Ki-67 as a prognostic marker in early-stage non-small cell lung cancer in Asian patients: a meta-analysis of published studies involving 32 studies

Literature search and study characteristics

We identified 2046 potentially relevant articles through the search strategy described
in Methods. As shown in Fig. 1, 2009 articles were excluded after the first screening based on the abstracts and/or
titles, and 37 articles remained after reviewing their full texts for relevance. Seven
articles were ultimately excluded, due to overlap with previously reported studies
(n?=?4) 16]–19], use of interventional treatments (n?=?1) 20], a lack of survival data (n?=?1) 21], or providing RFS other than OS/DFS in NSCLC (n?=?1) 22]. Additionally, two of the articles could be divided into two studies 23], 24]. Thus, a total of 30 eligible articles 5]–9], 23]–47] involving 32 studies were included in this meta-analysis. The flow diagram of the
study selection procedure is presented in Fig. 1.

Fig. 1. Flow diagram of the relevant studies selection procedure

As demonstrated in Table 1, 5600 patients with related clinical data from a total of 6178 patients were enrolled
in the 32 studies, which were published between 1993 and 2014. All 32 studies were
retrospective. Of the 32 studies, 11 were conducted in Japan, five in America, four
in China, four in Italy, two in Canada, two in Korea, and one each in Argentina, Brazil,
the Czech Republic, and Germany. The case size of each study varied from 44 to 494
(median, 156) patients. The age of the patients ranged from 19 to 89, and the overall
proportion of males was 66.11 %.

Table 1. Characteristics of studies included in the final meta-analysis of Ki-67 expression
and prognosis of NSCLC

All studies included information on disease stage, and the proportion of stages I?+?II
was 67.9 %. IHC was the only technique used to detect Ki-67 expression, using various
antibodies and cut-off values (range, 5–50 %), and 2503 (44.70 %) tissue samples had
‘high’ Ki-67 expression (Table 1).

Of the 32 studies, 19 provided HR and 95 % CI values directly, whereas in the other
13 studies, they were calculated from available data (n?=?6) or from Kaplan–Meier survival curves (n?=?7), as described by Tierney 48]. Of the 32 studies, 20 identified high Ki-67 expression as an indicator of poor prognosis,
whereas the remaining 12 studies showed no significant effect of high Ki-67 expression
on survival outcome.

Methodological quality of the studies

The results of the quality assessment of the included studies are shown in Table 1. Quality scores ranged from 24 to 36, with a median value of 33. All of the studies
satisfied most of the items and reported totals for the assay methods and confounders.

Correlation of high Ki-67 expression with OS in NSCLC

Of the 28 studies investigating the association between Ki-67 expression and OS, 14
involved Asian patients (n?=?2729) and 14 involved non-Asian patients (n?=?2287). The overall HR and 95 % CI for NSCLC patients was 1.59 (95 % CI 1.35–1.88,
P??0.001, n?=?5007), with significant heterogeneity (I
²
?=?74.8 %, P??0.001; Fig. 2, Table 2). Subgroup analyses showed that the risk was significant in both Asian and non-Asian
patients (HR 1.97, 95 % CI 1.43–2.71, P??0.001 and HR 1.37, 95 % CI 1.15–1.64, P?=?0.013, respectively) with significant heterogeneity (I
²
?=?82.1 %, P??0.001 and I
²
?=?74.0 %, P??0.001, respectively).

Fig. 2. The hazard ratio (HR) of Ki-67 expression associated with OS in all NSCLC patients.
HR??1 implied worse OS for the group with high Ki-67 expression

Table 2. HR values of OS and DFS of NSCLC subgroups

Next, subgroups including TNM stage (eight studies for stage I, eight for stages I–II,
seven for stages I–III, and one for stages III–IV) and type of NSCLC (10 studies for
ADC and two for non–ADC) were analyzed. The analyses indicated that high Ki–67 expression
was associated with a shorter OS in stage I, stages I–II, and stages I–III patients
(HR 1.85, 95 % CI 1.27–2.69, P?=?0.001; HR 1.72, 95 % CI 1.20–2.46, P?=?0.003; and HR 1.60, 95 % CI 1.21–2.12, P?=?0.001, respectively) with heterogeneity (I
²
?=?78.7 %, P??0.001; I
²
?=?76.1 %, P??0.001; and I
²
?=?36.5 %, P?=?0.001, respectively), but no association with shorter OS was observed in patients
in stages III–IV (HR 1.31, 95 % CI 0.68–2.53, P?=?0.42).

Another subgroup analysis (ADC vs. non–ADC) demonstrated that the ADC group showed
a significant association between high Ki–67 expression and shorter OS (HR 2.21, 95 %
CI 1.38–3.50, P??0.001). However, the association was not significant in the non-ADC group (HR 1.88,
95 % CI 0.88–4.01, P?=?0.105). Additionally, only Asian patients (vs. non-Asian patients) and the early-stage
group (stages I–II vs. advanced stage) in the ADC group demonstrated significant associations
between high Ki–67 expression and shorter OS. The combined HRs were 3.01, 95 % CI
1.96–4.02, P??0.001 and 3.30, 95 % CI 1.37–7.96, P?=?0.008, respectively. Non-Asian ADC patients and ADC patients at advanced stages
of the disease showed no significant association between high Ki–67 expression and
OS (HR 1.88, 95 % CI 0.88–4.01, P?=?0.359 and HR 1.51, 95 % CI 0.92–2.47, P?=?0.102, respectively).

