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2009 114: 4369-4372
Prepublished online September 15, 2009;
doi :10.1182/blood-2009-05-221689
The persistence of immunophenotypically normal residual bone marrow plasma cells at diagnosis identifies a good prognostic subgroup of symptomatic multiple myeloma patients
Bruno Paiva, Maria-Belén Vidriales, Gema Mateo, Jose J. Perez, Maria Angeles Montalbán, Anna Sureda, Laura Montejano, Norma C. Gutierrez, Alfonso Garcia de Coca, Natalia de las Heras, Maria Victoria Mateos, Maria Consuelo López-Berges, Raimundo Garcia-Boyero, Josefina Galende, Jose Hernandez, Luis Palomera, Dolores Carrera, Rafael Martinez, Javier de la Rubia, Alejandro Martin, Yolanda Gonzalez, Joan Blade, Juan Jose Lahuerta, Alberto Orfao, Jesus F. San-Miguel and on behalf of the GEM (Grupo Espaňol de MM)/PETHEMA (Programa para el Estudio de la Terapéutica en Hemopatias Malignas) Cooperative Study Groups
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From bloodjournal.hematologylibrary.org at MASARYKOVY UNIVERSITY on April 9, 2011. For personal use CLINICAL TRIALS AND OBSERVATIONS _on|y-_
Brief report
The persistence of immunophenotypically normal residual bone marrow plasma cells at diagnosis identifies a good prognostic subgroup of symptomatic multiple myeloma patients
*Bruno Paiva,1 *Maria-Belen Vidriales,1,2 Gema Mateo,1,2 Jose J. Perez,1 Maria Angeles Montalbän,3 Anna Sureda,4 Laura Montejano,3 Norma C. Gutierrez,12 Alfonso Garcia de Coca,5 Natalia de las Heras,6 Maria Victoria Mateos,1,2 Maria Consuelo Lopez-Berges,1 Raimundo Garcia-Boyero,7 Josefina Galende,8 Jose Hernandez,9 Luis Palomera,10 Dolores Carrera,11 Rafael Martinez,12 Javier de la Rubia,13 Alejandro Martin,14 Yolanda Gonzalez,15 Joan Blade,16 Juan Jose Lahuerta,3 Alberto Orfao,2,17 and Jesus F. San-Miguel1,2 on behalf of the GEM (Grupo Espahol de MMVPETHEMA (Programa para el Estudio de la Terapeutica en Hemopatias Malignas) Cooperative Study Groups
'Hospital Universitario de Salamanca, Salamanca; 2Centro de Investigation del Cancer, Salamanca; 3Hospital 12 de Octubre, Madrid; "Hospital Santa Creu I Sant Pau, Barcelona; 5Hospital Clinico Universitario de Valladolid, Valladolid; 6Complejo Hospitalario de Leon, Leon; 'Hospital General de Castellön, Castellön; BHospital del Bierzo, Ponferrada; 9Hospital General de Segovia, Segovia; "Hospital Lozano Blesa, Zaragoza; "Hospital Central de Asturias, Oviedo; 12Clinico San Carlos, Madrid; "Hospital La Fe, Valencia; "Hospital Virgen de la Concha, Zamora; 15Hospital Dr. Josep Trueta, Gerona; 16Hospital Clinic, Institut d'lnvestigacions Biomediques August Pi i Sunyer Barcelona, Barcelona; and 17Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain
Multiparameter flow cytometry immuno-phenotyping allows discrimination between normal (N-) and myelomatous (MM-) plasma cells (PCs) within the bone marrow plasma cell compartment (BMPCs). Here we report on the prognostic relevance of detecting more than 5% residual normal plasma cells from all bone marrow plasma cells (N-PCs/BMPCs) by multiparameter flow cytometry in a series of 594 newly diagnosed symptomatic MM patients, uniformly treated according to
Introduction _
