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Clinical significance of serum triple monoclonal components: A report of 6 cases and a review of the literature
Leukemia Research 38 (2014) 166–169
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Clinical significance of serum triple monoclonal components: A report of 6 cases and a review of the literature Salvatore Guastafierro a , Antonello Sica a , Rita Rosaria Parascandola a , Maria Giovanna Ferrara a , Anna Di Martino a , Luciano Pezone b , Umberto Falcone a,∗ a b
Division of Hematology, Second University of Naples, Naples, Italy Department of General Pathology, Second University of Naples, Naples, Italy
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: Triple monoclonal component Lymphoproliferative disorders Intracellular immunofluorescence Immunofixation Serum electrophoresis Cancer Diagnosis
a b s t r a c t A serum multiple monoclonal component (MC) is very rare. We here report 6 patients with 3 MCs. The triple MC was detected in all of them by immunofixation. 2/6 patients did not present hematological or oncological associated disease, while in the remaining 4, Waldenström macroglobulinaemia (2 cases), Polycythemia Vera and non-Hodgkin lymphoma were diagnosed. Of the 49 global patients reported in the literature (6 + 43), 64.6% had a lymphoproliferative disorder and only in 3 cases there was no associated disease. Therefore, the detection of such laboratory evidence should propel physicians to a deeper investigation. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The occurrence of multiple serum monoclonal components (MCs) is rare. We recently observed that a double MC is often associated with another disease, especially a hematological malignancy [1]. Serum triple MCs are even rarer, and very little is known about their clinical significance. We here report our experience with patients who have three serum MCs. In addition, we have reviewed all of the international literature concerning patients with serum triple MCs, focusing on the associated diseases. 2. Patients and methods Six patients (3M; 3F) with 3 serum MCs were included in this study. Four of these patients (#1, #2, #3, and #4 in Table 1) were discovered during our previous study on 34 patients with serum double monoclonal components observed from January 1996 to December 2011 [1]. Their ages ranged between 67 and 88 years (median age: 77.1). All patients underwent a routine laboratory work-up, bone marrow aspiration and biopsy, and skeleton X-ray. For patients in whom the diagnosis was made more recently, cytogenetic analysis and immunofluorescence studies were also performed. It is remarkable that in each case, the single or multiple MC was suspected—or evidenced—by high-resolution serum protein electrophoresis (on
∗ Corresponding author at: Via Guglielmo Marconi, 53, 81100 Caserta, Italy. Tel.: +39 0815666827; fax: +39 0815666827. E-mail addresses: [email protected], [email protected] (U. Falcone). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.10.020
agarose gel) and subsequently confirmed and characterized by immunofixation (IF). IF was performed using an agarose film and antisera monospecific for the heavy and light chains of human immunoglobulins (anti-␥, anti-␣, anti-, anti-␦, anti-, anti, and anti-). Antisera were tested for specificity against known cases of multiple myeloma. In patients #4, 5, and 6 (Table 1), an intracellular immunofluorescence study was also performed using fluorochrome-conjugated goat antibodies specific for human , ␥, or ␣ immunoglobulin heavy chains and or light chains. Briefly, 1 × 105 cells in 0.1 ml obtained from nucleated bone marrow cells, were spun onto glass slides by cytocentrifugation, fixed in an ethanol–acetic acid solution (1:20) at 20 ◦ C for 20 min, and rehydrated in PBS. The cytological preparations were sequentially incubated with 10 L of diluted tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate-conjugated antibodies to human heavy or light chains for 20 min at room temperature. After washing, the slides were examined with fluorescence microscopy using selective filters for rhodamine or fluorescein (Table 2).
3. Results Two of the 6 considered patients (patients #1 and #5 of Table 1) did not present hematological or oncological associated diseases. Specifically, they did not have hypercalcemia, osteolytic lesions, anemia, renal failure, or other evidence of myeloma, nor did they meet the accepted criteria for the diagnosis of Waldenström macroglobulinemia (WM), amyloidosis, lymphoma, or other chronic lymphoproliferative diseases. Moreover, they did not present autoimmune or infectious disorders. For the remaining 4/6 subjects (patients #2, #3, #4, and #6 of Table 1), the associated diseases were WM, WM, polycythemia vera, and non-Hodgkin
S. Guastafierro et al. / Leukemia Research 38 (2014) 166–169 Table 1 Patients with three serum monoclonal components.
167
Table 3 Immunofluorescence analysis of bone marrow plasma cells.
