Asymptomatic Monoclonal Gammopathies Research Articles

Defining Risk of MGUS and AMM Progression to Myeloma By Ig Heavy-Chain FISH

Interphase FISH analysis using probes for the immunoglobulin heavy chain constant region (IGHC) and variable regions (IGHV) revealed that plasma cells from healthy individuals and from patients with plasma cell (PC) dyscrasias had different patterns of signal-combinations of the two probes. We hypothesized that these patterns indicate diagnostic and prognostic significance.Patients and Methods: Bone marrow specimens from 39 healthy donors, 111 patients with MGUS, 89 patients with AMM, and 96 patients with MM were examined using interphase FISH with probes for IGHC and IGHV simultaneously (Tian et al. Gene Chr Can 2014) and immune staining for cytoplasmic Ig isotypes. For each specimen, the combination patterns of IGHC and IGHV probes were scored separately for Kappa and Lambda expressing PCs. In addition, gene expression profiles (GEP) of CD138+-purified PCs were analyzed. FISH were also performed with the probes to chromosome 1q21 (CKS1B), 1p13 (AHCYL1), 17p13 (TP53), and 17q12 (ERBB2) loci.Results: 1.IGHC and IGHV pattern diversity of PCs was present in all specimens, including healthy individuals. A bivariate distribution of polyclonal diversities among healthy individuals was calculated to create an ellipse defining the normal population pattern. Applying the ellipsoid definition to the patient populations, two subgroups could be distinguished in MGUS/AMM cases: one within the ellipse, representing patients with polyclonal PCs (without a dominant clone), while the other, who had a dominant clone with identical IGHC/IGHV FISH pattern, fell outside of the ellipse and was termed asymptomatic monoclonal gammopathy (Figure). With 99% confidence, 54 of 111 (48.7%) of MGUS and 66 of 89 (74.2%) of AMM were classified as monoclonal. In MM, 80 of 96 (86.0%) were stratified outside of the ellipse.2.The FISH-defined monoclonal subgroup (outside the ellipse) among AMM patients was at high risk of progressing to symptomatic myeloma; one year progression to MM requiring therapy was 26%, 2-year progression was 37%, and the 5-year progression rate was estimated at 64%. The subgroup with polyclonal PCs in AMM had significantly lower risk of disease progression (<10% in 2 years, p<0.0001). In univariate Cox regression analysis, besides FISH-defined monoclonality (HR 2.72, p<0.004), serum albumin (< 3.5 g/dL), GEP70 risk score (> -0.26), t(4;14), excess lambda light chain, and gain of chromosome1q21 region (>2) were significant progression factors of FISH-defined monoclonal patients (Hazard Ratios >2.2; p<0.005). In multivariate analysis, FISH-defined monoclonality (HR 2.15), age (>65 yr, HR 2.05), serum albumin (< 3.5 g/dL, HR 3.21) and having AMM (HR 3.02) remained significant predictors of early disease progression with an R2 of 0.434.Conclusion:We established a FISH predictive model on the basis of healthy bone marrow. Based on normal distribution of PC clonal diversities, patient subgroups with polyclonal PC and with monoclonal PC were detected in MGUS and AMM patients. The presence of a dominant clone in the bone marrow at the time of diagnosis identifies, among patients with MGUS and AMM, a subgroup at high-risk of early disease progression to myeloma requiring therapy. We are currently developing a model to identify with high accuracy patients will progress within 1, and 2 years.Figure: A 100(1-α)% prediction ellipse based on clonal distribution of healthy donors (left) defines the polyclonal PC region. MGUS, AMM, and MM patients with monoclonal PCs are outside this region. DisclosuresMorgan:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity’s Board of Directors or advisory committees; The Binding Site: Membership on an entity’s Board of Directors or advisory committees; MMRF: Membership on an entity’s Board of Directors or advisory committees.

Flow Cytometry Defined Cytoplasmic Immunoglobulin Index Is a Major Prognostic Factor for Progression of Asymptomatic Monoclonal Gammopathies to Clinical Multiple Myeloma

