- Email: [email protected]
More acute lymphoid leukemia than acute myeloid leukemia blasts are killed by rabbit antithymocyte globulin
Cytotherapy, 2019; 21: 1161 1165
SHORT REPORT
More acute lymphoid leukemia than acute myeloid leukemia blasts are killed by rabbit antithymocyte globulin
ROSY DABAS1, POONAM-DHARMANI KHAN1,2, MONICA MODI2, FAISAL M. KHAN1,2 & JAN STOREK1,2 1
Cumming School of Medicine, University of Calgary, Calgary, Canada, and 2Alberta Health Services, Alberta, Canada
Abstract Rabbit antithymocyte globulin (ATG, thymoglobulin), a polyclonal antibody, is used to prevent graft-versus-host disease (GVHD) and graft failure in the setting of allogeneic hematopoietic cell transplantation (HCT). Recent in vitro studies suggest that ATG also has anti-leukemic activity. Whether acute lymphoid leukemia (ALL) or acute myeloid leukemia (AML) is more sensitive to ATG is not known. We used primary cells from 12 B-ALL and 38 AML patients and measured ATGinduced complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC) at clinically relevant ATG concentrations (10 and 50 mg/L). At 50 mg/L, ALL blasts were killed to a greater degree than AML blasts by CDC (median 96% vs 50% dead cells, P = 0.001) as well as CIC (median 23% vs 11% apoptotic cells, P = 0.049). At 10 mg/L, the difference was significant for CDC but not CIC. In conclusion, the anti-leukemic activity of ATG, particularly CDC, is more potent for ALL than AML in vitro. If this applies in vivo, ATG-based GVHD prophylaxis may be particularly advantageous for ALL.
Key Words: acute lymphoid leukemia, acute myeloid leukemia, anti-thymocyte globulin, graft-versus-host disease, hematopoietic cell transplantation
Introduction Inclusion of polyclonal rabbit anti-thymocyte globulin (ATG, thymoglobulin) to myeloablative conditioning is known to reduce the risk of acute and chronic graftversus-host disease (GVHD) without increasing the risk of relapse [1 3]. ATG contains antibodies targeting a wide range of antigens expressed on both normal and malignant hematolymphoid cells, including T cells, B cells, natural killer cells, dendritic cells, plasma cells and leukemic stem cells and blasts [4]. Thus, in vitro, ATG kills leukemic stem cells and blasts [5 9]. This may be why ATG does not increase relapse, even though ATG is expected to impair the graft-versus-leukemia effect (GVL) [1 3]. ATG kills leukemic cells via both complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC, direct induction of apoptosis) [9]. Whether the
ATG-induced CDC or CIC is greater in acute lymphoid leukemia (ALL) than in acute myelogenous leukemia (AML) is unknown. We hypothesized greater cytotoxicity against ALL than AML because ATG is manufactured from the sera of rabbits immunized with human thymocytes, which consist of T cells and precursors, B cells and stromal cells, but few myeloid cells [10,11]. Here we evaluated this hypothesis.
Methods Patient population We studied heparin-anticoagulated blood specimens from patients with newly diagnosed (before induction chemotherapy) AML (n = 38) or B-cell ALL (B-ALL; n = 12) with circulating blasts. Patient characteristics are shown in Table I. The study was
Correspondence: Rosy Dabas, PhD, 3310 Hospital Drive NW, T2N 4N1 Calgary, Alberta, Canada. E-mail: [email protected] (Received 22 March 2019; accepted 23 August 2019) ISSN 1465-3249 Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2019.08.003
1162 R. Dabas et al. Table I. Patient characteristics. Number of patients
50
Age, median (range) 47.5 (20 98) Acute leukemia type (ALL/AML) (12/38) (23/15) AML subtype (M0 2/M4 5) ALL subtype (B cell/T cell) (12/0) % AML Blasts,a median (range) 65 (10 97) % ALL Blasts,a median (range) 65 (23 96) Cytogenetic risk status of AML (4/23/9/2) (Better/intermediate/poor/unknown)b Cytogenetic + molecular risk status of AML (6/5/14/9) (Better/intermediate/poor/unknown)b Cytogenetic risk status of ALL (Ph+/Ph /unknown) (6/5/1) M, male; F, female; Ph+, Philadelphia chromosome positive; Ph , Philadelphia chromosome negative. a Blasts were counted microscopically on Giemsa-stained smears by a clinical hematology lab. b National Comprehensive Cancer Network, Clinical Practice Guidelines in Oncology, Acute myeloid leukemia, Version 3.2017, Unknown cytogenetic + molecular risk status was defined as either cytogenetics not done/not available or minimum molecular test set (Flt3 internal tandem duplication + NPM1 mutation) not done/ not available.
