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In JoVE (3)

Other Publications (114)

Articles by Wan L. Lam in JoVE

 JoVE Biology

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

1Department of Cancer Genetics, BC Cancer Research Centre, 2Deeley Research Centre, BC Cancer Agency, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency


JoVE 870

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

 JoVE Biology

Methylated DNA Immunoprecipitation

1Department of Cancer Genetics and Developmental Biology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3These authors contributed equally., 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC, 5Photography/Video Production, Multi-Media Services, BC Cancer Agency, 6Department of Medical Genetics, Life Sciences Institute,, University of British Columbia - UBC


JoVE 935

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

 JoVE Biology

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC


JoVE 2763

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Other articles by Wan L. Lam on PubMed

Two-dimensional Display and Whole Genome Comparison of Bacterial Pathogen Genomes of High G+C DNA Content

High-resolution comparison of bacterial genomes facilitates the identification of the genetic changes responsible for clinically relevant phenotypes. For this purpose we have established a method for the display and comparison of high G+C bacterial genomes in two dimensions. Here we describe the application of two-dimensional bacterial genomic display to resolve the genomes of Bordetella pertussis, Mycobacterium avium and Mycobacterium tuberculosis, and its utility in strain comparison and detection of insertion and substitution mutations.

An Efficient Method for the Assessment of DNA Quality of Archival Microdissected Specimens

There will be an increasing need of methods for assessing the suitability of specimens for genetic-based assays as DNA markers become an integral part of molecular diagnosis. The targeting of specimens for specific analyses will require the ability to rapidly screen for DNA quality. Conventional methods such as Southern analysis and gene specific-polymerase chain reaction (PCR) often require quantities of material that represent a significant portion of the specimen, especially in microdissected samples. Here we describe a novel application of a commonly used PCR-based DNA-fingerprinting technology that requires minimal quantities of DNA to simultaneously assess multiple regions throughout the genome for DNA quality. Randomly amplified polymorphic DNA (RAPD) PCR generates DNA fragments of a broad size range with the product size reflecting the degree of sample fragmentation. Fourteen DNA samples extracted from cells microdissected from seven formalin-fixed, paraffin-embedded oral cancer biopsies were assessed for DNA quality using gene-specific PCR and RAPD-PCR. Although the more conventional assay required 2-ng DNA (or 300-cell equivalents) to examine DNA quality at a single locus, RAPD-PCR provided a more informative profile of DNA quality from the same microdissected archival specimens.

Two-dimensional Bacterial Genome Display: a Method for the Genomic Analysis of Mycobacteria

Annually, Mycobacterium tuberculosis is the cause of approximately three million deaths worldwide. It would appear that currently available therapies for this disease are inadequate. The identification of genes involved in mycobacterial virulence will facilitate the design of new prophylactic and therapeutic interventions. A method for high-resolution comparison of bacterial genomes has been developed to facilitate the identification of genes possibly involved in the virulence of clinically relevant mycobacteria. This 'two-dimensional bacterial genome display' (2DBGD) method utilizes two-dimensional DNA electrophoresis to separate, on the basis of size and G+C content, genomic fragments generated with different restriction endonucleases. The use of this method to identify genomic differences between species, strains and, most importantly, isogenic mutants of mycobacteria is reported. That 2DBGD can be used to identify differences resulting from either insertional mutagenesis using a gentamicin-resistance gene or from a frameshift mutation is demonstrated.

3p14 and 9p21 Loss is a Simple Tool for Predicting Second Oral Malignancy at Previously Treated Oral Cancer Sites

Treatment induces reactive changes that often resemble low-grade dysplasia at former oral cancer sites, complicating histopathological assessment. We tested a set of microsatellite markers shown previously to be predictive of progression for oral premalignant lesions for the ability to predict development of second oral malignancy (SOM). Sixty-eight oral leukoplakia at former cancer sites (with known outcome, 36 progressed to SOM) were evaluated for loss of heterozygosity at 19 loci on seven chromosome arms. 3p and/or 9p loss in these posttreatment leukoplakia was associated with a 26.3-fold increase in risk of developing SOM compared with those that retained both of these arms (P < 0.001), with 60% of cases with loss of heterozygosity developing SOM in 2 years. In contrast, histological diagnosis (moderate or severe dysplasia versus hyperplasia or mild dysplasia) had only a 1.7-fold increase in risk (P = 0.11). The identification of 3p and 9p loss in posttreatment lesions could serve as a simple and direct test for stratifying risk of SOM development.

