[Show abstract][Hide abstract] ABSTRACT: Monoclonal gammopathies (MGs) are hematological diseases characterized by high levels of a monoclonal immunoglobulin (Ig) or M-protein. Within this group are patients with more than one M-protein, referred to as double MGs (DMGs). The M-proteins in DMG patients may have different heavy chain (HC) isotypes that are associated with different light chains (LCs), or different HCs that are LC matched. In this study, we examined the clonal relatedness of the M-proteins in the latter type in a cohort of 14 DMG patients. By using PCR, we identified 7/14 DMG patients that expressed two Ig HC isotypes with identical Ig HC variable (IGHV), diversity (IGHD), joining (IGHJ), and complementarity determining region (HCDR3) sequences. Two additional DMG patients had two Ig transcripts using the same IGHV, IGHD and IGHJ genes but with slight differences in variable region or HCDR3 mutations. LC analysis confirmed that a single LC was expressed in 3/7 DMG patients with identical HC transcripts and in the two DMGs with highly similar transcripts. The PCR findings were confirmed by immunofluorescence for HC and LC expression. Clonally related HC-dissimilar/LC-matched DMGs may occur often and defines a new subtype of MG that may serve as a tool for studies of disease pathogenesis.
Preview · Article · Apr 2013 · Blood Cancer Journal
[Show abstract][Hide abstract] ABSTRACT: Epidemiological data have suggested that African Americans (AA) are twice as likely to be diagnosed with multiple myeloma (MM) as compared to European Americans (EA). Here, we have analyzed a set of cytogenetic and genomic data derived from AA and EA MM patients. We have compared the frequency of IgH translocations in a series of data from 115 AA patients from three studies and EA patients from the Eastern Cooperative Oncology Group (ECOG) studies E4A03 and E9487. We have also interrogated tumors from 45 AA and 196 EA MM patients for somatic copy number abnormalities associated with poor outcome. In addition, 35 AA and 178 EA patients were investigated for a transcriptional profile associated with high-risk disease. Overall, based on this cohort, genetic profiles were similar except for a significantly lower frequency of IgH translocations (40% vs. 52%; p=0.032) in AA patients. Frequency differences of somatic copy number aberrations were not significant after correction for multiple testing. There was also no significant difference in the frequency of high-risk disease based upon gene expression profiling. Our study represents the first comprehensive comparisons of the frequency and distribution of molecular alterations in MM tumors between AA and EA patients. (ECOG E4A03 is registered with ClinicalTrials.gov, number NCT00098475. ECOG E9487 is a companion validation set to the ECOG study E9486 and is registered with the National Institutes of Health, National Cancer Institute, Clinical Trials (PDQ), number EST-9486.).
[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is preceded by the asymptomatic pre-malignant state, monoclonal gammopathy of undetermined significance (MGUS). Although MGUS patients may remain stable for years, they are at increased risk of progressing to MM. A better understanding of the relevant molecular changes underlying the transition from an asymptomatic to symptomatic disease state is urgently needed. Our studies show for the first time that the CD147 molecule (extracellular matrix metalloproteinase inducer) may be having an important biological role in MM. We first demonstrate that CD147 is overexpressed in MM plasma cells (PCs) vs normal and pre-malignant PCs. Next, functional studies revealed that the natural CD147 ligand, cyclophilin B, stimulates MM cell growth. Moreover, when MM patient PCs displaying bimodal CD147 expression were separated into CD147(bright) and CD147(dim) populations and analyzed for proliferation potential, we discovered that CD147(bright) PCs displayed significantly higher levels of cell proliferation than did CD147(dim) PCs. Lastly, CD147-silencing significantly attenuated MM cell proliferation. Taken together, these data suggest that the CD147 molecule has a key role in MM cell proliferation and may serve as an attractive target for reducing the proliferative compartment of this disease.
