A deep intronic splice mutation of STAT3 underlies hyper IgE syndrome by negative dominance.

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients' primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

A recessive form of hyper-IgE syndrome by disruption of ZNF341-dependent STAT3 transcription and activity.

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

Masitinib for treatment of severely symptomatic indolent systemic mastocytosis: a randomised, placebo-controlled, phase 3 study.

BACKGROUND: Indolent systemic mastocytosis, including the subvariant of smouldering systemic mastocytosis, is a lifelong condition associated with reduced quality of life. Masitinib inhibits KIT and LYN kinases that are involved in indolent systemic mastocytosis pathogenesis. We aimed to assess safety and efficacy of masitinib versus placebo in severely symptomatic patients who were unresponsive to optimal symptomatic treatments. METHODS: In this randomised, double-blind, placebo-controlled, phase 3 study, we enrolled adults (aged 18-75 years) with indolent or smouldering systemic mastocytosis, according to WHO classification or documented mastocytosis based on histological criteria, at 50 centres in 15 countries. We excluded patients with cutaneous or non-severe systemic mastocytosis after a protocol amendment. Patients were centrally randomised (1:1) to receive either oral masitinib (6 mg/kg per day over 24 weeks with possible extension) or matched placebo with minimisation according to severe symptoms. The primary endpoint was cumulative response (≥75% improvement from baseline within weeks 8-24) in at least one severe baseline symptom from the following: pruritus score of 9 or more, eight or more flushes per week, Hamilton Rating Scale for Depression of 19 or more, or Fatigue Impact Scale of 75 or more. We assessed treatment effect using repeated measures methodology for rare diseases via the generalised estimating equation model in a modified intention-to-treat population, including all participants assigned to treatment minus those who withdrew due to a non-treatment-related cause. We assessed safety in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT00814073. FINDINGS: Between Feb 19, 2009, and July 15, 2015, 135 patients were randomly assigned to masitinib (n=71) or placebo (n=64). By 24 weeks, masitinib was associated with a cumulative response of 18·7% in the primary endpoint (122·6 responses of 656·5 possible responses [weighted generalised estimating equation]) compared with 7·4% for placebo (48·9 of 656·5; difference 11·3%; odds ratio 3·6; 95% CI 1·2-10·8; p=0·0076). Frequent severe adverse events (>4% difference from placebo) were diarrhoea (eight [11%] of 70 in the masitinib group vs one [2%] of 63 in the placebo group), rash (four [6%] vs none), and asthenia (four [6%] vs one [2%]). The most frequent serious adverse events were diarrhoea (three patients [4%] vs one [2%]) and urticaria (two [3%] vs none), and no life-threatening toxicities occurred. One patient in the placebo group died (unrelated to study treatment). INTERPRETATION: These study findings indicate that masitinib is an effective and well tolerated agent for the treatment of severely symptomatic indolent or smouldering systemic mastocytosis. FUNDING: AB Science (Paris, France).

Clinical potential of midostaurin in advanced systemic mastocytosis.

Advanced (Ad) systemic mastocytoses (SM) include aggressive SM (ASM) and mast cell leukemia (MCL) with or without an associated clonal hematological non-mast cell lineage disease (AHNMD). They are rare (<15%) but are associated with a poor prognosis due to rapid organ dysfunction. To date, responses to high-dose chemotherapy, cladribine, and imatinib were revealed to be suboptimal with a median survival time of 24 months. Midostaurin is a potent multikinase inhibitor including the most frequent KIT D816V mutation (>80%). We herein present a review of the most recent data of the use of midostaurin in AdSM. First, a multicenter Phase II study (CPKC412D2213) revealed an unprecedented overall response rate (ORR) of 69% regardless of KIT mutational status, with 38% of major response (MR) among 26 AdSM patients treated with midostaurin alone 200 mg daily. Second, a sponsor-initiated, multicenter, single-arm open Phase II study (CPKC412D2201) confirmed a high and durable ORR of 60% including 45% of MR among 89 AdSM patients. Finally, a French compassionate use program managed by the French Reference Centre for Mastocytosis allowed the treatment of almost a hundred AdSM patients to date in France since the CPKC412D2201 study closure. The outcome of the first 28 treated patients under cover of this on-going procedure revealed an ORR of 71% including 57% of MR. Most importantly, survival analysis revealed in comparison to a historical control cohort of AdSM patients who did not receive midostaurin a twofold lower risk of death (p=0.02) in midostaurin-treated patients. Side effects revealed were acceptable and manageable (mostly digestive). Midostaurin appears to be an effective and safe treatment of AdSM. However, its effect on the course of the AHNMD is less clear. For the future, combined therapy (hypomethylating agents, cladribine, mammalian target of rapamycin inhibitors, chemotherapy, and allogeneic bone marrow transplantation) may further improve long-term survival, particularly that of MCL and AdSM patients with AHNMD.

