Genome Microarray Research Articles

Differential adipose tissue gene expression profiles in abacavir treated patients that may contribute to the understanding of cardiovascular risk: a microarray study.

ObjectiveTo compare changes in gene expression by microarray from subcutaneous adipose tissue from HIV treatment naïve patients treated with efavirenz based regimens containing abacavir (ABC), tenofovir (TDF) or zidovidine (AZT).DesignSubcutaneous fat biopsies were obtained before, at 6- and 18–24-months after treatment, and from HIV negative controls. Groups were age, ethnicity, weight, biochemical profile, and pre-treatment CD4 count matched. Microarray data was generated using the Agilent Whole Human Genome Microarray. Identification of differentially expressed genes and genomic response pathways was performed using limma and gene set enrichment analysis.ResultsThere were significant divergences between ABC and the other two groups 6 months after treatment in genes controlling cell adhesion and environmental information processing, with some convergence at 18–24 months. Compared to controls the ABC group, but not AZT or TDF showed enrichment of genes controlling adherence junction, at 6 months and 18–24 months (adjusted p<0.05) and focal adhesions and tight junction at 6 months (p<0.5). Genes controlling leukocyte transendothelial migration (p<0.05) and ECM-receptor interactions (p = 0.04) were over-expressed in ABC compared to TDF and AZT at 6 months but not at 18–24 months. Enrichment of pathways and individual genes controlling cell adhesion and environmental information processing were specifically dysregulated in the ABC group in comparison with other treatments. There was little difference between AZT and TDF.ConclusionAfter initiating treatment, there is divergence in the expression of genes controlling cell adhesion and environmental information processing between ABC and both TDF and AZT in subcutaneous adipose tissue. If similar changes are also taking place in other tissues including the coronary vasculature they may contribute to the increased risk of cardiovascular events reported in patients recently started on abacavir-containing regimens.

Pseudogene INTS6P1 regulates its cognate gene INTS6 through competitive binding of miR-17-5p in hepatocellular carcinoma.

The complex regulation of tumor suppressive gene and its pseudogenes play key roles in the pathogenesis of hepatocellular cancer (HCC). However, the roles played by pseudogenes in the pathogenesis of HCC are still incompletely elucidated. This study identifies the putative tumor suppressor INTS6 and its pseudogene INTS6P1 in HCC through the whole genome microarray expression. Furthermore, the functional studies - include growth curves, cell death, migration assays and in vivo studies - verify the tumor suppressive roles of INTS6 and INTS6P1 in HCC. Finally, the mechanistic experiments indicate that INTS6 and INTS6P1 are reciprocally regulated through competition for oncomiR-17-5p. Taken together, these findings demonstrate INTS6P1 and INTS6 exert the tumor suppressive roles through competing for oncomiR-17-5p. Our investigation of this regulatory circuit reveals novel insights into the underlying mechanisms of hepatocarcinogenesis.

Acute cutaneous wounds treated with human decellularised dermis show enhanced angiogenesis during healing.

BackgroundThe influence of skin substitutes upon angiogenesis during wound healing is unclear.ObjectivesTo compare the angiogenic response in acute cutaneous human wounds treated with autogenic, allogenic and xenogenic skin substitutes to those left to heal by secondary intention.MethodsOn day 0, four 5mm full-thickness punch biopsies were harvested from fifty healthy volunteers (sites 1-4). In all cases, site 1 healed by secondary intention (control), site 2 was treated with collagen-GAG scaffold (CG), cadaveric decellularised dermis (DCD) was applied to site 3, whilst excised tissue was re-inserted into site 4 (autograft). Depending on study group allocation, healing tissue from sites 1-4 was excised on day 7, 14, 21 or 28. All specimens were bisected, with half used in histological and immunohistochemical evaluation whilst extracted RNA from the remainder enabled whole genome microarrays and qRT-PCR of highlighted angiogenesis-related genes. All wounds were serially imaged over 6 weeks using laser-doppler imaging and spectrophotometric intracutaneous analysis.ResultsInherent structural differences between skin substitutes influenced the distribution and organisation of capillary networks within regenerating dermis. Haemoglobin flux (p = 0.0035), oxyhaemoglobin concentration (p = 0.0005), and vessel number derived from CD31-based immunohistochemistry (p = 0.046) were significantly greater in DCD wounds at later time points. This correlated with time-matched increases in mRNA expression of membrane-type 6 matrix metalloproteinase (MT6-MMP) (p = 0.021) and prokineticin 2 (PROK2) (p = 0.004).ConclusionCorroborating evidence from invasive and non-invasive modalities demonstrated that treatment with DCD resulted in increased angiogenesis after wounding. Significantly elevated mRNA expression of pro-angiogenic PROK2 and extracellular matrix protease MT6-MMP seen only in the DCD group may contribute to observed responses.

