The biologic changes in recurrent prostate carcinoma following radiation therapy are not fully understood. The authors sought to determine the level of p53 protein overexpression and its association with cellular proliferation (Ki-67 labeling index), glutathione S-transferase-π (GST-π) expression, and other clinical pathologic findings in patients with locally persistent prostate carcinoma after radiation therapy.
The authors investigated p53 nuclear accumulation, cellular proliferation activity (Ki-67 labeling index by digital image analysis), and GST-π expression in 55 patients with persistent or recurrent prostate carcinoma after radiation therapy. All patients underwent salvage radical prostatectomy and bilateral pelvic lymphadenectomy following irradiation failure. The interval from radiation therapy to cancer recurrence ranged from 6 months to 17 years (mean, 3.8 years). Age at surgery ranged from 51 to 78 years (mean, 65 years). Mean follow-up after surgery was 5.7 years (range, 1–13 years).
p53 protein overexpression was associated with increased cell proliferation (Spearman rank correlation coefficient = 0.29, P = 0.03). A substantial proportion (62%) of recurrent cancer also showed GST-π immunoreactivity. No apparent correlation was observed between p53 protein overexpression, cellular proliferation (Ki-67 labeling index), or GST-π expression and Gleason score, pathologic stage, DNA ploidy, or patient outcome. There was an inverse correlation between GST-π expression and Gleason score (P = 0.06). The majority of prostate carcinomas (95%) were proliferative (mean Ki-67 labeling index, 7.0; range, 0–20), whereas concurrent prostatic intraepithelial neoplasia (PIN) had a lower Ki-67 labeling index (mean, 3.1; range, 0–11.5). Nineteen of 28 (68%) concurrent PIN demonstrated p53 immunoreactivity. A trend toward adverse clinical outcome was observed in patients with a higher Ki-67 labeling index in recurrent cancer.
The presence of multiple, separate foci of cancer in the prostate gland suggests a “field effect” in prostatic carcinogenesis.1–4 The p53 tumor suppressor gene plays an important role in genomic instability, cell cycle control and senescence, and DNA damage-induced apoptosis.5–10 Inactivation of the p53 gene is frequently encountered in many human cancers.5–6 Alteration of the p53 gene results in prolonged half-life with nuclear accumulation of the p53 protein. Inactivation of the p53 gene is an infrequent event in early stage prostate carcinoma.11–19 However, recent studies indicated that p53 nuclear accumulation was associated with adverse outcome after primary surgical or radiotherapeutic management and may be increased in recurrent prostate carcinoma after irradiation.15–19, 20–22 Furthermore, p53 mutations were associated with decreased sensitivity to DNA damaging agents, and loss of p53 function may be associated with radiation resistance.23–30 The relation between p53 protein overexpression and other biologic changes is uncertain following radiation therapy (RT) failure. In this study, we sought to determine the level of p53 protein overexpression and its association with cellular proliferation (Ki-67 labeling index), glutathione S-transferase-π (GST-π) expression, and other clinical pathologic findings in patients treated with salvage prostatectomy for locally persistent prostate carcinoma after RT.
MATERIALS AND METHODS
The study group consisted of 55 patients treated by salvage radical prostatectomy and bilateral pelvic lymphadenectomy between October 1967 and March 1996 at Mayo Clinic for biopsy-proven locally persistent or recurrent prostate carcinoma following RT. Informed consent was obtained from all patients. The interval from RT to cancer recurrence ranged from 6 months to 17 years (mean, 3.8 years). Age at surgery ranged from 51 to 78 years (mean, 65 years). Mean follow-up after surgery was 5.7 years (range, 1–13 years). After surgery, patients were evaluated quarterly for the first 2 years, semiannually for the next 3 years, and annually thereafter.31–32 Ten patients developed distant metastasis, 8 patients died of prostate carcinoma, and 6 patients died of other causes.
