Array-based Gene Expression Profiling Research Articles

Nuclear envelope protein lamin B receptor protects the genome from chromosomal instability and tumorigenesis.

Lamin B Receptor (LBR) is an inner nuclear membrane protein that assembles the nuclear envelope post mitosis. Here we show that LBR depletion induces mitotic defects accompanied by recurrent chromosomal losses. In addition, LBR knockdown results in nuclear aberrations such as nuclear blebs and micronuclei, with chromosomes showing higher frequency of losses, being enriched within the micronucleus. Furthermore, doxycycline-induced conditional depletion of LBR significantly increased tumor volumes that form within the subcutaneous xenografts of mice. Of note, the tumor-derived primary cells recapitulated chromosomal losses and gains, revealing a novel role for LBR as a tumor suppressor. Co-immunoprecipitation of LBR uncovered an association of LBR with telomere-associated factors. Interestingly, qPCR array-based gene expression profiling showed a significant upregulation of telomere repeat-binding factor 1 (TRF1) upon LBR depletion. Remarkably, TRF1 knockdown in the background of LBR depletion maintains chromosomal stability, unraveling a novel mechanism involving LBR and TRF in the maintenance of chromosomal stability in colorectal cancer cells.

Multifaceted array-based keloidal gene expression profiling reveals specific MDFI upregulation in keloid lesions.

Keloid lesions are characterized by mesenchymal cell proliferation and excessive extracellular matrix deposition. Previous microarray analyses have been performed to investigate the mechanism of keloid development. However, the molecular pathology that contributes to keloid development remains obscure. To explore the underlying essential molecules of keloids using microarrays. We performed microarray analyses of keloid and nonlesional skin tissues both invivo and invitro. Gene expression levels were compared between tissues and cells. Quantitative reverse transcription (qRT)-PCR and immunohistochemical staining were used to determine the expression levels of molecules of interest in keloid tissues. Several common molecules were upregulated in both keloid tissues and keloid-lesional fibroblasts. PTPRD and NTM were upregulated both invivo and invitro. The genes MDFI and ITGA4 were located at the centre of the gene coexpression network analysis using keloid tissues. qRT-PCR revealed significant expression levels of PTPRD and MDFI in keloid tissues. Immunopathological staining revealed that MDFI-positive cells, which have fibroblast characteristics, were located in the keloid-associated lymphoid tissue (KALT) portion of the keloid tissue. Our gene expression profiles of keloids could distinguish the difference between lesional tissue and cultured lesional fibroblasts, and MDFI was found to be commonly expressed in both tissues and cells. Thus, MDFI-positive cells, which were located in the KALT, may play an important role in keloid pathogenesis and thus might be useful for invitro keloid studies.

Integrated Analysis of DNA Copy Number Changes and Gene Expression Identifies Key Genes in Gastric Cancer.

This study was aimed at identifying differentially expressed genes (DEGs) with copy number changes in gastric cancer (GC) pathogenesis. Microarray data GSE33429, including array-based comparative genomic hybridization and gene expression profiles, were obtained. DEGs were screened between GC and adjacent noncancerous tissues. Genes located at Minimum Common Regions (MCRs) were identified, and overlapped genes between DEGs and genes with amplification or deletion were identified. Gene Ontology function and pathway enrichment analysis of DEGs were performed. A protein-protein interaction network for DEGs was built, and significant modules were mined from the network. Functional annotation of genes in modules was also performed. A total of 677 up- and 583 downregulated DEGs were identified, including 37 overexpressed genes located at gained MCRs and 28 downregulated genes located at deleted MCRs. In significant modules, upregulated genes with amplification, including DSN1 (MIS12 kinetochore complex component), MAPRE1 (microtubule-associated protein, RP/EB family, member 1), TPX2 (microtubule-associated), UBE2C (ubiquitin-conjugating enzyme E2C), and MYBL2 (v-myb avian myeloblastosis viral oncogene homolog-like 2), were associated with cell cycle, but downregulated genes with deletion, including UGT2B15 (UDP glucuronosyltransferase 2 family, polypeptide B15), UGT2B17 (UDP glucuronosyltransferase 2 family, polypeptide B17), ADH1B (alcohol dehydrogenase 1B), and ADH1A (alcohol dehydrogenase 1A), were related to metabolism. The identified genes DSN1, MAPRE1, TPX2, UBE2C, and MYBL2 located at gained MCRs and UGT2B15, UGT2B17, ADH1B, and ADH1A located at deleted MCRs may play an important role in GC progression through regulating cell cycle and metabolism.

Global expression profiling and pathway analysis in two different population groups in relation to high altitude.

High altitude (HA) is associated with number of stresses. Response of these stresses may vary in different populations depending upon altitude, duration of residency, ancestry, geographical variation, lifestyle, and ethnicities. For understanding population variability in transcriptome, array-based global gene expression profiling was performed on extracted RNA of male volunteers of two different lowland population groups, i.e., Indians and Kyrgyz, at baseline and day 7 of HA exposure (3200m). A total of 97 genes were differentially expressed at basal in Kyrgyz as compared to Indians (82 downregulated and 15 upregulated), and 196 were differentially expressed on day 7 of HA (118 downregulated and 78 upregulated). Ingenuity Pathway Analysis and gene ontology highlighted eIF2 signaling with most significant negative activation z score at basal in Kyrgyz compared to Indians with downregulation of various L- and S-ribosomal proteins indicating marked translational repression. On day 7, cAMP-mediated signaling is most enriched with positive activation z score in Kyrgyz compared to Indians. Plasma cAMP levels were higher in Kyrgyz on day 7 compared to Indians. Extracellular adenosine levels were elevated in both the groups upon HA, but higher in Kyrgyz compared to Indians. Valedictory qRT-PCR showed upregulation of ADORA2B and CD73 along with downregulation of ENTs in Kyrgyz compared to Indians indicating elevated levels of extracellular nucleotides mainly adenosine and activation of extracellular cAMP-adenosine pathway which as per literature triggers endogenous protective mechanisms under stress conditions like hypoxia. Thus, transcriptome changes at HA are population-specific, and it may be necessary to take care while interposing similar results in different populations.