Preview · Article · Mar 2012 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: Lenalidomide with dexamethasone is a standard induction treatment regimen for newly diagnosed myeloma (although a Federal Drug Administration indication is still absent). In the context of the Phase 3 clinical trial E4A03 (lenalidomide plus dexamethasone in low or high doses), we queried whether a fluorescence in situ hybridization (FISH)-based genetic classification into high risk (HR) and standard risk (SR) multiple myeloma (MM) would remain clinically significant. Of 445 E4A03 patients, 126 had FISH analysis; 21 were classified HR with t(4;14), t(14;16), or 17p13 deletions. Median survival follow-up approached 3 years. Patients with FISH data tended to be younger and healthier compared to the rest of the study population and, consequently, had superior overall survival (OS) results. Within the FISH cohort, shorter OS in the HR versus SR group (P = 0·004) corresponded to a hazard ratio of 3·48 [95% confidence interval: (1·42-8·53)], an effect also observed in multivariate analysis. Two-year OS rates were 91% for SR MM and 76% for HR MM. There was also evidence of interaction between risk status and treatment (P = 0·026). HR patients were less likely to attain good partial response (SR 46% and HR 30%, Odds Ratio = 2·0 [0·7-5·6]), but overall response rates were not different. FISH-based risk classification retained prognostic significance in patients receiving lenalidomide-based induction.
Full-text · Article · Sep 2011 · British Journal of Haematology
[Show abstract][Hide abstract] ABSTRACT: Detection of specific chromosomal abnormalities by FISH and metaphase cytogenetics allows risk stratification in multiple myeloma; however, gene expression profiling (GEP) based signatures may enable more specific risk categorization. We examined the utility of 2 GEP-based risk stratification systems among patients undergoing initial therapy with lenalidomide in the context of a phase 3 trial. Among 45 patients studied at baseline, 7 (16%) and 10 (22%), respectively, were high-risk using the GEP70 and GEP15 signatures. The median overall survival for the GEP70 high-risk group was 19 months versus not reached for the rest (hazard ratio = 14.1). Although the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk patients. The C-statistic for the GEP70, GEP15, and FISH based risk stratification systems was 0.74, 0.7, and 0.7, respectively. Here we demonstrate the prognostic value for GEP risk stratification in a group of patients primarily treated with novel agents. This trial was registered at www.clinicaltrials.gov as #NCT00098475.
[Show abstract][Hide abstract] ABSTRACT: DNA double-strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX has an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase-like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.
No preview · Article · May 2011 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: Waldenström's macroglobulinemia (WM) is a distinct clinicobiological entity defined as a B-cell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow (BM) and IgM paraprotein production. Cytogenetic analyses were historically limited by difficulty in obtaining tumor metaphases, and the genetic basis of the disease remains poorly defined. Here, we performed a comprehensive analysis in 42 WM patients by using a high-resolution, array-based comparative genomic hybridization approach to unravel the genetic mechanisms associated with WM pathogenesis. Overall, 83% of cases have chromosomal abnormalities, with a median of three abnormalities per patient. Gain of 6p was the second most common abnormality (17%), and its presence was always concomitant with 6q loss. A minimal deleted region, including MIRN15A and MIRN16-1, was delineated on 13q14 in 10% of patients. Of interest, we reported biallelic deletions and/or inactivating mutations with uniparental disomy in tumor necrosis factor (TNF) receptor-associated factor 3 and TNFalpha-induced protein 3, two negative regulators of the nuclear factor-kappaB (NF-kappaB) signaling pathway. Furthermore, we confirmed the association between TRAF3 inactivation and increased transcriptional activity of NF-kappaB target genes. Mutational activation of the NF-kappaB pathway, which is normally activated by ligand receptor interactions within the BM microenvironment, highlights its biological importance, and suggests a therapeutic role for inhibitors of NF-kappaB pathway activation in the treatment of WM.
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
Full-text · Article · Jul 2008 · Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
[Show abstract][Hide abstract] ABSTRACT: The immunoglobulin free light chain (FLC) assay is an invaluable tool for following patients with oligosecretory plasma cell dyscrasia. Baseline values have also been shown to be prognostic in all plasma cell disorders tested. A looming question, however, is the role it should play in following myeloma patients with disease that is measurable using serum and urine electrophoresis. We used the data and stored samples from a mature Eastern Cooperative Oncology Group clinical trial (E9486) to assess serum levels of FLC at baseline and after 2 months of alkylator-based therapy. For serial determinations, the absolute level of involved serum FLC or the difference of the involved and uninvolved FLC is preferred over the ratio of involved to uninvolved FLC. FLC response after 2 months of therapy was superior to early M-protein measurement to predict overall response. The ideal cut-point for FLC change appears to be between 40% and 50% reduction. The correlation between serial measurements of serum FLC and urine M-protein is inadequate to abolish the serial 24-hour urine protein. Although baseline values of FLC are prognostic in newly diagnosed myeloma patients, serial measurements do not appear to have added value in patients who have M-proteins measurable by electrophoresis.
