Ki-67: level of evidence and methodological considerations for its role in the clinical management of breast cancer: analytical and critical review

Early detection and improvements in systemic neoadjuvant and adjuvant therapies explain the observed decrease in mortality in breast cancer [ 53]. However, since chemotherapy is associated with adverse effects, it is important to be able to tailor treatment strategies for each patient. Companion diagnostic tests, such as HER2 or ER measurements, which are by essence predictive, are already key actors in daily therapeutic strategies. In parallel, non-associated tests, such as proliferation biomarkers, continue to be investigated in the hope of finding reliable tools to help to identify those women who are most likely to benefit from chemotherapy.

Ki-67 was significantly associated with DFS in multivariate analyses in seven RCTs and two meta-analyses with consistent HRs or relative risks (RRs) [ 27, 29, 32– 35, 39, 40, 83, 99, 100, 108, 109]. Although, more heterogeneous, similar results were reported in studies using samples from cohort studies. The HRs and RRs reported for Ki-67 in most of these studies were within the same ranges as those found for other validated prognostic markers (ER, HER2, uPA, node status, histological grade) (Table  5) [ 10, 64, 94, 100, 113].

The evidence reviewed here, with consistent results between the studies allowing the attribution of an LOE I-B, validates the use of Ki-67 as a prognostic factor for DFS in patients receiving adjuvant therapy. As none of the studies were specifically designed to assess Ki-67 as a prognostic factor, the LOE cannot be I-A. A LOE I defines a marker that is ready for clinical use, therefore, justifying its status as a biomarker as suggested by Diamandis [ 31]. This level is based on the hierarchical classification for medical utility of a biomarker proposed by Simon et al. in 2009 [ 98], an updated revision of the initial classification proposed by Hayes et al. in 1996 [ 52]. This differs dramatically from the LOE proposed by Colozza et al. [ 24] who suggested a level III or even IV. However, it should be emphasised that our conclusion is based on results from studies using samples from RCTs with central review of the marker; that was not the case in the review conducted by Colozza et al. that included studies published before 2004. This implies that standardization of the techniques and counting methods ensuring efficient and practical alternatives to centralized testing (i.e. automated staining and image analysis) will be necessary for everyday practice.

The results from the studies using samples from patients included in RCTs do not provide sufficient proof to conclude that Ki-67 is a predictive factor for short-term or long-term response to chemotherapy, since the study designs were not suitable for answering this question. The LOE is therefore II-B and a higher LOE will only be possible if suitably designed prospective studies are conducted. Nevertheless, an association between high Ki-67 expression at baseline and immediate response to hormonotherapy or chemotherapy in the neoadjuvant setting was reported in seven case series [ 14, 15, 73, 78, 82, 84, 90], two of them with pathological complete response (pCR) [ 73, 78]. The studies in the neoadjuvant chemotherapy setting analysed pCR as the endpoint. In contrast, the studies in the neoadjuvant hormonotherapy setting, used a clinical response endpoint. In breast cancer samples from women with incomplete pathological response after neoadjuvant therapy, the Ki-67 expression in the residual tumour was reported to be prognostic, irrespective of the original pre-treatment value [ 40, 59, 102].

Prognostic variables are needed in clinical practice. Histological grade can clearly distinguish between low and high risks tumours (grade 1 vs. grade 3) in terms of outcomes. However, about 40–50% of breast cancers are classified as grade 2 with a less well-defined risk. The histological grading system is constructed from a parameter of differentiation (glandular formation), nuclear appearance and a clear proliferation parameter (mitotic count). This explains why grade and Ki-67 index are closely linked, and why the grade is not always integrated in the multivariated models used for assessing Ki-67. The fact that such a link exists does not mean that the parameters are redundant and the use of Ki-67 index in a grade 2 population could be particularly useful to sub-classify them [ 2]. Patients with ER+ tumours are systematically treated by hormonotherapy today in the absence of contra-indications. It is possible that a Ki-67 assessment prior to deciding to propose additional adjuvant chemotherapy might be useful for a subset of ER+ patients with grade 2 tumours.

The choice of the cut-point has a major impact in practice, as it determines which patients are classified as ‘high Ki-67’, and therefore which have a poorer prognosis. These patients will generally receive more aggressive therapy. In the published studies reviewed, many different ways to select a cut-point were used, defining two or three subgroups. These include an arbitrary choice based either on the different cut-points proposed in the literature or the use of the “significant” mean value from an ‘in house’ series. In our review of studies using samples from RTCs, most arbitrary cut-points for adjuvant treatment choice were distributed between 5 and 34% with 10 or 20% being the most frequently used values (Table  2).

