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Increased expression of PSA mRNA during brachytherapy in peripheral blood of patients with prostate cancer
ADULT UROLOGY
INCREASED EXPRESSION OF PSA mRNA DURING BRACHYTHERAPY IN PERIPHERAL BLOOD OF PATIENTS WITH PROSTATE CANCER AYISHA SIDDIQUA, DAMODARAN CHENDIL, RANDALL ROWLAND, ALI S. MEIGOONI, MAHESH KUDRIMOTI, MOHAMMED MOHIUDDIN, AND MANSOOR M. AHMED
ABSTRACT Objectives. To determine the extent of iatrogenic tumor cell dissemination during brachytherapy by assessing prostate-specific antigen (PSA) mRNA expression in circulating prostate tumor cells using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The instrumentation used in the radioisotope seed placement of the prostate causes trauma to blood vessels and provides a vascular access for tumor cells that can lead to potential iatrogenic dissemination and systemic failure. Methods. Twenty-five patients treated for brachytherapy were recruited in the study. Controls included 4 normal men and 1 woman; case controls included 4 patients who underwent prostate biopsy for prostate cancer diagnosis. Peripheral blood (10 mL) was collected before, during, and after the brachytherapy procedure. Total RNA was isolated from mononuclear cells and phosphorus-32 RT-PCR was performed to analyze the mRNA expression of PSA and G6PDH genes. Results. Of 25 patients, 23 were negative for PSA mRNA expression and 2 were positive for PSA mRNA expression before brachytherapy. Of the 23 patients who were negative for PSA mRNA expression before treatment, 15 patients (65%) turned positive during or after brachytherapy and the remaining 8 patients remained negative throughout the treatment. Eight of the 25 patients developed rising serum PSA levels. Of these 8 patients, 1 (12.5%) did not have PSA mRNA expression in the peripheral blood before, during, or after brachytherapy; the remaining 7 patients who developed rising serum PSA levels had PSA mRNA expression after brachytherapy (P ⫽ 0.03). Conclusions. These findings strongly suggest that iatrogenic shedding of prostate cells occurs as a result of brachytherapy and raises the concern that these cells liberated at the time of brachytherapy increase the risk of metastatic deposits and results in systemic failure, as measured by serum PSA levels. UROLOGY 60: 270–275, 2002. © 2002, Elsevier Science Inc.
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rostate carcinoma is the second leading cause of cancer-specific death among men in the United States.1,2 Surgery, external beam radiotherapy, and interstitial implantation (brachytherapy) are all effective strategies used in the treatment of early-stage prostate cancer.3,4 Although the 5 and 10-year survival rates are excellent with any of Ayisha Siddiqua was the recipient of the student travel award for this study from the Radiation Research Society. This work was presented at the 48th Annual Meeting of the Radiation Research Society, April 2001, San Juan, Puerto Rico. From the Departments of Radiation Medicine and Surgery (Division of Urology), University of Kentucky, Lexington, Kentucky Reprint requests: Mansoor M. Ahmed, Ph.D., Department of Radiation Medicine, C15 UKMC, 800 Rose Street, University of Kentucky, Lexington, KY 40536 Submitted: September 17, 2001, accepted (with revisions): March 11, 2002
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© 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
these treatments, 15% to 25% will have failure in the early stages of cancer from both local and distant disease.5 The predominant pattern of failure after surgery or external radiotherapy appears to be due to local recurrence (50% to 70%). Although the overall results of brachytherapy have been excellent, a recent report from the M. D. Anderson Cancer Center indicated that the 5-year biochemical failure rate was 24% in patients with a prostate-specific antigen (PSA) level less than 10 ng/mL and 49% for those with a PSA level greater than 10 ng/mL.6 Although many studies have documented the incidence of biochemical relapse after prostate brachytherapy, few studies have indicated the pattern of failure. Stock et al.7 found that only 33% of patients with biochemical PSA relapse had local prostate failure as documented by prostate 0090-4295/02/$22.00 PII S0090-4295(02)01703-X
biopsies. This appears to differ substantially from the 51% positive biopsy rate in patients with biochemical PSA relapse after external beam radiotherapy. It is reasonable therefore to assume that a significantly higher proportion of biochemical relapse after prostate brachytherapy is a result of a higher incidence of systemic failure. It is conceivable that the insertion of multiple needles into the prostate gland during brachytherapy provides a vascular access for tumor cells, leading to iatrogenic dissemination of tumor cells that can potentially cause the development of metastatic foci and systemic failure. Two case studies have documented prostate tumors developing in the perineum after biopsy and prostate brachytherapy.8,9 This is direct clinical evidence demonstrating local iatrogenic-induced metastasis after prostatic manipulation by needles. Thus, the possibility exists that the disruption of the capillary network at brachytherapy will lead to hematogenous dissemination of prostate cells, leading to distant metastasis. Although the release of prostate cells has been documented after radical prostatectomy, transurethral resection of the prostate, and transrectal biopsy of the prostate, the release of cells after brachytherapy has not been evaluated. Thus, this study was undertaken to assess the incidence of circulating prostate cancer cells measured in the peripheral circulation of patients undergoing brachytherapy for the treatment of prostate cancer and to correlate this finding with the pattern of biochemical changes as measured by serum PSA levels. MATERIAL AND METHODS PATIENTS Twenty-five patients undergoing brachytherapy for prostate cancer were recruited for this study with informed consent approved by University of Kentucky Institutional Review Board. Four patients undergoing prostate biopsy who were negative for cancer were used as case controls to determine the iatrogenic effect of the instrumentation on the normal prostate on the PSA mRNA levels. Peripheral blood samples from 4 normal men and 1 woman were used as negative controls. Patients were followed up by sequential serum PSA measurements for assessment of failure. The follow-up ranged from 25 to 36 months (median 30).
