Systemic Mastocytosis Patients Research Articles

T-cell immune profile in blood of systemic mastocytosis: Association with disease features.

Systemic mastocytosis (SM) is a heterogeneous disease characterized by an expansion of KIT-mutated mast cells (MC). KIT-mutated MC display activated features and release MC mediators that might act on the tumour microenvironment and other immune cells. Here, we investigated the distribution of lymphocyte subsets in blood of patients with distinct subtypes of SM and determined its association with other disease features. We studied the distribution of TCD4+ and TCD4- cytotoxic cells and their subsets, as well as total NK- and B cells, in blood of 115 SM patients-38 bone marrow mastocytosis (BMM), 67 indolent SM (ISM), 10 aggressive SM (ASM)- and 83 age-matched healthy donors (HD), using spectral flow cytometry and the EuroFlow Immunomonitoring panel, and correlated it with multilineage KITD816V , the alpha-tryptasemia genotype (HαT) and the clinical manifestations of the disease. SM patients showed decreased counts (vs. HD) of TCD4- cytotoxic cells, NK cells and several functional subsets of TCD4+ cells (total Th1, Th2-effector memory, Th22-terminal effector and Th1-like Tregs), together with increased T-follicular-helper and Th1/Th17-like Treg counts, associated with different immune profiles per diagnostic subtype of SM, in multilineal versus MC-restricted KITD816V and in cases with a HαT+ versus HαT- genotype. Unique immune profiles were found among BMM and ISM patients with MC-restricted KITD816V who displayed HαT, anaphylaxis, hymenoptera venom allergy, bone disease, pruritus, flushing and GI symptoms. Our results reveal altered T- and NK-cell immune profiles in blood of SM, which vary per disease subtype, the pattern of involvement of haematopoiesis by KITD816V , the HαT genotype and specific clinical manifestations of the disease.

Diagnosis and management of systemic mastocytosis in a community hematology setting.

Systemic mastocytosis (SM) is a rare and potentially severe hematologic disorder characterized by the clonal proliferation of mast cells (MCs) into various organs. The clinical manifestations of advanced SM are caused by the uncontrolled release of cytokines and vasoactive amines from MC and disease-induced organ dysfunction. Patients with SM typically present with symptoms such as flushing, pruritus, diarrhea, and headaches, but outcomes following active treatment have not been well characterized. In this study, the clinical characteristics, treatment patterns, and natural history of an SM patient cohort diagnosed and treated within a community hematology network in the United States is described. We identified 105 patients who were diagnosed and managed in one of 19 community hematology clinics up to an index date of 1 October 2022. Data collection included patient and disease characteristics, baseline biochemistry and hematology, SM diagnostic criteria being met, biomarkers tested, CD2 and/or CD25 expression in MCs as well as serum tryptase levels at presentation. Data abstraction also included supportive care drugs and anticancer therapy used, treatment response, reason for discontinuation, and overall survival by disease subtype. A total of 105 SM patients were identified who met the inclusion criteria. The specific SM subtypes were indolent (47.6%), aggressive (9.5%), SM with an associated hematological neoplasm (19.0%), MC leukemia (1.9%), and subtype not documented (21.9%). Regardless of subtype, approximately 62% of patients did not receive SM-directed active therapy. Only 26% of patients with indolent systemic mastocytosis (ISM) received treatment compared to 65.6% with advanced subtypes. Relative to ISM cohort, the relative risk of death in patients with the advanced SM subtypes was approximately 15 times greater (hazard ratio = 15.0; 95% confidence interval: 3.3 to 66.5). SM patients present with multiple underlying symptoms, within various disease subtypes that are difficult to diagnose in a timely manner. As a result, many patients do not receive active drug therapy for their disease. Therefore, greater disease awareness is required as well as new tools for earlier disease detection.

Liquid Biopsy Enhances Diagnostic Workup of Hereditary Alpha-Tryptasemia (HaT) and Systemic Mastocytosis (SM) in Patients with Mast Cell-Mediated Symptoms and Elevated Basal Tryptase Levels

