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

Plamadeala, Victoria PhD*; Huang, Shuguang PhD*; McCreary, Suzanne M. BS*; Reitze, Nicholas J. BS*; Ewing, Amy L. BS*; Gabrin, Michael J. BS*; Bennett, Ana E. MD; Mulligan, Jude M. PhD; Wilson, Claire L. PhD; Wang, Dakun PhD*

Applied Immunohistochemistry & Molecular Morphology: April 2014 - Volume 22 - Issue 4 - p 308–316
doi: 10.1097/PDM.0000000000000031
Technical Article

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.

Supplemental Digital Content is available in the text.

*Precision Therapeutics, Pittsburgh, PA

Cleveland Clinic, Cleveland, OH

Almac Diagnostics, Craigavon, United Kingdom

Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Website, www.molecularpathology.com.

The authors declare no conflict of interest.

Reprints: Dakun Wang, PhD, Precision Therapeutics, 2516 Jane Street, Pittsburgh, PA 15237 (e-mail: dwang@ptilabs.com).

© 2014 Lippincott Williams & Wilkins, Inc.