A molecular diagnostic test used for treatment planning in cancer patients must be analytically robust and reproducible so as to enable consistent and accurate translation of each patient's tumor biology into clinically actionable information. The Oncotype DX Colon Cancer Assay generates a RS for individual patients from the reference normalized tumor expression level of 7 cancer-related genes. These genes were chosen from a refined list of 48 genes (from an original set of 761 candidate genes) which were consistently associated with colon cancer recurrence in four independent clinical development studies comprising more than 1800 patients . Among these 48 genes, two major pathways are represented; cell cycle and stroma-associated. The heart of the co-expressed stromal gene group is a collection of extra-cellular matrix (ECM) proteins including BGN, COL1A1, SPARC and CTHRC1. Cluster analysis from the development studies show these genes shared the lowest average distance between clusters (1-Pearson's R distance as low as 0.15) . Other subgroups observed in the stroma-associated gene cluster included the TGFβ signaling pathway (TGFBI, TGFB3, INHBA), early response genes (EGR1, GADD45B), the WNT pathway (SFRP2, SFRP4), and invasion related genes (PAI1, OPN, TIMP2, TIMP3) . Genes within the cell cycle group included checkpoint control genes and cell cycle regulation genes (such as CDC20, MCM2, MYBL2, CSE1L, MYC and MK167).
After identifying these biological pathways and genes as important markers of clinical outcome in stage II colon cancer, they were used to develop a test to enable translation of tumor biology into clinically useful information. Since biological heterogeneity was inherently embedded within the clinical validation of the Oncotype DX Colon Cancer Assay, any such variability is already incorporated into the prediction of disease recurrence provided by each individual patient's RS. However, the most important source of variability from the perspective of daily testing is assay process, or analytical variability. The 12 RT-qPCR assays and associated processes were therefore analytically validated using pre-defined performance criteria prior to clinical validation of the RS. Analytical validation for the Oncotype DX Colon Cancer Assay was patterned after the approach used for the widely accepted Oncotype DX Breast Cancer Assay [3–5]. At the time the Oncotype DX Breast Cancer Assay was analytically validated there was no widely accepted standard for multi-analyte RT-PCR tests. Therefore, methods commonly used to validate single-analyte laboratory tests were adapted for the purpose . Here, all 12 individual genes were shown to be linear over a 2,000 fold range, and 5 genes (ATP5E, MYC, GPX1, UBB and VDAC2) were linear over a 32,000 fold range. For all genes, the limit of detection was at a CT of 40, and the limit of quantitation at a CT of 36 or greater, providing uniformly high analytical sensitivity for all genes being reported. The sensitivity and accuracy of the Oncotype DX Colon Cancer Assay ensures robust reporting irrespective of the level of RNA expression, an attribute which may not be achieved with DNA microarrays given the lesser dynamic range of this platform [14, 15]. This may in part account for why assessments and validations of microarray systems have focused on precision or reproducibility, rather than accuracy [16–20].
The Oncotype DX Colon Cancer Assay is performed in a high-throughput process using multiple Tecan robotic workstations. In addition, multiple reagent lots and 7900 qPCR instruments provide potential sources of process variability. Therefore, all measureable analytical sources of variability were assessed to determine total system variability. Using two FPE RNA pools, an analysis of variance (ANOVA) model was applied to estimate the total analytical variability in CT measurements for each separate component of variance. The greatest source of analytical variability in CT (and therefore gene scores and RS) came from between-qPCR instruments and between-primer-probe lot components. However, even with these components the relative standard deviations (RSD) associated with each gene was still very small, and well within the pre-defined acceptance criterion of 10%. In fact, the upper bounds of the 95% confidence intervals on the RSD for all the genes in the 12-gene panel were within 10%. The high precision of the individual genes translates into a similarly high level of precision for the stromal gene group score (SD≤0.04), the cell cycle gene group scores (SD≤0.05) and the RS (SD≤1.38).
