Proliferation Marker Ki-67 in Early Breast Cancer
http://www.100md.com
《临床肿瘤学》
the Academic Department of Biochemistry and Breast Unit, Royal Marsden Hospital, London, UK
ABSTRACT
Molecular markers have been extensively investigated with a view to providing early and accurate information on long-term outcome and prediction of response to treatment of early breast cancer. Proliferation is a key feature of the progression of tumors and is now widely estimated by the immunohistochemical assessment of the nuclear antigen Ki-67. The expression of Ki-67 correlates with other measurements of proliferation, including S-phase and bromodeoxyuridine uptake. High Ki-67 is a sign of poor prognosis associated with a good chance of clinical response to chemotherapy, but its independent significance is modest and does not merit measurements in most routine clinical scenarios. However, its application as a pharmacodynamic intermediate marker of the effectiveness of medical therapy holds great promise for rapid evaluation of new drugs.
INTRODUCTION
Early breast cancer (EBC) has a highly variable prognosis and benefit from available therapies is unpredictable for the individual patient. Key factors such as tumor size, histological grade, vascular invasion, and nodal status are helpful, but increasing attention is being paid to the molecular features of the tumor. Estrogen receptor (ER) and Her-2–neu are now well established as predictive factors for treatment response and prognosis. Although not considered an obligatory marker, Ki-67 is also frequently measured both as a static marker of proliferative activity and, by making multiple measurements during treatment, as a possible dynamic intermediate or surrogate marker of treatment efficacy. We provide here an overview of the basic molecular biology of Ki-67 and its prognostic and predictive value at presentation of disease. In particular, we have focused on the evidence supporting the use of change in Ki-67 as an early predictor of treatment efficacy and as a prognostic factor of long-term outcome. The review is based on published evidence, identifying articles with searches of PubMed (key words: "Ki-67," "anti-Ki-67," "breast cancer," "prognostic," "proliferation") as well as references from relevant articles and from the authors’ personal experience. Other than when stated, we refer to Ki-67 as the presence of the Ki-67 antigen determined by immunohistochemistry.
BIOLOGY OF Ki-67
Nature, Sequence, and Function
Ki-67 was identified by Gerdes et al in 19911 as a nuclear nonhistone protein, shortly after the corresponding antibody was described by the same group2 in the city of Kiel (hence "Ki") after immunization of mice with the Hodgkin’s lymphoma cell line L428 (67 refers to the clone number on the 96-well plate in which it was found). The absence of Ki-67 in quiescent cells and its universal expression in proliferating tissues created great interest on its potential role as a marker of cell proliferation. A large number of studies (see references below for correlation with other proliferation markers) have confirmed this feature, and the expression of Ki-67 in resting cells has rarely been reported.3
The Ki-67 gene is on the long arm of human chromosome 10 (10q25).4 In 1993, Schluter et al published the complete sequence of the cDNA encoding for the protein.5 Two alternative mRNA species resulting from alternative splicing encode two isoforms of the protein. The "large" Ki-67 protein isoform has a calculated molecular mass of 359 kD, and the "small" isoform, a mass of 320 kD. The presence or absence of the sequence encoded by exon 7 of the gene differentiates one isoform from the other. The most outstanding feature when the sequence is analyzed is the presence of 16 repeated elements, the Ki-67 repeats," in the large exon 13 that makes up 70% and 79% of the open reading frame of the large and small species, respectively. These are concatenated sequences depicting an important similarity with 43% to 62% identical amino acids. Within these repeats, a sequence of 22 amino acids, the so-called Ki-67 motif, can be found. This motif is highly conserved between species and includes the epitope (FKEL) targeted by the original Ki-67 antibody. There is some evidence derived from murine and human cell lines that more postsplicing variants may exist with a consequent larger range of protein isoforms.6
The expression of Ki-67 varies in intensity throughout the cell cycle, and this has raised concern that it could lead to a misclassification of cycling cells as resting ones.3 Overall, evidence indicates that levels of Ki-67 are low during G1- and early S-phase and progressively increase to reach a maximum during mitosis. A rapid decrease in expression starts during anaphase and telophase.7 Some authors have found that the degree of expression in G1 can be minimal8-10 and may represent a handicap to Ki-67’s accuracy in identifying cells in this phase. This irregular presence of Ki-67 in G1 has been explained as the result of the differences between G1-phase in a previously dormant cell and G1 immediately following a previous cycle8 or even as a marker of different conditions of growth.9
The half-life of Ki-67 protein has been estimated at around 60 to 90 minutes.10,11 Differences of expression during the cell cycle do not seem to be due to the accumulation of nondegraded protein; rather, they seem largely to reflect variable de-novo synthesis.
The cellular appearance and location of the Ki-67 protein throughout the cell cycle is not homogeneous. During early G1, it is found as generally weakly staining discrete foci throughout karyoplasm.12 It progressively condenses during late G1 in larger perinucleolar granules.13,14 During S and G2 phases, it is mainly found associated with the nucleolar region in larger foci as well as with some heterochromatin regions. When the nuclear membrane disrupts during early mitosis, Ki-67 shows an intense expression associated with the surface of condensed chromosomes in the cytoplasm. This intensity rapidly disappears in anaphase-telophase.