Background and Objectives Hereditary alpha-Tryptasemia (HaT) and Systemic Mastocytosis (SM) both display elevated basal serum tryptase (BST) levels and share mast cell-mediated digestive, neuropsychiatric, cardiovascular, cutaneous and musculoskeletal symptoms. Lack of awareness and numerous differential diagnoses complicate and often delay diagnosis, while patients suffer from impaired quality of life and do not receive suitable treatment. HaT is defined by an autosomal dominant inherited amplification of an alpha-Tryptase encoding TPSAB1 gene and is found in 5-6% of the general population. SM is characterized by an activating cKIT D816V mutation that can be detected in 90% of patients. Currently, there is no test available to discriminate between HaT and SM. We aimed to establish a non-invasive blood test supporting the diagnostic workup of these still unrecognized entities. Methods We established a customized digital droplet (dd)PCR triplex assay to detect HaT and a highly sensitive ddPCR singleplex assay for cKIT D816V mutation. We designed a clinical assessment form to record the symptoms. We identified the frequency of HaT and cKIT D816V mutation in genomic DNA isolated from peripheral blood buffy coat samples in 112 patients presenting to our institution with suspected mast cell-mediated symptoms and highly normal (>8-11.4ng/ml) or elevated (>11.4 ng/ml) BST levels (prevalence cohort, Fig. 1). For symptom assessment, we performed blood testing and symptom assessment in these 112 patients and 34 additional patients presenting with suspected SM (n=146, symptom cohort, Fig. 2). As a control cohort we tested 62 age- and sex-matched patients presenting with BST levels < 6 ng/ml (thus not suspected to have HaT or SM). Results HaT accounted for 82 out of 112 cases (73.2%) of BST levels of 8 ng/ml or higher, while cKIT D816V indicating SM was identified in only 5 cases. In 28 patients (24,1%) no HaT genotype or cKIT D816V mutation was detected. One patient with negative cKIT D816V testing but clinical signs of SM was confirmed by bone marrow biopsy, increasing SM prevalence to 6/112 (5.4%). Interestingly the HaT and SM proportions increased to 91% (HaT) and 7.5% (SM) when higher BST cut-off level of 11.4 ng/ml and to 92,9% (HaT) and 28.6% (SM) when higher BST cut-off level of 20 ng/ml were used (Fig. 1). Of note, 21.4% of patients with BST > 20 ng/ml had both, HaT and SM, and no patient with BST >20 ng/ml was tested negative for HaT and SM. In the symptom cohort, we found significant differences in type and severity of symptoms between HaT, SM and the control group. The occurrence of digestive symptoms was up to twice as high in HaT or SM compared to the control group, for flatulence (HaT 83.5%; control group 53.2%) and diarrhea (SM 68.8%; control group 25.8%) or heartburn (SM 56.3%; control group 25.8%). Nausea was significantly more common in HaT (51.7%) than in SM patients (21.9%). HaT patients more frequently reported neuropsychiatric symptoms compared to control group and SM patients. This included memory impairment (HaT 45.9%; SM 23.3%; control group 16.1%) sleep disturbances (HaT 55.8%; SM 43.8%; control group 33.3%) and fatigue (HaT 67.4%; SM 50.0%; control group 37.7%). Conclusions Simultaneous, non-invasive testing for cKIT D816V and HaT in patients with suspected mast cell mediator symptoms allows discrimination of HaT from SM. Adding BST > 8 ng/ml and >11,4ng/ml to the diagnostic workup in symptomatic individuals increases the pretest probability to detect HaT to 73.8% and 91.0%. Diagnosed patients experience mental relieve through personal and medical acknowledgement of their symptoms and suitable treatment can be initiated. According to our results, HaT testing should be initiated in all individuals presenting with mast cell mediator symptoms and BST > 8 ng/ml, and cKIT testing should be initiated in patients with BST >11.4 ng/ml. In patients with BST >20 ng/ml, cKIT D816V positive blood testing or signs of advanced SM, bone marrow based diagnostic workup is still mandatory to cover diagnostic criteria for SM according to WHO.

Successful Management of D816V Mutated c-KIT Systemic Mastocytosis with Chronic Imatinib: A Case Report

First characterized in France in 1936, systemic mastocytosis (SM) is a rare disorder by an excessive proliferation and accumulation of abnormal mast cells in extracutaneous tissues (Nettleship et al., 1869). The release of large quantities of vasoactive mediators by these cells results in allergic- and anaphylactic-type reactions in one or more organ systems. Given the similarity of symptoms between allergic reactions and SM and the rarity of the condition, SM is often missed and misdiagnosed (Schwaab et al., 2020). Once considered a subtype of myeloproliferative neoplasms, mastocytosis was reclassified in the 2016 revision to the World Health Organization (WHO) due to advances in understanding of the disorder (Arber, et al., 2016). Presented below is a case of a severe systemic mastocytosis with an initially positive D816V mutated c-kit successfully managed with long-term imatinib use alongside intermittent bone marrow biopsies (BMB) to monitor what is now an indolent systemic mastocytosis. 69 year-old female with a past medical history of paroxysmal atrial fibrilation, hashimoto's thyroiditis, metabolic syndrome, and gastro-esophageal reflux (GERD) presents to clinic for routine management of what was previously severe systemic mastocytosis (SSM) and now is indolent systemic mastocytosis with the help of imatinib. To help control her ISM her medications include a daily histamine-1 blocker (ranitidine), a daily histamine-2 blocker (diphenhydramine), and a twice-daily histamine-1 antagonist (cetirizine). Originally evaluated in 2005 with a bone marrow biopsy (BMB) and found to have a Asp816Val (D816V) c-kit mutation, she was placed on imatinib with significant clinical improvement. This seemed to support that the D816V mutation may cause constitutional activation of the c-kit transmembrane protein and thus sensitivity to imatinib therapy. This despite the FDA labeling for imatinib as only to be used in SM patients without the common D816V mutation or for whom the mutational status is unknown (Reference ID: 4957210, 2001). Interestingly, after being placed on imatinib her repeat bone marrow biopsies in 2011 and 2015 indicated that the D816V mutation was no longer present in her CD117 positive mast cells but continued to benefit from imatinib therapy. This finding is suggestive of two unique mast cell populations as imatinib continued to help maintain a low tryptase level and decrease the severity of reactions. When reactions did occur, she treated them at one point with a cocktail of an histamine-2 blocker (ranitidine), histamine-1 blocker (diphenhydramine), and a mast cell stabilizer (cromolyn) and without requiring the use of epinephrine. As a part of her longitudinal observation, she received an additional BMB in 2022 that revealed a minimal residual monocytosis that persisted with less than 10% nucleated cells, an age-appropriate trilinear hematopoiesis, decreased iron stores without ringed sideroblast formation. Flow cytometry was indicative of a typical female karyotype with the population of aberrant mast cells that is consistent with the diagnosis of ISM. Importantly, no abnormalities were detected in the FLT3, IDH1, IDH2, NPM1 genes which, if abnormal, could obfuscate the apparent efficacy of imatinib treatment in the management of SM. Given the dearth of mastocytosis in the BMB and the absence of ongoing end-organ damage, the patient meets the definition of ISM. After the most recent BMB the patient altered her daily regimen due to gastric upset and found she could take of her histamine-1 blocker (diphenhydramine) once daily along with a twice daily regimen of her imatinib (split into two smaller daily doses), histamine-1 antagonist (cetirizine), her histamine-2 antagonist (famotidine), and citalopram (selective serotonin reuptake inhibitor or SSRI) with improved symptomatic control to this day. This case serves to illustrate the diagnostic and therapeutic challenges associated with SM, as well as the potential therapeutic use of imatinib in patients with D816V outside of its original FDA indication. Further characterization of the effect of imatinib on D816V mutated SM may warrant the consideration of expanding the FDA indications for imatinib as its role and efficacy outside of solely D816V negative c-kit MS.

