Leukemia (2014) 28, 166–173 & 2014 Macmillan Publishers Limited All rights reserved 0887-6924/14 www.nature.com/leu ORIGINAL ARTICLE Multiparameter flow cytometry for the identification of the Waldenstro¨m’s clone in IgM-MGUS and Waldenstro¨m’s Macroglobulinemia: new criteria for differential diagnosis and risk stratification B Paiva1,2, MC Montes1, R Garcı´a-Sanz1,2, EM Ocio1,2, J Alonso1, N de las Heras3, F Escalante3, R Cuello4, AG de Coca4, J Galende5, J Herna´ndez6, M Sierra7, A Martin1, E Pardal8,ABa´rez9, J Alonso10, L Suarez11, TJ Gonza´lez-Lo´ pez12, JJ Perez1, A Orfao2,13, M-B Vidrı´ales1,2 and JF San Miguel1,2 Although multiparameter flow cytometry (MFC) has demonstrated clinical relevance in monoclonal gammopathy of undetermined significance (MGUS)/myeloma, immunophenotypic studies on the full spectrum of Waldenstro¨m’s Macroglobulinemia (WM) remain scanty. Herein, a comprehensive MFC analysis on bone marrow samples from 244 newly diagnosed patients with an immunoglobulin M (IgM) monoclonal protein was performed, including 67 IgM-MGUS, 77 smoldering and 100 symptomatic WM. Our results show a progressive increase on the number and light-chain-isotype-positive B-cells from IgM-MGUS to smoldering and symptomatic WM (Po.001), with only 1% of IgM-MGUS patients showing 410% B cells or 100% light-chain-isotype-positive B-cells (Po.001). Complete light-chain restriction of the B-cell compartment was an independent prognostic factor for time-to progression in smoldering WM (median 26 months; HR: 19.8, P ¼ 0.001) and overall survival in symptomatic WM (median 44 months; HR: 2.6, P ¼ 0.004). The progressive accumulation of light-chain-isotype-positive B-cells accompanied the emergence of a characteristic Waldenstrom’s phenotype (CD22 þ dim/CD25 þ /CD27 þ /IgM þ ) that differed from other B-NHL by negative expression of CD5, CD10, CD11c or CD103. In contrast to myeloma, light-chain-isotype-positive plasma cells in IgM monoclonal gammopathies show otherwise normal antigenic expression. Our results highlight the potential value of MFC immunophenotyping for the characterization of the Waldenstro¨m’s clone, as well as for the differential diagnosis, risk of progression and survival in WM. Leukemia (2014) 28, 166–173; doi:10.1038/leu.2013.124 Keywords: Waldenstrom’s Macroglobulinemia; MGUS; flow cytometry; phenotype; plasma cells; survival INTRODUCTION there is some controversy to distinguish between smoldering WM The WHO defines Waldenstro¨m’s Macroglobulinemia (WM) as a and IgM-MGUS; some groups use the amount of serum 6 lymphoplasmacytic lymphoma associated with a monoclonal concentration of the M-protein and BM infiltration, while a immunoglobulin M (IgM) protein (regardless of size) and bone consensus panel defined BM disease involvement on histological examination as the main criterion.2 It is plausible to assume that marrow (BM) infiltration by small lymphocytes that may exhibit 7 plasma cell (PC) differentiation.1 As lymphoplasmacytic lymphoma similarly to multiple myeloma (MM), most (if not all) WM patients have eventually gone through the benign stages of IgM-MGUS infrequently involves the lymph nodes or other extramedullary and smoldering WM before developing clinical symptoms. sites, demonstration of BM infiltration (for example, through 2 Therefore, the identification of new objective criteria for the trephine biopsy) is therefore essential for the diagnosis of WM. differential diagnosis between these conditions, as well as more Many patients who fulfill the criteria for WM do not require accurate estimation of their risk of progression for optimal clinical immediate therapy because they are detected before developing management are mostly welcome, particularly in two settings: 3 disease-associated symptoms. These patients are classified as (i) to decide which patients are candidates for an early treatment smoldering WM, a clinically recognized entity with a cumulative intervention, and (ii) to optimize monitoring of premalignant probability of progression to symptomatic WM, amyloidosis or stages. However, the differential diagnosis among these patient lymphoma of 65% at 10 years.4 groups may be challenging as some asymptomatic cases can Smoldering WM patients have a greater risk of progression to present with markedly elevated IgM levels and BM infiltration, active disease than IgM-monoclonal gammopathy of undeter- while others show minimal tumor burden but require therapy due mined significance (MGUS) cases (18% at 10 years).5 Currently, to disease-related complications. Moreover, although serum IgM 1Hospital Universitario de Salamanca, Department of Hematology, Salamanca, Spain; 2IBSAL IBMCC (USAL-CSIC), Salamanca, Spain; 3Complejo Hospitalario de Leo´n, Leo´ n, Spain; 4Hospital Clı´nico Universitario de