Abstract 5725: HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia
Authors: Mc Dermott Sarah E. , Yoo Byunggil , Cheung Warren A. , Farrow Emily , Ge Bing , Gibson Margaret , Brown Patrick A. , Guest Erin , Pastinen Tomi , Farooqi Midhat S. 
Published: June 2022

Abstract
Background: KMT2A-rearrangement is a characteristic finding in approximately 70% of infant acute lymphoblastic leukemia (ALL) cases. We previously utilized a computational screen to find that KMT2A-r infant ALL demonstrated somatic allele specific expression for 431 genes when compared to non-KMT2A-r samples (Abstract Number 3649, AACR 2019). The gene that exhibited the most differential allele specific expression (ASE) pattern was HOXA9. Here, we follow up that analysis with manual examination of additional infant ALL samples.
Methods: We performed whole genome sequencing (WGS) and RNA sequencing (RNAseq) on peripheral blood or bone marrow samples from 48 infants with ALL (33 KMT2A-r cases and 15 non-KMT2A-r cases) treated on Children’s Oncology Group protocol AALL0631, at diagnosis (DX), at remission (MD), and at relapse (RL), if applicable. WGS data at MD was phased using a 1000 Genomes reference panel. HOXA9 expression was quantified in transcripts per million (TPM). Biallelic expression was manually calculated on the phased genome for samples with at least 1 germline single nucleotide polymorphism (SNP) within HOXA9 and at least 5 aligned RNAseq reads over the HOXA9 transcript. ASE was defined as minor allele fraction (MAF; allele fraction for the less prevalent allele) of ≤ 0.35. Whole genome bisulfite sequencing data and sequencing-derived copy number data were also analyzed to investigate possible explanations for HOXA9 ASE.
Results: We identified diagnostic samples from 21 patients, 9 KMT2A-AFF1, 9 KMT2A-MLLT1, and 3 non-KMT2A-r, that passed manual filtering. Data included 5 patients with paired DX and RL samples. Of the 15 KMT2A-r samples unable to be analyzed for ASE, 13 lacked a SNP for phasing and 2 had &lt; 5 reads. For the 12 non-KMT2A-r samples unanalyzable for ASE, 6 lacked a SNP and 6 had &lt; 5 reads. Average HOXA9 expression was 15.17 TPM (range 0.01 to 44.34) in KMT2A-r samples and 2.91 TPM (range 0 to 29.29) in non-KMT2A-r samples. ASE of HOXA9 (MAF ≤ 0.35) was observed in 14 samples, 72% of KMT2A-r (13 of 18 samples) and 33% of non-KMT2A-r (1 of 3 samples). RL samples were found to have HOXA9 ASE in all cases with ASE of HOXA9 in the DX sample. Of the 18 KMT2A-r samples, 6 of 9 with KMT2A-AFF1 and 7 of 9 with KMT2A-MLLT1 demonstrated HOXA9 ASE. Frequency of HOXA9 ASE at diagnosis was similar between patients known to later relapse versus those known to remain in remission. Allele specific methylation, mono-allelic deletion, or copy-neutral loss of heterozygosity (cn-LOH) for HOXA9 were not observed in KMT2A-r nor non-KMT2A-r samples.
Conclusion: Our expanded cohort supports the ASE of HOXA9 in KMT2A-r infant ALL. This phenomenon did not appear to segregate by KMT2A-r partner gene, nor could it be explained by allele specific methylation or copy number variation (e.g., deletion of one HOXA9 allele or cn-LOH). Overall, further investigation into a causal mechanism is needed.
Citation Format: Sarah E. Mc Dermott, Byunggil Yoo, Warren A. Cheung, Emily Farrow, Bing Ge, Margaret Gibson, Patrick A. Brown, Erin Guest, Tomi Pastinen, Midhat S. Farooqi. HOXA9 demonstrates allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5725.