Number Alterations Research Articles

Hippocampal tissue of patients with refractory temporal lobe epilepsy is associated with astrocyte activation, inflammation, and altered expression of channels and receptors

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Previous research has demonstrated several trends in human tissue that, undoubtedly, contribute to the development and progression of TLE. In this study we examined resected human hippocampus tissue for a variety of changes including gliosis that might contribute to the development and presentation of TLE. The study subjects consisted of six TLE patients and three sudden-death controls. Clinicopathological characteristics were evaluated by H&E staining. Immunohistological staining and Western blotting methods were used to analyze the samples. Neuronal hypertrophy was observed in resected epileptic tissue. Immunohistological staining demonstrated that activation of astrocytes was significantly increased in epileptic tissue as compared to corresponding regions of the control group. The Western blot data also showed increased CX43 and AQP4 in the hippocampus and downregulation of Kir4.1, α-syntrophin, and dystrophin, the key constituents of AQP4 multi-molecular complex. These tissues also demonstrated changes in inflammatory factors (COX-2, TGF-β, NF-κB) suggesting that these molecules may play an important role in TLE pathogenesis. In addition we detected increases in metabotropic glutamate receptor (mGluR) 2/3, mGluR5 and kainic acid receptor subunits KA1 (Grik4) and KA2 (Grik5) in patients’ hippocampi. We noted increased expression of the α1c subunit comprising class C L-type Ca2+ channels and calpain expression in these tissues, suggesting that these subunits might have an integral role in TLE pathogenesis. These changes found in the resected tissue suggest that they may contribute to TLE and that the kainic acid receptor (KAR) and deregulation of GluR2 receptor may play an important role in TLE development and disease course. This study identifies alterations in number of commonly studied molecular targets associated with astrogliosis, cellular hypertrophy, water homeostasis, inflammation, and modulation of excitatory neurotransmission in hippocampal tissues from TLE patients.

Dysfunctional Regulatory T Cells in Patients with Autoimmune Myasthenia Gravis: Therapeutic Targets? (PD6.006)

Objective: To investigate circulating regulatory T cell (Treg) numbers and suppressive function in patients with autoimmune myasthenia gravis (MG). Background Published studies examining the role of Tregs in the pathogenesis of MG have reported conflicting results. In these investigations, isolation of Tregs was based on expression of CD25 alone, which is also expressed on a subset of recently activated and memory T cells. More recently, low expression of the IL-7Rα chain (CD127) combined with high expression of CD25 has allowed for isolation of pure Treg populations with high suppressive functionality. Design/Methods: The expression of surface CD4, CD25, and CD127, and intracellular Foxp3 was analyzed by flow cytometry in 16 MG patients vs. 14 normal control subjects. The function of Tregs (CD4+CD25highCD127low/- cells) was examined by co-culture experiments with CD25-negative autologous responder T cells (Tresp), and their suppressive capacity was determined in T cell proliferation assays. Expression of intracellular Foxp3 was assessed by flow cytometry, and cytokine profiles from the T cell culture supernatants were analyzed. GM-CSF was added to T cell co-cultures and the effects on Tregs investigated. Results: No alteration in the relative numbers of Tregs within the peripheral CD4+ T cell pool was seen in MG patients. A reduced expression of Foxp3 within Tregs, and a defect in the ability of these cells to suppress Tresp cells was demonstrated for both polyclonal and AChR-induced T cell proliferation. Cytokine profiles revealed altered levels of IL-6, IL-17, IFN-γ, and IL-10 in MG patients. The demonstrated defects in Treg function/phenotype were partially reversed by in vitro exposure to GM-CSF. Conclusions: The suppressive function of Tregs is impaired in MG patients, likely playing a role in the immunopathogenesis of the disease. Enhancement of Treg function in vitro is feasible, and may represent a novel therapeutic strategy in MG. Supported by: The NIH (National Institute of Neurologic Disorders and Stroke, K08NS058800, and the Muscular Dystrophy Association (MDA) MNM and JRS; and National Institute of Allergy and Infectious Diseases, RO1 AI 058190, BSP. This project was also supported by the University of Illinois at Chicago (UIC) Center for Clinical and Translational Science (CCTS), Award Number UL1RR029879 from the National Center For Research Resources. Disclosure: Dr. Meriggioli has received personal compensation for activities with Athena Diagnostics, Talecris Biotherapeutics, Gliknik, and Celegene Corporation. Dr. Meriggioli receives patent payments for a patent on bispecific antibody coated dendritic cells. Dr. Meriggioli has received research support from the NIH/NINDS and the Muscular Dystrophy Association. Dr. Muthusamy has nothing to disclose. Dr. Rowin has nothing to disclose. Dr. Sheng has nothing to disclose. Dr. Prabhakar has received personal compensation for activities with Gliknik Inc. as a consultant.Dr. Prabhakar has received research support from NIH.

