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BRIEF REPORT
From Servicio de Hematología, Hospital
Clínico Universitario, Salamanca; Centro de Investigación
del Cáncer, Salamanca; Servicio de Citometría,
Universidad de Salamanca; Hospital Clinic i Provincial, Barcelona;
Hospital General, Segovia; Hospital Virgen Blanca, León; Hospital
Clínico Universitario, Valladolid; Hospital Clínico,
Madrid; and Hospital Clínico, Zaragoza, Spain.
Multiparametric immunophenotyping can be a sensitive method
for analyzing the plasma cell (PC) compartment in patients with multiple myeloma because it discriminates between myelomatous and
normal PCs. Using this approach, we compared the efficacy of high-dose
chemotherapy followed by autologous stem cell transplantation (ASCT)
with that of conventional chemotherapy. We found that ASCT provided a
significantly greater reduction in the level of residual tumor PCs and
with better recovery of normal PCs. This profile of coexistence of
normal PCs and myelomatous PCs resembled that observed in monoclonal
gammopathy of undetermined significance. We also found that
treatment-induced changes in the PC compartment correlated with disease
outcome. Thus, patients in whom at least 30% of gated PCs had a normal
phenotype after treatment had a significantly longer progression-free
survival (60 ± 6 months versus 34 ± 12 months;
P = .02).
(Blood. 2002;99:1853-1856) Immunophenotyping is an attractive technique
for evaluating changes in the plasma cell (PC) compartment in patients
with multiple myeloma (MM) because it allows discrimination between
myelomatous PCs (MM-PCs) and normal PCs (N-PCs).1-3 The
distinction between the 2 is based on the presence of phenotypic
aberrations in MM-PCs that are absent in N-PCs.3-6 Using
immunoglobulin (Ig)-gene rearrangement and sorting experiments, we
previously demonstrated the clonal and polyclonal nature of MM-PCs and
N-PCs, respectively. Moreover, dilutional experiments4
showed that multiparameter flow cytometry immunophenotyping has a
sensitivity of 10 A total of 87 patients with MM were included in the study.
Criteria for entering the study were the presence of MM-PCs with an
aberrant phenotype at diagnosis (in a series of 120 patients, 110 patients [92%] had such aberrancies); response to initial chemotherapy (thus, 23 patients [21%] with refractory disease were
excluded); and inclusion in the 94 Spanish Program for the Study and
Treatment of Hematological Malignancies (PETHEMA) trial. In this
protocol, after 4 cycles of vincristine, bis-chloroethylnitrosourea, melphalan, cyclophosphamide, and prednisone-vincristine,
bis-chloroethylnitrosourea, doxorubicin, and dexamethasone, patients
with a response were randomly assigned to receive either ASCT (with
melphalan [200 mg/m2 of body-surface area], 47 patients)
or 8 additional cycles of chemotherapy (40 patients). Subsequently, all
patients received maintenance therapy with interferon Immunophenotypic evaluation of the BMPC compartment was done at
diagnosis and after treatment (3 months after ASCT and 1 month after
the total 12 cycles of chemotherapy). The methods used for analyses of
PCs were described previously.4,10 We used a panel of
monoclonal antibodies in quadruple combinations (CD38/56/19/45, CD138/28/33/38, and CD20/117/138/38). The PC phenotypic aberrancies identified at diagnosis were used as patient-specific probes for follow-up analyses. To increase the sensitivity level of the technique, we used a 2-step acquisition procedure in which up to
2 × 106 cells were acquired through a specific "live
gate" drawn on SSC/CD38+++/CD138+
cells. In all cases, an FL1/FL2/FL3 isotype-matched negative control CD38 for antigen-positive cells was used to evaluate
specifically the PC autofluorescence level.
For data analysis, Paint-A-Gate PRO software (Becton Dickinson,
San Jose, CA) was used according to well-established
methods.4,10 Three variables were evaluated. Whole bone
marrow (BM) cellularity was assessed and the percentages of
phenotypically aberrant MM-PCs and N-PCs in the BM were determined. We
also measured MM-PCs and N-PCs in purified PCs selected through a
specific electronic gate and assessed the percentage of N-PCs among the
total PCs (ie, the percentage of N-PCs among total purified/gated BMPCs
[N-PCs/TPCs]). PCs were considered normal if they met the following
criteria on immunophenotyping: CD38+++, CD56 The significance of the differences observed between groups was
evaluated with Mann-Whitney U and Wilcoxon tests. Survival curves were plotted according to the Kaplan-Meier method and compared by using log-rank, Tarone, and Breslow tests.
