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Cell Proliferation in Prostate Cancer Patients with Lymph Node Metastasis

A Marker for Progression

Departments of Pathology [L. C.] and Urology [L. C.], Indiana University School of Medicine, Indianapolis, Indiana 46202 and the Division of Radiation Oncology [T. M. P.], the Departments of Pathology [T. J. S., D. M. R., D. G. B.], Urology [B. C. L., M. L. B., H. Z., D. G. B.] and the Section of Biostatistics [A. L. W., B. G. S.], Mayo Clinic, Rochester, Minnesota

	ABSTRACT

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The biological aggressiveness of lymph node-positive prostate cancer is closely linked to cancer volume in nodal metastases. We evaluated MIB-1 (Ki-67) labeling index and bcl-2 expression in primary cancer and matched nodal metastases from 138 node-positive patients treated with radical prostatectomy and bilateral pelvic lymphadenectomy between 1987 and 1992 at the Mayo Clinic. One hundred twenty-eight patients (93%) received androgen deprivation therapy within 90 days after radical prostatectomy. Mean patient age was 66 years (range, 51–78). The median follow-up was 6.7 years (range, 0.03–11). MIB-1 (Ki-67) labeling index was determined by digital image analysis, and nodal cancer volume was determined by the grid method. Systemic progression, defined as the presence of distant metastasis documented by biopsy or radiographic examination, was used as an outcome end point in the Cox proportional hazard models. MIB-1 labeling index in nodal metastases was predictive of systemic progression-free survival (P = 0.001). The 8-year systemic progression-free survival was 100% for those with MIB-1 labeling index <3.5% compared with 78% for those with MIB-1 labeling index 7.8%. MIB-1 labeling index correlated with Gleason score, DNA ploidy, and nodal cancer volume (P < 0.001, 0.04, and <0.001, respectively). After controlling for nodal cancer volume, MIB-1 labeling index remained significant in predicting systemic progression-free survival (P = 0.047). bcl-2 expression in the primary cancer and lymph node metastasis was associated with systemic progression-free survival in univariate analysis (P = 0.027 and 0.048, respectively) but was not significant after adjusting for nodal cancer volume (P = 0.52 and 0.17, respectively). Our data indicate that assessment of cell proliferation in nodal metastasis is predictive of clinical outcome in prostate cancer patients with regional lymph node metastasis.

	INTRODUCTION

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Prostate cancer is the most common noncutaneous malignancy and second leading cause of cancer-related mortality in the American male, accounting for 179,300 new cases and 37,000 deaths in 1999 (1) . About 10% of patients have pelvic lymph node metastases at the time of diagnosis (2 , 3) , and many received androgen deprivation (4) with radiotherapy (5) or surgery (6, 7, 8) . Despite the adverse prognostic significance of nodal involvement, the clinical outcome for these patients is variable (2) . Additional factors are needed to more accurately determine outcome in these patients. In this study, we sought to determine whether cell proliferation was associated with patient outcome.

	MATERIALS AND METHODS

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Patients.
The study group consisted initially of 269 patients who underwent radical prostatectomy and bilateral pelvic lymphadenectomy between January 1987 and December 1992 at Mayo Clinic (2) . All patients had regional lymph node metastasis, and none had clinical evidence of distant metastases at the time of surgery. The final study population was limited to 138 patients with sufficient tissue in the primary cancer and metastasis for immunostaining. One hundred twenty-eight patients (93%) received androgen deprivation therapy within 90 days after prostatectomy, and this consisted of orchiectomy or luteinizing hormone-releasing hormone agonist with or without an antiandrogenic agent. After surgery, patients were evaluated quarterly for the first 2 years, semiannually for 2 additional years, and annually thereafter. Follow-up examinations included physical examination, serum PSA³ measurement, chest radiography, and computerized tomography of the abdomen and pelvis as clinically indicated. A radionuclide bone scan was conducted at least annually or as clinically indicated. Serum PSA was measured using the Hybritech Tandem-R assay (Hybrityech, Inc., San Diego, CA) in all patients. In those followed after surgery at another institution, PSA determination was performed at Mayo Clinic by means of a mailed blood specimen; alternatively, patients were contacted annually, and additional medical information was obtained from the local physician, as necessary (2 , 6, 7, 8, 9, 10)

