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Clinicopathological Correlates of IgM Paraproteinemias
Original Contribution Clinicopathological Correlates of IgM Paraproteinemias Roger G. Owen,1,2 Liakat A. Parapia,2 Jean Higginson,3 Siraj A. Misbah,3 J. Anthony Child,1 Gareth J. Morgan,1 Andrew S. Jack1
Abstract IgM paraproteinemia is considered to be the major defining feature of Waldenström’s macroglobulinemia (WM), but it may also occur in other B-cell lymphoproliferative disorders. In this study we have reviewed the final pathological diagnosis of 106 patients with IgM paraproteinemia investigated in our laboratories between April 1993 and May 1999. In 22 of the 106 patients (20.8%), there was no clinical or laboratory evidence of an underlying lymphoproliferative disorder, and a diagnosis of monoclonal gammopathy of undetermined significance (MGUS) was therefore made. In 60 cases (56.6%), a diagnosis of WM was made, while in the remaining 24 patients, the final diagnosis was chronic lymphocytic leukemia (n = 10), diffuse large B-cell lymphoma (n = 5), extranodal marginal-zone lymphoma (n = 3), follicular lymphoma (n = 3), and mantle-cell lymphoma (n = 3). The median paraprotein concentration in patients with WM, MGUS, and "other" lymphoproliferative disorders was 13 g/L (range, 2-54), 6 g/L (range, 3-30), and 4.5 g/L (range, 3-61), respectively. It is clear that IgM paraproteins are demonstrable in all subtypes of peripheral B-cell disorders and, although paraprotein concentrations are generally higher in WM, there is considerable overlap. Immunophenotypic criteria are therefore essential for the accurate diagnosis of WM.
Clinical Lymphoma, Vol. 1, No. 1, 39-43, 2000 Key words: Waldenström’s macroglobulinemia, IgM paraprotein, Non-Hodgkin’s lymphoma
Introduction Waldenström’s macroglobulinemia (WM) is a chronic lymphoproliferative disorder characterized by bone marrow infiltration by a mixed population of lymphocytes, lymphoplasmacytoid cells, and plasma cells associated with IgM paraproteinemia.1-3 It accounts for approximately 2% of all hematological malignancies and has an annual incidence of six per 1,000,000 in white males, in whom the incidence is highest.2,4 The IgM paraproteinemia is considered to be the major defining feature of WM and it is responsible for many of the clinical features seen, such as hyperviscosity syndrome, cryoglobulinemia, and amyloidosis, although the latter appears to be rare.1-3,5,6 The IgM paraprotein may also have autoantibody activity, which may result in peripheral neuropathy, cold agglutinin hemolysis, immune thrombocytopenia (ITP), glomerulonephritis, acquired von Willebrand’s disease, and mixed cryoglobulinemia.2,3,6-9 IgM paraproteinemia may also, however, be seen in other B-cell lymphoproliferative disorders.10,11 In 1987, Kyle and 1Department
of Hematology, General Infirmary at Leeds of Hematology, Bradford Royal Infirmary 3Department of Immunology, General Infirmary at Leeds Submitted: Mar. 17, 2000; Revised: May 2, 2000; Accepted: May 22, 2000 Address for correspondence: R.G. Owen, MD, HMDS Laboratory, The General Infirmary at Leeds, Great George Street, Leeds, LS1 3EX Fax: 0113 392 6286; e-mail: [email protected] 2Department
Garton reviewed the diagnoses of 430 patients with IgM paraproteinemia investigated at the Mayo Clinic between 1956-1978. They diagnosed monoclonal gammopathy of undetermined significance (MGUS) in 56% and WM in 17% of cases, while the remaining cases had chronic lymphocytic leukemia (CLL), primary amyloidosis, "lymphoma," or "malignant lymphoproliferative disease."11 In this study we have attempted to further clarify this issue by utilizing the Revised European-American Lymphoma (REAL) classification12 and World Health Organization (WHO) criteria13 to reach a final diagnosis in 106 patients investigated with IgM paraproteinemia.
Patients and Methods One hundred six patients with IgM paraproteins were investigated in our regional hematopathology laboratory from April 1993 to May 1999. Sixty males and 46 females with a median age of 71 years (range, 32-91) were included in this analysis. IgM paraproteins were demonstrated and quantitated in each case using the Sebia Hydrasys system and typed by standard immunofixation techniques. The median IgM paraprotein concentration in these patients was 10 g/L (range, 261). IgMκ paraproteins were demonstrated in 78 cases (74%) while IgMλ paraproteins were demonstrated in 26 cases (25%). In the two remaining cases, both IgMκ and IgMλ paraproteins were demonstrable. The final pathological diagnosis in these two cases was MGUS.