ISOLATION OF MONONUCLEAR CELLS Ten milliliters of blood was collected before, during, and 1 hour after brachytherapy. Mononuclear cells were isolated using a Vacutainer CPT cell preparation tube (Becton Dickinson, Franklin Lake, NJ) containing sodium citrate gel and density gradient media. Sample processing was started immediately after withdrawing blood. From the pelleted cells, RNA isolation was performed.
RNA ISOLATION AND PHOSPHORUS-32 REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION Total RNA was isolated from the pelleted cells using TriZol reagent (Gibco, Gaithersburg, MD). One microgram of total UROLOGY 60 (2), 2002
RNA was reverse transcribed into cDNA. Following the conditions (temperature and MgCl2 concentration) described by Zippelius et al.10 with slight modifications, PCR was performed using 5 L of reverse-transcribed product with phosphorus 32 (32P)-5⬘-end-labeled primers specific for PSA gene (upper primer 735-755 [exon-3] and lower primer 892-912 [exon-4]).11,12 32P-RT-PCR products were run in 12% polyacrylamide gel electrophoresis, dried, and autoradiographed. A blank, negative control and a positive control (RNA extracted from prostate cancer cell line LNCaP) were used along with the patient samples. From the same RT product, 5 L was used to amplify the human G6PDH gene as an internal control using radiolabeled primers (sense primer: 5⬘-CCAAGACCATCCCCTATA-3⬘ and antisense: 5⬘-GGTGCCCTCCTCATACTGGAA-3⬘; GeneBank Accession No. X03674). 32PRT-PCR resulted in 178-bp and 90-bp product for PSA and G6PDH genes, respectively. Before patient analysis, the sensitivity of the 32P-RT-PCR technique was assessed in a normal female RNA sample (negative for PSA mRNA expression) contaminated with the RNA from PSA-positive control LNCaP cells. The radiolabeled PCR products were analyzed on 12% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. Using the BioRad gel documentation system and Multi-Analyst software, the intensity of the PSA and G6PDH bands were measured and densitometry values were calculated as the PSA/G6PDH percent ratio. All experiments were performed twice to confirm the presence of PSA mRNA expression.
STATISTICAL ANALYSIS Serum PSA measurements were obtained every 3 months after prostate brachytherapy. The American Society for Therapeutic Radiology and Oncology definition of biochemical failure was used to define relapse.13,14 PSA mRNA expression after brachytherapy was correlated with the serum PSA levels, grade, stage, and Gleason scores. The test of significance was performed using Student’s t test.
RESULTS SENSITIVITY OF PSA RNA DETECTION BY 32 P-RT-PCR A semiquantitative 32P-RT-PCR was used to analyze the sensitivity of PSA mRNA detection in total PSA-negative normal female RNA contaminated with the RNA from PSA-positive LNCaP control cells. By this assay, the presence of PSA mRNA expression was detected in as low as 10⫺7 g LNCaP RNA in 1 g of normal female RNA (Fig. 1A). This contamination simulates one tumor cell in 105 mononuclear cells. The PSA/G6PDH densitometry values were directly proportional to the level of contamination by LNCaP RNA. On the basis of these assay results, patient samples that showed a densitometric percent ratio value were labeled as follows: a percent ratio value of greater than 2% to 25% ⫽ ⫹ for PSA mRNA expression; greater than 25% to 50% ⫽ ⫹⫹; greater than 50% to 75% ⫽ ⫹⫹⫹; and greater than 75% to 100% ⫽ ⫹⫹⫹⫹. INCREASED PSA MRNA EXPRESSION SAMPLES COLLECTED DURING AND AFTER BRACHYTHERAPY All four normal male and female samples were negative for PSA mRNA expression. Samples col271
FIGURE 1. (A) Sensitivity of 32P-RT-PCR assay. One microgram of female RNA was contaminated with increasing concentrations of PSA-positive LNCaP RNA. Using this 32P-RT-PCR assay, the presence of PSA mRNA expression was detected in as low as 10⫺7 g LNCaP RNA in 1 g of normal female RNA. The 32P-RT-PCR products for PSA and G6PDH genes were run in 12% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. Using a gel documentation system, the intensity of the PSA and G6PDH bands were measured and densitometric values calculated as the percent PSA/G6PDH ratio. A bar graph below shows an increase in the percent PSA/G6PDH ratio as the LNCaP contamination increased. Error bars represent the mean of percent PSA/G6PDH ratio from two experiments. (B) Increased expression of PSA mRNA during brachytherapy. Patient samples were collected before, during, and after brachytherapy. Mononuclear cells were separated from the blood samples and RNA isolation performed. 32P-RT-PCR was performed for the PSA and G6PDH genes, and the samples were run in 12% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. LNCaP RNA was taken as a positive control. Patient samples that showed a densitometric ratio greater than 2% were considered positive for PSA mRNA expression. A bar graph below shows an increase in the percent PSA/G6PDH ratio during brachytherapy. Error bars represent the mean of the percent PSA/G6PDH ratio from two experiments.