Characterization of the Recurrent Translocation T(1;1)(p36.3;q21.1-2) in Non-Hodgkin Lymphoma by Multicolor Banding and Fluorescence in Situ Hybridization Analysis

Aberrations of chromosomal bands 1p36 and 1q11-q23 are among the most common chromosomal alterations in non-Hodgkin lymphoma (NHL). In this study, 16 cases of NHL showing recurrent unbalanced translocation t(1;1)(p36;q11-23) by G-band analysis were selected for further analysis. To delineate the exact breakpoints, multicolor band analysis for chromosome 1 (M-BAND1), and locus-specific fluorescence in situ hybridization (LS-FISH) using human genome designated BAC clones were performed. In all but one dicentric case, the breakpoint was determined to involve chromosomal bands 1p36.3 and 1q21.1-2. LS-FISH analysis for the TP73, MEL1, SKI, and CASP9 loci at 1p36, and the loci IRTA1, IRTA2, BCL9, AF1Q, JTB, and MUC1 at 1q21, verified the MBAND1 results and further delineated the breakpoints. In band 1p36, two hybridization patterns were observed, one involving deletions of MEL1, TP73, and SKI, but not CASP9, and the second involving a breakpoint telomeric to TP73. In region 1q21, four hybridization patterns were observed, the first involving duplication/translocation of all five genes; the second involving duplication/translocation of BCL9, AF1Q, JTB, and MUC1; the third involving duplication/translocation of AF1Q, JTB, and MUC1; and the fourth with a breakpoint telomeric to MUC1. Using an alpha-satellite probe for chromosome 1 (D1Z5), centromeric involvement in the unbalanced translocation t(1;1)(p36.3;q21.1-2) was excluded in all but the one dicentric case, that is, dic(1;1)(p36.3;q10). In conclusion, deletion of 1p36 and duplication of 1q21 through formation of an unbalanced translocation t(1;1)(p36.3;q21.1-2) is a non-random event in NHL, suggesting a deletion-duplication mechanism involved in lymphoma progression and justifying further systematic research.

Use of Complete Coverage Array Comparative Genomic Hybridization to Define Copy Number Alterations on Chromosome 3p in Oral Squamous Cell Carcinomas

Loss of 3p has been associated with oral cancer progression and is common in many cancers. However, regions of alteration on 3p are poorly defined. We have constructed a high-resolution chromosomal array using a tiling set of 535 human bacterial artificial chromosomes that provides near complete coverage of 3p. Array comparative genomic hybridization analysis of 20 microdissected oral squamous cell carcinomas showed multiple and recurrent segments of copy number changes. These include a deletion containing the FHIT gene; novel segments of copy decrease at 3p22, 3p24, and 3p26; and an unexpected approximately 0.7 Mbp segmental increase at 3p21. These data strongly support the value of using chromosomal array comparative genomic hybridization for detailed profiling of oral squamous cell carcinomas.

Novel Regions of Amplification on 8q Distinct from the MYC Locus and Frequently Altered in Oral Dysplasia and Cancer

Genetic studies aimed at identifying key alterations in oral cancers have focused on analysis of tumors, with few such studies using early oral premalignant lesions (OPLs) because of limitations in both sample availability and size. In this study, we used a randomly amplified polymorphic DNA (RAPD)-PCR approach to fingerprint DNA from microdissected normal and dysplastic cells and identified two recurrent genetic alterations on the long arm of chromosome 8 in OPLs, one mapping to 8q22 and the other to 8q24 near the MYC locus. We constructed a high-resolution bacterial artificial chromosome (BAC) comparative genomic hybridization array consisting of 166 overlapping BAC clones that spans about 52 Mbp, from 8q21 to 8q24. Hybridization of DNA from microdissected oral tumors to the array revealed alteration at 8q24, with amplification of the BAC containing MYC. Strikingly, at least two other novel regions of amplification at 8q22 were identified. Microsatellite analysis of 93 oral dysplasias and tumors confirmed the presence of one of the alterations at 8q22. Loss of heterozygosity (LOH) at D8S1830, mapping within one of the regions of amplification, was observed in high frequency in both OPLs and tumors. Of the 37 cases with LOH at D8S1830, 23 (62%) showed retention at D8S1793, which maps 1.6 Mbp centromeric to MYC. This is further support for the alteration at 8q22 being distinct from MYC. These data raise the possibility of additional oncogenes on 8q near the MYC locus that are potentially involved in OPL disease progression.

