Int. J. Radiation Oncology Biol. Phys., Vol. 52, No. 4, pp. 996 –1001, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/02/$–see front matter
PII S0360-3016(01)02720-1
CLINICAL INVESTIGATION
Central Nervous System
Ki-67: A PROGNOSTIC FACTOR FOR LOW-GRADE GLIOMA? BARBARA J. FISHER, M.D.,* ELITZA NAUMOVA, B.SC.,* CHRISTOPHER C. LEIGHTON, M.D.,* GEORGE N. NAUMOV, B.SC.,† NANCY KERKLVIET, B.SC.,‡ DAVID FORTIN, M.D.,§㛳 DAVID R. MACDONALD, M.D.,§㛳 J. GREGORY CAIRNCROSS, M.D.,§㛳 GLENN S. BAUMAN, M.D.,* ¶ AND LARRY STITT, PH.D. Departments of *Radiation Oncology and †Experimental Oncology, London Regional Cancer Centre and University of Western Ontario, London, Ontario, Canada; ‡Department of Pathology, London Health Sciences Hospital and University of Western Ontario, London, Ontario, Canada; §Department of Medical Oncology, London Regional Cancer Centre, London, Ontario, Canada; Departments of 㛳Clinical Neurological Sciences and ¶Epidemiology and Biostatistics, University of Western Ontario, London, Ontario, Canada Purpose: Immunohistochemical techniques were used to detect the expression of Ki-67, a nuclear proliferation marker, in 180 low-grade glioma tumor specimens to determine whether Ki-67 is a prognostic predictor of survival or tumor recurrence. Methods and Materials: A clinical database of 180 low-grade glioma patients (35 children aged 5%. An average Ki-67 value of >5% was prognostically significant for reduced cause-specific survival (CSS, p ⴝ 0.05) and a Ki-67 level >10% was strongly significant of a poor survival outcome (p ⴝ 0.009). Ki-67 was not prognostically significant for progression-free survival. Other prognostically significant factors for CSS included age (p ⴝ 0.05), Karnofsky performance status (p ⴝ 0.0001), radiation dose (p ⴝ 0.02), extent of surgical resection (biopsy vs. others, p ⴝ 0.004), and timing of radiation (p ⴝ 0.0005). Ki-67 did not remain an independent statistically significant factor for CSS on multivariate analysis. Age and Ki-67 positivity (both maximal and average values) directly correlated (i.e., advancing age was associated with a higher Ki-67 index). When the patient group was further subdivided by age and timing of RT (postoperative vs. deferred), the prognostic significance of Ki-67 for CSS was lost. Within the deferred RT subgroup, a maximal Ki-67 >2% was associated with a worsened CSS. Within the pediatric population, Ki-67-negative patients had a 5-year CSS and progression-free survival of 100%. The 5-year CSS and progression-free survival declined significantly to 84% and 67% for patients with tumors demonstrating any degree of Ki-67 positivity (p ⴝ 0.005 and p ⴝ 0.006, respectively). Conclusion: Ki-67 is a useful predictor of CSS in low-grade gliomas; however, it is not independent of other prognostic factors, particularly age. Although Ki-67 was not helpful in predicting which adult patients were likely to benefit from postoperative RT, the results of the present study indicate a possible utility in the selection of pediatric patients for RT and in the selection of poorer prognosis patients for clinical trials. © 2002 Elsevier Science Inc. Ki-67, Proliferation markers, Low-grade glioma, Radiotherapy.
INTRODUCTION
nostic subgroups of glioma patients have been described. Such methods include histologic classification (2), tumor grading (2), analysis of clinical variables (1, 3), and immunohistochemistry. Two of these methods, histologic classification and tumor grading, are based on light microscopic examination and thus are very subjective (4). The fourth method, immunohistochemical determination of prolifera-
The outcome of patients with low-grade glioma (LGG) is highly variable. It is important to develop methods of categorizing LGG patients into different prognostic subgroups to better define the optimal therapy for individual patients (1). To date a number of methods for predicting the progReprint requests to: Barbara J. Fisher, M.D., Department of Radiation Oncology, London Regional Cancer Centre, 790 Commissioners Rd. E., London, Ontario NA 4L6 Canada. Tel: 519685-8650; Fax: 519-685-8627; E-mail:
[email protected] Presented as an oral presentation at the ASTRO Meeting 2000.