lected from the 4 patients during prostate biopsy were negative for PSA mRNA expression. Of the 25 patients with prostate cancer, 23 were negative for PSA mRNA expression and 2 were positive for PSA mRNA expression before brachytherapy. Of the 23 patients who were negative for PSA mRNA expression before treatment, 15 (65%) turned positive during or after brachytherapy. However, the remaining 8 patients remained negative during and after brachytherapy. Two patients who were positive before treatment remained positive during and after treatment. An autoradiograph showing the highest PSA mRNA expression (⫹⫹⫹⫹) during brachytherapy is shown in Figure 1B. Most of the patients had a PSA mRNA expression level between 2% and 25%. CORRELATION OF CLINICAL DATA WITH PSA MRNA EXPRESSION IN PATIENTS AFTER BRACHYTHERAPY Of the 25 patients, 8 had rising serum PSA levels (biochemical relapse) as assessed by the American Society for Therapeutic Radiology and Oncology definition of biochemical relapse. Of the 15 patients who turned positive for PSA mRNA expression during or after brachytherapy, 7 (46.6%) had rising serum PSA levels. Of the 8 patients with neg272
ative PSA mRNA expression before, during, and after brachytherapy, 1 had a rising serum PSA level. This was statistically significant (P ⫽ 0.03; Table I). Elevation of post-treatment serum PSA levels did not correlate with stage, Gleason score, or pretreatment serum PSA level. Four patients who had pretreatment serum PSA levels of 4 ng/mL or less demonstrated PSA mRNA expression during or after the procedure. Of these 4 patients, 3 (75%) had rising serum PSA levels at last follow-up. Eighteen patients had pretreatment serum PSA levels between 4 and 10 ng/mL. Of these 18 patients, 12 demonstrated PSA mRNA expression during or after the procedure. Of these 12 patients, 4 (33%) had rising serum PSA levels. Three patients had pretreatment serum PSA levels greater than 10 ng/mL. Of these 3 patients, 1 (33%) had PSA mRNA expression during and after the procedure. Interestingly, none of these patients developed a rising serum PSA level. The pattern of relapse based on pretreatment PSA level was not significant. COMMENT Radical prostatectomy, brachytherapy, and external beam radiotherapy are currently used for the UROLOGY 60 (2), 2002
TABLE I. Comparative analysis of pretreatment, nadir, and current serum PSA level with PSA mRNA expression before, during, and after brachytherapy Pt. No.
Gleason Score
Pretreatment Serum PSA Level (ng/mL)
Before
During
After
PSA Nadir (ng/mL)
Biochemical Relapse
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
6 6 6 6 7 6 6 6 6 7 6 7 6 6 6 6 6 6 6 6 7 6 6 6 7
6.3 6 9.1 4.3 10 2.9 6.7 9.1 7 7.7 5.4 5.1 5 4 6.6 10.6 7 3.3 6.2 10.7 5.1 5.1 5.7 2.6 19
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫹⫹⫹⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺
⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺
1.0 N/A 0.2 0.4 0.1 0.8 0.1 0.1 1 0.9 0.7 0.9 0.7 0.5 0.2 2.1 0.1 0.9 0.7 ⱕ0.1 1.0 ⱕ0.1 1.0 1.2 0.2
No No No No No No No No Yes Yes Yes No No Yes No No No Yes Yes No Yes No No Yes No
PSA mRNA Expression
KEY: PSA ⫽ prostate-specific antigen; Pt. No. ⫽ patient number.
treatment of localized disease; however, the wide spectrum of biologic behavior exhibited by prostatic neoplasms poses a difficult problem in predicting the clinical course for an individual patient.15 Traditional prognostic markers, such as grade, clinical stage, and pretreatment serum PSA, are used extensively and are of some prognostic value for individual men.16,17 The pattern of posttreatment serum PSA values is now the reference standard to define the success of any treatment and a good predictor of eventual outcome. Studies have shown that patients undergoing radical prostatectomy have considerable shedding of tumor cells in the peripheral blood during surgery. Sokoloff et al.18 showed that circulating PSA mRNA was detected in 42 (62%) of 68 patients with Stage pT2 and pT3 disease, but they found no significant relationship between the preoperative positivity of circulating PSA mRNA and other pathologic parameters. On the contrary, Katz et al.19 noted that the staging accuracy of PSA mRNA expression was greater than that of other modalities such as digital rectal examination, transrectal ultrasonography, or computed tomography. They also demonstrated that circulating PSA mRNA expression was an independent factor in predicting extracapsular disease extenUROLOGY 60 (2), 2002
sion and was superior to any other established prognostic factors such as serum PSA, Gleason score, or clinical stage.20 Olsson et al.21 showed that circulating PSA mRNA was a significant predictor of PSA recurrence after radical prostatectomy. Ogawa et al.22 found that preoperative PSA mRNA was positive in the peripheral venous blood samples of 11 (48%) of 23 patients, and, in these patients, age, preoperative serum PSA value, and clinical or pathologic stage were not significantly associated with positive PSA mRNA expression. They also found that PSA mRNA was positive in the peripheral blood in 18 patients (78%) during radical prostatectomy. All 11 patients with positive preoperative PSA mRNA expression had positive PSA mRNA expression during radical prostatectomy. Seven (58%) of 12 patients with negative preoperative PSA mRNA expression had a positive conversion of intraoperative PSA mRNA.22 They found no significant association among the clinicopathologic parameters, positive intraoperative PSA mRNA expression, and positive conversion during radical prostatectomy.22 Because interstitial brachytherapy for prostate cancer is also an invasive procedure, the potential for similar patterns of tumor cell dissemination is present. To analyze whether shedding of tumor 273