Overexpression of LRP12, a Gene Contained Within an 8q22 Amplicon Identified by High-resolution Array CGH Analysis of Oral Squamous Cell Carcinomas

Chromosome 8q amplification is a common event observed in cancer. In this study, we used high-resolution array comparative genomic hybridization to resolve two neighboring regions on 8q that are both amplified in oral cancer. One region (at 8q24) contains the MYC oncogene, which is frequently overexpressed in many cancers, while the other region (at 8q22) represents a novel amplicon. The alignment of array comparative genomic hybridization profiles of 20 microdissected oral squamous cell carcinomas (OSCCs) revealed a approximately 5 Mbp region of frequent copy number alteration. This region harbors 16 known genes. Gene expression analysis comparing 15 microdissected OSCC with 16 normal epithelium samples revealed overexpression specific to LRP12 but not the neighboring genes, dihydropyrimidinase and FOG2, suggesting that LRP12 may function as an oncogene in oral tumors.

Methods for High Throughput Validation of Amplified Fragment Pools of BAC DNA for Constructing High Resolution CGH Arrays

The recent development of array based comparative genomic hybridization (CGH) technology provides improved resolution for detection of genomic DNA copy number alterations. In array CGH, generating spotting solution is a multi-step process where bacterial artificial chromosome (BAC) clones are converted to replenishable PCR amplified fragments pools (AFP) for use as spotting solution in a microarray format on glass substrate. With completion of the human and mouse genome sequencing, large BAC clone sets providing complete genome coverage are available for construction of whole genome BAC arrays. Currently, Southern hybridization, fluorescent in-situ hybridization (FISH), and BAC end sequencing methods are commonly used to identify the initial BAC clone but not the end product used for spotting arrays. The AFP sequencing technique described in this study is a novel method designed to verify the identity of array spotting solution in a high throughput manner.

A Tiling Resolution DNA Microarray with Complete Coverage of the Human Genome

We constructed a tiling resolution array consisting of 32,433 overlapping BAC clones covering the entire human genome. This increases our ability to identify genetic alterations and their boundaries throughout the genome in a single comparative genomic hybridization (CGH) experiment. At this tiling resolution, we identified minute DNA alterations not previously reported. These alterations include microamplifications and deletions containing oncogenes, tumor-suppressor genes and new genes that may be associated with multiple tumor types. Our findings show the need to move beyond conventional marker-based genome comparison approaches, that rely on inference of continuity between interval markers. Our submegabase resolution tiling set for array CGH (SMRT array) allows comprehensive assessment of genomic integrity and thereby the identification of new genes associated with disease.

OCGR Array: an Oral Cancer Genomic Regional Array for Comparative Genomic Hybridization Analysis

Genetic alterations have been recognized as important events in the carcinogenesis of oral squamous cell carcinoma (OSCC) and have been used as predictors of progression risk. In this study, we have designed an oral cancer-specific human bacterial artificial chromosome (BAC) array, called the oral cancer genomic regional array (OCGR), to detect and fine map copy number alterations in OSCC. This array contains a total of approximately 45 Mbp coverage of nine chromosomal regions reported to be involved in the progression of oral cancer. We demonstrate the detection of copy number alterations in 14 microdissected clinical specimens in each of the nine regions. These include both copy number increases and decreases. Although the number of regions selected for this first generation array is small, we observed multiple segmental changes. In some cases, we observed single BAC clone alterations at 7p11 and 11q13 which contain EGFR and cyclin D1 respectively highlighting the need for high resolution detection techniques. Array comparative genomic hybridization (CGH) complements traditional methods for detecting genetic alterations in OSCC (such as microsatellite and CGH analysis) by improving the detection of segmental copy number alterations to single BAC clone resolution. This work represents the first attempt at the construction of an oral cancer-specific CGH array.

