To better define the molecular basis of multiple myeloma (MM), we performed unsupervised hierarchic clustering of mRNA expression profiles in CD138-enriched plasma cells from 414 newly diagnosed patients who went on to receive high-dose therapy and tandem stem cell transplants. Seven disease subtypes were validated that were strongly influenced by known genetic lesions, such as c-MAF-and MAFB-, CCND1-and CCND3-, and MMSET-activating translocations and hyperdiploidy. Indicative of the deregulation of common pathways by gene orthologs, common gene signatures were observed in cases with c-MAF and MAFB activation and CCND1 and CCND3 activation, the latter consisting of 2 subgroups, one characterized by expression of the early B-cell markers CD20 and PAX5. A low incidence of focal bone disease distinguished one and increased expression of proliferation-associated genes of another novel subgroup. Comprising varying fractions of each of the other 6 subgroups, the proliferation subgroup dominated at relapse, suggesting that this signature is linked to disease progression. Proliferation and MMSET-spike groups were characterized by significant overexpression of genes mapping to chromosome 1q, and both exhibited a poor prognosis relative to the other groups. A subset of cases with a predominating myeloid gene expression signature, excluded from the profiling analyses, had more favorable baseline characteristics and superior prognosis to those lacking this signature. IntroductionMultiple myeloma (MM) is a malignancy of antibody-secreting, terminally differentiated B cells that home to and expand in the bone marrow, with symptoms related to anemia, immunosuppression, bone destruction, and renal failure. 1,2 Bone lesions developing adjacent to plasma cell foci result from the activation of osteoclasts and inactivation of osteoblasts. [3][4][5] Many of the pathogenetic mechanisms of this clinically heterogeneous malignancy have been unraveled by application of molecular genetics. [6][7][8] While sharing most of the genetic lesions seen in MM, 9-11 monoclonal gammopathy of undetermined significance (MGUS) rarely progresses to overt MM. 12 The universal activation of 1 of the 3 cyclin D genes is consistent with this being an initiating event in MM. 13 Nonhyperdiploid MM, present in 40%, is characterized by transcriptional activation of CCND1, CCND3, MAF, MAFB, or FGFR3/MMSET genes (resulting from translocations involving the immunoglobulin heavy chain locus). 8 While hyperdiploidy and CCND1 activation confer a favorable prognosis, MAF, MAFB, or FGFR3/MMSET activation and deletion of chromosomes 13 and 17 are associated with poor prognosis. [14][15][16][17][18][19][20][21][22][23][24][25] Although high-dose therapy has markedly improved MM prognosis, 26-28 individual patients' survival remains variable 29,30 and cannot be accurately predicted with current prognostic models. 31,32 In lymphoma and leukemia, microarray profiling has helped establish clinically relevant disease subclassifications. [33][34][35][36][37][38][39][40][41...

To molecularly define high-risk disease, we performed microarray analysis on tumor cells from 532 newly diagnosed patients with multiple myeloma (MM) treated on 2 separate protocols. Using log-rank tests of expression quartiles, 70 genes, 30% mapping to chromosome 1 (P < .001), were linked to early disease-related death. Importantly, most up-regulated genes mapped to chromosome 1q, and downregulated genes mapped to chromosome 1p. The ratio of mean expression levels of up-regulated to down-regulated genes defined a high-risk score present in 13% of patients with shorter durations of complete remission, event-free survival, and overall survival (training set: hazard ratio [HR], 5.16; P < .001; test cohort: HR, 4.75; P < .001). The high-risk score also was an independent predictor of outcome endpoints in multivariate analysis (P < .001) that included the International Staging System and high-risk translocations. In a comparison of paired baseline and relapse samples, the high-risk score frequency rose to 76% at relapse and predicted short postrelapse survival (P < .05). Multivariate discriminant analysis revealed that a 17-gene subset could predict outcome as well as the 70-gene model. Our data suggest that altered transcriptional regulation of genes mapping to chromosome 1 may contribute to disease progression, and that expression profiling can be used to identify high-risk disease and guide therapeutic interventions. (Blood.

The transcription factor IRF4 is required during an immune response for lymphocyte activation and the generation of immunoglobulin-secreting plasma cells 1-3 . Multiple myeloma, a malignancy of plasma cells, has a complex molecular etiology with several subgroups defined by gene expression profiling and recurrent chromosomal translocations 4,5 . Moreover, the malignant clone can sustain multiple oncogenic lesions, accumulating genetic damage as the disease progresses 6,7 . Current therapies for myeloma can extend survival but are not curative 8,9 . Hence, new therapeutic strategies are needed that target molecular pathways shared by all subtypes of myeloma. Using a loss-offunction, RNA-interference-based genetic screen we show here that IRF4 inhibition was toxic to myeloma cell lines, regardless of transforming oncogenic mechanism. Gene expression profiling and genome-wide chromatin immunoprecipitation analysis uncovered an extensive network of IRF4 target genes and identified MYC as a direct target of IRF4 in activated B cells and myeloma. Unexpectedly, IRF4 was itself a direct target of MYC transactivation, generating an autoregulatory circuit in myeloma cells. Though IRF4 is not genetically altered in most myelomas, they are nonetheless addicted to an aberrant IRF4 regulatory network that fuses the gene expression programs of normal plasma cells and activated B cells.Recently, we developed a genetic method to identify therapeutic targets in cancer in which small hairpin RNAs (shRNAs) that mediate RNA interference are screened for their ability to block cancer cell proliferation and/or survival 10 . Here we report the results of such an "Achilles heel" screen in multiple myeloma (Supplementary Table 3). We used myeloma cell lines from three molecular subtypes: KMS12 (CCND1 translocation), H929 (FGFR3/MMSET * Corresponding author: Louis M. Staudt, MD, PhD 9000 Rockville Pike Building 10, Room 4N114 Bethesda, MD 20892 301-402-1892 Fax: 301-496-9956 e-mail: lstaudt@mail.nih.gov Reprints and permissions information can be found at www.nature.com/reprints. Author contributions were as follows: Experimental design/discussion, A.L.S., V.N.N., J.E., L.M.S.; preparation and performance of experiments, A.L.S, N.C.T.E, L.L., V.N.N., S.D., X.Y., H.Z., Y.Z., B.C.; data analysis/interpretation, A.L.S., N.C.T.E., W.X., G.W., J.P., J.E., L.M.S.; manuscript preparation, A.L.S., L.M.S. New datasets have been deposited under GEO accessions GSE8958, GSE9067 (gene expression), and GSE9367 (ChIP-CHIP).Full Methods are available in the Supplementary Materials in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of this paper at www.nature.com/nature.The authors declare no competing financial interests. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2009 July 10. Published in final edited form as:Nature. (Fig.1a). Lymphoma cell lines were largely unaffected by IRF4 knockdown, with the exception of OCILy3, an activated B cell-like...

