Variant profiles of genes mapping to chromosome 16q loss in Wilms tumors reveals link to cilia-related genes and pathways.

BACKGROUND: Wilms tumor is the most common pediatric renal tumor and the fourth most common malignancy in children. Chromosome 16q deletion(del) or loss of heterozygosity (LOH) has been correlated with recurrence and overall poor prognosis, such that patients with 16qLOH and 1p allelic loss are treated with more aggressive chemotherapeutic regimens. METHODS: In the present study, we have compared the variant profiles of Wilms tumors with and without 16q del/LOH using both data available from the TARGET database (42 samples) and tumors procured from our legacy collection (8 samples). Exome-Seq data was analyzed for tumor specific variants mapping to 16q. Whole exome analysis was also performed. An unbiased approach for somatic variant analysis was used to detect tumor-specific, somatic variants. RESULTS: Of the 72 genes mapping to 16q, 42% were cilia-related genes and 28% of these were found to carry somatic variants specific to those tumors with 16qdel/LOH. Whole exome analyses further revealed that 30% of cilia-related genes across the genome carried alterations in tumors both with and without 16qdel/LOH. Additional pathway analyses revealed that many cilia-related pathway members also carried deleterious variant in these tumors including Sonic Hedgehog (SHh), Wnt, and Notch signaling pathways. CONCLUSIONS: The data suggest that cilia-related genes and pathways are compromised in Wilms tumors. The genes on chromosome 16q that carry deleterious variants in cilia-related genes may account for the more aggressive nature of tumors with 16q del/LOH.

Inactivation of Lgi1 in murine neuronal precursor cells leads to dysregulation of axon guidance pathways.

LGI1 mutations predispose to a rare epilepsy syndrome and when inactivated in mice leads to early onset seizures and premature death. Histopathology of the mature brain soon after birth shows cortical dysplasia in Lgi1 null mice with hypercellularity in the outer cortical layers. Here we show extensive gene expression changes in neuronal precursor cells from Lgi1 null mice compared with wild type mice. The most significantly dysregulated pathway involves canonical axon guidance signaling with multiple networks involved in cell movement, adhesion and invasion related to actin cytoskeleton reorganization. The Lgi1 null NPCs show increased cell motility in vitro compared with normal counterparts. Dysregulation of genes critical to cell movement/migration and critical transcription factors involved in early neuronal development is a prominent feature. These studies provide a critical mechanistic link to the observation of increased cellularity in the outer layers of the developing cortex in Lgi1 null mice.

Distinct signaling programs associated with progression of FGFR1 driven leukemia in a mouse model of stem cell leukemia lymphoma syndrome.

Constitutive activation of FGFR1 as a result of chromosome translocations is responsible for the development of a hematopoietic stem cell disorder that progresses to AML. We have developed a syngeneic mouse model of BCR-FGFR1 driven AML and used RNASeq to define gene expression signatures associated with disease progression. The development of the leukemic stem cells (LSC) is associated with a profound downregulation of specific transcription factors that normally maintain stem cell quiescence as well as cell adhesion and motility gene sets related to confinement to the stem cell niche. A prominent feature of the LSCs is the upregulation of genes involved in T-cell function, activation, migration and development. Despite this apparent T-cell priming in the LSCs, however, the majority of these genes are subsequently inactivated in the leukemic blast cells that derive from them. These studies provide insights into the molecular etiology of development and progression of FGFR1 driven AML.

Genomic analysis of racial differences in triple negative breast cancer.

Triple negative breast cancer (TNBC) is more prevalent in African Americans (AAs), has a more aggressive clinical course including a higher mortality rate and an increased occurrence of metastases. This study was designed to determine if racial differences at the molecular level might explain the more aggressive phenotype in AAs. Mutation profiling, was performed on 51 AA and 77 CA tumor/ normal pairs. Transcript expression analysis was performed on 35AA and 37CA. Genes with high frequency mutation rates such as MUC4 and TP53 were common to both racial populations, however genes that were less frequently mutated differed between the races suggesting that those cause the more aggressive nature of TNBC in AA women. JAK-Stat and HER2 signaling were unique to the AA and PTEN and mTOR were unique to the CA profiles. Many pathways identified by the mutational profiles were predicted to be down-regulated by the transcript expression profiles.

miR-339 Promotes Development of Stem Cell Leukemia/Lymphoma Syndrome via Downregulation of the BCL2L11 and BAX Proapoptotic Genes.

