Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry

Breast cancer is known to be a heterogeneous disease. Different subtypes exist which can be defined either by means of genetic array testing or based on approaches using immunohistochemical analyses [1]. In multigene tests especially, proliferation has a substantial impact on the prediction of the risk of recurrence [2, 3]. Likewise, in addition to the conventional histopathological parameters, the assessment of proliferation is one of the major factors for the treatment decisions in breast cancer patients [4]. A wide range of techniques is available to assess tumor cell proliferation such as calculating mitotic figures in stained tissue segments, flow cytometric analysis to determine the proportion of cells being in the S phase of the cell cycle, examination of thymidine-labeling index, proliferating cell nuclear antigen (PCNA), or cyclins E and D [5‐7].

Ki-67 is a nuclear protein being associated with cellular proliferation and was originally identified by Gerdes et al. [8] in the early 1980s, using a mouse monoclonal antibody directed against a nuclear antigen from a Hodgkin’s lymphoma-descended cell line. The most prevalent analysis method of Ki-67 antigen is the immunohistochemical evaluation. It was shown that Ki-67 nuclear antigen is expressed in certain phases of the cell cycle namely S, G1, G2, and M phases, but is nonexisting in G0 [9, 10]. In samples from normal breast tissue, it was found that Ki-67 is also expressed at low levels (<3 % of cells) in ER-negative cells, but not in ER-positive cells [11]. By means of immunostaining with the monoclonal antibody Ki-67, it is possible to assess the growth fraction of neoplastic cell populations. However, to date no standard operating procedure (SOP) or generally accepted cut-off definition for Ki-67 exists [12, 13]. For this reason, both the interlaboratory and the interstudy comparability of Ki-67 are limited [14‐16]. Therefore, Ki-67 is not implemented in standard routine pathology so far.

Nevertheless, this problem is known and several strategies for improvement have been initiated in Germany such as introduction of quality assurance conferences and pathologists’ participation in round robin tests. Regarding the evaluation of Ki-67, the round robin test started in the beginning of 2010 and includes the reproducibility of the Ki-67 index in the context of lymphoma diagnostics.

A meta-analysis involving 12,155 patients demonstrated that the Ki-67 positivity confers a higher risk of recurrence and a worse survival rate in patients with early breast cancer. Even though this meta-analysis could not scrutinize if Ki-67 had independent prognostic value beyond the standard clinico-pathological variables, it confirmed that high levels of Ki-67 are associated with worse prognoses [17]. Another meta-analysis investigating the proliferation markers and survival in early breast cancer included data from 32,825 patients and concluded that Ki-67 was associated with worse survival rates [7]. Nevertheless, it was assumed that this marker is not ready for routine use. The American Society of Clinical Oncology (ASCO) Tumor Marker Guidelines Committee did not advise the use of Ki-67 for prognosis in patients with newly identified breast cancer because of insufficient quality assurance [18]. Recently, the “International Ki-67 in Breast Cancer Working Group” published their recommendations based on current evidence concerning the evaluation and the interpretation of Ki-67, pursuing to increase the interlaboratory comparability and analytical validity of this marker in clinical practice, which is one of the most robust biomarkers measured by immunohistochemistry (IHC). They emphasized the potential of Ki-67 involving prognosis, prediction of relative response or deficiency to chemotherapy, and as a dynamic biomarker of the treatment effectiveness [19]. Furthermore, in the 2011 and in the 2013 St Gallen Consensus Conference, adding Ki-67 was recommended for the determination of proliferation and the differentiation of luminal A and B tumors [14‐16] as pioneered by Perou et al. [20], who obtained groundbreaking results with regards to intrinsic molecular breast cancer subtypes. In the 2013 St Gallen Consensus Conference, the majority of panelists voted Ki-67 for taking into account regarding the application of adjuvant chemotherapy in individual cases [16].

