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Journal of Clinical Oncology, Vol 22, No 11 (June 1), 2004: pp. 2133-2140 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.09.150 Ki-67 Staining Is a Strong Predictor of Distant Metastasis and Mortality for Men With Prostate Cancer Treated With Radiotherapy Plus Androgen Deprivation: Radiation Therapy Oncology Group Trial 92–02From the Departments of Radiation Oncology and Pathology, Fox Chase Cancer Center; Department of Biostatistics, American College of Radiology (RTOG), Philadelphia, PA; Department of Pathology, Baylor School of Medicine, Houston, TX; Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT; Department of Radiation Oncology, University of Western Ontario, London, ON, Canada; Department of Radiation Oncology, Medical College of Wisconsin, Milwaukee, WI; Department of Radiation Oncology, University of California San Francisco, San Francisco, CA; Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA; and the Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, MI Address reprint requests to Alan Pollack, MD, PhD, Department of Radiation Oncology, Fox Chase Cancer Center, 333 Cottman Ave, Philadelphia, PA 19111-2497; e-mail: A_Pollack{at}fccc.edu
PURPOSE: The Ki-67 staining index (Ki67-SI) has been associated with prostate cancer patient outcome; however, few studies have involved radiotherapy (RT) -treated patients. The association of Ki67-SI to local failure (LF), biochemical failure (BF), distant metastasis (DM), cause-specific death (CSD) and overall death (OD) was determined in men randomly assigned to short term androgen deprivation (STAD) + RT or long-term androgen deprivation (LTAD) + RT. PATIENTS AND METHODS: There were 537 patients (35.5%) on Radiation Therapy Oncology Group (RTOG) 92-02 who had sufficient tissue for Ki67-SI analysis. Median follow-up was 96.3 months. Ki67-SI cut points of 3.5% and 7.1% were previously found to be related to patient outcome and were examined here in a Cox proportional hazards multivariate analysis (MVA). Ki67-SI was also tested as a continuous variable. Covariates were dichotomized in accordance with stratification and randomization criteria. RESULTS: Median Ki67-SI was 6.5% (range, 0% to 58.2%). There was no difference in the distribution of patients in the Ki-67 analysis cohort (n = 537) and the other patients in RTOG 92-02 (n = 977) by any of the covariates or end points tested. In MVAs, Ki67-SI (continuous) was associated with LF (P = .08), BF (P = .0445), DM (P < .0001), CSD (P < .0001), and OD (P = .0094). When categoric variables were used in MVAs, the 3.5% Ki67-SI cut point was not significant. The 7.1% cut point was related to BF (P = .09), DM (P = .0008), and CSD (P = .017). Ki67-SI was the most significant correlate of DM and CSD. A detailed analysis of the hazard rates for DM in all possible covariate combinations revealed subgroups of patients treated with STAD + RT that did not require LTAD. CONCLUSION: Ki67-SI was the most significant determinant of DM and CSD and was also associated with OD. The Ki67-SI should be considered for the stratification of patients in future trials.
The quantification of the expression of the Ki-67 antigen using immunohistochemical methods has been shown to provide an estimate of the growth fraction.1–4 As typically slow-growing tumors, most prostate cancers treated with radiotherapy have been found to have relatively low Ki-67 staining indices (Ki67-SIs).5–7 However, there is a wide spectrum of values, with some cases having a Ki67-SI of more than 50% and others having no discernable activity. Moreover, Ki67-SI has consistently been a significant correlate of outcome for prostate cancer patients treated definitively with radical prostatectomy or radiotherapy (RT).5–15 The relationship of Ki67-SI to outcome is a continuous function; the higher the value, the greater the risk of biochemical or clinical failure. There are several reasons the Ki67-SI has not been adopted in clinical practice or clinical trials of prostate cancer patients. First, prior studies have involved relatively small numbers of patients. In terms of radiotherapy as primary treatment, there are also few studies to draw meaningful conclusions.5–7, 14 Second, there is little published on the relationship of Ki67-SI to outcome after the combination of RT plus short term androgen deprivation (STAD), 16 and no data are available on the relationship of Ki67-SI to outcome after RT plus long-term androgen deprivation (LTAD). Third, there is no consistently defined cut point that has been identified. In the analysis described here, archival diagnostic tissue samples from a large cohort of patients randomly assigned to receive either STAD + RT or LTAD + RT in Radiation Therapy Oncology Group (RTOG) protocol 92-02 were stained for Ki-67 using MIB-1 antibody. This analysis of Ki67-SI is one of the largest biomarker studies ever performed in patients with prostate cancer. The results lend credibility to the premise that Ki67-SI is a valuable independent correlate of prostate cancer patient outcome that should receive broader consideration in the pretreatment evaluative process, along with Gleason score, initial pretreatment prostate-specific antigen (iPSA) level, and T stage.
