BACKGROUND: Oncotype DX is a clinically validated, high-complexity, multianalyte 
reverse transcription-PCR genomic test that predicts the likelihood of breast 
cancer recurrence in early-stage, node-negative, estrogen receptor-positive 
breast cancer. The Recurrence Score (RS) provides a more accurate, reproducible 
measure of breast cancer aggressiveness and therapeutic responsiveness than 
standard measures. Individualized patient management requires strict performance 
criteria for clinical laboratory tests. We therefore investigated the analytical 
performance of the assay.
METHODS: Assays used a pooled RNA sample from fixed paraffin-embedded tissues to 
evaluate the analytical performance of a 21-gene panel with respect to 
amplification efficiency, precision, linearity, and dynamic range, as well as 
limits of detection and quantification. Performance variables were estimated 
from assays carried out with sample dilutions. In addition, individual patient 
samples were used to test the optimized assay for reproducibility and sources of 
imprecision.
RESULTS: Assay results defined acceptable operational performance ranges, 
including an estimated maximum deviation from linearity of <1 cycle threshold 
(C(T)) units over a > or =2000-fold range of RNA concentrations, with a mean 
quantification bias of 0.3% and CVs of 3.2%-5.7%. An analysis of study design 
showed that assay imprecision contributed by instrument, operator, reagent, and 
day-to-day baseline variation was low, with SDs of <0.5 C(T).
CONCLUSION: The analytical and operational performance specifications defined 
for the Oncotype DX assay allow the reporting of quantitative RS values for 
individual patients with an SD within 2 RS units on a 100-unit scale.