Double Hit Diffuse Large B-cell Lymphomas
Double Hit Diffuse Large B-cell Lymphomas
All of the aforementioned series utilized FISH to determine the presence of a myc rearrangement. FISH is expensive, time-consuming, and may not be available for rapid routine use in all laboratories. In addition, certain DLBCLs without evidence of myc rearrangement on FISH exhibit features of molecular Burkitt lymphoma by gene expression profiling, suggesting that FISH only captures a subset of patients with myc-driven DLBCL. Two years ago, the first experiences using immunohistochemical approach to assess myc protein expression in formalin-fixed paraffin-embedded tissue were published. In a series from Harvard, of 77 cases of DLBCL, 15 had high myc protein as defined by nuclear staining in greater than 50% of tumor cells. All myc rearranged cases had increased myc protein expression using this immunohistochemical assay; there were four additional cases that were negative on FISH but had increased protein expression. These cases had confirmed increased myc transcriptional activity by gene set enrichment analysis, suggesting alternative mechanisms for myc deregulation in these cases. Importantly, these cases had similar poor outcome, inferior to patients without this abnormality, as those cased detected by FISH.
Two groups independently validated these immunohistochemistry results, to define a group of "double hit" DLBCL which may be diagnosed with routine immunohistochemical methods in 2012. The group from Denmark evaluated 193 cases of DLBCL uniformly treated with R-CHOP therapy, and performed routine staining for myc, bcl-2 as well as markers allowing for cell of origin classification. In addition, FISH was performed for detection of myc rearrangement and for t. Eleven percent of cases had evidence of myc rearrangement by FISH, and six percent of cases were double hit by FISH. Poor outcome was only observed in the double hit cases. Moreover, 29% of cases had high expression of myc and bcl-2 on immunohistochemistry evaluation. These cases had similar inferior overall survival on multivariate analysis, controlled for clinical and molecular prognostic factors, specifically germinal center genotype vs. non germinal center genotype. These results were confirmed in a validation set.
Johnson and colleagues from British Columbia used a similar platform to evaluate prospective cases of DLBCL with immunohistochemical stains for bcl-2 and myc. In the training cohort, concurrent expression of myc (defined as 40% or more of tumor cells staining positive) and bcl-2 was found in 21% of cases. Increased myc was only predictive of outcome if increased bcl-2 was also present, and these results were also validated in an independent cohort after adjusting for clinical and molecular high risk features.
Subsequently, the R-CHOP consortium group performed a comprehensive gene expression analysis of 893 patients DLBCL treated with R-CHOP. Double hit DLBCL defined either by FISH or immunohistochemistry occurred in both GCB and ABC types of DLBCL, and conferred a similar poor prognosis. Interestingly, there was no difference in gene expression signatures between the GCB and ABC subptypes in the absence of MYC/BCL2 coexpression, suggesting that the poor prognosis of ABC subtype is largely driven by double hit status. Tumors with double hit status had downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. The authors concluded that MYC/BCL2 co-expression, rather than cell-of-origin classification, is the best predictor of prognosis in patients with DLBCL treated with R-CHOP.
In summary, the poor outcome in DLBCL observed with myc positive disease is largely due to "double hit" biology and it is the concurrent expression of bcl-2 and c-myc that is important for outcome. Concurrent overexpression of myc and bcl-2 is present in both germinal center and activated B-cell DLBCL suggesting heterogeneous molecular pathways may be responsible for myc deregulation, but the impact of these pathways results in a universally poor outcome with conventional therapy.
Use of Immunohistochemistry to Define Myc Positive Disease
All of the aforementioned series utilized FISH to determine the presence of a myc rearrangement. FISH is expensive, time-consuming, and may not be available for rapid routine use in all laboratories. In addition, certain DLBCLs without evidence of myc rearrangement on FISH exhibit features of molecular Burkitt lymphoma by gene expression profiling, suggesting that FISH only captures a subset of patients with myc-driven DLBCL. Two years ago, the first experiences using immunohistochemical approach to assess myc protein expression in formalin-fixed paraffin-embedded tissue were published. In a series from Harvard, of 77 cases of DLBCL, 15 had high myc protein as defined by nuclear staining in greater than 50% of tumor cells. All myc rearranged cases had increased myc protein expression using this immunohistochemical assay; there were four additional cases that were negative on FISH but had increased protein expression. These cases had confirmed increased myc transcriptional activity by gene set enrichment analysis, suggesting alternative mechanisms for myc deregulation in these cases. Importantly, these cases had similar poor outcome, inferior to patients without this abnormality, as those cased detected by FISH.
Two groups independently validated these immunohistochemistry results, to define a group of "double hit" DLBCL which may be diagnosed with routine immunohistochemical methods in 2012. The group from Denmark evaluated 193 cases of DLBCL uniformly treated with R-CHOP therapy, and performed routine staining for myc, bcl-2 as well as markers allowing for cell of origin classification. In addition, FISH was performed for detection of myc rearrangement and for t. Eleven percent of cases had evidence of myc rearrangement by FISH, and six percent of cases were double hit by FISH. Poor outcome was only observed in the double hit cases. Moreover, 29% of cases had high expression of myc and bcl-2 on immunohistochemistry evaluation. These cases had similar inferior overall survival on multivariate analysis, controlled for clinical and molecular prognostic factors, specifically germinal center genotype vs. non germinal center genotype. These results were confirmed in a validation set.
Johnson and colleagues from British Columbia used a similar platform to evaluate prospective cases of DLBCL with immunohistochemical stains for bcl-2 and myc. In the training cohort, concurrent expression of myc (defined as 40% or more of tumor cells staining positive) and bcl-2 was found in 21% of cases. Increased myc was only predictive of outcome if increased bcl-2 was also present, and these results were also validated in an independent cohort after adjusting for clinical and molecular high risk features.
Subsequently, the R-CHOP consortium group performed a comprehensive gene expression analysis of 893 patients DLBCL treated with R-CHOP. Double hit DLBCL defined either by FISH or immunohistochemistry occurred in both GCB and ABC types of DLBCL, and conferred a similar poor prognosis. Interestingly, there was no difference in gene expression signatures between the GCB and ABC subptypes in the absence of MYC/BCL2 coexpression, suggesting that the poor prognosis of ABC subtype is largely driven by double hit status. Tumors with double hit status had downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. The authors concluded that MYC/BCL2 co-expression, rather than cell-of-origin classification, is the best predictor of prognosis in patients with DLBCL treated with R-CHOP.
In summary, the poor outcome in DLBCL observed with myc positive disease is largely due to "double hit" biology and it is the concurrent expression of bcl-2 and c-myc that is important for outcome. Concurrent overexpression of myc and bcl-2 is present in both germinal center and activated B-cell DLBCL suggesting heterogeneous molecular pathways may be responsible for myc deregulation, but the impact of these pathways results in a universally poor outcome with conventional therapy.