Despite all this information about the nature, location, and sequence of Ki-67 protein, there is little known of its function beyond its being a protein phosphorylated via serine and threonine11 with a critical role in cell division. This has been concluded from the arrest of cell proliferation when Ki-67 is blocked either by microinjection of blocking antibodies13 or by inhibition of dephosphorylation.11
Determination of Ki-67
The original Ki-67 monoclonal antibody, when used for immunostaining was initially reported to stain proliferating cells in unfixed tissues but not formalin-fixed paraffin-embedded samples. In 1992, Cattoretti et al reported better success in staining Ki-67 in paraffin-embedded samples after development of the new antibodies MIB-1 and MIB-3. These antibodies seem to produce results equivalent to the original Ki-67 antibody targeting the same epitope (contained in the Ki-67 motif), as demonstrated by identical bands in Western immunoblots.15 Staining by MIB-1 and -3 of formalin-fixed paraffin-embedded samples is greatly enhanced by antigen retrieval (most frequently by microwave heating).16 Although several antibodies are now commercially available to stain Ki-67 in fresh and paraffin-embedded tissue, MIB-1 is the most widely used in recent studies (Fig 1). Ki-67 expression is usually estimated as the percentage of tumor cells positively stained by the antibody, with nuclear staining being the most common criterion of positivity. Currently, there are few data on the between-batch or between-laboratory variability in these measurements and whether the application of image analysis techniques (which have difficulties in tissues with heterogeneous cell populations such as breast cancer) will enhance the precision of assessment.
Ki-67 and Other Markers of Proliferation
Many studies have investigated the correlation between Ki-67 and other well-known markers of proliferation. A strong correlation has been found in most.
S-phase fraction as measured by flow cytometry,17-25 mitotic index,19,20,26-30 tyrosine kinase,20 and in vivo uptake of bromodeoxyuridine (a laborious but highly reliable procedure considered by many to be the gold standard of cell proliferation measurements)26,29,31-33 have each been regularly reported to correlate well with Ki-67.
The correlation of Ki-67 with proliferating cell nuclear antigen has been reported as positive,34 but weak,31 and the multiplicity of functions developed by proliferating cell nuclear antigen may account for this discrepancy.35 Studies of the correlation with DNA ploidy are also conflicting,20,21,33 and no correlation has been found with thymidilate synthase.28 A recently described marker of cell proliferation, the minichromosome maintenance protein 2 (Mcm-2), showed a strong correlation with Ki-67 although it seemed to be more sensitive in detecting cycling cells.36
Ki-67 in the Normal Breast
Few studies have reported results for Ki-67 on normal breast tissue. In samples from normal breast37,38 or in normal epithelium adjacent to fibroadenomas39 it is expressed at a very low level (< 3% of cells). One of the most interesting features found in these studies is the absence of Ki-67 positivity in the ER-positive population38 (ie, only ER-negative cells are proliferative in normal breast). This feature is lost in breast cancer where both markers are present in a higher percentage of cells and these populations overlap. There seems to be a progressively changing pattern from normal to cancerous tissue, with an intermediate degree of coincidence of both markers in noninvasive precancerous lesions.40
Ki-67 and Other Biologic Markers in Breast Cancer
Apoptosis, or programmed cell death, provides the major counterbalancing determinant of tumor growth to proliferation and has been consistently reported to be positively correlated with Ki-67,41 as shown in Figure 2. An index based on the Ki-67–apoptosis ratio (the cell turnover or growth index) has been developed to approximate the contribution that these two factors may make to tumor growth. While it is clear that this cannot accurately reflect growth dynamics, it may have utility as an early marker of response to treatment in primary therapy of breast cancer,42-44 though this remains to be rigorously demonstrated.
Bcl-2, an antiapoptotic protein, has been reported as being inversely correlated with baseline Ki-67.45 The oncogene p53 is frequently mutated or overexpressed in breast cancer, and those tumors presenting these changes show higher rates of proliferation as measured by Ki-67.19,31,45-48 Several studies have addressed the relation between Ki-67 and Her-2 expression, and although there seems to be a positive correlation,28,45,49 it is not always well-defined.22,50 The relationship with epidermal growth factor is similarly conflicting.22,45,50,51 ER status has been widely reported as being inversely correlated with Ki-67, with the higher rates of ER positivity shown in the least proliferating tumors17,19,20,31,34,45,46,52,53 and only a few studies have contradicted this.28,29,48
Of the commonly used pathological features of breast cancer probably the most robustly related to Ki-67 is histological grade with virtually no studies refuting this positive correlation.19-21,28,30,31,33,46,48,53-55 This is to be expected given that mitotic index is one of the three components of grade. Tumor size is not consistently linked to Ki-67 scores with some authors finding a positive relationship31,34,46,52,55 but others not.17,19,22,54
The strongest prognostic factor in EBC, lymph node status, has been intensively studied with regard to its correlation with Ki-67 in an attempt to find an easy marker of nodal involvement that would avoid unnecessary axillary surgery. In studies with more than 200 patients, there seems to be more evidence in favor46,52,56 than against54 a positive correlation (see below). The number of smaller studies favoring a lack of correlation is large.19,20,29,45,48 This may reflect the size of studies and/or the relative weakness in the relationship.
Ki-67 BASELINE MEASUREMENT: PROGNOSTIC VALUE
The search for useful tools to discriminate prognostic subgroups in patients affected by early breast cancer, and in particular, as noted above, in those without lymph node involvement, has led to an extensive study of Ki-67 in this setting. Data from 40 studies involving more than 11,000 patients have been analyzed. Those studies with a sample size greater than 200 patients have been summarized in Table 1.17,18,26-28,34,46,52,54,57-65
In this Table, the studies are presented as statistically significant (P, yellow box) or nonsignificant (N, pink box) for the relation between Ki-67 and clinical outcome. Results are considered in terms of disease-free survival (including metastasis free and local relapse free survival) and overall survival (including disease-specific survival) and the data are shown in three groups made according to the lymph node status (negative, positive, and mixed population). The statistical analysis is specified as univariate or multivariate.
There is strong evidence from these large studies, of the ability of Ki-67 to discriminate between good and bad prognostic groups in the node-negative population when considered as a single variable. While this significance is lost in the multivariate analyses in two of the studies,26,57 in most, it is maintained. In this node-negative group of patients, the large majority of studies failing to report a significant prognostic value of Ki-67 involved patient populations smaller than 200 (not depicted in the Table). In the lymph node–positive and mixed population, the results are less clear, but positive studies predominate.
