CD138-purified Plasma Cells Research Articles

LncRNA MALAT1 silencing represses CXCL12-induced proliferation, invasion, and homing behavior in multiple myeloma by inhibiting CXCR4

ABSTRACT Background: Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been identified in multiple myeloma (MM); its influence on the homing behavior in MM cells is unclear. This study elucidates the impact and mechanism by which MALAT1 affects the homing behavior in MM cells. Methods: Bone marrow was obtained from patients with MM. MALAT1 and C-X-C motif chemokine receptor 4 (CXCR4) expression in cluster of differentiation 138-purified (CD138) plasma cells were detected by qRT-PCR and Western blotting. MALAT1, CXCR4, and C-X-C motif chemokine ligand 12 (CXCL12) influences on MM cell viability, proliferation, and invasion was monitored by CCK-8, 5-Ethynyl-2'-deoxyuridine, and Transwell assays. qRT-PCR, Western blotting, and ELISA were utilized to detect homing effect-related proteins in MM cells, including intercellular adhesion molecule 1 (ICAM-1) and very late antigen-4 (VLA-4). Results: MALAT1 and CXCR4 were overexpressed in CD138-purified plasma cells from bone marrow of MM patients, associating with bone damage. MALAT1 upregulated CXCR4 in MM cells. MALAT1 overexpression enhanced MM cell viability, proliferation, and invasion, whereas CXCR4 silencing reversed them. CXCR4 silencing attenuated MALAT1 induction on ICAM-1 and VLA-4 expression in MM cells. CXCL12 upregulation intensified MM cell proliferation and invasion and ICAM-1 and VLA-4 expression in MM cells. MALAT1 silencing counteracted the impact of CXCL12. Conclusion: MALAT1 silencing may repress CXCL12 induction on MM cell proliferation, invasion, and homing behavior by inhibiting CXCR4. MALAT1 may be a promising target for MM treatment.

Molecular Long-Term Analysis of the GMMG-HD4 Trial in Multiple Myeloma-Patterns of Association of Chromosomal Aberrations with Response and Proliferation Determining Survival in Selecting Treatments in View of Limited Resources in Low- and Middle-Income Countries.

Based on the lack of differences in progression-free and overall survival after a median follow-up of 93 months in our HOVON-65/GMMG-HD4 trial (German part; n = 395) randomizing VAD induction (vincristin/adriamycin/dexamthasone)/tandem-transplantation/thalidomide-maintenance vs. PAD induction (bortezomib/adriamycin/dexamethasone)/tandem transplantation/bortezomib maintenance, we discern how chromosomal aberrations determine long-term prognosis by different patterns of association with proliferation and treatment-dependent response, whether responses achieved by different regimens are equal regarding prognosis, and whether subpopulations of patients could be defined as treatable without upfront "novel agents" in cases of limited resources, e.g., in low- or middle-income countries. Serum parameters and risk factors were assessed in 395 patients. CD138-purified plasma cells were subjected to fluorescence in situ hybridization (n = 354) and gene expression profiling (n = 204). We found chromosomal aberrations to be associated in four patterns with survival, proliferation, and response: deletion (del) del17p13, del8p21, del13q14, (gain) 1q21+, and translocation t(4;14) (all adverse) associate with higher proliferation. Of these, del17p is associated with an adverse response (pattern 1), and 1q21+, t(4;14), and del13q14 with a treatment-dependent better response (pattern 2). Hyperdiploidy associates with lower proliferation without impacting response or survival (pattern 3). Translocation t(11;14) has no association with survival but a treatment-dependent adverse response (pattern 4). Significantly fewer patients reach a near-complete response or better with "conventional" (VAD) vs. bortezomib-based treatment after induction or high-dose melphalan. These patients, however, show significantly better median progression-free and overall survival. Molecularly, patients responding to the two regimens differ in gene expression, indicating distinct biological properties of the responding myeloma cells. Patients with normal renal function (89.4%), low cytogenetic risk (72.5%), or low proliferation rate (37.9%) neither benefit in progression-free nor overall survival from bortezomib-based upfront treatment. We conclude that response level, the treatment by which it is achieved, and molecular background determine long-term prognosis. Chromosomal aberrations are associated in four patterns with proliferation and treatment-dependent responses. Associations with faster and deeper responses can be deceptive in the case of prognostically adverse aberrations 1q21+ and t(4;14). Far from advocating a return to "outdated" treatments, if resources do not permit state-of-the-art-treatment, normal renal function and/or molecular profiling identifies patient subpopulations doing well without upfront "novel agents".

P930: ISATUXIMAB, LENALIDOMIDE, BORTEZOMIB AND DEXAMETHASONE AS INDUCTION THERAPY FOR NEWLY-DIAGNOSED MULTIPLE MYELOMA PATIENTS WITH HIGH-RISK CYTOGENETICS: A SUBGROUP ANALYSIS FROM THE GMMG-HD7 TRIAL

Background: The multicenter phase III trial GMMG-HD7 (NCT03617731) demonstrated superior minimal residual disease (MRD) negativity rate after induction therapy in patients with transplant-eligible newly-diagnosed multiple myeloma (NDMM) by addition of the anti-CD38 monoclonal antibody isatuximab (Isa) to lenalidomide / bortezomib / dexamethasone (Isa-RVd), as compared to RVd alone (Goldschmidt H et al., 2021, ASH Annual Meeting). Aims: Here we present a subgroup analysis on patients with high-risk cytogenetics. Methods: Patients with transplant-eligible NDMM were equally randomized to receive three 42-day cycles of RVd (lenalidomide 25 mg/d p.o., d1–14 and d22-35; bortezomib 1.3 mg/m2 s.c. d1, 4, 8, 11, 22, 25, 29, 32; dexamethasone 20 mg/d d1-2, 4-5, 8-9, 11-12, 15, 22-23, 25-26, 29-30, 32-33) in both study arms. Isa was added to Isa-RVd as follows: 10 mg/kg i.v., cycle 1: d 1, 8, 15, 22, 29; cycles 2-3: d 1, 15, 29. Randomization for induction was stratified by Revised International Staging System. Primary endpoint of the trial was MRD negativity rate assessed by next-generation flow (NGF, cut-off 1x10-5) after induction therapy. Fluorescence in-situ hybridization (FISH) analysis was performed centrally on CD138-purified plasma cells. High-risk and ultra high-risk cytogenetics were defined as at least one or two of the following aberrations, respectively: del17p, t(4;14), t(14;16), gain1q21 (≥ 3 copies). Data cut-off for the present analysis was December 2021. Results: 660 patients (Isa-RVd: 331 and RVd: 329) were eligible for intention-to-treat analysis. The study met its primary endpoint, demonstrating superiority of NGF-MRD negativity rates with Isa-RVd compared to RVd (50.1% vs. 35.6%; odds ratio [OR]=1.82, 95% confidence interval [95% CI]: 1.33-2.48, p<0.001). High-risk cytogenetics were well balanced between the treatment arms. 264 of 584 (45.2%) and 82 of 580 (14.1%) evaluable patients had high-risk and ultra high-risk cytogenetics, respectively. Del17p, t(4;14), t(14;16) and gain 1q21 were present in 59 of 615 (9.6%), 67 of 613 (10.9%), 17 of 609 (2.8%) and 218 of 583 (37.4%) evaluable patients, respectively. Among patients with high-risk cytogenetics, MRD negativity rates were 50.4% (63/125) vs. 37.4% (52/139; OR=1.70, 95% CI: 1.04-2.79, p=0.03) with Isa-RVd vs. RVd. MRD negativity rates for ultra high-risk patients were 56.3% (27/48) vs. 44.1% (15/34; OR=1.63, 95% CI: 0.67-3.99, p=0.28) with Isa-RVd vs. RVd. Similar results were observed for Isa-RVd vs. RVd among the common major single high-risk cytogenetic features: del17p: 56.0% (14/25) vs. 35.3% (12/34), OR=2.33, 95% CI: 0.82-6.88; t(4;14): 57.6% (19/33) vs. 47.1% (16/34), OR=1.53, 95% CI: 0.58-4.06; t(14;16): 66.7% (6/9) vs. 50.0% (4/8), OR=2.00, 95% CI: 0.28-15.67; gain1q21: 48.2% (55/114) vs. 35.6% (37/104), OR=1.69, 95% CI: 0.98-2.92. Dividing evaluable patients in either standard risk (absence of any high-risk aberration; 320/578, 55.4%) vs. high-risk (exactly one high-risk aberration; 176/578, 30.4%) vs. ultra high-risk (≥2 high-risk aberrations; 82/578, 14.2%) yielded similar efficacy results. MRD negativity rates for Isa-RVd vs. RVd were 49.7% (86/173) vs. 36.7% (54/147; OR=1.70, 95% CI: 1.09-2.68) in standard risk patients, 46.7% (35/75) vs. 34.7% (35/101; OR=1.65, 95% CI: 0.90-3.05) in high-risk patients and 56.3% (27/48) vs. 44.1% (15/34; OR=1.63, 95% CI: 0.67-3.99) in ultra high-risk patients. Summary/Conclusion: Isa-RVd induction therapy is superior to RVd in patients with transplant-eligible NDMM and high-risk or ultra high-risk cytogenetics, consistent with the benefit observed in the overall trial population.

