Salvage Therapy with Azacitidine Increases Regulatory T Cells in Patients with AML or MDS and Early Relapse After Allogeneic Blood Stem Cell Transplantation
Authors: Schroeder Thomas , Fröbel Julia , Cadeddu Ron Patrick , Czibere Akos G. , Dienst Ariane , Zipperer Esther , Platzbecker Uwe , Bug Gesine , Uharek Lutz , Fenk Roland , Germing Ulrich , Kröger Nicolaus , Haas Rainer , Kobbe Guido 
Published: October 2019

Abstract
Abstract 1964

Introduction:
Treatment with azacitidine (Aza) and donor lymphocyte infusions (DLI) can induce sustained remissions in some patients (pts) with AML or MDS relapsing after allogeneic stem cell transplantation (allo-SCT). Meanwhile incidence and severity of GvHD seems to be relatively low when compared to historical data using DLI alone. As a potential mechanism murine models have suggested that Aza upregulates the transcription factor FoxP3 thereby expanding CD4+ regulatory T cells (Tregs). This has also been recently shown in 17 AML pts receiving Aza maintenance therapy following allo-SCT (Goodyear et al., 2012).

Patients and Methods:
To confirm and expand this knowledge we monitored CD4+CD25+FoxP3+ Tregs and lymphocyte subsets (CD3+; CD3+/CD4+; CD3+/CD8+; CD3−/CD56+; CD20+) by flow cytometry in 46 pts during salvage therapy with Aza (up to 8 cycles either 100 mg/m2/day d1-5 or 75 mg/m2/d d1-7) and DLI (envisaged on day 34/90/146) for relapse following allo-SCT. PB samples were obtained prior treatment (d0), after the 1st (d6), 2nd (d34), 4th (d90) and 6th cycle (d146). To assure a serial measurement only pts who had received at least 4 Aza cycles were eligible. Thereby 13 pts could be included, while 33 pts were excluded as a consequence of early drop-of resulting from progression or death (n=21), or due to missing samples (n=12).

Results:
Relapse of AML (n=8) or MDS (n=5) occurred in median 446 d (range:19–1688) following allo-SCT in these 13 pts. They received a median of 6 Aza cycles (range: 4–8). DLI were administered in all patients with a median number of 2 DLI per patient (range:1–4) resulting in a median total T cell dose of 5.0×106CD3+ cells/kg per patient (range:1–119). A CR rate of 62% (n=8) was observed in these 13 pts being overestimated in comparison to a CR rate of 33% (n=15) in the whole group due to positive selection of pts.
Prior to relapse 6 pts (46%) had suffered from aGvHD (Io 1 pt, IIo 1 pt, IIIo 3 pts, IVo 1 pt) and 2 pts (15%) from cGvHD (limited 1 pt, extensive 1 pt). At the beginning of Aza treatment 3 pts were still on immunosuppresion which could be tapered in all cases without GvHD flare. Following treatment with Aza aGvHD was observed in 5 pts (overall 38%, Io 3 pts, IIIo2 pts) in median 129 d (range: 20 – 253) following the 1st DLI, while cGvHD developed in 6 pts (overall 46%, limited 5 pts, extensive 1 pt).
In concordance with this rather mild presentation of GvHD, a 1.5-fold increase of Tregs was observed after 4 Aza cycles (d0: 8.23/μl vs. d90: 13.26/μl, p=0.0479). By grouping the pts on the basis of the median time to relapse (day 446), we observed a 3.2-fold increase of the absolute number (d0: 4.7/μl vs. d90: 14.8/μl, p=0.031) as well as an 1.9-fold increase of the frequency of Tregs (d0: 6.7% vs. d90: 12.9% of CD3+CD4+ cells, p=0.06) during treatment with Aza in the group of patients who relapsed early. On the other hand, in those patients who relapsed late the absolute number of Tregs (d0: 12.2/μl vs. d90: 11.9/μl, n. s.) was already higher and remained together with the Treg frequency (d0: 4.7% vs. d90 3.9%, n. s.) unchanged during treatment. Of interest, in those patients with early relapse only 1 pt developed aGvHD (Io), in contrast to 4 pts with aGvHD in those with late relapse. No significant changes were observed with regard to other lymphocyte subpopulations.

Conclusions:
We here demonstrate an intra-individual Treg expansion, which might be induced by Aza and may explain the low rate and mild presentation of GvHD observed following the combination of Aza and DLI. In line with the data of Goodyear et al. Aza-induced expansion of Tregs seems to be restricted to patients relapsing early after allo-SCT where the Treg repertoirs is still immature.

Disclosures:
Schroeder: Celgene: Travel support Other. Platzbecker:Celgene: Honoraria, Research Funding, Speakers Bureau. Bug:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, Travel support Other. Germing:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Kröger:Celgene: Research Funding. Kobbe:Celgene: Research Funding.