Introduction

Renal cell carcinoma (RCC) is one of the most prevalent urological malignancies worldwide1,2. The incidence of RCC, a highly aggressive disease, has steadily increased over the years. Approximately 30% of patients have metastases at first diagnosis, and another 20% of RCC patients with clinically localized disease will develop metastasis even after curative nephrectomy3. Although novel target therapies have been developed, most metastatic RCCs still eventually cause death4. Prediction models identifying patients with poor prognosis, who may benefit from early systematic therapy, are greatly needed. To date, the tumor, node, and metastasis (TNM) staging system is a widely used RCC prognostic predictor. However, it is inferior in accurately predicting the prognosis of RCC patients with diverse and complicated tumor backgrounds5. Therefore, novel biomarkers that can stratify patients with poor prognosis should be identified to precisely guide clinical decisions.

Ki-67, a proliferation marker that is expressed during the cell cycles of G1, S, G2, and M stages, except G0, is usually detected by immunohistochemical (IHC) staining6. Its strict association with cell proliferation and its co-expression with other well-known markers of proliferation indicate a pivotal role in cell division. Several studies recently reported that Ki-67 expression is associated with poor prognosis in several types of cancer7,8,9,10. However, the role of Ki-67 in the prognosis of RCC remains inconsistent. Ki-67 was considered an unfavorable prognostic marker in RCC in many studies11,12,13,14, but some studies reported that Ki-67 expression is prognostically irrelevant in RCC patients15,16. To obtain a more precise evaluation of the prognostic and clinicopathological value of Ki-67 expression in RCC, we performed a systematic review and meta-analysis to evaluate the prognostic value of Ki-67 quantitatively and explore the associations of Ki-67 with the clinicopathological features of RCC.

Results

Search Results

Our search strategy initially identified 1540 articles from the primary literature search. A total of 502 duplicate reports were excluded. After screening the titles and abstracts, 993 articles were excluded for various reasons such as non-human studies, letters, case reports, meeting records, reviews, and other obvious irrelevant studies. The remaining 45 articles were evaluated in full text. To avoid the heterogeneity caused by the detection method, studies without IHC evaluation were excluded. The remaining articles were further excluded for several reasons, such as no data available (hazard ratio [HR] and 95% confidence interval [CI]), low-quality studies17, and duplicate publication. Finally, 20 articles published from 2000 to 2016 with 5,398 patients satisfied the criteria for meta-analysis11,12,13,14,15,16,18,19,20,21,22,23,24,25,26,27,28,29,30,31. A flowchart of the study selection process is shown in Fig. 1.

Figure 1
figure 1

Flow chart of study selection.

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Characteristics of Studies

The main characteristics of the 20 studies are summarized in Table 1. Patients in these studies were all diagnosed with RCC with different tumor types and received radical or partial nephrectomy. Seven studies originated from the United States, three were from Finland, two were from Germany, two were from China, one was from France, one was from Japan, one was from Norway, one was from Italy, one was from Portugal, and one was from the United Kingdom. Among the studies, five studies were performed to analyze overall survival (OS), 11 studies were conducted to investigate cancer-specific survival (CSS), and six studies reported disease-free survival (DFS). Various clinicopathological data were reported in eight studies (TNM stage in four studies, pathological T stage in eight studies, metastasis in five studies, and Fuhrman grade in eight studies). All studies applied IHC staining to investigate Ki-67 expression. Positive Ki-67 expression was defined using different cutoff values among various studies, so we classified all the cases according to their original studies (positive or negative staining).

Table 1 Characteristics of eligible studies in the meta-analysis.
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Meta-Analysis