cells occurred in the peripheral blood, the sensitive 32 P-RT-PCR method was used to determine PSA mRNA expression as a marker of prostate cancer cells in the venous circulation. No PSA mRNA was detected in the blood from normal male or female blood samples or from the patients who underwent normal prostate ultrasound-guided biopsies. The 65% (15 of 23) positive rate for PSA mRNA expression during or after brachytherapy is similar to the rates reported after radical prostatectomy.22 Eight of these 23 patients remained negative for PSA mRNA expression before, during, and after brachytherapy. Of these 8 patients, 1 (12.5%) had a rising serum PSA level after treatment, indicating therapy failure; however, of the 15 patients who had PSA mRNA expression, 7 (46.6%) developed rising serum PSA levels. No significant correlation was found between PSA mRNA expression and the pretreatment clinicopathologic parameters of stage or time to PSA nadir. These results are similar to those reported by Olsson et al.21 in which circulating PSA mRNA was a significant predictor of PSA recurrence after radical prostatectomy but did not correlate with traditional pretreatment tumor characteristics. No statistically significant correlation between pretreatment serum PSA levels and detected PSA mRNA expression was observed, similar to the findings of Ogawa et al.22 after radical prostatectomy. They reported no significant association among the clinicopathologic parameters, positive intraoperative PSA mRNA, and positive conversion during radical prostatectomy. Several large studies have reported the results of biochemical PSA control after external beam radiotherapy and brachytherapy implantation of the prostate.6,23 Zagars et al.23 reported that 157 (22%) of 707 patients treated with external beam radiotherapy for localized prostate cancer developed biochemical serum PSA relapse at a median of 6.5 years. Eighty of these patients (51%) developed local recurrence, 24 (15%) developed distant disease, and the remaining patients had biochemical failure alone. Storey et al.,6 from the same institution, reported that biochemical serum PSA failure was seen in 37% (76 of 206) of patients after prostate brachytherapy at 5 years. The higher incidence of biochemical relapse in the latter group of patients is hard to explain, especially as these patients were probably at earlier stages with more organconfined disease. Stock et al.7 reported that only 33% of patients experiencing biochemical serum PSA relapse after brachytherapy had local prostatic failure, as documented by positive post-treatment biopsies, lower than the 51% rate of positive posttreatment biopsies reported in patients experiencing biochemical failure after external beam radiotherapy.23 Because local disease control in the 274
prostate itself does not appear to be the main factor in this biochemical relapse, it is reasonable to assume that much of this is due to a higher incidence of systemic failure. It is conceivable that the insertion of multiple needles into the prostate gland during brachytherapy provides a vascular access for tumor cells from trauma to blood vessels, leading to iatrogenic dissemination of tumor cells that can potentially lead to systemic failure. PSA bounce is a phenomenon that has been described after prostate brachytherapy and is reported to be observed in 35% of patients. PSA bounce also appears to be less frequent when the PSA nadir after treatment is lower (less than 4 ng/mL) and was not seen when the PSA nadir was less than 0.7 ng/mL.24 However, in our cohort of patients, a serum PSA rise was noted predominantly in patients expressing PSA mRNA, and the PSA nadir in these patients was low (less than 1.5 ng/mL). The prognostic significance of circulating prostate cancer cells as measured by RT-PCR for PSA mRNA after local therapy for prostate cancer has previously been reported by several investigators.25,26 Similar to our results, Philip et al.27 reported a poorer survival in patients found to have prostate cancer cells in the peripheral circulation in advanced disease. The higher incidence of biochemical failure in patients with PSA mRNA positivity is of concern, and strategies to reduce the impact of these circulating cells on the potential development of systemic disease need to be developed. However, these results need to be evaluated in a larger cohort of patients. CONCLUSIONS Our results suggest for the first time in published reports that a substantial number of patients undergoing brachytherapy have iatrogenic dissemination of prostate cancer cells in the peripheral blood caused by the insertion of needles in the prostate gland. Also, the detection of PSA mRNA in the peripheral circulation appears to have a significant correlation with biochemical failure after interstitial brachytherapy and needs additional study in a larger patient population. REFERENCES 1. Landis SH, Murray T, Bolden S, et al: Cancer statistics, 1999. CA Cancer J Clin 49: 8 –31, 1999. 2. Stamey TA, Yang N, Hay AR, et al: Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 317: 909 –916, 1987. 3. Fuks Z, Leibel SA, Wallner KE, et al: The effect of local control on metastatic dissemination in carcinoma of the prostate: long-term results in patients treated with 125I implantation. Int J Radiat Oncol Biol Phys 21: 537–547, 1991. 4. Kuban DA, el-Mahdi AM, and Schellhammer PF: I-125 interstitial implantation for prostate cancer: what have we learned 10 years later? Cancer 63: 2415–2420, 1989. UROLOGY 60 (2), 2002
