Joseph H. Butterfield

Establishment of an immature mast cell line from a patient with mast cell leukemia

A cell line showing many characteristics of immature mast cells has been established from the peripheral blood of a patient with mast cell leukemia. Cultured cells contain low levels of histamine, are stained metachromatically by toluidine blue, and contain chloroacetate esterase, aminocaproate esterase and tryptase activities. The cells lack T and B lymphocyte, as well as myeloid cell markers, and do not possess IgE receptors. Solid tumors of metachromatically positive cells have been successfully induced and serially passed in nude mice using 5-azacytidine transformed cells. This cell line may be useful for future studies of mast cells and their constituents.

Treatment of hypereosinophilic syndrome with imatinib mesilate

Patients with hypereosinophilic syndrome show persistent eosinophilia without recognised cause. We treated five such patients with 100 mg imatinib mesilate (formerly STI-571) daily; four male patients with normal serum interleukin 5 showed complete haematological responses; a female patient who did not respond had raised serum interleukin-5 concentrations. One patient developed leucopenia after 4 days of treatment; counts returned to normal when treatment was stopped. After 1 month, eosinophilia recurred; with further treatment for 2 days, eosinophil counts again became normal. All patients who responded stopped other treatments and reduced imatinib mesilate to 200 mg per week. This drug effectively controls eosinophilia in patients with hypereosinophilic syndrome and normal interleukin-5 concentrations.

Systemic mastocytosis in 342 consecutive adults: survival studies and prognostic factors

Clinical phenotype in systemic mastocytosis (SM) is markedly variable, which complicates prognostication and decision making regarding the choice and timing of therapy. In a retrospective study of 342 consecutive adult patients with SM seen at the Mayo Clinic between 1976 and 2007, disease subdesignation according to the World Health Organization (WHO) proposal was indolent (ISM) in 159 (46%), with associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) in 138 (40%), aggressive (ASM) in 41 (12%), and mast cell leukemia in 4 (1%). KITD816V was detected in bone marrow-derived DNA by allele-specific polymerase chain reaction (PCR) in 68% of 165 patients evaluated (ISM, 78%; ASM, 82%; SM-AHNMD, 60%; P = .03); JAK2V617F was detected in 4%, all in SM-AHNMD. Compared with those with nonindolent SM, life expectancy in ISM was superior and not significantly different from that of the age- and sex-matched US population. In addition, multivariable analysis identified advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess bone marrow blasts as independent adverse prognostic factors for survival. The current study validates the prognostic relevance of the WHO subclassification of SM and provides additional information of value in terms of both risk stratification and interpretation of clinical presentation and laboratory results.

Definitions, Criteria and Global Classification of Mast Cell Disorders with Special Reference to Mast Cell Activation Syndromes: A Consensus Proposal

Activation of tissue mast cells (MCs) and their abnormal growth and accumulation in various organs are typically found in primary MC disorders also referred to as mastocytosis. However, increasing numbers of patients are now being informed that their clinical findings are due to MC activation (MCA) that is neither associated with mastocytosis nor with a defined allergic or inflammatory reaction. In other patients with MCA, MCs appear to be clonal cells, but criteria for diagnosing mastocytosis are not met. A working conference was organized in 2010 with the aim to define criteria for diagnosing MCA and related disorders, and to propose a global unifying classification of all MC disorders and pathologic MC reactions. This classification includes three types of ‘MCA syndromes’ (MCASs), namely primary MCAS, secondary MCAS and idiopathic MCAS. MCA is now defined by robust and generally applicable criteria, including (1) typical clinical symptoms, (2) a substantial transient increase in serum total tryptase level or an increase in other MC-derived mediators, such as histamine or prostaglandin D2, or their urinary metabolites, and (3) a response of clinical symptoms to agents that attenuate the production or activities of MC mediators. These criteria should assist in the identification and diagnosis of patients with MCAS, and in avoiding misdiagnoses or overinterpretation of clinical symptoms in daily practice. Moreover, the MCAS concept should stimulate research in order to identify and exploit new molecular mechanisms and therapeutic targets.

