Abstract 2503: Identification of clinically relevant gene fusions in archived pediatric solid and liquid tumor samples using Arima-HiC sequencing
Authors: Farooqi Midhat S. , Sikkink Kristin , Reid Derek , Pastinen Tomi , Schmitt Anthony , Ahmed Atif 
Published: June 2022

Abstract
Gene fusions are known to have oncogenic effects in various cancers and serve as disease biomarkers. Obtaining gene-level resolution of these large structural variants is not possible with lower resolution karyotyping approaches and can be challenging using RNA sequencing approaches due to low transcript abundance or low-quality RNA. Furthermore, the ability of fluorescence in situ hybridization (FISH) techniques to detect fusions is limited by the targeted nature of these assays and subject to which probes were run. Here, we used a novel method, Arima-HiC sequencing, on 6 archived pediatric cancer samples to determine the technique’s effectiveness in detecting gene fusions. We first adapted the Arima Hi-C method for use with formalin-fixed paraffin-embedded (FFPE) tissue. We then selected four archived pediatric alveolar rhabdomyosarcoma (ARMS) tumors (FFPE archival period range: 8-12 years)—known to be fusion-positive via prior clinical testing—for Arima-HiC sequencing. Briefly, FFPE tissue scrolls were dewaxed, and the tissue rehydrated; it then underwent chromatin digestion, end-labeling, and proximity ligation prior to DNA purification per the Arima-HiC FFPE protocol. Purified DNA was next prepared as a short-read sequencing library and sequenced on a HiSeq 2500. The raw reads were aligned and deduplicated, and structural variants were called using the HiC-Breakfinder software. We additionally selected 2 non-FFPE cases, both pediatric leukemia cases with cryopreserved blasts (archival period range: 1-3 years), for Arima-HiC sequencing (as above). These cases had undergone standard of care cytogenetic (karyotyping, FISH) and 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 either a PAX3-FOXO1 (n=2) or PAX7-FOXO1 (n=2) gene fusion in each of our 4 ARMS cases, consistent with the original diagnostic cytogenetic finding. HiC data was additionally able to provide partner gene information (PAX3 or PAX7) in 2 of the 4 cases where this was unknown. We also test two cases of leukemia, one, a case of precursor B-cell acute lymphoblastic leukemia (B-ALL) in a 3-year-old male, and the other, a case of acute myeloid leukemia (AML) in a 2-year-old female. In the first case, we detected an EP300-ZNF384 gene fusion, which is a known, but rare, gene fusion seen in B-ALL. This finding is clinically relevant as ZNF384 fusions are associated with a more favorable prognosis. In the second case, we detected a SCN4B-MLLT10 gene fusion. This finding is also clinically relevant as MLLT10 fusions are rare in AML but indicate an unfavorable prognosis. In summary, this study demonstrates how Arima-HiC sequencing provides molecular diagnostic value in archived pediatric solid and liquid tumor specimens via the identification of clinically relevant gene fusions.
Citation Format: Midhat S. Farooqi, Kristin Sikkink, Derek Reid, Tomi Pastinen, Anthony Schmitt, Atif Ahmed. Identification of clinically relevant gene fusions in archived pediatric solid and liquid tumor samples using Arima-HiC sequencing [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 2503.