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E. Angelova et al.
Ph-like molecular and cytogenetic signatures are current- ly treated on frontline protocols with tyrosine kinase inhibitors, which have dramatically improved the out- come of this previously poor prognostic group.1-4 Outcomes of patients with T-ALL are generally inferior to those of their B-ALL counterparts, particularly in adults, and the molecular heterogeneity of T-ALL has only recently been uncovered using high-throughput molecular methods. Early T-cell precursor ALL (ETP- ALL) is a subset of T-ALL that was identified recently and found to include a sizeable proportion of patients with poor outcomes.5,6 In contrast to children, only 30- 40% of adults with ALL achieve long-term remission, and survival drops substantially in patients over 60 years of age.7,8 Despite advances in frontline treatment of adult ALL, the prognosis of patients who fail frontline and first salvage therapy is extremely poor9,10 and justifies the need to explore new therapeutic modalities.
approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center (MDACC) and conduct- ed in accordance with the Declaration of Helsinki.
Antibodies and reagents
For the flow cytometry analysis (details below), we used an allophycocyanin-conjugated anti-CD123 (IL-3 receptor α chain) antibody (clone 7G3; BD Pharmingen, BD Biosciences) accord- ing to the manufacturer’s recommendation. The humanized anti-CD123 antibody G4723A and a humanized non-targeting control antibody of the same IgG1 isotype and identical Fc sequences, were generated at ImmunoGen. IMGN632 and the control DGN549 antibody drug conjugate were produced via conjugation of DGN549 to the G4723A and the non-targeting IgG1 antibodies, respectively, as described previously.19
Multicolor/multiparameter flow cytometry analysis
The blast gate was defined on the basis of CD45dim expression and side-scatter characteristics and quantified as a percentage of total gated events. For analysis of CD123 expression, measure- ments included mean fluorescence intensity (MFI) on leukemic blasts (adjusted for background fluorescence using negative internal controls) and relative mean fluorescence intensity (RFI) ratio (leukemic blasts versus non-leukemic events). In patients’ samples, positive CD123 expression (CD123+) was defined as expression in ≥20% of leukemic blasts using MFI by compari- son to background fluorescence and fluorescence on non- leukemic gated events, respectively. Additional details are pro- vided in the Online Supplement.
Cytogenetics and molecular diagnostics
Conventional cytogenetics, fluorescence in situ hybridization, polymerase chain reaction-based molecular diagnostics, and next-generation sequencing-based mutation profiling were per- formed on bone marrow aspirate specimens as described previ- ously.20-22
Cell lines
B-ALL cell lines CRF-SB and JM-1 (from American Type Culture Collection) and KOPN-8, SEM, 380, TOM-1, and SD-1 (from Deutsche Sammlung von Mikroorganismen und Zellkulturen) were procured between 2000 and 2015; they were character- ized by the respective vendors using DNA profiling. Cytotoxicity was assessed using either a water-soluble tetra- zolium salt 8 (WST-8)-based cell viability assay (Dojindo Molecular Technologies) as described previously23 or the alamarBlue Cell Viability Reagent (Invitrogen). Further details are given in the Online Supplement.
In vitro evaluation of primary B-cell acute lymphoblas- tic leukemia samples
Bone marrow mononuclear cells from 11 newly diagnosed and 10 relapsed/refractory B-ALL patients were obtained from MDACC or ConversantBio. The number of CD123 antibody- binding sites per cell (ABC) was quantified by the BD QuantiBRITETM Fluorescence Quantitation Kit (BD Biosciences) using G4723A conjugated to R-phycoerythrin at a 1:1 ratio, as already described.24 Cell proliferation for samples treated with IMGN632 was assessed using the CellTiter-Glo® (Promega) or Cell TraceTM Violet stain (Invitrogen) techniques. Additional details are provided in the Online Supplement.
Statisticalanalysis
The statistical analysis methodology is described in the Online Supplement.
CD123, the interleukin-3 (IL-3) receptor α-chain, is the primary low-affinity subunit of the IL-3 receptor and pro- motes high-affinity binding to IL-3 when co-expressed with the β-subunit. IL-3 is mainly produced by T-lympho- cytes; it regulates the production of hematopoietic cells by stimulating cell cycle progression, differentiation, and inhi- bition of apoptosis. Early studies suggested that IL-3 plays a critical role in leukemogenesis through enabling leukemic cells to escape programmed cell death and grow autonomously.11 CD123 was previously reported to be expressed at a low level or to be absent on normal hematopoietic stem cells, but it is expressed at various lev- els in hematologic malignancies, including hairy cell leukemia,12 acute myeloid leukemia,13 blastic plasmacytoid dendritic cell neoplasm,14-16 and systemic mastocytosis.17 Differential overexpression of CD123 by neoplastic cells and their normal precursors has positioned this cell surface receptor as an attractive target of therapy.
The potential of CD123-targeted therapies in ALL remains largely unexplored. There are some data on CD123 expression in B-ALL, but only limited data for T- ALL.13,15,18 In this report, we present a comprehensive sin- gle-institution survey of CD123 expression in adult ALL and assess the correlation between CD123 expression and clinicopathological factors and outcomes. We also describe the in vitro impact of IMGN632, a conjugate of CD123-targeting antibody with a novel DNA-alkylating payload, in ALL cell lines and patients’ samples.
Methods
Study group
A total of 213 consecutive patients (183 with B-ALL, 30 with T-ALL) were identified and included in the study group. B-ALL patients were further subdivided into Ph+ (121/124 treatment- naïve) and Ph-negative (Ph–) (51/59 treatment-naïve) subsets based on cytogenetic, fluorescence in situ hybridization, and/or molecular detection of t(9;22)(q34.1;q11.2)/BCR-ABL1. In the T- ALL group, 19 patients were treatment-naïve and 11 presented with relapsed/refractory disease after prior treatment. T-ALL patients were subdivided into immunophenotypic subsets based on the expression of CD1a and sCD3: the subsets were early (CD1a−, sCD3−), thymic (CD1a+, sCD3−), or mature (CD1a−, sCD3+) T-ALL. Patients with ETP-ALL were defined as described previously.6 Additional details regarding the study group are provided in the Online Supplement. This study was
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haematologica | 2019; 104(4)