Background: Current diagnostic algorithms for systemic mastocytosis (SM) rely on the detection of KITD816V in blood to trigger subsequent bone marrow (BM) investigations.Methods: Here, we correlated the KITD816V mutational status of paired blood and BM samples from 368 adults diagnosed with mast cell activation syndrome (MCAS) and mastocytosis and determined the potential utility of investigating KITD816V in genomic DNA from blood‐purified myeloid cell populations to increase diagnostic sensitivity. In a subset of 69 patients, we further evaluated the kinetics of the KITD816V cell burden during follow‐up and its association with disease outcome.Results: Our results showed a high correlation (P < .0001) between the KITD816V mutation burden in blood and BM (74% concordant samples), but with a lower mean of KITD816V‐mutated cells in blood (P = .0004) and a high rate of discordant BM⁺/blood⁻ samples particularly among clonal MCAS (73%) and BM mastocytosis (51%), but also in cutaneous mastocytosis (9%), indolent SM (15%), and well‐differentiated variants of indolent SM (7%). Purification of different compartments of blood‐derived myeloid cells was done in 28 patients who were BM mast cell (MC)⁺/blood⁻ for KITD816V, revealing KITD816V‐mutated eosinophils (56%), basophils (25%), neutrophils (29%), and/or monocytes (31%) in most (61%) patients. Prognostically, the presence of ≥3.5% KITD816V‐mutated cells (P < .0001) and an unstable KITD816V mutation cell burden (P < .0001) in blood and/or BM were both associated with a significantly shortened progression‐free survival (PFS).Conclusions: These results confirm the high specificity but limited sensitivity of KITD816V analysis in whole blood for the diagnostic screening of SM and other primary MCAS, which might be overcome by assessing the mutation in blood‐purified myeloid cell populations.

Systemic mastocytosis (SM) is a rare clonal haematopoietic stem cell disease in which activating KIT mutations (most commonly KIT D816V) are present in virtually every (>90%) adult patient at similar frequencies among non-advanced and advanced forms of SM. The KIT D816V mutation is considered the most common pathogenic driver of SM. Acquisition of this mutation early during haematopoiesis may cause multilineage involvement of haematopoiesis by KIT D816V, which has been associated with higher tumour burden and additional mutations in other genes, leading to an increased rate of transformation to advanced SM. Thus, among other mutations, alterations in around 30 genes that are also frequently mutated in other myeloid neoplasms have been reported in SM cases. From these genes, 12 (i.e., ASXL1, CBL, DNMT3A, EZH2, JAK2, KRAS, NRAS, SF3B1, RUNX1, SF3B1, SRSF2, TET2) have been recurrently reported to be mutated in SM. Because of all the above, assessment of multilineage involvement of haematopoiesis by the KIT D816V mutation, in the setting of multi-mutated haematopoiesis as revealed by a limited panel of genes (i.e., ASXL1, CBL, DNMT3A, EZH2, NRAS, RUNX1 and SRSF2) and associated with a poorer patient outcome, has become of great help to identify SM patients at higher risk of disease progression and/or poor survival who could benefit from closer follow-up and eventually also early cytoreductive treatment.

Detection of KIT p.D816V is a cornerstone in the diagnosis and classification of mast cell activation syndromes (MCAS) and mastocytosis. However, KIT p.D816V may be undetected due to a low mutated-cell burden in the blood and bone marrow (BM) of many patients, particularly those without skin lesions. These findings underscore the need for ultrasensitive molecular techniques for the detection of KIT p.D816V in these clinical settings. Here, we evaluated, to our knowledge, for the first time, the sensitivity and specificity of a novel Flow-SuperRCA KIT p.D816V assay, compared with conventional allele-specific oligonucleotide quantitative polymerase chain reaction (ASOqPCR), through the analysis of 548 blood and BM samples from 337 adult patients with MCAS and mastocytosis. Our results demonstrated greater sensitivity of the new technique vs ASOqPCR (limit of detection: 0.001% vs 0.01% variant allele frequency), with higher rates of positivity for KIT p.D816V in whole blood and/or BM of 64% of patients with monoclonal MCAS and 55% of patients with BM mastocytosis (P< .0001). Notably, the sensitivity of the new assay went beyond that of ASOqPCR in purified BM mast cells (P< .0001). Additionally, clonality was newly identified in 18% of patients previously diagnosed with nonclonal MCAS, who presented with a unique cutaneous MCA-related symptomatic profile. These results confirm the high specificity and ultrasensitivity of the Flow-SuperRCA assay for the detection of KIT p.D816V, emerging as a well-suited whole-blood and whole-BM test for diagnostic screening and classification of patients with MCAS and mastocytosis. These findings further highlight the clonal nature of an unprecedently high fraction of patients who presented with anaphylaxis and MCAS, with important pathogenic, diagnostic, and clinical implications.

Background: Mast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM.Methods: Overall, we studied 115 SM patients ‐ 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)‐ and 32 healthy donors (HD). Spontaneous and in vitro‐stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated.Results: SM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1β, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ‐stimulated blood monocytes to produce IL1β and TGFβ. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non‐classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL⁺ DC was specifically encountered in BMM cases.Conclusion: These results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.

The anaerobic resorcinol degradation pathway in Azoarcus anaerobius is unique in that it uses an oxidative rather than a reductive strategy to overcome the aromatic ring stability in degradation of this compound, in a process that is dependent on nitrate respiration. We show that the pathway is organized in five transcriptional units, three of which are inducible by the presence of the substrate. Three σ ⁵⁴ -dependent promoters located upstream from the three operons coding for the main pathway enzymes were identified, which shared a similar structure with conserved upstream activating sequences (UASs) located at 103 to 111 bp from the transcription start site. Expression of the pathway is controlled by the bacterial enhancer-binding proteins (bEBPs) RedR1 and RedR2, two homologous regulators that, despite their high sequence identity (97%), have nonredundant functions: RedR2, the master regulator which also controls RedR1 expression, is itself able to promote transcription from two of the promoters, while RedR1 activity is strictly dependent on the presence of RedR2. The two regulators were shown to interact with each other, suggesting that the natural mode of activation is by forming heterodimers, which become active in the presence of the substrate after its metabolization to hydroxybenzoquinone through the pathway enzymes. The model structure of the N-terminal domain of the proteins is composed of tandem GAF and PAS motifs; the possible mechanisms controlling the activity of the regulators are discussed. IMPORTANCE Azoarcus anaerobius is a strict anaerobe that is able to use 1,3-dihydroxybenzene as the sole carbon source in a process that is dependent on nitrate respiration. We have shown that expression of the pathway is controlled by two regulators of almost identical sequences: the bEBPs RedR1 and RedR2, which share 97% identity. These regulators control three promoters with similar structure. Despite their sequence identity, the two bEBPs are not redundant and are both required for maximum pathway expression. In fact, the two proteins function as heterodimers and require activation by the pathway intermediate hydroxyhydroquinone. The structure of the domain sensing the activation signal resembles that of regulators that are known to interact with other proteins.