Ki-67: a review of utility in breast cancer
Tuesday, September 1st, 2015
Ki-67 is a protein found in proliferating cells that is identifiable by immunohistochemistry (IHC). Its prognostic and predictive value in breast cancer has been an area of avid research in recent literature and is increasingly shown to be of value. Identifying the presence of Ki-67 protein is now an accepted technique to differentiate hormone receptor (HR)-positive breast malignancies, and as a marker of prognosis in these tumours. It is also shown to have predictive value in neoadjuvant chemotherapy, and post-neoadjuvant endocrine therapy. Whilst it is not currently recommended as a routine investigation in the diagnosis of breast cancer, with standardisation of its methodology it has potential to become so.
Breast cancer is the most frequent cancer of women (excluding non-melanoma skin cancer) in Australia. Survival of breast cancer has improved significantly in recent decades, with five-year relative survival increasing from 72% in the mid-1980s to 89% by 2010.  Survival rates have improved as a result of developments in screening, treatment and also diagnosis.
It is currently an exciting era in diagnostic medicine, with rapidly increasing knowledge and research leading to increased availability of diagnostic techniques. Improved diagnostics are allowing us to classify tumours not only based on their anatomical location and pathological appearance, but also by molecular and genetic typing. This increasing complexity of diagnosis and subtyping is allowing for more individualised cancer treatments and better outcomes for patients. Immunohistochemistry is an area of diagnostics that has blossomed over the past two decades. One of the most frequent uses of diagnostic IHC is in breast pathology. IHC techniques may have prognostic and predictive value,  and contribute to the trend towards targeted and bespoke therapies. Numerous tests have now been developed and some have become a standard part of the diagnostic work-up, such as for oestrogen receptors (ER) and progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2).
Despite the improvements in diagnosis, there remains a group of patients whose risk of recurrence is indistinguishable based on current standard tests. This leads to potential overtreatment of patients who would not benefit from therapy, and potential under treatment of those who would.  Other tests include multi-gene predictors and urokinase plasminogen activator testing that also have proven benefit as prognostic factors, and possibly have predictive value. The Ki-67 protein is a marker of proliferation that has been known for over two decades and has been the subject of renewed study and reporting of late. However, its popularity and integration into practice has been somewhat controversial.
Ki-67 as a proliferation marker
Ki-67 is a unique protein that is found exclusively in proliferating cells. It is present in the nuclei of cells in the G1, S and G2 phases of cell division and peaks in mitosis. Cells in the G0 phase do not express Ki-67. It is present in all cells, both tumour and non-tumour, and its presence is a marker of growth fraction for a certain cell population.  Despite the numerous studies demonstrating its presence in proliferating cells, the exact role of Ki-67 in cell division is as yet unknown.  Ki-67 was first assessed for prognostic value in non-Hodgkin’s lymphoma, but is increasingly used in various malignancies,  most notably in breast cancer. It has now been proven that a higher fraction of stained nuclei is associated with worse prognosis, and healthy breast tissue exhibits low levels of Ki-67 (<3%). 
Counting mitoses, flow cytometry (for determining S-phase fraction), and IHC for Ki-67 are common techniques for determining growth fraction. Flow cytometry is not recommended in prognostication due to difficulty with methodology.  Logically, counting mitoses and Ki-67 should correlate highly but clinical studies have shown that only 51% of high Ki-67 expressing breast tumours have a high mitotic index.  Ki-67 and the other proliferation markers, despite showing promise, are not recommended as a routine part of breast cancer workup currently. 
