Diagnostic classification systems must always balance the use of data derived in a research setting with application of diagnostic techniques to clinical practice. For example, while gene-expression profiling (GEP) was the technique that established cell-of-origin subtypes of diffuse large B-cell lymphoma (DLBCL)1 and defined the entity of BCRABL1–like acute lymphoblastic leukemia (ALL),2 GEP is not widely available in most medical centers. Instead, immunohistochemistry and flow cytometry and/or next-generation sequencing technologies are used in practice to define DLBCL cell-of-origin and BCR-ABL1–like ALL, respectively. These surrogate techniques have some limitations and do not fully overlap with the “gold-standard” results of GEP,3 yet they provide critical information that is used in the clinical setting to inform prognosis and guide treatment decisions. Lymphomas with concurrent MYC and BCL2 rearrangements (so-called “double-hit” [DHIT] lymphomas) include cases that share morphology with DLBCL, but have more aggressive clinical behavior and are typically approached with more intensive therapy than other DLBCLs.4 For these reasons, a category of high-grade B-cell lymphoma with MYC and BCL2 (and/or BCL6) rearrangements, also known as DHIT lymphoma, was created in the most recent revised 4th edition World Health Organization classification of lymphomas. This has necessitated the application of fluorescence in situ hybridization (FISH) to evaluate for MYC, BCL2, and BCL6 rearrangements in DLBCL in order to identify patients with DHIT lymphoma who have poorer prognosis and may benefit from more aggressive therapy.4 Nevertheless, there remains uncertainty in defining the borders of DHIT lymphomas, as some DLBCL cases may show strong expression of MYC and BCL2 proteins yet lack dual rearrangements of these genes or vice versa. Moreover, amplification of MYC or BCL2 genes may be detected by FISH, and it is uncertain if gene amplification could be biologically equivalent to forced overexpression caused by rearrangement of these genes to immunoglobulin (IG) heavy chain loci. A recent study suggested that a signature defined by GEP may best define true DHIT lymphoma in terms of patient outcomes; however, only half of the cases with a DHIT signature had concurrent MYC and BCL2 rearrangements and would be diagnosed as DHIT lymphoma according to current diagnostic techniques.5
Dr. Brett Collinge and colleagues recently addressed issues surrounding the definition of DHIT lymphoma by correlating FISH and immunohistochemistry findings with GEP and MYC gene sequencing in 802 patients with DLBCL. They found that translocations of both MYC and BCL2 genes were strongly associated with increased mRNA and protein expression, which is expected, as translocations of MYC and BCL2 to IG gene loci are known to bring these genes in close proximity to regulatory elements, forcing their overexpression. They found that copy number gains of BCL2, but not MYC, produced aberrant protein expression, though rare cases with marked amplification of the MYC gene (numerous uncountable signals seen by FISH) were associated with protein overexpression. Moreover, only 3 percent of cases with copy-number gains of both MYC and BCL2 genes had a DHIT signature on GEP, and combinations of rearrangements and copy-number gains were not significantly associated with a DHIT signature. Thus, copy-number gains of MYC or BCL2 do not appear to be biologically equivalent to rearrangements detected by FISH in defining DHIT lymphoma. Additionally, the authors searched for MYC gene mutations in 442 tumors and found mutations of exon 2 in some cases that resulted in an amino acid substitution disrupting binding of the most common antibody (Y69) used to detect MYC by immunohistochemistry. Analogously, others have described mutations in the BCL2 gene that can result in negative BCL2 immunohistochemistry in some BCL2-translocated follicular lymphomas.6 These results underscore the fact that immunohistochemistry for MYC and BCL2 proteins does not substitute for FISH evaluation of rearrangement of these genes in diagnosing DHIT lymphomas.
In Brief
The work of Dr. Collinge and colleagues has refined our interpretation of FISH results in approaching a diagnosis of DHIT lymphoma. They confirm the current definition that requires dual MYC and BCL2 translocation identified by FISH, rather than increased gene copy number seen by FISH and reveal mechanisms of discordance between immunohistochemistry and FISH results. Their findings also highlight the fact that mere rearrangements of MYC and BCL2 are not fully adequate to define a biologic DHIT lymphoma entity revealed by GEP: Whereas 88 percent of FISH-defined DHIT lymphomas had a DHIT GEP signature, 103 (66%) of 155 tumors with DHIT GEP signatures were not captured by FISH. As the authors note, the current DHIT lymphoma category defined by the presence of dual gene rearrangements is an oversimplification that does not encompass all cases with shared DHIT biology, and likely misses a significant cohort of patients with DLBCL who may benefit from intensified therapy; it remains to be determined what mechanism drives the DHIT signature in those cases lacking MYC and BCL2 rearrangements. It is notable that GEP technology, first used to identify DLBCL cell-of-origin subsets more than 20 years ago,1 still has not penetrated into the diagnostic arena, even in the current era in which next-generation sequencing has become standard-of-care for classifying many hematologic neoplasms. The contribution of Dr. Collinge and colleagues adds to the body of work that supports adding GEP to our diagnostic armamentarium to define biologically meaningful entities more accurately and to better achieve personalized treatment of hematologic neoplasms.
Competing Interests
Dr. Hasserjian indicated no relevant conflicts of interest.