5. Prestidge BR: Radioisotopic implantation for carcinoma of the prostate: does it work better than it used to? Semin Radiat Oncol 8: 124 –131, 1998. 6. Storey MR, Landgren RC, Cottone JL, et al: Transperineal 125iodine implantation for treatment of clinically localized prostate cancer: 5-year tumor control and morbidity. Int J Radiat Oncol Biol Phys 43: 565–570, 1999. 7. Stock RG, Stone NN, Kao J, et al: The effect of disease and treatment-related factors on biopsy results after prostate brachytherapy: implications for treatment optimization. Cancer 89: 1829 –1834, 2000. 8. Teh BS, Chou CC, Schwartz MR, et al: Perineal prostatic cancer seeding following radioactive seed brachytherapy. J Urol 166: 212, 2001. 9. Wiersinga WJ, De Reijke TM, and Blank LE: Local tract metastasis of prostatic adenocarcinoma 8 years after (125)iodine brachytherapy. J Urol 166: 995, 2001. 10. Zippelius A, Lutterbuse R, Riethmuller G, et al: Analytical variables of reverse transcription-polymerase chain reaction-based detection of disseminated prostate cancer cells. Clin Cancer Res 6: 2741–2750, 2000. 11. Klobeck HG, Combriato G, Schulz P, et al: Genomic sequence of human prostate specific antigen (PSA). Nucleic Acids Res 17: 3981, 1989. 12. Schulz P, Stucka R, Feldmann H, et al: Sequence of a cDNA clone encompassing the complete mature human prostate specific antigen (PSA) and an unspliced leader sequence. Nucleic Acids Res 16: 6226, 1988. 13. Horwitz EM, Vicini FA, Ziaja EL, et al: The correlation between the ASTRO Consensus Panel definition of biochemical failure and clinical outcome for patients with prostate cancer treated with external beam irradiation. American Society of Therapeutic Radiology and Oncology. Int J Radiat Oncol Biol Phys 41: 267–272, 1998. 14. Hanlon AL, and Hanks GE: Scrutiny of the ASTRO consensus definition of biochemical failure in irradiated prostate cancer patients demonstrates its usefulness and robustness. American Society for Therapeutic Radiology and Oncology. Int J Radiat Oncol Biol Phys 46: 559 –566, 2000. 15. Gao CL, Dean RC, Pinto A, et al: Detection of circulating prostate specific antigen expressing prostatic cells in the bone marrow of radical prostatectomy patients by sensitive reverse transcriptase polymerase chain reaction. J Urol 161: 1070 –1076, 1999. 16. Wasson JH, Cushman CC, Bruskewitz RC, et al: A structured literature review of treatment for localized prostate
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cancer. Prostate Disease Patient Outcome Research Team. Arch Fam Med 2: 487– 493, 1993.. 17. Partin AW, and Oesterling JE: The clinical usefulness of prostate specific antigen: update 1994. J Urol 152: 1358 – 1368, 1994. 18. Sokoloff MH, Tso CL, Kaboo R, et al: Quantitative polymerase chain reaction does not improve preoperative prostate cancer staging: a clinicopathological molecular analysis of 121 patients. J Urol 156: 1560 –1566, 1996. 19. 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. 20. Ennis RD, Katz AE, de Vries GM, et al: Detection of circulating prostate carcinoma cells via an enhanced reverse transcriptase-polymerase chain reaction assay in patients with early stage prostate carcinoma. Independence from other pretreatment characteristics. Cancer 79: 2402–2408, 1997. 21. 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. 22. Ogawa O, Iinuma M, Sato K, et al: Circulating prostatespecific antigen mRNA during radical prostatectomy in patients with localized prostate cancer: with special reference to neoadjuvant hormonal therapy. Urol Res 27: 291–296, 1999. 23. Zagars GK, Pollack A, and von Eschenbach AC: Prostate cancer and radiation therapy—the message conveyed by serum prostate-specific antigen. Int J Radiat Oncol Biol Phys 33: 23–35, 1995. 24. Critz FA, Williams WH, Benton JB, et al: Prostate specific antigen bounce after radioactive seed implantation followed by external beam radiation for prostate cancer. J Urol 163: 1085–1089, 2000. 25. de la Taille A, Olsson CA, Buttyan R, et al: Blood-based reverse transcriptase polymerase chain reaction assays for prostatic specific antigen: long term follow-up confirms the potential utility of this assay in identifying patients more likely to have biochemical recurrence (rising PSA) following radical prostatectomy. Int J Cancer 84: 360 –364, 1999. 26. Mejean A, Vona G, Nalpas B, et al: Detection of circulating prostate derived cells in patients with prostate adenocarcinoma is an independent risk factor for tumor recurrence. J Urol 163: 2022–2029, 2000. 27. Kantoff PW, Halabi S, Farmer DA, et al: Prognostic significance of reverse transcriptase polymerase chain reaction for prostate-specific antigen in men with hormone-refractory prostate cancer. J Clin Oncol 19: 3025–3028, 2001.
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INCREASED EXPRESSION OF PSA mRNA DURING BRACHYTHERAPY IN PERIPHERAL BLOOD OF PATIENTS WITH PROSTATE CANCER AYISHA SIDDIQUA, DAMODARAN CHENDIL, RANDALL ROWLAND, ALI S. MEIGOONI, MAHESH KUDRIMOTI, MOHAMMED MOHIUDDIN, AND MANSOOR M. AHMED
ABSTRACT Objectives. To determine the extent of iatrogenic tumor cell dissemination during brachytherapy by assessing prostate-specific antigen (PSA) mRNA expression in circulating prostate tumor cells using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The instrumentation used in the radioisotope seed placement of the prostate causes trauma to blood vessels and provides a vascular access for tumor cells that can lead to potential iatrogenic dissemination and systemic failure. Methods. Twenty-five patients treated for brachytherapy were recruited in the study. Controls included 4 normal men and 1 woman; case controls included 4 patients who underwent prostate biopsy for prostate cancer diagnosis. Peripheral blood (10 mL) was collected before, during, and after the brachytherapy procedure. Total RNA was isolated from mononuclear cells and phosphorus-32 RT-PCR was performed to analyze the mRNA expression of PSA and G6PDH genes. Results. Of 25 patients, 23 were negative for PSA mRNA expression and 2 were positive for PSA mRNA expression before brachytherapy. Of the 23 patients who were negative for PSA mRNA expression before treatment, 15 patients (65%) turned positive during or after brachytherapy and the remaining 8 patients remained negative throughout the treatment. Eight of the 25 patients developed rising serum PSA levels. Of these 8 patients, 1 (12.5%) did not have PSA mRNA expression in the peripheral blood before, during, or after brachytherapy; the remaining 7 patients who developed rising serum PSA levels had PSA mRNA expression after brachytherapy (P ⫽ 0.03). Conclusions. These findings strongly suggest that iatrogenic shedding of prostate cells occurs as a result of brachytherapy and raises the concern that these cells liberated at the time of brachytherapy increase the risk of metastatic deposits and results in systemic failure, as measured by serum PSA levels. UROLOGY 60: 270–275, 2002. © 2002, Elsevier Science Inc.