approved by the Health Research Ethics Board of Alberta, and informed consents were obtained. Measuring CDC and CIC The assays have been described] Briefly, density gradient-separated mononuclear cells (MNCs) were incubated with a clinically relevant concentration of thymoglobulin (10 or 50 mg/L) as described previously [9]. Percent 7AAD+ (7-amino actinomycin D positive, ie, dead) or annexin V+ (apoptotic) cells among leukemic blasts were measured by flow cytometry. In ALL, the leukemic blasts were defined as cells that were CD45dim/neg, SSclow and lineageneg (CD117, CD33, CD41a, CD235a, CD14 and CD16). In AML other than the M4 or M5 subtypes (according to British-American-French classification), the leukemic blasts were defined as cells that were CD45dim/neg, side scatter low (SSclow) and lineageneg (negative for CD10, CD19, CD41a, CD235a, CD14 and CD16). For the M4 or M5 subtypes of AML, the leukemic blasts were defined as cells that are CD45dim/neg, SSclow/intermediate and lineageneg (negative for CD10, CD19, CD41a, CD235a, CD14 and CD16). In the CDC assay, MNCs were incubated for 15 min at 37˚C and 5% CO2 with ATG in the presence of 20% human active serum (containing complement). As a negative control, MNCs were incubated with ATG in the presence of 20% inactive serum (with heat-inactivated complement). As a positive control, MNCs were incubated with 300 mg/L ATG in the presence of 20% active serum. In the
case of 300 mg/L in the presence of active serum, cytotoxicity was determined among total MNCs. Additional controls were used as described previously [9]. After incubation and washing, cells were stained with 7AAD and the fluorochrome-conjugated antibodies identifying leukemic blasts (see the previous paragraph) and analyzed by flow cytometry. The readout was the adjusted (background subtracted) percent dead (7AAD+) cells among leukemic blasts. This was calculated as % 7AAD+ leukemic blasts treated with ATG in the presence of complement minus % 7AAD+ leukemic blasts treated with ATG in the absence of complement. In the CIC assay, MNCs were incubated for 4 h at 37˚C and 5% CO2 with ATG in medium (RPMI 1640) supplemented with 10% heat-inactivated fetal bovine serum. As a negative control, cells were incubated without ATG. As a positive control, cells were incubated for 4 h with ATG 300 mg/L or exposed to ultraviolet light for 10 min to induce apoptosis. Additional controls were used as described previously [9]. After incubation and washing, cells were stained with annexin V conjugated to Alexa Fluor 488, 7AAD and the fluorochrome-conjugated antibodies identifying leukemic blasts and analyzed by flow cytometry. The readout was the adjusted (background subtracted) percent apoptotic (annexin V+) cells among leukemic blasts. This was calculated as % annexin V+ leukemic blasts incubated with ATG minus % annexin V+ leukemic blasts incubated without ATG. Statistics The significance of the difference in the % 7AAD+ or annexin V+ AML versus ALL blasts was determined using the Mann-Whitney U test. P < 0.05 (twosided) was considered significant. Results In the CDC assay, ALL blasts were killed to a greater degree than AML blasts (Figure 1). At 10 mg/L ATG, median adjusted percent 7AAD+ cells was 22.6% among ALL blasts versus 2.3% among AML blasts (P = 0.001). At 50 mg/L ATG, it was 94.6% among ALL blasts versus 50.0% among AML blasts (P = 0.001). In the CIC assay, at 10 mg/L ATG, there was no significant difference between % annexin V+ ALL and AML blasts. At 50 mg/L, ALL blasts were killed to a greater degree than AML blasts (Figure 2). Median adjusted percent of annexin V+ cells was 23.1% among ALL blasts versus 10.1% among AML blasts (P = 0.049). At 300 mg/L ATG, median adjusted percent annexin V+ MNCs was 50.6% in ALL specimens versus 39.6% in AML specimens
ALL versus AML blasts with rabbit ATG treatment
1163
Figure 1. ATG induced CDC of AML versus ALL blasts. (Color version of figure is available online.).
(P = 0.741, not shown in Figure 2). Interestingly, with 10-min exposure to ultraviolet light, AML blasts were killed to a greater degree than ALL blasts. Specifically, the median adjusted percent annexin V+ MNCs was 41.8% in ALL versus 49.7% in AML specimens (P = 0.663, not shown in Figure 2).
Discussion In our study, we analyzed the cytotoxic effect of ATG on AML versus ALL blasts. ATG was more cytotoxic against ALL than AML blasts. We hypothesize that ATG has a higher affinity for or targets more antigens on ALL than AML blasts. Our
rationale for the latter hypothesis is that of all known antigens targetted by ATG, fewer are expressed on AML blasts (CD45, HLA Class I, CD184, CD11b and CD56) [4] than on B-ALL blasts (CD19, CD20, CD27, CD38, CD45, CD126, HLA Class I and II and CD138) [4,12,13]. Our study has limitations: (i) B-ALL but not Tcell ALL (T-ALL) blasts were studied. Although only »20% ALLs in Europe and North America are T-ALLs, this percent is higher in Eastern Asia. Thus, this is a significant limitation. (ii) The effect of ATG on blasts and not leukemic stem cells (LSCs) was studied because the immunophenotypic definition of AML LSCs is relatively accepted, but controversy remains regarding the immunophenotypic
Figure 2. ATG induced CIC of AML versus ALL blasts. (Color version of figure is available online.).