African-American Patients with Monoclonal Gammopathy Have a Reduced Risk of Transformation to Clinical Myeloma: Results of a Prospective Study SWOG S0120

Introduction:Both multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are associated with marked racial disparities; incidence of both MM and MGUS is increased nearly 3-fold in African American (AA) compared to Caucasian/European American (EA) cohorts. Current estimates of the risk of progression to clinical malignancy in MGUS and asymptomatic myeloma (AMM) are at 1% and 10% per year respectively, based on data from EA cohorts such as from the Olmstead county in Minnesota. Risk estimates from prospective studies in AA cohorts are urgently needed to guide optimal management of these patients.Methods:Between 2003 and 2011, 331 eligible patients with IgG/A monoclonal gammopathy were enrolled in a prospective observational trial (SWOG S0120). All patients underwent uniform staging evaluation at baseline and follow up monitoring for progression to clinical myeloma (CMM). This analysis is focused on the impact of race on clinical and biologic features and risk of progression.Results:Of 331 eligible patients, 57 (17%) were of AA descent. Clinical features of the AA cohort were comparable to the non-AA counterparts, with the exception of higher proportion of females (61% versus 43%; p=0.01) and hemoglobin < 12 g/dl (37% versus 23%; p=0.04) in the AA cohort. Among 126 patients with available data on gene expression profile (GEP) of CD138-purified plasma cells, the proportion of patients with GEP-defined subsets was similar between AA and non-AA cohorts. The risk of transformation to clinical malignancy in AA patients was significantly lower than in non-AA cohort (2 year risk 5% vs 15%; 5 year risk 13% versus 24%; log rank p 0.04). Differences in risk were evident for both MGUS (2 year risk 0% versus 2 %) and AMM (2 year risk 13% versus 25%). The proportion of patients with high risk GEP signature (GEP-70 gene risk > -0.26) in purified tumor cells was markedly lower in the AA cohort (0% versus 33%, p=0.01). Unbiased analysis of which genes in the AA cohort predict risk of progression is ongoing.Conclusions:Together these data provide the first prospective evidence that AA patients with myeloma precursor states carry significantly lower risk of progression to CMM compared to non-AA counterparts. This may be explained in part by the finding that precursor lesions in AA patients have markedly lower proportion of genomic changes (such as GEP70-risk signature derived predominantly from chromosome 1) previously associated with higher risk of malignancy in EA cohorts. The mechanisms underlying the transformation to CMM may therefore differ between AA and EA cohorts, which in turn may impact optimal management of these patients. DisclosuresBarlogie:Dana Farber Cancer Institute: Other: travel stipend; Celgene: Consultancy, Research Funding; ComtecMed- World Congress on Controversies in Hematology: Other: travel stipend; European School of Haematology- International Conference on Multiple Myeloma: Other: travel stipend; International Workshop on Waldenström's Macroglobulinemia: Other: travel stipend; Millenium: Consultancy, Research Funding; Myeloma Health, LLC: Patents & Royalties: : Co-inventor of patents and patent applications related to use of GEP in cancer medicine licensed to Myeloma Health, LLC; Multiple Myeloma Research Foundation: Other: travel stipend. Orlowski:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BioTheryX, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Poseida: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Millenium Pharmaceuticals: Consultancy, Research Funding.

Genomic Landscape and Its Prognostic Implications in Multiple Myeloma Using a Targeted Sequencing Approach

▪Next-generation sequencing (NGS) studies have shown that multiple myeloma is a heterogeneous disease with a complex subclonal architecture and few recurrently mutated genes. Only a minority of variants are potentially actionable and studies on their prognostic value are lacking. Therefore, strategies to investigate the landscape of chromosomal and gene lesions of a large number of myeloma samples in a robust fashion is needed. In this study, we developed a target-enrichment strategy to streamline simultaneous analysis of gene mutations, copy number changes and IGH translocations in multiple myeloma in a high-throughput fashion using NGS.We designed Agilent SureSelect cRNA pull down baits to target 246 genes implicated in myeloma, in lymphoid malignancies or other cancers, 2538 single nucleotide polymorphisms to detect copy number and allelic ratio at the single-gene and whole-genome level, and we tiled the whole IGH locus to detect IGH translocations and V(D)J rearrangements. As a pilot, we sequenced 13 myeloma cell lines and 10 control haematopoietic cells lines and validated its sensitivity and specificity. We have next applied this baitset to unmatched DNA from CD138-purified plasma cells from 426 patients at diagnosis, including 51 matched samples from a previous whole exome sequencing dataset. We sequenced at an average depth of 329x with 77% of the target region covered at >30x using Hiseq2000 machines (Illumina Inc.). We applied algorithms developed in-house to detect point mutations, insertion-deletions and IGH translocations, filtering out potential artifacts and germline variants. We then annotated as "oncogenic" all variants previously reported as somatic in cancer by the COSMIC database.418/426 patients had at least one variant, and overall we identified 2207 variants of which 667 were oncogenic. 212/246 evaluated genes were mutated at least once, but only 102 had at least one oncogenic variant. Furthermore, 417/667 (63%) oncogenic variants were accounted for by the top 8 driver genes previously identified (KRAS, NRAS, TP53, FAM46C, BRAF, DIS3, TRAF3, SP140). Additional findings of interest included clustered mutations in SF3B1, EGR1, IRF4, but in <5% of patients each. We identified IGH translocations in 127/375 patients, including 70 t(11;14) and 35 t(4;14). FISH validation suggested our pulldown approach had 93% sensitivity and 98% specificity. Given the high number of variables and patients, we looked at pair-wise interactions between genes and with karyotypic features. We found that BRAF variants are not mutually exclusive with KRAS or NRAS mutations, a finding with implications for pathogenesis and treatment with BRAF inhibitors. Furthermore, samples with IGH translocations, and t(4;14) in particular, had fewer mutations overall, were significantly enriched for mutations in SF3B1 and depleted of mutations in FAM46C, suggesting that the genomic landscape of myeloma may in part vary with karyotypic features.We then looked at prognostic models. While TP53 mutations had the strongest effect both on overall survival (OS) and progression free survival (PFS), we found a favorable impact of TRAF3 mutations on PFS, and a negative one of NRAS and SP140. More importantly, pairwise interactions served as a starting ground for a rationale subgroup analysis. For example, while FAM46C mutations had no effect on OS across the whole cohort, they predicted better survival in patients without IGH translocations (median 94 months for WT FAM46C vs. not reached for MUT, p=0.042 log-rank test). Similarly, we found a different magnitude of the negative impact of TP53 mutations on OS based on karyotype. Survival was shortened more than 8-fold in cases with both an IGH translocation and a TP53 mutation (median 89 months for WT TP53 vs. 11 for MUT, p=2e-10), while this effect was much less in the absence of an IGH translocation (94 months for WT vs. 50 for MUT, p=0.02).In conclusion, the large sample size and extent of sequencing provides further insight into the genomic landscape of myeloma and how this impacts the clinical phenotype confirming the utility of the targeted sequencing to both understand the biology as well as its clinical application. DisclosuresCampbell:14M genomics: Other: Co-founder and consultant.

