Screening for Hereditary Alpha-Tryptasemia in Subjects with Systemic Mastocytosis (SM) and Non-SM Mast Cell Activation Symptoms

Introduction Hereditary alpha-Tryptasemia (HαT) is a group of genetically defined traits that share increased copy number of TPSAB1 gene encoding for both the α- and β-alleles (Lyons et al 2018). Increased copy number (CN) of the α-tryptase coding sequence in TPSAB1 on one or both alleles represents the genetic base of HαT (Lyons et al 2016). HαT is characterized by mild elevation in serum tryptase levels and a variety of mast cell (MC) activation symptoms, including recurrent anaphylaxis. Prevalence in the general population is up to 5%, that increases up to 20% in systemic mastocytosis (SM); it has been suggested that HαT might be a germline variant predisposing to SM development. In SM, HαT correlated with higher incidence of mediator-related symptoms (Greiner G et al, Blood 2021). Due to its complexity, the assay for TPSAB1 CN is performed in few centers and the actual prevalence of HαT in selected subsets is still to be elucidated. To this end, we screened for HαT 2 groups of subjects, the first with MC activation symptoms and no evidence of SM, the second with diagnosis of SM according to WHO-2016. Methods Droplet digital PCR (ddPCR) was used to measure CN variation (CNV) in TPSAB1 by adapting standard CNV ddPCR protocol to genotype for both TPSAB1 and TPSB2 (Fig.1A). The high homology between α and β encoding isoforms and the presence of paralogous genes in a single locus makes TPSAB1 CNV detection very challenging. ddPCR was performed on genomic DNA with/without BamHI, using the PrimePCR ddPCR Copy Number reference AP3B1 (BioRad). Accuracy and precision of the ddPCR protocol was assessed by analyzing 10 samples in triplicate in 3 separate experiments. Data robustness and repeatability can be appreciated in Fig 1B. Results We studied 41 subjects with mediator-related symptoms and augmented basal serum tryptase (BST) (cohort 1) and 150 patients with ascertained diagnosis of mastocytosis (cohort 2). The BST threshold established for cohort 1 was equal or higher than 11 mcg/L. Median age was 64.7 yr, males 54%; median BST levels was 15.3 (range 12.3-21 mcg/L); 29% of the pts had history of anaphylaxis. In cohort 2, 134/150 (89.3%) pts had a diagnosis of SM, whereas 13/150 (8,6%) were Cutaneous Mastocytosis (CM). Among SM patients, 113(84.3%) presented with non-advanced SM variants. Advanced forms including aggressive SM (ASM) and SM with an associated hematological neoplasm (SM-AHN) were diagnosed in 6 (4.5%) and 8 (6%) respectively. In 3 pts, SM subtype was not available. Median age was 49 yr, males 55%; 41.7% of the pts had history of anaphylaxis. HαT was documented in 27 (65.9%) subjects in cohort 1, and 14 (9.3%) in cohort 2. In cohort 1, 3 α-tryptase (3α) copy number was observed more frequently (59.2% of HαT+ pts); conversely 3α and 2α-tryptase copy number were observed at a similar rate (42,8%) in cohort 2. HαT+ pts in cohort 1 presented significantly higher BST (17.1 vs 12.05 mgc/L, P<0.001), as previously reported (Greiner G et al, Blood 2021); however, occurrence of mediator related symptoms was comparable to HαT wt, 72% vs 71.4% , respectively; likewise for anaphylaxis (28% in HαT+ vs 33%). In cohort 2, BST levels were similar in HαT+ and HαT wt pts (24.6 and 24.3 mcg/L), as were anaphylaxis episodes (50% and 41%, respectively). A trend for lower MC burden in HαT+ as assessed by flow cytometry was demonstrated (% of bone marrow MC: 0.01% in HαT+ vs 0.07%) whereas no meaningful differences emerged regarding the symptom burden. In addition, a lower prevalence of KIT 816V mutation was observed in HαT+ (71.4% vs 89.5%; p=0.073). Conclusions In our study HαT+ was observed in around 10% of patients with SM, a prevalence lower than previously reported (Greiner et al, Blood 2021) and remarkably lower than in a selected cohort of subjects with raised BTL and history of mediator-released symptoms (66%). ddPCR represents a suitable method to investigate the presence of CNV in the α-tryptase coding sequence. Genetic testing for HαT+ should be considered in the diagnostic workout of patients presenting with anaphylaxis or MC mediator-related symptoms and no suspicion/evidence of SM. The clinical correlates of HαT in SM remain to be fully ascertained. Supported by IMH no.GR-2016-02362631 and AIRC, Mynerva project no21267 Figure 1 Figure 1. Disclosures Elena: CELGENE: Other: funding for meeting participation; PFIZER: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; GILEAD: Membership on an entity's Board of Directors or advisory committees. Vannucchi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Cladribine Therapy for Advanced and Indolent Systemic Mastocytosis: Mayo Clinic Experience in 42 Consecutive Cases

