Statistical Analysis Of Microarrays Research Articles

Nuclear factor IB is downregulated in vulvar squamous cell carcinoma (VSCC): Unravelling differentially expressed genes in VSCC through gene expression dataset analysis.

Vulvar squamous cell carcinoma (VSCC) comprises two distinct etiopathological subtypes: i) Human papilloma virus (HPV)-related VSCC, which arises via the precursor high grade squamous intraepithelial lesion (HSIL); and ii) HPV-independent VSCC, which arises via precursor, differentiated vulvar intraepithelial neoplasia (dVIN), driven by TP53 mutations. However, the mechanism of carcinogenesis of VSCC is poorly understood. The current study aimed to gain insight into VSCC carcinogenesis by identifying differentially expressed genes (DEGs) for each VSCC subtype. The expression of certain DEGs was then further assessed by performing immunohistochemistry (IHC) on whole tissue sections of VSCC and its precursors. Statistical analysis of microarrays was performed on two independent gene expression datasets (GSE38228 and a study from Erasmus MC) on VSCC and normal vulva. DEGs were identified that were similarly (up/down) regulated with statistical significance in both datasets. For HPV-related VSCCs, this constituted 88 DEGs, and for HPV-independent VSCCs, this comprised 46 DEGs. IHC was performed on VSCC (n=11), dVIN (n=6), HSIL (n=6) and normal vulvar tissue (n=7) with i) signal transducer and activator of transcription 1 (STAT1; an upregulated DEGs); ii) nuclear factor IB (NFIB; a downregulated DEG); iii) p16 (to determine the HPV status of tissues); and iv) p53 (to confirm the histological diagnoses). Strong and diffuse NFIB expression was observed in the basal and para-basal layers of normal vulvar tissue, whereas NFIB expression was minimal or completely negative in dVIN and in both subtypes of VSCC. In contrast, no discernable difference was observed in STAT1 expression among normal vulvar tissue, dVIN, HSIL or VSCC. By leveraging bioinformatics, the current study identified DEGs that can facilitate research into VSCC carcinogenesis. The results suggested that NFIB is downregulated in VSCC and its relevance as a diagnostic/prognostic biomarker deserves further exploration.

Differential and longitudinal immune gene patterns associated with reprogrammed microenvironment and viral mimicry in response to neoadjuvant radiotherapy in rectal cancer

BackgroundRectal cancers show a highly varied response to neoadjuvant radiotherapy/chemoradiation (RT/CRT) and the impact of the tumor immune microenvironment on this response is poorly understood. Current clinical tumor regression grading...

PF661 MOLECULAR PROFILING REVEALS HIGHLY DEREGULATED INSULIN GROWTH FACTOR BINDING PROTEINS IN PATIENTS WITH MYELOFIBROSIS AND RELATED NEOPLASMS

Background:The Insulin Growth Factor (IGF) system has been extensively studied in polycythemia vera (PV) showing erythroid progenitors to be hypersensitive to IGF1, levels of the IGF1‐binding protein – IGFBP‐1 to be fourfold increased but normal levels of IGF1 and IGFBP3. Furthermore, the stimulatory effect of IGFBP1 on erythroid progenitors in vitro provided a connection to overproduction of red blood cells in PV through IGF1. Most lately, increased IGF‐1R expression of mononuclear cells was found in patients with Philadelphia‐negative myeloproliferative neoplasms (MPN). In addition, the JAK2V617F mutation has been shown to result in IGF1 hypersensitivity in Ba/F3 cells providing a molecular basis for significance of IGF1 signaling in MPNs.Dysregulation of the IGF system is important in cancer development. We herein for the first time demonstrate several genes of IGF system to be markedly deregulated in MPNsAims:To study whole blood gene expression profiling in patients with MPNs.Methods:Gene expression microarrays were applied to whole blood from control subjects (n = 21) and patients with ET (n = 19), PV (n = 41), and PMF (n = 9). Affymetrix HG‐U133 2.0 Plus microarrays were used to generate gene expression profiles of 54.675 probe sets (38.500 genes). Total RNA was purified from whole‐blood, amplified to biotin‐labeled aRNA and hybridized to microarray chips. The statistical software R was applied to perform initial data processing and statistical analysis of microarray data.Results:20,439, 25,307, and 17,417 probe sets were significantly differentially expressed between control subjects and patients with ET, PV and PMF, respectively (FDR<0.05). Several of the insulin‐like growth factor (IGF) and insulin‐like growth factor binding protein (IGFBP) genes were significantly deregulated (Table 1). IGF1, IGF2, IGFALS, IGFBP2, and IGF2BP1 were significantly upregulated in all three disease entities of which IGFBP2 was progressively and significantly upregulated from ET over PV to PMF (FDR<0.05).Summary/Conclusion:In the present study we found several IGF‐related genes to be significantly deregulated in MPNs. Of note, IGF‐1 was significantly upregulated in all three subgroups of patients (ET, PV and PMF) and IGFBP1 was significantly downregulated in ET and PV patients. These observations are not in line with previous findings of normal IGF‐1 and highly elevated IGFBP1 levels in patients with PV. IGF‐1 has been shown to stimulate cell growth and up‐and down‐regulate the expression of anti‐ and pro‐apoptotic molecules, respectively, thereby protecting cancer cells from apoptosis. Enhanced IGF‐I gene expression might contribute to the chronic inflammatory state in MPN through enhancement of IL‐6 activity. It is intriguing to consider if IGF‐1 might also promote angiogenesis in MPN by stimulating VEGF secretion by e.g. megakaryocytes and monocytes similar to stimulation of VEGF‐secretion by e.g. myeloma cells. In conclusion, we have shown a number of genes in the IGF‐1 system to be deregulated in MPN, which altogether might contribute in driving clonal myeloproliferation, decreased apoptosis, bone marrow angiogenesis, immune evasion and inflammation as well. Future studies on the interplay between the transcriptome, IGF‐related proteins and IGF‐R expression levels might deliver new insight into the role of IGF‐system in the pathogenesis of MPNs and ultimately give the impetus to studies on the efficacy of inhibitors of IGF1 or IGF1R, which prohibit development of MPN‐phenotype in JAK2V617F mice.image