June 2000 • 39
CPC of IgM Paraproteinemias Table 1
Diagnostic Criteria Used in the Analysis of 106 Patients with IgM Paraproteinemia
Monoclonal Gammopathy of Undetermined Significance (MGUS): - No clinical evidence of an underlying lymphoproliferative disorder - Fewer than 5% plasma cells and fewer than 20% lymphocytes on bone marrow aspirate smears - No evidence of lymphoma or plasma cell infiltration on trephine biopsy sections - No clonal B cells detected by flow cytometry* Waldenström’s macroglobulinemia (WM): - Bone marrow infiltration by lymphocytes, lymphoplasmacytoid cells, and plasma cells with a diffuse, interstitial, or nodular pattern of infiltration - B-cell immunophenotype demonstrated by three-color flow cytometry or immunohistochemistry† - Absence of any clinical, morphological, or immunophenotypic features of other lymphoproliferative disorders Chronic lymphocytic leukemia (CLL): - Clonal peripheral blood lymphocytosis with the following immunophenotypic features: weak surface immunoglobulin, CD5+, CD19+, CD20+, CD23+ Mantle-cell lymphoma: - Bone marrow infiltration by irregular/cleaved lymphoid cells with the following immunophenotype: strong surface immunoglobulin, CD5+, CD19+, CD20+, CD23-‡ Follicular lymphoma: - Lymph node biopsy showing a follicular growth pattern and mixed population of centrocytes and centroblasts§ - CD20+, CD10+ immunophenotype demonstrated by flow cytometry and/or immunohistochemistry - Bcl-2 positivity demonstrated by immunohistochemistry Diffuse large B-cell lymphoma: - Lymph node/tissue infiltration by large lymphoid cells with a high rate of cell proliferation determined by Ki-67 immunostaining - B-cell immunophenotype (CD20+) demonstrated by flow cytometry and/or immunohistochemistry Extranodal marginal-zone lymphoma: - Lymphoid infiltration at a mucosa-associated lymphoid tissue (MALT) site with prominent lymphoepithelial lesionsll - CD5-, CD10-, CD20+, CD23- immunophenotype demonstrated by immunohistochemistry * Flow cytometry was performed in 15 of the 22 cases diagnosed with MGUS. No clonal B-cell populations were seen. † Immunophenotyping was performed in 57 of the 60 cases diagnosed with WM. In the remaining three patients, a diagnosis of WM was made on the basis of interstitial bone marrow infiltration with small lymphocytes and the absence of peripheral blood and lymph node involvement. Eighty-six percent of cases assessed were characterized by a CD19+, CD20+, CD5-, CD10-, CD23- immunophenotype. ‡ Three cases of mantle-cell lymphoma were diagnosed in this study. Diagnosis in these cases was made by morphological and immunophenotypic criteria. Evidence of the t(11;14) was not sought. § In one case, a diagnosis of follicular lymphoma was made solely on the basis of paratrabecular bone marrow infiltration by small, irregular centrocytes and larger centroblasts. ll Three cases of extranodal marginal-zone lymphoma were identified in this analysis. The diagnosis was made from gastric biopsies in two cases and a lung biopsy in the remaining case. Lymphoepithelial lesions were prominent in all cases.
Pathological diagnoses were made in each case according to REAL/WHO criteria,12,13 which are summarized in Table 1. MGUS is essentially a diagnosis of exclusion, and in these cases, a diagnosis was made when there was no evidence of plasma cell or lymphocytic infiltration on bone marrow aspirate and trephine biopsy specimens and if there was no clinical evidence of an underlying lymphoproliferative disorder. Computed tomography was not, however, routinely performed in all cases. Bone marrow aspirate samples in all cases, defined as MGUS, contained fewer than 5% plasma cells and fewer than 20% lymphocytes. Flow cytometry was also performed in most cases, and a diagnosis of MGUS was only made when no clonal B-cells were detected, ie, the kappa:lambda ratio did not fall outside our normal range (1.05-4.0:1). WM was defined as a clonal B-cell disorder characterized by bone marrow infil-
40 • June 2000
tration with lymphocytes, lymphoplasmacytoid cells, and plasma cells, but lacking any of the clinical, morphologic, and immunophenotypic features of other lymphoproliferative disorders. CLL and mantle-cell lymphoma were diagnosed according to standard immunophenotypic criteria, while diffuse large B-cell lymphoma, follicular lymphoma, and extranodal marginal-zone lymphoma were diagnosed on the basis of lymph node/tissue biopsies. Immunophenotype was determined in these cases by three-color flow cytometry (see below) and/or immunohistochemistry (see Table 1 for a more detailed description of the diagnostic criteria used in this analysis). Immunophenotyping was performed in 80 of the 84 (95%) cases diagnosed with B-cell lymphoproliferative disorders. Immunophenotype was determined in 69 of the 84
Roger G. Owen et al Final Pathological Diagnosis in 106 Patients with IgM Paraproteinemia No. of Patients
Median Paraprotein Concentration (g/L)
WM
60/106 (56.6%)
13.0 (range, 2-54)
MGUS
22/106 (20.8%)
6.0 (range, 3-30)
CLL
10/106 (9.4%)
3.0 (range, 3-6)
Diagnosis
Diffuse large B-cell lymphoma
5/106 (4.7%)
4.0 (range, 3-21)
Extranodal marginalzone lymphoma
3/106 (2.8%)
3.0 (range, 3-11)
Follicular lymphoma
3/106 (2.8%)
7.0 (range, 6-61)
Mantle-cell lymphoma
3/106 (2.8%)
8.0 (range, 6-10)
IgM paraproteins are demonstrable in all subtypes of peripheral (mature) B-cell disorders. Abbreviations: CLL = chronic lymphocytic leukemia; MGUS = monoclonal gammopathy of undetermined significance; WM = Waldenström's macroglobulinemia
(82%) cases by three-color flow cytometry. Briefly, B-cells were identified by virtue of their CD19 expression and side scatter characteristics and their immunophenotype was defined by using the following combination of antibodies (labeled with FITC/PE/Cy5, respectively): CD3 vs. CD3 vs. CD19, CD20 vs. CD5 vs. CD19, FMC7 vs. CD22 vs. CD19, CD11a vs. CD23 vs. CD19, CD10 vs. CD38 vs. CD19, and kappa vs. lambda vs. CD19. Immunophenotype was determined in an additional 11 cases by immunohistochemistry alone using an avidinbiotin-peroxidase technique and the following monoclonal antibodies: CD3, CD5, CD10, CD20, CD23, Bcl-2, and MIB-1. Unequivocal surface light chain restriction was demonstrated in all cases assessed by flow cytometry, and in each case the cell surface light chain corresponded to the serum paraprotein light chain. Immunophenotype was assessed by analysis of bone marrow specimens in 57 of the 60 cases of WM identified in this study (flow cytometry n = 52 and immunohistochemistry n = 5). The majority (86%) of cases analysed were characterized by a SIg+, CD19+, CD20+, CD22+, CD5-, CD10-, CD23- immunophenotype.