Delineation of a Minimal Region of Deletion at 6q16.3 in Follicular Lymphoma and Construction of a Bacterial Artificial Chromosome Contig Spanning a 6-megabase Region of 6q16-q21

Regional deletions of 6q are frequent karyotypic alterations in malignant lymphoma and are associated with an adverse clinical outcome. One such region of recurrent deletion is 6q16-q21; however, the specific genes affected have not been identified. Our objective in this study was to identify cases with deletion of 6q16-q21 in follicular lymphoma and to define a minimal region of deletion. A physical map of 6q16.2-q21 was constructed using map information from both sequence-based and bacterial artificial chromosome (BAC) fingerprint-based maps. Forty-three BAC clones spanning a 6-Mb region of 6q16.2-q21 were identified and obtained from the RP-11 library. Selected BACs were fluorescence-labeled and hybridized to a series of 34 follicular lymphomas with a regional 6q deletion detected by G banding. Twenty-four cases with deletion of the 6q16.3 region were detected. A minimal deletion of 2.3 Mb was defined. Our study has identified a limited region of deletion of 6q16.3 that may implicate four known genes in follicular lymphoma and possibly in other cancers. A BAC contig spanning a 6-Mb region has been anchored to the 6q16.2-q21 region. This map represents a useful resource for gene identification in this region, not only in lymphoma but also in other neoplasms with 6q alterations.

Genetic Alteration and Gene Expression Modulation During Cancer Progression

Cancer progresses through a series of histopathological stages. Progression is thought to be driven by the accumulation of genetic alterations and consequently gene expression pattern changes. The identification of genes and pathways involved will not only enhance our understanding of the biology of this process, it will also provide new targets for early diagnosis and facilitate treatment design. Genomic approaches have proven to be effective in detecting chromosomal alterations and identifying genes disrupted in cancer. Gene expression profiling has led to the subclassification of tumors. In this article, we will describe the current technologies used in cancer gene discovery, the model systems used to validate the significance of the genes and pathways, and some of the genes and pathways implicated in the progression of preneoplastic and early stage cancer.

SeeGH--a Software Tool for Visualization of Whole Genome Array Comparative Genomic Hybridization Data

Array comparative genomic hybridization (CGH) is a technique which detects copy number differences in DNA segments. Complete sequencing of the human genome and the development of an array representing a tiling set of tens of thousands of DNA segments spanning the entire human genome has made high resolution copy number analysis throughout the genome possible. Since array CGH provides signal ratio for each DNA segment, visualization would require the reassembly of individual data points into chromosome profiles.

Serial Analysis of Gene Expression Profiles of Developmental Stages in Non-small Cell Lung Carcinoma

Comprehensive Whole Genome Array CGH Profiling of Mantle Cell Lymphoma Model Genomes

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma with median patient survival times of approximately 3 years. Although the characteristic t(11;14)(q13;q32) is found in virtually all cases, experimental evidence suggests that this event alone is insufficient to result in lymphoma and secondary genomic alterations are required. Using a newly developed DNA microarray of 32 433 overlapping genomic segments spanning the entire human genome, we can for the first time move beyond marker based analysis and comprehensively search for secondary genomic alterations concomitant with the t(11;14) in eight commonly used cell models of MCL (Granta-519, HBL-2, NCEB-1, Rec-1, SP49, UPN-1, Z138C and JVM-2). Examining these genomes at tiling resolution identified an unexpected average of 35 genetic alterations per cell line, with equal numbers of amplifications and deletions. Recurrent high-level amplifications were identified at 18q21 containing BCL2, and at 13q31 containing GPC5. In addition, a recurrent homozygous deletion was identified at 9p21 containing p15 and p16. Alignment of these profiles revealed 14 recurrent losses and 21 recurrent gains as small as 130 kb. Remarkably, even the intra immunoglobulin gene deletions at 2p11 and 22q11 were detected, demonstrating the power of combining the detection sensitivity of array comparative genomic hybridization (CGH) with the resolution of an overlapping whole genome tiling-set. These alterations not only coincided with previously described aberrations in MCL, but also defined 13 novel regions. Further characterization of such minimally altered genomic regions identified using whole genome array CGH will define novel dominant oncogenes and tumor suppressor genes that play important roles in the pathogenesis of MCL.

