Azacitidine Induces Expansion of Regulatory T Cells and Tumour Specific CD8+ T Lymphocytes After Allogeneic Stem Cell Transplantation: A Strategy for Epigenetic Manipulation of a Graft-Versus-Leukemia Response

Abstract 324

Disease relapse and graft-versus-host disease (GVHD) remain the commonest causes of treatment failure after allogeneic stem cell transplantation (SCT) for Acute Myeloid Leukemia (AML). Azacitidine (AZA) possesses inherent anti-leukemic activity but also expands immunomodulatory T regulatory cells in animal models and up-regulates the expression of tumor antigens in vitro. Reasoning that AZA might selectively augment a graft-versus-leukemia (GVL) without a concomitant increase in GVHD we have studied the tolerability and immunological sequelae of AZA administration in patients who have undergone a reduced intensity allogeneic SCT for AML. All patients were transplanted using a conditioning regimen consisting of fludarabine (30 mg/m² IV × 5 days), melphalan (140 mg/m²IV), and alemtuzumab (10 mg IV × 5 days) with cyclosporine GVHD prophylaxis. Patients received AZA (36 mg/m²) × 5 days every 28 days commencing after sustained neutrophil and platelet engraftment at day +42 with the aim of administering monthly cycles of AZA until 12 months post-transplant. Numbers of CD4+CD25+CD127loFoxP3+ T regulatory cells were measured by flow cytometry. Tumor specific cytotoxic T lymphocytes (CTL) recognising members of the cancer testis antigen (CTA) family and WT1 were quantitated using a CD137 expression and enrichment assay. We report results on 27 patients (median age 59 years) who commenced treatment with AZA (follow-up 3–21 months). 11 patients were transplanted from an HLA identical related donor and 16 from a volunteer unrelated donor. Disease status at the time of transplant was: CR1 n=18; CR2 n=7; first relapse n=2. Post-transplant AZA was well tolerated and 24 patients tolerated at least three cycles of AZA. Haematological toxicity was modest with only two patients experiencing treatment delay for neutropenia, or thrombocytopenia. Three patients developed Grade 2 acute GVHD and no patient developed >Grade 2 acute GVHD. Two patients developed limited chronic GVHD. To date seven patients have relapsed at a median time of 6 months (4–15 months) post-transplant. Administration of AZA had no impact on absolute lymphocyte counts or CD8+ and CD4+ cell numbers compared with a control population of 17 patients transplanted using an identical conditioning regimen. However AZA administration was noted to increase the number of CD4+CD25+CD127loFoxP3+ regulatory T cells within the first 3 months post transplant compared with a time-matched control population (p=0.017) although there was no difference detectable at 6 or 12 months. AZA administration also induced a cytotoxic CD8+ T cell response to candidate tumor antigens MAGE, GAGE, RAGE and WT-1 in the peripheral blood. A CD8+ T cell response to these candidate tumor antigens was only detectable in 1/22 patients pre-transplant but circulating CTA- or WT1 specific CD8+ T cells were detected in 14/16 patients who had received at least six cycles of AZA with a frequency between 0.001–1.4% (mean 0.25%). In four patients with paired peripheral blood and bone marrow samples the size of the CD8+ T cell response was noted to be up to 100 fold greater in the bone marrow. Ex vivo characterisation of the CTA response using dextramers demonstrated the CD8+ T cell response to be effector memory (CD45RA-CCR7-) and functional assays confirmed by mobilization of CD107a and secretion of interferon-g, TNF-a and IL-2 in response to peptide. These data confirm the tolerability of adjunctive AZA post-transplant and its administration appears to be associated with a low incidence of both acute and chronic GVHD. The demonstration that AZA induces both early expansion of T regulatory cells and a tumor specific CD8+ CTL response highlight its potential use as a strategy to epigenetically manipulate a graft-versus-leukemia reaction after allogeneic SCT.