[Show abstract][Hide abstract] ABSTRACT: The Multiple Myeloma Research Consortium has established a tissue bank for the deposition of bone marrow samples from patients with multiple myeloma to be mailed and processed under good laboratory practices. To date, over 1,000 samples have been collected. At this time, limited information is available on shipped bone marrow aspirates in regards to cell viability, yield, purity, and subsequent RNA yield and quality. To test these determinants, we did a pilot study on behalf of the Multiple Myeloma Research Consortium where samples were drawn at Mayo Clinic Rochester (MCR) pooled and split into two equal aliquots. One-half of each sample was processed following good laboratory practices compliant standard operating procedures, immediately after sample procurement, at MCR. The CD138+ cells were stored at -80 degrees C as a Trizol lysate. The other half of the aspirate was sent overnight to Mayo Clinic Scottsdale where they were processed using identical standard operating procedures. The RNA was extracted and analyzed in a single batch at MCR. At both locations, samples were assayed for the following quality determinants: Viability was assessed using a three-color flow cytometric method (CD45, CD38, and 7-AAD). Cell counts were done to determine plasma cell recovery and post-sort purity determined by means of a slide-based immunofluorescent assay. RNA recovery and integrity was assessed using the Agilent Bioanalyzer. Lastly, gene expression profiles were compared to determine the signature emanating from the shipment of samples. Despite minor differences, our results suggest that shipment of samples did not significantly affect these quality determinants in aggregate.
[Show abstract][Hide abstract] ABSTRACT: The biological and clinical implications of p16 gene methylation in multiple myeloma (MM) are still unclear despite previous studies. In this comprehensive study, using methylation-specific PCR (MS-PCR), we show that p16 methylation is relatively common and occurs in monoclonal gammopathy of undetermined significance (MGUS; n=17), smoldering multiple myeloma (SMM; n=40), and MM (n=522) at a prevalence of 24%, 28%, and 34%, respectively. However, p16 methylation does not appear to affect gene expression level. In a large cohort of patients with long-term follow-up information (n=439), there was no difference in overall survival between patients with or without p16 methylation. We also found no association between p16 methylation and the main cytogenetic categories, although it was more common among patients with 17p13.1 deletions (p53 locus), a genetic progression event in MM. In addition, p16 methylation has no apparent effect on the cycle because there was also no difference in the plasma cell labeling index (a direct measurement of proliferation) between patients with and without p16 methylation. Our results question a major role for p16 methylation in the oncogenesis of the PC neoplasm, and we now believe p16 methylation may be a marker for overall epigenetic changes associated with disease progression, with no obvious direct biological or clinical consequences.
[Show abstract][Hide abstract] ABSTRACT: A specific role for increased level of expression of CKS1B, as a consequence of chromosome 1q21 copy number gain, has been postulated as both pathogenic, as well as a powerful clinical prognostic factor in multiple myeloma (MM). The purpose of this study is to determine the clinical associations and prognostic impact of copy number gain at chromosome 1q21 (with a bacteria artificial chromosome clone containing CKS1B) and CKS1B gene level of expression in MM. We studied the chromosome region 1q21 for copy number change in a cohort of myeloma patients treated by high-dose therapy with stem-cell rescue (HDT) (n = 159). A separate cohort of patients, treated by HDT was studied for CKS1B messenger RNA expression by gene expression profiling (n = 67). 1q21 gain was then correlated with clinical parameters and survival. Gain of 1q21 copy number was detected in about a third of MM and was associated with more proliferative disease and poor-risk cytogenetic categories such as t(4;14), and chromosome 13 deletion. Both 1q21 gain and increase gene expression level were significantly associated with reduced survival. However, neither is an independent prognostic marker in MM on multivariate Cox proportional hazard analysis.
