Prognostic Value of Circulating Plasma Cells in Monoclonal Gammopathy of Undetermined Significance
http://www.100md.com
《临床肿瘤学》
the Department of Internal Medicine, Division of Hematology, Mayo Clinic, Rochester, MN
ABSTRACT
PURPOSE: Monoclonal gammopathy of undetermined significance (MGUS) progresses to multiple myeloma or another related plasma cell disorder (PCD) at a rate of approximately 1% per year. Identification of patients with MGUS at high risk of progression will allow development of preventive strategies. We studied the prognostic value of circulating plasma cells (PCs) in patients with MGUS to predict progression.
PATIENTS AND METHODS: Patients were eligible for this retrospective analysis if they were seen at the Mayo Clinic between 1984 and 1997, were diagnosed with MGUS, and had an analysis of the peripheral blood for circulating PCs by the slide-based immunofluorescence method. Patients were observed for progression to another PCD.
RESULTS: Three hundred twenty-five patients were eligible and 63 (19%) had circulating PCs. Patients with circulating PCs were twice as likely (hazard ratio, 2.1) to experience progression to another PCD (most commonly myeloma), compared with those without circulating PCs (95% CI, 1.1 to 4.3; P = .03). In patients with circulating PCs, the median progression-free survival was 138 months compared with a median not yet reached for those without circulating PCs (P = .028). The median overall survival also was shorter for those with circulating PCs. Other factors with prognostic value were high levels of M protein and non–immunoglobulin G heavy-chain type.
CONCLUSION: The presence of circulating PCs, especially when combined with other known prognostic factors such as M protein concentration and immunoglobulin isotype, identify a group of individuals with MGUS at higher risk of progression to overt multiple myeloma.
INTRODUCTION
Symptomatic monoclonal gammopathies can present as multiple myeloma,1 amyloidosis, or Waldenstr?m macroglobulinemia. It is now recognized that these diseases often present first in an asymptomatic form. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma are two discrete phases that can precede overt myeloma. MGUS is present in more than 3% of the population and its incidence increases with age.2-6 MGUS is characterized by the presence of an M protein in the serum of 3 g/dL or less; no or small amounts of monoclonal light chains (Bence Jones protein) in the urine; bone marrow plasma cell content of less than 10%; and absence of skeletal lesions, anemia, hypercalcemia, or renal failure secondary to the plasma cell dyscrasia.7,8 The significance of this entity results from the risk of progression to clonal plasma cell disorders (PCDs) such as myeloma, primary systemic amyloidosis, and Waldenstr?m macroglobulinemia.
Data from large prospectively followed cohorts places the risk of progression of MGUS to myeloma or a related malignancy at 1% per year.6,9 Given that myeloma is incurable, prevention (or delay) of progression of MGUS to myeloma assumes clinical significance.10 This will only be feasible if the subset of patients with the greatest likelihood of progression (high-risk MGUS) can be accurately identified. Risk factors that seem to be promising include size of the M protein,6 the type of M protein,6 and higher bone marrow plasma cell percentage.11 Additional laboratory predictors of progression of MGUS to myeloma are needed to develop interventions targeting patients with high-risk MGUS.
Clonal plasma cells and B-lymphocytes can be detected in the peripheral blood of patients with PCDs by immunofluorescence microscopy and flow cytometry.12 We have demonstrated previously that the number of circulating plasma cells is a measure of disease activity13 and is an important, independent prognostic factor for survival in myeloma, smoldering myeloma,14,15 and primary amyloidosis.16 In this study, we evaluated the prognostic value of the presence of circulating plasma cells in patients with MGUS with respect to the risk of transformation to myeloma or another PCD and overall survival.
PATIENTS AND METHODS
Patient Selection and Characteristics
This study was initiated in 1985, and patients seen at the Mayo Clinic (Rochester, MN) through the end of 1997 were included for this analysis. Patients were included in this analysis if they had a diagnosis of MGUS and a study of the blood for circulating monoclonal plasma cells. Those who had the blood study after they experienced progression to myeloma or another PCD were excluded. A cutoff of 1997 was used for patient selection to allow at least a 5-year follow-up. Patients who experienced progression within 3 months of their peripheral-blood study were also excluded even though they had the clinical diagnosis of MGUS at the time of the blood study by use of conventional criteria. The criteria used for the diagnosis of MGUS were a monoclonal protein in the serum of 3 g/dL or less; no or small amounts of monoclonal light chains (Bence Jones protein) in the urine; absence of lytic bone lesions, anemia, hypercalcemia, or renal failure secondary to the plasma cell dyscrasia; and less than 10% plasma cells in the marrow if a bone marrow evaluation was performed. Patients who had anemia unrelated to their PCD were included. For those patients who had the blood plasma cell study performed on multiple occasions during the course of their disease, the results of the first test were used for this analysis. Approval for the study was obtained from the Mayo Clinic Institutional Review Board in accordance with federal regulations and the Declaration of Helsinki.
