ABSTRACT: The reverse transcriptase–polymerase chain reaction (RT-PCR) assay is an extremely sensitive technique for the detection of circulating cells expressing prostate-specific antigen (PSA) in prostate cancer patients. This article reviews the literature on the use of this technique as a preoperative parameter to predict both extraprostatic disease and PSA recurrence after radical prostatectomy. Despite the relative consensus regarding the increase in RT-PCR–positivity with tumor stage (ie, clinically localized vs metastatic prostate cancer), the use of RT-PCR as a clinical staging modality is controversial. To date, more than 16 institutions have evaluated the RT-PCR test in prostate cancer. Of these institutions, only two have reported the utility of RT-PCR as a staging modality and three have reported the utility of the test in predicting PSA recurrence. Before further conclusions are drawn regarding the clinical utility of RT-PCR in prostate cancer patients and its routine use is advocated, a larger patient population needs to be studied and followed for longer periods. [ONCOLOGY 13(2):187-193, 1999]
Prostate cancer is the second leading cause of cancer-related deaths in US men. It is estimated that 184,500 new cases of prostate cancer will be diagnosed in the United States in 1998, and over 39,200 deaths will result from this cancer. Recently, the National Cancer Institute has determined that the death rate from prostate cancer has decreased slightly; this may reflect favorable results achieved through early detection.
Unfortunately, the majority of patients with prostate cancer have extraprostatic spread at the time of diagnosis. Although nearly 60% of newly diagnosed cases of prostate cancer are predicted to be organ-confined based on digital rectal examination (DRE), serum prostatic specific antigen (PSA) levels, and histologic grading (Gleason score on biopsy), between 26% and 68% of these patients are ultimately found to have organ-confined disease on final pathologic analysis. Thus, more than one-third of men with clinically localized prostate cancer have extraprostatic disease at the time of surgery and are not curable by surgery alone.
Currently, preoperative staging of clinically localized prostate cancer is based on DRE findings, serum PSA level, and the results of prostate needle biopsy and radiologic explorations. Digital rectal examination frequently understages the extent of tumor. Imaging modalities, such as endorectal coil magnetic resonance imaging (MRI) and ultrasound, add little to the accuracy of preoperative staging.
The preoperative serum PSA value may be difficult to interpret, due to the volume of benign prostatic hyperplasia and the degree of tumor differentiation. Therefore, serum PSA levels are not sufficiently reliable to predict final pathologic stage on an individual basis in patients with localized prostate cancer.
Preoperative Gleason score determined from biopsy specimens strongly correlates with final pathologic stage in patients at either extreme of the scoring system (ie, those with Gleason scores from 2 to 4 or from 8 to 10). Unfortunately, prediction of pathologic stage is not as reliable for the more than 75% of men whose Gleason score falls between 5 and 7. Thus, improvements in existing preoperative staging techniques are clearly needed.
Recent advances in molecular biology have allowed for the detection of prostate cells in the peripheral blood. Through the use of reverse transcription followed by the polymerase chain reaction (RT-PCR) with primers specific for PSA messenger RNA (mRNA) or prostate-specific membrane antigen (PSMA) mRNA, circulating prostate cells can be identified.[7-36] This technique has been reported to detect cells in over 80% of patients with metastatic disease and in 40% of those with clinically localized prostate cancer.
There is a considerable debate over the clinical use of RT-PCR as a preoperative indicator of extraprostatic disease. This article will review the literature on the ability of RT-PCR to predict final pathologic stage and PSA recurrence after radical prostatectomy.
In 1992, Moreno et al reported the detection of circulating cells by RT-PCR for PSA in patients with metastatic prostate cancer. In 1994, Katz et al introduced the concept of molecular staging using RT-PCR technology. They found that the test could predict surgical failure (ie, capsular penetration, surgical margins, and seminal vesicle involvement) with high specificity and sensitivity.
Since the publication of the study by Katz et al, several teams have developed RT-PCR procedures to detect circulating cells and have evaluated these procedures in their patients.[13-36] To date, only one other team found a correlation between RT-PCR–positive results and pathologic stage.
At Columbia-Presbyterian Medical Center, ongoing investigations have studied the performance of RT-PCR in the staging of prostate cancer patients prior to radical prostatectomy.[7-12] The test’s sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and odds ratio for various surgical outcomes are summarized in Table 1. Patients with a positive RT-PCR assay had a 67% chance of having extraprostatic extension, a 49% likelihood of surgical failure, and a 60% likelihood of capsular penetration.