Acknowledgment—The authors thank Dr. David Ramsey for his assistance in obtaining the pathology specimens. Received Dec 28, 2000, and in revised form Oct 3, 2001. Accepted for publication Oct 10, 2001. 996
Ki-67: prognostic factor for LGG
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Table 1. Cox analysis of prognostic factors for overall survival Univariate
Age KPS Tumor location Timing of radiation Radiation dose Histologic features (astrocytoma vs. oligendroglioma/mixed glioma) Bulky residual tumor Ki-67†
Multivariate
Hazard ratio
p
Hazard ratio
p
3.95 0.33 1.69 2.81 0.69
0.057* 0.0001* 0.31 0.0005* 0.02* 0.88
7.89 0.26 1.75 4.31 0.31
0.0005* 0.00001* 0.30 0.0004* 0.002* 0.64
0.35 1.09
0.0004* 0.297
0.62 0.91
0.84 0.754
* Statistically significant. † Ki-67 as a continuous variable. Abbreviation: KPS ⫽ Karnofsky performance status.
tive activity with the monoclonal antibody to Ki-67, has been demonstrated to be clinically useful in distinguishing the biologic behavior of tumors (5) and correlates well with other proliferation markers, with mitotic activity, and with the histologic grade (6 –15). Ki-67 is a nuclear antigen that expresses itself during the proliferative phases of the cell cycle but not during the resting phase. The MIB-1 monoclonal antibody to Ki-67 recognizes this nuclear protein and therefore only stains cells undergoing active division. McKeevor et al. (16) concluded that Ki-67 as measured by the MIB-1 monoclonal antibody to Ki-67 was a superior predictor of survival compared with other markers of proliferation, such as proliferating cell nuclear antigen and bromodeoxyuridine. Montine et al. (10) found that a Ki-67 index of ⱖ7.5% in astrocytomas was associated with a higher histologic grade and poorer survival rate (p ⬍0.001) and was a better prognostic indicator than histologic grade (10). Ki-67 may have an advantage over these other methods because it is expressed throughout most of the cell cycle, thereby allowing the detection of a greater percentage of proliferating cells than does the mitotic count (6). The results of McKeevor et al. (16) and Montine et al. (10) notwithstanding, there is conflicting information in the literature concerning the utility of Ki-67 as a prognostic marker for gliomas in general and LGGs in particular. We previously published the clinical results of our policy of deferred radiotherapy (RT) for prognostically favorable LGGs (17). The ability of the MIB-1 antibody to detect Ki-67 on paraffin-embedded sections of archival materials allows the evaluation of proliferative potential in patients with long follow-up periods. The purpose of the present study was to investigate the possible prognostic relationship between the Ki-67 index and the clinical outcome of patients with LGGs, with specific reference to our policy of selective RT for symptomatic or bulky tumors (patients with bulky residual disease and astrocytic tumors were more likely to have had immediate postoperative treatment and those with complete resections and oligodendroglial or mixed glial pathologic features were more likely to have had RT deferred) (17). The specific question we wished to
answer was could Ki-67 be used clinically to ascertain which patients with a LGG by histologic criteria harbor a more aggressive tumor that would be best treated with early RT rather than surveillance. METHODS AND MATERIALS Statistical analysis The clinical factors extracted from the charts included age, gender, date of clinical presentation, presenting symptoms and signs, Karnofsky performance status, pre- and postoperative imaging findings, tumor location, type and extent of surgical resection, pathologic features, details of RT, date of diagnosis of tumor progression, details of additional treatment, and patient outcome. Statistical analysis was performed using BMDP version PC 90 (BMDP Statistical Software, Los Angeles, CA). Survival and progressionfree survival were obtained using the Kaplan–Meier product-limit method and were calculated from the date of surgery. Patients lost to follow-up were censored at the date of last contact. Potential prognostic factors and their effect on tumor progression and survival were analyzed using the Wilcoxon test. Univariate and multivariate analyses were performed using the Cox proportional hazards model (Table 1). p values were deemed significant if ⱕ0.05 and were based on two-tailed tests. Patients with unknown values were excluded for the analysis of that particular factor. Immunohistochemistry Paraffin blocks were obtained for 180 patients with LGG. The immunohistochemical technique for determining the Ki-67 index in tumors has been described by Cattoretto and Suurmeijer (18). For each patient, two representative nonconsecutive sections (4 m thick) were cut from paraffin blocks containing only tumor tissue (verified by the hospital pathology report). The formalin-fixed, paraffin-embedded sections were then deparaffinized and hydrated before an antigen retrieval process using citrate buffer (10 mM, pH 6.0). A streptavidin-biotin system based kit (LSAB2 Kit, Dako Corp., Carpinteria, CA) was used along with the