1164 R. Dabas et al. definition of ALL LSCs [14 15]. (iii) In vitro, but not in vivo, activity of ATG against leukemic cells was studied. Indirect evidence for high activity of ATG against ALL in vivo may be that among 44 ALL patients receiving ATG with conditioning, only 7 (16%) relapsed [16]. (iv) The small number of ALL patients in our study (n = 12) may not be representative of all ALL patients. Therefore, we cannot conclusively determine whether the higher degree of killing by ATG of ALL compared with AML blasts is applicable to most cases of B-ALL. B-ALL is known to respond in vivo to monoclonal antibodies, naked or conjugated to a toxin such as rituximab (anti-CD20) or inotuzumab (anti-CD22) [17 19]. In B-cell non-Hodgkin lymphoma, a combination of antibodies targetting both CD20 and CD22 may be more efficacious than targetting either antigen alone [20,21]. This has not been tested in ALL. Nevertheless, it is conceivable that the multiplicity of ALL antigens targeted by ATG is the reason for the antiALL effect of ATG in vitro and possibly also in vivo. To determine whether a certain subgroup of patients in our study had leukemic blasts more or less sensitive to ATG, for ALL, we split patients into those with versus without the Philadelphia (Ph) chromosome. There appeared to be no difference between these two groups. For AML, we split the patients into those whose AML blasts aberrantly expressed at least one vs no lymphoid antigen (CD2, CD4, CD7, or CD10). Although not statistically significant, the median percent killing by ATG via CDC (not CIC) was higher in the former group. Specifically, the median adjusted percent killed AML blasts in the CDC assay (at ATG concentration of 50 mg/L) was 51.3% in the cases with aberrant lymphoid marker expression versus 38.0% in the cases without aberrant lymphoid marker expression (data not shown). What is the relevance of our in vitro finding of ATG killing more ALL blasts than AML blasts to patients, given that so far, no study has shown a lower incidence of ALL or AML relapse with ATG compared with no ATG? We speculate that the “no decrease in relapse” in both ALL and AML patients could be due to different effects of pre-HCT versus post-HCT ATG exposure (area under the curve [AUC]) on relapse. The post-HCT AUC, but not the pre-HCT AUC, is expected to impair GVL effect. Consistent with that, a high pre-HCT AUC has been associated with decreased incidence of relapse in AML, whereas a high post-HCT AUC has been associated with an increased incidence of relapse [22,23]. Unfortunately, similar data do not exist for ALL. In an European Society for Blood and Marrow Transaplantation (EBMT) registry study, Czerw et al. [24] demonstrated no difference in outcomes for Ph-negative ALL patients
receiving ATG versus no ATG in terms of leukemia-free survival and overall survival. This might be attributed to the anti-GVL effect of ATG neutralizing the anti-leukemic effect of ATG. Together, these studies suggest that to harness the anti-leukemic effect of ATG in both ALL and AML patients without jeopardizing the GVL effect, ATG may need to be administered early during conditioning to achieve a high ratio of preHCT to post-HCT AUC. Overall, our study demonstrated that ATG has marked anti-ALL activity in vitro. Experiments in humanized mice and clinical trials are needed to determine whether ATG also has anti-leukemic activity in vivo. Declaration of Competing Interest Supported by grants from Alberta Innovates Health Solution (AIHS) and the Alberta Cancer Foundation (ACF). JS received research support from Sanofi. The other authors have no commercial, proprietary or financial interest in the products or companies described in this article. Author Contributions RD developed the cytotoxicity assays, ran the assays and analyzed the results. PDK provided input into assay development and interpretation. RD performed all statistical analyses included in the study. FMK and JS provided critical feedback. JS designed the study and supervised its conduct. RD wrote the manuscript. Acknowledgments We thank the patients for participating in research that would not benefit them but only future patients. We also thank the healthy volunteers involved in the study. This study could not have been conducted without the dedication of Mamta Kantharia, Jennifer LeBlanc, Lori Rackel and Laura Spilchen; numerous inpatient nurses; pharmacists, particularly Michelle Dowhan; and physicians, notably Dr. Michelle Geddes, Dr. Mona Shafey, Dr. Peter Duggan and Dr. Lynne Savoie. We also thank the staff of Calgary Laboratory Services, including Glenis Doiron. Finally, we thank Douglas Mahoney for invaluable feedback during this study. References [1] Walker I, Panzarella T, Couban S, Couture F, Devins G, Elemary M, et al. Pretreatment with anti-thymocyte globulin versus no anti-thymocyte globulin in patients with
ALL versus AML blasts with rabbit ATG treatment
[2]
[3]
[4] [5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
