Thomas Kielsgaard Kristensen

Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay.

The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation in peripheral blood in 81% of the mutation-positive cases with SM. The method also allows comparison of mutation-positive and mast cell fractions to determine whether the mutation is present in non-mast cells, a parameter that has recently been reported to be of prognostic importance in patients with indolent SM. Finally, the assay is suitable for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM.

Cutaneous manifestations in patients with mastocytosis: Consensus report of the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology.

Cutaneous lesions in patients with mastocytosis are highly heterogeneous and encompass localized and disseminated forms. Although a classification and criteria for cutaneous mastocytosis (CM) have been proposed, there remains a need to better define subforms of cutaneous manifestations in patients with mastocytosis. To address this unmet need, an international task force involving experts from different organizations (including the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology) met several times between 2010 and 2014 to discuss the classification and criteria for diagnosis of cutaneous manifestations in patients with mastocytosis. This article provides the major outcomes of these meetings and a proposal for a revised definition and criteria. In particular, we recommend that the typical maculopapular cutaneous lesions (urticaria pigmentosa) should be subdivided into 2 variants, namely a monomorphic variant with small maculopapular lesions, which is typically seen in adult patients, and a polymorphic variant with larger lesions of variable size and shape, which is typically seen in pediatric patients. Clinical observations suggest that the monomorphic variant, if it develops in children, often persists into adulthood, whereas the polymorphic variant may resolve around puberty. This delineation might have important prognostic implications, and its implementation in diagnostic algorithms and future mastocytosis classifications is recommended. Refinements are also suggested for the diagnostic criteria of CM, removal of telangiectasia macularis eruptiva perstans from the current classification of CM, and removal of the adjunct solitary from the term solitary mastocytoma.

Epidemiology of systemic mastocytosis in Denmark

Mastocytosis is a heterogeneous group of diseases characterized by abnormal proliferation of mast cells. Systemic mastocytosis (SM), in which abnormal mast cells are present in tissues beyond the skin, is divided into seven subcategories with varying degrees of severity and prognosis. Very little is known about the epidemiology of SM and its subcategories. This retrospective cohort study of 548 adults with SM diagnosed 1997–2010 was constructed using linked Danish national health registries. The most common subtype of mastocytosis was indolent SM (including urticaria pigmentosa) (n = 450; 82%), followed by SM with subtype unknown (n = 61; 11%), SM with associated clonal haematological non‐mast cell lineage disease (n = 24; 4%), aggressive SM (n = 8; 2%), and mast cell leukaemia (n = 5; 1%). The incidence rate for SM (all subtypes including urticaria pigmentosa) was 0·89 per 100 000 per year. Cumulative incidence was 12·46 per 100 000, and the 14‐year limited‐duration prevalence as of 1 January, 2011 was 9·59 per 100 000. This nationwide cohort from Denmark is the first population‐based epidemiological study of mastocytosis. In this cohort of patients aged 15 years and older, SM was found to be overall relatively rare with notable variation by subtype for patient characteristics, survival and epidemiological measures.

Sensitive KIT D816V mutation analysis of blood as a diagnostic test in mastocytosis

The recent progress in sensitive KIT D816V mutation analysis suggests that mutation analysis of peripheral blood (PB) represents a promising diagnostic test in mastocytosis. However, there is a need for systematic assessment of the analytical sensitivity and specificity of the approach in order to establish its value in clinical use. We therefore evaluated sensitive KIT D816V mutation analysis of PB as a diagnostic test in an entire case‐series of adults with mastocytosis. We demonstrate for the first time that by using a sufficiently sensitive KIT D816V mutation analysis, it is possible to detect the mutation in PB in nearly all adult mastocytosis patients. The mutation was detected in PB in 78 of 83 systemic mastocytosis (94%) and 3 of 4 cutaneous mastocytosis patients (75%). The test was 100% specific as determined by analysis of clinically relevant control patients who all tested negative. Mutation analysis of PB was significantly more sensitive than serum tryptase >20 ng/mL. Of 27 patients with low tryptase, 26 tested mutation positive (96%). The test is furthermore readily available and we consider the results to serve as a foundation of experimental evidence to support the inclusion of the test in diagnostic algorithms and clinical practice in mastocytosis. Am. J. Hematol. 89:493–498, 2014.

Circulating KITD816V mutation-positive non-mast cells in peripheral blood are characteristic of indolent systemic mastocytosis

It is presently accepted that the KIT D816V mutation is detectable in tissues with neoplastic mast cells in most patients with indolent systemic mastocytosis. In this study, neoplastic mast cells were detected in bone marrow, but not in peripheral blood, by flow cytometry in all 25 included cases of indolent systemic mastocytosis. However, the KIT D816V mutation was detected using mutation‐specific qPCR in both bone marrow and peripheral blood in all 25 cases, demonstrating for the first time that the KIT D816V mutation is consistently present in non‐mast cells in indolent systemic mastocytosis and that these cells are circulating in peripheral blood.