Molecular and Cytogenetic Evaluation in Systemic Mastocytosis

Systemic mastocytosis (SM) is a rare hematological neoplasm characterized by pleomorphic symptoms, related to mediators release from pathological mast cells. Tissue infiltration of liver, spleen, gastro-intestinal (GI) tract and osteolysis are present in aggressive disease. As KIT D816V mutation can be found in approximately 80% of patients, several additional somatic mutations of prognostic significance have been detected in a subset of patients. We report on clinical, cytogenetic and molecular characteristic of 160 patients with SM diagnosed in the last 15 years at our center. Fourteen (8.9%) patients had cutaneous mastocytosis, 85 (53.5%) had indolent SM (ISM), 12 (7.5%) had smoldering SM (SSM), 27 (17%) had aggressive SM (ASM), 17 (10.5%) had SM with an associated hematological neoplasm (AHN-SM) and 1 (0.6%) had mast cell leukemia. Symptoms were mostly related to mediators release: itching and blistering (69, 43%), anaphylaxis (49, 30%), diarrhea/bloating (19, 12%), osteoporosis/bone fracture (10, 6%), constitutional symptoms (8, 5%), bone pain and myalgia (16, 10%), anaphylaxis from Hymenoptera venom (24, 15%). While SM patients were mostly treated with antihistamines and membrane stabilizers, 29 patients needed a disease modifying therapy. Four patients with ISM were treated with Interferon (IFN) for bone lytic alterations, while 1 patient with ISM started midostaurin for uncontrolled symptoms. Other 11 patients with ASM were treated with midostaurin: 4 responded, while 7 needed multiple lines of therapy including allogeneic transplant in 4 cases. Eleven patients with ASM were treated with tyrosin kinase inhibitors (TKI) before midostaurin became available, while 2 elderly patients were treated with low dose cytarabine and TKI for concomitant acute leukemia. KIT D816V mutation was found in 111 of 131 (85%) evaluable patients with SM. Cytogenetics was normal in all but 2 patients with AHN-SM in which a chromosomal gain was reported. Among 6 patients with ISM, KIT mutation was found in only 2 cases. One patient had isolated KIT mutation and was treated with IFN for skin symptoms and bone fracture, and another patient had a DNMT3A associated mutation and was treated with imatinib. Moreover, 1 patient presented both DNMT3A and TET2 mutations and was treated with IFN for bone fracture. Among 12 ASM patients, 1 was negative for significant mutations: no disease control was achieved after target therapy, and bone marrow function slowly recovered after allogeneic transplant. The most frequently recorded mutations were SRSF2 and TET2: they were present in 4 patients each and recorded together in 3 cases. Two patients with SRSF2 and TET2 mutations died after multiple lines of therapy: 1 patient for infection and graft versus host disease after allogeneic transplant and 1 for progressive disease. Moreover, the latter had additional RUNX1 mutation. Two patients with concomitant SRSF2 and IDH1 mutations and TET2 mutation respectively responded to midostaurin, while 1 with DNMT3A mutation received multiple lines of therapy and died for pneumonia. One patient with ASXL1 mutation responded to chemotherapy after 2 previous lines of treatment. Among 4 patients with isolated KIT mutation, 2 responded to midostaurin, 1 obtained a partial control of symptoms after 4 lines of treatment and 1 suddenly died after achieving stable disease after 2 lines of therapy. Among 3 patients with SM associated to myelodyplastic syndrome, 1 was negative for KIT but positive for TP53 mutations and died for natural event after symptoms directed therapy and continuing recombinant erythropoietin. The other 2 KIT positive patients presented multiple concomitant mutations. Additional TET2 and SRSF2 mutations were recorded in a patient who died few weeks after diagnosis, while additional TET2, SRSF2, RUNX1 and KRAS mutations were recorded in a patient treated with midostaurine and 2CDA before successful allogeneic transplant. Molecular characterization of SM has uncovered a complex landscape of somatic mutations other than KIT D816V. Although the prognostic significance of some recurrent mutations has been reported, prospective correlation with clinical response may highlighted their role in the pathogenesis of disease and possibly elucidate mechanism of resistance to treatment. On the other hand, cytogenetics might no be considered mandatory except for AHN-SM