Results A diagnosis of WM was confirmed in 60 of the 106 (56.6%) cases assessed, while 22 (20.8%) were designated as MGUS (see Table 2). The median paraprotein concentration in these two groups was 13 g/L (range, 2-54) and 6 g/L (range, 3-30), respectively (Figure 1). In the remaining 24 cases the diagnosis was CLL (n = 10, 9.4%), diffuse large B-cell lymphoma (n = 5, 4.7%), extranodal marginal-zone lymphoma (n = 3, 2.8%), follicular lymphoma (n = 3, 2.8%), and mantle-cell lymphoma (n = 3, 2.8%). In these patients the median paraprotein concentration was 4.5 g/L (range, 3-61g/L). No cases of IgM multiple myeloma were identified in this analysis.
Figure 1 Distribution of IgM Paraproteins According to Disease Category 60
50 IgM Paraprotein Concentration (g/L)
Table 2
40
30
20
10
MGUS (n = 22)
WM (n = 60)
"OTHERS" (n = 24)*
The median IgM paraprotein concentration in patients diagnosed with WM, MGUS, and "other" B-cell lymphoproliferative disorders was 13 g/L, 6 g/L, and 4.5 g/L, respectively. There is, however, considerable overlap and defining entities on the basis of IgM concentration is clearly not appropriate. * 24 patients were diagnosed with B-cell lymphoproliferative disorders other than WM. These comprised CLL (n = 10), diffuse large B-cell lymphoma (n = 5), extranodal marginal-zone lymphoma (n = 3), follicular lymphoma (n = 3) and mantle-cell lymphoma (n = 3). Abbreviations: CLL = chronic lymphocytic leukemia; MGUS = monoclonal gammopathy of undetermined significance; WM = Waldenström's macroglobulinemia
Discussion There are currently no universally accepted diagnostic criteria for WM, a factor that has hindered progress in our understanding of the disorder.The majority of clinical studies to date have accepted the presence of an IgM paraprotein in the context of an apparently indolent lymphoproliferative disorder as sufficient evidence for a definitive diagnosis of WM.3 In this study we have reviewed the final pathological diagnosis in 106 patients with IgM paraproteinemia, and by applying the REAL/ WHO criteria (along with confirmatory immunophenotyping), we have demonstrated that IgM paraproteinemia is demonstrable in all subtypes of peripheral (mature) B-cell lymphoproliferative disorders. This is consistent with the data of Kyle and Garton who also found that a significant proportion of patients with IgM paraproteins had diagnoses other than MGUS and WM.11 In the latter study, patients were defined as having MGUS, WM, lymphoma, or "malignant lymphoproliferative disease" on the basis of their clinical features, paraprotein concentration, and the percentage of lymphocytes in the bone marrow. Fifty-six percent of patients were con-
June 2000 • 41
CPC of IgM Paraproteinemias sidered to have MGUS, 17% WM, 7% lymphoma, 5% CLL, 1% primary amyloidosis, and 14% "malignant lymphoproliferative disease." WM was, however, only diagnosed when the IgM paraprotein concentration was 30 g/L or more. Patients in whom marrow infiltration by lymphocytes and lymphoplasmacytoid cells were accompanied by less than 30 g/L of IgM paraprotein were designated as having "malignant lymphoproliferative disease." It is likely that many of these cases would have been designated WM in our study. The REAL/WHO classification criteria suggest that WM is a clinical syndrome occurring in most patients with lymphoplasmacytic lymphoma.12,13 We have recently shown that 89% of patients diagnosed with lymphoplasmacytic lymphoma have IgM paraproteins, while 4% have IgG paraproteins, and the remainder have no detectable monoclonal bands. A small minority of patients have both IgM and IgG monoclonal bands, while IgA paraproteins appear to be particularly rare in this setting.14 The prevalence and significance of IgM paraproteinemia in other B-cell lymphoproliferative disorders, however, is less well established. It has been suggested that the paraprotein concentration is useful in differentiating WM from other lymphoproliferative disorders. The French-American-British classification15 suggests that a paraprotein concentration of greater than 20 g/L is diagnostic of WM, while the Mayo Clinic group arbitrarily chose a concentration of 30 g/L to define WM in their analysis of 430 patients with IgM paraproteinemia.11 In the present study, we could not identify a paraprotein concentration that consistently differentiated WM from the other lymphoproliferative disorders. Paraprotein concentrations were indeed higher in patients with WM, but there was considerable overlap (see Figure 1). In addition, IgM paraprotein