Specimens.
The radical prostatectomy and bilateral pelvic lymphadenectomy specimens were examined by frozen section at operation and subsequently by permanent sections, as described previously (2 , 11, 12, 13) All histological evaluations were performed without knowledge of the clinical outcome. Briefly, the apex and base of the prostate were amputated or submitted as en face (shave) margins, and the prostate was serially sectioned perpendicular to the long axis of the gland from the apex to the tip of the seminal vesicles. After gross examination of the whole prostate slices, frozen sections were selected to encompass the cancer; the length, width, and height were determined by microscopic examination of frozen sections. The number of cancer sections submitted for frozen examinations from the radical prostatectomy specimens varied from 8 to 20 in this series, depending on cancer volume, prostate volume, and the preference of the pathologist. Approximately 14 prostate blocks were examined per case, and the method of sampling remained constant during the study period (2) .

The lymph nodes from the pelvic lymph node dissection were totally embedded. The median number of lymph nodes sampled was 14 (range, 4–33). Nodal cancer volume (size) was determined in permanent sections by the grid method (2) , and the total cancer volume of all positive nodes (nodal cancer volume) was used for statistical analysis.

The 1997 Tumor-Node-Metastasis system was used for pathological staging (14) . Grading of the primary cancer was performed according to the Gleason system (15) . Prostatectomy specimens were examined for DNA ploidy in all patients by flow cytometry with the Hedley technique (16) , and DNA histograms were classified as diploid, tetraploid, or aneuploid. (17)

Immunohistochemistry.
For inclusion in the study, adequate tissue from both the primary cancer and matched nodal metastases were required for immunostaining. Representative blocks of the primary cancer and paired lymph node metastases were selected from the same patient for immunostaining. Staining was performed on 6-µm, formalin-fixed, paraffin-embedded sections using the avidin-biotin complex technique. Primary monoclonal antibodies were used for evaluation of MIB-1 labeling index (Ki-67; Immunotech, Westbrook, ME; dilution, 1:50) and bcl-2 expression (Dako, Carpinteria, CA; dilution 1:20). 3,3-Diaminobenzidine was used as the chromogen, and 0.2% methyl green was used as the counterstain. Quantification of MIB-1 (Ki-67) labeling was performed using the CAS 200 digital image analyzer and proliferation index software programs (Becton Dickinson, Cellular Imaging Systems, San Jose, CA). At least 1000 cells were analyzed in each case. Immunoreactivity for bcl-2 was evaluated semiquantitatively on a 5% incremental scale ranging from 0 to 90%. All immunostains were evaluated without knowledge of clinical outcome. Positive and negative controls were run in parallel and gave appropriate results.

Statistical Analysis.
The number of cancer-specific deaths (8 events) was low; therefore, we focused on clinical systemic progression as a surrogate outcome variable. Progression was defined as the presence of distant metastasis documented by biopsy or radiographic examination as the end point for analysis. Survival was estimated using the Kaplan-Meier method. The Cox proportional hazards model was used to test for univariate associations of variables with systemic progression-free survival. Analysis of association of continuous variables with survival was performed using single degree of freedom (linear) terms in the Cox model. Nodal cancer volume was analyzed on the log base-2 scale because it was positively skewed. To avoid bias, no arbitrary cutoff points were chosen for continuous variables in the Cox regression analysis (18) . Continuous variables were split into three even groups (MIB-1) for the purpose of illustration and estimating survival rates at 8 years. The associations of MIB-1 labeling index and bcl-2 expression with clinical and pathological findings were assessed using the Spearman rank correlation coefficient. P < 0.05 was considered significant, and all Ps were two-tailed.

	RESULTS

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Patient characteristics are shown in Table 1 . The mean patient age was 66 years (range, 47–79), and the median follow-up was 6.7 years (range, 0.03–11). Twelve patients had distant metastasis (systemic progression), 8 died of prostate cancer, and 21 died of other causes. There was no significant difference in age, preoperative PSA concentration, surgical margin status, DNA ploidy, nodal cancer volume, systemic progression-free survival, and the length of follow-up between patients in the study group and those excluded from study because of lack of adequate tissue for immunostaining (data not shown). However, the study group had higher mean Gleason grade and more positive nodes than those excluded (P < 0.01).