Progression of Asymptomatic Monoclonal Gammopathies to Myeloma requiring treatment -Clinical Multiple Myeloma (CMM)- is an important issue in current clinical investigation toward secondary prevention, i.e. treating high-risk AMG. Several predictive models have been published, including one that incorporated gene expression profiling (GEP) of plasma cells (PC) where a GEP70 score ≥0.26 was linked to higher AMG-CMM progression in a multivariate model (Dhodapkar, Blood 2014). We have applied 2-parameter flow cytometry of DNA and cytoplasmic immunoglobulin (FDC) of bone marrow aspirates as part of baseline staging of all patients with plasma cell dyscrasia. A modification introduced in August 2006 on the doublet discrimination method increased accuracy and reproducibility of FDC results considerably and allowed for the detection of a plasma cell population with a low CI<2.8 as an independent predictor of PFS and OS in newly studies of newly diagnosed CMM treated with TT3b even in the context of GEP70 risk (Papanikolaou, ASH 2012). Realizing the importance of the FDC derived CI in CMM, we assessed whether FDC data from the observational AMG protocol S0120 could identify a population of patients with a high risk for progression to CMM. Out of 252 eligible patients, 130 had an FDC sample taken within 30 days prior to enrollment date and analyzed under the modified doublet discrimination method. FDC identified a light chain restricted population (LCR) in 121 patients. The number of distinct DNA stem lines in the flow cytometry assay, the percentage of LCR plasma cells (LCR%), their ploidy status and respective CI, were evaluated alone and in relation with clinical, laboratory and genetic parameters known to affect progression of AMG to CMM. For continuous FDC variables, the running log-rank statistics were used to determine the optimal cut-off points. In univariate analysis, the existence of at least two distinct DNA stem lines (HR: 3.3, P=0.002), a FDC LCR population of plasma cells >17% (HR: 6.76, P<0.001) and the presence of a LCR population with a CI<3.6 (HR: 6.42, P<0.001) (Figure 1) were statistically significant along with other clinical factors of established prognostic value. A M component> 3g/dL (HR: 12.5, P<0.001), an involved light chain level >10mg/dL (HR: 2.8, P=0.019), a GEP70 score ≥0.26 (HR: 8.22, P<0.001) and the presence of a LCR population with a CI < 3.6 (HR:4.15, P=0.002) survived in the multivariate analysis. To further confirm importance of a low CI to CMM progression, we compared the CI of S0120 with the TT3b patients. The CI was significantly lower in TT3b cases regardless of when the comparison was made for all patients or for strictly aneuploidy cases (P<0.0001) to exclude the possibility that the CI difference reflects the lower percentage of normal plasma cells found in CMM.In conclusion, FDC is an easily applicable, fast and low-cost test, which offers valuable prognostic information even in the era of gene expression profiling and other cytogenetic testing strategies. The identification of a low CI as a risk factor suggests that, progression of AMG to CMM is characterized by the emergence of a low immunoglobulin producing myeloma cell population. [Display omitted] DisclosuresHeuck:Celgene: Honoraria; Foundation Medicine: Honoraria; Millennium: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees. Dhodapkar:Celgene: Research Funding. Zangari:Norvartis: Membership on an entity’s Board of Directors or advisory committees; Onyx: Research Funding; Millennium: Research Funding. Morgan:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity’s Board of Directors or advisory committees; The Binding Site: Membership on an entity’s Board of Directors or advisory committees; MMRF: Membership on an entity’s Board of Directors or advisory committees.

Angiogenesis Associated with Alterations of the Bone Marrow Microenvironment Predicts Multiple Myeloma Progression to Symptomatic Disease in Mice and Humans

While multiple myeloma (MM) is almost invariably preceded by asymptomatic monoclonal gammopathy of undetermined significance (MGUS) and/or smoldering MM (SMM), the alterations of the bone marrow (BM) microenvironment that establish progression to symptomatic disease are circumstantial. Our aim was to identify changes in the BM microenvironment eliciting angiogenesis in MM.We have found that in Vk*MYC mice harboring oncogene-driven plasma cell proliferative disorder, the progression from asymptomatic to symptomatic MM was characterized by substantial modifications of the BM microenvironment, including a switch from a T helper type 1 (Th1) to a Th2 response, and accumulation of CD206+Tie2+ macrophages, which eventually favored angiogenesis and increased in microvessel density (MVD). Notably, MVD was also increased at diagnosis in the BM of MGUS and SMM patients that subsequently progressed to MM when compared with MGUS and SMM that remained quiescent.These findings suggest a multistep pathogenic process in MM, in which the immune system may contribute to angiogenesis and disease progression. They also suggest investigating MVD in asymptomatic patients as prognostic factor for the progression and outcome of this disease. DisclosuresNo relevant conflicts of interest to declare.

Clinicopathological significance and potential drug target of p15INK4B in multiple myeloma.

Multiple myeloma (MM) is a clonal malignancy characterized by the proliferation of malignant plasma cells in the bone marrow and the production of monoclonal immunoglobulin. In addition to genetic changes, gene hypermethylation is an alternative mechanism of tumor suppressor gene inactivation in MM. The cyclin-dependent kinase inhibitor 1 (CDKN2B or p15INK4B) gene lies adjacent to the tumor suppressor gene, cyclin-dependent kinase inhibitor 2 (CDKN2A), and is frequently mutated and deleted in a wide variety of tumors, including MM. However, there is a lack of systematic analysis of p15 epigenetic modification such as methylation in MM from different studies that can provide more powerful estimation of an effect. In this study, we have systematically reviewed the studies of p15INK4B promoter methylation in MM and quantified the association between p15INK4B promoter methylation and MM using meta-analysis methods. We observed that the frequency of p15INK4B methylation is significantly higher in MM patients than in normal healthy controls. The pooled odds ratio (OR) from ten studies including 394 MM and 99 normal individuals is 0.08, while confidence interval (CI) is 0.03–0.21 (P<0.00001). This indicates that p15INK4B inactivation through methylation plays an important role in the pathogenesis of MM. In addition, the frequency of p15INK4B methylation was significantly higher in patients with MM than in those with asymptomatic monoclonal gammopathy of undetermined significance. The pooled OR from four studies is 0.40, 95% CI =0.21–0.78 (P=0.007). These results suggest that silencing of p15INK4B gene expression by epigenetic modification such as promoter hypermethylation plays a role not only in the initiation of MM but also in plasma cell malignant transformation, disease progression, and development.