Target Expression, Preclinical Activity and Mechanism of Action of EM801: A Novel First-in-Class Bcma T-Cell Bispecific Antibody for the Treatment of Multiple Myeloma

Background. T-cell bispecific antibodies (TCBs) simultaneously binding CD3 on T-cells and individual tumor antigens, activate T-cells and destroy tumor antigen carrying cells. B-cell maturation antigen (BCMA), a surface antigen reported to be expressed on normal and malignant plasma cells (PCs), could represent a potentially promising target for TCBs in multiple myeloma (MM).TheAim of our study was to: i) assess expression of BCMA in normal and malignant PCs as well as cells of the bone marrow (BM) microenvironment by gene expression profiling and flow cytometry to validate it as potential clinical target for TCBs; ii) to evaluate activity of EM801 as member of a novel class of BCMA-TCBs in vitro on primary myeloma cells and in vivo in the H929-xenograft reconstituted NOG mouse model; and iii) to delineate its mechanism of action.Results.Expression. We investigated the expression of BCMA in CD138-purified PCs from BM aspirates obtained from 726 patients including MGUS (n=62), asymptomatic (n=59) and symptomatic MM (605), as well as different BM cellular subsets from healthy donors (n=10 PCs; plasmablasts, memory B-cells, T-cells, CD34+, CD14+, CD15+, n=5 each; n=8 mesenchymal stromal cells) using Affymetrix DNA-microarrays. BCMA expression was observed in malignant PC from 723/726 (99.5%) MGUS and MM patients, 10/10 normal PCs and 5/5 plasmablasts; gene expression of BCMA was undetectable in all other normal BM subsets. Using multiparameter flow cytometry, BCMA surface expression on malignant PCs was confirmed in 40/40 patients while being absent on normal BM cells. BCMA is thus a potential target in virtually all myeloma patients.Activity. In vitro, EM801 induced concentration dependent significant cell death in malignant plasma cells in BM-samples of 21/28 (75%) previously untreated and 8/10 (80%) relapsed/refractory MM patients in concentrations ranging from 10pM to 30nM. No or only minor unspecific toxicity on cells of the BM microenvironment was observed. In vivo efficacy of EM801 was studied in a subcutaneous H929 myeloma cell line xenograft model in NOG (NOD/Shi-scid/IL-2Rγnull) mice reconstituted with human PBMCs. Three doses of EM801, i.e. 0.026, 0.26 and 2.6 nM/kg, the same doses of a BCMAxCD3-(scFv)2 and two control groups were investigated (n=9 mice/group). Three weekly intravenous doses were given, starting on day 19 after tumor cell injection when tumor volumes were 293±135 mm3. On day 47, all mice from control groups had their tumors grown beyond 2000 mm3 and were euthanized for ethical reasons. In contrast, at 2.6 nM/kg (0.5 mg/kg) EM801 tumor regression was already observed after the second i.v. injection in 6/9 animals and the tumor regressed to 16±3 mm3 on day 47. BCMAxCD3-(scFv)2 bispecific antibody without Fc did not show any efficacy at all doses studied.Regarding the mechanism of action, we first demonstrated that EM801 effectively binds myeloma cells and T-cells with a strength of 1622±410 pN (5-10 fold of control) as measured by atomic force microscopy. Secondly, increasing concentrations (0.03-30nM) of EM801 led to progressive T-cell activation in primary BM samples, with significantly increased levels of CD69 (P<0.001), CD25 (P<0.001) and HLADR (P=0.001) expression in both CD4 and CD8 T-cells as compared to an unspecific TCB. Thirdly, EM801 induced significant secretion of interferon-γ (19-3000 pg/ml), granzyme B (68-2986 pg/ml), and perforin (145-3712 pg/ml) as measured by ELISA, together explaining the strong in vitro and in vivo activity of EM801.Conclusions. BCMA is selectively expressed at the RNA (723/726) and protein (40/40) levels on malignant PCs from virtually all MM patients, and thus represents a promising TCB-target. The novel BCMA-TCB EM801 was effective in vitro in 29/38 (76%) primary MM patients' BM samples at picomolar to low nanomolar concentrations, easily achievable in vivo in patients, as well as in the H929-xenograft reconstituted NOG mouse model at 0.5 mg/kg once a week. Neither in vitro (the BM microenvironment) nor in vivo the compound shows significant toxicity or side effects. EM801 confers cytotoxicity by effectively coupling T-cells with malignant PCs, inducing T-cell activation, secretion of interferon-γ, granzyme B and perforin, and thereby effectively killing malignant PCs. EM801 is thus a promising new compound for the treatment of multiple myeloma to be investigated in clinical phase I/II trials. DisclosuresSeckinger:EngMab AG: Research Funding; Takeda: Other: Travel grant. Neuber:EngMab AG: Research Funding. Vu:EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Strein:BB Biotech AG: Membership on an entity's Board of Directors or advisory committees; Novimmune SA: Membership on an entity's Board of Directors or advisory committees; EngMab AG: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Hundemer:EngMab AG: Research Funding. San Miguel:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Janssen-Cilag: Honoraria; Millennium: Honoraria; Novartis: Honoraria; Sanofi-Aventis: Honoraria; Onyx: Honoraria. Hose:Takeda: Other: Travel grant; EngMab AG: Research Funding. Paiva:Celgene: Consultancy; Janssen: Consultancy; Binding Site: Consultancy; BD Bioscience: Consultancy; EngMab AG: Research Funding; Onyx: Consultancy; Millenium: Consultancy; Sanofi: Consultancy.