The radical prostatectomy and bilateral pelvic lymphadenectomy specimens were examined as previously described.32 The TNM (tumor, lymph nodes, and metastasis) system was used for pathologic staging, and grading of the primary cancer was performed according to the Gleason system.33–35 Prostatectomy specimens were examined for deoxyribonucleic acid (DNA) ploidy by flow cytometry using the Hedley technique.36 DNA histograms were classified as diploid, tetraploid, and aneuploid. Immunostaining was performed on 6 µm formalin fixed, paraffin embedded sections using the avidin-biotin complex technique.37, 38 Primary monoclonal antibodies were used for evaluation of p53 (DO-7, Dako, Carpinteria, CA; dilution 1:100), Ki-67 antigen (MIB-1, Immunotech, Westbrook, ME; 1:50 dilution), and human glutathione S-transferase-π (GST-π) (Dako, 1:50 dilution). 3,3′-Diaminobenzidine (DAB) was used as the chromogen, and 0.2% methyl green was used as the counterstain. Microscopic fields with the highest degree of immunoreactivity were chosen for analysis. Quantification of MIB-1 immunostaining (for Ki-67 labeling index) was performed using the CAS 200 digital image analyzer and proliferation index software programs (Beckton Dickinson, Cellular Imaging Systems).38
Statistical analysis was performed using the Cox proportional hazards model, and survival curves were estimated using the Kaplan–Meier method, with distant-metastasis free and cancer specific survival as the endpoints. The association between p53 protein overexpression, Ki-67 labeling index, GST-π, and other clinical and pathologic findings were assessed as candidate predictors using Spearman rank correlation coefficients. A P value of <0.05 was considered significant, and all P values were two-sided.
The distribution of p53 nuclear accumulation, MIB-1, and GST-π expression in recurrent prostate carcinoma after RT is shown in Figure 1. Nuclear p53 accumulation was detected in 19 of 28 prostatic intraepithelial neoplasia (PIN) (68%) and 50 of 55 prostate carcinomas (91%), respectively (Fig. 2A and 2B). The mean Ki-67 labeling index was 0.40% (range, 0–1.4%), 3.1% (range, 0–11.5%), and 7% (range, 0–20%) in normal tissue, PIN, and prostate carcinoma, respectively (Fig. 2C and 2D). MIB-1 immunoreactive cells were not detected in 2 (4%) of 55 cases. The mean percentages of GST-π immunoreactive cells were 3.2% (range, 0–10%), 0.4% (range, 0–5%), and 6% (range, 0–40%) in normal tissue, PIN, and prostate carcinoma, respectively. Thirty-four (62%) of 54 recurrent cancers showed GST-π immunoreactivity (Fig. 2E and 2F). Two (7%) of 28 concurrent cases of PIN showed GST-π immunoreactivity.
Overexpression of p53 protein was associated with increased proliferative activity in prostate carcinoma cells (Fig. 3, P = 0.03), but there was no correlation between p53 protein overexpression, cellular proliferation (Ki-67 labeling index), or GST-π with Gleason score, pathologic stage, or DNA ploidy. GST-π expression in prostate carcinoma was lower in the presence of cancer with a Gleason score of 8–10, but this difference was only marginally significant (P = 0.06). With the Cox proportional hazards model, none of these candidate predictor markers was associated with distant metastasis free survival or cancer specific survival. Patients with a higher Ki-67 labeling index tended to have a worse prognosis. Five-year distant metastasis free survival was 91(± 6)% versus 73(± 10)% for those with Ki-67 labeling index <7% versus ≥7%, respectively (P = 0.8). Five-year cancer specific survival was 95(± 5)% versus 87(± 7)% for those with Ki-67 labeling index <7% versus ≥7%, respectively (P = 0.4). Five-year cancer specific survival was 86% versus 75% for those GST-π with ≥2% versus <2% in cancer, respectively (P = 0.96).
Radiation therapy (RT) is commonly used for the treatment of clinically localized prostate carcinoma.39 Although locally persistent or recurrent cancer may be associated with an adverse outcome,40 the contemporary use of post-RT prostate specific antigen (PSA) monitoring, transrectal ultrasound–guided prostatic biopsy,41 and early “salvage” intervention31–32 provides the opportunity to identify locally persistent cancer at an earlier and (potentially) more curable stage. The prognostic significance of various biomarkers in prostate carcinoma has been previously studied,11–18, 42 but little is known about the biologic characteristics associated with cancer that persist after RT. In this study, a large proportion of cancers showed p53 nuclear accumulation after RT, and overexpression of the p53 protein was associated with increased cellular proliferation of prostate carcinoma cells.