Abstract 5020: Genetic and epigenetic analysis of tumors in a zebrafish model of melanoma

Abstract The activating BRAF kinase mutation V600E occurs in ∼60% of human nevi and melanomas. A transgenic strain of zebrafish expressing BRAF-V600E under the control of the mitfa promoter on a p53 mutant background develops melanomas with 100% penetrance. Previously, we showed that a transposon-based vector, MiniCoopR, allows us to program melanocytes to express a candidate gene in a specialized zebrafish strain Tg(mitfa:BRAFV600E); mitfa-/-; p53-/-. This strain lacks melanocytes, but the lineage can be rescued by MiniCoopR injection at the 1-cell stage. This genetic system allows us to create cohorts of transgenic animals that express candidate genes and assay the effects on tumor development. Previous work identified two histone methyltransferases (HMTs), SETDB1 and SUV39H1, which cooperate with BRAF-V600E to accelerate the onset and severity of melanoma development. A number of HMTs display alterations in expression level or copy number in human cancers compared to normal tissues, and we tested a panel of methyltransferases using the MiniCoopR system. We hypothesize that HMTs promote tumorigenesis by altering the epigenetic program of melanocytes. In order to explore the effects on melanocyte specification and differentiation, we overexpressed and depleted candidate HMTs in zebrafish embryos and performed in situ hybridization for lineage-specific markers, such as crestin, sox10, mitfa, dct, and tyr. HMTs that exert specific developmental phenotypes were further characterized by performing chromatin immunoprecipitation (ChIP). Our goal is to correlate the processes affected in the developing embryo with the epigenetic program to elucidate the mechanism by which HMTs influence tumorigenesis. Additionally, we are developing a strain of zebrafish Tg(mitfa:BRAFV600E); mitfa-/-; alb-/-; p53-/-; Tg(mitfa:GFP) in which melanocytes rescued with MiniCoopR express GFP. These rescued melanocytes can be programmed to express candidate HMTs and allow us to visualize melanocyte and melanoma development in vivo. The work presented here will help correlate cancer phenotypes with specific chromatin-modifying enzymes that may be the basis for new drug development and biomarker discovery. 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 5020. doi:1538-7445.AM2012-5020

Abstract 4461: A prospective study of blood mitochondrial DNA copy number and risk of renal cell carcinoma

Abstract Background: Mitochondrial DNA (mtDNA) is vulnerable to mutations, and the number of copies of mtDNA per cell may increase to compensate for DNA damage. Recent studies have evaluated blood leukocyte mtDNA copy number and risk of various types of cancer, including renal cell carcinoma (RCC). Although alterations in mtDNA copy number have been associated with cancer risk, the direction of this association has been inconsistent between studies with prospective and retrospective blood collection. One case-control study found that low mtDNA copy number was associated with an increased risk of RCC; however, this association has not been investigated prospectively. Methods: We conducted a nested case-control study of RCC risk in relation to mtDNA copy number in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. A total of 230 cases and 469 controls matched on age, sex, race, date of blood collection and specimen type were included. Measurements of mtDNA copy number were performed in triplicate using a fluorescence-based QPCR assay. Assay results were highly reproducible, with a coefficient of variation of 5.3% for replicate QC samples from a single individual and an intraclass correlation coefficient of 0.69 for duplicate samples collected an average of 3.6 years apart from 45 controls. To evaluate risk of RCC in relation to mtDNA copy number, we calculated odds ratios (OR) and 95% confidence intervals (CI) using unconditional logistic regression models adjusted for all matching variables as well as history of hypertension, body mass index, smoking history, and family history of kidney cancer. Subjects were assigned to quartiles of mtDNA copy number based on the distribution among controls. Results: Median mtDNA copy number was higher for cases than for controls (131 vs. 121; P=0.001, Wilcoxon rank-sum test). Relative to subjects in the lowest quartile of mtDNA copy number, the ORs for subjects in the 2nd, 3rd, and 4th quartiles were 1.0 (95% CI 0.6-1.6), 1.5 (95% 0.9-2.3), and 1.9 (95% CI 1.2-3.1), respectively (Ptrend = 0.003). The association between mtDNA copy number and RCC was stronger among men (highest vs. lowest quartile, OR 2.4, 95% CI 1.3-4.4) than among women (OR 1.3, 95% CI 0.5-3.2; Pint = 0.24). Results were similar in conditional logistic regression analyses of matched sets, and when we restricted to subjects with mtDNA from buffy coat specimens and to cases diagnosed more than two years after sample collection. Conclusions: Results of this study, to our knowledge the first prospective investigation of mtDNA copy number and RCC risk, suggest that high mtDNA copy number is associated with an increased risk of RCC, particularly among men. Although our findings were inconsistent with prior case-control evidence, most prospective studies of other cancers (e.g., lung, pancreas, non-Hodgkin lymphoma) have reported positive associations. Replication of these findings in other prospective cohorts is needed. 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 4461. doi:1538-7445.AM2012-4461

