Abstract LB122: Arima-HiC sequencing accurately detects clinically-relevant structural variants in pediatric leukemia samples
Authors: Schmitt Anthony , Melnyk Shadi , Sikkink Kristin , Lansdon Lisa , Pastinen Tomi , Guest Erin , Farooqi Midhat 
Published: April 2023

Abstract
Genetic structural variants (SVs), especially those leading to gene fusions, are well-known oncogenic drivers. These SVs can produce overexpression or loss-of-function of certain genes, or generate chimeric fusion proteins. Thus, they serve as important disease biomarkers across several cancers and can also represent therapeutically targetable alterations. Detecting these SVs at a gene-level resolution can be challenging with lower resolution karyotyping approaches, or even RNA sequencing approaches, due to difficulties with culture, sample stability, low transcript abundance, or low-quality RNA. In addition, the ability of fluorescence in situ hybridization (FISH) techniques to detect gene fusions is limited by the targeted nature of these assays as they depend heavily on the design and selection of each probe. Here we use a novel method, Arima-HiC sequencing, that utilizes DNA to evaluate 12 pediatric leukemia samples and determine this assay’s effectiveness in detecting clinically-relevant SVs. We first selected 5 archived (cryopreserved) pediatric acute myeloid leukemia (AML) samples (archival period range: 1-4 years) known to be either fusion-positive (n=3) or fusion-negative (n=2) via prior clinical genetic testing (i.e., chromosomes, FISH, and/or microarray). All samples underwent Arima-HiC sequencing. Briefly, chromatin digestion, end-labeling, and proximity ligation were performed prior to DNA purification per the Arima-HiC protocol. Purified DNA was next prepared as a short-read sequencing library and sequenced on a HiSeq X. The raw reads were aligned and deduplicated, and SVs were called using HiC-Breakfinder software. For a discovery set, we then additionally selected 7 pediatric leukemia samples—6 precursor B-cell acute lymphoblastic leukemias (ALL) and 1 AML—for Arima-HiC sequencing (as above). These cases had undergone standard-of-care cytogenetic (karyotyping, FISH, microarray) and/or molecular (targeted cancer NGS sequencing panel) testing clinically, and a genetic driver/known gene fusion had not been identified. Using Arima-HiC sequencing, we identified the clinically-relevant SV in each of our 3 fusion-positive AML cases, consistent with the original diagnostic cytogenetic finding (n= 1 RUNX1-RUNX1T1 fusion, 1 CBFB-MYH11 fusion, and 1 CBFA2T3-GLIS2 fusion). The 2 fusion-negative AML cases were also negative for structural gene fusions by HiC data. In our discovery sample set, Arima-HiC sequencing was able to find clinically-relevant SVs that were not previously detected in 3 of 6 samples: a KMT2A-MLLT10 fusion was found in an AML case, and a ZNF384-EP300 fusion was found in each of 2 ALL cases. A ABHD17B-PTK2B fusion was found in another ALL case, although ABHD17B may be a novel partner PTK2B and is undergoing validation. Rearrangements involving KRAS and EGFR were detected in the final two ALL cases, and are also undergoing validation. Overall, this study demonstrates how Arima-HiC sequencing can provide diagnostic value in pediatric leukemia specimens via the identification of clinically relevant SVs.
Citation Format: Anthony Schmitt, Shadi Melnyk, Kristin Sikkink, Lisa Lansdon, Tomi Pastinen, Erin Guest, Midhat Farooqi. Arima-HiC sequencing accurately detects clinically-relevant structural variants in pediatric leukemia samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB122.