Analytical validation of SKY92 for the identification of high-risk Multiple Myeloma

Multiple myeloma is an incurable plasma cell cancer with a large variability in survival. Multiple myeloma patients classified high risk by the SKY92 gene expression classifier are at high risk of relapse and short survival. We performed analytical validation of the SKY92 assay with primary bone marrow specimens from 12 multiple myeloma patients and 7 reference cell line specimens. The SKY92 results were 100% concordant - with the reference and/or their expected result - for sensitivity, specificity, microarray stability and RLT buffer stability; The SKY92 results were 90% concordant for primary specimen stability, 96.4% for intermediate precision and 80%-100% for RNA stability. For the cell-line reproducibility the concordance was at least 92.9%, except for one near-cut point specimen. For the clinical specimen reproducibility, the concordance was 100%, except for two near-cut point specimens. Three independent laboratories were concordant in ≥ 77.8% and ≥ 92.9% of experiments (respectively patient specimens and cell lines). Statistical acceptance thresholds were developed as Delta ≤ 1.48 (change in SKY92 score) and standard deviation ≤ 0.45 (SD across SKY92 scores). Using the CLSI method of choice (EP05-A2/A3), restricted maximum likelihood or REML, the observed Deltas (0 - 1.14) and standard deviations (0.22-0.31) passed acceptance criteria. The analytical validation for the SKY92 assay as a prognostic molecular test for individual multiple myeloma patients is therefore successfully proven. The following Analytical Validation studies were designed and performed according to CLSI standards: Analytical Sensitivity, Precision, Repeatability, Intermediate Precision, Reproducibility, Microarray Stability, Sample Stability. SkylineDx, Rotterdam, The Netherlands