Analysis of the Blood for Monoclonal Plasma Cells
The presence and the quantity of monoclonal plasma cells in the blood were determined using a slide-based immunofluorescence microscopy technique as previously described.12,14,15 In brief, this technique allows identification of monoclonal plasma cells based on their classic morphology and confirmation of cytoplasmic immunoglobulin (cIg) light-chain restriction using staining for and light chains. Mononuclear cells were isolated from peripheral blood on Ficoll-Hypaque and were incubated with bromodeoxyuridine. BU-1 antibromodeoxyuridine monoclonal antibody was added to the cell spot on the fixed slides, and after incubation and washing, was detected using goat antimouse IgG labeled with rhodamine isothiocyanate. Simultaneously, lymphocytes and plasma cells were identified by the addition of monospecific anti- and anti- reagent labeled with fluorescein isothiocyanate (Tago, Burlingame, CA), and the slides were read in an epi-illumination fluorescence microscope (Zeiss, Thornwood, NY). Three different parameters were measured: the percentage of monoclonal plasma cells in peripheral blood, the absolute number of monoclonal plasma cells in blood (absolute PBPC), and the blood plasma cell labeling index. The blood plasma cell labeling index is determined as the proportion of labeled cells among at least 500 cIg-positive cells of the same light-chain isotype as the patient's monoclonal protein.
Statistical Methods
Patients were observed until death or, for those still alive, until June 2002. The major goal of the study was to assess the impact of the variables on progression-free and overall survival. Progression-free survival was defined as the duration between the peripheral-blood plasma cell (PBPC) determination and progression to smoldering myeloma, active myeloma, amyloidosis, or Waldenstr?m macroglobulinemia. Patients who did not experience progression were censored at the time of last follow-up. Overall survival was defined as the duration between the PBPC determination and death, with those alive censored at the last follow-up date. Treatment-free survival was similarly defined as the time from peripheral-blood examination to the start of therapy for any PCD. Nominal variables were compared using Fisher's exact test. A two-tailed P value less than .05 was considered significant. The product-limit method of Kaplan and Meier was used to estimate survival; the survival curves were compared by use of a log-rank test.17
RESULTS
Three hundred twenty-five patients with a diagnosis of MGUS seen at Mayo Clinic between 1984 and 1997 had an analysis of the peripheral blood for circulating plasma cells and form the study cohort. The median age at diagnosis of MGUS was 63 years (mean, 61 years; range, 30 to 87 years), and included 195 males (60%). Thirty-five of the 325 patients (11%) experienced progression to one of the PCDs during the follow-up period. The progression occurred at a median duration of 69 months (mean, 71 months; range, 3 to 179 months) from diagnosis and 30 months (mean, 39 months; range, 4 to 138 months) from the peripheral-blood evaluation. These included multiple myeloma (n = 21; 60%), smoldering myeloma (n = 11; 31%), amyloidosis (n = 2; 6%), and macroglobulinemia (n = 1; 3%). Two hundred eighty (86%) patients were alive at last contact or evaluation.
Sixty-three patients (19.4%) had detectable monoclonal plasma cells in the peripheral blood. The median percentage of plasma cells in these 63 patients was 1% (mean, 6.6%; range, 0.2% to 85%) and the median absolute number of plasma cells was 1.0 x 106/L (mean, 5.5 x 106/L; range, 0.1 to 130 x 106/L; Table 1). In forty-two (67%) of these patients, the absolute number of circulating plasma cells was categorized as high (defined as > 0.5 x 106 plasma cells/L). In most of the patients (n = 316; 97%) no bromodeoxyuridine-labeled plasma cells were seen on immunofluorescence microscopy. The presence of circulating plasma cells predicted for a shorter median progression-free survival. In patients with circulating plasma cells the median progression-free survival (from the peripheral-blood evaluation) was 138 months versus median not yet reached for those without circulating plasma cells (P = .028; Fig 1). Patients with circulating plasma cells also had a shorter median overall survival of 160 months versus median not yet reached at last follow-up (P = .019; Fig 2).
Given that many patients with smoldering myeloma are observed without any therapy for a considerable time, we analyzed if there was any difference in the treatment-free survival between those patients with circulating plasma cells compared with those without circulating plasma cells. Patients with circulating plasma cells had a median treatment-free survival of 138 months compared with median not yet reached for those without circulating plasma cells (P = .06). To rule out the possibility of having selected patients late in the course of their disease, we examined if there was a correlation between the time from diagnosis of MGUS to peripheral-blood evaluation and the test result. The median time to testing among those with circulating plasma cells was 13 months (mean, 33.6 months) compared with 8 months (mean, 33.1 months) in those without circulating plasma cells (P = .9).
In 255 patients (78%) the test for blood plasma cells was performed within 5 years of their diagnosis of MGUS. In the remaining patients, the test became available more than 5 years after the diagnosis of MGUS, but at a time when the patient was still classified as MGUS. Among those patients who had the test within 5 years of diagnosis; 175 (69%) had it within a year of diagnosis, 29 (11%) had it at more than 1 to 2 years, 22 (9%) had it at more than 2 to 3 years, and 29 (11%) had it at more than 3 to 5 years from diagnosis. We also separately analyzed these 255 patients with respect to the role of circulating plasma cells in predicting progression. The median overall survival (160 months v not reached; P = .02) remained shorter for those with circulating plasma cells, as did median progression-free survival (138 months v not reached; P = not significant). Similarly, an analysis of the remaining 70 patients demonstrated a shorter median progression-free survival (66 months v not reached; P = .002) and no difference in the overall survival (P = .5).
To eliminate any effect of a long interval between the diagnosis and testing for circulating plasma cells, we performed a similar analysis confined to the patients who had the test done within 9 months of diagnosis (n = 161; approximately 50% of the study cohort). Both the median progression-free survival (hazard ratio, 3.0; 95% CI, 1.1 to 8.8; P = .04) and overall survival (hazard ratio, 3.3; 95% CI, 1.4 to 7.8; P = .008) estimates were significantly inferior for those with circulating plasma cells compared with patients without this finding.