BRD4 Proteolysis Targeting Chimera (PROTAC) ARV-825, Causes Sustained Degradation of BRD4 and Modulation of Chemokine Receptors, Cell Adhesion and Metabolic Targets in Leukemia Resulting in Profound Anti-Leukemic Effects

Background: Mutational or non-mutational epigenetic events that aberrantly modify the chromatin regulatory machinery to enhance oncogene expression are a hallmark of myeloid malignancies. BRD4, a member of the bromodomain and extra terminal domain (BET) family, is a transcriptional coactivator that co-occupies super enhancer complexes associated with transcription of oncogenes (MYC, SOX2, NF-kB etc.) - and apoptosis regulators (Bcl-2, Bcl-XL, etc.) and has been validated as a target in AML therapy1.ARV-825 is a hetero-bifunctional PROteolysis TArgeting Chimera (PROTAC) that recruits BRD4 to the E3 ubiquitin ligase cereblon and leads to efficient and sustained degradation of BRD4 resulting in down-regulation of MYC2.Objectives: We examined the anti-leukemic effect of ARV-825 against AML cell lines, primary AML blasts and a mouse model of disseminated leukemia. Since tumor-stroma interactions driven by oncogene activation play a major role in resistance to AML therapy, we tested whether ARV-825 could overcome stroma-mediated drug resistance. As MYC harmonizes nutrient acquisition by cancer cells through regulation of the metabolites antiporter systems (SLC7A11, SLC5A5) 3, we profiled changes in a few important amino acids and organic acids in AML cell in response to ARV-825.Results: The IC50s for all tested cell lines and primary AML cells at 72 hours were in the low nanomolar range (2-50 nM) and 10-100 times lower than JQ1, a small molecule BRD4 inhibitor. ARV-825 induces sustained BRD4 degradation accompanied by down-regulation of targets such as MYC, anti-apoptotic BCL-2 family molecules and an increase in apoptosis and DNA damage4.In an in vitro tumor-stroma co-culture model including hypoxic conditions, bone marrow derived mesenchymal stromal cells (MSCs) protected OCI-AML3 cells from cytarabine ( 55.4% apoptosis with vs. 35.0% without MSCs in normoxia and 50.6% vs. 32.8%, respectively, in hypoxia), but no such protection was observed against ARV-825 (58.7% apoptosis vs. 55.2% in normoxia and 57.4% vs. 58.3%, respectively, in hypoxia). Mass cytometry based proteomic analysis (CyTOF) (Fig. 1), immunoblotting and flow cytometry showed that among apoptotic, cell adhesion and signaling proteins, MYC, CD44 and surface CXCR4 were the most down-regulated proteins in AML cells. The functional relevance of surface CXCR4 down regulation was confirmed in migration assays against the CXCR4 ligand SDF-1. Phosphorylation of CXCR4 by PIM1 kinase is necessary for surface expression of CXCR4, ARV-825 treatment reduced PIM1 levels and phosphorylation of CXCR4 in AML cells while overexpression of PIM1 or Myc reversed the phenomena. Quantitative PCR and immunoblotting analysis confirmed the transcriptional down regulation of total CD44 and CD44 variants 8-10 (2-fold change treated vs. untreated). As a functional correlate of CD44 variants, mass spectrometry based intracellular metabolomics and flow cytometry confirmed reduction in cysteine uptake and increased reactive oxygen species (ROS) generation (Fig. 2). Additional metabolic readouts using the Sea horse system revealed inhibition of mitochondrial respiration as indicated by decreased in oxygen consumption rate and production of ATP upon treatment of ARV-825. Furthermore, array based gene expression profiling showed down-regulation of additional amino acid transporters and the Wnt/β-catenin pathway.Finally, in a mouse model of human leukemia, the leukemia burdens were significantly lower in the ARV-825 treated mice as confirmed by luciferase imaging, flow cytometry, spleen size and survived longer compared to control mice (p=0.0005) (Fig.3).Conclusion: ARV-825 has substantial, broad anti-AML activity and importantly modulates the tumor microenvironment and metabolism to overcome stroma-mediated drug resistance. Together, our data suggest that ARV-825 is an effective in targeting BET family of proteins for the treatment of AMLReference:1. Nature 2011; 478(7370): 524-528. doi: 10.1038/nature103342. Chem Biol 2015; 22(6): 755-763. doi: 10.1016/j.chembiol.2015.05.0093. Cancer Res 2015; 75(9): 1782-1788. doi: 10.1158/0008-5472.CAN-14-37454. 604(675): ASH 2015,San franscisco,USA. [Display omitted] DisclosuresLorenzi:Erytech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. Qian:Arvinas, LLC: Employment. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Novel mouse model recapitulates genome and transcriptome alterations in human colorectal carcinomas.