Differentiating asymptomatic monoclonal gammopathy (AMG including MGUS and AMM) from clinical multiple myeloma (CMM) by gene expression profiling of purified plasma cells (PC-GEP).

8604 Background: Previous research indicated that PC from AMG and CMM could not be distinguished at the GEP level. We reported that a GEP70 risk score could identify a subset of AMG patients at hig...

Serial exome analysis of disease progression in premalignant gammopathies

Serial exome analysis of disease progression in premalignant gammopathies

Clinical, genomic, and imaging predictors of myeloma progression from asymptomatic monoclonal gammopathies (SWOG S0120)

AbstractAll cases of clinical myeloma (CMM) are preceded by an asymptomatic monoclonal gammopathy (AMG), classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM). We analyzed data from AMG patients (n = 331) enrolled in a prospective, observational clinical trial (S0120). Baseline data from clinical variables, gene expression profiles (GEP) of purified tumor cells, and findings of magnetic resonance imaging (MRI) were correlated with the risk of progression to CMM requiring therapy. GEP of purified tumor cells revealed that all molecular subtypes of CMM are also represented in the AMG phase. An increased risk score (>-0.26) (based on a 70-gene signature, GEP70) was an independent predictor of the risk of progression to CMM. Combination of elevated serum free light chain, M-spike, and GEP70 risk score identified a subset with high risk (67% at 2 years) of progression to CMM requiring therapy. Importantly, absence of these factors in AMM patients predicted low risk similar to MGUS. Detection of multiple (>1) focal lesions by MRI also conferred an increased risk of progression. These data demonstrate that signatures associated with high-risk CMM impact disease risk and support inclusion of genomic analysis in the clinical management of AMGs. This trial was registered at www.clinicaltrials.gov as # NCT00900263.Key Points

Plasma Circulating Proteasomes As Biomarkers Along Natural History Of Asymptomatic Monoclonal Gammopathies

BackgroundMonoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic plasma cell dyscrasias with a heterogeneous probability to progress to symptomatic multiple myeloma (MM). Reliable markers for progression to MM are vital to advance the understanding of myeloma precursor disease and for the development of intervention trials designed to delay/prevent MM. The Mayo Clinic and Spanish PETHEMA have proposed models to stratify patient risk based on clinical parameters. At the current time, no molecular biomarkers have been established to determine risk of transformation. Based on the fact that MM tumor cells are highly sensitive to proteasome inhibition and that circulating proteasomes (cProt) have been detected in the blood of MM patients, we conducted a prospective clinical study designed to characterize patterns of cProt in peripheral blood from MGUS, SMM and MM patients. Patients and MethodsNinety two patients diagnosed with asymptomatic monoclonal gammopathies (39 MGUS and 53 SMM; median age 63 years; 46M/47F) were studied. This group was compared to normal sera from healthy donors (n=6) and untreated patients with recent diagnosed MM (n=38). Initial baseline demographics, clinical and laboratory data were collected. MGUS patients were classified according to Mayo Clinic risk score (M-protein, monoclonal isotype and serum FLC), while SMM could be stratified according to PETHEMA (malignant bone marrow plasma cell (BMPC) percentage and immunoparesis) and Mayo system (BMPC infiltration, serum M-protein and serum FLC). Plasma and bone marrow supernatant samples were collected at diagnosis and frozen to -80ºC. In 58 MGUS and SMM cases, sequential plasma samples at 6 months and 1 year were also analyzed. Chymotrypsin-like, caspase-like, and trypsin-like activities from cProt were assayed by continuously monitoring the production of 7-amino-4-methylcoumarin (AMC) from fluorogenic peptides by plasma. Briefly, samples were activated with SDS (for chymotrypsin-like and caspase-like) or 10% Tween-20 (for trypsin-like). The reaction wells contained 30 μL assay buffer (25 mmol/L HEPES), 10 μL activated sample, and 10 μL of the prospective fluorogenic peptide-AMC substrate. To measure the fluorescence release of free AMC with time, the SpectraMax M5 (Molecular Devices) instrument was used with a read interval of 1 min during 30 min at 37ºC. All samples were performed by triplicate. Enzymatic activities were quantified (pmol AMC/s/mL plasma) by generating a standard curve of AMC. ResultsMGUS patients had zero (38.5%), one (41%) or two risk factors (20.5%) according to the Mayo Clinic model. In contrast, 49% of the patients with SMM were classified as high-risk according to the PETHEMA model, versus 69.8% with 2 or 3 risk factors in the Mayo Clinic model. Chymotrypsin activity levels in plasma were statistically correlated with serum M-protein concentration and total IgG concentration (p<0.001). Chymotrypsin-like activity was differentially expressed in plasma across the different groups of patients (p=0.009; Figure 1). Particularly, SMM and MM showed higher levels than healthy controls and MGUS patients. In SMM, patients with highest-risk of transformation showed a higher levels of this chymotrypsin-like activity than the other groups (p=0.02). When only IgG SMM and MGUS patients were considered, a correlation with immunoparesis (reduction of IgM and IgA), BMPC infiltration, relative lower hemoglobin levels and higher FLC ratio (p<0.05) was observed. Caspase-like activity was also associated with diagnosis, showing higher levels in symptomatic and SMM patients than healthy donors and MGUS (p=0.016) (Figure 2) and correlated with IgG and serum M-protein (p=0.01 and p=0.006). In contrast, trypsin-like levels were negatively correlated along the spectrum of tumoral mass in the four groups (p=0.004) (Figure 3). Bone marrow supernatant chymotrypsin activity was higher in symptomatic MM than MGUS patients (p=0.004), with a trend for caspase. [Display omitted] [Display omitted] [Display omitted] ConclusionChymotrypsin-and caspase-like activity of circulating proteasome in asymptomatic gammopathies is related to tumoral mass and immunoparesis degree. MGUS patients are close to healthy individuals, with SMM not so different than symptomatic patients. Prognostic significance of these findings after longer follow-up is warranted. Disclosures:No relevant conflicts of interest to declare.

