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Open Access Highly Accessed Research article

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

Egil S Blix12*, Jonathan M Irish34, Anne Husebekk5, Jan Delabie6, Lise Forfang7, Anne M Tierens6, June H Myklebust7 and Arne Kolstad8

Author Affiliations

1 Department of Oncology, University Hospital of North Norway, Tromsø, Norway

2 Immunology Research group, Institute of Medical Biology, University of Tromsø, Tromsø, Norway

3 Department of Medicine, Oncology Division, Stanford University, Stanford, CA 94305, USA

4 Department of Microbiology and Immunology, Baxter Laboratory of Genetic Pharmacology, Stanford University, Stanford, CA 94305, USA

5 Department of Laboratory Medicine, University Hospital North Norway, Tromsø, Norway

6 Department of Pathology, Division of Cancer Medicine and Surgery, Oslo University Hospital, Oslo, Norway

7 Institute for Cancer Research, Oslo University Hospital and Centre for Cancer Biomedicine, Oslo, Norway

8 Department of Oncology, Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway

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BMC Cancer 2012, 12:478  doi:10.1186/1471-2407-12-478

Published: 16 October 2012

Abstract

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.

Keywords:
B-cell lymphoma; B-cell receptor; CD40; Phospho-flow cytometry; Cell signaling