Summary Background Lenalidomide plus dexamethasone is a reference treatment for patients with newly diagnosed myeloma. The combination of the proteasome inhibitor bortezomib with lenalidomide and dexamethasone has shown significant efficacy in the setting of newly diagnosed myeloma. We aimed to study whether the addition of bortezomib to lenalidomide and dexamethasone would improve progression-free survival and provide better response rates in patients with previously untreated multiple myeloma who were not planned for immediate autologous stem-cell transplant. Methods In this randomised, open-label, phase 3 trial, we recruited patients with newly diagnosed multiple myeloma aged 18 years and older from participating Southwest Oncology Group (SWOG) and National Clinical Trial Network (NCTN) institutions (both inpatient and outpatient settings). Key inclusion criteria were presence of CRAB (C=calcium elevation; R=renal impairment; A=anaemia; B=bone involvement) criteria with measurable disease (measured by assessment of free light chains), Eastern Cooperative Oncology Group (ECOG) performance status of 0–3, haemoglobin concentration 9 g/dL or higher, absolute neutrophil count 1 × 103 cells per mm3; or higher, and a platelet count of 80 000/mm3 or higher. We randomly assigned (1:1) patients to receive either an initial treatment of bortezomib with lenalidomide and dexamethasone (VRd group) or lenalidomide and dexamethasone alone (Rd group). Randomisation was stratified based on International Staging System stage (I, II, or III) and intent to transplant (yes vs no). The VRd regimen was given as eight 21-day cycles. Bortezomib was given at 1·3 mg/m2 intravenously on days 1, 4, 8, and 11, combined with oral lenalidomide 25 mg daily on days 1–14 plus oral dexamethasone 20 mg daily on days 1, 2, 4, 5, 8, 9, 11, and 12. The Rd regimen was given as six 28-day cycles. The standard Rd regimen consisted of 25 mg oral lenalidomide once a day for days 1–21 plus 40 mg oral dexamethasone once a day on days 1, 8, 15, and 22. The primary endpoint was progression-free survival using a prespecified one-sided stratified log rank test at a significance level of 0·02. Analyses were intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00644228. Findings Between April, 2008, and February, 2012, we randomly assigned 525 patients at 139 participating institutions (264 to VRd and 261 to Rd). In the randomly assigned patients, 21 patients in the VRd group and 31 in the Rd group were deemed ineligible based mainly on missing, insufficient, or early or late baseline laboratory data. Median progression-free survival was significantly improved in the VRd group (43 months vs 30 months in the Rd group; stratified hazard ratio [HR] 0·712, 96% CI 0·56–0·906; one-sided p value 0·0018). The median overall survival was also significantly improved in the VRd group (75 months vs 64 months in the Rd group, HR 0·709, 95% CI 0·524–0·959; two-sided p value 0·025). The rates of overall response (partial response or better) were...

F18-fluorodeoxyglucose positron emission tomography (FDG-PET) is a powerful tool to investigate the role of tumor metabolic activity and its suppression by therapy for cancer survival. As part of Total Therapy 3 for newly diagnosed multiple myeloma, metastatic bone survey, magnetic resonance imaging, and FDG-PET scanning were evaluated in 239 untreated patients. All 3 imaging techniques showed correlations with prognostically relevant baseline parameters: the number of focal lesions (FLs), especially when FDG-avid by PET-computed tomography, was positively linked to high levels of ␤-2-microglobulin, C-reactive protein, and lactate dehydrogenase; among gene expression profiling parameters, high-risk and proliferation-related parameters were positively and low-bone-disease molecular subtype inversely correlated with FL. The presence of more than 3 FDG-avid FLs, related to fundamental features of myeloma biology and genomics, was the leading independent parameter associated with inferior overall and event-free survival. Complete FDG suppression in FL before first transplantation conferred significantly better outcomes and was only opposed by gene expression profilingdefined high-risk status, which together accounted for approximately 50% of survival variability (R 2 test). Our results provide a rationale for testing the hypothesis that myeloma survival can be improved by altering treatment in patients in whom FDG suppression cannot be achieved after induction