the Grupo Espahol de MM 2000 (GEM2000) protocol. Our results show that symptomatic MM patients with more than 5% N-PCs/BMPCs (n = 80 of 594; 14%) have a favorable baseline clinical prospect, together with a significantly lower frequency of high-risk cytogenetic abnormalities and higher response rates. Moreover, this group of patients had a significantly longer progression-free survival (median, 54 vs 42 months, P = .001) and overall survival (median, not reached vs
89 months, P = .04) than patients with less than or equal to 5% N-PCs/BMPCs. Our findings support the clinical value of detecting residual normal PCs in MM patients at diagnosis because this reveals a good prognostic category that could benefit from specific therapeutic approaches. This trial was registered at www.clinicaltri-als.gov as NCT00560053. (Blood. 2009; 114:4369-4372)
Multiparameter flow cytometry (MFC) immunophenotyping allows discrimination between myelomatous plasma cells (MM-PCs) and normal/reactive PC (N-PCs).14 This has been used for the differential diagnosis between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM),3 the identification of high-risk MGUS and smoldering MM (SMM) patients4 minimal residual disease investigation,58 definition of prognostic antigenic profiles,1 and identification of new therapeutic targets.2 Typically, in symptomatic MM patients at diagnosis, most bone marrow plasma cells (BMPCs) are clonal, and N-PCs from all BMPCs are only detected in a minority of MM cases.3-9 By contrast, 82% of MGUS and 40% of SMM patients show more than 5% residual normal plasma cells from all bone marrow plasma cells (N-PCs/BMPCs), in association with a low risk of progression to symptomatic MM.4 Here we report on the results of a prospective analysis of the prognostic impact of the presence of more than 5% N-PCs/BMPCs at diagnosis in a large series of uniformly treated symptomatic MM patients.
Methods
Patients
The study included 594 untreated, symptomatic MM patients diagnosed according to the International Myeloma Working Group criteria10 and uniformly treated following the Spanish GEM2000 protocol (VBMCP/ VBAD X6 plus autologous stem cell transplantation [ASCT]).16 Written informed consent was obtained in accordance with the Declaration of Helsinki, as well as Institutional Review Board approval from each participating hospital. Response was assessed using the European Group for Blood and Marrow Transplant criteria.1112 At the study endpoint, 380 patients (64%) relapsed/progressed and 188 (32%) died, with a median follow-up of 54 months; median progression-free survival (PFS) and overall survival (OS) were 44 and 97 months, respectively. It is the current standard of clinical practice in MM to report cytogenetic results on purified PCs,13 but unfortunately this was not part of the initial workload of the GEM2000 protocol. Therefore, we used a complementary series of 501 patients with complete cytogenetic/fluorescence in situ hybridization data
Submitted May 14, 2009; accepted August 18, 2009. Prepublished online as Blood First Edition paper, September 15, 2009; DOI 10.1182/blood-2009-05-221689.
The online version of this article contains a data supplement.