Correlation of high Ki-67 expression with OS in NSCLC using different cut-off values

Subgroup analysis demonstrated that the risks between Ki–67 expression and OS were
not significant using different Ki-67 cut–off values (10 %, 25 %, 50 %). The pooled
HRs and 95 % CIs were as follows: 1.80 (95 % CI 1.20–2.70) vs. 1.53 (95 % CI 1.28–1.84)
for a cut–off value of 10 %, 1.57 (95 % CI 1.27–1.95) vs. 1.60 (95 % CI 1.22–2.08)
for a cut–off value of 25 %, and 1.56 (95 % CI 1.30–1.86) vs. 1.72 (95 % CI 1.27–2.33)
for a cut–off value of 50 % with significant heterogeneities (Additional file 4: Table S2, Additional file 5: Figure S1, Additional file 6: Figure S2 and Additional file 7: Figure S3).

Correlation between high Ki-67 expression and DFS in NSCLC

The pooled HR and 95 % CI for DFS provided in eight studies was 2.21, 95 % CI 1.43–3.43,
P??0.001, with heterogeneity (I
²
?=?75.3 %, P??0.001; Fig. 3, Table 2). Subgroup analysis showed that the risk in Asian patients was higher than that in
non-Asian patients, and the combined HRs and 95 % CIs were as follows: HR 2.78, 95 %
CI 1.78–4.34, P??0.001 and HR 1.83, 95 % CI 1.09–3.06, P?=?0.022, respectively. Further subgroup analysis indicated that the very early stage
(stage I) showed the highest risk, when compared with stages I–II or I–III, with the
following combined HRs and 95 % CIs: HR 4.31, 95 % CI 2.37–7.84, P??0.001; HR 1.51, 95 % CI 1.02–2.23, P?=?0.038; and HR 2.02, 95 % CI 0.97–4.20, P??0.06, respectively.

Fig. 3. The hazard ratio (HR) of Ki-67 expression associated with DFS in all NSCLC patients.
HR??1 implied worse OS for the group with high Ki-67 expression

Association between high Ki-67 expression and the clinicopathological characteristics
of NSCLC

In this meta-analysis, clinicopathological features, such as age, gender, smoking
habits, pathological type, lymph node status, and tumor differentiation grade, as
impacted by increased Ki-67 expression were compared on the basis of the 32 studies.
The results of the meta-analysis showed significant associations between high Ki-67
expression and being male, smoking habits, being a non-ADC patient, higher tumor stage
(T
_2-4
) and poorer differentiation grade (moderate or poor); the combined ORs and 95 % CIs
were as follows: OR 1.89, 95 % CI 1.53–2.33, P??0.001; OR 2.20, 95 % CI 1.72–2.82, P??0.000; OR 1.88, 95 % CI 1.60–2.22, P??0.001; OR 1.46, 95 % CI 1.13–1.88, P?=?0.004; and OR 1.47, 95 % CI 1.15–1.88, P?=?0.002, respectively. Moreover, significant associations between Ki–67 and gender
(male), being a non-ADC patient, higher tumor stage, and poorer differentiation were
seen only in Asian NSCLC patients. The combined ORs and 95 % CIs were as follows:
OR 2.18, 95 % CI 1.67–2.81, P??0.001; OR 2.22, 95 % CI 1.82–2.70; OR 1.47, 95 % CI 1.12–1.94, P?=?0.006; and OR 1.50, 95 % CI 1.15–1.94, P?=?0.002, respectively (Table 3).

Table 3. OR values for NSCLC subgroups according to clinical characteristics

There was no significant association between Ki–67 expression and age (60 vs. 60)
or lymph node status (N
_1–3
vs. N
₀
); the combined ORs and 95 % CIs were OR 1.08, 95 % CI 0.85–1.37, P?=?0.553 and OR 1.01, 95 % CI 0.83–1.22, P?=?0.927, respectively (Table 3).

Sensitivity analysis

Sensitivity analysis showed that the pooled HRs of OS and DFS were similar to those
calculated after one study was removed and the rest were reanalyzed (Additional file
8: Figure S4 and Additional file 9: Figure S5). Moreover, the HR remained unchanged (HR 1.86, 95 % CI 1.44–2.28, P??0.001 and HR 2.74, 95 % CI 1.25–4.22, P??0.001, respectively) after the ‘trim and fill’ method was used (Additional file
10: Figure S6 and Additional file 11: Figure S7). Additionally, we report the combined HR and 95 % CI results of the fixed
effects model: pooled HR 1.86, 95 % CI 1.44–2.28, P??0.001 for OS and pooled HR 1.52, 95 % CI 1.08–1.96, P??0.001 for DFS. These values were consistent with the random-effects model. Both
analyses support the reliability of our results.

Publication bias

Begg’s test indicated no publication bias among the studies included in the current
meta-analysis regarding the HRs of OS and DFS, with P values of 0.395 and 0.902, respectively. Egger’s test indicated no publication bias
for DFS (P?=?0.34), but it showed seemingly significant publication bias for OS after assessing
the funnel plot (P??0.001; Fig. 4).

Fig. 4. Funnel Plots of Begg’s and Egger’s were used to detect publication bias on OS and
DFS. Begg’s funnel plots showed seemingly publication bias on OS (A) while Egger’s
funnel plots showed no publication bias on OS in all NSCLC. It showed no publication
bias on DFS in Begg’s test (C) and Egger’s test (D)