The Oncotype DX Colon Cancer Assay is a genomic test for patients with newly diagnosed stage II colon cancer, launched in January 2010 by Genomic Health. The test is a validated diagnostic assay based on an individual patient's colon tumor expression of 12 genes, which quantifies the likelihood of recurrence in stage II colon cancer following surgery. The result from the assay is a continuous recurrence score value from 0 to 100 that corresponds to a specific likelihood of colon cancer recurrence 3 years after surgery. A lower score corresponds to a lower risk of recurrence, and a higher score corresponds to a higher risk of recurrence. The Recurrence Score result provides additional information on recurrence risk beyond traditional clinical and pathological characteristics such as tumor stage (T-stage), mismatch repair (MMR) status, number of lymph nodes examined, tumor grade and lymphovascular invasion. The Oncotype DX Colon Cancer Assay has the greatest utility for the stage II colon cancer patients with T3 and MMR proficient tumors. The Oncotype DX assay is a non-invasive test that is performed on a small amount of the tissue removed during surgery, which means no additional invasive procedure is required. After the surgical procedure, the tissue sample is fixed (usually in formalin) and embedded in paraffin so it can be preserved for diagnostic testing. To perform an Oncotype DX test, a pathologist will send the tumor block or several thin sections of the tissue sample to Genomic Health. Genomic Health uses a laboratory process known as RT-PCR to measure the expression of the 12-gene panel.
Technical feasibility Prior to selecting genes for the Oncotype DX Colon Cancer Assay, feasibility studies were conducted to optimize the Genomic Health platform for quantitative assessment of gene expression from fixed paraffin-embedded (FPE) colon tumor tissue. These studies in FPE colon tumor tissue identified (1) the optimal method for reliably extracting RNA and measuring gene expression by quantitative RT-PCR technology, (2) the requirement for review of each case by a pathologist for manual microdissection to remove normal colon tissue adjacent to the tumor, and (3) reference genes for normalization of gene expression. The use of carefully selected reference genes to normalize gene expression in the context of sources of pre-analytical variability such as time of fixation and block age is a critical feature of this technology. The findings from these feasibility studies provided the technical foundation for subsequent studies.
Genes To identify the genes for the Oncotype DX Colon Cancer Assay, four large, independent development studies were conducted which included patients treated with surgery alone and patients treated with surgery followed by adjuvant 5-FU/LV chemotherapy. Colon tumor samples from a total of 1,851 stage II and stage III colon cancer patients from the NSABP studies C-01/C-02, C-04, and C-06 and a cohort of patients treated at the Cleveland Clinic were utilized for identifying genes that were consistently associated with risk of recurrence. 761 candidate genes were studied in the NSABP C01/C02 (surgery alone) and NSABP C04 (surgery+5FU/LV) development studies. Based on the results of these studies, the gene list was refined to 375 genes, which were then studied in the Cleveland Clinic study (surgery alone) and NSABP C06 (surgery+5FU/LV). Of the initial large pool of candidate genes, 48 genes were found to have a consistent and statically significant association with recurrence-free interval in three of the four development studies. Importantly, the multiple large independent studies enabled identification of clinically relevant genes with confidence, with fewer than two of the 48 recurrence-associated genes expected to be false discoveries. The 7 cancer related genes are Ki-67, C-MYC, MYBL2, FAP, BGN, INHBA, and GADD45B.
Development and validation Analyses across these four development studies enabled the definition of a multi-gene assay for prediction of tumor recurrence in stage II colon cancer patients following surgery. Based on modeling and assessment of laboratory analytical performance, a 12-gene assay, including seven cancer-related genes and five reference genes, was defined, yielding a colon cancer Recurrence Score result, scaled from 0 to 100, based upon reference-normalized expression of the seven cancer-related genes. The seven cancer-related genes include two key biologic pathways (cell cycle genes and stromal genes) associated with recurrence in the development studies, as well as an individual gene GADD45B (associated with cellular response to stress). Higher expression of the cell cycle group, including Ki67, is associated with better outcome in stage II colon cancer following surgery, and this result is consistent with reports in the literature, including studies by the NSABP. Cell cycle genes are well-known to produce various effects in different biological contexts, ranging from promotion of proliferation to induction of cell senescence or apoptosis. Higher expression of the stromal gene group is associated with poor outcome in colon cancer, which is consistent with observations in the literature that stromal genes are associated with induction of tumor cell proliferation, tumor invasion, and metastasis. The cell cycle gene group and the stromal gene group are included in the calculation of the Recurrence Score result as an average of the three genes in each group. GADD45B is an individual gene used for calculation of the Recurrence Score result and is known to be a marker of genotoxic stress which may be indicative of tumor-stroma interactions and thus more aggressive tumor behavior. Following gene identification and algorithm development, a process for conducting the assay, including all steps from receipt of the tumor specimen to generation of the assay result, was finalized and analytically validated prior to conducting clinical validation studies. Analytical validation of Oncotype DX test ensures that the assay will give accurate, precise and reproducible results across different reagent lots, operators, and patient samples. Standardization of the assay is critical to ensuring that the Oncotype DX assay yields reliable results which clinicians can use to informing patient management treatment decisions.
Clinical validation
… excerpt ends here. Continue reading the full article.