P
rostate carcinoma is the second leading cause of cancer-specific death among men in the United States.1,2 Surgery, external beam radiotherapy, and interstitial implantation (brachytherapy) are all effective strategies used in the treatment of early-stage prostate cancer.3,4 Although the 5 and 10-year survival rates are excellent with any of Ayisha Siddiqua was the recipient of the student travel award for this study from the Radiation Research Society. This work was presented at the 48th Annual Meeting of the Radiation Research Society, April 2001, San Juan, Puerto Rico. From the Departments of Radiation Medicine and Surgery (Division of Urology), University of Kentucky, Lexington, Kentucky Reprint requests: Mansoor M. Ahmed, Ph.D., Department of Radiation Medicine, C15 UKMC, 800 Rose Street, University of Kentucky, Lexington, KY 40536 Submitted: September 17, 2001, accepted (with revisions): March 11, 2002
270
© 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
these treatments, 15% to 25% will have failure in the early stages of cancer from both local and distant disease.5 The predominant pattern of failure after surgery or external radiotherapy appears to be due to local recurrence (50% to 70%). Although the overall results of brachytherapy have been excellent, a recent report from the M. D. Anderson Cancer Center indicated that the 5-year biochemical failure rate was 24% in patients with a prostate-specific antigen (PSA) level less than 10 ng/mL and 49% for those with a PSA level greater than 10 ng/mL.6 Although many studies have documented the incidence of biochemical relapse after prostate brachytherapy, few studies have indicated the pattern of failure. Stock et al.7 found that only 33% of patients with biochemical PSA relapse had local prostate failure as documented by prostate 0090-4295/02/$22.00 PII S0090-4295(02)01703-X
biopsies. This appears to differ substantially from the 51% positive biopsy rate in patients with biochemical PSA relapse after external beam radiotherapy. It is reasonable therefore to assume that a significantly higher proportion of biochemical relapse after prostate brachytherapy is a result of a higher incidence of systemic failure. It is conceivable that the insertion of multiple needles into the prostate gland during brachytherapy provides a vascular access for tumor cells, leading to iatrogenic dissemination of tumor cells that can potentially cause the development of metastatic foci and systemic failure. Two case studies have documented prostate tumors developing in the perineum after biopsy and prostate brachytherapy.8,9 This is direct clinical evidence demonstrating local iatrogenic-induced metastasis after prostatic manipulation by needles. Thus, the possibility exists that the disruption of the capillary network at brachytherapy will lead to hematogenous dissemination of prostate cells, leading to distant metastasis. Although the release of prostate cells has been documented after radical prostatectomy, transurethral resection of the prostate, and transrectal biopsy of the prostate, the release of cells after brachytherapy has not been evaluated. Thus, this study was undertaken to assess the incidence of circulating prostate cancer cells measured in the peripheral circulation of patients undergoing brachytherapy for the treatment of prostate cancer and to correlate this finding with the pattern of biochemical changes as measured by serum PSA levels. MATERIAL AND METHODS PATIENTS Twenty-five patients undergoing brachytherapy for prostate cancer were recruited for this study with informed consent approved by University of Kentucky Institutional Review Board. Four patients undergoing prostate biopsy who were negative for cancer were used as case controls to determine the iatrogenic effect of the instrumentation on the normal prostate on the PSA mRNA levels. Peripheral blood samples from 4 normal men and 1 woman were used as negative controls. Patients were followed up by sequential serum PSA measurements for assessment of failure. The follow-up ranged from 25 to 36 months (median 30).
ISOLATION OF MONONUCLEAR CELLS Ten milliliters of blood was collected before, during, and 1 hour after brachytherapy. Mononuclear cells were isolated using a Vacutainer CPT cell preparation tube (Becton Dickinson, Franklin Lake, NJ) containing sodium citrate gel and density gradient media. Sample processing was started immediately after withdrawing blood. From the pelleted cells, RNA isolation was performed.
RNA ISOLATION AND PHOSPHORUS-32 REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION Total RNA was isolated from the pelleted cells using TriZol reagent (Gibco, Gaithersburg, MD). One microgram of total UROLOGY 60 (2), 2002
RNA was reverse transcribed into cDNA. Following the conditions (temperature and MgCl2 concentration) described by Zippelius et al.10 with slight modifications, PCR was performed using 5 L of reverse-transcribed product with phosphorus 32 (32P)-5⬘-end-labeled primers specific for PSA gene (upper primer 735-755 [exon-3] and lower primer 892-912 [exon-4]).11,12 32P-RT-PCR products were run in 12% polyacrylamide gel electrophoresis, dried, and autoradiographed. A blank, negative control and a positive control (RNA extracted from prostate cancer cell line LNCaP) were used along with the patient samples. From the same RT product, 5 L was used to amplify the human G6PDH gene as an internal control using radiolabeled primers (sense primer: 5⬘-CCAAGACCATCCCCTATA-3⬘ and antisense: 5⬘-GGTGCCCTCCTCATACTGGAA-3⬘; GeneBank Accession No. X03674). 32PRT-PCR resulted in 178-bp and 90-bp product for PSA and G6PDH genes, respectively. Before patient analysis, the sensitivity of the 32P-RT-PCR technique was assessed in a normal female RNA sample (negative for PSA mRNA expression) contaminated with the RNA from PSA-positive control LNCaP cells. The radiolabeled PCR products were analyzed on 12% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. Using the BioRad gel documentation system and Multi-Analyst software, the intensity of the PSA and G6PDH bands were measured and densitometry values were calculated as the PSA/G6PDH percent ratio. All experiments were performed twice to confirm the presence of PSA mRNA expression.
STATISTICAL ANALYSIS Serum PSA measurements were obtained every 3 months after prostate brachytherapy. The American Society for Therapeutic Radiology and Oncology definition of biochemical failure was used to define relapse.13,14 PSA mRNA expression after brachytherapy was correlated with the serum PSA levels, grade, stage, and Gleason scores. The test of significance was performed using Student’s t test.
