with node-negative breast cancer, and therefore the potential for these women to develop recurrent disease and thus benefit from adjuvant therapy.

Molecular Detection of Prostate Epithelial Cells from the Surgical Field and Peripheral Circulation during Radical Prostatectomy

Purpose Prostate cancer progression despite organ confined pathological assessment has been reported in a variable number of men after radical retropubic prostatectomy. To study this phenomenon, we used the prostate specific antigen (PSA) reverse transcriptase-polymerase chain reaction assay. Materials and Methods We prospectively assayed the peripheral venous blood before, during and after surgical manipulation as well as the intraoperative field blood for PSA reverse transcriptase-polymerase chain reaction-positive cells in 22 men undergoing radical retropubic prostatectomy. Results PSA reverse transcriptase-polymerase chain reaction-positive cells were identified in 20 of the 22 operative field samples (91 percent) and 4 of 16 (25 percent) had evidence of intraoperative hematogenous dissemination (p = 0.046). No significant association was identified among Gleason score, pathological stage and the PSA reverse transcriptase-polymerase chain reaction result. Conclusions Our results suggest that tumor cell spillage and less frequently hematogenous dissemination may be associated with operative manipulation of the prostate during radical retropubic prostatectomy and may potentially represent mechanisms of failure after radical retropubic prostatectomy.