Whole-exome analysis of foetal autopsy tissue reveals a frameshift mutation in OBSL1, consistent with a diagnosis of 3-M Syndrome.

BackgroundWe report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants.ResultsWhole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings.ConclusionsOur study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a recurrent set of clinical symptoms.

TRIB2 as a biomarker for diagnosis and progression of melanoma.

Malignant melanoma is the most deadly form of skin cancer. There is a critical need to identify the patients that could be successfully treated by surgery alone and those that require adjuvant treatment. In this study, we demonstrate that the expression of tribbles2 (TRIB2) strongly correlates with both the presence and progression of melanocyte-derived malignancies. We examined the expression of TRIB2 in addition to 12 previously described melanoma biomarkers across three independent full genome microarray studies. TRIB2 expression was consistently and significantly increased in benign nevi and melanoma, and was highest in samples from patients with metastatic melanoma. The expression profiles for the 12 biomarkers were poorly conserved throughout these studies with only TYR, S100B and SPP1 showing consistently elevated expression in metastatic melanoma versus normal skin. Strikingly we confirmed these findings in 20 freshly obtained primary melanoma tissue samples from metastatic lesions where the expression of these biomarkers were evaluated revealing that TRIB2 expression correlated with disease stage and clinical prognosis. Our results suggest that TRIB2 is a meaningful biomarker reflecting diagnosis and progression of melanoma, as well as predicting clinical response to chemotherapy.

Case of 7p22.1 Microduplication Detected by Whole Genome Microarray (REVEAL) in Workup of Child Diagnosed with Autism.

Introduction. More than 60 cases of 7p22 duplications and deletions have been reported with over 16 of them occurring without concomitant chromosomal abnormalities. Patient and Methods. We report a 29-month-old male diagnosed with autism. Whole genome chromosome SNP microarray (REVEAL) demonstrated a 1.3 Mb interstitial duplication of 7p22.1 ->p22.1 arr 7p22.1 (5,436,367–6,762,394), the second smallest interstitial 7p duplication reported to date. This interval included 14 OMIM annotated genes (FBXL18, ACTB, FSCN1, RNF216, OCM, EIF2AK1, AIMP2, PMS2, CYTH3, RAC1, DAGLB, KDELR2, GRID2IP, and ZNF12). Results. Our patient presented features similar to previously reported cases with 7p22 duplication, including brachycephaly, prominent ears, cryptorchidism, speech delay, poor eye contact, and outburst of aggressive behavior with autism-like features. Among the genes located in the duplicated segment, ACTB gene has been proposed as a candidate gene for the alteration of craniofacial development. Overexpression of RNF216L has been linked to autism. FSCN1 may play a role in neurodevelopmental disease. Conclusion. Characterization of a possible 7p22.1 Duplication Syndrome has yet to be made. Recognition of the clinical spectrum in patients with a smaller duplication of 7p should prove valuable for determining the minimal critical region, helping delineate a better prediction of outcome and genetic counseling

TLR4/NF-κB-responsive microRNAs and their potential target genes: a mouse model of skeletal muscle ischemia-reperfusion injury.

Background. The aim of this study was to profile TLR4/NF-κB-responsive microRNAs (miRNAs) and their potential target genes in the skeletal muscles of mice following ischemia-reperfusion injury. Methods. Thigh skeletal muscles of C57BL/6, Tlr4 −/−, and NF-κB −/− mice isolated based on femoral artery perfusion were subjected to ischemia for 2 h and reperfusion for 0 h, 4 h, 1 d, and 7 d. The muscle specimens were analyzed with miRNA arrays. Immunoprecipitation with an argonaute 2- (Ago2-) specific monoclonal antibody followed by whole genome microarray was performed to identify mRNA associated with the RNA-silencing machinery. The potential targets of each upregulated miRNA were identified by combined analysis involving the bioinformatics algorithm miRanda and whole genome expression. Results. Three TLR4/NF-κB-responsive miRNAs (miR-15a, miR-744, and miR-1196) were significantly upregulated in the muscles following ischemia-reperfusion injury. The combined in silico and whole genome microarray approaches identified 5, 4, and 20 potential target genes for miR-15a, miR-744, and miR-1196, respectively. Among the 3 genes (Zbed4, Lrsam1, and Ddx21) regulated by at least 2 of the 3 upregulated miRNAs, Lrsam1 and Ddx21 are known to be associated with the innate immunity pathway. Conclusions. This study profiled TLR4/NF-κB-responsive miRNAs and their potential target genes in mouse skeletal muscle subjected to ischemia-reperfusion injury.