In exploratory analyses, various factors including age, M protein concentration, type of immunoglobulin, hemoglobin level, serum albumin concentration, serum beta2-microglobulin, creatinine, and presence of circulating plasma cells were evaluated for their prognostic value with regard to risk of progression. In a univariate analysis only three of these factors were found to be significant predictors for progression: presence of circulating plasma cells, higher M protein concentration, and non-IgG subtype (Table 2; Figs 3 and 4). We also evaluated a scoring system using these three variables to evaluate the combined effect of these variables on progression-free survival. One point each was given for the presence of circulating plasma cells, non-IgG heavy chain, and an M protein of 2 g/L. The combined scores stratified the cohort into four groups with differing progression-free survival (P < .0001 by log-rank test; Fig 5).
DISCUSSION
Circulating clonal cells, both B lymphocytes and plasma cells, can be detected in some patients within each of the plasma cell proliferative disorders including MGUS, smoldering or indolent myeloma, active multiple myeloma, and amyloidosis.13,18-21 The number of circulating plasma cells has been documented to be of prognostic significance for survival in myeloma,15 for progression in smoldering myeloma,14 and for survival in amyloidosis.16 In this study, we extend these findings to MGUS by demonstrating that circulating plasma cells are an independent predictor of progression of MGUS and a prognostic factor for overall survival.
Various methods have been used for detection of clonal cells in the circulation with differing sensitivities and specificities. Flow cytometry using antibodies against CD38 or CD138 identifies plasma cell populations for additional study.12 Use of antibodies to and light chain provides additional information regarding clonality based on light-chain restriction. In this study, we used a slide-based immunofluorescence method for detection of plasma cells in the peripheral blood that has been studied extensively, is reproducible, and has a high degree of specificity.13-15,18,22,23 Although other methods such as allele specific oligonucleotide–polymerase chain reaction are more sensitive at detecting circulating tumor cells,24 the immunofluorescence method has been in use in our practice for 19 years, can be performed in the clinical laboratory, and provides results rapidly enough to be useful in making clinical decisions. The use of the same method over many years was necessary to determine whether the blood plasma cells predicted progression in MGUS.
In this study, circulating plasma cells were detected in nearly one fifth of patients, comparable to that found in patients with amyloidosis16,18,22 but lower than that in newly diagnosed myeloma, with which 80% of patients have detectable circulating plasma cells.15 When the absolute number of plasma cells in the blood is evaluated, patients with MGUS and circulating plasma cells have a lower number compared with that seen in myeloma (1 v 4.4 x 106/L).
The presence of circulating plasma cells seems also to have value in predicting the risk of progression for patients with smoldering myeloma. In a study of 57 patients observed during a 12-month period, 64% of the patients who had abnormal PBPCs (defined as 3 x 106/L circulating plasma cells), 3% of cIg-positive cells were plasma cells, or labeling index > 0.5% had progression to active myeloma.14 During the same time frame only six of 43 patients (14%) without abnormal PBPCs experienced disease progression.14
Clonal circulating cells in patients with plasma cell dyscrasias are not confined to morphologically identifiable plasma cells. Several authors have reported on the presence of clonal B lymphocytes in these patients. Increased numbers of immunoglobulin-secreting cells were reported in patients with plasma cell dyscrasias by Shimizu et al21 as early as 1980. B lymphocytes with immunoglobulin gene rearrangements identical to that seen in the marrow myeloma cells have been demonstrated in the peripheral blood25 and have been correlated with tumor volume and disease activity in patients with myeloma.26 As observed with the presence of circulating plasma cells, the presence of these cells has also been associated with a poorer prognosis in these patients.27 Isaksson et al28 has demonstrated a prognostic significance for clonal B cells in the peripheral blood in patients with MGUS. In a study of 57 patients, the presence of clonal B cells as determined by immunofluorescence microscopy was associated with a 3.5-fold higher risk of transformation in patients with MGUS. It is possible that these clonal lymphocytes represent precursor cells for the malignant plasma cells.
Patients with MGUS have differing propensities to progress into overt myeloma that requires therapy, and the risk of progression is difficult to predict at the time of initial diagnosis. Large groups of prospectively observed patients with MGUS have shed some light on potential prognostic factors. Kyle et al6 studied 1,384 patients with MGUS, evaluated at the Mayo Clinic from 1960 to 1994, and observed them for a total of 11,009 person-years (median, 15.4 years; range, 0 to 35 years). During follow-up, 115 patients (8%) developed multiple myeloma, primary amyloidosis, lymphoma, macroglobulinemia, plasmacytoma, or chronic lymphocytic leukemia. The cumulative probability of progression to one of these disorders was 10% at 10 years, 21% at 20 years, and 26% at 25 years. In addition, 32 patients had an increase in M protein value to more than 3 g/dL or the percentage of bone marrow plasma cells to more than 10% without development of symptomatic multiple myeloma. Factors associated with higher risk of progression included higher M protein levels and IgA or IgM subgroups. In another series of patients with MGUS reported by Cesana et al,11 the cumulative probability of progression at 10 and 15 years from diagnosis was 14% and 30%, respectively. A higher risk of progression was seen in those with IgA or IgM M protein, higher M protein levels, Bence Jones proteinuria, higher bone marrow plasma cell percentage, and elevated erythrocyte sedimentation rate. Higher levels of M protein and IgA M protein were also identified as risk factors for progression by Gregersen et al29 in their study of 1,247 Danish patients.
