Abstract
The integrin heterodimer CD49d/CD29 (a.k.a. Very Late Antigen-4, VLA-4) mediates cell-cell and cell-matrix interaction through the binding of its ligands VCAM-1 and fibronectin. VLA-4 can be present on the cell surface at different conformation states that affect the binding affinity for the ligands. In chronic lymphocytic leukemia (CLL), higher VLA-4 levels, as determined by measuring the expression of CD49d chain by flow cytometry, have been demonstrated to associate with a worse prognosis, in keeping with the role of VLA-4 as key molecule favoring CLL cell localization in protective niches of bone marrow and lymph nodes. Given the emerging clinical relevance of VLA-4 evaluation in CLL, both in the setting of the conventional chemo-immunotherapy and the novel drugs targeting the BCR pathway, here we describe the flow cytometric approaches followed by us to quantify the CD49d expression levels and the VLA-4 activation status in CLL cells.
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References
Hemler ME, Elices MJ, Parker C, Takada Y (1990) Structure of the integrin VLA-4 and its cell-cell and cell-matrix adhesion functions. Immunol Rev 114:45–65
Eksioglu-Demiralp E, Alpdogan O, Aktan M, Firatli T, Ozturk A, Budak T et al (1996) Variable expression of CD49d antigen in B cell chronic lymphocytic leukemia is related to disease stages. Leukemia 10:1331–1339
Zucchetto A, Bomben R, Dal Bo M, Bulian P, Benedetti D, Nanni P et al (2006) CD49d in B-cell chronic lymphocytic leukemia: correlated expression with CD38 and prognostic relevance. Leukemia 20:523–525
Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F et al (2008) Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood 111:865–873
Bulian P, Shanafelt TD, Fegan C, Zucchetto A, Cro L, Nuckel H et al (2014) CD49d is the strongest flow cytometry-based predictor of overall survival in chronic lymphocytic leukemia. J Clin Oncol 32:897–904
Shanafelt TD, Geyer SM, Bone ND, Tschumper RC, Witzig TE, Nowakowski GS et al (2008) CD49d expression is an independent predictor of overall survival in patients with chronic lymphocytic leukaemia: a prognostic parameter with therapeutic potential. Br J Haematol 140:537–546
Dal Bo M, Bulian P, Bomben R, Zucchetto A, Rossi FM, Pozzo F et al (2016) CD49d prevails over the novel recurrent mutations as independent prognosticator of overall survival in chronic lymphocytic leukemia. Leukemia 30:2011–2018
Tissino E, Benedetti D, Herman SE, ten Hacken E, Ahn IE, Chaffee KG et al (2017) Inside-out VLA-4 integrin activation is maintained in ibrutinib-treated chronic lymphocytic leukemia expressing CD49d: clinical relevance. Hematol Oncol 35:109–110
Hartmann TN, Grabovsky V, Wang W, Desch P, Rubenzer G, Wollner S et al (2009) Circulating B-cell chronic lymphocytic leukemia cells display impaired migration to lymph nodes and bone marrow. Cancer Res 69:3121–3130
Rose DM, Han J, Ginsberg MH (2002) Alpha4 integrins and the immune response. Immunol Rev 186:118–124
Ruoslahti E (1991) Integrins. J Clin Invest 87:1–5
Zucchetto A, Benedetti D, Tripodo C, Bomben R, Dal BM, Marconi D et al (2009) CD38/CD31, the CCL3 and CCL4 chemokines, and CD49d/vascular cell adhesion molecule-1 are interchained by sequential events sustaining chronic lymphocytic leukemia cell survival. Cancer Res 69:4001–4009
Burger JA, Ghia P, Rosenwald A, Caligaris-Cappio F (2009) The microenvironment in mature B-cell malignancies: a target for new treatment strategies. Blood 114:3367–3375
Arana E, Harwood NE, Batista FD (2008) Regulation of integrin activation through the B-cell receptor. J Cell Sci 121:2279–2286
Liu YJ, Arpin C (1997) Germinal center development. Immunol Rev 156:111–126
Chigaev A, Waller A, Amit O, Halip L, Bologa CG, Sklar LA (2009) Real-time analysis of conformation-sensitive antibody binding provides new insights into integrin conformational regulation. J Biol Chem 284:14337–14346
de Rooij MF, Kuil A, Geest CR, Eldering E, Chang BY, Buggy JJ et al (2012) The clinically active BTK inhibitor PCI-32765 targets B-cell receptor- and chemokine-controlled adhesion and migration in chronic lymphocytic leukemia. Blood 119:2590–2594
Herman SE, Mustafa RZ, Jones J, Wong DH, Farooqui M, Wiestner A (2015) Treatment with ibrutinib inhibits BTK- and VLA-4-dependent adhesion of chronic lymphocytic leukemia cells in vivo. Clin Cancer Res 21:4642–4651
Acknowledgment
This work was supported in part by the Associazione Italiana Ricerca Cancro (AIRC), Investigator Grants IG-17622; Progetto Giovani Ricercatori no. GR-2011-02346826, Ministero della Salute (MdS), Rome, Italy; 5 × 1000_2010_MdS to the Centro di Riferimento Oncologico of Aviano; Ricerca clinica, traslazionale, di base, epidemiologica e organizzativa, Regione Friuli Venezia Giulia (“Linfo-Check” Project), Trieste, Italy; Associazione Italiana contro le Leucemie, linfomi e mielomi (AIL), Venezia Section, Pramaggiore Group, Italy; PMU research fund-FFF-E-17/25-130-HAH.
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Zucchetto, A., Tissino, E., Chigaev, A., Hartmann, T.N., Gattei, V. (2019). Methods for Investigating VLA-4 (CD49d/CD29) Expression and Activation in Chronic Lymphocytic Leukemia and Its Clinical Applications. In: Malek, S. (eds) Chronic Lymphocytic Leukemia. Methods in Molecular Biology, vol 1881. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8876-1_8
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DOI: https://doi.org/10.1007/978-1-4939-8876-1_8
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