There are difficulties in assessing the possible prognostic uses of Ki-67 since virtually all of these studies are retrospective and involve a highly heterogeneous population. The use of different antibodies, techniques and scoring protocols without a standard minimum number of cells to be counted may account for some of the differences between studies. The lack of an optimal cutoff point for the definition of prognostic subgroups is probably the main obstacle to a synthesis of the results for extrapolation to clinical practice. Overall it seems that Ki-67 does carry independent prognostic significance from routinely measured pathological features but this is modest and does not merit routine inclusion in the work-up of primary breast cancer.
Ki-67 AND PREDICTION OF TREATMENT RESPONSE
The association of Ki-67 and response to medical treatment is clearer. Five out of six studies reporting the value of Ki-67 to predict response (clinical and/or pathological) to chemotherapy in early or locally advanced breast cancer found that higher Ki-67 was associated with better response66-70 but one found no association.71 In contrast no significant relationship between Ki-67 score and response to treatment has been reported for neoadjuvant endocrine treatment.44,72,73 This may explain why inconsistent results have been reported when mixed chemotherapy and hormonal therapy have been used, with both negative24,45 and positive74 results reported.
It is important to note that while high scores of Ki-67 are associated with higher chance of response to chemotherapy, high Ki-67 is a marker of poor prognosis overall. However, good clinical response, particularly pathological complete response, is associated with good long-term prognosis. This suggests that stratification according to Ki-67 levels might improve the prognostic significance of clinical response in neoadjuvant chemotherapy. To our knowledge this approach has not been undertaken.
CHANGES IN Ki-67 THROUGH TREATMENT: CLINICAL SIGNIFICANCE
The availability of minimally invasive tumor sampling techniques in EBC (ie, fine-needle aspirate, core-cut biopsy) has prompted investigators to study whether changes in Ki-67 score occur after a variable time of treatment and can provide a better predictive or prognostic value than baseline measurements. If so, these early changes might provide valuable intermediate markers of treatment benefit, particularly in relation to drug development.
Studies with MCF-7 xenograft tumors in nude mice with estrogen deprivation75 or antiestrogen hormonal treatments76 provide encouraging proof of principle. Each of these treatments results in a rapid decrease of Ki-67 within 1 week of treatment. The study by Johnston et al demonstrated a complex relation between Ki-67 decrease, changes in apoptosis, and tumor regression following estrogen withdrawal compared with tamoxifen (Fig 3). Estrogen withdrawal induced a profound decrease in Ki-67 (approximately five-fold), accompanied by an increase in apoptosis (approximately four-fold), and this resulted in tumor regression. With tamoxifen, lesser reductions in Ki-67 (approximately two-fold) and increases in apoptosis (approximately three-fold) were seen, and these were associated with no more than stabilization of tumor size. These findings suggest that yet smaller changes in Ki-67 and apoptosis could lead to slowing of tumor growth but still be associated with progressive disease. The implications of these findings for clinical studies are discussed below.
Before assuming that change in any biomarker is due to an intervention, the intrinsic variability of the marker without the mediating intervention should be assessed. Few reports have addressed this issue. Our studies on the reproducibility of Ki-67 measurements in core-cut biopsies suggest that a change in Ki-67 score of at least 32% to 50% between two determinations is required to consider the difference statistically different for an individual patient, and attributable to treatment effect.77,78 These individual requirements do not apply in the same way to populations but are important to take into consideration in the statistical powering of these studies. Further data on the variability in the measurements of Ki-67 are provided in the placebo arms of several short-term presurgical studies,79-82 and these largely support our estimates of variability. However, different biopsy sites, staining, and counting procedures will affect this variability.
In the clinic, effective hormonal treatments suppress Ki-67 levels in both short-term (< 6 weeks)42,79-87 and long-term (eg, 12 weeks) studies.44,72,73,88-90 This has also been shown with withdrawal of hormone replacement therapy.91 As would be expected, these changes are only seen in hormone receptor–positive tumors.80-82,91,92 Reduced Ki-67 levels with hormonal therapy are illustrated in Figure 4, which shows the substantial falls in Ki-67 at 2 weeks and 12 weeks in 56 patients treated with anastrozole before surgery.93 At 2 weeks, only four patients did not show a reduction in Ki-67 levels. The geometric means of reduction were 76% and 82% at 2 and 12 weeks, respectively. There were some patients who showed increases in Ki-67 between 2 and 12 weeks, and it is possible that this is an early indication of resistance to the therapy. It is, however, difficult to distinguish these relatively modest increases in individual patients from the between-sample variability that is discussed above.
The reduction in Ki-67 with endocrine therapy probably reflects the well-described cytostatic effect of the drugs.94 The changes in Ki-67 occur early and well before significant tumor regression is seen. This suggests that treatment selection of a less proliferative cell population probably plays little part in the changes.
Ki-67 decreases also occur with chemotherapy and mixed chemohormonal treatments.24,45,66,67,70,74,77,78,95,96 We have found only one study failing to show this,69 and our own work suggests this may be related to the timing of the second biopsy.97 The reduced proliferation with chemotherapy may be at least partly due to increased apoptosis in actively proliferating cells98 such that the residual population would be enriched for Ki-67–negative cells. The relationship of decrease in Ki-67 during cytotoxic chemotherapy over 2 to 3 months with long-term outcome has been addressed in two studies,45,95 which found in a multivariate analysis that a decrease in Ki-67 of more than 25% and a residual score less than 10% each predicted a longer disease-free survival.