Hypereosinophilic syndrome: A multicenter, retrospective analysis of clinical characteristics and response to therapy

BACKGROUND Hypereosinophilic syndrome (HES) is a heterogeneous group of rare disorders defined by persistent blood eosinophilia > or =1.5 x 10(9)/L, absence of a secondary cause, and evidence of eosinophil-associated pathology. With the exception of a recent multicenter trial of mepolizumab (anti-IL-5 mAb), published therapeutic experience has been restricted to case reports and small case series. OBJECTIVE The purpose of the study was to collect and summarize baseline demographic, clinical, and laboratory characteristics in a large, diverse cohort of patients with HES and to review responses to treatment with conventional and novel therapies. METHODS Clinical and laboratory data from 188 patients with HES, seen between January 2001 and December 2006 at 11 institutions in the United States and Europe, were collected retrospectively by chart review. RESULTS Eighteen of 161 patients (11%) tested were Fip1-like 1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) mutation-positive, and 29 of 168 patients tested (17%) had a demonstrable aberrant or clonal T-cell population. Corticosteroid monotherapy induced complete or partial responses at 1 month in 85% (120/141) of patients with most remaining on maintenance doses (median, 10 mg prednisone equivalent daily for 2 months to 20 years). Hydroxyurea and IFN-alpha (used in 64 and 46 patients, respectively) were also effective, but their use was limited by toxicity. Imatinib (used in 68 patients) was more effective in patients with the FIP1L1-PDGFRA mutation (88%) than in those without (23%; P < .001). CONCLUSION This study, the largest clinical analysis of patients with HES to date, not only provides useful information for clinicians but also should stimulate prospective trials to optimize treatment of HES.

Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes

Mast cells (MC), blood basophils (Ba) and moncoytes (Mo) are of haemopoietic origin. Lineage‐relationships and transdifferentiation between MC and Mo, or MC and Ba, have been considered, based on common expression of antigens. In this study, comparative phenotypic analyses on MC, Ba and Mo and on respective cell lines were performed using monoclonal antibodies (mAb) to previously defined and novel CD antigens (CD1–130). By cluster analysis, the overall (all 130 CD) phenotypic relationships (given as similarity indices, SI), between primary cells (MC, Ba and Mo) and corresponding cell lines (HMC‐1, KU‐812, U937) were 0.716, 0.779 and 0.757, respectively. When primary cells were compared, lower SI values were found (MC versus Ba, 0.509; MC versus Mo, 0.625; Mo versus Ba, 0.698). More distant relationships were found between MC versus Ba and MC versus Mo, compared with Ba versus Mo, for adhesion receptor (R)‐, complement R‐ and cytokine R profiles. Analysis of cytokine R revealed most significant dissimilarities between MC versus Ba and MC versus Mo (SI < 0.2). Moreover, in contrast to other CD subgroups and other lineages, MC and HMC‐1 differed from each other in cytokine R expression (SI = 0.286). Cytokine R detectable on HMC‐1 but not MC were granulocyte–macrophage colony‐stimulating factor (GM‐CSFR)α(CD116), CD40, Apo‐1/FAS(CD95) and gp130(CD130). Cytokine R detectable on Ba but not MC, were interleukin‐3 (IL‐3)Rα(CD123), IL‐1RII(CD121b), IL‐2Rα(CD25) and CD40. In summary, MC, Ba and Mo display a unique CD profile with MC being the most distantly related cell. The most significant mismatch within a given lineage is the loss of cytokine R on mature MC as compared with normal myeloid progenitors and HMC‐1 cells.