Clinical, genomic, and imaging predictors of malignancy: Analysis of the first U.S. cooperative group prospective clinical trial in asymptomatic monoclonal gammopathies (SWOG S0120).

8515 Background: Asymptomatic monoclonal gammopathies (AMG) are the most common plasma cell dyscrasia classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM). Clinical outcome in AMGs can be highly variable and there is an unmet need to identify newer clinical, genomic and imaging parameters from prospective studies to guide patient management. Methods: We analyzed clinical, genomic and imaging data from AMG patients (n=334) enrolled in a prospective observational clinical trial (S0120) conducted under the auspices of SWOG. Baseline data from clinical variables, as well as gene expression profiles of purified tumor cells and the findings on magnetic resonance imaging (MRI) were correlated with the risk of progression to symptomatic myeloma (MM) requiring therapy. Results: In addition to serum M spike, percent bone marrow plasma cells and the ratio of involved/uninvolved serum free light chains, the level of serum beta-2-microglobulin was associated with an increased risk of progression to clinical myeloma requiring therapy. Gene expression profiles (GEP) of purified tumor cells revealed that all of the known molecular GEP subtypes of human MM are also represented in the precursor phase. An increased risk score (&gt; - 0.26) based on a 70-gene signature (GEP70) was an independent predictor of the risk of progression to clinical MM requiring therapy. The presence of focal lesions on MRI also conferred an increased risk of disease progression but was not independent of other clinical and genomic features. Conclusions: These data represent the first comprehensive evaluation of clinical, genomic and imaging features of AMGs in the context of a prospective US-cooperative group trial, and demonstrate that while all genetic subtypes of MM have a precursor phase, genetic signatures previously associated with high risk myeloma also predict the risk of progression to clinical malignancy requiring therapy. These findings suggest the need to integrate both clinical assessment of tumor bulk and genomic properties of tumor cells in the clinical management of these patients. Clinical trial information: NCT00900263.

Differential diagnosis of monoclonal gammopathy of undetermined significance.

Monoclonal gammopathy of undetermined significance (MGUS) is an asymptomatic plasma cell disorder occurring in 4.2% of adults > 50 years of age, which can progress into symptomatic diseases either through proliferation of the plasma cell clone, giving rise to multiple myeloma and other lymphoplasmacellular neoplasms, or through organ damage caused by the monoclonal protein, as seen in light-chain amyloidosis and related conditions. Differential diagnosis of asymptomatic and symptomatic monoclonal gammopathies is the determinant for starting therapy. The criteria for determining end-organ damage should include markers of organ injury caused by the monoclonal protein. Patient assessment and optimal follow-up are now performed using risk stratification models that should also take into account the risk of developing AL amyloidosis. Patients with low-risk MGUS (approximately 40% of all MGUS patients) need limited assessment and very infrequent follow-up. The ongoing development of novel molecular biomarkers and advanced imaging techniques will improve the identification of high-risk patients who may benefit from early therapeutic intervention through innovative clinical trials.

DNA Flow Cytometry and Metaphase Cytogenetics Can Predict Progression of Asymptomatic Monoclonal Gammopathies (AMG) to Symptomatic Multiple Myeloma (MM).