Ki-67 as a surrogate genetic marker
Ki-67 and mitotic rate are both considered markers of cell proliferation, however Ki-67 is considered a superior prognostic marker.  One reason it can be used for prognostication is that it may act as a surrogate for genetically different tumours. Patients with ER-positive tumours, like other malignancies, are known to display a great variance in behaviour, including response to therapy. This occurs because tumours display a heterogeneous mix of gene expression grade index.  To improve prognostication and therapy recommendations, breast malignancies were genetically subclassed into five subtypes (luminal A, luminal B, HER2-enriched, basal-like, and normal breast-like). Of most interest is the differentiation between luminal A and luminal B, which (by one author’s definition) are both ER-positive and HER2-negative tumours but display contrasting behaviour.  Luminal B tumours typically have worse outcomes and demonstrate higher proliferation. Genetic typing showed certain genes (such as CCNB1, MK167, and MYBL2) have higher expression in luminal B tumours.  Given that genetic testing is expensive, and hence impractical, as a routine test in some settings,  Ki-67 can be used as a surrogate measure. This phenomenon has been studied wherein the combined prognostic value (IHC4) of ER status, PR status, Ki-67, and HER2 was shown to be of similar prognostic value to a more expensive 21-gene test.  Very recent Australian data shows that when tumours are divided into luminal A and B with the use of Ki-67 “the 15-year breast cancer specific survival was 91.7% [and] 79.4%” respectively.  This confirms the clinical variation in these tumours. These figures were only in lymph node-negative breast cancer treated with breast-conserving surgery and postoperative radiotherapy.
Ki-67 has been accepted to differentiate between luminal B and luminal A tumours without additional genetic testing. [12,13] The best cut-off score to differentiate ER-positive HER2-negative tumours is currently thought to be around 14%. At or above this figure, a tumour can be regarded as luminal B subtype and hence having a poorer prognosis. However Ki-67 is also associated with “younger age at diagnosis, higher grade, larger tumor size, positive lymph node involvement, and lymphovascular invasion.’  This is echoed in other large preclinical trials. 
A high Ki-67 is also shown to be associated with poorer ten-year relapse-free survival and breast cancer specific survival. This has been demonstrated in node-positive tumours, node-negative tumours, those treated with tamoxifen as the only agent, and those who are treated with combination therapy of tamoxifen and a chemotherapeutic agent. [6,10] A large retrospective Australian study has confirmed that Ki-67 appears to have significant mortality prediction. In their experience, a Ki-67 cut-off of 10% yielded the highest sensitivity and specificity, and at this level patient mortality rose from 3% in the low-Ki-67 group to 22%, and 15-year survival increased from 3% to 22%. Of note, this study did not differentiate luminal A and luminal B, and this did not exclude ER-negative tumours, nor-HER2-negative tumours, and so only looked at outcomes based on Ki-67. Interestingly, all HER2-positive tumours were high-Ki-67 tumours. The difference in the Ki-67 cut-off when compared with the 14% from previous trials is likely explained by the lack of inter-laboratory validation. The poorer 15-year survival of the high-Ki-67 tumours, compared with Pathmanathan’s  study, can be partially explained by the inclusion of HER2-positive tumours and triple negative tumours, which are known to have poorer prognosis.
Aleskandarany et al.  in their larger study confirmed the variation between luminal tumour but also suggest that there is little prognostic value in Ki-67 in subcategorising HER2-positive and triple negative tumours. Further, they revealed that “ [a high Ki-67 is] associated with premenopausal status, larger tumor size, definite vascular invasion, and lymph node involvement”, thus in non-luminal tumours may be selecting a patient group with other predictors of poor prognosis.
Ki-67 predictive value
Studies regarding the predictive value of the test are not yet as convincing as for prognostication, but continue to be an area of continued research and debate.
There are potential roles for Ki-67 in directing therapy in primary chemotherapy, neoadjuvant chemotherapy, neoadjuvant endocrine therapy, and in radiotherapy case selection. Chang et al.  suggested that tumours with a high Ki-67 are likely to respond more favourably to chemotherapeutic agents in the primary setting and that Ki-67 as a marker may be measured temporally during treatment to assess response. This study, however, had a small sample size and a single therapeutic regime, making it difficult to adopt in clinical practice.
Viale,  in his large retrospective review, showed that Ki-67 did not predict the relative efficacy of neoadjuvant chemoendocrine therapy in node-negative hormone receptor (HR)-positive tumours. However, this does not imply that Ki-67 has no role in directing adjuvant chemotherapy in other groups of breast malignancy. This has been further studied in a group of high risk breast malignancies by Denkert et al.  Denkert’s group demonstrated that Ki-67 predicts response to neoadjuvant chemotherapy in HR-positive, HR-negative, HER2-negative, and triple negative groups. It also shows an effect on disease free survival (DFS), and overall survival (OS) in HR-positive and HER-negative groups. This study also reveals that Ki-67 percentage is a continuum and subsets may not be simply broken down into ‘high’ and ‘low’; rather, multiple cut-off points may be required for a single tumour type and a variation of cut points required based on the studied endpoint (e.g. DFS or pathological response) and different tumours. To achieve this, further trials recording information prospectively will be necessary.
