Butterfield, Joseph H.

The syndrome of episodic angioedema with eosinophilia, first reported over 40 years ago, is a hypereosinophilic disorder that, uniquely, is not associated with end-organ pathology. However, patients develop a constellation of symptoms that include angioedema, urticaria, fatigue, and fever. Episodes are accompanied by massive hypereosinophilia and weight gain. Type II serum cytokine levels (IL-5, IL-13, IL-9, and IL-10) show cyclic variations peaking at or just prior to the peak of eosinophilia and an abnormal Th2 cell phenotype has been reported. Attacks may occur with predictable regularity and have been described in both adults and children. Glucocorticoid therapy reliably reverses symptoms with accompanying diuresis, defervesce, and normalization of the eosinophil count. In this report, a patient who had the syndrome of episodic angioedema with eosinophilia exceeding 20 years is reported. He has had no end-organ damage to date. Testing for the CHIC 2 deletion, a surrogate for the FIP1L1-PDGFRA fusion, was negative. Use of imatinib mesylate, initially as a steroid-sparing agent, and subsequently as a maintenance medication, plus low-dose prednisone has provided long-term control of hypereosinophilia and all clinical manifestations.

Abstract Background: The FIP1L1-PDGFRA fusion, an imatinib-sensitive molecular target, is detected in a subset of cases with moderate-to-severe eosinophilia (eosinophil count ≥1500/μL). Using 2 FISH strategies to detect loss of the CHIC2 locus as a surrogate marker of the FIP1L1-PDGFRA fusion, we previously found 14% of cases to be CHIC2-deleted when studying bone marrow smears from 81 adult patients with moderate-to-severe, non-reactive eosinophilia at our institution (Blood 2004, 104:3038–3045). Since then, the FISH test has been made available for routine clinical use both within our institution, and to external physicians as a mail-in test, for the workup of patients with eosinophilic disorders, including systemic mast cell disease (SMCD). Study design: FISH data, subsequent to the test’s introduction into routine clinical practice (from Nov. 2003), were retrospectively analyzed for CHIC2-deletion. Data from the initial pilot study of 81 patients was not used in this analysis (Table). The imatinib treatment analysis, however, included all CHIC2-deleted patients treated at our institution, including those from the pilot study (Table). Results: A total of 741 patients were studied in the post-pilot phase. Of these, 20 cases were found to be CHIC2-deleted, with an overall prevalence of 2.7% (Table). Of the 219 patients seen at our institution, 13 (all male) were found to be CHIC2-deleted (Table) (prevalence=5.9%), and of these, 10 have been treated with imatinib (100 mg/day starting dose) to date. Nine patients are evaluable for response - all achieved a complete clinical and histological remission (7 patients were tested by FISH post-treatment - all achieved cytogenetic remission). One patient received concomitant corticosteroids for co-morbidities and could not be evaluated for response. After a median follow-up of 24 months (range 17 to 39 months), all 9 patients continue to be in remission, with a normal eosinophil count. At last follow-up, one patient had discontinued imatinib and the others were on maintenance therapy at the following dose: 100mg/wk (n=1), 100mg BIW (n=1), 100mg TIW (n=2), 50mg QOD (n=1), and 100 mg/day (n=3). Conclusions: FIP1L1-PDGFRA prevalence in non-selected patients with eosinophilia is lower than anticipated from prior studies. Patients with FIP1L1-PDGFRA exhibit durable responses to imatinib therapy. It may be feasible to maintain some patients in remission on an empirically derived, intermittent schedule of low-dose imatinib. Pilot Data Post-pilot Data (n=741) Total Mayo samples Mayo samples Mail-in samples Number of Patients 81 138 603 822 Number CHIC2-deleted 11 2 18 31 Prevalence 14% 1.4% 3.0% 3.8% Number Imatinib treated 8 2 n/a 10