Abstract 2345: Amplification of mouse chromosome 4 in chemically induced and invasive mouse lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer death in the United States and is ranked second only to bladder cancer in proportion of cases due to past occupational exposures. Though the genetic changes associated with lung cancer are not well understood, the pattern of mutations observed in lung adenocarcinoma from tobacco exposed patients is distinct from that of lung adenocarcinoma from unexposed patients. We used Spectral Karyotyping (SKY), mapping with fluorescently labeled genomic clones (FISH), comparative genomic hybridization (CGH), expression array, real time polymerase chain reaction and Western blot to analyze 15 primary adenocarcinoma and 9 pairs of high and low invasive cell cultures to detect molecular changes. The medial portion of chromosome 4 was deleted in 67% of all of the cell strains. Duplication of the proximal region of chromosome 4 occurred in 22% of the spontaneously-occurring high-invasive cells strains and 83% of the chemically-induced high-invasive cell cultures. Mouse chromosome 1 and 15 were amplified in 90% of the high-invasive cell strains. FISH mapping further narrowed the region of deletion of chromosome 4 to 39.6 centimorgans (cM) and the region of duplication to 10-35 cM. Expression array and real time PCR analysis demonstrated increased expression of the cell cycle inhibitory factor p16 and the apoptotic factor Cathepsin D in all of the tumor cell strains. The expression of cyclooxygenase 2 (COX-2), Kruppel like factor-4 (KLF4), Cyclin E and c-myc was significantly higher in the high-invasive cells strains compared to the low-invasive cell strains. Western blotting showed increased Cox-2, Cyclin E, KLF4 and c-myc proteins and decreased expression of Cathepsin D. In addition, the KLF4 protein was higher in the chemically-induced cell strains compared to the spontaneously-occurring cell strains while COX-2 was higher in the spontaneously-occurring cell strains. The KLF4 protein was identified in the tumor cell strains at the expected size of 55 KD as well as a smaller 45 KD form. Coordinated expression of KLF4 and c-myc is important in the induction of a stem cell phenotype and may be important in chemical carcinogenesis. The higher COX-2 in the spontaneous tumors indicates the importance of inflammation in spontaneous tumor induction. The homologous linkage groups on human chromosomes 9p21, 1p36, 9q and 8q are altered in asbestos-induced human lung adenocarcinoma. Alteration in copy number and expression of these genes may play a functional role in lung cancer development. 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 2345. doi:1538-7445.AM2012-2345

La frequenza delle anomalie dentarie nei pazienti affetti da cheilognatopalatoschisi

ObjectivesThe purpose of this study was to investigate dental abnormalities in children with cleft lip and palate (CLP) in order to identify the most prevalent anomalies and the teeth most frequently affected. Materials and methodsWe analyzed orthopanoramics X-rays (OPT), computed tomography Dental Scans, clinical records, and dental casts from 18 consecutive patients (10 boys, 8 girls; mean age 11.2 years, range: 6 to 18), with CLP (complete or incomplete, mono- or bilateral) and recorded the presence of the following anomalies: supernumerary teeth, agenesis, shape and size anomalies, ectopic and dystopic eruptions, rotations and retained teeth. ResultsThe most frequent abnormalities were: agenesis (prevalence: 22.5%), ectopic and dystopic eruption (19.3%), rotations (17.7%), shape and size anomalies (16.1%), and retained teeth (14.5%). The most frequently affected teeth were the lateral (45.1%) and central incisors (32.2%), the cuspids (14.5%), and the second bicuspid (6.4%). Lateral incisors were usually missing (39.2%) or retained (21.4%), while central incisors were more often rotated (50%) or deformed (25%). The cuspids were ectopic (66.6%) or retained (23%), while the bicuspids were missing (25%), retained (25%), or presented alterations in number (25%) or shape/size (25%). ConclusionsEach subject presented at least one dental anomaly. This high frequency indicates that identification of these anomalies is fundamental for appropriate management of cleft lip and palate.

