A search of PubMed was done, using the search terms “Ki-67”, “prognostic”, “predictive”, “adjuvant”, “neoadjuvant”, “metastatic”, and “breast cancer”. Only studies published in English between January, 1983, and July, 2009, which included more than 40 patients, were considered for this review.
ReviewKi67 in breast cancer: prognostic and predictive potential
Introduction
Decisions about the adjuvant therapy of early breast cancer are affected by a complex interplay of factors that predict the prognosis and the efficacy of therapeutic options. Guidelines stratify patients into prognostic subsets and suggest preferred treatment protocols on the basis of reported estimates of efficacy. The leading parameters that define treatment recommendations for early breast cancer are oestrogen-receptor (ER), progesterone-receptor (PgR), and human epidermal growth-factor (HER2) status. Many other markers have also been assessed, including Ki67, the use of which has been explored as a prognostic or predictive marker in breast cancer and other malignant diseases. Although some pathologists report Ki67 in addition to other biological markers in breast cancer (figure), the existing guidelines of the American Society of Clinical Oncology do not include Ki67 in the list of required routine biological markers.1
A report from the St Gallen International Expert Consensus on the primary therapy of early breast cancer outlines guidelines for endocrine and chemotherapy treatment. In addition to traditional parameters, such as stage, grade, and endocrine status, the panel recommends the use of proliferation markers (eg, Ki67 index and mitosis) and multigene assays when choosing the appropriate systemic treatment.2 This new direction is consistent with the TailorX study, a large randomised study led by the Eastern Cooperative Oncology Group (ECOG), and the Mindact trial, coordinated by the Breast International Group (BIG), both of which are assessing the role of different multigene assays in determining the benefit of chemotherapy in addition to endocrine treatment in node-negative hormone-positive tumours. The Oncotype Dx gene test is a commercially available multigene assay in which five of the 16 genes reflect the proliferative status of the tumour. These specific genes, which include Ki67, are heavily weighted in the formula used to calculate the test's recurrence score.3
In light of new data, we have investigated whether there is now evidence that could change guidelines to include Ki67 in the standard pathological assessment of early breast cancers. This review updates the current knowledge on Ki67 and assesses the evidence in the published work about the prognostic and predictive role of this marker.
The Ki67 antigen was originally identified by Gerdes and colleagues4 in the early 1980s, by use of a mouse monoclonal antibody against a nuclear antigen from a Hodgkin's lymphoma-derived cell line. This non-histone protein was named after the researchers' location, Ki for Kiel University, Germany, with the 67 label referring to the clone number on the 96-well plate.4
The complete gene locus of the Ki67 protein, comprising a 74 basepair (bp) 5′ region and a 264 bp 3′ region, has been sequenced and aligned to a continuous sequence of 29 965 bp length located on chromosome 10q25-ter. The gene is organised in 15 exons with sizes from 67 to 6845 bp and in 14 introns with sizes from 87 to 3569 bp. Three introns contain homologue copies of “Alu-repeats”. Exon 13 at the “centre” of this gene contains 16 homologous segments of 366 bp (Ki67 repeats), each including a highly conserved motif of 66 bp (Ki67 motif). Two Ki67 protein isoforms with molecular weights of 345 and 395 kDa have been identified.5, 6, 7 The protein is found mainly in the nucleolar cortex and in the dense fibrillar components of the nucleolus during interphase; during mitosis it becomes associated with the periphery of the condensed chromosomes.8, 9, 10 The half-life of the Ki67 antigen has been shown to be about 1–1·5 h, regardless of the cell position in the cell cycle.11, 12 Studies have identified the involvement of Ki67 in the early steps of polymerase I dependent rRNA synthesis;13, 14 although it seems that the protein has an important function in cell division its exact role is still obscure and there is little published work on its overall function.
Ki67 expression varies throughout the different cell-cycle phases. Cells express the antigen during G1, S, G2, and M phases, but not during the resting phase G0.15 Ki67 levels are low in the G1 and S phases and rise to their peak level in mitosis. Later in the mitotic phase (anaphase and telophase), a sharp decrease in Ki67 levels occurs.16
Initially, pathologists only tested fresh or frozen tissue for Ki67 because of the reduced immunostaining caused by fixation. Over time, several antibodies, such as MM-1, Ki-S5, and SP-6, have been assessed on paraffin sections after antigen retrieval. Different groups have used different antibodies.17, 18, 19 MIB-1 has been validated more frequently;1, 20 however, no large “head-to-head” study comparing paraffin findings to the original test on the frozen tissue has been done. In daily practice, Ki67 is most often measured on paraffin sections by an immunohistochemical method, using the MIB-1 antibody.
