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Originally published as JCO Early Release 10.1200/JCO.2007.15.4120 on April 28 2008 © 2008 American Society of Clinical Oncology. Prognostic Value of Immunophenotyping in Multiple Myeloma: A Study by the PETHEMA/GEM Cooperative Study Groups on Patients Uniformly Treated With High-Dose Therapy
From the Hospital Universitario de Salamanca; Centro de Investigación del Cáncer (CIC, IBMCC USAL-CSIC); Servicio General de Citometría, Universidad de Salamanca; Hospital 12 de Octubre; Clínico San Carlos, Madrid; Clínic Universitari Barcelona; Hospital Sant Pau Barcelona; Hospital La Fé; Clínico Universitario, Valencia; Hospital Germans Trias i Pujol Badalona; Hospital de Galdakao; Hospital Morales Messeguer, Murcia; and the Hospital Lozano Blesa, Zaragoza, Spain Corresponding author: Jesús F. San Miguel, Servicio de Hematología, Hospital Universitario de Salamanca, Paseo San Vicente 58-182, 37007 Salamanca, SPAIN; e-mail: sanmigiz{at}usal.es
Purpose To analyze the prognostic impact of immunophenotyping in patients with multiple myeloma (MM). Patients and Methods We have prospectively analyzed the prognostic impact of antigenic markers, assessed by multiparametric flow cytometry, in a series of 685 newly diagnosed MM patients that were uniformly treated according to the GEM 2000 protocol. Results Our results show that expression of both CD19 and CD28 as well as the absence of CD117 were associated with a significantly shorter progression free-survival (PFS) and overall survival (OS). Interestingly, the CD28 expression correlated with t(14;16) and del(17p), while CD117-negative patients were associated with t(4;14) and del(13q). Simultaneous assessment of CD28 and CD117 antigens allowed stratification of patients with MM into three risk categories: poor risk (CD28 positive CD117 negative), intermediate (either both markers negative or both positive), and good risk (CD28 negative CD117 positive), with PFS rates of 30, 37, and 45 months, respectively (P = .01), and OS rates of 45, 68, and not reached, respectively (P = .0001). Conclusion To the best of our knowledge, this is the first prospective analysis in which the prognostic impact of a relatively high number of antigenic markers has been simultaneously analyzed in a large series of uniformly treated patients, showing that the expression of several antigens (particularly CD28 and CD117) on bone marrow plasma cells from patients with MM can help to identify patients at high risk of progression.
Multiple myeloma (MM) is a devastating disease with a highly heterogeneous outcome, with survival durations ranging from a few months to longer than 10 years. Multiparameter flow cytometry immunophenotyping has been increasingly used for diagnostic and prognostic evaluation of hematologic malignancies.1-5 In MM, immunophenotypic analysis is restricted to research purposes and to differential diagnosis of unusual atypical cases.1,6-9 Some studies suggest that markers associated with a more immature PC phenotype (CD20, CD45, sIg)10-12 and the expression of certain cancer testis on myelomatous PC13-14 correlate with a poor outcome. Also, downregulation of CD56 and a higher expression of CD44 have been associated with an extramedullary spreading of malignant PC,15,16 and expression of CD28 has been related to disease activity.17-21 However, frequent discrepancies have been reported, which may be due to an inappropriate study design (small series of patients and not uniformly treated) and technical pitfalls (single v multiparameter labelings, use of different clones of monoclonal antibody conjugates and distinct criteria for definition of positivity). As a consequence, the clinical and prognostic value of immunophenotyping in MM remains questionable. We have prospectively analyzed the prognostic impact of a relatively high number of antigenic markers in a series of 685 newly diagnosed patients with MM uniformly treated according to the Spanish GEM/PETHEMA 2000 protocol. Our results show that expression of several antigens (particularly CD28 and CD117) on bone marrow PC from MM patients can help to identify patients at high risk of progression.