Mast cell sarcoma: new cases and literature review.

Mast cell sarcoma (MCS) is a rare form of mastocytosis characterized by the presence of solid tumor(s) comprising malignant mast cells that harbor destructive infiltration capability and metastatic potential. Here, we present an extensive literature review and report on 23 cases of MCS, including 3 new cases from the French National Reference Center for Mastocytosis. From our analysis, it appears that MCS can occur at any age. It can manifest de novo or, to a lesser extent, may evolve from a previously established mastocytosis. Bone tumor is a frequent manifestation, and symptoms of mast cell activation are rare. Histological diagnosis can be difficult because MCS is frequently composed of highly atypical neoplastic mast cells and can thus mimic other tumors. Unexpectedly, the canonical KIT D816V mutation is found in only 21% of MCS; therefore, complete KIT gene sequencing is required. The prognosis of patients with MCS is poor, with a median survival time of less than 18 months, and progression to mast cell leukemia is not unusual. Because conventional chemotherapies usually fail, the role of targeted therapies and bone marrow transplantation warrants further investigation in such aggressive neoplasms.

Mastocytosis among elderly patients: A multicenter retrospective French study on 53 patients.

Mastocytosis is a heterogeneous group of diseases with a young median age at diagnosis. Usually indolent and self-limited in childhood, the disease can exhibit aggressive progression in mid-adulthood. Our objectives were to describe the characteristics of the disease when diagnosed among elderly patients, for which rare data are available.The French Reference Center conducted a retrospective multicenter study on 53 patients with mastocytosis >69 years of age, to describe their clinical, biological, and genetic features.The median age of our cohort of patients was 75 years. Mastocytosis variants included were cutaneous (n = 1), indolent systemic (n = 5), aggressive systemic (n = 11), associated with a hematological non-mast cell disease (n = 34), and mast cell leukemia (n = 2). Clinical manifestations were predominantly mast cell activation symptoms (75.5%), poor performance status (50.9%), hepatosplenomegaly (50.9%), skin involvement (49.1%), osteoporosis (47.2%), and portal hypertension and ascites (26.4%). The main biological features were anemia (79.2%), thrombocytopenia (50.9%), leucopenia (20.8%), and liver enzyme abnormalities (32.1%). Of the 40 patients tested, 34 (85%), 2 (5%), and 4 (10%) exhibited the KIT D816V mutant, other KIT mutations and the wild-type form of the KIT gene, respectively. Additional sequencing detected significant genetic defects in 17 of 26 (65.3%) of the patients with associated hematological non-mast cell disease, including TET2, SRSF2, IDH2, and ASLX1 mutations. Death occurred in 19 (35.8%) patients, within a median delay of 9 months, despite the different treatment options available.Mastocytosis among elderly patients has a challenging early detection, rare skin involvement, and/or limited skin disease; it is heterogeneous and has often an aggressive presentation with nonfortuitous associated myeloid lineage malignant clones, and thus a poor overall prognosis.

Telangiectasia macularis eruptiva perstans (TMEP): A form of cutaneous mastocytosis with potential systemic involvement.