Development and Validation of a Diagnostic Tool for the Timely Diagnosis of Patients with Systemic Mastocytosis

Background: Systemic mastocytosis (SM), a rare, clonal mast cell disease associated with heterogenous clinical presentation, is characterized by severe, debilitating and often unpredictable skin, gastrointestinal, neurocognitive, and systemic symptoms (including life-threatening anaphylaxis), long-term co-morbidities, and in Advanced SM (AdvSM), organ damage due to mast cell infiltration. SM consists of 2 distinct groups of variants, advanced and non-advanced: AdvSM includes aggressive SM (ASM), SM with an associated hematologic neoplasm (SM-AHN), and mast cell leukemia (MCL); non-AdvSM includes indolent SM (ISM) and smoldering SM (SSM). Arriving at a definitive diagnosis may take years and is one of the challenges of SM management. To aid clinicians in the identification of SM, we developed and validated a diagnostic algorithm using data from select community hematology practices in the United States. Methods: A sample of 209 patients (SM and control) was obtained from the Quality Cancer Care Alliance (QCCA), a network of 19 community oncology and hematology practices. Data collection consisted of patient characteristics, laboratory parameters, and signs and symptoms at presentation. General linear models (GLM) with a logit link function were used in a backwards elimination process with the p value set at < 0.05 to identify patient factors at presentation that were associated with a diagnosis of SM. A Dx scoring algorithm (range: 0-26) was then derived from the final model coefficients, with the intent of correctly differentiating between SM and non-SM patients. A receiver operating characteristic (ROC) curve analysis was then done to measure the Dx accuracy of the algorithm. Results: Data from 105 SM and 104 non-SM control (diagnosed with hematological cancers) patients were collected from QCCA. The SM cohort included patients with different SM subtypes, including: ISM (47.6%); ASM (9.5%); SM-AHN (19.0%); MCL (1.9%); and subtype not documented (21.9%). The factors identified as being predictive ofa correct diagnosis of SM were 1) patient age, 2) lymph node status, 3) absolute neutrophil count, and 4-7) the following symptoms within 30 days of presentation: diarrhea, rash, skin lesions, and unintended weight loss. In the algorithm, patients were assigned scores for each diagnostic factor they possessed [Figure 1]. The final score was then associated with an overall percent likelihood of a positive SM diagnosis. The area under the ROC curve was 0.89 (95%CI 0.84-0.93), indicating good predictive accuracy. Patients with a total score of ≥ 9 units were found to be at highest risk for a positive SM diagnosis with the associated sensitivity, specificity, and positive predictive values of 84.8%, 76.0%, and 82.7%, respectively. Conclusions: This model indicates that the presence or absence of 7 characteristics may help determine whether patients are at increased risk of having SM. Use of this diagnostic algorithm may help facilitate earlier detection of SM by raising clinical suspicion to facilitate appropriate clinical workup, which would allow the timely initiation of effective targeted therapies for this rare disease. Additional application and external validation of the algorithm in a new sample of 162 patients managed through another community oncology network is ongoing.

Performance and Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real Time PCR for KIT D816V Mutation Detection: A Nationwide Pilot Study By the RIMA (Rete Italiana Mastocitosi) Association

Systemic Mastocytosis (SM) is a rare hematological neoplasm, but its incidence is probably underestimated because of the heterogeneity of clinical symptoms and presentation that may cause diagnostic difficulties especially outside reference centers. Detection of the activating D816V KIT mutation in the bone marrow or peripheral blood is one of the minor criteria for the diagnosis of SM, but it poses some challenges since in the indolent forms (the most frequent) the mast cell burden is very low. For this reason, the National Comprehensive Cancer Network and the European Competence Network on Mastocytosis (ECNM) recommend a high-sensitivity PCR-based assay, such as Allele-Specific Oligonucleotide-Real Time Quantitative PCR (ASO-qPCR) or ddPCR, while Sanger and Next Generation Sequencing are considered not adequate. Accurate quantitation of allele burden (AB) is also desirable since it offers diagnostic (an AB³10% qualifies as a B-finding according to the latest WHO classification) and prognostic information and might be used to monitor response to tyrosine kinase inhibitors. Despite the widespread routine availability of real time and digital PCR technologies, a map of labs across Italy offering KIT D816V testing with adequate sensitivity is lacking, nor have cooperative efforts aimed to check lab performance and reproducibility of diagnostic results ever been performed. Based on these premises, RIMA undertook, with the sponsorship of the GIMEMA Working Party on Chronic Myeloproliferative Neoplasms, a project aimed to: i) build a nationwide network of competent reference laboratories performing KIT D816V mutation testing; ii) promote harmonization of local procedures and ensure adequate proficiency and cross-comparability of results. The pilot phase of this project involved 7 labs (L) spread across the country (Milan, Bologna, Verona, Florence, Rome, Naples, Pavia) serving one or more reference clinical centers for the diagnosis and management of SM. First, a survey aimed to assess the techniques and procedures in use in each participating lab was conducted. Five labs (L2-L6) used a commercial ddPCR assay (KIT p.D816V c.2447A>T, Assay ID dHsaCP2000023; Bio-Rad); one lab (L7) used a home brew ASO-qPCR assay; one lab (L1) used a commercial semi-quantitative real time PCR-based assay (PlentiPlex Mastocytosis D816V kit; Pentabase). All labs performed analyses in duplicate or triplicate using DNA as input material, with amounts variable from 50 to 250ng/replicate. Reported limit of detection (LoD) was 0.0003% for the home brew method and 0.01% for the commercial methods. Subsequently, a round robin test to evaluate and compare performance in terms of sensitivity and inter-lab reproducibility was carried out. Mutated and wild-type DNA isolated from HMC-1.2 and HL-60 cells, respectively, was mixed and diluted to mimic different D816V ABs, from 10% down to 0.01%. Dilutions were externally assessed by the UK Wessex Genomics Laboratory Service, using an accredited ddPCR assay with a limit of detection (LoD) of 0.01%. Of note, the sixth dilution (D6) was scored as slightly below their LoD (0.008%; 3 positive droplets only). Identical batches of blinded vials were then distributed and analyzed in parallel by the 7 participating labs according to their own routine protocols. Positivity/negativity as scored by L1-7 and AB values as scored by L2-7 are detailed in Table 1. All the evaluated methods proved highly accurate in the detection and, for ddPCR and ASO-qPCR, in the quantitation of the KIT D816V (R 2 between 0.988 and 0.998). A very high degree of concordance was achieved, for all dilutions, across different labs and methods (CV between 0.07 and 0.8). D6 was scored borderline by L1 and positive by all other labs. This pilot experience demonstrates that the evaluated PCR-based methods may reliably identify and quantitate the KIT D816V mutation down to an AB of 0.01% with a high degree of cross-comparability of results, thus assisting clinicians in the diagnosis and monitoring of SM patients. To the best of our knowledge, this is the first cooperative effort aimed to assess the performance and reproducibility of KIT D816V mutation testing in SM. Involvement of additional Italian labs is already planned, and further implementation within the ECNM will be proposed. Definition of common SOPs, uniform sample requirements and web-based reporting following the GIMEMA LabNet model will be pursued.