The development of myeloid and lymphoid neoplasms related to overexpression of FGFR1 kinases as a result of chromosome translocations depends on the promotion of a stem cell phenotype, suppression of terminal differentiation, and resistance to apoptosis. These phenotypes are related to the stem cell leukemia/lymphoma syndrome (SCLL), which arises through the effects of the activated FGFR1 kinase on gene transcription, which includes miRNA dysregulation. In a screen for miRNAs that are directly regulated by FGFR1, and which stimulate cell proliferation and survival, we identified miR-339-5p, which is highly upregulated in cells carrying various different chimeric kinases. Overexpression of miR-339-5p in SCLL cell types enhances cell survival and inhibition of its function leads to reduced cell viability. miR-339-5p overexpression protects cells from the consequences of FGFR1 inactivation, promoting cell-cycle progression and reduced apoptosis. Transient luciferase reporter assays and qRT-PCR detection of endogenous miR-339-5p expression in stably transduced cell lines demonstrated that BCR-FGFR1 can directly regulate miR-339-5p expression. This correlation between miR-339-5p and FGFR1 expression is also seen in primary human B-cell precursor acute lymphoblastic leukemia. In a screen to identify targets of miR-339-5p, we identified and verified the BCL2L11 and BAX genes, which can promote apoptosis. In vivo, SCLL cells forced to overexpress miR-339-5p show a more rapid onset of disease and poorer survival compared with parental cells expressing endogenous levels of miR-339-5p. Analysis of human primary B-cell precursor ALL shows a significant higher expression of miR339-5p compared with the two cohorts of CLL patient samples, suggesting direct roles in disease progression and supporting the evidence generated in mouse models of SCLL.Significance: Proapoptiotic genes that are direct targets of miR-339-5p significantly influence promotion and aggressive development of leukemia/lymphomas associated with FGFR1 overexpression. Cancer Res; 78(13); 3522-31. ©2018 AACR.

The niacin/butyrate receptor GPR109A suppresses mammary tumorigenesis by inhibiting cell survival.

GPR109A, a G-protein-coupled receptor, is activated by niacin and butyrate. Upon activation in colonocytes, GPR109A potentiates anti-inflammatory pathways, induces apoptosis, and protects against inflammation-induced colon cancer. In contrast, GPR109A activation in keratinocytes induces flushing by activation of Cox-2-dependent inflammatory signaling, and the receptor expression is upregulated in human epidermoid carcinoma. Thus, depending on the cellular context and tissue, GPR109A functions either as a tumor suppressor or a tumor promoter. However, the expression status and the functional implications of this receptor in the mammary epithelium are not known. Here, we show that GPR109A is expressed in normal mammary tissue and, irrespective of the hormone receptor status, its expression is silenced in human primary breast tumor tissues, breast cancer cell lines, and in tumor tissues of three different murine mammary tumor models. Functional expression of this receptor in human breast cancer cell lines decreases cyclic AMP production, induces apoptosis, and blocks colony formation and mammary tumor growth. Transcriptome analysis revealed that GPR109A activation inhibits genes, which are involved in cell survival and antiapoptotic signaling, in human breast cancer cells. In addition, deletion of Gpr109a in mice increased tumor incidence and triggered early onset of mammary tumorigenesis with increased lung metastasis in MMTV-Neu mouse model of spontaneous breast cancer. These findings suggest that GPR109A is a tumor suppressor in mammary gland and that pharmacologic induction of this gene in tumor tissues followed by its activation with agonists could be an effective therapeutic strategy to treat breast cancer.

Molecular mechanism of SLC5A8 inactivation in breast cancer.

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS(G12V)) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.

A systematic evaluation of miRNA:mRNA interactions involved in the migration and invasion of breast cancer cells.

In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the invasiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in invasive cell lines when compared to normal and less invasive cell lines. Transfection of miR-200c, miR-205, and miR-375 mimics into MDA-MB-231 cells led to the inhibition of in vitro cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that miR-200c plays a pivotal role in regulating the invasiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer invasiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

Analysis of wilms tumors using SNP mapping array-based comparative genomic hybridization.