Accurate whole genome sequencing as the ultimate genetic test.

Featured Article: Drmanac R, Sparks AB, Callow MJ, Halpern AL, Burns NL, Kermani BG, et al. Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays. Science 2010;327:78–81.4 Even 30 years ago, it was obvious that Sanger sequencing had limited throughput, and a more efficient process could replace many tedious gene and genome mapping projects. It would take until the mid-2000s for massively parallel sequencing (MPS)5 technologies to demonstrate they could overtake the Sanger sequencing hegemony. Our paper was not the first description of a viable MPS technology, but it firmly established that human whole genome sequencing (WGS) could be done affordably (<US$5000 in reagent cost), with high accuracy (<1 error in 100 kb), and with high throughput, thus heralding the arrival of personal genome sequencing. This work was the result of a long effort that started in Serbia in 1987 with a proposal for sequencing by hybridization (SBH) on dot-blot DNA arrays. SBH on random bead microarrays prepared by emulsion PCR, in which a micron-sized bead replaced a millimeter-sized DNA dot, offered a first MPS solution (www.rdrmanac.com). The US Department of Energy and NIST grants funded most of these initial efforts. After the events of September 11, 2001, the NIH received an increase in biodefense funding, and our US$2.3 million grant for ligation-based MPS technology on genomic DNA microarrays was awarded in 2003 by …

Exceptional complex chromosomal rearrangements in three generations.

We report an exceptional complex chromosomal rearrangement (CCR) found in three individuals in a family that involves 4 chromosomes with 5 breakpoints. The CCR was ascertained in a phenotypically abnormal newborn with additional chromosomal material on the short arm of chromosome 4. Maternal karyotyping indicated that the mother carried an apparently balanced CCR involving chromosomes 4, 6, 11, and 18. Maternal transmission of the derivative chromosome 4 resulted in partial trisomy for chromosomes 6q and 18q and a partial monosomy of chromosome 4p in the proband. Further family studies found that the maternal grandmother carried the same apparently balanced CCR as the proband's mother, which was confirmed using the whole chromosome painting (WCP) FISH. High resolution whole genome microarray analysis of DNA from the proband's mother found no evidence for copy number imbalance in the vicinity of the CCR translocation breakpoints, or elsewhere in the genome, providing evidence that the mother's and grandmother's CCRs were balanced at a molecular level. This structural rearrangement can be categorized as an exceptional CCR due to its complexity and is a rare example of an exceptional CCR being transmitted in balanced and/or unbalanced form across three generations.

Immunomodulatory Effects of theAgaricus blazeiMurrill-Based Mushroom Extract AndoSan in Patients with Multiple Myeloma Undergoing High Dose Chemotherapy and Autologous Stem Cell Transplantation: A Randomized, Double Blinded Clinical Study

Forty patients with multiple myeloma scheduled to undergo high dose chemotherapy with autologous stem cell support were randomized in a double blinded fashion to receive adjuvant treatment with the mushroom extract AndoSan, containing 82% of Agaricus blazei Murrill (19 patients) or placebo (21 patients). Intake of the study product started on the day of stem cell mobilizing chemotherapy and continued until the end of aplasia after high dose chemotherapy, a period of about seven weeks. Thirty-three patients were evaluable for all study endpoints, while all 40 included patients were evaluable for survival endpoints. In the leukapheresis product harvested after stem cell mobilisation, increased percentages of Treg cells and plasmacytoid dendritic cells were found in patients receiving AndoSan. Also, in this group, a significant increase of serum levels of IL-1ra, IL-5, and IL-7 at the end of treatment was found. Whole genome microarray showed increased expression of immunoglobulin genes, Killer Immunoglobulin Receptor (KIR) genes, and HLA genes in the Agaricus group. Furthermore, AndoSan displayed a concentration dependent antiproliferative effect on mouse myeloma cells in vitro. There were no statistically significant differences in treatment response, overall survival, and time to new treatment. The study was registered with Clinicaltrials.gov NCT00970021.

