High frequencies of chromosomal aberrations in multiple myeloma and monoclonal gammopathy of undetermined significance in direct chromosome preparation

Although many cases of multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are cytogenetically normal, interphase fluorescence in situ hybridization (FISH) analyses reveal aberrations in the majority of the cases. Most likely, non-neoplastic cells are more prone to divide in culture than neoplastic cells. Direct chromosome preparations (DCP) would be one way to circumvent this methodological problem. We have investigated 47 bone marrow samples from 39 patients by DCP. A median of 58 metaphases (range 9-158) was analysed per sample. Interphase FISH analyses using probes to detect IGH rearrangements, -13/13q-, +3, +7, and +11 were also performed. Abnormal karyotypes were detected in 15 (63%) of 24 MM and in 4 (50%) of eight MGUS/smouldering MM (SMM) cases that could be successfully cytogenetically analysed. Age, sex, or degree of bone marrow plasma cell (PC) infiltration did not influence the karyotypic patterns (P > 0.05). However, the frequencies of aberrant karyotypes varied in relation to the Colcemide concentrations used - 7% (30 ng/ml) versus 69% and 67% (100 and 200 ng/ml, respectively) (P = 0.01). Combining the G-banding and FISH results, abnormalities were detected in 29 of 31 (94%) MM and in six of eight (75%) MGUS/SMM patients. Thus, cytogenetic and FISH analyses after DCP using 100-200 ng Colcemide/ml identified aberrations in most MM/MGUS/SMM, irrespective of PC percentages.     

Kappa/lambda ratio on peripheral blood lymphocytes and bone marrow plasma cell labeling index in monoclonal gammopathies

Bone marrow plasma cell labeling index (L.I.), kappa/lambda ratio, CD4+ and CD8+ subpopulations were studied in patients with smoldering multiple myeloma (SMM), active myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). The loss of the light chain isotype suppression (LCIS) and a high labeling index (L.I.) allowed us to distinguish all MM patients from SMM patients. On the contrary, a low L.I. and a normal kappa/lambda ratio were found in most kappa and lambda MGUS. A significant difference in the kappa/lambda ratio was noted between MGUS and K-SMM. A significant decrease in CD4+ and a significant increase in CD8+ were not observed in patients with LCIS. The kappa/lambda ratio together with the plasma cell L.I. could be useful parameters for distinguishing active MM from SMM, especially in the cases with a border line L.I.     

Clonal circulating cells are common in plasma cell proliferative disorders: a comparison of monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and active myeloma

The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45- CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).     

Sialyltransferase activity and sialic acid levels in multiple myeloma and monoclonal gammopathy

A significant elevation of peripheral blood mononuclear cell sialyltransferase activity (STA) was observed in 14 patients with multiple myeloma (MM), and compared to 7 patients with monoclonal gammopathy of undetermined significance (MGUS) and to 10 controls. Serum sialyltransferase was significant higher in MM patients as compared to controls. It was also higher than in MGUS patients, but the difference here was not statistically significant. STA was also determined in mononuclear bone marrow cells of 5 patients with MM (with 50 to 100% plasma cells in the bone marrow aspirate) and found to be 19 times higher than that of bone marrow mononuclear cells from 6 patients with non-malignant disorders (with less than 1% plasma cells in the bone marrow aspirate). No significant differences were observed in peripheral blood mononuclear cell sialic acid levels between MM, MGUS and controls.     