Our meta-analysis demonstrated that high Ki-67 expression in RCC was associated with poor OS (fixed-effect model, HR = 1.95; 95% CI: 1.44–2.64; p < 0.001; I2 = 0.0%, p = 0.594; Fig. 2A), CSS (fixed-effect model, HR = 1.67; 95% CI: 1.47–1.89; p < 0.001; I2 = 2.7%, p = 0.417; Fig. 2B), and DFS (fixed-effect model, HR = 2.56; 95% CI: 1.79–3.67; p < 0.001; I2 = 0.0%, p = 0.598; Fig. 2C). To explore the source of heterogeneity, meta-regression analysis on CSS was conducted by ethnicity, tumor extent at time of diagnosis, counting method, cutoff of staining, follow up time. The results showed that ethnicity (p = 0.889), tumor extent at time of diagnosis (p = 0.415), counting method (p = 0.858), cutoff of staining (p = 0.305), and follow up time (p = 0.467) were not significant contributors to heterogeneity. Furthermore, subgroup analysis stratified by ethnicity, tumor extent at time of diagnosis, histopathological subtype, counting method, cutoff of staining, and follow up time was also performed. With regard to ethnicity, high Ki-67 expression was correlated with poor OS (HR = 1.95; 95% CI: 1.44–2.64; p < 0.001), CSS (HR 1.67; 95% CI: 1.47–1.89; p < 0.001), and DFS (HR = 2.63; 95% CI: 1.73–4.01; p < 0.001) in Caucasian patients, as well as with poor DFS (HR = 2.38; 95% CI: 1.18–4.80; p = 0.015) but not with CSS (HR = 1.88; 95% CI: 0.41–8.67; p = 0.417) in Asian patients (Table 2). Regarding the tumor extent, high Ki-67 expression was associated with poor OS (HR 2.01; 95% CI: 1.46–2.77; p < 0.001) and CSS (HR 1.80; 95% CI: 1.52–2.31; p < 0.001) but not with DFS (HR = 2.11; 95% CI: 0.74–6.50; p = 0.178) for all stages of RCC; with poor DFS (HR = 2.63; 95% CI: 1.79–3.85; p < 0.001) but not with OS (HR 1.40; 95% CI: 0.52–3.76; p = 0.504) and CSS (HR = 2.29; 95% CI: 0.85–6.19; p = 0.104) for localized RCC; and with poor CSS (HR = 1.49; 95% CI: 1.22–1.82; p < 0.001) for metastatic RCC. For histopathological subtype, high Ki-67 expression was correlated with poor CSS (HR = 1.67; 95% CI: 1.47–1.89; p < 0.001) and DFS (HR = 3.66; 95% CI: 2.00–6.72; p < 0.001) for ccRCC. With respect to counting method, high Ki-67 expression was correlated with poor OS (HR = 1.86; 95% CI: 1.22–2.82; p = 0.004), CSS (HR 1.66; 95% CI: 1.44–1.90; p < 0.001), and DFS (HR = 2.57; 95% CI: 1.73–3.79; p < 0.001) for eyeball counting, as well as with poor OS (HR = 2.05; 95% CI: 1.32–3.19; p = 0.001) and CSS (HR = 1.73; 95% CI: 1.26–2.38; p = 0.001) for formal counting. In the cutoff of staining subgroup analysis, high Ki-67 expression was correlated with poor DFS (HR = 7.18; 95% CI: 1.91–26.99; p = 0.004) but not with CSS (HR = 1.26; 95% CI: 0.66–2.41; p = 0.476) when the cutoff value was less than 10%. Studies with a cutoff value greater than or equal to 10% showed that high Ki-67 expression was associated with poor OS (HR = 1.86; 95% CI: 1.22–2.82; p = 0.004), CSS (HR = 1.90; 95% CI: 1.45–2.49; p < 0.001), and DFS (HR = 2.36; 95% CI: 1.62–3.43; p < 0.001). Additionally, high Ki-67 expression was associated with poor CSS (HR 1.59; 95% CI: 1.38–1.84; p < 0.001) and DFS (HR = 2.45; 95% CI: 1.54–3.91; p < 0.001) in patients with follow up time less than 60 months; with poor OS (HR = 1.95; 95% CI: 1.44–2.64; p < 0.001), CSS (HR 1.93; 95% CI: 1.22–3.03; p = 0.005), and DFS (HR = 2.73; 95% CI: 1.55–4.82; p = 0.001) in patients with follow up time greater than or equal to 60 months.

Figure 2
figure 2

Forest plots of studies evaluating the association between Ki-67 expression and prognostic outcomes of RCC patients: (A) effect of Ki-67 overexpression on OS, (B) CSS, and (C) DFS. HR: hazard ratio; CI: confidence interval; OS: overall survival; CSS: cancer-specific survival; DFS: disease-free survival; RCC: renal cell carcinoma. HR > 1 implies unfavorable prognosis for patients with high Ki-67 expression.

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Table 2 Subgroup analysis of pooled HR for RCC patients with Ki-67 overexpression.
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In the comprehensive analyses of the role of Ki-67 expression in RCC as a biomarker, we investigated the relationship between elevated Ki-67 expression and clinicopathological characteristics. As shown in Table 3, high Ki-67 expression was significantly related to TNM stage (III/IV vs. I/II: risk ratio [RR] 2.03; 95% CI: 1.68–2.44; p < 0.001), pathological T stage (T3/T4 vs. T1/T2: RR = 1.67; 95% CI: 1.35–2.06; p < 0.001), metastasis (yes vs. no: RR = 2.15; 95% CI: 1.77–2.62; p < 0.001), and Fuhrman grade (III/IV vs. I/II: RR = 1.77; 95% CI: 1.20–2.60; p = 0.004). Some significant interstudy heterogeneity was observed in pathological T stage and Fuhrman grade, but analyses on TNM stage and metastasis did not exhibit significant heterogeneity.