No indication of aberrant neutrophil extracellular trap release in indolent or advanced systemic mastocytosis.

In disease states with chronic inflammation, there is a crosstalk between mast cells and neutrophil granulocytes in the inflamed microenvironment, which may be potentiated by tryptase. In systemic mastocytosis (SM), mast cells are constitutively active and tryptase is elevated in blood. Mast cell activation in SM leads to symptoms from various organs depending on where the active mast cells reside, for example, palpitations, flush, allergic symptoms including anaphylactic reactions, and osteoporosis. Whether neutrophil function is altered in SM is not well understood. In the current study, we assessed nucleosomal citrullinated histone H3 (H3Cit-DNA) as a proxy for neutrophil extracellular trap release in plasma from 55 patients with indolent and advanced SM. We observed a strong trend towards a correlation between leukocyte count, eosinophil count and neutrophil count and H3Cit-DNA levels in patients with advanced SM but not in indolent SM; however, no differences in H3Cit-DNA levels in SM patients compared with healthy controls. H3Cit-DNA levels did not correlate with SM disease burden, tryptase levels, history of anaphylaxis or presence of cutaneous mastocytosis; thus, there is no evidence of a general neutrophil extracellular trap release in SM. Interestingly, H3Cit-DNA levels and leukocyte counts were elevated in a subgroup of SM patients with aberrant mast cell CD2 expression, which warrants further investigation. In conclusion, we found no evidence of global increase in neutrophil extracellular trap release in SM.

Salivary Cystatin D Interactome in Patients with Systemic Mastocytosis: An Exploratory Study.

Mastocytosis, a rare blood disorder characterized by the proliferation of clonal abnormal mast cells, has a variegated clinical spectrum and diagnosis is often difficult and delayed. Recently we proposed the cathepsin inhibitor cystatin D-R26 as a salivary candidate biomarker of systemic mastocytosis (SM). Its C26 variant is able to form multiprotein complexes (mPCs) and since protein-protein interactions (PPIs) are crucial for studying disease pathogenesis, potential markers, and therapeutic targets, we aimed to define the protein composition of the salivary cystatin D-C26 interactome associated with SM. An exploratory affinity purification-mass spectrometry method was applied on pooled salivary samples from SM patients, SM patient subgroups with and without cutaneous symptoms (SM+C and SM-C), and healthy controls (Ctrls). Interactors specifically detected in Ctrls were found to be implicated in networks associated with cell and tissue homeostasis, innate system, endopeptidase regulation, and antimicrobial protection. Interactors distinctive of SM-C patients participate to PPI networks related to glucose metabolism, protein S-nitrosylation, antibacterial humoral response, and neutrophil degranulation, while interactors specific to SM+C were mainly associated with epithelial and keratinocyte differentiation, cytoskeleton rearrangement, and immune response pathways. Proteins sensitive to redox changes, as well as proteins with immunomodulatory properties and activating mast cells, were identified in patients; many of them were involved directly in cytoskeleton rearrangement, a process crucial for mast cell activation. Although preliminary, these results demonstrate that PPI alterations of the cystatin D-C26 interactome are associated with SM and provide a basis for future investigations based on quantitative proteomic analysis and immune validation.