As we demonstrate in this study, presence of circulating plasma cells identifies a group of patients with MGUS who are at an increased risk of early progression to myeloma or a related disorder. It is unclear if the presence of circulating plasma cells denotes a group that is biologically different, or whether it reflects evaluation at a later stage in the natural history of this disorder. Lack of correlation between the time from diagnosis and the likelihood of finding circulating plasma cells would favor the former. However, serial studies for circulating plasma cells in patients with MGUS would be required to answer this question conclusively. Isaksson et al,28 in their study of circulating clonal B cells in MGUS, found emergence of clonal abnormalities before transformation; these abnormalities, which were not detected at initial evaluations, favored the latter hypothesis. The biologic basis for the movement of plasma cells from the marrow to the peripheral circulation is not clear. Given the important role of bone marrow microenvironment in the biology of myeloma,30 understanding the basis of this migration will provide additional insight into the transition from MGUS to myeloma. Differential expression of various adhesion molecules at different stages of the disease may play a role. The migration of plasma cells may also be related to the proliferative state of the malignant plasma cells.
In conclusion, we have demonstrated the potential prognostic value for circulating plasma cells in patients with MGUS. This study shows that the presence of circulating plasma cells in patients with MGUS has prognostic value even when the analysis is restricted to patients with recently diagnosed MGUS. We did not find evidence that the presence of circulating plasma cells was merely a marker for duration of disease. However, MGUS is asymptomatic and there are no clinical or laboratory tests to determine how long patients have had the condition when it is first diagnosed. Thus, even if the adverse prognostic effect conferred by the presence of circulating plasma cells is because it serves as a marker for disease duration, the finding still has major clinical and biologic significance. This study again confirms the value of the M protein concentration and the type of immunoglobulin as predictors of progression in MGUS. A scoring system using these three factors seems to stratify patients according to their risk of progression, and may be a clinically useful tool. These patients seem to be at a higher risk of progression and need closer follow-up. They may also be candidates for clinical trials evaluating novel preventive therapies.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by grant Nos. CA62242, CA85818, CA93842, CA65125, and CA100080 from the National Cancer Institute, Bethesda, MD. S.V.R. is supported by the Multiple Myeloma Research Foundation and the Goldman Philanthropic Partnerships. S.V.R. and R.F. are also supported by Leukemia and Lymphoma Society Translational Research Awards.
Presented in part at the Annual Meeting of the American Society of Hematology, December 6-9, 2003, San Diego, CA.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
REFERENCES
Kyle RA, Gertz MA, Witzig TE, et al: Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 78:21-33, 2003
Kyle RA, Finkelstein S, Elveback LR, et al: Incidence of monoclonal proteins in a Minnesota community with a cluster of multiple myeloma. Blood 40:719-724, 1972
Saleun JP, Vicariot M, Deroff P, et al: Monoclonal gammopathies in the adult population of Finistere, France. J Clin Pathol 35:63-68, 1982
Axelsson U: A 20-year follow-up study of 64 subjects with M-components. Acta Med Scand 219:519-522, 1986
Kyle RA, Therneau T, Rajkumar SV, et al: Prevalence of monoclonal gammopathy of undetermined significance (MGUS) among Olmsted County, MN residents 50 years of age. Blood 102:934a, 2003 (abstr 3476)
Kyle RA, Therneau TM, Rajkumar SV, et al: A long-term study of prognosis in monoclonal gammopathy of undetermined significance. N Engl J Med 346:564-569, 2002
Kyle RA, Therneau TM, Rajkumar SV, et al: Long-term follow-up of 241 patients with monoclonal gammopathy of undetermined significance: The original Mayo Clinic series 25 years later. Mayo Clin Proc 79:859-866, 2004
Kyle RA, Rajkumar SV: Monoclonal gammopathies of undetermined significance. Hematol Oncol Clin North Am 13:1181-1202, 1999
Gregersen H, Mellemkjaer L, Ibsen JS, et al: Cancer risk in patients with monoclonal gammopathy of undetermined significance. Am J Hematol 63:1-6, 2000
Kyle RA, Rajkumar SV: Multiple myeloma. N Engl J Med 351:1860-1873, 2004
Cesana C, Klersy C, Barbarano L, et al: Prognostic factors for malignant transformation in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. J Clin Oncol 20:1625-1634, 2002
Witzig TE, Kimlinger TK, Ahmann GJ, et al: Detection of myeloma cells in the peripheral blood by flow cytometry. Cytometry 26:113-120, 1996
Witzig TE, Gonchoroff NJ, Katzmann JA, et al: Peripheral blood B cell labeling indices are a measure of disease activity in patients with monoclonal gammopathies. J Clin Oncol 6:1041-1046, 1988
Witzig TE, Kyle RA, O'Fallon WM, et al: Detection of peripheral blood plasma cells as a predictor of disease course in patients with smouldering multiple myeloma. Br J Haematol 87:266-272, 1994
Witzig TE, Gertz MA, Lust JA, et al: Peripheral blood monoclonal plasma cells as a predictor of survival in patients with multiple myeloma. Blood 88:1780-1787, 1996
Pardanani A, Witzig TE, Schroeder G, et al: Circulating peripheral blood plasma cells as a prognostic indicator in patients with primary systemic amyloidosis. Blood 101:827-830, 2003
