134P Characterizing sarcoma molecular landscape by nanopore adaptive sampling sequencing

Pathological diagnosis of sarcoma is based on histopathological features and immunohistochemical staining, as well as fluorescence in situ hybridization (FISH), Sanger sequencing, massively parallel DNA sequencing, and methylation-based arrays. With this approach, up to 80–85% of sarcoma cases are classified. Nanopore sequencing is a rapid, point of care method for characterizing tumors' molecular landscape including single nucleotide variations, copy number alterations (CNA), translocations and methylation. Using low coverage whole genome Nanopore sequencing of DNA, we identify CNA and methylation patterns in sarcomas. Using methylation-based deconvolution, we identified different sarcoma types. We apply adaptive sampling Nanopore sequencing to characterize sarcoma molecular landscape and thus better classify sarcoma, identify driver mutations, and help plan blood-based tumor markers. The sarcoma panel includes introns and exons of sarcoma related genes, tumor suppressors and oncogenes, spanning 10% of the genome. All patients signed an informed consent to participate in the study. Fresh tissue was flash frozen, and DNA extracted using Qiagen DNeasy Blood & Tissue Kit; 3 ug of DNA underwent sarcoma panel Nanopore adaptive sampling sequencing in Promethion2 solo. Two cases were tested; a synovial and an undifferentiated spindle cell sarcoma. Sarcoma panel adaptive sampling led to 30x coverage. In the synovial sarcoma sample, the methylation pattern and a SYT translocation concurred with the diagnosis. Additionally, we found an NF2 mutation with loss of heterozygosity. We confirmed the SYT translocation using a PCR reaction and the NF2 mutation concurs with a FoundationOne test. In the undifferentiated spindle cell sarcoma sample, we found Ewing's sarcoma methylation pattern and a FUS-ERG translocation. We validated the FUS translocation using FISH. Sarcoma panel Nanopore adaptive sampling can diagnose rare sarcoma types, such as FUS translocated Ewing's sarcoma, and aid in characterizing the molecular landscape. Furthermore, Nanopore analysis may facilitate blood-based tumor markers. Additional testing of a diverse sarcoma cohort is needed to evaluate the clinical utility of this approach.