Alterations in number and functions of lymphocytes after exposure to chronic mild stress: the effect of desipramine

Alterations in number and functions of lymphocytes after exposure to chronic mild stress: the effect of desipramine

Research Article: Correlation of nucleolar morphology to transformed properties of Simian Virus 40 T antigen expressing cells

Abstract. Alterations in nucleolar morphology and number have been routinely noted by pathologists and tumor biologists. More recently, cancer patient prognosis has been correlated to increases in nucleolar size, number and morphology in tumor sections. This study investigated the nucleolar changes in the well-characterized SV40 T antigen transformation model by assessing B23-positive nucleoli. B23, or nucleophosmin, is essential in rRNA processing and p53 regulation via the sequestration of p19ARF. By using B23 immunofluorescence, we found that cells expressing T antigen manifest significant increases in the number, size and the number of large nucleoli present. The total number of nucleoli is increased significantly in both spontaneously immortalized C57Bl/6 mouse embryo fibroblasts and T antigen transformed cell lines compared to the primary cells; however, the properties of growth rate, saturation density and tumorigenesis correlated to significant increases in nucleolar size, not the total number of ...

Systemic immune cell response in rats after pulmonary exposure to manganese-containing particles collected from welding aerosols

Welding fume inhalation affects the immune system of exposed workers. Manganese (Mn) in welding fume may induce immunosuppressive effects. The goal was to determine if Mn in welding fume alters immunity by reducing the number of circulating total leukocytes and specific leukocyte sub-populations. Sprague-Dawley rats were treated by intratracheal instillation (ITI) with either a single dose (2.00 mg/rat) or repeated doses (0.125 or 2.00 mg/rat for 7 weeks) with welding fumes that contained different levels of Mn. Additional rats were treated by ITI once a week for 7 weeks with the two doses of manganese chloride (MnCl2). Bronchoalveolar lavage was performed to assess lung inflammation. Also, whole blood was recovered, and the number of circulating total leukocytes, as well as specific lymphocyte subsets, was determined by flow cytometry. The welding fume highest in Mn content significantly increased lung inflammation, injury, and production of inflammatory cytokines and chemokines compared to all other treatment groups. In addition, the same group expressed significant decreases in the number of circulating CD4+ and CD8+ T-lymphocytes after a single exposure, and significant reductions in the number of circulating total lymphocytes, primarily CD4+ and CD8+ T-lymphocytes, after repeated exposures (compared to control values). Repeated MnCl2 exposure led to a trend of a reduction (but not statistically significant) in circulating total lymphocytes, attributable to the changes in the CD4+ T-lymphocyte population levels. The welding fume with the lower concentration of Mn had no significant effect on the numbers of blood lymphocytes and lymphocyte subsets compared to control values. Evidence from this study indicates that pulmonary exposure to certain welding fumes cause decrements in systemic immune cell populations, specifically circulating T-lymphocytes, and these alterations in immune cell number are not dependent exclusively on Mn, but likely a combination of other metals present in welding fume.

Synaptic activity in X-linked mental retardation: a thorny issue

Synaptic activity in X-linked mental retardation: a thorny issue

Abstract B27: Integrative analysis identifies GAS41 as a novel oncogene in NSCLC, localizing to the 12q15 amplicon

Abstract Background: The application of a multidimensional integrated analysis of recurring genomic and expression alterations can identify new insights into the molecular mechanisms involved in the pathogenesis of NSCLC. Distinguishing the key mechanisms and causal events driving tumorigenesis will lead not only to a better understanding of lung cancer phenotypes and biology, but also new molecular markers and therapeutic targets. Using this approach, we identified the chromosomal region at 12q13-15, and more specifically Glioma amplified sequence 41 (GAS41) to be frequently amplified and overexpressed in NSCLC. A putative transcription factor, amplification of GAS41 has been reported in dedifferentiated liposarcomas and in the earliest stages of glioma and astrocytoma. While the oncogene MDM2 has long been believed to be the driver of this amplicon, we hypothesize GAS41 is an oncogene capable of driving selection of the 12q15 amplicon, and not merely a passenger event. Methods: An integrative genomics approach, examining 261 NSCLC tumors (169 adenocarcinomas (AC) and 92 squamous cell carcinomas (SqCC)) profiled for copy number and gene expression alterations was used to identify novel candidate oncogenes in NSCLC. Recurrent DNA amplifications were identified using the GISTIC algorithm and integrated with gene expression data to identify genes frequently amplified and overexpressed. Genes were classified as overexpressed if the fold change between tumor and matched non-malignant tissues was greater than 2 fold. The functional significance of GAS41 was assessed by lentiviral knockdown and ectopic overexpression in lung cancer cell lines with and without GAS41 amplification, and human bronchial epithelial cells respectively. In vitro assays measuring proliferation, anchorage independent growth, senescence and apoptosis were used to assess the phenotypic effect of gene dosage manipulation. Survival analysis was performed using the Cox regression model for multiple independent cohorts. Results: GAS41 is gained or amplified in over 20% of NSCLC tumors, with similar frequencies of amplification in both AC (26%) and SqCC (24%). Although frequently co-amplified with MDM2, amplification of GAS41 was observed to occur in the absence of MDM2 amplification. Overexpression of GAS41 in human bronchial epithelial cells abrogated senescence, whereas knockdown reduced cell proliferation, impaired colony formation and induced cellular senescence only in lung cancer cell lines with amplification. Western blotting revealed increased p21, cleaved PARP and reduced levels of phospho-p53 in knockdown lines as compared to empty vector controls, suggesting GAS41 is implicated in the regulation of the p21-p53 pathway. Consistent with in vitro results, patients expressing high levels of GAS41 displayed poorer outcomes compared to those with lower levels of GAS41. Conclusions: Our findings reveal GAS41 as a candidate oncogene frequently amplified and overexpressed in NSCLC, both in the presence and absence of MDM2 amplification. Gene dosage manipulation resulted in distinct phenotypic changes characteristic of oncogenes, and thus implicate amplification of GAS41 as a novel mechanism of NSCLC tumorigenesis.