Histamine elevation in familial Mediterranean fever: A study from the Juvenile Inflammatory Rheumatism cohort

BackgroundFamilial Mediterranean Fever (FMF) is the most frequent monogenic autoinflammatory disease (AID). Some patients have persistent symptoms despite colchicine intake. Mast cells (MC) are innate immune cells involved in inflammatory conditions including AID. Their activation is responsible for various symptoms such as abdominal pain, bloating and pruritus. ObjectiveOur objective was to evaluate features of a systemic MC activation in FMF adult patients. MethodsFMF adult patients prospectively filled a MC activation survey and usual MC mediators (tryptase and histamine in whole blood, plasma and urine) were measured. They were compared with a healthy control group (HC) and a systemic mastocytosis (SM) group. When digestive biopsies were realized during follow-up, MC infiltration in digestive mucosa was analyzed in FMF, in comparison with SM, Crohn disease (CD) and normal biopsies. ResultsForty-four FMF patients, 44 HC and 44 SM patients were included. Thirty-one (70%) FMF patients had symptoms of mast cell activation, versus 14 (32%) in the HC group (p = 0.0006). Thirty (68%) FMF patients had at least one elevated MC mediator: mainly whole blood histamine, in 19 (43%) and urinary histamine, in 14 (32%), which were significantly higher than in HC subjects. MC infiltration was comparable in FMF digestive biopsies, biopsies of CD and normal biopsies but was lower than in SM biopsies. ConclusionFMF patients show frequent symptoms of MC activation and an increase of blood or urinary histamine never described before in this disease. This suggests an implication of MC and possibly basophils in FMF pathophysiology.

Systemic mastocytosis in adults: 2023 update on diagnosis, risk stratification and management.

Systemic mastocytosis (SM) results from clonal proliferation of mast cells (MC) in extracutaneous organs. The major criterion is presence of multifocal MC clusters in the bone marrow and/or extracutaneous organs. Minor diagnostic criteria include elevated serum tryptase level, MC CD25/CD2/CD30 expression, and presence of activating KIT mutations. Establishing SM subtype as per the International Consensus Classification/World Health Organization classification systems is an important first step. Patients either have indolent/smoldering SM (ISM/SSM) or advanced SM, including aggressive SM (ASM), SM with associated myeloid neoplasm (SM-AMN), and mast cell leukemia. Identification of poor-risk mutations (i.e., ASXL1, RUNX1, SRSF2, NRAS) further refines the risk stratification. Several risk models are available to help assign prognosis in SM patients. Treatment goals for ISM patients are primarily directed toward anaphylaxis prevention/symptom control/osteoporosis treatment. Patients with advanced SM frequently need MC cytoreductive therapy to reverse disease-related organ dysfunction. Tyrosine kinase inhibitors (TKI) (midostaurin, avapritinib) have changed the treatment landscape in SM. While deep biochemical, histological and molecular responses have been documented with avapritinib treatment, its efficacy as monotherapy against a multimutated AMN disease component in SM-AMN patients remains unclear. Cladribine continues to have a role for MC debulking, whereas interferon-α has a diminishing role in the TKI era. Treatment of SM-AMN primarily targets the AMN component, particularly if an aggressive disease such as acute leukemia is present. Allogeneic stem cell transplant has a role in such patients. Imatinib has a therapeutic role only in the rare patient with an imatinib-sensitive KIT mutation.

Proteomic and transcriptomic screening demonstrates increased mast cell–derived CCL23 in systemic mastocytosis

Systemic mastocytosis (SM) is a heterogeneous group of mast cell-driven diseases diagnosed by bone marrow sampling. However, there are a limited number of available blood disease biomarkers. Our aim was to identify mast cell-derived proteins that could potentially serve as blood biomarkers for indolent and advanced forms of SM. We performed a plasma proteomics screening coupled with single-cell transcriptomic analysis in SM patients and healthy subjects. Plasma proteomics screening identified 19 proteins upregulated in indolent disease compared to healthy, and 16 proteins in advanced disease compared to indolent. Among these, 5 proteins, CCL19, CCL23, CXCL13, IL-10, and IL-12Rβ1, were higher in indolent relative to healthy and in advanced disease compared to indolent. Single-cell RNA sequencing demonstrated that CCL23, IL-10, and IL-6 were selectively produced by mast cells. Notably, plasma CCL23 levels correlated positively with known markers of SM disease severity, namely tryptase levels, percentage bone marrow mast cell infiltration, and IL-6. CCL23 is produced predominantly by mast cellsin SM, and CCL23 plasma levels are associated withdisease severity, correlating positively with established markers ofdisease burden, thus suggesting that CCL23 is a specificSMbiomarker. In addition, the combination of CCL19, CCL23, CXCL13, IL-10, and IL-12Rβ1 may be useful fordefining disease stage.

Symptomatology and Diagnostic Journey of Patients Diagnosed with Systemic Mastocytosis in The US Oncology Network

Systemic mastocytosis (SM) is a rare disorder characterized by severe and unpredictable symptoms across multiple systems. Understanding symptoms and the diagnostic journeys of patients with SM provides insight into clinical management. Here we describe the symptomatology and diagnostic journey of SM patients, focusing on smoldering/indolent (non-advSM), diagnosed in The US Oncology Network (USON).

Capitalizing on paradoxical activation of the mitogen-activated protein kinase pathway for treatment of Imatinib-resistant mast cell leukemia.