Abstract Background: Current therapy in adult systemic mastocytosis (SM) includes observation alone, topical therapy for cutaneous disease, symptomatic non-cytoreductive therapy and cytoreductive therapy. The latter involves several drugs whose individual merit in the treatment of SM has not been well characterized. Patients and Methods: Diagnosis of SM was according to the 2001 WHO criteria (World Health Organization Classification of Tumours of Hematopoietic and Lymphoid Tissues, p 1–351. Lyon, France: IARC Press; 2001). Response to cytoreductive therapy was assessed by consensus criteria (Eur J Clin Invest.2007;37:435). The study population was selected from a series of 342 adult SM patients referred to our institution and seen between 1964 and 2008. KITD816V mutation analysis was performed by DNA sequencing. Results: A total of 134 study patients received treatment that included at least one cytoreductive drug as either first-line (n=105) or second-line (n=29) therapy. Interferon-alpha with or without prednisone (n=58), hydroxyurea (n=43), imatinib mesylate (n=42) and 2-chlorodeoxyadenosine (n=26) were the most frequently used cytoreductive agents accounting for 120 of the 134 study cases; the remaining 14 patients received other cytoreductive drugs, none of which were effective. i. Interferon-alfa (IFN-a) with or without prednisone IFN-a with or without prednisone (24 and 34 patients, respectively) was given to 12 patients with indolent SM (ISM), 14 aggressive SM (ASM), and 32 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD). For all 58 patients receiving therapy, complete (CR), major (MR), and partial (PR) response/regression were achieved in 3, 7, and 14 patients, respectively, for an overall response rate (ORR) of 41%. ORR in ISM, ASM, and SM-AHNMD was 50%, 43%, and 38%, respectively. Median duration of response was 12 months (range, 1–117 months). Anemia and elevated ESR were significantly associated with inferior ORR; 29% vs. 56% (p=0.04) and 14% vs. 57% (p=0.01), respectively. BM detection of KITD816V did not appear to affect response rates (p=0.4). ii. Hydroxyurea (HU) HU was given to 2 ASM, 40 SM-AHNMD and 1 mast cell leukemia (MCL) patients. The drug was used as first-line therapy in 30 patients. MR and PR were achieved in 1 and 8 SM-AHNMD patients, respectively (ORR=21%). Median duration of response was 37 months (range, 5–84 months). iii. Imatinib mesylate (IM) in patients with SM and associated eosinophilia IM was given to 22 SM patients who had associated eosinophilia (SM-eos): 2 had ISM, 2 ASM, and 18 SM-AHNMD. The drug was used as first-line therapy in 9 patients. Ten of the 22 patients with SM-eos were known to harbor the FIP1L1-PDGFRA fusion mutation and all responded to IM (ORR=100%). None of 5 FIP1L1-PDGFRA-negative SM-eos patients had a response to IM. In the remaining 7 patients with unknown mutation status, 4 patients had a response to IM (1 CR, 1 MR, and 2 PR). iv. IM in patients with SM not associated with eosinophilia IM was administered to 20 SM patients without associated eosinophilia (7 ISM, 2 ASM, 10 SM-AHNMD, and 1 MCL). Response was documented in 4 (20%) patients (1 CR, 1 MR, and 2 PR) including 1 with ISM, 2 with ASM and 1 with SM-AHNMD. Median duration of response was 19.5 months (range, 9–69 months). Two patients developed interstitial pneumonitis. v. 2-Chlorodeoxyadenosine (2-CdA) 2-CdA was given to 10 ISM, 3 ASM, and 13 SM-AHNMD patients; the drug was used as first-line therapy in 8 patients. CR, MR and PR were achieved in 1, 7, and 4 patients, respectively (ORR=46%). ORR in ISM, ASM, and SM-AHNMD was 50%, 33%, and 46%, respectively. Median duration of response was 11 months (range, 3–74 months). Presence of leukocytosis (WBC > 10 x 109/L), monocytosis (absolute monocyte count > 0.9 x 109/L) or circulating immature myeloid cells was significantly associated with inferior response; 13% vs. 61% (p=0.02); 11% vs. 69% (p=0.006) and 0% vs. 71%, (p=0.001), respectively. Response was not affected by BM detection of KITD816V (p=1.0). Conclusions: Both IFN-a and 2-CdA are reasonable treatment options, with comparable efficacy, for symptomatic SM including the subcategories of ISM, ASM and SM-AHNMD. IFN-a response was significantly better in the absence of anemia or elevated ESR. 2-CdA response was similarly better in the absence of leukocytosis, monocytosis or circulating immature myeloid cells. The therapeutic value of IM was limited to FIP1L1-PDGFRA-positive disease.

