Erming Tian

Gene expression profiles in primary ovarian serous papillary tumors and normal ovarian epithelium: identification of candidate molecular markers for ovarian cancer diagnosis and therapy.

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‐β receptor III, platelet‐derived growth factor receptor α, 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.

Prognostic impact of cytogenetic and interphase fluorescence in situ hybridization-defined chromosome 13 deletion in multiple myeloma: early results of total therapy II.

Summary. Cytogenetic abnormalities of chromosome 13 (CA 13) and those detected by fluorescence in situ hybridization (FISH 13) have both been associated with poor prognosis in multiple myeloma (MM) patients. The prognostic implications of CA, FISH 13 and other standard laboratory parameters were examined in the first 231 patients enrolled in Total Therapy II, an intensive cytotoxic chemotherapy programme with tandem autotransplants. Three‐year projections of event‐free survival (EFS) and overall survival (OS) were 71% and 77% respectively. CA 13 was detected in 14% and significantly correlated with FISH 13 (present in 51%), tumour burden, proliferative activity and lactic dehydrogenase (LDH). Both EFS and OS were significantly shorter in patients with CA 13, FISH 13, LDH ≥ 190 U/l, β2 microglobulin ≥ 4 mg/l and C reactive protein ≥ 4·0 mg/l; other CA was an additional risk factor for OS. Two‐thirds of CA 13 patients were identified by FISH 13 and plasma‐cell‐labelling index (PCLI) ≥ 0·4%; however, PCLI failed to identify additional risk groups in FISH subsets. Although present in considerably fewer patients, CA 13 imparted more rapid relapse (61% at 3 years) and death (43% at 3 years) than FISH 13 (38% and 35%; P = 0·02 and 0·1 respectively) and should be part of the initial work‐up of patients with MM.

Control of Autophagic Cell Death by Caspase-10 in Multiple Myeloma

We performed a loss-of-function RNA interference screen to define therapeutic targets in multiple myeloma, a genetically diverse plasma cell malignancy. Unexpectedly, we discovered that all myeloma lines require caspase-10 for survival irrespective of their genetic abnormalities. The transcription factor IRF4 induces both caspase-10 and its associated protein cFLIPL in myeloma, generating a protease that does not induce apoptosis but rather blocks an autophagy-dependent cell death pathway. Caspase-10 inhibits autophagy by cleaving the BCL2-interacting protein BCLAF1, itself a strong inducer of autophagy that acts by displacing beclin-1 from BCL2. While myeloma cells require a basal level of autophagy for survival, caspase-10 tempers this response to avoid cell death. Drugs that disrupt this vital balance may have therapeutic potential in myeloma.

Genomic instability in multiple myeloma: Evidence for jumping segmental duplications of chromosome arm 1q

Multiple myeloma (MM) is a malignant plasma cell disorder characterized by complex karyotypes and chromosome 1 instability at the cytogenetic level. Chromosome 1 instability generally involves partial duplications, whole‐arm translocations, or jumping translocations of 1q, identified by G‐banding. To characterize this instability further, we performed spectral karyotyping and fluorescence in situ hybridization with probes for satII/III (1q12), BCL9 (1q21), and IL6R (1q21) on the karyotypes of 44 patients with known 1q aberrations. In eight patients, segmental duplication of 1q12–21 and adjacent bands occurred on nonhomologous chromosomes. In five cases, the 1q first jumped to a nonhomologous chromosome, after which the 1q12–21 segment again duplicated itself 1–3 times. In three other cases, segmental duplications occurred after the 1q first jumped to a nonhomologous chromosome, where the proximal adjacent nonhomologous chromosome segment was duplicated prior to the 1q jumping or inserting itself into a new location. These cases demonstrate that satII/III DNA sequences are not only associated not only with the duplication of adjacent distal chromosome segments after translocation, but are also associated with the duplication and jumping/insertion of proximal nonhomologous chromosome segments. We have designated this type of instability as a jumping segmental duplication.

Evi27 encodes a novel membrane protein with homology to the IL17 receptor

Evi27 is a common site of retroviral integration in BXH2 murine myeloid leukemias. Here we show that integration at Evi27 occurs in a CpG island ∼6 kb upstream from a novel gene (designated Evi27) with homology to the IL17 receptor (Il17r) and that proviral integrations result in increased expression of the Evi27 protein on the cell surface. The human EVI27 homolog was also cloned and mapped to chromosome 3p21. Multiple Evi27 isoforms were detected at the RNA and protein level in both human and mouse, indicating that Evi27 expression is complex. Some of the isoforms are shown to likely represent secreted soluble forms of the protein produced by intron incorporation or by proteolytic cleavage. In the mouse, highest Evi27 expression occurs in liver and testes with lower expression in kidney and lung. In humans, EVI27 is expressed at high levels in the kidney, with moderate levels in the liver, brain, and testes. Within hematopoietic cells, Evi27 expression is restricted. Northern and Western analysis showed that Evi27 is expressed in selected T-cell, B-cell and myeloid cell lines. These results suggest that Evi27 expression is tightly regulated during hematopoietic differentiation. Collectively, these studies identify a new member of the cytokine receptor family whose increased and uncoordinated expression may lead to myeloid leukemia by altering Evi27s normal ability to control the growth and/or differentiation of hematopoietic cells.