Patients A total of 685 untreated symptomatic patients with MM22 were included in this study, all were treated with the GEM 2000 protocol (six alternating cycles of VBCMP/VBAD followed by high-dose therapy: melphalan 200 mg/m2 supported by autologous stem-cell transplant). Table 1 presents patient characteristics. At the time this was written, 422 patients (62%) relapsed/progressed and 269 patients (39%) died, with a median follow-up for survivors of 48 months. The median progression-free survival (PFS) and overall survival (OS) were 37 months (95% CI, 33 to 41 months) and 67 months (95% CI, 57 to 76 months), respectively. Response23 to induction chemotherapy (before autologous transplantation) showed: 12% complete remissions (CR) with negative immunofixation (IF), 12% near CR (negative electrophoresis but positive IF), 70% with partial remission (PR), and 6% with progression. At day +100 after transplantation, 36% of patients were in CR with negative IF, 17% in near CR, 43% in PR, and 3% in progression.
Immunophenotypic Studies Immunophenotypic studies were performed at diagnosis on bone marrow samples obtained after informed consent, and referred within 24 hours to two central laboratories (Hospital Universitario de Salamanca, 70% of samples; Hospital 12 Octubre de Madrid, 30% of samples). Both laboratories used a previously common protocol as part of the work package of the Spanish Myeloma Network. Erythrocyte-lysed whole bone marrow (BM) samples were stained using the following surface four-color combinations (FITC/PE/PerCP or PerCP-Cy-5.5/APC): CD38/CD56/CD19/CD45, CD138/CD28/CD33/CD38, and CD20/CD117/CD138/CD38. An aliquot just labeled with CD38-APC was used as negative control. Data acquisition was performed on a FACSCalibur flow cytometer (BD Biosciences, San José, CA) according to well-established methods.24 Information on  3 x 103 PC was recorded for further analysis. In samples containing  10% PC, a second acquisition step was required in order to analyze 3 x 103 PC, as previously reported.6,24,25 Analysis was performed on selected PC, using the Paint-A-Gate-PRO software (BD Biosciences), as previously reported.24 Monoclonal antibodies combinations were designed to distinguish between myelomatous and normal PC based on either aberrant expression of markers (ie, asynchronous expression of CD19, CD20, and/or CD45) and/or the expression of an antigen at abnormally high levels (ie, overexpression of CD28, CD33, CD56, and/or CD117).24 Therefore, the results here reported specifically correspond to myelomatous PC. The antigenic profile of myelomatous PC was assessed based on: presence or absence of antigen expression; pattern of antigen expression (homogeneous v heterogeneous); and percentage of PC showing positive expression for a given antigen. For further statistical analyses, cases were grouped into two categories: positive cases, which included those showing an either bright or weak, but uniform positivity; and negative cases (Table 2). Cases with an heterogeneous pattern of expression (from negative to positive) were considered to be positive if more than 20% of the PC displayed a level of expression above that of the control level; otherwise, they were considered to be negative.
The number of bone marrow PC by morphology was 43% (range, 5% to 96%). As expected, multiparameter flow cytometry showed lower percentages of BM PC than morphology (median, 13%; range, 1% to 95%), probably due to a dilutional effect.
Fluorescence in Situ Hybridization Analysis
Statistical Analyses
Frequency of Antigen Expression Table 2 summarizes the frequencies and patterns of antigen expression found in the 685 patients with MM. The results show that the antigenic profile of myelomatous PC is highly diverse (Table 2). Myelomatous PC from a minority of patients retained surface antigens typically associated with early B-cell maturation stages: CD19 expression was found in 4% of cases (heterogeneous pattern of expression); while CD20 and CD45 were detected in 17% and 27% of the cases, respectively, (homogeneous dim or bright expression; Table 2). Bright expression of CD56, a marker involved in the anchorage of PC to stromal structures, was present in 60% of patients with MM. Among the remaining patients, 15% showed dim reactivity for CD56 while the other cases expression of this antigen on BM PC was partially or completely negative (Table 2). Positivity for CD117, a tyrosine kinase receptor absent in normal PC, was detected in one third of all myeloma patients (32%). Other antigens such as CD28 and CD33, whose expression is typically associated with non-B-cell hematopoietic lineages (activated T-lymphocytes and myeloid cells, respectively) were observed in 36% and 18% of the patients, respectively.