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Ki-67 mouse antihuman antibody (MMI, Novacastra, Newcastle-upon-Tyne, England). Nonspecific staining was blocked by incubating in a universal blocking solution (Dako Corp.) for 30 min at room temperature. The slides were then incubated with the Ki-67 primary antibody overnight at a 1/150 dilution. The appropriate negative and positive controls were run in parallel with the test slides. After immunohistochemical staining with Ki-67, the sections were examined using a Nikon Diaphot TMD microscope with a color CCD camera (Sony Corp., DXC-950, Japan) attached, allowing semiautomated computer analysis. Proliferating tumor cells during late G1, S, M, and G2 phases of the cell cycle were positively identified by the presence of red nuclear staining. Nonproliferating neoplastic tumor cells were identified by the distinct blue nuclear counterstain. Five random observation views were obtained (at 200⫻ magnification) from each section (total of 10 observation views per patient). All Ki-67 positive cells and the total number of cells were quantified for each observation view using Northern Eclipse image analysis software (Northern Eclipse, Toronto, Canada). In the present study, the Ki-67 proliferation index was assessed using an image analysis system to eliminate observer subjectivity. The measurements were then exported to a Microsoft Excel spreadsheet (Microsoft Corp., Edmond, WA). For each patient, the proliferation index was calculated by obtaining the ratio of Ki-67 positive cells to the total number of tumor cells per observation view multiplied by 100% and averaged over the 10 observations. The maximal Ki-67 value was also recorded. RESULTS Demographics Tumor specimens were collected for the study population, which consisted of 180 patients with a diagnosis of LGG (104 males and 76 females; 35 children and 145 adults [age ⬎18 years]). The Karnofsky performance status at the time of diagnosis was estimated to be ⱖ70 for 119 patients. Of the 180 patients, 131 had cerebral tumors, 7 had brainstem gliomas, 15 had cerebellar tumors, and 27 had tumors located at other sites. Thirty-one patients underwent gross complete resection, 113 subtotal resection, and 36 biopsy only. Eighty patients received immediate postoperative RT. The median dose was 45 Gy in 25 fractions. RT was deferred until tumor progression in 100 patients. The average Ki-67 value demonstrated a stronger correlation with survival than did the maximal Ki-67 value, and therefore the average Ki-67 value was used for this analysis (Table 2). Of the 180 specimens, 128 (71%) were Ki-67 positive (i.e., the percentage of Ki-67 staining cells was ⬎0). Only 7.7% of the tumor specimens had a Ki-67 level of ⱖ5%. Univariate analysis of the cause-specific survival (CSS) and progression-free survival (PFS) was performed using the Wilcoxon model at various cutoff points for Ki-67 positivity at 1% intervals between 0% and 10%. For CSS, a Ki-67 level of ⱖ2% positivity was of borderline statistical
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Table 2. Average Ki-67 values and univariate survival analysis (all subjects): Ki-67 as a discrete variable p value Branch point for Ki-67 positivity Negative (Ki-67 ⫽ 0) vs. positive (Ki-67 ⬎ 0) ⬎2% vs. ⱕ2% ⬎5% vs. ⱕ5% ⬎10% vs. ⱕ10%
% Tumors positive above cut point (n ⫽ 180)
CSS (Wilcoxon model)
PFS (Wilcoxon model)
70.3
0.594
0.696
18 8 3
0.055* 0.047* 0.009*
0.643 0.339 0.07
Abbreviations: CSS ⫽ cause-specific survival; PFS ⫽ progression-free survival. * Statistically significant.
significance (p ⫽ 0.055) but reached true statistical significance at a level of ⱖ5% positivity (p ⫽ 0.047, Fig. 1). A Ki-67 index of ⱖ10% was strongly significant at p ⫽ 0.009 for CSS (Table 2). The Ki-67 index was less directly related to PFS and was of only borderline significance at a level of ⱖ10%. When the patient population of 180 patients was subdivided into two subgroups—immediate postoperative RT vs. deferred RT, and Ki-67 indexes were reanalyzed, the statistical significance of the average Ki-67 value for CSS was lost (Tables 3 and 4). Table 3 illustrates the univariate (Wilcoxon) analysis of the average Ki-67 levels in relation to CSS and PFS for the immediate postoperative RT group, a group of patients with poorer prognostic factors and survival. Table 4 presents the results for the better prognostic group who had their postoperative RT deferred. An average Ki-67 index of ⬎2% was statistically significant for CSS in the deferred RT group (p ⫽ 0.04 Wilcoxon) and a Ki-67 value of ⬎5 was even more prognostic (p ⫽ 0.014).