Changes in the PC compartment in patients treated with ASCT
compared with patients given chemotherapy
In patients with MM, ASCT is considered to provide higher response rates and longer survival than chemotherapy,11-14 but in most studies of this issue, response to treatment was based on standard criteria. Thus, it would be useful to have additional indications of the superiority of ASCT. Because MM results from expansion of malignant PCs, we hypothesized that use of sensitive techniques to assess changes occurring in this tumor cell compartment could provide additional insights into the differences between these 2 treatment methods. We found that, in comparison with chemotherapy, ASCT produced a significantly higher reduction in the percentage of residual MM-PCs together with a greater recovery of normal BMPCs. This PC profile resembled that observed in patients with monoclonal gammopathy of undetermined significance, in whom N-PCs and MM-PCs coexist, rather than that in patients with MM at diagnosis, in whom almost all BMPCs (> 95%) are phenotypically aberrant.1 We also correlated the immunophenotypic response to treatment (no MRD) with the electrophoretic response at the same time point. Almost all the patients who had only an objective response (on electrophoresis) had MRD (44 of 48 patients [92%]). Among the 39 patients who had complete remission (on electrophoresis), about half (20 patients) still had MRD. When immunofixation was used to define complete remission, 74% of patients had results consistent with those of immunophenotypic studies (MRD, 60%; and no MRD, 14%), but 14% of patients were found to have MRD on phenotyping though immunofixation assessment detected no MRD, whereas the remaining 12% had no MRD on phenotyping but did have MRD by immunofixation. Effect of changes in the PC compartment on PFS In all 87 patients, we evaluated the effect on PFS of changes observed in the PC compartment induced by treatment by analyzing BM samples obtained 3 months after ASCT or after 12 cycles of chemotherapy. As shown in Figure 1, patients with low levels of residual disease after treatment (< 0.12% MM-PCs) had a trend toward an improved PFS (P = .1). Patients with a high percentage of N-PCs ( 0.14%) had a slightly
longer median PFS (P = .1). However, analysis of
purified/gated PCs was the most powerful variable for discriminating
between different risk categories. Thus, patients in whom at least 30%
of gated PCs had a normal phenotype had a significantly longer PFS than
patients with less than 30% N-PCs (median PFS, 60 ± 6 months
versus 34 ± 12 months; P = .02).
It is known that the percentage of BMPCs assessed by conventional morphologic analysis has limited prognostic value, probably because of the patchy pattern of BM infiltration and possible dilution of the BM sample with blood, which can produce bias. These 2 factors might also affect the precision of flow cytometry-based immunophenotypic approaches for measuring MM-PCs and N-PCs with respect to overall BM cellularity. The new variable we used here (analysis of PC subsets in purified/gated BMPCs) is likely to be less influenced by sampling errors and could represent a more objective indicator for disease monitoring in patients with MM. Although the cut-off level of at least 30% for N-PCs/TPCs showed the highest predictive value in discriminating among MM patients at different risks of relapse, higher cut-off levels might be more accurate for specific assessment of patients undergoing ASCT, since the percentage of N-PCs/TPCs was significantly higher in this group of patients. Recovery of N-PCs after treatment may lead to recovery of noninvolved
Igs. As shown in Table 3, patients with
an increase of more than 50% in the level of noninvolved Igs had a
significantly higher percentage of N-PCs and N-PCs/TPCs, together with
a lower percentage of MM-PCs, than patients with an increase in
polyclonal Igs of less than 50% (P < .05). However, the
level of Ig recovery did not distinguish 2 different prognostic groups
(P = .8). It is possible that recovery of Ig requires more
time than recovery of PCs, and perhaps the prognostic importance of Ig
recovery must be evaluated at later time points.
In summary, our results show that multiparameter flow cytometry immunophenotyping is a rapid, easily standardized, sensitive method for evaluating the PC compartment in patients with MM and that it may offer valuable prognostic information.
We thank Ramón García-Sanz for statistical assistance, Mark Anderson for collaboration in the English language, and the physicians who referred patients for these studies.
Submitted June 5, 2001; accepted October 17, 2001.
Supported in part by a grant from Dirección General de Enseñanza Superior (PM97-0161), Ministerio de Educación y Cultura; and MM JEVITT SA firm and Fondo de Investigación Sanitaria (FIS 96/0397), Ministerio de Sanidad y Consumo, Madrid, Spain.