concentrations do not appear to have prognostic significance in WM6,16,17; they appear to be a continuous variable and do not correlate with the extent of bone marrow disease.18 Our analysis and that of Kyle and Garton11 also demonstrate the extreme rarity of IgM multiple myeloma despite the fact that IgM MGUS appears to be relatively common. No cases of myeloma were diagnosed in a total of 536 patients with IgM paraproteins included in these two studies. It is, therefore, more appropriate to consider IgM MGUS as occult lymphoma rather than a precursor of myeloma. Indeed, we have recently demonstrated that approximately 8% of patients with WM have a documented antecedent history of MGUS. It is clear that if IgM paraproteins are demonstrable in most peripheral B-cell disorders and that paraprotein concentrations are of limited diagnostic value in individual patients, then immunophenotypic criteria are needed for the accurate diagnosis of WM. We have recently assessed the immunophenotypic profile of 111 cases of WM (including 55 of the cases included in this analysis) by three-color flow cytometry and immunohistochemistry. We found that 90% of these cases were defined by the phenotype CD19+, CD20+, CD5-, CD10-, CD23- and that the majority of these cases were also IgD- by immunohistochemistry, while the plasma cell component was almost invariably CD138+.6 These results are in keeping with those of
42 • June 2000
Matutes et al, who also demonstrated a CD5-, CD23- immunophenotype in the majority of 25 cases of lymphoplasmacytic lymphoma assessed by flow cytometry.19 A higher incidence of CD5 and CD23 expression has, however, been demonstrated recently by Papamichael and colleagues in their analysis of 126 patients with immunocytoma assessed by immunohistochemical methods.20 The CD19 + , CD20 + , CD5 - , CD10 - , CD23 - immunophenotype is characteristic of postgerminal center B-cells21, which suggests that WM may arise from marginal-zone memory B-cells. This hypothesis is also supported by immunoglobulin variable region sequence analysis, as somatic hypermutation is demonstrable in most cases without intraclonal variation.22-25 This data suggests a possible relationship between WM and marginal-zone lymphoma, particularly of splenic and nodal types, and merits further investigation. In this regard, it is interesting to note that in their recent analysis of 124 patients with non-MALT marginal-zone lymphoma, the Lyon group found that a significant minority of their patients had IgM paraproteinemia.26 In conclusion, we have clearly demonstrated that IgM paraproteinemia is demonstrable in all subtypes of B-cell lymphoproliferative disorders and that paraprotein concentrations are of limited diagnostic value in this setting. We therefore propose that phenotypic criteria (CD19 + , CD20 + , CD5 - , CD10 - , CD23-) are used along with clinical and morphological assessment to accurately define WM in future clinical trials.
Acknowledgement R.G. Owen is supported by the Annette Fox Leukaemia Research Fund (Bradford).
References 1.
Waldenstrom J. Incipient myelomatosis or essential hyperglobulinemia with fibrinogenopenia - a new syndrome? Acta Med Scand 1944; 117:216247. 2. Dimopoulos MA, Alexanian R. Waldenström’s macroglobulinemia. Blood 1994; 83:1452-1459. 3. Dimopoulos MA, Panayiotidis P, Moulopoulos LA, et al. Waldenström’s macroglobulinemia: clinical features, complications, and management. J Clin Oncol 2000; 18:214-226. 4. Herrinton LJ, Weiss NS. Incidence of Waldenstrom’s macroglobulinemia. Blood 1993; 82:3148-3150. 5. Gertz MA, Kyle RA, Noel P. Primary systemic amyloidosis: a rare complication of immunoglobulin M monoclonal gammopathies and Waldenstrom’s macroglobulinemia. J Clin Oncol 1993; 11:914-920. 6. Owen RG, Parapia LA, Richards SJ, et al. Waldenstrom’s macroglobulinemia: development of diagnostic criteria and identification of prognostic factors. J Clin Oncol (submitted). 7. Owen RG, Lubenko A, Savage J, et al. Autoimmune thrombocytopenia in Waldenstrom’s macroglobulinemia. Am J Hematol (submitted). 8. Stone MJ. Studies on monoclonal antibodies. The specificity and binding properties of a Waldenstrom macroglobulin with anti-γ activity. J Lab Clin Med 1973; 81:393-409. 9. Stone MJ, Fedak JE. Studies on monoclonal antibodies II. Immune complex (IgM-IgG) cryoglobulinemia: the mechanism of cryoprecipitation. J Immunol 1974; 113:1377-1385. 10. Moore DF, Migliore PJ, Shullenberger CC, et al. Monoclonal macroglob-