Plots from a control sample run prior to and during the clinical validation study demonstrate consistent stability for each gene assay. During the clinical validation study, less than 2% of the qPCR plates had to be repeated because of failure to meet both qPCR positive and negative control specifications. Such a well-controlled assay process is an important element of any prognostic or predictive molecular test performed in a clinical reference laboratory used for treatment planning.
The two principal co-expressed gene groups in the Oncotype DX Colon Cancer Assay have long been known as important in cancer progression. The expression levels of an ECM protein (BGN), a fibroblast specific integral membrane serine protease (FAP), and a TGFβ family member (INHBA) are aggregated in the colon RS algorithm to form the SGS, where higher expression is associated with a higher risk of recurrence. Dvorak first described cancers as wounds that do not heal , and it is now generally accepted that activated stroma represents a "wound healing response" that can promote tumor growth, cell migration, invasion and angiogenesis [22–27]. In the same way that tissue regeneration during wound healing involves a complex relationship between the host and the microenvironment, tumorgenesis is also dependant on extra-cellular interactions and signals from the stroma. High amounts of stroma have been associated with poor clinical outcome in patients with colon cancer [28, 29], but as demonstrated by clinical validation of the Oncotype DX Colon Cancer Assay, the level of activation and associated gene expression within the stroma is strongly associated with risk of recurrence. GADD45B is entered into the algorithm as an individual gene, although it tends to associate with the larger stromal group on cluster analysis . Interestingly, GADD45B is believed to stimulate BGN expression .
The second co-expressed gene group that proved to be clinically informative as a component of the colon cancer RS was the cell cycle gene group. In contrast to the Oncotype DX Breast Cancer Assay, where higher expression of cell cycle genes (STK15, MYBL2, MK167 and CCNB1) is associated with increased risk of recurrence [1, 2], higher expression of the colon cell cycle genes (such as CDC20, MCM2, MYBL2, CSE1L, MYC and MK167), was found to correlate with a lower risk of recurrence . This is consistent with other reported evidence that cell cycle gene expression correlates with a good prognosis in colon cancer [31–35]. Garrity et al. reported only a weak correlation in colon cancer between MK167 levels and S-phase (the standard measure of proliferation) , indicating that expression of this gene may not signify rapidly dividing tumors. Instead, increased expression of these cell cycle checkpoint and control genes may represent tightened control of various stages of the cell cycle in response to DNA damage or misalignment of chromosomes during mitosis. APC is mutated in 80% of sporadic colon carcinomas  by either allelic loss or mutations in the multi cluster region (MCR), and there appears to be an interdependence of the two hits . Homozygous deletions of APC are very rare, and residual APC activity is associated with difference biological characteristics depending on the type of mutation and its associated truncated protein (N-APC) [38–41]. For example, different APC mutations have been shown to result in various levels of β-catenin activation [38, 39, 42, 43] and thus different growth advantages . APC is also involved in chromosomal segregation, whereby it localizes to the ends of microtubules within the kinetochore and forms a complex with checkpoint proteins [35, 44]. Some N-APC mutants have been shown to impair spindle checkpoint and contribute to mis-segregation of chromosomes [41, 45–47]. With tighter cell cycle control and the ability (albeit a modest one) to undergo apoptosis or mitotic catastrophe in response to such mitotic errors, a tumor could reduce its abundance of aneuploidy and chromosomal instability (CIN) . Given that CIN is associated with poor outcome [49–52] it could explain how high expression of cell cycle and checkpoint genes (such as CDC20, MCM2, MYBL2, CSE1L, MYC and MK167) were correlated with a lower risk of recurrence . Since APC mutations are much rarer in breast cancer, it is likely that cell cycle genes (and specifically cell cycle control genes) do not harbor the same prognostic information as they do for colon cancer. This possible connection in colon cancer between different types of APC mutations, cell cycle control gene expression, CIN and prognosis warrants further investigation.