LAUSR

Acute myeloid leukemia (AML) is the overall most common form of acute leukemia, characterized by a high relapse frequency and low long-term survival rate especially for adults. Even though children with AML have a better prognosis than adults, patients with recurrent AML - independent of age - show poorer overall survival, accelerated disease progression and they often do not respond to conventional treatment. Therefore, a more personalized treatment approach is required to prolong event free survival (EFS) and expand treatment options for relapse and primary resistant (R/PR) AML patients. During the last decade, AML research has largely been focused on improving diagnostic and prognostic tools in AML by investigating data derived from whole genome- and whole exome sequencing (WGS and WES, respectively), gene panels or differential expression analyses of individual genes and gene-fusions. Nevertheless, studies on relapsing AML incorporating WGS data are rare, although necessary to investigate the full repertoire of genetic aberrations underlying AML progression and therapy resistance. To this end, we applied a combination of WGS (WES for a subset of cases) and RNA-seq of longitudinal samples from 48 adult and 21 pediatric R/PR AML cases from the Nordic countries. These comprised tumor samples collected at diagnosis (n=49) and relapse (n=76), as well as PR specimens (n=6). Normal bone marrow (BM) derived stromal cells were cultivated from leukemic BM as a source of patient-matched constitutional DNA, while CD34+ BM cells from five healthy donors were used as a source of normal control RNA. Our findings reveal recurrent relapse specific mutations in CSF1R (2.9% of relapse cases) not previously reported in de novo AML, suggesting the use of receptor tyrosine kinase inhibitors as a novel therapeutic option for a subset of AML relapse cases. Further, we report specific differences in the mutational spectrum between adult and pediatric R/PR AML. In adults, we detected higher mutational frequencies of, for instance, ARID1A (6.3%), H3F3A (6.3%) and MGA (10.4%) compared with previous AML studies of only specimens from initial diagnosis, while these mutations were not seen in pediatric AML. In contrast, internal tandem duplications (ITDs) in UBTF were detected solely in pediatric relapsing AML (n=3 [14.3%]). IKZF1 was more frequently mutated in pediatric R/PR AML (14.3%) than previously reported (0.5-2.7%; Bolouri et al., Nat Med. 2018; Shiba et al., Br J Haematol, 2016). Also, differential gene expression analysis identified IKZF1 as downregulated in pediatric chemotherapy resistant samples in comparison with treatment responsive counterparts, independent of IKZF1 mutational status. By investigating differential gene expression patterns of longitudinal samples, we found lower expression of the complement inhibitor CR1/CD35 at relapse compared to their patient matched diagnostic samples in both adults and children. Additionally, IL1R1, encoding a key regulator of inflammation and immune response, was upregulated in both adult and pediatric diagnosis specimens from cases with short EFS, indicating a pronounced role of chronic inflammation during disease progression and AML cell survival. Finally, our findings reveal overexpression of GLI2 and SGMS2 among samples associated with short EFS. Overexpression of these genes may prevent excessive cell proliferation while increasing stemness and dormancy, leading to increased chemotherapy resistance and shorter EFS. Taken together, our results emphasize the advantage of applying a combination of WGS and RNA-seq, to be able to gain a more complete picture of alterations, including mutations, gene fusions and copy number alterations combined with gene expression analysis, when attempting to characterize AML at relapse. This is the first study of both adult and pediatric AML incorporating WGS and RNA-seq analyses on sequential AML samples. Knowledge gathered from this study has provided critical new insights into the biologic basis of this complex disease and will hopefully help to pave the way for improved and individualized treatment strategies. No relevant conflicts of interest to declare.