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PDF file, 871K, S1 Chromogenic staining (DAB, brown) of paraffin-embedded DLBCL cell lines DHL4, and Toledo, untreated and BCR crosslinked, with anti-pLYN (Y396), anti-pSYK (Y323), and anti-pBTK (Y551); S2 Schematic illustration of Tissuequest analysis workflow. Individual cells are first identified by finding the nuclei (DAPI); S3 Qualitative Western blot of whole cell lysates for untreated (BCR-) and crosslinked (BCR+) cell lines; S4 S4 Chromogenic staining (DAB, brown) of two paraffin-embedded patient DLBCL specimens with anti-pLYN (Y396), anti-pSYK (Y323), and anti-pBTK (Y551); S5 (A) Schematic illustration of Tissuequest workflow extended to subcellular localization of FOXO1. In this example there are 83% of all cells that are positive for FOXO1 and 67% of the cells show cytoplasmic FOXO1 staining (67%). The ratio of these percentages is used to calculate the Fcyt score; S6 (A) Chromogenic staining (DAB, brown) of paraffin-embedded DLBCL cell lines DHL4, and Toledo, untreated and BCR crosslinked, with anti- FOXO1; S7 (A) IHC staining of paraffin-embedded DLBCL cell lines U2932, DHL6 and Toledo, untreated and crosslinked, with anti-pAKTsub (red) and DAPI (blue); S8 (A) Comparison of immunofluorescent images evaluated by an expert using a 3-tier system (0 - negative, 1 - weak positive, 2 - strong positive); S9 Comparison of FOXO1 cytoplasmic localization and BCR signaling in primary DLBCL tumors.

PDF file - 462K, Table S1: pLYN, pSYK and pBTK levels in DLBCL cell lines; S2 DLBCL cell line classification

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 FOXO1 on BCR-cross-linked formalin-fixed paraffin-embedded (FFPE) DLBCL cell lines as a model system and on two clinical cohorts of FFPE DLBCL specimens (n = 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 among DLBCL with qIF evidence of active BCR signaling compared with those without (P = 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.