Table 3 Meta-analysis of the association between high Ki-67 expression and clinicopathological features of RCC.
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Sensitivity Analysis

A sensitivity analysis was conducted by sequential omission of individual studies. Results are shown in supplementary materials (Sensitivity analysis). The pooled HR of OS, CSS, and DFS were not significantly changed, suggesting the robustness of the results.

Publication Bias

Begg’s and Egger’s tests, as well as funnel plots, were conducted to estimate publication bias in the present meta-analysis. As shown in Fig. 3, the funnel plots indicated that the included studies had no evident asymmetry. Furthermore, the results from Begg’s test (P value) and Egger’s test (intercept with corresponding 95% CI, P value) for the included studies assessing the survival outcomes were PBegg’s = 0.806, intercept 0.51 with 95% CI: −2.97 to 3.99, PEgger’s = 0.673 (OS); PBegg’s = 0.161, intercept 0.99 with 95% CI: −0.07 to 2.06, PEgger’s = 0.065 (CSS); and PBegg’s = 0.133, intercept 2.02 with 95% CI: −1.19 to 5.23, PEgger’s = 0.156 (DFS). These findings suggested that significant publication bias did not exist in our meta-analysis.

Figure 3: Funnel plots and Begg’s and Egger’s tests for the evaluation of potential publication bias.
figure 3

(A) Overall survival, (B) cancer-specific survival, and (C) disease-free survival.

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Discussion

First described in 1991 by Gerdes et al.32, Ki-67, a nuclear protein, is a famous marker of cell proliferation. It is expressed throughout the cell cycle in proliferating but not quiescent (G0) cells, so it has been used as a proliferation marker in many cancers. Recently, Ki-67 has drawn increasing attention as an attractive prognostic prediction marker and potential therapeutic target in malignant neoplasms. Several studies suggested that Ki-67 is significantly associated with the prognosis of bladder cancer, breast cancer, lung cancer, upper urinary tract urothelial carcinomas, cervical cancer, and lymphoma6,9,33,34,35,36. However, the prognostic and clinicopathological values of Ki-67 remain ambiguous in RCC. Therefore, we conducted this meta-analysis to resolve the remaining controversy and reach a reasonable conclusion.

In this study, we focused exclusively on validating Ki-67 IHC expression and evaluated the prognostic values of Ki-67 IHC expression in RCC. We concluded that high Ki-67 expression predicted unfavorable prognosis for patients with RCC. In particular, RCC patients with high Ki-67 expression exhibited poor OS, CSS, and DFS. Subgroup analysis revealed the following results. (1) In terms of ethnicity, high Ki-67 expression was correlated with poor OS, CSS, and DFS in Caucasian patients, as well as with poor DFS but not significantly with CSS in Asian patients. (2) Regarding the tumor extent, high Ki-67 expression was associated with poor OS and CSS but not significantly with DFS for all stages of RCC; with poor DFS but not significantly with OS and CSS for localized RCC; and with poor CSS for metastatic RCC. (3) For histopathological subtype, high Ki-67 expression was correlated with poor CSS and DFS for ccRCC. (4) In the cutoff of staining subgroup analysis, high Ki-67 expression was correlated with poor DFS but not significantly with CSS when the cutoff value was less than 10%. Studies with a cutoff value greater than or equal to 10% showed that high Ki-67 expression was associated with poor OS, CSS, and DFS. Subgroup analysis revealed that RCC patients with high Ki-67 expression presented a relatively unfavorable survival outcome, even though some association did not reach statistical significance. The absence of a significant association was possibly due to the relatively limited studies in the subgroups.

Our results also suggested that RCC patients with high Ki-67 expression were likely to have a higher TNM stage, pathological T stage, positive metastasis, and a higher Fuhrman grade. The biological mechanism of Ki-67 may partly explain its prognostic and clinicopathological significance in patients with RCC. Ki-67 is a well-known cell proliferation biomarker in many tumors and plays a critical role in mitosis by regulating chromatin recombinant. Ki-67 has been considered a good molecular surrogate of the aggressive behavior and therapy response for survival outcome assessment in several cancers including RCC28. In addition to the commonly applied proliferative activity marker, Ki-67 may be associated with promotion of epithelial-to-mesenchymal transition (EMT)37,38. EMT induction is a key process for development and progression of malignant tumors.