Extracellular Matrix-Related Genes Are Deregulated in Peripheral Blood from Patients with Myelofibrosis and Related Neoplasms

Introduction: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) which include essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) are characterized by varying degrees of bone marrow fibrosis and endothelial proliferation. We and others have previously reported that these stromal alterations are reflected by increased serum levels of matrix derived metabolites, striated collagens type I/III and basement membrane components. The existence of a prefibrotic seromarker profile in MPNs is further evidenced by reports on increased serum levels of matrix metalloproteinase-3 (MMP-3) and decreased tissue inhibitor of metalloproteinase- I (TIMP-I). Using whole blood gene expression profiling, we aimed to provide a comprehensive gene signature of extracellular matrix-related proteins in MPNs with particular focus on genes associated with the regulation of major stromal proteins and MMPs.Methods: Gene expression profiling was performed on whole blood from 21 control subjects, 19 patients with ET, 41 patients with PV, and 9 patients with PMF. RNA was converted to biotin labeled amplified RNA (aRNA) using the MessageAmpTM III RNA amplification kit, and fragmented aRNA was hybridized to Affymetrix HG-U133 Plus 2.0 microarray chips recognizing 54,675 probe sets (38,500 genes). The R statistical software was applied to perform data preprocessing and statistical analysis of microarray data.Results: We identified 20,439, 25,307, and 17,417 probe sets that were differentially expressed between controls and patients with ET, PV, and PMF, respectively (FDR£0.05). These genes included 116 genes encoding extracellular matrix and adhesion molecules (ECM) important for cell-cell and cell-matrix interactions. These genes are represented on the Qiagen Human ECM panel, and in addition, all remaining collagen genes have been included. In patients with ET, COL1A1, COL1A2, COL3A1, COL4A2, COL4A5, LAMA2, LAMB1, MMP1, MMP7, MMP11, MMP12, MMP14, AND TIMP3 were among the 42 upregulated ECM genes (FDR<0.05). In patients with PV, 53 ECM genes were upregulated including COL1A1, COL1A2, COL3A1, COL4A2, LAMA2, LAMB1, MMP1, MMP7, MMP8, MMP9, MMP11, MMP12, MMP14, and TIMP3 (FDR<0.05). In PMF, COL1A2, COL3A1, COL4A2, COL4A5, LAMA2, MMP1, MMP8, MMP9, MMP14, and TIMP3 were among the 26 upregulated genes (FDR<0.05) (Table 1). 17, 14, and 13 ECM genes were significantly downregulated in ET, PV, and PMF, respectively (FDR<0.05) (data not shown). ITGA7, ITGB3, and MMP1 were significantly upregulated from ET over PV to PMF, whereas ITGAL, SPG7, and TGFBI were significantly downregulated from ET over PV to PMF. ADAMTS8, ADAMTS13, COL10A1, COL14A1, COL1A2, COL29A1, COL3A1, COL4A2, COL6A1, ITGA7, ITGB3, ITGB5, LAMA2, MMP1, MMP14, NCAM1, THBS2, and TIMP3 were significantly upregulated in both ET, PV, and PMF (FDR<0.05). COL4A3BP, COL6A2, ITGA4, ITGA5, ITGAL, ITGB1, PECAM1, SPG7, and TGFBI were significantly downregulated in both ET, PV, and PMF (FDR<0.05). In table 2a-b are shown the 10 most significantly up- and downregulated genes.Discussion and conclusions: Bone marrow fibrosis and endothelial proliferation in MPNs are elicited due to the release of fibrogenic and angiogenic growth factors primarily from hyperproliferating megakaryocytes. The connective tissue components of the bone marrow in MPNs include type III collagen, which is deposited in the early disease stages (ET/PV) as “reticulin fibrosis” being accompanied and substituted by mature Van Giesson positive collagen (type I collagen) in the advanced myelofibrosis stage. Increased endothelial cell proliferation is followed by the development of continuous sheets of basement membrane material beneath endothelial cells as assessed by increased deposition of type IV collagen and laminin. Using whole blood gene expression profiling, we provide evidence that abnormal extracellular matrix metabolism is reflected in the gene signature of peripheral blood cells from patients with MPNs. Further studies are needed to determine whether these changes represent local bone marrow fibrogenesis and/or systemic disease manifestations. DisclosuresHasselbalch:Novartis: Research Funding.