Early changes in Ki-67 have also been found to correlate positively with clinical and/or pathologic response in EBC with both hormone therapy and chemotherapy.24,45,66,70,72,77,88-90,95 In these studies, the timing of the second biopsy varied. The relationship to clinical and/or pathologic response within 2 weeks is particularly useful since these changes preceded clinical response, while the changes at 12 to 16 weeks are usually concordant with clinical response rather than predictive.
This issue has been recently supported by a report on the results of the IMPACT trial.93,99,100 This trial compared 12 weeks of neoadjuvant treatment with anastrozole or tamoxifen or the combination of both drugs in postmenopausal women with ER-positive EBC, followed after surgery by the same regimen as adjuvant treatment. Ki-67 decrease as determined in a biopsy taken 2 weeks after initiation of treatment predicted the better long-term outcome seen in the parallel adjuvant trial with anastrozole.101
The possibility that changes in Ki-67 might predict long-term efficacy (after 10-14 days of treatment) has opened a new scenario for clinical research in EBC. This so-called "short-term preoperative" setting refers to the time between diagnosis of primary operable EBC and the surgery itself, an interval when treatment is not usually administered. Thus, drugs with established safety and low toxicity may be tested and compared against placebo using a decrease in Ki-67 as the primary end point. This model has been exploited in the assessment of several treatments; these have been mainly hormonal,42,79-84 but have more recently included inhibitors of signal transduction pathways.102,103 Most of these showed a significant drop in Ki-67 in the treatment arms compared with placebo or no treatment.
Different treatments have also been compared in this short-term setting to obtain rapid information on potential differences in efficacy.42,81,82,87 Comparative studies between drugs require their having similar pharmacokinetics or the use of schedules to compensate for different pharmacokinetics; they should also have a mechanism of action that similarly affects proliferation (or another end point such as apoptosis). These approaches could also allow the differential effectiveness of drugs on subgroups of patients to be assessed leading to the identification of targets to select optimal therapy. For example, a recent report by Ellis et al found differences in the Ki-67 response to letrozole and tamoxifen depending on whether tumors overexpressed Her-1 and/or Her-2,89 and these results paralleled differential clinical response in the same subgroups.104 These differences had been previously suggested in a pooled analyses of several smaller studies in the short-term preoperative setting.105 The effect on Ki-67 of Her-2–targetted therapy combined with endocrine therapy would seem to be a rational approach to evaluating efficacy.
While the above rationale and examples of the use of early changes in Ki-67 suggest that there will be a growing interest in its use as an intermediate marker of benefit, there remain challenges in the handling of the derived data that need to be met to allow its widespread use. One complication is the non-normal distribution of the data, which logarithmic transformation may also not normalize.89 It is also not clear that the proportional reduction in Ki-67 is the most relevant measure for predicting outcome. The arguments made above suggest that proportional reduction may be an appropriate parameter for predicting benefit in the adjuvant setting, but that the residual (on-treatment) level of Ki-67 may be a better predictor of response and/or absolute long-term outcome since this is more likely to relate to the growth rate of the persistent disease. Currently available and future data sets should examine these possibilities.
SUMMARY AND CONCLUSIONS
The Ki-67 protein, measured with immunohistochemical techniques, is a useful marker of cell proliferation. The suggested lack of accuracy to identify some cells entering the cell cycle in early G0 has not substantially affected the ability of Ki-67 to identify the proliferating population of cells in comparison with other markers of proliferation. Compared with these other markers, Ki-67 staining is easy to perform, economical, and more reproducible. Correlation between Ki-67 mRNA levels and the presence of the protein identified by immunohistochemistry has not been yet fully studied106; this will be required to allow appropriate interpretation of Ki-67 expression as determined with new diagnostic tools, including expression microarrays.
Definitions of cut-offs for Ki-67 vary and hamper comparisons between studies: it may be more helpful to consider Ki-67 as a continuous variable. In multivariate analyses, Ki-67 retains prognostic significance in lymph node–negative patients, but its clinical utility seems slight. Instead, a key role for Ki-67 in EBC may relate to the measurable changes in its expression during treatment. The lack of a close agreement between decreases in Ki-67 and tumor response in some clinical trials possibly results from the need for major reductions in proliferation and/or increases in apoptosis to lead to the regression of a fast-growing tumor. These decreases in proliferation may nonetheless be indicative of benefit from the treatment. There is now a strong rationale for the future design of trials using short-term changes in Ki-67 as a marker of treatment efficacy. However, for analysis of Ki-67 to reach its potential either as a baseline or as a pharmacodynamic measurement, greater attention will need to be paid to standardized methodologies, approaches to scoring, statistical analysis, and between-laboratory quality assessment.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank our numerous clinical and laboratory colleagues who have contributed to the studies we have conducted leading to the development of this review.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Detre S, Salter J, Barnes DM, et al: Time-related effects of estrogen withdrawal on proliferation- and cell death-related events in MCF-7 xenografts. Int J Cancer 81:309-313, 1999
Johnston SR, Boeddinghaus IM, Riddler S, et al: Idoxifene antagonizes estradiol-dependent MCF-7 breast cancer xenograft growth through sustained induction of apoptosis. Cancer Res 59:3646-3651, 1999
Assersohn L, Salter J, Powles TJ, et al: Studies of the potential utility of Ki67 as a predictive molecular marker of clinical response in primary breast cancer. Breast Cancer Res Treat 82:113-123, 2003
Ellis PA, Smith IE, Detre S, et al: Reduced apoptosis and proliferation and increased Bcl-2 in residual breast cancer following preoperative chemotherapy. Breast Cancer Res Treat 48:107-116, 1998