Pt #
Sex (age)
Serum MCs (IF)
Urine MCs (IF)
Associated disease
Combination of class-specific antibodies
Plasma cells in the bone marrow (%)
1 2 3 4 5 6
♀ (88) ♂ (67) ♂ (70) ♂ (77) ♀ (82) ♀ (79)
IgG IgG IgM IgM IgM IgM IgM IgM IgM IgG IgG IgA IgG IgG IgA IgG IgG IgM
None None
None WM WM PV None NHL
RITC-conjugated
FITC-conjugated
Pt #4
Pt #5
Pt #6
Anti-␥ Anti-␥ Anti-␣ Anti-␥ Anti-␥ Anti-␣ Anti-␣ Anti- Anti-
Anti-␣ Anti- Anti- Anti- Anti- Anti- Anti- Anti- Anti-
0 0 0 70 30 100 <1 0 0
0 0 0 25 75 0 100 0 0
0 0 0 70 30 0 0 100 <1
+ + + + + + + + +
lymphoma, respectively. In patients #4 and #6, the 3 MCs were detected before diagnosis of the hematologic malignancy, whereas in patients #2 and #3, the two diagnoses were concomitant. Serum electrophoresis showed three different picks in the ␥-region only in one case (patient #6). In 2/6 cases (patients #1 and 2), only one localized band was detected, whereas 2 bands were observed in 3/6 patients (patients #3, 4, and 5). The triple MC was detected and characterized by IF in all 6 patients (Fig. 1A–F). The 18 MCs detected in our patients were 8 IgG, 8 IgM, and 2 IgA. There was no significant difference in the and chain distribution. Intracellular immunofluorescence staining (Table 3) demonstrated that 3 MCs were produced by 3 different plasma cell populations because there was no simultaneous detection of immunoglobulins of different classes in their cytoplasm. In patient #4, 100% of plasma cells positive for ␣ heavy chains were simultaneously positive for light chains but negative for light chains; moreover, 70% of ␥positive plasma cells reacted with anti- chain antibodies and 30% with anti- chain antibodies. In patient #5, 100% of plasma cells positive for ␣-chains were also positive for -chains but negative for -chains; in addition, 75% and 25% of ␥-positive plasma cells reacted with anti- and anti- antibodies, respectively. In patient #6, 100% of plasma cells that were -positive were simultaneously -positive, but -negative; furthermore, 70% and 30% of ␥-positive plasma cells were - and -positive, respectively. Bone marrow infiltration by plasma cells ranged between 1% and 4%. In the 2 WM patients (patients #2 and 3 in Table 1), lymphoplasmacytoid cells were also observed (13.8% and 12%, respectively). 4. Discussion
Fig. 1. (A–F) Immunofixation detecting and characterizing the triple MC in the reported patients (1–6).
The simultaneous occurrence of 3 different serum monoclonal components (MCs) in one patient is rare, and its exact incidence is not known. Pruzanski [32] reported 1 triclonal component (TC) out of 789 monoclonal gammopathies, whereas Kyle [21] reported 1 TC and 57 biclonal components out of 3447 patients with serum MCs. Most of the reported cases in the English literature (Table 4) concern a single patient, and only one author [13] described 4 cases of serum TC. To the best of our knowledge, this represents the first description of 6 consecutive cases. Four of these patients had a hematological malignancy, and in particular, 3/4
Table 2 Bone marrow plasma cells and biochemical values in patients with serum triple paraprotein. Pt #
PC (%)
Hgb (g/dl)
Crea (mg/dl)
2 MG (mg/l)
LDH (U/l)
Ca (mg/dl)
SP (g/dl)
IgG (mg/dl)
IgA (mg/dl)
IgM (mg/dl)
1 2 3 4 5 6
3.0 3.2a 1.0a 4.0 3.3 4.0
9.8 12.9 14.5 17.7 10 12.7
1.6 1.2 1.1 1.0 0.9 1.0
7.4 2.5 3.2 5 2.7 2.9
370 355 320 530 290 530
8.3 9.5 10.1 9.1 8.8 9.1
7.7 7.7 9.1 8.4 7.4 7.3
2532 646 1352 2727 822 1690
117 107 421 563 595 250
498 1275 1688 86 22 2090
a
Patients # 2 and 3 had WM and in their bone marrow also lymphoplasmacytoid cells were present (13.8%, and 12%, respectively).
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S. Guastafierro et al. / Leukemia Research 38 (2014) 166–169
Table 4 Cases reported in the international literature and associated diseases. Pt#
Ref. (Year)
Sex (age)
Serum MCs
Associated disease
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
[2] (2009) [3] (2006) [3] [4] (1998) [4] [5] (1997) [6] (1996) [7] (1996) [8] (1995) [9] (1995) [10] (1993) [11] (1993) [12] (1993) [13] (1992) [13] (1992) [13] (1992) [13] (1992) [14] (1992) [15] (1987) [16] (1987) [17] (1986) [18] (1985) [19] (1982) [20] (1981) [21] (1981) [22] (1980) [23] (1980) [24] (1979) [25] (1979) [26] (1979) [27] (1979) [28] (1978) [29] (1976) [30] (1976) [30] (1976) [31] (1975) [32] (1974) [33] (1973) [34] (1972) [34] (1972) [35] (1972) [36] (1969) [37] (1967)
♂ (64) ♀ (68) ♀ (50) ♂ (81) ♂ (75) ♂ (79) ♂ (75) ♀ (39) ♀ (?) ♂ (56) ♂ (92) ♂ (68) ♀ (64) ♀ (95) ♀ (87) ♂ (93) ♂ (78) ♀ (64) ♀ (84) ♀ (70) ♂ (50) ♀ (60)
IgG IgG IgG IgM IgM IgA IgM IgM IgG IgM IgG IgG IgM IgM IgG IgM IgG IgA IgM IgG IgM IgG IgG IgA IgM IgA IgG IgG2 IgG4 IgG1 /IgG3 hybrid IgG IgG IgA IgM IgG IgA IgM IgG IgA IgM IgG IgA IgM IgG IgG IgM IgM IgG IgM IgG IgG IgM IgG IgA IgG IgA IgA IgG IgA IgM IgM IgG IgG IgM IgG IgA IgM IgM IgG IgM IgG2 IgM/IgA hybrid IgM IgG IgA IgA IgG IgG IgG IgG IgG IgM IgG IgA IgM IgG IgG IgG IgA IgM IgG IgG IgM? IgM IgG IgA IgM IgG IgG IgG IgG IgG IgM IgM IgA IgG IgA IgM IgM IgM IgG4 IgG IgG IgA IgG IgG IgA IgG IgG IgA IgM IgM IgM IgG IgA IgM IgG IgA IgM
O. tsutsugamushi infection NHL NHL NHL NHL WM, MM NHL Teratoma; rheumatoid arthritis NHL MM Adenocarcinoma of the esophagus NHL; AL NHL Breast cancer; NHL? NHL ? ? NHL ALS-like disorder NHL AIDS? NHL; Sjögren’s syndrome; autoimmune thyroiditis NHL WM Atypical lymphoproliferative process MM MM WM; Sjögren’s syndrome Type I disgammaglobulinaemia (immunodeficiency syndrome) NHL Malignant histiocytosis Felty’s syndrome WAS NHL WM; cryoglobulinemia NHL AL Primary immunodeficiency MM MM WM WM None
? (62) ♀ (79) ♀ (64) ♂ (69) ♂ (68) ♀ (67) ♂ (5) ♀ (66) ♂ (31) ? (?) ♂? (5mos) ♂ (56) ♀ (58) ♂ (86) ♀ (62) ? (6 mos) ? (?) ? (?) ? (?) ♀ (64) ♂ (52)
had a lymphoproliferative disease. Two of the 6 patients showed no associated disease. Of the 49 (6 + 43) total reported patients, 31 (63.26%) had a lymphoproliferative disease. The most frequently reported MC association was IgG IgA IgM (12 cases). In most of the reported cases, as well as in 5/6 of our patients, only one or two of the 3 MCs were evidenced by serum protein electrophoresis. One of our patients had PV. The association of MC and chronic myeloproliferative diseases has only occasionally been reported. A retrospective study [38] comparing 164 PV and 218 TE patients with 500 healthy subjects showed no statistically significant difference in the occurrence of MC in the two groups. The occurrence of single or multiple MCs has been reported in association with several infectious diseases (e.g., HIV, HCV). Park et al. [2] reported 18 patients with scrub typhus and MG. These patients showed a serum MC during the acute phase and the early convalescence. Nine of 18 had 2 MCs, and 1/18 had 3 MCs. In 3 patients, the MC was already present before antibodies against Orientia tsutsugamushi appeared. In all patients, the MC disappeared within 7–15 weeks after therapy with doxycycline. In the patient with the serum triple MC, the disappearance of the paraproteins was observed while the titer of specific antibodies was still high. During the early phase of infection by O. tsutsugamushi, increased levels of IL-6 and IL-10 and plasma cell growth and differentiation are observed. A TC has also been described in immunodeficiency. In this case, it could be ascribed to the hyperstimulation of a small number of functionally
available plasma cell clones or to repeated infections. Regarding the source of the 3 different serum MCs, several hypotheses could be made. The MCs could result from the activity of one or more plasma cell clones. The origin from a single clone has been described in a few cases [4,12,26]. To determine the origin of these paraproteins, several techniques can be used: molecular studies, idiotypic specificity determination, intracellular immunofluorescence staining, and electron microscopy. In 6/6 of our patients, serum MCs were produced by at least 2 different plasma cell clones, as shown by the simultaneous presence of paraproteins with and light chains in each patient. In fact, in B lymphocytes, light chain gene rearrangement begins only after the light chain gene has been silenced. Thus, the simultaneous synthesis of and chains in a single cell must be considered as not possible. In 3/6 patients (patients #4, 5, and 6), intracellular immunofluorescence staining allowed us to demonstrate that the 3 serum MCs were produced by 3 different plasma cell clones. Specifically, IgG and IgA MCs (pt #4 and 5) and IgM and IgG MCs (pt #6) were produced by 2 distinct plasma cell populations that could represent two unrelated cell clones or, alternatively, 2 subclones deriving from a common precursor. In patient #1, in whom a specific study was not performed, it is conceivable that IgM and IgG were produced by 2 distinct plasma cell subclones with a common precursor, even if their origin from a single plasma cell clone frozen in the switchphase cannot be excluded. Up to 87% of double MCs with different
S. Guastafierro et al. / Leukemia Research 38 (2014) 166–169
heavy chains (HCs) but matched light chains have been reported. Without a molecular analysis, the clonal relationship is uncertain. Very recently, Tschumper et al. [39], using Ig HC variable gene PCR, observed that the two HC variable regions were clearly distinct and therefore genuinely biclonal in only 2/8 patients with biclonal multiple myeloma (HC-dissimilar/LC-matched Igs by IF). This result suggests that HC-dissimilar/LC-matched double MCs may occur more often than believed. In conclusion, based on our experience and the cases reported in the international literature, 3 serum MCs must be carefully evaluated because they rarely have been described in healthy individuals. In fact, they are more often associated with oncohematological diseases, and particularly with lymphoproliferative conditions. Therefore, even in the absence of clinical manifestations, the detection of such laboratory evidence should encourage physicians to conduct a more thorough investigation. Conflict of interest All authors have no conflict of interest to report. Acknowledgements None. No funding to declare. Contributions: S.G. and U.F. provided the conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the article, revised it critically for important intellectual content, and final approval of the version to be submitted; A.S., R.R.P. M.G.F., A.D.M., and L.P. supplied the acquisition of data; revised the manuscript critically for the final approval. References [1] Guastafierro S, Ferrara MG, Sica A, Parascandola RR, Santangelo S, Falcone U. Serum double monoclonal components and hematological malignancies: only a casual association? Review of 34 cases. Leuk Res 2012;36:1274–7. [2] Park DS, Cho JH, Lee JH, Lee KE. Clinical course of monoclonal and oligoclonal gammopathies in patients infected with Orientia tsutsugamushi. Am J Trop Med Hyg 2009;81:660–4. ´ [3] Colovic´ N, Miljic´ P, Colovic´ M, Milosevic-Jovci c´ N. Multiple M components in two patients with splenic lymphoma with villous lymphocytes. Ann Hematol 2006;85:51–4. [4] Saito N, Hirai K, Torimoto Y, Taya N, Kohgo Y, Takemori N, et al. Plural immunoglobulin synthesis in a single cell: an ultrastructural study of two cases with three M-proteins. Ultrastruct Pathol 1998;22:421–9. [5] Grosbois B, Jégo P, de Rosa H, Ruelland A, Lancien G, Gallou G, et al. Triclonal gammopathy and malignant immunoproliferative syndrome. Rev Med Interne 1997;18:470–3. [6] Gemignani F, Marchesi G, Di Giovanni G, Salih S, Quaini F, Nobile-Orazio E. Low-grade non-Hodgkin B-cell lymphoma presenting as sensory neuropathy. Eur Neurol 1996;36:138–41. [7] Lolin YI, Chow J, Wickham NW. Monoclonal gammopathy of unknown significance and malignant paraproteinemia in Hong Kong. Am J Clin Pathol 1996;106:449–56. [8] Burnett JR, Crooke MJ, Romeril KR. Triclonal gammopathy in a woman with nonHodgkin’s lymphoma. N Z Med J 1995;108:192–3. [9] Milosevic-Jovcic N, Dovezenski N, Jovanovic L, Rolovic Z, Gotic M, Radosevic N, et al. Three monoclonal IgG components, an IgG4(lambda), an IgG2(kappa) and an IgG1/IgG3 (kappa) Gm(f,b) hybrid, in a single myeloma patient. Eur J Haematol 1995;54:288–95. [10] Dupont C, Brulin C, Pinel A, Saveuse H, Lesur G, Rouveix E, et al. Triclonal gammopathy and cancer of the esophagus with PTH-like syndrome. Ann Med Interne (Paris) 1993;144:75–6. [11] Lafforgue P, Senbel E, Figarella-Branger D, Boucraut J, Horschowsky N, Pellissier JF, et al. Systemic amyloidosis AL with temporal artery involvement revealing lymphoplasmacytic malignancy in a man presenting as polymyalgia rheumatica. Ann Rheum Dis 1993;52:158–60.
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[12] Murata T, Fujita H, Harano H, Hukawa M, Kanamori H, Matsuzaki M, et al. Triclonal gammopathy (IgA kappa, IgG kappa, and IgM kappa) in a patient with plasmacytoid lymphoma derived from a monoclonal origin. Am J Hematol 1993;42:212–6. [13] Tenenbaum N, Meignan S, Vincent JP. Bi- and tri-clonal gammopathies and the elderly. Ann Med Interne (Paris) 1992;143:89–93. [14] Fujita H, Matsuzaki M, Murata T, Noguchi T, Hashimoto Y, Tanabe J, et al. Three M-components (IgG kappa, IgA kappa, IgM kappa) in a patient with non-Hodgkin’s lymphoma. Rinsho Ketsueki 1992;33:384–90. [15] Crawford J, Eye MK, Cohen HJ. Evaluation of monoclonal gammopathies in the “well” elderly. Am J Med 1987;82:39–45. [16] Köhler M, Daus H, Köhler C, Schlimmer P, Wernert N, Scheurlen PG. Lymphocytic plasmocytoid lymphoma with a three-banded gammopathy: reactivity of one of these paraproteins with cytomegalovirus. Blut 1987;54:25–32. [17] Ray RA, Schotters SB, Jacobs A, Rodgerson DO. Triclonal gammopathy in a patient with plasma cell dyscrasia. Clin Chem 1986;32:205–6. [18] Berg AR, Weisenburger DD, Linder J, Armitage JO. Lymphoplasmacytic lymphoma. Report of a case with three monoclonal proteins derived from a single neoplastic clone. Cancer 1986;57:1794–7. [19] Hoshizaki H, Katsura K, Niki Y, Sasaki M, Takaoka S. A case of lymphoplasmacytic lymphoma with 3 coexisting M-components (IgM-K, IgM-L and IgG-K) in serum. Acta Haematol Jpn 1982;45:920–8. [20] Tung E, Kuan TK, Litman GW, Wang AC. Three monoclonal immunoglobulins, an IgG2(kappa), an IgM(kappa) and an IgM/A hybrid, in one patient. I. Isolation and characterization. Immunology 1981;44:257–63. [21] Kyle RA, Robinson RA, Katzmann JA. The clinical aspects of biclonal gammopathies. Review of 57 cases. Am J Med 1981;71:999–1008. [22] Bouvet JP, Liacopoulos P, Pillot J, Banda R, Tung E, Wang AC. Three Mcomponents IgA lambda + IgG kappa n + IgG kappa h in one patient (DA): lack of shared idiotypic determinants between IgA and IgG, and the presence of an unusual kappa h chain of 30,000 M.W. J Immunol 1980;125:213–20. [23] Dworsky E, Sletten K, Harboe M, Wetteland P. Structural studies of three IgG kappa proteins from a patient with multiple myeloma. Scand J Immunol 1980;12:281–7. [24] Hopper JE, Haren JM, Kmiecik TE. Evidence for shared idiotypy expressed by the IgM, IgG, and IgA serum proteins of a patient with a complex multiple paraprotein disorder. J Immunol 1979;122:2000–6. [25] Bushell AC, Whicher JT, Yuille T. The progressive appearance of multiple urinary Bence-Jones proteins and serum paraproteins in a child with immune deficiency. Clin Exp Immunol 1979;38:64–9. [26] Krueger RG, Fair DS, Kyle RA. Monoclonal IgM, IgA and IgG in the serum of a single individual: immunofluorescence identification of cells producing the immunoglobulins. Eur J Immunol 1979;9:602–6. [27] Sibille M, Cordier JF, Pasquier J. Malignant histiocytosis with triclonal gammopathy. Nouv Presse Med 1979;8:1352. [28] Abraham GN. Human triclonal anti-IgG gammopathy. I. Iso-electric focusing characteristics of the IgG, IgA and IgM anti-IgG and their heavy and light chains. Immunology 1978;35:429–36. [29] Radl J, Dooren LH, Morell A, Skvaril F, Vossen JM, Uittenbogaart CH. Immunoglobulins and transient paraproteins in sera of patients with the Wiskott–Aldrich syndrome: a follow-up study. Clin Exp Immunol 1976;25:256–63. [30] Afifi N, Peaud PY, Rouhier D, Bothier F, Cordier JF, Creyssel R, et al. Sept observations de gammopathies biclonales et triclonales au cours d’hemopathies