Valladolid, Valladolid, Spain; 5Hospital Comarcal del Bierzo, PonferradaLeo´ n, Spain; 6Hospital General de Segovia, Segovia, Spain; 7Hospital Virgen de la Concha, Zamora, Spain; 8Hospital Virgen del Puerto, Plasencia, Spain; 9Hospital Nuestra Sen˜ ora de Sonsoles, A´ vila, Spain; 10Hospital Rio Carrio´ n, Palencia, Spain; 11Hospital Santos Reyes, Aranda de Duero, Spain; 12Complejo Asistencial Universitario de Burgos, Burgos, Spain and 13Servicio General de Citometrı´a and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain. Correspondence: Professor JF San Miguel, IBSAL-Hospital Universitario de Salamanca, Paseo de San Vicente 58–182, Salamanca 37007, Spain. E-mail: [email protected] Received 12 March 2013; revised 10 April 2013; accepted 12 April 2013; accepted article preview online 24 April 2013; advance online publication, 14 May 2013 Immunophenotyping of IgM monoclonal gammopathies B Paiva et al 167 concentration is increased in WM, it may often do not exceed MFC immunophenotypic studies 2 3 g/dl, thus showing considerable overlap with IgM-MGUS; Erythrocyte-lysed whole-BM samples were immunophenotyped using similar variability has also been reported for the levels of BM 4-color combinations of the following fluorochrome-conjugated—fluores- infiltration.8–13 Finally, the differential diagnosis of WM may also cein isothiocyanate/phycoerythrin/peridinin chlorophyll protein-cyanin 5.5 be challenged by other diseases, which may also show an IgM (PerCPCy5.5)/allophycocyanin—monoclonal antibodies: cytoplasmatic Ig paraprotein (for example, IgM-MM, marginal zone, mantle cell or lambda light chain (CyIgl)/cytoplasmatic Ig kappa CyIgk/CD19/CD38, follicular lymphoma). CyBcl2/CD103/CD19/CD38, surface membrane IgM (SmIgM)/CD25/CD19/ CD38, SmIgM/CD27/CD19/CD38 CD11c/CD5/CD19/CD38, CD22/CD23/ In recent years, multiparameter flow cytometry (MFC) immuno- CD19/CD38, CD138/CD25/CD19/CD38, HLA-DR/CD11b/CD19/CD38, FMC7/ phenotyping has demonstrated to be of great value in the 14 CD24/CD19/CD38, CD45RA/CD45RO/CD19/CD38, CD38/CD10/C19/CD20, differential diagnosis of B-cell lymphoproliferative disorders, as CD38/CD56/CD19/CD45. Thus, a total of 23 different phenotypic markers, well as for baseline prognostication of patients with (non-IgM) including SmIgM, CyIgl and CyIgk were systematically evaluated in both MGUS and MM.15,16 Its use in WM was strongly recommended in BM mature B cells and PCs identified as CD19 þ /SSClow and CD38hi/SSCint the Second International WM Workshop in 2003,2 but immuno- cells, respectively, after careful exclusion of B-cell precursors (CD19 þ / hi low phenotypic studies on patients with IgM monoclonal CD38 /SSC ). A stain-and-then-lyse direct immunofluorescence techni- gammopathies remain limited, focused only on patients with que was used to evaluate all surface markers; for the staining of CyIgl, the most aggressive forms of the disease, and no consensus has CyIgk and CyBcl2, the Fix & Perm reagent kit (DAKO, Glostrup, Denmark) was used, strictly following the recommendations of the manufacturer. The been reached so far about the phenotypic characteristics of the 17–23 evaluation of SmIgM, CyIgl and CyIgk was performed after washing serum Waldenstro¨m’s clone. Ig. In all cases, one tube stained with CD19-PerCPCy5.5 and CD38-APC was Here, we report on a comprehensive immunophenotypic used for specific evaluation of baseline autofluorescence levels of B cells analysis of a large series of newly diagnosed IgM-MGUS, smolde- and PCs, respectively (negative control). ring and symptomatic WM patients. Our results show that MFC is a Data acquisition was performed in a FACSCalibur (4-color) flow valuable technique to assess the tumor burden and the degree of cytometer (Becton Dickinson Biosciences, BDB, San Jose, CA, USA) using clonality in patients’ BM aspirate, providing invaluable information the FACSDiva 6.1 software (BDB), and a two-step acquisition procedure. In 4 for the differential diagnosis of IgM-MGUS and prognostication of the first step, information about 5 Â 10 events corresponding to the WM. In addition, the specific phenotypic characteristics of clonal B whole-sample cellularity was stored; while in the second step, data about CD19 þ or CD38 þ gated events was selectively stored, for a minimum of cells and PCs are also described in detail. 106 leukocytes/tube. Data analysis was performed using the Infinicyt software (Cytognos, Salamanca, Spain). PATIENTS AND METHODS Patients Statistical analysis A total of 244 newly diagnosed patients with an IgM monoclonal The Mann–Whitney U test was used to estimate the statistical significance gammopathy—classified as IgM-MGUS (n ¼ 67), smoldering WM (n ¼ 77) of differences observed between groups. Smoldering WM patients who and symptomatic WM (n ¼ 100)—who were referred