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.

Summary Acute respiratory infections (ARI) are a common reason for seeking medical attention and the threat of pandemic influenza will likely add to these numbers. Using human viral challenge studies with live rhinovirus, respiratory syncytial virus, and influenza A, we developed peripheral blood gene expression signatures that distinguish individuals with symptomatic ARI from uninfected individuals with > 95% accuracy. We validated this “acute respiratory viral” signature - encompassing genes with a known role in host defense against viral infections - across each viral challenge. We also validated the signature in an independently acquired dataset for influenza A and classified infected individuals from healthy controls with 100% accuracy. In the same dataset, we could also distinguish viral from bacterial ARIs (93% accuracy). These results demonstrate that ARIs induce changes in human peripheral blood gene expression that can be used to diagnose a viral etiology of respiratory infection and triage symptomatic individuals.

Using fluorescence in situ hybridization we investigated amplification of chromosome band 1q21 (Amp1q21) in more than 500 untreated patients with monoclonal gammopathy of undetermined significance (MGUS; n ‫؍‬ 14), smoldering multiple myeloma (SMM; n ‫؍‬ 31), and newly diagnosed MM (n ‫؍‬ 479) as well as 45 with relapsed MM. The frequency of Amp1q21 was 0% in MGUS, 45% in SMM, 43% in newly diagnosed MM, and 72% in relapsed MM (newly diagnosed versus relapsed MM, P < .001). Amp1q21 was detected in 10 of 12 patients whose disease evolved to active MM compared with 4 of 19 who remained with SMM (P < .001). Patients with newly diagnosed MM with Amp1q21 had inferior 5-year event-free/ overall survival compared with those lacking Amp1q21 (38%/52% versus 62%/78%, both P < .001). Thalidomide improved 5-year EFS in patients lacking Amp1q21 but not in those with Amp1q21 (P ‫؍‬ .004). Multivariate analysis including other major predictors revealed that Amp1q21 was an independent poor prognostic factor.Relapsed patients who had Amp1q21 at relapse had inferior 5-year postrelapse survival compared with those lacking Amp1q21 at relapse (15% versus 53%, P ‫؍‬ .027). The proportion of cells with Amp1q21 and the copy number of 1q21 tended to increase at relapse compared with diagnosis. Our data suggest that Amp1q21 is associated with both disease progression and poor prognosis. (Blood.

To identify genetic events underlying the genesis and progression of multiple myeloma (MM), we conducted a high-resolution analysis of recurrent copy number alterations (CNAs) and expression profiles in a collection of MM cell lines and outcome-annotated clinical specimens. Attesting to the molecular heterogeneity of MM, unsupervised classification using nonnegative matrix factorization (NMF) designed for array comparative genomic hybridization (aCGH) analysis uncovered distinct genomic subtypes. Additionally, we defined 87 discrete minimal common regions (MCRs) within recurrent and highly focal CNAs. Further integration with expression data generated a refined list of MM gene candidates residing within these MCRs, thereby providing a genomic framework for dissection of disease pathogenesis, improved clinical management, and initiation of targeted drug discovery for specific MM patients.

The aims of this study were to assess the feasibility of prospective pharmacogenomics research in multicenter international clinical trials of bortezomib in multiple myeloma and to develop predictive classifiers of response and survival with bortezomib. Patients with relapsed myeloma enrolled in phase 2 and phase 3 clinical trials of bortezomib and consented to genomic analyses of pretreatment tumor samples. Bone marrow aspirates were subject to a negative-selection procedure to enrich for tumor cells, and these samples were used for gene expression profiling using DNA microarrays. Data quality and correlations with trial outcomes were assessed by multiple groups. Gene expression in this dataset was consistent with data published from a single-center study of newly diagnosed multiple myeloma. Response and survival classifiers were developed and shown to be significantly associated with outcome via testing on independent data. The survival classifier improved on the risk stratification provided by the Interna-

We have developed an enhanced form of reduced representation bisulfite sequencing with extended genomic coverage, which resulted in greater capture of DNA methylation information of regions lying outside of traditional CpG islands. Applying this method to primary human bone marrow specimens from patients with Acute Myelogeneous Leukemia (AML), we demonstrated that genetically distinct AML subtypes display diametrically opposed DNA methylation patterns. As compared to normal controls, we observed widespread hypermethylation in IDH mutant AMLs, preferentially targeting promoter regions and CpG islands neighboring the transcription start sites of genes. In contrast, AMLs harboring translocations affecting the MLL gene displayed extensive loss of methylation of an almost mutually exclusive set of CpGs, which instead affected introns and distal intergenic CpG islands and shores. When analyzed in conjunction with gene expression profiles, it became apparent that these specific patterns of DNA methylation result in differing roles in gene expression regulation. However, despite this subtype-specific DNA methylation patterning, a much smaller set of CpG sites are consistently affected in both AML subtypes. Most CpG sites in this common core of aberrantly methylated CpGs were hypermethylated in both AML subtypes. Therefore, aberrant DNA methylation patterns in AML do not occur in a stereotypical manner but rather are highly specific and associated with specific driving genetic lesions.

Monoclonal gammopathy of undetermined significance (MGUS) can progress to multiple myeloma (MM).