View larger version (129K): [in this window] [in a new window]   Fig. 1. MIB-1 immunostaining in the lymph node metastasis from prostate cancer.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Kaplan-Meier curves (systemic progression-free survival) according to MIB-1 labeling index in the lymph node metastasis. To avoid bias, no arbitrary cutoff points were chosen for continuous variables in the Cox regression analysis, and patients were divided into three even groups according to MIB-1 labeling index for the purpose of illustration. Numbers within parentheses represent number of patients still under observation at 3, 5, and 7 years.

	DISCUSSION

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In this report, we found that cell proliferation of nodal metastases determined by digital image analysis of MIB-1 labeling index was significantly associated with systemic progression-free survival in prostate cancer patients with regional lymph node metastasis, and this finding was independent of nodal cancer volume. Patients with higher MIB-1 labeling indices had worse prognosis than those with a lower index. Furthermore, MIB-1 labeling index in nodal metastases was correlated with Gleason score, DNA ploidy, and nodal cancer volume. Our data indicate that cell proliferation in nodal metastasis is closely associated with the biological behavior of prostate cancer and that digital image analysis of MIB-1 labeling index in nodal metastases provides useful prognostic information regarding patient outcome.

In a previous study, we found that nodal cancer volume was the best predictor of systemic progression-free survival in prostate cancer patients among several classical clinical and pathological factors (2) . The risk of distant metastasis in patients with regional metastasis increased proportionally with increasing nodal cancer volume when treated by radical prostatectomy and immediate androgen deprivation (2) . Cher et al. (19) demonstrated that lymph node-positive patients with lower cellular proliferative fraction in lymph node metastases had significant survival advantage over those with higher proliferative fraction, and our results are similar. However, in their study, histological grade and pathological stage of the primary cancer were not significant in predicting outcome, suggesting that the behavior of lymph node-positive prostate cancer was more closely linked to the biological aggressiveness of the metastatic cancer rather than the primary cancer (19) . They hypothesized that patients with a high proliferative fraction in lymph node metastases have increased nodal cancer volume and were more likely to have distant metastasis with decreased survival than those whose cancer had a low proliferative rate.

In addition to cellular proliferation, other factors may be important in cell cycle regulation, tumor progression, and therapeutic response. For example, the bcl-2 oncoprotein inhibits apoptosis and prolongs the duration of cell survival. Its overexpression may be a predictor of clinical outcome in patients with localized prostate cancer (20 , 21) , and it may be associated with emergence of the androgen-independent state (22, 23, 24) . In the present study, bcl-2 expression was associated with other previously established prognostic factors (e.g., nodal cancer volume), but an independent association with systemic progression-free survival was not demonstrated conclusively. However, the statistical test of associations was of borderline significance (P = 0.17) for patient outcome, which suggested that further study of this factor may be of value.

The present study may have potential limitations. Although this relatively large population of node-positive patients had extended follow-up, the number of outcome events (i.e., distant metastases) were limited. Consequently, some variables that were inconclusive because of limited statistical power may attain statistical significance if the sample size is increased. We used clinical (systemic) progression as an end point in this study; however, most studies use biochemical progression as an end point. The clinical utility of biochemical progression for evaluation of treatment outcome is yet to be established (25 , 26) . At the Mayo Clinic, most lymph node-positive patients treated with radical prostatectomy also underwent immediate adjuvant hormonal therapy (2 , 6, 7, 8, 9, 10) . Therefore, our findings strictly apply to this treatment strategy, and the results reported herein should be interpreted cautiously when compared with other studies with different therapeutic regimens.

In summary, the biological aggressiveness of lymph node-positive prostate cancer is closely linked to cancer volume in the nodal metastases. However, the assessment of cellular proliferation in nodal metastasis provides significant prognostic information in prostate cancer patients treated by radical prostatectomy, bilateral pelvic lymphadenectomy, and androgen deprivation.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Supported in part by Grant #IRG-84-002-16 from the American Cancer Society (to. L. C.).

¹ To whom requests for reprints should be addressed, at Department of Pathology, Indiana University School of Medicine, UH 3465, 550 North University Boulevard, Indianapolis, IN 46202. E-mail: lcheng{at}iupui.edu

² Present address: Bostwick Laboratories, 6722 Patterson Avenue, Richmond, VA 23226.

³ The abbreviation used is: PSA, prostate-specific antigen.

Received 4/22/99; revised 7/12/99; accepted 7/15/99.

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