Abstract 2915 Background:AMG is the most common plasma cell dyscrasia, currently classified as either monoclonal gammopathy of undetermined significance (MGUS) or asymptomatic multiple myeloma (AMM), based on the level of monoclonal immunoglobulin (M-protein), bone marrow plasmacytosis and other criteria defined by the International Myeloma Working Group. While information is available on the impact of clinical variables such as bone marrow plasmacytosis, free light chains, isotype and M-protein on the hazard of progression to symptomatic MM (MM), little is known about the value of the karyotype and DNA content, as determined by DNA/cIg flow cytometry, on the risk of progression from AMG to MM. Methods:Patients from the Myeloma Institute for Research and Therapy (MIRT) with AMG that were enrolled in a prospective observational clinical trial were evaluated. All patients underwent detailed clinical staging at entry and were followed at pre-specified intervals per protocol. Cox proportional hazards regression was used to model univariate and multivariate associations of baseline features with progression to MM. The number of distinct DNA stem lines in the flow cytometry assay, their percentages, respective DNA Indices (DI), cytoplasmic Immunoglobulin Indices (cIgI), and percent of cells in S phase were evaluated alone and in relation to the karyotype report at baseline. A DI between 0.99 and 1.01 referred to diploidy, lesser than 0.99 to hypodiploidy and more than 1.01 hyperdiploidy. Results:Data from 267 eligible MIRT patients with AMG were analyzed. Of these patients 99% (265/267) had performed DNA/cIg flow cytometry and had a karyotype report at diagnosis. Cytogenetic abnormalities were detected in 20 of the 265 patients from whom data were available. From the 265 patients from whom DNA/cIg flow cytometry data were available, no abnormal clones were identified in 14% (37/265), one clone was identified in 95 patients (36%), two clones in 122 patients (46%), three clones in 10 (4%), and in 1 patient 4 clones were identified. Most patients with abnormal DNA content had hyperdiploid clones (132/243 patients). The second most frequent finding was diploid DI, in 39% (104/243) of patients; 3% (7/243) had a hypodiploid DI. The median DI was 1.01 (0.9–2.02) and median cIg was 7 (1–50). Interestingly, the median cIgI value in AMG was more than twice that of its value (3.4, 1–22) in Total Therapy 3 MM patients (p=0.001). In univariate analysis of the parameters in this study, the presence of an abnormal karyotype (p=0.032, HR=2.62), the number of DNA/cIg clones (p=0.016, HR=1.69) and the percentage of the dominant clone (p=0.003, HR=1.03) were significantly related to progression to MM. Ploidy by DNA/cIg analysis, the S-phase fraction, and cIg did not reach statistical significance (p=0.863, p=0.132 and p=0.240, respectively). In multivariate analysis, only the number of abnormal clones (p=0.013, HR=1.78) retained statistical significance, while the percentage of the dominant clone neared significance (p=0.070, HR=1.02). Using running log rank tests we were able to identify optimal cut-points for the percentage of the dominant clone and the number of clones (12% and 2 clones respectively). From these, a risk score was obtained which identifies three distinct groups with 3-yr MM progression probabilities of 12%, 30% and 67% (p<0.001) (Figure 1). Conclusions:Abnormal metaphase cytogenetics and DNA/cIg flow cytometry have a prognostic value in the prediction of progression of AMG to MM. Hyperdiploidy is the dominant finding in AMG, however, its presence or absence does not predict progression. Clonal heterogeneity, as portrayed through DNA/cIg flow cytometry analysis, with the number of abnormal clones and the percentage of the dominant clone were major prognostic factors for progression to MM. Taken together they identify three distinct subgroups with a low (12%), moderate (30%) and high (67%) probability of 3-year time to progression to MM. [Display omitted] Disclosures:Dhodapkar:Celgene: Research Funding; KHK: Research Funding.

Bone Marrow Biopsy May Be Required During the Initial Evaluation of Individuals with Asymptomatic Monoclonal Gammopathy