Recently, Nejat et al described the stratification of RT-PCR PSA results according to preoperative serum PSA levels (£ 10 ng/mL vs > 10 ng/mL). As shown in Table 2, this stratification can improve staging: 91% of patients with RT-PCR that was positive for PSA and a preoperative serum PSA level > 10 ng/mL had pT3 disease, as compared with 36% of patients with a negative RT-PCR for PSA (chi-square test = 17.2; P = .001). If PSA stratification was not used, 60% of patients with RT-PCR positive for PSA had pT3 disease vs 21% of those with RT-PCR negative for PSA.
In our literature review, we divided clinical studies according to the type of RT-PCR protocol used, as well as the molecular target (PSA, PSMA, or hK2) (Table 3). The initial clinical report of Moreno et al describing the detection of circulating prostate cells involved patients with D0 to D3 disease. The RT-PCR assay was positive in one-third of the cases, indicating the presence of circulating prostatic cells.
Additional studies examining patients with known metastatic prostate cancer indicated that RT-PCR detected circulating cells in the peripheral blood or bone marrow in 31% to 100% of patients (Table 3). The majority of studies consistently failed to detect PSA-expressing cells in control populations, as shown in the negative control column in Table 3.
Reverse transcriptase–polymerase chain reaction analyses of venous blood from patients with clinically localized prostate cancer (T1-T2) identify hematogenous PSA-expressing cells in 0% to 81% of patients prior to radical prostatectomy. As shown in Table 3, most groups have reported a gradual increase in RT-PCR–positivity (regardless of RT-PCR target or sample source) related to increasing tumor stage (ie, clinically localized vs metastatic prostate cancer).
In the literature, 1,158 RT-PCR assays for PSA in peripheral blood have been carried out, 757 in patients with pT1 to pT2 disease and 401 in patients with pT3+ disease. Overall, 23% (174/757) of patients with organ-confined disease and 38% (151/401) of those with extraprostatic disease were RT-PCR–positive. All but two groups reported no advantage to using RT-PCR as a clinical modality at this time.
Recently, Grasso et al described their experience using a nested RT-PCR assay for PSA and PSMA. They found a statistical difference in RT-PCR–positivity between patients with pT2 disease and those with pT3 disease (37.5% and 81.5% respectively; P = .001). Their PSA/PSMA nested RT-PCR had an odds ratio of 7.3 (95% confidence interval [CI], 2.3 to 23.4) for predicting tumor extraprostatic extension, consistent with the Columbia University data (odds ratio: 5.5; 95% CI, 3.2 to 9.3).
Possible Reasons for Differences Among Institutions
There is much debate in the field over the validity and clinical significance of the detection of circulating prostate cells using PCR technology. The great disparity in the PCR techniques performed at different laboratories could account for the varying results reported in different studies. After 1995, seven academic institutions established a PCR consortium to accrue at least 300 patients undergoing radical prostatectomy and assess the role of RT-PCR in staging and prediction of recurrence. This study confirmed specimen-related processing, technical, and clinical assay performance variability issues, indicating that equivalent technical performance does not equate with clinical performance.
Besides differences in assays, there are differences regarding selection criteria for patients (such as patient age, preoperative serum PSA, and a lack of standardized conditions for collection of samples), technical specifications for running the assay (such as varying mRNA harvesting conditions, unique primers, and distinct PCR conditions) and pathologic proceedings. Differences in these parameters could prevent comparisons of results from different studies from being made (see Table 4).
1. Parker SL, Tong T, Bolden S, et al: Cancer statistics, 1998. CA Cancer J Clin 48:10-11, 1998.
2. Stephenson RA, Stanford JL: Population-based prostate cancer trends in the United States: Patterns of change in the area of prostate-specific antigen. World J Urol 15: 331-335, 1997.
3. Carter HB, Coffey DS: Prostate cancer: The magnitude of the problem in the United States, in Coffey DS, Resnick FA, Karr JP (eds): A Multidisciplinary Analysis of Controversies in the Management of Prostate Cancer, chap 1, pp 1-7. New York, Plenum Press, 1988.
4. Partin AW, Yoo J, Carter B, et al: The use of prostatic specific antigen, clinical stage and Gleason score to predict pathological stage in men with localized prostatic cancer. J Urol 150:110-114, 1993.
5. Rifkin MD, Zerhouni EA, Gatsonis CA, et al: Comparison of magnetic resonance imaging and ultrasound in staging early prostate cancer. N Engl J Med 323:621-626, 1990.