Roger G. Owen et al ulinemia in malignant lymphoma. Ann Int Med 1970; 72:43-47. 11. Kyle RA, Garton JP. The spectrum of IgM monclonal gammopathy in 430 cases. Mayo Clin Proc 1987; 62:719-731. 12. Harris NL, Jaffe ES, Stein H, et al. A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group. Blood 1994; 84:1361-1392. 13. Harris NL, Jaffe ES, Diebold J, et al. World Health Organization classification of neoplastic diseases of the hematopoietic and lymphoid tissues: report of the Clinical Advisory Committee meeting – Airlie House, Virginia, November 1997. J Clin Oncol 1999; 17:3835-3849. 14. Owen RG, Parapia LA, Richards SJ, et al. Paraprotein status in lymphoplasmacytoid lymphoma/immunocytoma: do all cases correspond to Waldenström’s macroglobulinaemia? Brit J Haematol 1999; 105 (suppl 1): 89a (Abstract #239). 15. Bennett JM, Catovsky D, Daniel MT, et al. Proposals for the classification of chronic (mature) B and T lymphoid leukaemias. French-American-British (FAB) Cooperative Group. J Clin Pathol 1989; 42:567-584. 16. Facon T, Brouillard M, Duhamel A, et al. Prognostic factors in Waldenstrom’s macroglobulinemia: a report of 167 cases. J Clin Oncol 1993; 11:1553-1558. 17. Gobbi PG, Bettini R, Montecucco C, et al. Study of prognosis in Waldenstrom’s macroglobulinemia: a proposal for a simple binary classification with clinical and investigational utility. Blood 1994; 83:29392945. 18. Bartl R, Frisch B, Mahl G, et al. Bone marrow histology in Waldenstrom’s macroglobulinaemia. Clinical relevance of subtype recognition. Scand J Haematol 1983; 31:359-375. 19. Matutes E, Owusu-Ankormah K, Morilla R, et al. The immunological pro-
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file of B-cell disorders and proposal of a scoring system for the diagnosis of CLL. Leukemia 1994; 8:1640-1645. Papamichael D, Norton AJ, Foran JM, et al. Immunocytoma: a retrospective analysis from St. Bartholomews Hospital – 1972 to 1996. J Clin Oncol 1999; 17:2847-2853. De Wolf-Peeters C, Pittaluga S, Dierlamm J, et al. Marginal zone B-cell lymphomas including mucosa-associated lymphoid tissue type lymphoma (MALT), monocytoid B-cell lymphoma and splenic marginal zone cell lymphoma and their relation to the reactive marginal zone. Leuk Lymphoma 1997; 26:467-478. Wagner SD, Martinelli V, Luzzatto L. Similar patterns of Vkappa gene usage but different degrees of somatic mutation in hairy cell leukemia, prolymphocytic leukemia, Waldenstrom’s macroglobulinemia, and myeloma. Blood 1994; 83:3647-3653. Aoki H, Takishita M, Kosaka M, et al. Frequent somatic mutations in D and/or JH segments of Ig gene in Waldenstrom’s macroglobulinemia and chronic lymphocytic leukemia (CLL) with Richter’s syndrome but not in common CLL. Blood 1995; 85:1913-1919. Stevenson F, Sahota S, Zhu D, et al. Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes. Immunol Rev 1998; 162:247-259. Ivanovski M, Silvestri F, Pozzato G, et al. Somatic hypermutation, clonaldiversity, preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in hepatitis C virus-associated immunocytomas. Blood 1998; 91:2433-2442. Berger F, Felman P, Thieblemont C, et al. Non-MALT marginal zone B-cell lymphomas: a description of clinical presentation and outcome in 124 patients. Blood 2000; 95:1950-1956.
June 2000 • 43
Abstract IgM paraproteinemia is considered to be the major defining feature of Waldenström’s macroglobulinemia (WM), but it may also occur in other B-cell lymphoproliferative disorders. In this study we have reviewed the final pathological diagnosis of 106 patients with IgM paraproteinemia investigated in our laboratories between April 1993 and May 1999. In 22 of the 106 patients (20.8%), there was no clinical or laboratory evidence of an underlying lymphoproliferative disorder, and a diagnosis of monoclonal gammopathy of undetermined significance (MGUS) was therefore made. In 60 cases (56.6%), a diagnosis of WM was made, while in the remaining 24 patients, the final diagnosis was chronic lymphocytic leukemia (n = 10), diffuse large B-cell lymphoma (n = 5), extranodal marginal-zone lymphoma (n = 3), follicular lymphoma (n = 3), and mantle-cell lymphoma (n = 3). The median paraprotein concentration in patients with WM, MGUS, and "other" lymphoproliferative disorders was 13 g/L (range, 2-54), 6 g/L (range, 3-30), and 4.5 g/L (range, 3-61), respectively. It is clear that IgM paraproteins are demonstrable in all subtypes of peripheral B-cell disorders and, although paraprotein concentrations are generally higher in WM, there is considerable overlap. Immunophenotypic criteria are therefore essential for the accurate diagnosis of WM.