[Show abstract][Hide abstract] ABSTRACT: Fluorescence in situ hybridization (FISH) is more sensitive than conventional cytogenetics for recognizing chromosomal changes. Several FISH-detected abnormalities have been associated with inferior prognosis, including deletion of chromosomes 17 and 13 (Delta13) and t(4;14)(p16.3;q32). We analyzed the prognostic value of FISH testing in 238 patients who received high-dose therapy between January 1990 and September 2001. All patients had pretransplantation cytoplasmic immunoglobulin FISH done on cytospin slides from bone marrow aspirates for t(11;14), t(4;14), and -17(p13.1) (TP53). Time to progression and overall survival were significantly shorter for patients with t(4;14) and those with -17(p13.1) but were not affected by t(11;14). Overall survival was significantly shorter for patients with both t(4;14) and Delta13 abnormalities than for those with Delta13 alone (26.8 vs 18.8 months). In a multivariable analysis of the effect of Delta13 and t(4;14), the risk ratio for t(4;14) was greater than for Delta13 (2.6 vs 1.5). For high-dose therapy patients, -17(p13) and t(4;14) have clinical importance for estimating time to progression and overall survival. The presence of t(4;14) identifies a subset of patients whose time to progression is only 8.2 months. These patients receive minimal benefit from autologous stem cell transplantation and are candidates for novel therapeutic approaches.
[Show abstract][Hide abstract] ABSTRACT: Nonrandom recurrent chromosomal abnormalities are ubiquitous in multiple myeloma (MM) and include, among others, translocations of the immunoglobulin heavy chain locus (IgH). IgH translocations in MM result in the up-regulation of oncogenes, and include more commonly t(11;14)(q13;q32), t(4;14)(p16;q32), and t(14;16)(q32;q23). Based on the recurrent nature of these translocations and their finding since the early stages of the plasma cell (PC) disorders, we hypothesized that they would confer biologic and clinical variability. In addition, deletions of 13q14 and 17p13 have also been associated with a shortened survival. We used cytoplasmic Ig-enhanced interphase fluorescent in situ hybridization to detect deletions (13q14 and 17p13.1), and translocations involving IgH in 351 patients treated with conventional chemotherapy entered into the Eastern Cooperative Oncology Group clinical trial E9486/9487. Translocations were frequently unbalanced with loss of one of the derivative chromosomes. The presence of t(4; 14)(p16;q32) (n = 42; 26 vs 45 months, P <.001), t(14;16)(q32;q23) (n = 15; 16 vs 41 months, P =.003), - 17p13 (n = 37; 23 vs 44 months, P =.005), and - 13q14 (n = 176; 35 vs 51 months, P =.028) were associated with shorter survival. A stratification of patients into 3 distinct categories allowed for prognostication: poor prognosis group (t(4;14)(p16;q32), t(14; 16)(q32;q23), and - 17p13), intermediate prognosis (- 13q14), and good prognosis group (all others), with median survivals of 24.7, 42.3, and 50.5 months, respectively (P <.001). This molecular cytogenetic classification identifies patients into poor, intermediate, and good risk categories. More importantly it provides further compelling evidence that MM is composed of subgroups of patients categorized according to their underlying genomic aberrations.
[Show abstract][Hide abstract] ABSTRACT: The t(11;14)(q13;q32) results in up-regulation of cyclin D1 and is the most common translocation detected in multiple myeloma, where it is also associated with a lymphoplasmacytic morphology. We performed an interphase fluorescent in situ hybridization (FISH) study to determine the clinical and biologic significance of the abnormality when testing a large cohort of myeloma patients. Bone marrow slides from multiple myeloma patients entered into the Eastern Cooperative Oncology Group phase III clinical trial E9486 and associated laboratory correlative study E9487 were analyzed using interphase FISH combined with immune-fluorescent (cytoplasmic immunoglobulin-FISH) detection of clonal plasma cells. We used FISH probes that hybridize to the 14q32 and 11q13 chromosomal loci. The t(11;14)(q13;q32) was correlated with known biologic and prognostic factors. Of 336 evaluable patients, 53 (16%) had abnormal FISH patterns compatible with the t(11;14)(q13;q32). These patients appeared to be more likely to have a serum monoclonal protein of less than 10 g/L (1 g/dL) (28% vs 15%, P =.029) and a lower plasma cell labeling index (P =.09). More strikingly, patients were less likely to be hyperdiploid by DNA content analysis (n = 251, 14% vs 62%, P <.001). Patients with the t(11;14)(q13;q32) appeared to have better survival and response to treatment, although this did not reach statistical significance. Multiple myeloma with the t(11;14)(q13;q32) is a unique subset of patients, not only characterized by cyclin D1 up-regulation and a lymphoplasmacytic morphology, but is also more frequently associated with small serum monoclonal proteins and is much less likely to be hyperdiploid. These patients do not have a worsened prognosis as previously thought.
