138 InXtroduction Oswald Steward Reeve-Irvine research center, departments of anatomy & nurobiology, nurobiology & behavior, and neurosurgery, university of california at irvine, Irvine, CA 92697 Introduction Monoclonal gammopathy of unknown signifi cance (MGUS) affects up to 2% of persons aged 50 years or over, and about 3% of those older than 70 years. The aim of this chapter is to highlight some of the features of this disease process and its relationship to aging. It will focus on (1) epidemiology, (2) biol- ogy of MGUS and the role of various cytokines in the pathophysiology, (3) correlation of the biology of MGUS with aging, (4) diagnosis and follow-up of patients with MGUS, and (5) natural history and predictors of progression in patients with MGUS. Epidemiology The monoclonal gammopathies are a group of dis- orders associated with proliferation of a single clone (monoclonal) of plasma cells. They are characterized by the secretion of an immunologically homogenous monoclonal protein (M-protein, M-component, M-spike, or paraprotein). Each M-protein consists of two heavy (H) polypeptide chains of the same class and subclass, and two light (L) chains of the same type. The heavy chains are IgG, IgM, IgA, IgD, and IgE, while the light-chain types are kappa (κ), and lambda (λ). In contrast, a polyclonal gammopa- thy is characterized by an increase in one or more heavy chains, and in both types of light chains, and is usually associated with an infl ammatory or reac- tive process [1]. The term monoclonal gammopathy of unknown signifi cance (MGUS) was fi rst coined by Biological and clinical signifi cance of monoclonal gammopathy Arati V. Rao, Harvey Jay Cohen Kyle et al., to replace the term benign monoclonal gammopathy, which was misleading because, at diagnosis, it is not known if the disease process will remain stable and asymptomatic, or evolve into symptomatic multiple myeloma (MM) [2]. The International Myeloma Working Group has defi ned MGUS as the presence of a monoclonal protein in patients without the evidence of multiple myeloma, amyloidosis, Waldenström macroglob- ulinemia (WM), or any other B-cell lymphoprolif- erative disorder. More specifi cally it is defi ned as an M-spike of Ͻ3.0 g/dL or trace or no light chains in a 24-hour urine collection, less than 10% plasma cells in the bone marrow, and no related organ or tis- sue impairment, i.e., no lytic bone lesions, and the absence of anemia, hypercalcemia, and renal insuf- fi ciency [1]. As for most cancers, especially hemato- logic malignancies, this condition demonstrates an increased incidence with age and affects up to 2% of persons у50 years and about 3% of those older than 70 years [3]. A screening study conducted in Sweden demonstrated MGUS in 0.1–0.2% of persons aged 30–49 years, 1.1–2.0% of persons 50–79 years, and in 5.7% of persons 80–89 years [4]. In a cluster of cases of MM, Kyle et al. were able to detect an M-protein in 15 of 1200 persons 50 years or older (1.25%) [3], and in France, 303 of 17 968 persons 50 years or older (1.7%) had an M-protein [5]. Crawford et al. have reported that 10% of 111 persons older than 80 years had an M-protein ranging in concentration from 0.2 to 1.8 g/dL [6]. This has also been reiterated by Cohen et al., who found that 3.6% of 816 persons 70 years or older had an M-protein [7]. As with 11 Blood Disorders in the Elderly, ed. Lodovico Balducci, William Ershler, Giovanni de Gaetano. Published by Cambridge University Press. © Cambridge University Press 2008. Monoclonal gammopathy 139 MM, the incidence of MGUS is higher in African– Americans than in whites, and in one study the prevalence of an M-protein was 8.4% in 916 African– Americans [7]. In contrast, the incidence of MGUS is only about 2.7% in elderly Japanese patients [8]. The monoclonal protein in MGUS is most commonly IgG (73%), followed by IgM (14%) and IgA (11%). The light chains in MGUS most commonly involve the κ molecules (62%) [9]. MGUS is frequently a single abnormality, but it may be associated with many other diseases, as would be expected in the elderly populations. Most common associations have been with the B-cell lymphoproliferative disorders like chronic lym- phocytic leukemia, non-Hodgkin lymphoma, and hairy-cell leukemia [10]. One prospective study showed that MGUS was detected in 1.1% of patients with solid tumors referred for systemic chemother- apy [11]. A third of patients with chronic neutrophilic leukemia, which is a rare disorder characterized by persistent leukocytosis of mature neutrophils, have an elevated M-protein [12]. It has also been seen in Gaucher’s disease [13], myelofi brosis, hepatitis C infection, HIV infection [14], rheumatoid arthri- tis [15], and other related disorders. Interestingly, MGUS has been observed after liver, renal, and bone-marrow transplantation, and in these patients the development of an M-protein correlated with the presence of a viral infection, e.g., cytomegalovi- rus infection [16,17]. The gammopathies can be further classifi ed as: • benign (IgG, IgM, IgA, IgD) • associated with malignancies that are not known to produce monoclonal proteins • idiopathic Bence-Jones proteinuria [18,19] • biclonal gammopathies [20] • triclonal gammopathies [21] Idiopathic Bence-Jones proteinuria is a condition in which patients excrete large amounts of mono- clonal light chains (Bence-Jones protein) and follow a benign course. A small series of seven patients revealed no evidence of malignant plasma-cell dis- order, and no serum M-protein, but urine light-chain excretion of Ͼ1 g/day [19]. In all these patients the plasma cell labeling index was low. After a follow-up of 7–28 years, three of the seven patients, developed MM, while two other patients developed asympto- matic MM, and evolving MM. One patient devel- oped primary amyloidosis after 12 years, and two patients continued to have stable levels of Bence- Jones proteins. The authors suggested that patients with idiopathic Bence-Jones proteinuria should be monitored regularly and indefi nitely. Of patients with a gammopathy of unknown signifi cance, 3–4% have a biclonal gammopathy, characterized by the production of two different M-proteins. This may be due to the proliferation of two different clones of plasma cells each producing an unrelated monoclonal protein, or it may result from a single clone of plasma cells producing two M-proteins. In a series of 57 biclonal gammopathy patients, the most common diagnoses were biclonal gammopathy of undetermined signifi cance (65%), multiple myeloma (16%), and lymphoproliferative disease (19%) [20]. The clinical features and response to therapy were similar to patients with monoclonal gammopathy. Of note, serum protein electrophore- sis (SPEP) may produce only a single band on the acetate strip, and the biclonal gammopathy may be recognized only by immunofi xation. Triclonal gammopathy has also been reported, and these patients may also have underlying lym- phoproliferative disorder, or a nonhematologic condition causing production of three different immunoglobulins [21]. Biology of MGUS and the role of various cytokines It is well known that germinal-center B cells uniquely modify the DNA of immunoglobulin (Ig) genes through sequential rounds of somatic hyper- mutation, antigen selection, and IgH switch recom- bination. Post-germinal-center plasmablasts can generate plasmablasts that have successfully com- pleted somatic hypermutation and IgH switching before migrating to the bone marrow, where stromal cells enable terminal differentiation into plasma 140 Arati V. Rao, Harvey Jay Cohen cells. MGUS and MM are both characterized by the accumulation of transformed plasmablasts or plasma cells in the bone marrow [22,23]. However, MGUS is less proliferative than MM, with Ͻ1% cells synthesizing DNA [24]. Gene expression profi ling data has demonstrated a higher level of cyclin D1, D2, or D3 mRNA in patients with MGUS and sub- sequently with MM, when compared to normal plasma cells [25]. This allows the plasma cells to be more susceptible to proliferative stimuli, with selec- tive expansion, after interacting with bone-marrow stromal cells that produce interleukin 6 (IL-6) and other cytokines. There is also some evidence that the Rb protein which controls the cell-cycle restriction point from G1 to S phase might be dysregulated due to methylation of p16, which can inhibit cyclinD/ CDK4 and thus prevent phosphorylation of Rb [26]. This, along with deletion of chromosome 13, may be the earliest change in MGUS that allows progres- sion to MM [24]. In addition, activating mutations of N-ras and K-ras are absent with MGUS but are seen in 30–40% patients with MM [27]. Role of IL-6, IL-6R, IL-1β, and TNF-α The function, differentiation, and survival of