Abstract 4492: Characterization of the T-Cell receptor repertoire in patients with acute myeloid leukemia treated with 5-Azacytidine plus chemotherapy
Authors: Beckford John , Fulton Noreen , Odenike Toyosi , Stock Wendy , Alachkar Houda 
Published: September 2020

Acute Myeloid Leukemia (AML) is a hematological malignancy with a 5 year overall survival of less than 30%. DNA hypomethylating agents such as 5-Azacytidine (5-Aza) have been increasingly used in AML therapy. The effect of hypomethylating agents on T-cell driven anti-leukemia responses remains unclear. For anti-leukemia responses, T-cells must express a T-cell receptor (TCR) with specificity for cancer antigens. TCR recombination, the rearrangement of V, D (beta only) and J segments across the TCR alpha and beta loci into a mature TCR locus, shapes the antigen specificity of TCRs. DNA methylation across the TCR loci may limit the rearrangement of particular V, D, J segments and impact TCR repertoire composition. Whether 5-Aza can alter TCR loci methylation patterns, increase TCR diversity and enhance T-cell driven anti-leukemia responses is unknown.

In a cohort of 23 patient with AML, we performed TCR-seq on pre- (diagnostic) and post-therapy (day 5 post 5-Aza) peripheral blood RNA samples obtained from 13 responders and 10 non-responders. Illumina compatible libraries were generated from patient samples using a 5'RACE like approach and sequenced on the MiSeq platform, generating 300x2bp reads. Reads were processed and analyzed using MiXCR and VDJTools, respectively. GraphPad Prism 8 (Two-sided T test) was used for statistical comparisons, P values &lt; 0.05 were considered significant after adjusting for multiple comparisons using the Bonferroni-Dunn method.

We obtained an average of 1,146,043 (SD± 767,216) reads and identified an average of 15,732 (SD ± 17,388) clones per sample. We compared pre- and post-therapy TCR diversity among both outcome groups using the Shannon diversity index. TCR diversity did not significantly change following 5-Aza treatment (4827 vs 6159, P= 0.55). Also, TCR diversity was not significantly different between outcome groups pre- and post-therapy. We designated clones as minor (&lt; 10 reads) or major (&gt; 10 reads). We compared pre- and post-therapy minor clone TRBV exon patterns and discovered a 2.2 fold increase in unique clones containing TRBV16 among responders post therapy (P = 0.017). We also observed a 2.5 fold increase in unique clones containing TRBV7-4 in non-responders post therapy (P = 0.034). There were no significant exon pattern changes in non-stratified analysis. When pre-therapy minor clone exon patterns were compared between outcome groups, a higher frequency of unique clones containing TRBV12-2 in responders versus non-responders was observed (2.8 fold, P &lt; 0.001). In addition, we observed a higher frequency of major clones containing TRBV12-5 among responders post therapy (2.4 fold, P = 0.0558). In the pre-therapy samples, there was a higher frequency of clones containing TRBV5-4 among responders compared to non-responders (2.7 fold, P =0.045).

In conclusion, changes in TCR repertoire features such as the utilization of specific V exons may associate with response to 5-Azacytidine therapy.

Citation Format: John Beckford, Noreen Fulton, Toyosi Odenike, Wendy Stock, Houda Alachkar. Characterization of the T-Cell receptor repertoire in patients with acute myeloid leukemia treated with 5-Azacytidine plus chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4492.