Dysfunctional telomeres promote genomic instability and metastasis in the absence of telomerase activity in oncogene induced mammary cancer

Telomerase is a ribonucleoprotein that maintains the ends of chromosomes (telomeres). In normal cells lacking telomerase activity, telomeres shorten with each cell division because of the inability to completely synthesize the lagging strand. Critically shortened telomeres elicit DNA damage responses and limit cellular division and lifespan, providing an important tumor suppressor function. Most human cancer cells express telomerase which contributes significantly to the tumor phenotype. In human breast cancer, telomerase expression is predictive of clinical outcomes such as lymph node metastasis and survival. In mouse models of mammary cancer, telomerase expression is also upregulated. Telomerase overexpression resulted in spontaneous mammary tumor development in aged female mice. Increased mammary cancer also was observed when telomerase deficient mice were crossed with p53 null mutant animals. However, the effects of telomerase and telomere length on oncogene driven mammary cancer have not been completely characterized. To address these issues we characterized neu proto-oncogene driven mammary tumor formation in G1 Terc-/- (telomerase deficient with long telomeres), G3 Terc-/- (telomerase deficient with short telomeres), and Terc+/+ mice. Telomerase deficiency reduced the number of mammary tumors and increased tumor latency regardless of telomere length. Decreased tumor formation correlated with increased apoptosis in Terc deficient tumors. Short telomeres dramatically increased lung metastasis which correlated with increased genomic instability, and specific alterations in DNA copy number and gene expression. We concluded that short telomeres promote metastasis in the absence of telomerase activity in neu oncogene driven mammary tumors.

Human Obesity Reduces the Number of Hepatic Leptin Receptor (Ob-R) Expressing NK Cells

Objective. In the industrialized world, obesity is an increasing socioeconomic health problem. Obese subjects have a higher risk of developing several types of cancer. NK cells are an integral component of the innate immune system, able to destruct tumor cells. The adipokine leptin plays a crucial role in the development of obesity and its related diseases. Peripheral leptin signaling is modulated by the liver. Methods. The aim of this study was to evaluate the number of hepatic NK cells (CD56+) and the number of leptin-receptor positive (Ob-R+) cells in the livers of five normal-weight and five obese humans. Livers were removed during autopsy and accurately defined sections were stained immunohistochemically and CD56+, Ob-R+, and double-positive cells were quantified. Results. Results revealed a dramatic reduction of NK cells and Ob-R-expressing NK cells in the livers of obese individuals. Conclusions. The present study demonstrates, for the first time, body-weight-dependent numbers of hepatic NK cells. This supports the hypothesis of obesity-associated alterations of immune cell numbers in different human organs.

Enhanced Induction of Regulatory Macrophages Upon Azathioprine/Infliximab Combination Treatment In Vitro

G A A b st ra ct s to determine whether Treg defects resulted from either increased apoptosis or decreased maintenance of Foxp3 expression in Tregs. Methods/Results: WASP-deficient DCs led to decreased In Vitro generation of adaptive Tregs. More importantly, upon transfer of Foxp3cells into recipient mice, there was a marked reduction in the generation of Foxp3+ inducible Tregs in WASP/RAG DKO mice compared to RAG KO controls. The defects in WT Treg function in chimeric mice could not be explained by alterations in the maintenance or apoptosis of Foxp3+ donor cells in WASP/RAG DKO mice compared to RAG KO mice. Employing an In Vitro suppression assay, WT Tregs functioned normally when MLN DCs isolated from either WT or WASP-deficient mice were used as APCs. In contrast, there was aberrant Treg function when DCs were isolated from WASP/RAG DKO mice. Conclusions: WASP deficiency in innate immune cells leads to defective Treg generation and function In Vivo. Similarly, there was reduced generation of WT Tregs In Vitro in the presence of WASPdeficient DCs. However, Treg function was compromised only when DCs were harvested from WASP/RAG DKO mice but not WASP-deficient mice, suggesting an intrinsic aberrancy in WASP-deficient DCs unmasked in a lymphopenic setting. In summary, colitis in chimeric mice may result from DC-driven alterations in Treg number and function.