Ellis studied Ki-67 in the neoadjuvant endocrine therapy setting, and reported that it has limited role in pre-treatment biopsies, but its post- neoadjuvant treatment value predicts relapse-free survival.  Ellis suggests that when Ki-67 and ER status are combined post-surgery, a low value is correlated with low levels of relapse, and states that therapy beyond continuation of endocrine agent is likely unnecessary. In contrast, poor biomarker profile post-surgery is associated with significantly earlier relapse, more typical of ER-negative tumours; patients should be “offered all adjuvant treatments”. 
Ki-67 also has predictive value outside of HR-positive tumours. There is evidence showing that in HR-negative tumours, a Ki-67 >20% is a predictor for clinical and pathological response in the neoadjuvant setting with anthracycline-based chemotherapy.  It showed that patients with HR-negative status and Ki-67 >20% were much more likely to respond to their prescribed regime. However the authors did not give the absolute variation in response based on Ki-67, and did not test with a variety of agents or protocols to see if IHC could be used to recommend a particular agent.
Another role for Ki-67 in the neoadjuvant chemotherapy setting is in reviewing the response to therapy. A number of authors have shown that Ki-67 percentage often decreases after adjuvant therapies, and that reduction may correlate with pathological response and DFS.  Dowsett and colleagues [23,24] measured Ki-67 both at baseline and two-week post-neoadjuvant endocrine therapy. These authors suggest that the Ki-67 after two weeks of neoadjuvant therapy is of greater prognostic value than at baseline. They hypothesised that a great change in Ki-67 should also be predictive of outcome, but the trial failed to show this.
Despite the scarcity of high-quality data the latest St Gallens consensus supports the use of Ki-67 in defining luminal B tumours and states, “For patients with luminal B (HER2-negative) disease, the majority of the panel considered chemotherapy to be indicated. Chemotherapy regimens for luminal B (HER2-negative) disease should generally contain anthracyclines and… taxanes”.  This suggests that some groups have already adopted Ki-67 as a significant predictive factor in the management of HR-positive tumours.
Barriers to Ki-67 being used as a routine component of breast cancer workup
When Ki-67 staining is performed, nuclei display brown pigmentation. The area of greatest staining is used for counting, and the fraction of nuclei stained by the antibody is used to determine a percentage score. Ki-67 score is the first IHC marker that requires exact quantification to assess its benefit and there is currently no standardised methodology to do this. [25,26] This has led to a broad range of recommendations regarding the minimum number of cells analysed to accurately ascertain the percentage.  There are also many antibodies that are commercially available which may display subtle variances in result.  Further variations may also be seen based on the method of counting, i.e. computer aided versus human analysis.  The lack of a standard method to ascertain the percentage in a reproducible way combined with the other variances in techniques leads to inter/ intra operator and laboratory variances. These have made it currently difficult to incorporate Ki-67 into routine use. 
Other IHC assays have been validated in the field of breast malignancies, such as for HER2,  and have led to more concrete recommendations.  Validation involves standardised recommendations for numerous factors including tissue handling, fixation, assay selection, comparison to standards, and ensuring inter and intra-laboratory concordance.  Further, this has been complimented by the development of HER2 in-situ hybridisation (ISH) to assess the underlying gene expression, which may be superior or complimentary.  These advancements are yet to be achieved in Ki-67 analysis. Validation and standardisation of Ki-67 in a similar way has been called for by many authors, and if achieved will increase confidence in results and may allow for it to be used as part of routine testing. 
The renewed interest in Ki-67 in breast malignancies has proved its prognostic value, particularly in subgrouping HR-positive HER2- negative breast cancers. There is now increasing evidence to show that it may have a predictive role, with most evidence pointing to its role in both directing neoadjuvant chemotherapy and in assessing tumours post-neoadjuvant therapy to help direct further adjuvant
therapy. Ki-67, along with other commonly used IHC assays and genetic testing are facilitating a move away from previously crude methods of treatment to increasingly tailored treatment solutions for our patients. Once standardised, Ki-67 may provide a cost-effective contribution to this trend.
Conflict of interest
K Parthasarathi: email@example.com
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