This study is the first comprehensive analysis on the associations between Ki-67 expression and prognostic and clinicopathological significance in patients with RCC, but several limitations should be acknowledged. First, all included studies measured Ki-67 expression via IHC, but the criteria to determine the positive or negative expression of Ki-67 were inconsistent in different studies, which may potentially contribute to heterogeneity. Therefore, a more unified standard should be defined in the future. Second, a remarkable heterogeneity of studies was observed in certain categories of analysis. The heterogeneity was probably caused by differences in factors such as the patients’ characteristics (ethnicity, nationality, gender, age, and tumor stage and grade), variation in cutoff values for Ki-67 expression, and different durations of follow up. Third, relatively few studies were extracted in some subgroup analyses, which might render premature results. With more eligible studies published in the future, an update is necessary to achieve a more reliable result. Finally, research with positive results is potentially more likely to be submitted and published than work with negative results, which could cause publication bias39, although this bias was not detected in the present analysis.

To conclude, despite the limitations listed above, this meta-analysis suggested the prognostic and clinicopathological importance of Ki-67 expression in RCC. The results demonstrated that high Ki-67 expression was associated with poor prognosis and advanced clinicopathological features, which could potentially serve as risk stratification markers and even therapeutic targets in RCC. However, more large-scale, multicenter prospective studies with standardized methods and long-term follow up are needed to verify our results.

Methods

Search Strategy

This meta-analysis was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)40.

A systematic literature search was performed in the electronic databases PubMed, Web of Science, and Embase on April 20, 2016 using the following search strategy: (“Ki67” or “Ki-67” or “MIB-1”) and (“carcinoma” or “neoplasm” or “tumor” or “cancer” or “malignancy”) and (“kidney” or “renal”) and (“prognosis” or “prognostic” or “survival” or “outcome” or “mortality”). Moreover, we manually searched the reference lists of relevant literature.

Selection Criteria

Studies were included based on the following criteria: (1) the association of Ki-67 with the prognostic value in RCC should be described, (2) studies detected Ki-67 protein expression by IHC, and (3) studies reported survival outcomes (OS, CSS, or DFS) with HR and 95% CI. Exclusion criteria were as follows: (1) non-English papers; (2) case reports, letters, commentaries, meeting records, or review articles; (3) the study focused on animal models or cancer cells; (4) the study investigated the survival outcomes of RCC by the combination of Ki-67 with other IHC marker(s); (5) the study did not analyze Ki-67 protein expression, clinical features, and survival outcome; and (6) the study lacked sufficient data for obtaining HR and 95% CI. All evaluations were independently performed by three individual researchers to ensure the accurate inclusion of studies. For duplicate studies, we only retrieved the most informative and recent studies for further analyses.

Data Extraction

The data of the eligible studies were extracted independently with a predefined form. Discrepancies in data extraction were resolved by discussion. The following data were extracted: surname of the first author, publication year, origin of the studied population, study design, extent of tumor, histopathological subtype, number of patients, gender, patient’s age, cutoff value, follow-up time, and effect estimates, namely, HR of Ki-67 expression for OS, CSS, or DFS, as well as their 95% CI (Table 1). If the HR and 95% CI were not directly available, we calculated HRs and their 95% CI based on the methods reported by Tierney et al.41.

Quality Assessment

The quality of the included studies was evaluated using the Newcastle–Ottawa scale, which was recommended by the Cochrane Non-Randomized Studies Methods Working Group17. Each study can be assessed by eight methodology items with a score ranging from 0 to 9. The high scores indicated high quality. We considered studies with scores of 6 or more as high quality for the meta-analysis. Only high-quality studies were included in further analysis to assure the quality of this meta-analysis.

Statistical Analysis

Pooled HR and RR with 95% CI were used to evaluate the association of Ki-67 expression with RCC prognosis and clinicopathological characteristics, respectively. An observed HR > 1 indicated poor prognosis for patients with high Ki-67 expression. An observed RR > 1 implied advanced clinicopathological characteristics in the high Ki-67 expression group. A heterogeneity test of pooled HR and RR was conducted using Cochran’s Q test and Higgins I-squared statistic. A P value of less than 0.1 was considered significant. The I2 values >50% is considered as a measure of severe heterogeneity42. A random-effect model was used when heterogeneity was observed (p < 0.1); otherwise, a fixed-effect model was used. The reasons for inter-study heterogeneity were also explored by using meta-regression analysis. To obtain a more precise evaluation of heterogeneity, we only performed meta-regression analysis on CSS which had sufficient included studies (n ≥ 10). Publication bias was assessed by funnel plot visual inspection and then statistically evaluated by Begg’s43 and Egger’s tests44. The statistical processes were performed by Stata 12.0 software (StatCorp, College Station, TX, USA), and p < 0.05 was considered statistically significant.

Additional Information

How to cite this article: Xie, Y. et al. Prognostic and clinicopathological role of high Ki-67 expression in patients with renal cell carcinoma: a systematic review and meta-analysis. Sci. Rep. 7, 44281; doi: 10.1038/srep44281 (2017).

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