Introduction

Radical prostatectomy (RP) is associated with an effective and durable local disease control for patients with clinically localized prostate cancer (PCa) [1]. Nevertheless, up to 30 % of patients experience biochemical recurrence (BCR) despite assumed effective local control [1]. Accurate evaluation of BCR risk status after initial treatment would guide physicians in the selection of high-risk patients who could benefit from adjuvant multimodal therapy and closer follow-up and spare those who would suffer from the side effects of such additional treatment without any benefit.

Molecular biomarkers may provide a better understanding of the biology of an individual’s tumor and may help stratify the heterogeneous patient population undergoing RP into risk categories that could help clinical decision-making regarding adjuvant therapy and follow-up scheduling. Ki-67 is an established marker of cell proliferation that is present during the G1, S, G2, and M stages of the cell cycle [2]. Previous single-center smaller studies have shown the strong prognostic value of Ki-67 in patients with PCa [3]. Despite such studies that add to our knowledge, no marker is used for individual treatment recommendation in PCa. This is largely due to the lack of external validation in large multicenter studies as part of a phased biomarker research program [4, 5]. Therefore, we sought to validate the prognostic value of Ki-67 in patients treated with RP for clinically localized PCa.

Patients and methods

Patient selection and data collection

This was an institutional-review-board-approved study, with all participating sites providing institutional data sharing agreements prior to the initiation of the study. Prior to analysis, the database was closed and the final data set was produced. The study cohort included 3,294 patients from eight European and North American centers with PCa treated with RP between 2000 and 2011. Patients with preoperative prostate-specific antigen (PSA) >50 ng/ml (n = 15), missing preoperative PSA (n = 57), surgical margin status (n = 13), lymph node status (n = 54), and RP Gleason score (n = 32) were excluded from the analysis. A total of 3,123 patients were considered for analysis. None of the patients received preoperative radiotherapy, hormonal treatment, or chemotherapy. No patient had distant metastatic disease evidence found at the time of RP.

Pathological evaluation and immunochemistry

All surgical specimens were processed according to standard pathologic procedures as outlined elsewhere [6]. Genitourinary pathologists assigned pathologic stage, which was reassigned according to the 2007 American Joint Committee on Cancer (AJCC) tumor, node, and metastasis (TNM) staging system when necessary. Lymphatic tissue removed during RP was submitted for histological examination. Positive pathological margin was defined as tumor cells in contact with the inked surface of the prostatectomy specimen.

Immunostaining was performed in a single laboratory on tissue slides. Immunostaining was performed on the Dako Autostainer (Carpinteria, CA) using mouse monoclonal antibody MIB-1 which is used in clinical applications to determine the Ki-67 labeling index (1:200 dilution; Dako). We used bright-field microscopy imaging coupled with advanced color detection software (Automated Cellular Imaging System, Clarient, Inc., CA). Ki-67 labeling index was considered to be high when samples demonstrated ≥20 % reactivity.

Follow-up

Follow-up (FU) was performed according to institutional protocols in agreement with local guidelines at the time. Generally, patients were seen postoperatively quarterly for the first year, semiannually in the second year, and annually thereafter. Digital rectal examination and PSA evaluation were performed at each visit. The primary endpoint BCR was defined as PSA value >0.2 ng/ml on two consecutive visits. The date of BCR was attributed to the day of the first PSA. In case of lymph node metastasis, immediate adjuvant androgen deprivation therapy was initiated. No patient received immediate postoperative radiotherapy.

Statistical analysis

Associations of Ki-67 with categorical variables were assessed using Chi-square test. Differences in continuous variables were analyzed using the Mann–Whitney U test. BCR-free survival curves were generated using the Kaplan–Meier method; log-rank test was applied for pairwise comparison of survival. Univariable and multivariable Cox regression models addressed the association of Ki-67 with BCR after RP. All p values were two-sided, and statistical significance was defined as a p < 0.05. Statistical analyses were performed using SPSS 17.0 statistical software (Chicago, IL).

Results

Descriptive characteristics

Baseline characteristics of the 3,123 patients are listed in Table 1. Ki-67-positive status was observed in 762 (24.4 %) patients. There was a significant association between Ki-67 status and lymph node involvement (LNI) (p = 0.039). None of the other clinicopathological characteristics was associated with Ki-67 status.

Table 1 Association of clinicopathologic variables and Ki-67 status
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Association of Ki-67 with biochemical recurrence after RP

Median follow-up for patients who did not experience disease recurrence was 44 months (range 3–144). Six hundred and twenty-one (19.9 %) patients experienced BCR within a median follow-up of 21 months (range 1–135). The estimated 3-year biochemical-free survival was 85 % for patients with negative Ki-67 status and 82.1 % for patients with positive Ki-67 status (log-rank test, p = 0.014) (Fig. 1).

