Development and Validation Of Highly Sensitive Mrdx BCR-ABL Test For Monitoring Deep Molecular Response In Patients With Chronic Myeloid Leukemia

Treatment of chronic myeloid leukemia (CML) patients with tyrosine kinase inhibitors (TKI) has led to progressively lower levels of disease burden and higher rates of complete molecular responses. Very few assays with an analytical sensitivity of 4.5-logs (0.00316% BCR-ABL/ABL) have been validated, and assay standardization at these levels has been problematic (EUTOS, 2007). In order to have an International Scale (IS) standardized assay that can precisely and accurately detect 4.5 log reductions in BCR-ABL/ABL ratios, the test system design must be tightly controlled to ensure this critical performance metric can be reproducibly met. The MolecularMD MRDx^TM BCR-ABL Test was developed and validated to be an accurate, reproducible, and highly sensitive IS-standardized solution to measure minimal residual disease (MRD) in CML patients using either PAXgene Blood RNA or EDTA blood collection tubes.

The MRDx Test is a one-step quantitative real-time polymerase chain reaction (RT-qPCR) test that allows for quantitation of BCR-ABL e13/14a2 (b2/3a2) transcripts (covering ≥ 95% of CML patients) and ABL transcripts in RNA extracted from peripheral blood. To achieve a sensitivity of 4.5 logs, several one step enzyme systems were evaluated against RNA samples that spanned the potential dynamic range of the assay (MR1.0 to MR5.0: MR1.0=1 log molecular response (MR), or 10% IS; MR5.0=5 log MR, or 0.001% IS). The one step enzyme system with the best sensitivity and precision was chosen based on the performance of three distinct lots. In addition, in order to better control for the variation of the reverse transcription, in vitro transcribed RNA calibrators were developed to create a standard curve for copy number determination rather than the more commonly used plasmid calibrators that may not accurately reflect the same processing as the patient sample RNA.

The limit of detection (LOD) for blood drawn into PAXgene Blood RNA tubes was validated by testing a dilution series created from a baseline CML patient blood sample diluted into non-diseased subject blood. Creating this type of dilution series allows for the determination of the LOD in the actual clinical sample matrix as compared using cell line dilution series that have much higher BCR-ABL and ABL copy numbers than routine patient specimens. Based on the analysis of each level of sample tested over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.7 (Mean=MR4.9, 95% CI=MR5.2 to MR4.7) using the more conservative upper bound of the 95% confidence interval. The precision was evaluated based on the standard deviation of the log₁₀ BCR-ABL/ABL ratio and was found to be ≤ 0.20 SD at MR5.0 and above. The LOD for blood drawn into EDTA blood collection tubes was validated by creation of a dilution series from a baseline CML patient RNA sample diluted into RNA isolated from blood of non-diseased subjects. Based on the number of samples with detectable BCR-ABL in at least 95% of replicates over a multi-day, multi-operator, and multi-instrument study, the LOD of the MRDx BCR-ABL Test was determined to be MR4.9 (Mean=MR5.0, 95% CI=MR5.1 to MR4.9) using the more conservative upper bound of the 95% confidence interval with precision being ≤ 0.22 SD at MR5.0 and above.

The accuracy of the BCR-ABL copy numbers, ABL copy numbers, and BCR-ABL/ABL ratio using the MRDx BCR-ABL Test was evaluated for ten patient samples using one step droplet digital PCR (ddPCR) as a reference method. For the BCR-ABL copy numbers, ABL copy numbers, and the BCR-ABL/ABL ratio, the bias of the MRDx Test relative to ddPCR by Bland-Altman method was 0.053, 0.030, and 0.023 respectively. The MRDx BCR-ABL Test reports on the IS with a conversion factor of 1.0 by use of the WHO International Standard 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR; secondary standards were created for bi-annual monitoring of the assay conversion factor to ensure accurate reporting of patient results on the IS. Confirmation of the conversion factor was done by an independent laboratory using different real-time PCR instruments and multiple technicians.

Based on the validation data, the MRDx BCR-ABL Test is an accurate, reproducible, and highly sensitive IS-standardized solution to the growing need for a reliable and robust quantitative BCR-ABL assay that can be used for the monitoring of minimal residual disease in CML patients.