Abstract 5067The incidental finding of a monoclonal gammopathy during workup for various conditions or in the context of a routine check-up is increasingly common. Several “patients” are then referred for diagnostic evaluation of their monoclonal gammopathy and additional workup is needed. It has been proposed that a bone marrow (BM) aspirate and biopsy is indicated when the monoclonal protein (M-protein) is ≥1.5 g/dL, when abnormalities are noted in the complete blood cell count, serum creatinine level, serum calcium level, or radiographic bone survey, in individuals with non-IgG monoclonal gammopathy and in those with an abnormal serum free light chain (FLC) ratio. The aim of this study was to identify factors that could aid in the evaluation of individuals presenting with asymptomatic monoclonal gammopathy and in whom invasive diagnostic testing with a bone marrow biopsy is considered.Thus, we analyzed our database and identified patients who were referred to the Department of Clinical Therapeutics of the University of Athens, Greece, for evaluation of asymptomatic monoclonal gammopathy and in whom a BM trephine biopsy, a serum and urine protein electrophoresis (SPEP) with immunofixation and quantitative immunoglobulins were performed. SPEPs were scanned and M-protein was measured using imaging analysis software. Patients with a monoclonal M-protein ≥ 3 g/dl (30 g/L), i.e. those diagnosed with asymptomatic/smoldering myeloma (SMM) or Waldenstrom’s macorglobulinemia based on the standard criteria, were not included in the analysis. Clonality of BM plasma cells or lymphoplasmacytes was assessed by immunohistochemistry. Patients who eventually were diagnosed with plasma cell related conditions (i.e. amyloidosis, peripheral neuropathy, dermatoses, etc.) were also excluded from the analysis.Our analysis included 161 patients: 53% were females, median age was 64 year (range 33–89 years), 53% had a monoclonal IgG protein, 15.5% had a monoclonal IgA protein, 24% a monoclonal IgM protein and 2.5% had only a monoclonal light chain, while 4% had a biclonal protein. In 64% of patients the monoclonal light chain was kappa and in 37% was lambda. The median serum M-protein was 0.948 g/dl (range 0.1–2.99 g/dl); 52% of patients had an M-protein of <1 g/dl and 79% of <2 g/dl. Immunoparesis of at least one of the uninvolved immunoglobulins was present in 38% of cases and of both of the uninvolved immunoglobulins in 6%. Median BM infiltration by monoclonal plasma cells or lymphoplasmacytes was 15%. In 66.5% of individuals there was a BM infiltration of ≥10% by monoclonal plasma cells or lymphoplasmacytes, while in 10% of the studied cases the BM infiltration was ≥50%.A significant correlation of the size of M-protein and of the infiltration of the BM was found (R=0.592, p<0.001). However, 27% of patients with M-protein <0.5 g/dl had ≥10% clonal plasma cells or lymphoplasmacytes in their BM biopsies. The respective rates were 46% for those with M-protein <1 g/dl, 54% for those with M-protein 1.5 g/dl and 58% for those with M-protein <2 g/dl.Ninety per cent of those who had immunoparesis of at least one of the uninvolved immunoglobulins had ≥10% clonal plasma cells or lymphoplasmacytes. A BM infiltration of ≥10% was more frequent in individuals with a monoclonal IgG or IgA protein (72% and 80%, respectively) vs. 45% of those with a monoclonal IgM protein (p=0.015). Light chain isotype, age and gender were not predictive of the degree of BM plasma cell infiltration.In multivariate analysis, immunoparesis of at least one of the uninvolved immunoglobulins (OR: 6.45, 95% CI: 2.32–18, p<0.001), an IgG or IgA monoclonal protein (OR: 2.67, 95% CI: 1.1–6.4, p=0.028) and an M-protein of ≥1 g/dl (OR: 5.4, 95% CI: 2.23–13) were independently associated with the presence of ≥10% of clonal infiltration in BM biopsy. By combining the above risk factors we found that in those who had all three, 97% had ≥10% clonal cells in the BM biopsy, while in those with 0–1 of the above factors the probability to find ≥10% clonal cells was 43%. These findings indicate that even patients with low risk for BM infiltration by clonal plasma cells, may be diagnosed as SMM when a BM biopsy is performed.In conclusion, our data on a large number of individuals with asymptomatic monoclonal gammopathy who underwent a BM biopsy may indicate that the latter exam may provide useful information and could be included in the standard initial workup of these individuals. Disclosures:No relevant conflicts of interest to declare.

Bortezomib for ITP: First Report of Successful Treatment of Refractory ITP In a Patient with Otherwise Asymptomatic Monoclonal Gammopathy

AbstractAbstract 4667Current concepts of the pathophysiology of primary immune thrombocytopenia (ITP) reflect a complex interplay between antiplatelet antibodies, cytotoxic T-lymphocytes (CTL), T regulatory cells, and impaired bone marrow megakaryopoiesis. The mechanisms of ITP in the presence of monoclonal gammopathy of unknown significance (MGUS), a form of secondary ITP, are less well understood, yet treatment has been essentially the same as for primary ITP. Inhibition of antiplatelet CTL responses through apoptosis induction in clonal antiplatelet-antibody producing plasma cells and proteasome inhibition in CTLs may be achieved with bortezomib. However, bortezomib may cause transient isolated thrombocytopenia via presumed inhibition of marrow thrombopoiesis or enhanced platelet clearance peripherally.We used bortezomib in a 55 year-old female with IgG lambda monoclonal gammopathy and ITP refractory to previous treatment with corticosteroids, intravenous immunoglobulin, splenectomy, danazole, and rituximab, none of which induced a durable increase in her platelet counts to hemostatic concentrations. She suffered from repeated episodes of prolonged epistaxis, and when the platelet count fell below 10K/mm3, she experienced a life-threatening gastrointestinal hemorrhage. Otherwise, she had no anemia, her creatinine and calcium remained normal throughout, and skeletal surveys showed no evidence for myeloma. Repeated bone marrow examinations, reviewed at our institution, demonstrated lambda monotypic plasma cells always below 10%, and a normal female karyotype on metaphase cytogenetics. Her thrombocytopenia and bleeding episodes improved transiently on dexamethasone/IMiD combinations (thalidomide followed by lenalidomide), with concurrent reduction in total IgG by almost 50% (M-spike was not measured prior to IMiDs), however she became intolerant to thalidomide due to peripheral neuropathy and experienced diarrhea on lenalidomide. Her platelets decreased and her epistaxis worsened as her m-spike increased on dexamethasone single agent. We thus focused our treatment approach on more robust suppression of the monoclonal protein and initiated a course of weekly bortezomib (1.3 mg/m2), with dexamethasone (20 mg) on the day of and the day after bortezomib. After one month of therapy, the platelet count normalized, and she had no further bleeding episodes. The bortezomib dose was reduced to 1 mg/m2 and dexamethasone reduced to 20 mg weekly after 2 months, with persistently normal platelet counts thereafter. Her ITP resolution on bortezomib was accompanied by a decrease of her serum M-protein from 1.04 to 0.39 g/dL.This case illustrates that bortezomib can be remarkably effective in refractory ITP with concurrent monoclonal gammopathy. Assuming similar pathophysiology, it might also have efficacy in primary refractory ITP. This case also raises the question of whether refractory ITP in the presence of a clonal plasma cell disorder should be considered as a myeloma defining symptom. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CDK2 Phosphorylation of Smad2 Disrupts TGF-β Transcriptional Regulation in Resistant Primary Bone Marrow Myeloma Cells