In the last years, several multigene tests of risk assessment in early breast cancer have been developed including different proliferation-related genes—among others Ki-67—to optimize the treatment and avoid unnecessary chemotherapy. Two large ongoing prospective randomized multicenter studies, called TAILORx (Trial for Assigning IndividuaLized Options for Treatment Rx) [21] using Oncotype DX^® [22], and MINDACT (Microarray in Node-Negative Disease May Avoid ChemoTherapy) [23] using Mammaprint^® [24] investigate the role of these multigene assays in identifying the benefit of chemotherapy in addition to the endocrine treatment in node-negative early breast cancer [25]. Furthermore, the St Gallen Conference 2011 and 2013 considered IHC as state of the art for clinical routine [14, 16].

The aim of this study was to evaluate the routine use and value of Ki-67 as a prognostic marker in breast cancer. Data from a regional population-based cancer registry (Tumor Centre Regensburg, Bavaria, Germany) were analyzed. Associations between Ki-67, clinical and histopathological parameters were evaluated. This study included data from six institutes for pathology including the university hospital and private institutions, and investigated the use of Ki-67 in the routine clinical setting of different certified and noncertified breast cancer centers.

To our knowledge, this is one of the largest retrospective studies analyzing the data from a high-quality clinical cancer registry on routine use and prognostic significance of Ki-67 in breast cancer patients. Our study was performed using the data of a clinical cancer registry which reflects the routine care in different institutions. First of all, it was found that Ki-67 is determined in a large proportion of tumors from patients with primary breast cancer. This suggests that the parameter is used in clinical routine although not recommended in national guidelines. The study demonstrated that the Ki-67 showed an association with the common histopathologic parameters. The effect was clearly seen in the association between Ki-67 and grading. This result reinforces the assumption of a similar behavior of these two parameters, both associated with proliferation. Similarly higher tumor stages and higher nodal status were associated with higher Ki-67 quartiles indicating that the more aggressive the tumor is the higher is the percentage of cells positively stained for Ki-67. Notably, in multivariable analysis, Ki-67 was a self-reliant parameter in respect of DFS and OS. Patients with tumors that had a high-Ki-67-labeling index (Ki-67 > 25 %) had both worse DFS and OS than patients with tumors that had low-Ki-67-labeling index (Ki-67 < 25 %). Furthermore, it was found that Ki-67 values between 0 and 15 % are not linearly related to DFS and OS, while Ki-67 values >15 % seem to have a linear relationship. Consequently, this study confirms that high-Ki-67-labeling index is a prognostic parameter for DFS as well as for OS. As to Ki-67 categories, the P-values of all the three categories in the fourth quartile (Ki-67: 26–35, 36–45 and >45 %) were statistically significant, compared to the first category [Ki-67 ≤ 15 % (median)] which raises the question of an optimal Ki-67 cut-off point. This should be a subject of further research. Even though the gene expression profiling is already commercially available to analyze tumor characteristics, currently this method is not likely to be widely adopted in clinical routine work because of high cost and lack of evidence from prospective trials. Previous studies were able to demonstrate that a prognostic model, the IHC 4 score, using ER, PR, HER2, and Ki-67 provides similar prognostic information to that in the 21-gene Genomic Health recurrence score [26]. Therefore, the use of Ki-67 and its correlation with the routine clinical and histopathological parameters were evaluated in a large data set. Currently, neither in the St Gallen nor in the ASCO recommendations nor in the German interdisciplinary S3 Guidelines for the Diagnosis, Treatment and Follow-up of Breast Cancer Ki-67 has been introduced as a routine parameter. However, our data showed that Ki-67 is used to a considerable extent in routine clinical work. In contrast to Ki-67, histological grading has been one of the most commonly used parameters for therapy decision-making for a long time. Grading describes differentiation as well as proliferation in various tumors. The most relevant problem using this factor is reproducibility among different institutions [27]. Elston and Ellis therefore modified the Bloom and Richardson grading system and designed the Nottingham combined histologic grade [28]. Their classification system was an important landmark in terms of achieving reproducibility of grading assessments [29]. Among classical histopathological parameters, grading was strongly correlated to Ki-67-labeling indices. This correlation was proven in various former studies [30‐36]. These findings are in accordance to our results. A further powerful correlation was noted in steroid receptor status and Ki-67 corresponding with previous studies. ER status has been largely identified as being inversely correlated with Ki-67, with the higher rates of ER positivity shown in the lowest proliferating tumors [35, 37‐40]. Moreover, it could be demonstrated that high levels of Ki-67 are associated with HER2/-neu positivity according to former studies [41]. Viale et al. [42] concluded that higher values of Ki-67-labeling index were associated with adverse prognostic factors. In their univariate analyses, high (>11 %) Ki-67-labeling index was associated with larger tumors, higher tumor grade, peritumoral vascular invasion, and HER-2 positivity (each P < 0.01). Vascular and lymphatic invasion were also associated with higher Ki-67 values as previously described by Jacquemier et al. [43]. Consequently, one of the major findings of this study was that Ki-67 was associated with all the commonly used histopathologic parameters confirming the results of similar studies regarding clinical work [44]. One of the main questions was whether Ki-67 provides prognostic information in routine use. In former studies, Ki-67 has also been valued as a prognostic factor being associated with breast cancer outcomes [3, 30, 36, 45‐49]. Ki-67 previously has been already considered as a biomarker for therapeutic decision [13, 50]. Colozza et al. [51] reviewed the role of proliferation markers as prognostic and predictive tools in early breast cancer. All of the chosen studies correlated statistically significant with DFS and OS in multivariate analysis. However, the selected studies enclosed, had a rather smaller number of patients ranging from 127 to 707 patients, and were limited to designated tumor stages. Two meta-analyses including studies performed between 2006 and 2011 elucidate the prognostic role of Ki-67 in breast cancer. It is notable that in both meta-analyses, the included studies applied different eligibility criteria, study design, methods for analyzing Ki-67, and cut-off points. The meta-analysis of de Azambuja et al. [17] investigated the prognostic value of Ki-67 only in univariate analyses for both DFS and OS. Concerning DFS, data from 38 studies (including 10,954 patients) were comprised and a fixed effect HR of 1.88 (95 % CI 1.75–2.02) was found. In terms of OS, 35 studies were incorporated (including 9,472 patients) and a HR of 1.89 (1.74–2.06) was identified. In the meta-analysis by Stuart-Harris et al. [7], both univariate and multivariate analyses were reported. The pooled adjusted HRs were 2.05 (1.80–2.33) for DSF and 1.88 (1.55–2.27) for OS in univariate analyses, and 1.76 (1.56–1.98) for DFS and 1.42 (1.14–1.77) for OS in multivariate analyses. However, these data include merely data from clinical studies and study populations, but no data reflecting the routine medical care.

