Kittur, Jaya

Abstract Background Cytogenetic abnormalities at diagnosis are relatively uncommon in essential thrombocythemia (ET). In the current study of 818 consecutive patients with ET who were fully annotated for karyotype, we describe the spectrum and prevalence of cytogenetic abnormalities at diagnosis, followed by a comprehensive assessment of phenotypic and molecular correlates and prognostic relevance. Methods The study cohort consisted of 818 consecutive patients with ET that were diagnosed according to the World health Organization 2016 criteria and underwent evaluation between 1967-2021. In order to minimize the inadvertent inclusion of patients with masked polycythemia vera, JAK2 mutated cases with hemoglobin (Hb) level >16 g/dL in women and 16.5 g/dL in men were excluded; similarly, cases with anemia defined by sex adjusted Hb level of <11 g/dL in women and <12.5 g/dL in men were also excluded, in order to avoid inadvertent inclusion of patients with prefibrotic myelofibrosis. Cytogenetic studies were performed either at or within one year of diagnosis and reported according to the International System for Human Cytogenetic Nomenclature. Disease status and survival information was updated in May 2021. JMP Pro 16.0.0 software package, SAS Institute, Cary, NC was utilized for all analyses. Results Prevalence and spectrum of cytogenetic abnormalities Karyotype was normal in 755 patients (92%), showed loss of Y chromosome (-Y) in 16 (2%), and showed abnormalities other than -Y in 47 (5.7%); most common abnormalities included del(20q) (n=10, 21%), trisomy 9 (n=8, 17%), trisomy 8 (n=2, 4%), del(5q) (n=2, 4%), and del(3p) (n=2, 4%). Other sole cytogenetic abnormalities were identified in 18 (38%) patients. Phenotypic and molecular correlates Abnormal karyotype, other than -Y, in comparison with normal karyotype was associated with older age (median age; 63 vs 58 years, p=0.02), lower hemoglobin level (p=0.003), and a higher incidence of arterial thrombosis prior to/at diagnosis (25% vs 13%; p=0.03). 603 patients were annotated for driver mutations; abnormal/normal/-Y frequencies were 78%/60%/71% for JAK2, 22%/26%/14% CALR, 0%/3%/0% MPL and 0%/10% /14% triple negative (p=0.31). NGS information was available in 226 patients and showed absence of ASXL1 mutation in all patients with abnormal karyotype vs 8/211 (4%) with normal karyotype vs 2/4 (50%) with -Y (p<0.0001). Disease transformation and overall-survival. At a median follow-up of 9.6 years (range; 0.01-49.4 years), a total of 96 patients (12%) underwent fibrotic transformation: 6 (13%) with abnormal karyotype, 89 (12%) with normal karyotype and 1 (6%) with -Y (p=0.77). Leukemic transformation rates were also similar with respective frequencies of 4%, 3% and 0% (p=0.71). Abnormal karyotype and -Y were associated with inferior survival with median of 12 years (range; 0.1-34) and 9 years (range; 0.01- 19.9), respectively, compared to 21 years (range; 0.01-49.4) for normal karyotype (p<0.0001) (Figure). In univariate analysis, risk factors for overall survival included abnormal karyotype (p=0.001), - Y (p=0.004), age >60 years (p<0.0001), leukocytosis >11 x10 9/L (p<0.0001), male gender (p=0.0003), and history of thrombosis (p=0.001). During multivariable analysis, abnormal karyotype other than -Y (p=0.003), age >60 years (p<0.0001), leukocytosis >11 x10 9/L (p=0.001), and male gender (p=0.01) remained significant. Additional analysis suggested individual prognostic impact for del(20q) (p=0.04) and also for trisomy 9 (p=0.09) and other abnormalities (p=0.07), with borderline significance. Conclusion The current study confirms the association of abnormal karyotype in ET with older age, lower hemoglobin level, and history of arterial thrombosis, and its mutual exclusivity with ASXL1 mutations. Our observation regarding the independent adverse impact of abnormal karyotype other than -Y, on overall survival, in the absence of association with fibrotic or leukemic transformation, requires clarification from additional studies, which should also investigate the effect of specific abnormalities. Figure 1 Figure 1. Disclosures Szuber: Novartis: Honoraria.