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 U.S.C. section 1734.
Reprints: Jesús F. San Miguel, Servicio de Hematología, Hospital Clínico Universitario, Po San Vicente 58-182, 37007 Salamanca, Spain; e-mail: sanmigiz{at}gugu.usal.es.
1. Ocqueteau M, Orfao A, Almeida J, et al. Immunophenotypic characterization of plasma cells from monoclonal gammopathy of undetermined significance patients: implications for the differential diagnosis between MGUS and multiple myeloma. Am J Pathol. 1998;152:1655-1665[Abstract]. 2. Rawstron AC, Owen RG, Davies FE, et al. Circulating plasma cells in multiple myeloma: characterization and correlation with disease stage. Br J Haematol. 1997;97:46-55[CrossRef][Medline] [Order article via Infotrieve].
3.
Harada H, Kawano MM, Huang N, et al.
Phenotypic difference of normal plasma cells from mature myeloma cells.
Blood.
1993;81:2658-2663 4. Almeida J, Orfao A, Ocqueteau M, et al. High-sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma. Br J Haematol. 1999;107:121-131[CrossRef][Medline] [Order article via Infotrieve]. 5. Ocqueteau M, Orfao A, Garcia-Sanz R, Almeida J, Gonzalez M, San Miguel JF. Expression of the CD117 antigen (c-Kit) on normal and myelomatous plasma cells. Br J Haematol. 1996;95:489-493[Medline] [Order article via Infotrieve].
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Pope B, Brown RD, Gibson J, Yuen E, Joshua D.
B7-2-positive myeloma: incidence, clinical characteristics, prognostic significance, and implications for tumor immunotherapy.
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2000;96:1274-1279 7. Yaccoby S, Barlogie B, Epstein J. Mature myeloma cells are proliferative with self-renewal potential. Blood. 1997;92:588a. 8. Yaccoby S, Barlogie B, Epstein J. Primary plasma cells growing in SCDI-hu mice: a model for studying the biology and treatment of myeloma and its manifestations. Blood. 1998;92:29082913.
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Alternating combination VCMP/VBAP chemotherapy versus melphalan/prednisone in the treatment of multiple myeloma: a randomized multicentric study of 487 patients.
J Clin Oncol.
1993;11:1165-1171 10. Orfao A, Ciudad J, Almeida J, San Miguel JF. Residual disease detection of leukemia. In: Stewart C,Nicholson JKA, eds. Immunophenotyping. New York, NY: Wiley-Liss; 2000:239-259.
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Barlogie B, Jagannath S, Vesole D, et al.
Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma.
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A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroup Francaise du Myelome.
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Impact on survival of high-dose therapy with autologous stem cell support in patients younger than 60 years with newly diagnosed multiple myeloma: a population-based study.
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© 2002 by The American Society of Hematology.
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  A. C. Rawstron, A. Orfao, M. Beksac, L. Bezdickova, R. A. Brooimans, H. Bumbea, K. Dalva, G. Fuhler, J. Gratama, D. Hose, et al. Report of the European Myeloma Network on multiparametric flow cytometry in multiple myeloma and related disorders Haematologica, March 1, 2008; 93(3): 431 - 438. [Abstract] [Full Text] [PDF]
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 E. Perez-Persona, M.-B. Vidriales, G. Mateo, R. Garcia-Sanz, M.-V. Mateos, A. G. de Coca, J. Galende, G. Martin-Nunez, J. M. Alonso, N. de las Heras, et al. New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smoldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells Blood, October 1, 2007; 110(7): 2586 - 2592. [Abstract] [Full Text] [PDF]
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M.-V. Mateos, J.-M. Hernandez, M.-T. Hernandez, N.-C. Gutierrez, L. Palomera, M. Fuertes, J. Diaz-Mediavilla, J.-J. Lahuerta, J. de la Rubia, M.-J. Terol, et al. Bortezomib plus melphalan and prednisone in elderly untreated patients with multiple myeloma: results of a multicenter phase 1/2 study Blood, October 1, 2006; 108(7): 2165 - 2172. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||||
Top
Abstract
Introduction
4 to 10
2
and dexamethasone. The 2 arms were well balanced according to
prognostic features, including the percentage of bone marrow PCs
(BMPCs) at diagnosis (Table 
ie,
MM-PCs were not detected at a sensitivity limit of 10