*B.P. and M.-B.V. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
© 2009 by The American Society of Hematology
BLOOD, 12 NOVEMBER 2009 • VOLUME 114, NUMBER 20
4369
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4370     PAIVAetal 0n'y- BLOOD, 12 NOVEMBER 2009 • VOLUME 114, NUMBER 20
Table 1. Patient demographics and baseline characteristics of symptomatic MM patients
	% of normal PC from all BMPC (N-PC/BMPC)		
Patient demographics and baseline characteristics	s 5% (n = 514)	> 5% (n = 80)	P
Male/female*	283 (55%)/231 (45%)	48 (60%)/32 (40%)	NS
Age.y Subtype of MM*	58 (32-70)	58 (35-70)	NS
IgG	304 (59%)	37 (46%)	.06
IgA	123 (24%)	17(22%)	NS
Bence-Jones protein	82(16%)	22 (27%)	.04
Nonsecretory ISS*	5(1%)	4 (5%)	NS
Stage I	180 (35%)	38 (48%)	.03
Stage II	226 (44%)	30 (37%)	NS
Stage III	108 (21%)	12(15%)	NS
ß2-microglobulin, mg/Lt	3.2 (0.09-41)	2.5 (0-24)	.009
Hemoglobin, g/l_t	106(10-167)	121 (60-168)	< .001
Albumin, g/dl_t	3.6 (0.3-7)	3.8(1.8-6.4)	.02
Platelet count, x109/l_t	207 (6-990)	241 (58-486)	< .001
M-component, g/dl_t	4 (0-47)	2 (0-22)	< .001
Immunoparesis, 1 or 2 Ig*	427 (83%)	34 (42%)	.003
Plasma cells by morphology, %t	40% (1-100%)	17 (1-95)	< .001
Plasma cells by MFC, %f	13(0.1-90)	1.6(0.05-22)	< .001
Plasma cells in S-phase, %t	1.4 (0-15)	2.0(0-15)	NS
CD117+ cases by MFC, n (%)*	175 (34)	39 (49)	.04
Patient demographics and baseline characteristics of symptomatic MM patients grouped according to the percentage of N-PC/BMPC.
MM indicates multiple myeloma; N-PC/BMPC, normal plasma cells from all bone marrow plasma cells; PC, plasma cells; BMPC, bone marrow plasma cells; NS, statistically not significant (P > .05); ISS, international staging system; and MFC, multiparameter flow cytometry. *Results expressed as percentage of cases. tResults expressed as median (range).
from immunomagnetic-enriched plasma cells, with a rather short follow-up, which precludes survival analysis. Information of serum-free light chain (sFLC) was also extracted from this series.
MFC immunophenotypic studies
Erythrocyte-lysed whole BM samples were stained using a 4-color direct immunofluorescence technique, previously described in detail.168 The following monoclonal antibody combinations (fluorescein isothiocyanate/ phycoerythrin/peridinin chlorophyll protein-Cy5.5/allophycocyanin) were used to identify aberrant antigen expression in PCs: CD38/CD56/CD19/ CD45, CD138/CD28/CD33/CD38, and CD20/CD117/CD138/CD38.1"-814 Acquisition was performed in a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest program (BD Biosciences). 'A4-6-8 Information was recorded for at least 3 X 103 BMPCs/tube. The Paint-A-Gate PRO program (BD Biosciences) was used for data analysis, following the recommendations of the European Myeloma Network.14
Statistical analyses
The x2 and Mann-Whitney U tests were used to estimate statistical significant differences. Survival curves were plotted using the Kaplan-
Meier method, assessing differences with the log-rank test. For the multivariate analysis, the Cox regression proportional hazard model (stepwise regression) was used. Statistical analyses were carried out with SPSS (Version 15.0; SPSS Inc).
Results and discussion
Two groups have identified the presence on N-PCs in MM patients at diagnosis in a small series.9-15 However, neither the number of these cells nor the clinical and prognostic value was analyzed. Here, 80 of 594 newly diagnosed MM patients (14%) had more than 5% N-PCs/BMPCs by MFC, all of them with CRAB features (increased calcium, renal insufficiency, anaemia, or bone lesions). Interestingly, the presence of more than 5% N-PCs/BMPCs was associated with characteristics related to a favorable prognosis1619 (Table 1): lower £2 microglobulin (P)2M (P = .01) and M-component serum levels (P < .001), lower BMPC infiltration by both morphology (P < .001) and MFC (P < .001), together with
Table 2. Cytogenetics
	% of normal PC fron	l all BMPC (N-PC/BMPC)	
Cytogenetics	s 5% (n = 439)	> 5% (n = 62)	P
IgH translocations	187 (43%)	8(13%)	< .001
t(4;14)	66 (15%)	0 (0%)	.002
t(11;14)	79 (18%)	8(13%)	NS
t(14;16)	18 (4%)	0 (0%)	NS
Others	35 (8%)	0 (0%)	.009
Del (13q)	180 (41%)	3 (5%)	< .001
Del (17p)	36 (8%)	2 (3%)	NS
High-risk: any t(4; 14), t(14;16), or del(17p)-	114 (26%)	2 (3%)	.006
Information on patient cytogenetics corresponds to a parallel series of not uniformly treated MM patients, not to the GEM2000 series reported in the present paper. Results expressed as percentage of cases. NS indicates not statistically significant.