Abstract 3894: Copy number alterations in urothelial carcinomas: Their clinicopathological significance and correlation with DNA methylation alterations

Abstract Purpose: The aim of this study was to clarify the genetic backgrounds underlying the clinicopathological characteristics of urothelial carcinomas (UCs). Experimental Design: We analyzed copy number alterations by array comparative genomic hybridization analysis using a high-resolution (244K) oligonucleotide array, DNA methylation alterations on a genome-wide scale using bacterial artificial chromosome array-based methylated CpG island amplification, and DNA methylation status on C-type CpG islands using bisulfite modification in 49 samples of UCs of the urinary bladder, ureter and renal pelvis. The clinicopathological significance of copy number alterations and the correlations between alterations of copy number and those of DNA methylation were examined. Results: Losses of 2q33.3-q37.3, 4p15.1-q26 and 5q13.3-q35.2 and gains of 7p11.2-q11.23 and 20q13.11-q13.31 were correlated with higher histological grade, and gain of 7p21.2-p21.1 was correlated with deeper invasion. Losses of 6q14.1-q27 and 17p13-q11.1 and gains of 19q13.11-q13.12 and 20q13.12-q13.33 were correlated with lymph vessel involvement. Loss of 16p12.2-p12.1 and gains of 3q26.31-q29 and 20q13.2-q13.33 were correlated with vascular involvement. Losses of 5q14.1-q23.2, 6q14.1-q27, 8p22-p21.3, 11q13.5-q14.1 and 15q11.2-q22.2 and gains of 3q27.3-q29, 7p11.2-q11.22, 10p13-p12.33, 12q24.31 and 19q13.11-q13.2 were correlated with the development of aggressive non-papillary UCs. Losses of 1p33-p31.1, 2q22.1-q23.1, 10q11.23-q21.1 and 15q21.3 were correlated with tumor recurrence. Unsupervised hierarchical clustering analysis based on copy number alterations clustered UCs into three subclasses, clusters A, B1 and B2: copy number alterations associated with genome-wide DNA hypomethylation, regional DNA hypermethylation on C-type CpG islands associated with chromosomal losses, and genome-wide DNA hypo- and hypermethylation, were accumulated in clusters A, B1 and B2, respectively. Conclusions: There is a need to explore tumor-related genes that may encode potential therapeutic targets at chromosomal loci where copy number alterations are correlated with clinicopathological parameters reflecting the malignant potential of UCs. Chromosomal loci where copy number alterations are correlated with UC recurrence may be potential indicators of prognosis. Both genetic and epigenetic events appear to accumulate during urothelial carcinogenesis, reflecting the clinicopathological diversity and histological heterogeneity of UCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3894. doi:10.1158/1538-7445.AM2011-3894

Abstract 3911: Functional copy number alterations (CNAS) in oral cancer

Abstract Background: Alterations in DNA copy number can functionally affect genes and produce a transformed cancer phenotype. Discoevery of novel cancer genes can be facilitated by identifying genomic regions undergoing frequent, significant alteration. Array comparative genomic hybridization techniques have generated vast amounts of raw copy number data requiring computational analysis to identify aberrations likely to be functional. In this analysis, we present data from a comprehensive computational approach designed to robustly map chromosomal aberrations in oral cavity squamous cell carcinoma (OCSCC). Methods: DNA was extracted from 31 microdissected OCSCC frozen tumors and hybridized with reference DNA on the Agilent 1M copy number array, containing nearly 1 million probes across the genome at high resolution. The RAE computational framework was used to identify CNAs. In this analysis, segmentation of probe data is first performed using paired reference HapMap data and individual-tumor thresholds to account for stromal admixture and tumor heterogeneity. Scores (single-copy gain, amplification, hemizygous loss, homozygous deletion) are then assigned, followed by soft discrimination thresholding and significance testing with false discovery rate correction. Regions of interest (ROIs) are then agnostically identified as regions of significant (FDR<0.01) alteration, expanded to adjacent loci to account for spatial and amplitude uncertainty. Finally, described copy number polymorphisms were excluded. Results: 49 ROIs were identified, 27 amplification regions (chromosomes 3,4,5,7,8,9,11,16,18,20,21) and 22 deletion regions (chromosomes 2,3,4,5,7,8,9,10,13,15,16,18,19). ROIs ranged in prevalence from 6.5-77.4% of samples, and encompassed 0-483 genes (total=2799, median=4), and 0-13 microRNAs (total=77, median=0). Eleven ROIs were focal intragenic CNAs. Four ROIs contained no named genes or microRNAs. Hierarchical clustering identified 3 clusters, based on escalating levels of CNA. Complete gene details of the 49 ROIs will be presented. Conclusions: We report significant regions of CNA in oral cancer, by applying a robust computational framework to identify recurrent chromosomal aberrations in OCSCC, which represent highly significant candidate functional events. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2011-3911

