Abstract 243: Long-read sequencing of pediatric cancer genomes identifies multiple clinically relevant variants
Authors: Yoo Byunggil , Cheung Warren , Pushel Irina , Lansdon Lisa , Bi Chengpeng , Guest Erin , Pastinen Tomi , Farooqi Midhat 
Published: April 2023

Abstract
Genetic characterization of pediatric cancer samples clinically for diagnostic, prognostic, or therapeutic information currently relies on an assortment of methods that complement and partially overlap one another, such as fluorescence in situ hybridization (FISH), karyotyping, microarray, and sequencing. This need for multiple tests results in longer analysis time, increased total costs, and higher consumption of sometimes limited tumor material. Long-read sequencing (lrSeq) using HiFi technology, i.e., circular consensus sequencing of reads 12-16kb in length, is able to provide sequence level changes with highly accurate base quality (&gt;99%), as well as information on copy number variants, structural variants, and methylation status, all fully phased. Here, we assess the ability of lrSeq to detect clinically relevant genetic alterations (CRGAs) in pediatric cancer samples. We selected 10 patient cases of solid and liquid tumors with known CRGAs from prior clinical testing. These included 3 cases of T-cell acute lymphoblastic leukemia (T-ALL): one with t(9;17) involving NOTCH1, one with t(7;7) involving TRB, and one with biallelic CDKN2A deletion; 2 high-grade glioma (HGG) cases: one with t(2;9) causing a WDCP-NTRK2 fusion, and one with a TP53 mutation; 2 cases of acute myeloid leukemia (AML): one with biallelic mutations in CEPBA and one with a KMT2A-rearrangement (KMT2A-r; t(v;11q23.3)); 1 case of B-cell ALL with KMT2A-r, t(v;11q23.3); 1 neuroblastoma (NBL) case with regional amplifications of chromosome 12 including MDM2; and 1 case of anaplastic large cell lymphoma (ALCL) with an NPM1-ALK fusion (t(2;5)). DNA from these samples was sequenced on the PacBio Sequel II System to a target depth of 25x. HiFi reads were processed using the PacBio Human WGS Workflow. Methylation prediction was made from Primrose and 5mC modification probability of CpG sites were computed by pb-CpG-tools. We then assessed phased CpG methylation in known imprinted genes.We found that lrSeq detected translocations in 6 out of 6 cases, providing precise break ends and often clarifying unclear cytogenetic findings (such as partner genes). HiFi sequencing also detected 3 of 3 sequence alterations in CEBPA and TP53; biallelic deletion of CDKN2A; and known regions with loss of heterozygosity (LOH) in 2 out of 2 cases. However, it had difficulty detecting all amplified regions of chromosome 12 in the case of NBL, such as the one containing MDM2. Finally, we were able to detect methylation differences in a phased manner by parental haplotype in known imprinting regions across the genome. The difference in 5mC modification probability between the two alleles of the known imprinted genes was 81.5. Our results demonstrate that lrSeq can detect multiple CRGAs across several different types of genetic variants and pediatric cancers, bringing us closer to the promise of an all-in-one genetic test for cancer characterization.
Citation Format: Byunggil Yoo, Warren Cheung, Irina Pushel, Lisa Lansdon, Chengpeng Bi, Erin Guest, Tomi Pastinen, Midhat Farooqi. Long-read sequencing of pediatric cancer genomes identifies multiple clinically relevant variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 243.