Clinical Lymphoma, Vol. 1, No. 1, 39-43, 2000 Key words: Waldenström’s macroglobulinemia, IgM paraprotein, Non-Hodgkin’s lymphoma
Introduction Waldenström’s macroglobulinemia (WM) is a chronic lymphoproliferative disorder characterized by bone marrow infiltration by a mixed population of lymphocytes, lymphoplasmacytoid cells, and plasma cells associated with IgM paraproteinemia.1-3 It accounts for approximately 2% of all hematological malignancies and has an annual incidence of six per 1,000,000 in white males, in whom the incidence is highest.2,4 The IgM paraproteinemia is considered to be the major defining feature of WM and it is responsible for many of the clinical features seen, such as hyperviscosity syndrome, cryoglobulinemia, and amyloidosis, although the latter appears to be rare.1-3,5,6 The IgM paraprotein may also have autoantibody activity, which may result in peripheral neuropathy, cold agglutinin hemolysis, immune thrombocytopenia (ITP), glomerulonephritis, acquired von Willebrand’s disease, and mixed cryoglobulinemia.2,3,6-9 IgM paraproteinemia may also, however, be seen in other B-cell lymphoproliferative disorders.10,11 In 1987, Kyle and 1Department
of Hematology, General Infirmary at Leeds of Hematology, Bradford Royal Infirmary 3Department of Immunology, General Infirmary at Leeds Submitted: Mar. 17, 2000; Revised: May 2, 2000; Accepted: May 22, 2000 Address for correspondence: R.G. Owen, MD, HMDS Laboratory, The General Infirmary at Leeds, Great George Street, Leeds, LS1 3EX Fax: 0113 392 6286; e-mail: [email protected] 2Department
Garton reviewed the diagnoses of 430 patients with IgM paraproteinemia investigated at the Mayo Clinic between 1956-1978. They diagnosed monoclonal gammopathy of undetermined significance (MGUS) in 56% and WM in 17% of cases, while the remaining cases had chronic lymphocytic leukemia (CLL), primary amyloidosis, "lymphoma," or "malignant lymphoproliferative disease."11 In this study we have attempted to further clarify this issue by utilizing the Revised European-American Lymphoma (REAL) classification12 and World Health Organization (WHO) criteria13 to reach a final diagnosis in 106 patients investigated with IgM paraproteinemia.
Patients and Methods One hundred six patients with IgM paraproteins were investigated in our regional hematopathology laboratory from April 1993 to May 1999. Sixty males and 46 females with a median age of 71 years (range, 32-91) were included in this analysis. IgM paraproteins were demonstrated and quantitated in each case using the Sebia Hydrasys system and typed by standard immunofixation techniques. The median IgM paraprotein concentration in these patients was 10 g/L (range, 261). IgMκ paraproteins were demonstrated in 78 cases (74%) while IgMλ paraproteins were demonstrated in 26 cases (25%). In the two remaining cases, both IgMκ and IgMλ paraproteins were demonstrable. The final pathological diagnosis in these two cases was MGUS.
June 2000 • 39
CPC of IgM Paraproteinemias Table 1
Diagnostic Criteria Used in the Analysis of 106 Patients with IgM Paraproteinemia
Monoclonal Gammopathy of Undetermined Significance (MGUS): - No clinical evidence of an underlying lymphoproliferative disorder - Fewer than 5% plasma cells and fewer than 20% lymphocytes on bone marrow aspirate smears - No evidence of lymphoma or plasma cell infiltration on trephine biopsy sections - No clonal B cells detected by flow cytometry* Waldenström’s macroglobulinemia (WM): - Bone marrow infiltration by lymphocytes, lymphoplasmacytoid cells, and plasma cells with a diffuse, interstitial, or nodular pattern of infiltration - B-cell immunophenotype demonstrated by three-color flow cytometry or immunohistochemistry† - Absence of any clinical, morphological, or immunophenotypic features of other lymphoproliferative disorders Chronic lymphocytic leukemia (CLL): - Clonal peripheral blood lymphocytosis with the following immunophenotypic features: weak surface immunoglobulin, CD5+, CD19+, CD20+, CD23+ Mantle-cell lymphoma: - Bone marrow infiltration by irregular/cleaved lymphoid cells with the following immunophenotype: strong surface immunoglobulin, CD5+, CD19+, CD20+, CD23-‡ Follicular lymphoma: - Lymph node biopsy showing a follicular growth pattern and mixed population of centrocytes and centroblasts§ - CD20+, CD10+ immunophenotype demonstrated by flow cytometry and/or immunohistochemistry - Bcl-2 positivity demonstrated by immunohistochemistry Diffuse large B-cell lymphoma: - Lymph node/tissue infiltration by large lymphoid cells with a high rate of cell proliferation determined by Ki-67 immunostaining - B-cell immunophenotype (CD20+) demonstrated by flow cytometry and/or immunohistochemistry Extranodal marginal-zone lymphoma: - Lymphoid infiltration at a mucosa-associated lymphoid tissue (MALT) site with prominent lymphoepithelial lesionsll - CD5-, CD10-, CD20+, CD23- immunophenotype demonstrated by immunohistochemistry * Flow cytometry was performed in 15 of the 22 cases diagnosed with MGUS. No clonal B-cell populations were seen. † Immunophenotyping was performed in 57 of the 60 cases diagnosed with WM. In the remaining three patients, a diagnosis of WM was made on the basis of interstitial bone marrow infiltration with small lymphocytes and the absence of peripheral blood and lymph node involvement. Eighty-six percent of cases assessed were characterized by a CD19+, CD20+, CD5-, CD10-, CD23- immunophenotype. ‡ Three cases of mantle-cell lymphoma were diagnosed in this study. Diagnosis in these cases was made by morphological and immunophenotypic criteria. Evidence of the t(11;14) was not sought. § In one case, a diagnosis of follicular lymphoma was made solely on the basis of paratrabecular bone marrow infiltration by small, irregular centrocytes and larger centroblasts. ll Three cases of extranodal marginal-zone lymphoma were identified in this analysis. The diagnosis was made from gastric biopsies in two cases and a lung biopsy in the remaining case. Lymphoepithelial lesions were prominent in all cases.