Kaplan E, Meier P: Non-parametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958
Witzig TE, Dhodapkar MV, Kyle RA, et al: Quantitation of circulating peripheral blood plasma cells and their relationship to disease activity in patients with multiple myeloma. Cancer 72:108-113, 1993
Boccadoro M, Omede P, Massaia M, et al: Human myeloma: Several subsets of circulating lymphocytes express plasma cell-associated antigens. Eur J Haematol 40:299-304, 1988
Omede P, Boccadoro M, Gallone G, et al: Multiple myeloma: Increased circulating lymphocytes carrying plasma cell-associated antigens as an indicator of poor survival. Blood 76:1375-1379, 1990
Shimizu K, Murate T, Kunii A: Circulating immunoglobulin-secreting cells in patients with plasma cell dyscrasia. Blood 55:590-594, 1980
Witzig TE, Kyle RA, Greipp PR: Circulating peripheral blood plasma cells in multiple myeloma. Curr Top Microbiol Immunol 182:195-199, 1992
Witzig TE, Gertz MA, Pineda AA, et al: Detection of monoclonal plasma cells in the peripheral blood stem cell harvests of patients with multiple myeloma. Br J Haematol 89:640-642, 1995
Billadeau D, Van Ness B, Kimlinger T, et al: Clonal circulating cells are common in plasma cell proliferative disorders: A comparison of monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and active myeloma. Blood 88:289-296, 1996
Cassel A, Leibovitz N, Hornstein L, et al: Evidence for the existence of circulating monoclonal B-lymphocytes in multiple myeloma patients. Exp Hematol 18:1171-1173, 1990
Chiu EK, Ganeshaguru K, Hoffbrand AV, et al: Circulating monoclonal B lymphocytes in multiple myeloma. Br J Haematol 72:28-31, 1989
Osterborg A, Nilsson B, Bjorkholm M, et al: Blood clonal B cell excess at diagnosis in multiple myeloma: Relation to prognosis. Eur J Haematol 38:173-178, 1987
Isaksson E, Bjorkholm M, Holm G, et al: Blood clonal B-cell excess in patients with monoclonal gammopathy of undetermined significance (MGUS): Association with malignant transformation. Br J Haematol 92:71-76, 1996
Gregersen H, Mellemkjaer L, Ibsen JS, et al: The impact of M-component type and immunoglobulin concentration on the risk of malignant transformation in patients with monoclonal gammopathy of undetermined significance. Haematologica 86:1172-1179, 2001
Roodman GD: Role of the bone marrow microenvironment in multiple myeloma. J Bone Miner Res 17:1921-1925, 2002(Shaji Kumar, S. Vincent R)
ABSTRACT
PURPOSE: Monoclonal gammopathy of undetermined significance (MGUS) progresses to multiple myeloma or another related plasma cell disorder (PCD) at a rate of approximately 1% per year. Identification of patients with MGUS at high risk of progression will allow development of preventive strategies. We studied the prognostic value of circulating plasma cells (PCs) in patients with MGUS to predict progression.
PATIENTS AND METHODS: Patients were eligible for this retrospective analysis if they were seen at the Mayo Clinic between 1984 and 1997, were diagnosed with MGUS, and had an analysis of the peripheral blood for circulating PCs by the slide-based immunofluorescence method. Patients were observed for progression to another PCD.
RESULTS: Three hundred twenty-five patients were eligible and 63 (19%) had circulating PCs. Patients with circulating PCs were twice as likely (hazard ratio, 2.1) to experience progression to another PCD (most commonly myeloma), compared with those without circulating PCs (95% CI, 1.1 to 4.3; P = .03). In patients with circulating PCs, the median progression-free survival was 138 months compared with a median not yet reached for those without circulating PCs (P = .028). The median overall survival also was shorter for those with circulating PCs. Other factors with prognostic value were high levels of M protein and non–immunoglobulin G heavy-chain type.
CONCLUSION: The presence of circulating PCs, especially when combined with other known prognostic factors such as M protein concentration and immunoglobulin isotype, identify a group of individuals with MGUS at higher risk of progression to overt multiple myeloma.
INTRODUCTION
Symptomatic monoclonal gammopathies can present as multiple myeloma,1 amyloidosis, or Waldenstr?m macroglobulinemia. It is now recognized that these diseases often present first in an asymptomatic form. Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma are two discrete phases that can precede overt myeloma. MGUS is present in more than 3% of the population and its incidence increases with age.2-6 MGUS is characterized by the presence of an M protein in the serum of 3 g/dL or less; no or small amounts of monoclonal light chains (Bence Jones protein) in the urine; bone marrow plasma cell content of less than 10%; and absence of skeletal lesions, anemia, hypercalcemia, or renal failure secondary to the plasma cell dyscrasia.7,8 The significance of this entity results from the risk of progression to clonal plasma cell disorders (PCDs) such as myeloma, primary systemic amyloidosis, and Waldenstr?m macroglobulinemia.
Data from large prospectively followed cohorts places the risk of progression of MGUS to myeloma or a related malignancy at 1% per year.6,9 Given that myeloma is incurable, prevention (or delay) of progression of MGUS to myeloma assumes clinical significance.10 This will only be feasible if the subset of patients with the greatest likelihood of progression (high-risk MGUS) can be accurately identified. Risk factors that seem to be promising include size of the M protein,6 the type of M protein,6 and higher bone marrow plasma cell percentage.11 Additional laboratory predictors of progression of MGUS to myeloma are needed to develop interventions targeting patients with high-risk MGUS.
