Event In Multiple Myeloma Research Articles

Risk Factors of Thromboembolic Events in Multiple Myeloma Treated with IMiDs-Based Therapy While on LMWH Prophylaxis,

Abstract 3340 Background.Immunomodulator drugs (IMiDs) are promising oral agents in Multiple Myeloma (MM); and MM that cannot benefit from novel agents, including IMiDs, only have 9 months survival. On the other hand IMiDs are associated with an increased risk of thromboembolic events (TE) in MM, and must be stopped when TE occurs with a potential shortened life expectancy. Although DVT (Deep Venous Thrombosis), including PE (Pulmonary Embolism), was primarily observed, arterial events were also described. Guidelines have proposed LWMH for VTE prophylaxis for patients that displayed greater than 2 risk factors of VTE. Studies have showed a decrease incidence of TE since a prophylaxis was mandatory; however TE remained despite use of LMWH. We sought to characterize and study the incidence of TE in MM treated with an IMiDs-based regimen and having LMWH as TE prophylaxis, and to determine risk factors for patients to develop TE. Method.MMVAR/IFM 2005–04 is a large multicenter, prospective, randomized, open-label, phase 3, EBMT and IFM combined study that compared VTD to TD for MM patients in first progression after autologous transplantation. Treatment comprised 8 cycles of bortezomib 1.3 mg/m2 IV bolus on days 1, 4, 8 and 11 of a 21-days cycle and then on days 1, 8, 15 and 22 of a 42-days cycle for 4 more cycles. In both arms, oral thalidomide was administered at 200 mg/day for 1 year with dexamethasone at 40 mg/day for 4 days every 3 weeks for 1 year. A TE prophylaxis was mandatory in both arms using enoxaparin 40 mg/day during one year. TTP was the primary end point. Response was assessed by EBMT criteria. Adverse events were graded by the NCI-CTCAE, Version 3.0. Results.The MMVAR trial was stopped because of superiority of VTD over TD at first interim analysis, as 157 relapsed were recorded out of 267 patients randomized in arm VTD (n=135) and arm TD (n=132), respectively. With a median follow-up of 27 months, the probability of achieving CR and CR+PR during the first year, the median TTP and PFS were greater in the VTD arm, although it did not translate into a better OS, yet. In the VTD and TD arms, the mean number of treatment cycles for the 12 cycles was 7.56 vs 9.93, respectively. Treatment was discontinued due to toxicity in 48 patients, including 8 (17%) related to occurrence of TE, and 33 patients died during the treatment period. There were 24 (8.9%) TE recorded, 12 in either arm; we have then decided to pool the 2 groups for the subsequent analysis. The characteristics of the MM with TE were not different from the overall population, 14 male/10 female, median age (range) was 63 (42–76) with 25% patients older than 65. The median (min-max) time from start of MMVAR to occurrence of TE was 3.4 months (0.3–11.9), not different in either arm. TE occurred in 16 (66%) vs. 8 (33%) patients in the first 4 months and after 4 months, respectively. 15 (62.5%) pts had at least some tumor burden reduction (minor response and better) at time of occurrence of TE, while 12.5% had progression of MM, 12.5% stable disease, and 12.5% were non evaluable. All TE occurred while pts were on LMWH prophylaxis since the initiation of the study treatment but one. The occurrence of TE impacted the treatment of MM as 16 (67%) pts did stop their IMiDs-based treatment, either transiently for 8 (33%) pts or definitely for 8 (33%) pts. The doses of the IMiDs were reduced for 5 pts when IMiDs were reintroduced. Overall only 5 pts had no change applied to their MM treatment while TE occurred. The occurrence of TE might have impacted response rate, CR rate, and the survival end points, TTP, PFS and OS. An update of this sub analysis will be presented at ASH with multivariate analysis to determine risk factors for occurrence of TE while on LMWH prophylaxis. Conclusion.Although LMWH is recommended to patients with high risk of TE, the optimal dose and duration of LMWH remains to be determined. More studies are needed to determine risk factors of TE in MM patients treated with IMiDs-based regimen, and to guide physician in their routine practice with the optimal TE prophylaxis.On behalf of the Myeloma Subcommittee of the Chronic Leukemia Working Party of the EBMT (European Group for Blood and Marrow Transplantation) and the IFM (Intergroupe Francophone du Myélome). Disclosures:Leleu:Amgen: Honoraria; Roche: Research Funding; Janssen Cilag: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; LeoPharma: Honoraria, Research Funding; Novartis: Research Funding. Masszi:Centocor Ortho Biotech Research & Development: Research Funding. Hajek:Merck:; Janssen: Honoraria; Celgene: Honoraria.

Epigenetic Dysregulation of the Tissue Inhibitor of Metalloproteinases-2 Gene in Multiple Myeloma

Abstract 5089 Background:Multiple myeloma (MM) is a B-cell neoplasm characterized by the accumulation of malignant plasma cells in the bone marrow (BM) as well as abundant BM angiogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene expression. There is increasing evidence for a role of the tissue inhibitor of metalloproteinases 2 (TIMP2) gene as a tumor suppressor. Overexpression of TIMP2 may result in decreased invasive potential, suppression of tumor growth and vascularization as well as inhibition of angiogenesis. We could previously show that TIMP2 may become hypermethylated in association with transcriptional silencing in indolent and aggressive non-Hodgkin's lymphomas. Recently, downregulation of TIMP2 was also reported in MM, and this is thought to contribute to increased BM angiogenesis. In this study, we determined the methylation status of the promoter-associated CpG island of TIMP2 in primary MM samples and investigated correlations between TIMP2 methylation and clinical parameters. Methods:Methylation of the promoter-associated CpG island of TIMP2 was analyzed by methylation-specific polymerase chain reaction (MSP) in samples from 76 MM patients (median age 65 years [range 40–94]; 44 males, 32 females; 71 BM samples, 5 peripheral blood samples). Overall survival curves were plotted according to the method of Kaplan and Meier and compared using the log-rank test. Correlations between variables were analyzed using the Fisher’s exact two-sided test and independent t-test, respectively. Results:MSP analysis revealed that there was aberrant methylation of the TIMP2 promoter region in 4/76 MM patient samples. We found an association of TIMP2 hypermethylation with plasma cell leukemia (p<0.0001) and decreased platelet counts (257.1 G/l vs. 111.5 G/l; p=0.04). There was a trend towards an inferior overall survival in MM cases with a hypermethylated TIMP2 gene, but this difference was not statistically significant (p=0.11). Conclusions:Our study shows that promoter hypermethylation of TIMP2 is a novel epigenetic event in MM that may contribute to disease progression and could serve as a prognostic biomarker. Further studies are warranted to elucidate the functional consequences of epigenetic dysregulation of TIMP2 in the pathogenesis of MM. Additionally, the increasing evidence for the role of DNA methylation changes in MM may serve as a basis for the use of epigenetically targeted therapeutic approaches in malignant plasma cell disorders. Disclosures:Herman:MDxHealth: Consultancy.