lymphocytaires chroniques. Lyon Med 1976;236:117–24. [31] Grubb AO, Zettervall OH. Immunochemical evidence for a common variable region in three immunoglobulin classes in the same individual. Proc Natl Acad Sci USA 1975;72:4115–8. [32] Pruzanski W, Underdown B, Silver EH, Katz A. Macroglobulinemia-myeloma double gammopathy. A study of four cases and a review of the literature. Am J Med 1974;57:259–66. [33] Danon F, Seligmann M. Serum monoclonal immunoglobulins in childhood. Arch Dis Child 1973;48:207–11. [34] Ottó S, Puskás E, Medgyesi GA, Gergely J. Diclonal and multiple gammopathies. Haematologia (Budap) 1972;6:471–87. [35] Harboe M, Hannestad K, Sletten K. Oligoclonal macroglobulinaemia. Scand J Immunol 1972;1:13–26. [36] Sanders JH, Fahey JL, Finegold I, Ein D, Reisfeld R, Berard C. Multiple anomalous immunoglobulins. Clinical, structural and cellular studies in three patients. Am J Med 1969;47:43–59. [37] Jensen K, Jensen KB, Olesen H. Three M-components in serum from an apparently healthy person. Scand J Haematol 1967;4:485–8. [38] Randi ML, Tison T, Ruzzon E, Pacquola E, Girolami A. Association of monoclonal gammopathy and polycythemia vera or essential thrombocythemia: study of a large cohort of patients. Ann Hematol 2003;82:214–7. [39] Tschumper RC, Dispenzieri A, Abraham RS, Henderson KJ, Jelinek DF. Blood Cancer J. 2013;3:e112.
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Clinical significance of serum triple monoclonal components: A report of 6 cases and a review of the literature Salvatore Guastafierro a , Antonello Sica a , Rita Rosaria Parascandola a , Maria Giovanna Ferrara a , Anna Di Martino a , Luciano Pezone b , Umberto Falcone a,∗ a b
Division of Hematology, Second University of Naples, Naples, Italy Department of General Pathology, Second University of Naples, Naples, Italy
a r t i c l e
i n f o
Article history: Received 11 June 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: Triple monoclonal component Lymphoproliferative disorders Intracellular immunofluorescence Immunofixation Serum electrophoresis Cancer Diagnosis
a b s t r a c t A serum multiple monoclonal component (MC) is very rare. We here report 6 patients with 3 MCs. The triple MC was detected in all of them by immunofixation. 2/6 patients did not present hematological or oncological associated disease, while in the remaining 4, Waldenström macroglobulinaemia (2 cases), Polycythemia Vera and non-Hodgkin lymphoma were diagnosed. Of the 49 global patients reported in the literature (6 + 43), 64.6% had a lymphoproliferative disorder and only in 3 cases there was no associated disease. Therefore, the detection of such laboratory evidence should propel physicians to a deeper investigation. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction The occurrence of multiple serum monoclonal components (MCs) is rare. We recently observed that a double MC is often associated with another disease, especially a hematological malignancy [1]. Serum triple MCs are even rarer, and very little is known about their clinical significance. We here report our experience with patients who have three serum MCs. In addition, we have reviewed all of the international literature concerning patients with serum triple MCs, focusing on the associated diseases. 2. Patients and methods Six patients (3M; 3F) with 3 serum MCs were included in this study. Four of these patients (#1, #2, #3, and #4 in Table 1) were discovered during our previous study on 34 patients with serum double monoclonal components observed from January 1996 to December 2011 [1]. Their ages ranged between 67 and 88 years (median age: 77.1). All patients underwent a routine laboratory work-up, bone marrow aspiration and biopsy, and skeleton X-ray. For patients in whom the diagnosis was made more recently, cytogenetic analysis and immunofluorescence studies were also performed. It is remarkable that in each case, the single or multiple MC was suspected—or evidenced—by high-resolution serum protein electrophoresis (on
∗ Corresponding author at: Via Guglielmo Marconi, 53, 81100 Caserta, Italy. Tel.: +39 0815666827; fax: +39 0815666827. E-mail addresses: [email protected], [email protected] (U. Falcone). 0145-2126/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.leukres.2013.10.020
agarose gel) and subsequently confirmed and characterized by immunofixation (IF). IF was performed using an agarose film and antisera monospecific for the heavy and light chains of human immunoglobulins (anti-␥, anti-␣, anti-, anti-␦, anti-, anti, and anti-). Antisera were tested for specificity against known cases of multiple myeloma. In patients #4, 5, and 6 (Table 1), an intracellular immunofluorescence study was also performed using fluorochrome-conjugated goat antibodies specific for human , ␥, or ␣ immunoglobulin heavy chains and or light chains. Briefly, 1 × 105 cells in 0.1 ml obtained from nucleated bone marrow cells, were spun onto glass slides by cytocentrifugation, fixed in an ethanol–acetic acid solution (1:20) at 20 ◦ C for 20 min, and rehydrated in PBS. The cytological preparations were sequentially incubated with 10 L of diluted tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate-conjugated antibodies to human heavy or light chains for 20 min at room temperature. After washing, the slides were examined with fluorescence microscopy using selective filters for rhodamine or fluorescein (Table 2).
3. Results Two of the 6 considered patients (patients #1 and #5 of Table 1) did not present hematological or oncological associated diseases. Specifically, they did not have hypercalcemia, osteolytic lesions, anemia, renal failure, or other evidence of myeloma, nor did they meet the accepted criteria for the diagnosis of Waldenström macroglobulinemia (WM), amyloidosis, lymphoma, or other chronic lymphoproliferative diseases. Moreover, they did not present autoimmune or infectious disorders. For the remaining 4/6 subjects (patients #2, #3, #4, and #6 of Table 1), the associated diseases were WM, WM, polycythemia vera, and non-Hodgkin
S. Guastafierro et al. / Leukemia Research 38 (2014) 166–169 Table 1 Patients with three serum monoclonal components.
167
Table 3 Immunofluorescence analysis of bone marrow plasma cells.
Pt #
Sex (age)
Serum MCs (IF)
Urine MCs (IF)
Associated disease
Combination of class-specific antibodies
Plasma cells in the bone marrow (%)
1 2 3 4 5 6
♀ (88) ♂ (67) ♂ (70) ♂ (77) ♀ (82) ♀ (79)
IgG IgG IgM IgM IgM IgM IgM IgM IgM IgG IgG IgA IgG IgG IgA IgG IgG IgM
None None
None WM WM PV None NHL
RITC-conjugated
FITC-conjugated
Pt #4
Pt #5
Pt #6
Anti-␥ Anti-␥ Anti-␣ Anti-␥ Anti-␥ Anti-␣ Anti-␣ Anti- Anti-
Anti-␣ Anti- Anti- Anti- Anti- Anti- Anti- Anti- Anti-
0 0 0 70 30 100 <1 0 0
0 0 0 25 75 0 100 0 0
0 0 0 70 30 0 0 100 <1
+ + + + + + + + +
lymphoma, respectively. In patients #4 and #6, the 3 MCs were detected before diagnosis of the hematologic malignancy, whereas in patients #2 and #3, the two diagnoses were concomitant. Serum electrophoresis showed three different picks in the ␥-region only in one case (patient #6). In 2/6 cases (patients #1 and 2), only one localized band was detected, whereas 2 bands were observed in 3/6 patients (patients #3, 4, and 5). The triple MC was detected and characterized by IF in all 6 patients (Fig. 1A–F). The 18 MCs detected in our patients were 8 IgG, 8 IgM, and 2 IgA. There was no significant difference in the and chain distribution. Intracellular immunofluorescence staining (Table 3) demonstrated that 3 MCs were produced by 3 different plasma cell populations because there was no simultaneous detection of immunoglobulins of different classes in their cytoplasm. In patient #4, 100% of plasma cells positive for ␣ heavy chains were simultaneously positive for light chains but negative for light chains; moreover, 70% of ␥positive plasma cells reacted with anti- chain antibodies and 30% with anti- chain antibodies. In patient #5, 100% of plasma cells positive for ␣-chains were also positive for -chains but negative for -chains; in addition, 75% and 25% of ␥-positive plasma cells reacted with anti- and anti- antibodies, respectively. In patient #6, 100% of plasma cells that were -positive were simultaneously -positive, but -negative; furthermore, 70% and 30% of ␥-positive plasma cells were - and -positive, respectively. Bone marrow infiltration by plasma cells ranged between 1% and 4%. In the 2 WM patients (patients #2 and 3 in Table 1), lymphoplasmacytoid cells were also observed (13.8% and 12%, respectively). 4. Discussion
Fig. 1. (A–F) Immunofixation detecting and characterizing the triple MC in the reported patients (1–6).
The simultaneous occurrence of 3 different serum monoclonal components (MCs) in one patient is rare, and its exact incidence is not known. Pruzanski [32] reported 1 triclonal component (TC) out of 789 monoclonal gammopathies, whereas Kyle [21] reported 1 TC and 57 biclonal components out of 3447 patients with serum MCs. Most of the reported cases in the English literature (Table 4) concern a single patient, and only one author [13] described 4 cases of serum TC. To the best of our knowledge, this represents the first description of 6 consecutive cases. Four of these patients had a hematological malignancy, and in particular, 3/4
Table 2 Bone marrow plasma cells and biochemical values in patients with serum triple paraprotein. Pt #
PC (%)
Hgb (g/dl)
Crea (mg/dl)
2 MG (mg/l)
LDH (U/l)
Ca (mg/dl)
SP (g/dl)
IgG (mg/dl)
IgA (mg/dl)
IgM (mg/dl)
1 2 3 4 5 6
3.0 3.2a 1.0a 4.0 3.3 4.0
9.8 12.9 14.5 17.7 10 12.7
1.6 1.2 1.1 1.0 0.9 1.0
7.4 2.5 3.2 5 2.7 2.9
370 355 320 530 290 530
8.3 9.5 10.1 9.1 8.8 9.1
7.7 7.7 9.1 8.4 7.4 7.3
2532 646 1352 2727 822 1690
117 107 421 563 595 250
498 1275 1688 86 22 2090
a
Patients # 2 and 3 had WM and in their bone marrow also lymphoplasmacytoid cells were present (13.8%, and 12%, respectively).
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Table 4 Cases reported in the international literature and associated diseases. Pt#
Ref. (Year)
Sex (age)
Serum MCs
Associated disease
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
[2] (2009) [3] (2006) [3] [4] (1998) [4] [5] (1997) [6] (1996) [7] (1996) [8] (1995) [9] (1995) [10] (1993) [11] (1993) [12] (1993) [13] (1992) [13] (1992) [13] (1992) [13] (1992) [14] (1992) [15] (1987) [16] (1987) [17] (1986) [18] (1985) [19] (1982) [20] (1981) [21] (1981) [22] (1980) [23] (1980) [24] (1979) [25] (1979) [26] (1979) [27] (1979) [28] (1978) [29] (1976) [30] (1976) [30] (1976) [31] (1975) [32] (1974) [33] (1973) [34] (1972) [34] (1972) [35] (1972) [36] (1969) [37] (1967)
♂ (64) ♀ (68) ♀ (50) ♂ (81) ♂ (75) ♂ (79) ♂ (75) ♀ (39) ♀ (?) ♂ (56) ♂ (92) ♂ (68) ♀ (64) ♀ (95) ♀ (87) ♂ (93) ♂ (78) ♀ (64) ♀ (84) ♀ (70) ♂ (50) ♀ (60)
IgG IgG IgG IgM IgM IgA IgM IgM IgG IgM IgG IgG IgM IgM IgG IgM IgG IgA IgM IgG IgM IgG IgG IgA IgM IgA IgG IgG2 IgG4 IgG1 /IgG3 hybrid IgG IgG IgA IgM IgG IgA IgM IgG IgA IgM IgG IgA IgM IgG IgG IgM IgM IgG IgM IgG IgG IgM IgG IgA IgG IgA IgA IgG IgA IgM IgM IgG IgG IgM IgG IgA IgM IgM IgG IgM IgG2 IgM/IgA hybrid IgM IgG IgA IgA IgG IgG IgG IgG IgG IgM IgG IgA IgM IgG IgG IgG IgA IgM IgG IgG IgM? IgM IgG IgA IgM IgG IgG IgG IgG IgG IgM IgM IgA IgG IgA IgM IgM IgM IgG4 IgG IgG IgA IgG IgG IgA IgG IgG IgA IgM IgM IgM IgG IgA IgM IgG IgA IgM
O. tsutsugamushi infection NHL NHL NHL NHL WM, MM NHL Teratoma; rheumatoid arthritis NHL MM Adenocarcinoma of the esophagus NHL; AL NHL Breast cancer; NHL? NHL ? ? NHL ALS-like disorder NHL AIDS? NHL; Sjögren’s syndrome; autoimmune thyroiditis NHL WM Atypical lymphoproliferative process MM MM WM; Sjögren’s syndrome Type I disgammaglobulinaemia (immunodeficiency syndrome) NHL Malignant histiocytosis Felty’s syndrome WAS NHL WM; cryoglobulinemia NHL AL Primary immunodeficiency MM MM WM WM None
? (62) ♀ (79) ♀ (64) ♂ (69) ♂ (68) ♀ (67) ♂ (5) ♀ (66) ♂ (31) ? (?) ♂? (5mos) ♂ (56) ♀ (58) ♂ (86) ♀ (62) ? (6 mos) ? (?) ? (?) ? (?) ♀ (64) ♂ (52)
had a lymphoproliferative disease. Two of the 6 patients showed no associated disease. Of the 49 (6 + 43) total reported patients, 31 (63.26%) had a lymphoproliferative disease. The most frequently reported MC association was IgG IgA IgM (12 cases). In most of the reported cases, as well as in 5/6 of our patients, only one or two of the 3 MCs were evidenced by serum protein electrophoresis. One of our patients had PV. The association of MC and chronic myeloproliferative diseases has only occasionally been reported. A retrospective study [38] comparing 164 PV and 218 TE patients with 500 healthy subjects showed no statistically significant difference in the occurrence of MC in the two groups. The occurrence of single or multiple MCs has been reported in association with several infectious diseases (e.g., HIV, HCV). Park et al. [2] reported 18 patients with scrub typhus and MG. These patients showed a serum MC during the acute phase and the early convalescence. Nine of 18 had 2 MCs, and 1/18 had 3 MCs. In 3 patients, the MC was already present before antibodies against Orientia tsutsugamushi appeared. In all patients, the MC disappeared within 7–15 weeks after therapy with doxycycline. In the patient with the serum triple MC, the disappearance of the paraproteins was observed while the titer of specific antibodies was still high. During the early phase of infection by O. tsutsugamushi, increased levels of IL-6 and IL-10 and plasma cell growth and differentiation are observed. A TC has also been described in immunodeficiency. In this case, it could be ascribed to the hyperstimulation of a small number of functionally
available plasma cell clones or to repeated infections. Regarding the source of the 3 different serum MCs, several hypotheses could be made. The MCs could result from the activity of one or more plasma cell clones. The origin from a single clone has been described in a few cases [4,12,26]. To determine the origin of these paraproteins, several techniques can be used: molecular studies, idiotypic specificity determination, intracellular immunofluorescence staining, and electron microscopy. In 6/6 of our patients, serum MCs were produced by at least 2 different plasma cell clones, as shown by the simultaneous presence of paraproteins with and light chains in each patient. In fact, in B lymphocytes, light chain gene rearrangement begins only after the light chain gene has been silenced. Thus, the simultaneous synthesis of and chains in a single cell must be considered as not possible. In 3/6 patients (patients #4, 5, and 6), intracellular immunofluorescence staining allowed us to demonstrate that the 3 serum MCs were produced by 3 different plasma cell clones. Specifically, IgG and IgA MCs (pt #4 and 5) and IgM and IgG MCs (pt #6) were produced by 2 distinct plasma cell populations that could represent two unrelated cell clones or, alternatively, 2 subclones deriving from a common precursor. In patient #1, in whom a specific study was not performed, it is conceivable that IgM and IgG were produced by 2 distinct plasma cell subclones with a common precursor, even if their origin from a single plasma cell clone frozen in the switchphase cannot be excluded. Up to 87% of double MCs with different
S. Guastafierro et al. / Leukemia Research 38 (2014) 166–169
heavy chains (HCs) but matched light chains have been reported. Without a molecular analysis, the clonal relationship is uncertain. Very recently, Tschumper et al. [39], using Ig HC variable gene PCR, observed that the two HC variable regions were clearly distinct and therefore genuinely biclonal in only 2/8 patients with biclonal multiple myeloma (HC-dissimilar/LC-matched Igs by IF). This result suggests that HC-dissimilar/LC-matched double MCs may occur more often than believed. In conclusion, based on our experience and the cases reported in the international literature, 3 serum MCs must be carefully evaluated because they rarely have been described in healthy individuals. In fact, they are more often associated with oncohematological diseases, and particularly with lymphoproliferative conditions. Therefore, even in the absence of clinical manifestations, the detection of such laboratory evidence should encourage physicians to conduct a more thorough investigation. Conflict of interest All authors have no conflict of interest to report. Acknowledgements None. No funding to declare. Contributions: S.G. and U.F. provided the conception and design of the study, acquisition of data, analysis and interpretation of data, drafting the article, revised it critically for important intellectual content, and final approval of the version to be submitted; A.S., R.R.P. M.G.F., A.D.M., and L.P. supplied the acquisition of data; revised the manuscript critically for the final approval. References [1] Guastafierro S, Ferrara MG, Sica A, Parascandola RR, Santangelo S, Falcone U. Serum double monoclonal components and hematological malignancies: only a casual association? Review of 34 cases. Leuk Res 2012;36:1274–7. [2] Park DS, Cho JH, Lee JH, Lee KE. Clinical course of monoclonal and oligoclonal gammopathies in patients infected with Orientia tsutsugamushi. Am J Trop Med Hyg 2009;81:660–4. ´ [3] Colovic´ N, Miljic´ P, Colovic´ M, Milosevic-Jovci c´ N. Multiple M components in two patients with splenic lymphoma with villous lymphocytes. Ann Hematol 2006;85:51–4. [4] Saito N, Hirai K, Torimoto Y, Taya N, Kohgo Y, Takemori N, et al. Plural immunoglobulin synthesis in a single cell: an ultrastructural study of two cases with three M-proteins. Ultrastruct Pathol 1998;22:421–9. [5] Grosbois B, Jégo P, de Rosa H, Ruelland A, Lancien G, Gallou G, et al. Triclonal gammopathy and malignant immunoproliferative syndrome. Rev Med Interne 1997;18:470–3. [6] Gemignani F, Marchesi G, Di Giovanni G, Salih S, Quaini F, Nobile-Orazio E. Low-grade non-Hodgkin B-cell lymphoma presenting as sensory neuropathy. Eur Neurol 1996;36:138–41. [7] Lolin YI, Chow J, Wickham NW. Monoclonal gammopathy of unknown significance and malignant paraproteinemia in Hong Kong. Am J Clin Pathol 1996;106:449–56. [8] Burnett JR, Crooke MJ, Romeril KR. Triclonal gammopathy in a woman with nonHodgkin’s lymphoma. N Z Med J 1995;108:192–3. [9] Milosevic-Jovcic N, Dovezenski N, Jovanovic L, Rolovic Z, Gotic M, Radosevic N, et al. Three monoclonal IgG components, an IgG4(lambda), an IgG2(kappa) and an IgG1/IgG3 (kappa) Gm(f,b) hybrid, in a single myeloma patient. Eur J Haematol 1995;54:288–95. [10] Dupont C, Brulin C, Pinel A, Saveuse H, Lesur G, Rouveix E, et al. Triclonal gammopathy and cancer of the esophagus with PTH-like syndrome. Ann Med Interne (Paris) 1993;144:75–6. [11] Lafforgue P, Senbel E, Figarella-Branger D, Boucraut J, Horschowsky N, Pellissier JF, et al. Systemic amyloidosis AL with temporal artery involvement revealing lymphoplasmacytic malignancy in a man presenting as polymyalgia rheumatica. Ann Rheum Dis 1993;52:158–60.
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