Abstract 3822: DNA copy number changes in the PI3K signaling pathway genes in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is associated with poor prognosis and a high mortality rate when compared to the other breast cancer subtypes. These tumors lack the expression of ER, PR and HER2 receptors, and do not respond well to the currently available adjuvant therapies. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions. Alterations of genes in this pathway have been shown to predict responsiveness to cancer targeted therapies. In this study, five genes involved in this signaling pathway, PIK3CA, AKT1, AKT2, PTEN and FGFR, were evaluated for DNA copy number changes in two sets of TNBC specimens. Methods: TNBC samples were obtained from the Hospital Nossa Senhora das Gracas, Curitiba, PR, Brazil. Two sets of TNBC samples were analyzed: the first set was composed of 30 formalin fixed-paraffin embedded tissue specimens and were analyzed for the PIK3CA, AKT1, AKT2, PTEN and FGFR genes by FISH. Locus-specific probes were prepared from BAC contigs for each gene and a centromeric probe for chromosome 3 was used as a control for copy number. The second set was composed of 17 frozen samples and were analyzed by array-CGH using a 44K-Agilent array platform. Real-Time PCR (TaqMan assay), was used to validate the array-CGH data for the above five genes. Results: FISH: PTEN loss and FGFR3 amplification were the most common findings occurring in 56% and 41% of the TNBC samples respectively. PIK3CA, AKT2 and AKT1 were amplified in 31%, 25% and 9.5% of the samples respectively. Array-CGH/Real time PCR: DNA copy number changes were observed by array-CGH in all, but one TNBC sample. The gene with highest frequency of amplification detected by array-CGH was PIK3CA (amplified in 77% of the cases). These findings were confirmed in 70% of the cases. FGFR2, AKT2 and PTEN were amplified in 35%, 24% and 24% of the cases respectively, These findings were confirmed for FGFR2 in 83% of the samples and for PTEN in all the samples. AKT2 amplification was only confirmed in 25% of the samples. Finally AKT1 was observed amplified in by array-CGH in 6% of the cases, which was confirmed by Real-time PCR.Conclusions: Based on our initial results, we conclude that TNBC presents DNA copy number alterations in genes involved in the PIK3 signaling pathway. Upon validation of these alterations in a larger number of cases, with correlation to prognostic parameters, their potential use as therapeutic targets can be considered for further investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2011-3822

Abstract 5354: Characterisation of the cell line HC-AFW1 derived from a paediatric hepatocellular carcinoma

Abstract Paediatric hepatocellular carcinoma (HCC) has an incidence of 1.5 cases per million children and is linked to viral hepatitis in endemic regions. In Europe childhood HCC mostly arises in otherwise healthy liver and has an unknown ethiology. Current treatment results of HCC are dissatisfactory due to advanced disease at diagnosis and lacking response to chemotherapy. To improve the research of childhood HCC and to address specific therapeutic approaches a preclinical model for childhood HCC is needed. So far, there has been one existing cell line, HepG2, which has lately been discussed controversially as being hepatoblastoma. We established a cell line HC-AFW1 from the tumor of a 5 year old boy. The original tumor histology was characteristic as transitional liver cell tumor including hepatoblastoma and HCC morphology. These histological findings were confirmed through several reference pathology laboratories worldwide. The cell line HC-AFW1 was established by xenotransplantation of original tumor into immuno-deficient mice and re-culturing in vitro. The cell line is in sable culture now for over 8 months with a doubling time of 40 h. Cultured cells present epithelial characteristics and express hepatoma proteins like AFP, glypican 3, E-cadherin, CD10, CD326, HepPar1, Vimentin and Desmin. Catenin beta shows a deletion of 49 amino acids in the exon 3 involving the phosphorylation sites of GSK3 and was detected in cell nuclei. Cytogenetic analysis revealed large anomalies in the chromosomal map. This involves chromosomal aberrations found in hepatoblastoma as well as in adult HCC. Several alterations of gene copy numbers were detected by genome wide SNP array. Among different tested drugs cisplatin and irinotecan showed an effective inhibition of tumor cell growth in an MTT assay at concentration below 5 µg/ml. Transplantation of HC-AFW1 cells into NOD/SCID mice resulted in fast growing dedifferentiated tumors with high levels of serum AFP. This xenotransplants as well as stable cultured cells solely consist of HCC components suggesting a selection of HCC and degeneration of formally hepatoblastoma tumor parts. Therefore, HC-AFW1 is the first paediatric HCC-derived cell line and represents a valuable tool to investigate the biology and therapeutic strategies in childhood HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5354. doi:10.1158/1538-7445.AM2011-5354