Patient Characteristics There were 1,514 analyzable patients in RTOG protocol 92-02, 17 with 761 in the STAD + RT arm and 753 in the LTAD + RT arm. Tissue was available for Ki67-SI analysis in 537 patients (35.4%), with 257 randomly assigned to the STAD + RT arm and 280 to the LTAD + RT arm. For the study cohort, median age was 70 years (range, 43 to 88 years), and median iPSA was 20.2 ng/mL (range, 0.1 to 295.0 ng/mL). There were 359 patients (67%) with an iPSA of  30 ng/mL and 237 patients (44%) with T2c disease. An institutional Gleason score was available in four patients, with scores of 2 to 6 in 192 patients (39%). Median follow-up for the parent cohort (n = 1,514) was 101.1 months, and for the Ki67-SI available test cohort (n = 537) was 96.3 months.
Treatment Characteristics
Ki-67 Staining
End Points The Ki67-SI was defined as the percentage of tumor cells that displayed nuclear MIB-1 staining. Whenever possible, 2,000 tumor cells were counted using an eyepiece grid.
Statistics
The Ki67-SI was determined in 537 of 1,514 patients in the parent cohort. Analyses were performed to determine if the Ki67-SI test cohort was representative of the 977 cases in which Ki67-SI was not available. Table 1 shows that there was no statistically significant difference in pretreatment characteristics between the test cohort and the remaining cases in the parent cohort. Table 2 displays univariate comparisons of the test cohort to the Ki67-SI absent group in terms of the end points of OD, CSD, DM, LF and BF. No significant differences between the groups were identified.
Median Ki67-SI was 6.5, with a range of 0 to 58.2. Based on prior analyses of prostate cancer patients treated with RT ± STAD at The University of Texas M.D. Anderson Cancer Center6 and in RTOG trial 86-10,16 Ki67-SI was dichotomized at 3.5 (n = 121) versus > 3.5% (n = 416) and 7.1 (n = 290) versus > 7.1 (n = 247); these cut points have previously been shown to be predictive of outcome. Because the 3.5% cut point was not found to be significantly related to any of the end points listed in Table 2, the discussion to follow focuses on the 7.1% cut point.
As shown in Table 3, pretreatment characteristics and assigned treatment were generally well distributed between those who had a Ki67-SI
The most clinically important, as well as the most significant, association was between Ki67-SI and progression to DM. The data validate that when the Ki67-SI is > 7.1% there is a substantially increased risk of distant metastasis; however, the relationship is a continuous function. The results in Table 6 illustrate how the relative risk of distant metastasis increases as the Ki67-SI cut point increases up to between 9% and 10%. The optimal cut point for the patient population participating in RTOG protocol 92-02 seems to be in this range. In support of these findings, Table 7 shows that the significance of Ki67-SI in the multivariate analyses was strengthened when it was used as a continuous variable. For this analysis iPSA was also included as a continuous variable, as the relationships for iPSA as a continuous variable were stronger as well. As a continuous variable, Ki67-SI was significantly associated with all of the end points tested, except for LF.
Another analysis was performed to determine if Ki67-SI was useful in defining which patients in RTOG protocol 92-02 who were treated with STAD + RT would not require LTAD + RT. This was accomplished by testing the relationship of all possible prognostic subgroups (n = 16) based on the dichotomized variables of Gleason score (2 to 6 v 7 to 10), iPSA ( 30 v > 30 ng/mL), T stage (T2c v T3-T4) and Ki67-SI 7.1% v > 7.1%. Table 8 displays the estimated (or predicted) relative risk and CIs of distant metastasis from the Cox model for the 16 possible groups for patients randomly assigned to receive STAD + RT. To compensate for the multiple CIs, a Bonferroni correction was used to establish a new  level. The new level was computed with the formula: (adjusted) = (old)/n, where the new level is the probability that must be achieved to be significant given the number of comparisons, the old is the level that formerly defined significance, and n is the number of comparisons (ie, CIs) reported. Thus, with an original level of 0.05 and 16 comparisons, the new level is 0.003, which translates to a 99.7% CI.