Clarke RB, Laidlaw IJ, Jones LJ, et al: Effect of tamoxifen on Ki67 labelling index in human breast tumours and its relationship to oestrogen and progesterone receptor status. Br J Cancer 67:606-611, 1993
Dowsett M, Dixon JM, Horgan K, et al: Antiproliferative effects of idoxifene in a placebo-controlled trial in primary human breast cancer. Clin Cancer Res 6:2260-2267, 2000
Dowsett M, Bundred NJ, Decensi A, et al: Effect of raloxifene on breast cancer cell Ki67 and apoptosis: A double-blind, placebo-controlled, randomized clinical trial in postmenopausal patients. Cancer Epidemiol Biomarkers Prev 10:961-966, 2001
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Robertson JF, Nicholson RI, Bundred NJ, et al: Comparison of the short-term biological effects of 7alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)-nonyl]estra-1,3,5, (10)-triene-3,17beta-diol (Faslodex) versus tamoxifen in postmenopausal women with primary breast cancer. Cancer Res 61:6739-6746, 2001
Johnston SR, MacLennan KA, Sacks NP, et al: Modulation of Bcl-2 and Ki-67 expression in oestrogen receptor-positive human breast cancer by tamoxifen. Eur J Cancer 30A:1663-1669, 1994
Bajetta E, Celio L, Di Leo A, et al: Effects of short-term pre-operative tamoxifen on steroid receptor and Ki-67 expression in primary breast cancer: An immunocytochemical study. Int J Oncol 12:853-858, 1998
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ABSTRACT
Molecular markers have been extensively investigated with a view to providing early and accurate information on long-term outcome and prediction of response to treatment of early breast cancer. Proliferation is a key feature of the progression of tumors and is now widely estimated by the immunohistochemical assessment of the nuclear antigen Ki-67. The expression of Ki-67 correlates with other measurements of proliferation, including S-phase and bromodeoxyuridine uptake. High Ki-67 is a sign of poor prognosis associated with a good chance of clinical response to chemotherapy, but its independent significance is modest and does not merit measurements in most routine clinical scenarios. However, its application as a pharmacodynamic intermediate marker of the effectiveness of medical therapy holds great promise for rapid evaluation of new drugs.
INTRODUCTION
Early breast cancer (EBC) has a highly variable prognosis and benefit from available therapies is unpredictable for the individual patient. Key factors such as tumor size, histological grade, vascular invasion, and nodal status are helpful, but increasing attention is being paid to the molecular features of the tumor. Estrogen receptor (ER) and Her-2–neu are now well established as predictive factors for treatment response and prognosis. Although not considered an obligatory marker, Ki-67 is also frequently measured both as a static marker of proliferative activity and, by making multiple measurements during treatment, as a possible dynamic intermediate or surrogate marker of treatment efficacy. We provide here an overview of the basic molecular biology of Ki-67 and its prognostic and predictive value at presentation of disease. In particular, we have focused on the evidence supporting the use of change in Ki-67 as an early predictor of treatment efficacy and as a prognostic factor of long-term outcome. The review is based on published evidence, identifying articles with searches of PubMed (key words: "Ki-67," "anti-Ki-67," "breast cancer," "prognostic," "proliferation") as well as references from relevant articles and from the authors’ personal experience. Other than when stated, we refer to Ki-67 as the presence of the Ki-67 antigen determined by immunohistochemistry.
BIOLOGY OF Ki-67
Nature, Sequence, and Function
Ki-67 was identified by Gerdes et al in 19911 as a nuclear nonhistone protein, shortly after the corresponding antibody was described by the same group2 in the city of Kiel (hence "Ki") after immunization of mice with the Hodgkin’s lymphoma cell line L428 (67 refers to the clone number on the 96-well plate in which it was found). The absence of Ki-67 in quiescent cells and its universal expression in proliferating tissues created great interest on its potential role as a marker of cell proliferation. A large number of studies (see references below for correlation with other proliferation markers) have confirmed this feature, and the expression of Ki-67 in resting cells has rarely been reported.3
The Ki-67 gene is on the long arm of human chromosome 10 (10q25).4 In 1993, Schluter et al published the complete sequence of the cDNA encoding for the protein.5 Two alternative mRNA species resulting from alternative splicing encode two isoforms of the protein. The "large" Ki-67 protein isoform has a calculated molecular mass of 359 kD, and the "small" isoform, a mass of 320 kD. The presence or absence of the sequence encoded by exon 7 of the gene differentiates one isoform from the other. The most outstanding feature when the sequence is analyzed is the presence of 16 repeated elements, the Ki-67 repeats," in the large exon 13 that makes up 70% and 79% of the open reading frame of the large and small species, respectively. These are concatenated sequences depicting an important similarity with 43% to 62% identical amino acids. Within these repeats, a sequence of 22 amino acids, the so-called Ki-67 motif, can be found. This motif is highly conserved between species and includes the epitope (FKEL) targeted by the original Ki-67 antibody. There is some evidence derived from murine and human cell lines that more postsplicing variants may exist with a consequent larger range of protein isoforms.6
The expression of Ki-67 varies in intensity throughout the cell cycle, and this has raised concern that it could lead to a misclassification of cycling cells as resting ones.3 Overall, evidence indicates that levels of Ki-67 are low during G1- and early S-phase and progressively increase to reach a maximum during mitosis. A rapid decrease in expression starts during anaphase and telophase.7 Some authors have found that the degree of expression in G1 can be minimal8-10 and may represent a handicap to Ki-67’s accuracy in identifying cells in this phase. This irregular presence of Ki-67 in G1 has been explained as the result of the differences between G1-phase in a previously dormant cell and G1 immediately following a previous cycle8 or even as a marker of different conditions of growth.9
The half-life of Ki-67 protein has been estimated at around 60 to 90 minutes.10,11 Differences of expression during the cell cycle do not seem to be due to the accumulation of nondegraded protein; rather, they seem largely to reflect variable de-novo synthesis.
