Augmented expression of Ki-67 is correlated with clinicopathological characteristics and prognosis for lung cancer patients: an up-dated systematic review and meta-analysis with 108 studies and 14,732 patients

As shown in Fig. 1, two investigators read the full text and considered 108 studies in 95 articles [18, 25, 32‐124] consisting of 14,732 patients as applicable. The baseline characteristics of the included articles are indicated in Table 1. Ninety-six studies included data regarding OS, and nineteen studies included data regarding DFS. The number of cohorts of each study ranged from 32 to 778. In terms of the study region, 62 studies were from Asia, 30 were from Europe, and 15 were from America. In total, 104 studies consisted of 14,596 NSCLC patients: of these, 28 studies reported on ADC patients, and 10 studies reported on SQC patients. All the studies detected Ki-67 reactivity using IHC.

In total, 95 studies with 13,678 lung cancer patients investigated the impact of Ki-67 expression on OS (Table 2). The pooled HR of the total population for OS was 1.122 (95%CIs: 1.089–1.156, Z = 7.56, P < 0.001; I² = 78.20%, P < 0.001, Figs. 2, 3 and 4), showing that a high Ki-67 level indicates worse outcome for lung cancer patients. Furthermore, the correlation of high Ki-67 expression with DFS in 3127 lung cancer patients was then analyzed (Table 3). For the total study population, worse DFS (HR = 1.894, 95%CIs: 1.456–2.463, Z = 4.76, P < 0.001, Fig. 5) was observed among patients with high expression of Ki-67, while the heterogeneity using the random effects model was obvious (I² = 78.30%, P < 0.001). To investigate the source of heterogeneity, subgroup analyses of publication year, region, histological type, sample size, cut-off value of Ki-67 and estimated method for HR determination were performed. From the subgroup analysis of OS, no heterogeneity was found in the small cell lung cancer (SCLC) group (I² = 22.30%, P = 0.277). Next, a reduction in heterogeneity was observed after performing subgroup analysis of DFS according to the study region, especially in the studies from America (I² = 8.40%, P = 0.351) and Asia (I² = 25.60%, P = 0.179). As indicated in the subgroup of cutoff value, there was a low degree of heterogeneity in both Ki-67 low expression (I² = 25.30%, P = 0.196) and high expression groups (I² = 19.40%, P = 0.287). Furthermore, we also performed meta-regression analysis to explore the original of the heterogeneity in the studies. Consistent with the subgroup analysis, the results revealed that the regions and cut-off values might be the potential bias for the heterogeneity (P = 0.017 and P = 0.022, respectively). Altogether, we concluded that the different regions and inconsistent cut-off values might have contributed to the heterogeneity in the results of analyses for DFS. The regression also revealed that the heterogeneity originated from regions and inconsistent cut-off values (Table 4).

An association of Ki-67 expression with age in 2506 lung cancer patients was identified using the fixed effects model in 19 studies, and higher Ki-67 expression was found to be more common in older patients (OR = 1.246, 95%CIs: 1.039–1.494; Z = 2.37, P = 0.018, I² = 0.00%, P = 0.967, Table 5 and Additional file 1: Figure S1). Subsequently, the results revealed significant differences in Ki-67 level between male and female (OR = 1.874, 95%CIs: 1.385–2.535; Z = 4.07, P < 0.001, I² = 69.70%, P < 0.0001, Additional file 1: Figure S1B). Meta-analysis of 15 studies including 2152 lung cancer patients revealed a positive association between high Ki-67 level and smoking history (OR = 3.087, 95%CIs: 2.504–3.806, Z = 10.56, P < 0.001; I2 = 39.40%, P = 0.064, Additional file 1: Figure S1C). According to the histological type, a pooled OR of 0.397 (95%CIs: 0.236–0.667) indicated that Ki-67 expression was significantly higher in ADC compared with that in SQC (Z = 3.49, P < 0.001; I² = 81.20%, P < 0.001, Additional file 2: Figure S2A). Next, tumor differentiation was considered. The results from 11 studies enrolling 1731 lung cancer patients showed that an elevated Ki-67 level was associated with poor differentiation, with a pooled OR of 1.993 (95%CIs:1.262–3.146, Z = 2.96, P = 0.003; I² = 66.30%, P = 0.001, Additional file 2: Figure S2B). A total of 13 studies with 1851 individuals were analyzed in this meta-analysis, and the results showed that a higher Ki-67 level was positively associated with the pathologic stage III/IV with a low degree of heterogeneity (OR = 1.867, 95%CIs: 1.498–2.327, Z = 5.56, P < 0.001; I² = 23.1%, P = 0.210, Additional file 2: Figure S2C). A trend toward positive correlation was found between a high Ki-67 level and larger tumor size in 12 studies based on 1707 lung cancer patients, with a pooled OR of 1.436 (95%CIs:1.127~ 1.290,, Z = 2.93, P = 0.003; I² = 0.00%, P = 0.876, Additional file 3: Figure S3A). Twenty-three studies comprising 2994 cases were used for meta-analysis of Ki-67 expression and lymph node metastasis, and the pooled OR indicated that a high Ki-67 level was significantly correlated with lymph node metastasis positive (OR = 1.653, 95%CIs: 1.285–2.127, Z = 3.91, P < 0.0001; I² = 46.70%, P = 0.008, Additional file 3: Figure S3B). The association of Ki-67 expression and TNM stage was then incorporated into the meta-analysis. Eight studies with 736 patients showed a trend for correlation between Ki-67 overexpression and advanced TNM stages, with a pooled OR of 1.50 (95%CIs:1.053~ 2.126, Z = 2.25, P = 0.024; I² = 36.90%, P = 0.134, Additional file 3: Figure S3C). In the meta-analysis, no association between Ki-67 and tumor stage was observed in lung cancer patients (OR = 1.287, 95%CIs:0.882–1.877, Z = 1.31, P = 0.191; I² = 55.30%, P = 0.013). Additionally, analysis of four selected studies using the random effects model did not reveal any significance for the association between Ki-67 expression and metastasis (OR = 2.609, 95%CIs: 0.667–10.204, Z = 1.38, P = 0.168) or invasion (OR = 0.993, 95%CIs: 0.511–1.930, Z = 0.02, P = 0.984; I² = 14.20%, P = 0.312).

