The Complexity and Challenges of Translating the Dark Zone Signature into Immunohistochemistry in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma/High-grade B-cell lymphoma with MYC and BCL2 rearrangement (DLBCL/HGBCL-MYC/BCL2) is a distinct disease entity with worse prognosis and is usually diagnosed with identification of MYC and BCL2 rearrangement by fluorescence in situ hybridization (FISH) analysis. Recent progress in gene expression analysis has identified a dark zone signature (DZsig), which characterizes a gene expression profile (GEP) for DLBCL/HGBCL-MYC/BCL2. Notably, DZsig includes both MYC/BCL2-double-hit and non-double-hit cases with DLBCL, and high-grade and Burkitt morphologies; importantly, the latter group is present in numbers that are comparable to or greater than the former group. Although GEP analysis can identify cases exhibiting HGBCL-like biology regardless of double-hit status, GEP analysis will not be globally applied to DLBCL cases due to cost and equipment constraints. Therefore, simpler surrogate approaches for detecting DZsig, such as immunohistochemistry (IHC), are desired. Here, we attempted to develop an immunohistochemical approach that can serve as an alternative to GEP or FISH assays utilizing 287 tumors with DLBCL morphology, regardless of double-hit status. Our strategy for detecting DZsig by IHC is based on a two-step algorithm, which involves applying the Hans classifier and antibodies to MYC, ALOX5, and LMO2. The DZ-IHC algorithm had a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 76.2%, 95.5%, 59.3%, and 98.1%, respectively. The 5-year overall survival (OS) rates for immunohistochemical DZ (iDZ)-positive, iDZ-negative subtypes in GCB-type and non-GCB-type tumors were 55%, 82%, and 69%, respectively (p < 0.01), which is similar to that reported using the GEP assay. To date, this represents the largest cohort evaluated for DZsig selection by IHC; nevertheless, the algorithm is not yet adequate for routine application owing to its suboptimal PPV and further improvements are needed for clinical implementation.