haematological malignancies undergoing haemopoietic cell transplantation from unrelated donors: a randomised, controlled, open-label, phase 3, multicentre trial. Lancet Oncol 2016;17:164–73. Chang YJ, Wang Y, Mo XD, Zhang XH, Xu LP, Yan CH, et al. Optimal dose of rabbit thymoglobulin in conditioning regimens for unmanipulated, haploidentical, hematopoietic stem cell transplantation: Long-term outcomes of a prospective randomized trial. Cancer 2017;123:2881–92. Bacigalupo A, Lamparelli T, Barisione G, Bruzzi P, Guidi S, Alessandrino PE, et al. Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplant-related mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol Blood Marrow Transplant 2006;12: 560–5. Mohty M. Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond. Leukemia 2007;21:1387–94. Liu H, Qin Y, Wang X, Yang K, Zhu J, Zha C, et al. Polyclonal rabbit antithymocyte globulin induces apoptosis and has cytotoxic effects on human leukemic cells. Clin Lymphoma Myeloma Leuk 2012;12:345–54. Ayuk F, Maywald N, Hannemann S, Larsen U, Zander A, Kroger N. Comparison of the cytotoxicity of 4 preparations of anti-T-cell globulins in various hematological malignancies. Anticancer Res 2009;29:1355–60. Grullich C, Ziegler C, Finke J. Rabbit anti T-lymphocyte globulin induces apoptosis in peripheral blood mononuclear cell compartments and leukemia cells, while hematopoetic stem cells are apoptosis resistant. Biol Blood Marrow Transplant 2009;15:173–82. Dabas R, Dharmani-Khan P, Modi M, Van Slyke T, Luider J, Morris D, et al. Anti-thymocyte globulin’s activity against acute myeloid leukemia stem cells. Bone Marrow Transplantation 2018. Dabas R, Lee R, Servito MT, Dharmani-Khan P, Modi M, Van Slyke T, et al. Antithymocyte globulin at clinically relevant concentrations kills leukemic blasts. Biology of Blood and Marrow Transplantation 2016;22:815–24. Akashi K, Richie LI, Miyamoto T, Carr WH, Weissman IL. B lymphopoiesis in the thymus. J Immunol 2000;164:5221–6. Isaacson PG, Norton AJ, Addis BJ. The human thymus contains a novel population of B lymphocytes. Lancet 1987;2: 1488–91. Zand MS, Vo T, Huggins J, Felgar R, Liesveld J, Pellegrin T, et al. Polyclonal rabbit antithymocyte globulin triggers B-cell and plasma cell apoptosis by multiple pathways. Transplantation 2005;79:1507–15. Storek J, Mohty M, Boelens JJ. Rabbit Anti-T Cell Globulin in Allogeneic Hematopoietic Cell Transplantation. Biol Blood Marrow Transplant 2015;21:959–70. le Viseur C, Hotfilder M, Bomken S, Wilson K, R€ ottgers S, Schrauder A, et al. In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
1165
maturation have stem cell properties. Cancer Cell 2008;14:47–58. Bernt KM, Armstrong SA. Leukemia stem cells and human acute lymphoblastic leukemia. Semin Hematol 2009;46:33–8. Daly A, Savoie ML, Geddes M, Chaudhry A, Stewart D, Duggan P, et al. Fludarabine, Busulfan, antithymocyte globulin, and total body irradiation for pretransplantation conditioning in acute lymphoblastic leukemia: excellent outcomes in all but older patients with comorbidities. Biology of Blood and Marrow Transplantation 2012;18:1921–6. Thomas DA, O’Brien S, Faderl S, Garcia-Manero G, Ferrajoli A, Wierda W, et al. Chemoimmunotherapy with a modified hyper-CVAD and rituximab regimen improves outcome in de novo Philadelphia chromosome-negative precursor B-lineage acute lymphoblastic leukemia. J Clin Oncol 2010;28:3880–9. Hoelzer D, Huettmann A, Kaul F, Sebastian I, Nadja J, Martin M, et al. Immunochemotherapy with rituximab improves molecular CR rate and outcome in CD20+ B-lineage standard and high risk patients; results of 263 CD20+ patients studied prospectively in GMALL study 07/2003. Blood 2010;116. 170-170. Kantarjian HM, DeAngelo DJ, Stelljes M, Liedtke M, Stock W, G€ okbuget N, et al. Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia. N Engl J Med 2016;375:740–53. Weber T, Botticher B, Mier W, Sauter M, Kr€amer S, Leotta K, et al. High treatment efficacy by dual targeting of Burkitt’s lymphoma xenografted mice with a (177)Lu-based CD22specific radioimmunoconjugate and rituximab. Eur J Nucl Med Mol Imaging 2016;43:489–98. Kraeber-Bodere F, Pallardy A, Maisonneuve H, Campion L, Moreau A, Soubeyran I, et al. Consolidation anti-CD22 fractionated radioimmunotherapy with (90)Y-epratuzumab tetraxetan following R-CHOP in elderly patients with diffuse large B-cell lymphoma: a prospective, single group, phase 2 trial. Lancet Haematol 2017;4:e35–45. Dabas R, Jamani K, Kangarloo SB, Dharmani-Khan P, Williamson TS, Ousia S, et al. Antirelapse effect of pretransplant exposure to rabbit antithymocyte globulin. Blood Advances 2019;3:1394. Admiraal R, Nierkens S, de Witte MA, Petersen EJ, Fleurke GJ, Verrest L, et al. Association between anti-thymocyte globulin exposure and survival outcomes in adult unrelated haemopoietic cell transplantation: a multicentre, retrospective, pharmacodynamic cohort analysis. Lancet Haematol 2017;4:e183–91. Czerw T, Labopin M, Giebel S, Socie G, Volin L, Fegueux N, et al. Anti-thymocyte globulin improves survival free from relapse and graft-versus-host disease after allogeneic peripheral blood stem cell transplantation in patients with Philadelphia-negative acute lymphoblastic leukemia: An analysis by the Acute Leukemia Working Party of the EBMT. Cancer 2018;124:2523–33.