Prevention of fatal side effects during cancer therapy of cancer patients with high-dosed pharmacological inhibitors is to date a major challenge. Moreover, the development of drug resistance poses severe problems for the treatment of patients with leukemia or solid tumors. Particularly drug-mediated dimerization of RAF kinases can be the cause of acquired resistance, also called "paradoxical activation." In the present work we re-analyzed the effects of different tyrosine kinase inhibitors (TKIs) on the proliferation, metabolic activity, and survival of the Imatinib-resistant, KIT V560G, D816V-expressing human mast cell (MC) leukemia (MCL) cell line HMC-1.2. We observed that low concentrations of the TKIs Nilotinib and Ponatinib resulted in enhanced proliferation, suggesting paradoxical activation of the MAPK pathway. Indeed, these TKIs caused BRAF-CRAF dimerization, resulting in ERK1/2 activation. The combination of Ponatinib with the MEK inhibitor Trametinib, at nanomolar concentrations, effectively suppressed HMC-1.2 proliferation, metabolic activity, and induced apoptotic cell death. Effectiveness of this drug combination was recapitulated in the human KIT D816V MC line ROSAKIT D816V and in KIT D816V hematopoietic progenitors obtained from patient-derived induced pluripotent stem cells (iPS cells) and systemic mastocytosis patient samples. In conclusion, mutated KIT-driven Imatinib resistance and possible TKI-induced paradoxical activation can be efficiently overcome by a low concentration Ponatinib and Trametinib co-treatment, potentially reducing the negative side effects associated with MCL therapy.

MPN-182 Droplet Digital PCR for Non-Invasive Detection of the KIT D816V Mutation in the Peripheral Blood of Patients With Suspected Systemic Mastocytosis.

Systemic mastocytosis (SM) is a rare and underdiagnosed hematologic neoplasm. Detection of the D816V KIT mutation in the bone marrow (BM) is one of the minor criteria for the diagnosis of SM and requires sensitive methods like ASO-qPCR. In patients with suspected SM, non-invasive pre-screening of peripheral blood (PB) by ASO-qPCR has been shown to detect the D816V mutation with a concordance with BM results of 92% in indolent SM (ISM) patients with skin lesions and of 66% in those without skin lesions. However, ASO-qPCR is not standardized, and commercial kits are not available. Droplet digital PCR (ddPCR) might be a valuable alternative to ASO-qPCR. To validate a commercially available ddPCR assay for the detection and quantitation of the KIT D816V mutation and to evaluate its potential as a non-invasive screening tool in patients with suspected SM. To assess specificity and calculate the limit of blank, PB samples from 30 healthy donors (HDs) were used. The limit of detection was determined by mixing KIT D816V-mutated (HMC-1.2) and unmutated (HMC-1.1) cell lines in order to mimic different allele burdens, from 50% down to 0.01%. Accuracy was investigated by analyzing, in parallel, 35 samples from patients with confirmed or suspected ISM by ASO-qPCR and ddPCR. Concordance between ddPCR results in BM and PB was assessed in 60 matched PB/BM samples. ddPCR was performed using 50 ng/µL of genomic DNA with the KIT p.D816V human mutation assay on a QX200 instrument (Bio-Rad). No KIT D816V-positive events were detected in any of the HD samples. In HMC 1.2 cell dilutions, KIT D816V mutation could be detected down to 0.01% allele burden. Comparison of ddPCR and ASO-qPCR revealed very high concordance in mutation detection and quantitation. All patients positive for KIT D816V in the BM also tested positive in the PB by ddPCR. Moreover, ddPCR revealed high concordance in mutation quantitation between BM and PB, even at very low variant allele frequencies. ddPCR is an attractive alternative to ASO-qPCR for KIT D816V mutation detection and quantification in SM and may reliably detect the D816V mutation non-invasively in PB in patients with suspected SM.

Patient-reported outcomes among patients with systemic mastocytosis in routine clinical practice: Results of the TouchStone SM Patient Survey.

Systemic mastocytosis (SM) is a rare clonal neoplasm driven by KIT D816V and other mutations. Data were collected from the patient perspective on disease burden and included an SM-specific symptom assessment tool. US adults aged 18 years and older with a self-reported SM diagnosis completed an online TouchStone SM Patient Survey of 100 items, including the 12-item Short-Form Health Survey, the Indolent Systemic Mastocytosis Symptom Assessment Form, and the Work Productivity and Activity Impairment Questionnaire, as well as questions about SM diagnosis, the impact of SM on daily activities, work impairment, and health care use. The results were analyzed using descriptive statistics. Fifty-six individuals completed the survey (89% women; median age, 48 years; mean time since diagnosis, 6.7 years), reporting indolent SM (66%), aggressive SM (9%), smoldering SM (5%), and unknown SM subtype (18%). Over a 1-year recall, respondents reported seeking emergency care for anaphylaxis (30%) and taking three or more prescription medications (52%) for SM. Over one half of patients (54%) reduced their work hours because of SM, and 64% avoided leaving home because of symptoms. A majority of respondents (93%) had experienced ≥10 SM-related symptoms, noting that the most bothersome were anaphylactic episodes (18%), abdominal/stomach pain (16%), diarrhea/loose stools (13%), and fatigue (11%). Whereas an Indolent Systemic Mastocytosis Symptom Assessment Form-derived total symptom score of 28 is used to indicate moderate-to-severe symptoms, the mean total symptom score was 52.7. Mental and physical component summary scores from the 12-item Short-Form Health Survey were below population norms. Patients who were surveyed reported substantial symptom burden and unmet needs because of SM, as evidenced by seeking emergency care and reporting bothersome symptoms, poor quality of life, and reduced work hours and productivity. The objective of this research was to understand the burden and unmet needs in the rare disease of systemic mastocytosis (SM) to guide future care. Fifty-six patients completed an online survey containing questions about their diagnosis, medications, health care use, quality of life, and SM symptoms. The results demonstrated that SM is associated with severe and burdensome symptoms, anaphylactic events, emergency department visits, use of multiple medications, reduced ability to work, and poor physical and psychological quality of life. These findings suggest the need for future advances to address unmet needs in patients affected by SM.

