Molecular Subtyping of Diffuse Large B-Cell Lymphoma Using a Novel Quantitative Reverse Transcription-polymerase Chain Reaction Assay.

Ta, Robert; Santini, Chris; Gou, Patricia; Lee, Greg; Tai, Yu Chuan; O'Brien, Cathal; Fontecha, Marcel; Grant, Cliona; Bacon, Larry; Finn, Stephen; Vandenberghe, Elisabeth; Quinn, Fiona; Dua, Rajiv; Flavin, Richard

Diffuse Large B-cell Lymphoma (DLBCL), is a heterogeneous disease. Cell-of-origin classification in DLBCL has identified Activated B-cell (ABC) and Germinal Center B-Cell (GCB) as two major subtypes. ABC subtype patients show reduced overall survival with standard therapies. We report development of a qRT-PCR-based Lymphoma cell-of-origin (LCOO) assay to determine ABC, GCB, and Unclassifiable (UC) subtypes in formalin-fixed, paraffin embedded tissue (FFPET) DLBCL samples. We trained the LCOO classifier on two DLBCL cohorts with validation done using an analytical grade assay in an independent cohort of 60 FFPET DLBCL samples. In the validation cohort, LCOO classification was 88.1%, 84.7%, and 84.7% concordant, respectively with microarray, Immunohistochemistry (Hans), and Lymphoma Subtyping Test (LST; NanoString Technologies, Inc. Seattle, WA, USA). Importantly, LCOO and LST assays commonly assigned subtypes in 17/18 (94.4%) ABC and 34/38 (89.5%) GCB DLBCL samples from this cohort. Progression-free survival (PFS) and overall survival (OS) of ABC and GCB subtypes, as classified by all platforms, were not significantly different in validation cohort. LCOO classification using publicly available microarray gene expression from two independent data sets (414 fresh frozen (FF) and 474 FFPET DLBCL biopsies) showed significantly worse outcome for ABC subtype compared to GCB subtype. In conclusion, a sensitive, reproducible, LCOO assay developed on an easy to standardize qRT-PCR platform could be an important clinical tool for DLCBL cell-of-origin classification.