BACKGROUND: Telangiectasia macularis eruptiva perstans (TMEP) has not been fully characterized. OBJECTIVE: We sought to estimate the frequency and clinical characteristics of TMEP in a cohort of adult patients with cutaneous mastocytosis, and to assess the presence of systemic involvement. METHODS: We included all consecutive patients evaluated for cutaneous mastocytosis in 2 centers: the Mastocytosis Competence Center of the Midi-Pyrénées from May 2006 to December 2013, and the French Reference Center for Mastocytosis from January 2008 to September 2013. Skin phenotype, histopathology, presence of KIT mutation in the skin, and assessment of systemic involvement according to World Health Organization (WHO) criteria were prospectively investigated. RESULTS: Of 243 patients with cutaneous mastocytosis, 34 (14%) were given a diagnosis of TMEP. The diagnosis of systemic mastocytosis was established in 16 patients (47%) with TMEP. Three patients (9%) had aggressive systemic mastocytosis (C-findings according to WHO). In all, 32 patients (94%) exhibited at least 1 mast cell activation-related symptom. LIMITATIONS: Patient recruitment was undertaken at 2 referral centers with expertise in the diagnosis and treatment of mastocytosis so that the clinical findings and incidence of systemic involvement may be overestimated in comparison with the overall population of patients with TMEP. CONCLUSION: TMEP accounts for about 14% of patients with cutaneous mastocytosis. The disease manifests as mast cell activation symptoms in almost all patients and can be associated with systemic involvement in about 50% of cases.

A French National Survey on Clotting Disorders in Mastocytosis.

Mastocytosis is characterized by a clonal mast cell proliferation with organ infiltration and uncontrolled degranulation. Although not characteristic and poorly explained, some patients develop clotting abnormalities. We retrospectively identified patients with established diagnosis of mastocytosis and related clotting abnormalities (clinical and/or biological) using the national French Reference Centre for Mastocytosis database. From our cohort of 14 adult patients with clotting abnormalities (median age 46 years [range 26-75]), 4 had a presentation suggestive of a primary hemostasis disorder alone (by their symptoms and/or abnormal clotting tests [PFA, von Willebrand's disease [vWD] screening]) and 10 had a laboratory impairment of secondary hemostasis. Among these, 7 had bleeds characteristic of a coagulation cascade disorder (severe/life-threatening in 5 and mild in 2 patients). Clotting abnormalities were of variable severity, typically related to intense crisis of degranulation, such as anaphylactic reactions, and/or to severe organ infiltration by mast cells. Importantly, classical hemostatic management with platelet transfusion, fresh frozen plasma, or vitamin K infusions was unsuccessful, as opposed to the use of agents inhibiting mast cell activity, particularly steroids. This illustrates the crucial role of mast cell mediators such as tryptase and heparin, which interfere both with primary (mainly via inhibition of von Willebrand factor) and secondary hemostasis. There was interestingly an unusually high number of aggressive mastocytosis (particularly mast cell leukemia) and increased mortality in the group with secondary hemostasis disorders (n = 5, 36% of the whole cohort). Mast cell degranulation and/or high tumoral burden induce both specific biologic antiaggregant and anticoagulant states with a wide clinical spectrum ranging from asymptomatic to life-threatening bleeds. Hemostatic control is achieved by mast cell inhibitors such as steroids.

Long-term efficacy and safety of cladribine (2-CdA) in adult patients with mastocytosis.

Mastocytosis (M) is a clonal myeloid-disabling disorder for which no curative therapy is currently available. Cladribine (2-chlorodeoxyadenosine [2-CdA]) is a synthetic purine analog cytoreductive treatment, for which efficacy is mostly reported in advanced M. Here we report, with a long-term follow-up period (>10 years) efficacy and safety in 68 adult patients with M (36 [53%] had indolent M and 32 [47%] had advanced M) treated by 2-CdA (0.14 mg/kg in infusion or subcutaneously, days 1-5; repeated at 4-12 weeks until 1 to 9 courses). Median 2-CdA courses number was 3.7 (1-9). The overall response rate was 72% (complete remission [R]/major/partial R: 0%/47%/25%) and according to indolent/advanced M was 92% (major/partial R: 56%/36%) and 50% (major/partial R: 37.5%/12.5%), respectively. Clinical improvement was observed for 10 of 11 mediator release and 6 of 7 mast cell infiltration-related symptoms including urticaria pigmentosa and organomegaly (P < .02). Serum tryptase levels decreased (P = .01). Median durations of response were 3.71 (0.1-8) and 2.47 (0.5-8.6) years for indolent and aggressive M, respectively. The most frequent grade 3/4 toxicities were lymphopenia (82%), neutropenia (47%), and opportunistic infections (13%). 2-CdA appears to provide a significant efficacy with some toxicity in various M subtypes, mostly in indolent M, refractory to multiple symptomatic therapies.