NPM1 mutation is a stable marker for minimal residual disease monitoring in acute myeloid leukaemia patients with increased sensitivity compared to WT1 expression.

Mutation in the NPM1 gene occurs in 60% of acute myeloid leukaemia (AML) patients with normal karyotype. NPM1 mutation is potentially a superior minimal residual disease (MRD) marker compared to WT1 gene overexpression by being specific to the malignant clone, although experimental evidence published so far includes very limited numbers of relapsed cases. Also, the stability of the NPM1 mutation has been questioned by reports of the mutation being lost at relapse. In the present study we compared NPM1 mutation and WT1 overexpression as MRD markers in 20 cases of relapsed AML. The 20 patients experienced a total of 28 morphological relapses. Karyotypic evolution was detected in 56% of relapses. All relapses were accompanied by high levels of NPM1 mutation, along with high WT1 mRNA levels, thus demonstrating complete stability of both markers during relapse. Detectable NPM1 mutation following a period of morphological remission was accompanied by a morphological relapse in all cases. In contrast, WT1 expression was detected in 33% of the NPM1 mutation negative samples. This background WT1 expression produced by non‐leukaemia cells was highly variable, both between and within patients, and limited the de facto sensitivity of the WT1 expression analysis. The present study therefore provides important experimental evidence demonstrating that NPM1 mutation is superior to WT1 overexpression as marker of MRD in NPM1‐mutated AML, even in the presence of extensive karyotypic evolution.

Anaphylaxis caused by mosquito allergy in systemic mastocytosis

In the summer of 1996, a 56-year-old man was bitten on his arm by a mosquito in his garden in Bavaria, Germany. About 15 min later he had diarrhoea, felt nauseous, and lost consciousness. He was bitten again by a mosquito in May, 2001, and August, 2001. Following the bite in August, 2001, the reaction developed rapidly, and he immediately lost consciousness and went into cardiac arrest before the ambulance arrived. Because of delayed resuscitation, he had hypoxic brain damage to the basal ganglia, resulting in spastic tetraplegia. Red-brown maculo papular skin lesions were seen on his upper legs and a skin biopsy showed increased mast cell numbers. In 2006, he was bitten a fourth time by a mosquito. Despite immediate adminis tration of rescue medi cation consisting of epinephrine, H1-antihistamine, and corticosteroid, he again had a severe reaction and cardiac arrest. In 2012, the patient was referred to the depart ment of dermatology and allergy, Charite—Universitatsmedizin Berlin, Germany, for further investigation. On the basis of the history of maculopopular skin lesions and increased mast cell numbers on skin biopsy, we suspected systemic mastocytosis. WHO diagnostic criteria for systemic mastocytosis were confi rmed. Despite having only slightly raised serum tryptase of 11·5 μg/L (normal range <11·4 μg/L), bone marrow exam ination showed spindle shaped mast cells expressing CD25, and the typical Kit-mutation (D816V) was detected by PCR of peripheral blood leucocytes. From the pat ient’s description of the appearance of the mosquitoes that bit him, and knowledge of the geographic region where the incidents occurred, Culex pipiens was identifi ed by an expert from the Bernhard Nocht Institute, Hamburg, Germany, as the most likely of the 100 known mosquito species in central Europe to be responsible for inducing such reactions. As an alternative possibility, the patient’s skin prick test reaction and basophil release in response to Aedes communis, another common European species, was also tested. Total serum IgE of 48·4 kU/L was within normal range (<100 kU/L). ImmunoCAP (ThermoFisher, Uppsala, Sweden) failed to detect mosquito-specifi c IgE. Measurement of serum Culex pipiens-specifi c IgE by indirect ELISA showed no diff erence between the patient and four healthy controls (patient 0·067 optical density; controls 0·049, 0·05, 0·106 and 0·082; blank 0·062). To test for functional reactivity to mosquito allergens, skin prick testing was done on the patient and in healthy controls. Additionally, the patient showed a very strong basophil activation response to Culex pipiens and a weaker response to Aedes communis (appendix). Previous studies have shown that desensitisation treatment of individuals with strong local immediate or delayed reactions to mosquito bites are eff ective and safe. However we did not consider immunotherapy as a therapeutic option in this patient because of his un predictable and severe reactions to mosquito bites. Instead he is taking desloratadine 5 mg daily prophylactically and is meticulously avoiding mosquitoes. He is also equipped with emergency medication including epinephrine, H1-antihistamine, and cortico steroid. The patient was instructed to carry two epinephrine autoinjectors at all times. We have also off ered him prophylactic treatment with omalizumab during the mosquito season (usually April–October). Omalizumab had a protective eff ect against severe anaphylactic reactions in a masto cytosis patient who had had several near-fatal anaphylactic reactions to bee stings. Anaphylaxis to mosquito bite has been previously reported but at present remains unexplained. The unique nature of this case of a grade IV allergic reaction to mosquito bites is that a clonal mast cell disorder was identifi ed as concomitant disease without remark able increase in serum tryptase. Unidentifi ed mosquito allergy could be an underestimated cause of anaphylaxis, especially in combination with occult systemic mastocytosis.