Human colorectal carcinomas are defined by a nonrandom distribution of genomic imbalances that are characteristic for this disease. Often, these imbalances affect entire chromosomes. Understanding the role of these aneuploidies for carcinogenesis is of utmost importance. Currently, established transgenic mice do not recapitulate the pathognonomic genome aberration profile of human colorectal carcinomas. We have developed a novel model based on the spontaneous transformation of murine colon epithelial cells. During this process, cells progress through stages of pre-immortalization, immortalization and, finally, transformation, and result in tumors when injected into immunocompromised mice. We analyzed our model for genome and transcriptome alterations using ArrayCGH, spectral karyotyping (SKY), and array based gene expression profiling. ArrayCGH revealed a recurrent pattern of genomic imbalances. These results were confirmed by SKY. Comparing these imbalances with orthologous maps of human chromosomes revealed a remarkable overlap. We observed focal deletions of the tumor suppressor genes Trp53 and Cdkn2a/p16. High-level focal genomic amplification included the locus harboring the oncogene Mdm2, which was confirmed by FISH in the form of double minute chromosomes. Array-based global gene expression revealed distinct differences between the sequential steps of spontaneous transformation. Gene expression changes showed significant similarities with human colorectal carcinomas. Pathways most prominently affected included genes involved in chromosomal instability and in epithelial to mesenchymal transition. Our novel mouse model therefore recapitulates the most prominent genome and transcriptome alterations in human colorectal cancer, and might serve as a valuable tool for understanding the dynamic process of tumorigenesis, and for preclinical drug testing. © 2016 Wiley Periodicals, Inc.

DNA methylation and gene expression profiles show novel regulatory pathways in hepatocellular carcinoma.

BackgroundAlcohol is a well-known risk factor for hepatocellular carcinoma (HCC), but the mechanisms underlying the alcohol-related hepatocarcinogenesis are still poorly understood. Alcohol alters the provision of methyl groups within the hepatic one-carbon metabolism, possibly inducing aberrant DNA methylation. Whether specific pathways are epigenetically regulated in alcohol-associated HCC is, however, unknown. The aim of the present study was to investigate the genome-wide promoter DNA methylation and gene expression profiles in non-viral, alcohol-associated HCC. From eight HCC patients undergoing curative surgery, array-based DNA methylation and gene expression data of all annotated genes were analyzed by comparing HCC tissue and homologous cancer-free liver tissue.ResultsAfter merging the DNA methylation with gene expression data, we identified 159 hypermethylated-repressed, 30 hypomethylated-induced, 49 hypermethylated-induced, and 56 hypomethylated-repressed genes. Notably, promoter DNA methylation emerged as a novel regulatory mechanism for the transcriptional repression of genes controlling the retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22, RDH16), iron homeostasis (HAMP), one-carbon metabolism (SHMT1), and genes with a putative, newly identified function as tumor suppressors (FAM107A, IGFALS, MT1G, MT1H, RNF180).ConclusionsA genome-wide DNA methylation approach merged with array-based gene expression profiles allowed identifying a number of novel, epigenetically regulated candidate tumor-suppressor genes in alcohol-associated hepatocarcinogenesis. Retinol metabolism genes and SHMT1 are also epigenetically regulated through promoter DNA methylation in alcohol-associated HCC.Due to the reversibility of epigenetic mechanisms by environmental/nutritional factors, these findings may open up to novel interventional strategies for hepatocarcinogenesis prevention in HCC related to alcohol, a modifiable dietary component.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0077-1) contains supplementary material, which is available to authorized users.

English

The uncontrolled proliferation of hematopoietic cells with no capacity to differentiate into mature blood cells leads to leukemia. Though considerable amount of work has been done in understanding the molecular basis and gene expression profiles of hematologic malignancies viz., chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML), the role of various underlying genes and mechanisms predisposing the disease are poorly understood. To develop the early diagnosis, preventive and therapeutic strategies, identification of population specific novel mutations and candidate genes are required. Micro array based gene expression profiling was performed for total of 18 samples (4 from each subtype of leukemia that is, CLL, CML, ALL, AML and 2 controls) from Indian population using single color hybridization. The expression of all genes presented in terms of fold variation was subjected to F-test. The microarray data of genes showing differential regulation with respect to the control samples have been obtained from total 50, 238 probes covering 14,992 genes on Agilent’s Human 8X60K Array. The experiment was conducted with expectation to have similar patterns of result in terms of gene expression but it demonstrates statistically significant relationship only among CML and ALL which are of myeloid and lymphoid origin, respectively, in contrast to other combinations. Gene expression profiles of four subtypes of leukemia were compared to each other to ascertain the overall association and significance of genes for occurrence of different types of leukemiawhich would guide in the development of common probable biomarkers for leukemias followed by effective diagnosis, prognosis and treatment. Based on their geomean fold values, the highly upregulated genes found in this study are listed.   Keywords: Leukemia, microarray, gene expression profiling, fold variation, lymphoid, myeloid, geomean fold.

Tumor suppressive mechanisms in hepatocellular carcinoma through inhibition of IL-6 signaling

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor in the liver and the third leading cause of cancer death worldwide (696,000 deaths, 9.2%). Although significant progress has been made in the HCC field, the molecular mechanisms and signaling pathways underlying HCC development and progression are still poorly understood. This is probably the case because HCC is very heterogeneous in terms of clinical presentation, genomic alterations and gene expression patterns. In a recent study, we applied an integrative approach of high-resolution array-based comparative genomic hybridization (arrayCGH) and gene expression profiling coupled with patient prognosis to identify tumor 'driver' genes which have genomic aberrations in HCC clinical specimens and functionally contribute to tumor progression. This analysis led to the identification of 10 potential cancer driver genes. Six of these genes were located on chromosome 8 p which is associated with poor outcome. Importantly, the six genes on chromosome 8 p included the tumor suppressor gene DLC1 that had been shown previously to be involved in HCC development. Next we performed clonogenicity, cell migration assays and xenograft mouse models to analyze the five additional genes' for tumor suppressive properties. These experiments confirmed the tumor suppressor function of two additional tumor suppressor genes on chromosome 8 p, SH2D4A and SORBS3. Furthermore, functional analysis revealed that SH2D4A and SORBS3 function in a convergent manner to inhibit the IL-6 pathway. Thereby, SORBS3 inhibits the IL-6 pathway via activation of the estrogen receptor alpha and SH2D4A via inhibition of STAT3 signaling. Taken together, in this study we identified two novel tumor suppressor genes on chromosome 8 p which functionally cooperate to inhibit IL-6 signaling.

