Quantitative Immunoshistochemistry Identifies B-Cell Receptor Signaling and AKT Activity in Diffuse Large B-Cell Lymphoma
Agata M Bogusz, Richard HG Baxter, Treeve Currie, Papiya Sinha, Jeffery L Kutok, Scott J Rodig. Brigham and Women's Hospital, Boston, MA; Yale University, New Haven, CT
Background: Diffuse large B cell lymphoma (DLBCL) is the most common type on non-Hodgkin lymphoma. The prognosis is poor indicating the need for more individualized therapeutic approaches. B cell receptor (BCR) mediated signaling and PI3K/AKT signaling are implicated in the pathogenesis of DLBCL. Recently, the BCR-downstream kinases SYK and BTK and AKT have emerged as potential therapeutic targets. Application of these targeted therapies requires quantitative assessment of the activity of distinct signal transduction networks in clinical specimens.
Design: We employed quantitative immunohistochemical (qIHC) analysis in formalin-fixed paraffin embedded DLBCL cell lines and a cohort of 60 patient specimens on a tissue microarray using antibodies to phosphorylated forms of proximal BCR related kinases LYN, SYK, BTK. We also evaluated the subcellular localization of AKT-regulated transcription factor FOXO1, and examined signal integration between the BCR and AKT signaling.
Results: We identified a robust protein signature underlying BCR signaling in DLBCL patient specimens. Active BCR signaling was successfully detected in more than 50% of the examined tumors. Further analysis of distal BCR signaling via PI3K/AKT pathway revealed a survival-associated signal: cytoplasmic localization of the forkhead transcription factor FOXO1 was seen in 52% of all tumors and was associated with active BCR signature in 60-70% of positive specimens. Nuclear exclusion of FOXO1 was independent of BCR signaling in a subset of DLBCL tumors suggesting that constitutive AKT activation may mediate survival in these cases. Cytoplasmic localization of FOXO1 correlated with evidence of AKT activation in the majority of the DLBCL cell lines, providing a robust surrogate marker for AKT activity in patient specimens.
Conclusions: These results reveal that a large proportion of DLBCLs manifest active BCR signaling that translates to AKT activation and cytoplasmic FOXO1 localization. We conclude that qIHC provides a framework for assessing the integrity and activity of the BCR pathway in DLBCL biopsy samples that will assist in patient selection for appropriate targeted therapy.
Tuesday, March 20, 2012 1:00 PM
Poster Session IV # 200, Tuesday Afternoon