Comprehensive Genomic and Transcript Profiling of CBL Gene in Childhood AML: A Report from Children's Oncology Group Studies AAML03P1, AAML0531 and COG/NCI Target AML Initiative
Authors: Chin Diana , Kutny Matthew A. , Grim Jonathan , Gerbing Robert B. , Miller Kristen , Farrar Jason E. , Guidry Auvil Jaime M. , Smith Malcolm A. , Gerhard Daniela S. , Davidsen Tanja M. , Gesuwan Patee , Hermida Leandro C. , Marra Marco A , Mungall Andrew J. , Moore Richard , Long William , Ma Yussanne , Zong Stuart , Kolb E. Anders , Gamis Alan S. , Alonzo Todd A. , Meshinchi Soheil 
Published: October 2019

Abstract
The Casitas B-Lineage Lymphoma (CBL) gene encodes for an E3 ubiquitin ligase that targets activated receptor tyrosine kinases for degradation. Mutations of the CBL gene have been described in juvenile myelomonocytic leukemia (JMML) but less is known about mutations and variants of CBL in de novo AML. We previously reported that somatic mutations of CBL are rare in pediatric AML. In this report we present a comprehensive evaluation of genomic and transcript variants of CBL including novel deletion events as well as transcript variants which, in combination with somatic mutations, account for over 6% of pediatric AML with extreme association with inv(16) and favorable outcome.
Initial assessment of CBL transcript in a cohort of 100 patients identified previously reported deletion of exon 8 (CBL ΔE8, N=2) associated with CBL splice mutations as well as a novel whole exon 8 and 9 deletion variant (CBL ΔE8+9, N=3) without identifiable underlying somatic alterations. Long distance PCR, as well as custom Nanostring CNV array evaluation revealed a genomic deletion underlying this transcript variant. Subsequent whole genome sequencing as part of COG/NCI TARGET AML initiative, identified discrete genomic deletions of 1998, 3588 and 6189 bp across exon 8 and 9, leading to the generation of this novel variant.
We evaluated the functional consequence of the novel CBL ΔE8+9 deletion variant by expressing it in IL3-dependent Ba/F3 cell line. Compared to control cells, Ba/F3 cells expressing CBL ΔE8+9 demonstrated cytokine independent growth.
A comprehensive profiling of CBL variants was conducted in 796 pediatric de novo AML patients by transcript profiling (transcript variants) or by exome capture sequencing (somatic mutations including point mutations and smaller indels). All patients were treated on Children's Oncology Group studies AAML03P1 (N=167) and AAML0531 (N=629) and presence of CBL variants was correlated with disease characteristics and clinical outcome. Of the 796 patient specimens tested, 50 patients (6.3%) had one of 3 distinct CBL variants; transcript variant (N=28), somatic mutation (N=14), or dual transcript variant and somatic mutation (N=8). All cases of CBL ΔE8+9 were associated with a corresponding genomic deletion. Out of 14 cases of CBL ΔE8 and 1 case of CBL ΔE9, only 4 cases (27%) had a splice site mutation identified as the underlying mechanism of splice variant.
Presence of CBL variants was correlated with clinical characteristics and outcome. Those with CBL variants had a significantly higher prevalence of inv(16) compared with CBL wild type (WT) (37% vs. 13%, p<0.001). This association differed by CBL variant type; 44% transcript variants and 50% dual variants had inv(16) compared to 14% somatic mutations and 13% CBL WT (p<0.001). NPMc+ was more prevalent in those with CBL somatic mutations (29%) than transcript variant (4%), dual variant (0%) or CBL WT (8%) (p=0.035). Similarly, genetic risk groups differed between CBL variants vs. WT (Low risk 70% vs 39%, p=<0.001; Standard risk 22% vs. 46%, p=0.001; High risk 8% vs. 15%, p=0.196). Clinical characteristics including gender, age, race and ethnicity were not significantly different. FAB morphologic assessment revealed an enrichment for the M4 subtype in CBL variant vs. WT (53% vs. 23%, P<.001) which is likely accounted for by the association of inv(16) with this morphologic group.
Patients with CBL variants had a 100% clinical remission rate by end of induction II compared to 89% for CBL WT patients (p=0.014). Survival from study entry was similar between CBL mutant vs. WT patients (5 year OS 72% vs. 66%, p=0.24; 5 year EFS 61% vs. 50%, p=0.11). Due to the strong association of CBL mutation with core binding factor leukemia, we assessed whether CBL variant was prognostic of outcome within this favorable risk group, but there was no significant difference in outcomes.
Variants of the CBL gene in pediatric AML include genetic mutations with and without whole exon deletions. These CBL variants are highly associated with low risk AML but do not provide independent risk prognosis. The cooperating events of CBL variants in core binding factor leukemia deserve greater study. Our initial analysis of the transcript variants in a cell line model suggest that these large exon 8+9 deletions represent important oncogenic events.
The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative.

Disclosures
No relevant conflicts of interest to declare.