The reference group used for comparison was comprised of the most favorable patients randomly assigned to the LTAD + RT arm with an iPSA 30 ng/mL, Gleason score 2 to 6, T stage T2c, and Ki67-SI 7.1. All CIs constructed were compared to the reference group. There were four prognostic groups of patients on the STAD + RT arm that had a similar risk of developing distant metastasis as those in the LTAD + RT reference group. The four favorable STAD + RT groups were: (1) PSA 30 ng/mL, Gleason score 2 to 6, stage T2c, and Ki67-SI 7.1%; (2) PSA > 30, Gleason score 2 to 6, stage T2c, and Ki67-SI 7.1%; (3) iPSA 30 ng/mL, Gleason score 7 to 10, stage T2c, and Ki67-SI 7.1%, and (4) iPSA > 30 ng/mL, Gleason score 7 to 10, stage T2c, and Ki67-SI 7.1%). When the STAD + RT patients in these four relatively favorable groups were pooled (n = 59) and compared to LTAD + RT patients with the same prognostic features (n = 63), the 5-year distant metastasis rates were not different (5.2% v 3.6%; P = .69; Gray's test). The 5-year metastasis rates for the pooled unfavorable STAD + RT (n = 173) and the LTAD + RT (n = 201) were statistically different at 19.2% and 10.7% (P = .01; Gray's test), respectively. There seems to be no statistically significant difference in the incidence of distant metastasis when patients with the more favorable features are treated with STAD + RT or LTAD + RT.
The immunohistochemical determination of the proportion of cells staining positive for the nuclear expression of the proliferation antigen Ki-67 has long been recognized as a correlate of growth fraction.1–4 For patients with prostate cancer, the vast majority of papers have shown that Ki-67 staining of prostate tumor tissue, usually from prostatectomy specimens is predictive of patient outcome. Of the relatively few reports of Ki-67 staining of pretreatment diagnostic tissue from patients treated definitively for local-regional prostate cancer with radiotherapy, all have shown that this measure of growth fraction is related to patient outcome.5–7,14 However, the prior studies have involved small numbers of patients, and there is no clear definition of a cut point that could be applied clinically on a routine basis. The analysis described here is one of the largest multi-institutional prostate cancer biomarker studies reported. The Ki67-SI, when used as a continuous variable, was found to be the strongest predictor of distant metastasis, cause-specific death and overall death. The higher the Ki67-SI, the greater the risk of an adverse event. The strongest association was observed between the Ki67-SI and distant metastasis. In order to illustrate the impact of Ki67-SI in univariate analysis and to define a Ki67-SI cut point with clinical applicability, the 3.5%6,7,16 and 7.1%16 cut points were tested. These cut points have been observed previously to correlate with patient outcome, including biochemical failure and distant metastasis. The search for a consistent end point has been problematic. The 3.5% cut point was confirmed in two different populations; one consisting of relatively favorable patients treated with radiation alone at M.D. Anderson Cancer Center6 and the other consisting of locally advanced patients enrolled in RTOG protocol 86-10.16 In the current study of patients from RTOG protocol 92-02, the 3.5% cut point was not associated with patient outcome. However, the 7.1% Ki67-SI cut point, which was derived from a prior analysis of RTOG protocol 86-10, 16 was strongly predictive of distant metastasis and cause-specific death in the RTOG 92-02 cohort. An investigation of different cut points in RTOG 92-02 revealed a relative risk of distant metastasis of 3.5 to 3.8 with a Ki67-SI of 9 to 10 (Table 6), suggesting that 7.1% is not the optimal cut point in this population. Thus, the most appropriate cut point is still clouded by some inconsistency between databases, which is probably related in part to the prognostic composition and distribution of Ki67-SI values of the patients analyzed. The distribution of Ki67-SI values might be skewed in studies with small patient numbers. The current analysis of about 500 cases with well-defined, mostly high risk features, is by far the most important to date. Although a Ki67-SI of 9 to 10 appears to be optimal in the RTOG 92-02 cohort as a predictor of the DM, the substantiation