Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma.

Blix ES, Irish JM, Husebekk A, Delabie J, Forfang L, Tierens AM, Myklebust JH, Kolstad A
BMC Cancer. 2012 12: 478

PMID: 23072591 · PMCID: PMC3519597 · DOI:10.1186/1471-2407-12-478

BACKGROUND - Knowledge about signaling pathways in malignant cells may provide prognostic and diagnostic information in addition to identify potential molecular targets for therapy. B-cell receptor (BCR) and co-receptor CD40 signaling is essential for normal B cells, and there is increasing evidence that signaling via BCR and CD40 plays an important role in the pathogenesis of B-cell lymphoma. The aim of this study was to investigate basal and induced signaling in lymphoma B cells and infiltrating T cells in single-cell suspensions of biopsies from small cell lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) and marginal zone lymphoma (MZL) patients.

METHODS - Samples from untreated SLL/CLL and MZL patients were examined for basal and activation induced signaling by phospho-specific flow cytometry. A panel of 9 stimulation conditions targeting B and T cells, including crosslinking of the B cell receptor (BCR), CD40 ligand and interleukins in combination with 12 matching phospho-protein readouts was used to study signaling.

RESULTS - Malignant B cells from SLL/CLL patients had higher basal levels of phosphorylated (p)-SFKs, p-PLCγ, p-ERK, p-p38, p-p65 (NF-κB), p-STAT5 and p-STAT6, compared to healthy donor B cells. In contrast, anti-BCR induced signaling was highly impaired in SLL/CLL and MZL B cells as determined by low p-SFK, p-SYK and p-PLCγ levels. Impaired anti-BCR-induced p-PLCγ was associated with reduced surface expression of IgM and CD79b. Similarly, CD40L-induced p-ERK and p-p38 were also significantly reduced in lymphoma B cells, whereas p-p65 (NF-κB) was equal to that of normal B cells. In contrast, IL-2, IL-7 and IL-15 induced p-STAT5 in tumor-infiltrating T cells were not different from normal T cells.

CONCLUSIONS - BCR signaling and CD40L-induced p-p38 was suppressed in malignant B cells from SLL/CLL and MZL patients. Single-cell phospho-specific flow cytometry for detection of basal as well as activation-induced phosphorylation of signaling proteins in distinct cell populations can be used to identify aberrant signaling pathways.

MeSH Terms (21)

CD40 Antigens CD40 Ligand CD79 Antigens Cluster Analysis Extracellular Signal-Regulated MAP Kinases Flow Cytometry Humans Interleukins Leukemia, Lymphocytic, Chronic, B-Cell Lymphoma, B-Cell Lymphoma, B-Cell, Marginal Zone Models, Biological Phospholipase C gamma Phosphoproteins Phosphorylation Receptors, Antigen, B-Cell Signal Transduction STAT5 Transcription Factor STAT6 Transcription Factor T-Lymphocytes Transcription Factor RelA

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