Establishment of a PML-RARa Copy Number Estimator for Prototype Xpert ® PML-RARa Assay

Statement of the Problem: Acute Promyelocytic Leukemia (APL) represents 10-15% of Acute Myeloid Leukemia (AML). The PML-RARa fusion transcript is expressed in more than 95% of APL patients. Three PML-RARa isoforms (bcr1, bcr2, and bcr3) are identified in 90-95% of PML-RARa positive cases. Prototype Xpert ® PML-RARa, an automated cartridge-based assay for measuring PML-RARa fusion transcript levels (bcr1, bcr2, and bcr3), is standardized to quantify the amount of PML-RARA relative to ABL1 control gene based on delta Ct in peripheral blood. Since PML-RARa level is crucial for diagnosis and ongoing therapeutic monitoring in APL, it can be useful to obtain the PML-RARa copy number (CN). The aim of this work is to develop PML-RARa CN estimator and to compare %PML-RARa/ABL1 reporting between delta Ct-based and CN-based methods. Methodology & Theoretical Orientation: Four levels of PML-RARa (bcr1, bcr2, and bcr3) and ABL1 IVT-RNA panels as well as two lots of prototype Xpert ® PML-RARa assay were used to generate standard curves for CN and %CN reporting. The samples with spiked-in bcr1 IVT-RNA and APL clinical samples containing PML-RARa fusion transcript were examined to evaluate the CN and %CN between two lots of the prototype Xpert ® PML-RARa assay and to compare the delta Ct-based and CN-based methods for reporting %PML-RARa/ABL1. Findings: Linearity was demonstrated in Ct vs CN input for PML-RARa (R ²>0.98) and ABL1 (R ²>0.97). Less than 2-fold difference was exhibited for CN and %CN across two different lots. Less than 2-fold difference was observed in %PML-RARa/ABL1 reporting between delta Ct-based and CN-based approaches. Conclusion & Significance: A PML-RARa copy number estimator for prototype Xpert® PML-RARa assay was established, which will provide helpful information for diagnosis and prognosis of APL. (Product in development. Not for use in diagnostic procedures. Not reviewed by any regulatory body.)