Resistance to growth suppression by TGF-beta1 is common in cancer; however, mutations in this pathway are rare in hematopoietic malignancies. In multiple myeloma, a fatal cancer of plasma cells, malignant cells accumulate in the TGF-beta-rich bone marrow due to loss of both cell cycle and apoptotic controls. Herein we show that TGF-beta activates Smad2 but fails to induce cell cycle arrest or apoptosis in primary bone marrow myeloma and human myeloma cell lines due to its inability to activate G(1) cyclin-dependent kinase (CDK) inhibitors (p15(INK4b), p21(CIP1/WAF1), p27(KIP1), p57(KIP2)) or to repress c-myc and Bcl-2 transcription. Correlating with aberrant activation of CDKs, CDK-dependent phosphorylation of Smad2 on Thr(8) (pT8), a modification linked to impaired Smad activity, is elevated in primary bone marrow myeloma cells, even in asymptomatic monoclonal gammopathy of undetermined significance. Moreover, CDK2 is the predominant CDK that phosphorylates Smad2 on T8 in myeloma cells, leading to inhibition of Smad2-Smad4 association that precludes transcriptional regulation by Smad2. Our findings provide the first direct evidence that pT8 Smad2 couples dysregulation of CDK2 to TGF-beta resistance in primary cancer cells, and they suggest that disruption of Smad2 function by CDK2 phosphorylation acts as a mechanism for TGF-beta resistance in multiple myeloma.

Smoldering multiple myeloma and monoclonal gammopathy of undetermined significance

Smoldering multiple myeloma (SMM) consists of the presence of a serum M protein of 30 g/L or more and/or 10% or more bone marrow plasma cells (BMPCs), with no clinical manifestations or symptoms of myeloma. It accounts for approximately 10% of all myelomas, and the median time to progression to a symptomatic multiple myeloma ranges from 2 to 3 years. The main factors for progression are the plasma cell mass (M-protein size and percent of BMPCs), the spinal MRI pattern, the plasma cell proliferative index, and the variant of SMM ("evolving" vs "nonevolving"). Although treatment with thalidomide is promising (based on the results of two phase II trials), outside the context of a clinical trial, a watch-and-wait approach with clinical evaluation every 4 months is recommended until evident symptomatic disease progression occurs. Patients with monoclonal gammopathy of undetermined significance (MGUS) have a serum M protein lower than 30 g/L and a proportion of BMPCs of less than 10%, with no clinical findings or symptoms attributable to the monoclonal gammopathy. MGUS has a high prevalence, and its annual rate of malignant transformation is 1%, such that the actuarial probability of progression to a symptomatic monoclonal gammopathy at 25 years of follow-up is as high as 40%. The factors associated with a higher probability of malignant transformation are a relatively high plasma cell mass, immunoglobulin A M-protein type, and the "evolving" variant. It is recommended that patients with MGUS are monitored annually. Importantly, patients with asymptomatic monoclonal gammopathies must not be treated before the development of overt multiple myeloma.

Zoledronate for early-stage, untreated myeloma: An interim report of a randomized study

