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RAS pathway alterations in pediatric AML
patients: unique patient number (UPN) 57 and UPN 415 also had a heterozygous deletion. Additionally patient UPN 57 with variant allele frequency (VAF) 0.83 had non- sense mutations in the remaining allele, while UPN 50 with VAF 0.94 had 17q uniparental disomy (UPD) (Online Supplementary Figure S2). UPN 105 and UPN 333 had two or three different CN regions in NF1, respectively with partially homozygous deletions. Other two patients (UPN 262 and 367) had two types of mutations each. However, it was not clear whether these alterations were mono- allelic or bi-allelic (UPN 262, VAF 0.28 and 0.26; UPN 367, VAF 0.28 and 0.08). Thus, we concluded that at least four patients (UPN 50, UPN 57, UPN 105, and UPN 333) had bi-allelic NF1 inactivation. Next, on the basis of the VAF of each mutation, we estimated whether NF1 mutations were somatic or germline. If a mutation is a heterozygous germline mutation, then the VAF is around 0.5.36 We con- sidered mutations in UPN 262 (VAF 0.28 and 0.26) and UPN 367 (VAF 0.28 and 0.08) as somatic mutations. Regarding UPN 50 (VAF 0.94) and UPN 57 (VAF 0.83), it was impossible to predict whether these mutations were somatic or germline because their VAF were high owing to their co-existence with heterozygous deletion or UPD. On the contrary, these two mutations were determined as somatic in the COSMIC v90 (URL: https://cancer.sanger.ac.uk/cosmic). R1241X detected in UPN 57 was previously observed in adult AML and E1561X detected in UPN 50 was previously detected in non-hematological malignancies.37,38
PTPN11 mutations were detected in 15 (4.6%) patients (Figure 1). Of these, 14 were located in exon 3 or exon 13,
which are known mutation hotspots in AML and JMML (Figure 2).39 As previously observed,39 codon 76 represent- ed a mutational hot spot (four of 15, 27%) with three dif- ferent amino acid substitutions (Figure 2), and 13 of the 15 mutations have been reported as somatic mutations.39-41 Although the remaining two mutations (V45L and T493I) have not been confirmed as somatic mutations, V45L was earlier detected in lung carcinoma and showed an associ- ation with activation of protein-tyrosine phosphatase.41 However, T493I has not been reported in any hematolog- ical or other disease. These two variants have not been reported as SNP on any database such as COSMIC v90, ClinVar, mutations taster, Ensembl GRCh37, or db SNP (URL: https://www.ncbi.nlm.nih.gov/snp/, https://www.ncbi.nlm.nih.gov/snp/, http://grch37.ensem- bl.org/index.html, https://www.ncbi.nlm.nih.gov/clin- var/, and http://mutationtaster.org/); therefore, we identi- fied these as novel disease-causing mutations.
CBL mutations were found in six (1.8%) patients (Figure 1). Among these, four were deletions or insertions and deletions in exon 8 and two were missense mutations in exon 9. Five of these mutations were in the linker region or the RING finger domain which were previously report- ed as the affected regions in myeloid malignancies with CBL mutations (Figure 2).12,13,18 None of the six mutations have been reported as SNP or germline mutations in any online databases or previous reports.42 CBL mutations especially missense mutations were shown to exhibit a strong association with 11q-acquired UPD.18 11q UPD was detected in only one patient with a missense mutation (UPN 97) by CN analysis (Online Supplementary Figure S2).
Figure 1. Molecular and cytogenetic aberrations in 80 pediatric acute myeloid leukemia patients with RAS pathway alterations. Each column displays the cytogenetic aber- ration pattern and clinical status of an individual sample. Orange indicates RAS pathway and other genetic alterations. Blue indicates chromosomal aberrations. Purple indi- cates gene expression. Gray indicates clinical outcome. Blanks indicate the absence of the chromosomal aberration, genetic alteration, or prognostic event. CR: complete remission.
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