Colon Cancer Assay Underlying Technology

The Oncotype DX Colon Cancer Assay Process

As a multi-gene diagnostic assay, the Oncotype DX Colon Cancer Assay is designed to support individualized colon cancer treatment planning. The assay provides an individualized, quantitative assessment of colon cancer recurrence risk, increasing confidence that each stage II treatment plan is tailored to the individual patient.

The Oncotype DX Colon Cancer Assay is performed in the licensed Genomic Health laboratory where the assay was developed. First, each patient tumor specimen is reviewed at Genomic Health by a board-certified anatomic pathologist and marked for manual micro-dissection of tumor tissue. Second, RNA is extracted from the enriched colon cancer tumor specimen and purified. Third, the RNA is analyzed using a technique called real-time RT-PCR (reverse transcriptase-polymerase chain reaction). Fourth, laboratory results are subject to a rigorous process of quality control. Finally, the quantitative Recurrence Score result is calculated from the gene expression results, and the report is generated.

Advantages of RT-PCR
The Oncotype DX Colon Cancer Assay 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 Cancer Assay 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 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.
1. Optimization of methods for quantifying gene expression in formalin-fixed, paraffin-embedded tissue (FPET).
2. Selection of 761 candidate genes from the human genome.
3. Testing of candidate genes to identify an optimal gene panel for clinical validation.
4. Prospective clinical validation of the 12-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 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 76 cancer-related genes used for the Oncotype DX Colon Cancer Assay 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 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 12-gene panel and Recurrence Score result calculation.
The Oncotype DX Colon Cancer Assay gene panel and Recurrence Score result calculation were validated in a large, independent, multicenter clinical trial (QUASAR). The Recurrence Score contains gene groups consistently identified in the 1,851 patients from the development studies, and the validation of the colon cancer Recurrence Score in QUASAR provides strong support for biological relevance. The endpoints and analysis plan were prospectively defined. The results of these studies were presented in the general session for Colorectal Cancer at ASCO 2009.

• The 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 Recurrence Score are representative of the two key biological pathways (cell cycle genes and stromal genes) identified in the 4 development studies.
• In clinical studies, the Recurrence Score demonstrated a consistent and significant relationship to colon cancer recurrence

12-Gene Panel Used to Calculate 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 (Bertagnolli, J Clin Oncol 2010).