Gene Mutations Detected by Whole-Exome Sequencing and Recurrent Cytogenetic Abnormalities Are Independent Events in Multiple Myeloma

Abstract 1816Multiple Myeloma (MM) is characterized by several recurrent chromosomal abnormalities, some of them driving the outcome of the patients, especially t(4;14), del(17p), and hyperdiploidy. On the other hand, recent data based on whole genome sequencing showed that MM is characterized by many gene mutations, some of them being recurrent. In order to try to reconcile these two types of genetic abnormalities, we performed whole exome sequencing (WES) in 53 newly-diagnosed patients characterized by high or low risk at the chromosomal level. We selected 16 patients with del(17p) in at least 60% of the clonal plasma cells, 5 patients with del(12p), including 3 with associated t(4;14), 2 patients with t(14;16), 1 patient with both del(17p), del 12p), and t(4;14), considered as the poor risk group (24 patients), 24 patients with hyperdiploidy (including chromosome 5 gain), and 2 patients with a normal SNParray profile, considered as the good risk group (26 patients, and 3 patients with hyperdiploidy and either del(17p) and/or del(12p), considered as the uncertain risk group. A total of 3621 non-silent mutations were observed through the 53 tumor genomes. The median number of mutations per patient was 79 (31–462). We did not observe differences in the number of mutations according to cytogenetic risk. Regarding specific gene mutations, 376 genes presented 2 mutations, and 128 genes presented at least 3 mutations (3–16). Comparison with recently published sequencing data (Chapman, Nature, 2011) revealed only a few common mutated genes (NRAS 26%, KRAS 23%, TP53 13%, BRAF 11%, and FAM46C 10%). In contrast, many recurrently mutated genes were identified in this series, but not in the published one. This could be related to the relatively low number of sequenced cases in both series. Very interestingly, quite a high number of strictly identical mutations involving many genes were observed in different patients (139 cases), suggesting that those mutations are more probably driver rather than passenger events. To conclude, in this large comprehensive study, we did not find any significant correlation between recurrent chromosomal changes and gene mutations, suggesting that these two events occur independently. We cannot address the issue of the prognostic value of the gene mutations because of the low number of patients sequenced so far. This question will definitely have to be addressed in future larger series on newly diagnosed patients with MM. Disclosures:Facon:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees. Munshi:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees.

Promoter methylation of the bone morphogenetic protein 6 gene in multiple myeloma

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, are multifunctional signaling molecules that have become of increasing interest in cancer research. Recent observations suggest that alterations in BMPs and BMP signaling are associated with tumorigenesis and disease progression in various types of malignancies. This study investigated the methylation status of the BMP6 gene promoter in various types of plasma cell proliferative disorders by combined bisulfite restriction analysis. While BMP6 methylation was not detected in any samples from monoclonal gammopathies of undetermined significance, intramedullary multiple myeloma (MM), plasma cell leukemia or solitary plasmacytoma, both case studies and cell line studies showed that multiple extramedullary plasmacytoma (MEP) consistently carried a methylated BMP6 promoter. The BMP6 methylation-positive MEP was an aggressive form of MM with extremely high levels of serum lactate dehydrogenase (LDH). Bisulfite sequencing analysis confirmed intensive methylation at CpG sites of the BMP6 promoter region. The methylation of BMP6 was correlated with decreased levels of mRNA transcripts. Expression of BMP6 was restored by the demethylating agent 5-aza-2'-deoxycytidine, suggesting that the methylation is associated with transcriptional silencing. Our study implied that BMP6 promoter methylation is not a common event in MMs, but occurs in aggressive MEP. These findings warrant further investigation to clarify whether BMP6 methylation together with elevated LDH could be a marker of poor prognosis in MEP patients who should be considered for early intensive treatment.

Practical management of adverse events in multiple myeloma: Can therapy be attenuated in older patients?

The current standard of care for elderly patients with newly diagnosed multiple myeloma is melphalan and prednisone (MP) in combination with either bortezomib (VMP) or thalidomide (MPT), with lenalidomide plus dexamethasone increasingly being employed. The addition of bortezomib or thalidomide to the established MP regimen significantly improves outcomes and prolongs survival in elderly and transplant-ineligible patients. However, these benefits are accompanied by increases in treatment-related adverse events (AEs), which may be particularly pronounced in older individuals. Patients receiving bortezomib as part of a VMP regimen commonly experience transient and cyclical thrombocytopenia and neutropenia, along with gastrointestinal AEs. Fortunately, these AEs can be managed with appropriate supportive care and, when necessary, adjustments in dose. Peripheral neuropathy (PN) is the most important side effect of bortezomib, and although it is reversible in a high proportion of patients, it affects their quality of life. Furthermore, PN can require temporary or permanent withholding of bortezomib, which will reduce treatment efficacy. PN is also a common adverse effect of thalidomide; thromboembolic events are also a key concern, requiring thromboprophylaxis in patients receiving thalidomide in combination. For lenalidomide in combination with dexamethasone, the most clinically important adverse effects are hematologic toxicity (particularly neutropenia) and thromboembolic events. Recent phase III studies in newly diagnosed elderly patients are providing further insight into the most appropriate treatment regimens to maximize outcomes and minimize toxicity in individual patients. Of note, once-weekly bortezomib dosing (in combination with MP ± T) was shown to reduce the incidence of peripheral neuropathy and gastrointestinal events compared with twice-weekly dosing, while maintaining efficacy. Elderly patients may be less able to withstand the AEs associated with newer treatment regimens and combinations of multiple drugs, and may experience greater declines in quality of life and, subsequently, reduced treatment adherence. It is therefore critical that these patients are closely monitored and any emergent AEs promptly and appropriately managed. For very elderly, frail patients, tailored therapy, reduced intensity regimens, and adverse event management are necessary to encourage treatment adherence and reduce discontinuation. This article will provide practical guidance on the management of bortezomib-, thalidomide-, and lenalidomide-associated AEs, to maximize treatment feasibility and active drug delivered, and thus help minimize toxicity and maximize outcomes.

