Nicholas J. Dickens

A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value

To obtain a comprehensive genomic profile of presenting multiple myeloma cases we performed high-resolution single nucleotide polymorphism mapping array analysis in 114 samples alongside 258 samples analyzed by U133 Plus 2.0 expression array (Affymetrix). We examined DNA copy number alterations and loss of heterozygosity (LOH) to define the spectrum of minimally deleted regions in which relevant genes of interest can be found. The most frequent deletions are located at 1p (30%), 6q (33%), 8p (25%), 12p (15%), 13q (59%), 14q (39%), 16q (35%), 17p (7%), 20 (12%), and 22 (18%). In addition, copy number-neutral LOH, or uniparental disomy, was also prevalent on 1q (8%), 16q (9%), and X (20%), and was associated with regions of gain and loss. Based on fluorescence in situ hybridization and expression quartile analysis, genes of prognostic importance were found to be located at 1p (FAF1, CDKN2C), 1q (ANP32E), and 17p (TP53). In addition, we identified common homozygously deleted genes that have functions relevant to myeloma biology. Taken together, these analyses indicate that the crucial pathways in myeloma pathogenesis include the nuclear factor-κB pathway, apoptosis, cell-cycle regulation, Wnt signaling, and histone modifications. This study was registered at http://isrctn.org as ISRCTN68454111.

Homozygous Deletion Mapping in Myeloma Samples Identifies Genes and an Expression Signature Relevant to Pathogenesis and Outcome

Purpose: Myeloma is a clonal malignancy of plasma cells. Poor-prognosis risk is currently identified by clinical and cytogenetic features. However, these indicators do not capture all prognostic information. Gene expression analysis can be used to identify poor-prognosis patients and this can be improved by combination with information about DNA-level changes. Experimental Design: Using single nucleotide polymorphism–based gene mapping in combination with global gene expression analysis, we have identified homozygous deletions in genes and networks that are relevant to myeloma pathogenesis and outcome. Results: We identified 170 genes with homozygous deletions and corresponding loss of expression. Deletion within the “cell death” network was overrepresented and cases with these deletions had impaired overall survival. From further analysis of these events, we have generated an expression-based signature associated with shorter survival in 258 patients and confirmed this signature in data from two independent groups totaling 800 patients. We defined a gene expression signature of 97 cell death genes that reflects prognosis and confirmed this in two independent data sets. Conclusions: We developed a simple 6-gene expression signature from the 97-gene signature that can be used to identify poor-prognosis myeloma in the clinical environment. This signature could form the basis of future trials aimed at improving the outcome of poor-prognosis myeloma. Clin Cancer Res; 16(6); 1856–64

MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

The t(4;14) translocation leads to the simultaneous overexpression of two genes, FGFR3 and MMSET, in myeloma plasma cells. The findings of this study suggest that MMSET is implicated in the pathogenesis of the t(4;14) translocation. Background The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Design and Methods The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. Results We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival.

XBP1s levels are implicated in the biology and outcome of myeloma mediating different clinical outcomes to thalidomide-based treatments

Immunoglobulin production by myeloma plasma cells depends on the unfolded protein response for protein production and folding. Recent studies have highlighted the importance of IRE1alpha and X box binding protein 1 (XBP1), key members of this pathway, in normal B-plasma cell development. We have determined the gene expression levels of IRE1alpha, XBP1, XBP1UNSPLICED (XBP1u), and XBP1SPLICED (XBP1s) in a series of patients with myeloma and correlated findings with clinical outcome. We show that IRE1alpha and XBP1 are highly expressed and that patients with low XBP1s/u ratios have a significantly better overall survival. XBP1s is an independent prognostic marker and can be used with beta2 microglobulin and t(4;14) to identify a group of patients with a poor outcome. Furthermore, we show the beneficial therapeutic effects of thalidomide in patients with low XBP1s/u ratios. This study highlights the importance of XBP1 in myeloma and its significance as an independent prognostic marker and as a predictor of thalidomide response.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material. Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis. Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor. Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Genetic Factors Underlying the Risk of Thalidomide-Related Neuropathy in Patients With Multiple Myeloma

PURPOSEnTo indentify genetic variation that can modulate and predict the risk of developing thalidomide-related peripheral neuropathy (TrPN).nnnPATIENTS AND METHODSnWe analyzed DNA from 1,495 patients with multiple myeloma. Using a custom-built single nucleotide polymorphism (SNP) array, we tested the association of TrPN with 3,404 SNPs. The SNPs were selected in predicted functional regions within 964 genes spanning 67 molecular pathways thought to be involved in the pathogenesis, treatment response, and adverse effects associated with myeloma and its therapy. Patient cases and controls were derived from two large clinical trials that compared thalidomide with conventional-based treatment in myeloma patients (Medical Research Council Myeloma-IX and HOVON-50/GMMG-HD3).nnnRESULTSnWe report TrPN associations with SNPs-ABCA1 (rs363717), ICAM1 (rs1799969), PPARD (rs2076169), SERPINB2 (rs6103), and SLC12A6 (rs7164902)-where we show cross validation of the associations in both trials. To investigate whether TrPN SNP associations were related to exposure to thalidomide only or general drug-related peripheral neuropathy, we performed a second analysis on patients treated with vincristine. We report SNPs associated with vincristine neuropathy, with a seemingly distinct underlying genetic mechanism.nnnCONCLUSIONnOur results are consistent with the hypothesis that an individuals risk of developing a peripheral neuropathy after thalidomide treatment can be mediated by polymorphisms in genes governing repair mechanisms and inflammation in the peripheral nervous system. These findings will contribute to the development of future neuroprotective strategies with thalidomide therapy and the better use of this important compound.