Wilms tumor (WT) has been a model to study kidney embryogenesis and tumorigenesis and, although associated with hereditary, cancer predisposition syndromes, the majority of tumors occur sporadically. To analyze genetic changes in WT we have defined copy number changes and loss of heterozygosity in 56 Wilms tumors using high resolution oligonucleotide arrays at a average resolution of ~12 Kb. Consistent deletions were seen on chromosomes 1p, 4q, 7p, 9q, 11p, 11q, 14q, 16q, and 21q. High frequency gains were seen for 1q and lower frequency gains were seen on 7q and chromosomes 8, 12 and 18. The high resolution provided by the SNP mapping arrays has defined minimal regions of deletion for many of these LOH events. Analysis of CNAs by tumor stage show relatively stable karyotypes in stage 1 tumors and more complex aCGH profiles in tumors from stages 3-5.

Integration of transcript expression, copy number and LOH analysis of infiltrating ductal carcinoma of the breast.

BACKGROUND: A major challenge in the interpretation of genomic profiling data generated from breast cancer samples is the identification of driver genes as distinct from bystander genes which do not impact tumorigenesis. One way to assess the relative importance of alterations in the transcriptome profile is to combine parallel analyses that assess changes in the copy number alterations (CNAs). This integrated analysis permits the identification of genes with altered expression that map within specific chromosomal regions which demonstrate copy number alterations, providing a mechanistic approach to identify the 'driver genes'. METHODS: We have performed whole genome analysis of CNAs using the Affymetrix 250K Mapping array on 22 infiltrating ductal carcinoma samples (IDCs). Analysis of transcript expression alterations was performed using the Affymetrix U133 Plus2.0 array on 16 IDC samples. Fourteen IDC samples were analyzed using both platforms and the data integrated. We also incorporated data from loss of heterozygosity (LOH) analysis to identify genes showing altered expression in LOH regions. RESULTS: Common chromosome gains and amplifications were identified at 1q21.3, 6p21.3, 7p11.2-p12.1, 8q21.11 and 8q24.3. A novel amplicon was identified at 5p15.33. Frequent losses were found at 1p36.22, 8q23.3, 11p13, 11q23, and 22q13. Over 130 genes were identified with concurrent increases or decreases in expression that mapped to these regions of copy number alterations. LOH analysis revealed three tumors with whole chromosome or p arm allelic loss of chromosome 17. Genes were identified that mapped to copy neutral LOH regions. LOH with accompanying copy loss was detected on Xp24 and Xp25 and genes mapping to these regions with decreased expression were identified. Gene expression data highlighted the PPARα/RXRα Activation Pathway as down-regulated in the tumor samples. CONCLUSION: We have demonstrated the utility of the application of integrated analysis using high resolution CGH and whole genome transcript analysis for detecting driver genes in IDC. The high resolution platform allowed a refined demarcation of CNAs and gene expression profiling provided a mechanism to detect genes directly impacted by the CNA. This is the first report of LOH integrated with gene expression in IDC using a high resolution platform.

Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease.

BACKGROUND: Studies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing. RESULTS: We present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells. CONCLUSIONS: The findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.

Copy number and gene expression alterations in radiation-induced papillary thyroid carcinoma from chernobyl pediatric patients.

BACKGROUND: Following exposure to radiation during the Chernobyl fallout tragedy, papillary thyroid carcinoma (PTC) increased significantly in individuals who were children at the time of the accident. We have used two high-throughput, whole genome platforms to analyze radiation-induced PTCs from pediatric patients from the Chernobyl region. METHODS: We performed comparative genomic hybridization using Affymetrix 50K Mapping arrays and gene expression profiling on 10 pediatric post-Chernobyl PTCs obtained from patients living in the region. We performed an overlay analysis of these two data sets. RESULTS: Many regions of copy number alterations (CNAs) were detected including novel regions that had never been associated with PTCs. Increases in copy numbers were consistently found on chromosomes 1p, 5p, 9q, 12q, 13q, 16p, 21q, and 22q. Deletions were observed less frequently and were mapped to 1q, 6q, 9q, 10q, 13q, 14q, 21q, and 22q. Gene expression analysis revealed that most of the altered genes were also perturbed in sporadic adult PTC; however, 141 gene expression changes were found to be unique to the post-Chernobyl tumors. The genes with the highest increases in expression that were novel to the pediatric post-Chernobyl tumors were TESC, PDZRN4, TRAa/TRDa, GABBR2, and CA12. The genes showing the largest expression decreases included PAPSS2, PDLIM3, BEXI, ANK2, SORBS2, and PPARGCIA. An overlay analysis of the gene expression and CNA profiles was then performed. This analysis identified genes showing both CNAs and concurrent gene expression alterations. Many of these are commonly seen in sporadic PTC such as SERPINA, COL8A, and PDX, while others were unique to the radiation-induced profiles including CAMK2N1, AK1, DHRS3, and PDE9A. CONCLUSIONS: This type of analysis allows an assessment of gene expression changes that are associated with a physical mechanism. These genes and chromosomal regions are potential markers for radiation-induced PTC.

