Leukemia (2011) 25, 697–706 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE Competition between clonal plasma cells and normal cells for potentially overlapping bone marrow niches is associated with a progressively altered cellular distribution in MGUS vs myeloma B Paiva1,2,MPe´rez-Andre´s2,3, M-B Vı´driales1,2, J Almeida2,3, N de las Heras4, M-V Mateos1,2,LLo´pez-Corral1,2, NC Gutie´rrez1,2, J Blanco1, A Oriol5, MT Herna´ndez6, F de Arriba7, AG de Coca8, M-J Terol9, J de la Rubia10, Y Gonza´lez11, A Martı´n12, A Sureda13, M Schmidt-Hieber2,3, A Schmitz14, HE Johnsen14, J-J Lahuerta15, J Blade´16, JF San-Miguel1,2 and A Orfao2,3 on behalf of the GEM (Grupo Espan˜ol de MM)/PETHEMA (Programa para el Estudio de la Terape´utica en Hemopatı´as Malignas) cooperative study groups and the Myeloma Stem Cell Network (MSCNET) 1Servicio de Hematologı´a, Hospital Universitario de Salamanca, Salamanca, Spain; 2Servicio de Hematologia, Centro de Investigacio´n del Ca´ncer (CIC, IBMCC USAL-CSIC), Salamanca, Spain; 3Servicio General de Citometrı´a and Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain; 4Servicio de Hematologı´a, Complejo Hospitalario de Leo´n, Leo´n, Spain; 5Servicio de Hematologı´a, Hospital Universitari Germans Trias i Pujol, Badalona, Spain; 6Servicio de Hematologı´a, Hospital Universitario de Canarias, Tenerife, Spain; 7Servicio de Hematologı´a, Hospital Morales Meseguer, Murcia, Spain; 8Servicio de Hematologı´a, Hospital Clı´nico Universitario de Valladolid, Valladolid, Spain; 9Servicio de Hematologı´a, Hospital Clinico Universitario de Valencia, Valencia, Spain; 10Servicio de Hematologı´a, Hospital La Fe, Valencia, Spain; 11Servicio de Hematologı´a, Hospital Josep Trueta, Girona, Spain; 12Servicio de Hematologı´a, Hospital Virgen de la Concha, Zamora, Spain; 13Servicio de Hematologı´a, Hospital Santa Creu I Sant Pau, Barcelona, Spain; 14Servicio de Hematologia, Service of Hematology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark; 15Servicio de Hematologı´a, Hospital 12 de Octubre, Madrid, Spain and 16Servicio de Hematologı´a, Hospital Clı´nic, IDIBAPS, Barcelona, Spain Disappearance of normal bone marrow (BM) plasma cells (PC) progressionFE1 and 10% per year for monoclonal gammo- predicts malignant transformation of monoclonal gammopathy pathy of undetermined significance (MGUS) and smoldering of undetermined significance (MGUS) and smoldering myeloma myeloma (SMM), respectively1–4Fto symptomatic multiple (SMM) into symptomatic multiple myeloma (MM). The homing, þ myeloma (MM) with median overall survival rates of 3–7 behavior and survival of normal PC, but also CD34 hemato- 5,6 7–14 poietic stem cells (HSC), B-cell precursors, and clonal PC years. Despite the different tumor mass and clinical largely depends on their interaction with stromal cell-derived behavior of the disease, clonal PC from MGUS, SMM and MM factor-1 (SDF-1) expressing, potentially overlapping BM stromal patients show highly similar and largely overlapping genetic cell niches. Here, we investigate the distribution, phenotypic profiles.15–17 In addition, no clear phenotypic differences have characteristics and competitive migration capacity of these cell been reported so far among clonal PC from MGUS, SMM and populations in patients with MGUS, SMM and MM vs healthy MM,18 except for a few molecules involved in the interaction adults (HA) aged 460 years. Our results show that BM and 19 peripheral blood (PB) clonal PC progressively increase from between PC and their microenvironment. MGUS to MM, the latter showing a slightly more immature We have previously shown that the proportion of normal PC immunophenotype. Of note, such increased number of clonal within the bone marrow (BM) PC compartment (normal PC/BM PC is associated with progressive depletion of normal PC, þ PC) is an efficient single parameter for discrimination between B-cell precursors and CD34 HSC in the BM, also with a parallel MGUS and MM;20 at diagnosis, most MGUS cases (480%) increase in PB. In an ex vivo model, normal PC, B-cell 21 þ display 45% normal PC/BM PC, whereas the great majority precursors and CD34 HSC from MGUS and SMM, but not 22 MM patients, were able to abrogate the migration of clonal PC (485%) of symptomatic MM show o5% normal PC/BM PC. into serial concentrations of SDF-1. Overall, our results show Additionally, the presence of 45% normal PC/BM PC at that progressive competition and replacement of normal BM diagnosis is associated with both a lower risk of progression of cells by clonal PC is associated with more advanced disease in MGUS and SMM,21 and a favorable outcome in MM.22 In turn, a patients with MGUS, SMM and MM. progressive decrease in the serum levels of uninvolved Leukemia (2011) 25, 697–706; doi:10.1038/leu.2010.320; immunoglobulins (Ig) is observed from MGUS to SMM and published online 21 January 2011 21,22 Keywords: monoclonal gammopathies; malignant transformation; MM. Altogether, these results suggest that disappearance of bone marrow niche competition; plasma cells normal BM PC followed