Smoldering multiple myeloma: natural history and recognition of an evolving type

Patients with smoldering multiple myeloma (SMM) meet the diagnostic criteria of multiple myeloma (MM) but are asymptomatic. Between January 1978 and July 2001, 53 patients (median age 63 years) were diagnosed with SMM. The median serum M-protein and proportion of bone marrow plasma cells were 36 g/l and 27% respectively. Two subsets of SMM were identified: (i) evolving SMM (n = 22), characterized by a progressive increase in serum M-protein, a previously recognized monoclonal gammopathy of undetermined significance (MGUS) and a significant higher proportion of IgA type and (ii) non-evolving SMM (n = 26) with stable M-protein that abruptly increases when symptomatic MM develops. Thirty-four patients developed symptomatic MM. The median time to progression in the overall series was 3.2 years and the only feature associated with a shorter time to progression was the evolving versus non-evolving type (1.3 vs. 3.9 years respectively, P = 0.007). The pattern of progression consisted of anaemia, lytic bone lesions or both, without renal failure, hypercalcaemia or extramedullary plasmacytomas. Fifty-seven per cent of patients that required chemotherapy showed no or minimal response. The median survival from diagnosis and from progression was 8.2 and 3.5 years respectively.     

Monoclonal gammopathy of undetermined significance and smoldering multiple myeloma

Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic disorders characterized by monoclonal plasma cell proliferation in the bone marrow in the absence of end-organ damage. Updated diagnostic criteria for these disorders, risk-stratification models to determine prognosis, and the current management of these two entities are discussed in this review.     

Differentiation of monoclonal gammopathy of undetermined significance and multiple myeloma using flow cytometric characteristics of plasma cells

BACKGROUND AND OBJECTIVES: The differential diagnosis between multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) may be uncertain in some cases; this problem is reflected by discrepancies between different classification systems with an accordance in only 2/3 of cases. We studied whether flow-cytometric characteristics of plasma cells (PC) can be used for the differentiation between MGUS and MM. DESIGN AND METHODS: Patients were divided into 3 groups: Group A included 13 myeloma patients with a plasma cell infiltration of the bone marrow of 10-30%, serum M-protein < or = 3.5 g/dL (IgG) or < or = 2 g/dl (IgA) and without bone lesions in conventional radiography. Group B consisted of 53 patients who fulfilled the Durie and Salmon diagnostic criteria including at least one major criterion, and group C individuals with MGUS (n=17). The ratio of immunophenotypically normal (i.e. CD19(+)/CD56(-)) to all bone marrow plasma cells (BMPC), the number of peripheral blood PC (PBPC), the percentage of BMPC in S-phase and the DNA content of BMPC were analyzed. RESULTS: All individuals with MGUS and no patient with MM in group A or group B had a ratio of phenotypically normal to all BMPC > or = 20%. The median of monoclonal PBPC was 0/microL (range 0-2/ microL) in MGUS, 1/microL (range 0-30/microL) in MM group A and 2.4/microL (range 0-211/microL) in MM group B. The median percentage of BMPC in S-phase was 1.6% both in MGUS and in group A and 3% in group B. Aneuploidy was found in 12%, 11% and 41% in MGUS, group A and group B, respectively. INTERPRETATION AND CONCLUSIONS: The ratio of immunophenotypically normal to all BMPC was the only flow-cytometric parameter for the differentiation of MGUS and MM group A (p<0.0005). The other parameters were significantly different between MGUS and MM group B, but not group A.     

Measurement of apoptosis and proliferation of bone marrow plasma cells in patients with plasma cell proliferative disorders

The proliferative rate of malignant plasma cells, as measured by the plasma cell labelling index (PCLI), is an important prognostic factor in multiple myeloma (MM); however, the PCLI alone is probably Inadequate to describe tumour growth because it ignores the idea that myeloma cells may have a reduced rate of apoptosis. The aims of this study were to develop a flow cytometric method to measure the apoptosis index of fresh marrow plasma cells and develop a plasma cell growth index (PCGI) that related both proliferation and apoptosis to disease activity. Marrow aspirates were obtained from 91 patients with plasma cell disorders and the plasma cells in apoptosis were identified by either 7-amino actinomycin-D (7-AAD) or annexin V-FITC three-colour flow cytometry. The median plasma cell apoptotic index (PCAI) for patients with monoclonal gammopathy of undetermined significance (MGUS), smouldering or indolent myeloma (SMM/IMM), and new multiple myeloma (MM) was 5.2, 3.4 and 2.4, respectively (P=0.03, MGUS v MM). The median PCLI for these same patient groups was 0.0, 0.2 and 0.6, respectively (P<0.001, MGUS v MM). The paired PCLI and PCAI for each sample were used to derive the PCGI=2 + [PCLI-(O.1)(PCAI)]. The median PCGI for patients with inactive disease (MGUS, SMM/IMM or amyloidosis) was 1.8 compared to 2.4 for those with active disease (new or relapsed MM) (P<0.001). These results suggest that a decrease in the PCAI may be a factor in the progression from MGUS to SMM to overt MM.     

