Analytical validation of the Oncotype DX genomic diagnostic test for recurrence prognosis and therapeutic response prediction in node-negative, estrogen receptor-positive breast cancer.
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).


CONCLUSIONS
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.
Cronin
Maureen
M
Genomic Health, Inc., Redwood City, CA 94063, USA. mcronin@genomichealth.com
Sangli
Chithra
C
Liu
Mei-Lan
ML
Pho
Mylan
M
Dutta
Debjani
D
Nguyen
Anhthu
A
Jeong
Jennie
J
Wu
Jenny
J
Langone
Kim Clark
KC
Watson
Drew
D
D014408:Biomarkers, Tumor / Q000032:analysis; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000592:standards; D015894:Genome, Human; D006801:Humans; D016013:Likelihood Functions; D025202:Molecular Diagnostic Techniques / Q000592:standards; D009364:Neoplasm Recurrence, Local / Q000175:diagnosis; D009376:Neoplasms, Hormone-Dependent / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D011379:Prognosis; D011960:Receptors, Estrogen / Q000378:metabolism*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction
D014408:Biomarkers, Tumor; D011960:Receptors, Estrogen