Bone Marrow Mast Cells in Systemic Mastocytosis Exhibit Aberrations in Histamine, Size and Granularity

Anaphylaxis is a feature of clonal mast cell disorders (cMCDs), including systemic mastocytosis (SM), monoclonal mast cell activation syndrome (MMAS) and monoclonal mast cells of unknown significance (MMUS), as well as idiopathic mast cell activation syndrome (iMCAS) [1]. Studies by others and our group [2, 3] revealed an aberrant activated phenotype of bone marrow mast cells (BMMCs) in non-advanced cMCDs with a significantly higher expression of FcεRI in patients with cMCDs and anaphylaxis. Regarding iMCAS, we are unaware of any study suggesting the presence of intrinsic quantitative or qualitative abnormalities in BMMCs. Therefore, we sought to analyse the size and granularity of BMMCs and to assess the intracellular content of histamine (ICH) and membrane-bound histamine (MBH) in cMCDs and iMCAS. After BM biopsy, we enrolled and classified 48 patients. We determined the forward scatter area (FSC-A) and side scatter area (SSC-A). As shown in Figure 1A, BMMCs of patients with cMCDWVA were the largest compared (highest FSC-A) to non-clonal patients with WVA (p = 0.0006), iMCAS (p = 0.032), CM (p = 0.055) and controls (p = 0.002). There were significant differences between cMCDANA− and WVA (p = 0.015), CM (p = 0.042) and controls (p = 0.020). The granularity (SSC-A) was the highest in patients with cMCDWVA and higher than in WVA (p = 0.008), iMCAS (p = 0.006), CM (p = 0.043) and controls (p = 0.021). BMMCs from patients with cMCDANA− were more granulated in comparison to those with iMCAS (p = 0.0490). No significant difference in size or granularity was observed between patients with cMCDWVA and cMCDANA− or between patients with iMCAS and controls. As shown in Figure 1B, ICH was the lowest in patients with cMCDWVA and was lower compared to patients with cMCDANA− (p = 0.0270), iMCAS (p = 0.020), patients with CM (p = 0.007) and controls (p = 0.030). The results of the MBH have a similar distribution as compared to the ICH, that is, patients with a cMCD have fewer MCs with MBH. Patients with a cMCDWVA have the lowest number of MCs with MBH as compared to patients with WVA (p < 0.0001), iMCAS (p < 0.0001), CM (p = 0.0017) and controls (p = 0.001). The MBH of cMCDANA− was also lower than that of iMCAS (p = 0.0079) and patients with WVA (p = 0.0399). MBH in cMCDWVA was significantly lower than in cMCDANA− (p = 0.0268). Spearman correlation analysis revealed a weak inverse correlation between the surface marker CD63 and ICH (r = −0.3873, p = 0.0196) and a moderate inverse correlation between CD63 and MBH (r = −0.5205, p = 0.0006). No significant correlation was found between KIT allele burden and ICH (r = 0.3355, p = 0.2027), nor between bST and ICH (r = −0.2877, p = 0.0937). Additional information about patients' inclusion, study methods and findings is available at https://doi.org/10.5281/zenodo.14747070. The underlying cause of the elevated risk of anaphylaxis in cMCDs and iMCAS remains unclear. However, it can be related to an augmented burden of MCs and/or an activated aberrant phenotype. We observed a higher bST in patients with cMCDs. In line with Gülen et al. [4], the median bST tended to be lower in patients with cMCDWVA compared to cMCDANA−. Patients with iMCAS exhibited bST comparable with other non-clonal disorders. BMMCs from patients with cMCDs displayed an aberrant activated status with larger and more granulated cells. This study does not support the postulation that MCs from females are more granulated than those from males [5]. However, experiments on rodents might not be extrapolated to humans. Of note, BMMCs from patients with iMCAS did not exhibit this aberrant activation state and had a size and granularity comparable to non-clonal patients. BMMCs of patients with cMCDs overall had the lowest ICH and MBH. Giannetti et al. [6] reported that patients with cMCDs have significantly higher amounts of histamine metabolites in a 24-h urine collection. This might suggest that the lower ICH in patients with cMCDs is attributable to the continuous release. However, the reduced intracellular histamine seems to be an intrinsic feature, as no correlation was found between ICH and readouts of MC degranulation. Once more, in patients with iMCAS, no significant differences in ICH were observed when compared to non-clonal patients. MBH of BMMCs is lower in patients with cMCDs, with a significant difference between those with and without anaphylaxis. MCs can retain mediators over extended periods and express HR1, HR2, HR3 and HR4 [7]. The hypothesis of a few studies conducted thus far is that MCs can induce auto- and paracrine effects via their histamine receptors (HRs) by presenting histamine [8]. Additionally, Daniela et al. [9] demonstrated that histamine release by MCs was inhibited by exogenous histamine. It is postulated that the reduced MBH on BMMCs from patients with cMCDs, especially in those with anaphylaxis, may result in a diminished immunomodulatory effect of this amine. It is plausible that some patients may have hereditary alpha-tryptasemia (HaT). A limitation of the study is that we did not perform TPASB1 genotyping. However, it is unlikely that this can have influenced our results since HaT does not seem associated with intrinsic abnormalities in BMMCs. In conclusion, the reduced intracellular histamine content and MBH observed in the larger and more granulated aberrantly activated BMMCs in cMCDs is an intrinsic property. MBH was significantly lower in patients with cMCDs and anaphylaxis when compared with cMCDs without anaphylaxis. Furthermore, no significant morphological differences were observed between males and females. Finally, BMMCs of patients with iMCAS demonstrated no morphological or phenotypic differences compared to non-clonal subjects. Conceptualization: V.S. and M.B.; experiments: M.B., J.E., C.M. and M.V.H.; clinical data acquisition: M.B., A.T., V.S., A.V., K.V., M.-B.M. and E.D.B.; data analysis: M.B., J.E. and V.S. writing of the manuscript: M.B., J.E. and V.S.; review of the manuscript: all authors; and final approval: all authors. Vito Sabato is a senior clinical researcher at the Research Foundation Flanders/Fonds Wetenschappelijk Onderzoek (FWO: 1804518N). Jessy Elst is a senior postdoctoral fellow at the Research Foundation Flanders/Fonds Wetenschappelijk Onderzoek (FWO: 1280325N). Informed consent was obtained from all patients. The authors declare no conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.