Interpreting aCGH-defined karyotypic changes in gliomas using copy number status, loss of heterozygosity and allelic ratios.

We have used SNP mapping arrays to simultaneously record copy number changes, loss of heterozygosity and allele ratios (ploidy) in a series of 13 gliomas. This combined analysis has defined novel amplification events in this tumor type involving chr1:241544532-243005121 and chr18:54716681-54917277 which contain the AKT3 and ZNF532 genes, respectively. The high resolution of this analysis has also identified homozygous deletions involving chr17:25600031-26490848 and Chr19:53883612-55061878. Throughout the karyotypes of these tumors, the combined analysis revealed counter intuitive relationships between copy number and LOH that requires reinterpretation of the significance of copy number gains and losses. It was not uncommon to observe copy number gains that were associated with loss of heterozygosity as well as copy number losses that were not. These events appeared to be related to ploidy status in the tumors as determined using allelic ratio calculations. Overall, this analysis of gliomas provides evidence for the need to perform more comprehensive interpretation of the CGH data beyond copy number analysis alone to evaluate the significance of individual events in the karyotypes.

Reexpression of LGI1 in glioma cells results in dysregulation of genes implicated in the canonical axon guidance pathway.

The LGI1 gene suppresses invasion in glioma cells and predisposes to epilepsy. In a gene expression array comparison between parental cells and T98G cell clones forced to express LGI1, we demonstrate that the canonical axon guidance pathway is the most significantly affected. In particular, aspects of axon guidance that involve reorganization of the actin cytoskeleton, which is also involved in cell movement and invasion, were affected. Analysis of actin fiber organization using fluorescence microscopy demonstrated that different T98G cell clones expressing the exogenous LGI1 gene show high levels of stress fibers compared with controls. Since stress fiber formation is associated with loss of cell mobility, we used scratch wound assays to demonstrate that LGI1-expressing clones show a significant reduction in cell mobility. LGI1 reexpression also resulted in loss of the PDGFRA and EGFR proteins, suggesting a rapid turnover of these receptors despite increased mRNA levels for PDGFRA. LGI1 suppression of invasion is associated with loss of ERK/MAPK1 activation. LGI1 is a secreted protein, and when the culture supernatant from cells expressing FLAG- and GFP-tagged proteins were applied to parental T98G cells, ERK/MAPK1 phosphorylation and cell mobility was suppressed, demonstrating that the LGI1 protein acts as a suppressive agent for cell movement in this assay. These observations support a previous suggestion that LGI1 can reduce cellular invasion in in vitro assays and, as a secreted agent, may be developed as a means of treating metastatic cancer. In addition, this observation provides a mechanistic link for LGI1's common role in metastasis and epilepsy development.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.

BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

Universal Reference RNA is not a representative normal sample for oligonucleotide microarray studies.

Translational research has been defined as the scientific study using human material that will ultimately generate patient specific data. A major caveat in human directed study is the availability of high quality and quantities of patient derived homogeneous cells for analysis. Whereas there exist sources for which tumor tissue and blood samples can be made available, the same cannot be said for normal tissue. The absence of normal control tissue has led to the creation of pooled cell lines and tissues for purchase known as "reference RNA". Although initially created for purposes of standardization, the difficulty associated with acquiring normal tissue has led some investigators to use sources of universal pooled RNA for comparative analysis with clinical tissue specimens. In order to study the effects of using Universal Reference RNA on expression profiling experiments we have evaluated the performance of universal RNA compared to RNA obtained from a purified population of colon epithelial cells in defining a set of altered transcripts in colon cancer.