New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smoldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells

Monoclonal gammopathy of uncertain significance (MGUS) and smoldering multiple myeloma (SMM) are plasma cell disorders with a risk of progression of approximately 1% and 10% per year, respectively. We have previously shown that the proportion of bone marrow (BM) aberrant plasma cells (aPCs) within the BMPC compartment (aPC/BMPC) as assessed by flow cytometry (FC) contributes to differential diagnosis between MGUS and multiple myloma (MM). The goal of the present study was to investigate this parameter as a marker for risk of progression in MGUS (n = 407) and SMM (n = 93). Patients with a marked predominance of aPCs/BMPC (> or = 95%) at diagnosis displayed a significantly higher risk of progression both in MGUS and SMM (P< .001). Multivariate analysis for progression-free survival (PFS) selected the percentage aPC/BMPC (> or = 95%) as the most important independent variable, together with DNA aneuploidy and immunoparesis, for MGUS and SMM, respectively. Using these independent variables, we have identified 3 risk categories in MGUS (PFS at 5 years of 2%, 10%, and 46%, respectively; P< .001) and SMM patients (PFS at 5 years of 4%, 46%, and 72%, respectively; P < .001). Our results show that multiparameter FC evaluation of BMPC at diagnosis is a valuable tool that could help to individualize the follow-up strategy for MGUS and SMM patients.     

Circulating monoclonal B lymphocytes in multiple myeloma

Peripheral blood mononuclear cells from 28 patients with multiple myeloma (MM) and nine patients with monoclonal gammopathy of unknown significance (MGUS) were studied by immunoglobulin gene analysis. Clonal immunoglobulin gene rearrangements in peripheral blood mononuclear cells (PBIGRA) were demonstrated in 10 of the 28 MM patients (36%). Bone marrow and peripheral blood mononuclear cells were studied simultaneously in five of these 10 patients, and identical gene rearrangements were demonstrated in both. The incidence of such gene arrangements was higher in patients with active disease (cases at presentation or relapsed = 10/19 [47%]) compared to remission status (0/9) and higher in untreated (47%) compared to treated patients (11%) (P less than 0.05). Patients with this phenomenon had higher serum calcium levels (P less than 0.001), and higher bone marrow plasma cell counts (P less than 0.05). Serum creatinine and beta 2-microglobulin were also higher but did not reach statistical significance. None of the patients with monoclonal gammopathy of uncertain significance had gene arrangements. Our findings confirm that circulating B lymphocytes are part of the malignant clone in MM and their presence correlates with high tumour volume.     

Acquired Fanconi syndrome is an indolent disorder in the absence of overt multiple myeloma

Adult-acquired Fanconi syndrome (FS) is a rare complication of monoclonal gammopathy. We retrospectively reviewed 32 patients diagnosed with adult-acquired FS between April 1968 and June 2002 at Mayo Clinic (Rochester, MN). At diagnosis, most patients had monoclonal gammopathy of undetermined significance (MGUS) or smoldering multiple myeloma (SMM), with a median creatinine level of 176.8 microM (2.0 mg/dL; range, 79.56-327.08 microM [0.9-3.7 mg/dL]) and evidence of osteomalacia. During the average 65 months (range, 2-238 months) of follow-up, 5 patients developed end-stage renal disease (ESRD) and only 1 of 14 patients with MGUS transformed to multiple myeloma (MM). Also, 14 deaths occurred, with only 1 from ESRD but 4 from alkylator-related leukemia or myelodysplastic syndrome. Chemotherapy offered little benefit on renal functions of MGUS or SMM patients. In conclusion, FS associated with monoclonal gammopathy does not appear to confer an additional risk of subsequent evolution to MM. ESRD occurs late in the disease process.     

