Expanded Natural Killer Cells For The Control Of Minimal Residual Disease In Ph+ Acute Lymphoblastic Leukemia Patients
Authors: Torelli Giovanni Fernando , Peragine Nadia , Raponi Sara , Mariglia Paola , Pauselli Simona , De Propris Maria Stefania , Vitale Antonella , Guarini Anna , Foá Robin 
Published: October 2019

Abstract
The management of Ph+ acute lymphoblastic leukemia (ALL) patients has profoundly changed during the last decade. Virtually all adult Ph+ ALL patients, including the elderly where the abnormality accounts for over 50% of cases, can obtain a complete remission (CR) with the use of TK inhibitors (and steroids) without systemic chemotherapy (Vignetti M et al. Blood 2007;109:3676; Foà R et al. Blood 2011;118:6521). Most patients remain, however, minimal residual disease (MRD)-positive. The possibility of targeting MRD via an immune-mediated control is particularly appealing in these patients. Previous studies have shown that natural killer (NK) cells with killing activity against autologous blasts may be expanded from ALL patients in CR (Torelli GF et al. Haematologica 2005;90:785). NK cell recognition of malignant targets is regulated by activating and inhibitory receptors. The major receptors with activating functions are NKG2D, DNAM-1 and the natural cytotoxicity receptors (NCRs) (NKp30, NKp44 and NKp46). MIC-A/B and ULBPs are ligands for NKG2D, PVR and Nec-2 for DNAM-1, while NCRs are orphan receptors. The pathways of NK-ALL recognition are unclear. Possible differences in NK cell killing susceptibility and in the expression of NK cell activating ligands among subgroups of ALL patients have been suggested.
The aims of this study were: 1) to analyze the pathways of NK-ALL recognition, with particular attention to Ph+ samples; and 2) to verify whether differences in NK cell activating receptor ligand expression among molecularly-defined subgroups of patients correlate with the susceptibility to recognition and killing by NK cells activated and expanded under GMP conditions.
PBMCs were collected from 23 healthy donors and 3 adult Ph+ ALL patients in CR. NK cells were enriched and cultured for 14 days in the presence of irradiated autologous feeder cells, autologous plasma, IL-2 and IL-15. The expression of the activating receptors NKG2D, DNAM-1 and NCRs was then analyzed. Samples from 46 newly diagnosed adult ALLs, median age 34 years (18-74), were also investigated: 39 patients had B-ALL - 15 BCR-ABL+, 7 MLL-AF4+, 2 E2A-PBX1+, 15 negative - and 7 had T-ALL. The expression of the NKG2D and DNAM-1 ligands on ALL blasts was analyzed. The cytotoxic activity of ex vivo expanded NK cells against primary ALL blasts was determined in a 51Cr release assay. For blocking experiments, NK cells were pre-treated with the anti-NKG2D or anti-DNAM-1 neutralizing mAbs.
NK cells from healthy donors and from Ph+ ALL patients could be expanded respectively 33.2±15.2 and 39.1±19.3 fold. Expanded NK cells were represented by a homogenous population displaying a high expression of CD56 and CD16, in the absence of CD3. DNAM-1, NKG2D, NKp30 and NKp44 activating receptors presented a significantly increased expression after expansion from healthy donors (DNAM-1 p=.0007; NKG2D p=.0004; NKp30 p=.05; NKp44 p=.001). DNAM-1 and NKG2D showed a significantly increased expression after expansion also from Ph+ ALL patients (DNAM-1 p=.0012; NKG2D p=.045), while the expression of NCRs was not tested on these samples. The phenotypic analysis performed within molecularly-defined subgroups of ALL revealed that Ph+ cases presented an overall high surface expression of NKG2D and DNAM1 ligands. In particular, when compared to ALLs carrying no known molecular markers, Ph+ samples showed significantly higher levels of ULBP-1, ULBP-3 and MIC-B (p=.008, p=.026 and p=.033, respectively). In line with the phenotypic results, primary blasts from Ph+ ALL (n=5) appeared significantly more susceptible to NK-dependent lysis than B-ALL without molecular aberrations (n=6) (p=.007). When cytotoxic assays were performed in the presence of neutralizing mAbs, the NK cell killing potential was significantly inhibited by anti-DNAM-1 (p=.006), suggesting a pathway of recognition of ALL blast cells in the setting of the Nec-2/DNAM-1 interaction.
The high expression of ligands for activating receptors in Ph+ ALL cases, together with the highest levels of susceptibility to NK cell-mediated lysis by this subgroup of ALL, point to a possible therapeutic use of autologous NK cells activated and expanded ex vivo. Management of Ph+ ALL patients - particularly the elderly - with TK inhibitors plus an immune-based strategy aimed at controlling/eradicating MRD without the use of systemic chemotherapy and/or transplant program is worthy of being investigated.

Disclosures:
No relevant conflicts of interest to declare.