6. Partin AW, Carter HB, Chan DW, et al: Prostate specific antigen in the staging of localized prostate cancer: Influence of tumor differentiation, tumor volume and benign hyperplasia. J Urol 143:747-752, 1990.
7. Katz AE, Olsson CA, Raffo AJ, et al: Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay. Urology 43:765-775, 1994.
8. Katz AE, de Vries GM, Benson MC, et al: The role of the reverse-transcriptase polymerase chain reaction assay for prostate-specific antigen in the selection of patients for radical prostatectomy. Urol Clin North Am 23:541-549, 1996.
9. Olsson CA, de Vries GM, Benson MC, et al: The use of RT-PCR for prostate-specific antigen assay to predict potential surgical failures before radical prostatectomy: Molecular staging of prostate cancer. Br J Urol 77: 411-417, 1996.
10. Olsson CA, de Vries GM, Raffo AJ, et al: Preoperative reverse transcriptase polymerase chain reaction for prostate specific antigen predicts treatment failure following radical prostatectomy. J Urol 155:1557-1562, 1996.
11. Nejat RJ, Katz AE, Olsson CA: The role of reverse transcriptase-polymerase chain reaction for staging patients with clinically localized prostate cancer. Semin Urol Oncol 16:40-45, 1998.
12. Nejat RJ, Katz AE, Benson MC, et al: Enhanced RT-PCR for PSA combined with serum PSA predicts pathologic stage and outcome in 300 radical prostatectomy patients. J Urol 159:291A, 1998.
13. Grasso YZ, Gupta MK, Levin HS, et al: Combined nested RT-PCR assay for prostate-specific antigen and prostate-specific membrane antigen in prostate cancer patients: Correlation with pathological stage. Cancer Res 58:1456-1459, 1998.
14. Wood DP, Banerjee M: Presence of circulating prostate cells in the bone marrow of patients undergoing radical prostatectomy is predictive of disease-free survival. J Clin Oncol 15: 3451, 1997.
15. Israeli RS, Miller WH, Su SL, et al: Sensitive nested reverse transcriptase polymerase chain reaction detection of circulating prostate tumor cells: Comparison of prostate specific membrane antigen and prostate specific antigen based assays. Cancer Res 54:6306-6310, 1994.
16. Seiden MV, Kantoff PW, Krithivas K, et al: Detection of circulating tumor cells in men with localized prostate cancer. J Clin Oncol 12:2634-2639, 1994.
17. Jaakkola S, Vornanen T, Leinonen J, et al: Detection of prostatic cells in peripheral blood: Correlation with serum concentrations of prostate specific antigen. Clin Chem 41:182-186, 1995.
18. Loric S, Dumas F, Eschwege P, et al: Enhanced detection of hematogenous circulating prostatic cells in patients with prostate adenocarcinoma by using nested reverse transcription polymerase chain reaction assay based on prostate-specific membrane antigen. Clin Chem 41:1698-1704, 1995.
19. Sokoloff MH, Tso CL, Kaboo R, et al: Quantitative polymerase chain reaction does not improve preoperative cancer staging: A clinicopathological molecular analysis of 121 patients. J Urol 156:1560-1566, 1996.
20. Ghossein RA, Rosai J, Scher HI, et al: Prognostic significance of detection of prostate-specific antigen transcripts in the peripheral blood of patients with metastatic androgen-independent prostatic carcinoma. Urology 50: 100-105, 1997.
21. Kawakami M, Okaneya T, Furihata K, et al: Detection of prostate cancer cells circulating in peripheral blood by reverse transcription-PCR for hKLK2. Cancer Res 57:4167-4170, 1997.
22. Corey E, Arfman EW, Oswin MM, et al: Detection of circulating prostate cells by reverse transcriptase-polymerase chain reaction of human glandular kallikrein (hK2) and prostate-specific antigen (PSA) messages. Urology 50:184-188, 1997.
23. Corey E, Arfman EW, Liu AY, et al: Improved protocol for reverse transcriptase polymerase chain reaction protocol with exogenous internal competitive control for prostate specific antigen mRNA in blood and bone marrow. Clin Chem 43:443-452, 1997.
24. Gomella LG, Raj GV, Moreno JG: Reverse transcriptase polymerase chain reaction for prostate specific antigen in the management of prostate cancer. J Urol 158:326-337, 1997.