Identification and Validation of IMiD-14 Model Predictive of IMiD Resistance in Multiple Myeloma

Abstract BACKGROUND: Immunomodulatory derivatives (IMiDs) have become an integral part of multiple myeloma (MM) treatment, both in newly diagnosed and relapsed refractory patients. Long-term treatment with IMiDs is inevitably associated with relapse and emerging resistance. The precise mechanisms of IMiDs resistance are not entirely clear. Gene-expression profiling (GEP) can be used to elucidate resistance/response signature associated with IMiDs. PATIENTS AND METHODS: GEP of paired samples of CD138-purified plasma cells, obtained from patients prior to and 48 hours after single-agent therapy with thalidomide (42 newly diagnosed MM), lenalidomide (18 relapsed/refractory MM), or pomalidomide (18 relapsed/refractory MM) were compared. Samples were hybridized to Affymetrix expression arrays. Affymetrix U133Plus2.0 arrays were used in the pomalidomide study and Affymetrix U95Av2 arrays were used for thalidomide and lenalidomide pharmacogenomics studies. Among 176 genes that significantly changed pre- and post IMiD treatment (P&lt;0.05 by SAM analysis, and with change in same direction), 14 genes were selected that had P values &lt;0.05 based on univariate Cox regression analysis for PFS in thalidomide arm of TT 2 trial. These 14 genes were combined to create a continuous score based upon each patient's difference between the average log2-scale expression of the 4 prognosis-unfavorable (hazard ratio [HR] &gt; 1) and the 10 prognosis-favorable (HR &lt; 1) genes. An optimal cutoff for this IMiDs-based risk score (hereafter referred to as IMiD14) was then established with the running log-rank test, so that patients with scores higher than the cutoff were considered as IMiDs resistant patients. RESULTS: The IMiDs resistant group in TT2 (Thalidomide arm) training set exhibited significantly poor PFS (P &lt; 0.001) and inferior OS (P &lt; 0.001). Next, we examined whether IMiD14 model had outcome discriminatory power in several independent test cohorts treated with IMiDs. In validation cohort TT3a (induction with VT(thalidomide)D-PACE, and maintenance with VTD in the 1st year followed by TD for 2 years), there were significant differences in outcome between patients whom IMiD14 model predicted to have IMiDs resistance versus others, 3-year PFS and OS rates for the predicted IMiDs resistant patients were 63% and 71%, whereas for the rest of patients the corresponding rates were 87% and 90% (PFS P = 0.01, OS P &lt; 0.001). In validation cohort TT3b (induction with VT(thalidomide)D-PACE, and maintenance with VRD for 3 years), 3-year PFS and OS rates for the predicted IMiDs resistant patients were 62% and 70%, whereas for the rest of patients the corresponding rates were 80% and 85% (PFS P = 0.002, OS P = 0.003). The discriminatory power of IMiD14 was also independently confirmed in the validation cohort HOVON65/GMMG-HD4 VAD arm (induction with vincristine, doxorubicin, and dexamethasone, and maintenance with thalidomide for 2 years); 3-year PFS and OS rates for the predicted IMiDs resistant patients were 16% and 38%, versus 54% and 82%, respectively for the rest of the patients (PFS P &lt; 0.001, OS P &lt; 0.001). CONCLUSION: We have identified IMiD14 gene expression signature that accurately predicts resistance to IMiD treatment and outcome in multiple myeloma patients. These predictive genes could provide novel targets for therapeutic intervention and are being evaluated in prospective datasets. Figure 1. Figure 1. Disclosures Barlogie: Celgene: Honoraria, Research Funding; Millennium: Honoraria, Research Funding; Novartis: Consultancy. Sonneveld:Janssen-Cilag, Celgene, Onyx, Karyopharm: Honoraria, Research Funding; novartis: Honoraria. Usmani:Celgene: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau; Millennium: Honoraria, Speakers Bureau; Janssen Oncology: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Pharmacyclics: Research Funding; Array BioPharma: Honoraria, Research Funding.

Insulin like growth factor binding protein 7 (IGFBP7) expression is linked to poor prognosis but may protect from bone disease in multiple myeloma.

BackgroundInsulin like growth factor binding protein 7 (IGFBP7) is a secreted protein binding insulin like growth factor 1 (IGF-1), insulin, vascular endothelial growth factor A (VEGFA), and activin A. It antagonizes bone morphogenetic proteins and is involved in the tumour propagation of solid as well as haematological malignancies. Its role in multiple myeloma (MM) is not defined so far. We therefore aim here to investigate its prognostic and pathophysiological role in MM.MethodsThe clinical significance of IGFBP7 gene expression was investigated by gene expression profiling in two independent cohorts (n = 948) of newly-diagnosed MM patients. Methylation of the IGFBP7 promoter was analysed by pyrosequencing and treatment of MM cell lines with 5-aza-2-deoxycytidine. The impact of IGFBP7 on MM cells was studied by CCK-8 assay, BrdU assay and flow cytometry, respectively. IGFBP7 expression in bone marrow stromal cells (BMSCs) was studied by quantitative RT-PCR. For osteoblast development, immortalized and primary human BMSCs were cultured in osteogenic differentiation medium for 7–14 days in the presence of recombinant human IGFBP7 and/or activin A.ResultsMedian IGFBP7 expression is significantly lower in CD138-purified plasma cells from individuals with MGUS and MM, compared to normal bone marrow plasma cells. IGFBP7 gene expression in MM cells is regulated by methylation, shown by pyrosequencing and exposure to demethylating agents (5-aza-2-deoxycytidine). High expression of IGFBP7 in MM cells is associated with adverse survival in two independent cohorts of 247 and 701 newly-diagnosed MM patients treated with high-dose therapy and autologous stem cell transplantation. IGFBP7 is associated with prognostically adverse chromosomal aberrations (t(4;14) and gain of 1q21), MMSET expression, and higher myeloma cell proliferation. In vitro, IGFBP7 overcomes activin A induced osteoblast suppression and promotes osteogenesis. MM cells downregulate IGFBP7 in stromal cells, possibly contributing to the osteoblast suppression found in MM. Conversely, higher IGFBP7 expression is associated with a lower probability of myeloma bone disease.ConclusionsOur data indicate that IGFBP7 expression is a marker for a specific methylation pattern in myeloma, linked to translocation t(4;14) associated MMSET expression, showing clinical features of adverse prognosis with absence of myeloma bone disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0105-1) contains supplementary material, which is available to authorized users.

BH3 Profiling Identifies Bcl-2 Dependency in Multiple Myeloma and Predicts Sensitivity to BH3 Mimetics

Despite significant improvement in the treatment of Multiple Myeloma (MM), patients still experience relapse and the disease remains incurable. Cellular fate decision in response to drug therapy is mainly determined by the interactions among BCL-2 family members. Thus anti-apoptotic BCL-2 family proteins represent an attractive target for therapy. BH3 profiling is a functional assay that identifies the tumor cell’s addiction to anti-apoptotic members, such as BCL-2, BCL-XL or MCL-1. In the present study, we determined the BCL-2, BCL-XL and MCL-1 dependency of both MM cell lines and primary myeloma samples, and then tested the ability of BH3 profiling to predict the in vitrosensitivity to BH3 mimetics.First, the BCL-2, BCL-XL or MCL-1 dependence of MM cell lines (n=11) was measured using BH3 profiling. After cell permeabilization, mitochondria were exposed to standardized amounts of peptides derived from the BH3 domains of BH3-only proteins. For instance, the BAD BH3-only peptide binds with high affinity to BCL-2 and BCL-XL, whereas HRK and MS1 peptides bind with high affinity only to BCL-XL and MCL-1, respectively. The cytochrome c release induced by each peptide was quantified by FACS analysis. Dependence on individual anti-apoptotic proteins was found to be very heterogeneous from one cell line to another. Most cell lines (7/11) were found to be MCL-1 dependent. Five cell lines were found to be Bcl-XL dependent and 2 cell lines BCL-2 dependent. Notably, 3 cell lines showed co-dependence to both MCL-1 and BCL-XL anti-apoptotic proteins.We then determined the in vitro sensitivity of MM cell lines to the BH3 mimetics ABT-199 (BC-2 specific) and ABT-263 (BCL-2, BCL-XL and BCL-w specific). Only 3 cell lines out of 11 were found sensitive to ABT-199 and 263 (LD50<100 nM). Interestingly, as we have previously found in other cancers, BH3 profiling predicted sensitivity to ABT-199 and 263 treatment.We then determined the mitochondrial priming of primary plasma cells obtained from 11 MM patients. BH3 profiling was performed on CD138-purified plasma cells. As observed with MM cell lines, patient samples showed heterogeneous dependency to anti-apoptotic proteins: MCL-1 (n=3), BCL-2 (n=2), Bcl-XL (n=2). Of note, mitochondria of 5 patient samples were found to be independent of BCL-2, BCL-XL or MCL-1. Cells were cultured with/without ABT-199 and 263 during 16 hours and then cell death was assessed by flow cytometry after annexin V staining. Two and 4 samples were found sensitive (LD50 < 100 nM) to ABT-199 and ABT-263, respectively. Once again, BH3 profiling with the BAD peptide correlated with in vitro sensitivity to ABT-199 and 263.To conclude, Multiple Myeloma is a heterogeneous disease regarding its dependence to anti-apoptotic proteins, and cannot be considered as monolithically MCL-1 or BCL-2 dependent. Rather, this determination must be made individually. BH3 profiling allows the identification of a BCL-2 dependent subset of MM patients and predicts ABT-199 in vitro sensitivity. It is of clinical interest to identify BCL-2 dependency in MM due to the current development of the oral BH3 mimetic ABT-199 that showed very promising results in BCL-2 dependent malignancies. DisclosuresOff Label Use: The presentation discuss the sensitivity of myeloma cells to the Bcl-2 specific BH3 mimetic ABT-199. Le Gouill:Roche: Consultancy; Janssen: Consultancy. Letai:Abbvie: Consultancy.