Imported African histoplasmosis in an immunocompetent patient 40 years after staying in a disease-endemic area.

Histoplasmosis caused by Histoplasma capsulatum var. duboisii is a rare disease outside central and western Africa. In Europe, all cases are imported. We report a case of an African histoplasmosis with isolated pulmonary involvement in a non-immunocompromised patient that occurred 40 years after his stay in a disease-endemic area. The patient was given itraconazole. (18)F-fluoro-2-deoxy-d-glucose positron emission tomography-computed tomography was used to assess evolution during treatment. The outcome for the patient was favorable.

Association of mastocytosis with inflammatory joint diseases: a series of 31 patients.

OBJECTIVES: We studied the clinical phenotypes and tolerance to treatments in a series of patients affected by both inflammatory joint diseases and mastocytosis. METHODS: This retrospective multicenter study was conducted on behalf of 3 networks focused on mastocytosis, pediatric, and adults' inflammatory joint diseases. Patients who displayed both mastocytosis and inflammatory joint diseases were included. RESULTS: A total of 31 patients were included. They had spondyloarthritis (SpA) (16 patients), rheumatoid arthritis (6 patients), juvenile idiopathic arthritis (2 patients), and undifferentiated arthritis (7 patients). The median ages at diagnosis of arthritis and mastocytosis were 44 and 40.5 years, respectively. Disease-modifying anti-rheumatic drugs (DMARDs) were required in 22 patients, comprising mostly methotrexate (13 patients), salazopyrin (8 patients), anti-tumor-necrosis-factor agents (7 patients), and corticosteroids (9 patients). They were well tolerated. Adverse events occurred in 2/24 patients receiving non-steroidal anti-inflammatory drugs. The prevalence of SpA among the 600 patients included in the mastocytosis cohort was 2.33%, which is significantly higher than the prevalence of SpA in the French population (p < 0.001). CONCLUSIONS: This study suggests that mastocytosis is associated with a higher prevalence of SpA than expected, and that DMARDs, notably anti-TNFα agents, are well tolerated in patients with mastocytosis. Mast cells might be involved in the development of SpA.

Peripheral blood 8 colour flow cytometry monitoring of hairy cell leukaemia allows detection of high-risk patients.

Although purine analogues have significantly improved the outcome of hairy cell leukaemia (HCL) patients, 30-40% relapse, illustrating the need for minimal residual disease (MRD) markers that can aid personalized therapeutic management. Diagnostic samples from 34 HCL patients were used to design an 8-colour flow cytometry (8-FC) tube for blood MRD (B/RD) analysis (188 samples) which was compared to quantitative IGH polymerase chain reaction (Q-PCR) on 83 samples and to qualitative consensus IGH PCR clonality analysis on 165 samples. Despite heterogeneous HCL phenotypes at diagnosis, discrimination from normal B lymphocytes was possible in all cases using a single 8-FC tube, with a robust sensitivity of detection of 10(-4) , comparable to Q-PCR at this level, but preferable in terms of informativeness, simplicity and cost. B/RD assessment of 15 patients achieving haematological complete remission after purine analogues was predictive of a clinically significant relapse risk: with a median follow-up of 95 months; only one of the nine patients with reproducible 8-FC B/RD levels below 10(-4) (B/RD(neg) ) relapsed, compared to 5/6 in the B/RD(pos) group (P = 0.003). These data demonstrate the clinical interest of a robust 8-FC HCL B/RD strategy that could become a surrogate biomarker for therapeutic stratification and new drug assessment, which should be evaluated prospectively.

Liquid chromatography-tandem mass spectrometry assay for therapeutic drug monitoring of the tyrosine kinase inhibitor, midostaurin, in plasma from patients with advanced systemic mastocytosis.