RESULTS SENSITIVITY OF PSA RNA DETECTION BY 32 P-RT-PCR A semiquantitative 32P-RT-PCR was used to analyze the sensitivity of PSA mRNA detection in total PSA-negative normal female RNA contaminated with the RNA from PSA-positive LNCaP control cells. By this assay, the presence of PSA mRNA expression was detected in as low as 10⫺7 g LNCaP RNA in 1 g of normal female RNA (Fig. 1A). This contamination simulates one tumor cell in 105 mononuclear cells. The PSA/G6PDH densitometry values were directly proportional to the level of contamination by LNCaP RNA. On the basis of these assay results, patient samples that showed a densitometric percent ratio value were labeled as follows: a percent ratio value of greater than 2% to 25% ⫽ ⫹ for PSA mRNA expression; greater than 25% to 50% ⫽ ⫹⫹; greater than 50% to 75% ⫽ ⫹⫹⫹; and greater than 75% to 100% ⫽ ⫹⫹⫹⫹. INCREASED PSA MRNA EXPRESSION SAMPLES COLLECTED DURING AND AFTER BRACHYTHERAPY All four normal male and female samples were negative for PSA mRNA expression. Samples col271
FIGURE 1. (A) Sensitivity of 32P-RT-PCR assay. One microgram of female RNA was contaminated with increasing concentrations of PSA-positive LNCaP RNA. Using this 32P-RT-PCR assay, the presence of PSA mRNA expression was detected in as low as 10⫺7 g LNCaP RNA in 1 g of normal female RNA. The 32P-RT-PCR products for PSA and G6PDH genes were run in 12% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. Using a gel documentation system, the intensity of the PSA and G6PDH bands were measured and densitometric values calculated as the percent PSA/G6PDH ratio. A bar graph below shows an increase in the percent PSA/G6PDH ratio as the LNCaP contamination increased. Error bars represent the mean of percent PSA/G6PDH ratio from two experiments. (B) Increased expression of PSA mRNA during brachytherapy. Patient samples were collected before, during, and after brachytherapy. Mononuclear cells were separated from the blood samples and RNA isolation performed. 32P-RT-PCR was performed for the PSA and G6PDH genes, and the samples were run in 12% polyacrylamide gel electrophoresis. The gel was dried and autoradiographed. LNCaP RNA was taken as a positive control. Patient samples that showed a densitometric ratio greater than 2% were considered positive for PSA mRNA expression. A bar graph below shows an increase in the percent PSA/G6PDH ratio during brachytherapy. Error bars represent the mean of the percent PSA/G6PDH ratio from two experiments.
lected from the 4 patients during prostate biopsy were negative for PSA mRNA expression. Of the 25 patients with prostate cancer, 23 were negative for PSA mRNA expression and 2 were positive for PSA mRNA expression before brachytherapy. Of the 23 patients who were negative for PSA mRNA expression before treatment, 15 (65%) turned positive during or after brachytherapy. However, the remaining 8 patients remained negative during and after brachytherapy. Two patients who were positive before treatment remained positive during and after treatment. An autoradiograph showing the highest PSA mRNA expression (⫹⫹⫹⫹) during brachytherapy is shown in Figure 1B. Most of the patients had a PSA mRNA expression level between 2% and 25%. CORRELATION OF CLINICAL DATA WITH PSA MRNA EXPRESSION IN PATIENTS AFTER BRACHYTHERAPY Of the 25 patients, 8 had rising serum PSA levels (biochemical relapse) as assessed by the American Society for Therapeutic Radiology and Oncology definition of biochemical relapse. Of the 15 patients who turned positive for PSA mRNA expression during or after brachytherapy, 7 (46.6%) had rising serum PSA levels. Of the 8 patients with neg272
ative PSA mRNA expression before, during, and after brachytherapy, 1 had a rising serum PSA level. This was statistically significant (P ⫽ 0.03; Table I). Elevation of post-treatment serum PSA levels did not correlate with stage, Gleason score, or pretreatment serum PSA level. Four patients who had pretreatment serum PSA levels of 4 ng/mL or less demonstrated PSA mRNA expression during or after the procedure. Of these 4 patients, 3 (75%) had rising serum PSA levels at last follow-up. Eighteen patients had pretreatment serum PSA levels between 4 and 10 ng/mL. Of these 18 patients, 12 demonstrated PSA mRNA expression during or after the procedure. Of these 12 patients, 4 (33%) had rising serum PSA levels. Three patients had pretreatment serum PSA levels greater than 10 ng/mL. Of these 3 patients, 1 (33%) had PSA mRNA expression during and after the procedure. Interestingly, none of these patients developed a rising serum PSA level. The pattern of relapse based on pretreatment PSA level was not significant. COMMENT Radical prostatectomy, brachytherapy, and external beam radiotherapy are currently used for the UROLOGY 60 (2), 2002
TABLE I. Comparative analysis of pretreatment, nadir, and current serum PSA level with PSA mRNA expression before, during, and after brachytherapy Pt. No.
Gleason Score
Pretreatment Serum PSA Level (ng/mL)
Before
During
After
PSA Nadir (ng/mL)
Biochemical Relapse
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
6 6 6 6 7 6 6 6 6 7 6 7 6 6 6 6 6 6 6 6 7 6 6 6 7
6.3 6 9.1 4.3 10 2.9 6.7 9.1 7 7.7 5.4 5.1 5 4 6.6 10.6 7 3.3 6.2 10.7 5.1 5.1 5.7 2.6 19
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
⫺ ⫺ ⫹⫹⫹⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺
⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫹ ⫹⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺
1.0 N/A 0.2 0.4 0.1 0.8 0.1 0.1 1 0.9 0.7 0.9 0.7 0.5 0.2 2.1 0.1 0.9 0.7 ⱕ0.1 1.0 ⱕ0.1 1.0 1.2 0.2
No No No No No No No No Yes Yes Yes No No Yes No No No Yes Yes No Yes No No Yes No
PSA mRNA Expression
KEY: PSA ⫽ prostate-specific antigen; Pt. No. ⫽ patient number.
