Chung, Tae-Hoon

Abstract Chromosome 13 (Δ13) abnormalities are found in greater than 50% of patients with Multiple Myeloma (MM). MM is most commonly defined by chromosome 13 monosomy or 13q loss (85%). Interstitial deletions comprise the remaining 15%. Many studies have revealed that Δ13 in MM are associated with poor survival and reduced response to therapy. Genes mapping to chromosome 13 may be involved in pathogenesis and/or progression of the disease due to loss of function from gene mutation or from epigenetic effects such as haploinsufficiency. In this study, array-based comparative genomic hybridization coupled with microarray technology (aCGH) is used to detect gene copy number loss on chromosome 13 from nine MM patient samples. Whole genome long-oligo microarrays constructed by Agilent Technologies were used which contain 40,000 genes that span the human genome with an average spatial resolution of ~75 kb. Using genomic DNA isolated from MM patients with interstitial deletions on chromosome 13, DNA was amplified, labeled and hybridized with a differentially labeled normal DNA reference to determine gene/genomic copy number changes. Arrays were analyzed to search for the minimum region of loss based upon single copy loss for a series of nearby mapping transcripts. A common region of loss of 2.2 Mb, at 13q14.2 was detected. Additionally, we investigated the correlation between genomic copy number change and the expression level for MM patients in the13q region. From an independent gene expression data set whose expression measurements were conducted with Affymetrix HG-U133A v2 microarrays, data was selected that corresponded to the samples used for current aCGH. Expression values from MM samples were divided by the mean expression values from 12 normal bone marrow samples for each gene and the resulting values were treated as surrogate ratios between MM and normal samples. Probes from both microarrays were then aligned according to their chromosomal positions and merged if their chromosomal positions overlapped. Composite chromosomal maps were generated that displayed the expression levels and copy number changes. The maps were used to differentiate chromosomal regions in 13q where copy number changes and expression levels show high correlation and regions where such correlation was not observed. Although the number of probes sampled in the expression microarray was much smaller than those in aCGH microarray, a chromosomal region of great interest, that encompasses 13q14.2, arose naturally from this analysis. Although the mechanism by which loss of 13q effects tumorigenesis in MM could be a haploinsufficiency model, we are not ruling out the presence of a tumor suppressor gene in this region. We are evaluating candidate tumor suppressor genes in the region for loss of function by mutational analysis and hypermethylation studies.

Abstract Chromosome 13 deletion (Δ13) is one the most common genetic abnormalities observed in multiple myeloma (MM) and confers a poor prognosis. Studies to identify critical tumor suppressor genes in MM till date have lack adequate resolution, and the molecular phenotype associated with Δ13 has not been established. In this study we seek to establish a molecular profile for Δ13 and to finely map the minimal common region of deletion (CDR) on chromosome 13 by using gene expression profiling (GEP) and array comparative genomic hybridization (aCGH). GEP was performed on RNA from purified plasma cells of 72 newly diagnosed MM and 50 human myeloma cell lines (HMCLs) using the Affymetrix U133A chip and U133plus chip (Affymetrix, Santa Clara, CA) respectively. Patients were assigned TC classes which correlates with underlying genetic subtypes. In addition, aCGH was performed on 79 MM samples (36 with GEP data) and 50 myeloma cell lines (HMCLs; 48 with GEP data) using a platform utilizing 60-mer oligonucleotides (Human Genome CGH 44B Oligo Microarrays, Agilent Technologies), which have a resolution of about 70Kb. Raw data was extracted using the Feature Extraction 8.1 and visualized using CGH Analytics 3.2 (Agilent Technologies). To define a signature that reflects the biological consequences of Δ13 and not its close association with some genetic subtypes of MM (example, t(4;14) and t(14;16)), we selected a training cohort consisting of cases belonging to D1, D2, 11q13 and none TC class such that cases with and without Δ13 are balanced for ploidy and TC classes (n=28). A 152-gene Δ13 signature was identified. Its specificity was confirmed by leave-one-out cross validation using the K-nearest neighbor (KNN) class prediction algorithm (predictive accuracy of 100% in the training cohort). It was subsequently validated in a validation cohort that includes t(4;14) and t(14;16) that were not use to derive this gene signature, maintaining a high predictive accuracy (88%). This gene-set may therefore reflect core transcriptional consequences of Δ13 and is enriched for genes involved in the cell cycle and apoptosis. Using aCGH, 20 patients have Δ13 (mostly whole chromosome) all involving the 13q14–q21 region. Of the 32 HMCLs with Δ13 (whole chromosome or interstitial), 22 have mono-allelic loss of RB1 whereas 4 had bi-allelic loss. Besides RB1, bi-allelic loss of several other genes in the 13q14–q21 region (CYSLTR2, CDADC1, ITM2B, PCDH9) was also observed. As the CDR is large and contains a number of genes, we utilized a set of criteria (bi-allelic loss in HMCLs, within 13q14–q21 CDR, gene expression significantly correlated with copy number, and loss could explain molecular phenotype) to narrow down the potential candidates. Only RB1 and ITM2B fulfilled these criteria. Furthermore, RB protein levels correlated well with mRNA levels and DNA copy suggesting that even in absence of mutations resulting in bi-allelic loss, mono-allelic loss of RB1 could be tumorigenic through a haploinsufficiency mechanism.