The use of data-derived ‘optimal’ cut-points can result in serious bias due to different patient populations in each series. It should be stressed that transforming continuous variables, such as the Ki-67 index, into two categories can lead to a loss of power of the biomarker [ 88, 95, 108, 109]. In addition, this is unrealistic at the individual level, since it suggests that patients, who have tumours with Ki-67 levels close to the cut-point but on either side of the cut-point, are very different, whereas in reality they are probably very similar. Technically it is not necessary for statistical analysis to have a binary variable, and it has been show that a model with continuous values provides more information [ 95]. In clinical practice, one way of expressing the results is to use two cut-point values which define a central ‘grey’ area between the low and high values. For patients whose Ki-67 level falls in this grey area, other factors could be considered in the decision to offer chemotherapy or not. This is the approach adopted by the St Gallen International Expert Consensus who recommended the use of Ki-67 to measure proliferation [ 47, 48, 95]; women with ER+ tumours and ‘high’ Ki-67 (i.e. >30%) should receive chemo-hormonotherapy, those with ‘low’ Ki-67 (≤15%) (luminal A tumours) should receive hormonotherapy alone and the ‘intermediate’ level (16–30%) is not decisive for therapeutic decision.

Ki-67 expression is detected by immunohistochemical techniques on histological slides. Molecular testing using RT-QPCR on fixed-paraffin embedded tissue samples is also feasible [ 28] but not used in practice. Both techniques give quantitative results but the qualitative aspect of tumour heterogeneity is only accessible on histology slides. Comparative studies are in progress but the results are not yet available. Moreover, both techniques, as for all biomarkers, need standardized pre-analytical conditions which require cooperation between radiologists, surgeons, and pathologists. Most laboratories use MIB-I or SP6 antibodies for immunohistochemistry that provide highly comparable results, although SP6 appears to be better suited for image analysis [ 116]. However, the methods of antigen retrieval from paraffin-embedded samples, the concentrations of antibodies, the time of incubation, as well as the amplification reagents vary and may significantly influence the final results [ 114, 116]. We observed this variation in the studies analysed in this review (Table  2). Automatic immunostaining was reported to be used in only three of the published studies, despite the fact that most laboratories are nowadays equipped with such systems [ 88, 108, 109]. Also, the way samples are treated immediately after collection and the way they are stored may affect the final results, but generally only sparse information on this was provided in most of studies reported. In general, all studies reported using a negative control. However, there was no standardized positive control for staining calibration. Some studies used tonsil tissue, while others used known highly positive breast cancer tissue. The intensity of nuclear staining that was considered to be positive also varied; in some cases any staining was taken as positive, whereas in others positivity required ‘marked’ staining. Some studies reported using ‘hot spots’ (or areas of intense staining) for the assessment, whereas others used fields with different intensity of staining giving the result as a mean value. Significant variation in the number of fields examined, the number of tumour cells counted or estimated, the use of a graticule for counting or the use of automated counting systems was also seen. Some studies reported a double reading of all slides, or of a certain percentage of slides.

Due to limits in histological quantitative analysis and tumour heterogeneity, leading to inter/intra-observer variations on grade scoring, some grade 2 tumours are mis-classified as grade 1, and also some grade 1 tumours are mis-classified as grade 2. The assessment of Ki-67 levels in these borderline cases provides additional information to clinicians. Similar overlap exists between grade 2 and grade 3 tumours, but without a significant impact on therapeutic decision. In view of the inaccuracies expected in the Ki-67 index values, partly due to the heterogeneity of the techniques as discussed above, and partly due to tumour heterogeneity, it may be useful to generalize automated quantitative image analysis, to report both ‘hot spots’ and mean Ki-67 values, and to expand the 16–30% intermediate level of St Gallen to 11–30% [ 47]. This wider intermediate level would ensure a better identification of tumours with low and high levels of Ki-67. The risk of making an error when assessing a Ki-67 score <10 or >30% will be low in routine practice, but is to be expected for the intermediate level between 11 and 30%, requiring, therefore, double assessment or automated image analysis.

Reporting key details are essential to assess the reliability of the study results. Initiatives such as the CONSORT guidelines have been shown to improve the quality of reporting for RCTs [ 74, 75]. In a similar way, the REMARK guidelines were developed to improve reporting of prognostic studies and their results [ 71]. Mallett et al. [ 68] reported in 2010 the results from an analysis of reports of prognostic tumour marker studies published in 2006 and 2007 using the REMARK score. The aim of their study was to assess if the publication of the guidelines had had an immediate impact on the quality of the reported studies. Although most of the studies reported the number of patients in the analyses (98%), only just over half reported the number of eligible patients (56%) and excluded patients (54%). Only 36% of the reports clearly defined the outcomes analysed. The authors concluded that although good reporting is essential for the interpretation and clinical application of prognostic studies, the standards of reporting in 2006 and 2007 were poor. They called for a wider use of the REMARK guidelines to help improve reporting and enhance prognostic research. The results of our review show that articles published prior to the publication of REMARK in 2005 had a lower range of REMARK scores ( n = 9; 6–13) than those published after ( n = 9; 10–18) which suggests that the quality of reporting has improved.