We developed and validated quantitative bioanalytical liquid chromatography-tandem mass spectrometry assay for the protein kinase inhibitor, midostaurin. Plasma samples were pre-treated using a protein precipitation with methanol containing midostaurin-d5 as an internal standard. After centrifugation, 5µL of the supernatant was injected into the chromatographic system. The system consisted of a 3.5µm particle bonded octadecyl silica column, with gradient elution using a mixture of 0.1% (v/v) formic acid in acetonitrile and 10mM ammonium formate in water with 0.1% formic acid. The analyte was quantified using the selected reaction-monitoring mode of a triple quadrupole mass spectrometer equipped with a heated electrospray interface. The assay was validated in a 75-2500ng/mL calibration range. For quality control, within-day and between-day precisions were 1.2-2.8%, and 1.2-6.9%, respectively. The ß-expectation tolerance limit (accuracy) met the limits of acceptance ±15% (±20% for the LLQ). The drug was sufficiently stable under all relevant analytical conditions. The assay has successfully been used to assess drug levels for therapeutic drug monitoring in patients presenting advanced systemic mastocytosis and treated with the promising midostaurin.

Gastrointestinal manifestations in mastocytosis: a study of 83 patients.

BACKGROUND: Mastocytosis is a heterogeneous disease characterized by mast cell accumulation in 1 or more organs. Gastrointestinal manifestations of systemic mastocytosis have been previously studied in small cohorts of patients, and no specific histologic description is available. OBJECTIVE: We sought to assess the clinical and pathologic features of gastrointestinal manifestations in patients with mastocytosis. METHODS: Medical history and gastrointestinal symptoms of patients with mastocytosis (n = 83) were compared with those of matched healthy subjects (n = 83) by means of patient questionnaire. Data were analyzed for epidemiologic, clinical, biological, and genetic factors associated with gastrointestinal symptoms for patients with mastocytosis. A comparative analysis of gastrointestinal histology from patients with mastocytosis (n = 23), control subjects with inflammatory bowel disease (n = 17), and healthy subjects (n = 19) was performed. RESULTS: The following gastrointestinal symptoms occurred more frequently and were more severe in patients with mastocytosis than in healthy subjects: bloating (33% vs 7.2%, P < .0001), abdominal pain (27.3% vs 4.8%, P < .0001), nausea (23% vs 8.4%, P = .02), and diarrhea (33.85% vs 1.2%, P < .0001). Patients with mastocytosis had a significantly higher incidence of personal history of duodenal ulcer (P = .02). Wild-type (WT) c-Kit was associated with diarrhea (P = .03). Specific histologic lesions were present in patients with mastocytosis but were not correlated with clinical symptoms. CONCLUSION: Gastrointestinal manifestations in patients with mastocytosis are highly prevalent and often severe. Clinical symptoms do not correspond to histologic findings, are nonspecific, and can simulate irritable bowel syndrome.

Mast cell leukemia.

Mast cell leukemia (MCL) is a very rare form of aggressive systemic mastocytosis accounting for < 1% of all mastocytosis. It may appear de novo or secondary to previous mastocytosis and shares more clinicopathologic aspects with systemic mastocytosis than with acute myeloid leukemia. Symptoms of mast cell activation-involvement of the liver, spleen, peritoneum, bones, and marrow-are frequent. Diagnosis is based on the presence of ≥ 20% atypical mast cells in the marrow or ≥ 10% in the blood; however, an aleukemic variant is frequently encountered in which the number of circulating mast cells is < 10%. The common phenotypic features of pathologic mast cells encountered in most forms of mastocytosis are unreliable in MCL. Unexpectedly, non-KIT D816V mutations are frequent and therefore, complete gene sequencing is necessary. Therapy usually fails and the median survival time is < 6 months. The role of combination therapies and bone marrow transplantation needs further investigation.

Mast Cell Leukaemia: c-KIT Mutations Are Not Always Positive.

Mast cell leukemia (MCL) is a rare and aggressive disease with poor prognosis and short survival time. D816V c-KIT mutation is the most frequent molecular abnormality and plays a crucial role in the pathogenesis and development of the disease. Thus, comprehensive diagnostic investigations and molecular studies should be carefully carried out to facilitate the therapeutic choice. A MCL patient's case with rare phenotypic and genotypic characteristics is described with review of major clinical biological and therapeutic approaches in MCL.