Abstract Background: Systemic mastocytosis (SM) and hypereosinophilic syndrome (HES) are JAK2V617F-negative orphan diseases without effective long-term therapeutic options. Both of these diseases share clinical and laboratory features with FIP1L1-PDGFRA-positive chronic eosinophilic leukemia (CEL). SM is associated with KITD816V or other KIT mutations. HES is currently not molecularly characterized. The exquisite sensitivity of FIP1L1-PDGFRA to inhibition by imatinib mesylate (IM) underlies the efficacy of this agent in treating FIP1L1-PDGFRA-positive CEL. In contrast, KITD816V is resistant to inhibition by IM, and is only moderately sensitive to dasatinib (cellular IC50=micromolar concentrations), which may explain the relatively disappointing clinical responses in SM with these agents. TG101348 is an orally bioavailable JAK2-selective inhibitor that is currently being tested in a Phase 1 clinical trial for the treatment of myelofibrosis. The aim of the current study was to evaluate TG101348 (relative to the tyrosine kinase inhibitors IM, dasatinib, and sorafenib) for its ability to: inhibit growth of leukemic cell lines that carry KITD816V and FIP1L1-PDGFRA; and inhibit the in vitro growth of eosinophil colonies derived from progenitor cells from HES patients. Methods: The following cell lines were used for in vitro experiments: HMC-1, a mast cell leukemia line (KITD816V-positive); EOL-1, a CEL derived line (FIP1L1-PDFRA-positive), Ba/F3 T674I, a Ba/F3 line that express the IM-resistant FIP1L1-PDFRAT674I mutation, and HEL, a human erythroleukemia line (JAK2V617F-positive). Cell proliferation assays were performed in triplicate using the XTT assay (Leukemia. 2007 21:1658). Drug concentrations in these experiments ranged from 2.9 × 10−12 M to 10−4 M. JAK Inhibitor I (Calbiochem) is a non-selective JAK inhibitor tool compound. Eosinophil colonies were obtained by plating PBMCs from healthy controls or HES patients in methylcellulose, in the presence of IL-3, IL-5, and GM-CSF. Effects of IM and TG101348 on eosinophil colony growth were studied in parallel experiments. Results: Consistent with prior observations, the HMC-1 cell line was resistant to inhibition by IM (IC50>10 mM), but was moderately sensitive to dasatinib (Table). In contrast, TG101348 inhibited HMC-1 growth at nanomolar concentrations, similar to its effect on HEL cells that harbor the JAK2V617F mutation. TG101348 inhibited HMC-1 growth 53-fold and 4-fold more potently than IM and dasatinib, respectively (Figure). Sorafenib, a multikinase inhibitor that reportedly has activity against KIT kinase (enzyme IC50=68 nM), had only a limited effect on HMC-1 growth (IC50=4000 nM). In contrast to HMC-1, EOL-1 growth was inhibited by IM, dasatinib, sorafenib, and TG101348 at picomolar concentrations (Table). The T674I mutation (in the context of FIP1L1-PDGFRA) corresponds to the T315I mutation in BCR-ABL – it occurs infrequently in CEL and is usually found in the context of transformation to acute leukemia. Growth of Ba/F3 cells harboring FIP1L1-PDFGRA-T674I has been shown to be inhibited by sorafenib (confirmed in this report; Table) and nilotinib, at nanomolar concentrations. TG101348 is less potent at inhibiting growth of these cells, with cellular IC50 of ~2 mM – pharmacokinetic data from the ongoing Phase I study of TG101348 shows that this concentration is achievable in plasma with once daily dosing. Drug Cellular IC50 HMC-1 (nM) EOL-1 (pM) Ba/F3 T674I (nM) HEL (nM) Imatinib 18800 4 10700 - Dasatinib 1300 <1 30900 - Sorafenib 4000 2 16 - Jak Inhibitor I 926 30 4300 ? TG101348 355 <1 1950 300 Figure Figure Conclusions: The JAK2-selective inhibitor TG101348 is a potent inhibitor of KITD816V and FIP1L1-PDGFRA, warranting clinical trials using this drug in SM and FIP1L1- PDGFRA-positive CEL. Data regarding TG101348 effects on signaling intermediates in leukemic cell lines and on eosinophil colony growth will be presented at the meeting – data from the latter experiments may indicate a therapeutic role for TG10134 in the treatment of patients with HES.

