Single base substitution and insertion/deletion mutational signatures in adult core binding factor acute myeloid leukemia

(SBSsigns) of the types of somatic single nucleotide variants (SNVs) and their ﬂ anking nucleotides, ID signatures (IDsigns) are de ﬁ ned nucleotides and the presence of repetitive/ microhomology regions (https://cancer.sanger.ac.uk/signatures/). or enzymatic deamina- of 5-methylcytosine sequencing -positive

TO THE EDITOR Single base substitutions (SBSs) and insertions/deletions (indels; IDs) arise through several mechanisms such as errors during DNA replication/repair and exposures to mutagens, with the different mutational processes occasionally generating specific mutational signatures. SBS signatures (SBSsigns) result from recurring trinucleotide patterns of the transition/transversion types of somatic single nucleotide variants (SNVs) and their flanking nucleotides, whereas ID signatures (IDsigns) are defined according to size, nucleotides affected, and the presence of repetitive/ microhomology regions (https://cancer.sanger.ac.uk/signatures/). Some signatures are associated with underlying etiologic factors, e.g. SBS7 and ID13 in UV-associated melanoma and SBS4 and ID3 in smoking-induced lung cancer [1,2], whereas others are linked to inherent defects of DNA recombination, replication, and repair (SBS6 and ID1) or caused by spontaneous or enzymatic deamination of 5-methylcytosine to thymine (SBS1) (https://cancer.sanger. ac.uk/signatures/).
Whether SBS18 is overrepresented also in adult RUNX1:: RUNX1T1-positive AML is unknown. In fact, our knowledge of SBSsigns is rudimentary-and non-existing as regards IDsigns-in adult core binding factor (CBF) AML, which consists of cases positive for either RUNX1::RUNX1T1 or CBFB::MYH11 [inv(16) (p13q22)/t(16;16)(p13;q22)] [7]. The only publication to date addressing SBSsigns in adult CBF AML reported a high frequency of SBS1 [8], a clock-like signature that accumulates with age (https://cancer.sanger.ac.uk/signatures/). To ascertain if SBS18 is a common mutational signature in adult CBF AML, we performed WGS of ten cases with RUNX1::RUNX1T1 and ten with CBFB::MYH11, focusing not only on SBSsigns but also on IDsigns. All patients had de novo AML, thus excluding those previously exposed to chemoand/or radiotherapy that could have affected the mutational signatures. The cases were selected based on the availability of good quality DNA from both diagnosis and remission. The median age of the patients was 51.5 years (range 19-74 years) and the female/male ratio was 1:1.5. All genetic analyses were performed at the Department of Clinical Genetics and Pathology, Office for Medical Services, Region Skåne, Lund, Sweden. The basic clinical and genetic features of the CBF AMLs are summarized in Supplementary Table 1 and data on WGS of paired diagnostic/ remission samples and bioinformatic analyses are provided in Supplementary Information.
The average sequencing depths of the WGS varied from 29× to 57× per sample (median 40×) and the Q30 value was 96.23%, with 2 × 150 bp read length. The WGS analyses confirmed the RUNX1:: RUNX1T1 and CBFB::MYH11 gene fusions in all cases and also revealed that the genomic breaks clustered within introns 6 of RUNX1 and 1 of RUNX1T1 and within introns 5 of CBFB and 33 of MYH11, respectively (Supplementary Table 1). No other chimeric genes were detected. All chromosomal gains and losses previously found by conventional G-banding were identified by WGS except for two subclonal trisomies in one case (Supplementary Tables 1  and 2). WGS also identified 32 copy number abnormalities (≤10 Mb) and five uniparental isodisomies, all of which undetectable by chromosome banding analyses (Supplementary Table 2). None of the cases displayed any signs of chromothripsis.
In conclusion, our findings suggest that the etiologies/mechanisms underlying transitions/transversions, SBSsigns, and IDsigns are similar in the two CBF AML types ( Fig. 1 and Supplementary  Figs. 1 and 4). Unfortunately, the etiologies of many of the common SBSsigns and IDsigns in the RUNX1::RUNX1T1-and CBFB:: MYH11-positive cases are presently unknown. However, those with known or suspected origins can be dichotomized into i) spontaneous DNA changes/errors (SBS1, ID1, and ID2) and ii) associations with external agents, gene mutations, and ROS (SBS5, SBS18, SBS32, SBS88, and ID9). The lower frequency of SBS18 in adult vs. pediatric AML, despite being among the most common SBSsigns in the adult cases ( Fig. 1 and Supplementary Fig. 3), may be explained by the fact that the SBS18 frequency did not increase with age in our patient cohort, whereas several other SBSsigns did (Fig. 2). In a recent study of pediatric AML, SBS18 was related to intrinsic ROS mechanisms that may have been induced already during fetal development [5]. Thus, if SBS18 occurs early on during the leukemogenic process of CBF AML, it would be more pronounced in childhood than in adult cases because the latter would have accumulated other age-related SBSsigns resulting in a relatively lower proportion of SBS18. Further studies of SBS18 and ROS-induced DNA damage in adult and childhood CBF AML are needed to clarify this issue.

DATA AVAILABILITY
The dataset generated during the current study will be made available in the EGA-SE depository upon its completion. Until then, the data are available from the corresponding author upon request through the following https://doi.org/10.17044/ scilifelab.17082971 (WGS dataset). Supplementary information is available at Leukemia's website.