Results of a Prospective Clinical Trial of Pre-DLI Lymphoreduction Using Oral Fludarabine In Patients with Mixed Chimerism Post Allogeneic Transplant.
Authors: Shaw Bronwen E. , Byrne Jenny , Das-Gupta Emma , Ethell Mark , Figueroa Daniel , Madrigal J. Alejandro , Morgan Gareth J , Tulpule Sameer , Potter Michael , Russell Nigel H. 
Published: October 2019

Abstract
Abstract 1299
Donor leukocyte infusions (DLI) are frequently used following reduced intensity conditioned (RIC) transplants to convert mixed donor chimerism (MC) to full donor chimerism (FDC). In part, due to the significant correlation, which several studies have shown between persisting MC and an increased risk of disease relapse. There are few factors which have been consistently shown to predict for responsiveness. We recently reported response rates (conversion to FDC) of approximately 50% in patients with MC. Patients with a high peripheral blood lymphocyte count pre-DLI were significantly less likely to respond than patients with a low lymphocyte count (33% compared to 89%) (Shaw BE et al, BBMT 2007;13(5):550-9). Based on these and other data we hypothesised that the use of a lymphoreducing agent pre-DLI in those with a high lymphocyte count would enhance DLI responsiveness and hence improve clinical outcomes.
We instituted a prospective pilot trial (CCR2942) to investigate this hypothesis. Inclusion criteria were: 1. mixed chimerism in whole blood (< 95% donor), 2. previous reduced intensity transplant for a haematological malignancy or failure to respond to a previous dose of DLI and 3. a lymphocyte count of >1.0 × 109/l. Patients consenting to the trial received three doses of oral fludarabine as an outpatient at 25mg/m2 on day -7, -6 and -5. DLI was given on day 0. The starting dose of DLI for patients with MC alone was 5 × 105 CD3/kg (unrelated donor) or 1 × 106 CD3/kg (sibling donor). Higher doses were used in those with evidence of both disease and MC. Samples were collected at various time points for Ki67 and T cell subset analysis.
To date, 15 patients have been entered onto the trial and 13 have reached the study end point (day 90 chimerism) (1 early death due to leukaemia relapse, 1 currently too early for assessment). Each received a single dose of DLI. The disease categories were: AML (8), ALL (1), MDS (2), T-PLL (1), HD (1), DLBCL (1) and MCL (1). Only 2 patients had co-existing evidence of disease (morphological relapse of AML, 2% positive by immunophenotyping in T-PLL). The mean age was 51 years (range: 22–66), 9 males and 6 females. 11 HLA-matched sibling donors and 4 10/10 allele matched unrelated donors. The majority of patients (11) had conditioning with fludarabine, melphalan and alemtuzumab, with cyclosporine post transplant.
The median time to DLI post-transplant was 7.2 months (range: 3–11). The median percentage of donor chimerism pre-DLI was 85% (range: 18–93). The median lymphocyte count pre-DLI was 1.3 (range: 1.0–2.7). Of the 13 eligible patients, 9 (69%) have responded to a single dose of DLI (CR=8 (>95%), PR=1 (chimerism 94% donor at study end)). Three patients developed GvHD – grade 1 (liver), grade III (liver/GI), grade IV (liver/skin). GVHD resolved completely in all cases and all patients achieved FDC. 3 patients reactivated a virus (CMV, 2 × EBV), although not all required treatment.
The chimerism level pre-transplant was associated with response, with those below the median having a trend towards a worse response rate (p=0.052, Fishers exact test). As only 2 patients had measurable disease at study entry, this parameter was not statistically assessed, however the patient with a frank relapse of AML progressed rapidly despite DLI, suggesting that this strategy may not be sufficiently aggressive in that setting.
We analysed the patterns in T cell subsets (CD8, CD4 and Tregs). There was a significant decrease in the absolute numbers of CD8 and CD4 cells between day -7 and day 0, while the absolute number of Tregs was not significantly changed. Interestingly, we found significantly higher absolute counts at all time points in both CD8 and Treg subsets in the DLI responders, compared to non-responders (unpaired t-test, mean: 13.66 vs 0.03, p<0.0001 and mean: 0.018 vs 0.008, p<0.004 respectively). There were no significant differences in CD4 counts.
In conclusion, in patients with MC, the use of pre-DLI lymphoreduction results in good response rates, superior to a historical cohort. The incidence of GVHD and adverse events is low. Preliminary finding suggest that both CD8 and Treg subsets may play a role in responsiveness. For patients with very low levels of donor chimerism or frank disease relapse an increased intensity of pre-DLI chemotherapy is likely to be necessary. Larger patient numbers and randomised studies are required to investigate the efficacy of this approach further.

Disclosures:
No relevant conflicts of interest to declare.