Microarray transposon tracking for the mapping of conditionally essential genes in Campylobacter jejuni.

Although whole genome approaches to the study of bacteria have grown substantially in the past decade, there is still a need for quick and easy methods for the determination of which genes are essential for the growth of these bacteria under specific growth conditions. There are numerous methods to accomplish this depending on the resources and equipment available, each with their own advantages and disadvantages. Here we describe a method we successfully employed to map the essential genes of Campylobacter jejuni using a microarray transposon tracking approach where we constructed a saturated transposon mutant library in the C. jejuni strain NCTC11168 and used a genomic microarray approach to identify genes lacking transposon insertions under standard laboratory growth conditions. With a fully saturated library, the absence of transposon insertions can be used as an indicator of a gene essential for the survival and growth for the conditions used for the mutant library.

Atrial Fibrillation, Neurocognitive Decline and Gene Expression After Cardiopulmonary Bypass.

OBJECTIVE Atrial fibrillation and neurocognitive decline are common complications after cardiopulmonary bypass. By utilizing genomic microarrays we investigate whether gene expression is associated with postoperative atrial fibrillation and neurocognitive decline.METHODS Twenty one cardiac surgery patients were prospectively matched and underwent neurocognitive assessments pre-operatively and four days postoperatively. The whole blood collected in the pre-cardiopulmonary bypass, 6 hours after-cardiopulmonary bypass, and on the 4th postoperative day was hybridized to Affymetrix Gene Chip U133 Plus 2.0 Microarrays. Gene expression in patients who developed postoperative atrial fibrillation and neurocognitive decline (n=6; POAF+NCD) was compared with gene expression in patients with postoperative atrial fibrillation and normal cognitive function (n=5; POAF+NORM) and patients with sinus rhythm and normal cognitive function (n=10; SR+NORM). Regulated genes were identified using JMP Genomics 4.0 with a false discovery rate of 0.05 and fold change of >1.5 or <-1.5.RESULTS Eleven patients developed postoperative atrial fibrillation. Six of these also developed neurocognitive decline. Of the 12 patients with sinus rhythm, only 2 developed neurocognitive decline. POAF+NCD patients had unique regulation of 17 named genes preoperatively, 60 named genes six hours after cardiopulmonary bypass, and 34 named genes four days postoperatively (P<0.05) compared with normal patients. Pathway analysis demonstrated that these genes are involved in cell death, inflammation, cardiac remodeling and nervous system function.CONCLUSION Patients who developed postoperative atrial fibrillation and neurocognitive decline after cardiopulmonary bypass may have differential genomic responses compared to normal patients and patients with only postoperative atrial fibrillation, suggesting common pathophysiology for these conditions. Further exploration of these genes may provide insight into the etiology and improvements of these morbid outcomes.

Comparative analysis of temporal gene expression patterns in the developing ovary of the embryonic chicken.

Many genes participate in the process of ovarian germ cell development, while the combined action mechanisms of these molecular regulators still need clarification. The present study was focused on determination of differentially expressed genes and gene functions at four critical time points in chicken ovarian development. Comparative transcriptional profiling of ovaries from embryonic day 5.5 (E5.5), E12.5, E15.5 and E18.5 was performed using an Affymetrix GeneChip chicken genome microarray. Differential expression patterns for genes specifically depleted and enriched in each stage were identified. The results showed that most of the up- and downregulated genes were involved in the metabolism of retinoic acid (RA) and synthesis of hormones. Among them, a higher number of up- and downregulated genes in the E15.5 ovary were identified as being involved in steroid biosynthesis and retinol metabolism, respectively. To validate gene changes, expressions of twelve candidate genes related to germ cell development were examined by real-time PCR and found to be consistent with the of GeneChip data. Moreover, the immunostaining results suggested that ovarian development during different stages was regulated by different genes. Furthermore, a Raldh2 knockdown chicken model was produced to investigate the fundamental role of Raldh2 in meiosis initiation. It was found that meiosis occurred abnormally in Raldh2 knockdown ovaries, but the inhibitory effect on meiosis was reversed by the addition of exogenous RA. This study offers insights into the profile of gene expression and mechanisms regulating ovarian development, especially the notable role of Raldh2 in meiosis initiation in the chicken.