Interferon-α Treatment of Six Patients with the Idiopathic Hypereosinophilic Syndrome

Table. SI Units and Drug The hypereosinophilic syndrome encompasses a spectrum of disorders having the following common criteria: 1) persistent eosinophilia (>1500 eosinophils/mm3) for at least 6 months (or death before 6 months with signs and symptoms of this syndrome); 2) no evidence of parasitic, allergic, or other recognized causes of eosinophilia after comprehensive evaluation; and 3) signs and symptoms of organ system involvement or dysfunction that can be directly related to eosinophilia or are otherwise unexplained in the clinical setting [1-3]. Hardy and Anderson [4] in 1968 suggested that many syndromes having in common marked eosinophilia and organ dysfunction could be grouped together as the hypereosinophilic syndrome. Historically, survival in patients with untreated hypereosinophilic syndrome has been poor, with neurologic (64%), skin (56%), and cardiovascular (54%) systems most often affected [3]. Recently, clinical case reports [5-7] suggested that interferon- may be an effective treatment for patients with the hypereosinophilic syndrome who are resistant to glucocorticoids and hydroxyurea. We examined the effects of interferon- in six patients with the hypereosinophilic syndrome, five of whom were unresponsive to or intolerant of conventional therapy. Methods Men or nonpregnant women 18 years of age or older meeting criteria for the hypereosinophilic syndrome [1-3] were candidates for inclusion in this phase I study. This study was approved by the Institutional Review Board of the Mayo Clinic. After giving informed consent, patients were carefully screened to exclude secondary causes of eosinophilia. Screening entailed taking a general medical history and examination; obtaining bone marrow biopsy specimens (if not previously done); and thoroughly reviewing previous laboratory reports, radiographs, clinical studies, and records of previous or current therapy. Interferon- 2B (Intron A, Schering-Plough Research Institute, Kenilworth, New Jersey) was administered daily as a single subcutaneous injection. The initial dose of interferon- ranged from 1.0 to 6.25 106 U/d and was individualized for each patient depending on clinical status and tolerance. As the eosinophil count decreased, concurrent medications were tapered and, if possible, discontinued. Peripheral blood counts (including hemoglobin, platelet count, and leukocyte count and differential) were measured daily during initial dose escalation and every 3 to 4 weeks thereafter. The goal was to decrease the total eosinophil count by 90% or to fewer than 1000 cells/mm3. Each patient received interferon- for a minimum of 9 months, and a bone marrow biopsy specimen was obtained again after each patient completed at least 9 months of therapy. Serum levels of eosinophil major basic protein were determined in each patient using a specific double-antibody radioimmunoassay [8]. Patient Histories Figure 1 shows levels of blood eosinophils and interferon- dosages for patients 1 to 6. Figure 1. Summary of blood eosinophil levels and interferon- dosages for all six patients. Patient 1 A 42-year-old white man presented in August 1991 with fatigue, cough, and eosinophilia (leukocyte count, 18.2 109/L [18 200/mm3]; 54% eosinophils). He had had no previous response to or had been intolerant of prednisone, hydroxyurea, etoposide, and a combination of cyclosporine and prednisone; he developed cognitive dysfunction, visual loss, and probable central nervous system demyelination (identified by magnetic resonance imaging scan). When he was seen at our clinic in August 1992, he complained of weakness and visual blurring associated temporally with high eosinophil counts. His medications were prednisone (20 mg/d) plus one aspirin per day. His physical examination showed a 1 to 2 out of 6 systolic murmur at the lower left sternal border, which radiated to the cardiac apex, and hypertension of 160 to 170/100 to 110 mm Hg. The bone marrow specimen showed approximately 30% eosinophils. His serum vitamin B12 and IgE levels were normal. An echocardiogram showed metallic brightness of the anterior mitral leaflet and mitral regurgitation and suggested the presence of an additional tissue layer laterally and inferiorly from the tip of the posterior mitral leaflet to the apex. An electrocardiogram showed evidence of left ventricular hypertrophy with QRS widening. Test results from methacholine inhalation challenge indicated the presence of reactive airway disease. The leukocyte count was 17.5 109/L (17 500/mm3) with 52% eosinophils, 25.5% segmented neutrophils, 1.5% bands, 2% basophils, and 16.5% lymphocytes. The hemoglobin level was 135 g/L, and the platelet count was 133 109/L. The patient was started on subcutaneous interferon- (1 MU/d) in August 1992, and his dosage was increased as tolerated to 3.5 MU/d at 14 weeks. The total leukocyte count and percentage of eosinophils decreased to 5.8 109/L (5800/mm3) and 