Whole Blood Transcriptional Profiling Reveals Highly Deregulated Atherosclerosis Genes in Myeloproliferative Cancer

Introduction: The Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are associated with a huge comorbidity burden, including an increased risk of cardiovascular diseases. Recently, chronic inflammation has been suggested to be the driving force for clonal evolution and disease progression in MPNs but also potentially having an impact upon the development of accelerated (premature) atherosclerosis, which is recorded in several other inflammatory diseases. Using whole blood gene expression profiling we have previously reported massive deregulation of inflammation genes and genes involved in oxidative stress. Herein, we extend our studies to explore the landscape of atherosclerosis genes, which have not been investigated previously in MPNs but may add novel important information in regard to deregulation of these genes of potential importance for the development of premature atherosclerosis and accordingly the heavy cardiovascular disease burden in MPNs.Methods:Global gene expression profiling was performed on 21 control subjects, 19 patients with ET, 41 patients with PV, and 9 patients with PMF. Gene expression profiles were generated using Affymetrix HG-U133 2.0 Plus microarrays recognizing 54.675 probe sets (38.500 genes). Total RNA was purified from whole blood, amplified to biotin-labeled aRNA, and hybridized to microarray chips. The R statistical software was applied to perform data preprocessing and statistical analysis of microarray data.Results:We identified 20,439, 25,307, and 17,417 probe sets that were differentially expressed between controls and patients with ET, PV, and PMF, respectively (FDR£0.05). These genes included 84 genes represented on the Qiagen Human Atherosclerosis gene panel. In patients with ET, FABP3, FN1, LIF, LPL, PDGFB, PDGFRB, PPARD, SERPINE1, and TGFB2 were among the 28 upregulated genes, and APOE, BCL2, ITGA5, KLF2, NFKB1, and SELPLG were among the 17 downregulated genes. In patients with PV, 39 atherosclerosis genes were upregulated including VCAM1, FN1, PDGFRB, LPL, TGFB2, SERPINE1, PDGFB, and LIF, and 17 genes were downregulated including APOE, ITGA5, KLF2, NFKB1, SELPLG, and BCL2. In patients with PMF, PDGFB, HBEGF, LIF, PDGFRB, SERPINE1, TGFB2, and VWF were among the 24 upregulated genes, and KLF2, BCL2, IL1R2, NFKB1, ITGB2, ITGA5, IFNAR2, and SELPLG were among the 22 downregulated genes. BCL2L1, MMP1, PDGFA, PTGS1, and THBS4 were progressively significantly upregulated, whereas BCL2 were progressively significantly downregulated from ET over PV to PMF (all FDR<0.05). The top 15 significantly up- and downregulated genes are shown in Table 1 and 2, respectively.Discussion and conclusions: Atherosclerosis is a chronic inflammatory disease, which involves several circulating blood cells, including both leukocytes and platelets. Taking into account that chronic inflammation today is considered of utmost importance in MPN-pathogenesis and MPNs carry an increased risk of chronic inflammatory diseases, such as inflammatory bowel diseases, age related macular degeneration and chronic kidney diseases, which all are associated with early development of atherosclerosis, we speculated if whole blood gene expression profiling of all the inflammatory cells (granulocytes, monocytes, platelets etc) being involved in the atherosclerotic process might unravel deregulation of atherosclerosis genes and perhaps depict a particular MPN-atherosclerosis gene signature. Indeed, in the present study we have found that several genes of importance for the development of atherosclerosis are highly deregulated and a subset of 6 genes (BCL2L1, MMP1, PDGFA, PTGS1, and THBS4) were progressively significantly upregulated, in the biological “ inflammatory ” continuum from the early cancer stages (ET/PV) to the advanced myelofibrosis stage, whereas BCL2 was progressively and significantly downregulated. In conclusion, we have for the first time shown massive deregulation of atherosclerosis genes in MPNs, likely reflecting the inflammatory state in MPNs in association with in vivo activation of the cell types (leukocytes, platelets, endothelial cells) being deeply involved in the atherosclerotic process. The significance of our observations should be further explored and confirmed in other MPN-cohorts and the impact of cytoreductive therapy (e.g interferon-alpha2) and statins upon deregulated genes as well. [Display omitted] DisclosuresHasselbalch:Novartis: Research Funding.