hematopoietic cells are governed by the presence of certain cytokines. These cytokines in turn require expression of an appropriate cellular receptor to exert their many biologic effects. The develop- ment of MGUS and MM is dependent upon differ- ent cytokines like granulocyte colony-stimulating factor (G-CSF), interferon alpha (IFN-α), leukemia inhibitory factor (LIF), IL-11, tumor necrosis fac- tor alpha (TNF-α), and IL-6. IL-6, a multifunctional cytokine, has been thoroughly investigated in MM and MGUS and may possess the most biologic and clinical signifi cance [28]. The primary function of IL-6 is to stimulate the differentiation of mature B cells into plasma cells and also to allow proliferation of plasmablasts in the bone marrow [29]. In addition, IL-6 is known to inhibit fas- and dexamethesone- induced plasma-cell apoptosis in vitro [30,31]. Initial clinical observations have suggested that high serum IL-6 levels correlate with advanced disease, aggressive disease, and chemotherapy refractoriness [32]. Multiple studies have been per- formed to demonstrate that IL-6 stimulates pro- liferation of myeloma cells in vitro, and anti-IL-6 antibodies or IL-6 antisense oligonucleotides can inhibit IL-6-stimulated growth of myeloma cells. The autocrine and paracrine functions of IL-6 in myeloma, along with IL-6 transgenic mouse mod- els, has also been studied. The expression of IL-6 receptors (IL-6R) by myeloma cells, and responses in patients treated with anti-IL-6 antibodies, have also been examined in order to study the role of IL-6 in the pathophysiology of MGUS and MM [33,34]. More recently, similar fi ndings have also been con- fi rmed in patients with MGUS. Sati et al. developed a dual-color fl uorescence in-situ hybridization (FISH) technique to investigate the expression of IL-6 mRNA in bone-marrow cells of patients with MM, with MGUS, and in healthy bone-marrow donors [35]. The IL-6 protein could be detected by direct immunofl uorescence in all plasma cells from all patients with MM, and in those with MGUS, with lower levels of expression in patients with MGUS than in those with MM. However, neither the IL-6 mRNA nor protein could be detected in normal plasma cells from healthy subjects. These data demonstrated that patients with MGUS and MM express the IL-6 mRNA, and support the hypothesis of autocrine synthesis of IL-6 in these patients. Most investigators, however, agree that the contribution of autocrine IL-6 is minimal, and there are emerging data that the paracrine secretion of IL-6 is the major factor in the pathogenesis of MGUS, MM, and other monoclonal gammopathies [36]. IL-6 production has been detected by Th2 T cells, monocytes, endothelial cells, fi broblasts, and bone- marrow stromal cells, and the latter is probably the major source of IL-6 in monoclonal gammopathies [37]. This has been confi rmed by Klein et al., who attributed the high production of IL-6 to adher- ent cells of the bone-marrow microenvironment by demonstrating a spontaneous proliferation of myeloma cells in vitro. Recombinant IL-6 was able to amplify this proliferation, and anti-IL-6 antibod- ies were able to inhibit these cells [28]. Monoclonal gammopathy 141 Of note, the signaling of IL-6 is mediated via a spe- cifi c heterodimer receptor made up of an α chain of 80 kD (IL-6R) and a β transducer chain of 130 kD (gp130). A remarkable feature of the IL-6 receptor is the agonist role of its soluble form (sIL-6R), which is able to bind IL-6 with an affi nity similar to that of membrane IL-6R. Also, the IL-6/sIL-6R complex is able to bind and activate the gp130 transducer chain [38,39]. Stasi et al. investigated the clinical sig- nifi cance of serum sIL-6R in 81 patients with MGUS, and 164 patients with MM, and found higher levels of sIL-6R in the MM patients. In a univariate analy- sis, sIL-6R was a signifi cant but weak prognostic indicator, and higher levels were associated with shorter survival [40]. The relationship of IL-6 to other cytokines like TNF-α and IL-1β has also been well studied. TNF-α and IL-1β are potent inducers of IL-6 production and play a role in paracrine secretion of IL-6 [41]. These two cytokines, especially IL-1β, are potent osteoclast- activating factors and play a role in the development of lytic lesions (Fig. 11.1). Lacy et al. performed in-situ hybridization for IL-1β using bone-marrow aspirates from 51 patients with MM, 7 with smol- dering myeloma, 21 with MGUS, and 5 healthy subjects [42]. IL-1β mRNA was detected in plasma cells from a majority of patients with MM (49 of 51) and smoldering myeloma, but only 5 of 21 patients with MGUS, and none of the normal sub- jects had any detectable IL-1β mRNA. This contrast in cytokine expression of IL-6 and IL-1β between patients with MGUS and MM has also been demon- strated by Donovan et al. [43]. TNF-α plays a role in the production of IL-6 in a dose-dependent fashion. Blade et al. mea sured serum levels of IL-6 and TNF-α in 38 healthy subjects and 100 patients with MGUS. IL-6 levels were signifi cantly higher in MGUS than in healthy controls (p Ͻ 0.0001). Similarly, TNF-α levels were signifi cantly higher in MGUS than in control populations (p ϭ 0.015) [44]. More recently, the role of adhesion molecules in the biology of myeloma has been studied [45]. Normal B cells are able to home to certain tissues due to the presence of surface adhesion molecules. Myeloma cells may express a variety of surface adhesion molecules such as NCAM (CD56), ICAM (CD54), HCAM (CD44), and others. A recent study has also demonstrated impaired osteoblastogenesis in myeloma, thought to be due to increased levels of cytokines like IL-1β, TNF-α, and IL-6 which in MGUS MM-Low LI IL-6 IL-1b MM-High LI M-protein adhesion molecules lytic bone lesions paracrine IL-6 OAF IL-1b Figure 11.1 Role of IL-1β and IL-6 in the transition of MGUS to MM. MM, multiple myeloma; MGUS, monoclonal gammopathy of unknown signifi cance; LI, labeling index; OAF, osteoclast-activating factor. Adapted from Lacy MQ et al., Blood 1999; 93: 300–5 [42]. 142 Arati V. Rao, Harvey Jay Cohen turn led to upregulation of ICAM-1 [46]. It has also been hypothesized that acquisition of NCAM expres- sion in myeloma is a malignancy-related phenom- enon. NCAM (CD56) is strongly expressed on most myeloma cells but is not found on normal plasma cells. In one study, CD56 expression in high density was present in 43 of 57 patients with untreated MM and in none of 23 patients with MGUS [47]. IL-6 has been shown to increase HCAM (CD44) gene expres- sion and cause overexpression of all CD44 variant exons [48]. It is thought that these adhesion mol- ecules play a role in cell-to-cell contact between myeloma cells and marrow stromal cells, and this maybe leads to the homing of myeloma cells to the bone marrow and to the development of osteolytic bone lesions, and may also play a crucial role in myeloma cell survival. IL-6 and bone-marrow angiogenesis There has been a study which demonstrated that vascular endothelial growth factor (VEGF) expressed and secreted by myeloma cells stimulates the expres- sion of IL-6 by microvascular endothelial cells and the bone-marrow stromal cells. In turn, IL-6 stimu- lates the expression of VEGF, which as we know is a potent stimulator of angiogenesis [49]. Numerous studies have now demonstrated that marrow angio- genesis parallels tumor progression and correlates with tumor growth and metastatic potential in mul- tiple myeloma patients. We also have evidence that bone-marrow angiogenesis progressively increases along the spectrum of plasma-cell disorders, from MGUS to advanced myeloma. This has been studied in the bone-marrow samples of 400 patients (76 with MGUS) by immunohistochemical staining for CD34 to identify microvessels, and compared to normal bone-marrow samples [50]. The median microvessel density per ϫ 400 high-power fi eld was 1.3 in controls, 3 in MGUS, 11 in newly diagnosed MM, and 20 in relapsed MM. Higher-grade angiogenesis was noted with more advanced disease, and this correlated with the bone-marrow plasma-cell percentage, bone-mar- row plasma-cell labeling index, and survival. Role of HHV-8 Human herpesvirus 8 (HHV-8), also known as Kaposi sarcoma-associated herpesvirus (KSHV), was originally described after isolation from a patient with Kaposi sarcoma [51]. In these patients it has been isolated from primary sarcoma cells as well as B cells, macrophages, and dendritic cells. HHV-8 has also been shown to be associated with systemic Castleman disease, and primary effu- sion lymphoma where it is localized to just the malignant cells. The viral genome encodes a large number of homologs of cellular genes, includ- ing genes functioning in cell regulation (cyclin D), control of apoptosis (bcl-2, death effector domain