A Combined Survival Model Integrating Gene Expression and Alternative Splicing Events Provides Higher Predicative Power for Risk Stratification

AbstractAbstract 1929Considerable efforts have been spent evaluating impact of global gene expression profile on clinical outcome. Although significant correlation has been described between outcome and expressed gene signature, the overall predictability of such models have reached a plateau. Biologically this is expected as gene function is modulated at multiple levels. Besides the change in level of expression, post-transcriptional changes such as alternate splicing alter specificity of gene function and may affect the eventual outcome. Although various genes have normal alternate spliced form, dysregulation of alternative splicing that alters protein function has been implicated in number of disease processes including cancer. We have observed significant level of dysregulated splicing events in multiple myeloma (MM). We hypothesize that a combined model that includes dysregulated splicing events, besides level of expressed genes, may provide superior survival model in MM. To develop a combined model we have hybridized RNA isolated from CD138+ purified MM cells collected at the time of diagnosis from 170 newly-diagnosed patients treated homogeneously in tandem transplantation IFM trials, 23 MM cell lines and 6 Healthy donors on Affymetrix Exon 1.0 ST GeneChip arrays. Exon array not only provides an accurate measure of expression levels for genes, but also allows simultaneous identification of alternative splicing events. Pre-processing and normalization methods in aroma, affymetrix and robust multichip analysis model in FIRMA, followed by t-tests with Benjamini-Hochberg multiple hypothesis corrections were used respectively to identify differential expression and alternative splicing. We identified 1454 differentially expressed genes and 759 differential splicing events between healthy donors and MM patients, and 5476 differentially expressed genes and 4012 differential splicing events between healthy donors and MM cell lines. There are 1071 differentially expressed genes and 286 alternative spliced exons shared between MM samples and MM cell lines.Univariate survival analysis using FIRMA scores of exons identified a total of 89 genes with more than 10 alternative splicing events between healthy donors and MM patients associated with survival with Cox proportional hazard model and log-rank tests. We have now built 3 different survival models considering: 1) gene expression only, 2) alternative splicing only, and 3) a combined model integrating gene expression and alternative splicing events. We utilized refined regularized variable selection methods to handle these high-dimensional feature space. Our analysis suggests that composite model using gene expression and alternative splicing information performs significantly better than the gene expression only model in identifying high-risk patients, when the data were divided in median or quartiles. Specifically, the difference in overall survival is 32.6 months to 38.5 months using the median survival, and 18 months vs 23 months for median event free survival. We are currently in the process of validating the combined model. Our data suggests the need for inclusion of modifiers of transcriptome to develop a comprehensive model that will have higher predicative power for risk stratification as well as for selection of therapeutic intervention. Disclosures:Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Munshi:Millennium Pharmaceuticals: Honoraria, Speakers Bureau.

Production of TRAIL by Multiple Myeloma Cells: a Potential Prediction Marker for Skeletal-Related Events

AbstractAbstract 2975 Introduction:Skeletal complications including pathologic bone fractures, skeletal related plasmacytoma, bone pain and hypercalcemia are major clinical problems for patients with multiple myeloma (MM). Osteoclastgenesis are reportedly triggered by receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by osteoprotegerin (OPG). Since OPG is an inhibitor of RANKL, a possible role of TRAIL as an osteoclast inducer is suggested. We have previously reported that TRAIL was produced from myeloma cells and it correlated with skeletal-related events (SRE) at the 51th ASH meeting. In the present study, we analyzed a total of 56 cases with MM and examined their urine deoxypyridinorine (U-DPD) concentration, and the expression of DKK-1, MIP-1a by their MM cells, revealing that TRAIL is a powerful and independent predictor of SRE. Materials and Methods:Myeloma cells were purified from bone marrow samples from 56 MM patients using CD138-immunomagnetic beads. Plasma cells from MGUS patients were also evaluated. All samples were obtained under informed consent according to Helsinki Declaration. TRAIL, DKK1 and MIP1a RNA expression in purified MM cells was analyzed using real time PCR. Serum concentrations of TRAIL from MM patients and healthy donors were determined with ELISA. We also examined concentrations of TRAIL in bone marrow plasma from MM and MGUS patients. MM patients were given Skeletal-Related Event Score (SRE score) according to skeletal complications and therapeutic events such as irradiation and operation. Osteoclasts were generated using peripheral blood mononuclear cells (PBMC), M-CSF, sRANK-L, and the murine pre-osteoclastic cell line RAW264.7. Numbers of viable osteoclasts and RAW264.7 cells were determined upon treatment with rTRAIL. Results:TRAIL expression in myeloma cells from MM patients was 5-fold greater than that in plasma cells from MGUS patients (p<0.01). No correlation was observed between TRAIL expression levels in myeloma cells and serum TRAIL concentrations. Serum TRAIL concentrations in healthy donors were not significantly different from those in MM patients. However, TRAIL expression levels in myeloma cells correlated to TRAIL concentrations in corresponding bone marrow plasma samples (p<0.05). Univariate analysis revealed that there was a significant correlation of TRAIL expression levels with serum calcium levels (p<0.05), U-DPD concentrations (p<0.05) and SRE scores (p<0.01). DKK1 expression levels in myeloma cells had no correlation with serum calcium (p=0.20), U-DPD (p=0.26), or SRE scores (p=0.12), while MIP1a expression levels correlated with serum calcium (p<0.01) and U-DPD (p<0.05) levels, but not with SRE scores (p=0.53). Summary of statistical analysis is shown in table 1. Although PBMC-derived osteoclasts constitutively expressed TRAIL receptors, DR4 and DR5, rTRAIL did not trigger apoptosis in osteoclasts or RAW264.7 cells. Conclusions:We propose that MM patients with high TRAIL expression levels in MM cells tend to have greater chances of skeletal complications. Since serum TRAIL concentrations did not correlate with TRAIL RNA expression in myeloma cells, increase of TRAIL in bone marrow microenvironment should be important to develop bone destruction. TRAIL is an important predictable factor for SREs, serum calcium, and U-DPD. Since DKK-1 suppresses osteoblast differentiation rather than manipulating osteoclastgenesis, DKK1 expression may not be capable enough of reflecting SRE events. At this time, how MM cells survive from TRAIL expression, which is associated with apoptosis, is not known. We hypothesize that MM cells are protected by their adhesion to stromal cells as previously reported (J. Immunol. 180:1545-55, 2008), while osteoclasts may be directly activated by TRAIL. Since direct induction of apoptosis of osteoblast by TRAIL is also reported, taken our findings together, TRAIL may be a key molecule inducing SRE in MM patients. Our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Germinal Center Specific Activation of K-Ras, Common In Multiple Myeloma, Is Selected Against and Is Not Sufficient to Initiate Plasma Cell Transformation In Mice