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BLOOD,12NOVEMBER2009-VOLUME114,NUMBER20 0nly-  PROGNOSTIC VALUE OF MFC DETECTING NORMAL PC IN MM 4371
Table 3. Response to therapy
EBMT criteria	s 5% N =	PC/BMPC	> 5% N	= PC/BMPC	P	
	After induction	After ASCT	After induction	After ASCT	After induction	After ASCT
CR	56(11%)	168 (33%)	17 (21%)	51 (64%)	.01	< .001
nCR	61 (12%)	99 (19%)	19 (24%)	8 (10%)	.005	< .001
PR or less	397 (77%)	247 (48%)	44 (55%)	21 (26%)	< .001	< .001
Results are expressed as number (percentage) of cases. N = PC indicates normal plasma cells; BMPC, bone marrow plasma cells; and ASCT, autologus stem eel transplantation.
higher hemoglobin levels (P < .001), among other features (Table 1). In addition, immunoparesis (reduction below the lower normal limit in the levels of one or 2 uninvolved Ig) was significantly less frequent among patients with more than 5% N-PCs/BMPCs than in those with less than or equal to 5% N-PCs/BMPCs (42% vs 83%, respectively; P = .003), probably reflecting higher levels of residual N-PCs. Concerning the MM-PC immunophenotypic profile, the only antigen showing significant differences was CD 117, a phenotypic marker associated with a favorable outcome,1-20-21 with cases holding more than 5% N-PCs/BMPCs showing a higher incidence of positive CD117 expression (49% vs 34%, respectively; P = .04). In the complementary series of patients with complete cytogenetic/ftuorescence in situ hybridization information (Table 2), the frequency of cases with more than 5% N-PCs/BMPCs (12%; Table 1) was almost identical, which excludes a bias in patient selection. Interestingly, patients with more than 5% N-PCs/BMPCs had a lower incidence of del (13q) (5% vs 41%; P < .001) and IgH translocations (13% vs 43%; P < .001) than cases with less than or equal to 5% N-PCs/BMPCs. Moreover, whereas the incidence of t(ll;14), which has little influence on survival, was similar in both group of patients (13% vs 18%; not significant), t(4;14) was not detected in cases with more than 5% N-PCs/BMPCs, although it represented 15% of cases with less than or equal to 5% N-PCs/BMPCs (P = .002). In addition, when
high-risk cytogenetic cases [t(4;14), t(4;16), or del (17p)] were grouped, we founded a significantly lower frequency in cases with more than 5% N-PCs/BMPCs (3% vs 26%, respectively; P = .006). Moreover, considering patients with sFLC data available (N = 261) at diagnosis, cases with more than 5% N-PCs/BMPCs presented a higher frequency of normal sFLC ratio than those with less than or equal to 5% N-PCs/BMPCs (10 of 33, 30%; vs 16 of 228, 7%; P < .001).