25. Noguchi M, Miyajima J, Itoh K, et al: Detection of circulating tumor cells in patients with prostate cancer using prostate specific membrane-derived primers in the polymerase chain reaction. Int J Urol 4:374-379, 1997.
26. de Cremoux P, Ravery V, Podgorniak MP, et al: Value of the preoperative detection of prostate-specific-antigen-positive circulating cells by nested RT-PCR in patients submitted to radical prostatectomy. Eur Urol 32:69-74, 1997.
27. Thiounn N, Saporta F, Flam TA, et al: Positive prostate-specific antigen circulating cells detected by reverse transcriptase-polymerase chain reaction does not imply the presence of prostatic micrometastases. Urology 50:245-250, 1997.
28. Ellis WJ, Vessella RL, Corey E, et al: The value of a reverse transcriptase polymerase chain reaction assay in preoperative staging and follow-up of patients with prostate cancer. J Urol 159:1134-1138, 1998.
29. Vessela RL, Lange PH, Blumenstein BA, et al: Multicenter RT-PCR PSA clinical trial for preoperative staging of prostate cancer. J Urol 159:292A, 1998.
30. Eschwege P, Dumas F, Blanchet P, et al: Cellules circulantes et cancer de prostate: Interêt en pratique quotidienne. Séminaires d’uro-néphrologie (France) 23:287, 1997.
31. Van Nguyen C, Song W, Scardino PT, et al: RT-PCR for PSA and hK2: Implications for staging and patient management in men undergoing radical prostatectomy. J Urol 159:292A, 1998.
32. Hedican SP, Nelson JB, Marshke P, et al: Evaluation of preoperative prostate cancer staging and postoperative detection of recurrence utilizing the reverse transcriptase polymerase chain reaction (RT-PCR). J Urol 159:292A, 1998.
33. Moreno JG, Croce CM, Fisher R, et al: Detection of hematogenous micrometastasis in patients with prostate cancer. Cancer Res 52:6110-6112, 1992.
34. Melchior SW, Corey E., Ellis WJ, et al: Early tumor cell dissemination in patients with clinically localized carcinoma of the prostate (CAP). J Urol 155:554A, 1996.
35. Ignatoff JM, Oefelein MG, Watkin W, et al: Prostate specific antigen reverse transcriptase-polymerase chain reaction assay in preoperative staging of prostate cancer. J Urol 158:1870-1875, 1997.
36. Slawin KM, O’Hara SM, Song W et al: Comparison of results obtained using different RT-PCR PSA assay methods on a single set of clinical specimens. J Urol 155:417A, 1996.
37. Schellhammer PF: Radical prostatectomy: Patterns of local failure and survival in 67 patients. Urology 31:191-197, 1988.
38. Ohori M, Wheeler TM, Kattan MW, et al: Prognostic significance of positive surgical margins in radical prostatectomy specimens. J Urol 154:1818-1824, 1995.
39. Partin AW, Pound CR, Clemens JQ, et al: Serum PSA after anatomic radical prostatectomy: The Johns Hopkins experience after 10 years. Urol Clin North Am 20:713-725, 1993.
40. Trapasso JG, de Kernion JB, Smith RB, et al: The incidence and significance of detectable levels of serum prostate specific antigen after radical prostatectomy. J Urol 152:1821-1825, 1994.
41. Zincke H, Oesterling JE, Blute ML, et al: Long-term (15 years) results after radical prostatectomy for clinically localized (stage T2c or lower) prostate cancer. J Urol 152:1850-1857, 1994.
42. Pound CR, Partin AW, Epstein JI, et al: Prostate-specific antigen after anatomic radical retropubic prostatectomy: Patterns of recurrence and cancer control. Urol Clin North Am 24:395-406, 1997.
43. Humphrey PA, Frazier HA, Vollmer RT, et al: Stratification of pathologic features on radical prostatectomy specimens that are predictive of elevated initial postoperative serum prostate-specific antigen levels. Cancer 71:1821-1827, 1993.
44. Epstein JI, Partin AW, Saugageot J, et al: Prediction of progression following radical prostatectomy: A multivariate analysis of 721 patients with long term follow-up. Am J Surg Pathol 20:286-292, 1996.
45. Paradis V, Echwege P, Loric S, et al: CD44 expression in prostate cancers is associated with blood dissemination. J Urol 159: 299A, 1998.
46. Eschwege P, Loric S, Paradis V, et al: Blood-borne prostate cells detection marks adhesion defect and recurrence in cancer. Eur Urol 33(suppl 1):36, 1998.