Genetic associations with thalidomide mediated venous thrombotic events in myeloma identified using targeted genotyping

A venous thromboembolism (VTE) with the subsequent risk of pulmonary embolism is a major concern in the treatment of patients with multiple myeloma with thalidomide. The susceptibility to developing a VTE in response to thalidomide therapy is likely to be influenced by both genetic and environmental factors. To test genetic variation associated with treatment related VTE in patient peripheral blood DNA, we used a custom-built molecular inversion probe (MIP)-based single nucleotide polymorphism (SNP) chip containing 3404 SNPs. SNPs on the chip were selected in functional regions within 964 genes spanning 67 molecular pathways thought to be involved in the pathogenesis, treatment response, and side effects associated with myeloma therapy. Patients and controls were taken from 3 large clinical trials: Medical Research Council (MRC) Myeloma IX, Hovon-50, and Eastern Cooperative Oncology Group (ECOG) EA100, which compared conventional treatments with thalidomide in patients with myeloma. Our analysis showed that the set of SNPs associated with thalidomide-related VTE were enriched in genes and pathways important in drug transport/metabolism, DNA repair, and cytokine balance. The effects of the SNPs associated with thalidomide-related VTE may be functional at the level of the tumor cell, the tumor-related microenvironment, and the endothelium. The clinical trials described in this paper have been registered as follows: MRC Myeloma IX: ISRCTN68454111; Hovon-50: NCT00028886; and ECOG EA100: NCT00033332.

Abstract 4737: Genetic determinants associated with multiple myeloma risk in three large population sets

Classic epidemiological approaches aimed at identifying environmental exposures associated with aetiology of Multiple Myeloma (MM) have failed to identify a common cause. The majority of the studies aimed at identifying genetic risk in MM have been underpowered and limited in coverage. We have taken a candidate gene approach by assaying 3,404 single nucleotide polymorphism (SNPs) selected in approximately 983 candidate genes on a “Bank on a Cure” (BOAC) Targeted Genotyping assay, focusing on coding SNPs and SNPs in regulatory regions. SNPs were genotyped using DNA extracted from peripheral blood. 2595 presenting MM cases were derived from clinical trials held within the UK (1228), US (697) and the Netherlands (670). Genotype data were available from large population control datasets, which were used to examine 1809 SNPs for association with MM risk. The control population sets consisted of the UK Wellcome Trust Case-Control Consortium 2 (WTCCC2) study with 3,000 individuals from the 1958 British Birth Cohort and the UK Blood Service collections, genotyped on both the Illumina 1.2M Duo (Human1-2M-DuoCustom_v1) and the Affymetrix SNP 6.0 array; 2350 US Caucasian controls from the Nurses’ Health Study (NHS), genotyped on the Illumina 550K chip, and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO), genotyped on Illumina 317K plus 240K; and 5974 Dutch >55yrs old population controls from the Erasmus Rotterdam Health for the Elderly (ERGO) study, genotyped on the Illumina 550K array. We have also used fluorescence in situ hybridization (FISH) status for 702 of the UK cases to perform a subset analysis for hyperdiploidy and IgH translocations, two of the major myeloma pathogenic subgroups. Quality control measures were performed on the datasets to protect against artificial effects, induced by population stratification, cross platform genotyping and low genotyping quality (95% call rate and HWE (p Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4737.

Deletion of 16q identified by FISH is an independent adverse prognostic marker in multiple myeloma

Chromosomal translocations lead to oncogene activation in ansignificant number of haematological malignancies. Those involvingnthe immunoglobulin heavy chain locus, IGH, at chromosome bandn14q32 are frequently observed in B-cell malignant proliferation. Ansmall number have been described in B-cell precursor acutenlymphoblastic leukaemia (BCP-ALL). However, their biological andnclinical significance is currently unknown. Detailed fluorescence innsitu hybridisation (FISH) and molecular studies were carried out onna series of BCP-ALL patients with chromosomal abnormalitiesninvolving 14q32. Novel and recurrent translocations affecting different chromosomes were highlighted. Refined FISH mappingnidentified putative IGH partner genes at, or flanking, the translocationnbreakpoints. Four translocations: two previously reported,nt(14;19)(q32;q13), t(8;14)(q11;q32), and two novel, t(14;14)(q11;q32)/ninv(14)(q11q32) and t(14;20)(q32;q13), were identified. Molecularnanalyses showed that four different members of the CAATT enhancernbinding protein (CEBP) gene family were involved: CEBPA (19q13,nn59), CEBPD (8q11, n58), CEBPE (14q11, n53) and CEBPB (20q13,nn52). One patient with a t(14;19)(q32;q13) was observed to involventhe fifth family member CEBPG (19q13, n51). Breakpoints werenlocated within the 30 untranslated region (UTR) of CEBPA and eithern30 UTR or 50 of CEBPE, whereas breakpoints in 8q11 were B30 kbncentromeric of CEBPD. Where material was available, over-expressionnof target genes was shown by quantitative real-time PCR.nOverall, this study has demonstrated for the first time theninvolvement of five members of the same gene family in a singlensubtype of haematological disease. It has indicated that transcriptionalnupregulation of CEBP gene family members, by juxtapositionnto IGH, is important in BCP-ALL: a mechanism in complete contrastnto that involving CEPBA in acute myeloid leukaemia.