Development and Validation of a Highly Sensitive e1a2 (p190) BCR-ABL Test to Determine Complete Molecular Response (Minimal Residual Disease Negative) As a Primary Endpoint for Patients with Newly Diagnosed Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL)

In Ph+ ALL, the absence of detectable disease has shown prognostic value for a reduced risk of relapse and improved survival. However, as the level of undetectable disease is determined by the lower limit of detection of the test in use, standardization of such an endpoint for a drug regulatory submission is critical. To date, no tyrosine kinase inhibitor (TKI) has received approval for the newly diagnosed Ph+ ALL adult patient population in the US.

Takeda (Millennium Pharmaceuticals, Inc.) is conducting a phase 3, randomized, open-label, multicenter efficacy study comparing ponatinib versus imatinib, administered in combination with reduced-intensity chemotherapy, in participants with newly diagnosed Ph+ ALL (NCT03589326). The primary endpoint for this study is minimal residual disease (MRD)-negative complete remission (CR), where MRD-negativity is defined as a BCR-ABL:ABL raw ratio of ≤0.01% (MR4.0) in bone marrow aspirate samples at the end of induction. Patients who achieve post-induction ponatinib or imatinib maintained MRD-negative CR will potentially delay or avoid stem cell transplantation.

Previously, for the purposes of initiating and monitoring treatment free remission or discontinuation of TKI therapy in chronic phase CML patients, we developed and validated the MRDx® BCR-ABL Test which is an FDA authorized test for the quantitative detection of BCR-ABL e13a2 or e14a2 transcripts. This test will be used in this study and reports BCR-ABL:ABL levels on the International Scale (IS) with traceability to the World Health Organization (WHO) first International Genetic Reference Panel and with a limit of detection below 0.0032% (i.e., MR4.5). Similarily, for assessment of the e1a2 (p190) BCR-ABL:ABL transcripts, we developed and validated a one-step reverse transcription, quantitative polymerase chain reaction (RT-qPCR) test in order to accurately and precisely assess all clinical decision points and disease levels for this study. Because of the lack of available reference material for e1a2, a droplet digital PCR (ddPCR) based test was co-developed to quantify e1a2 BCR-ABL copy numbers in bone marrow aspirates, as well as in peripheral blood samples (to allow assessment of concordance). e1a2 in vitro transcribed RNA calibrators assign copy numbers to determine the e1a2 BCR-ABL:ABL raw % ratios of unknown samples. The e1a2 RT-qPCR test exceeded an analytical sensitivity of MR4.5 (0.0032% raw ratio of BCR-ABL:ABL) with a dynamic linear range from MR4.5 to MR1.0. The test also includes cell line derived RNA assay controls formulated to 10%, 0.1% and 0.01% BCR-ABL:ABL, necessary for decision points in the clinical trial.

Validation studies included limit of blank, limit of detection (LOD), limit of quantification, assay range, analytical specificity, repeatability, reproducibility (multi-day, multi-operator, and multi-instrument), and accuracy by comparison to a reference method (ddPCR). The validation of the e1a2 RT-qPCR test with bone marrow aspirate samples was conducted with 1 µg RNA inputs per well and LOD was also verified with 0.5 µg RNA input per well.

In conclusion, the validated e1a2 RT-qPCR test allows for accurate standardization of BCR-ABL:ABL measurement across multiple centers in an international Phase 3 study. The e1a2 RT-qPCR test data will be used to assess the primary endpoint in the first registrational trial to be conducted in newly diagnosed Ph+ ALL adult patients.