Fig. 1
figure 1

Kaplan–Meier curves: biochemical recurrence-free survival in patients treated with radical prostatectomy for prostate cancer according to Ki-67 status

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Tables 2 and 3 summarize the univariable and multivariable cox regression analyses. For the entire cohort, preoperative PSA, RP Gleason sum, LNI, positive surgical margins, extracapsular extension, and seminal vesicle invasion were associated with BCR in univariable and multivariable analyses; Ki-67-positive status was also associated with BCR in both analyses (p = 0.015 and p = 0.019, respectively) (Table 2).

Table 2 Univariable and multivariable Cox regression analyses for prediction of biochemical recurrence in patients treated with radical prostatectomy for prostate cancer with Ki-67 status (overall analysis)
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Table 3 Multivariable Cox regression analyses for prediction of biochemical recurrence according Ki-67 status in subgroups of patients treated with radical prostatectomy
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Multivariable analyses in subgroup of patients revealed a significant association of Ki-67-positive status and BCR only in patients without LNI (p = 0.004), without surgical margins positivity (p = 0.047), or with Gleason sum = 7 (p = 0.015).

Discussion

In the present study, we investigated the significance of Ki-67 status in the prediction of BCR in a large international cohort of patients treated with RP after adjusting for the effects of standard clinic-pathologic features. We confirmed previous studies that demonstrated the usefulness of Ki-67 in PCa [3, 7]. After RP, most studies demonstrated Ki-67 as an independent predictor of BCR [8–23]; however, conclusions from these studies were limited by their relatively small size and single-center nature (Table 4). Similarly, after radiation therapy, Ki-67 has been shown to have prognostic value with regards to disease progression, distant metastasis, and disease-related mortality [24].

Table 4 Studies on Ki-67 as predictive biomarker of BCR after radical prostatectomy on multivariable analysis
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We demonstrated that Ki-67 was associated with LNI but none of the other clinic-pathologic features. Such association has been already reported by Revelos et al. [15]. Interestingly, in multivariable analyses performed in subgroup of patients, Ki-67 was not significantly associated with BCR in patients with unfavorable pathologic features such as LNI, surgical positive margins, extracapsular extension or seminal invasion, or Gleason score sum >7. Ki-67 does not seem to add any statistical and/or clinical value in patients who have already pathologic features of biologically and clinically aggressive disease. In agreement with this, Gunia et al. reported a significant contribution of Ki-67 only in patients with favorable characteristics (PSA <20 ng/ml, Gleason score <7 (4 + 3), no seminal invasion, and/or no LNI) [21]. Similarly, Zellweger et al. [25] demonstrated that Ki-67 assessed in prostate needle biopsies was only associated with BCR in patients with low-risk disease. Accordingly, in the present study, Ki-67 was associated with BCR in patients with favorable pathologic criteria such as negative surgical margin and no LNI.

Relevance of a biomarker in patient decision guidance lies in its capacity to classify patients into risk groups with corresponding recommendations [26]. Adjuvant therapy after RP is generally discussed in patients with unfavorable pathologic features, while patients with no positive surgical margin and no LNI could erroneously consider cured. Even in this group with favorable pathologic results, some patients will experience disease recurrence [27–29]. To consider all patients for adjuvant therapy would be inappropriate and lead to overtreatment. Therefore, validated new prognostic biomarkers could be useful in the identification of an individuals’ risk of disease recurrence allowing informed decision-making regarding multimodal therapy and risk-adjusted follow-up scheduling. Our findings indicate that Ki-67 could be helpful to identify patients who should be considered candidates for early adjuvant treatment. However, adjuvant therapy after RP remains a controversial topic that needs further evaluations [1]. Likely, the benefit of these therapies would be in patients who are at increased risk based on molecular features reflecting the inherent biological potential of the cells rather than relying on pathologic features alone.

Incorporation of informative biomarkers in clinical practice has been disappointing to the present day [26]. Ki-67 is a marker of proliferation expressed during G1, S, G2, and M phases of cell cycle but not in G0 and early G1 phases [2]. Despite its easy quantification by immunostaining on paraffin-embedded tissues and relevant results in the published literature, the use of Ki-67 in clinical setting is still limited. Many reasons can preclude the clinical use of new tissue biomarkers in PCa [30]. The variability of cutoff values to define positive status, study and limited cohorts, and lack of prospective studies could explain why Ki-67 is not yet a widespread tissue biomarker in clinical practice [3]. Therefore, further large and multicenter studies with established protocols are needed to consider this promising biomarker in the decision-making process in PCa.

To the best of our knowledge, this study investigated the association of Ki-67 with BCR after RP in the largest international multi-institutional cohort so far. However, our study is limited by its retrospective nature. Another limitation is due to the use of immunohistochemical techniques. Therefore, we used established staining protocols and automated scoring systems based on bright-field microscopy imaging coupled with advanced color detection software to reduce the number of related variables.

Conclusion

Ki-67 positive status is an independent and significant prognostic marker of BCR after RP. Its significance is particularly relevant in patients with favorable pathologic criteria. Its use could be informative in the identification of high-risk patients who may benefit from adjuvant therapy and/or closer follow-up despite assumed local curative surgery.