KIT D816V mutation burden does not correlate to clinical manifestations of indolent systemic mastocytosis

BACKGROUND Clinical manifestations of indolent systemic mastocytosis (ISM) comprise mediator-related symptoms, anaphylaxis, and osteoporosis. A new sensitive method for KIT D816V mutation detection allows quantification of the level of mutation-positive cells. OBJECTIVE To investigate whether the fraction of KIT D816V positive cells in peripheral blood (PB) or bone marrow (BM) aspirate in adult patients with ISM correlates with clinical manifestations of the disease. METHODS We included 48 adult patients with ISM (28 females/20 males) from our center in whom the KIT D816V mutation level in both BM aspirate and PB was analyzed. For each patient, the severity of mediator-related symptoms (skin, gastrointestinal, musculoskeletal, and neuropsychiatric) and episodes of anaphylaxis were evaluated by interview and medical record files. Bone mineral density was determined by using dual-energy x-ray absorptiometry. RESULTS Median fraction (range) of KIT D816V positive cells was 0.6 (0.01%-90%) in BM and 0.3 (0.003%-49%) in PB. Mutation level did not differ between patients with none/mild symptoms and patients with moderate/severe symptoms, patients with and without anaphylaxis, or patients with osteoporosis/osteopenia and normal bone mineral density. No significant differences in clinical profile were detected in patients with different levels of mutation except for an indication of longer disease duration and age in patients with highest mutation levels. CONCLUSION To our knowledge, this is the first report on the clinical impact of the fraction of KIT D816V mutation positive cells in ISM, which in the present study does not seem to correlate with clinical manifestations of the disease.

Association of inclusion body myositis with T cell large granular lymphocytic leukaemia

SEE HOHLFELD AND SCHULZE-KOOPS DOI101093/BRAIN/AWW053 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Inclusion body myositis and T cell large granular lymphocytic leukaemia are rare diseases involving pathogenic cytotoxic CD8+ T cells. After encountering four patients with both disorders, we prospectively screened 38 patients with inclusion body myositis for the presence of expanded large granular lymphocyte populations by standard clinical laboratory methods (flow cytometry, examination of blood smears, and T cell receptor gene rearrangements), and performed muscle immunohistochemistry for CD8, CD57, and TIA1. Most (22/38; 58%) patients with inclusion body myositis had aberrant populations of large granular lymphocytes in their blood meeting standard diagnostic criteria for T cell large granular lymphocytic leukaemia. These T cell populations were clonal in 20/20 patients and stably present on follow-up testing in 15 patients a median of 350 days later. T cell aberrant loss of CD5 or gain of expression of CD16 and CD94 were common (19/42, 45%). In comparison, 2/15 (14%) age-matched patients with dermatomyositis, polymyositis, or necrotizing myopathy, and 0/20 (0%) age-matched healthy subjects had large granular lymphocyte expansions, with none of these patients having T cell aberrant expression of CD5, CD16 or CD94. Reduced blood CD4/CD8 ratio, increased blood CD8 count, and lymphocytosis were additional biomarkers highly correlated with flow cytometry-measured large granular lymphocyte expansions. Cross-sectional data suggested more aggressive disease in patients with such expansions than without. Muscle immunohistochemistry demonstrated invasion of large granular lymphocytes into muscle in 15/15 inclusion body myositis patients but in only 1/28 patients with dermatomyositis or polymyositis. The extent of CD8+ and CD57+ cells in inclusion body myositis muscle correlated with the size of blood large granular lymphocyte populations. Myofibre-invading cells expressed CD57, a marker of persistent T cell exposure to antigen and T cell aggressiveness. In many patients with inclusion body myositis, the autoimmune T cell expansion has evolved into a neoplastic-like or overtly neoplastic disorder, perhaps contributing to its relative refractoriness to immune-directed therapies previously reported.

Adult-onset systemic mastocytosis in monozygotic twins with KIT D816V and JAK2 V617F mutations

To the Editor: We present clinical, histopathologic, and mutational findings and clinical course over 30 years in a pair of monozygotic twins with adult-onset systemic mastocytosis (SM) both carrying a somatic KIT D816Vand Janus kinase 2 (JAK2) V617F mutation. Pregnancy and labor were reportedly uneventful, and the parents were described as healthy. Both twins were healthy during childhood and adolescence. From the age of 38 (twin A) and 40 (twin B) years, they gradually developed reddish-brown, flat skin elements on the femora and thighs spreading within a few years to the abdomen and arms, with intermittent pruritic exacerbations triggered by friction or heat. In both twins, periodic, colicky abdominal pains and diarrhea were described. No anaphylactic episodes were reported. At the age of 50 years, a synchronous evaluation was performed. On objective examination, the twins were identical