Pathological diagnoses were made in each case according to REAL/WHO criteria,12,13 which are summarized in Table 1. MGUS is essentially a diagnosis of exclusion, and in these cases, a diagnosis was made when there was no evidence of plasma cell or lymphocytic infiltration on bone marrow aspirate and trephine biopsy specimens and if there was no clinical evidence of an underlying lymphoproliferative disorder. Computed tomography was not, however, routinely performed in all cases. Bone marrow aspirate samples in all cases, defined as MGUS, contained fewer than 5% plasma cells and fewer than 20% lymphocytes. Flow cytometry was also performed in most cases, and a diagnosis of MGUS was only made when no clonal B-cells were detected, ie, the kappa:lambda ratio did not fall outside our normal range (1.05-4.0:1). WM was defined as a clonal B-cell disorder characterized by bone marrow infil-
40 • June 2000
tration with lymphocytes, lymphoplasmacytoid cells, and plasma cells, but lacking any of the clinical, morphologic, and immunophenotypic features of other lymphoproliferative disorders. CLL and mantle-cell lymphoma were diagnosed according to standard immunophenotypic criteria, while diffuse large B-cell lymphoma, follicular lymphoma, and extranodal marginal-zone lymphoma were diagnosed on the basis of lymph node/tissue biopsies. Immunophenotype was determined in these cases by three-color flow cytometry (see below) and/or immunohistochemistry (see Table 1 for a more detailed description of the diagnostic criteria used in this analysis). Immunophenotyping was performed in 80 of the 84 (95%) cases diagnosed with B-cell lymphoproliferative disorders. Immunophenotype was determined in 69 of the 84
Roger G. Owen et al Final Pathological Diagnosis in 106 Patients with IgM Paraproteinemia No. of Patients
Median Paraprotein Concentration (g/L)
WM
60/106 (56.6%)
13.0 (range, 2-54)
MGUS
22/106 (20.8%)
6.0 (range, 3-30)
CLL
10/106 (9.4%)
3.0 (range, 3-6)
Diagnosis
Diffuse large B-cell lymphoma
5/106 (4.7%)
4.0 (range, 3-21)
Extranodal marginalzone lymphoma
3/106 (2.8%)
3.0 (range, 3-11)
Follicular lymphoma
3/106 (2.8%)
7.0 (range, 6-61)
Mantle-cell lymphoma
3/106 (2.8%)
8.0 (range, 6-10)
IgM paraproteins are demonstrable in all subtypes of peripheral (mature) B-cell disorders. Abbreviations: CLL = chronic lymphocytic leukemia; MGUS = monoclonal gammopathy of undetermined significance; WM = Waldenström's macroglobulinemia
(82%) cases by three-color flow cytometry. Briefly, B-cells were identified by virtue of their CD19 expression and side scatter characteristics and their immunophenotype was defined by using the following combination of antibodies (labeled with FITC/PE/Cy5, respectively): CD3 vs. CD3 vs. CD19, CD20 vs. CD5 vs. CD19, FMC7 vs. CD22 vs. CD19, CD11a vs. CD23 vs. CD19, CD10 vs. CD38 vs. CD19, and kappa vs. lambda vs. CD19. Immunophenotype was determined in an additional 11 cases by immunohistochemistry alone using an avidinbiotin-peroxidase technique and the following monoclonal antibodies: CD3, CD5, CD10, CD20, CD23, Bcl-2, and MIB-1. Unequivocal surface light chain restriction was demonstrated in all cases assessed by flow cytometry, and in each case the cell surface light chain corresponded to the serum paraprotein light chain. Immunophenotype was assessed by analysis of bone marrow specimens in 57 of the 60 cases of WM identified in this study (flow cytometry n = 52 and immunohistochemistry n = 5). The majority (86%) of cases analysed were characterized by a SIg+, CD19+, CD20+, CD22+, CD5-, CD10-, CD23- immunophenotype.
Results A diagnosis of WM was confirmed in 60 of the 106 (56.6%) cases assessed, while 22 (20.8%) were designated as MGUS (see Table 2). The median paraprotein concentration in these two groups was 13 g/L (range, 2-54) and 6 g/L (range, 3-30), respectively (Figure 1). In the remaining 24 cases the diagnosis was CLL (n = 10, 9.4%), diffuse large B-cell lymphoma (n = 5, 4.7%), extranodal marginal-zone lymphoma (n = 3, 2.8%), follicular lymphoma (n = 3, 2.8%), and mantle-cell lymphoma (n = 3, 2.8%). In these patients the median paraprotein concentration was 4.5 g/L (range, 3-61g/L). No cases of IgM multiple myeloma were identified in this analysis.
