Azacitidine-induced reconstitution of the bone marrow T cell repertoire is associated with superior survival in AML patients

Abstract

Hypomethylating agents (HMA) like azacitidine are licensed for the treatment of acute myeloid leukemia (AML) patients ineligible for allogeneic hematopoietic stem cell transplantation. Biomarker-driven identification of HMA-responsive patients may facilitate the choice of treatment, especially in the challenging subgroup above 60 years of age. Since HMA possesses immunomodulatory functions that constitute part of their anti-tumor effect, we set out to analyze the bone marrow (BM) immune environment by next-generation sequencing of T cell receptor beta (TRB) repertoires in 51 AML patients treated within the RAS-AZIC trial. Patients with elevated pretreatment T cell diversity (11 out of 41 patients) and those with a boost of TRB richness on day 15 after azacitidine treatment (12 out of 46 patients) had longer event-free and overall survival. Both pretreatment and dynamic BM T cell metrics proved to be better predictors of outcome than other established risk factors. The favorable broadening of the BM T cell space appeared to be driven by antigen since these patients showed significant skewing of TRBV gene usage. Our data suggest that one course of AZA can cause reconstitution to a more physiological T cell BM niche and that the T cell space plays an underestimated prognostic role in AML.Trial registration: DRKS identifier: DRKS00004519.

Fig. 1. Global immune metrics of the entire cohort.

The figure displays the bone marrow (BM) T cell receptor repertoire richness (A), clonality (B), Simpson (C), and Shannon indices (D) as measures for the repertoire diversity for the healthy control group, as well as the AML samples before treatment initiation (AML day 0) and after the first course of azacitidine (AML day 15).

Fig. 2. AML patients with the most diverse bone marrow (BM) T cell receptor beta (TRB) repertoires at diagnosis harbor favorable prognosis compared to the other patients.

Applying the third quartile of the measured Shannon indices as cut-off we identified a group of AML patients with particularly diverse TRB repertoires (A), who had fewer bone marrow blasts (B) and displayed prolonged event-free (C), and overall survival (D).

Fig. 3. AML patients who experience a richness boost of their bone marrow (BM) T cell receptor beta (TRB) repertoires after the first course of azacitidine have superior prognosis.

Applying the third quartile of the measured richness as cut-off we identified AML patients with extremely rich TRB repertoires after azacitidine treatment (A), without any association to the measured blast count (B), and longer event-free (C), as well as overall survival (D) compared to the other patients.

Fig. 4. AML patients experiencing a richness boost after azacitidine (AZA) treatment display skewed TRBV gene usage.

Principal component analyses (PCA) of TRBV and TRBJ gene usage comparing AML patients with increased T cell repertoire richness after azacitidine to AML patients without immunological response (A, B) revealed TRBV skewing. Similarly, patients with particular diverse T cell repertoires at diagnosis showed differential TRBV (C) and TRBJ gene usage (D).

Fig. 5. Bone marrow T cell receptor beta (TRB) cluster analysis using the GLIPH2 algorithm.

Applying the GLIPH2 algorithm we identified TRB clusters sharing antigen specificity with relevant overlap between healthy donors (HD) and AML samples (A). Thereby, B displays exemplary clonal space analysis for four AML patients pre- and post-azacitidine and the contribution of AML specific clusters as well as clusters shared with HD.

Fig. 6. Favorable T cell receptor beta (TRB) clusters with low generation probability (low pGen) are enriched in patients with T cell richness boost after azacitidine.

Comparing TRB clusters determined with the GLIPH2 algorithm, we identified clusters with low generation probability (−log generation probability >29,89) which were only shared between healthy donors (HD) and AML patients on day 15 after azacitidine (AZA) or enriched in post-AZA samples compared to pretreatment samples (day 0; A). These low generation probability clusters were enriched in AML patients with elevated T cell repertoire richness after AZA (B).