Mrvil, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1

Ecotropic MuLVs induce myeloid leukemia in BXH2 mice by insertional mutagenesis of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by viral tagging as common sites of viral integration in BXH2 leukemias. Previous studies showed that a frequent common integration site in BXH2 leukemias is the Nf1 tumor suppressor gene. Unexpectedly, about half of the viral integrations at Nf1 represented a previously undiscovered defective nonecotropic virus, termed MRV. Because other common integration sites in BXH2 leukemias encoding proto-oncogenes contain ecotropic rather than MRV viruses, it has been speculated that MRV viruses may selectively target tumor suppressor genes. To determine if this were the case, 21 MRV-positive BXH2 leukemias were screened for new MRV common integration sites. One new site, Mrvi1 was identified that was disrupted by MRV in two of the leukemias. Ecotropic virus did not disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, suggesting that Mrvi1 is specifically targeted by MRV. Mrvi1 encodes a novel protein with homology to Jaw1, a lymphoid restricted type II membrane protein that localizes to the endoplasmic reticulum. MRV integration occurs at the 5′ end of the gene between two differentially used promoters. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias. Like Jaw1, which is downregulated during lymphoid differentiation, Mrv1 is downregulated during monocytic differentiation of BXH2 leukemias. Taken together, these data suggest that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of a gene important for myeloid cell growth and/or differentiation. Experiments are in progress to test whether Mrvi1 is a tumor suppressor gene.

An intermediate-risk multiple myeloma subgroup is defined by sIL-6r; levels synergistically increase with incidence of SNP rs2228145 and 1q21 amplification

IL-6 signaling can be enhanced through transsignaling by the soluble IL-6 receptor (sIL-6r), allowing for the pleiotropic cytokine to affect cells it would not ordinarily have an effect on. Serum levels of sIL-6r can be used as an independent prognostic indicator and further stratify the GEP 70-gene low-risk group to identify an intermediate-risk group in multiple myeloma (MM). By analyzing more than 600 MM patients with ELISA, genotyping, and gene expression profiling tools, we show how the combination of 2 independent molecular genetic events is related to synergistic increases in sIL-6r levels. We also show that the rs2228145 minor allele is related to increased expression levels of an IL-6r splice variant that purportedly codes exclusively for a sIL-6r isoform. Together, the SNP rs2228145 minor allele C and amplification of chromosome 1q21 are significantly correlated to an increase in sIL-6r levels, which are associated with lower overall survival in 70-gene low-risk disease, and aid in identification of the intermediate-risk MM group.

c-Met signaling promotes IL-6-induced myeloma cell proliferation

Objectives:  Hepatocyte growth factor (HGF) is a constituent of the myeloma microenvironment and is elevated in sera from myeloma patients compared to healthy individuals. Increased levels of serum HGF predict a poor prognosis. It has previously been shown by us and others HGF can act as a growth factor to myeloma cells in vitro although these effects have been moderate. We therefore wanted to investigate if HGF could influence the effects of interleukin (IL)‐6.

Clonal selection and double-hit events involving tumor suppressor genes underlie relapse in myeloma.

To elucidate the mechanisms underlying relapse from chemotherapy in multiple myeloma, we performed a longitudinal study of 33 patients entered into Total Therapy protocols investigating them using gene expression profiling, high-resolution copy number arrays, and whole-exome sequencing. The study illustrates the mechanistic importance of acquired mutations in known myeloma driver genes and the critical nature of biallelic inactivation events affecting tumor suppressor genes, especially TP53, the end result being resistance to apoptosis and increased proliferation rates, which drive relapse by Darwinian-type clonal evolution. The number of copy number aberration changes and biallelic inactivation of tumor suppressor genes was increased in GEP70 high risk, consistent with genomic instability being a key feature of high risk. In conclusion, the study highlights the impact of acquired genetic events, which enhance the evolutionary fitness level of myeloma-propagating cells to survive multiagent chemotherapy and to result in relapse.

Evidence for a novel mechanism for gene amplification in multiple myeloma: 1q12 pericentromeric heterochromatin mediates breakage‐fusion‐bridge cycles of a 1q12∼23 amplicon

Gene amplification is defined as a copy number (CN) increase in a restricted region of a chromosome arm, and is a mechanism for acquired drug resistance and oncogene activation. In multiple myeloma (MM), high CNs of genes in a 1q12∼23 amplicon have been associated with disease progression and poor prognosis. To investigate the mechanisms for gene amplification in this region in MM, we performed a comprehensive metaphase analysis combining G‐banding, fluorescence in situ hybridization, and spectral karyotyping in 67 patients with gain of 1q. In six patients (9%), evidence for at least one breakage‐fusion‐bridge (BFB) cycle was found. In three patients (4%), extended ladders of 1q12∼23 amplicons were identified. Several key structures that are predicted intermediates in BFB cycles were observed, including: equal‐spaced organization of amplicons, inverted repeat organization of amplicons along the same chromosome arm, and deletion of sequences distal to the amplified region. The 1q12 pericentromeric heterochromatin region served as both a recurrent breakpoint as well as a fusion point for sister chromatids, and ultimately bracketed both the proximal and distal boundaries of the amplicon. Our findings provide evidence for a novel BFB mechanism involving 1q12 pericentromeric breakage in the amplification of a large number of genes within a 1q12∼23 amplicon.