Abstract Introduction: The utility of patient reported symptoms and serum tryptase levels in distinguishing those with systemic mastocytosis (SM) versus mast cell activation syndrome (MCAS) versus those not meeting formal diagnostic criteria for SM or MCAS has not been systematically examined. Methods: This study was approved by our institutional review board. Patients were referred for suspected SM based on symptoms of mast cell activation, osteopenia, skin rash, etc., or had an established diagnosis of SM. All patients were given a Mastocytosis Symptom Assessment Form (MastSAF) to complete at their initial evaluation. The MastSAF is comprised of 36 symptom questions to be graded on a scale of 0 (absent) to 10 (worst imaginable), and is organized around 9 symptom clusters: gastrointestinal (8 questions), constitutional (3 questions), musculoskeletal (5 questions), cutaneous (4 questions), neuropsychological (6 questions), genitourinary (3 questions), respiratory (4 questions), angioedema (1 question), and cardiovascular (2 questions). All patients underwent bone marrow biopsy and serum tryptase level assessment. SM was diagnosed by 2008 WHO criteria. In the presence of characteristic symptoms, if clonal or abnormal mast cells were not identified, and if serum/urine mast cell mediator levels were increased, then non-SM associated, non-monoclonal MCAS was diagnosed. Results: A total 53 patients were studied. 1) SM: Of 28 patients, 13 had indolent SM (ISM), 9 aggressive SM (ASM) and 6 SM with associated hematological disease (SM-AHD) The median total symptom score was 47 (range 8-159). The median (range) for SM subgroups was: ISM 51 (9-159), ASM 55 (19-157) and SM-AHD 27 (8-131) (p=0.2). The normalized median score for individual symptom categories (total median score/no. of symptoms per category) in order of severity was cutaneous 1.9, gastrointestinal 1.8, constitutional 1.7, neuropsychological 1.3, respiratory 1.3, musculoskeletal 0.9, cardiovascular 0.3, genitourinary 0.3, and angioedema 0. Symptom severity was not significantly different among the 3 SM subgroups, except for constitutional symptoms (median score ASM 9, ISM 4, SM-AHD 2.5, p=0.02). The median (range) tryptase level was 48.4 ng/mL (8.8-282); four patients (14%) had a baseline level <20 ng/mL. The median (range) tryptase level among SM subgroups was: ISM 46.9 (8.8-225), ASM 103 (29.4-282), and SM-AHD 43.5 (28.5-233) (p=0.1). When considering patients with tryptase level ≥50 versus <50 ng/mL, symptom scores were not significantly higher in the former group with the exception of constitutional (p=0.02) and genitourinary symptoms (p=0.04). 2) MCAS: 15 patients The median total symptom score was 127 (range 2-248). The normalized median score for individual symptom categories in order of severity was neuropsychological 4.5, musculoskeletal 4.2, cutaneous 4.0, constitutional 3.3, gastrointestinal 2.1, respiratory and cardiovascular 2.0 each, genitourinary 1.7, and angioedema 1.0. The median (range) serum tryptase level at referral was 12.7 ng/mL (1.7-25.8); five patients (33%) had a baseline level >20 ng/mL. 3) Neither SM/MCAS: 10 patients The median total symptom score was 119 (range 45-177). The normalized median score for individual symptom categories in order of severity was musculoskeletal 4.2, gastrointestinal 3.6, constitutional 3.3, neuropsychological 3.3, cutaneous 3.0, cardiovascular and genitourinary 2.0 each, respiratory 1.3, and angioedema 0. The median (range) serum tryptase level at referral (n=9) was 4.8 ng/mL (2.9-6.6). 4) Comparison: MCAS vs. SM: Symptom scores were significantly higher in MCAS as compared to SM (p<0.05), except for genitourinary and respiratory symptoms, which were not significantly different. ‘Neither SM/MCAS’ vs. SM: Symptom scores (total, gastrointestinal, constitutional, musculoskeletal, cutaneous, and neuropsychological) were significantly higher in the former group (p<0.05). Other symptom scores were not significantly different. Conclusions: The spectrum and severity of patient reported symptoms was broadly similar among WHO subcategories of SM, and serum tryptase level had limited if any correlation with symptom scores. Despite the significantly higher overall symptom burden in MCAS versus SM, tryptase levels in the former group were significantly lower with values >30 ng/mL unusual. Despite overlap, the top ranked symptoms in the 3 groups were different. Disclosures No relevant conflicts of interest to declare.

