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V. Madan et al.
Figure 1. Mutational landscape of t(8;21) acute myelogenous leukemia (AML). Matrix displays individual somatic mutations detected in diagnosis and relapse AML with RUNX1-RUNX1T1 fusion. Mutational frequencies are illustrated on the left and bar graphs on the right depict absolute number and type of mutations. The annotation bars at the bottom display patient information including the cytogenetic aberrations detected in karyotype analysis. NA: information not available.
along with matched germline (complete remission) DNA (Online Supplementary Table S3). We achieved a mean depth of 100x (range 64-271x); an average 68% of nucleotides were covered by at least 20 reads (Online Supplementary Figure S1). We identified 55 somatic muta- tions (in 52 genes) in newly-diagnosed cases and 76 muta- tions (in 69 genes) at relapse, which included recurrent mutations in KIT, TET2, DHX15 and MGA (Online Supplementary Table S4). We assessed the stability of muta- tions in diagnosis and relapse samples analyzed using whole exome sequencing. Overall, the disease evolution followed the pattern reported previously for AML,35 as the founding clone or a subclone at diagnosis survived the therapy, gained additional mutations, and expanded at relapse (Online Supplementary Figure S2). Interestingly, all four mutations of TET2 (in 3 cases) were acquired at relapse (Online Supplementary Table S4).
Further to uncovering the repertoire of co-operating mutations in t(8;21) AML, we analyzed mutational status of 530 genes (Online Supplementary Table S1) in 76 newly- diagnosed and 19 relapse t(8;21) AML cases using target- ed-exome sequencing (Online Supplementary Table S3). In this cohort, the mean sequencing coverage across targeted bases was 96x (range 76-319x), with 70% of bases covered greater than 20x (Online Supplementary Figure S1).
Overall, we observed that the mutations of KIT were most frequent at both diagnosis and relapse in our cohort, along with recurring alterations in ASXL2, MGA, DHX15, TET2 and FLT3 genes (Figure 1, Online Supplementary Table
S5 and Online Supplementary Figure S3). Somatic mutations of ASXL2 were detected in 20% of newly-diagnosed (17 of 86) and 10% of relapsed (3 of 29) cases while ASXL1 was mutated at a much lower frequency (2% of newly- diagnosed cases and 7% of relapsed cases) (Figure 1, Online Supplementary Table S5 and Online Supplementary Figure S3). Mutations of DHX15, a RNA helicase, occurred exclusively at Arg222 residue in 7 cases. MGA and TET2 genes predominantly harbored nonsense and frameshift mutations spread throughout the transcript (Figure 1, Online Supplementary Figure S3 and Online Supplementary Table S5).
ASXL2 was the second most frequently mutated gene, and interestingly, all alterations were truncating mutations located in exons 11 and 12 (Figure 1 and Online Supplementary Figure S3). Unlike alterations of ASXL1, which are recurrent in several hematologic diseases, recent reports5,8,9 have highlighted the incidence of ASXL2 mutations specifically in the t(8;21) subtype of AML. In this study, we aimed to investigate the consequences of deficiency of ASXL2 on hematopoietic development using a mouse model.
Impaired hematopoiesis in Asxl2-deficient mice
Our RT-PCR analysis showed that Asxl2 is expressed in a broad range of murine hematopoietic cell types (Online Supplementary Figure S4A). To investigate the physiological role of ASXL2 in steady-state hematopoiesis, we used gene- trap mice (referred to as Asxl2 KO mice) described previous-
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haematologica | 2018; 103(12)