Analytical Performance of a Formalin-fixed Paraffin-embedded Tissue-based 634-probe Prognostic Assay for Predicting Outcome of Patients With Stage II Colon Cancer

Abstract

A formalin-fixed paraffin-embedded tissue-based prognostic assay to assess the risk for recurrence in stage II colon cancer has recently been clinically validated. This study describes the analytical performance and quality control measures of the assay. The reportable range was determined to be [-1.129, 1.414] in risk score units. The accuracy was evaluated with a split sample comparison within the production lab and between the production lab and a reference lab. The concordance between the replicates within the production lab was 79% (95% confidence interval, 64%-91%). There was no evidence of bias, and the concordance was 78% (95% confidence interval, 61%-90%) between the labs. The lab-to-lab concordance was further evaluated by simulating risk scores from the full reportable range. The simulation suggested a higher concordance. The sensitivity study demonstrated that the percentage of tumor tissue did not impact the risk score and that RNA concentration of 9.5 ng/μL was a conservative determination of the analyte lower limit of quantification. From the precision study, the repeatability and reproducibility estimates were 0.1267 and 0.0548 in risk score units, respectively. Furthermore, multifaceted quality control measures were implemented, such as proper tissue processing steps, high-risk and low-risk controls, nontemplate control, and a gene expression-based classifier to evaluate the cDNA amplification kit, a key reagent in the assay. In conclusion, this study demonstrates the strong analytical performance of the assay and further supports its use as an objective standardized prognostic test for stage II colon cancer.

Association Between Results of a Gene Expression Signature Assay and Recurrence-Free Interval in Patients With Stage II Colon Cancer in Cancer and Leukemia Group B 9581 (Alliance)

“…12 Institutional review board approval and patient informed consent were required at each participating institution. The study excluded patients with high-risk features, and the overall risk of recurrence observed in the study was 14.6% at 5 years.…”

“…6 The analytical properties of this assay have also been reported. 12 The assay was developed and intended for use on FFPE, a commonly used, standard method of tumor tissue preservation.…”