Abstract 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.

Summary We describe our single institution experience with cladribine therapy in 42 patients with systemic mastocytosis (SM): 22 advanced (adv‐SM; median age 65 years, 68% males) and 20 indolent/smouldering SM (ISM/SSM; median age 56 years, 45% males); subcategories included eight aggressive, 13 associated with another haematological neoplasm, one mast cell leukaemia, 17 ISM and three SSM. Overall/major response rates were 77%/45% for adv‐SM and 70%/60% for ISM/SSM, and median (range) duration of response 10 (4–75) and 46 (4–140) months respectively. A >50% reduction in bone marrow mast cell burden and serum tryptase level was documented in 63% and 67% of patients with adv‐SM and 50% and 46% with ISM/SSM respectively. The presence of KIT proto‐oncogene, receptor tyrosine kinase ( KIT ) D816V predicted response in adv‐SM: 17 (90%) of 19 with and none of three without the mutation responded ( P < 0·01). Treatment‐emergent adverse events were mostly limited to transient cytopenias: Grade 3/4 neutropenia, thrombocytopenia, or lymphopenia occurred in 27%, 27% and 27% of patients with adv‐SM, and 5%, 5% and 30% with ISM/SSM respectively. The present study provides practical information that might be considered when making treatment choices between cladribine and newer KIT‐targeted therapies and identifies the absence of KIT D816V as a potential marker of cladribine resistance in advanced SM; the latter observation needs confirmation in a larger study.

SummaryInstitutional database search (1999–2020) for acute myeloid leukaemia (AML) identified 109 cases of myeloid sarcoma (MS), of which 19 were isolated and presented de novo. The latter displayed longer survival (median 78 months), compared to MS with synchronous intramedullary AML (n = 32; median 16 months) and de novo AML without MS (n = 729; median 22 months; P = 0·13). However, the difference in survival was no longer apparent after accounting for bone marrow cytogenetic risk status (P = 0·67). Treatment‐induced MS tumour resolution was not affected by the presence of intramedullary disease (P = 0·61). The current study clarifies the prognosis of de novo isolated MS, in the context of AML.

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&gt;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 &lt;1 30900 - Sorafenib 4000 2 16 - Jak Inhibitor I 926 30 4300 ? TG101348 355 &lt;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.

7030 Background: JAK2V617F occurs in approximately 50% of patients with essential thrombocythemia. Qualitative studies of mutation analysis have previously reported an association between JAK2V617F and advanced age, higher hemoglobin level, higher leukocyte count, and lower platelet count. A possible association with thrombotic complication has also been considered. Methods: Allele-specific, quantitative PCR analysis for JAK2V617F was performed on 176 patients with ET, using genomic DNA from archived bone marrow, which was collected within one year (n=72), between 1 and 5 years (n=64), or after 5 years (n=40) of diagnosis. Results: JAK2V617F was detected in 96 patients (55%), in whom mutant allele burden ranged from 1% to 100% (median 6.3%). Neither mutational frequency (p=0.37) nor mutant allele burden (p=0.62) was affected by the timing of bone marrow sample collection. Presence of JAK2V617F was significantly associated with higher hemoglobin level (p<0.0001), lower platelet count (p=0.001), higher leukocyte count (p=0.008), increased incidence of venous thrombosis occurring after diagnosis (p=0.02), and older age at diagnosis (p=0.03). All but age retained significance in multivariable analysis. In mutation-positive patients (n=96), JAK2V617F allele burden clustered between 1% and 22% in 94 cases, in whom it correlated directly and significantly with platelet and leukocyte counts, palpable splenomegaly at diagnosis, and venous thrombosis occurring after diagnosis. Conclusions: JAK2V617F allele burden imparts additional phenotypic effects in ET. No significant financial relationships to disclose.