The cellular appearance and location of the Ki-67 protein throughout the cell cycle is not homogeneous. During early G1, it is found as generally weakly staining discrete foci throughout karyoplasm.12 It progressively condenses during late G1 in larger perinucleolar granules.13,14 During S and G2 phases, it is mainly found associated with the nucleolar region in larger foci as well as with some heterochromatin regions. When the nuclear membrane disrupts during early mitosis, Ki-67 shows an intense expression associated with the surface of condensed chromosomes in the cytoplasm. This intensity rapidly disappears in anaphase-telophase.
Despite all this information about the nature, location, and sequence of Ki-67 protein, there is little known of its function beyond its being a protein phosphorylated via serine and threonine11 with a critical role in cell division. This has been concluded from the arrest of cell proliferation when Ki-67 is blocked either by microinjection of blocking antibodies13 or by inhibition of dephosphorylation.11
Determination of Ki-67
The original Ki-67 monoclonal antibody, when used for immunostaining was initially reported to stain proliferating cells in unfixed tissues but not formalin-fixed paraffin-embedded samples. In 1992, Cattoretti et al reported better success in staining Ki-67 in paraffin-embedded samples after development of the new antibodies MIB-1 and MIB-3. These antibodies seem to produce results equivalent to the original Ki-67 antibody targeting the same epitope (contained in the Ki-67 motif), as demonstrated by identical bands in Western immunoblots.15 Staining by MIB-1 and -3 of formalin-fixed paraffin-embedded samples is greatly enhanced by antigen retrieval (most frequently by microwave heating).16 Although several antibodies are now commercially available to stain Ki-67 in fresh and paraffin-embedded tissue, MIB-1 is the most widely used in recent studies (Fig 1). Ki-67 expression is usually estimated as the percentage of tumor cells positively stained by the antibody, with nuclear staining being the most common criterion of positivity. Currently, there are few data on the between-batch or between-laboratory variability in these measurements and whether the application of image analysis techniques (which have difficulties in tissues with heterogeneous cell populations such as breast cancer) will enhance the precision of assessment.
Ki-67 and Other Markers of Proliferation
Many studies have investigated the correlation between Ki-67 and other well-known markers of proliferation. A strong correlation has been found in most.
S-phase fraction as measured by flow cytometry,17-25 mitotic index,19,20,26-30 tyrosine kinase,20 and in vivo uptake of bromodeoxyuridine (a laborious but highly reliable procedure considered by many to be the gold standard of cell proliferation measurements)26,29,31-33 have each been regularly reported to correlate well with Ki-67.
The correlation of Ki-67 with proliferating cell nuclear antigen has been reported as positive,34 but weak,31 and the multiplicity of functions developed by proliferating cell nuclear antigen may account for this discrepancy.35 Studies of the correlation with DNA ploidy are also conflicting,20,21,33 and no correlation has been found with thymidilate synthase.28 A recently described marker of cell proliferation, the minichromosome maintenance protein 2 (Mcm-2), showed a strong correlation with Ki-67 although it seemed to be more sensitive in detecting cycling cells.36
Ki-67 in the Normal Breast
Few studies have reported results for Ki-67 on normal breast tissue. In samples from normal breast37,38 or in normal epithelium adjacent to fibroadenomas39 it is expressed at a very low level (< 3% of cells). One of the most interesting features found in these studies is the absence of Ki-67 positivity in the ER-positive population38 (ie, only ER-negative cells are proliferative in normal breast). This feature is lost in breast cancer where both markers are present in a higher percentage of cells and these populations overlap. There seems to be a progressively changing pattern from normal to cancerous tissue, with an intermediate degree of coincidence of both markers in noninvasive precancerous lesions.40
Ki-67 and Other Biologic Markers in Breast Cancer
Apoptosis, or programmed cell death, provides the major counterbalancing determinant of tumor growth to proliferation and has been consistently reported to be positively correlated with Ki-67,41 as shown in Figure 2. An index based on the Ki-67–apoptosis ratio (the cell turnover or growth index) has been developed to approximate the contribution that these two factors may make to tumor growth. While it is clear that this cannot accurately reflect growth dynamics, it may have utility as an early marker of response to treatment in primary therapy of breast cancer,42-44 though this remains to be rigorously demonstrated.
Bcl-2, an antiapoptotic protein, has been reported as being inversely correlated with baseline Ki-67.45 The oncogene p53 is frequently mutated or overexpressed in breast cancer, and those tumors presenting these changes show higher rates of proliferation as measured by Ki-67.19,31,45-48 Several studies have addressed the relation between Ki-67 and Her-2 expression, and although there seems to be a positive correlation,28,45,49 it is not always well-defined.22,50 The relationship with epidermal growth factor is similarly conflicting.22,45,50,51 ER status has been widely reported as being inversely correlated with Ki-67, with the higher rates of ER positivity shown in the least proliferating tumors17,19,20,31,34,45,46,52,53 and only a few studies have contradicted this.28,29,48
Of the commonly used pathological features of breast cancer probably the most robustly related to Ki-67 is histological grade with virtually no studies refuting this positive correlation.19-21,28,30,31,33,46,48,53-55 This is to be expected given that mitotic index is one of the three components of grade. Tumor size is not consistently linked to Ki-67 scores with some authors finding a positive relationship31,34,46,52,55 but others not.17,19,22,54
The strongest prognostic factor in EBC, lymph node status, has been intensively studied with regard to its correlation with Ki-67 in an attempt to find an easy marker of nodal involvement that would avoid unnecessary axillary surgery. In studies with more than 200 patients, there seems to be more evidence in favor46,52,56 than against54 a positive correlation (see below). The number of smaller studies favoring a lack of correlation is large.19,20,29,45,48 This may reflect the size of studies and/or the relative weakness in the relationship.