AbstractAbstract 137Activating mutations in Ras (N- and K-) are the most common mutations known in multiple myeloma (MM), and are associated with advanced clinical stage and poor outcomes. We re-sequenced coding regions of highly expressed cytokine signaling candidate genes from MM patient samples and found rare novel mutations. Still, the most common recurrent somatic mutations we found affected N and K-Ras. Previous studies of MM patient samples suggest that Ras mutations may be late/progression events, but the role of Ras activation in MM pathogenesis has not been tested directly. Toward this end, we generated double-transgenic mice by crossing Lox-stop-lox(LSL)- K-ras G12D knock-in mice (K-ras+/G12D) with Cγ1-Cre knock-in mice (Cγ1+/Cre) expressing the Cre recominase specifically in germinal center (GC) B-cells. K-ras+/+/Cγ1+/Cre mice served as negative controls. We found 58% of unstimulated K-ras+/G12D/Cγ1+/Cre mice developed fatal double-positive CD4/8 T-cell lymphomas (n=12) and 42% developed lung adenocarcinomas (n=12). The median survival of these mice with lymphomas was 19 weeks, compared to the 300 day end point of unstimulated K-ras+/+/Cγ1+/Cre control mice. Tumor cells from these mice demonstrated successful recomination of the K-Ras locus. In contrast, no evidence of multiple myeloma or monoclonal gammopathy was found by serum ELISA, SPEP, flow cytometry or histologic examination. We isolated B-cell subsets from K-ras+/G12D/Cγ1+/Cre mice and documented successful recombination in splenic germinal center cells (B220+, GL-7+) but not in bone marrow plasma cells. Together, these data suggest that Ras activation by “leaky” off-target Cre expression was sufficient to initiate tumorigenesis in two non-B-cell compartments, but that activation of the K-rasG12D allele in germinal center B-cells was associated instead with clearance of recombined cells. We conclude that activating Ras mutations are disease progression events in MM and that earlier mutations are required before primary plasma cells are transformed. This novel mouse model will be useful for identifying and testing early genetic events that drive MM development in cooperation with Ras. Disclosures:No relevant conflicts of interest to declare.

Breast plasmacytoma

Breast plasmacytoma (BP) is extremely rare. The published data on this manifestation include predominantly case reports and do not provide any statistical information. To identify clinical signs and radiological features of BP. Five patients with BP were retrospectively identified in the pathological and radiological databases of the years 1997-2009 at our institution. Additionally, 48 patients were collected from the literature in the period from 1988 to date. Therefore, our study involves 53 patients. The prevalence of BP in our institution was 1.5% of all identified cases with plasmacytoma and 0.2% of the cases with breast cancer. In 8 of 53 patients (15%), primary BP was diagnosed, and in 45 patients (85%) involvement of the breast was a secondary event of the multiple myeloma (MM). Clinically, 83% of the patients presented with breast lumps. BP was diagnosed incidentally in 9%. In 8% of the cases clinical signs were not reported. On mammography, intramammary round or oval masses were most commonly found (89%). They were solitary in 66% of the cases and multiple in 34%. Median size was 21 mm, ranging from 8 to 90 mm. In 9% of the identified cases BP manifested as diffuse infiltration of the breast. In 2% no abnormalities were identified on mammography. On ultrasound, identified lesions were homogeneously echo-poor or hypoechoic, less frequently mixed hypo- to hyperechoic. Treatment of BP is the same as for other localizations of plasmacytoma and is based on chemotherapy and/or radiotherapy. Primary BP had a better prognosis than breast involvement as a secondary event in MM. In conclusion, BP does not have specific radiological or clinical features and can be misdiagnosed as primary breast carcinoma or even as a benign process. However, BP should be considered in the differential diagnosis of breast disorders, especially in patients with MM.

Upregulation of TRAIL in Myeloma Cells Correlates with Skeletal-Related Events.

Abstract 2844Poster Board II-820 Introduction:Skeletal complications including bone fracture, bone pain and hypercalcemia are major clinical events in patients of multiple myeloma (MM). Osteoclastgenesis is known to be induced by free receptor activator of nuclear factor kappa β ligand (RANKL) and inhibited by dimerization of RANKL and osteoprotegerin (OPG). OPG is also known as a soluble inhibitor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL); therefore, a possible role of TRAIL as an osteoclast inducer is suggested, although the association of TRAIL with bone lesions in MM is a matter of debate. We thus investigated the expression of TRAIL mRNA in purified MM cells and analyzed its association with skeletal-related events. Patients and Methods:MM cells were purified from bone marrow samples from 40 MM patients by CD138-immunomagnetic beads (Miltenyi Biotech, Paris, France). TRAIL mRNA expression in purified MM cells was analyzed by real time PCR(ABI PRISM 7700 Sequence Detector, Applied Biosystems). Simultaneous analysis of serum TRAIL concentrations, analyzed by ELISA (Diaclone, Cedex, France), and TRAIL mRNA-expression levels was also performed in 23 cases. Each of these patients was given a score called skeletal-related event score (SRE score) according to the skeletal complications (pathological fracture, bone-associated plasmacytoma, >12mg/dL hypercalcemia, and receiving an pathological fracture-related operation or radiation therapy). Results:Significant association (p=0.0006) was seen between TRAIL mRNA expression levels and the SRE score (Fig.1). Serum calcium levels also had significant association to TRAIL mRNA expression levels (p=0.0050). On the other hand, no association of TRAIL mRNA with hemoglobin (p=0.3970) and platelets (p=0.9401) was seen. Serum TRAIL concentrations in MM cases, which were equivalent to those in healthy individuals, did not correlate to TRAIL mRNA expression levels in purified MM cells (p=0.4094). Conclusions:The data suggest that MM patients with high TRAIL expression in MM cells tend to have more skeletal complications, which may be mediated by increased osteoclastgenesis. Since serum TRAIL concentrations did not correlate with TRAIL mRNA levels in MM cells, increased TRAIL expression in bone marrow microenvironment could be important. Despite of previous reports suggesting TRAIL-induced apoptosis of hematopoietic cells, the observed high TRAIL expression did not correlate with anemia or thrombocytopenia in our cases. Although mechanisms regulating TRAIL expression in MM cells and protection from TRAIL-induced apoptosis remain to be determined, our findings may introduce a new strategy targeting TRAIL to reduce skeletal events in MM. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

