Residual Mast Cells Can Explain Persistent Molecular Positivity in Difference from Normal Flow Cytometric-Defined MRD Negative Core Binding Factor AML

Introduction: Core binding factor acute myeloid leukemia (CBF-AML) accounts for 30% of pediatric and 15% adult AMLs and is defined by fusions involving RUNX1 or CBFB. RT-PCR is commonly used to monitor measurable residual disease (MRD) in patients with CBF-AML. However, it has been reported that these fusions can be detected in patients during complete remission (CR) or after hematopoietic cell transplant (HCT). This raises the question of if these patients should be considered MRD positive, given they appear otherwise disease free. We utilized flow cytometric cell sorting and fluorescent in situ hybridization (FISH) to determine the source of persistent CBF fusions. Methods: Flow cytometry was used to isolate mast cells (CD117 bright/CD34 negative) from 13 patients with CBF-AML fusions (8 RUNX1:: RUNX1T1 and 5 CBFB:: MYH11) alongside progenitor cells (CD34+/CD117 heterogeneous, 12 patients) and T cells (CD3+, 11 patients) as available for FISH analysis. These patients were fusion positive by RT-PCR but had no evidence of MRD by difference from normal multidimensional flow cytometry (ΔN), the gold standard for determination of residual disease in Children's Oncology Group-sponsored clinical trials for AML. Cells were sorted from 6 additional patients with a history of RUNX1:: RUNX1T1 CBF-AML who were MRD negative by ΔN but without RT-PCR results available. Results: In 11/13 patients, the mast cell fraction was positive for the CBF fusion but all other sorted cell fractions tested were negative. In the remaining two specimens, all cell fractions were negative, likely due to FISH sensitivity compared to RT-PCR. Both CBF fusions exhibited the same pattern, with fusions detected only in the mast cell fractions (7 RUNX1:: RUNX1T1 and 4 CBFB:: MYH11). The additional 6 patients who were MRD negative by ΔN but without RT-PCR data followed the same pattern, with RUNX1:: RUNX1T1 detected by FISH in the mast cells (6/6 patients) and not in other sorted populations (5/6 patients sorted for progenitor and T cells). Two patients had a clinical history of CBF-AML and systemic mastocytosis (SM). ΔN revealed no evidence of residual AML but showed abnormal mast cells with expression of CD2 and CD25, consistent with abnormalities associated with SM. FISH analysis of sorted mast cells revealed the presence of the RUNX1:: RUNX1T1 fusion, while progenitor and T cells were both negative. In all other patients the mast cells showed no phenotypic abnormalities, demonstrating that