SHORT REPORT
More acute lymphoid leukemia than acute myeloid leukemia blasts are killed by rabbit antithymocyte globulin
ROSY DABAS1, POONAM-DHARMANI KHAN1,2, MONICA MODI2, FAISAL M. KHAN1,2 & JAN STOREK1,2 1
Cumming School of Medicine, University of Calgary, Calgary, Canada, and 2Alberta Health Services, Alberta, Canada
Abstract Rabbit antithymocyte globulin (ATG, thymoglobulin), a polyclonal antibody, is used to prevent graft-versus-host disease (GVHD) and graft failure in the setting of allogeneic hematopoietic cell transplantation (HCT). Recent in vitro studies suggest that ATG also has anti-leukemic activity. Whether acute lymphoid leukemia (ALL) or acute myeloid leukemia (AML) is more sensitive to ATG is not known. We used primary cells from 12 B-ALL and 38 AML patients and measured ATGinduced complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC) at clinically relevant ATG concentrations (10 and 50 mg/L). At 50 mg/L, ALL blasts were killed to a greater degree than AML blasts by CDC (median 96% vs 50% dead cells, P = 0.001) as well as CIC (median 23% vs 11% apoptotic cells, P = 0.049). At 10 mg/L, the difference was significant for CDC but not CIC. In conclusion, the anti-leukemic activity of ATG, particularly CDC, is more potent for ALL than AML in vitro. If this applies in vivo, ATG-based GVHD prophylaxis may be particularly advantageous for ALL.
Key Words: acute lymphoid leukemia, acute myeloid leukemia, anti-thymocyte globulin, graft-versus-host disease, hematopoietic cell transplantation
Introduction Inclusion of polyclonal rabbit anti-thymocyte globulin (ATG, thymoglobulin) to myeloablative conditioning is known to reduce the risk of acute and chronic graftversus-host disease (GVHD) without increasing the risk of relapse [1 3]. ATG contains antibodies targeting a wide range of antigens expressed on both normal and malignant hematolymphoid cells, including T cells, B cells, natural killer cells, dendritic cells, plasma cells and leukemic stem cells and blasts [4]. Thus, in vitro, ATG kills leukemic stem cells and blasts [5 9]. This may be why ATG does not increase relapse, even though ATG is expected to impair the graft-versus-leukemia effect (GVL) [1 3]. ATG kills leukemic cells via both complement-dependent cytotoxicity (CDC) and complement-independent cytotoxicity (CIC, direct induction of apoptosis) [9]. Whether the
ATG-induced CDC or CIC is greater in acute lymphoid leukemia (ALL) than in acute myelogenous leukemia (AML) is unknown. We hypothesized greater cytotoxicity against ALL than AML because ATG is manufactured from the sera of rabbits immunized with human thymocytes, which consist of T cells and precursors, B cells and stromal cells, but few myeloid cells [10,11]. Here we evaluated this hypothesis.
Methods Patient population We studied heparin-anticoagulated blood specimens from patients with newly diagnosed (before induction chemotherapy) AML (n = 38) or B-cell ALL (B-ALL; n = 12) with circulating blasts. Patient characteristics are shown in Table I. The study was
Correspondence: Rosy Dabas, PhD, 3310 Hospital Drive NW, T2N 4N1 Calgary, Alberta, Canada. E-mail: [email protected] (Received 22 March 2019; accepted 23 August 2019) ISSN 1465-3249 Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2019.08.003
1162 R. Dabas et al. Table I. Patient characteristics. Number of patients
50
Age, median (range) 47.5 (20 98) Acute leukemia type (ALL/AML) (12/38) (23/15) AML subtype (M0 2/M4 5) ALL subtype (B cell/T cell) (12/0) % AML Blasts,a median (range) 65 (10 97) % ALL Blasts,a median (range) 65 (23 96) Cytogenetic risk status of AML (4/23/9/2) (Better/intermediate/poor/unknown)b Cytogenetic + molecular risk status of AML (6/5/14/9) (Better/intermediate/poor/unknown)b Cytogenetic risk status of ALL (Ph+/Ph /unknown) (6/5/1) M, male; F, female; Ph+, Philadelphia chromosome positive; Ph , Philadelphia chromosome negative. a Blasts were counted microscopically on Giemsa-stained smears by a clinical hematology lab. b National Comprehensive Cancer Network, Clinical Practice Guidelines in Oncology, Acute myeloid leukemia, Version 3.2017, Unknown cytogenetic + molecular risk status was defined as either cytogenetics not done/not available or minimum molecular test set (Flt3 internal tandem duplication + NPM1 mutation) not done/ not available.