1p21 deletions are strongly associated with 1q21 gains and are an independent adverse prognostic factor for the outcome of high-dose chemotherapy in patients with multiple myeloma

Deletions involving chromosome 1p are frequent events in multiple myeloma (MM). As karyotyping and single nucleotide polymorphism-based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we investigated the prevalence and prognostic significance of del(1p21) in 203 MM patients undergoing high-dose therapy and autologous SCT. 1p21 status was also evaluated in 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 41 patients with plasma cell leukemia (PCL). FISH combined with cytoplasmic light chain detection (cIg-FISH) detected hemizygous 1p21 deletions in 18% of the MM, 34% of PCL but none of the MGUS cases. The presence of 1p21 deletions was correlated with 1q21(CKS1B) amplification (P=0.01), and del17p(TP53) (P=0.05) but not with del(13q), t(11;14) or t(4;14). Patients with 1p21 deletions had significantly shorter progression-free survival (PFS; median 14.2 vs 25.4 months, P<0.001) and overall survival (OS; median 39.4 vs 82.3 months, P=0.001) than those without such deletions. In multivariate analysis, del(1p21) was an independent risk factor for PFS (P= 0.003) and OS (P=0.013) after adjusting for del(13q), del(p53), t(4;14) and 1q21 amplifications. Our results indicate that del(1p21) is an independent poor prognostic factor associated with disease progression in MM.

The role of bisphosphonates in multiple myeloma

Multiple myeloma (MM) is the second most common hematologic malignancy and the most common malignancy to involve bone. More than 85% of patients with MM have bone involvement, which can be devastating. Bisphosphonate therapy is the mainstay of treatment for MM bone disease; it has decreased the frequency of skeletal events in MM and delayed their development. Further, the toxicity of these drugs is low and generally manageable. Whether bisphosphonates have any antitumor effects in MM patients (in contrast to what has been reported in preclinical models) is unclear and requires further study. Although bisphosphonates have been extremely effective for treating MM bone disease, they do not completely inhibit the development of skeletal events, but only decrease them significantly. Other antiresorptive agents now being developed may further enhance the quality of life for MM patients when used in combination with bisphosphonates.

Interim Analyses of the MMRC Reference Collection Identifies Recurrent Genomic-Level Events in Multiple Myeloma and Demonstrates That CDKN2C/p18 Deletion Is the Pre-Eminent Copy Number Alteration in Poor Prognosis Disease

The Multiple Myeloma Research Consortium (MMRC) Genomics Initiative was instigated to harness the power of multiple genomic approaches to further the understanding of multiple myeloma. To date, 137 samples from patients with newly diagnosed and relapsed myeloma (out of an estimated final total of 250 by the end of the year) have been subjected to expression profiling and array comparative genomic hybridization. To identify regions of recurrent copy number alteration with a high degree of confidence, we have used the Genomic Identification of Significant Targets In Cancer (GISTIC) algorithm, which detects such regions and assigns a probability to each. Application of GISTIC to the MMRC collection identified 14 significant regions of amplification and 15 significant regions of deletion. The algorithm further detects peaks of copy number change that contribute to each region's significance. Of genes that were expressed in our dataset, a total of 64 across the genome were found to lie within the boundaries of significant amplification peaks and 30 were found to lie within significant deletion peaks. Given the likelihood that this gene collection is highly enriched for genes important in the pathogenesis of myeloma and potential therapeutic targets, it has been prioritized for further validation with the amplified genes submitted for arrayed RNAi and the deleted genes expedited for re-sequencing. In order to define a poor prognosis group, we developed and applied a model based on the gene expression dataset of Shaughnessy and colleagues. To identify pathways activated in poor prognosis disease, we applied Gene Set Enrichment Analysis with the Molecular Signatures Database. This demonstrated enrichment of multiple canonical pathways within the poor prognosis group, including those associated with proliferation, cell cycle progression and DNA repair. A search for recurrent copy number events associated with poor prognosis disease revealed that by far the most significant event, and the only one surviving correction for multiple hypothesis testing, was mono- or bi-allelic deletion of CDKN2C (p18). Interestingly, the poor prognosis group also demonstrated higher expression of p18 in those samples without bi-allelic deletion and of CDKN2A (p16). Deletion and increased expression of p18 in high proliferation index myeloma samples has previously been described. However, our analyses demonstrate the pre-eminence of p18 loss for defining poor prognosis disease compared to other recurrent copy number changes. Furthermore, it expands on work in other tumors demonstrating feedback mechanisms between cyclin dependent kinase inhibitor pathways, suggesting a more complex model of interplay between the pathways than previously described. In conclusion, the MMRC reference collection is proving an important tool in the fight to understand key genetic events in multiple myeloma and would ultimately be anticipated to contribute towards development of improved therapy for the disease.

Loss of p53 Is a Marker of Progression in Plasma Cell Neoplasias and Is a Negative Prognostic Factor in Relapsed Disease.

