Underlying Technology
The Oncotype DX Assay Process
As a multi-gene diagnostic assay, the Oncotype DX assay is designed to support individualized treatment planning. The assay provides a quantitative assessment of the likelihood of chemotherapy benefit and distant recurrence, which may increase confidence that the treatment plan is tailored to the individual patient.
The Oncotype DX assay is performed in the licensed Genomic Health laboratory where the assay was developed. First, RNA is extracted from the breast cancer tumor specimen and purified. Next, the RNA is analyzed using a technique called real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Finally, the Recurrence Score result is calculated from the gene expression results.
Advantages of RT-PCR
The Oncotype DX assay analyzes the expression of a panel of 21 genes from a tumor specimen using a technique called RT-PCR. A high-throughput, real-time RT-PCR method was developed to analyze the expression of select genes simultaneously. It is sensitive, specific, highly reproducible and has a wide dynamic range. RT-PCR is a mature technology that is routinely utilized in several clinical applications including viral load testing for HIV.
To quantify gene expression, RNA is extracted from formalin-fixed, paraffin-embedded (FPET) tumor tissue and subjected to DNase I treatment. Total RNA content is measured and the absence of DNA contamination is verified. Reverse transcription is performed and is followed by quantitative TaqMan® (Roche Molecular Systems, Inc.) RT-PCR reactions in 384-well plates. The expression of each of 16 genes is measured in triplicate and then normalized relative to a set of five reference genes.
The Oncotype DX assay standardized testing methods have been optimized to minimize variability due to:
- Tissue preparation method: FPET vs. fresh frozen
- Tumor block age, storage and variability in preparation
- Heterogeneity within and between FPET blocks
- Heterogeneity with respect to enriched tumor and non-tumor areas within an FPET block
Oncotype DX Assay Development
The Oncotype DX assay was developed in four steps. Each of these steps is described in detail below.
- Optimization of methods for quantifying gene expression in formalin-fixed, paraffin-embedded tissue (FPET)
- Selection of 250 candidate genes from the human genome
- Testing of candidate genes to identify an optimal gene panel for clinical validation
- Prospective clinical validation of the 21-gene panel and Recurrence Score result calculation
Step 1. Optimization of methods for quantifying gene expression in formalin-fixed, paraffin-embedded tissue
The ability to work with FPET samples is critical in the U.S., as this is the standard method for tumor preservation and storage. When tissue is preserved in paraffin, the RNA is fragmented. However, the relative ratio of RNA between genes is unchanged. By utilizing RT-PCR techniques, the expression of most genes—relative to a set of reference genes—can be measured. To develop the Oncotype DX assay, Genomic Health researchers optimized RT-PCR technology 1) for high-throughput, real-time quantitation of specific RNA in FPET, and 2) to be reproducible regardless of the variability inherent in tumor blocks.
Step 2. Selection of 250 candidate genes from the human genome
Genomic Health researchers relied on numerous sources to identify 250 candidate genes—those possibly associated with breast cancer tumor behavior—from among the approximately 25,000 genes in the human genome.
Step 3. Testing of candidate genes to identify an optimal gene panel for clinical validation
The 250 candidate genes were analyzed in a total of 447 patients from three independent clinical studies in order to identify a panel of genes strongly correlated with distant recurrence-free survival. The selection of the 16 cancer genes used for the Oncotype DX assay was based on the results of the three clinical trials, which demonstrated a consistent and strong statistical link between these genes and distant breast cancer recurrence. Five reference genes were identified to normalize the expression of these cancer-related genes. In addition, these studies were the basis for the Recurrence Score result calculation, which combines the gene expression data from this gene panel into a single result.
Step 4. Prospective clinical validation of the 21-gene panel and Recurrence Score result calculation
The Oncotype DX assay gene panel and Recurrence Score result calculation were validated in a large, independent, multicenter clinical trial (NSABP Study B-14) and in a large population-based case-control study in breast cancer patients at Northern California Kaiser Permanente. The endpoints and analysis plan were prospectively defined. The results of these studies were included in the Best of Oncology session at ASCO 2004 and the results of the NSABP B-14 clinical validation study were published in The New England Journal of Medicine (December 30, 2004).
21-Gene Panel Used to Calculate Recurrence Score Result
The Oncotype DX assay gene panel was selected from the published literature, a genomics database and experiments based on DNA arrays performed on fresh-frozen tissue. The Recurrence Score result algorithm was derived from 3 studies of breast cancer patients and then was clinically validated in multiple independent studies.
- The Recurrence Score result is calculated from the expression of 16 cancer-related genes and 5 reference genes used to normalize the expression of those cancer genes
- In clinical studies, the 16 cancer genes demonstrated a consistent statistical link to distant breast cancer recurrence, as well as robust predictive power regarding chemotherapy benefit
21-Gene Panel Used to Calculate Recurrence Score Result:
16 cancer genes and
5 reference genes from
3 studies