Identification of genes involved in squamous cell carcinoma of the lung using synchronized data from DNA copy number and transcript expression profiling analysis.

Lung cancer is the leading cause of cancer deaths in the world and squamous cell carcinoma (SqCC) is the second most common in this group. Genomic DNA copy number alterations are fundamental genetic events in the development and progression of SqCC as well as other epithelial-derived cancers. The ability to identify tumor suppressor genes (TSGs) and oncogenes that are affected during tumor initiation and progression could facilitate the identification of novel molecular targets for therapeutic intervention and provide diagnostic biomarkers. Despite the association of many genetic alterations in lung cancer the molecular mechanisms of tumor progression remain ambiguous since often too many candidates are revealed using conventional genetic microarray analysis. To overcome this limitation, we have identified genes in SqCC which show concordant gene expression changes defined using microarray analysis with DNA copy number alterations defined by BAC-array comparative genomic hybridization (aCGH) in the same tumors. An in-house overlay algorithm was used to synchronize the data resulting from the two analyses. Although the expression levels of many genes were altered when compared to normal controls, those which correlated with copy number changes were far fewer, providing a manageable number for biological studies. We identified over 2000 genes which displayed both gene expression alterations and mapped to BACs which demonstrated a corresponding loss or gain. A further stringent statistical analysis identified minimal regions of overlap for losses or gains which displayed a coincident decrease or increase in the expression of genes mapping to those regions. Consistent gains involved 3q23-q29, 5p15.1-q11.1 and chromosomes 18 and 20, while consistent losses involved 3p26.3-p12.3, 9p24.3-q34.3, and chromosomes 17 and 19. The concordance finding between these two approaches suggests that DNA copy number alterations can directly influence gene expression patterns that impact on tumorigenesis in SqCC of the lung.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples.

BACKGROUND: Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. METHODS: Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. RESULTS: The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. CONCLUSION: The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.

Candidate glioblastoma development gene identification using concordance between copy number abnormalities and gene expression level changes.

Copy number abnormalities (CNAs) in tumor cells are presumed to affect expression levels of genes located in region of abnormality. To investigate this relationship we have surveyed the losses, gains and amplifications in 30 glioblastomas using array comparative genome hybridization and compared these data with gene expression changes in the same tumors using the Affymetrix U133Plus2.0 oligonucleotide arrays. The two datasets were overlaid using our in-house overlay tool which highlights concordance between CNAs and expression level changes for the same tumors. In this survey we have highlighted genes frequently overexpressed in amplified regions on chromosomes 1, 4, 11, and 12 and have identified novel amplicons on these chromosomes. Deletions of specific regions on chromosomes 9, 10, 11, 14, and 15 have also been correlated with reduced gene expression in the regions of minimal overlap. In addition we describe a novel approach for comparing gene expression levels between tumors based on the presence or absence of chromosome CNAs. This genome wide screen provides an efficient and comprehensive survey of genes which potentially serve as the drivers for the CNAs in GBM.

The application of microarray technology to the analysis of the cancer genome.

The identification of genetic events that are involved in the development of human cancer has been facilitated through the development and application of a diverse series of high resolution, high throughput microarray platforms. Essentially there are two types of array; those that carry PCR products from cloned nucleic acids (e.g. cDNA, BACs, cosmids) and those that use oligonucleotides. Each has advantages and disadvantages but it is now possible to survey genome wide DNA copy number abnormalities and expression levels to allow correlations between losses, gains and amplifications in tumor cells with genes that are over- and under-expressed in the same samples. The gene expression arrays that provide estimates of mRNA levels in tumors have given rise to exon-specific arrays that can identify both gene expression levels, alternative splicing events and mRNA processing alterations. Oligonucleotide arrays are also being used to interrogate single nucleotide polymorphisms (SNPs) throughout the genome for linkage and association studies and these have been adapted to quantify copy number abnormalities and loss of heterozygosity events. To identify as yet unknown transcripts tiling arrays across the genome have been developed which can also identify DNA methylation changes and be used to identify DNA-protein interactions using ChIP on Chip protocols. Ultimately DNA sequencing arrays will allow resequencing of chromosome regions and whole genomes. With all of these capabilities becoming routine in genomics laboratories, the idea of a systematic characterization of the sum genetic events that give rise to a cancer cell is rapidly becoming a reality.