Table 1. Samples to p53 deletionsp53deletionFISHaCGHFISH and aCGHMGUS142--SMM108--Newly diagnosed MM-182-Relapsed/refractory MM6215619HMCLs-48-Total31238619Background: Inactivation of p53 by mutation or allelic loss is a rare event in multiple myeloma (MM) at the time of disease diagnosis and believed to be more common in the late stages of the disease. Here we defined the prevalence of p53 deletions in monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), newly diagnosed MM, relapsed MM and human myeloma cell lines (HMCLs). Indirect mechanisms of p53 inactivation such as amplification of MDM2, or deletion of CDKN2A (p14ARF) were also investigated.Patients and Methods: A total of 631 MM samples and 48 HMCLs were analyzed for p53 abnormalities (Table 1) and compared with a cohort of plasma cell leukemia (PCL) previously published by us. Interphase fluorescence in situ hybridization (FISH) and highresolution array-based comparative genomic hybridization (aCGH) were used to detect p53 deletions. MDM2 amplification and p14ARF loss were evaluated by aCGH. Overall survival (OS) was estimated through Kaplan Meier method.Results: The prevalence of p53 deletions was 1.4%, 3.7%, 8.9%, 20% and 87.5% in MGUS, SMM, newly diagnosed MM, relapsed MM and HMCLs respectively (Figure 1). Of interest patients in first relapse showed a p53 deletion prevalence of 18% in comparison with 33% for patients in second relapse or more. We obtained p53 deletion status in 8 patients noted to have deletion at the time of relapse and in whom we had previous samples stored. In 7 of 8 cases deletions had been acquired (absent in the previous samples). The median OS for relapsed MM patients with or without p53 deletion were 4.2 months and 37.8 months respectively (p <0.001). MDM 2 amplification and p14ARF loss was detected in 0.4% and 2% of new diagnosed MM, 0.6% and 5.1% of relapsed MM and 2% and 29% of HMCLs respectively.Discussion: Emergence of p53 deletion/mutations denotes progression genetic events in MM. Importantly we show for the first time the overriding importance of p53 deletions as prognostic markers in relapsed MM. A molecular staging system that is based on the presence of p53 inactivation may be more powerful than classifying patients based on the loss of p53. Other abnormalities involving p53 pathway regulators like MDM2 gain and p14ARF loss are infrequent events in MM, even in advanced disease. [Display omitted]

Erythropoiesis-Stimulating Agents Do Not Adversely Affect Long-Term Outcomes Nor Increase the Risk of Thromboembolic Events in Multiple Myeloma Patients Treated in the Phase III VISTA Trial.

Introduction: Treatment of myeloma-associated anemia (both disease and treatment induced) includes erythropoiesis-stimulating agents (ESA) and/or red-blood-cell transfusions (RBCT). Limited data from patient subsets in retrospective studies have suggested that ESA may have a detrimental effect on outcomes, including reduced time-to- progression (TTP) and overall survival (OS), in patients with multiple myeloma (MM). Furthermore, ESA may increase the risk of deep-vein thrombosis (DVT) and pulmonary embolism (PE), especially in patients receiving immunomodulatory-based regimens and/or anthracyclines with glucocorticoids. Since the impact of ESA use on long-term outcomes and thromboembolic events in MM has not been extensively evaluated, we conducted a sub-analysis of the prospective multi-center, randomized, phase III VISTA trial in frontline MM (San Miguel et al. Blood 2007), to assess the potential impact of ESA use on TTP, OS and rates of DVT/PE.Methods: Patients were randomized to receive nine 6-week cycles of bortezomib (1.3 mg/m2 on days 1, 4, 8, 11, 22, 25, 29, and 32, cycles 1–4, and days 1, 8, 22, and 29, cycles 5–9) plus melphalan (9 mg/m2) and prednisone (60 mg/m2) administered on days 1–4 of each cycle (VMP; n=340) or melphalan–prednisone (MP; n=337) alone. No protocol-specified antithrombotic prophylaxis was required. Baseline characteristics, including age, sex and disease characteristics, were similar between ESA and non-ESA groups.Results: Median Hb level at the time of ESA initiation was 9.75 g/dl in the VMP arm and 9.30 g/dl in the MP arm; consistent with current guidelines that ESA should not be initiated until Hb is <10 g/dl. The incidence of treatment-emergent anemia (defined as Hb < 8.0 g/dl) was lower in the VMP arm (23%) than the MP arm (33%), and fewer patients in the VMP versus MP arm were treated with ESA (30% vs 39%, respectively; erythropoietins 20% vs 24% and darbepoietin 11% vs 18%, respectively), or RBCT (26% vs 35%, respectively), potentially reflecting greater anti-myeloma activity with VMP. Median TTP was similar between patients who received ESA and those who did not in both treatment groups (Table). While one-year OS rates were similar, 2-year OS rates appeared higher for patients receiving ESA (Table). TE complications were low in both treatment arms and were not affected by ESA use (3% vs 2% for VMP, and 3% vs 1% for MP, for patients receiving or not receiving ESA, respectively).Conclusions: Our post-hoc analysis from a large, well-controlled multicenter phase III trial in frontline MM shows that ESA use did not adversely impact long-term outcomes with VMP or MP, and may be associated with a survival benefit. Furthermore, ESA use did not appear to increase the risk of TE complications with VMP or MP. These data suggest that ESA can be safely administered with VMP/MP for the treatment of anemia in frontline MM patients. Prospective, randomized studies are needed to further investigate the relationship between ESA and RBCT use, other agents and long-term outcomes in MM patients.Table. TTP and OS rates by ESA and RBCT use and per treatmentVMP (n=340)MP (n=337)+ ESA (n=102)− ESA (n=238)+ ESA (n=131)− ESA (n=206)TTP, months (95%CI)19.9 (18.9, NE)NE (18.3, NE)15.0 (13.5, 21.8)17.5 (14.7, 19.0)1-year survival rate % (95% CI)92.0 (86.6, 97.3)87.8 (83.5, 92.0)82.6 (76.0, 89.2)81.4 (75.9, 86.9)2-year survival rate % (95% CI)86.7 (77.9, 95.4)80.8 (73.1, 88.4)77.3 (68.5, 86.1)65.4 (55.7, 75.2)+ RBCT (n=87)− RBCT (n=253)+ RBCT (n=117)− RBCT (n=220)TTP, months (95%CI)NE (24.0, NE)21.7 (18.9, NE)14.1 (10.8, 16.6)18.0 (15.2, 20.0)1-year survival rate % (95% CI)80.9 (72.4, 89.3)91.8 (88.4, 95.3)71.0 (62.7, 79.4)87.7 (83.2, 92.2)2-year survival rate % (95% CI)67.2 (50.4, 83.9)88.3 (83.8, 92.8)58.3 (47.4, 69.2)76.1 (66.9, 85.3)NE=not evaluable

Loss of Chromosome 1p21 Band Is Associated with Disease Progression and Poor Survival in Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplantation.