Background Systemic mastocytosis (SM) is formally classified as advanced, smoldering (SSM), or indolent (ISM). Advanced SM indicates the presence of an associated non-mast cell lineage hematologic neoplasm (SM-AHN) or at least one "C finding" (i.e. features of organopathy from mast cell infiltration); the latter defines aggressive SM (ASM). Both advanced SM and ISM might be associated with MC mediator symptoms (MC-MSs) or urticaria pigmentosa (UP). Current drug therapy for SM is primarily directed at reversing C findings or alleviating symptoms/UP, and includes cladribine, midostaurin, and avapritinib. The former has a long track-record of safety while the latter two are recent additions that target the associated KIT mutation, but with concerning gastrointestinal and neurological side effects. We retrospectively reviewed our experience with cladribine therapy in SM, in order to maintain a proper perspective in making treatment choices. Methods The current study includes 42 patients with SM (22 advanced and 20 indolent/smoldering), recruited from the Mayo Clinic SM database, based on documentation of treatment with cladribine. Conventional criteria were used for diagnosis and classification (Blood 2016;127:2391) and definitions of response (Eur J Clin Invest. 2007 Jun;37(6):435); in the latter regard, we utilized modified Valent criteria that incorporated a minimum of one-month duration to qualify as response. Results Advanced SM: 22 patients (median age 65 years, range 48-80; males 68%) had advanced SM, including 13 SM-AHN, 8 ASM, and 1 mast cell leukemia. Median (range) BM MC burden was 30% (10%-90%), serum tryptase 193 ng/ml (21-1370), hemoglobin 10.6 g/dL (7-15), leukocytes 6 x 10(9)/L (0.5-63), and platelets 115 x 10(9)/L (8-320). KITD816V was detected in 86% of 19 evaluated and abnormal karyotype in 14% of 21 evaluated. 50% of patients had palpable hepatomegaly, 59% palpable splenomegaly, 100% MC-MSs, and 14% UP; 87% displayed at least one C finding. Median time from BM biopsy confirmed diagnosis to initiation of cladribine therapy was 1.5 months (range 0.1-17). 55% of patients received at least 3 cycles of treatment. Overall response (OR) was documented in 17 (77%) patients with similar OR rates in ASM (88%) vs SM-AHN (69%; p=0.32), with median time to response of 3.7 months (range 0.4-9). OR included 45% major response (MR) and 32% partial response (PR). 17 patients were evaluated for BM MC response with 44% showing >50% reduction, in MCs, including 19% with complete resolution. Organomegaly response was complete in 36% and partial in 27%. Tryptase response of >50% was documented in 32%. Cladribine side effects were limited to cytopenias with 36% showing grade 3 or 4 myeloid cytopenia. Median duration of response was 6 months (range 1-67). Six patients, including 5 with SM-AHN, progressed to either AML or MCL. Among the 5 patients who did not respond to cladribine, one with ASM was successfully treated with midostaurin. Indolent or smoldering SM: 20 patients (median age 56 years, range 36-73; males 45%) had ISM (n=17) or SSM (n=3). Median (range) BM MC burden was 13% (5%-60%) and serum tryptase level 49 ng/ml (14-1630). KITD816V was detected in 95% of 19 evaluated patients and abnormal karyotype in 12% of 17 evaluated. All patients displayed MC-MSs and 46% UP. Median time from BM biopsy confirmed diagnosis to initiation of cladribine therapy was 4 months (range 0-92). 85% of patients received at least 3 cycles of cladribine therapy. OR was documented in 14 (70%) patients, including 60% complete or major regression of symptoms or UP, with median time to maximal response of 8.5 months (range 1-98); response rates in patients with UP were similar with 57% complete or major regression; 10 patients were evaluable for BM MC response with 50% showing >50% reduction in MC burden. Serum tryptase response was evaluated in 13 patients with 46% showing >50% reduction in levels. Treatment-emergent cladribine side effects were limited to cytopenias with only 3 (15%) patients experiencing grade 3 or 4 myeloid cytopenia and (n=3; 15%) and 6 (30%) experiencing grade 3 or 4 lymphopenia. Conclusions: The current study confirms the favorable side effect profile and long-term safety of cladribine as a first-line drug of choice for both indolent and advanced SM. Response to cladribine therapy was evident in all aspects of the disease, including MC-associated organopathy, UP, MC-MSs, serum tryptase level and bone marrow MC burden. Disclosures Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding. Al-Kali: Novartis: Research Funding; Astex: Other: Research support to institution.