High Throughput Sequencing-Based Pathogen Discovery In Multiple Myeloma

BackgroundMultiple myeloma is a malignancy of antibody-secreting, terminally differentiated B cells with a predilection for the bone marrow. Considering that microbial infections are common triggers for antibody production, we hypothesize that known and/or previously undiscovered viruses may be present in myeloma tissues and may influence myeloma prognosis, even if the viruses themselves do not cause myeloma.Virus-harboring tissues should presumably contain both human and pathogenic DNA/RNA, but viruses may not be detected if they are present at frequencies that are too low for detection by standard assays. Therefore, high-throughput sequencing, followed by computational subtraction of human sequence reads should result in the identification of non-human (and candidate pathogenic) sequences. The Multiple Myeloma Research Consortium has generated such a dataset, thus providing a unique opportunity to address this question. MethodsWe have developed a computational subtraction pipeline, PathSeq, for pathogen discovery. It consists of 3 main modules. Quality filtering is carried out in the “pre-subtraction module”. In the “subtraction module”, the quality filtered reads are analyzed by alignment to a series of human genome and transcriptomes databases using the BWA aligner and BLAST. In the “post-subtraction module”, residual nucleotide and conceptually translated sequences are aligned to microbial databases. We also generate large contigs composed of unmapped reads that do not possess significant alignment similarity to any sequence in the reference databases. Such contigs may be suggestive of novel microorganisms.Whole genome sequencing data obtained from an initial set of 26 tumor-normal sample pairs (reported in Chapman et al, Nature 2010) were analyzed by PathSeq. In addition, analysis of mRNA sequencing data from 84 tumors is ongoing. Comparative marker selection analysis was also performed on array-based gene expression profiling data from 20 of the initial 26 tumors. ResultsPathSeq analyses revealed the presence of Epstein-Barr virus (EBV) sequence reads in 7 cases and human herpesvirus 6B (HHV-6B) reads in 2 cases. In most instances, the viral sequences were found in both tumor and normal samples from the same patient. The lowest viral-human read ratio was 1 in 1 billion, demonstrating the sensitivity of the PathSeq algorithm for pathogen identification.Comparative marker selection analysis comparing gene expression profiles between the 7 tumors with EBV reads with 13 tumors without viral sequences revealed a tentative virus associated signature, with SHISA2 (which encodes a protein that modulates FGF and Wnt signaling) as the gene whose expression is most significantly associated with virus positivity. ConclusionsWe have described the identification of EBV and HHV6B sequences in DNA samples obtained from myeloma tissues. Our data are preliminary and it is possible that the viral sequences are simply vestiges of non-active herpesviral infection present in the tumor microenvironment. Therefore, these findings need to be confirmed by our ongoing PathSeq analysis of RNA sequencing data. In addition, efforts are ongoing to identify novel pathogens, as described above.The translational utility of knowledge about the influence of host-viral relationships on the prognosis of myeloma, if they exist, transcends the need to prove causation. Therefore if viral sequences are found in RNA sequencing data, future plans will include comparison of our data to published prognosis-related gene expression signatures in myeloma. Disclosures:No relevant conflicts of interest to declare.

Abstract 806: RNA sequencing based prognostic indexes show a high degree of correlation with historic array based gene expression measurements in multiple myeloma.

Abstract The Multiple Myeloma Research Foundation (MMRF) and the Multiple Myeloma Research Consortium (MMRC) have pioneered efforts towards understanding the genomic underpinnings of multiple myeloma. The MMRC centers have submitted over 1900 tumor samples to the MMRC tissue bank, from which >400 have been used in the Multiple Myeloma Genomics Initiative (MMGI), which is a multi-institutional comprehensive study to unveil the complete genomic blueprint of myeloma with public data access. In the initial phase 238 myeloma tumors were analyzed for somatic copy number alterations using array-based comparative genomic hybridization (aCGH) and array-based gene expression profiling (GEP). The large majority of this reference collection was also analyzed by array-based methylation profiling. We have also performed whole genome or exome sequencing on >200 normal/tumor pairs. These studies led to several key discoveries including global hypomethylation, homozygous deletion of UTX, novel mutations in FAM46C (13%) and DIS3/RRP44 (11%), and potentially therapeutically actionable BRAF mutations (4%). To improve our understanding of the transcriptional activities in myeloma, we have performed RNA-sequencing on 84 myeloma patient samples and 65 human myeloma cell lines for which whole genome or exome sequencing data is also available. RNA-seq has a number of advantages over GEP including improved dynamic range and the detection of exon and isoform levels, allele specific expression, and fusion transcripts. There are a number of robust gene expression classifications and indexes with prognostic value for myeloma samples tested by GEP. Within the cohort of 84 patient samples there are 42 samples with matched GEP data. Using this subgroup we tested if the current GEP methods could be directly moved to RNA-seq data by matching the current probesets to genes. We then compared the results across the patient set for each index or classification. For classifications we determined that the TC classification could be directly transitioned to RNA-seq data with 100% concordance. For the indexes we showed a strong correlation for the proliferation index (R2=0.971) and the NFKB index (R2=0.961) but only a moderate correlation for the 70-gene index (R2=0.761). The decreased correlation in the 70-gene index is clearly due to the large number of probesets used, which are associated with genes that are clearly not expressed by RNA-seq. Integration of the DNA and RNA sequencing data showed that 42% of the mutations detected in the DNA are expressed. Interestingly we observed the progressive loss of wildtype FAM46C alleles, which are often heterozygously mutated and expressed in patients but is exclusively homozogous in cell lines. Additional integrated analysis will improve out understanding of myelomagenesis and might lead to new and improved classification and prognostic models. Citation Format: Jonathan Keats, Venkata Yellapantula, Winnie Liang, Ahmet Kurdoglu, Angela Baker, Joan Levy, Todd Golub, David Craig, John Carpten. RNA sequencing based prognostic indexes show a high degree of correlation with historic array based gene expression measurements in multiple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 806. doi:10.1158/1538-7445.AM2013-806