Interferon-alfa2 Treatment of Patients with Polycythemia Vera and Related Neoplasms Impacts Deregulation of Oxidative Stress Genes and Antioxidative Defence Mechanisms. Potential Implications of IFN-Alfa Induced Changes in TP53, NRF2 and CXCR4 for Genomic Instability and CD34+ Mobilisation

Introduction: The Philadelphia-negative myeloproliferative neoplasms are associated with chronic inflammation and accumulation of reactive oxygen species (ROS). Whole blood transcriptional profiling studies have most recently identified deregulation of several oxidative and anti-oxidative stress genes. Amongst the genes significantly downregulated is TP53 and the NFE2L2 or Nrf2 gene, the latter having a key role in the regulation of the oxidative stress response and in modulating both migration and retention of hematopoietic stem cells (HSCs). During MPN-disease progression, the HSC pool is steadily expanding with the egress of CD34+cell from stem cell niches into the circulation. In addition to Nrf2 several other genes are involved in this process, including CXCR4, which is also significantly downregulated in MPNs. Interferon-alpha2 (IFN) is recognized as a highly efficacious and promising agent in the treatment of MPNs. Taken into account that chronic inflammation with ROS accumulation is likely involved in the pathogenesis of MPNs, ultimately implying the induction of an altered redox balance of pivotal significance for stem cell mobilization in MPNs, we herein report the first study on the impact of IFN upon oxidative stress and anti-oxidative defence genes.Methods: The HG-U133 Plus 2.0 microarray was used to profile expression of 38.500 genes in whole blood from ET (n=19), PV (n=41), PMF (n=9) and controls (n=21) (data set 1) and in ET (n=8), PV (n=21), and PMF (n=4) before and during treatment with IFN (data set 2). All patients received treatment with IFN, in the large majority in a dosage ranging from 45-90 ug x 1 sc/week. Total RNA was purified from whole blood amplified to biotin-labeled RNA, and hybridized to microarrays. The R statistical software was applied to perform data preprocessing and statistical analysis of microarray data.Results: Single gene analysis of 154 genes found to be included in previous studies focusing on deregulation of oxidative stress genes in various diseases were chosen for further analysis. In response to treatment with IFN, 19 genes were upregulated in ET including CXCR4 and TP53, and 11 genes were downregulated including FOXO3, PRDX2, and PRDX6. In patients with PV, ATOX1, CXCR4, SEPP1, and TP53 were among the 29 upregulated genes, and FOXO3 and PRDX2 were among the 14 downregulated genes. In response to treatment with IFN, 2 genes were upregulated in PMF which were CYBB and MSRA, and 9 genes were downregulated including PRDX2, and FOXO3 (all P<0.05). In a recent microarray study (data set 1), we have shown significant downregulation of genes associated with oxidative stress including ATM, CYBA, NRF2, PTGS1, SIRT2, TTN, and significant upregulation of AKR1B1, CCND1, DEFB122, GPX8 in ET, PV, and PMF vs controls. These genes were not significantly deregulated during IFN-treatment in any of the disease entities. Similarly, the significant downregulation of the NFR2 and the CXCR4 genes before IFN-treatment was no longer present after IFN treatment.Discussion and Conclusions: The present study has shown IFN to have a major impact upon deregulated oxidative stress genes and antioxidative defence genes, implying a gene signature that mostly corresponds to decreased oxidative stress and enhancement of antioxidative defence genes. In regard to TP53 - being significantly downregulated before IFN treatment - this gene was significantly upregulated in ET and PV and no longer deregulated in PMF during IFN treatment. Downregulation of TP53 implies genomic instability due to an increased burden of oxidative stress upon the genome, which accordingly is reduced by IFN treatment. In addition to being the master regulator of the antioxidant response, Nrf2 also has a major role for normal stem cell function. Thus, our findings of a change in the Nrf2 gene expression from being significantly downregulated before IFN exposure to not being deregulated during treatment with IFN may imply an improvement in the antioxidant defence mechanisms against increased oxidative stress and accumulation of ROS. In conclusion, our findings of a major impact of IFN upon several oxidative stress genes and antioxidative defence genes may imply an IFN mediated dampening of genotoxic damage to hematopoietic cells and stromal cells as well, thereby ultimately diminishing the risk of additional mutations that might drive the malignant clone towards myelofibrotic and leukemic transformation. [Display omitted] DisclosuresHasselbalch:Novartis: Research Funding.

Integrative Bioinformatic Analysis of Transcriptomic Data Identifies Conserved Molecular Pathways Underlying Ionizing Radiation-Induced Bystander Effects (RIBE)

Ionizing radiation-induced bystander effects (RIBE) encompass a number of effects with potential for a plethora of damages in adjacent non-irradiated tissue. The cascade of molecular events is initiated in response to the exposure to ionizing radiation (IR), something that may occur during diagnostic or therapeutic medical applications. In order to better investigate these complex response mechanisms, we employed a unified framework integrating statistical microarray analysis, signal normalization, and translational bioinformatics functional analysis techniques. This approach was applied to several microarray datasets from Gene Expression Omnibus (GEO) related to RIBE. The analysis produced lists of differentially expressed genes, contrasting bystander and irradiated samples versus sham-irradiated controls. Furthermore, comparative molecular analysis through BioInfoMiner, which integrates advanced statistical enrichment and prioritization methodologies, revealed discrete biological processes, at the cellular level. For example, the negative regulation of growth, cellular response to Zn2+-Cd2+, and Wnt and NIK/NF-kappaB signaling, thus refining the description of the phenotypic landscape of RIBE. Our results provide a more solid understanding of RIBE cell-specific response patterns, especially in the case of high-LET radiations, like α-particles and carbon-ions.

