Detection of c-Kit Mutation in Peripheral Blood vs. Bone Marrow Aspirates in Patients with Systemic Mastocytosis: Comparison Study of Pathological and Clinical Laboratory Findings in Patients with and without Detectable c-Kit Mutation in the Peripheral Blood.
Authors: Maric Irina , Robyn Jamie , Fu Weiming , Stoddard Jennifer , Metcalfe Dean D. , Noel Pierre 
Published: October 2019

Abstract
The identification of the KIT D816V mutation in patients with systemic mastocytosis (SM) has lately gained a major prognostic significance, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. Imatinib was shown to be ineffective in patients carrying KIT D816V mutation, but effective in cases with some other c-kit mutations. Therefore, it is of paramount importance to correctly identify SM patients with KIT D816V mutation. However, the reported frequency of the KIT D816V mutation in SM patients is highly variable in the literature (30%-over 95%). It has been suggested that such variability is due to patient selection, sensitivity of the molecular methods used to detect the mutation or the source of the tested specimen (peripheral blood (PB) vs. bone marrow (BM) aspirate). To date, there has been no systematic study comparing PB and BM mutational findings in SM patients. In this study, we performed mutational analysis of both PB and BM samples in SM patients and compared the results with pathological, clinical laboratory and flow cytometric findings in patients with and without a detectable c-kit mutation in PB. We analyzed in parallel BM aspirates and PB from 55 patients who came to our clinic for evaluation of SM. After diagnostic workup (physical evaluation, measurement of serum tryptase level, study of BM biopsy, flow cytometric analysis of mast cells and mutational analysis by RT-PCR/RFLP), 46 of 55 patients were diagnosed with SM using the WHO diagnostic criteria. Nine patients did not fulfill the WHO diagnostic criteria for SM and all tested negative for c-kit mutation. Out of 46 patients diagnosed with SM, all but two patients (44/46; 95.6%) tested positive for KIT D816V mutation in the BM aspirate, but only 9/46 patients (19.5%) had the mutation detectable in the PB. Two patients who tested negative for KIT D816V mutation in the BM were shown to carry different c-kit mutation by sequencing. No tested patients carried the FIP1L1-PDGFRa fusion gene. 42/46 patients (91%) fulfilled major WHO pathological criteria for diagnosis of SM (dense mast cell aggregates in the BM biopsy). The other 9% had increased atypical spindle-shaped mast cells in the BM biopsy without dense aggregates. Flow cytometric analysis of PB showed no significant increase in circulating mast cells in patients with a detectable KIT D816V mutation in PB (average less than 0.01% mast cells). Comparison of patients with and without a detectable PB mutation showed more extensive BM biopsy involvement by mast cells in PB positive patients (average 45% vs. 15%), higher average serum tryptase levels (266 ng/ml vs. 85 ng/ml) and higher average PB absolute eosinophil counts (710 vs. 234/uL). Flow cytometric analysis of BM mast cells showed that 100% of KIT D816V positive patients had aberrant CD25 expression on mast cells. CD2 expression was more variable, but comparable in both groups of patents (67% vs. 69%). We conclude that the source of the specimen for c-kit analysis is of crucial importance for correct diagnosis, and recommend that all patients with suspected SM should always have BM aspirates tested for the KIT D816V mutation. PB testing yields falsely negative results in over 80% of cases and identifies only SM patients with a markedly increased mast cell burden.