Myelomagenesis: capturing early microenvironment changes

Plasma cell neoplasms result from the clonal expansion of terminally differentiated, immunoglobulin heavy-chain class switched B cells that typically secrete a monoclonal immunoglobulin. The 2008 World Health Organization (WHO) classification of plasma cell neoplasms encompasses a broad spectrum of disorders, from the precursor disorder monoclonal gammopathy of undetermined significance (MGUS) to plasma cell leukemia. The classification includes, in addition to precursor lesion MGUS, plasma cell myeloma, plasmacytoma, immunoglobulin deposition diseases, and osteosclerotic myeloma. Plasma cell myeloma is further divided into symptomatic plasma cell myeloma or multiple myeloma (MM), asymptomatic smoldering myeloma (SMM), non-secretory myeloma, and plasma cell leukemia. Although histopathologic cut-off criteria are incorporated into the classification schema, distinction between MGUS, SMM, and MM depends primarily on the presence or absence of end-organ damage, as defined by "CRAB" criteria (hypercalcemia, renal insufficiency, anemia, lytic bone lesions, or a combination of these). Systematic evaluation of pathogenetic differences between MGUS and MM should offer invaluable insights into early myelomagenesis. Given the complex, intertwined nature of the malignant plasma cell and its surroundings, multiple pathogenetic mechanisms play a critical role in interactions between neoplastic cells and their microenvironment. Understanding the events leading to end-organ damage, like anemia and bone remodeling, is a critical part of investigating early myelomagenesis and should provide us with better tools for early identification and treatment of these patients.     

Bone marrow angiogenesis and progression in multiple myeloma

Tumour growth is angiogenesis-dependent. We found a high correlation between the extent of bone marrow angiogenesis, evaluated as microvessel area, and the proliferating (S-phase) fraction of marrow plasma cells, evaluated as labelling index (LI), in patients with multiple myeloma (MM) and in those with monoclonal gammopathies of undetermined significance (MGUS). Angiogenesis itself was significantly associated with active as opposed to non-active MM and MGUS. The highest microvessel area accompanied rapidly progressive MM with the highest LI. When a cut-off value of 2% or greater of the microvessel area was used, most patients with active MM were classified correctly. The risk of active disease in patients with MM increased in parallel with the microvessel area. A causal relationship between plasma cell growth, activity phase in MM and marrow angiogenesis is suggested. Since angiogenesis proceeds in step with the enlargement of plasma cell tumours and the activity phase in MM, its measurement could be a useful prognostic marker in patients with plasma cell proliferative disorders.     

Molecular imaging in myeloma precursor disease

Multiple myeloma (MM) is consistently preceded by its pre-malignant states, monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering multiple myeloma (SMM). By definition, precursor conditions do not exhibit end-organ disease (anemia, hypercalcemia, renal failure, skeletal lytic lesions, or a combination of these). However, new imaging methods are demonstrating that some patients in the MGUS or SMM category are exhibiting early signs of MM. Although MGUS/SMM patients are currently defined as low-risk versus high-risk based on clinical markers, we currently lack the ability to predict the individual patient's risk of progression from MGUS/SMM to MM. Given that the presence of gross lytic bone lesions is a hallmark of MM, it is reasonable to believe that less severe bone changes defined by more sensitive imaging may be predictive of MM progression. Indeed, since bone disease is such an essential aspect of MM, imaging techniques directed at the detection of early bone lesions, have the potential to become increasingly more useful in the setting of MGUS/SMM. Current guidelines for the radiological assessment of MM still recommend the traditional skeletal survey, although its limitations are well documented, especially in early phases of the disease when radiographs can significantly underestimate the extent of bone lesions and bone marrow involvement. Newer, more advanced imaging modalities, with higher sensitivities, including whole-body low-dose computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) are being employed. Also various imaging techniques have been used to provide an assessment of bone involvement and identify extra-osseous disease. This review emphasizes the current state of the art and emerging imaging methods, which may help to better define high-risk versus low-risk MGUS/SMM. Ultimately, improved imaging could allow more tailored clinical management, and, most likely play an important role in the development of future treatment strategies for high-risk precursor disease.     

