Ki-67 Assays, Including IHC4 and PEPI
Proliferation is a hallmark of cancer cells.9 Ki-67, a nuclear nonhistone protein whose expression varies in intensity throughout the cell cycle, has been used as a measurement of tumor cell proliferation.10 Two large meta-analyses have demonstrated that high Ki-67 expression in breast tumors is independently associated with worse disease-free and overall survival rates.11,12 Ki-67 expression has also been used to classify HR-positive tumors as luminal A or B. After classifying tumor subtypes based on intrinsic gene expression profiling, Cheang and colleagues determined that a Ki-67 cut point of 13.25% differentiated luminal A and B tumors.13 However, the ideal cut point for Ki-67 remains unclear, as the sensitivity and specificity in this study was 77% and 78%, respectively. Others have combined Ki-67 with standard ER, PR, and HER2 testing. This immunohistochemical 4 (IHC4) score, which weighs each of these variables, was validated in postmenopausal patients from the ATAC (Arimidex, Tamoxifen, Alone or in Combination) trial who had ER-positive tumors and did not receive chemotherapy.14 The prognostic information from the IHC4 was similar to that seen with the 21-gene recurrence score (Oncotype DX), which is discussed later in this article. The key challenge with Ki-67 testing currently is the lack of a validated test methodology and intra-observer variability in interpreting the Ki-67 results.15 Recent series have suggested that Ki-67 be considered as a continuous marker rather than a set cut point.16 These issues continue to impact the clinical utility of Ki-67 for decision-making for adjuvant chemotherapy.
Ki-67 and the preoperative endocrine prognostic index (PEPI) score have been explored in the neoadjuvant setting to separate postmenopausal women with endocrine-sensitive versus intrinsically resistant disease and identify patients at risk for recurrent disease.17 The on-treatment levels of Ki-67 in response to endocrine therapy have been shown to be more prognostic than baseline values, and a decrease in Ki-67 as early as 2 weeks after initiation of neoadjuvant endocrine therapy is associated with endocrine-sensitive tumors and improved outcome. The PEPI score was developed through retrospective analysis of the P024 trial18 to evaluate the relationship between post-neoadjuvant endocrine therapy tumor characteristics and risk for early relapse. The score was subsequently validated in an independent data set from the IMPACT (Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen) trial.19 Patients with low pathological stage (0 or 1) and a favorable biomarker profile (PEPI score 0) at surgery had the best prognosis in the absence of chemotherapy. On the other hand, higher pathological stage at surgery and a poor biomarker profile with loss of ER positivity or persistently elevated Ki-67 (PEPI score of 3) identified de novo endocrine-resistant tumors that are higher risk for early relapse.20 The ongoing Alliance A011106 ALTERNATE trial (ALTernate approaches for clinical stage II or III Estrogen Receptor positive breast cancer NeoAdjuvant TrEatment in postmenopausal women, NCT01953588) is a phase 3 study to prospectively test this hypothesis.
21-Gene Recurrence Score (Onco type DX Assay)
The 21-gene Oncotype DX assay is conducted on paraffin-embedded tumor tissue and measures the expression of 16 cancer related genes and 5 reference genes using quantitative polymerase chain reaction (PCR). The genes included in this assay are mainly related to proliferation (including Ki-67), invasion, and HER2 or estrogen signaling.21 Originally, the 21-gene recurrence score assay was analyzed as a prognostic biomarker tool in a prospective-retrospective biomarker substudy of the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 clinical trial in which patients with node-negative, ER-positive tumors were randomly assigned to receive tamoxifen or placebo without chemotherapy.22 Using the standard reported values of low risk (< 18), intermediate risk (18–30), or high risk (≥ 31) for recurrence, among the tamoxifen-treated patients, cancers with a high-risk recurrence score had a significantly worse rate of distant recurrence and overall survival.21 Inferior breast cancer survival in cancers with a high recurrence score was also confirmed in other series of endocrine-treated patients with node-negative and node-positive disease.23–25
The predictive utility of the 21-gene recurrence score for endocrine therapy has also been evaluated. A comparison of the placebo- and tamoxifen-treated patients from the NSABP B-14 trial demonstrated that the 21-gene recurrence score predicted benefit from tamoxifen in cancers with low- or intermediate-risk recurrence scores.26 However, there was no benefit from the use of tamoxifen over placebo in cancers with high-risk recurrence scores. To date, this intriguing data has not been prospectively confirmed, and thus the 21-gene recurrence score is not used to avoid endocrine therapy.