Abstract Hyperdiploid MM (H-MM), characterized by recurrent trisomies constitute about 50% of MM, yet very little is known about its pathogenesis and oncogenic mechanisms. Studies in leukemia and solid tumors have shown gene dosage effect of aneuploidy on gene expression. To determine the possible gene dosage effect and deregulated cellular program in H-MM we undertook a gene expression study of CD138-enriched plasma-cell RNA from 53 hyperdiploid and 37 non-hyperdiploid MM (NH-MM) patients using the Affymetrix U133A chip (Affymetrix, Santa Clara, CA). Gene expression data was analyzed using GeneSpring 7 (Agilent Technologies, Palo Alto, CA). Genes differentially expressed between H-MM and NH-MM were obtained by t-test (p<0.01). The majority of the differentially expressed genes (57%) were under-expressed in H-MM. Genes located on the commonly trisomic chromosomes were mostly (but not always) over-expressed in H-MM and constitute 76% of over-expressed genes. Chromosome 1 contained the most differentially expressed genes (17%) followed by chromosome 12 (9%), and 19 (8%). To examine the relationship of gene copy number to gene expression, we examined the expression of genes on chromosomes 9 and 15 in subjects with 2 copies (15 normal control and 20 NH-MM) and 3 copies (12 H-MM) of each chromosome as detected by interphase FISH. We then derived a ratio of the mean expression of each gene on these chromosomes between patients with 3 copies and 2 copies of the chromosome. If a simple relationship exists between gene expression and gene copy number, one would expect the ratio of expression of most genes on these two chromosomes to be about 1.5 in H-MM compared to NH-MM. However, many genes have ratios either higher than 2 or lower than 0.5. Furthermore, when the heterogeneity of cells with underlying trisomies is taken into consideration by correcting the ratio for the number of cells with trisomies, the actual ratio is always lower than the expected ratio. When the expression of genes on a chromosome was compressed to a median value, this value was always higher in the trisomic chromosomes for H-MM compared to NH-MM. The data suggests that although gene dosage influence gene expression, the relationship is complex and some genes are more gene dosage dependent than others. Amongst the differentially expressed genes with known function, 33% are involved in mRNA translation/protein synthesis. Of note, 37 of the top 100 differentially expressed genes are involved in these processes. In particular, 60 ribosomal protein (RP) genes are significantly (p<0.05) upregulated in H-MM. This signature in H-MM is not associated with increase proliferation as measured by PCLI. This predominant signature suggests that deregulated protein synthesis may be important for the biology of H-MM. Many of these RPs are involved in the synthesis of product of oncogenic pathways (e.g. MYC, NF-KB pathways) and may mediate the growth and survival of tumor cells. It is therefore possible that these tumor cells may be sensitive to the disruption of mRNA translation/protein synthesis. Targeting the mTOR pathway with rapamycin may therefore be useful for treatment of H-MM.