Idiopathic anaphylaxis: Diagnosis and management

Introduction: Idiopathic anaphylaxis (IA) is a diagnosis of exclusion and is based on the inability to identify a causal relationship between a trigger and an anaphylactic event, despite a detailed patient history and careful diagnostic assessment. The prevalence of IA among the subset of people who experienced anaphylaxis is challenging to estimate and varies widely, from 10 to 60%; most commonly noted is ∼20% in the adult anaphylactic population. Comorbid atopic conditions, such as food allergy, allergic rhinitis, and asthma, are present in up to 48% of patients with IA. Improved diagnostic technologies and an increased understanding of conditions that manifest with symptoms associated with anaphylaxis have improved the ability to determine a more accurate diagnosis for patients who may have been initially diagnosed with IA. Methods: Literature search was conducted on PubMed, Google Scholar and Embase. Results: Galactose-α-1,3-galactose (α-gal) allergy, mast cell disorders, and hereditary a-tryptasemia are a few differential diagnoses that should be considered in patients with IA. Unlike food allergy, when anaphylaxis occurs within minutes to 2 hours after allergen consumption, α-gal allergy is a 3‐6-hour delayed immunoglobulin E‐mediated anaphylactic reaction to a carbohydrate epitope found in red meat (e.g., beef, lamb, pork). The more recently described hereditary α-tryptasemia is an inherited autosomal dominant genetic trait caused by increased germline copies of tryptase human gene alpha-beta 1 (TPSAB1), which encodes α tryptase and is associated with elevated baseline serum tryptase. Acute management of IA consists of carrying an epinephrine autoinjector to be administered immediately at the first signs of anaphylaxis. Long-term management for IA with antihistamines and other agents aims to potentially reduce the frequency and severity of the anaphylactic reactions, although the evidence is limited. Biologics are potentially steroid-sparing for patients with IA; however, more research on IA therapies is needed. Conclusion: The lack of diagnostic criteria, finite treatment options, and intricacies of making a differential diagnosis make IA challenging for patients and clinicians to manage.

Cutaneous mastocytosis in an infant: Case report and clinicopathological correlation

Mastocytosis is a disorder of clonal proliferation of the mast cells, which can be cutaneous or systemic. Abnormal mast cell infiltration comprising multifocal compact clusters or cohesive aggregates can affect one or more organ systems. Cutaneous mastocytosis is a relatively uncommon condition in the pediatric population. We report a case of 9 month infant presented with multiple papular and vesicular skin rashes since 6 months of age. On clinical examination Darier’s sign was negative. The serum tryptase levels were within normal limits. Clinical differential diagnoses were benign cephalic histiocytosis vs cutaneous mastocytosis. Skin biopsy revealed a mononuclear cell infiltrate in the papillary dermis reaching up to the dermo-epidermal junction. Toluidine blue staining highlighted the metachromatic granules. CD117, CD30 IHC stains were positive which confirmed the diagnosis of cutaneous mastocytosis. This case is presented to highlight the histomorphology and the special stains in cases of mastocytosis.

Utility of serum tryptase in Emergency Department patients with possible anaphylaxis

BACKGROUND Diagnosing anaphylaxis is often straightforward but can be challenging if the presentation is atypical. In patients with atypical symptoms suspected to be due to an acute allergic reaction, s-tryptase can give additional diagnostic information. Measuring s-tryptase is also helpful in diagnosing mastocytosis. Obtaining s-tryptase levels has been done in the emergency department (ED) at Landspitali since 2011. The aim of this study was to evaluate the benefit of obtaining s-tryptase levels in the ED. METHODS With institutional review board approval, all cases where s-tryptase level was obtained in ED patients from 2011 – 2018 were retrospectively reviewed. A database was collected including information on patient demographics, presenting symptoms, treatment, diagnosis, s-tryptase level and follow up. RESULTS A total of 214 patients had a s-tryptase level measured. Serum tryptase was elevated (>12 µg/L) in 36 cases. Females were 131 and average age 40.6 years. Of the patients, 86.4% had skin or mucosal symptoms, 48.1% cardiovascular symptoms, 49.5% respiratory symptoms and 36.0% had gastrointestinal symptoms. An allergist reviewed 126 returning patients and 65 were considered to have had an episode of anaphylaxis. Of those 65 were 4 patients which did not meet the diagnostic criteria for anaphylaxis but had raised s-tryptase levels. Sensitivity of s-tryptase measurement was 40.9% and specificity 97.1%. CONCLUSIONS Obtaining a s-tryptase level from ED patients with possible anaphylaxis seems to be useful in atypical cases. The measurement is specific but not sensitive. No cases of mastocytosis were identified in the patient cohort.