AbstractNerve growth factor (NGF ) can influence mast cell development and function in murine rodents by interacting with its receptors on mast cells. We now report the identification of mRNA transcripts of full-length tyrosine kinase-containing trkA, trkB, and trkC neurotrophin receptor genes in HMC-1 human mast cell leukemia cells. Although HMC-1 cells lacked p75 mRNA, they expressed transcripts for the exon-lacking splice variant of trkA (trkAI), truncated trkB (trkB.T1), and truncated trkC. By flow cytometry, HMC-1 cells exhibited expression of TrkA, TrkB, and TrkC receptor proteins containing full-length tyrosine kinase domains. NGF stimulation of HMC-1 cells induced tyrosine phosphorylation of TrkA protein, increased expression of the early response genes c-fos and NGF1-A, and activation of ERK-mitogen–activated protein (MAP) kinase, results which indicate that TrkA receptors in HMC-1 cells are fully functional. Highly purified populations of human lung mast cells expressed mRNAs for trkA, trkB and trkC, whereas preparations of human umbilical cord blood-derived mast cells expressed mRNAs for trkA and trkC, but not trkB. Moreover, preparations of human umbilical cord blood-derived immature mast cells not only expressed mRNA transcript and protein for TrkA, but exhibited significantly higher numbers of chymase-positive cells after the addition of NGF to their culture medium for 3 weeks. In addition, HMC-1 cells expressed mRNAs for NGF, brain-derived neurotrophic factor (BDNF ), and neurotrophin-3 (NT-3), the cognate ligands for TrkA, TrkB, and TrkC, whereas NGF and BDNF transcripts were detectable in human umbilical cord blood mast cell preparations. Taken together, our findings show that human mast cells express a functional TrkA receptor tyrosine kinase and indicate that NGF may be able to promote certain aspects of mast cell development and/or maturation in humans. Our studies also raise the possibility that human mast cells may represent a potential source for neurotrophins.