Molecular Analysis of Primary Cutaneous Aggressive T-Cell Lymphomas: the Epidermotropic CD8+, the Pleomorphic CD8+ and the Gamma Delta Subsets.

AbstractAbstract 2713Primary cutaneous T-cell lymphomas (CTCL) are usually characterized by a T-helper CD4+ immunophenotype and show an indolent clinical course; on the contrary, cases harbouring a CD8+ cytotoxic immunophenotype exhibiting epidermotropism (AECTCL) or pleomorphic morphology (PTL-NOS), as well as cases with gamma-delta + (CGDL) immunophenotype, display a very different course. Clinically, these tumours are very aggressive, with poor outcome in adults. Chemotherapy, preferably based on intensive CHOP-like regimens, represents the mainstay of treatment, when possible followed by stem cell transplantation. Histologically, tumour cells constitute a population of pleomorphic medium-large size elements. In these rare CTCL subtypes few reports have been published dealing with oncogenomic investigations. With this study, by array-based comparative genomic hybridization (a-CGH) and gene expression profiling (a-GEP), we aimed to explore genomic alterations possibly involved in tumorigenesis of aggressive CTCL referred to our department. We focused on 9 cases of CD8+ AECTCL, 3 cases of CD8+ PTL-NOS and 4 cases of CGDL, who were extensively investigated by a-CGH; a-GEP was applied in 5 cases of AECTCL to further expose genomic profile of neoplastic cells. Among AECTCL patients, immunophenotype was always typical and EBV was demonstrated only in one case. Results were assessed by statistical analysis to reveal more significant common chromosomal aberrations. By a-CGH investigations, we found the presence of extensive gains and losses of both large and small chromosomal regions; copy number gains were more frequent than losses. In CD8+ cases, we observed gains of 3p21.33-p21.2 in 10 out of 12 cases, 6p21.2-p21.1 in 8/12, 7q11.23 in 9/12, 7q21.2-q22.1 in 10/12, 7q36.1-q36.3 in 7/12, 8q24.3 in 8/12, 11pter in 11/12, 11q12.3-q13.2 in 10/12, 16p13.3 in 10/12, 17q in 10/12, trisomy 19 (as a mosaic aberration) in 12/12, and trisomy 22 in 8/12; losses involved 4q12-q22.2 in 8/12, 9p21.3 in 10/12 (homozygous in 7) and 14q11.2 in 9/12. In CGDL, recurrent copy number alterations (observed in 50% to 75% of cases) were gains of 2q13-qter, 17q21.3, 19 (trisomy, as mosaic) and 22q11.21, along with losses of 4q12-q22, 6q23.3 and 9p21.3. Summarizing, most of our genomic results have been previously described in other cutaneous lymphomas: gains of 3p21 in diffuse large B-cell lymphomas (DLBCL), of 7q21 in mycosis fungoides (MF), of 8q24 in PTL-NOS and in Sézary syndrome (SS), of 11q22-q13 in DLBCL, of 16p in DLBCL and in angioimmunoblastic T-cell lymphoma (AITL), of 19p in AITL and natural killer lymphomas, trisomy 22 in AITL, PTL-NOS and SS, gain of 17q in SS and in ALCL, MF and PTL-NOS. However, we underline the loss of p21.3, determining CDKN2A deletion, as a common alteration in these three CTCL entities. We also found other common anomalies in AECTCL and PTL-NOS, with particular interest with regard to gain of 17q, 19p13 and 19q13.11-q32, which associate with the JAK/STAT signaling pathway activation. Other alterations in AECTCL cases involve c-MYC (8q24), CCND1/CDK4–6 (11q13) and IL21R (16p13.3). Worth mentioning, GEP analysis in AECTCL confirmed altered expression of CDKN2A, JAK3 and STAT6 genes. Although most genetic aberrations detected in our study have already been described in other lymphoproliferative disorders, we conclude that the combination of aberrations appears characteristic in these aggressive disorders. Disclosures:No relevant conflicts of interest to declare.