Tenofovir disoproxil fumarate: toxicity, toxicokinetics, and toxicogenomics analysis after 13 weeks of oral administration in mice.

Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir that exhibits activity against HIV and hepatitis B. The goals of this study were to evaluate the molecular mechanism of TDF-induced toxicity in mice after 13 weeks of daily oral administration (50-1000 mg/kg) by correlating transcriptional changes with plasma drug levels and traditional toxicology end points. Plasma levels and systemic exposure of tenofovir increased less than dose proportionally and were similar on days 1 and 91. No overt toxicity was observed following the completion of TDF administration. The kidneys of TDF-treated mice were histopathologically normal. This result is consistent with the genomic microarray results, which showed no significant differences in kidney transcriptional levels between TDF-treated animals and controls. In liver, after 4 and 13 weeks, cytomegaly was observed in mice treated with 1000 mg/kg of TDF, but mice recovered from this effect following cessation of administration. Analysis of liver transcripts on day 91 reported elevated levels of Cdkn1a in TDF-treated animals compared with controls, which may have contributed to the inhibition of liver cell cycle progression.

Employing social network analysis for disease biomarker detection.

Detection of disease biomarkers in general and cancer biomarkers in particular is an important task which has received considerable attention in the area of in silico genomic experiments. We describe a new approach for detecting cancer biomarkers based on genomic microarray data; it is characterised by employing Social Network Analysis (SNA) techniques. Through social interaction perspective, we can have genes as actors in a social network, where similarities between genes can be described as connections between these actors. The correct determination of biomarkers out of huge genomic data dramatically decreases the number of features. It is also possible to achieve the same or better classification performance compared to using the whole data. The minimum number of biomarkers can be researched further biologically to reduce the numerous time-consuming in vitro experiments. Results of the conducted experiments with selected biomarkers are promising and efficient.

Sema6A and Mical1 control cell growth and survival of BRAFV600E human melanoma cells.

We used whole genome microarray analysis to identify potential candidate genes with differential expression in BRAFV600E vs NRASQ61R melanoma cells. We selected, for comparison, a peculiar model based on melanoma clones, isolated from a single tumor characterized by mutually exclusive expression of BRAFV600E and NRASQ61R in different cells. This effort led us to identify two genes, SEMA6A and MICAL1, highly expressed in BRAF-mutant vs NRAS-mutant clones. Real-time PCR, Western blot and immunohistochemistry confirmed preferential expression of Sema6A and Mical1 in BRAFV600E melanoma. Sema6A is a member of the semaphorin family, and it complexes with the plexins to regulate actin cytoskeleton, motility and cell proliferation. Silencing of Sema6A in BRAF-mutant cells caused cytoskeletal remodeling, and loss of stress fibers, that in turn induced cell death. Furthermore, Sema6A depletion caused loss of anchorage-independent growth, inhibition of chemotaxis and invasion. Forced Sema6A overexpression, in NRASQ61R clones, induced anchorage-independent growth, and a significant increase of invasiveness. Mical1, that links Sema/PlexinA signaling, is also a negative regulator of apoptosis. Indeed, Mical-1 depletion in BRAF mutant cells restored MST-1-dependent NDR phosphorylation and promoted a rapid and massive NDR-dependent apoptosis. Overall, our data suggest that Sema6A and Mical1 may represent new potential therapeutic targets in BRAFV600E melanoma.

Genomic Microarray Alterations Add Prognostic Power to the IPSS-R in MDS with Normal Karyotype