Impact of Antigen Expression on Patients’ Survival
On analyzing the prognostic impact of different antigen combinations, we found that based on the simultaneous assessment of CD28 and CD117 expression three different risk group categories could be identified: good prognosis, CD28-negative CD117-positive patients, with a median PFS and OS of 45 months and not reached, respectively; poor prognosis, CD28-positive CD117-negative patients, with a median PFS and OS of 30 and 45 months, respectively; and an intermediate prognosis subgroup (CD28-negative/CD117-negative and CD28-positive/CD117-positive patients) with median PFS and OS of 37 and 68 months, respectively (Table 3 and Fig 2).
Relationship Between the Pattern of Antigen Expression and Disease Characteristics Expression of CD28 was associated with a significant higher frequency of advanced International staging system (ISS) stages, high PC proliferative activity (S phase 2.5%), nonhyperdiploidy DNA cell content, as well as with higher frequency of t(4;14) and del(17p) (Table 4). In turn, lack of CD117 expression was associated with higher levels of BM infiltration, greater frequencies of anemia, renal impairment (creatinine 2 mg/dL), elevated β2-microglobulin, advanced ISS, nonhyperdiploid cases, IgH translocations—including t(11;14) and t(4;14)—and del(13q) (Table 4). Interestingly, among patients with advanced clinical stage (ISS 3), the presence of a CD28-positive CD117-negative phenotype identified a subgroup of patients with shorter survival as compared with that of patients without this adverse phenotypic signature (PRF at 3 years, 26% v 44%; P = .10; OS at 3 years, 40% v 58%; P = .04). Regarding the correlation of high-risk cytogenetics [t(4,14); t(4;16); or del(17p)], with immunophenotypic profiles, it should be noted that within this cohort of high-risk patients only 7% displayed what we have considered a favorable antigenic profile (CD28 negative CD117 positive), while 93% expressed either CD28-positive or CD117-negative antigens (although only 25% coexpressed both). Moreover, within this high-risk cytogenetics group, the CD28-positive CD117-negative profile did not influence disease outcome.
On analyzing the influence of antigen expression on the response to therapy, before and after transplantation, any of the antigens tested showed significant influence. However, interestingly none of the patients that were in progression at day +100 after transplantation (n = 18) showed the good prognosis CD28-negative CD117-positive immunophenotypic pattern. A multivariate analysis of prognostic factors for survival was performed in the whole series of patients (n = 685) and subsequently in cases with available cytogenetic information (n = 231). In the whole series, variables showing an independent adverse impact on OS were: more than 10% BM PC by flow cytometry (P = .03), anemia (< 10 g/dL; P = .0001), high S phase of PC (> 2.5%; P = .007), advanced age (> 60 years; P = .03), ISS stage (3; P = .03), and a CD28-positive CD117-negative phenotype (P = .01). For PFS, the same variables except the CD28 and CD117 immunophenotype, were selected in the multivariate analysis. Once cytogenetic information was included, the antigen expression lost their independent prognostic value for OS, and prognostic factors included t(4;14) (P = .001), S phase of PC (P = .003), advanced ISS (P = .004) and age older than 60 years (P = .003). As far as PFS, only t(4;14) (P = .0001) and del(17p) (P = .03) retained their independent value in the Cox proportional hazards model.