approved by the Health Research Ethics Board of Alberta, and informed consents were obtained. Measuring CDC and CIC The assays have been described] Briefly, density gradient-separated mononuclear cells (MNCs) were incubated with a clinically relevant concentration of thymoglobulin (10 or 50 mg/L) as described previously [9]. Percent 7AAD+ (7-amino actinomycin D positive, ie, dead) or annexin V+ (apoptotic) cells among leukemic blasts were measured by flow cytometry. In ALL, the leukemic blasts were defined as cells that were CD45dim/neg, SSclow and lineageneg (CD117, CD33, CD41a, CD235a, CD14 and CD16). In AML other than the M4 or M5 subtypes (according to British-American-French classification), the leukemic blasts were defined as cells that were CD45dim/neg, side scatter low (SSclow) and lineageneg (negative for CD10, CD19, CD41a, CD235a, CD14 and CD16). For the M4 or M5 subtypes of AML, the leukemic blasts were defined as cells that are CD45dim/neg, SSclow/intermediate and lineageneg (negative for CD10, CD19, CD41a, CD235a, CD14 and CD16). In the CDC assay, MNCs were incubated for 15 min at 37˚C and 5% CO2 with ATG in the presence of 20% human active serum (containing complement). As a negative control, MNCs were incubated with ATG in the presence of 20% inactive serum (with heat-inactivated complement). As a positive control, MNCs were incubated with 300 mg/L ATG in the presence of 20% active serum. In the
case of 300 mg/L in the presence of active serum, cytotoxicity was determined among total MNCs. Additional controls were used as described previously [9]. After incubation and washing, cells were stained with 7AAD and the fluorochrome-conjugated antibodies identifying leukemic blasts (see the previous paragraph) and analyzed by flow cytometry. The readout was the adjusted (background subtracted) percent dead (7AAD+) cells among leukemic blasts. This was calculated as % 7AAD+ leukemic blasts treated with ATG in the presence of complement minus % 7AAD+ leukemic blasts treated with ATG in the absence of complement. In the CIC assay, MNCs were incubated for 4 h at 37˚C and 5% CO2 with ATG in medium (RPMI 1640) supplemented with 10% heat-inactivated fetal bovine serum. As a negative control, cells were incubated without ATG. As a positive control, cells were incubated for 4 h with ATG 300 mg/L or exposed to ultraviolet light for 10 min to induce apoptosis. Additional controls were used as described previously [9]. After incubation and washing, cells were stained with annexin V conjugated to Alexa Fluor 488, 7AAD and the fluorochrome-conjugated antibodies identifying leukemic blasts and analyzed by flow cytometry. The readout was the adjusted (background subtracted) percent apoptotic (annexin V+) cells among leukemic blasts. This was calculated as % annexin V+ leukemic blasts incubated with ATG minus % annexin V+ leukemic blasts incubated without ATG. Statistics The significance of the difference in the % 7AAD+ or annexin V+ AML versus ALL blasts was determined using the Mann-Whitney U test. P < 0.05 (twosided) was considered significant. Results In the CDC assay, ALL blasts were killed to a greater degree than AML blasts (Figure 1). At 10 mg/L ATG, median adjusted percent 7AAD+ cells was 22.6% among ALL blasts versus 2.3% among AML blasts (P = 0.001). At 50 mg/L ATG, it was 94.6% among ALL blasts versus 50.0% among AML blasts (P = 0.001). In the CIC assay, at 10 mg/L ATG, there was no significant difference between % annexin V+ ALL and AML blasts. At 50 mg/L, ALL blasts were killed to a greater degree than AML blasts (Figure 2). Median adjusted percent of annexin V+ cells was 23.1% among ALL blasts versus 10.1% among AML blasts (P = 0.049). At 300 mg/L ATG, median adjusted percent annexin V+ MNCs was 50.6% in ALL specimens versus 39.6% in AML specimens
ALL versus AML blasts with rabbit ATG treatment
1163
Figure 1. ATG induced CDC of AML versus ALL blasts. (Color version of figure is available online.).
(P = 0.741, not shown in Figure 2). Interestingly, with 10-min exposure to ultraviolet light, AML blasts were killed to a greater degree than ALL blasts. Specifically, the median adjusted percent annexin V+ MNCs was 41.8% in ALL versus 49.7% in AML specimens (P = 0.663, not shown in Figure 2).
Discussion In our study, we analyzed the cytotoxic effect of ATG on AML versus ALL blasts. ATG was more cytotoxic against ALL than AML blasts. We hypothesize that ATG has a higher affinity for or targets more antigens on ALL than AML blasts. Our
rationale for the latter hypothesis is that of all known antigens targetted by ATG, fewer are expressed on AML blasts (CD45, HLA Class I, CD184, CD11b and CD56) [4] than on B-ALL blasts (CD19, CD20, CD27, CD38, CD45, CD126, HLA Class I and II and CD138) [4,12,13]. Our study has limitations: (i) B-ALL but not Tcell ALL (T-ALL) blasts were studied. Although only »20% ALLs in Europe and North America are T-ALLs, this percent is higher in Eastern Asia. Thus, this is a significant limitation. (ii) The effect of ATG on blasts and not leukemic stem cells (LSCs) was studied because the immunophenotypic definition of AML LSCs is relatively accepted, but controversy remains regarding the immunophenotypic
Figure 2. ATG induced CIC of AML versus ALL blasts. (Color version of figure is available online.).
