Genetic Heterogeneity of t(8;21)(q22;q22.1) Acute Myeloid Leukemia Revealed By High-Throughput Targeted Sequencing

Background: Recently, a comprehensive high-throughput sequencing study of pediatric and adult core-binding factor (CBF) AML [t(8;21), n=85; inv(16), n=80] provided novel aspects of the genetic heterogeneity and disease biology of t(8;21) AML (Faber et al. Nat Genet 2016). In addition to well-established variants in genes encoding for proteins in tyrosine kinase (TK)/RAS signaling, epigenetic regulation (ER), and in the cohesin complex (CC), novel candidate genes were identified.

Aims: We aimed to validate and to further extend recent findings by comprehensive profiling of the mutational spectrum of t(8;21) AML using a high-throughput targeted sequencing (HTS) approach.

Methods: HTS was performed of the entire coding region of 244 genes with evidence in hematological malignancies. Pretreatment peripheral blood or bone marrow specimens of 143 adult t(8;21) AML patients (pts) were analyzed. 114/143 pts were enrolled in seven prospective AMLSG treatment trials. Libraries (total probes size: 1.36 Mbp) were prepared using SureSelectXT custom solutions (Agilent). Paired-end sequencing was carried out on a HiSeq platform (Illumina).

Results: A mean on-target sequencing depth of 900x was obtained for all pts. Coding variants without known SNP annotation and with a minimum variant allele fraction (VAF) of 5% were called. In sum 756 mutations (missense: 70%; indels: 19%; nonsense: 9%; other: 2%) were detected with an average of 5.3 per pt (SD: ±2.5). 99% of pts harbored at least 1 mutation and 90% 3 or more. In line with previous studies, activating mutations in TK/RAS signaling pathways represented recurrent events: KIT (34/143; 24%), NRAS (24/143; 17%), FLT3 (15/143; 10%; point mutations only), and KRAS (4/143; 3%).In addition to these mutations conferring proliferative clonal advantage, a striking enrichment of mutations was observed in genes involved in histone modification and DNA methylation, ASXL2 (22/143; 15%), ASXL1 (21/143; 15%), KDM6A (14/143; 10%), EZH2 (11/143; 8%), CREBBP (8/143; 6%), SRCAP (8/143; 6%), TET2 (19/143; 13%) and DNMT3A (9/143; 6%), emphasizing their contribution to impairing differentiation in this leukemia subtype. Further, frequent aberrations were found in the cohesin genes that are involved in sister chromatid segregation during mitosis and DNA repair: RAD21 (17/143; 12%), SMC1A (10/143; 7%), STAG2 (5/143; 3%), and SMC3 (5/143; 3%). In general, mutations in genes belonging to the same functional subgroups were rarely co-occurring suggesting functional redundancy without additional synergistic effect. We also identified recurrent variants in previously detected candidate genes outside these functional clusters. A remarkably high incidence of loss of function mutations was found in the ZBTB7A gene(27/143; 19%), encoding for a transcription factor guiding hematopoietic lineage fate. Recurrent mutations were also observed in CCND2 (14/143; 10%) , involved in hematopoietic cell proliferation, MGA (11/143; 8%), a negative regulator of MYC, as well as DHX15 (10/143; 7%), associated with ribosome biogenesis and spliceosome function. When focusing on the clonal hierarchy we found that the median VAF in genes belonging to ER and CC (0.31; range 0.3-0.91; 0.35, range 0.05-0.96; respectively) was higher than in genes associated with TK/RAS signaling (0.16, range 0.05-0.53) suggesting that mutations affecting the epigenetic state and differentiation occur earlier than those impairing proliferation during development of t(8;21) AML.

Conclusion: Using a comprehensive sequencing approach we could further delineate the molecular pattern of t(8;21) AML that despite shared genetic lesions is remarkably different to inv(16) AML. Here, mutation clusters in genes involved in TK/RAS signaling, ER and CC were confirmed as well as novel t(8;21)-associated gene mutations such as in ZBTB7A and CCND2 that play an essential role in regulation of hematopoietic cell proliferation and differentiation. Further analyses including sample size extension as well as correlation of findings with clinical parameters are ongoing.