The analysis of the antigenic profile of malignant cells has been shown to be a valuable tool for both the diagnosis and the identification of prognostic markers in different hematologic diseases.27-29 In contrast, in MM attempts directed to investigate the prognostic value of the immunophenotypic characteristics of PC have frequently lead to conflicting results, probably due to technical pitfalls or short series of heterogeneously treated patients. In this study, we have prospectively analyzed the prognostic impact of seven relevant antigenic markers, assessed by multiparameter flow cytometry, in a series of 685 newly diagnosed patients with MM uniformly treated according to the GEM 2000 protocol. To the best of our knowledge, this is the largest prospective trial designed with this goal. Our results show that three individual markers (CD19, CD28, and CD117) may afford prognostic information. Moreover, the combinations of CD28 and CD117 allow patient stratification into three risk categories: poor risk CD28-positive CD117-negative patients (23%), intermediate CD28-negative CD117-negative and CD28-positive CD117-negative patients (56%), and good risk CD28-negative CD117-positive myeloma patients (21%). Therefore, we can identify one fourth of patients with MM who would not benefit from autologous stem-cell transplantation, and in which alternative therapeutic strategies based on novel agents should be encouraged. In contrast, a similar proportion of patients who are CD28 negative CD117 positive enjoy a prolonged survival with ASTC. According to the results of this study, immunophenotypical assessment of patients with MM at diagnosis should include evaluation of the CD19, CD28, and CD117 expression. In order to specifically assess the expression of these markers on myelomatous plasma cells, they should be combined with CD38, CD138, and CD45, as well as CD56, in order to better discriminate between normal and abnormal plasma cells.44 In our study, the individual antigen showing more clear influence on disease outcome was CD28, a costimulatory molecule involved in T-cell activation. Interestingly, recent reports21 suggest that CD28 could be involved in dendritic cell–PC interactions within the bone marrow stroma, which could play an important role in myeloma cell survival. This is supported by the fact that the addition of anti-CD28 monoclonal antiobody to myelomatous PC cultures induces apoptosis.30 Several small studies, including our own, had already suggested that expression of CD28 is associated with an increased myelomatous PC proliferation, tumoral expansion, and treatment failure15,19,31; moreover it is associated with a nonhyperdiploidy MM, which is also a poor prognostic feature.31 This association has been confirmed in this study; in addition, herein, to our knowledge, we report for the first time the association between CD28 expression and both t(4;14) and del(17p), as well as advanced ISS and a high proliferative PC index. The c-kit receptor (CD117) is a tyrosine kinase involved in cell growth that is absent in normal PC but can be detected in one third of patients with myeloma. Information on the prognostic impact of CD117 in MM is scant. Although our initial data,32 as well as other small studies,33,34 suggest that c-kit–positive MM might represent a poor-risk category, subsequent data from our group,31 as well as from Bataille et al,35 indicate that patients with CD117-positive MM could have a better outcome. Once again, the sample size and treatment heterogeneity may account for such discrepancies. In any case, this study convincingly shows that CD117 expression is associated with a significantly longer PFS and OS, and that together with CD28 it probably represents the prognostically most important combination of phenotypic markers in MM. Moreover, we show some relevant associations between this marker and several characteristics of the disease. Thus, patients lacking CD117 expression showed high BM PC infiltration, more frequent renal impairment, anemia, and advanced clinical stage. In line with our previous report,36 CD117-negative patients were associated with a higher frequency of nonhyperdiploidy and del(13q). In addition, we have now found that this latter cohort of patients is associated with a higher frequency of t(4;14). CD56 (NCAM) is an adhesion molecule involved in anchoraging of myelomatous PC to the BM stroma, and its absence of expression in BM PC from MM patients is associated with extramedullary spreading, aggressive disease, and adverse