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MicroRNAs (miRNAs) are noncoding RNAs that regulate global gene expression. miRNAs often act synergistically to repress target genes, and their dysregulation can contribute to the initiation and progression of a variety of cancers. The clinical relationship between global expression of miRNA and mRNA in cancer has not been studied in detail. We used whole-genome microarray analyses of CD138-enriched plasma cells from 52 newly diagnosed cases of multiple myeloma to correlate miRNA expression profiles with a validated mRNA-based risk stratification score, proliferation index, and predefined gene sets. In stark contrast to mRNAs, we discovered that all tested miRNAs were significantly up-regulated in high-risk disease as defined by a validated 70-gene risk score (P < 0.01) and proliferation index (P < 0.05). Increased expression of EIF2C2/AGO2, a master regulator of the maturation and function of miRNAs and a component of the 70-gene mRNA risk model, is driven by DNA copy number gains in MM. Silencing of AGO2 dramatically decreased viability in MM cell lines. Genome-wide elevated expression of miRNAs in high-risk MM may be secondary to deregulation of AGO2 and the enzyme complexes that regulate miRNA maturation and function. DICER1 | expression profile | multiple myeloma | risk stratification | system biology M icroRNAs (miRNAs) belong to a class of noncoding small RNAs with mature sequences that contain ≈22 nucleotides (1). As repressors of gene expression, miRNAs can bind to the 3′ untranslated region (3′UTR) of an mRNA and inhibit its translation or induce its degradation (1).Dysregulation of miRNA is involved in cancer initiation and progression (2, 3), and miRNA expression profiles have prognostic implications (4-6). Inhibiting miRNA has proved effective in vivo (7) and therefore could be a novel therapeutic strategy for cancer (8, 9). To date, few studies have investigated the roles of miRNA in multiple myeloma (MM), a plasma cell dyscrasia that homes to and expands in the bone marrow and produces disease manifestations that include osteolytic bone destruction with hypercalcemia, anemia, immunosuppression, and end-organ damage (10). Several miRNAs have been implicated in survival and growth of myeloma cells and myeloma tumor growth. For instance, miR-21 is a target of Stat3 and thus a critical component in IL-6/Stat3-dependent survival and growth pathways of myeloma cells (11). In addition, IL-6 inhibitor SOCS1 and p53 pathway component p300-CBPassociated factor are targets of multiple miRNAs, including miR-106b-25 cluster, miR-32, miR-181a/b, and miR-19a/b (12); suppression of these miRNAs inhibited myeloma tumor growth in nude mice (12).Applying miRNA expression profiles, Pichiorri et al. (12) identified miRNAs that were differentially expressed in plasma cells of healthy donors, subjects with a benign precursor to MM (monoclonal gammopathy of undetermined significance), and patients with MM. In other miRNA expression profiling studies, Roccaro et al. (13) To further investigate the potential involvement o...

Total Therapy 2 examined the clinical benefit of adding thalidomide up-front to a tandem transplant regimen for newly diagnosed patients with multiple myeloma. When initially reported with a median follow-up of 42 months, complete response rate and event-free survival were superior among the 323 patients randomized to thalidomide, whereas overall survival was indistinguishable from that of the 345 patients treated on the control arm. With further follow-up currently at a median of 72 months, survival plots segregated 5 years after initiation of therapy in favor of thalidomide (P ‫؍‬ .09), reaching statistical significance for the one third of patients exhibiting cytogenetic abnormalities (CAs; P ‫؍‬ .02), a wellrecognized adverse prognostic feature. The duration of complete remission was also superior in the cohort presenting with CAs such that, at 7 years from onset of complete remission, 45% remained relapse-free as opposed to 20% on the control arm (P ‫؍‬ .05). These observations were confirmed when examined by multivariate analysis demonstrating that thalidomide reduced the hazard of death by 41% among patients with CA-positive disease (P ‫؍‬ .008). Because two thirds of patients without CAs have remained alive at 7 years, the presently emerging separation in favor of thalidomide may eventually reach statistical significance as well. (Blood. 2008;112:3115-3121)

Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

The Total Therapy 3 trial 2003-33 enrolled 303 newly diagnosed multiple myeloma patients and was noted to provide superior clinical outcomes compared with predecessor trial Total Therapy 2, especially in gene expression profiling (GEP)-defined low-risk disease. We report here on the results of successor trial 2006-66 with 177 patients, using bortezomib, lenalidomide, and dexamethasone maintenance for 3 years versus bortezomib, thalidomide, and dexamethasone in year 1 and thalidomide/dexamethasone in years 2 and 3 in the 2003-33 protocol. Overall survival (OS) and event-free survival (EFS) plots were super-imposable for the 2 trials, as were onset of complete response and complete response duration (CRD), regardless of GEP risk. GEP-defined highrisk designation, pertinent to 17% of patients, imparted inferior OS, EFS, and CRD in both protocols and, on multivariate analysis, was the sole adverse feature affecting OS, EFS, and CRD. Mathematical modeling of CRD in low-risk myeloma predicted a 55% cure fraction (P < .001). Despite more rapid onset and higher rate of CR than in other molecular subgroups, CRD was inferior in CCND1 without CD20 myeloma, resembling outcomes in MAF/ MAFB and proliferation entities. IntroductionTotal Therapy 3 (2003-33; TT3) used, for newly diagnosed multiple myeloma (MM), 2 cycles of VTD-PACE (bortezomib, thalidomide, and dexamethasone and 4-day continuous infusions of cisplatin, doxorubicin, cyclophosphamide, and etoposide) as induction before and consolidation therapy after melphalan-based tandem transplantation, which was followed by 3 years of intended maintenance with VTD in year 1 and thalidomide/dexamethasone in years 2 and 3. 1-3 Results revealed superior outcomes, especially in gene expression profiling (GEP)-defined low-risk MM, compared with predecessor phase 3 trial Total Therapy 2 (TT2), which randomized patients up-front to receive or not receive thalidomide during induction, consolidation, and maintenance phases. 4 In further analyses examining the timeliness of completion of intended protocol steps, we concluded that the improved results in TT3 versus TT2 were attributable to the incorporation, up-front, of bortezomib in TT3. 3 To validate these findings and bortezomib pharmacogenomic data, 5 a successor trial 2006-66 enrolled another 177 patients. The trials were identical in design, except that the maintenance phase in 2006-66 applied 3 years rather than 1 year of bortezomib and used lenalidomide instead of thalidomide. with monthly cycles of bortezomib 1.0 mg/m 2 on days 1, 4, 8, and 11, whereas thalidomide was given continuously at 100 mg/day and dexamethasone 20 mg on days 1 to 4 and 8 to 11; in years 2 and 3, thalidomide was continued at 100 mg/day and dexamethasone was limited to monthly pulses of 20 mg on days 1 to 4. In 2006-66, bortezomib, lenalidomide, and dexamethasone (VRD) was given for 3 years, composing monthly cycles of bortezomib 1.0 mg/m 2 on days 1, 4, 8, and 11 in year 1 followed by weekly administration in years 2 and 3; lenalidomide was administered...