Prendergast et al. studied 18 patients with locally recurrent prostate carcinoma after RT, and found that 72% had p53 nuclear immunoreactivity; among 5 patients with available pre-RT biopsies, all had p53 immunoreactivity.20 The immunohistochemical findings correlated with single strand conformation polymorphism and DNA sequencing analysis.20 This observation suggested that p53 alterations may be present prior to RT and may serve as a pretherapy marker for cancer recurrence. Retention of proliferating cell nuclear antigen (PCNA) immunoreactivity in post-RT prostatic biopsies correlated with local cancer recurrence.43 In the current series, most patients (96%) had histologically evident prostate carcinoma that was proliferative, as demonstrated by MIB-1 immunostaining. The mean Ki-67 labeling index in recurrent prostate carcinoma was increased (mean, 7.0%), compared with those from prostatectomy series without prior RT at our institution (mean, 2.7%; unpublished data). Patients with higher cellular proliferative rates also had increased p53 protein overexpression, which suggested that these tumors were biologically active.
Prior studies demonstrated the prognostic importance of post-RT DNA ploidy and Gleason grade as factors associated with outcome for patients with locally recurrent prostate carcinoma.32 For such patients, locally persistent cancer tended to have increased DNA content44 and may32, 45, 46 or may not47 be more poorly differentiated. Nonetheless, the biologic nature of cancers that persist at the primary site despite RT administered with curative intent is not fully understood.
Glutathione S-transferase π (GST-π) is a detoxifying enzyme that inactivates reactive oxygen species by conjugation with glutathione (Toffoli et al.48 and Pacelli et al., unpublished data). Most prostate carcinomas do not express GST-π, and loss of GST-π expression was considered a phenotype associated with malignant transformation.49, 50 Previous studies demonstrated that GST-π immunoreactivity was greatest in benign epithelium, with a progressive decrease from the malignant cells populating the primary cancer to those found in lymph node metastasis; a high percentage of immunoreactive cells in benign prostatic epithelium correlated with better patient outcome (Pacelli et al., unpublished data). In the current study, a substantial proportion (62%) of patients with recurrent cancer had GST-π immunoreactivity. Cancers with a higher Gleason score had diminished GST-π immunoreactivity, which did not, however, reach statistical significance (P = 0.06). These results should be interpreted cautiously because of the limited sample size and events.
The current investigation had several limitations. It provided a description of certain post-RT biologic cancer characteristics in a select group of patients with cancer recurrence clinically confined to the primary site of origin, and who underwent a second curative attempt with a surgical salvage procedure. Therefore, caution should be used in interpreting these results. The study design did not allow determination of whether the biologic characteristics of cancer changed over time (i.e., time-dependent cancer “clonal evolution”)44, 46 or whether cancer recurrence was an expression of an innately aggressive tumor (i.e., tumor “selection”) prone to recurrence.51 Indeed, the observation that p53 immunoreactivity was also present in the pre-RT specimens of patients with post-RT cancer recurrence20 and the association of abnormal p53 protein expression with an adverse patient outcome14–19 may suggest that these patients had unfavorable cancer types prior to the inception of RT. Additional study is necessary to obtain conclusive evidence supportive of the “clonal evolution” or “selection” hypotheses. Analysis of the time course of p53 positivity rates after RT would provide insights into the molecular mechanisms of radiation-induced changes, and assessment of p53 mutations by DNA sequencing may yield additional information.
In summary, patients selected for salvage prostatectomy had recurrent prostate carcinoma that remained biologically aggressive after RT. Increased p53 nuclear accumulation correlated with cellular proliferative activity, both of which were found in a high proportion of study patients. Additional study is required to clarify the causality and significance of these observations.