In general, scoring systems are based on the percentage of tumour cells stained by the antibody. In one method, the pathologist examines the stained section with a standard light microscope 40× objective, using a 10×10 graticule, and the Ki67 score is defined as the percentage of total number of tumour cells with nuclear staining. This requires counting at least 1000 tumour cells with nuclear staining in ten high-powered fields (×40).21
In a clinical laboratory this approach has limitations. Some pathologists estimate the percentage of nuclei staining, whereas others count several hundred consecutive nuclei in different areas of tumours to give an overall average index. However, estimating the percentage of cells is poorly reproducible and manual counting is tedious. Therefore, automated readers have been used for scoring large series of samples.22 This method can also be applied on fine-needle aspiration (FNA) specimens.23 An important concern is that automated methods might count non-malignant nuclei, whereas a manual count would more likely exclude this potential error.
Tissue microarray technology has been introduced into pathology laboratories during the past decade. There are concerns with the scoring reliability of tissue microarrays, because of tumour heterogeneity. To overcome this issue, some experts have recommended the use of multiple cores.24, 25 However, studies have shown that tissue microarrays yield reliable and reproducible results even if only one 0·6 mm tissue sample per tumour is used.26 One study has shown that the relation between the Ki67 index and prognosis is valid for the primary tumour, but not for axillary lymph-node metastases, because they have a lower mitotic activity compared with the primary lesion.27
Healthy breast tissue can express low levels of Ki67 (<3%). Several investigators have reported that steroid-receptor expression and Ki67 antigen are detected in separate cell populations in healthy human breast epithelium, with Ki67 expressed exclusively in ER-negative cells. ER-positive cells did not proliferate in healthy human breast tissue. This separation between steroid-receptor expression and proliferation does not exist in malignant tissue.28 A correlation has been described between expression of Ki67 and breast density as well as with precancerous lesions.29, 30
Fabian and colleagues31 showed that Ki67 expression decreased when letrozole, an aromatase inhibitor, was given concomitantly with hormone replacement therapy in a chemoprevention study of high-risk women.
In ductal carcinoma in situ (DCIS), about 40% of tumours express high levels of Ki67. Increased levels are associated with higher grade lesions, comedo necrosis, and the presence of microinvasion. Hence, it is not surprising that Ki67 is a predictor for recurrence in DCIS.32, 33
The WHO classification of breast carcinoma describes 16 rare epithelial subtypes.34 Ki67 expression is one of the parameters used to characterise the immunoprofile of these different subtypes. For example, the lipid-rich and sebaceous breast carcinomas typically present with high Ki67 levels.35, 36 Invasive lobular cancer, which represents 5–15% of the invasive breast tumours, usually shows a low Ki67 index, and some investigators consider this an explanation for the behaviour of lobular carcinoma.37
The current most widely used Nottingham grading system for breast cancer combines nuclear grade, tubular formation, and mitotic rate. Both Ki67 and mitotic index are markers of cell proliferation. Ki67 is expressed in all cell-cycle phases except in G0, the resting phase, and proponents advocate its use as a prognostic marker superior to mitotic rate. Although the measurement of mitotic count is subject to preanalytical fixation factors, these can be overcome by optimum fixation of biopsy tissue.38, 39
Many studies have shown good correlation between grade and proliferative markers;40, 41, 42, 43 however, a Finnish study reported the opposite. The researchers divided 265 patients with breast cancer according to Ki67 fraction and mitotic index, reporting discordance between values for Ki67 and mitotic index. The group with high Ki67 levels and a low mitotic index showed a similar outcome to that of patients with low Ki67 levels and a low mitotic index. Those patients with cancers showing low Ki67 levels and a high mitotic index had an unfavourable prognosis similar to the group with high Ki67 levels and a high mitotic index. It is important to emphasise that these subgroups were very small and need to be validated in larger cohorts.44 Because mitotic rate varies with fixation and methodology, there might be some merit to recommending a more reliable test for proliferative rate. Ki67 expression might be more reproducible, but further studies are necessary before any recommendations about the relative use of grade and Ki67 can be made.
Section snippets
Prognostic role
Many studies have investigated the possible use of Ki67 as a prognostic marker for breast cancer. The study by Cheang and colleagues45 describes an immunopanel of ER, PgR, HER2, and Ki67 that can segregate the luminal A and B subtypes in a similar manner to that defined by a 50-gene expression profile. Luminal breast cancers with Ki67 levels of at least 14% were assigned to the luminal B category and had a worse prognosis for both breast cancer recurrence and death compared with tumours with
Predictive role
The data on Ki67 as a predictive marker is scarce and based on various different laboratory and statistical methods. Three central questions need answers and clarifications: can Ki67 serve as a tool to identify patients who especially benefit from chemotherapy or endocrine therapy?; Can Ki67 identify patients who benefit from a particular chemotherapy agent?; Can Ki67 predict a benefit of a specific endocrine treatment?
In this review, we have included all the reported studies that involved more
Conclusions
The use of Ki67 as a predictive and prognostic marker in breast cancer has been widely investigated. Similar to the issues of quality testing with other prognostic or predictive markers, such as HER2 or ER, the reported studies of Ki67 use various methods for assessing and scoring Ki67, which makes comparisons difficult. An accurate analysis of the usefulness of Ki67 will depend on consistent, reproducible, and valid scores in large cohorts. Efforts should be made to develop a standard
Search strategy and selection criteria
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