Clonal plasma cells and B-lymphocytes can be detected in the peripheral blood of patients with PCDs by immunofluorescence microscopy and flow cytometry.12 We have demonstrated previously that the number of circulating plasma cells is a measure of disease activity13 and is an important, independent prognostic factor for survival in myeloma, smoldering myeloma,14,15 and primary amyloidosis.16 In this study, we evaluated the prognostic value of the presence of circulating plasma cells in patients with MGUS with respect to the risk of transformation to myeloma or another PCD and overall survival.
PATIENTS AND METHODS
Patient Selection and Characteristics
This study was initiated in 1985, and patients seen at the Mayo Clinic (Rochester, MN) through the end of 1997 were included for this analysis. Patients were included in this analysis if they had a diagnosis of MGUS and a study of the blood for circulating monoclonal plasma cells. Those who had the blood study after they experienced progression to myeloma or another PCD were excluded. A cutoff of 1997 was used for patient selection to allow at least a 5-year follow-up. Patients who experienced progression within 3 months of their peripheral-blood study were also excluded even though they had the clinical diagnosis of MGUS at the time of the blood study by use of conventional criteria. The criteria used for the diagnosis of MGUS were a monoclonal protein in the serum of 3 g/dL or less; no or small amounts of monoclonal light chains (Bence Jones protein) in the urine; absence of lytic bone lesions, anemia, hypercalcemia, or renal failure secondary to the plasma cell dyscrasia; and less than 10% plasma cells in the marrow if a bone marrow evaluation was performed. Patients who had anemia unrelated to their PCD were included. For those patients who had the blood plasma cell study performed on multiple occasions during the course of their disease, the results of the first test were used for this analysis. Approval for the study was obtained from the Mayo Clinic Institutional Review Board in accordance with federal regulations and the Declaration of Helsinki.
Analysis of the Blood for Monoclonal Plasma Cells
The presence and the quantity of monoclonal plasma cells in the blood were determined using a slide-based immunofluorescence microscopy technique as previously described.12,14,15 In brief, this technique allows identification of monoclonal plasma cells based on their classic morphology and confirmation of cytoplasmic immunoglobulin (cIg) light-chain restriction using staining for and light chains. Mononuclear cells were isolated from peripheral blood on Ficoll-Hypaque and were incubated with bromodeoxyuridine. BU-1 antibromodeoxyuridine monoclonal antibody was added to the cell spot on the fixed slides, and after incubation and washing, was detected using goat antimouse IgG labeled with rhodamine isothiocyanate. Simultaneously, lymphocytes and plasma cells were identified by the addition of monospecific anti- and anti- reagent labeled with fluorescein isothiocyanate (Tago, Burlingame, CA), and the slides were read in an epi-illumination fluorescence microscope (Zeiss, Thornwood, NY). Three different parameters were measured: the percentage of monoclonal plasma cells in peripheral blood, the absolute number of monoclonal plasma cells in blood (absolute PBPC), and the blood plasma cell labeling index. The blood plasma cell labeling index is determined as the proportion of labeled cells among at least 500 cIg-positive cells of the same light-chain isotype as the patient's monoclonal protein.
Statistical Methods
Patients were observed until death or, for those still alive, until June 2002. The major goal of the study was to assess the impact of the variables on progression-free and overall survival. Progression-free survival was defined as the duration between the peripheral-blood plasma cell (PBPC) determination and progression to smoldering myeloma, active myeloma, amyloidosis, or Waldenstr?m macroglobulinemia. Patients who did not experience progression were censored at the time of last follow-up. Overall survival was defined as the duration between the PBPC determination and death, with those alive censored at the last follow-up date. Treatment-free survival was similarly defined as the time from peripheral-blood examination to the start of therapy for any PCD. Nominal variables were compared using Fisher's exact test. A two-tailed P value less than .05 was considered significant. The product-limit method of Kaplan and Meier was used to estimate survival; the survival curves were compared by use of a log-rank test.17
RESULTS
Three hundred twenty-five patients with a diagnosis of MGUS seen at Mayo Clinic between 1984 and 1997 had an analysis of the peripheral blood for circulating plasma cells and form the study cohort. The median age at diagnosis of MGUS was 63 years (mean, 61 years; range, 30 to 87 years), and included 195 males (60%). Thirty-five of the 325 patients (11%) experienced progression to one of the PCDs during the follow-up period. The progression occurred at a median duration of 69 months (mean, 71 months; range, 3 to 179 months) from diagnosis and 30 months (mean, 39 months; range, 4 to 138 months) from the peripheral-blood evaluation. These included multiple myeloma (n = 21; 60%), smoldering myeloma (n = 11; 31%), amyloidosis (n = 2; 6%), and macroglobulinemia (n = 1; 3%). Two hundred eighty (86%) patients were alive at last contact or evaluation.