Abstract 2980: Clinico-pathological correlation of UBE2C alterations in colorectal carcinoma and efficacy of UBE2C inhibition by proteosome inhibitor as a viable therapeutic target

Abstract Substantial evidence implicates UBE2C in several human cancers including colorectal carcinoma. We sought to determine the prognostic value of the UBE2C alterations in colorectal cancer (CRC), UBE2C signaling in CRC cell lines and effect of protesome inhibitors in modulating UBE2C expression in vitro and in vivo nude mice xenografts. We examined UBE2C expression by immunohistochemistry and UBE2C gene copy number by FISH in CRC.The oncogenic role of UBE2C in CRC cell lines was explored and potential therapeutic value of Bortezomib in treating CRC by inhibiting UBE2C was also investigated using in vivo and in vitro analysis. UBE2C dysregulation was seen in a large percentage of colorectal carcinomas: 45.9% at the protein level; 30.7 % at the gene copy number and UBE2C alteration in 61.5 % of the CRC. UBE2C dysregulation occurs early in the progression from adenoma to carcinoma sequence and was associated with proliferative marker Ki-67(p=0.0023), accumulation of cyclin A (p=0.0175) and B1 (p=0.0026). UBE2C expression was associated with a poor overall survival (p=0.0267) in all CRC patients and interestingly, in the early stage CRC subgroup, UBE2C expression was an independent (RR 3.67 95% C.I 1.15-14.03) prognostic marker.Thus, the above findings show the potential of targeting UBE2C enzyme in early stage CRC as well as in pre neoplastic adenomatous lesions. In CRC cell lines, UBE2C depletion resulted in suppression of cellular growth and accumulation of cyclin A and B1, whereas over overexpression of UBE2C promoted cell proliferation. Bortezomib treatment of CRC cells and xenograft downregulated the expression of UBE2C at mRNA as well as protein level, inhibited cell proliferation resulted in accumulation of cyclin A and cyclin B1 and reduced tumor growth. Altogether, our results establish UBE2C as a key molecule in colorectal carcinogenesis and identify a subgroup of CRC patients with high UBE2C alterations showing a poor overall survival. We have also shown for the first time the efficacy of Bortezomib in inhibiting UBE2C expression at the protein as well as the mRNA level and stabilization of cyclin A and B1. These studies may have important implications for future preclinical and clinical studies in CRC aimed at determining the usefulness of a novel strategy for treating CRC with inhibitors of proteasome pathways, either alone or in combination with other agents. Further work is ongoing to check if treatment with the UBE2C siRNA sensitize the cells to Bortezomib treatment or other commonly used colorectal cancer therapies and if Bortezomib-induced tumor regression comparable to conventional chemotherapeutics like oxaliplatin or 5FU treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2980. doi:10.1158/1538-7445.AM2011-2980

Effect of chromium and cobalt ions on primary human lymphocytes in vitro

Cobalt-chromium (Co-Cr) alloy metal-on-metal hip resurfacing is increasingly common among younger more active patients suffering from osteoarthritis. Recent reports have increased awareness of metal ions leaching from metallic articulations; this ion exposure may have adverse effects on the immune system. As previous studies reported alterations in lymphocyte number and function in patients with Co-Cr implants, we investigated effects of clinically relevant concentrations of Cr6+ and Co2+ on primary human lymphocytes in vitro. Here, both resting and activated (anti-CD3 ± anti-CD28 antibodies) primary human lymphocytes were exposed to Cr6+ or Co2+ (0.1–100 µM). Following 24 or 48 h of exposure, cell viability, proliferation, cytokine [interferon-γ (IFNγ and interleukin-2 (IL-2)] release, and apoptosis (with and without pre-treatment of cells with a caspase-3 inhibitor) were assessed. Exposure to 10 and 100 µM Cr6+ significantly decreased cell viability and increased apoptosis in both resting and activated lymphocytes. Cell proliferation and cytokine release were also significantly reduced in activated lymphocytes following exposure. The exposure of resting lymphocytes to 100 µM Co2+ resulted in significant decreases in cell viability accompanied by a significant increase in apoptosis. Activated lymphocytes also showed this response after exposure to 100 µM Co2+; in fact, activated cells were significantly more sensitive to Co2+ toxicity. Exposure to 10 µM Co2+ led to significant decreases in cell proliferation and cytokine release, but no significant increase in apoptosis, in activated cells. The results indicate that exposure to high concentrations of metal ions initiate apoptosis that results in decreased lymphocyte proliferation. IL-2 release is inhibited by both metal ions at concentrations that are not overtly toxic. However, metal ion concentrations not directly cytotoxic to lymphocytes may affect events at a molecular level, thereby impeding lymphocyte proliferation. Hence, this may contribute to altered immune system function in patients with Co-Cr implants.