Evaluation of Methods for Gene Selection in Melanoma Cell Lines

A major objective in microarray experiments is to identify a panel of genes that are associated with a disease outcome or trait. Many statistical methods have been proposed for gene selection within the last fifteen years. While the comparison of some of these methods has been done, most of them concentrated on finding gene signatures based on two groups. This study evaluates four gene selection methods when the outcome of interested is continuous in nature. We provide a comparative review of four methods: the Statistical Analysis of Microarrays (SAM), the Linear Models for Microarray Analysis (LIMMA), the Lassoed Principal Components (LPC), and the Quantitative Trait Analysis (QTA). Comparison is based on the power to identify differentially expressed genes, the predictive ability of the genelists for a continuous outcome (G2 checkpoint function), and the prognostic properties of the genelists for distant metastasis-free survival. A simulated dataset and a publicly available melanoma cell lines dataset are used for simulations and validation, respectively. A primary melanoma dataset is used for assessment of prognosis. No common genes were found among the genelists from the four methods. While the SAM was generally the best in terms of power, the QTA genelist performed the best in the prediction of the G2 checkpoint function. Identification of genelists depends on the choice of the gene selection method. The QTA method would be preferred over the other approaches in predicting a quantitative outcome in melanoma research. We recommend the development of more robust statistical methods for differential gene expression analysis.

IDENTIFICATION OF BIOMARKERS FOR TUBERCULOSIS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

IDENTIFICATION OF BIOMARKERS FOR TUBERCULOSIS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

MGMT-STP27 Methylation Status as Predictive Marker for Response to PCV in Anaplastic Oligodendrogliomas and Oligoastrocytomas. A Report from EORTC Study 26951

The long-term follow-up results from the EORTC-26951 trial showed that the addition of procarbazine, CCNU, and vincristine (PCV) after radiotherapy increases survival in anaplastic oligodendrogliomas/oligoastrocytomas (AOD/AOA). However, some patients appeared to benefit more from PCV treatment than others. We conducted genome-wide methylation profiling of 115 samples included in the EORTC-26951 trial and extracted the CpG island hypermethylated phenotype (CIMP) and MGMT promoter methylation (MGMT-STP27) status. We first show that methylation profiling can be conducted on archival tissues with a performance that is similar to snap-frozen tissue samples. We then conducted methylation profiling on EORTC-26951 clinical trial samples. Univariate analysis indicated that CIMP+ or MGMT-STP27 methylated tumors had an improved survival compared with CIMP- and/or MGMT-STP27 unmethylated tumors [median overall survival (OS), 1.05 vs. 6.46 years and 1.06 vs. 3.8 years, both P < 0.0001 for CIMP and MGMT-STP27 status, respectively]. Multivariable analysis indicates that CIMP and MGMT-STP27 are significant prognostic factors for survival in presence of age, sex, performance score, and review diagnosis in the model. CIMP+ and MGMT-STP27 methylated tumors showed a clear benefit from adjuvant PCV chemotherapy: the median OS of CIMP+ samples in the RT and RT-PCV arms was 3.27 and 9.51 years, respectively (P = 0.0033); for MGMT-STP27 methylated samples, it was 1.98 and 8.65 years. There was no such benefit for CIMP- or for MGMT-STP27 unmethylated tumors. MGMT-STP27 status remained significant in an interaction test (P = 0.003). Statistical analysis of microarray (SAM) identified 259 novel CpGs associated with treatment response. MGMT-STP27 may be used to guide treatment decisions in this tumor type.

Identification of polo-like kinase 1 (PLK1) in aggressive prostate cancer by paradigm analysis.

