Characterization of Genetic Findings Underlying Aggressive B-Cell Lymphomas with Atypical Results on MYC Break-Apart Fluorescence in Situ Hybridization (FISH) Analysis

Introduction: MYC rearrangements (MYCr) are important distinguishing features of disease in diffuse large B-cell lymphoma (DLBCL) and high-grade B-cell lymphoma (HGBCL). Identification of MYCr in the clinical laboratory most often relies on fluorescence in situ hybridization (FISH) using MYC break-apart (BAP) and MYC/IGH dual color, dual fusion (DF) strategies. In 7.5% of cases, BAP analysis reveals unbalanced patterns of unclear significance. We used whole-genome sequencing (WGS) to characterize the genomic underpinnings of these atypical results in DLBCL/HGBCL. Methods: We initially surveyed the International cytogenetic community to characterize the breadth of interpretation practices for atypical MYC BAP results. Following IRB approval, we then subjected 14 DLCBL/HGBCL specimens sampled between 2019 and 2021 to WGS analysis. These included 7 cases with atypical MYC BAP findings without an IGH partner on FISH and 7 control specimens with MYCr with (n=1) and without (n=6) an IGH partner. FISH analysis was performed with BAP MYC (comprising a red (R) and a green (G) probe which respectively hybridize 5’ and 3’ to MYC (Abbott Laboratories, Des Plaines, IL, USA). MYC/IGH DF probe sets (Abbott Laboratories) were also performed. Cases with loss of a G (R-fusion (RF)-type pattern) or R (G-fusion (GF)-type pattern) signal on BAP analysis were deemed unbalanced in contrast to typical balanced RGF-type patterns. DNA was extracted from formalin-fixed, paraffin embedded (FFPE) sections using Qiagen AllPrep FFPE kits. Extracted DNA quality control was performed on an Agilent bioanalyzer. NEB ultraII libraries were created following a modified Covaris fragmentation protocol designed to capture larger inserts (Murphy S, et al, 2021). Libraries were multiplexed on an Illumina NovaSeq S4. Mapping to the GRCh38 reference genome and structural rearrangement (SR) calling were performed with BIMA 3.1.5/SVAtools FFPEseq pipeline (Murphy S, et al, 2021). The mean range of unique mapped reads was 492M. Bridge coverage adjusted for library insert length and pipeline estimated tumor percent was 39.8X (18.5X-72X). Results: Out of 54 survey responders (from ≥31 different institutions and ≥ 4 different countries), 36% and 42% respectively reported interpreting RF- and GF-type patterns as a MYCr, 58% and 56% reported RF and GF-type patterns as equivocal and 6% and 2% respectively reported RF and GF-patterns as negative for a MYCr. WGS confirmed a MYCr in all control cases with a balanced pattern on BAP FISH (Table 1). MYCr partners in this group included previously recognized loci (IGH (n=1), IGL (n=2), ZCCHC7 (n=1), RFTN1 (n=1), DMD (n=1) and BCL11A (n=1)). We identified an underlying SR involving the MYC region with a relative gain of genomic material 5’ of this gene in all 5 cases with a RF-type pattern, in line with BAP signal patterns. Four cases involved putative fusion events juxtaposing MYC to 1) TG gene, 2) an intergenic region upstream of TG, 3) to IRF8 and 4) to SPAG1 while the 5th case involved a copy number (CN) gain of MYC. WGS also reconciled BAP FISH results for the 2 cases with a GF-type pattern, both with SRs leading to a gain of the 3’ MYC BAP FISH probe-binding sequence. While the first juxtaposed MYC with ACTB, the second involved a CN loss including MYC and the 5’ FISH probe-binding sequence. MYCr partners in cases with RF- and GF-type patterns were previously unreported in DLBCL/HGBCL. Myc expression level by immunohistochemistry was ≥ 40% in 4/5 and 2/2 cases with RF- and GF-type patterns, respectively. Conclusion: In line with unresolved underlying genomic mechanisms, atypical MYC BAP FISH results are variably interpreted in clinical laboratories. Nonetheless, accurate interpretation is important for diagnostic and therapeutic purposes. We demonstrate that unbalanced patterns on BAP without an IGH partner on DF analysis often, but not always, involve true MYCr. Given that the MYC partner genes in these cases have not been previously described, their clinical and biologic significance remains uncertain. WGS may serve to elucidate these atypical findings obtained with MYC BAP analysis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal