**5. Cytogenetic studies on MGUS and SMM**

MGUS, SMM and MM present common chromosomal abnormalities [46-49] whose prevalence and relative association between these diagnostic groups have been controversial for years. The development of new techniques and methodologies has helped to define new biomarkers and elucidate the pathogenetic mechanisms of progression, characterized as a multistep process from the precursor state to myeloma.

The first step in the pathogenesis is likely an abnormal response to antigenic stimulation, mediated possibly by aberrant expression of toll-like receptors and overexpression of inter‐ leukin (IL) 6 receptors and IL-1β. This then results in the development of primary cytogenetic abnormalities, either hyperdiploidy or immunoglobulin heavy chain translocations [36]. Hyperdiploid tumors, which include about 50% of MM tumors, often have multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21; also, a substantially lower prevalence of immunoglobulin heavy chain translocations and monosomy of chromosome 13 compared with nonhyperdiploid tumors. Trisomies of these same chromosomes also occur in premalig‐ nant MGUS tumors [47].

(1,860 studied patients). The t(14;20) patients had a short median survival of only 14.4 months [50]. It has been determined that these three translocations produce cyclin D2 enhance‐

D group cyclin Directly

D group cyclin Directly

D group cyclin Indirectly

MAF translocation

MAF translocation

**MGUS:** Monoclonal gammopathy of undetermined significance; **SMM:** Smoldering multiple myeloma

**Table 2.** Translocations into the immunoglobulin heavy chain locus in MGUS and SMM patients

group

group

**Group Deregulated Gene Cell Level Consequence**

CCND1 Enhance cyclin D1

Monoclonal Gammopathy of Undetermined Significance

CCND3 Enhance cyclin D3

FGFR-3 and MMSET Enhance cyclin D2

c-MAF upregulation Enhance cyclin D2

MAFB upregulation Enhance cyclin D2

(normally B-cells express cyclin D2 and cyclin D3 but

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not cyclin D1)

Using interphase FISH, Chiecchio et al [50] performed a study to evaluate chromosome 13 deletion (delta 13), deletion of TP53 (17p13), ploidy status and immunoglobulin heavy chain translocations. They found that 50% of MGUS patients carried one of the primary immuno‐ globulin heavy chain translocations and the remaining patients displayed a hyperdiploid karyotype. Thus 72/189 (42%) MGUS, 70/127 (63%) SMM, and 223/338 (57%) of MM cases were hyperdiploid. When the individual incidences of the specific translocations were compared, only t(4;14) was significantly less frequent in MGUS. The authors propose that ploidy status and immunoglobulin heavy chain rearrangements were early events delineating different pathogenic pathways [50]. The study revealed a significantly lower frequency of delta 13 in the pre-malignant conditions than in MM. Delta 13 was rare in MGUS (25%) and SMM (34%) compared to MM (47%).Translocations directly involve cyclin D genes (CCND1 and CCND3) suggesting a possible role of delta 13 in the progression of the disease, specifically in these genetic sub-groups [50]. In MGUS, the greatest variation in the proportion of abnormal plasma cells carrying the abnormality was seen for delta 13 and 16q23 deletion. The presence and time of occurrence of delta 13 depend on the presence of specific concurrent abnormalities: earlier

ment (Table 2).

**Translocation**

t(11;14)(q13;q32)

t(6;14)(p21;q32)

t(4;14)(p16;q32)

t(14;16)(q32;q23)

t(14;20)(q32;q11) (5% of MGUS patients)

(50%) [50]

[50-53]

**(Prevalence %) [References]**

(15%-25% of MGUS/SMM patients)

(1% of MGUS/SMM patients) [50]

(3%-5% of MGUS/SMM patients) [53, 55]

(0%-1.5% of SMM patients) [50, 55]

(2%-5% of MGUS patients) (13% of SMM patients) [48, 52-54]

IgH translocated MGUS

It has been well established that each translocation subgroup found in MM tumors is associated with deregulation of a D group cyclin either directly, such as occurs with the t(11;14) (cyclin D1) and t(6;14) (cyclin D3) or indirectly, such as occurs with the t(4;14) or in the MAF translo‐ cation group [47]. All these translocations have also been reported in MGUS (Table 2).

The first studies that showed structural chromosomal changes in MGUS and performed fluorescence *in situ* hybridization experiments (FISH) found 14q32 and 13q14 abnormalities [51, 52]. Subsequent studies have determined that approximately 50% of SMM show primary translocations involving the immunoglobulin heavy chain locus leading to the dysregulation of oncogenes including the Cyclin D, FGFR3/MMSET and MAF genes [46, 48, 53] (see Table 2). There is evidence of an immunoglobulin light chain-λ translocation in MGUS associated with a prevalence of 10% in MGUS/SMM [53].

Ross et al [55] found that cases characterized by t(4;14), t(14;16), particularly the t(14;20), can be stable as either MGUS or SMM for years before progression occurs. It has been shown that t(4;14), t(14;16) and t(14;20) translocations are associated with a poor prognosis in MM


**MGUS:** Monoclonal gammopathy of undetermined significance; **SMM:** Smoldering multiple myeloma

**Table 2.** Translocations into the immunoglobulin heavy chain locus in MGUS and SMM patients

between groups. On the other hand, the Salamanca model is a superior model, in particular, to identify a truly high-risk MGUS population; however, its main disadvantages are invasive‐

The biological events related to progression from normal plasma cells to MM precursor disease and to MM involve many overlapping oncogenic steps that differently affect each individual [42]. Several authors discuss the very early and partially overlapping molecular pathogenic events that are shared by MGUS, and how they are associated to progression at the MGUS to

MGUS, SMM and MM present common chromosomal abnormalities [46-49] whose prevalence and relative association between these diagnostic groups have been controversial for years. The development of new techniques and methodologies has helped to define new biomarkers and elucidate the pathogenetic mechanisms of progression, characterized as a multistep

The first step in the pathogenesis is likely an abnormal response to antigenic stimulation, mediated possibly by aberrant expression of toll-like receptors and overexpression of inter‐ leukin (IL) 6 receptors and IL-1β. This then results in the development of primary cytogenetic abnormalities, either hyperdiploidy or immunoglobulin heavy chain translocations [36]. Hyperdiploid tumors, which include about 50% of MM tumors, often have multiple trisomies involving chromosomes 3, 5, 7, 9, 11, 15, 19, and 21; also, a substantially lower prevalence of immunoglobulin heavy chain translocations and monosomy of chromosome 13 compared with nonhyperdiploid tumors. Trisomies of these same chromosomes also occur in premalig‐

It has been well established that each translocation subgroup found in MM tumors is associated with deregulation of a D group cyclin either directly, such as occurs with the t(11;14) (cyclin D1) and t(6;14) (cyclin D3) or indirectly, such as occurs with the t(4;14) or in the MAF translo‐ cation group [47]. All these translocations have also been reported in MGUS (Table 2).

The first studies that showed structural chromosomal changes in MGUS and performed fluorescence *in situ* hybridization experiments (FISH) found 14q32 and 13q14 abnormalities [51, 52]. Subsequent studies have determined that approximately 50% of SMM show primary translocations involving the immunoglobulin heavy chain locus leading to the dysregulation of oncogenes including the Cyclin D, FGFR3/MMSET and MAF genes [46, 48, 53] (see Table 2). There is evidence of an immunoglobulin light chain-λ translocation in MGUS associated

Ross et al [55] found that cases characterized by t(4;14), t(14;16), particularly the t(14;20), can be stable as either MGUS or SMM for years before progression occurs. It has been shown that t(4;14), t(14;16) and t(14;20) translocations are associated with a poor prognosis in MM

ness (it requires a bone marrow aspirate), technical complexity and high cost.

**5. Cytogenetic studies on MGUS and SMM**

process from the precursor state to myeloma.

116 Multiple Myeloma - A Quick Reflection on the Fast Progress

with a prevalence of 10% in MGUS/SMM [53].

MM transition [43-45].

nant MGUS tumors [47].

(1,860 studied patients). The t(14;20) patients had a short median survival of only 14.4 months [50]. It has been determined that these three translocations produce cyclin D2 enhance‐ ment (Table 2).

Using interphase FISH, Chiecchio et al [50] performed a study to evaluate chromosome 13 deletion (delta 13), deletion of TP53 (17p13), ploidy status and immunoglobulin heavy chain translocations. They found that 50% of MGUS patients carried one of the primary immuno‐ globulin heavy chain translocations and the remaining patients displayed a hyperdiploid karyotype. Thus 72/189 (42%) MGUS, 70/127 (63%) SMM, and 223/338 (57%) of MM cases were hyperdiploid. When the individual incidences of the specific translocations were compared, only t(4;14) was significantly less frequent in MGUS. The authors propose that ploidy status and immunoglobulin heavy chain rearrangements were early events delineating different pathogenic pathways [50]. The study revealed a significantly lower frequency of delta 13 in the pre-malignant conditions than in MM. Delta 13 was rare in MGUS (25%) and SMM (34%) compared to MM (47%).Translocations directly involve cyclin D genes (CCND1 and CCND3) suggesting a possible role of delta 13 in the progression of the disease, specifically in these genetic sub-groups [50]. In MGUS, the greatest variation in the proportion of abnormal plasma cells carrying the abnormality was seen for delta 13 and 16q23 deletion. The presence and time of occurrence of delta 13 depend on the presence of specific concurrent abnormalities: earlier when t(4;14) or t(14;16) was present, later with t(14;20), and even later with t(11;14) or t(6;14).This data suggests a possible role of delta 13 in the transition from MGUS to MM specifically in cases with t(11;14) or t(6;14). Chromosome 13 deletion on its own probably does not affect prognosis [50].

of clonal plasma cells carrying a given abnormality supporting the hypothesis that the number of genetically abnormal plasma cell increases from high-risk SMM to active MM [49]. In a later study López-Corral et al [64] have performed for the first time a comprehensive high-resolu‐ tion analysis of genomic imbalances by high-density 6.0 S SNP-array in 20 MGUS, 20 SMM and 34 MM patients to search for the genetic lesions that may be involved in the transformation from MGUS to MM. Their results showed a progressive increase in the incidence of copy number abnormalities from MGUS to SMM and to MM. The study shows for the first time the different copy number and loss of heterozygosity profiles present at three stages of monoclonal gammopathy evolution: MGUS, SMM and MM. There were significantly more copy number

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119

alterations in MM than in MGUS patients, values for SMM being intermediate [64].

ability of the tumor cell to interact with the bone marrow microenvironment [66].

[65] (see Fig. 1).

**6. Clinical management**

In summary, it has been proposed that the pathogenesis of MGUS and MM can be considered as occurring in three phases [6]. First, partially overlapping genetic events common to MGUS and MM include at a minimum primary immunoglobulin heavy chain translocations, hyper‐ diploidy, and del13 that lead directly or indirectly to dysregulation of a CCND gene; second, the transition from MGUS to MM is associated with increased MYC expression and sometimes K-RAS mutations, but can also include del13 in t(11;14) tumors; third, additional progression of the MM tumor seems to be associated with other events. For example, increased proliferation and genomic instability, and decreased dependence on the bone marrow microenvironment, sometimes including extramedullary spread of disease, can be associated with late MYC rearrangements that often involve an immunoglobulin locus, activating mutations of the nuclear factor-κB pathway, deletion or mutation of TP53, and inactivation of p18INK4c or RB

As mentioned above, the UK Myeloma Forum and the Nordic Myeloma Study Group have proposed guidelines for the management of MGUS [28]. They suggest that is essential that patients should be monitored not only by laboratory testing but also clinically. Low risk patients (serum IgG <1.5 g/dL; IgA or IgM <1.0 g/dL; normal free light chain ratio in the absence of symptoms such as anemia or renal dysfunction) can be monitored in the primary-care setting

Taking into account that the majority of MM plasma cell are quiescent, it has been suggested that the growth of the tumor is restricted to a specialized subpopulation of cells [43]. In this sense, the bone marrow microenvironment plays an essential role in the pathogenesis of MM. The bone marrow microenvironment in which MGUS and MM cells live is composed of extracellular matrix and different types of cells, e.g., stromal cells, osteoclasts, osteoblasts, immune cells (T lymphocytes, dendritic cells), other hematopoietic, cells and their precursors, and vascular endothelial cells. Reciprocal positive and negative interactions among these cells are mediated by a variety of adhesion molecules, cytokines, and receptors [65]. MAF translo‐ cations dysregulate expression of a MAF transcription factor that causes increased expression of many genes, including CCND2 and adhesion molecules that are thought to enhance the

We have treated previously in this chapter, that MGUS progresses to MM at annual frequency of 1% [2], however little is known about the proportion of patients whose MM has evolved from this precursor condition. Zhan F et al [56] developed a gene-expression profiling study in which 52 genes differentially expressed in MGUS and MM identifying and validating a MGUS-like MM with favorable clinical features and longer survival.

Point mutations, such as N-RAS, K-RAS, MYC up-regulation, and gain or loss of chromosome 1q or 1p, also seem to correlate with disease progression from myeloma precursor disease, MGUS and SMM [57]. Rasmussen et al [58] found a high prevalence of activating RAS mutations in MM (31%) compared with MGUS (5%) and suggest that these mutations may facilitate the transition from MGUS to MM in a subset of patients. Only N-RAS mutation was found in MGUS. At present, RAS mutations are the major genetic difference between MGUS and MM [43].

In a case report, Chiecchio et al [59] describe the clinicopathological and genetic findings of a young patient initially diagnosed with SMM: loss of 1p and a rearrangement of MYC were first observed in a small population of plasma cells one year prior to the clinical diagnosis of MM, but these subclones increased rapidly in size to become the major population suggesting that they were directly involved in the transformation [59].

MicroRNA is a novel class of short non-coding RNA molecules regulating a wide range of cellular functions through translational repression of their target genes. Recently, epigenetic dysregulation of tumor-suppressor microRNA genes by promoter DNA methylation has been implicated in human cancers, including MM [60]. It has been reported that MGUS and MM patients seem to upregulate miR-21, miR-106b, miR-181a, and miR-181b; which are microRNA involved in B-cell and T-cell lymphocyte differentiation as well as oncogene regulation [61]. Recently, Jones et al [62] have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for MM. The combination of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients [62].

In the progression process to malignant condition it also seems to be important the proportion of clonal plasma cell with specific genetic abnormalities in every diagnostic group. In fact, López-Corral et al [63] observed a significant difference in MGUS compared with SMM, and in SMM compared with MM, suggesting that the progression from MGUS to SMM and eventually to MM involves a clonal expansion of genetically abnormal plasma cell. This result was found for immunoglobulin heavy chain translocations, 13q and 17p deletions, and 1q gains. In other recent study, López-Corral et al [49] analyzed the genomic characteristics by FISH, Single-nucleotide polymorphism arrays and gene expression profile finding that the overexpression of four SNORD genes (SNORD25, SNORD27, SNORD30 and SNORD31) was correlated with shorter time progression to symptomatic MM. However, they failed to find chromosomal lesions associated to risk of progression, observing an increase in the proportion of clonal plasma cells carrying a given abnormality supporting the hypothesis that the number of genetically abnormal plasma cell increases from high-risk SMM to active MM [49]. In a later study López-Corral et al [64] have performed for the first time a comprehensive high-resolu‐ tion analysis of genomic imbalances by high-density 6.0 S SNP-array in 20 MGUS, 20 SMM and 34 MM patients to search for the genetic lesions that may be involved in the transformation from MGUS to MM. Their results showed a progressive increase in the incidence of copy number abnormalities from MGUS to SMM and to MM. The study shows for the first time the different copy number and loss of heterozygosity profiles present at three stages of monoclonal gammopathy evolution: MGUS, SMM and MM. There were significantly more copy number alterations in MM than in MGUS patients, values for SMM being intermediate [64].

Taking into account that the majority of MM plasma cell are quiescent, it has been suggested that the growth of the tumor is restricted to a specialized subpopulation of cells [43]. In this sense, the bone marrow microenvironment plays an essential role in the pathogenesis of MM. The bone marrow microenvironment in which MGUS and MM cells live is composed of extracellular matrix and different types of cells, e.g., stromal cells, osteoclasts, osteoblasts, immune cells (T lymphocytes, dendritic cells), other hematopoietic, cells and their precursors, and vascular endothelial cells. Reciprocal positive and negative interactions among these cells are mediated by a variety of adhesion molecules, cytokines, and receptors [65]. MAF translo‐ cations dysregulate expression of a MAF transcription factor that causes increased expression of many genes, including CCND2 and adhesion molecules that are thought to enhance the ability of the tumor cell to interact with the bone marrow microenvironment [66].

In summary, it has been proposed that the pathogenesis of MGUS and MM can be considered as occurring in three phases [6]. First, partially overlapping genetic events common to MGUS and MM include at a minimum primary immunoglobulin heavy chain translocations, hyper‐ diploidy, and del13 that lead directly or indirectly to dysregulation of a CCND gene; second, the transition from MGUS to MM is associated with increased MYC expression and sometimes K-RAS mutations, but can also include del13 in t(11;14) tumors; third, additional progression of the MM tumor seems to be associated with other events. For example, increased proliferation and genomic instability, and decreased dependence on the bone marrow microenvironment, sometimes including extramedullary spread of disease, can be associated with late MYC rearrangements that often involve an immunoglobulin locus, activating mutations of the nuclear factor-κB pathway, deletion or mutation of TP53, and inactivation of p18INK4c or RB [65] (see Fig. 1).

## **6. Clinical management**

when t(4;14) or t(14;16) was present, later with t(14;20), and even later with t(11;14) or t(6;14).This data suggests a possible role of delta 13 in the transition from MGUS to MM specifically in cases with t(11;14) or t(6;14). Chromosome 13 deletion on its own probably does

We have treated previously in this chapter, that MGUS progresses to MM at annual frequency of 1% [2], however little is known about the proportion of patients whose MM has evolved from this precursor condition. Zhan F et al [56] developed a gene-expression profiling study in which 52 genes differentially expressed in MGUS and MM identifying and validating a

Point mutations, such as N-RAS, K-RAS, MYC up-regulation, and gain or loss of chromosome 1q or 1p, also seem to correlate with disease progression from myeloma precursor disease, MGUS and SMM [57]. Rasmussen et al [58] found a high prevalence of activating RAS mutations in MM (31%) compared with MGUS (5%) and suggest that these mutations may facilitate the transition from MGUS to MM in a subset of patients. Only N-RAS mutation was found in MGUS. At present, RAS mutations are the major genetic difference between MGUS

In a case report, Chiecchio et al [59] describe the clinicopathological and genetic findings of a young patient initially diagnosed with SMM: loss of 1p and a rearrangement of MYC were first observed in a small population of plasma cells one year prior to the clinical diagnosis of MM, but these subclones increased rapidly in size to become the major population suggesting

MicroRNA is a novel class of short non-coding RNA molecules regulating a wide range of cellular functions through translational repression of their target genes. Recently, epigenetic dysregulation of tumor-suppressor microRNA genes by promoter DNA methylation has been implicated in human cancers, including MM [60]. It has been reported that MGUS and MM patients seem to upregulate miR-21, miR-106b, miR-181a, and miR-181b; which are microRNA involved in B-cell and T-cell lymphocyte differentiation as well as oncogene regulation [61]. Recently, Jones et al [62] have developed a biomarker signature using microRNAs extracted from serum, which has potential as a diagnostic and prognostic tool for MM. The combination

In the progression process to malignant condition it also seems to be important the proportion of clonal plasma cell with specific genetic abnormalities in every diagnostic group. In fact, López-Corral et al [63] observed a significant difference in MGUS compared with SMM, and in SMM compared with MM, suggesting that the progression from MGUS to SMM and eventually to MM involves a clonal expansion of genetically abnormal plasma cell. This result was found for immunoglobulin heavy chain translocations, 13q and 17p deletions, and 1q gains. In other recent study, López-Corral et al [49] analyzed the genomic characteristics by FISH, Single-nucleotide polymorphism arrays and gene expression profile finding that the overexpression of four SNORD genes (SNORD25, SNORD27, SNORD30 and SNORD31) was correlated with shorter time progression to symptomatic MM. However, they failed to find chromosomal lesions associated to risk of progression, observing an increase in the proportion

of miR-1246 and miR-1308 can distinguish MGUS from myeloma patients [62].

MGUS-like MM with favorable clinical features and longer survival.

that they were directly involved in the transformation [59].

not affect prognosis [50].

118 Multiple Myeloma - A Quick Reflection on the Fast Progress

and MM [43].

As mentioned above, the UK Myeloma Forum and the Nordic Myeloma Study Group have proposed guidelines for the management of MGUS [28]. They suggest that is essential that patients should be monitored not only by laboratory testing but also clinically. Low risk patients (serum IgG <1.5 g/dL; IgA or IgM <1.0 g/dL; normal free light chain ratio in the absence of symptoms such as anemia or renal dysfunction) can be monitored in the primary-care setting

recommended tests for monitoring include serum protein electrophoresis, serum total im‐ munoglobulin, complete blood count, creatinine, urea, electrolytes and serum calcium. In addition, it should be evaluated using bone marrow cytogenetic and FISH with bone imaging studies. Nevertheless, it is important to highlight that sometimes it will be neces‐ sary to perform Magnetic Resonance Imaging or Positron Emission Tomography-Comput‐ ed Tomography, instead of traditional x-rays. Patients with unexplained anemia or kidney failure should be evaluated with a full bone scan that also include cytogenetic and FISH. Korde et al [57] reported that is critical to recognize that in a disease such as MM, where defining criteria rely on the presence or absence of end-organ damage, diag‐ nosis is only as good as the tools and technology able to detect end-organ damage. For instance, in SMM or high-risk MGUS patients suspicious to harbor bone disease, imaging evaluation may be better served by obtaining magnetic resonance imaging or Positron Emission Tomography-Computed Tomography rather than traditional skeletal surveys. International Myeloma Working Group members recommend for intermediate-risk and high-risk MGUS patients should have a bone marrow aspirate and biopsy with both con‐ ventional cytogenetics and FISH [32]. If available, a plasma cell labeling index and a search for circulating plasma cells in the peripheral blood using flow cytometry are use‐ ful. Patients with IgM isotype should have a computational tomography scan of the ab‐ domen since asymptomatic retroperitoneal lymph nodes may be present. If there is evidence of MM or Waldeström macroglobulinemia, lactate dehydrogenase, 2-microglo‐ bulin, and C-reactive protein levels should be measured. If the results of these tests are satisfactory, International Myeloma Working Group recommend patients should be fol‐ lowed with serum protein electrophoresis and complete blood cell count in 6 months and

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In clinical practice, patients with MGUS are followed clinically without treatment until progression. However, the existence of easily identifiable precursor states represents an opportunity for chemoprevention [67]. However, it must be weighed that benefits achieved by treating a precursor state is greater than a potential for therapeutic toxicity. Recently, Korde

Bhattacharyya et al [68] reported a clinic case of IgM-MGUS associated with cryoglobulinemia and cold agglutinin disease, which was treated with immunotherapy and was successful (Table 3). Immunochemotherapy, consisting of rituximab (375 mg/m2, day 1), fludarabine (25 mg/m2, days 2-4), and cyclophosphamide (250 mg/m2, days 2-4), was administered every 4 weeks up to three times as a first-line treatment followed by three cycles of monthly rituximab treatment. Extensive skin lesions with livedo reticularis entirely disappeared prior to initiation

Pepe et al [69] studied 100 patients affected by MGUS, grouped according to the presence (group A, 50 patients) or absence (group B) of vertebral fractures and/or osteoporosis. Group A was treated with alendronate (70 mg/weekly) plus calcium and cholecalciferol for 18

et al [57] revised early treatment strategies for MGUS and SMM.

of the second cycle in association with the declined serum level of IgM.

then annually for life [32].

**7. Management**

**Figure 1.** Model for molecular pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). TR1, the initial transition to a recognizable tumor involves two mostly non-overlapping path‐ ways (IgH translocations versus multiple trisomies) that include primary events associated with dysregulated cyclin D expression in MGUS and MM. TR2, the transition from MGUS to MM is associated with increase MYC expression and sometimes with activating mutations of K-RAS or chromosome 13 deletion. Early and late progression events for symptomatic MM tumors are shown. Reproduced with permission from Kuehl WM and Bergsagel PL. Molecular pathogenesis of multiple myeloma and its premalignant precursor. J Clin Invest. 2012;122(10):3456-63. doi:10.1172/ JCI61188. Copyright from the American Society for Clinical Investigation.

at intervals of 3-4 months initially for the first year and then lengthened to 6-12 months based on the patient`s clinical history, laboratory results and comorbid conditions. Should be checked for serum protein electrophoresis, complete blood count, calcium, and serum creatinine every 6 months and if they are stable, every 2 to 6 years. There is also an alternative strategy suggesting that screening should be performed only if there is an increase in symptoms associated with MM. International Myeloma Working Group members suggest that the patients with low risk-MGUS should be followed during 6 months after the diagnosis of MGUS [32]. On the other hand, they specified that a bone marrow examination should be required if the patients had any CRAB features.

UK Myeloma Forum and the Nordic Myeloma Study Group recommend that patients with high-risk MGUS (IgG ≥1.5 g/dL; IgA or IgM >1.0 g/dL; IgD or IgE at any level) should be referred to a hematology specialist 3-4 times per year as a minimum [28]. The recommended tests for monitoring include serum protein electrophoresis, serum total im‐ munoglobulin, complete blood count, creatinine, urea, electrolytes and serum calcium. In addition, it should be evaluated using bone marrow cytogenetic and FISH with bone imaging studies. Nevertheless, it is important to highlight that sometimes it will be neces‐ sary to perform Magnetic Resonance Imaging or Positron Emission Tomography-Comput‐ ed Tomography, instead of traditional x-rays. Patients with unexplained anemia or kidney failure should be evaluated with a full bone scan that also include cytogenetic and FISH. Korde et al [57] reported that is critical to recognize that in a disease such as MM, where defining criteria rely on the presence or absence of end-organ damage, diag‐ nosis is only as good as the tools and technology able to detect end-organ damage. For instance, in SMM or high-risk MGUS patients suspicious to harbor bone disease, imaging evaluation may be better served by obtaining magnetic resonance imaging or Positron Emission Tomography-Computed Tomography rather than traditional skeletal surveys. International Myeloma Working Group members recommend for intermediate-risk and high-risk MGUS patients should have a bone marrow aspirate and biopsy with both con‐ ventional cytogenetics and FISH [32]. If available, a plasma cell labeling index and a search for circulating plasma cells in the peripheral blood using flow cytometry are use‐ ful. Patients with IgM isotype should have a computational tomography scan of the ab‐ domen since asymptomatic retroperitoneal lymph nodes may be present. If there is evidence of MM or Waldeström macroglobulinemia, lactate dehydrogenase, 2-microglo‐ bulin, and C-reactive protein levels should be measured. If the results of these tests are satisfactory, International Myeloma Working Group recommend patients should be fol‐ lowed with serum protein electrophoresis and complete blood cell count in 6 months and then annually for life [32].

#### **7. Management**

at intervals of 3-4 months initially for the first year and then lengthened to 6-12 months based on the patient`s clinical history, laboratory results and comorbid conditions. Should be checked for serum protein electrophoresis, complete blood count, calcium, and serum creatinine every 6 months and if they are stable, every 2 to 6 years. There is also an alternative strategy suggesting that screening should be performed only if there is an increase in symptoms associated with MM. International Myeloma Working Group members suggest that the patients with low risk-MGUS should be followed during 6 months after the diagnosis of MGUS [32]. On the other hand, they specified that a bone marrow examination should be required if

**Figure 1.** Model for molecular pathogenesis of monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM). TR1, the initial transition to a recognizable tumor involves two mostly non-overlapping path‐ ways (IgH translocations versus multiple trisomies) that include primary events associated with dysregulated cyclin D expression in MGUS and MM. TR2, the transition from MGUS to MM is associated with increase MYC expression and sometimes with activating mutations of K-RAS or chromosome 13 deletion. Early and late progression events for symptomatic MM tumors are shown. Reproduced with permission from Kuehl WM and Bergsagel PL. Molecular pathogenesis of multiple myeloma and its premalignant precursor. J Clin Invest. 2012;122(10):3456-63. doi:10.1172/

UK Myeloma Forum and the Nordic Myeloma Study Group recommend that patients with high-risk MGUS (IgG ≥1.5 g/dL; IgA or IgM >1.0 g/dL; IgD or IgE at any level) should be referred to a hematology specialist 3-4 times per year as a minimum [28]. The

the patients had any CRAB features.

JCI61188. Copyright from the American Society for Clinical Investigation.

120 Multiple Myeloma - A Quick Reflection on the Fast Progress

In clinical practice, patients with MGUS are followed clinically without treatment until progression. However, the existence of easily identifiable precursor states represents an opportunity for chemoprevention [67]. However, it must be weighed that benefits achieved by treating a precursor state is greater than a potential for therapeutic toxicity. Recently, Korde et al [57] revised early treatment strategies for MGUS and SMM.

Bhattacharyya et al [68] reported a clinic case of IgM-MGUS associated with cryoglobulinemia and cold agglutinin disease, which was treated with immunotherapy and was successful (Table 3). Immunochemotherapy, consisting of rituximab (375 mg/m2, day 1), fludarabine (25 mg/m2, days 2-4), and cyclophosphamide (250 mg/m2, days 2-4), was administered every 4 weeks up to three times as a first-line treatment followed by three cycles of monthly rituximab treatment. Extensive skin lesions with livedo reticularis entirely disappeared prior to initiation of the second cycle in association with the declined serum level of IgM.

Pepe et al [69] studied 100 patients affected by MGUS, grouped according to the presence (group A, 50 patients) or absence (group B) of vertebral fractures and/or osteoporosis. Group A was treated with alendronate (70 mg/weekly) plus calcium and cholecalciferol for 18


54 patients with MGUS and osteopenia or osteoporosis [70]. They also demonstrated that increase bone mineral density in patients with bone loss with the theoretical added benefit of reducing fractures although it was not observed that the progression can be delayed or

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123

There are two ongoing studies, in the first, the aim is to assess whether omega-3 fatty acids reduce activated NF-κB levels in peripheral blood lymphocytes [71]. Omega-3 supplementa‐ tion will be initiated at three 1250 mg capsules daily for the first month. If dose is well tolerated, it will be increased to six 1250 mg capsules daily for 30 days and finally to nine 1250 mg capsules daily. Treatment period is 12 months (study design nonrandomized). No study results posted on clinicaltrials.gov [71]. In the second study, the aim is to test whether green tea extract reduces the M-protein concentration [72]. Patients receive oral green tea catechin extract (Polyphenon E) daily on days 1-28. Treatment repeats every 28 days for up to 6 courses in the absence of disease progression or unacceptable toxicity. No study results posted on clinical‐

Golombick et al [73] investigated the effect of curcumin on plasma cells and osteoclasts in patients with MGUS (see Table 3). Twenty-six patients with MGUS were randomized into two groups (single-blind, randomized, crossover pilot). The pilot study found that curcu‐ min may decrease both serum M-protein (in patients with levels of >20 g/L) and urinary Ntelopeptide of type I collagen bone turnover marker in patients with MGUS. Recently, Golombick et al [74] performed a randomized, double-blind placebo-controlled crossover 4 g curcumin study and an open-label extension study using an 8 g curcumin. 19 MGUS and 17 SMM were randomized into two groups: one received 4 g curcumin and the other 4 g placebo, crossing over at 3 months. 25 patients completed the 4 g crossover study and 18 the 8 g extension study. In some patients curcumin therapy decreased the free light-chain ratio

Curcumin is the most active component in *Curcuma longa* or turmeric (tropical plant native to southern and southeastern tropical Asia). Curcumin has been shown to downregulate IL-6 and nuclear factor-κB; to inhibit osteoclastogenesis and to reduce bone turnover; suppresses proliferation and induces apoptosis in MM cells [75] and inhibits osteoclastogenesis through the suppression of RANKL signaling [76]. Nevertheless, it is known that curcumin inhibits IL-12 production in dendritic cells, thereby dampening the Th1 response [77]. This suggests that may have an immunosuppressive effect. However, Rajkumar [78] indicated that finding reported by Golombick [74] is a modest decrease in free light chain levels by 25-50% in one quarter of the patients, reason why he disagrees with curcumin as a preventive or therapeutic strategy in MGUS (Table 3). Rajkumar also indicated that using risk stratification model approximately 50% of all MGUS patients are considered low-risk MGUS, and have a lifetime risk of progression of only 2%. Therefore, he recommends that focus should be put on

There is an increased interest in identifying biomarkers that can predict patients who will inevitably progress to symptomatic MM. These include genetic and/or epigenetic targets and microenvironment and/or its interaction with tumor cells, which may change the future of

prevented.

trials.gov [72].

and uDPYD (a marker of bone resorption).

preventive strategies in patients with high-risk SMM.

**MGUS:** Monoclonal gammopathy of undetermined significance; **SMM:** Smoldering multiple myeloma; **NA:** Not available.

#### **Table 3.** Therapy on patients with MGUS

months, and group B was treated with calcium and cholecalciferol. Treatment with alendro‐ nate could lead to a significant reduction in fracture risk in MGUS patients with skeletal fragility. During the whole period of investigation, eight patients in group A developed MM and therefore were not able to continue the study. A further 12 patients included in group A did not want to take the drugs prescribed. Additionally, the author indicated that this study has some limitations, mainly because of the lack of a real control group (longitudinal‐ ly followed for the entire observation period) and the lack of morphometric evaluation of vertebral fractures at 18 months. Another similar study was administered zoledronic acid to 54 patients with MGUS and osteopenia or osteoporosis [70]. They also demonstrated that increase bone mineral density in patients with bone loss with the theoretical added benefit of reducing fractures although it was not observed that the progression can be delayed or prevented.

There are two ongoing studies, in the first, the aim is to assess whether omega-3 fatty acids reduce activated NF-κB levels in peripheral blood lymphocytes [71]. Omega-3 supplementa‐ tion will be initiated at three 1250 mg capsules daily for the first month. If dose is well tolerated, it will be increased to six 1250 mg capsules daily for 30 days and finally to nine 1250 mg capsules daily. Treatment period is 12 months (study design nonrandomized). No study results posted on clinicaltrials.gov [71]. In the second study, the aim is to test whether green tea extract reduces the M-protein concentration [72]. Patients receive oral green tea catechin extract (Polyphenon E) daily on days 1-28. Treatment repeats every 28 days for up to 6 courses in the absence of disease progression or unacceptable toxicity. No study results posted on clinical‐ trials.gov [72].

Golombick et al [73] investigated the effect of curcumin on plasma cells and osteoclasts in patients with MGUS (see Table 3). Twenty-six patients with MGUS were randomized into two groups (single-blind, randomized, crossover pilot). The pilot study found that curcu‐ min may decrease both serum M-protein (in patients with levels of >20 g/L) and urinary Ntelopeptide of type I collagen bone turnover marker in patients with MGUS. Recently, Golombick et al [74] performed a randomized, double-blind placebo-controlled crossover 4 g curcumin study and an open-label extension study using an 8 g curcumin. 19 MGUS and 17 SMM were randomized into two groups: one received 4 g curcumin and the other 4 g placebo, crossing over at 3 months. 25 patients completed the 4 g crossover study and 18 the 8 g extension study. In some patients curcumin therapy decreased the free light-chain ratio and uDPYD (a marker of bone resorption).

