Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma.
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.
Department of Oncology, University Hospital of North Norway, Tromsø, Norway. egil.blix@uit.no
Egil S
ES
Blix
Jonathan M
JM
Irish
Anne
A
Husebekk
Jan
J
Delabie
Lise
L
Forfang
Anne M
AM
Tierens
June H
JH
Myklebust
Arne
A
Kolstad
D019013:CD40 Antigens / Q000378:metabolism; D023201:CD40 Ligand / Q000378:metabolism; D051925:CD79 Antigens / Q000378:metabolism; D016000:Cluster Analysis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007378:Interleukins / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism / Q000473:pathology; D016393:Lymphoma, B-Cell / Q000378:metabolism* / Q000473:pathology; D018442:Lymphoma, B-Cell, Marginal Zone / Q000378:metabolism / Q000473:pathology; D008954:Models, Biological; D051966:Phospholipase C gamma / Q000378:metabolism; D010750:Phosphoproteins / Q000145:classification / Q000378:metabolism*; D010766:Phosphorylation; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism; D050800:STAT6 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism / Q000473:pathology; D051996:Transcription Factor RelA / Q000378:metabolism
D019013:CD40 Antigens; D051925:CD79 Antigens; D007378:Interleukins; D010750:Phosphoproteins; D011947:Receptors, Antigen, B-Cell; D050799:STAT5 Transcription Factor; D050800:STAT6 Transcription Factor; D051996:Transcription Factor RelA; D023201:CD40 Ligand; D048049:Extracellular Signal-Regulated MAP Kinases; D051966:Phospholipase C gamma