Human Cancer Biology Quantitative Immunofluorescence Reveals the Signature of Active B-cell Receptor Signaling in Diffuse Large B-cell Lymphoma

Purpose:B-cell receptor (BCR)–mediated signaling is important in the pathogenesis of a subset of diffuse large B-cell lymphomas (DLBCL) and the BCR-associated kinases SYK and BTK have recently emerged as potential therapeutic targets. We sought to identify a signature of activated BCR signaling in DLBCL to aid the identification of tumors that may be most likely to respond to BCR-pathway inhibition. Experimental Design: We applied quantitative immunofluorescence (qIF) using antibodies to phosphorylated forms of proximal BCR signaling kinases LYN, SYK, and BTK and antibody to BCR-associated transcription factor FOXO1onBCR-cross-linked formalin-fixedparaffin-embedded (FFPE)DLBCLcell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n 1⁄4 154). Results: A robust signature of active BCR signaling was identified and validated in BCR-cross-linked DLBCL cell lines and in 71/154 (46%) of the primary DLBCL patient specimens. Further analysis of the primary biopsy samples revealed increased nuclear exclusion of FOXO1 amongDLBCLwith qIF evidence of active BCR signaling compared with those without (P 1⁄4 0.004). Nuclear exclusion of FOXO1 was also detected in a subset of DLBCL without evidence of proximal BCR signaling suggesting that alternative mechanisms for PI3K/AKT activation may mediate FOXO1 subcellular localization in these cases. Conclusion: This study establishes the feasibility of detecting BCR activation in primary FFPE biopsy specimens of DLBCL. It lays a foundation for future dissection of signal transduction networks in DLBCL and provides a potential platform for evaluating individual tumors in patients receiving novel therapies targeting the BCR pathway. Clin Cancer Res; 18(22); 6122–35. 2012 AACR.