Characterization of a case series of Mastocytosis in a health care center in Cali, Colombia

Mastocytosis is a group of rare diseases, which correspond to neoplasms of the myeloid lineage. In Colombia there are only case reports and so far there are no studies of greater extension. We conducted a case series in which an active search was made for patients with a diagnosis of mastocytosis, either cutaneous (CM) or systemic (SM), from the total number of consultations between June 2004 and June 2019 in the reference hemato-oncologic center ("mastocytosis"). A total of 4 cases of CM and 3 cases of SM were identified. The most frequent clinical manifestations were skin lesions, which were present in 100% of patients; of these hyperpigmented macules were the most frequent findings. Serum tryptase (TS) levels were found to be elevated in 67% (2/3) of patients with DM. Both TS levels and mean absolute eosinophils were higher in patients with MS. In this case series we found a higher frequency of extracutaneous involvement, and in general a very poor response to the management. The findings of this series are comparable to those reported in world literature.

Characteristics and Laboratory Findings of Food-Induced Anaphylaxis in Children: Study in an Asian Developing Country

<b><i>Introduction:</i></b> Food allergy is the major cause of pediatric anaphylaxis. Characteristics and triggers may be different in different geographical regions. Studies focusing on food-induced anaphylaxis (FIA) in Asian developing countries are limited. Our study aimed to study characteristics of FIA in a tertiary care center in an Asian developing country. <b><i>Methods:</i></b> Retrospective review of pediatric anaphylaxis admission and outpatient visit at a tertiary care hospital in Bangkok, Thailand during 2008–2018 was performed. Data regarding clinical presentation, place reaction occurred, time of onset, investigations (serum tryptase, specific immunoglobulin E, and skin test), treatment, and follow-up periods were collected. <b><i>Results:</i></b> One hundred seventy-four anaphylaxis admission records of which 61 episodes of FIA were retrieved. Data from outpatients visit consisted of 17 patients. Most patients were male (76.7%). The median age was 7.1 years (interquartile range 1.9–12.4). The major causes of FIA were shrimp/shellfish (37%), wheat (15.1%), and cow’s milk (11%). Food causing anaphylaxis varied according to age-group: infants had anaphylactic reactions to egg, wheat, and cow’s milk, preschools to wheat and peanut, and older children to shrimp/shellfish. Cutaneous manifestations occurred in all patients, followed by lower respiratory tract symptoms (83.6%) and gastrointestinal symptoms (50.8%). There was no biphasic anaphylaxis reported. Elevated serum tryptase was found in only 4 patients (7%). <b><i>Conclusion:</i></b> Recognizing characteristics of pediatric FIA is crucial. The common causes of FIA in our study in Asian children were egg in infants, wheat and peanut in preschool children, and shrimp/shellfish in school-age children and adolescents. Skin manifestation presented in all patients with FIA.

Association of Elevated Serum Tryptase with Cutaneous Photodamage and Skin Cancers

<b><i>Introduction:</i></b> Mast cells and their major protein, the serine proteinase tryptase, can be involved in cutaneous photodamage and carcinogenesis. The serum test of tryptase (S-tryptase) measures total tryptase protein (active tryptase and inactive protryptases), and S-tryptase is elevated in a variety of diseases, for example, in mastocytosis and α-tryptasemia. <b><i>Objectives:</i></b> The objective of this study is to study whether S-tryptase is a marker of cutaneous photodamage and carcinogenesis. <b><i>Methods:</i></b> Adult subjects (<i>n</i> = 399, aged 21–79) evaluated to be at risk for skin cancers were recruited at the dermatological policlinic and examined for photodamage severity, mole count, actinic keratoses (AKs), skin cancers, and immunosuppression (IS). A blood sample was analyzed for S-tryptase using the ImmunoCAP® Tryptase fluoroenzymeimmunoassay. <b><i>Results:</i></b> There was no difference in S-tryptase between non-IS (<i>n</i> = 321) and IS (<i>n</i> = 78) subjects or between genders. S-tryptase correlated slightly to photodamage and AKs in 321 non-IS subjects, and this association can be related, in part, to the age of subjects. In 34 subjects, S-tryptase was elevated (≥13.5 ng/mL), and in 20 males, but not in 14 females, the photodamage level was significantly (<i>p</i> = 0.031) more severe than in 179 males with normal S-tryptase. In contrast, there were more frequently subjects (<i>n</i> = 12) with past or present skin cancer (basal or squamous cell carcinoma or melanoma) in 14 females with elevated S-tryptase than in 186 female controls. So far, no explanation has been found for the elevated S-tryptase. <b><i>Conclusion:</i></b> There are significant associations between elevated S-tryptase and skin carcinogenesis, but the molecular mechanisms are unclear and gender differences can exist.