Concerning response to therapy (Table 3), patients with more than 5% compared with those with less than or equal to 5% N-PCs/BMPCs showed higher rates of complete remission after induction (21% vs 11%, respectively; P < .001), and at day 100 after ASCT (64% vs 33%, respectively; P < .001). Accordingly, cases with more than 5% N-PCs/BMPCs were more frequently minimal residual disease negative at day 100 after ASCT (68% vs 37%; P < .001) and also showed a faster and more complete recovery of the polyclonal PC population at day 100 after ASCT, compared with less than or equal to 5% N-PCs/BMPCs cases (mean ratio of N-PCs/BMPCs of 87 vs 64%, respectively; P < .001). Differences in response rates translated into distinct survival, as cases with more than 5% N-PCs/BMPCs showed a better outcome than patients with less than or equal to 5% N-PCs/BMPCs with significant differences in both PFS (median, 54 vs 42 months; P = .001) and OS (median, not reached vs 89 months, P = .04)
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(Figure 1). Therefore, the presence of more than 5% N-PCs/ BMPCs emerges as a new prognostic factor for symptomatic MM patients, similar to what has previously been found in both MGUS and SMM.4 Other baseline significant factors for survival in the univariate analysis were: low hemoglobin (< 100 g/L; P < .008), higher calcium (> 10 mg/dL; P < .04), increased creatinine (> 2 mg/dL; P = .002) levels, higher percentages of BMPCs by microscopy (> 30% BMPCs; P < .006) and MFC (> 15%BMPCs; P ^ .007), advanced disease (International Staging System [ISS] stage III; P < .004), and high-risk cytogenetics (P < .001). Despite the association found between the presence of more than 5% N-PCs/BMPCs and immunoparesis, it should be emphasized that the latter variable did not appear to have a significant impact on survival, suggesting that it cannot be used as a surrogate marker to replace immunophenotyping. Multivariate analysis performed in cases with cytogenetic information (N = 176) showed only cytogenetics as an independent prognostic factor for PFS (P < .001) and OS (P = .002). Because cytogenetic information was only available for a subset of patients, a new multivariate analysis was performed for the whole series. With respect to PFS, both the ISS disease stage (relative risk of progression [RR] = 1.4; P = .03) and the percentage of N-PCs/BMPCs (RR = 1.6; P = .008) were selected as independent prognostic factors, whereas for OS hemoglobin levels (RR = 1.6; P = .01) together with the ISS disease stage (RR = 2.1; P = .003) fitted the model.
In conclusion, our results show that in symptomatic MM patients the identification of a significant number of residual normal PCs at diagnosis (> 5% N-PCs/BMPCs) reveals a specific subgroup of patients with a unique biologic signature and prolonged survival who could benefit from specific therapeutic approaches.
References _
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Acknowledgments
We thank all members of the GEM/PETHEMA group for their cooperation.
This work was supported by the Cooperative Research Thematic Network (RTICs; RD06/0020/0006), MM Jevitt, SL firm, and Instituto de Salud Carlos III/ Subdireccion General de Investigation Sanitaria (FIS: PI060339, 02/0905, 01/0089/01-02, and PI060339).
Authorship
Contribution: J.F.S.-M., J.J.L., and A.O. conceived the idea, and together with M.-B.V., designed the study protocol; B.P., M.-B.V., G.M., J.J.P., M.A.M., M.C.L.-B., and L.M. analyzed the flow cytometry data; A.S., N.C.G., A.G.d.C, N.d.l.H., M.V.M., R.G.-B., J.G., J.H., L.P., D.C., R.M., J.d.l.R., A.M., Y.G., J.J.L., and J.B. contributed with provision of study material or patients; B.P., M.-B.V., and G.M. collected and assembled data; B.P, M.-B.V., and J.F.S.-M. analyzed and interpreted data; B.P. and M.-B.V. performed statistical analysis; B.P, M.-B.V, and J.F.S.-M. wrote the manuscript; and all authors reviewed and approved the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
A complete list of GEM Cooperative Study Group participants appears in the online "Appendix" (available on the Blood website; see the Supplemental Materials link at the top of the online article).
Correspondence: Jesus F. San-Miguel, Hospital Universitario de Salamanca, Paseo de San Vicente 58-182, 37007 Salamanca, Spain; e-mail: sanmigiz@usal.es.
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