outcome;16,37,38 although, this has not been confirmed by immunohistochemistry in another small cohort of patients.39 In this series, downregulation of CD56 in myelomatous PC correlates with a tendency to short PFS, but not different OS. Different groups have shown that the absence of expression of CD45 on MM PC is associated with disease progression and adverse prognostic factors.12,40 This could be related to the potential control that CD45 plays over the receptors of two major growth factors for myeloma cells, interleukin-6 and insulinlike growth factor-1.35 However, this data (poor prognosis for CD45-negative patients) would contradict the observation of Robillard et al18 showing that the proliferating PC compartment, which is theoretically involved in disease progression, is included within the CD45-positive bright PC fraction.18 Moreover, Menke et al41 have found that the expression, and not the absence, of the 180-kDa isoform of CD45, recognized by CD45RO antibodies, correlated with poor survival in patients with MM. Of note, we did not found any prognostic influence for CD45 expression in our large series of patients with MM. CD19 is a marker acquired at the very early stages of B-cell differentiation, present during B-cell maturation, and still expressed in the great majority of normal BM PC. By contrast, only a small proportion (< 5%) of patients with MM express this antigen.24,35,36 Our study shows that despite the small number of CD19-positive patients (n = 30), these patients have a dismal outcome as compared with the CD19-negative patients, with approximately 1 and 2 years shorter PFS and OS, respectively. Moreover, the majority of CD19-positive patients did not expressed CD117, a feature that also confers a worse prognosis to patients with MM. In contrast, the CD20 antigen did not show any influence on disease outcome. Expression of myeloid markers, such as CD13 or CD33, in myelomatous PC is intriguing. Some studies have previously reported that patients with MM with this phenotype tend to have a poor prognosis, but once again, the series of patients were very small.42,43 In this study, we have observed that although the outcome of CD33-positive patients was slightly worse than that of CD33-negative patients, differences did not reach statistical significance. Cytogenetics was more important than immunophenotyping as prognostic factor in the multivariate analysis. However, only a small proportion of patients (< 25%) are at high risk by FISH cytogenetics [t(4,14); t(4;16); or del(17p)] because del13p by itself did no have influence on disease outcome.45 In the rest of the patients the combination CD28 positive CD117 negative would allows the discrimination of two different prognostic subgroups. Altogether, these findings suggest that antigenic expression of PC, and particularly the CD28-positive CD117-negative immunophenotype, as well as expression of CD19, may contribute to identify patients with myeloma with high risk of progression and short survival.
The author(s) indicated no potential conflicts of interest.
Conception and design: Juan J. Lahuerta, Alberto Orfao, Jesús F. San Miguel Provision of study materials or patients: Maria-Belén Vidriales, Juan J. Lahuerta, Maria V. Mateos, Norma Gutierrez, Laura Rosiñol, Laura Montejano, Joan Bladé, Rafael Martínez, Javier de la Rubia, Joaquín Díaz-Mediavilla, Anna Sureda, José M. Ribera, José M. Ojanguren, Felipe de Arriba, Luis Palomera, Maria J. Terol Collection and assembly of data: Gema Mateo, Maria-Belén Vidriales, Maria V. Mateos, Norma Gutierrez Data analysis and interpretation: Gema Mateo, M. Angeles Montalbán, Maria-Belén Vidriales, Norma Gutierrez Manuscript writing: Gema Mateo, Maria-Belén Vidriales, Jesús F. San Miguel Final approval of manuscript: Alberto Orfao, Jesús F. San Miguel
We thank M. Anderson and M.J. Rodrigo for their editorial assistance.
published online ahead of print at www.jco.org on April 28, 2008. Supported by the Cooperative Research Thematic Network (RD06/0020/0006), MM Jevitt Foundation Grant No. QAG7, and Instituto de Salud Carlos III/Subdirección General de Investigación Sanitaria (PI060339 and 02/0905). Presented in part at the 49th Annual Meeting of the American Society of Hematology December 8-11, 2007. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test was used to estimate differences between groups. Survival curves were plotted according to the Kaplan-Meier method, and the log-rank test was used to estimate the statistical significance of the differences. P 
