Waldenstršms Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma that is characterized by immunoglobulin-M (IgM) secreting B-cells that infiltrate the bone marrow, spleen and lymph nodes. The WM tumor compartment displays notable heterogeneity and is comprised of small lymphocytes, plasmacytoid lymphocytes and plasma cells in varying fractions that can be identified by flow cytometry. It has been recently shown that the WM cell profile can shift over time from predominantly monotypic B-lymphocytic cells to a more homogenous plasma cell population in response to treatment with various chemoimmunotherapeutics (Barakat et al, Am J Clin Pathol, 2011). This shift in cell populace is reflected by loss of characteristic B-lymphocyte surface antigens (CD19, CD20) and acquisition/overexpression of plasma cell markers (CD38, CD138), which can be detected by flow cytometry. WM is a relatively rare cancer and there is a corresponding scarcity of reliable preclinical models with only 3 molecularly validated human WM cell lines in existence. Although similar to one another, these human-IgM secreting models, namely BCWM.1 (developed from a newly diagnosed patient), MWCL-1 (developed from a relapsed/refractory patient) and RPCI-WM1 (developed in our laboratory from a highly refractory terminal WM patient) may represent distinct subclasses of WM. We investigated if the clinical variability at the time of development of these models had any phenotypic implications through altered expression of surface proteins.
A comprehensive flow cytometric surface antigen analysis was performed using the BCWM.1, MWCL-1 and RPCI-WM1 cell lines. Surface antigens present on progenitor, immature, activated germinal center and memory B-cells along with those present on plasmablasts and plasma cells were examined. Presence of stem cell markers was also examined. Briefly, antibodies for CD5, 10, 11c, 13, 14, 16, 19, 20, 22, 23, 24, 25, 27, 28, 30, 32, 34, 35, 38, 39, 40, 43, 45, 45RA, 45RO, 52, 54, 56, 62L, 66b, 69, 70, 73, 79b, 80, 86, 90, 101, 105, 110, 111, 117, 123, 127, 133, 134, 135, 137, 138, 154, 184, 197, 202b, 243, 252, 268, 272, 278, 279, 309, 338, FMC7, HLADR, Kappa and Lambda were utilized. Antibodies for determining presence of intracellular CD247, 289, Kappa and Lambda were also used.
We observed notable differences between the immunophenotypic profiles of RPCI-WM1, BCWM.1 and MWCL-1. These variances are reflective of the dominating malignant B-cell population present at the time in the index patient from whom the cell lines were respectively established. By normalizing the mean fluorescent intensity signals to MESF values, we observed 14 differentially expressed antigens between BCWM.1 and MWCL-1, 23 CD antigens differentially altered between RPCI-WM1 vs. BCWM.1 and 29 surface markers differentially expressed in RPCI-WM1 relative to MWCL-1 cells (MESF fold change >2). Expression of 12 representative antigens across all WM models is shown in Table 1.
This is the first evaluation of the 3 most well established WM models. Our investigation displays important variations in surface protein expression and highlight biological distinction that may develop during the course of WM and/or exposure to therapeutics. This analysis validates that the RPCI-WM1 cell line was derived from a more advanced WM clone with predominantly plasmacytoid features that developed under ongoing therapeutic pressure and is consistent with the clinical phenotype of the index patient (rituximab, bortezomib and chemotherapy resistant). Understanding the significance of the distinct immunophenotypic profiles of existing WM models can (a) further improve the biology of WM, (b) impact of therapeutics on the WM clone and (c) engagement or disengagement of therapeutics targets (as is the case of CD20) during the disease course, which can help guide rational development of therapeutic strategies.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.