BackgroundDisease karyotype is the parameter with the strongest prognostic impact in the IPSS-R. Around 50% of MDS patients have a normal karyotype. Conventional G-banding cytogenetics has a resolution of 5-10 Mb. SNP microarrays is a genomic array with a higher resolution than cytogenetics and allows the detection of small copy number alterations (CNA) and absence of heterozygosity (AOH).Our hypothesis is that in MDS patients with normal karyotype small cryptic alterations might exist, undetectable by conventional karyotyping, but with an impact on patient’s outcome. Our aim is to study a series of MDS patients with normal karyotype belonging to the IWG-PM consortium (MDS Foundation) by SNP microarrays. Herein, we describe the preliminary findings as recruitment is ongoing and complete results will be presented at the meeting.Patients and methodsWe have recruited microarray data of 125 MDS patients with normal karyotype from centers belonging to the IWG-PM of the MDS Foundation. All cases included were studied with SNP array from Affymetrix®.We collected clinical, biological and microarray data and we studied their impact on overall survival (OS) and acute myeloid leukemia (AML) evolution in 73 patients. The median age was 67.8 years (21.8-92.1), 51.2% were male and the most frequent subtype according 2008 WHO classification was refractory cytopenia with multilineage dysplasia (41.6%), followed by refractory anemia with excess of blasts-1 (RAEB-1) and -2, 18.4% and 16.8%, respectively. Median values at diagnosis were, for hemoglobin: 9.3g/dL, leukocytes count: 3.6x109/L, platelet count: 139x109/L, percentage of polymorphonuclear cells: 47%, and bone marrow blasts: 3.6%. Nineteen patients were analyzed with their matched germ-line control sample.The statistical analysis was performed with SPSS and R software. As level of significance, 5% (two-sided) was chosen. No adjustment for multiple testing was applied.ResultsFifty nine cases were found to be altered by microarrays (47.2%), with a median number of alterations of 1 (range: 1-8). CNA were detected in 36.8% of cases, in 8 cases CNA were larger than 5 Mb. AOH were detected in 18.4% of cases. The median size of the affected genome by CNA was 116 Kb, adding up CNA plus AOH was 24745.05Kb. There were significant differences, in distribution of FAB diagnosis and median percentage of bone marrow blasts, in patients altered by SNP arrays in comparison to patients with normal findings by this technique.The impact on the outcome has been studied in 73 patients. The median OS was 35.6 months. The results of SNP array analysis showed that alterations detected by microarrays had no significant impact on outcome (P=0.237 for OS and P=0.807 for AML evolution), but the median OS is higher in cases with a "real" (neither visible by cytogenetics nor submicroscopic alterations) normal karyotype (53.48 vs. 25.90 months). Of note, the presence of AOH has a strong impact on OS (53.48 vs. 16.13 months, P=0.004), less on AML (P=0.086); also its size, greater than 25Mb, has an impact on OS (P=0.012) and AML evolution (P=0.050).To compensate for differing risk distributions, we performed analyses adjusting for IPSS-R. Adjusted for risk, the microarray result adds prognostic power on OS (P=0.010) and AML evolution (P=0.059). As it was observed, the presence of AOH had prognostic impact on OS (P=0.008) and AML evolution (P=0.017). The affected genome size showed a significant impact on OS (P=0.006) and AML (P=0.024), four categories were defined: 0 Kb, >0-500 Kb, >500-20000 Kb and >20000 Kb.ConclusionsMDS patients with normal karyotypes and alterations detected by genomic microarrays have lower OS and shorter time to AML compared to those without CNA. While not significant in marginal analysis, it can be demonstrated if adjustment for IPSS-R is performed. Alterations and, particularly, AOH have an independent prognostic impact and add prognostic power to IPSS-R.AcknowledgmentsFinancial support: This work was supported, in part, by a grant from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras, Obra Social “La Caixa”, Celgene Spain and NHRI-EX103-10003NI,Taiwan.Special thanks to Dr. Al-Ali, Dr. Bäsecke, members of Grupo Español de SMD (GESMD) and MDS Foundation. DisclosuresNo relevant conflicts of interest to declare.

TSLP-Induced Alterations of Multiple Signaling Pathways in Primary CRLF2 B-ALL Xenografts