Blastic Plasmacytoid Dendritic Cell Neoplasm: Clinical, Immunohistochemical and Molecular Evaluation of 23 Cases with Primary Cutaneous Involvement,

Abstract 3565Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, haematologic malignancy. The disease may occur at any age, with strong male predominance. Skin presentation is very common. Histologically, tumour cells constitute a uniform population of round or more pleomorphic medium size elements. Clinically this tumour is very aggressive, with poor outcome in adults (median survival 13 months). Chemotherapy (CT), preferably with acute leukemia-type regimens, is the mainstay of treatment, when possible associated with stem cell transplantation (SCT). Few studies investigated copy number changes by array-based comparative genomic hybridization (array-CGH) and gene expression profiling, highlighting a peculiar set of chromosomal losses involving regulatory genes.The present study focused on 23 cases of BPDCN, that were extensively investigated cytogenetically (array-CGH and SNP6 array) and immunohistochemically. Clinico-pathologic findings and molecular data were analyzed in order to identify prognostic markers and possible clinical subsets.At onset 12 cases had diffuse, 7 multiple non contiguous and 4 localized skin involvement. Cases 16 and 18 were father and his daughter. Specific therapy was administered in 20 cases: 10 ALL-type CT; 4 NHL-type CT; 2 mono-CT with 1 radiotherapy; 2 radiotherapy alone; 2 SCT. Ten patients relapsed, in a median time of 10 months: 9 cases received further CT and 2 underwent allo-SCT. At last follow-up, 11 patients had died of disease and 2 of therapy-related causes. Median overall survival (OS) was 21 months. At last follow-up 10 patients were alive, 6 disease-free (8 to 28 months) and 4 with disease.Immunophenotype was typical (CD4, CD56, BDCA2, Tcl-1, and CD123) in all but three patients: 1 negative both for CD4 and CD56, 1 for CD56 and 1 for CD4. All cases expressed CD45RA, 19 CD68/KP1, 10 CD2 and/or CD7 and, when tested, for BDCA2/CD303.T-cell and B-cell receptor genes were germline with no evidence of EBV infection.We observed chromosomal imbalances in all patients. Loss of CDKN2A locus (9p21.3) occurred in 15/23 patients, and in 5 cases a biallelic loss was found. FISH confirmed 9p21.3 loss in all 5 cases. Of note, patients with biallelic loss of locus 9p21.3, in respect to those with hemizygous loss, had reduced survival probability (hazard ratio 11.98; confidence interval [CI] 1.21–118.96; log-rank test, p=0.0349). Median OS was 20 and 35 months and median time to relapse 11 and 15 months, respectively. Furthermore, loss of RB1 (13q13.1-q14.3) and CDKN1B (12p13.2-p13.1) locus were observed in 13 cases. An additional, yet unreported, cytogenetic alteration was the loss of 7p12 in 5 of our patients. This is the locus of IKZF1 gene, which encodes the transcription factor Ikaros. Of note, biallelic and monoallelic deletions of IKZF1 has been shown to be associated with chemoresistance and very poor outcome in a subset of BCR-ABL1 positive acute lymphoblastic leukemias. Array-CGH and SNP6 in our series confirmed that BPDCN is characterized by a complex pattern of genomic losses, predominantly affecting chromosomes 9 (71%), 13 (61%), 12 (57%), 5 (19%), 7 (19%), 14 (14%) and 15 (14%).In conclusion, this is the largest reported series of BPDCN patients, confirming high aggressiveness of the disease. We found negative correlation between age and time to relapse (p=0.0053) and a lower rate of bone marrow involvement in the group of patients with localized skin disease (2 of whom in CR after local skin radiotherapy). Furthermore, we observed absence of CD4 and/or CD56 expression in 3/23 cases. We are the first to report reduced OS probability among patients with homozygous loss of locus 9p21.3 in comparison to those with hemizygous loss, and the possible loss of 7p12-IKZF1 gene in BPDCN. Disclosures:No relevant conflicts of interest to declare.

Gene Copy Number Aberrations Are Associated with Survival in Histologic Subgroups of Non-small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) is characterized by a multitude of genetic aberrations with unknown clinical impact. In this study, we aimed to identify gene copy number changes that correlate with clinical outcome in NSCLC. To maximize the chance to identify clinically relevant events, we applied a strategy involving two prognostically extreme patient groups. Short-term (<20 month; n = 53) and long-term survivors (>58 month; n = 47) were selected from a clinically well-characterized NSCLC patient cohort with available fresh frozen tumor specimens. The samples were analyzed using high-resolution single-nucleotide polymorphism array technology to assess gene copy number variations and array-based gene expression profiling. The molecular data were combined with information on clinical parameters. Genetic aberrations were strongly associated with tumor histology. In adenocarcinoma (n = 50), gene copy number gains on chromosome 8q21-q24.3 (177 genes) were more frequent in long-term than in short-term survivors. In squamous cell carcinoma (n = 28), gains on chromosome 14q23.1-24.3 (133 genes) were associated with shorter survival, whereas losses in a neighboring region, 14q31.1-32.33 (110 genes), correlated with favorable outcome. In accordance with copy number gains and losses, messenger RNA expression levels of corresponding genes were increased or decreased, respectively. Comprehensive tumor profiling permits the integration of genomic, histologic, and clinical data. We identified gene copy number gains and losses, with corresponding changes in messenger RNA levels that were associated with prognosis in adenocarcinoma and squamous cell carcinoma of the lung.

Effect of Long-term Storage in TRIzol on Microarray-Based Gene Expression Profiling

Although TRIzol is widely used for preservation and isolation of RNA, there is suspicion that prolonged sample storage in TRIzol may affect array-based gene expression profiling (GEP) through premature termination during reverse transcription. GEP on Illumina arrays compared paired aliquots (cryopreserved or stored in TRIzol) of primary samples of multiple myeloma (MM) and acute myeloid leukemia (AML). Data were analyzed at the "probe level" (a single consensus value) or "bead level" (multiple measurements provided by individual beads). TRIzol storage does not affect standard probe-level comparisons between sample groups: different preservation methods did not generate differentially expressed probes (DEP) within MM or AML sample groups, or substantially affect the many DEPs distinguishing between these groups. Differences were found by gene set enrichment analysis, but these were dismissible because of instability with permutation of sample labels, unbalanced restriction to TRIzol aliquots, inconsistency between MM and AML groups, and lack of biological plausibility. Bead-level comparisons found many DEPs within sample pairs, but most (73%) were <2-fold changed. There was no consistent evidence that TRIzol causes premature reverse transcription termination. Instead, a subset of DEPs were systematically due to increased signals in TRIzol-preserved samples from probes near the 5' end of transcripts, suggesting better mRNA preservation with TRIzol. TRIzol preserves RNA quality well, without a deleterious effect on GEP. Samples stored frozen with and without TRIzol may be compared by GEP with only minor concern for systematic artifacts. The standard practice of prolonged sample storage in TRIzol is suitable for GEP.