Functional imaging with dual-energy computed tomography for supplementary non-invasive assessment of mast cell burden in systemic mastocytosis

Systemic mastocytosis (SM) is characterized by multifocal accumulation of neoplastic mast cells (MCs), predominately affecting the bone marrow (BM). Imaging with computed tomography (CT) is used for assessment of bone mineral density and structure. However, the value of functional imaging with dual-energy CT (DECT) and the assessment of virtual-non-calcium attenuation values (VNCa-AV) for visualization of BM disease burden in SM has not yet been assessed. DECT of the axial skeleton was performed in 18 patients with SM (indolent SM [ISM], n = 6; smoldering SM [SSM]/advanced SM [AdvSM], n = 12) and 18 control subjects. VNCa-AV were obtained in 5 representative vertebraes per patient and correlated with laboratory, morphologic and molecular parameters. VNCa-AV strongly correlated with quantitative BM MC infiltration (r = 0.7, R2 = 0.49, P = 0.001) and serum tryptase levels (r = 0.7, R2 = 0.54, P < 0.001). Mean VNCa-AV were significantly higher in SSM/AdvSM as compared to ISM (− 9HU vs. − 54HU, P < 0.005) and controls (− 38HU, P < 0.005). Nine of 10 (90%) patients with a VNCa-AV > − 30HU and 7/7 (100%) patients with a VNCa-AV > − 10HU had SSM or AdVSM. BM VNCa-AV provide information about the MC burden of SM patients and correlate with SM subtypes. DECT may therefore serve as a supplementary tool for SM diagnosis, subclassification and monitoring in a one-stop-shop session.

Clinical Outcomes of Adults with Systemic Mastocytosis: A 15-Year Multidisciplinary Experience.

Simple SummaryThe term mastocytosis denotes a heterogeneous group of rare disorders characterized by abnormal accumulation and activation of mast cells in various organs. Based on the WHO classification, mastocytosis can essentially be split into indolent and advanced systemic disease. Indolent mastocytosis is the most prevalent variant in adults. In our retrospective study, we aimed to analyze the clinical outcomes of patients with mastocytosis attending our center over the last 15 years. We found that most patients in our cohort suffered from the indolent variant of mastocytosis. Furthermore, we determined the overall prevalence of mastocytosis in our region to be one case per 10,000 adults. In addition, overall survival was significantly better in indolent mastocytosis patients compared to patients with advanced disease. Therefore, awareness of differences in the prognoses of patients with indolent and advanced mastocytosis is important.Systemic mastocytosis (SM) is a rare, clonal, clinically heterogeneous disorder of the mast cells (MCs), and mainly affects adults. The present study aims to describe the clinical and laboratory features as well as the outcomes of SM. A 15-year retrospective study was conducted on 195 consecutive SM patients (aged ≥ 18 years) diagnosed in 2006–2020 at the Multidisciplinary Mastocytosis Center at Karolinska University Hospital. Patients with indolent SM (ISM) represented the most common SM variant (88.2%). Furthermore, the frequencies of aggressive SM and SM with associated non-mast-cell hematological neoplasm were 4.1% and 7.7%, respectively. The prevalence of SM in the adult population of the Stockholm region was estimated to be 10.6/100,000 inhabitants, and the mean incidence of SM cases in the Stockholm region was 0.77/100,000 people per year. In this series, tryptase levels were below 20 ng/mL in 51 patients (26%). Osteoporosis was present in 21.9% of all cases. Interestingly, there was no progression from ISM to advanced SM variants in our study. Furthermore, overall survival was significantly better in ISM patients compared to advanced SM patients (p < 0.0001). Our data suggest that the early recognition and correct diagnosis of SM has prognostic significance.

Altered innate immune profile in blood of systemic mastocytosis patients.

BackgroundMast 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.MethodsOverall, 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.ResultsSM 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.ConclusionThese 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.

Comprehensive Analysis of Acquired Genetic Variants and Their Prognostic Impact in Systemic Mastocytosis.

Simple SummarySystemic mastocytosis (SM) is a clonal haematopoietic stem cell disease typically characterized by the expansion and accumulation of neoplastic mast cells carrying the activating KIT D816V as a driver mutation. Multilineage involvement of haematopoiesis by this KIT mutation, particularly in a multi-mutated context, also involving other genes (e.g., SRSF2, ASXL1, DNMT3A, RUNX1, EZH2, CBL and NRAS) found to be frequently mutated in other myeloid neoplasms, have recently emerged as a genetic background associated with malignant transformation of SM. Therefore, assessment of multilineage involvement of haematopoiesis by KIT D816V and additional mutations in genes known to be associated with the prognosis of SM have become of great help to identify good vs. poor-prognosis SM patients who could benefit from a closer follow-up and, eventually, also early cytoreductive treatment.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.