Sixty-three patients (19.4%) had detectable monoclonal plasma cells in the peripheral blood. The median percentage of plasma cells in these 63 patients was 1% (mean, 6.6%; range, 0.2% to 85%) and the median absolute number of plasma cells was 1.0 x 106/L (mean, 5.5 x 106/L; range, 0.1 to 130 x 106/L; Table 1). In forty-two (67%) of these patients, the absolute number of circulating plasma cells was categorized as high (defined as > 0.5 x 106 plasma cells/L). In most of the patients (n = 316; 97%) no bromodeoxyuridine-labeled plasma cells were seen on immunofluorescence microscopy. The presence of circulating plasma cells predicted for a shorter median progression-free survival. In patients with circulating plasma cells the median progression-free survival (from the peripheral-blood evaluation) was 138 months versus median not yet reached for those without circulating plasma cells (P = .028; Fig 1). Patients with circulating plasma cells also had a shorter median overall survival of 160 months versus median not yet reached at last follow-up (P = .019; Fig 2).
Given that many patients with smoldering myeloma are observed without any therapy for a considerable time, we analyzed if there was any difference in the treatment-free survival between those patients with circulating plasma cells compared with those without circulating plasma cells. Patients with circulating plasma cells had a median treatment-free survival of 138 months compared with median not yet reached for those without circulating plasma cells (P = .06). To rule out the possibility of having selected patients late in the course of their disease, we examined if there was a correlation between the time from diagnosis of MGUS to peripheral-blood evaluation and the test result. The median time to testing among those with circulating plasma cells was 13 months (mean, 33.6 months) compared with 8 months (mean, 33.1 months) in those without circulating plasma cells (P = .9).
In 255 patients (78%) the test for blood plasma cells was performed within 5 years of their diagnosis of MGUS. In the remaining patients, the test became available more than 5 years after the diagnosis of MGUS, but at a time when the patient was still classified as MGUS. Among those patients who had the test within 5 years of diagnosis; 175 (69%) had it within a year of diagnosis, 29 (11%) had it at more than 1 to 2 years, 22 (9%) had it at more than 2 to 3 years, and 29 (11%) had it at more than 3 to 5 years from diagnosis. We also separately analyzed these 255 patients with respect to the role of circulating plasma cells in predicting progression. The median overall survival (160 months v not reached; P = .02) remained shorter for those with circulating plasma cells, as did median progression-free survival (138 months v not reached; P = not significant). Similarly, an analysis of the remaining 70 patients demonstrated a shorter median progression-free survival (66 months v not reached; P = .002) and no difference in the overall survival (P = .5).
To eliminate any effect of a long interval between the diagnosis and testing for circulating plasma cells, we performed a similar analysis confined to the patients who had the test done within 9 months of diagnosis (n = 161; approximately 50% of the study cohort). Both the median progression-free survival (hazard ratio, 3.0; 95% CI, 1.1 to 8.8; P = .04) and overall survival (hazard ratio, 3.3; 95% CI, 1.4 to 7.8; P = .008) estimates were significantly inferior for those with circulating plasma cells compared with patients without this finding.
In exploratory analyses, various factors including age, M protein concentration, type of immunoglobulin, hemoglobin level, serum albumin concentration, serum beta2-microglobulin, creatinine, and presence of circulating plasma cells were evaluated for their prognostic value with regard to risk of progression. In a univariate analysis only three of these factors were found to be significant predictors for progression: presence of circulating plasma cells, higher M protein concentration, and non-IgG subtype (Table 2; Figs 3 and 4). We also evaluated a scoring system using these three variables to evaluate the combined effect of these variables on progression-free survival. One point each was given for the presence of circulating plasma cells, non-IgG heavy chain, and an M protein of 2 g/L. The combined scores stratified the cohort into four groups with differing progression-free survival (P < .0001 by log-rank test; Fig 5).
DISCUSSION
Circulating clonal cells, both B lymphocytes and plasma cells, can be detected in some patients within each of the plasma cell proliferative disorders including MGUS, smoldering or indolent myeloma, active multiple myeloma, and amyloidosis.13,18-21 The number of circulating plasma cells has been documented to be of prognostic significance for survival in myeloma,15 for progression in smoldering myeloma,14 and for survival in amyloidosis.16 In this study, we extend these findings to MGUS by demonstrating that circulating plasma cells are an independent predictor of progression of MGUS and a prognostic factor for overall survival.
Various methods have been used for detection of clonal cells in the circulation with differing sensitivities and specificities. Flow cytometry using antibodies against CD38 or CD138 identifies plasma cell populations for additional study.12 Use of antibodies to and light chain provides additional information regarding clonality based on light-chain restriction. In this study, we used a slide-based immunofluorescence method for detection of plasma cells in the peripheral blood that has been studied extensively, is reproducible, and has a high degree of specificity.13-15,18,22,23 Although other methods such as allele specific oligonucleotide–polymerase chain reaction are more sensitive at detecting circulating tumor cells,24 the immunofluorescence method has been in use in our practice for 19 years, can be performed in the clinical laboratory, and provides results rapidly enough to be useful in making clinical decisions. The use of the same method over many years was necessary to determine whether the blood plasma cells predicted progression in MGUS.
In this study, circulating plasma cells were detected in nearly one fifth of patients, comparable to that found in patients with amyloidosis16,18,22 but lower than that in newly diagnosed myeloma, with which 80% of patients have detectable circulating plasma cells.15 When the absolute number of plasma cells in the blood is evaluated, patients with MGUS and circulating plasma cells have a lower number compared with that seen in myeloma (1 v 4.4 x 106/L).
