How the Oncotype DX® Colon Recurrence Score Works
The Oncotype DX Colon Recurrence Score is designed to support individualized colon cancer treatment planning. The test provides an individualized, quantitative assessment of colon cancer recurrence risk. This increases confidence that Stage II and Stage III colon cancer treatment plans are tailored to each individual patient.
The Oncotype DX Colon Recurrence Score was developed by and is performed in the licensed Genomic Health® laboratory. Each patient tumor specimen is reviewed at Genomic Health by a board-certified anatomic pathologist and marked for manual micro-dissection of tumor tissue. Then RNA is extracted from the enriched colon cancer tumor specimen and purified. Next the RNA is analyzed using a technique called real-time RT-PCR (reverse transcriptase-polymerase chain reaction). The laboratory results are then subject to a rigorous process of quality control. Finally, the quantitative Colon Recurrence Score result is calculated from the gene expression results, and the report is generated.
How the Test Was Developed
The Oncotype DX Colon Recurrence Score was developed in four steps, which are described below.
- Optimization of methods for quantifying gene expression in formalin-fixed, paraffin-embedded tissue (FPET).
- Selection of 761 candidate genes from the human genome.
- Testing of candidate genes to identify an optimal gene panel for clinical validation.
- Prospective clinical validation of the 12-gene panel and Colon 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. To develop the Oncotype DX Colon Recurrence Score, 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. 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.
Step 2. Selection of 761 candidate genes from the human genome
Genomic Health researchers relied on numerous sources to identify 761 candidate genes—those possibly associated with colon 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 761 candidate genes were analyzed in a total of 1,851 patients in order to identify a panel of genes strongly correlated with the likelihood of colon cancer recurrence. The selection of the 7 cancer-related genes used for the Oncotype DX Colon Recurrence Score was based on the results of four developmental studies conducted by the NSABP and Cleveland Clinic Foundation (CCF) in collaboration with Genomic Health, which demonstrated a consistent and strong statistical link between these genes and colon cancer recurrence. Five reference genes were identified to normalize the expression of these cancer-related genes. In addition, these studies form the basis for the calculation providing Colon Recurrence Score result, which combines the gene expression data from this gene panel into a single result.
Step 4. Prospective clinical validation of the 12-gene panel and Colon Recurrence Score algorithm
The Oncotype DX Colon Recurrence Score gene panel and resulting Colon Recurrence Score algorithm were first validated in stage II patients from a large, independent, multicenter clinical trial (QUASAR). Since then several trials have confirmed the Colon Recurrence Score value in stage II patients. The NSABP C-07 trial has validated the Oncotype DX Colon Recurrence Score for stage III disease and further confirms the assay’s value in stage II patients.
- In clinical studies, the Colon Recurrence Score result demonstrated a consistent and significant relationship to colon cancer recurrence.
- The Colon Recurrence Score result is calculated from the expression of 7 cancer-related genes, and 5 reference genes used to normalize the expression of the cancer-related genes.
- The gene groups included in the Colon Recurrence Score result are representative of the two key biological pathways (cell cycle genes and stromal genes) identified in the 4 development studies.
12-Gene Panel Used to Calculate Colon Recurrence Score Result
How is MMR Assessed?
Tumor MMR status can be ascertained in two different ways: immunohistochemistry (IHC) to identify protein expression of known proteins in the MMR pathway or DNA-based PCR analysis to assess the presence of microsatellite instability. Genomic Health, Inc. provides MMR testing using IHC to detect the protein expression of two MMR-related genes (MLH1 and MSH2). Both methods, IHC and PCR, have been shown to be highly concordant, with concordance rates of up to 97% reported in the literature.
Advantages of RT-PCR
The Oncotype DX Colon Recurrence Score analyzes the expression of a panel of 12 genes from a formalin-fixed paraffin embedded tumor (FPET) 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. This method is sensitive, precise, reproducible, and has a wide dynamic range. RT-PCR is a mature technology that is routinely utilized in several clinical applications, including the broadly adopted Oncotype DX Breast Recurrence Score as well as viral load testing for HIV.
To quantify gene expression, RNA is extracted from manually micro-dissected, 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 7 cancer-related genes is measured in triplicate and then normalized relative to a set of five reference genes.
The Oncotype DX Colon Recurrence Score 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
Why RT-PCR was chosen for the Oncotype DX Colon Recurrence Score:
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- Precise, accurate, and highly reproducible over a wide dynamic range
- Minimizes variability that may result from:
- Tissue preparation method, type of fixative, and fixation time
- Tumor block age, storage, and variability in preparation
- Sample heterogeneity