5006 Background: Integrative analysis that combines expression data, copy number variation, sequence, and epigenetic data with thousands of known biological interactions can help identify strong biological associations and novel “genotype to phenotype” associations. Methods: We performed multi-dataset differential expression analysis with Statistical Analysis of Microarrays (SAM) and gene set enrichment analysis (GSEA) across 10 publicly-available PCa microarray datasets to identify genes and pathways differentially expressed between local (n=471) and metastatic (n=220) PCa. We used PARADIGM, an integrated pathway analysis method, on PCa tumor samples with mRNA expression, copy number variation, and TMPRSS2:ERG fusion status data to infer differential fusion gene-related “activities” for pathway features (genes, protein complexes, etc) within a “Superimposed Pathway” representing a comprehensive collection of genetic interactions currently containing 20,314 known interactions among 16,362 concepts representing 6916 proteins, 7345 complexes, 1449 families, 55 RNAs, 15 miRNAs and 582 processes. Results: Out of the 7571 genes tested (those genes having data in two or more studies), the meta-analysis on gene expression revealed 210 (1.8% FDR) positively associated with PCa metastasis and 403 (0.94% FDR) negatively associated genes (threshold was p&lt;0.001, 2-tailed Student’s t-test). GSEA highlighted cell proliferation, cell cycle control, and DNA damage repair pathways with metastatic tumors. The PARADIGM analysis identified a network containing 914 features connected by 1137 edges. PLK1 was identified as both highly expressed in metastatic PCa and as one of the fourteen hubs in the largest (596-feature) sub-network identified by PARADIGM. PLK1 was also associated with high Gleason Sum and recurrent disease in independent local PCa datasets. Conclusions: Using an approach pioneered by members of our SU2C/PCF supported PCa Dream Team, integrated analysis across multiple PCa datasets associates PLK1 activity with aggressive PCa and suggests it may provide a novel treatment target for at least a genetic sub-set of advanced PCa.

Multiwalled Carbon Nanotube-Induced Gene Signatures in the Mouse Lung: Potential Predictive Value for Human Lung Cancer Risk and Prognosis

Concerns over the potential for multiwalled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n = 160) exposed to 0, 10, 20, 40, or 80 μg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 d postexposure. By using pairwise statistical analysis of microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5-fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at d 56 postexposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at d 56 postexposure to 10 μg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n = 256) and test set (n = 186). Furthermore, both gene signatures were associated with human lung cancer risk (n = 164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace.

High-throughput arrays identify distinct genetic profiles associated with lymph node involvement in rectal cancer.

Preoperative clinical diagnosis of lymph node involvement guides treatment decisions for rectal cancer. Unfortunately, clinical staging still suffers from a lack of accuracy. The aim of this study was to evaluate objective genetic differences in primary rectal cancers with and without associated lymph node metastasis. cDNA microarrays were generated from fresh-frozen tumors. Normalized data underwent global unsupervised hierarchical clustering analysis, and discriminating genes were mapped. Top discriminating genes were compared between stage II and III rectal cancers by use of an empirical Bayes 2 group t test with the Statistical Analysis of Microarrays and the Reproducibility-Optimized Test Statistic software separately to guide data reduction and deal with the difficulties of simultaneous statistical inference. Ingenuity Pathways Analysis software was used to analyze discriminating genes in terms of function and biological processes. Fifty-five patients with stage II and 22 patients with stage III rectal adenocarcinomas not treated with chemoradiation were included. Two major unsupervised clusters emerged representing stage II and III cancers. In 1 cluster, 11 of 12 patients (92%) had stage III cancer; in the other cluster, 54 of 65 patients (83%) had stage II (p < 0.001). Five significantly differentially expressed genes characterized the stage III cluster: interleukin-8, 3-hydroxy-3-methylglutaryl coenzyme A synthase, carbonic anhydrase, ubiquitin, and cystatin (all p < 0.05). Of the 12 patients with differential expression of the 5 marker genes, only one had stage II cancer. Fifty-four of 55 stage II patients clustered with alternative expression patterns of the predictor genes. Differentially expressed genes are related to cancer-associated processes, pathways, and networks. The identified gene signatures have not yet been validated in independent patient populations. Distinct gene expression signatures from primary rectal adenocarcinomas can help differentiate the presence or absence of lymph node metastases. These data are informative, and validation of this gene signature may provide a novel approach for more appropriate individualized treatment selection.