The presence of circulating plasma cells seems also to have value in predicting the risk of progression for patients with smoldering myeloma. In a study of 57 patients observed during a 12-month period, 64% of the patients who had abnormal PBPCs (defined as 3 x 106/L circulating plasma cells), 3% of cIg-positive cells were plasma cells, or labeling index > 0.5% had progression to active myeloma.14 During the same time frame only six of 43 patients (14%) without abnormal PBPCs experienced disease progression.14
Clonal circulating cells in patients with plasma cell dyscrasias are not confined to morphologically identifiable plasma cells. Several authors have reported on the presence of clonal B lymphocytes in these patients. Increased numbers of immunoglobulin-secreting cells were reported in patients with plasma cell dyscrasias by Shimizu et al21 as early as 1980. B lymphocytes with immunoglobulin gene rearrangements identical to that seen in the marrow myeloma cells have been demonstrated in the peripheral blood25 and have been correlated with tumor volume and disease activity in patients with myeloma.26 As observed with the presence of circulating plasma cells, the presence of these cells has also been associated with a poorer prognosis in these patients.27 Isaksson et al28 has demonstrated a prognostic significance for clonal B cells in the peripheral blood in patients with MGUS. In a study of 57 patients, the presence of clonal B cells as determined by immunofluorescence microscopy was associated with a 3.5-fold higher risk of transformation in patients with MGUS. It is possible that these clonal lymphocytes represent precursor cells for the malignant plasma cells.
Patients with MGUS have differing propensities to progress into overt myeloma that requires therapy, and the risk of progression is difficult to predict at the time of initial diagnosis. Large groups of prospectively observed patients with MGUS have shed some light on potential prognostic factors. Kyle et al6 studied 1,384 patients with MGUS, evaluated at the Mayo Clinic from 1960 to 1994, and observed them for a total of 11,009 person-years (median, 15.4 years; range, 0 to 35 years). During follow-up, 115 patients (8%) developed multiple myeloma, primary amyloidosis, lymphoma, macroglobulinemia, plasmacytoma, or chronic lymphocytic leukemia. The cumulative probability of progression to one of these disorders was 10% at 10 years, 21% at 20 years, and 26% at 25 years. In addition, 32 patients had an increase in M protein value to more than 3 g/dL or the percentage of bone marrow plasma cells to more than 10% without development of symptomatic multiple myeloma. Factors associated with higher risk of progression included higher M protein levels and IgA or IgM subgroups. In another series of patients with MGUS reported by Cesana et al,11 the cumulative probability of progression at 10 and 15 years from diagnosis was 14% and 30%, respectively. A higher risk of progression was seen in those with IgA or IgM M protein, higher M protein levels, Bence Jones proteinuria, higher bone marrow plasma cell percentage, and elevated erythrocyte sedimentation rate. Higher levels of M protein and IgA M protein were also identified as risk factors for progression by Gregersen et al29 in their study of 1,247 Danish patients.
As we demonstrate in this study, presence of circulating plasma cells identifies a group of patients with MGUS who are at an increased risk of early progression to myeloma or a related disorder. It is unclear if the presence of circulating plasma cells denotes a group that is biologically different, or whether it reflects evaluation at a later stage in the natural history of this disorder. Lack of correlation between the time from diagnosis and the likelihood of finding circulating plasma cells would favor the former. However, serial studies for circulating plasma cells in patients with MGUS would be required to answer this question conclusively. Isaksson et al,28 in their study of circulating clonal B cells in MGUS, found emergence of clonal abnormalities before transformation; these abnormalities, which were not detected at initial evaluations, favored the latter hypothesis. The biologic basis for the movement of plasma cells from the marrow to the peripheral circulation is not clear. Given the important role of bone marrow microenvironment in the biology of myeloma,30 understanding the basis of this migration will provide additional insight into the transition from MGUS to myeloma. Differential expression of various adhesion molecules at different stages of the disease may play a role. The migration of plasma cells may also be related to the proliferative state of the malignant plasma cells.
In conclusion, we have demonstrated the potential prognostic value for circulating plasma cells in patients with MGUS. This study shows that the presence of circulating plasma cells in patients with MGUS has prognostic value even when the analysis is restricted to patients with recently diagnosed MGUS. We did not find evidence that the presence of circulating plasma cells was merely a marker for duration of disease. However, MGUS is asymptomatic and there are no clinical or laboratory tests to determine how long patients have had the condition when it is first diagnosed. Thus, even if the adverse prognostic effect conferred by the presence of circulating plasma cells is because it serves as a marker for disease duration, the finding still has major clinical and biologic significance. This study again confirms the value of the M protein concentration and the type of immunoglobulin as predictors of progression in MGUS. A scoring system using these three factors seems to stratify patients according to their risk of progression, and may be a clinically useful tool. These patients seem to be at a higher risk of progression and need closer follow-up. They may also be candidates for clinical trials evaluating novel preventive therapies.
Authors' Disclosures of Potential Conflicts of Interest
The authors indicated no potential conflicts of interest.
NOTES
Supported by grant Nos. CA62242, CA85818, CA93842, CA65125, and CA100080 from the National Cancer Institute, Bethesda, MD. S.V.R. is supported by the Multiple Myeloma Research Foundation and the Goldman Philanthropic Partnerships. S.V.R. and R.F. are also supported by Leukemia and Lymphoma Society Translational Research Awards.
Presented in part at the Annual Meeting of the American Society of Hematology, December 6-9, 2003, San Diego, CA.
Authors' disclosures of potential conflicts of interest are found at the end of this article.
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