Abstract Introduction: The analysis of what goes awry in a malignant cell has focused primarily on mutations of protein-encoding genes and their regulatory sequences. However, recent work on microRNA (miRNA) has shed light on the possible involvement of miRNA genes in human disease. miRNA are small “noncoding” or “non-messenger” RNAs of about 21–25 nucleotides in length that function as regulators of gene expression essentially by pairing to the mRNA of protein-coding genes to initiate mRNA degradation or repression of translation. We are interested in characterizing miRNA expression profiles in human myeloma cell lines (HMCLs) and myeloma patient samples. Multiple myeloma (MM) is a plasma cell tumor characterized by frequent chromosomal translocations. Materials and methods: The expression of miRNA in HMCLs, primary MM cells (hyperdiploid samples) and normal plasma cells was determined using human microRNA chips. The GenoExporerTM Human microRNA chips were developed by GenoSensor (Tempe, AZ) and the microarray contains 226 human microRNA sequences in addition to control sequences. The DNA oligo probes are synthesized and immobilized on the chips (1”x 3” standard glass microslide). The probes are designed based on the active mature miRNA sequences and some of their flanking sequences. Our analysis included seven myeloma patients (hyperdiploid MM), eight different HMCLs (MM1, 8226 with t(14;16); SKMM2, U266, INA-6 with t(11;14) and H929, KMS11, UTMC-2 with t(4;14)), and eight normal human plasma cells. The normal human plasma cells were collected at Mayo Clinic Rochester from bone samples of patients undergoing orthopedic surgery. For the assay 5–10 μg of total RNA per sample were used at a concentration of 1 μg/μL. The RNA is directly labeled with biotin and used as a target for the on-chip hybridization assays. A streptavidin-Alexa dye is used to stain the hybridized targets and the fluorescent signals are captured and analyzed. The gene signal intensities were normalized to tRNA signal intensity. Results: The primary MM cells and HMCLs displayed a distinctive miRNA expression profile compared with normal plasma cells. Using the GeneSpring 7 (Agilent Technologies, Palo Alto, CA) for gene expression analysis we have identified miRNA genes with significant variation in expression levels between tumor and normal samples including miR-125b, miR-133a, miR1, and miR-124a (p<0.01). Recently defined algorithms were used to identify putative targets for the miRNA genes of interest such as leukemia inhibitory factor (LIF) as targets of miR-125b and Stat3 and angiopoietin-1 precursor as targets of miR-124a (Lewis et al. 2003). Of note, miR-15 and miR-16, previously identified to be downregulated in CLL, were expressed at low levels in some MM patients and HMCLs, but not in the normal plasma cells. Further analysis will be carried out to validate the data from miRNA profiling using northern blot analysis or real-time RT-PCR to measure expression levels of miRNA genes and that of their target genes. Conclusion: Analysis of miRNA expression pattern (along with aCGH and gene expression profiling studies) will undoubtedly refine our understanding of the various genotypic subtypes of multiple myeloma.

Abstract Genetic abnormalities are important in the pathobiology of myeloma and established prognostic factors. High-density aCGH allow global unbiased detection of genomic gains and losses. In this study, we attempt to identify survival critical genomic aberrations by correlating aCGH-defined abnormalities with survival in 2 different cohorts of patients. aCGH was performed in 64 MM patients treated with high dose therapy from the Mayo Clinic using Agilent’s 44k DNA microarray genechip. In addition, a publicly available aCGH dataset performed in 67 MM patients treated with total therapy from the University of Arkansas Medical School using the lower density Agilent 22k chip (Human 1A v2) was also analyzed. Base 2 logarithms of the ratios between red and green intensities (log-ratios) were treated as a primary measure of copy number ratio between tumor and normal samples. To detect the regions of genomic aberration, values of a sliding window of 15 consecutive log-ratios were compared with those of an artificial ‘reference’ chromosome using the Wilcoxon rank sum test. The reference chromosome, constructed for each sample, was assembled by collecting log-ratio values close to zero. For this, 15 randomly sampled log-ratios from autosomal regions whose p-value of Wilcoxon signed rank test against 0 was larger than 0.05 were recursively accepted until the number of log-ratios in the assembled reference chromosome reached 1000. The p-values and their corresponding false discovery rates (q-values) for the copy number gain or loss were calculated independently for each sliding window and assigned to the center position of corresponding sliding window. The survival difference was examined between samples that had gained or lost a chromosomal region (sliding window) and samples that had not using the method of Kaplan-Meier and the statistical significance was assessed by the log-rank test. The sliding window was identified as gained or lost if P-value < 10 −4 and Q-value < 10[suo]-3. Sliding windows whose gains or losses led to poor survival with log-rank test p-value < 0.5, corresponding q-value < 0.5, and happened to ≥ 10% of all samples were retained. Several regions of genomic gains and losses were significantly associated with poorer survival with 5 deletion hotspots common between the 2 datasets (Table). We validated the prognostic significance of these survival critical loci using FISH in an independent dataset of 169 patients treated with high dose therapy. Clustering of these survival critical loci revealed clusters of MM characterized by low, intermediate or high number of these abnormalities, which are independent of ploidy and translocations subtypes. These subgroups have significantly different survival. In addition, the clustering analysis suggests the chromosome 13 loss is an early event whereas chromosome 1 abnormalities are late events. Our strategy therefore identified novel regions of genomic aberration that are important prognostically. However, it is likely that these regions are manifestation of genomically unstable tumors with complex abnormalities that have poorer prognosis. Chr Start End Length Cytoband 1 74,137,824 75,972,544 1,834,720 p31.1 1 62,433,123 84,792,809 22,359,686 p31.3 – p22.3 20 4,100,452 6,189,410 2,088,958 p13 – p12.3 20 6,717,170 13,142,205 6,425,035 p12.3 – p12.1 20 4,708,668 10,566,333 5,857,665 p13 – p12.2