6680 Background: We have recently demonstrated that “prophylactic” administration of pamidronate is not useful to prevent or delay disease progression in patients with untreated, early-stage myeloma, although this treatment may reduce the number of cases developing skeletal events, thus possibly changing the clinical manifestations of the disease at progression (Musto et al, Leuk Lymphoma 2003; 44: 1545 and J Clin Oncol 2003; 21: 3177). No data are available about the effect of zoledronate (ZOL), a third generation bisphosphonate, in this specific setting of patients. Methods: Starting on June, 2001, all our patients with asymptomatic monoclonal gammopathy, fulfilling the diagnostic criteria of stage IA, IIA or “smouldering” myeloma, are invited to participate into an on-going clinical, randomized trial comparing one-year administration of ZOL alone vs simple observation. The aim of this study was to establish whether ZOL may modify the natural history of the disease, in terms of rate, time and type of progression, in these otherwise untreated patients. As of December, 2003, sixty-two patients, out of a planned accrual of 120 patients, have been randomized (thrity-one per arm) to receive or not ZOL (Zometa, Novartis Pharmaceuticals. Origgio, Italy) for one year or until progression, on an out-patient basis, at the dose of 4 mg as 15' i.v. single monthly infusion. Results: No significant adverse events have been so far recorded. In the observational arm two patients were lost at follow-up after six and twenty months, respectively. Asymptomatc hypocalcemia occurred in four of ZOL-treated patiens. After a median follow-up of 17 months there have been three (9.6%) progressions requiring treatment in the ZOL group and five (17.2%) within the controls (p.n.s.). Median time-to-progression was 17 and 14.3 months, respectively (p.n.s.). Bone lesions and/or hypercalcemia at the time of progression were observed in 4/5 of controls, and in 1/3 of ZOL-treated patients. Conclusions: In this interim analysis, a moderate trend in favour of ZOL-arm was seen. The final evaluation of our trial, including a larger number of patients with prolonged follow-up, is required to clarify whether the use of ZOL may be clinically useful in initial stages of myeloma. No significant financial relationships to disclose.

Zoledronate for early-stage, untreated myeloma: An interim report of a randomized study

6680 Background: We have recently demonstrated that “prophylactic” administration of pamidronate is not useful to prevent or delay disease progression in patients with untreated, early-stage myeloma, although this treatment may reduce the number of cases developing skeletal events, thus possibly changing the clinical manifestations of the disease at progression (Musto et al, Leuk Lymphoma 2003; 44: 1545 and J Clin Oncol 2003; 21: 3177). No data are available about the effect of zoledronate (ZOL), a third generation bisphosphonate, in this specific setting of patients. Methods: Starting on June, 2001, all our patients with asymptomatic monoclonal gammopathy, fulfilling the diagnostic criteria of stage IA, IIA or “smouldering” myeloma, are invited to participate into an on-going clinical, randomized trial comparing one-year administration of ZOL alone vs simple observation. The aim of this study was to establish whether ZOL may modify the natural history of the disease, in terms of rate, time and type of progression, in these otherwise untreated patients. As of December, 2003, sixty-two patients, out of a planned accrual of 120 patients, have been randomized (thrity-one per arm) to receive or not ZOL (Zometa, Novartis Pharmaceuticals. Origgio, Italy) for one year or until progression, on an out-patient basis, at the dose of 4 mg as 15' i.v. single monthly infusion. Results: No significant adverse events have been so far recorded. In the observational arm two patients were lost at follow-up after six and twenty months, respectively. Asymptomatc hypocalcemia occurred in four of ZOL-treated patiens. After a median follow-up of 17 months there have been three (9.6%) progressions requiring treatment in the ZOL group and five (17.2%) within the controls (p.n.s.). Median time-to-progression was 17 and 14.3 months, respectively (p.n.s.). Bone lesions and/or hypercalcemia at the time of progression were observed in 4/5 of controls, and in 1/3 of ZOL-treated patients. Conclusions: In this interim analysis, a moderate trend in favour of ZOL-arm was seen. The final evaluation of our trial, including a larger number of patients with prolonged follow-up, is required to clarify whether the use of ZOL may be clinically useful in initial stages of myeloma. No significant financial relationships to disclose.

The Pathogenesis of Acquired von Willebrand's Disease in A Patient with Asymptomatic Monoclonal Gammopathy; Developed Malignant Lymphoma at 30 Months after The Bruising Onset

The Pathogenesis of Acquired von Willebrand's Disease in A Patient with Asymptomatic Monoclonal Gammopathy; Developed Malignant Lymphoma at 30 Months after The Bruising Onset

The Laboratory Diagnosis of Plasma Cell Myeloma and Related Disorders

Plasma cell myeloma constitutes the commonest primary malignant tumor affecting the skeleton of the adult. It is also one of several forms of monoclonal gammopathy that include Waldenström's macroglobulinemia, the heavy chain diseases, "benign" (nonprogressive, asymptomatic) monoclonal gammopathy, and primary amyloidosis. The diagnosis of classic or advanced myeloma rests on the findings of sheets of plasmacytic cells in a bone marrow biopsy sample or aspirate; a serum monoclonal immunoglobulin usually of the IgG or IgA class or Bence Jones protein in the urine; and evidence of multiple lytic lesions in x-ray views of the skeleton. Pitfalls in the diagnosis of earlier or unusual forms of myeloma require a careful application of classic diagnostic criteria or of criteria established by clinical cooperative groups.

The Evolution of Asymptomatic Monoclonal Gammopathies.

Twenty cases of "asymptomatic" monoclonal gammopathies were detected by routine electrophoresis in patient's sera or in blood donors and were followed over 3-14 years. Four cases have shown a malignant evolution-two evolved toward Waldenström's macroglobulinemia after 3 years and two could be classified as myeloma 3 and 7 years, respectively, after detection of the monoclonal protein. The remaining cases were still "asymptomatic" 4 years later (7 cases), 7-9 years later (8 cases) and 14 years later (1 case). A malignant evolution occurred in approximately 20% of cases.