Evolution of the 21-gene Assay Oncotype DX®
and fragmentation of RNA over time during storage, Cronin et al. managed to develop a robust, high-throughput, realtime, reverse- transcriptase polymerase chain reaction (RT-PCR) analysis of formalin- fixed paraffin-embedded (FFPE) tumour tissue that could also be used for archival tissue blocks.3,4
As a second step, 250 candidate genes were selected from published literature, genomic databases and experiments based on DNA arrays performed on fresh-frozen tissue. Expression of these genes was measured by RT-PCR in tumour samples of 447 patients with node- negative, oestrogen-receptor (ER)-positive breast cancer from three independent clinical studies. Individual clinical data and results of gene expression analyses were correlated to identify prognostic genes.5–7 The 16 genes with the highest correlation to distant recurrence after 10 years were selected for final model building and validation.8 The panel includes genes associated with proliferation, invasion, ER, the human epidermal growth factor HER2neu and three other genes (see Figure 1).9
Relative expression of these genes is measured
in relation to the average expression of five reference genes. An algorithm was designed to calculate a numerical Recurrence Score (RS) ranging between 1 and 100 (see Figure 1). A low RS value corresponds to a low probability of distant recurrence at 10 years, whereas a higher score is associated with a higher probability.
Validation of the Recurrence Score in Node-negative, ER-positive Breast Cancer
The Oncotype DX assay validation study was conducted utilising tumour specimens from patients who had been prospectively enrolled within the NSABP B-14 study.8
Figure 1: Oncotype DX (Genomic Health, Redwood City, CA) Recurrence Score – Genes and Algorithm
Proliferation
Ki-67 STK15 Survivin Cyclin B1 MYBL2
Invasion
Stromelysin 3 Cathepsin L2
HER-2
GRB7 HER-2
CD68
Reference
Beta-actin GAPDH RPLPO GUS TFRC
Category
Low risk
RS (0–100)
RS <18
Intermediate risk RS ≥18 and <31 High risk
RS ≥31
Oestrogen
ER PR
BcI2 SCUBE2 GSTM1 BAG1
RS = +0.47 x HER-2 group score –0.34 x ER group score +1.04 x proliferation group +0.10 x invasion group score +0.05 x CD68 –0.08 x GSTM1 –0.07 x BAG1
RS = Recurrence Score. Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Sparano J, et al., Development of the 21-Gene Assay and Its Application in Clinical Practice and Clinical Trials, J Clin Oncol, Vol. 26, No 5 (February 10), 2008: 721–728.
Figure 2: Rate of Distant Recurrence as a Continuous Function of the Recurrence Score
Low-risk group
30 35 40
The B-14 study included 2,617 women with
node-negative, ER-positive breast cancer treated with tamoxifen for five years. RT-PCR was successfully performed in 668 of 675 cases. In these cases tumour blocks contained sufficient material to perform the test. The Kaplan-Meier estimate for distant recurrence at 10 years for the corresponding patients was 15%, indicating a highly representative node-negative, ER-positive patient population. Based on the results of previous studies, three risk groups were defined: a low risk of recurrence at 10 years after surgery for an RS <18, an intermediate risk for an RS of 18–30 and a high risk for an RS ≥31 (see Figure 1). The distribution of patients in these groups was 51, 22 and 27%, respectively. The RS proved to be a reliable predictor for the probability of distant recurrence at 10 years after surgery. Only 6.8% (95% confidence interval [CI] 4.0–9.6%) of patients with a low RS had distant recurrence at 10 years, 14.3% (95% CI 8.3–20.3%) of those in the intermediate group and 30.5% (95% CI 23.6–37.4%; p<0.001 compared with the low RS group) of those with a high RS. The proportion of patients without distant recurrence in the low RS group was 93% – significantly higher than the figure of 69.5% in the high-risk group. The RS was also significantly correlated with the relapse-free interval and OS (p<0.001 for both). In a multivariate Cox model, RS predicted distant recurrence independently of age and tumour size (p<0.001). RS is a continuous variable for predicting distant recurrence at 10 years (see Figure 2). The probability of distant recurrence at 10 years increases continuously with increasing scores. For RS >50, the likelihood of distant recurrence increases only slightly with further increases of the RS (see Figure 2).
Population-based External Validation of the Prognostic Significance of the Recurrence Score
For further validation, a case–control study was performed in 4,964 patients diagnosed with node-negative breast cancer who did not
EUROPEAN ONCOLOGY
25 20 15
10 5 0
0 5 10 15 20 25 30 Recurrence Score
The dashed curves indicate the 95% confidence interval. The rug plot on top of the x-axis shows the Recurrence Score for individual patients in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-14 study.8
receive adjuvant chemotherapy.10 The analysis included 220 cases and
570 individually matched controls alive at the date of death of their matched patients. After adjusting for tumour size and grade, the RS correlated with the risk of breast cancer death in ER-positive patients regardless of tamoxifen treatment. At 10 years after surgery, the risk of breast cancer death in ER-positive tamoxifen-treated patients was 2.8% (95% CI 1.7–3.9%), 10.7% (95% CI 6.3–14.9%) and 15.5% (95% CI 7.6–22.8%) in the low, intermediate and high RS groups, respectively.
The results of both the B-14 validation study and the external validation study were the basis for regulatory Clinical Laboratory Improvement Amendments (CLIA) approval of Oncotype DX as a diagnostic test for ER-positive, lymph-node-negative breast cancer treated with tamoxifen.
Recurrence Score as a Predictor of the Additional Benefit of Chemotherapy
The results of a neoadjuvant study hinted towards a correlation between RS and response to chemotherapy.11
Patients with locally advanced breast cancer received chemotherapy with doxorubicin and paclitaxel
37
35 40 45 50
Intermediate- risk group
High-risk group
Rate of distant recurrence at 10 years (% of patients)
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