Abstract Waldenström macroglobulinemia (WM) is a lymphoid neoplasm associated with accumulation of lymphoplasmacytic cells in the bone marrow and an elevated monoclonal IgM in the serum. Although the genetic and molecular mechanisms behind the pathogenesis of WM are not well understood, losses of the long arm of chromosome 6 have been found as the only recurrent genomic aberration. These deletions, mostly starting at 6q21, are observed in upwards of 50% of patients and involve the majority of the clonal cells. To better characterize pathologic gene expression signatures in different types of WM at the molecular level (6q deleted versus not), we examined gene expression level changes of more than 20000 human gene probes in bone marrow tissues of clinically proven WM patients. We used Affymetrix HG-U133A v2 chips (n = 24). Clonal cells were purified using a combination of anti-CD138 and anti-CD19 Miltenyi™ magnetic microbeads. Based on cIgM-FISH results, we classified samples into two groups - 6q normal (ND) group and 6q deletion (D) group. We then proceeded to identify genes that showed differential expression between the two groups. We selected 165 differentially expressed genes (DEG) using SAM, and underwent further investigation of biological annotations of them using Gene Ontology™ (GO) terms. Among165 DEG, only 32 showed high level of expression in ND group compared to D group while 133 showed high level of expression in D group. As expected, all genes that reside in 6q region showed under-expression in D group, constituting 53% (17 genes) of genes under-expressed in D group (32 genes). According to GO analysis, the 6q-deletion-induced under-expression affects many biological processes, and is associated with a transcriptional upregulation of downstream genetic cascades. This likely indicates release of regulatory control by a tumor suppressor gene. Genes involved in cell growth and maintenance, cell communication and morphogenesis are also over-expressed in D group. Interestingly, a gene that is involved in innate immune response and inflammatory response shows under-expression in D group but is not accounted for by other mechanisms. In similar vein, a gene that is involved in negative regulation of cytokine biosynthesis showed over-expression in D group. We think these transcriptional characteristics can provide basic framework for differentiating subtypes of WM and search for genes associated with WM pathogenesis