Fig. 1. Overall survival for adult and pediatric population combined (n ⫽ 180): Ki-67 ⱕ5% vs. Ki-67 ⬎5%.
Ki-67: prognostic factor for LGG
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Table 3. Average Ki-67 levels and univariate survival analysis (immediate postoperative radiation patients only, n ⫽ 80) p value
Branch point for Ki-67 positivity Negative (Ki-67 ⫽ 0) vs. positive (Ki-67 ⬎ 0) ⱕ2% vs. ⬎2% ⱕ5% vs. ⬎5%
CSS (Wilcoxon model)
% Tumors positive above cut point (n ⫽ 80)
65
0.554
0.826
24 10
0.369 0.303
0.671 ⬎0.99
PFS (Wilcoxon model)
Abbreviations as in Table 2.
A total of 81 patients had progression after treatment. Twenty-eight of these patients developed malignant transformation. Ten of the malignant transformations were in Ki-67–negative patients and 18 in Ki-67–positive patients. This difference was of borderline statistical significance (p ⫽ 0.054) Adult vs. pediatric patients Sixty percent of the pediatric patient group had Ki-67– positive tumors. No statistical significance was found between CSS and Ki-67 positivity in the pediatric group, but borderline statistical significance was found in relation to PFS (p ⫽ 0.055, Fig. 2). The 5-year PFS for the 14 Ki-67– negative pediatric patients was 100% but dropped to 67% for the 21 Ki-67–positive patients (Ki-67 ⬎0). No statistically significant relationship was found between Ki-67 positivity and survival in the adult population of 145 patients. The correlation between age and Ki-67 values was examined. A Pearson correlation coefficient of the log values of both average Ki-67 values was statistically significant (p ⫽ 0.038). A Spearman rank correlation coefficient was performed to measure the association between age and the
Table 4. Average Ki-67 values and univariate survival analysis (deferred radiation patients only, n ⫽ 99) p value Branch point for Ki-67 positivity Negative (Ki-67 ⫽ 0) vs. positive (Ki-67 ⬎ 0) Ki-67 ⱕ2% vs. ⬎2% Ki-67 ⱕ5% vs. ⬎5%
% Tumors positive above cut point (n ⫽ 99)
CSS (Wilcoxon model)
65
0.55
0.83
24
0.37
0.67
10
0.30
⬎0.99
Abbreviations as in Table 2.
PFS (Wilcoxon model)
Fig. 2. Pediatric LGG population: Ki-67–negative patients (n ⫽ 14) vs. Ki-67–positive patients (n ⫽ 21).
