A Tale of Two Validations: Molecular Diagnostic Assays for the Detection of Clinically Significant Alterations

Abstract

Laboratory developed tests (LDTs) have become an integral part of laboratory medicine with applications to a variety of complex genetic testing and are at the frontlines of clinical medicine. Proper validation and implementation of LDTs is essential to ensure accurate and high quality results to aid in not only the diagnostic work-up but also for prognostic information and identification of therapeutic options for patients. With numerous techniques and technologies applied to LDTs, it is important to understand which ones are the best methods to apply to a given clinical problem. To best learn this, two validations were performed for two different genetic assays: first a real-time PCR SNP genotyping assay for Apolipoprotein L1 (ApoL1) variant alleles (G1 and G2 alleles) to assess the risk of developing chronic kidney disease (CKD) and secondly a comprehensive next generation sequencing (NGS) assay, OncoPlex version 6 (OncoPlex v6), to detect somatic mutations through an expanded panel of 340 genes. Both validations proved excellent performance characteristics. The accuracy of the real-time PCR genotyping assay had an average of 99.4% and a perfect inter-run and intra-run reproducibility of 100%. Using a simple real-time PCR methodology could potentially determine the patient’s risk for CKD before they progress to the advance stages of the disease. OncoPlex v6 demonstrated exceptional performance characteristics with approximately a 2.5-fold increase in average sequencing coverage and a 3.7-fold increase in percent on-target sequencing. The accuracy and precision of OncoPlex v6 across all classes of alterations was above 97% and 92% respectively, with the majority of variants not identified either present at low variant allele fractions or demonstrating low-level copy gains or losses. In addition to excellent performance characteristics, the modular capture chemistry of OncoPlex v6 demonstrates improvements over prior versions by reduced capture cost, as well as superior sequencing quality and faster turnaround time. Both assays are capable of providing critical information that can impact prognosis, diagnosis, and treatment options.