Expression of Cereblon (CRBN) Is Associated with Disease Stage, Genetic Subgroups and Specific Micro-RNAs in Multiple Myeloma

AbstractAbstract 1820 Introduction:Since the introduction of thalidomide into the treatment of multiple myeloma (MM) immunomodulatory drugs (IMiDs) have become an essential treatment modality for the management of MM. Recently, cereblon (CRBN) has been identified as the thalidomide binding protein disrupting the E3 ubiquitin ligase complex composed by CRBN, DDB1 and Cul4 (Ito et al., Science 2010). Moreover, CRBN is essential for the antimyeloma activity of lenalidomide and pomalidomide (Zhu et al.; Blood 2011). However, the genetic and epigenetic mechanisms by which CRBN is regulated are not understood so far. Therefore, we aimed to determine if CRBN expression associates with clinically relevant subgroups and response to therapy with lenalidomide. In addition, we investigated additional regulatory layers by identifying key microRNAs (miRNAs) correlated with CRBN expression. Methods:CRBN expression was measured by real-time PCR (qPCR) using CD138 purified plasma cells from 17 patients with a monoclonal gammopathy of undetermined significance (MGUS) and 139 patients with MM. CRBN expression was normalized to GUSB (used as internal control) and to the expression levels of CRBN in normal plasma cells (n=4). MiRNA expression profiles were generated using Agilent mirRNA-arrays on patients with the highest and lowest CRBN mRNA expression levels, respectively (n=42). All patients were characterized by a comprehensive set of FISH probes for the presence of recurring cytogenetic abnormalities. Results:CRBN expression was variable in the investigated samples (median: 0.717; range: 0.078 – 5.285). In patients with MGUS (median: 0.833) CRBN expression was significantly (p=0.01) lower as compared to patients with a MM (median: 0.673). CRBN expression was associated with cytogenetic subgroups (p<0.05), with patients harboring a translocation t(11;14) and gains at 9q34 presenting with the highest CRBN expression levels and patients with a deletion(del) 17p13 or gains at 1q21 with the lowest CRBN expression levels. Out of 526 miRNAs that passed the detection threshold, 28 miRNAs were significantly associated with CRBN expression (p<0.005; >2-fold change). A total of 21 miRNAs were positively correlated and seven miRNAs were negatively correlated with high CRBN expression levels. Of particular interest, miRNAs with conserved target sites in the CRBN 3′-UTR (miRNA -23a and let-7g) were represented in the signature. Summary:The expression of CRBN is variable in plasma cell disorders with decreasing CRBN expression levels in MM compared to the precursor state MGUS and normal plasma cells. Moreover, CRBN is lower expressed in poor cytogenetic subgroups (in particular del17p13 and gains at 1q21). This might provide a novel mechanism underlying the adverse clinic outcome in this patient group. Finally, the differential expression of miRNAs with putative CRBN target sites may uncover an important regulatory mechanism. Disclosures:No relevant conflicts of interest to declare.

Pharmacogenomic Study of Carfilzomib in Relapsed/Refractory Multiple Myeloma Suggests That Carfilzomib Is a More Potent Proteasome Inhibitor Than Bortezomib

Abstract 5015 BACKGROUND:The introduction of bortezomib (BOZ) – the first FDA-approved proteasome inhibitor (PI) – as a therapeutic option for multiple myeloma (MM) has significantly improved patient outcomes in the last decade. However, relapsing patients often have BOZ-resistant disease. Carfilzomib (CFZ), a next-generation PI, recently received FDA approval for the treatment of patients with relapsed and/or refractory MM with prior history of BOZ treatment. The present study is the first post-CFZ phamacogenomic study in patients with relapsed/refractory MM, and was performed as part of the previously reported UARK Phase II CFZ compassionate use trial (registered with ClinicalTrials. gov as NCT00999414). METHODS:Gene expression profiling (GEP) analyses were performed on samples from 20 patients, all of whom had received prior treatment with BOZ and at least 1 immunomodulatory agent. CD138-purified plasma cells were collected from patients prior to and 24 hours after the second dose of CFZ. Ten patients received 20 mg/m2/day, and 10 patients received 27 mg/m2/day CFZ. Similar GEP analyses were done on pre- and 48-hr post a BOZ test dose (1. 0mg/m2) paired samples available on 25 previously treated patients on Total Therapy 6 (TT6) trial (received >1 cycle of prior treatment, 14 with previous BOZ treatment), and 142 newly diagnosed patients on Total Therapy 3A (TT3A) clinical trial. All samples were hybridized to Affymetrix U133Plus2. 0 arrays according to the manufacturer’s recommendations. RESULTS:Eight patients (40%; 4 patients in each dosing group) achieved responses of PR or better after 1 cycle of CFZ. At the false discovery rate (FDR) of 0. 05 by paired significance analysis of microarrays (SAM), expression levels of 113 probesets (104 genes) significantly changed in response to CFZ treatment, with higher expression values observed post CFZ. Among these 104 genes, 22 (21%) were proteasome-related genes (PSMA1, PSMA4, PSMA5, PSMA7, PSMB2, PSMB3, PSMB4, PSMB5, PSMB6, PSMB7, PSMC1, PSMC3, PSMC4, PSMC5, PSMD1, PSMD2, PSMD3, PSMD4, PSMD6, PSMD11, PSMD13, PSMD14). Additionally, expression-based proliferation indices (Zhan, Blood 2006) were significantly lower (p<0. 05) for the post CFZ samples. Using the same statistical analysis, no probesets were significantly changed in response to the BOZ test dose in the 25 patients treated on TT6. Additionally, the GEP-based proliferation indices in response to BOZ were also unchanged. The same results were obtained when comparison was limited to the 14 TT6 patients with prior BOZ treatment. In a similar analysis of samples from 142 TT3A patients (newly diagnosed patients with no prior BOZ exposure), expression of 51 probesets (40 genes) significantly changed (FDR=0. 05) in response to BOZ treatment. Among those 40 genes, only 3 (7. 5%) were proteasome-related (PSMA6, PSMC2, PSMD14). PSMD14 is the only proteasome-related gene overlapping between the CFZ and the BOZ studies at FDR=0. 05. PSMB2, PSMD4, and PSMA7 were also changed when a higher FDR threshold (FDR=0. 15) was used for TT3A. Above results suggest that the proteasome subunits targeted by these 2 PIs could be different. Higher expression of proteasome genes following test doses of proteasome inhibitors may reflect a cellular response to the inhibition of a critical pathway. Alternatively, it may reflect selection of cells less sensitive to the inhibitor. CONCLUSION:This is the first-in-human pharmacogenomic study comparing CFZ with BOZ and reporting on their differential GEP features. We have compared CFZ GEP effects (all patients with prior BOZ exposure) to BOZ GEP effects on patients with prior BOZ exposure or without prior BOZ exposure. The study results indicate that CFZ leads to a more global effect on the proteasomal degradation pathway compared with BOZ, and likely has more potent proteasome inhibitory activity than BOZ. This effect may in part reflect CFZ's irreversible inhibitory activity, whereas BOZ is a reversible inhibitor. Disclosures:Barlogie:Onyx: Research Funding. Usmani:Onyx: Consultancy, Research Funding.