Analysis of 128 cases of multiple myeloma and 31 cases of monoclonal gammapathies of undetermined significance

Monoclonal immunoglobulin from 138 patients with multiple myeloma (MM) showed that 68 patients were of IgG type, 23 of IgA type,33 of BJ type, 6 of IgD type, one of IgM type, 5 of nonsecreting type and the remaining 2 of IgA and IgG double monoclonal type. Urinary Bence Jones protein was tested with concentrated urine specimen with a positive rate of 75%. Classification of light chain revealed the ratio of oK/L being 1.09:1. The difference of distribution of K or L among IgG, IgA BJ types was no significant. 31 cases with monoclonal gammapathies of undetermined significance (MGUS) were also analyzed in this study. There were some difficulties in the differential diagnosis of MGUS and MM,but a significant difference was present in the plasma cell percentage of bone marrow smear of the two groups. Dynamic investigation in this series showed that patients with percentage of plasma cell less than 10% persisting for a long duration are likely to have MGUS only.     

A validated FISH trisomy index demonstrates the hyperdiploid and nonhyperdiploid dichotomy in MGUS

Two major genetic categories of multiple myeloma (MM) exist. Hyperdiploid MM (48 to 74 chromosomes, median 53 chromosomes) is associated with trisomies especially of chromosomes 3, 7, 9, 11, 15, and 19, whereas the nonhyperdiploid (< 48 chromosomes or more than 74 chromosomes) MM is associated with primary translocations such as t(11;14), t(4;14), and t(14;16). Whether this dichotomy exists in monoclonal gammopathy of undetermined significance (MGUS) is uncertain due to limitations of current methods in the study of ploidy. This is especially true in MGUS where the number of clonal plasma cells is small. In this study, we derived a fluorescent in situ hybridization (FISH)-based trisomy index from pooled cytogenetic data (karyotype analysis) from 2 large cohorts of patients with MM with abnormal karyotype, and then validated it in 2 independent cohorts of patients who had known ploidy status either by karyotyping or DNA content measurement using flow cytometry. Using the criteria of 2 or more trisomies from a 3-chromosome combination, hyperdiploid myeloma can be detected with high specificity. Applying this index on 28 patients with smoldering multiple myeloma (SMM) or MGUS (11 SMM, 17 MGUS) who had normal karyotype, 11 cases of hyperdiploid SMM/MGUS were detected. This percentage (40%) is remarkably similar to the percentage of hyperdiploid MM reported in the literature, suggesting that hyperdiploid MM may originate early during disease evolution.     

Bone marrow of patients with active multiple myeloma: Angiogenesis and plasma cell adhesion molecules LFA-1, VLA-4, LAM-1, and CD44

Bone marrow plasma cells and stromal cells in multiple myeloma (MM) have been shown to be capable of releasing cytokines with angiogenic properties. Plasma cells can also express adhesion molecules controlling their adhesive interactions with endothelial cells. In the present study, we have evaluated by immunohistochemistry the extent of angiogenesis in the bone marrow of: a) 51 patients with active and non-active MM; b) 25 patients with monoclonal gammopathy of undetermined significance (MGUS). Plasma cells were investigated by flow cytometry for the expression of the adhesion molecules LFA-1, VLA-4, LAM-1, and CD44. The results showed that, while angiogenesis was very low or absent in patients with MGUS and non-active MM, it increased markedly in those with active MM. The highest detectability of plasma cell adhesion molecules, except LAM-1, was also found in these patients. The functional significance of these findings is unknown. Their consistent occurrence in the bone marrow of active myeloma patients, however, strongly suggests that more frequent adhesive interactions between plasma cells and their microvasculature underlie tumor dissemination.