Abstract 4822: Analysis of somatic copy number alterations associated with poor prognosis and progression of multiple myeloma in African Americans

Abstract The plasma cell malignancy Multiple Myeloma (MM) is a rare but deadly form of cancer with 5 year survival rates of ∼30%. The disease exacts an especially heavy burden on African American (AA) patients. In fact, African Americans are twice a likely to be diagnosed with and die from MM as compared to Caucasian Americans. Although socio-economic factors likely influence this disparity, the role of biology has not been ruled out. We have profiled ∼250 MM tumors for somatic copy number alterations using array-based Comparative Genomic Hybridization (aCGH) and Gene Expression Profiling as part of the Multiple Myeloma Genomics Initiative. To determine if somatic alterations may play a role in the disparity in incidence and/or severity of MM in AA, we compared the frequency of specific chromosomal aberrations in genes and genomic regions previously found to be involved in progression and poor outcome in MM among a cohort of one hundred ninety-six (196) patients including 16 African Americans and 180 Caucasians. The frequency of occurrence (expressed as a percentage) was determined for each of the following aberrations: 1p deletion, homozygous deletion of p18, chromosome 13 deletion, and chromosome 13 monosomy. No significant difference was detected in the frequency of chromosome 13 deletion and chromosome 13 monosomy between the two groups. However, significant differences were observed for chromosome 1p alterations. Homozygous deletion at the p18 locus at 1p occurred in 12.5% (2/16) of tumors from AA patients and 2.8% (5/180) of CA patient tumors (p = 0.045). We further stratified tumors by hyperdiploid or non-hyperdiploid status using a-CGH profiles to determine ploidy status. Most significantly, p18 homozygous deletion was found in 25% (2/8) of AA patients with non-hyperdiploid tumors and in only 1.2% (1/83) of CA patients with non-hyperdiploid tumors (p=3.2 × 10-4). A nominally significant difference among non-hyperdiploid patients was also present for overall 1p deletion, which was observed in 2/8 (25%) of AA non-hyperdiploid tumors and 33/83 (40%) of CA with non-hyperdiploid tumors (p=0.05). No differences were observed for 1p deletion or p18 homozygous deletion among hyperdiploid tumors between AA and CA patients. These results suggest that deletions at 1p are more common among African American MM patients. Furthermore, homozygous deletion encompassing the p18 locus is more significantly common among AA MM patients, especially AA patients with non-hyperdiploid tumors. As 1p deletion and p18 homozygous deletion have been linked to poor outcome, our results suggest that the increased frequency of these alterations in tumors from AA patients may in part explain the disparity in unfavorable outcomes seen among AA MM patients 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 4822.

Abstract 1175: PCR array-based gene expression profiling identifies CD70 and CD74 as molecular markers and potential therapeutic targets for human melanoma

Abstract Melanoma is one of the most prevalent cancers. While early detection and surgical excision can cure melanoma, patients with advanced disseminated disease have a very poor prognosis due to its extremely aggressive growth characteristics and high resistance to chemotherapy and radiation. Improvement of our understanding of the mechanisms that lead to progression of metastatic melanoma, as well as development of novel therapeutic strategies are urgently required for patients. In our study, we applied PCR arrays and bioinformatics analysis to evaluate gene expression of metastatic melanoma cells and normal skin cells. We used the Human Inflammatory Cytokine and Receptors RT2 Profiler™ PCR Array to analyze the differential expression of genes in metastatic melanoma cell lines (A375) and primary normal epidermal melanocytes (NHEM), which are the precursor cell of melanoma. We found the 43 genes were highly expressed in A375 which were upregulated by more than 20 fold in comparison to NHEM. We next selected the top-ten differentially expressed genes (CD70, IL-1β, MGLL, SERPINA3, MDK, IL13RA2, CD74, VEGFA, CXCL1, and IL8), whose expression levels in A375 cells were &amp;gt; 250-fold higher than in the primary normal NHEM, for further validation. The results from our RT-PCR, immunoblot, immunocytochemistry (ICC) and tissue microarray (TMA) analysis showed that CD70, SERPINA3, IL13RA2 and CD74 genes were highly expressed at mRNA and protein levels in various melanoma cell lines and metastatic melanoma tumor tissues. A much lower level of expression of each gene was detected in normal human skin cell lines and normal skin tissues. These results thus demonstrated that the four genes identified by our PCR array are likely of molecular markers and should be pursued as therapeutic targets for melanoma treatment. To further verify their potentials as potential melanoma therapeutic targets, we also evaluated the effects of siRNAs targeting CD70 and CD74 genes and the monoclonal antibodies against CD70 and CD74 proteins for regulation of melanoma growth. Treatment with CD70 or CD74-specific siRNAs and also monoclonal antibodies dramatically inhibited cell growth in various melanoma cell lines, indicating that CD70 and CD74 are also attractive molecular targets in the treatment of human metastatic melanoma. Our findings have important implications in understanding the metastatic process of melanoma and discovering novel therapeutic strategies for melanoma treatment. The molecular markers and therapeutic targets developed in this study will have a direct and immediate translational application. If successful, our study will represent a significant advance in the diagnosis and treatment of melanoma. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the Melanoma SPORE grant from the NCI, P50, CA093459). 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 1175.

