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The objective of this study was to examine the association between the immunohistochemical Ki67 labeling index (IHC Ki67), Ki67 mRNA expression level, and first-generation gene signatures in a cohort of breast cancer patients. We assessed associations between IHC Ki67 and first-generation gene signatures in a panel of 39 tumor samples, using an oligonucleotide microarray. Gene expression analyses included Ki67 alone (MKi67), 21-gene signature, mitosis kinome score signature, and genomic grade index. Correlation coefficients were calculated by Spearman's rank correlation test. In all cases, IHC Ki67, MKi67, and three genetic markers were highly correlated (ρ, 0.71–0.97). Estrogen receptor (ER)-positive cases showed strong correlations between IHC Ki67 and other signatures (ρ, 0.79–0.83). The ER-negative cases showed slightly lower correlations (ρ, 0.58–0.73). In ER-positive cases, the low IHC Ki67 group showed significantly longer relapse-free survival than the high IHC Ki67 group (P = 0.007). This difference was confirmed by multivariate analysis. Our data indicate that IHC Ki67 shows similar predictive power for proliferation in ER-positive cancers as genomic markers. Further study of IHC Ki67 is needed to define prognostic factors and predictive factors for chemotherapy using central laboratory assessment. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02319.x, 2012)
Immunohistochemical assessment of Ki67 labeling index (IHC Ki67) has been described as a prognostic and predictive marker for breast cancer. However, Ki67 is not included in routine clinical decision-making due to a lack of clarity regarding how Ki67 measurement and thresholds should influence clinical decisions.
According to the recent St. Gallen consensus conference, the Ki67 labeling index is chiefly important in the distinction between ‘luminal A’ and ‘luminal B (HER2-negative)’ subtypes, and Ki67 was described as a predictive marker for chemotherapy. However, guidelines of the American Society of Clinical Oncology do not include Ki67 in the list of required routine biological markers. Recent studies have addressed the use of IHC in breast cancer to include assessment of the proliferation marker Ki67, leading to more refined definitions of good and poor prognosis in estrogen receptor (ER)-positive cancers.[4-6]
Several genetic prognostic markers have been described for breast cancer, and at least two of them (the 21-gene recurrence score and the 70-gene prognostic signature) are commercially available and increasingly used in clinical practice.[7-12] These markers were developed from and validated in a mixed cohort of patients, including both ER-positive and ER-negative cancers as well as low and high histological grade tumors.
However, use of genetic markers is 50 times more expensive than IHC Ki67 and histological grading. Assessment using IHC Ki67 and histological grading is also predictive for prognosis and sensitivity to chemotherapy, and perhaps to endocrine therapy. These features are also associated with the first-generation genomic prognostic assays, which invariably include many genes that capture clinical phenotype information.[13, 14]
One of our co-authors previously showed that genetic markers and Ki67 mRNA expression (mRNA Ki67) had similar predictive power for both prognosis and sensitivity to chemotherapy. However, few clinical datasets provide information on correlations between genomic markers, mRNA Ki67, and IHC Ki67 in the same cohort of patients with survival data.
We hypothesized that IHC Ki67 had similar predictive power for prognosis as some first-generation gene signatures and mRNA Ki67 alone. The objective of this study was to explore associations between IHC Ki67, mRNA Ki67, and various first-generation gene signatures in a cohort of patients who had long-term follow-up for survival.
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Our data show that IHC Ki67 had similar predictive power for proliferation as genomic markers with multiple genes and using only one gene (Ki67). Furthermore, IHC Ki67 was more informative in ER-positive cancers than in ER-negative cancers.
Our data suggest that histological grade might still be a useful prognostic marker. Across the five markers, there were consistent significant differences between grades 3 and 1 and between grades 3 and 2. However, in stepwise multivariate analysis, grade was excluded and IHC Ki67 was retained, indicating that the two parameters have similar prognostic power. The other covariates, including PgR status, tumor size, nodal status, and type of surgery, may have distinct prognostic power independent of proliferative markers such as histological grade and IHC Ki67. Iwamoto et al. reported similar results indicating that clinical variables remained predictive of survival or chemotherapy response independent of genetic markers.
As a well-established cell proliferation marker in breast cancer, IHC Ki67 is an excellent candidate biomarker for luminal B tumors in ER-positive cases. Two meta-analyses have shown a statistically significant association between high Ki67 expression and increased risk of breast cancer relapse and death.[20, 21] However, lack of clarity regarding Ki67 measurement procedures and cut-off points has hindered clinical application of IHC Ki67.
Our data suggest that IHC Ki67 is correlated closely with first-generation genomic markers. Other recent studies confirm the conclusion that IHC Ki67 has similar predictive power for proliferation as genomic markers. Cuzick and colleagues reported that four standard IHC assays (ER, PgR, HER2, and Ki67) carried out in a high quality laboratory can provide prognostic information similar to that provided by the 21-gene signature, in endocrine-treated ER-positive breast cancer patients, using material from the ATAC trial. This approach has wide applicability and could extend the circumstances in which improved prognostic information is routinely available. Thus far, only IHC assays for ER, PgR, and HER2 have been widely adopted.
Williams and colleagues reported that assays using IHC markers of proliferation, such as Ki67, are easy and quick to carry out, relatively inexpensive, and correlate closely with the 21-gene signature. However, further research using validated methods is necessary before widespread adoption in clinical laboratory settings. If validated, IHC Ki67 expression at $US30 per test represents an economical means of testing.
In some cases, we obtained discordant results between IHC Ki67 and the 21-gene score and other genomic markers. When multiple different genomic prognostic tests, IHC Ki67, and histological grade are used clinically and applied to the same case, discordant results can increase confusion in clinical decision making. When multiple predictors are applied to the same case, despite similar predictive performances, discordant risk predictions frequently occur. Currently, in such cases, we cannot identify the superior prognostic marker or predictor of sensitivity to chemotherapy.
Limitations of our study include small sample size and methods of collection of samples. We collected 10 samples from cases in four categories. Our study might include bias of collection. Also, genomic markers with multiple genes were assessed by average gene expression based on data normalized using the MAS5 algorithm, and log 2-converted mRNA gene expression data, a departure from the original methods used in similar published reports.
For determining prognosis and predicting sensitivity to chemotherapy, gene expression profiling (by, for example, the 21-gene or 70-gene signatures) remains the gold standard. Nevertheless, we suggest that IHC Ki67 could emerge as a cost-effective alternative, defined and validated for worldwide clinical diagnostic use.
In conclusion, our data show that IHC Ki67 shows predictive power for proliferation similar to that of genomic markers in ER-positive cancers. It is possible that IHC Ki67 could replace genetic markers for predicting prognosis and sensitivity to chemotherapy in the clinic. However, a lack of clarity regarding IHC Ki67 measurement and cut-off points hinders clinical application. A prospective clinical trial of IHC Ki67 is needed to assess its prognostic value and ability to predict sensitivity to chemotherapy using central laboratory assessment.