To identify potential publication bias, Begg’s test and funnel plots were used. No publication bias was found in the analysis for OS (p = 0.444, Fig. 6a) and DFS (P = 0.246, Fig. 6b). Moreover, there was no publication bias among any of the analyses used to correlate Ki-67 expression and clinicopathological characteristics (all P > 0.05, Table 5, Additional file 4: Figure S4, Additional file 5: Figure S5, Additional file 6: Figure S6 and Additional file 7: Figure S7).

The databases that we searched included PubMed, Web of Science, EMBASE, and Chinese datasets (WanFang, China National Knowledge Infrastructure and Chinese VIP) until June 1, 2017. The key words identifying the articles were as follows: (Ki-67 OR Ki67 OR MIB-1 OR “proliferative index” OR “proliferative activity” OR “mitotic index” OR “labeling index” OR “mitotic count” OR “proliferative marker” OR “mitotic figure” OR “mitotic activity”) AND (Cancer OR carcinoma OR adenocarcinoma OR tumour OR tumor OR malignanc* OR neoplas*) AND (Lung OR pulmonary OR respiratory OR respiration OR aspiration OR bronchi OR bronchioles OR alveoli OR pneumocytes OR “air way”).

Publications were included if they met the following inclusion criteria: (1) the patients enrolled had been diagnosed with lung cancer; (2) the results for the study included the correlation between Ki-67 and overall survival (OS) or disease-free survival (DFS); (3) the samples used in the studies were human lung tissue, serum or sputum but not animals or cell lines; (4) the techniques used to measure the expression level of Ki-67 in cancer tissue or tumors of the patients were immunohistochemistry (IHC), PCR/RT-PCR, ELISA or western blotting; (5) the study provided hazard ratios (HRs) and their 95% confidence intervals (CIs) or sufficient information for estimating these parameters; (6) the article was fully written in English or Chinese; and (7) the sample size was larger than 30. Studies were excluded if they met the following exclusion criteria:(1) if they included animal experiments or cell lines or were pre-clinical studies, meta-analyses, reviews, comments, conference abstracts, letters or case reports; (2) articles in languages other than English or Chinese; and (3) studies did not include the key information for survival analyses such as HRs and 95%CIs. To avoid data duplication, when the same patient cohort was reported in different publications or the same article was found in different journals, only the most recent and complete publication was included.

All the articles were independently reviewed and selected by two investigators. Discrepancies were resolved by discussion and arbitrated by a third investigator. The following information was extracted from each publication: first author’s name, year of publication year, pathology type, tumor stage, number of patients, sample type, cut-off value of Ki-67, determination assay, method to extract HR and survival type. Additionally, we also obtained the clinicopathological characteristics of the lung cancer patients in the included studies including age (old/young), gender (male/female), histological type (adenocarcinoma/squamous carcinoma, ADC/SQC), smoking status (smoker/non-smoker), differentiation (poor/well or moderate), pathologic stage (III-IV/I-II), tumor size (large/small), tumor stage (T3–4/T1-T2), metastasis (yes/no), lymph node (N1-Nx/N0), TNM stage (T3–4/T1-T2) and invasion (yes/no). Especially, the tumor stage was used to describe the size and extent of tumor. And the TNM stage were used to define the progression of cancer based on the size and extension tumor, lymphatic involvement and metastasis status. Based on the Newcastle Ottawa Scale (NOS) criteria [142], studies with NOS scores higher than 6 are considered high-quality studies, whereas those with NOS scores less than 5 are defined as low-quality studies. This study was strictly performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [143] and the PRISMA checklist were also provided in the Additional file 8.

HR and 95%CIs were used to measure the relationship between Ki-67 expression and prognosis of lung cancer patients. The most accurate determination was made when the study directly provided the HRs and 95%CIs. The multivariate HRs were calculated by using the Cox proportional hazards model, which could independently predict the survival outcome for the lung cancer patients. We preferentially chose the multivariate values when the study provided both univariate and multivariate HRs. If the above data were not available, we used the Engauge Digitizer version 4.1 to extract the survival rates from KM curves and estimated the HR according the method as Tierneyet al. described [144, 145]. Moreover, we calculated the HR from the original survival data that the study provided using SPSS. An observed HR > 1 indicated worse prognosis for the lung cancer patients with high expression of Ki-67, if the 95%CIs for the overall HR was not 1, we considered the prognostic effect of Ki-67 on to be statistically significant. Odds ratio (OR) with 95%CIs were used to analyze the degree of association between Ki-67 level and clinicopathological characteristics. Heterogeneity was determined using the χ² and inconsistency (I²) tests [146]. I² > 50% or P < 0.1 indicated substantial heterogeneity among the studies, in which case a random effects model was applied; otherwise, we utilized the fixed effects model. Then, subgroup analysis and meta-regression analysis were used to investigate any source of heterogeneity. Moreover, publication bias was assessed using Begg’s test and funnel plots, and P-values < 0.05 indicated statistically significant publication bias [147].