Hi-C FFPE Sequencing for Detection of Fusions and Rearrangements that are Diagnostic, Prognostic, and Therapeutic Biomarkers in Lymphoma

Introduction/Background/Significance: Lymphoma diagnosis relies on histologic examination, immuno-profiling, and often fluorescence in situ hybridization (FISH). Because FISH is performed in a panel of single- or dual-target test formats and analyzed through microscopy and manual image analysis, assessment of multiple target biomarkers can be cumbersome and expensive. As additional biomarkers for diagnosis, subtyping, prognosis, and therapeutic decision making become available, target panels will become larger, posing additional challenges to laboratories. Furthermore, many guideline recommended rearrangements are cryptic to FISH testing complicating accurate diagnosis.

Materials and Methods/Case Presentation/Objective: Hi-C sequencing from FFPE samples is used to overcome limitations of FISH testing. FFPE tissue was processed using the Arima HiC+ for FFPE kit followed by NGS sequencing to capture long-range interactions across the genome. A custom bioinformatics pipeline was used to identify translocations, amplifications, deletions, and complex rearrangements, with breakpoints. Additional clinical testing by IHC, FISH, and/or NGS were available for comparison.

Results/Description/Main Outcome Measures: From a cohort of 226 Non-Hodgkin's Lymphoma cases, a final set of 197 clinically relevant aberrations were detected. Hi-C sequencing detected 98% of these variants including 68 variants not targeted by, and/or not detected by FISH. The novel variants included those targeted by FISH (e.g. MYC, BCL2, BCL6) or not targeted but relevant for diagnosis or subtyping (e.g. CCND1, IRF4, MALT1, etc.), prognosis (e.g. TP53, CKDN2A/B, etc.), or therapeutic opportunity.

Conclusions: Hi-C sequencing of FFPE samples provides a high-resolution genome wide approach to detect all cytogenomic biomarkers recommended by clinical testing guidelines and emerging biomarkers in an unbiased manner.