10%, respectively; however, because of thrombocytopenia (63 109/L), the dosage of interferon- was subsequently decreased. He has continued receiving a maintenance interferon- dosage of 1.5 to 2.0 MU/d for 23 months; his total leukocyte count stabilized at 3.7 109/L (3700/mm3) with 9% eosinophils. During therapy with interferon-, the serum level of eosinophil major basic protein decreased from 9020 to 3174 ng/mL (normal level, 538 144 ng/mL). He discontinued prednisone therapy. Since then, his clinical symptoms have included paroxysmal cough and dyspnea unresponsive to glucocorticoids; pansinusitis shown by computed tomographic scanning; and recurrent abdominal discomfort, vomiting, and diarrhea. He has fixed visual and neurologic deficits, particularly of fine motor movements, neither of which have progressed since he started interferon- therapy. Patient 2 A 23-year-old white man presented to our clinic in July 1990 with chest and abdominal pain and gastric erosions necessitating treatment with a histamine-2 (H2)-receptor antagonist and transfusion of 4 units of packed red blood cells. Physical examination showed splenomegaly. An echocardiogram in October 1990 showed trivial mitral regurgitation and a decreased cardiac ejection fraction (45%). The serum vitamin B12 level was more than 1480 pmol/L (normal level, 150 to 750 pmol/L). Peripheral blood counts (leukocyte count, 31.3 109/L [31 300/mm3]; 68% eosinophils) and bone marrow eosinophils were increased. Cytogenetic studies showed no apparent abnormal clones on this or numerous subsequent bone marrow aspirates. Initial therapy with various dosages of prednisone and hydroxyurea was ineffective. Splenectomy was done to relieve thrombocytopenia. He was hospitalized in August 1991 for progressive leukocytosis and eosinophilia (leukocyte count, 216 109/L [216 000/mm3]; 41% eosinophils). At this time, he was given intravenous methylprednisolone (2 g daily for 4 days), interferon- (5 MU/d subcutaneously), vincristine (2 mg/wk intravenously), therapeutic leukapheresis, and transfusions with platelets and packed red blood cells. He remained platelet- and transfusion-dependent for 3 months. Subsequently, he required interferon- (5 to 6 MU/d five days per week) and aspirin (one tablet daily); prednisone (10 to 20 mg every other day) was tapered and discontinued. Another echocardiogram in December 1991 showed left ventricular enlargement, an ejection fraction of 53%, and an immobile posterior leaflet of the mitral valve with moderate-to-severe mitral regurgitation. In August 1992, there was a decrease in the degree of mitral regurgitation and improvement in the ejection fraction (62%) but no change in appearance of the posterior leaflet of the mitral valve. His wife became pregnant while he was taking interferon-, and she gave birth to a healthy baby boy. After 15 months of treatment with interferon-, the patient suddenly had midthoracic myelopathic symptoms. A magnetic resonance imaging scan showed spinal cord compression by extradural masses at T7, T10, and L3. Open biopsy showed that the masses were granulocytic sarcomas (chloromas), and radiation therapy was administered. After radiation therapy, the dosage of interferon- was increased to 6.5 MU/d for 5 of 7 days. His total leukocyte count decreased to 6.6 109/L (6600/mm3) with 12% eosinophils. During treatment with interferon-, his serum level of eosinophil major basic protein decreased from 25 687 to 10 320 ng/mL. He continued to have joint and muscle pain in the lower extremities. An area of hypesthesia developed in the right cheek. Six months later, he had documented recurrence of chloromas at several spinal cord levels and intracranial areas. A second course of radiation therapy was begun; however, the patient became septic and died in June 1993. Patient 3 A 32-year-old white man had eosinophilia (leukocyte count, 20 109/L [20 000/mm3]; 25% eosinophils) in June 1989 several months after a flu-like illness. He felt well until December 1989 when he developed recurrent episcleritis, dizziness, dysphonia, and progressive fatigue. In June 1990, he developed waxing and waning mouth ulcers, a penile ulcer, and a fine erythematous rash on the thighs. He also developed furuncles on his legs and arms that grew Staphylococcus aureus. His skin lesions were partially responsive to prednisone and colchicine. A physical examination at our clinic in December 1990 showed splenomegaly; no cardiac abnormalities were evident. The glans penis was occupied by a yellow dried exudate from a chronic ulceration. Furunculoid lesions were present on the elbows and left calf. A tiny aphthous-type oral ulcer was present. Bone marrow aspiration showed 60% eosinophils; levels of peripheral blood eosinophils were 6000 to 12 000/mm3. An echocardiogram and the level of serum IgE were normal. Treatment with interferon- (6.25 MU/d) was started in January 1991, and after 8 weeks of therapy, his penile and mucosal ulcers healed. These ulcers have not recurred; he is less fatigued, and he has been able to