B-cell acute lymphoblastic leukemia (B-ALL) with genetic defects leading to overexpression of CRLF2 (CRLF2 B-ALL) is associated with a high relapse rate and poor prognosis. CRLF2 B-ALL comprises approximately half of the high risk B-ALL characterized by a gene expression profile that is similar to that of Philadelphia chromosome-positive ALL (Ph-like B-ALL). In pediatric patients, CRLF B-ALL occurs 5 times more frequently among children of Hispanic and Native American ethnicity and is a major contributor to health disparities in ALL. CRLF2 (cytokine related ligand factor 2) together with the IL-7 receptor alpha chain, forms a receptor complex that is activated by the cytokine, TSLP (Thymic Stromal Lymphopoietin). Activation of CRLF by TSLP leads to downstream JAK-STAT5 and mTOR pathway phosphorylation even in CRLF2 B-ALL harboring activating JAK mutations. We found that primary human marrow (BM) stromal cells express TSLP (RT-PCR and ELISA) and thus provide an in vivo source of TSLP to activate CRLF2 B-ALL cells. Our next step was to develop a xenograft model system to identify the in vivo CRLF2-mediated gene expression profile and disease mechanisms that might contribute to poor prognosis. Unlike most other cytokines, mouse TSLP is species-specific and thus does not activate the human CRLF2 receptor complex. We engineered immune-deficient NOD/SCID IL-2Rγ null (NSG) mice to express normal serum levels (~20 pg/ml) of human TSLP (hTSLP+ mice), as well as control mice that lack human TSLP (hTSLP– mice). Primary human CRLF2 B-ALL were injected into hTSLP+ and hTSLP– mice and expanded for 10 weeks in vivo. Whole genome microarray was performed on CRLF2 B-ALL cells isolated by magnetic separation from the BM of hTSLP+ and hTSLP- xenograft mice. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in hTSLP+ as compared to hTSLP- mice, confirming hTSLP activity in the hTSLP+ xenograft mice. Microarray identified 280 genes that are upregulated and 281 genes that are downregulated (> 1.7 fold; p<.05) in vivo in leukemia cells from hTSLP+ as compared to hTSLP– mice. GSEA and Ingenuity Pathway Analysis of these data show increased RAS pathway activation and altered glucose metabolism in CRLF2 B-ALL from hTSLP+ as compared to hTSLP– mice. In addition to increased mTOR pathway activation, these gene expression data implicate altered glucose metabolism and increased RAS pathway activation as potential contributors to the poor prognosis in CRLF2 B-ALL. The hTSLP+ CRLF2 B-ALL xenograft mice described here provide a novel preclinical model for studying disease mechanisms and identifying therapies to target signaling pathways activated by TSLP in CRLF2 B-ALL. DisclosuresNo relevant conflicts of interest to declare.

Prostaglandin E Synthase Is Upregulated By Gas6 during Cancer-Induced Venous Thromboembolism

Venous thromboembolism (VTE) is the most common morbid complication related to cancer and its treatments. Although malignancies are characterized by a hypercoagulable state leading to VTE, the pathophysiology of this state has not been well studied. Growth arrest specific 6 (Gas6) is a protein that has pro-coagulant properties. Gas6 deficient mice develop smaller venous thrombi as compared to wild type mice, and express less tissue factor in the endothelium when challenged with thrombotic stimuli. We hypothesize that Gas6 may be involved in cancer-induced venous thrombosis. In order to test this hypothesis, venous thrombi were induced in wild type (WT) and Gas6 null (-/-) mice injected with M27 murine lung cancer cell lines. Thrombus size was measured using ultrasonography, thrombus weight and histology. We observed that WT mice with cancer developed larger thrombi than their healthy counterparts (p<0.05). However, these larger thrombi induced by cancer were not seen in Gas6-/- mice, suggesting that Gas6 has a pathophysiologic role in promoting malignancy associated VTE. Whole genome microarray analysis was then used to identify differential gene expression in WT and Gas6-/- endothelial cells co-cultured with M27 murine lung carcinoma cells. Microarray analysis revealed that prostaglandin E synthase (PTGES) was increased in WT endothelial cells but not in Gas6-/- cells co-cultured with M27. These results were confirmed using real-time PCR and immunofluorescence staining (p<0.05). In WT endothelial cells, PTGES expression was regulated through ERK1/2 phosphorylation. We also show that co-culture of WT endothelial cells with M27 augments the secretion of PGE2, the enzymatic product of PTGES. PGE2 activates platelets in vitro after binding to its receptor, EP3. In vivo, EP3 receptor antagonism reversed the effect of cancer-induced thrombosis in WT mice. These results show that Gas6, through upregulation of PGE2, contributes to cancer-induced VTE. DisclosuresNo relevant conflicts of interest to declare.

Antitumor effects of tyropeptin‐boronic acid derivatives. Gene expression analyses of RPMI8226 cells treated with tyropeptin‐boronic acid derivatives using Agilent human whole genome microarray

Antitumor effects of tyropeptin‐boronic acid derivatives. Gene expression analyses of RPMI8226 cells treated with tyropeptin‐boronic acid derivatives using Agilent human whole genome microarray