by impaired secretion of normal Igs is associated with malignant transformation of MGUS and SMM as well as disease progression in MM. Serum Ig levels are tightly regulated in humans and they Introduction largely depend on Ig production and secretion by long-living BM and mucosa-associated lymphoid tissue-derived PC.23,24 Plasma cell (PC) disorders include an heterogeneous spectrum Recently produced normal PC, which have left secondary of diseases, from pre-malignant conditions with variable rates of lymphoid tissues through peripheral blood (PB), usually need to migrate into BM niches and adhere to CXCL12 (SDF-1)- Correspondence: Professor A Orfao, Centro de Investigacio´n del expressing BM stromal cells to become long-living BM ´ Cancer, Avda. Universidad de Coimbra S/N (Campos Miguel de PC.23,24 SDF-1-expressing BM stromal cells are also crucial for Unamuno), Salamanca 37007, Spain. 25 F E-mail: [email protected] early pro-B and together with interleukin-7-expressing 25 26,27 Received 21 October 2010; revised 30 November 2010; accepted cells Fpotentially also for later pre-B-cell development 10 December 2010; published online 21 January 2011 into immature/transitional and naive B-cells that will exit the BM Altered bone marrow homing to PC niches in MG B Paiva et al 698 through PB.28 These findings suggest that due to the limited were studied, with paired BM and PB samples available in number of BM PC niches, normal PC as well as normal B-cell 6 of these latter 20 HA analyzed. For BM control samples, no precursors25 could compete with clonal PC for BM PC niches; in significant differences in their compositions were observed turn, CXCL12-CXCR4 signaling appears to be also a key between subjects who were younger and those older than component of the CD34 þ hematopoietic stem cell (HSC) 55 years (Supplementary Table 1). niche.29,30 Interestingly, recent results show that in normal All control and patient samples were collected after informed individuals, both CD34 þ HSC and PC recirculate between consent was given by each individual, according to the local the BM and the PB.23,31,32 Accordingly, it can be hypothesized ethical committees and the Helsinki Declaration. that BM B-cell precursors, CD34 þ HSC and PC are under a tight and continuous regulation by the SDF-1/CXCR4 axis, Multiparameter flow cytometry immunophenotypic with a certain degree of competition among them, for the studies same BM niches. In this competition, an advantage could be Approximately 1 ml of EDTA-anti-coagulated PB/case was expected for the malignant clone based on its numerical immunophenotyped using a direct eight-color immunofluores- expansion. An additional factor to be considered is the cence stain-and-then-lyse technique,32,37 with the following advanced age of these patients as SDF-1-expressing BM niches combination of monoclonal antibodies (Pacific blue (PB)/ undergo age-induced impairment in their ability to support anemonia majano cyan (AmCyan)/fluorescein isothiocyanate/ 33 normal hematopoiesis, which could be partly explained by a phycoerythrin (PE)/peridinin chlorophyll protein–cyanin 5.5 progressive increase in the number of adipocytic deposits in (PerCP–Cy5.5)/PE–cyanin 7 (PE–Cy7)/allophycocyanin/alexa- 34,35 the BM. Thus, progressive replacement of normal precursors fluor 700 (AF700)): CD20/CD45/surface IgM (sIgM) or sIgl/ and normal PC by the malignant clone could contribute to sIgG, sIgA or sIgk/CD19/CD10/CD27/CD38. In all PB samples explain the occurrence of cytopenias and hypogammaglobuli- analyzed, the following maturation-associated B-cell subsets nemia in MM patients. were identified32 in addition to circulating plasmablasts/PC In this study, we analyze the distribution and competitive (CD10À/CD27 þþ/CD38 þþ): (i) immature (CD10 þ /CD27À/ þ migrating capacity of B-cell precursors, CD34 HSC, normal CD38 þ ), (ii) naive (CD10À/CD27À/CD38À), (iii) memory non- PC and clonal PC in the BM and PB of patients with PC disorders switched (CD10À/CD27 þ /CD38À/IgM þ ) and (iv) memory vs healthy adults (HA) aged 460 years. Taken together, our switched (CD10À/CD27 þ /CD38À/IgMÀ) B-cells. Because of results show that progressive competition and replacement of their different patterns of expression of CD38, CD19 and normal BM cells by clonal PC is associated with the malignant CD45, and their unique light scatter characteristics,22,38 this transformation of MGUS and SMM and progression of sympto- multiparameter eight-color staining also allowed detection of matic MM. circulating PB clonal PC, as clearly different from normal circulating PB plasmablasts/PC. In a subset of 13 cases (2 MGUS, 2 SMM and 9 MM), a more detailed phenotypic Materials and methods characterization of PB vs BM clonal PC was performed using the following antibody combinations: (i) CD19/CD45/sIgl/sIgG Patients, controls and samples plus sIgA/CD138/CD27/sIgk/CD38; (ii) CD19/CD45/CD20/CD22/ Overall a total of 206 patients with clonal PC disorders were CD138/CD27/CD56/CD38; (iii) CD19/CD45/FMC7/CD24/CD138/ prospectively studied.