Abstract 4527: The HER3-EREG axis and its role in colorectal cancer aggression

Abstract Yearly, 50,000 Americans die of colorectal cancer metastases to liver and lung. EGFR-targeted agents improve survival in a subset of metastatic colorectal cancer (mCRC) cases, and overexpression of the HER-family ligands amphiregulin (AREG) and epiregulin (EREG) correlates with EGFR-targeted agent susceptibility. Here we present evidence of association between EREG overexpression and disease aggression by showing that knockdown of EREG or HER3 impede anchorage independent growth in the HCT 116 CRC cell line. We also find that a HER3-EREG axis gene signature groups mCRC tumors into prognostically distinct groups. Snap-frozen CRC hepatic metastases (65) with patient matched normal liver, as well as 71 primary CRC tumors with patient matched normal colonic mucosa were analyzed via qRT-PCR for receptors (HER1-4) and ligands (AREG, and EREG). Among the mCRC cases HER2, HER3, AREG, and EREG were expressed at higher levels than unmatched normal mucosa (p&amp;lt;0.005). HER2, AREG, and EREG were also overexpressed in mCRC compared to unmatched primary CRC tissue (p&amp;lt;0.005). Expression of AREG and EREG were highly correlated (Rs=0.90), and higher EREG mRNA expression was associated with markedly decreased post-hepatectomy overall survival (OS) (p=0.018). In a panel of 9 CRC cell lines HCT 116 had the highest EREG expression. Using the pLKO.1 viral vector, shRNAs targeting EREG, and HER3 were introduced to HCT 116 cells. Empty vector was used as a control. mRNA levels for HER3 and EREG were knocked down by 87% and 73% respectively and protein downregulation was confirmed by western blot. Knockdown and control cells were grown in 0.35% soft agar at a density of 5000 cells per well in 6 well plates. Knockdowns impeded anchorage independent growth with a mean colony count (&amp;gt;50 um diameter) of 170 for HER3, and 285 for EREG as compared to 530 for the empty vector (p&amp;lt;0.005). RNA from 3 distinct sets of HER3, EREG, and empty vector infections was assayed on Agilent Human GE 4x44K v2 microarrays. Unsupervised Principle Component Analysis (PCA) suggested broad concordance in the impact of EREG or HER3 knockdown upon the expression profile. Statistical Analysis of Microarrays (SAM) comparing HER3, and EREG knockdowns to empty vector control revealed 205, and 432 differentially expressed genes with at least a 1.5 fold-change and a false discovery rate of 0%. When HER3 and EREG differential gene lists were compared, 95 overlapping genes with 100% directional agreement were observed. For 51 mCRC cases RNA was available for microarray analysis. A predictor of HER3-EREG activity was constructed from 250 genes that discriminate HER3/EREG knockdown and empty vector control cells and are generally variable in tumors. The predictor was used to assign a HER3-EREG activity score to each mCRC case. This score is significantly associated with OS (p=0.005) with the top 50% of patients by HER3-EREG score demonstrating 5 year OS of 23% as compared to 49% for cases in the lower half of HER3-EREG activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4527. doi:1538-7445.AM2012-4527

Pooled shRNA screen for sensitizers to inhibition of the mitotic regulator polo-like kinase (PLK1)

RNAi screening holds the promise of systemizing the search for combination therapeutic strategies. Here we performed a pooled shRNA library screen to look for promising targets to inhibit in combination with inhibition of the mitotic regulator polo-like kinase (PLK1). The library contained ~4,500 shRNAs targeting various signaling and cancer-related genes and was screened in four lung cancer cell lines using both high (IC80) and low (IC20) amounts of the PLK1 inhibitor GSK461364. The relative abundance of cells containing individual shRNAs following drug treatment was determined by microarray analysis, using the mock treatment replicates as the normalizing reference. Overall, the inferred influences of individual shRNAs in both high and low drug treatment were remarkably similar in all four cell lines and involved a large percentage of the library. To investigate which functional categories of shRNAs were most prominent in influencing drug response, we used statistical analysis of microarrays (SAM) in combination with a filter for genes that had two or more concordant shRNAs. The most significant functional categories that came out of this analysis included receptor tyrosine kinases and nuclear hormone receptors. Through individual validation experiments, we determined that the two shRNAs from the library targeting the nuclear retinoic acid receptor gene RARA did indeed silence RARA expression and as predicted conferred resistance to GSK461364. This led us to test whether activation of RARA receptor with retinoids could sensitize cells to GSK461364. We found that retinoids did increase the drug sensitivity and enhanced the ability of PLK1 inhibition to induce mitotic arrest and apoptosis. These results suggest that retinoids could be used to enhance the effectiveness of GSK461364 and provide further evidence that RNAi screens can be effective tools to identify combination target strategies.

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

Brain region‐specific gene expression changes after chronic intermittent ethanol exposure and early withdrawal in C57BL/6J mice

Neuroadaptations that participate in the ontogeny of alcohol dependence are likely a result of altered gene expression in various brain regions. The present study investigated brain region-specific changes in the pattern and magnitude of gene expression immediately following chronic intermittent ethanol (CIE) exposure and 8 hours following final ethanol exposure [i.e. early withdrawal (EWD)]. High-density oligonucleotide microarrays (Affymetrix 430A 2.0, Affymetrix, Santa Clara, CA, USA) and bioinformatics analysis were used to characterize gene expression and function in the prefrontal cortex (PFC), hippocampus (HPC) and nucleus accumbens (NAc) of C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA). Gene expression levels were determined using gene chip robust multi-array average followed by statistical analysis of microarrays and validated by quantitative real-time reverse transcription polymerase chain reaction and Western blot analysis. Results indicated that immediately following CIE exposure, changes in gene expression were strikingly greater in the PFC (284 genes) compared with the HPC (16 genes) and NAc (32 genes). Bioinformatics analysis revealed that most of the transcriptionally responsive genes in the PFC were involved in Ras/MAPK signaling, notch signaling or ubiquitination. In contrast, during EWD, changes in gene expression were greatest in the HPC (139 genes) compared with the PFC (four genes) and NAc (eight genes). The most transcriptionally responsive genes in the HPC were involved in mRNA processing or actin dynamics. Of the few genes detected in the NAc, the most representatives were involved in circadian rhythms. Overall, these findings indicate that brain region-specific and time-dependent neuroadaptive alterations in gene expression play an integral role in the development of alcohol dependence and withdrawal.