Amplifications involving chromosome 1q and deletions involving 1p are frequent events in multiple myeloma (MM). The pathogenesis and clinical significance of these anomalies are largely unknown. As karyotyping and SNP based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we investigated the prevalence and prognostic significance of del(1p21) in a cohort of 235 MM patients undergoing highdose therapy and autologous stem cell transplant at our institution. Fluorescence in situ hybridization combined with cytoplasmic light chain detection (cIg-FISH) was used to evaluate clonal plasma cells for 1p21 deletion as well as other myeloma-associated chromosomal abnormalities. In addition, 1p21 status was evaluated in 16 patients with of monoclonal gammopathy of undetermined significance (MGUS) and 41 patients with plasma cell leukemia (PCL). CIg-FISH detected hemizygous 1p21 deletions in 18% of the MM, 34% of PCL but none of the MGUS cases. In MM, the median percentage of clonal plasma cells harboring del(1p21) was 55% (range 20–95%). The presence of 1p21 deletions was strongly correlated with 1q21amplification (p=0.01), and marginally correlated with del(p53) (p=0.05) or t(4;14) (p= 0.06), but not with del(13q) or t(11;14). There was no association between del (1p21) and other biological factors including age, gender, Hb, albumin, C-reactive protein, beta-2 microglobulin level, isotype or bone marrow plasmacytosis. The median follow-up in this cohort was 36 months. Patients with 1p21 deletions had significantly shorter progression-free (median 14.2 vs. 25.4 months, p<0.001) and overall survivals (median 39.4 vs. 82.3 months, p=0.001) than those without such deletions. In multivariate analysis, del(1p21) was an independent risk factor for progression free (p= 0.003) and overall survivals (p=0.013) after adjusting for del(13q), del(p53), t(4;14) and 1q21 amplification. 1q21 amplification was significant risk factor in univariant analysis for either progression-free or overall survivals, but did not remain as an independent risk factor in multivariant model adjusting for other genetic risk factors. Our results indicate that del(1p21) is an independent poor prognostic factor associated with disease progression in MM. We recommend include this genetic aberration as part of a routine work-up in the risk-stratification of MM patients receiving autologous stem cell transplantation.

Epigenetic Inactivation of the Secreted Frizzled-Related Protein Gene Family in Multiple Myeloma.

Multiple myeloma (MM) is a B-cell neoplasm that is characterized by the accumulation of malignant plasma cells in the bone marrow. Previous molecular studies have largely focused on acquired genetic aberrations in MM. There is increasing evidence that in addition to genetic aberrations epigenetic processes play a major role in carcinogenesis. Aberrant methylation of CpG islands near gene promoter regions is the most widely studied epigenetic abnormality in human malignancies and is associated with loss of gene function. This epigenetic event acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function. The Wnt pathway has been recognized to be essential for normal organ development, and a role for Wnt signal transduction at several stages of lymphocyte differentiation and in the self-renewal of hematopoietic stem cells could be demonstrated. Alterations in the Wnt pathway have been shown to contribute to the pathogenesis of various human malignancies. Epigenetic silencing of the family of secreted frizzled-related proteins (SFRPs), which act as Wnt antagonists, was recently reported in several solid tumors and in acute lymphoblastic leukemia. In order to investigate the potential role of abnormal Wnt signaling in MM, we determined the methylation status of the promoter-associated CpG islands of SFRP-1, 2, 4 and 5 in the MM cell lines U266, LP-1, RPMI-8226 and OPM-2. Methylation-specific polymerase chain reaction (MSP) analysis revealed that promoter hypermethylation of the SFRP genes was a frequent event in MM cell lines (SFRP-1: 2/4, SFRP-2: 2/4, SFRP-4: 1/4 and SFRP-5: 3/4). Aberrant methylation of SFRP-1 and SFRP-2 in MM cell lines was associated with transcriptional silencing, as determined by real-time reverse transcriptase polymerase chain reaction. Treatment of cell lines that carry a hypermethylated SFRP-1 and SFRP-2 gene, respectively, with the demethylating agent 5-aza-2′-deoxycytidine resulted in gene reexpression. We then analyzed by MSP the methylation status of SFRP-1, 2, 4 and 5 in 76 specimens obtained from MM patients. The frequency of aberrant methylation among the primary patient samples was 38.2% (29/76) for SFRP-1, 59.2% (45/76) for SFRP-2, 2.6% (2/76) for SFRP-4 and 7.6% (6/76) for SFRP-5. There was a high incidence of concomitant hypermethylation of SFRP-1 and SRFP-2. We conclude that promoter hypermethylation of the SFRP genes is a novel epigenetic event in MM that may contribute to aberrant activation of the Wnt pathway. Further studies are warranted to elucidate the functional consequences of aberrant Wnt signaling by downregulation of the SFRP genes in the pathogenesis of MM. Additionally, the increasing evidence for the important role of DNA methylation changes in malignant plasma cell disorders may serve as a basis for the use of epigenetically targeted therapeutic approaches in MM in the future.

Chromosome 1p21 Deletion Defines a Subgroup of Multiple Myeloma with Poor Clinical Outcomes Independent of del (13q), del(p53), t(4;14) or 1q21 Amplification.

Amplifications involving chromosome 1q and deletions involving 1p are frequent events in multiple myeloma (MM). The pathogenesis and clinical significance of these anomalies is largely unknown but CKS1B amplification at 1q21 detected in 30–40% of MM patients is associated with disease progression. As karyotyping and SNP based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we used FISH combined with cytoplasmic light chain detection (cIg-FISH) to investigate the prevalence and prognostic significance of del(1p21) in a cohort of 186 MM patients undergoing autologous stem cell transplant. CIg-FISH detected hemizygous 1p21 deletions in 18% of the cases. The median percentage of clonal plasma cells harboring del(1p21) was 55% (range 20–95%). The presence of 1p21 deletions was strongly correlated with CKS1B amplification (p=0.004), t(4;14) (p= 0.027), and del(p53) (p=0.04), but not with del(13q) or t(11;14). There was no association between del (1p21) and other biological factors including age, gender, Hb, albumin, C-reactive protein, beta-2 microglobulin level, isotype or bone marrow plasmacytosis. Patients with 1p21 deletions had significantly shorter progression-free (median 10.5 vs. 25.4 months, p=0.0001) and overall survivals (median 33.9 months vs. not reached, p=0.001) than those without such deletions. On multivariate analysis, del(1p21) was an independent risk factor for progression free (p< 0.0001) and overall survivals (p=0.0005) after adjusting for other genetic risk factors including del(13q), del(p53), t(4;14) and CKS1B amplification. Our results indicate that del(1p21) is a novel genetic risk factor and warrant inclusion of this genetic aberration in the risk-stratification of MM. Further studies are required to identify candidate tumor suppressor gene(s) at the 1p21 locus and explore their role in the molecular pathogenesis of MM.