Bortezomib-Based Induction Therapy Followed by Autologous Stem Cell Transplantation and Maintenance Therapy with Bortezomib Improves Outcome In Myeloma Patients with Gain 1q21 and t(4;14) - a Subgroup Analysis of the HOVON-65/GMMG-HD4 Trial

Bortezomib-Based Induction Therapy Followed by Autologous Stem Cell Transplantation and Maintenance Therapy with Bortezomib Improves Outcome In Myeloma Patients with Gain 1q21 and t(4;14) - a Subgroup Analysis of the HOVON-65/GMMG-HD4 Trial

Gene Expression Profiling (GEP) of CD138-Purified Plasma Cells: Modeling for High LDH and Cytogenetic Abnormalities as Independently Adverse Features of Multiple Myeloma (MM) In Total Therapy (TT) Protocols

AbstractAbstract 2985 Background:The prognosis of patients with MM is best captured by GEP-defined risk, distinguishing ~15% of patients with a median overall survival (OS) in TT2 of ~2yr as opposed to >10yr in low-risk disease. Serum LDH elevation has remained an independent adverse feature, along with the presence of metaphase cytogenetic abnormalities (CA). Here we examined whether GEP features within CD138-selected plasma cells can identify MM-associated serum LDH elevation and CA. Patients and Methods:The training set consisted of 621 cases and the test set consisted of 325 TT patients who had both baseline clinical and GEP data from whole genome Affymetrix U133Plus2.0 microarrays on CD138-enriched plasma cells. Using the training data and the scoring approach described in Shaughnessy et al (2007), we defined a GEP score based on 50 genes to predict serum LDH >190U/L and a GEP score with 15 genes to predict the presence of CA. The gene scores were then tested in univariate and multivariate Cox regression models to assess their clinical utilities in both training and test sets. Results:In the training set, sensitivity and specificity of the GEP 50-gene score for serum LDH >190 were 65% and 76%, respectively; those of the GEP 15-gene score predicting CA were 75% and 78%, respectively. While both GEP scores were significantly associated with overall and event-free survival (p < .001), they were not selected in multivariate stepwise Cox regression analysis. However, the gene scores seem comparable to the clinical variables they try to predict. When serum LDH >190 and CA were replaced by the GEP scores in multivariate models, both gene scores were significant at the .1 level, and the R-squared (R2) statistic decreased by only 2.4% (Table 1). In the test set, sensitivity and specificity of the GEP 50-gene score for serum LDH >190 were 59% and 71%, respectively. Those of the GEP 15-gene score for CA were 58% and 70%, respectively. The GEP scores behaved similarly overall as in the training set with the LDH gene score showing a little higher association than the CA gene score (p<.01 for the GEP CA score and p<.001 for the GEP LDH score). In multivariate stepwise Cox regression, only CA was selected, and when CA was replaced with the GEP CA score (or GEP LDH score), the R-squared statistic decreased by 4.6% (or 1.9%). Conclusions:In summary, we conclude that the GEP scores for LDH and CA are highly comparable to their corresponding clinical variables in terms of survival prediction capability. Our future research will focus on refining our gene selection procedure to achieve higher prediction accuracy, studying the molecular features of selected genes and, based on GEP data, building a predictive model that is at least as powerful as if only clinical variables were used in the model.Table 1Overall survival analysis in training setUnivariate Association with Overall SurvivalVariablen/N (%)HR (95% CI)P-valueR2Any cytogenetic abnormality203/612ü(33%)2.42ü(1.79,3.29)<.00115.1%LDH >= 190 U/L167/612ü(27%)2.35ü(1.73,3.20)<.00113.1%GEP 15-gene score for CA247/612ü(40%)1.98ü(1.46,2.70)<.00110.5%GEP 50-gene score for LDH >=190U/L216/612ü(35%)2.55ü(1.87,3.46)<.00117.3%Multivariate Stepwise Cox Regression AnalysisVariablen/N (%)HR (95% CI)P-valueCumulative R2GEP 70 gene risk88/612ü(14%)2.27ü(1.53,3.35)<.00119.7%LDH >= 190 U/L167/612ü(27%)1.68ü(1.21,2.33)0.00224.7%Any cytogenetic abnormality203/612ü(33%)1.84ü(1.31,2.57)<.00128.0%Creatinine >= 2 mg/dL47/612ü(8%)2.20ü(1.43,3.38)<.00130.8%Albumin < 3.5 g/dL178/612ü(29%)1.52ü(1.08,2.15)0.01631.9%GEP TP53 deletion85/612ü(14%)2.13ü(1.40,3.22)<.00133.0%GEP MF subgroup37/612ü(6%)1.74ü(1.04,2.92)0.03532.7%Multivariate Cox Regression Analysis after Replacing LDH >=190U/L and CA Clinical Variables with GEP ScoresVariablen/N (%)HR (95% CI)P-valueCumulative R2GEP 70 gene risk88/612ü(14%)2.20ü(1.43,3.40)<.00119.7%Creatinine >= 2 mg/dL47/612ü(8%)2.48ü(1.62,3.80)<.00124.0%GEP 50-gene score for LDH >=190U/L216/612ü(35%)1.39ü(0.95,2.02)0.08627.1%Albumin < 3.5 g/dL178/612ü(29%)1.56ü(1.11,2.19)0.01028.5%GEP TP53 deletion85/612ü(14%)2.32ü(1.53,3.51)<.00129.3%GEP 15-gene score for CA247/612ü(40%)1.58ü(1.12,2.23)0.00930.4%GEP MF subgroup37/612ü(6%)1.76ü(1.05,2.95)0.03230.3% Disclosures:No relevant conflicts of interest to declare.

Gene expression profiling (GEP) analysis of CD138-purified plasma cells (PC) obtained from MRI-defined focal lesions (FL) under CT guidance: Applying the 70 gene risk model and comparison with data generated on random samples (RS) from multiple myeloma (MM) treated with total therapy 2, 3, 4, and 5 (TT2, TT3, TT4, and TT5).

