Erming Tian

BACKGROUND: Myeloma cells may secrete factors that affect the function of osteoblasts, osteoclasts, or both. METHODS: We subjected purified plasma cells from the bone marrow of patients with newly diagnosed multiple myeloma and control subjects to oligonucleotide microarray profiling and biochemical and immunohistochemical analyses to identify molecular determinants of osteolytic lesions. RESULTS: We studied 45 control subjects, 36 patients with multiple myeloma in whom focal lesions of bone could not be detected by magnetic resonance imaging (MRI), and 137 patients in whom MRI detected such lesions. Different patterns of expression of 57 of approximately 10,000 genes from purified myeloma cells could be used to distinguish the two groups of patients (P<0.001). Permutation analysis, which adjusts the significance level to account for multiple comparisons in the data sets, showed that 4 of these 57 genes were significantly overexpressed by plasma cells from patients with focal lesions. One of these genes, dickkopf 1 (DKK1), and its corresponding protein (DKK1) were studied in detail because DKK1 is a secreted factor that has been linked to the function of osteoblasts. Immunohistochemical analysis of bone marrow-biopsy specimens showed that only myeloma cells contained detectable DKK1. Elevated DKK1 levels in bone marrow plasma and peripheral blood from patients with multiple myeloma correlated with the gene-expression patterns of DKK1 and were associated with the presence of focal bone lesions. Recombinant human DKK1 or bone marrow serum containing an elevated level of DKK1 inhibited the differentiation of osteoblast precursor cells in vitro. CONCLUSIONS: The production of DKK1, an inhibitor of osteoblast differentiation, by myeloma cells is associated with the presence of lytic bone lesions in patients with multiple myeloma.

Bone marrow plasma cells (PCs) from 74 patients with newly diagnosed multiple myeloma (MM), 5 with monoclonal gammopathy of undetermined significance (MGUS), and 31 healthy volunteers (normal PCs) were purified by CD138(+) selection. Gene expression of purified PCs and 7 MM cell lines were profiled using high-density oligonucleotide microarrays interrogating about 6800 genes. On hierarchical clustering analysis, normal and MM PCs were differentiated and 4 distinct subgroups of MM (MM1, MM2, MM3, and MM4) were identified. The expression pattern of MM1 was similar to normal PCs and MGUS, whereas MM4 was similar to MM cell lines. Clinical parameters linked to poor prognosis, abnormal karyotype (P =.002) and high serum beta(2)-microglobulin levels (P =.0005), were most prevalent in MM4. Also, genes involved in DNA metabolism and cell cycle control were overexpressed in a comparison of MM1 and MM4. In addition, using chi(2) and Wilcoxon rank sum tests, 120 novel candidate disease genes were identified that discriminate normal and malignant PCs (P <.0001); many are involved in adhesion, apoptosis, cell cycle, drug resistance, growth arrest, oncogenesis, signaling, and transcription. A total of 156 genes, including FGFR3 and CCND1, exhibited highly elevated ("spiked") expression in at least 4 of the 74 MM cases (range, 4-25 spikes). Elevated expression of these 2 genes was caused by the translocation t(4;14)(p16;q32) or t(11;14)(q13;q32). Thus, novel candidate MM disease genes have been identified using gene expression profiling and this profiling has led to the development of a gene-based classification system for MM.

With the goal of identifying genes with a differential pattern of expression between ovarian serous papillary carcinomas (OSPCs) and normal ovarian (NOVA) epithelium and using this knowledge for the development of novel diagnostic and therapeutic markers for ovarian cancer, we used oligonucleotide microarrays with probe sets complementary to 12,533 genes to analyze the gene expression profiles of 10 primary OSPC cell lines, 2 established OSPC cell lines (UCI-101, UCI-107) and 5 primary NOVA epithelial cultures. Unsupervised analysis of gene expression data identified 129 and 170 genes that exhibited >5-fold upregulation and downregulation, respectively, in primary OSPC compared to NOVA. Genes overexpressed in established OSPC cell lines had little correlation with those overexpressed in primary OSPC, highlighting the divergence of gene expression that occurs as a result of long-term in vitro growth. Hierarchical clustering of the expression data readily distinguished normal tissue from primary OSPC. Laminin, claudin 3, claudin 4, tumor-associated calcium signal transducers 1 and 2 (TROP-1/Ep-CAM, TROP-2), ladinin 1, S100A2, SERPIN2 (PAI-2), CD24, lipocalin 2, osteopontin, kallikrein 6 (protease M), kallikrein 10, matriptase (TADG-15) and stratifin were among the most highly overexpressed genes in OSPC compared to NOVA. Downregulated genes in OSPC included transforming growth factor-beta receptor III, platelet-derived growth factor receptor alpha, SEMACAP3, ras homolog gene family member I (ARHI), thrombospondin 2 and disabled-2/differentially expressed in ovarian carcinoma 2 (Dab2/DOC2). Differential expression of some of these genes, including claudin 3, claudin 4, TROP-1 and CD24, was validated by quantitative RT-PCR and flow cytometry on primary OSPC and NOVA. Immunohistochemical staining of formalin-fixed, paraffin-embedded tumor specimens from which primary OSPC cultures were derived further confirmed differential expression of CD24 and TROP-1/Ep-CAM markers on OSPC vs. NOVA. These results, obtained with highly purified primary cultures of ovarian cancer, highlight important molecular features of OSPC and may provide a foundation for the development of new type-specific therapies against this disease.

Reciprocal chromosomal translocations, which are mediated by errors in immunoglobulin heavy chain (IgH) switch recombination or somatic hypermutation as plasma cells are generated in germinal centers, are present in most multiple myeloma (MM) tumors. These translocations dysregulate an oncogene that is repositioned in proximity to a strong IgH enhancer. There is a promiscuous array of nonrandom chromosomal partners (and oncogenes), with the 3 most frequent partners (11q13 [cyclin D1]; 4p16 [FGFR3 and MMSET]; 16q23 [c-maf]) involved in nearly half of MM tumors. It is now shown that a novel t(6;14)(p21;q32) translocation is present in 1 of 30 MM cell lines and that this cell line uniquely overexpresses cyclin D3. The cloned breakpoint juxtaposes gamma 4 switch sequences with 6p21 sequences that are located about 65 kb centromeric to the cyclin D3 gene. By metaphase chromosome analysis, the t(6;14) (p21;q32) translocation was identified in 6 of 150 (4%) primary MM tumors. Overexpression of cyclin D3 messenger RNA (mRNA) was identified by microarray RNA expression analysis in 3 of 53 additional primary MM tumors, each of which was found to have a t(6;14) translocation breakpoint by interphase fluorescence in situ hybridization analysis. One tumor has a t(6;22)(p21;q11) translocation, so that cyclin D3 is bracketed by the IgL and IgH breakpoints. These results provide the first clear evidence for primary dysregulation of cyclin D3 during tumorigenesis. It is suggested that the initial oncogenic event for most MM tumors is a primary immunoglobulin translocation that dysregulates cyclin D1, cyclin D3, and other oncogenes to provide a proliferative stimulus to postgerminal center plasma cells.