Allogeneic NK cells eradicate myeloblasts but not neoplastic mast cells in systemic mastocytosis associated with acute myeloid leukemia
Authors: Celalettin Ustun, Sarah Williams, Sasha Skendzel, Behiye Kodal, Michel Arock, Jason Gotlib, Daniel A. Vallera, Sarah Cooley, Martin Felices, Daniel Weisdorf, Jeffrey Miller
Journal: American Journal of Hematology (2017)

Systemic mastocytosis (SM) with an associated hematologic malignancy (SM-AHN) is classified as a specific variant of SM by the World Health Organization (WHO).1 In some of these patients, AHN is diagnosed as acute myeloid leukemia (AML),2 often with the KIT D816V mutation.3 These patients have been treated with therapeutic regimens targeting AML, yet, these conventional therapies generally been ineffective in the treatment of the SM component of the disease.2, 4 It was reported that the lymphoid cells seen in this setting were an admixture of T and B lymphocytes, while natural killer (NK) cells were virtually absent in the bone marrow (BM) of patients with SM.5 Allogeneic hematopoietic cell transplantation (HCT) can cure some of these patients, documenting the anti-neoplastic potency of lymphocyte (and or NK cell) mediated graft-versus-mast cell activity.2 Whether NK cells have a cytolytic effect on neoplastic mast cells in SM has not been reported. We present two patients with SM-AML who received haploidentical NK-cell investigational therapy6 for relapsed/refractory AML. SM component was diagnosed later in the phase of their therapy by our center. A 61-year-old male diagnosed with normal karyotype AML did not respond to standard induction (cytarabine and idarubicin, 7 & 3) or reinduction chemotherapy (mitoxantrone, etoposide and cytarabine, MEC; Figure 1A–C). Before starting an NK-cell based relapsed refractory AML trial, we noticed atypical mast cells in the patient's BM biopsy in addition to 15% myeloblasts (Figure 1D). These mast cells had aberrant coexpression of CD2 and CD25. Serum tryptase level was elevated (33 ng/mL), and KIT D816V mutation positive. The patient was diagnosed with SM by WHO criteria (1 major and ≥ 2 minor criteria; Figure 1D). At day 0, allogeneic haploidentical NK cells (7.9 × 106/kg) enriched by depletion of CD3+ and CD19+ cells were infused after lymphodepleting preparative regimen (fludarabine and cyclophosphamide). Interleukin 15 (IL-15, 2 mcg/kg/day) was administered subcutaneously (days 1–5 and 8–12) to promote NK cell expansion in vivo. At day7, 11% of circulating blood leukocytes were donor NK cells. At day 14 and 28 after therapy, his BM biopsy was hypocellular with marked reduction in the myeloblasts populations. However, mast cells persisted and confirmed with mast cell tryptase immunohistochemical staining (Figure 1E,F). He received HCT from HLA full-matched sibling at day 60. Immunohistochemical stains of BM trephine sections of patient 1 (A–F) and patient 2 (G–I), labeled with an anti-tryptase tryptase highlighting mast cells, (A) at the time of diagnosis, original magnification ×50 oil, (B) day 14 of induction chemotherapy, original magnification ×50 oil, (C) day 13 of reinduction chemotherapy, original magnification ×50 oil, (D) scattered mast cells prior to NK cell therapy, original magnification ×10, (E) rare persistent mast cells on day 14, original magnification ×10, and (F), slightly increased 28 following therapy, original magnification ×10. In patient 2, (G) increased mast cells prior to NK cell therapy, original magnification ×10×, (H) persistently increased mast cells two weeks after NK cell therapy, original magnification ×10, (I) postmortem evaluation of the spleen with scattered mast cells five weeks after NK cell therapy and two weeks after allogeneic HCT, original magnification ×10. A 56-year-old man was diagnosed with secondary AML (preceding myelodysplastic syndrome treated with 5-azacytidine) with del(5q) and monosomy7 failed induction and reinduction chemotherapies (7 & 3; MEC) and received an experimental haploidentical NK-cell therapy followed by TCRα/β depleted HCT from the same donor. In addition to persistent myeloblasts (5%), there were dense mast cell infiltrates, including some areas where the mast cells were spindling (Figure 1G) in his pre-treatment BM. Aberrant coexpression of CD2 and CD25 could not be definitively demonstrated. Serum tryptase was elevated (43 ng/mL), KIT mutation was negative by next generation sequencing. The patient