In this study, it was confirmed that Ki-67 is as a prognostic factor in breast cancer patients in a routine setting. A 2010 published review article concluded increasing evidence that Ki-67 is a valuable prognostic marker but as to its predictive role its applicability is limited [12]. No robust evidence was found that Ki-67 can serve as a tool to identify patients who will benefit from a specific chemotherapy or endocrine treatment. Nevertheless, the validation of Ki-67 as a predictive factor was not the subject of the present study. This topic has been investigated in recent studies with inconsistent results. Contrary to the prognostic markers, predictive markers shall support in decision making of certain therapies as they appreciate the potential to respond to a therapy [52‐54]. Various studies have explored the predictive value of Ki-67-labeling index of which some of them reported an association between high-pretreatment Ki-67-labeling index and better responses to chemotherapy in the neoadjuvant setting [11, 55‐58] whereas other studies found no such association [47, 57, 59‐61].

In conclusion, for the first time, a large cohort of a clinical cancer registry was analyzed assuring the data of clinical routine conditions. The current study demonstrated that Ki-67 is widely applied in routine clinical work. Ki-67 was associated with common histopathological parameters, but was shown to be an independent prognostic parameter for DFS and OS in breast cancer patients. These findings underline the importance of Ki-67 as a prognostic parameter. Therefore, future work in this field is called for; it should focus on the standardization of Ki-67 assessment in routine clinical settings and on the role of Ki-67 in treatment decisions.