NMP1 IHC is a Sensitive Method for Detecting Low-Level Residual Disease in Post-Induction Marrows: Comparison of NPM1 IHC, NPM1 RT-PCR, and Flow Cytometric Studies

Summary: Per NCCN guidelines for acute myeloid leukemia (AML), assessment of minimal residual disease (MRD) is recommended upon completion of induction chemotherapy at day (D) 30-35. The low aspirate cellularity and lack of CD34 or CD117 expression in many NPM1-mutated AML (NPM1mut AML) makes residual disease assessment challenging. Molecular testing has superior sensitivity but a longer turnaround time and limited accessibility. Our study aims to evaluate the utility of NPM1 IHC in NPM1mut AML post induction.  NPM1mut AML cases undergoing bone marrow (BM) evaluation at D30-35 between 5/1/2020 to 7/1/ 2024 with concurrent FC and NPM1 RT-PCR testing were evaluated. Variable cell types positive on NPM1 IHC (polyclonal antibody PA1-46356) were semi-quantified, and the results were compared to NPM1 RT-PCR (sensitivity 0.0001) and to FC analysis. We identified 20 NPM1mut AML cases (17 type A, 3 type D) undergoing BM evaluation for MRD at D30-35. The cellularity ranged between 5% to 70% (median 37%). All cases had less than 5% blasts by manual differential count. FC was negative for abnormal blast population in 19/20 cases (16 by MRD FC and 3 by standard sensitivity FC) and was suspicious in 1 case (MRD FC 0.3%). 8/20 cases had positive staining for NPM1 in various cell types including large mononuclear cells (i.e. blasts), intermediate mononuclear cells (i.e. myelocytes and metamyelocytes), and megakaryocytes. All IHC positive cases had residual mutated NPM1 by RT-PCR ranging between 0.0013 and 1.7577 copies (median 0.0105). 4/8 positive cases had NPM1 level >0.01 by PCR, of which 2 had easily identifiable positive cells (>10) by IHC and 2 had only rare positive cells (less than 5). This might be attributed to the small core size or the marked hypocellularity of the marrow in the latter 2 cases. IHC negative cases (12/20) were either negative (4/12) for NPM1 RT-PCR or had a very low level (8/12) residual NPM1 RT-PCR (less than 0.0013). NPM1 IHC provides a sensitive means of MRD assessment in post-induction BMs. Utilizing RT-PCR as a gold standard, NPM1 IHC can detect residual disease with a 100% specificity and 50% sensitivity at any NPM1 RT-PCR level, and 100% sensitivity at a level of higher than 0.0013. NPM1 IHC demonstrated greater sensitivity than MRD FC analysis. Therefore, NPM1 IHC can be a valuable tool for quick assessment of residual disease in post-induction NPM1mut AML to identify patients for closer follow-up or additional therapies. Nathanael Bailey, Nidhi Aggarwal, Katelyn Davis, Erika Moore, Sara Monaghan, Bryan Rea, and Majd Jawad