Correlation of hypoxic signalling to histological grade and outcome in cartilage tumours

The molecular mechanisms underlying the progression of central chondrosarcoma are so far poorly understood. The aim of this study was to identify genes involved in the progression of these tumours by comparison of gene expression and correlation of expression profiles to histological grade and clinical outcome. Array-based gene expression profiling of 19 chondrosarcoma samples was performed. Beside differences in the expression of cartilage matrix molecules, high-grade chondrosarcoma showed enhanced expression of the matrix metalloproteinase MMP-2 and of the hypoxia-inducible molecule galectin 1. Immunohistochemical analysis of galectin 1 and of further hypoxia-associated proteins was performed on 68 central and peripheral tumour samples. Hypoxia-inducible factor 1alpha (HIF-1alpha) activation was significantly elevated in high-grade central chondrosarcoma. A negative correlation of carbonic anhydrase IX expression to metastasis-free survival was independent of histological grade. The expression patterns identified in this study point towards a substantial role for angiogenic and hypoxic signalling in chondrosarcoma progression. The constitutive activation of the transcription factor HIF-1alpha in high-grade chondrosarcoma could play a central role in the regulation of cell metabolism and vascularization in these tumours and may, for this reason, represent a potential target for chondrosarcoma therapy.

Homozygous deletion of CDKN2A/2B is a hallmark of iron-induced high-grade rat mesothelioma

In humans, mesothelioma has been linked to asbestos exposure, especially crocidolite and amosite asbestos, which contain high amounts of iron. Previously, we established a rat model of iron-induced peritoneal mesothelioma with repeated intraperitoneal injections of iron saccharate and an iron chelator, nitrilotriacetate. Here, we analyze these mesotheliomas using array-based comparative genomic hybridization (aCGH) and gene expression profiling by microarray. Mesotheliomas were classified into two distinct types after pathologic evaluation by immunohistochemistry. The major type, epithelioid mesothelioma (EM), originated in the vicinity of tunica vaginalis testis, expanded into the upper peritoneal cavity and exhibited papillary growth and intense podoplanin immunopositivity. The minor type, sarcomatoid mesothelioma (SM), originated from intraperitoneal organs and exhibited prominent invasiveness and lethality. Both mesothelioma types showed male preponderance. SMs revealed massive genomic alterations after aCGH analysis, including homozygous deletion of CDKN2A/2B and amplification of ERBB2 containing region, whereas EMs showed less genomic alterations. Uromodulin was highly expressed in most of the cases. After 4-week treatment, iron deposition in the mesothelia was observed with 8-hydroxy-2′-deoxyguanosine formation. These results not only show two distinct molecular pathways for iron-induced peritoneal mesothelioma, but also support the hypothesis that oxidative stress by iron overload is a major cause of CDKN2A/2B homozygous deletion.

The Role of microRNA Genes in Breast Cancer Progression and Metastasis.

Abstract MicroRNAs influence tumor progression and metastasis via action as metastasis promoting genes (for example mir-373 and mir-520) or metastasis suppressor genes (for example mir-335). To identify additional microRNAs capable of driving breast cancer metastasis in vivo, we employed a functional genomics screen using the '4T1' syngeneic (Balb/c) mouse model of breast cancer progression. Approximately 400 microRNA sequences were subcloned into the pMSCV_puro retroviral expression vector and the resultant microRNA expression library transduced into fluorescent non-metastatic 67NR-pMSCV_mCherry and mildy-metastatic 66cl4-pMSCV_mCherry cells and selected with puromycin. The transduced cell lines were then implanted into the mammary fat pads of 20 syngeneic Balb/c mice per cell line. Following establishment of primary tumors, the primary tumors and secondary organs (lung, liver, spine, femurs), including any overt metastatic deposits, were dissected under a fluorescent stereomicroscope and the microRNA inserts amplified from the integrated retroviral genome by PCR.In an alternative approach, we have studied two populations of MDA-MB-231 human breast cancer cells that exhibit different behavior in vitro and in vivo to elucidate processes involved in cellular invasion and metastasis. A late-passage MDA-MB-231 (231variant) population formed invasive clusters when embedded in Matrigel, whereas early-passage (P3) MDA-MB-231 (231) cells did not. Moreover, 231v cells were more motile than 231 cells and showed enhanced tumorigenicity when grown as subcutaneous xenografts in nude mice, whereas proliferation and apoptotic response were indistinguishable between the two lines in vitro. Molecular characterization of the two cell populations cultured in 3D by array-based gene expression profiling revealed elevated expression of NF kappa B target genes in 231v cells (IL1A, IL1B, IL6, PTX3, NR4A2, HMOX1, FLIP). MicroRNA array analysis of 3D cultures also revealed marked induction (approximately 8-fold) of the related NF kappa B target microRNAs, mir-146a and mir-146b (Figure 1). Further evaluation of a panel of human breast cancer cell lines revealed that mir-146a and mir-146b were dramatically elevated in metastatic human breast cancer cell lines that display both epithelial-to-mesenchymal transition and high NF kappa B activity (MDA-MB-231, Hs578T, MDA-MB-436), compared to non-metastatic lines. Experiments to elucidate the function of mir-146a and mir-146b during breast cancer progression are underway. Figure 1: TaqMan qPCR analysis of mir-146a and mir-146b expression in 231 and 231v cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6142.