Curcumin is the most active component in *Curcuma longa* or turmeric (tropical plant native to southern and southeastern tropical Asia). Curcumin has been shown to downregulate IL-6 and nuclear factor-κB; to inhibit osteoclastogenesis and to reduce bone turnover; suppresses proliferation and induces apoptosis in MM cells [75] and inhibits osteoclastogenesis through the suppression of RANKL signaling [76]. Nevertheless, it is known that curcumin inhibits IL-12 production in dendritic cells, thereby dampening the Th1 response [77]. This suggests that may have an immunosuppressive effect. However, Rajkumar [78] indicated that finding reported by Golombick [74] is a modest decrease in free light chain levels by 25-50% in one quarter of the patients, reason why he disagrees with curcumin as a preventive or therapeutic strategy in MGUS (Table 3). Rajkumar also indicated that using risk stratification model approximately 50% of all MGUS patients are considered low-risk MGUS, and have a lifetime risk of progression of only 2%. Therefore, he recommends that focus should be put on preventive strategies in patients with high-risk SMM.

months, and group B was treated with calcium and cholecalciferol. Treatment with alendro‐ nate could lead to a significant reduction in fracture risk in MGUS patients with skeletal fragility. During the whole period of investigation, eight patients in group A developed MM and therefore were not able to continue the study. A further 12 patients included in group A did not want to take the drugs prescribed. Additionally, the author indicated that this study has some limitations, mainly because of the lack of a real control group (longitudinal‐ ly followed for the entire observation period) and the lack of morphometric evaluation of vertebral fractures at 18 months. Another similar study was administered zoledronic acid to

**MGUS:** Monoclonal gammopathy of undetermined significance; **SMM:** Smoldering multiple myeloma; **NA:** Not

**Drug [References] Treatment**

Alendronate plus calcium and cholecalciferol

*vs.*

[68]

calcium and cholecalciferol [69]

Rituximab, fludarabine, and cyclophosphamide

Curcumin vs.

placebo [73]

Curcumin vs.

placebo [74]

available.

Zoledronic acid [70] 4 mg, i.v. at 0, 6,

**scheme**

122 Multiple Myeloma - A Quick Reflection on the Fast Progress

and 12 months

70 mg/weekly, at 18 months

Every 4 weeks up to three times followed by three cycles of monthly rituximab treatment

4 g/day and an open-label 8 g curcumin extension study, oral, at 3 months

**Table 3.** Therapy on patients with MGUS

4 g/day oral 26 MGUS

**Nº of patients (age or study/control)**

54 MGUS and osteopenia or osteoporosis (50-91 years; median=67 years)

100 MGUS With presence or absence (control) vertebral fractures and/or osteoporosis

(50/50)

1 MGUS associated with cryoglobulinemia and cold agglutinin disease

(17/9)

19 MGUS 17 SMM (12/13)

**Benefit Observations**

Reducing fractures. 48 patients completed the study.

with time.

take the drugs.

Reducing fractures. 8 patients developed MM

NA

NA

Decreases M-protein and skin lesions disappeared.

Decreases bone resorption and Mprotein (12-30%) of patients with M-protein

Decreasing free light chain and marker of bone resorption.

>20 g/L

Some patients showed adverse effects. Progression of MGUS does not diminish

12 patients did not want to

Curcumin may benefit some but not all patients with MGUS and SMM.

> There is an increased interest in identifying biomarkers that can predict patients who will inevitably progress to symptomatic MM. These include genetic and/or epigenetic targets and microenvironment and/or its interaction with tumor cells, which may change the future of

disease progression [65]. Dynamic changes in tumor and microenvironment, cell immunophe‐ notype, mRNA and protein expression, should offer insight into disease progression [57, 78].

**References**

[1] Kyle RA, Therneau TM, Rajkumar SV, Larson DR, Plevak MF, Offord JR, Dispenzieri A, Katzmann JA, Melton LJ 3rd. Prevalence of monoclonal gammopathy of undeter‐

Monoclonal Gammopathy of Undetermined Significance

http://dx.doi.org/10.5772/56138

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[2] Kyle RA, Therneau TM, Rajkumar SV, Offord JR, Larson DR, Plevak MF, Melton LJ 3rd. A long-term study of prognosis in monoclonal gammopathy of undetermined

[3] Kyle RA, Rajkumar SV. Monoclonal gammopathy of undetermined significance. Br J

[5] Kyle RA. Sequence of testing for monoclonal gammopathies. Arch Pathol Lab Med.

[6] Attaelmannan M, Stanley L. Understanding and identifying monoclonal gammopa‐

[7] Waldeström J. Studies on conditions associated with disturbed gamma globulin for‐

[8] Kyle, RA. Monoclonal gammopathy of undetermined significance: natural history in

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[10] Kyle RA, Finkelstein S, Elveback LR, Kurland LT. Incidence of monoclonal proteins in a Minnesota community with a cluster of multiple myeloma. Blood. 1972;40(5):

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[13] Axelsson U, Bachmann R, Hällén J. Frequency of pathological proteins (M-compo‐ nents) om 6,995 sera from an adult population. Acta Med Scand. 1966;179(2):235-47.

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[15] Landgren O, Gridley G, Turesson I, Caporaso NE, Goldin LR, Baris D, Fears TR, Hoover RN, Linet MS. Risk of monoclonal gammopathy of undetermined signifi‐

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719-24.

#### **8. Conclusion**

In conclusion, it is crucial to follow up cases of MGUS carefully, including their systematic recording as a fundamental contribution to understand the evolution of this pathology and its malignant transformation process. This will be critical to develop better biomarkers that contribute to understand the evolution and malignant transformation of MGUS. These efforts should lead to the development of new, more effective management and treatment strategies.