proteins), cell–cell interaction, immunoregulation, and cytokine signaling, especially IL-6 [52]. IL-6, which is considered an important growth factor for myeloma, and a biologically active homolog to human IL-6, termed vIL-6, has been identifi ed in the HHV-8 genome [53]. This vIL-6 binds to gp130 directly, suggesting that this molecule may directly activate IL-6R signal transduc tion without binding to the IL-6R α chain. HHV-8 also contains the viral homolog for interferon regulatory factor (vIRF), which has been detected in patients with multiple myeloma. Fibroblasts transfected with vIRF develop into stromal tumors when injected into nude mice, thus suggesting vIRF has properties of a viral onco- gene [54]. Rettig et al. have demonstrated vIL-6 RNA transcripts in cultured KSHV-infected bone-marrow dendritic cells, thus suggesting a role in producing paracrine stimulation of plasma-cell growth and the possibility of transformation of MGUS to MM [55]. However, a follow-up study refuted these fi ndings by using PCR analysis for multiple regions of the HHV-8 genome and serologic studies on patients with MM and found no role of HHV-8 in the etiol- ogy of MM [56]. Ablashi et al. performed serologic assays (whole-virus ELISA) to detect IgG antibody to HHV-8 in 362 patients with MGUS and 110 patients with MM. Only 7.8% of the MGUS sera contained HHV-8 antibody to lytic proteins, and no differ- ences were noted in the distribution of antibody Monoclonal gammopathy 143 to HHV-8 in sera from MGUS patients who pro- gressed to MM. The seroprevalence of HHV-8 in MGUS (7.8%), MM (5.4%), and healthy donors (5.9%) was similar, thus arguing for the lack of epi- demiologic evidence of HHV-8 in the pathogenesis of MM. Currently, it is unclear if MGUS patients with HHV-8 infection will progress and go on to develop overt MM. Relationship between aging and the development of monoclonal gammopathy The prevalence fi ndings discussed previously in the Epidemiology section suggest that there may be some fundamental changes that occur with the process of aging that make individuals more sus- ceptible to developing a monoclonal gammopathy. Animal models have provided some clues in under- standing the pathophysiology of age-related mono- clonal gammopathy. In aging C57BL/KaLwRij mice, 80% of aged animals will develop a monoclonal gammopathy that is essentially indistinguishable from an MGUS in humans [57,58]. Also, plasma-cell dyscrasias such as MGUS, MM, or WM are rarely seen in C57BL/KaLwRij mice less than two years old. It is hypothesized that these animals may have a dysregulated immune system that predisposes them to develop a monoclonal gammopathy. Radl has demonstrated his fi ndings in the C57BL/KaLwRij mice as an imbalance between a failing T-cell com- partment (due to an involuted thymus) with an oth- erwise intact B-cell compartment [57]. The loss of a balanced T-cell/B-cell dichotomy in the immune system may lead to a restriction of the B-cell reper- toire and thus to excessive B-cell clonal prolifera- tion, excessive immunoglobulin production, and ultimately to the development of a monoclonal gammopathy. More recently, Ellis et al. have demonstrated that the relative numbers of the CD30ϩ T-cell subset and levels of CD30 expression are elevated in activated lymphocytes from normal aged individuals (у60 years) and in MGUS patients, when compared to younger controls [59]. Peripheral blood lymphocytes from MGUS patients and age-matched controls produced comparable levels of IL-6 when activated with anti-CD3 plus IL-2, and costimulation with a soluble form of CD30 ligand (sCD30L/CD8alpha) augmented anti-CD3 inducible IL-6 production similarly in both groups. However, peripheral blood lymphocytes from MGUS patients also produced measurable IL-6 when activated with sCD30L/ CD8alpha alone. This capability was associated with the unique presence of CD30ϩ T cells in the peripheral blood of MGUS patients. Furthermore, a higher percentage of activated MGUS T cells express CD30 when activated by incubation with idiotype- expressing autologous serum than those activated by anti-CD3 plus IL-2. These results indicate that quantitative alterations in CD30ϩ T cells accom- pany aging and MGUS, and that these cells may con- tribute to the chronic activation of B cells though the production of IL-6. In addition to the above murine data, there is also a wealth of data to indicate that IL-6 gene expres- sion, along with tissue and serum levels of IL-6, all increase with age. As indicated before, IL-6 is the chief cytokine implicated in the development of MM [60–64]. Early observations demonstrated an age-associated rise in IL-6 in autoimmune prone mice [63]. However, subsequent studies have dem- onstrated a similar age-associated increase in IL-6 in “normal” (without any disease) mice. Similarly, an age-associated increase in IL-6 has been described in healthy older humans and in older adults with coincident age-associated diseases like Alzheimer dementia, osteoporosis, and lymphoproliferative disorders [65]. One proposed mechanism for this age-associated increase in IL-6 is the reduced infl u- ence of normally inhibiting sex steroids on endog- enous IL-6. The ability of estrogen to repress IL-6 expression has been studied in human endometrial stromal cells and from observations that menopause or oophorectomy resulted in increased IL-6 levels [66,67]. Similarly, dihydrotestosterone also inhib- its IL-6, albeit to a lesser extent than estrogen [68]. In one study orchiectomy induced bone-marrow 144 Arati V. Rao, Harvey Jay Cohen IL-6 protein and mRNA expression and led to increased replication of bone-marrow osteoclast progenitors, which was prevented by administration of IL-6-neutralizing antibody or implantation of a slow-release form of testosterone [69]. Thus it seems likely that at the time of menopause or andropause, IL-6 gene expression is not that tightly regulated, leading to inappropriate expression in some tis- sues and a rise in serum levels. This age-associated rise in IL-6 is of physiologic consequence, render- ing an individual susceptible to a number of disease processes induced by pro-infl ammatory signals, including MM osteoclast stimulation, lymphopro- liferative disorders, decreased functional status, and frailty [70]. One might hypothesize that in an older patient with MGUS, the rise in IL-6 levels may potentiate the usual molecular changes seen in an aging individual, like decreased immune surveil- lance, decreased DNA repair, telomere shortening, and decreased chromosomal stability, and thus lead to development of MM (Fig. 11.2). Diagnosis and follow-up of patients with MGUS The initial workup for a patient with MGUS should include a complete history and physical examina- tion, complete blood count, blood fi lm, serum elec- trolytes, blood urea nitrogen, serum creatinine, and calcium. High-resolution agarose gel serum protein electrophoresis (SPEP) is the recommended method for detection of an M-protein [1]. An M-protein is usually seen as a dense discrete band on the agarose gel electrophorectic strip or as a tall, narrow spike or peak in the beta or gamma regions, or rarely in the alpha 2 region of the densitometer tracing (Fig. 11.3). A polyclonal increase in the immunoglobulins will produce a broad band that is limited to the gamma region. An M-protein may be present even when the total protein concentration, beta- and gamma- globulin levels, and quantitative serum immu- noglobulin levels are all within normal limits. A small M-protein may be concealed in the normal Age Time Oncogene activation Free radicals Carcinogens Viruses Initiation MGUS ↓ Immune surveillance DNA repair Telomere shortening Microenvironment imbalance ↓ Chromosomal stability ↑ Apoptosis resistance HHV-8 activation IL-6 IL-6 Host resistance Growth factors Promotion MM Figure 11.2 Relationship between age, HHV-8, and IL-6 in the pathogenesis of MGUS and MM. Adapted from Cohen HJ et al. in Balducci L et al., eds, Comprehensive Geriatric Oncology (London: Taylor and Francis, 2004), 194–203 [71]. Monoclonal gammopathy 145 Albumin a1 a2 bg Albumin a1 a2 bg Albumin a1 a2 bg Normal Se r u m IgG-kappa Se r u m kappa Bence-Jones Protein Urine AHS G A M K L AHS G A M K L AHS G A M K L EP IFE Figure 11.3 Serum and urine monoclonal protein. EP, electrophoresis; IFE, immunofi xation. Adapted from Wu J et al., Clin Geriatr 2005; 13: 18–24 [72]. 146 Arati V. Rao, Harvey Jay Cohen beta or gamma areas and thus easily overlooked [73]. Immunofi xation should be performed when a peak or band is seen on SPEP, and this confi rms the presence of and type of M-protein [74,75]. Immunofi xation can detect a serum M-protein of 0.02 g/dL and a urine M-protein of 0.004 g/dL. Urine protein electrophoresis (UPEP) is also important in the evaluation of monoclonal gam- mopathy. Ideally, immunofi xation of a 24-hour urine sample is recommended but it can be performed on a random sample or the fi rst morning specimen. It is not uncommon for a patient to have a normal SPEP with no M-protein, but for urine immunofi xation to show a monoclonal light chain [76]. Other stud- ies might include a bone-marrow aspiration and biopsy, a radiological skeletal bone survey, quanti- tative serum immunoglobulins, and 24-hour urine collection for protein quantitation and electro- phoresis. Light chains may not be detected in the urine because of reabsorption by the proximal renal tubules. Also, there is variation in glomerular fi ltra- tion and tubular function, and this is relevant in patients with non-secretory myeloma, solitary plas- macytoma, or primary systemic amyloidosis. These issues may be circumvented by measuring serum free light chains, which has been shown to be a very sensitive method of detecting and monitoring light chains [77]. The quantitative immunoassays can detect less than 0.1 mg/dL κ and λ chains compared to 15–50 mg/dL by immunofi xation, and 50–200 mg/ dL by SPEP [78]. It is important to differentiate a patient with MGUS from one with MM or WM. Most patients with MGUS are asymptomatic, and may be diag- nosed incidentally by their primary-care physicians performing a workup for anemia or other related conditions. The size of the M-protein is helpful, and has been a matter of debate, with the International Myeloma Working Group using the level of 3 g/dL as the cutoff value [1]. Patients with MGUS do not have any signs or symptoms from related organ and tissue damage, as cited above. Most patients with MM or WM have a reduction in polyclonal or background immunoglobulins, while only 30% of patients with MGUS have a decrease in polyclonal immunoglobulins. The morphologic appearance of the bone marrow might help in differentiation, and this was illustrated in a study where bone-marrow aspirates from 154 patients were examined by blinded cytologists [79]. These patients underwent bone-marrow aspiration as part of a workup for sus- pected myeloma. The single morphologic charac- teristic that strongly differentiated MM from MGUS was the presence of large nucleoli in the plasma cells of patients with MM. Higher percentage of plasma cells (mean 48% in MM vs. 10% in MGUS), irregu- lar cytoplasmic contour of plasma cells, presence of cartwheel chromatin and vacuolization, more ani- socytosis and plasma cells in clusters were features more prominent with MM bone-marrow specimens. Of note, the popular and commonly used beta-2 microglobulin level is thought not to be useful in differentiating normal individuals from those with MGUS or with early MM [80]. Plasma cells in MM are phenotypically distinct from their normal counterparts, and this has been studied by fl ow cytometry in patients with MGUS and MM [81]. The clonal plasma cells of patients with MGUS show a phenotypic profi le similar to that of myelo- matous plasma cells (CD38ϩ, CD56ϩ, and CD19Ϫ), although the proportion of phenotypically normal plasma cells is higher in patients with MGUS than in those with myeloma. Thus, there are actually two populations of plasma cells in persons with MGUS: one is normal and polyclonal (CD38ϩ, CD56Ϫ, CD19ϩ), and the other is clonal and has an abnor- mal immunophenotype (CD38ϩ, CD56ϩ, CD19Ϫ). This study also demonstrated that the proportion of bone-marrow plasma cells that was polyclonal (as assessed by fl ow cytometry of bone-marrow aspirate with the use of four monoclonal antibodies – CD38 or CD138, CD56, CD19, and CD45) was the best single factor for distinguishing between MGUS and multiple myeloma. Only 1.5% of patients with MM had more than 3% of normal plasma cells, whereas 98% of patients with MGUS had more than 3%. Conventional cytogenetics is not very useful in differentiating MGUS from MM, because of the low number of cells in metaphase in MGUS. It is now thought that MGUS patients already have Monoclonal gammopathy 147 the chromosomal characteristics of a plasma-cell malignancy, and this was confi rmed in a study in which interphase FISH was performed on bone- marrow plasma cells of 36 patients with MGUS. Chromosomal abnormalities were identifi ed in 53% patients, with gains in chromosomes 3, 11, 7, and 18 most commonly seen [82]. The deletion of chro- mosome 13q is a clinically relevant feature in MM, and one study utilized interphase FISH to demon- strate deletion of the 13q14 locus in 45% patients with MGUS [83]. This was confi rmed by a long-term follow-up study (median follow-up 30 months) using conventional cytogenetics and interphase/ metaphase FISH in 18 asymptomatic, untreated MGUS patients [84]. Deletion of 13q14 was identi- fi ed in fi ve of these patients, and all fi ve progressed to MM 6 to 12 months after identifi cation of the 13q anomaly. The authors concluded that the extent of 13q deletion does not vary with clinical outcome, and plays a crucial role in the pathogenesis of MM by conferring a proliferative advantage to clonal plasma cells. However, these results must be interpreted with caution since transition from MGUS to MM can also occur in patients with normal karyotype, as sug- gested by two patients in the same study. Another tool in distinguishing the two conditions might be the presence and amount of circulating plasma cells that can be seen in MM, MGUS, and smoldering myeloma. However, a recent study of 327 patients with MGUS has suggested that patients who had detectable plasma cells in the peripheral blood had a shorter median progression-free survival, shorter median survival, and shorter time to initia- tion of any therapy for progressive disease [85]. Finally, the plasma-cell labeling index, which measures the synthesis of DNA, is a useful test for differentiating MGUS from MM. This was evalu- ated in one study of 80 patients (59 MM, 20 MGUS, 1 plasma-cell leukemia) where plasma-cell pro- liferation analysis was performed after bromode- oxyuridine (BRD-URD) incorporation and double immunoenzymatic labeling on cytological smears [86]. The BRD-URD is incorporated into the nucleus of cells synthesizing DNA. The plasma-cell labeling index (percentage of cells in S phase) was 0.25 for MGUS, 0.4 for stage I MM, 2.4 for stage III MM, and 3.7 for plasma-cell leukemia. While there was no correlation between the labeling index and beta-2 microglobulin levels between the MGUS and MM patients, there was a correlation of these variables within the different stages of MM. Thus, making the right diagnosis of MGUS is important, as these patients need regular follow-up in order to detect the development of the malig- nant form, i.e., MM or other related disorders. It has been suggested by Kyle & Rajkumar that if a patient has a serum M-protein value of Ͻ1.5 g/dL and no other features suggestive of a plasma-cell dyscrasia, (i.e., no anemia, hypercalcemia, renal failure) a bone-marrow examination or skeletal bone survey is not required and an SPEP should be repeated annu- ally [87]. Patients with M-protein levels between 1.5 and 2.5 g/dL who are asymptomatic should have additional studies performed, including quantita- tive immunoglobulins and 24-hour UPEP, but do not need a bone-marrow biopsy or skeletal survey. The SPEP should be repeated every 3–6 months for a year and if stable the duration between the tests can be increased to 6–12 months and then annu- ally or if any symptoms occur. If the M-protein level is Ͼ2.5 g/dL, complete workup, including quan- titative immunoglobulins, 24-hour UPEP, bone- marrow aspiration and biopsy, and skeletal bone survey, must be performed. Given the data we have from recent studies, it might be useful to check serum free light chains in patients with M-protein Ͼ1.5 mg/dL and use it along with the kappa/lambda free light chain ratio to risk-stratify patients with MGUS in order to predict progression to more malignant disease (Table 11.1) [88]. If the M-protein is IgM, a bone-marrow aspiration and biopsy is indi- cated to rule out WM or any other lymphoprolifera- tive disorder. If all the studies are normal, the SPEP can be repeated every 2–3 months for a year, and if stable can be repeated at 6- to 12-month intervals. It is unusual for a serum monoclonal protein to dis- appear during long-term follow-up. Formerly, if the M-protein remained stable for 3–5 years, the process was assumed to be benign and additional follow-up was not mandatory. However, the most recent data [...]... 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TNF-α, and IL-6 which in MGUS MM-Low LI IL-6 IL-1b MM-High LI M-protein adhesion molecules lytic bone lesions paracrine IL-6 OAF IL-1b Figure 11.1 Role of IL-1β and IL-6 in the transition of MGUS