Abstract In an attempt to identify novel genetic abnormalities in multiple myeloma (MM) we analyzed 68 MM patient samples and 42 human myeloma cell lines (HMCL) with high-resolution (60kb) array-based comparative genomic hybridization (aCGH). We concentrated on bi-allelic genomic deletions with the expectation that these regions might contain novel tumor suppressor genes and identified a common abnormality at 14q32. Fine mapping of the 14q32 microdeletion by PCR demonstrated a 45 kb minimal deletion encompassing two genes; TRAF3, a negative regulator of the non-canonical NF-kB pathway, and AMN. To determine the frequency of copy number abnormalities of this region a cohort of 161 patients were screened with a FISH probe mapping to the minimally deleted region. This identified 7 (4.4%) patients with bi-allelic deletion and 19 (11.8%) patients with a single copy of the target region. Sequencing of all TRAF3 coding exons in 62 patients and 42 HMCL identified inactivating mutations in 9 patient samples and 4 HMCL. Overall, the incidence of TRAF3 inactivation, bi-allelic deletion or mutation, is 7/42 (16.7%) in HMCL and 12/62 (19.4%) in patients, making it one of the most common genetic abnormalities identified in MM. Importantly, dysregulation of NF-kB pathways is a common event in MM, but the mechanism for this dysregulation is largely unknown. Although TRAF3 is a negative regulator of the non-canonical NF-kB pathway it has not previously been implicated in cancer. Therefore, we tested the tumor suppressor function of TRAF3 by reintroducing TRAF3 into HMCL harboring TRAF3 abnormalities using an adenovirus gene transfer system. Expression of TRAF3 in HMCL with TRAF3 abnormalities rapidly reversed cellular processing of NFKB2 from p100 (an inactive isoform) to p52 (active) and additionally led to a 60% reduction in cellular proliferation (range, 10–89%) associated with Go/G1 cell cycle arrest and induction of cell death by apoptosis. At 48 hrs post infection the proportion of cells entering S-phase or G2/M declined by 31% (range, 8.1–56.1) and the proportion undergoing apoptosis increased by 28% (range 11.7–47.4%). In contrast, over-expression of TRAF3 in control HMCL without TRAF3 abnormalities had no effect. Overall, TRAF3 is identified as a novel tumor suppressor that regulates the non-canonical NF-kB pathway and is inactivated in ~ 20% of MM patients. Given the involvement of the NF-kB pathway in multiple human malignancies and the near constitutive expression of TRAF3 we propose that inactivation of TRAF3, a novel tumor suppressor identified for the first time in this study, may occur in other cancers. In this regard, we have recently identified a Burkitt lymphoma cell line that does not express TRAF3 and thus has constitutive processing of p100 to p52. The clinical and therapeutic consequences of this finding and its frequency in other tumor types are currently the focus of further investigation. In conclusion, the abnormalities identified in this study represent the first mutations identified that cause constitutive NF-kB activation in MM.

Abstract Adrenocortical carcinoma (ACC) is a rare endocrine malignancy accounting for between 0.02% and 0.2% of all cancer deaths. Surgical removal offers the only current potential for cure. Unfortunately, ACC has undergone metastatic spread in 40% to 70% of patients at the time of diagnosis. Standard chemotherapy with mitotane is often ineffective with intolerable side effects. The modern molecular technology of comparative genomic hybridization allows the examination of DNA for chromosomal alterations, which can lend biological insight into cancer processes. Genomes of 25 ACC clinical samples were queried on the Agilent 44K Human Genome comparative genomic hybridization array detecting regions of chromosomal gain and loss within the tumor population. Commonly shared amplifications appearing in ≥50% of tumors at P ≤ 10−4 include regions within chromosomes 5, 7, 12, 16q, and 20. Deleted genomic regions within ACC include portions of chromosomes 1, 3p, 10q, 11, 14q, 15q, 17, and 22q. Genomic aberrations in regions associated with differential survival (P ≤ 0.05) and presence in ≥20% of tumors include amplifications of 6q, 7q, 12q, and 19p. Deletions within stratified survival groups include localized regions within 3, 8, 10p, 16q, 17q, and 19q. Statistical analysis of this genetic landscape reveals a set of chromosomal aberrations strongly associated with survival in an accumulation-dependent fashion. These regions may hold prognostic indicators and offer therapeutic targets that can be used to develop novel treatments for aggressive tumors. [Mol Cancer Ther 2008;7(2):425–31]

Abstract Hyperdiploid multiple myeloma (H-MM) is the most common form of myeloma. In this gene expression profiling study, we show that H-MM is defined by a protein biosynthesis signature that is primarily driven by a gene dosage mechanism as a result of trisomic chromosomes. Within H-MM, four independently validated patient clusters overexpressing nonoverlapping sets of genes that form cognate pathways/networks that have potential biological importance in multiple myeloma were identified. One prominent cluster, cluster 1, is characterized by high expression of cancer testis antigen and proliferation-associated genes. Tumors from these patients were more proliferative than tumors in other clusters (median plasma cell labeling index, 3.8; P < 0.05). Another cluster, cluster 3, is characterized by genes involved in tumor necrosis factor/nuclear factor-κB signaling and antiapoptosis. These patients have better response to bortezomib as compared with patients within other clusters (70% versus 29%; P = 0.02). Furthermore, for a group of patients generally thought to have better prognosis, a cluster of patients with short survival (cluster 1; median survival, 27 months) could be identified. This analysis illustrates the heterogeneity within H-MM and the importance of defining specific cytogenetic prognostic factors. Furthermore, the signatures that defined these clusters may provide a basis for tailoring treatment to individual patients. [Cancer Res 2007;67(7):2982–9]