title: Quantitative immunofluorescence reveals the signature of active B-cell receptor signaling in diffuse large B-cell lymphoma.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
authors.forename: Agata M
authors.initials: AM
authors.lastname: Bogusz
authors.forename: Richard H G
authors.initials: RH
authors.lastname: Baxter
authors.forename: Treeve
authors.initials: T
authors.lastname: Currie
authors.forename: Papiya
authors.initials: P
authors.lastname: Sinha
authors.forename: Aliyah R
authors.initials: AR
authors.lastname: Sohani
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: Kutok
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Rodig
mesh_terms: D000328:Adult; D000077329:Agammaglobulinaemia Tyrosine Kinase; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D016403:Lymphoma, Large B-Cell, Diffuse / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D015398:Signal Transduction; D000072377:Syk Kinase; D046888:Tissue Array Analysis; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D011947:Receptors, Antigen, B-Cell; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000606896:SYK protein, human; D000072377:Syk Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt