Beta-2-Microglobulin Is an Independent Predictor of Progression in Asymptomatic Multiple Myeloma

Beta-2-Microglobulin Is an Independent Predictor of Progression in Asymptomatic Multiple Myeloma Davide Rossi, MD 1 ; Marco Fangazio, MD 1 ; Lorenzo De Paoli, MD 1 ; Alessia Puma, MD 1 ; Paola Riccomagno, MD 1 ; Valeria Pinto, MD 1 ; Paola Zigrossi, MD 1 ; Antonio Ramponi, MD 2 ; Guido Monga, MD 2 ; and Gianluca Gaidano, MD, PhD 1 BACKGROUND: Although serum beta-2 microglobulin (B2M) represents a key variable for symptomatic multiple myeloma (MM) prognostication, its role in predicting the risk of progression of asymptomatic MM to symptomatic disease has not been explored. METHODS: This study was bases on a consecutive series of 148 patients with asymptomatic MM and explored the cumulative probability of progression to symptomatic MM as the primary endpoint. RESULTS: In univariate analysis, a serum B2M level >2.5 mg/l was associated with an increased probability of disease progression (5-year risk, 64.5%; P <.001) along with serum monoclonal component (smc) (P <.001), urinary monoclonal component (umc) (P <.001), and bone marrow plasma cells (BMPCs) (P <.001). In multivariate analysis, serum B2M was selected as an independent predictor of progression (hazard ratio, 3.30; P ¼.002). Serum B2M was combined with smc, umc, and BMPC to create a risk-stratification model based on 4 groups with different risk of progression: very low (5-year risk, 0%), low-intermediate (5-year risk, 19.6%), high-intermediate (5-year risk, 60.7%), and high (5-year risk, 80.7%). The model that included serum B2M along with smc, umc, and BMPC was able to predict disease progression better than the model that was based on smc, umc, and BMPC without serum B2M (C statistics, 0.760 vs 0.726). CONCLUSIONS: The current results indicated that 1) serum B2M is an independent predictor of asymptomatic MM progression, and 2) serum B2M adds prognostic information when combined with the most widely used prognosticators of asymptomatic MM progression. Cancer 2010;116:2188 200. VC 2010 American Cancer Society. KEYWORDS: multiple myeloma, asymptomatic, beta-2 microglobulin, progression, prognosis. Asymptomatic multiple myeloma (MM) is a plasma cell-proliferative disorder characterized by a bone marrow plasma cell (BMPC) percentage 10% and/or a serum monoclonal component (smc) 3 g/dl and by the absence of end organ damage. 1,2 Although it is projected that up to 80% of patients with asymptomatic MM will progress to symptomatic disease and require treatment, the risk of progression is not uniform across patients. In fact, the category that is recognized as asymptomatic MM includes both patients who harbor an indolent disease similar to monoclonal gammopathy of undetermined significance (MGUS) and patients who have a rapidly progressive disease and might benefit from very close follow-up. 3-15 To date, few clinical predictors of asymptomatic MM progression have been identified, including BMPC percentage, smc level, and serum free light chain (FLC) burden measured either by the serum FLC ratio or by Bence-Jones (BJ) proteinuria. 3-15 At least 3 predictive models have been derived from the combination of these variables, allowing the stratification of patients with asymptomatic MM into groups with a distinct risk of progression. 8,12,15 Beta-2 microglobulin (B2M) is a serum marker of tumor burden in lymphoid malignancies, including MM. 16-23 Although B2M represents a key variable of the International Staging System (ISS) for symptomatic MM, 22 its role in asymptomatic MM has not been explored in detail. In fact, the ISS database did not include patients with asymptomatic Corresponding author: Davide Rossi, MD, Division of Hematology, Department of Clinical and Experimental Medicine, Amedeo Avogadro University of Eastern Piedmont, Via Solaroli 17, 28100 Novara, Italy; Fax: (011) 39-0321-620421; rossidav@med.unipmn.it 1 Division of Hematology, Department of Clinical and Experimental Medicine, Amedeo Avogadro University of Eastern Piedmont and Maggiore della Carita Hospital, Novara, Italy; 2 Division of Pathology, Department of Medical Sciences, Amedeo Avogadro University of Eastern Piedmont and Maggiore della Carita Hospital, Novara, Italy The first author designed the study, interpreted data, performed statistical analysis, and drafted the article; the last author supervised the study design and data interpretation and drafted the article; the second author contributed to data collection and statistical analysis; the third, fourth, fifth, sixth, and seventh authors collected and revised clinical data; and the eighth and ninth authors collected and revised pathologic data. All authors read and approved the article. DOI: 10.1002/cncr.24959, Received: May 26, 2009; Revised: July 14, 2009; Accepted: August 17, 2009, Published online March 2, 2010 in Wiley InterScience (www.interscience.wiley.com) 2188 Cancer May 1, 2010

B2M in Asymptomatic Multiple Myeloma/Rossi et al MM, 22 and none of the studies on the prognosis for patients with asymptomatic MM focused on B2M. 3-15 Therefore, in the current study, we investigated a cohort of patients who had asymptomatic MM to verify 1) whether B2M levels predict progression to symptomatic MM and 2) how B2M interacts with currently known predictors of progression to symptomatic MM. MATERIALS AND METHODS Patients This study was based on a consecutive series of 148 patients with asymptomatic MM. All patients were referred to our center for the screening of monoclonal gammopathy from June 1998 through June 2008. The database was updated for the analysis in February 2009. The median follow-up of patients who remained alive was 48 months. No patient was lost at follow-up. All patients provided informed consent in accordance with local institutional review board requirements and the Declaration of Helsinki. The diagnosis of asymptomatic MM was according to the International Myeloma Working Group criteria 1 andrequiredbmpcs 10% and/or an smc level 3 g/dl in the absence of end organ damage defined according to calcium elevation, renal failure, bone disease and anemia (CRAB) criteria; amyloidosis; hyperviscosity; or recurrent infections. The following clinical variables were recorded at presentation: age, sex, percentage of BMPCs on bone marrow biopsy, smc type, smc size, type of light chain, urinary immunofixation, urinary monoclonal component (umc), polyclonal immunoglobulin (Ig) reduction, a previous history of MGUS, complete blood count, serum albumin, C-reactive protein, serum B2M, serum creatinine, creatinine clearance calculated with the Cockcroft- Gault formula, serum calcium, and skeleton x-ray. All 148 patients were complete for the data listed above. Serum B2M levels were quantified by nephelometry (Siemens Healthcare Diagnostics, Milan, Italy; reference range, 1.8-2.3 mg/l). By using the 2.3 mg/l upper limit of normal (ULN) of our laboratory, 55 of 148 patients (37.2%) with asymptomatic MM had abnormal B2M levels. Quantification of serum M-protein was performed by densitometry of the monoclonal peak on electrophoresis for IgG monoclonal proteins. Immunochemical measurement of the total Ig isotype level was used for IgA monoclonal proteins migrating in the beta band on serum electrophoresis. Quantification of urinary light chain excretion was performed directly on a 24-hour urine collection. The proportion of BMPCs was estimated on bone marrow biopsy as reported previously. 1,12 BMPC percentage was assessed on bone marrow aspirates and on paraffin-embedded bone marrow biopsy specimens that were stained by hematoxylin and eosin with a differential count of 500 cells. In addition, immunohistochemistry was applied to paraffin sections of the bone marrow biopsy specimens with the use of antibodies directed against CD138. The bone marrow aspirate, biopsy, and immunohistochemistry stains were reviewed independently by 1 hematopathologist (A.R.) and by 1 hematologist (D.R.). BMPC percentage estimates from all 3 methods were combined to provide the final proportion of BMPCs. 12 All patients underwent regular follow-up every 3 to 4 months. During follow-up, the following variables were recorded: 1) the date of progression to symptomatic MM, 2) the date of last follow-up, and 3) the date of death. Statistical Analysis The primary endpoint of the study was progression to symptomatic MM, which was defined according to International Myeloma Working Group criteria. 1 The time to progression was measured from the date of asymptomatic MM diagnosis to either the date of progression to symptomatic MM, or the date of last follow-up, or the date of death. The cumulative probability of progression to symptomatic MM was calculated by using Kaplan-Meier analyses in which data from patients who had died were censored. 24 Kaplan-Meier curves were compared with the use of log-rank statistics. The cumulative incidence of progression to symptomatic MM was calculated by accounting for death as a competing risk 25 and was compared across groups with the Gray test for equality. 26 Cox proportional hazards regression was used to build a multivariate model. 27 Because the hazard ratio derived from Cox analysis measures the magnitude of risk rather than a model s ability to accurately classify patients, the Harrell C statistic was used to further evaluate the discriminatory value of the prognostic models in terms of progression to symptomatic MM. 28 The C statistic estimates the proportion of correct prediction of the model (a C statistic of 1 indicates perfect discrimination, and a C statistic of 0.5 is equivalent to chance). 28 The standard error of the C statistic was calculated based on 3000 bootstrap samples. The stratification of continuous clinical variables was based on the best predictive cutoff value or an usual limit of normal. The best cutoff values for B2M, BMPC percentage, smc, umc, hemoglobin, calcium, creatinine, creatinine clearance, and C-reactive protein were selected according to receiver operating curve (ROC) analysis using disease progression as a state variable and according to the Cancer May 1, 2010 2189

Table 1. Clinical Characteristics of Patients With Asymptomatic Multiple Myeloma a Variable No. of Patients/ Total No. (%) Median [Range] a Median age [range], y 67 [57-73] Men:women 75:73 Type of monoclonal component IgG 113/148 (76.4) IgA 32/148 (21.6) Light chain 3/148 (2) j 85/148 (57.4) k 63/148 (42.6) Serum monoclonal component, g/dl 1.1 [0.8-1.7] IgG, g/dl 1.2 [0.8-1.8] IgA, g/dl 0.9 [0.6-1.5] Positive urinary immunofixation 27/148 (18.2) Urinary monoclonal component, mg/24 h b 98 [44.525] Polyclonal immunoglobulin reduction 114/148 (77) Bone marrow plasma cells, % 15 [12-20] Albumin, g/dl 4.3 [4-4.5] Beta-2-microglobulin, mg/l 2.0 [1.5-2.5] C-reactive protein, mg/l 0.3 [0.1-0.4] Hemoglobin, g/dl 13.8 [12.7-14.5] Platelet count, 10 9 /L 220 [178-283] White blood cells, 10 9 /L 6.0 [4.9-7.2] Absolute neutrophil count, 10 9 /L 3.3 [2.5-4.5] Absolute lymphocyte count, 10 9 /L 1.9 [1.5-2.4] Calcium, mg/dl 9.4 [9-9.7] Creatinine, mg/dl 1.0 [0.9-1.1] Creatinine clearance, ml/min 72 [58-97] Previous MGUS 32/148 (21.6) ISS I 128/148 (86.5) II 18/148 (12.2) III 2/148 (1.4) Progression to symptomatic disease 29/148 (19.5) Symptoms at progression Calcium elevation 4/29 (13.8) Renal failure 5/29 (17.2) Anemia 14/29 (48.3) Bone lesion 13/29 (44.8) IgG indicates immunoglobulin G; IgA, immunoglobulin A; MGUS, monoclonal gammopathy of undetermined significance; ISS, International Staging System. a Ranges (in square brackets) indicate the 25th through 75th percentiles for continuous variables. b This was calculated only for patients who had positive urinary immunofixation results. Youden index, which was calculated using the sensitivity and specificity derived from ROC analysis. 29 Categorical variables were compared by using chi-square tests or Fisher exact tests, as appropriate. Continuous variables were compared by using the Mann-Whitney test or the Kruskal-Wallis test, as appropriate. All statistical tests were 2-sided, and statistical significance was defined as a P value <.05. The analysis was performed with Statistical Package for the Social Sciences (SPSS) software (version 16.0; SPSS, Inc., Chicago, Ill) and, in the competing risk analysis, with the R statistical package (available at: http:// www.r-project.org/ accessed May 25, 2009). RESULTS Characterization of the Asymptomatic Multiple Myeloma Cohort Clinical features at diagnosis of the patients with asymptomatic MM (n ¼ 148) are reported in Table 1. The median age at diagnosis was 67 years. In total, 75 patients were men, and 73 patients were women. Of 148 patients, 2190 Cancer May 1, 2010

B2M in Asymptomatic Multiple Myeloma/Rossi et al 113 (76.4%) had IgG, 32 patients had (21.6%) IgA, and 3 patients had light chain smc (2%). The light chain type was j in 85 patients (57.4%) and k in 63 patients (42.6%). The median smc levels was 1.1 g/dl. Concentrations of uninvolved Igs were reduced in 114 of 148 patients (77%). Positive urinary immunofixation was documented in 27 of 148 patients (18.2%). Among the patients who had positive urinary immunofixation, the median umc level was 98 mg per 24 hours. The median hemoglobin concentration was 13.8 g/dl, the median serum B2M level was 2.0 mg/l, the median serum albumin level was4.3g/dl,andthemedianserumc-reactiveprotein level was 0.3 mg/l. On bone marrow biopsy, the median BMPC percentage was 15%. A previous history of MGUS was recorded in 32 of 148 patients (21.6%). According to ISS criteria, 128 of 148 patients (86.5%) patients had stage I disease, 18 of 148 patients (12.2%) had stage II disease, and 2 of 148 patients (1.4%) had stage III disease. During 484 cumulative person-years of follow-up (range, 0.5-10.1 person-years), 4 of 148 patients died (median follow-up of patients who remained alive, 4 years). During this period, 29 of 148 patients progressed to symptomatic MM, accounting for a 5-year probability of progression of 29.5% (95% confidence interval, [CI], 19.6%-39.4%). Serum Beta-2-Microglobulin Is an Independent Predictor of Asymptomatic Multiple Myeloma Progression Clinical features at diagnosis were evaluated for their impact on the risk of progression to symptomatic MM. According to the Youden index, the best cutoff values were 2.5 mg/l for B2M, 1.5 g/dl for smc, 500 mg per 24 hours for umc, 20% for BMPC percentage, 13.5 g/dl for hemoglobin, 10.0 mg/dl for calcium, 0.9 mg/dl for creatinine, 70 ml per minute for creatinine clearance, and 1.0 mg/l for C-reactive protein. On the basis of these cutoff values, at diagnosis, 40 of 148 patients (27%) patients had B2M >2.5 mg/dl, 57 of 148 patients (38.5%) had smc >1.5 g/dl, 8 of 148 patients (5.4%) had umc >500 mg per 24 hours, 46 of 148 patients (31.1%) had a BMPC percentage 20%, 63 of 148 patients (42.5%) had hemoglobin 13.5 g/dl, 19 of 148 patients (12.8%) had calcium >10 mg/dl, 98 of 148 patients (66.2%) had creatinine >0.9 mg/dl, 71 of 148 patients (48%) had a creatinine clearance <70 ml per minute, and 11 of 148 patients (7.4%) had C-reactive protein >1.0 mg/l. Among the clinical features at diagnosis, univariate analysis identified B2M >2.5 mg/l as a risk factor of progression to symptomatic MM (cumulative probability at 5 years, 64.5%; hazard ratio [HR], 3.85; 95% CI, 1.85-8.01; P <.001) (Table 2, Fig. 1A). Other variables that were associated with a significantly increased risk of progression to symptomatic MM included smc >1.5 g/dl (cumulative probability at 5 years, 49.1%; HR, 5.76; P <.001), umc >500 mg per 24 hours (cumulative probability at 5 years, 68.7%; HR, 6.60; P <.001), a BMPC percentage 20% (cumulative probability at 5 years, 50.2%; HR, 5.77; P <.001), hemoglobin 13.5 g/dl (cumulative probability at 5 years, 43.5%; HR, 2.24; P ¼.035), and creatinine >0.9 mg/dl (cumulative probability at 5 years, 36.3%; HR, 2.89; P ¼.031) (Table 2; Figs. 1B-D, 2).These associations also remained significant after correcting for death as a competing risk (Table 2). Clinical variables that were not associated with progression to symptomatic MM (P.05 in all analyses) were age, sex, smc type, type of light chain, polyclonal Ig reduction, albumin, C-reactive protein, platelets, white blood cell count, absolute neutrophil count, absolute lymphocyte count, calcium, creatinine clearance, and a previous history of MGUS (Table 2). The independent predictive value of B2M for the risk of progression to symptomatic MM was assessed in a Cox multivariate analysis. Variables that were entered into the multivariate analysis along with B2M were smc and BMPC percentage. Multivariate analysis selected B2M >2.5 mg/l (HR, 3.30; 95% CI, 1.57-6.91; P ¼.002) as an independent predictor of progression to symptomatic MM along with smc >1.5 g/dl (HR, 3.71; 95% CI, 1.46-9.39; P ¼.006) and a BMPC percentage 20% (HR, 3.96; 95% CI, 1.75-8.97; P ¼.001) (Table 3). Beta-2-Microglobulin Synergizes With Other Risk Factors in Predicting Asymptomatic Multiple Myeloma Progression When treated as a categorical variable, B2M >2.5 mg/dl was not associated with any 1 of the variables correlated most significantly with progression by univariate analysis, including smc >1.5 g/dl (P ¼.324), umc >500 mg per 24 hours (P ¼.034), or BMPC percentage 20% (P ¼.068). This observation documents the finding that B2M >2.5 mg/dl did not have colinearity with smc, umc, or BMPC percentage and, thus, that B2M did not behave as a reciprocal surrogate of smc, umc, or BMPC percentage. On this basis, we used bivariate log-rank analysis to test how B2M >2.5 mg/l interacted with smc >1.5 g/dl, umc >500 mg per 24 hours, and BMPC Cancer May 1, 2010 2191

Table 2. Clinical Variables at Asymptomatic Multiple Myeloma (MM) Diagnosis That Predicted Progression to Symptomatic MM in Univariate Analysis Variable No. of Events/ No. of Patients Cumulative Probability at 5 years 6 SE, % a HR 95% CI Cox P b Gray P c Age <60 y 5/41 15.6 8.4.088 Age 60 y 24/107 34.3 6.3 2.31 0.88-6.08 Women 12/73 28.9 8.2.286 Men 17/75 28.7 6.0 1.49 0.71-3.13 Type of serum monoclonal component IgG 18/113 24.3 5.6.076 IgA 9/32 35.6 10.2 2.07 0.92-4.65 Type of light chain j 19/85 35.5 7.5.325 k 10/63 19.6 6.2 1.47 0.68-3.16 Serum monoclonal component.5 g/dl 6/91 11.1 6 <.001 d <.001 d Serum monoclonal component >1.5 g/dl 23/57 49.1 7.6 5.76 2.34-14.18 Urinary monoclonal component 500 mg/24 h 24/140 26.2 5.2 <.001 d <.001 d Urinary monoclonal component >500 mg/24 h 5/8 68.7 17.8 6.60 2.49-17.49 No polyclonal immunoglobulin reduction 2/34 36.2 26.155 Polyclonal immunoglobulin reduction 27/114 30 5.2 2.84 0.67-12.03 Bone marrow plasma cells <20% 9/102 17.7 5.9 <.001 d <.001 d Bone marrow plasma cells 20% 20/46 50.2 8.7 5.77 2.62-12.69 Albumin >3.5 g/dl 27/140 27.6 5.1.257 Albumin 3.5 g/dl 2/8 60 29.7 2.30 0.54-9.78 Beta-2-microglobulin 2.5 mg/l 14/108 16.7 4.5 <.001 d <.001 d Beta-2-microglobulin >2.5 mg/l 15/40 64.5 12.1 3.85 1.85-8.01 C-reactive protein 1.0 mg/l 25/137 24.9 4.8.303 C-reactive protein >1.0 mg/l 4/11 53.3 19 1.74 0.60-5.02 Hemoglobin >13.5 g/dl 12/85 19.4 5.3.035 d.036 d Hemoglobin 13.5 g/dl 17/63 43.5 9.6 2.24 1.05-4.66 Platelets >15010 9 /L 26/131 29.7 5.6.838 Platelets 15010 9 /L 3/17 20.7 10.7 0.88 0.26-2.92 White blood cells >410 9 /L 25/134 28.2 5.4.366 White blood cells 410 9 /L 4/14 41.8 13.4 1.63 0.56-4.70 Absolute neutrophil count >210 9 /L 26/131 30.5 5.7.721 Absolute neutrophil count 210 9 /L 3/17 17.6 9.2 0.80 0.24-2.66 Absolute lymphocyte count >210 9 /L 12/58 32.3 8.4.959 Absolute lymphocyte count 210 9 /L 17/90 25.7 6.2 0.98 0.46-2.05 Calcium 10 mg/dl 24/129 27.3 5.4.226 Calcium >10 mg/dl 5/19 32.4 12.2 1.82 0.69-4.80 Creatinine 0.9 mg/dl 5/50 13.4 7.3.031 d.023 d Creatinine >0.9 mg/dl 24/98 36.3 6.5 2.89 1.10-7.58 Creatinine clearance 70 ml/min 14/77 25.4 6.5.406 Creatinine clearance <70 ml/min 15/71 32.8 8.2 1.36 0.65-2.82 No previous MGUS 21/116 27.5 5.8.407 Previous MGUS 8/32 33.8 11.5 1.41 0.62-3.19 ISS I 24/128 26.9 5.3.187 II-III 5/20 41 15.6 1.91 0.72-5.03 SE indicates standard error; HR, hazard ratio; CI, confidence interval; IgG indicates immunoglobulin G; IgA, immunoglobulin A; MGUS, monoclonal gammopathy of undetermined significance; ISS, International Staging System. a Calculated by using the Kaplan-Meier method. b Calculated by using Cox analysis. c Calculated by using the Gray test after adjusting for death as a competing risk. d Statistically significant P value. 2192 Cancer May 1, 2010

B2M in Asymptomatic Multiple Myeloma/Rossi et al Figure 1. These survival curves illustrate the cumulative probability of asymptomatic multiple myeloma (MM) progression to symptomatic disease according to beta-2 microglobulin (BM2) and other clinical risk factors. The cumulative probability of asymptomatic MM progression to symptomatic disease is illustrated according to levels of (A) B2M, (B) serum monoclonal component (smc), (C) urinary monoclonal component (umc) and (D) the percentage of bone marrow plasma cells (BMPC). percentage 20% in predicting progression of asymptomatic MM. B2M identified a subgroup of patients with asymptomatic MM who had a higher probability of progression despite being characterized at diagnosis by favorable predictors. This observation was reproducible in all favorable risk categories. Within the stratum of asymptomatic MM that harbored favorable predictors, B2M >2.5 mg/l segregated a group of patients with asymptomatic MM who had higher cumulative probability of progression despite being characterized by smc 1.5 g/ dl (P ¼.010), umc 500 mg per 24 hours (P ¼.003), or BMPC percentage <20% (P ¼.009) (Table 4, Fig. 3). In addition, B2M discriminated unfavorable risk categories within the stratum of patients with asymptomatic MM who had poor prognosticators. Indeed, patients who had the highest cumulative probability of progression were identified by the combination of B2M >2.5 mg/dl with smc >1.5 g/dl (P ¼.009) or a BMPC percentage 20% (P ¼.013) (Table 4, Fig. 3). These associations Figure 2. These survival curves illustrate the cumulative probability of asymptomatic multiple myeloma progression to symptomatic disease according to serum creatinine. Cancer May 1, 2010 2193

remained significant after correcting for death as a competing risk (Table 4). Because of the low number of patients, the stratum of patients with umc >500 mg/dl was not powered enough to detect a difference in the risk of progression according to B2M. Beta-2-Microglobulin Improves the Risk Stratification of Asymptomatic Multiple Myeloma The burdens of smc, BMPC and FLC measured in serum or estimated indirectly by BJ proteinuria are among the most widely recognized risk factors for asymptomatic Table 3. Clinical Variables at Asymptomatic Multiple Myeloma (MM) Diagnosis That Predicted Progression to Symptomatic MM in Multivariate Analysis Variable HR 95% CI P a Serum monoclonal component 1.5 g/dl.006 Serum monoclonal component >1.5 g/dl 3.71 1.46-9.39 Beta-2-microglobulin 2.5 mg/l.002 Beta-2-microglobulin >2.5 mg/l 3.30 1.57-6.91 Bone marrow plasma cells <20%.001 Bone marrow plasma cells 20% 3.96 1.75-8.97 HR indicates hazard ratio; CI, confidence interval. a Calculated by using Cox analysis. MM progression. 8,12,15 Consistent with previous observations, smc, umc, and BMPC percentage were selected as predictors of progression in our asymptomatic MM cohort. When combined into a model, smc, umc, and BMPC percentage allowed us to stratify patients into 3 risk categories (Fig. 4A). B2M contributed toward segregating patients with asymptomatic MM who had no risk of progression and patients with asymptomatic MM who were projected to progress in virtually all cases. Three lines of evidence suggest that B2M may improve the risk stratification model defined by smc, umc, and BMPC percentage. First, B2M >2.5 mg/l adds prognostic information within the low-risk group characterized by smc 1.5 g/dl, umc 500 mg per 24 hours, and BMPC percentage <20% and within the high-risk group defined by smc >1.5 g/dl, and/or umc >500 mg per 24 hours, and/or BMPC percentage 20% (Table 5). Conversely, the prognosis of intermediate-risk patients who harbored smc >1.5 g/dl, or umc >500 mg per 24 hours, or BMPC 20% was not affected significantly by B2M (Table 5). Second, the risk stratification model that included B2M >2.5 mg/l along with smc >1.5 g/dl, umc Table 4. Bivariate Analysis of the Interaction Between Beta-2-Microglobulin and Serum Monoclonal Component, Urinary Monoclonal Component, and Bone Marrow Plasma Cell Percentage in Predicting the Progression of Asymptomatic Multiple Myeloma Variable No. of Events/ No. of Patients Cumulative Probability at 5 Years 6 SE, % a Log-Rank P b Gray P c Serum monoclonal component 1.5 g/dl B2M 2.5 mg/l 2/69 1.7 1.7.010.011 B2M >2.5 mg/l 4/22 46.2 23 Serum monoclonal component >1.5 g/dl B2M 2.5 mg/l 12/39 36.4 8.6.009.020 B2M >2.5 mg/l 11/18 80.3 11.9 Urinary monoclonal component 500 mg/24 h B2M 2.5 mg/l 13/105 16.2 4.6.003.006 B2M >2.5 mg/l 11/35 60 13.5 Urinary monoclonal component >500 mg/24 h B2M 2.5 mg/l 1/3 32.3 27.2.107.128 B2M >2.5 mg/l 4/5 100 21.9 Bone marrow plasma cells <20% B2M 2.5 mg/l 4/79 8 4.2.009.012 B2M >2.5 mg/l 5/23 52.1 18.1 Bone marrow plasma cells 20% B2M 2.5 mg/l 10/29 36.7 10.2.013.028 B2M >2.5 mg/l 10/17 77.7 13.1 SE indicates standard error; B2M, beta-2-microglobulin. a Calculated by using the Kaplan-Meier method. b Calculated by using the log-rank test. c Calculated by using the Gray test after adjusting for death as a competing risk. 2194 Cancer May 1, 2010

B2M in Asymptomatic Multiple Myeloma/Rossi et al Figure 3. These survival curves illustrate the interaction between beta-2 microglobulin (B2M) and other clinical risk factors on the cumulative probability of asymptomatic multiple myeloma (MM) progression to symptomatic disease. A B2M level >2.5 mg/l segregated a group with asymptomatic MM that had a higher cumulative probability of progression within the strata of patients characterized by (A) a serum monoclonal component (smc) 1.5 g/dl (P ¼.010; pairwise comparison) and smc >1.5 g/dl (P ¼.009; pairwise comparison), (B) a urinary monoclonal component (umc) 500 mg per 24 per hours (P ¼.003; pairwise comparison), and (C) bone marrow plasma cells (BMPC) <20% (P ¼.009; pairwise comparison) and BMPC 20% (P ¼.013; pairwise comparison). Cancer May 1, 2010 2195

Figure 4. Risk stratification models were based on serum monoclonal component, urinary monoclonal component, and bone marrow plasma cells with or without beta-2 microglobulin. (A) Patients were assigned 1 point for meeting each of the following criteria: serum monoclonal component <1.5 mg/l, urinary monoclonal component >500 mg per 24 hours, and bone marrow plasma cells 20%. The cumulative probability of asymptomatic multiple myeloma (MM) progression to symptomatic disease is represented for patients who had 0 (low risk), 1 (intermediate risk), or >1 (high risk) risk factors. (B) Patients were assigned 1 point for meeting each of the following criteria: beta-2 microglobulin >2.5 mg/l, serum monoclonal component <1.5 mg/l, urinary monoclonal component >500 mg per 24 hours, and bone marrow plasma cells 20%. The cumulative probability of asymptomatic MM progression to symptomatic disease is represented for patients who had 0 (low risk), 1 (low-intermediate risk), 2 (high-intermediate risk), or >2 (high risk) risk factors. Table 5. The Addition of Beta-2-Microglobulin to Prognostic Categories Defined According to Serum Monoclonal Component, Urinary Monoclonal Component, and Bone Marrow Plasma Cells Prognostic Category No. of Events/ No. of Patients Cumulative Probability at 5 Years 6 SE, % a Log-Rank P b Gray P c Low-risk (smc 1.5 g/dl, and umc 500 mg/24 h, and BMPC <20%) Beta-2-microglobulin 2.5 mg/l 0/57 0.005.005 Beta-2-microglobulin >2.5 mg/l 2/16 40 25.3 Intermediate-risk (smc >1.5 g/dl, or umc >500 mg/24 h, or BMPC 20%) Beta-2-microglobulin 2.5 mg/l 5/32 5.4 7.3.091.096 Beta-2-microglobulin >2.5 mg/l 3/10 70.8 24.1 High-risk (smc >1.5 g/dl, and/or umc >500 mg/24 h, and/or BMPC 20%) Beta-2-microglobulin 2.5 mg/l 9/19 55 13.034.076 Beta-2-microglobulin >2.5 mg/l 10/14 89 10.2 SE indicates standard error; smc, serum monoclonal component, umc, urinary monoclonal component; BMPC, bone marrow plasma cells; NA, not assessable. a Calculated by using the Kaplan-Meier method. b Calculated by using the log-rank test. c Calculated by using the Gray test after adjusting for death as a competing risk. >500 mg per 24 hours, and BMPC percentage 20% allowed us to segregate 4 groups of patients with asymptomatic MM who had different risks of progressing to symptomatic disease (Fig. 4B, Table 6). The cumulative probability of progressing to symptomatic MM at 5 years was 0% for the very low risk group (0 risk factors), 19.6% for the low-intermediate risk group (1 risk factor), 60.7% for the high-intermediate risk group (2 risk factors), and 80.7% for the high-risk group (3 or 4 risk factors; P <.001) (Fig. 4B). After correcting for death as a competing 2196 Cancer May 1, 2010

B2M in Asymptomatic Multiple Myeloma/Rossi et al Table 6. Clinical Characteristics of Patients With Asymptomatic Multiple Myeloma According to Risk Category Variable No. of Patients/Total No. (%) or Median [Range] a P b Very Low Risk: 0 Risk Factors Low-Intermediate: 1 Risk Factor High-Intermediate: 2 Risk Factors High Risk: 3-4 Risk Factors Age [range], y 64 [52-71] 67 [53-74] 68 [62-72] 72 [66-79].031 c Men 24/57 (42.1) 24/48 (50) 16/28 (57.1) 11/15 (73.3).156 Type of monoclonal component IgG 41/57 (71.9) 41/48 (85.4) 22/28 (78.6) 9/15 (60).327 IgA 15/57 (26.3) 7/48 (14.6) 5/28 (17.9) 5/15 (33.3) Light chain 1/57 (1.8) 0 1/28 (3.6) 1/15 (6.7) j 34/57 (59.6) 23/48 (47.9) 19/28 (67.9) 9/15 (60).365 K 23/57 (40.4) 25/48 (52.1) 9/28 (32.1) 6/15 (40) Serum monoclonal component, g/dl 0.8 [0.6-1.0] 1.3 [0.8-1.7] 1.8 [1.5-2.1] 1.9 [1.5-2.6] <.001 c Serum monoclonal component, >1.5 g/dl 0 (0) 22/48 (45.8) 22/28 (78.6) 13/15 (86.7) <.001 c Positive urinary immunofixation 6/57 (10.5) 5/48 (10.4) 9/28 (32.1) 7/15 (46.7).001 c Median urinary monoclonal component, mg/24 h 0 [0-0] 0 [0-0] 0 [0-39] 0 [0-525].001 c Urinary monoclonal component >500 mg/24 h 0 (0) 0 (0) 3/28 (10.7) 5/15 (33.3) <.001 c Polyclonal immunoglobulin reduction 40/57 (70.2) 36/48 (75) 25/28 (89.3) 13/15 (86.7).118 Bone marrow plasma cells, % 13 [12-15] 15 [13-18] 25 [17-34] 35 [22-50] <.001 c Bone marrow plasma cells 20% 0 (0) 10/48 (20.8) 21/28 (75) 15/15 (100) <.001 c Albumin, g/dl 4.3 [4.1-4.5] 4.2 [4.0-4.4] 4.3 [4.0-4.5] 4.1 [3.6-4.4].202 Beta-2-microglobulin, mg/l 1.7 [1.4-2.0] 2.0 [1.4-2.7] 2.3 [2.0-2.7] 3.1 [2.7-3.9] <.001 c Beta-2-microglobulin >2.5 mg/l 0 (0) 16/48 (33.3) 10/28 (35.7) 14/15 (93.3) <.001 c C-reactive protein, mg/l 0.2 [0.1-0.4] 0.3 [0.1-0.5] 0.2 [0.1-0.4] 0.3 [0.1-0.5].155 Hemoglobin, g/dl 14.1 [13.1-14.8] 13.5 [12.7-14.3] 14.0 [12.4-14.5] 13.0 [11.8-13.8].025 c Platelets, 10 9 /L 222 [187-287] 209 [169-280] 231 [178-293] 182 [130-289].339 White blood cells, 10 9 /L 6.0 [4.7-7.1] 6.0 [4.9-7.2] 5.9 [4.9-8.4] 5.8 [3.9-6.3].552 Absolute neutrophil count, 10 9 /L 3.2 [2.3-4.7] 3.5 [2.6-4.5] 3.2 [2.9-4.4] 2.9 [2.6-3.8].682 Absolute lymphocyte count, 10 9 /L 1.9 [1.4-2.4] 1.9 [1.5-2.2] 2.0 [1.6-2.8] 1.8 [1.2-2.4].401 Calcium, mg/dl 9.5 [9.1-9.7[ 9.4 [9.0-9.8] 9.3 [9.1-9.9] 9.3 [8.9-9.4].311 Creatinine, mg/dl 1.0 [0.9-1.1] 1.0 [0.9-1.1] 1.0 [0.9-1.1] 1.2 [1.1-1.4].003 c Previous MGUS 10/57 (17.5) 10/48 (20.8) 9/28 (32.1) 3/15 (20).488 ISS II-III 1/57 (1.8) 9/48 (18.8) 4/28 (14.3) 6/15 (40).001 c IgG indicates immunoglobulin G; IgA, immunoglobulin A; MGUS, monoclonal gammopathy of undetermined significance; ISS, International Staging System. a Ranges (in square brackets) indicate the 25th through 75th percentiles for continuous variables. b P values were calculated using the chi-square test for categorical variables and the Kruskal-Wallis test for continuous variables. c Statistically significant P value. risk, the 5-year cumulative incidence of progression to symptomatic MM for the very low risk, low-intermediate risk, high intermediate risk, and high-risk groups was 0%, 19.6%, 60.7%, and 75.3%, respectively (P <.001). Third, based on the C statistic, the model that included B2M along with smc, umc, and BMPC percentage (C statistic, 0.760; standard error [SE], 0.045) allowed us to predict asymptomatic MM progression better than the model that was based on smc, umc, and BMPC percentage without B2M (C statistics, 0.726; SE, 0.049). DISCUSSION In this report, we document our findings that 1) B2M is an independent predictor of asymptomatic MM progression, and 2) B2M adds prognostic information when combined with the most widely used prognosticators of asymptomatic MM progression. Although B2M is a well established prognostic factor in symptomatic MM, 22 its role in asymptomatic disease is poorly characterized. Our data demonstrate that B2M is an independent predictor of asymptomatic MM progression and that patients who present with a B2M level >2.5 mg/l have an adjusted 3.3-fold increase in the risk of progression to symptomatic disease. To date, none of the previous studies on asymptomatic MM focused on B2M, 3-15 and only 2 reports included B2M among baseline variables. 7,10 In the study by Moulopoulos et al, 7 patients with asymptomatic MM who presented with B2M >2.5 mg/l had a short time to progression to symptomatic disease. However, because of the low numbers of patients (n ¼ 38) in that study, the increased risk of progression indicated by B2M elevation did not reach statistical significance. 7 In the study by Cesana et al 10 that included both MGUS and asymptomatic MM, B2M elevation did not emerge as a risk factor Cancer May 1, 2010 2197

for progression to symptomatic disease. However, it should be underscored that the criteria for the definition of asymptomatic MM and for progression to symptomatic disease used by Cesana et al 10 did not fulfill currently accepted criteria for these conditions. 1 In addition, data on baseline B2M were lacking in a significant proportion of patients. 10 Along with tumor mass, B2M also reflects renal function. In our cohort, as expected, B2M displayed a strong correlation with serum creatinine (Spearman q ¼ 0.529; P <.001) and creatinine clearance (Spearman q ¼ 0.404; P <.001). Despite this strong association, we could exclude the possibility that the prognostic value of B2M is led by renal function, because: 1) creatinine clearance, which is the standard measure of renal function in adults, was not associated with asymptomatic MM progression when treated either as a categorical variable (Table 2) or as a continuous variable (HR, 0.99; 95% CI, 0.98-1.01; P ¼.404); 2) in a multivariate analysis that included smc, BMPC percentage, and creatinine rather that B2M, creatinine was not selected as an independent predictor of progression (P ¼.327); 3) based on C statistics, the model that included B2M along with smc, umc, and BMPC percentage (C statistic, 0.760; SE, 0.045) allowed us to predict asymptomatic MM progression better than the model that was based on creatinine along with smc, umc, and BMPC percentage without B2M (C statistic, 0.735; SE, 0.049). smc, BMPC percentage, and FLC burden, measured in serum or estimated by BJ proteinuria, are among the most widely recognized risk factors of asymptomatic MM progression. 8,12,15 Our results document that B2M improves the risk stratification model defined by smc, umc, and BMPC percentage by adding information both in the low-risk stratum and in the high-risk stratum of patients with asymptomatic MM. In fact, B2M contributes to identifying a group of patients who have asymptomatic MM with a very favorable prognosis who are not expected to progress and a group of high-risk patients with asymptomatic MM virtually all of whom are projected to progress. Different technical methods for measuring B2M as well different reference ranges exist across laboratories. The application of a standardized value of B2M would allow use of a 2.5 mg/l cutoff in other asymptomatic MM series that used different technical methods for measuring B2M. The standardized value of B2M could be obtained by dividing the original value expressed in mg/l by the ULN of the laboratory. Accordingly, the 2.5 mg/l cutoff value for B2M in our series corresponds to a standardized cutoff value of 1.1-fold of the ULN. The prognostic role of unbalanced light chain production in asymptomatic MM has been described at the urinary level and, more recently, at the serum level. 8,15 Because serum FLC levels have entered the routine clinical practice only recently, this information was not available for the current analysis. On this basis, in our cohort, the baseline serum FLC burden was estimated indirectly by measuring BJ proteinuria. Our data confirmed that an excess of light chains is a risk factor for asymptomatic MM progression. In MM, magnetic resonance imaging (MRI) allows the characterization of patterns of bone marrow involvement by tumor plasma cells and allows the observation of changes in the pattern of microcirculation within the bone. 7-9,30 Abnormal bone marrow appearance on an MRI can be detected in a fraction of patients with asymptomatic MM and reportedly can predict an increased risk of progression to symptomatic disease. 7-9,30 In our survival analysis, the presence of MRI abnormalities was associated with a higher (although not significant) probability of progression (cumulative probability of progression at 5 years, 50.1% vs 23.9%; P ¼.110). IgA isotype has been suggested as another possible parameter that correlates with a higher risk of progression to asymptomatic MM. 8,10-12 In our study, similar to what was reported by Kyle et al, 12 the IgA isotype in asymptomatic MM indicated a 15% higher cumulative probability of progression compared with the IgG isotype in asymptomatic MM. This difference was at the limit of significance (P ¼.076). The finding that this value does not reach statistical significance may be because of the statistical power allowed by our sample size. Cytogenetics, either conventional or molecular, is 1 of the strongest prognostic factors in patients with symptomatic MM. 31 In the context of asymptomatic MM, the prognostic role of cytogenetics remains to be established and is suggested by: 1) the higher risk of progression among patients with asymptomatic MM who have cytogenetic abnormalities 32 ; 2) the higher prevalence of 13q deletion and 1q gain among patients who have progressive, asymptomatic MM 33 ; and 3) clonal evolution upon progression. 34 The current standard of care for asymptomatic MM is observation without therapy until the patient develops disease progression or symptoms. 1 Although it is projected that most patients with asymptomatic MM will progress to symptomatic disease, the risk of progression is 2198 Cancer May 1, 2010

B2M in Asymptomatic Multiple Myeloma/Rossi et al not uniform across patients. Prognostic models for predicting asymptomatic MM progression, 8,12,15 including the model described in the current study, would allow the identification of low-risk patients who harbor a very indolent disease similar to MGUS and high-risk patients who might benefit from close follow-up and who may be optimal candidates for experimental therapies aimed at delaying the progression of MM. 35-41 CONFLICT OF INTEREST DISCLOSURES The authors declare no competing financial interests. This study was supported by Progetto Integrato Oncologia 2006, Ministero della Salute, Rome, Italy; Ricerca Sanitaria Finalizzata, Regione Piemonte, Torino, Italy; Progetto Alfieri, Fondazione CRT, Torino, Italy; and Novara-AIL Onlus, Novara, Italy. REFERENCES 1. The International Myeloma Working Group. 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