[Show abstract][Hide abstract] ABSTRACT: Chromosome 13 abnormalities (Delta13) have been associated with an unfavorable prognosis in patients with multiple myeloma (MM). The significance of this has been unresolved because of diverse methods of detection and heterogeneous groups of patients. We conducted a study of Delta13 in patients entered into the Eastern Cooperative Oncology Group trial E9486/E9487. Patients with newly diagnosed MM (median follow-up of survivors >100 months) were studied for Delta13, using bone marrow samples obtained at study enrollment. We used interphase fluorescence in situ hybridization with the probes LSI13 (Rb)/D13S319 with simultaneous immunofluorescence detection of bone marrow plasma cells (PCs). We detected Delta13 in 176 of 325 (54%) evaluable patients. Patients with Delta13 were more likely to have a serum monoclonal protein at a concentration < or =1 g/dl (22 versus 13%; P = 0.04), light-chain-only MM (19.3 versus 10.8%; P = 0.04), gamma light chain (42 versus 28%; P = 0.027), stage III (56 versus 42%; P = 0.014), and be female (60 versus 50%; P = 0.087). The PC labeling index and Delta13 correlated (P = 0.03). Patients with Delta13 were less likely to respond to treatment (74 versus 63%; P = 0.041) and had a significantly shorter median overall survival (34.9 versus 51 months; P = 0.021). The association of Delta13 and survival remained an independent prognostic variable in a regression model. Among patients with Delta13, those receiving IFN had a worse overall survival that those not receiving the medication (P = 0.03). The presence of Delta13 is an important and independent adverse prognostic factor in newly diagnosed MM and is associated with specific biological features.
[Show abstract][Hide abstract] ABSTRACT: Deletions of the long arm of chromosome 13 (13q-) are observed in patients with multiple myeloma (MM), are rarely observed in the monoclonal gammopathy of undetermined significance (MGUS) and have been associated with a worsened prognosis in MM. However, no minimally deleted region in the 13q arm has been defined at 13q, and consequently no tumor suppressor genes have yet been identified that are important for disease pathogenesis. We attempted to characterize these chromosome 13q deletions at the molecular cytogenetic level. We studied 351 newly diagnosed patients, entered into the E9486/E9487 clinical study of the Eastern Cooperative Oncology Group. Fluorescent in situ hybridization (FISH) combined with immune fluorescent detection (cIg-FISH) of clonal plasma cells (PC) and cytomorphology were used to analyze interphase, bone marrow (BM) cell, cytospin slides. We simultaneously used DNA probes for the following locus specific probes (LSI); LSI 13 (Rb) and D13S319, which hybridize to 13q14. We subsequently studied distal deletions using the D13S25 probe (13q14.3) and a subtelomeric probe (13qSTP) for the 13q-arm (D13S327) in 40 cases with documented LSI 13 (Rb)/D13S319 deletion and 40 without deletion of these loci. Of 325 evaluable patients, we found 13q deletions in 176 (54%) using LSI 13 (Rb) and D13S319 probes. Of 40 patients with LSI 13 (Rb)/D13S319 deletions, 34 (85%) had coexistent deletion of both D13S25/13qSTP. These results indicate that chromosome 13 deletions in MM involve loss of most if not all of the 13q arm perhaps even indicating monosomy. In six cases the 13qSTP signal was conserved, but D13S25 was lost indicating large interstitial deletions involving 13q14. In 39 of the 40 cases without LSI 13 (Rb)/D13S319 deletions, the normal pattern of two pairs of signals was observed for D13S25/13qSTP. Deletions involving 13q14 are very common in MM as detected by cIg-FISH. These deletions appear to predominantly involve loss of large segments of the 13q arm or monosomy 13, and only occasionally represent an interstitial deletion.