Transcriptional profiling and network analysis of the murine angiotensin II-induced abdominal aortic aneurysm.

We sought to characterize temporal gene expression changes in the murine angiotensin II (ANG II)-ApoE-/- model of abdominal aortic aneurysm (AAA). Aortic ultrasound measurements were obtained over the 28-day time-course. Harvested suprarenal aortic segments were evaluated with whole genome expression profiling at 7, 14, and 28 days using the Agilent Whole Mouse Genome microarray platform and Statistical Analysis of Microarrays at a false discovery rate of <1%. A group of angiotensin-treated mice experienced contained rupture (CR) within 7 days and were analyzed separately. Progressive aortic dilatation occurred throughout the treatment period. However, the numerous early expression differences between ANG II-treated and control were not sustained over time. Ontologic analysis revealed widespread upregulation of inflammatory, immune, and matrix remodeling genes with ANG II treatment, among other pathways such as apoptosis, cell cycling, angiogenesis, and p53 signaling. CR aneurysms displayed significant decreases in TGF-β/BMP-pathway signaling, MAPK signaling, and ErbB signaling genes vs. non-CR/ANG II-treated samples. We also performed literature-based network analysis, extracting numerous highly interconnected genes associated with aneurysm development such as Spp1, Myd88, Adam17 and Lox. 1) ANG II treatment induces extensive early differential expression changes involving abundant signaling pathways in the suprarenal abdominal aorta, particularly wide-ranging increases in inflammatory genes with aneurysm development. 2) These gene expression changes appear to dissipate with time despite continued growth, suggesting that early changes in gene expression influence disease progression in this AAA model, and that the aortic tissue adapts to prolonged ANG II infusion. 3) Network analysis identified nexus genes that may constitute aneurysm biomarkers or therapeutic targets.

Angiotensin Converting Enzyme Exerts System Control Over Fuel Handling In Skeletal Muscle

Using a genetical metabolomics approach we assessed whether altered energy supply in locomoter muscle underlies the elevated aerobic performance of human genotypes containing a silencer region (I‐allele) of the major regulatory enzyme of vasoconstriction, angiotensin converting enzyme (ACE).Extensor muscle, m. vastus lateralis, of ACE‐II/ID genotypes holding the I‐allele demonstrated a trend for elevated capillarity compared to ACE‐DD counterparts lacking the I‐allele (311.0 vs. 279.7 mm‐2, n=20, p=0.10). In untrained subjects, maximal oxygen uptake during bicycle exercise was lower in ACE‐DD genotypes (44.9 vs. 47.8 ml O2/min/kg). Exhaustive aerobic exercise selectively reduced low density lipoproteins in serum of ACE‐II/ID genotypes (−14%) but was spared in ACE‐DD (+6%, p=0.25). By contrast, non‐polar metabolites in exercised muscle, comprising tentatively identified LDL‐derived glycerophosphocholine species, were depleted in ACE‐DD genotypes (q=2.8%; statistical analysis of microarrays). The interaction effect of exercise and genotype on lipidic muscle metabolites was maintained in trained subjects (p=0.03, ANOVA). The observations indicate that elevated import of serum lipids into exercised muscle underlies the enhanced aerobic exercise performance of I‐allelic genotypes for this major checkpoint of vascular perfusion.

System Level Changes in Gene Expression in Maturing Bladder Mucosa

Bladder problems clinically present early in life as birth defects that often lead to kidney failure and late in life as overactive bladder, incontinence and related disorders. We investigated the transcriptome of mouse bladder mucosa at juvenile and adult stages by microarray to identify the pathways associated with normal, healthy growth and maturation. We hypothesized that understanding these pathways could be key to achieving bladder regeneration or reawakening normal function in the elderly population. RNA was isolated from the mucosa at 3, 6, 20 and 30 weeks postnatally. Affymetrix® Mouse 430 v2 arrays were used to profile the expression of approximately 45,000 genes. The software program Statistical Analysis of Microarrays was used to identify genes that significantly changed during the time course. No genes were significantly up-regulated during maturation. However, 66 well annotated genes demonstrated a statistically significant downward trend, of which 10 of 10 were confirmed by quantitative polymerase chain reaction. The main functions affected by age were transcription, regulation of cellular processes, neurogenesis, blood vessel development and cell differentiation. Notable genes included collagens, Mmp2, SPARC and several transcription factors, including Crebbp, Runx1, Klf9, Mef2c, Nrp1, Pex1 and Tcf4. These molecules were indirectly regulated by inferred Tgfb1 and Egf growth factors. Analysis of gene promoter regions for overrepresented upstream transcription factor binding sites identified specificity protein 1 and epidermal growth factor receptor-specific transcription factor as potentially major transcriptional regulators driving maturation related changes. These findings identify a coherent set of genes that appear to be down-regulated during urothelial maturation. These genes may represent an attractive target for bladder regeneration or for treating age related loss of function.