8126 Background: We have reported on the superior performance of RS-based GEP to distinguish 85% with low-risk (LO) and 15% with high-risk (HI) MM (Shaughnessy, Blood 2008). MM sometimes presents as macrofocal disease so that RS is uninformative. Here we report on a comparison of concurrent FL and RS GEP data from 69 patients, 3 of whom were enrolled in TT2, 22 in TT3, 40 in TT4 and 4 in TT5. Methods: GEP risk scores were compared, heat-map hierarchical clustering was applied to determine the relative vicinity of RS and FL samples, and overall survival (OS) and event-free survival (EFS) were compared applying the RS-derived 70-gene model. Results: Congruence in risk designation applied to 56 patients with LO/LO and 4 with HI/HI features, whereas 5 displayed RS-LO/FL-HI and 4 displayed RS-HI/FL-LO. Unsupervised hierarchical clustering placed 46 patients' RS and FL next to each other. OS and EFS comparisons were performed according to tertile 70-gene risk scores with RS and FL samples: OS and EFS were significantly inferior in the 12 patients exhibiting top-tertile risk scores in both RS and FL samples with 3-yr estimates of 73% and 49% compared to the remaining 59 patients with 3-yr OS and EFS estimates of 95% each (p<0.001). Conclusions: Genomic features in FL contributed to outcome prediction. Whether FL-GEP in macro-focal disease, absent RS-GEP, has similar independent outcome implications awaits additional accrual and follow-up of such patients. No significant financial relationships to disclose.

Cytogenetic Abnormalities and Appearance of Multiple Myeloma in Whole Body-MRI.

Abstract 4868 IntroductionIn magnetic resonance imaging (MRI) multiple myeloma (MM) presents with circumscribed focal lesions or diffuse infiltration of bone marrow. To identify genetic mechanisms influencing the growth pattern, whole-body MRI of 99 patients with asymptomatic and 114 patients with symptomatic MM were evaluated retrospectively by two experienced radiologists. The pattern was analyzed in the spine and focal lesions were counted in the whole body differentiating intra-osseous and soft tissue lesions as well as osseus tumors affecting cortical bone. Cytogenetic analysis was performed using interphase fluorescence in-situ hybridization (iFISH) on CD138-purified monoclonal plasma cells acquired by unilateral bone marrow aspiration for the following aberrations: t(4;14), t(11;14), t(14;16), deletions 13q14 and 17p13, as well as gain of 1q21. Statistical analysis was performed to address the following questions: i) Is there a significant correlation of chromosomal abnormalities with the presentation of MM in MRI ii) Is there an association of the occurrence of affection of cortical bone with cytogenetic aberrations. As a number of more than 7 focal lesions in the axial skeleton has been shown to be an adverse prognostic factor for patients with symptomatic MM, we performed a search for an optimal cut-off point in number of focal lesions in whole body MRI with respect to progression free survival and overall survival. As event for progression free survival initiation of treatment for asymptomatic MM and progression after the first line of treatment for symptomatic MM was defined. ResultsCorrelation of the presentation of MM in MRI with common chromosomal abnormalities was found neither concerning focal or diffuse infiltration patterns nor an affection of cortical bone, potentially leading to instability.A search for the optimal cut-off point led to a number of more than one and more than 8 focal lesions in whole body MRI for asymptomatic and symptomatic MM respectively.The only significant prognostic factors for progression of asymptomatic MM into symptomatic disease were the presence per se and a number of more than one focal lesion or diffuse infiltration in MRI. For symptomatic myeloma a number of more than 8 focal lesions was the only significant prognostic factor for overall survival (HR 4.87; p-value <0.001). In symptomatic disease the factors t(4;14), gain of 1q21 and a diffuse infiltration pattern (for overall survival) and gain of 1q21 (for progression free survival) lost statistical significance after adjustment of p-values because of multiple testing. ConclusionIn our cohort of 213 patients the most important risk factors for overall survival were focal lesions above a cut-off point of 1 and 8 for asymptomatic and symptomatic MM, respectively. No correlation of the appearance of MM in MRI with the presence of cytogenetic abnormalities in iFISH analysis was found. We therefore conclude that the infiltration pattern in MRI is not associated with cytogenetic abnormalities and that the number of focal lesions in whole body MRI is an important and independent risk factor for patients with multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Gene expression profiling (GEP) of cd 138-purified plasma cells (pc) in previously treated multiple myeloma (PTMM): Validating prognostic models developed in newly diagnosed MM (NDMM)

8598 Background: We have previously reported on the strong discriminatory power in NDMM of GEP-derived 70-gene risk in CD138-purified plasma-cells developed in 351 patients enrolled in TT2 and validated in 441 patients enrolled in two TT3 trials and in PTMM treated with single agent high-dose dexamethasone or bortezomib. Here we report on the overall survival outcomes in 137 patients with PTMM. Methods: Treatment regimens included further autotransplantation and novel agent combinations. The purpose of the investigation was to determine whether PTMM OS was also governed by GEP features, such as high-risk (HR) score, proliferation score (PS), proliferation (PR) molecular subgroup, gain/amplification of chromosome 1q (amp1q) and deletion of the short arm (del1p) and TP53 deletion. Results: Compared with NDMM, PTMM was characterized by greater proportions with HR (32% v 16%, p&lt;0.001), PS (20% v 10%, p=0.002) and PR (24% v 11%, p &lt; 0.001); no difference was observed for TP53 deletion (21% v 30%, p=0.11); amp1q/del1p was more common in PTMM (17% v 9%, p=0.01). OS was 60% at 8yr in NDMM compared to a median of only 2.4yr in PTMM. According to HR, 4-yr OS estimates were 80% for LR v 37% for HR in NDMM (p&lt;0.0001) and 52% for LR v 24% for HR in PTMM (p&lt;0.01). On multivariate analysis of both standard prognostic factors and GEP HR, OS in PTMM was adversely affected by HR status (HR=2.00, p=0.047) and albumin &lt;3.5g/dL (HR=2.66, P=0.013), accounting for a cumulative R2 value of 20%. Conclusions: GEP-derived high-risk features are more prevalent in PTMM relative to NDMM in terms of HR, PS, PR and amp1q/del1p status. As in NDMM, HR status confers poor OS in PTMM. Consistent with serial sampling where LR to HR transformation routinely occurs, these data support the concept that HR in PTMM is derived from clonal evolution. Treatment strategies that presume the existence of underlying HR cells even in LR disease, should be pursued. [Table: see text]

Use of bortezomib (BOR) pharmacogenomics (PG) to identify mechanisms of drug resistance and predict survival in multiple myeloma (MM) treated with total therapy 3 (TT3)