Figure 1 Distribution of IgM Paraproteins According to Disease Category 60
50 IgM Paraprotein Concentration (g/L)
Table 2
40
30
20
10
MGUS (n = 22)
WM (n = 60)
"OTHERS" (n = 24)*
The median IgM paraprotein concentration in patients diagnosed with WM, MGUS, and "other" B-cell lymphoproliferative disorders was 13 g/L, 6 g/L, and 4.5 g/L, respectively. There is, however, considerable overlap and defining entities on the basis of IgM concentration is clearly not appropriate. * 24 patients were diagnosed with B-cell lymphoproliferative disorders other than WM. These comprised CLL (n = 10), diffuse large B-cell lymphoma (n = 5), extranodal marginal-zone lymphoma (n = 3), follicular lymphoma (n = 3) and mantle-cell lymphoma (n = 3). Abbreviations: CLL = chronic lymphocytic leukemia; MGUS = monoclonal gammopathy of undetermined significance; WM = Waldenström's macroglobulinemia
Discussion There are currently no universally accepted diagnostic criteria for WM, a factor that has hindered progress in our understanding of the disorder.The majority of clinical studies to date have accepted the presence of an IgM paraprotein in the context of an apparently indolent lymphoproliferative disorder as sufficient evidence for a definitive diagnosis of WM.3 In this study we have reviewed the final pathological diagnosis in 106 patients with IgM paraproteinemia, and by applying the REAL/ WHO criteria (along with confirmatory immunophenotyping), we have demonstrated that IgM paraproteinemia is demonstrable in all subtypes of peripheral (mature) B-cell lymphoproliferative disorders. This is consistent with the data of Kyle and Garton who also found that a significant proportion of patients with IgM paraproteins had diagnoses other than MGUS and WM.11 In the latter study, patients were defined as having MGUS, WM, lymphoma, or "malignant lymphoproliferative disease" on the basis of their clinical features, paraprotein concentration, and the percentage of lymphocytes in the bone marrow. Fifty-six percent of patients were con-
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CPC of IgM Paraproteinemias sidered to have MGUS, 17% WM, 7% lymphoma, 5% CLL, 1% primary amyloidosis, and 14% "malignant lymphoproliferative disease." WM was, however, only diagnosed when the IgM paraprotein concentration was 30 g/L or more. Patients in whom marrow infiltration by lymphocytes and lymphoplasmacytoid cells were accompanied by less than 30 g/L of IgM paraprotein were designated as having "malignant lymphoproliferative disease." It is likely that many of these cases would have been designated WM in our study. The REAL/WHO classification criteria suggest that WM is a clinical syndrome occurring in most patients with lymphoplasmacytic lymphoma.12,13 We have recently shown that 89% of patients diagnosed with lymphoplasmacytic lymphoma have IgM paraproteins, while 4% have IgG paraproteins, and the remainder have no detectable monoclonal bands. A small minority of patients have both IgM and IgG monoclonal bands, while IgA paraproteins appear to be particularly rare in this setting.14 The prevalence and significance of IgM paraproteinemia in other B-cell lymphoproliferative disorders, however, is less well established. It has been suggested that the paraprotein concentration is useful in differentiating WM from other lymphoproliferative disorders. The French-American-British classification15 suggests that a paraprotein concentration of greater than 20 g/L is diagnostic of WM, while the Mayo Clinic group arbitrarily chose a concentration of 30 g/L to define WM in their analysis of 430 patients with IgM paraproteinemia.11 In the present study, we could not identify a paraprotein concentration that consistently differentiated WM from the other lymphoproliferative disorders. Paraprotein concentrations were indeed higher in patients with WM, but there was considerable overlap (see Figure 1). In addition, IgM paraprotein concentrations do not appear to have prognostic significance in WM6,16,17; they appear to be a continuous variable and do not correlate with the extent of bone marrow disease.18 Our analysis and that of Kyle and Garton11 also demonstrate the extreme rarity of IgM multiple myeloma despite the fact that IgM MGUS appears to be relatively common. No cases of myeloma were diagnosed in a total of 536 patients with IgM paraproteins included in these two studies. It is, therefore, more appropriate to consider IgM MGUS as occult lymphoma rather than a precursor of myeloma. Indeed, we have recently demonstrated that approximately 8% of patients with WM have a documented antecedent history of MGUS. It is clear that if IgM paraproteins are demonstrable in most peripheral B-cell disorders and that paraprotein concentrations are of limited diagnostic value in individual patients, then immunophenotypic criteria are needed for the accurate diagnosis of WM. We have recently assessed the immunophenotypic profile of 111 cases of WM (including 55 of the cases included in this analysis) by three-color flow cytometry and immunohistochemistry. We found that 90% of these cases were defined by the phenotype CD19+, CD20+, CD5-, CD10-, CD23- and that the majority of these cases were also IgD- by immunohistochemistry, while the plasma cell component was almost invariably CD138+.6 These results are in keeping with those of
42 • June 2000
Matutes et al, who also demonstrated a CD5-, CD23- immunophenotype in the majority of 25 cases of lymphoplasmacytic lymphoma assessed by flow cytometry.19 A higher incidence of CD5 and CD23 expression has, however, been demonstrated recently by Papamichael and colleagues in their analysis of 126 patients with immunocytoma assessed by immunohistochemical methods.20 The CD19 + , CD20 + , CD5 - , CD10 - , CD23 - immunophenotype is characteristic of postgerminal center B-cells21, which suggests that WM may arise from marginal-zone memory B-cells. This hypothesis is also supported by immunoglobulin variable region sequence analysis, as somatic hypermutation is demonstrable in most cases without intraclonal variation.22-25 This data suggests a possible relationship between WM and marginal-zone lymphoma, particularly of splenic and nodal types, and merits further investigation. In this regard, it is interesting to note that in their recent analysis of 124 patients with non-MALT marginal-zone lymphoma, the Lyon group found that a significant minority of their patients had IgM paraproteinemia.26 In conclusion, we have clearly demonstrated that IgM paraproteinemia is demonstrable in all subtypes of B-cell lymphoproliferative disorders and that paraprotein concentrations are of limited diagnostic value in this setting. We therefore propose that phenotypic criteria (CD19 + , CD20 + , CD5 - , CD10 - , CD23-) are used along with clinical and morphological assessment to accurately define WM in future clinical trials.
Acknowledgement R.G. Owen is supported by the Annette Fox Leukaemia Research Fund (Bradford).
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