Ki-67 BASELINE MEASUREMENT: PROGNOSTIC VALUE
The search for useful tools to discriminate prognostic subgroups in patients affected by early breast cancer, and in particular, as noted above, in those without lymph node involvement, has led to an extensive study of Ki-67 in this setting. Data from 40 studies involving more than 11,000 patients have been analyzed. Those studies with a sample size greater than 200 patients have been summarized in Table 1.17,18,26-28,34,46,52,54,57-65
In this Table, the studies are presented as statistically significant (P, yellow box) or nonsignificant (N, pink box) for the relation between Ki-67 and clinical outcome. Results are considered in terms of disease-free survival (including metastasis free and local relapse free survival) and overall survival (including disease-specific survival) and the data are shown in three groups made according to the lymph node status (negative, positive, and mixed population). The statistical analysis is specified as univariate or multivariate.
There is strong evidence from these large studies, of the ability of Ki-67 to discriminate between good and bad prognostic groups in the node-negative population when considered as a single variable. While this significance is lost in the multivariate analyses in two of the studies,26,57 in most, it is maintained. In this node-negative group of patients, the large majority of studies failing to report a significant prognostic value of Ki-67 involved patient populations smaller than 200 (not depicted in the Table). In the lymph node–positive and mixed population, the results are less clear, but positive studies predominate.
There are difficulties in assessing the possible prognostic uses of Ki-67 since virtually all of these studies are retrospective and involve a highly heterogeneous population. The use of different antibodies, techniques and scoring protocols without a standard minimum number of cells to be counted may account for some of the differences between studies. The lack of an optimal cutoff point for the definition of prognostic subgroups is probably the main obstacle to a synthesis of the results for extrapolation to clinical practice. Overall it seems that Ki-67 does carry independent prognostic significance from routinely measured pathological features but this is modest and does not merit routine inclusion in the work-up of primary breast cancer.
Ki-67 AND PREDICTION OF TREATMENT RESPONSE
The association of Ki-67 and response to medical treatment is clearer. Five out of six studies reporting the value of Ki-67 to predict response (clinical and/or pathological) to chemotherapy in early or locally advanced breast cancer found that higher Ki-67 was associated with better response66-70 but one found no association.71 In contrast no significant relationship between Ki-67 score and response to treatment has been reported for neoadjuvant endocrine treatment.44,72,73 This may explain why inconsistent results have been reported when mixed chemotherapy and hormonal therapy have been used, with both negative24,45 and positive74 results reported.
It is important to note that while high scores of Ki-67 are associated with higher chance of response to chemotherapy, high Ki-67 is a marker of poor prognosis overall. However, good clinical response, particularly pathological complete response, is associated with good long-term prognosis. This suggests that stratification according to Ki-67 levels might improve the prognostic significance of clinical response in neoadjuvant chemotherapy. To our knowledge this approach has not been undertaken.
CHANGES IN Ki-67 THROUGH TREATMENT: CLINICAL SIGNIFICANCE
The availability of minimally invasive tumor sampling techniques in EBC (ie, fine-needle aspirate, core-cut biopsy) has prompted investigators to study whether changes in Ki-67 score occur after a variable time of treatment and can provide a better predictive or prognostic value than baseline measurements. If so, these early changes might provide valuable intermediate markers of treatment benefit, particularly in relation to drug development.
Studies with MCF-7 xenograft tumors in nude mice with estrogen deprivation75 or antiestrogen hormonal treatments76 provide encouraging proof of principle. Each of these treatments results in a rapid decrease of Ki-67 within 1 week of treatment. The study by Johnston et al demonstrated a complex relation between Ki-67 decrease, changes in apoptosis, and tumor regression following estrogen withdrawal compared with tamoxifen (Fig 3). Estrogen withdrawal induced a profound decrease in Ki-67 (approximately five-fold), accompanied by an increase in apoptosis (approximately four-fold), and this resulted in tumor regression. With tamoxifen, lesser reductions in Ki-67 (approximately two-fold) and increases in apoptosis (approximately three-fold) were seen, and these were associated with no more than stabilization of tumor size. These findings suggest that yet smaller changes in Ki-67 and apoptosis could lead to slowing of tumor growth but still be associated with progressive disease. The implications of these findings for clinical studies are discussed below.
Before assuming that change in any biomarker is due to an intervention, the intrinsic variability of the marker without the mediating intervention should be assessed. Few reports have addressed this issue. Our studies on the reproducibility of Ki-67 measurements in core-cut biopsies suggest that a change in Ki-67 score of at least 32% to 50% between two determinations is required to consider the difference statistically different for an individual patient, and attributable to treatment effect.77,78 These individual requirements do not apply in the same way to populations but are important to take into consideration in the statistical powering of these studies. Further data on the variability in the measurements of Ki-67 are provided in the placebo arms of several short-term presurgical studies,79-82 and these largely support our estimates of variability. However, different biopsy sites, staining, and counting procedures will affect this variability.
In the clinic, effective hormonal treatments suppress Ki-67 levels in both short-term (< 6 weeks)42,79-87 and long-term (eg, 12 weeks) studies.44,72,73,88-90 This has also been shown with withdrawal of hormone replacement therapy.91 As would be expected, these changes are only seen in hormone receptor–positive tumors.80-82,91,92 Reduced Ki-67 levels with hormonal therapy are illustrated in Figure 4, which shows the substantial falls in Ki-67 at 2 weeks and 12 weeks in 56 patients treated with anastrozole before surgery.93 At 2 weeks, only four patients did not show a reduction in Ki-67 levels. The geometric means of reduction were 76% and 82% at 2 and 12 weeks, respectively. There were some patients who showed increases in Ki-67 between 2 and 12 weeks, and it is possible that this is an early indication of resistance to the therapy. It is, however, difficult to distinguish these relatively modest increases in individual patients from the between-sample variability that is discussed above.
