Clinical use of genome-wide hi-c sequencing for assessment of structural variants in diffuse large B-cell lymphoma

Abstract Introduction: Diffuse large B-cell lymphoma (DLBCL) is a common and aggressive B-cell malignancy with heterogeneous genomic findings, including a variety of fusions and copy number alterations. However, standardization among laboratories for using cytogenetic techniques is lacking and is often targeted, thus precluding a genome-wide analysis. Herein, we present a case of DLBCL with unresolved fluorescence in situ hybridization (FISH) findings while also providing additional genomic findings that may facilitate therapy selection. Case details and methods: Case details are that of a 70-year-old male with DLBCL with a non-GC phenotype. FISH revealed an IGH rearrangement to an unknown partner while MYC revealed copy number gain, likely indicating polysomy of chromosome 8. The patient was treated with polatuzumab vedotin-rituximab-cyclophosphamide, doxorubicin, and prednisone (pola-R-CHP) but subsequently progressed through therapy. To assess the IGH rearrangement and identify additional genomic events, whole-genome Hi-C sequencing was performed from 5 micron formalin-fixed, paraffin embedded (FFPE) sections and cross-linked chromatin was digested using a restriction enzyme. The 5'-overhangs were filled and digested ends were labeled with biotinylated nucleotide. Spatially proximal digested ends of DNA were ligated, which was then purified, fragmented, and enriched. DNA libraries were then prepared and sequenced by paired-end sequencing. Structural variant (SV) analysis was performed using HiCUP and hic_breakfinder. SV were then manually curated and processed to create a single VCF file. Results: Whole-genome Hi-C sequencing revealed a fusion of IGH to an intergenic partner on chromosome 10, resulting in a t(14;10). Copy number variant analysis revealed gains of chromosomes 4, 7, 8, and 15q with loss at 5p. Interestingly, an 18 Mb tandem amplification on chromosome 17 resulted in amplification of PRKCA, CD79B, CLTC, MSI2, and HLF. Conclusion: Hi-C sequencing was able to define the partner of the IGH gene rearrangements to be an intergenic region of chromosome 10, revealing that this was likely not related to the pathogenesis in this case. Furthermore, Hi-C sequencing found copy number aberrations and a tandem amplification of CD79B, a key regulator in B-cell signaling. Accordingly, this finding is associated with a more aggressive phenotype in DLBCL, as was observed in this patient. Coincidentally, the patient was already appropriately treated with pola-R-CHP as polatuzumab vedotin is an antibody-drug conjugate targeting CD79b. Herein, we demonstrated that whole-genome Hi-C sequencing is a robust technology that facilitates SV analysis on FFPE in DLBCL. Accordingly, this technique is sensitive and specific as it can refine unclear FISH findings while potentially providing additional genomic biomarkers, ultimately guiding therapy selection and/or clinical trial enrolment.