Translating tumor biology into personalized treatment planning: analytical performance characteristics of the Oncotype DX Colon Cancer Assay

Abstract

Background: The Oncotype DX Colon Cancer Assay is a new diagnostic test for determining the likelihood of recurrence in stage II colon cancer patients after surgical resection using fixed paraffin embedded (FPE) primary colon tumor tissue. Like the Oncotype DX Breast Cancer Assay, this is a high complexity, multi-analyte, reverse transcription (RT) polymerase chain reaction (PCR) assay that measures the expression levels of specific cancer-related genes. By capturing the biology underlying each patient's tumor, the Oncotype DX Colon Cancer Assay provides a Recurrence Score (RS) that reflects an individualized risk of disease recurrence. Here we describe its analytical performance using pre-determined performance criteria, which is a critical component of molecular diagnostic test validation.

Results: All analytical measurements met pre-specified performance criteria. PCR amplification efficiency for all 12 assays was high, ranging from 96% to 107%, while linearity was demonstrated over an 11 log2 concentration range for all assays. Based on estimated components of variance for FPE RNA pools, analytical reproducibility and precision demonstrated low SDs for individual genes (0.16 to 0.32 CTs), gene groups (≤ 0.05 normalized/aggregate CTs) and RS (≤ 1.38 RS units).

Conclusions: Analytical performance characteristics shown here for both individual genes and gene groups in the Oncotype DX Colon Cancer Assay demonstrate consistent translation of specific biology of individual tumors into clinically useful diagnostic information. The results of these studies illustrate how the analytical capability of the Oncotype DX Colon Cancer Assay has enabled clinical validation of a test to determine individualized recurrence risk after colon cancer surgery.

Figure 1

RS algorithm. Shown are gene names, their associated gene group and the algorithm for calculation of the Recurrence Score.

Figure 2

Variability chart (box-plots) for RT positive controls run prior to and during clinical validation stratified by gene. A standard RNA template was run across several 7900 instruments, using various primer-probe lots prior to and during clinical validation. The same RNA was included on every RT plate at the same concentration as test samples, and was used as an RT control. Nuclease-free water was used as the RT negative control. Standard deviations in aggregate cycle threshold measurements between samples ranged from 0.19 to 0.33, showing a highly controlled process.

Figure 3

Histogram of PCR positive controls (RNaseP) run prior to and during the clinical validation study. An RNaseP TaqMan assay, with gDNA as the template, was distributed across twelve wells of a 384-well qPCR plate and used as the qPCR positive control. A pre-specified acceptance criterion of 1%CV was applied. During the clinical validation study, only twenty eight samples (2%) failed this 1% CV specification, demonstrating a highly controlled process.