FISH Diagnostics of Double Hit or Triple Hit B-Cell Lymphomas

Abstract 1599

Fluorescence in situ hybridization (FISH) is currently a widely used and most objective technology in identifying double hit or triple hit lymphomas (DHL/THL) that consist of a spectrum of genetically and pathologically heterogeneous B-cell neoplasms. Accurate detection with appropriate FISH probes or panels helps diagnosis, risk stratification and therapy selection, particularly in commonly seen aggressive lymphomas such as Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL. We retrospectively look into 147 DHL/THL cases out of 927 consecutive MYC FISH positive B-cell lymphomas tested in our lab in the past four and half years to analyze typical and atypical findings to attempt to improve detection rates and correctly interpret atypical FISH results. FISH probes (Vysis) for MYC breakapart (ba), MYC/IGH translocation, BCL2/IGH translocation, BCL6 ba and CCND1/IGH translocation were applied to paraffin embedded tissue, lymph node biopsy, bone marrow and peripheral blood specimens. Among 927 cases, MYC and MYC/IGH were both ordered in 627 cases, MYC ba was ordered without MYC/IGH in 252, MYC/IGH without MYC ba in 48 cases, BCL2 was ordered in 379 cases, BCL6 in 280, BCL2 and BCL6 in 277, CCND1 in 104, BCL2 and CCND1 in 94, BCL6 and CCND1 in 90, and BCL2, BCL6 and CCND1 in 90. Of the 147 cases, 12 were MYC and CCND1 positive with a diagnosis of mantle cell lymphoma (MCL); one of these cases was THL with BCL6 rearranged and its 5' probe deleted. Most common is BCL2 or BCL6 positive seen in 88 and 20 cases respectively. 24 cases were THLs with MYC and both BCL2 and BCL6 positive. In 91 cases with MYC ba and MYC/IGH both probes performed, 55 were positive for MYC ba but negative for MYC/IGH (60%) compared to 34 positives for both MYC ba and MYC/IGH (37.4%) which is significant as reported in literature that more cases with DHL/THL showed MYC rearrangements with non-IGH or non-IG partner genes unlike the majority of the Burkitt lymphomas. Interestingly, in one DHL and one THL, MYC ba was negative but MYC/IGH was positive, suggesting a cryptic/complex insertional translocation reported by other groups and our lab. For breakapart probes, a deletion of the flanking sequences concomitant with gene rearrangement is relatively common. In 147 DHL/THL cases, MYC ba positive with a deletion was seen in 11 (7 for 3' and 4 for 5'), and BCL6 ba positive with a deletion was seen in 6 (2 for 3' and 4 for 5'). One DHL showed 5' MYC probe amplification. Based on our experience concurring with studies by other groups, we suggest: 1) FISH testing in aggressive B-cell lymphomas include BCL2 and BCL6 in the probe panel as IHC results may not be as sensitive and reliable as DNA testing, 2) both MYC ba and MYC/IGH probes should be tested simultaneously to increase detection rates by minimizing false negative results caused by cryptical insertion translocation of IGH promoter/enhancer sequences into the MYC gene (locus) or vice versa, and to help differentiate MYC/IGH translocation from MYC/non-IGH or non-IG rearrangement seen more often in non-BL including DHL/THL, 3) CCND1 may be needed in the panel when transformed MCL is a diagnostic concern, and 4) atypical FISH results should be analyzed and interpreted correctly such as rearrangements with a flanking probe deletion and/or amplification. Next generation sequencing may delineate all of these atypical genetic abnormalities evidenced by FISH and/or cytogenetic studies and undetected genomic alterations as well.