1164 R. Dabas et al. definition of ALL LSCs [14 15]. (iii) In vitro, but not in vivo, activity of ATG against leukemic cells was studied. Indirect evidence for high activity of ATG against ALL in vivo may be that among 44 ALL patients receiving ATG with conditioning, only 7 (16%) relapsed [16]. (iv) The small number of ALL patients in our study (n = 12) may not be representative of all ALL patients. Therefore, we cannot conclusively determine whether the higher degree of killing by ATG of ALL compared with AML blasts is applicable to most cases of B-ALL. B-ALL is known to respond in vivo to monoclonal antibodies, naked or conjugated to a toxin such as rituximab (anti-CD20) or inotuzumab (anti-CD22) [17 19]. In B-cell non-Hodgkin lymphoma, a combination of antibodies targetting both CD20 and CD22 may be more efficacious than targetting either antigen alone [20,21]. This has not been tested in ALL. Nevertheless, it is conceivable that the multiplicity of ALL antigens targeted by ATG is the reason for the antiALL effect of ATG in vitro and possibly also in vivo. To determine whether a certain subgroup of patients in our study had leukemic blasts more or less sensitive to ATG, for ALL, we split patients into those with versus without the Philadelphia (Ph) chromosome. There appeared to be no difference between these two groups. For AML, we split the patients into those whose AML blasts aberrantly expressed at least one vs no lymphoid antigen (CD2, CD4, CD7, or CD10). Although not statistically significant, the median percent killing by ATG via CDC (not CIC) was higher in the former group. Specifically, the median adjusted percent killed AML blasts in the CDC assay (at ATG concentration of 50 mg/L) was 51.3% in the cases with aberrant lymphoid marker expression versus 38.0% in the cases without aberrant lymphoid marker expression (data not shown). What is the relevance of our in vitro finding of ATG killing more ALL blasts than AML blasts to patients, given that so far, no study has shown a lower incidence of ALL or AML relapse with ATG compared with no ATG? We speculate that the “no decrease in relapse” in both ALL and AML patients could be due to different effects of pre-HCT versus post-HCT ATG exposure (area under the curve [AUC]) on relapse. The post-HCT AUC, but not the pre-HCT AUC, is expected to impair GVL effect. Consistent with that, a high pre-HCT AUC has been associated with decreased incidence of relapse in AML, whereas a high post-HCT AUC has been associated with an increased incidence of relapse [22,23]. Unfortunately, similar data do not exist for ALL. In an European Society for Blood and Marrow Transaplantation (EBMT) registry study, Czerw et al. [24] demonstrated no difference in outcomes for Ph-negative ALL patients
receiving ATG versus no ATG in terms of leukemia-free survival and overall survival. This might be attributed to the anti-GVL effect of ATG neutralizing the anti-leukemic effect of ATG. Together, these studies suggest that to harness the anti-leukemic effect of ATG in both ALL and AML patients without jeopardizing the GVL effect, ATG may need to be administered early during conditioning to achieve a high ratio of preHCT to post-HCT AUC. Overall, our study demonstrated that ATG has marked anti-ALL activity in vitro. Experiments in humanized mice and clinical trials are needed to determine whether ATG also has anti-leukemic activity in vivo. Declaration of Competing Interest Supported by grants from Alberta Innovates Health Solution (AIHS) and the Alberta Cancer Foundation (ACF). JS received research support from Sanofi. The other authors have no commercial, proprietary or financial interest in the products or companies described in this article. Author Contributions RD developed the cytotoxicity assays, ran the assays and analyzed the results. PDK provided input into assay development and interpretation. RD performed all statistical analyses included in the study. FMK and JS provided critical feedback. JS designed the study and supervised its conduct. RD wrote the manuscript. Acknowledgments We thank the patients for participating in research that would not benefit them but only future patients. We also thank the healthy volunteers involved in the study. This study could not have been conducted without the dedication of Mamta Kantharia, Jennifer LeBlanc, Lori Rackel and Laura Spilchen; numerous inpatient nurses; pharmacists, particularly Michelle Dowhan; and physicians, notably Dr. Michelle Geddes, Dr. Mona Shafey, Dr. Peter Duggan and Dr. Lynne Savoie. We also thank the staff of Calgary Laboratory Services, including Glenis Doiron. Finally, we thank Douglas Mahoney for invaluable feedback during this study. References [1] Walker I, Panzarella T, Couban S, Couture F, Devins G, Elemary M, et al. Pretreatment with anti-thymocyte globulin versus no anti-thymocyte globulin in patients with
ALL versus AML blasts with rabbit ATG treatment
[2]
[3]
[4] [5]
[6]
[7]
[8]
[9]
[10] [11]
[12]
[13]
[14]