Coordinate Interstitial Deletion of Retinoblastoma (RB1) and Neurobeachin (NBEA) Is a Recurring Event in Multiple Myeloma.

Monoallelic chromosome 13 deletion detected by cytogenetics predicts poor patient survival in multiple myeloma (MM), but the genes responsible have not been conclusively identified. To this end, we performed array comparative genomic hybridization (aCGH) using a novel, human chromosome 13 oligonucleotide array (Nimblegen) with 385,272 probes and median probe spacing of 60 base pairs. The dense coverage, and the use of germline DNA collected from each patient as internal controls for DNA copy number polymorphisms, enabled unprecedented map resolution of somatic DNA gains and losses on chromosome 13. Array CGH was performed on genomic DNA isolated from CD138+ bone marrow plasma cells purified from 20 patients with MM, monoclonal gammopathy of undetermined significance (MGUS), or amyloidosis. Visual analysis of the aCGH data identified 4 patients with chromosome 13 interstitial deletions that were confirmed using a circular binary segment algorithm (Nimblegen). Monosomy chromosome 13 was detected in 5 patients by cytogenetics, and as expected, appeared normal by aCGH due to data normalization. We also performed an unsupervised analysis of the data, which identified 49 genes with DNA copy number decreases. Both methods identified copy number decreases at 13q14 commonly affected in MM and MGUS patients and thought to harbor a relevant tumor suppressor. Three of the 4 patients with interstitial deletions at 13q14 had striking regions of DNA copy loss whose minimally deleted region was defined by a patient with a small deletion spanning exon 20 of RB1, encoding part of the functionally important ‘pocket domain' responsible for binding E2F transcription factors. We found RB1 protein levels in MM cell lines correlated with RB1 genomic copy number, and therefore considered the model that RB1 haploinsufficiency contributes to MM. However, we found Rb1 heterozygous (HET) and wild type (WT) mice had indistinguishable steady-state B, T and myeloid compartments in addition to plasma cell induction in response to sheep red blood cell stimulation. Disease burden was similar in HET vs. WT Rb1 mice in a model of NRAS induced tumorigenesis. These results suggest other genomic events cooperate with RB1 copy number loss in MM. Unexpectedly, we found the 3 patients that had an interstitial deletion of RB1 at 13q14 concomitantly harbored a separate interstitial loss at 13q13. Every patient with DNA copy number loss of RB1 also had DNA copy loss within 13q13 (5 patients who lost the entire chromosome and 3 patients with interstitial deletions). The minimally deleted region at 13q13 mapped to the 5′ end of Neurobeachin (NBEA), which encodes a Protein Kinase A (PKA) anchoring protein. We detected NBEA transcripts at low levels in normal human plasma cells. NBEA transcripts and protein were robustly expressed in 3/5 MM cell lines. This is the first report of coordinate copy number loss of RB1 and NBEA on chromosome 13 in MM. Taken together, our data suggest that chromosome 13 deletions in MM may target protein dose level of RB1 and at least one other gene, likely NBEA. Our data provide a novel rationale for future studies to examine the biological consequences of coordinate loss of NBEA and RB1.

A Simple and Reliable Method To Verify the Authenticity and Purity of Human Myeloma Cell Lines.

The availability of human myeloma cell lines (HMCLs) has drastically benefited out understanding of multiple myeloma (MM). They represent essential resources to the myeloma research community at large who use them to identify and more importantly validate oncogenic events in MM. Furthermore, they represent the front line tools in efforts to identify novel therapeutic agents for MM patients. However, the myeloma research community is not immune to problems of cell line contamination (mixed populations) or systemic errors where HMCLs are labeled incorrectly. As the community evolves towards the development of tailor medicine it is essential that our basic tools are both pure and correct. Otherwise, a potentially beneficial therapeutic agent designed for a specific patient subset maybe lost simply because of a simple laboratory mix-up at some point over the years. To that end we have developed a simple PCR based method that can be used to validate the purity and authenticity of HMCLs. First two reference collections of over 50 HMCL maintained at the NCI and Mayo Clinic Arizona were compared using microsatellite fingerprinting. This process identified several previously unnoticed discrepancies. First, in both labs several cell lines that reportedly originated from different patients had the same fingerprints. These include the following unexpected pairs; ANBL-6 and DP–6, CAG and ARP–1, KMS–11 and KMS–20, KMS–11 and KMS–21PE/BM, JIM3 and KPMM2. Second, both KP–6 and KAS6/1 showed a level of cross-contamination with ANBL–6. All of the discrepant cell lines were requested from the original contributor and those that were available were retested. After this step we had identified unique fingerprints for ANBL–6, DP–6, KMS–11, and KMS–20. Furthermore, pure populations of KP–6 and KAS6/1 were confirmed. However, CAG and ARP–1 still had an identical fingerprint. Since the use of microsatellite fingerprinting is an expensive task and the identification of cross-contamination is not very robust we set out to develop better method. We decided to use a simple qualitative PCR method, as the binomial presence or absence of a band makes the interpretation of the fingerprint extremely easy. Furthermore, the high sensitivity of a qualitative PCR can pick up a small amount of contamination in the culture that can be lost in microsatellite analysis. This method involves a multiplex PCR screen that simultaneously interrogates 10 regions of the human genome. The screen is divided into two reactions, one with a panel of highly polymorphic copy number variations (CNV) that are often completely absent in some individuals, and a second one based on known biologic events present in the cell lines. The integration of the results from the CNV and Biological Fingerprints will identify each HMCL as a unique entity. We validated the methodology by confirming that this qualitative screen could pick up the cross–contamination identified in the original KP–6 and KAS6/1 samples. As our understanding of myeloma increases and we strive to develop new therapies for specific biological entities it will be essential that groups confirm both the authenticity and purity of their test cultures. We hope that widespread use of this simple screening methodology will significantly improve the quality and reproducibility of assays carried out in labs around the world.