The reduction in Ki-67 with endocrine therapy probably reflects the well-described cytostatic effect of the drugs.94 The changes in Ki-67 occur early and well before significant tumor regression is seen. This suggests that treatment selection of a less proliferative cell population probably plays little part in the changes.
Ki-67 decreases also occur with chemotherapy and mixed chemohormonal treatments.24,45,66,67,70,74,77,78,95,96 We have found only one study failing to show this,69 and our own work suggests this may be related to the timing of the second biopsy.97 The reduced proliferation with chemotherapy may be at least partly due to increased apoptosis in actively proliferating cells98 such that the residual population would be enriched for Ki-67–negative cells. The relationship of decrease in Ki-67 during cytotoxic chemotherapy over 2 to 3 months with long-term outcome has been addressed in two studies,45,95 which found in a multivariate analysis that a decrease in Ki-67 of more than 25% and a residual score less than 10% each predicted a longer disease-free survival.
Early changes in Ki-67 have also been found to correlate positively with clinical and/or pathologic response in EBC with both hormone therapy and chemotherapy.24,45,66,70,72,77,88-90,95 In these studies, the timing of the second biopsy varied. The relationship to clinical and/or pathologic response within 2 weeks is particularly useful since these changes preceded clinical response, while the changes at 12 to 16 weeks are usually concordant with clinical response rather than predictive.
This issue has been recently supported by a report on the results of the IMPACT trial.93,99,100 This trial compared 12 weeks of neoadjuvant treatment with anastrozole or tamoxifen or the combination of both drugs in postmenopausal women with ER-positive EBC, followed after surgery by the same regimen as adjuvant treatment. Ki-67 decrease as determined in a biopsy taken 2 weeks after initiation of treatment predicted the better long-term outcome seen in the parallel adjuvant trial with anastrozole.101
The possibility that changes in Ki-67 might predict long-term efficacy (after 10-14 days of treatment) has opened a new scenario for clinical research in EBC. This so-called "short-term preoperative" setting refers to the time between diagnosis of primary operable EBC and the surgery itself, an interval when treatment is not usually administered. Thus, drugs with established safety and low toxicity may be tested and compared against placebo using a decrease in Ki-67 as the primary end point. This model has been exploited in the assessment of several treatments; these have been mainly hormonal,42,79-84 but have more recently included inhibitors of signal transduction pathways.102,103 Most of these showed a significant drop in Ki-67 in the treatment arms compared with placebo or no treatment.
Different treatments have also been compared in this short-term setting to obtain rapid information on potential differences in efficacy.42,81,82,87 Comparative studies between drugs require their having similar pharmacokinetics or the use of schedules to compensate for different pharmacokinetics; they should also have a mechanism of action that similarly affects proliferation (or another end point such as apoptosis). These approaches could also allow the differential effectiveness of drugs on subgroups of patients to be assessed leading to the identification of targets to select optimal therapy. For example, a recent report by Ellis et al found differences in the Ki-67 response to letrozole and tamoxifen depending on whether tumors overexpressed Her-1 and/or Her-2,89 and these results paralleled differential clinical response in the same subgroups.104 These differences had been previously suggested in a pooled analyses of several smaller studies in the short-term preoperative setting.105 The effect on Ki-67 of Her-2–targetted therapy combined with endocrine therapy would seem to be a rational approach to evaluating efficacy.
While the above rationale and examples of the use of early changes in Ki-67 suggest that there will be a growing interest in its use as an intermediate marker of benefit, there remain challenges in the handling of the derived data that need to be met to allow its widespread use. One complication is the non-normal distribution of the data, which logarithmic transformation may also not normalize.89 It is also not clear that the proportional reduction in Ki-67 is the most relevant measure for predicting outcome. The arguments made above suggest that proportional reduction may be an appropriate parameter for predicting benefit in the adjuvant setting, but that the residual (on-treatment) level of Ki-67 may be a better predictor of response and/or absolute long-term outcome since this is more likely to relate to the growth rate of the persistent disease. Currently available and future data sets should examine these possibilities.
SUMMARY AND CONCLUSIONS
The Ki-67 protein, measured with immunohistochemical techniques, is a useful marker of cell proliferation. The suggested lack of accuracy to identify some cells entering the cell cycle in early G0 has not substantially affected the ability of Ki-67 to identify the proliferating population of cells in comparison with other markers of proliferation. Compared with these other markers, Ki-67 staining is easy to perform, economical, and more reproducible. Correlation between Ki-67 mRNA levels and the presence of the protein identified by immunohistochemistry has not been yet fully studied106; this will be required to allow appropriate interpretation of Ki-67 expression as determined with new diagnostic tools, including expression microarrays.
Definitions of cut-offs for Ki-67 vary and hamper comparisons between studies: it may be more helpful to consider Ki-67 as a continuous variable. In multivariate analyses, Ki-67 retains prognostic significance in lymph node–negative patients, but its clinical utility seems slight. Instead, a key role for Ki-67 in EBC may relate to the measurable changes in its expression during treatment. The lack of a close agreement between decreases in Ki-67 and tumor response in some clinical trials possibly results from the need for major reductions in proliferation and/or increases in apoptosis to lead to the regression of a fast-growing tumor. These decreases in proliferation may nonetheless be indicative of benefit from the treatment. There is now a strong rationale for the future design of trials using short-term changes in Ki-67 as a marker of treatment efficacy. However, for analysis of Ki-67 to reach its potential either as a baseline or as a pharmacodynamic measurement, greater attention will need to be paid to standardized methodologies, approaches to scoring, statistical analysis, and between-laboratory quality assessment.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
Acknowledgment
We thank our numerous clinical and laboratory colleagues who have contributed to the studies we have conducted leading to the development of this review.
NOTES
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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