Cytoreductive therapy in 108 adults with systemic mastocytosis: Outcome analysis and response prediction during treatment with interferon-alpha, hydroxyurea, imatinib mesylate or 2-chlorodeoxyadenosine.

Cytoreductive therapy in systemic mastocytosis (SM) includes several drugs whose individual merit has not been well characterized. We retrospectively studied 108 Mayo Clinic patients who met the 2008 WHO diagnostic criteria for SM and received at least one cytoreductive drug. The numbers of patients who were evaluable for response to treatment with interferon‐alpha with or without prednisone (IFN‐α), hydroxyurea (HU), imatinib mesylate (IM) or 2‐chlorodeoxyadenosine (2‐CdA) were 40, 26, 22, and 22, respectively. The corresponding overall (major) response rates, according to recently published consensus criteria, were 53% (18%), 19% (0%), 18% (9%), and 55% (37%). The respective overall response rates in indolent SM, aggressive SM and SM associated with another clonal hematological nonmast cell lineage disease (SM‐AHNMD) were 60%, 60%, 45% for IFN‐α, 0, 0, 21% for HU, 14%, 50%, 9% for IM and 56%, 50%, 55% for 2‐CdA. The absence of mast cell mediator release symptoms in IFN‐α‐treated patients and presence of circulating immature myeloid cells in 2‐CdA‐treated patients predicted inferior response. TET2 mutational status did not influence treatment response. Although the major response rates with these four cytoreductive agents were still suboptimal and HU was mainly used in patients with SM‐AHNMD, the current study favors 2‐CdA or IFN‐α as first‐line current therapy in SM and identifies patients who are likely to respond to such therapy. Am. J. Hematol., 2009.

Pathogenesis and classification of eosinophil disorders: a review of recent developments in the field

Eosinophils and their products play an essential role in the pathogenesis of various reactive and neoplastic disorders. Depending on the underlying disease, molecular defect and involved cytokines, hypereosinophilia may develop and may lead to organ damage. In other patients, persistent eosinophilia is accompanied by typical clinical findings, but the causative role and impact of eosinophilia remain uncertain. For patients with eosinophil-mediated organ pathology, early therapeutic intervention with agents reducing eosinophil counts can be effective in limiting or preventing irreversible organ damage. Therefore, it is important to approach eosinophil disorders and related syndromes early by using established criteria, to perform all appropriate staging investigations, and to search for molecular targets of therapy. In this article, we review current concepts in the pathogenesis and evolution of eosinophilia and eosinophil-related organ damage in neoplastic and non-neoplastic conditions. In addition, we discuss classifications of eosinophil disorders and related syndromes as well as diagnostic algorithms and standard treatment for various eosinophil-related disorders.

Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c-kit proto-oncogene

The human mast cell line (HMC)‐1 cell line is growth‐factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC‐1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC‐1, named HMC‐1560 and HMC‐1560,816, with different phenotypes and designated by the locations of specific mutations in the c‐kit proto‐oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing™ method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c‐kit gene causing an amino acid substitution of Gly‐560 for Val. In contrast, only HMC‐1560,816 cells have the c‐kitV816 mutation found in mast cell neoplasms causing an Asp→Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3′‐kinase (PI 3‐kinase) in both variants of HMC‐1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC‐1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.