PF237 THE PROGNOSTIC IMPACT OF LYMPHOCYTE REPERTOIRE RECOVERY AFTER INDUCTION TREATMENT OF CHILDHOOD AML
Authors: Svaton M. , Novakova M. , Reznickova L. , Darzentas N. , Stuchly J. , Svenkrtova A. , Zaliova M. , Zuna J. , Mejstrikova E. , Kotrova M. , Reigl T. , Hrusak O. , Stary J. , Trka J. , Fronkova E. 
Published: June 2019

Background:

Next‐generation sequencing (NGS) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements enables detailed analysis of lymphocyte antigen repertoire composition. We have shown previously that the diversity of IG heavy chain (IGH) rearrangements after induction treatment predicts relapse of childhood acute lymphoblastic leukaemia (ALL), independently of minimal residual disease (MRD) level (Kotrova, Blood, 2015). The evaluation of such prognostic markers in childhood acute myeloid leukaemia (AML) is difficult due to the rarity and heterogeneity of the disease.

Aims:

Our aim was to find out whether the B and T cell lymphocyte repertoire diversity after two induction blocks (day 56) correlates with the outcome or with other risk factors in childhood AML.

Methods:

Sixty childhood AML patients treated with BFM‐AML04 and BFM‐AML12 protocols were enrolled in the study based on the availability of DNA at d56 of treatment. Patients with Down syndrome AML or M3 subtype were excluded. The study cohort was enriched for patients with relapse (n = 30). 29 of 60 patients had a translocation involving the KMT2A (MLL) gene. 41 (68%) patients reached complete remission (CR) at d28, 15 (25%) by d56 and 4 (7%) reached CR later. Median follow‐up of the patients without event was 3.2 years.

The sequencing libraries for IGH, TR beta (TRB) and gamma (TRG) rearrangements were prepared from bone marrow (BM) DNA using 2‐step PCR with multiplex target‐specific primers in 1st round and sequencing adaptors and individual barcodes in 2nd round. Final libraries were sequenced on MiSeq (Illumina) employing the 2 × 250 bp kit, and the sequencing data were analysed using the ARResT/Interrogate platform. Flow cytometry (FC) for lymphocyte subpopulations and MRD was performed using 4 to 8‐colour antibody staining.

Results:

Patients who achieved CR at d28 had better relapse‐free survival (RFS) compared to patients with later remission, however no such correlation was observed for overall survival (OS). The diversity of IGH rearrangements correlated strongly with B lymphocyte counts according to flow cytometry. CR status (d28 vs d56) had no impact on IGH, TRB or TRG repertoire composition at d56. Low IGH repertoire diversity measured as Shannon entropy was associated with poorer RFS (35.6 ± 8 vs. 86.2 ± 9%). This is retained also in the favourable subgroup with CR at d28 (51.5 ± 9% vs 90 ± 9%). FC evaluation of B lymphocyte counts provides similar prediction, but IGH diversity assessment is more sensitive and provides detailed sequence data of IGH rearrangements. IGH diversity was higher in patients with MLL translocation, however even in this group patients with relapse tend to have lower diversity.

Summary/Conclusion:

B lymphocyte regeneration in BM at d56, that can be effectively and sensitively determined by sequencing of IG/TR repertoire, possibly reflects condition and ability of native immune system under antileukaemic treatment and appears to be a promising prognostic factor predicting relapse in childhood AML.

Supported by AZV 16–32568A, PRIMUS/17/MED/11 and LO1604.