treatment of localized disease; however, the wide spectrum of biologic behavior exhibited by prostatic neoplasms poses a difficult problem in predicting the clinical course for an individual patient.15 Traditional prognostic markers, such as grade, clinical stage, and pretreatment serum PSA, are used extensively and are of some prognostic value for individual men.16,17 The pattern of posttreatment serum PSA values is now the reference standard to define the success of any treatment and a good predictor of eventual outcome. Studies have shown that patients undergoing radical prostatectomy have considerable shedding of tumor cells in the peripheral blood during surgery. Sokoloff et al.18 showed that circulating PSA mRNA was detected in 42 (62%) of 68 patients with Stage pT2 and pT3 disease, but they found no significant relationship between the preoperative positivity of circulating PSA mRNA and other pathologic parameters. On the contrary, Katz et al.19 noted that the staging accuracy of PSA mRNA expression was greater than that of other modalities such as digital rectal examination, transrectal ultrasonography, or computed tomography. They also demonstrated that circulating PSA mRNA expression was an independent factor in predicting extracapsular disease extenUROLOGY 60 (2), 2002
sion and was superior to any other established prognostic factors such as serum PSA, Gleason score, or clinical stage.20 Olsson et al.21 showed that circulating PSA mRNA was a significant predictor of PSA recurrence after radical prostatectomy. Ogawa et al.22 found that preoperative PSA mRNA was positive in the peripheral venous blood samples of 11 (48%) of 23 patients, and, in these patients, age, preoperative serum PSA value, and clinical or pathologic stage were not significantly associated with positive PSA mRNA expression. They also found that PSA mRNA was positive in the peripheral blood in 18 patients (78%) during radical prostatectomy. All 11 patients with positive preoperative PSA mRNA expression had positive PSA mRNA expression during radical prostatectomy. Seven (58%) of 12 patients with negative preoperative PSA mRNA expression had a positive conversion of intraoperative PSA mRNA.22 They found no significant association among the clinicopathologic parameters, positive intraoperative PSA mRNA expression, and positive conversion during radical prostatectomy.22 Because interstitial brachytherapy for prostate cancer is also an invasive procedure, the potential for similar patterns of tumor cell dissemination is present. To analyze whether shedding of tumor 273
cells occurred in the peripheral blood, the sensitive 32 P-RT-PCR method was used to determine PSA mRNA expression as a marker of prostate cancer cells in the venous circulation. No PSA mRNA was detected in the blood from normal male or female blood samples or from the patients who underwent normal prostate ultrasound-guided biopsies. The 65% (15 of 23) positive rate for PSA mRNA expression during or after brachytherapy is similar to the rates reported after radical prostatectomy.22 Eight of these 23 patients remained negative for PSA mRNA expression before, during, and after brachytherapy. Of these 8 patients, 1 (12.5%) had a rising serum PSA level after treatment, indicating therapy failure; however, of the 15 patients who had PSA mRNA expression, 7 (46.6%) developed rising serum PSA levels. No significant correlation was found between PSA mRNA expression and the pretreatment clinicopathologic parameters of stage or time to PSA nadir. These results are similar to those reported by Olsson et al.21 in which circulating PSA mRNA was a significant predictor of PSA recurrence after radical prostatectomy but did not correlate with traditional pretreatment tumor characteristics. No statistically significant correlation between pretreatment serum PSA levels and detected PSA mRNA expression was observed, similar to the findings of Ogawa et al.22 after radical prostatectomy. They reported no significant association among the clinicopathologic parameters, positive intraoperative PSA mRNA, and positive conversion during radical prostatectomy. Several large studies have reported the results of biochemical PSA control after external beam radiotherapy and brachytherapy implantation of the prostate.6,23 Zagars et al.23 reported that 157 (22%) of 707 patients treated with external beam radiotherapy for localized prostate cancer developed biochemical serum PSA relapse at a median of 6.5 years. Eighty of these patients (51%) developed local recurrence, 24 (15%) developed distant disease, and the remaining patients had biochemical failure alone. Storey et al.,6 from the same institution, reported that biochemical serum PSA failure was seen in 37% (76 of 206) of patients after prostate brachytherapy at 5 years. The higher incidence of biochemical relapse in the latter group of patients is hard to explain, especially as these patients were probably at earlier stages with more organconfined disease. Stock et al.7 reported that only 33% of patients experiencing biochemical serum PSA relapse after brachytherapy had local prostatic failure, as documented by positive post-treatment biopsies, lower than the 51% rate of positive posttreatment biopsies reported in patients experiencing biochemical failure after external beam radiotherapy.23 Because local disease control in the 274
prostate itself does not appear to be the main factor in this biochemical relapse, it is reasonable to assume that much of this is due to a higher incidence of systemic failure. It is conceivable that the insertion of multiple needles into the prostate gland during brachytherapy provides a vascular access for tumor cells from trauma to blood vessels, leading to iatrogenic dissemination of tumor cells that can potentially lead to systemic failure. PSA bounce is a phenomenon that has been described after prostate brachytherapy and is reported to be observed in 35% of patients. PSA bounce also appears to be less frequent when the PSA nadir after treatment is lower (less than 4 ng/mL) and was not seen when the PSA nadir was less than 0.7 ng/mL.24 However, in our cohort of patients, a serum PSA rise was noted predominantly in patients expressing PSA mRNA, and the PSA nadir in these patients was low (less than 1.5 ng/mL). The prognostic significance of circulating prostate cancer cells as measured by RT-PCR for PSA mRNA after local therapy for prostate cancer has previously been reported by several investigators.25,26 Similar to our results, Philip et al.27 reported a poorer survival in patients found to have prostate cancer cells in the peripheral circulation in advanced disease. The higher incidence of biochemical failure in patients with PSA mRNA positivity is of concern, and strategies to reduce the impact of these circulating cells on the potential development of systemic disease need to be developed. However, these results need to be evaluated in a larger cohort of patients. CONCLUSIONS Our results suggest for the first time in published reports that a substantial number of patients undergoing brachytherapy have iatrogenic dissemination of prostate cancer cells in the peripheral blood caused by the insertion of needles in the prostate gland. Also, the detection of PSA mRNA in the peripheral circulation appears to have a significant correlation with biochemical failure after interstitial brachytherapy and needs additional study in a larger patient population. REFERENCES 1. Landis SH, Murray T, Bolden S, et al: Cancer statistics, 1999. CA Cancer J Clin 49: 8 –31, 1999. 2. Stamey TA, Yang N, Hay AR, et al: Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 317: 909 –916, 1987. 3. Fuks Z, Leibel SA, Wallner KE, et al: The effect of local control on metastatic dissemination in carcinoma of the prostate: long-term results in patients treated with 125I implantation. Int J Radiat Oncol Biol Phys 21: 537–547, 1991. 4. Kuban DA, el-Mahdi AM, and Schellhammer PF: I-125 interstitial implantation for prostate cancer: what have we learned 10 years later? Cancer 63: 2415–2420, 1989. UROLOGY 60 (2), 2002
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