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Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins

Leukemia volume 27, pages 241–245 (2013)Cite this article

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Tumor-derived cell lines provide valuable models to assess disease characteristics but require careful assessment with regard to their representation of parental disease, and here genetic imprints from recent advances in genomic sequencing of primary tumor cells may prove highly relevant. In the rare B-cell malignancy Hairy Cell Leukemia (HCL), a number of cell lines have been generated, but evidence linking cell lines to primary tumor remains incomplete. These include Hair-M,1 HCLL-7876,2 EH,3 Eskol,4 HC-1(ref. 5) and HCLv-07,6 examined to date for ‘tumor’-associated CD20 phosphorylation,7 interferon-α oncogene regulation,8 CD11c gene expression9 and HCL cell adhesion and migration.10 Here we have assessed these HCL cell lines further by examining the phenotype in more detail, by comparing B-cell receptor (BCR) function and by evaluating the BRAF mutation. The BRAF V600E mutation is present near universally in typical HCL.11, 12, 13 However, a few cases lack this signature mutation, associated with tumor use of the IGHV4-34 gene,14 and in a solitary HCL case expressing CD5.15 In HCL variant (HCLv) disease, the absence of the BRAF V600E mutation is a consistent feature.12, 13, 14

Notably, four of the cell lines (HCLL-7876=Eskol>HC-1>Hair-M) were positive for ANXA1 expression by western blot analysis for the 38-kDa-size protein (Figures 1a–c), using the antibody ‘Purified Mouse anti-Annexin I’, clone 29/Annexin I17 (BD Transduction Laboratories, Oxford, UK). Positive controls for ANXA1 expression included four primary s-HCL tumor samples (s-HCL1/14/15/17) and two healthy PBMNC (peripheral blood mononuclear cells) preparations (non-B-cell positive). ANXA1 expression was also gauged by immunostaining of acetone-fixed cytospin preparations, using the same antibody with anti-mouse immunoglobulin (Ig)-peroxidase and diaminobenzidine substrate for detection, and hematoxylin staining for nuclei. The level of ANXA1 expression in the HCLL-7876 HCL cell line was lower than that seen in primary s-HCL tumor cells by both western blot analysis (Figure 1b) and immunostaining (Figure 1c). However, ANXA1 expression was a consistent feature of the bulk of cells in the selected HCLL-7876 cell line, with ANXA1 staining both nuclear and cytoplasmic protein as found in primary s-HCL cells (Figure 1c) and confirming western blot data. Separate studies also report convergence of results for detection of ANXA1 with western blots and immunostaining.23, 24 It is noteworthy that in extensive immunohistochemical staining for ANXA1 and other diagnostic markers in HCL cases showing weak or focal staining were scored as positive.18 These criteria affirm ANXA1 expression in the HCLL-7876 cell line, and indicate that its expression in the other three cell lines (Eskol, HC-1 and Hair-M) can also be considered positive. Two cell lines (EH and HCLv-07) were clearly negative for ANXA1 (Figures 1a and b) by western blot analysis, an expected phenotype for HCLv-07. ANXA1 immunostaining in the EH cell line confirmed its absence (Figure 1c). As EH is CD5+ (Figures 1a and d), it is unclear from these data whether it associates with ‘atypical’ HCL15 or the rare CD5+ HCLv cases.20, 21, 22

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Acknowledgements

This work was funded by Leukaemia and Lymphoma Research (UK). Cell lines were obtained from DMSZ (HC-1) or were kind gifts from The Danish Cancer Society/Bill Harvey (Hair-M and Eskol), Professor J Cawley at Liverpool (HCLL-7876), Dr Carl Shelley at Harvard (EH) and Dr M Sasaki at Juntendo University, Tokyo (HCLv-07).

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  1. Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK

    N J Weston-Bell, D Hendriks, G Sugiyarto, F Forconi & S S Sahota

  2. Department of Rheumatology and Clinical Immunology, Section Immunology, University Medical Center, Groningen, The Netherlands

    N A Bos

  3. Department of Hematology, University Medical Center, Groningen, The Netherlands

    H C Kluin-Nelemans

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Weston-Bell, N., Hendriks, D., Sugiyarto, G. et al. Hairy cell leukemia cell lines expressing annexin A1 and displaying B-cell receptor signals characteristic of primary tumor cells lack the signature BRAF mutation to reveal unrepresentative origins. Leukemia 27, 241–245 (2013). https://doi.org/10.1038/leu.2012.163

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