Abstract Background: Blood eosinophilia results either from autonomous expansion of a myeloid stem/progenitor cell, or as a response to cytokines that promote growth and differentiation of eosinophil progenitors. A subset of patients with hypereosinophilic syndrome/idiopathic eosinophilia (HES/IE) display abnormal T-cell immunophenotype and/or clonal rearrangements of the T-cell receptor (TCR), operationally assigned the term “lymphocyte variant” HES/IE. Information on the prevalence, FIP1L1-PDGFRA status, and clinical course of such patients is limited. Methods: Consecutive patients with eosinophilia were identified by querying our institutional electronic database from 1995 to 2004. Clonal rearrangements of the γchain of the TCR were identified by polymerase chain reaction followed by gel electrophoresis in all cases, and confirmed by Southern Blot analysis of the gene coding for the β chain of the TCR, using standard methods. FIP1L1-PDGFRA was screened by fluorescence in situ hybridization. Results: We studied a total of 205 consecutive patients with eosinophilia defined as an absolute eosinophil count (AEC) of > 600/μL, who also had a bone marrow study performed. Of these, TCR gene rearrangements studies were performed in 99 patients (AEC range of 650 to 38,000/μL) including 3 children and 96 adults (age range 18 to 88 years; 58 males). Among these 99 patients, 14 (~14%) had a demonstrable T-cell clone in peripheral blood, bone marrow aspirate, lymph node, and/or skin. Six of the 14 patients with a demonstrable T-cell clone were found to have a concurrent T-cell malignancy at the time of evaluation including T-cell large granular lymphocytic (T-LGL) leukemia - 1 case and peripheral T-cell lymphoma - 5 cases. The other 8 (~8%) patients otherwise fulfilled the working diagnosis of “lymphocyte variant” HES/IE (median AEC=2045/μL, range 720/μL to 9600/μL). The median duration of eosinophilic prodrome, prior to identification of the occult T-cell clone was 5 years (range 3 months to 11 years), and virtually all had dominant skin involvement presenting as dermatitis (5 cases) or episodic angioedema with eosinophilia (EAE) (2 cases). Five of 7 patients tested had an elevated serum IgE level, and a similar proportion displayed an abnormal T-cell immunophenotype (most commonly, aberrant CD5 and/or CD7 expression). Six of the 8 patients with an occult T-cell clone were evaluated for FIP1L1-PDGFRA and tested negative. Two patients were effectively treated with low-dose oral cyclophosphamide or methotrexate (follow-up duration of 3+ and 6+ years, respectively) whereas imatinib treatment was ineffective in another 2 patients. Two patients (25%) transformed into cutaneous T-cell lymphoma after 3 to 8 years of eosinophilic prodrome. Conclusion: The current study suggests that the presence of a T-cell clone in idiopathic eosinophilia is a marker/harbinger of a primarily skin-based peripheral T-cell lymphoma that responds to T cell-directed therapy but not imatinib therapy, which is consistent with the absence of FIP1L1-PDGFRA in such cases.

Objective: A growing number of cancer antineoplastic agents can cause life-threatening acute infusion reactions. Because previous studies have not studied these reactions from the perspective of patients, this study was undertaken with that objective in mind. Methods: Patients who had an acute infusion reaction were interviewed based on the Leventhal model. Once saturation of content was achieved, interviews were transcribed and analyzed with qualitative methodology. Results: Twenty-one patients were enrolled. Most were women (n = 15); the median age was 58 years, and paclitaxel was the most common inciting agent. Three themes emerged. First, these reactions are frightening; patients made remarks such as “I was just thinking oh my God, I am dying.” Second, prior education about these reactions seemed to mitigate this fear, “Basically everything the nurses told me potentially could happen, like happened. So, I was prepared.” Third, when health-care providers were prompt and attentive during the reaction, patients described less fear with future chemotherapy, “So no, I’m really not fearful about going in tomorrow because I know they’ll be there and they’ll be watching me.” Conclusion: These reactions evoke fear which can be mitigated with education prior to and with prompt responsiveness during the acute infusion reaction.

Abstract The neutral protease tryptase has been isolated from a human mast cell line, HMC-1. The HMC-1 line was established from the peripheral blood of a patient with mast cell leukemia and maintained as continuously proliferating clones in vitro and as solid mast cell tumors in nude mice. HMC-1-derived tryptase was purified by sequential chromatography on Dowex 1, DEAE 5 PW, and heparin-agarose. Purified tryptase has an apparent molecular weight of 150,000, as determined by molecular sieve HPLC, but migrates as a doublet of bands of 32/35,000 on SDS-PAGE gels. Maximal enzymatic activity was observed at pH 8.5. Cleavage of tosyl-L-arginine methyl ester by purified tryptase was inhibited by dansyl-L-glutamyl-glycyl-L-arginine chloromethyl ketone 2 HCI, HgCI2, tosyl-L-lysine chloromethyl ketone, leupeptin, and PMSF but not by benzamidine, aprotinin, tosyl-L-phenylalanine chloromethyl ketone, soybean trypsin inhibitor, human plasma, ovomucoid inhibitor, or lima bean trypsin inhibitor. Microsequencing of purified tryptase yielded an amino terminal sequence that was identical to that previously reported for human pituitary-derived tryptase.