of the Ki67-SI 7.1% cut point, supports the use of this cut point for the present. The accurate determination of prognosis before treatment is of the utmost import for patients with prostate cancer. Treatment options are numerous and appropriate selection is based on risk stratification. This is particularly true when the recommendation is for LTAD + RT, because of the pronounced side effects and diminution of quality of life22,23 from LTAD. In addition to the side effects attributable to LTAD as a single agent, there is also evidence emerging that there is an increase in radiotherapy-related rectal toxicity when androgen deprivation is combined with RT.24,25 The ability to accurately segregate patients into risk groups has been greatly enhanced by the inclusion of iPSA into models of patient outcome, along with the classical factors of Gleason score and T stage. The promise of biomarkers is that risk stratification will become even more precise and possibly reveal mechanistic relationships in the process. The addition of the Ki67-SI to iPSA, Gleason score, and T stage identified subgroups of patients treated with STAD + RT who had the same DM rate as those with the most favorable prognostic attributes who were treated with LTAD + RT (Table 8); there was no apparent advantage to the use of LTAD in these subgroups. These findings should be considered hypothesis-generating since the numbers of patients in the individual subgroups was small (broad CIs), and it is possible that unknown prognostic factors, aside from iPSA, Gleason score, and T stage, were unevenly distributed. In summary, the analysis of the Ki67-SI in pretreatment diagnostic prostate biopsy samples from RTOG protocol 92-02 is one of the largest biomarker studies of prostate cancer patients enrolled onto a multi-institutional trial. The results demonstrate a very strong relationship between the Ki67-SI and DM, CSD and OD, which was more significant than the conventional prognostic factors of iPSA, Gleason score and T stage. Although the data presented suggest that a Ki67-SI > 7.1% is associated with a high risk of DM, the optimal cut point has not been firmly established.
The authors indicated no potential conflicts of interest.
Supported in part by grant CA-06927 from the National Cancer Institute and the US Department of Defense, US Army Medical Research Grant PC020427 and the Pennsylvania Department of Health. The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Cancer Institute or the US Department of Defense. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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17. Hanks JE, Pajak TF, Porter A, et al: Phase III trial of long term adjuvant androgen deprivation after neoadjuvant hormonal cytoreduction and radiotherapy in locally advanced carcinoma of the prostate: the Radiation Therapy Oncology Group Protocol 92-02. J Clin Oncol21:3972–3978, 2003 18. Cox J, Grignon D, Kaplan R, et al: Consensus statement: Guidelines for PSA following radiation therapy. Int J Radiat Oncol Biol Phys37:1035–1041, 1997[CrossRef][Medline] 19. Cox DR, Oakes D: Analysis of Survival Data, Analysis of Survival Data. London, UK, Champman and Hall, 1984 20. Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc53:447–457, 1958 21. Kalbfleisch JD, Ross LP: The statistical analysis of failure time data. New York, NY, John Wiley & Sons, 1980 22. van Andel G, Kurth KH: The impact of androgen deprivation therapy on health related quality of life in asymptomatic men with lymph node positive prostate cancer. Eur Urol44:209–214, 2003[CrossRef][Medline] 23. Higano CS: Side effects of androgen deprivation therapy: Monitoring and minimizing toxicity. Urology61:32–38, 2003[CrossRef][Medline] 24. Sanguineti G, Agostinelli S, Foppiano F, et al: Adjuvant androgen deprivation impacts late rectal toxicity after conformal radiotherapy of prostate carcinoma. Br J Cancer86:1843–1847, 2002[Medline] 25. Feigenberg SJ, Hanlon AL, Horwitz EM, et al: Androgen deprivation increases late morbidity in prostate cancer patients treated with 3D conformal radiation therapy. Int J Radiat Oncol Biol Phys57:S176, 2003 (suppl 2) Submitted September 26, 2003; accepted March 15, 2004. This article has been cited by other articles:
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 tests and Cox proportional hazards models.