received reduced intensity conditioning (fludarabine, cyclophosphamide, two fractions of total body irradiation totaling 400 cGy followed by infusion of the CD3-,CD19-NK cellular product (3.5 × 107/kg after overnight culture with 1000 IU/mL IL-2) from his haploidentical donor followed by IL-2 (9 × 106 u/day) subcutaneously qod for six doses. Fourteen days after NK cell infusion, a BM biopsy showed no increase in myeloblasts, but persistence of the expanded mast cells population. (Figure 1H). At day 7 and 14, circulating leukocytes were 16% and 11%, respectively. Serum tryptase remained high at 87.3 ng/mL. The patient underwent allogeneic haploidentical HCT after antithymocyte globulin at day 20 but died with neurologic toxicity and sepsis two weeks after the allogeneic HCT. Postmortem examination of the spleen demonstrated persistent mast cells infiltration by immunohistochemistry (Figure 1I). To evaluate if neoplastic mast cells could efficiently activate NK cells, we tested NK cell in vitro activity against two established human mast cell lines harboring no KIT mutation (HMC1.1) or KIT mutation (ROSA KIT D816V) and compared activation by the HL-60 AML cell line. Normal donor peripheral blood mononuclear cells (PBMCs) were incubated with the target cell line ± IL-15 to induce NK cell activation and priming (Figure 2). Degranulation, via surface CD107a measurement, and inflammatory cytokine production, via intracellular IFNγ measurement, were assessed on the gated NK cell population by flow cytometry. While incubation with HL-60 cells as targets yielded NK cell degranulation without (12.32% ± 4.07%) or with IL-15 (21.07% ± 5.75%), incubation with the mast cell lines HMC1.1 or ROSA KIT D816V yielded negligible CD107a expression with (0.69% ± 0.08% and 1.31% ± 0.31%) or without (1.61% ± 0.54% and 2.96% ± 1.42%) stimulatory IL-15 (Figure 2A). Cytokine production was more evident in the IL-15-treated group with HL-60 cells inducing 3.5-fold greater IFNγ compared with incubation with either mast cell line (HL-60: 7.48% ± 1.5% vs. HMC1.1: 1.19% ± 0.51%, and ROSA KIT D816V: 2.09% ± 0.64%; Figure 2B). Taken together these data indicate that AML cells induce NK cell activation more than mast cells. PBMCs were isolated by Ficoll gradient and placed in culture for 4 h with mast cell (HMC1.1 or ROSA KIT D816V) and AML cell (HL-60) lines in the presence or absence of IL-15 (10 ng/mL) at a 2:1 effector (PBMC) to target (HMC1.1/ROSA KITD816V/HL-60) ratio. Cells were then stained with anti-CD107a-FITC, anti-IFNγ-BV421 (intracellular), anti-CD56-PE-Cy7, and anti-CD3-PE-CF594. Cells were then run in BD LSRII flow cytometer and data was analyzed with Flowjo software. CD107a (A) and IFNγ (B) were then evaluated within the CD56 + CD3- NK cell population. Bars depict mean ± SEM. N = 3 We could not obtain long-term results from NK cell therapy in these patients because of early death and transplantation. However, these clinical data support the contention that AML-directed chemotherapy and immunosuppressive pre-NK cell therapy are ineffective in eliminating neoplastic mast cells.2 More importantly, we documented that NK cells, supported by either IL-2 or IL-15, do not effectively decrease the neoplastic mast cell burden, although NK cells are active in depleting AML.6 The mechanism how mast cells may evade NK cell recognition remains unclear. We are evaluating strategies to better understand the mechanisms and engage NK cells to mast cells by targeting CD33. The authors thank to Dr. Joseph H. Butterfield for graciously providing HMC1.1 cells and Dixie Lewis for her contribution in case report preparation. Authors have no conflict of interest to disclose relevant to this study Conceived the concept: Ustun, Miller Clinical data collection: Ustun, Skendzel Data analysis: Ustun Literature search: Ustun, Skendzel Writing article: Ustun, Miller, Skendzel, Williams, Gotlib, Vallera, Weisdorf, Cooley, Kodal, Felices, Arock Editing article: Ustun, Miller, Skendzel, Williams, Gotlib, Vallera, Weisdorf, Cooley, Kodal, Felices, Arock Conceived the concept: Miller Preclinical data analysis: Miller, Gotlib, Vallera, Weisdorf, Arock Clinical data collection: Skendzel Pathologic data collection and analysis: Williams Providing figure: Williams, Cooley, Kodal, Felices Clinical data: Gotlib, Vallera, Weisdorf Performed experiments: Cooley, Kodal, Felices Analyzing clinical data: Arock Providing cell lines: Arock