haematological malignancies undergoing haemopoietic cell transplantation from unrelated donors: a randomised, controlled, open-label, phase 3, multicentre trial. Lancet Oncol 2016;17:164–73. Chang YJ, Wang Y, Mo XD, Zhang XH, Xu LP, Yan CH, et al. Optimal dose of rabbit thymoglobulin in conditioning regimens for unmanipulated, haploidentical, hematopoietic stem cell transplantation: Long-term outcomes of a prospective randomized trial. Cancer 2017;123:2881–92. Bacigalupo A, Lamparelli T, Barisione G, Bruzzi P, Guidi S, Alessandrino PE, et al. Thymoglobulin prevents chronic graft-versus-host disease, chronic lung dysfunction, and late transplant-related mortality: long-term follow-up of a randomized trial in patients undergoing unrelated donor transplantation. Biol Blood Marrow Transplant 2006;12: 560–5. Mohty M. Mechanisms of action of antithymocyte globulin: T-cell depletion and beyond. Leukemia 2007;21:1387–94. Liu H, Qin Y, Wang X, Yang K, Zhu J, Zha C, et al. Polyclonal rabbit antithymocyte globulin induces apoptosis and has cytotoxic effects on human leukemic cells. Clin Lymphoma Myeloma Leuk 2012;12:345–54. Ayuk F, Maywald N, Hannemann S, Larsen U, Zander A, Kroger N. Comparison of the cytotoxicity of 4 preparations of anti-T-cell globulins in various hematological malignancies. Anticancer Res 2009;29:1355–60. Grullich C, Ziegler C, Finke J. Rabbit anti T-lymphocyte globulin induces apoptosis in peripheral blood mononuclear cell compartments and leukemia cells, while hematopoetic stem cells are apoptosis resistant. Biol Blood Marrow Transplant 2009;15:173–82. Dabas R, Dharmani-Khan P, Modi M, Van Slyke T, Luider J, Morris D, et al. Anti-thymocyte globulin’s activity against acute myeloid leukemia stem cells. Bone Marrow Transplantation 2018. Dabas R, Lee R, Servito MT, Dharmani-Khan P, Modi M, Van Slyke T, et al. Antithymocyte globulin at clinically relevant concentrations kills leukemic blasts. Biology of Blood and Marrow Transplantation 2016;22:815–24. Akashi K, Richie LI, Miyamoto T, Carr WH, Weissman IL. B lymphopoiesis in the thymus. J Immunol 2000;164:5221–6. Isaacson PG, Norton AJ, Addis BJ. The human thymus contains a novel population of B lymphocytes. Lancet 1987;2: 1488–91. Zand MS, Vo T, Huggins J, Felgar R, Liesveld J, Pellegrin T, et al. Polyclonal rabbit antithymocyte globulin triggers B-cell and plasma cell apoptosis by multiple pathways. Transplantation 2005;79:1507–15. Storek J, Mohty M, Boelens JJ. Rabbit Anti-T Cell Globulin in Allogeneic Hematopoietic Cell Transplantation. Biol Blood Marrow Transplant 2015;21:959–70. le Viseur C, Hotfilder M, Bomken S, Wilson K, R€ ottgers S, Schrauder A, et al. In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
1165
maturation have stem cell properties. Cancer Cell 2008;14:47–58. Bernt KM, Armstrong SA. Leukemia stem cells and human acute lymphoblastic leukemia. Semin Hematol 2009;46:33–8. Daly A, Savoie ML, Geddes M, Chaudhry A, Stewart D, Duggan P, et al. Fludarabine, Busulfan, antithymocyte globulin, and total body irradiation for pretransplantation conditioning in acute lymphoblastic leukemia: excellent outcomes in all but older patients with comorbidities. Biology of Blood and Marrow Transplantation 2012;18:1921–6. Thomas DA, O’Brien S, Faderl S, Garcia-Manero G, Ferrajoli A, Wierda W, et al. Chemoimmunotherapy with a modified hyper-CVAD and rituximab regimen improves outcome in de novo Philadelphia chromosome-negative precursor B-lineage acute lymphoblastic leukemia. J Clin Oncol 2010;28:3880–9. Hoelzer D, Huettmann A, Kaul F, Sebastian I, Nadja J, Martin M, et al. Immunochemotherapy with rituximab improves molecular CR rate and outcome in CD20+ B-lineage standard and high risk patients; results of 263 CD20+ patients studied prospectively in GMALL study 07/2003. Blood 2010;116. 170-170. Kantarjian HM, DeAngelo DJ, Stelljes M, Liedtke M, Stock W, G€ okbuget N, et al. Inotuzumab ozogamicin versus standard therapy for acute lymphoblastic leukemia. N Engl J Med 2016;375:740–53. Weber T, Botticher B, Mier W, Sauter M, Kr€amer S, Leotta K, et al. High treatment efficacy by dual targeting of Burkitt’s lymphoma xenografted mice with a (177)Lu-based CD22specific radioimmunoconjugate and rituximab. Eur J Nucl Med Mol Imaging 2016;43:489–98. Kraeber-Bodere F, Pallardy A, Maisonneuve H, Campion L, Moreau A, Soubeyran I, et al. Consolidation anti-CD22 fractionated radioimmunotherapy with (90)Y-epratuzumab tetraxetan following R-CHOP in elderly patients with diffuse large B-cell lymphoma: a prospective, single group, phase 2 trial. Lancet Haematol 2017;4:e35–45. Dabas R, Jamani K, Kangarloo SB, Dharmani-Khan P, Williamson TS, Ousia S, et al. Antirelapse effect of pretransplant exposure to rabbit antithymocyte globulin. Blood Advances 2019;3:1394. Admiraal R, Nierkens S, de Witte MA, Petersen EJ, Fleurke GJ, Verrest L, et al. Association between anti-thymocyte globulin exposure and survival outcomes in adult unrelated haemopoietic cell transplantation: a multicentre, retrospective, pharmacodynamic cohort analysis. Lancet Haematol 2017;4:e183–91. Czerw T, Labopin M, Giebel S, Socie G, Volin L, Fegueux N, et al. Anti-thymocyte globulin improves survival free from relapse and graft-versus-host disease after allogeneic peripheral blood stem cell transplantation in patients with Philadelphia-negative acute lymphoblastic leukemia: An analysis by the Acute Leukemia Working Party of the EBMT. Cancer 2018;124:2523–33.