8538 Background: The prognosis of patients with MM is best captured by gene expression profiling (GEP) analysis of CD138-purified plasma cells (PC), distinguishing a high-risk group of 15% with dismal survival using a 70-gene baseline risk model (BLR). Translational research in TT3 was designed to investigate whether short-term BOR-induced GEP alterations could advance our understanding of BOR's novel mechanism of action. Methods: PG studies were performed as part of two TT3 trials (TT3a, n=303; TT3b, n=177), obtaining PC prior to and 48hr after a BOR test-dose (1.0mg/m2), which was accomplished in 142 patients in TT3a (training set) and 127 in TT3b (test set). Among 1051 genes significantly altered post-BOR in TT3a, 80 were identified as being significantly associated with EFS. A continuous risk score was calculated and an optimal cut-point for EFS separation determined. The independent prognostic power of the binary risk score was tested in TT3b. Multivariate analyses (MV) were employed to determine post-BOR risk (PBR) in relationship to standard prognostic variables and BLR. Results: The discriminatory power in TT3a (3-yr OS: 95% v 45%, p&lt;0.0001; 3-yr EFS: 90% v 35%, p&lt;0.0001) was confirmed in TT3b (18-mo OS: 100% v 65%, p=0.0004; 18-mo EFS: 95% v 45%, p&lt;0.0001). Evaluating PBR in the context of BLR, 12/26 in TT3a and 7/21 in TT3b deemed as having low BLR had high PBR; conversely, 8/126 in TT3a and 14/106 in TT3b deemed as having high BLR had low PBR. In the context of our 8 molecular subgroup model, high PBR was over-represented in the Proliferation (PR) subgroup (7/15 in TT3a, 8/18 in TT3b) and absent in the Low Bone disease (LB) group (0/28). On MV, PBR was an independent adverse variable for both OS and EFS in TT3a (OS: HR=3.17, p=0.006, R2=55%; EFS: HR=4.40, p&lt;0.001, R2=48%) and in TT3b (OS: HR=13.00, p=0.002, R2=48%; EFS: HR=15.57, p&lt;0.001, R2=55%). Proteasome genes ranked first among those differentially up-regulated by BOR. Conclusions: PG identified a powerful 80-gene PBR model with unprecedented prognosis-discriminating power, dispelling BLR from MV analysis by altering BLR designation mainly from low to high risk. High PBR (18%) could be traced to up-regulation of proteasome genes, the target of BOR. [Table: see text]

Immunoglobulin isotypes in multiple myeloma: laboratory correlates and prognostic implications in total therapy protocols

Immunoglobulin isotypes in multiple myeloma: laboratory correlates and prognostic implications in total therapy protocols

Impact of Chromosomal Abnormalities Del(13), T(4;14), and Del(17p) and Prior Treatment on Outcomes in Patients with Relapsed or Refractory Multiple Myeloma Treated with Lenalidomide

The chromosomal abnormalities of del(13), t(4;14), and del(17p) are associated with poor progression-free survival (PFS) and shorter overall survival (OS) in newly diagnosed multiple myeloma (MM) treated with traditional chemotherapy. In patients with relapsed or refractory MM, a recent study demonstrated that lenalidomide (Revlimid®) can overcome poor prognosis conferred by del13q and t(4;14) but not del17p13 (Bahlis et al 2007). Here, we performed a retrospective analysis of medical records obtained from 49 clinical centers participating in the French Autorisation Temporaire d'Utilisation program. Patients with relapsed or refractory MM received dexamethasone 40 mg orally (days 1–4; 9–12 and 17–20 for 4 cycles, then days 1–4 beginning with cycle 5) and lenalidomide 25 mg orally on days 1–21 of a 28 day cycle. CD138-purified plasma cells were analyzed with fluorescent in-situ hybridization (FISH) for del(13), t(4;14), and del(17p) at diagnosis. Response and disease progression endpoints were evaluated using the European Group for Blood and Marrow Transplantation criteria. A multivariate analysis was performed to assess the impact of the following 7 variables on outcomes: any chromosomal change, prior bortezomib use, prior thalidomide use, prior transplant, progression on thalidomide, age, and number of lines of previous therapy. In total, 207 patients were included in the analysis; the median number of treatment cycles was 5 (range, 1–22). Most patients in the current study had received prior thalidomide (87%) or bortezomib (81%). The overall response rate (ORR) was 59%, including 7% complete response and 14% very good partial response. Median progression-free survival (PFS) and overall survival (OS) were 9.6 months and 15.1 months, respectively. These values are comparable to the recently published phase III trials (Weber et al., 2007; Dimopoulos et al., 2007), despite the higher median number of prior therapies in this analysis (5 vs. 3). Overall, 41% of patients had del(13), 14% had t(4;14) and 5% had del(17p). The ORR was significantly lower, and PFS and OS significantly shorter, in patients with del(13) compared with patients without del(13) (ORR: 43% vs. 71%, P<0.001; PFS: 5.0 months vs. 12.5 months, P<0.0001; OS: 10.4 months vs. 17.4 months, P=0.001). A similar pattern was observed in patients with t(4;14) versus patients without t(4:14) (ORR 39% vs. 62%, p=0.04; PFS 5.5 months vs. 10.6 months, p<0.01; OS 9.4 months vs. 15.4 months, p=0.005). Multivariate analysis identified hemoglobin (<10 g/dL), progression on thalidomide, and del(13) as independent predictors of reduced PFS (Table). There was a trend towards reduced PFS with prior bortezomib use and number of prior therapies. Age, sex, prior transplant, prior thalidomide use, and t(4;14) did not affect PFS. The results from the analysis indicate that del(13), progression on thalidomide, and hemoglobin levels ≥10 g/dL have a significant impact on outcomes in heavily pre-treated patients with relapsed or refractory MM. Randomized trials are needed to further assess these findings.

High-Dose Therapy with Peripheral Blood Stem Cell Transplantation for Patients with Multiple Myeloma: Prognostic Impact of Chromosomal Aberrations and Correlation with the ISS-Score

Multiple Myeloma (MM) is a malignant lymphoproliferative B-cell disease characterized by the accumulation of monoclonal plasma cells in the bone marrow. Acquired genomic aberrations have been shown to significantly impact response to chemotherapy and survival in MM. The aim of our study was to assess the clinical relevance of genomic abnormalities in 306 MM patients treated with high-dose chemotherapy (HDCT) and peripheral stem cell transplantation (PBSCT) in our center.We analyzed 171 males and 135 females with a median age of 60 years (range 25 – 73 years). According to the international staging system (ISS), MM patients were classified as stage I (46.6%), stage II (36.1%) and stage III (17.4%) at the onset of chemotherapy. All patients underwent frontline HDCT with 200 melphalan mg/m2 and PBSCT according or in analogy to the GMMG-HD3- or GMMG-HD4-trials. Interphase-FISH-analysis was performed on CD138-purified plasma cells using probes for chromosomes 1q21, 6q21, 8p21, 9q34, 11q23, 13q14.3, 15q22, 17p13, 19q13, and 22q11, as well as IgH-translocations t(4;14)(p16.3;q32.3) and t(11;14)(q13;q32.3). For the entire group, the median overall survival (OS) and progression-free survival (PFS) after HDCT was 6.4 and 2.2 years, respectively.Table 1. Chromosomal abnormalities with significant results (a-level=0.05) on PFS or OS (univariate analysis, unadjusted p-values)Aberration yesvs.noFrequency %3-year PFS %P value3-year OS %P valuedel(8p21)1926 vs. 370.0158 vs. 780.02del(13q14.3)4623 vs. 53<0.00165 vs. 830.03del(17p13)1022 vs. 400.0244 vs. 82<0.001t(4;14)1410 vs. 42<0.00154 vs. 810.04+1q213520 vs. 46<0.00167 vs. 850.002+19q135449 vs. 220.0376 vs. 730.92In a first step, we analyzed the prognostic impact of each individual chromosomal aberration on PFS and OS (Table 1). After adjustment for the ISS-score, del(8p21), del(13q14.3), del(17p13), t(4;14) as well as gains of 1q21 and 19q13 preserved significant impact on PFS, while del(17p13), t(4;14) and gain of 1q21 were of statistical significance for OS, indicating that these chromosomal aberrations give prognostic information in addition to the ISS-score. Subsequently, we performed a multivariate analysis including all the chromosomal aberrations analyzed. While del(17p13) and gain of 1q21 showed significant results on OS, del(13q14.3), del(22q11) and gain of 15q22 were significant for PFS.In conclusion, our results show that the heterogeneity seen in the clinical course of MM patients after HDCT can be correlated with distinct chromosomal aberrations. This analysis may have implications for the risk-adopted management of patients with MM.