Randomized Phase III BMT CTN Trial of Calcineurin Inhibitor–Free Chronic Graft-Versus-Host Disease Interventions in Myeloablative Hematopoietic Cell Transplantation for Hematologic Malignancies

PURPOSE Calcineurin inhibitors (CNI) are standard components of graft-versus-host disease (GVHD) prophylaxis after hematopoietic cell transplantation (HCT). Prior data suggested that CNI-free approaches using donor T-cell depletion, either by ex vivo CD34 selection or in vivo post-transplant cyclophosphamide (PTCy) as a single agent, are associated with lower rates of chronic GVHD (cGVHD). METHODS This multicenter phase III trial randomly assigned patients with acute leukemia or myelodysplasia and an HLA-matched donor to receive CD34-selected peripheral blood stem cell, PTCy after a bone marrow (BM) graft, or tacrolimus and methotrexate after BM graft (control). The primary end point was cGVHD (moderate or severe) or relapse-free survival (CRFS). RESULTS Among 346 patients enrolled, 327 received HCT, 300 per protocol. Intent-to-treat rates of 2-year CRFS were 50.6% for CD34 selection (hazard ratio [HR] compared with control, 0.80; 95% CI, 0.56 to 1.15; P = .24), 48.1% for PTCy (HR, 0.86; 0.61 to 1.23; P = .41), and 41.0% for control. Corresponding rates of overall survival were 60.1% (HR, 1.74; 1.09 to 2.80; P = .02), 76.2% (HR, 1.02; 0.60 to 1.72; P = .95), and 76.1%. CD34 selection was associated with lower moderate to severe cGVHD (HR, 0.25; 0.12 to 0.52; P = .02) but higher transplant-related mortality (HR, 2.76; 1.26 to 6.06; P = .01). PTCy was associated with comparable cGVHD and survival outcomes to control, and a trend toward lower disease relapse (HR, 0.52; 0.28 to 0.96; P = .037). CONCLUSION CNI-free interventions as performed herein did not result in superior CRFS compared with tacrolimus and methotrexate with BM. Lower rates of moderate and severe cGVHD did not translate into improved survival.

Rash in Patients after T-Cell-Depleted Peripheral Blood Stem Cell Transplantation: Eosinophilia and Pruritus Do Not Distinguish Acute Graft-Versus-Host Disease from Drug Rash

Background: The incidence and severity of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (alloHSCT) is decreased with T-cell depletion. However, ~14% of these T-cell depleted (TCD) recipients will have aGVHD with skin involvement (Barba et al. 2017). Furthermore, alloHSCT recipients are at increased risk for drug eruptions and infectious exanthems (Byun et al. 2011). While histopathologic differences have been reported between aGVHD and non-aGVHD rash in TCD recipients (Fischer et al. 2015), skin biopsies alone are insufficient to determine rash etiology. As such, distinguishing inflammatory non-aGVHD rashes from aGVHD of the skin after TCD HSCT remains challenging and relies on clinical presentation. While peripheral eosinophilia is seen in both aGVHD and drug hypersensitivity, rashes that present with concomitant eosinophilia after HSCT are often suspected to have a drug-induced etiology. We sought to assess the incidence and features of aGVHD and non-aGVHD rashes within 1 year after TCD alloHSCT, as well as common etiologies of non-aGVHD rash. These findings may guide clinicians in earlier diagnosis and management of non-aGVHD rash. Methods: Using a clinical research database, 243 adult patients were identified who received alloTCD peripheral blood stem cell transplantation (PBSCT) at a single institution between 2008 and 2011. All patients had CD34+ hematopoietic progenitor cells selected using: the Isolex 300i Magnetic Cell, followed by additional T-cell rosetting with neuraminidase-treated sheep erythrocytes or using the CliniMACS CD34+ Reagent System (Table 1). Given decreased incidence of aGVHD with Isolex versus CliniMACS (Barba et al. 2017), we established Isolex and CliniMACS TCD groups and assessed aGVHD and non-aGVHD rash within these two CD34+ selection modalities. To identify non-aGVHD patients with skin rash, charts were reviewed from date of HSCT through 1 year post HSCT by review of dermatology visit notes or by extraction of International Classification of Diseases-9 (ICD-9) codes for skin lesion or rash (Table 2); skin infections and chronic GVHD rashes were excluded. Acute GVHD was diagnosed with histological confirmation when clinically indicated. Rash characteristics, including pruritus and peripheral eosinophilia at onset of rash, were collected from charts of both non-aGVHD and aGVHD rash patients. Results: Among 243 TCD PBSCT transplant recipients, 152 patients (63%) were identified with skin rash within 1 year after HSCT. Of these patients, 43 had aGVHD rash and 109 had non-aGVHD rash. The majority of aGVHD rashes had skin stage III aGVHD regardless of CD34+ selection method (Table 3). For patients with non-aGVHD rash, etiologies included inflammatory conditions (Table 4). TCD by Isolex led to non-aGVHD rash development at a median onset of 60 days and aGVHD rash at a median onset of 71 days; while TCD by CliniMACS led to non-aGVHD rash development at a median onset of 56.5 days and aGVHD rash at a median onset of 75 days. Of patients who had recorded complete blood counts at onset of rash, elevated percent eosinophilia (% eosinophils >7%) was present in 10% of Isolex and 5% of CliniMACS non-aGVHD rashes versus 3% of Isolex and 4% of CliniMACS aGVHD rashes. Peripheral eosinophilia was not associated with aGVHD versus non-aGVHD skin rash post TCD HSCT (p≥0.99) nor when separated by CD34+ selection method (Isolex p=0.673; CliniMACS p≥0.99). While non-aGVHD skin rash patients had higher incidence of pruritus compared to aGVHD skin rash, pruritus was not a significant predictor of aGVHD versus non-aGVHD skin rash (p=0.20) nor when separated by CD34+ selection modality (Isolex p=0.188; CliniMACS p=0.469). Conclusions: In our case series of 243 TCD PBSCT recipients of whom 152 had skin rashes, over three-quarters of all non-aGVHD skin rashes with clear etiologies were attributed to drug eruptions. Our results suggest that the commonly utilized feature of peripheral eosinophilia may not be helpful in differentiating between aGVHD and drug rashes after TCD alloHSCT. Additionally, pruritus at rash onset was not helpful in distinguishing cause of rash as due to aGVHD or non-aGVHD after TCD alloHSCT. Based on these data, if clinical scenario supports GVHD associated rash, the presence of peripheral eosinophilia or pruritus should not delay initiation of therapy for GVHD. Disclosures No relevant conflicts of interest to declare.

Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow

Introduction Gene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery. Methods Twenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests. Results BM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p<0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p<0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p<0.05; CD36 22% vs. 11%, p<0.05; CD62P 16% vs. 3%, p<0.05; GPA 16% vs. 4%, p<0.05; CD41/61 19% vs. 3%, p<0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p<0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p<0.05) and on day 0 (p<0.05) and 1 (p<0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p<0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p<0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p<0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p<0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with >50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p<0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p<0.05) and ACD-A (88% vs. 68 ± 0.7%, p<0.05). Conclusions SCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. Disclosures Luo: bluebird bio Inc.: Employment. Pierciey: bluebird bio: Employment.