average value. Ki-67 was also statistically significant (p ⫽ 0.024)— older age was associated with a higher Ki-67 value. Prognostic factors Cox univariate and multivariate analyses were performed on the 180 patients. Age, Karnofsky performance status ⱕ70, RT timing, and RT dose were statistically significant for overall survival on univariate and multivariate analysis (Table 1). Bulky residual tumor was only significant on the univariate analysis. Tumor location and the Ki-67 index (as a continuous variable) were not statistically significant. None of the above factors were prognostically significant for PFS. DISCUSSION The relative radiosensitivity of proliferating cells compared with nonproliferating cells has long been known (19, 20). The Ki-67 index is an indicator of the percentage of proliferating cells within a tumor and therefore a higher Ki-67 index theoretically indicates a more proliferative tumor. One might expect that highly proliferative tumors might be associated with shorter postoperative progressionfree intervals and with higher intrinsic radioresponsiveness, although not necessarily higher radiocurability. This study was conducted to determine whether the Ki-67 index can identify patients with poorer outcomes who might benefit from more aggressive treatment or entry into clinical trials. Univariate analysis of our group of 180 LGG patients revealed that the average Ki-67 index was a significant prognostic indicator for CSS, but not PFS. PFS may have been affected by the variability in the diagnosis of progression and the variability of the initial treatment and therefore CSS may be the more reliable end point. Our treatment policy, previously reported by Leighton et al. (17), involved
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selecting patients for postoperative RT on the basis of adverse prognostic factors such as residual tumor bulk and histologic findings. The postoperative RT group therefore represented a group with poorer prognostic features than the deferred RT group (17). The Ki-67 index was not useful in identifying patients who progressed despite RT. Within the deferred RT group, for which RT administration was not a confounding factor, the maximal Ki-67 did seem to identify a subgroup of patients with poorer CSS for whom more aggressive treatment might be of benefit. Attempts to correlate the Ki-67 labeling index with patient outcome have yielded mixed results. Several studies have reported associations between Ki-67 and tumor survival or tumor progression (4, 16, 22, 23). However two large series did not demonstrate a correlation between Ki-67 and clinical outcome or indicated a loss of prognostic utility after adjusting for other patient factors (24, 25). There are a number of reasons why different authors have reported contradictory results in terms of the prognostic utility of Ki-67, including different techniques of determining the Ki-67 index, interobserver subjectivity, the heterogeneity of Ki-67 within the tumor specimen (12, 26), and the small numbers of patients analyzed in some of the studies. Some studies have been histologically specific and others contained a histologically mixed population of LGGs. Heesters et al. (27) found that the Ki-67 index was lower in oligodendrogliomas than in astrocytomas but was more prognostically significant of the latter. A MIB-LI (MIB-1 labeling index) ⬍10% was of prognostic value on univariate analysis for both histologic types but on multivariate analysis was significant only for astrocytomas. This may also explain the variability between cutoff points in different studies demonstrating survival. For example, Schiffer et al. (28) evaluated 40 astrocytomas and found that a MIB1-LI of 8% was statistically different in terms of survival (p ⫽ 0.0066) and MIB-1 remained significant as an independent factor on multivariate analysis. Coons et al. (29) found that a cutoff Ki-67 index of 5% was statistically significant in 81 oligodendrogliomas and mixed gliomas. Montine et al. (10) noted a similar finding to ours, that patients with a Ki-67 index ⱖ3% were at risk of shorter survival. Tumor growth and response are complex processes and thus the determination of proliferation rate alone may not be sufficient to predict either prognosis or RT response. Because the Ki-67
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index has not been shown to correlate with tumor invasiveness (21), it may be necessary to look at multiple predictive factors measuring tumor invasion, apoptosis, angiogenesis, and so forth or perhaps at new molecular markers to have better prognostic information. Two smaller studies have looked at the relation between Ki-67 and RT primarily in fibrillary astrocytomas (i.e., no oligodendroglioma element). Hilton et al. (24) reported on 96 fibrillary astrocytoma patients, 58 of whom received postoperative RT. He found no difference in survival between the irradiated and unirradiated patients and no correlation between Ki-67 and survival in either the radiated or the unirradiated cohort, nor in the population as a whole. Similarly, Mastronardi et al. (30) studied 115 fibrillary astrocytomas and 15 mixed gliomas and found that Ki-67 did not predict for either survival or RT response. Ki-67 increased with increasing tumor grade but showed no association with either of the other clinicopathologic parameters. It appeared to have prognostic utility in univariate analysis but not after adjusting for patient age and tumor grade. Numerous studies (4, 15, 17) have demonstrated a positive correlation between the Ki-67 proliferation index and histologic grade of fibrillary astrocytomas but uniform methods in the quantification of the proliferation index were lacking. Ki-67 values for 23 subtotally resected pilocytic astrocytomas correlated with the biologic behavior of the tumor as measured by clinical and neuroradiologic followup. The mean MIB-LI tended to be lower in the group with progressive tumor, although the difference was not significant. Those tumors with negative MIB-1 were unlikely to show progression of residual tumor after partial resection (16). Our pediatric patient group yielded similar results in that the MIB-1 negative tumors had 100% survival and PFS rates. In conclusion, an average Ki-67 index of ⬎5% was predictive of CSS but not PFS in our LGG population. A maximal Ki-67 index of ⬎2% indicated a poorer CSS within the deferred RT subgroup. The independent prognostic utility of Ki-67 has not yet been proved, but its relationship to CSS may help identify a poorer prognostic group for entry into clinical trials of new therapies. Our results suggest the prognostic utility of this test may be higher in pediatric patients.
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