High-resolution Chromosome Arm 5p Array CGH Analysis of Small Cell Lung Carcinoma Cell Lines

Genomic amplification of regions on chromosome arm 5p has been observed frequently in small cell lung cancer (SCLC), implying the presence of multiple oncogenes on this arm. Although conventional comparative genomic hybridization (CGH) detects gross chromosomal copy number changes, gene discovery requires a higher-resolution approach in order to identify regions of alteration precisely. To identify candidate genes on this chromosome arm, we developed a high-resolution, 10-clone-per-megabase bacterial artificial chromosome CGH array for 5p and examined a panel of 15 SCLC cell lines. Utilization of this CGH array has allowed the fine-mapping of breakpoints to regions as small as 200 kb in a single experiment. In addition to reporting our observations of aberrations at the well-characterized SKP2 and TERT loci, we describe the identification of microdeletions that have escaped detection by conventional screens and the identification TRIO and ANKH as novel putative oncogenes.

Involvement of Multiple Developmental Genes on Chromosome 1p in Lung Tumorigenesis

Lung cancer is the leading cause of cancer death in North America. Despite advances in lung cancer treatment, the overall 5 year survival rate for those diagnosed with the disease is bleak presumably due to the late stage of diagnosis. Owing to the difficulty of early detection, preneoplastic specimens are rare. However, studying both preinvasive and invasive stages of disease is necessary to fully understand lung cancer progression. Aberration of chromosome arm 1p is common in lung and other cancers. In this study, we used a genomic array with complete tiling coverage of 1p to profile preinvasive and invasive squamous non-small cell lung carcinoma samples. With this technology, multiple novel submegabase alterations were identified. Three of the 1p alterations harbored genes belonging to gene families known to be involved in cancer development through either the Wnt or the Notch developmental pathways. Our finding of a 0.4 Mb amplified region at 1p36.12 containing WNT4 in preinvasive lung cancer, coupled with the identification of three additional alterations in invasive tumors that also contain genes related to the Notch and Wnt pathways, strongly suggests an intricate role of these pathways in early and late stages of lung cancer development. Furthermore, ectopic expression of DVL1, LRP8 and Notch2 in malignant lung tissue validates the biological impact of these genetic alterations. Importantly, this implication of pathways known only to be activated in fetal lung development lends support to the proposed model of lung cancer ontology whereby tumors arise from dysregulated pleuripotent stem cells.

An Integrated MBAND and Submegabase Resolution Tiling Set (SMRT) CGH Array Analysis of Focal Amplification, Microdeletions, and Ladder Structures Consistent with Breakage-fusion-bridge Cycle Events in Osteosarcoma

Osteosarcoma (OS) is characterized by chromosomal instability and high-copy-number gene amplification. The breakage-fusion-bridge (BFB) cycle is a well-established mechanism of genomic instability in tumors and in vitro models used to study the origins of complex chromosomal rearrangements and cancer genome amplification. However, until now, there have been no high-resolution cytogenetic or genomic array studies of BFB events in OS. In the present study, multicolor banding (mBAND) FISH and submegabase resolution tiling set (SMRT) array comparative genomic hybridization (CGH) were used to identify and map genomic signatures of BFB events in four OS cell lines and one patient tumor. The expected intermediates associated with BFB-dicentric chromosomes, inverted duplications, and intra- and interchromosomal amplifications-were identified. mBAND analysis provided detailed mapping of rearrangements in 1p, 6p, and 8q and showed that translocation junctions were often in close proximity to fragile sites. More detailed mBAND studies of OS cell line MG-63 revealed ladderlike FISH signals of equally spaced interchromosomal coamplifications of 6p21, 8q24, and 9p21-p22 in a homogeneously staining region (hsr). Focal amplifications that concordantly mapped to the hsr were localized to discrete genomic intervals by SMRT array CGH. The complex amplicon structure in this hsr suggests focal amplifications immediately adjacent to microdeletions. Moreover, the genomic regions in which there was deletion/amplification had a preponderance of fragile sites. In summary, this study has provided further support for the role of the BFB mechanism and fragile sites in facilitating gene amplification and chromosomal rearrangement in OS.

Alteration of AKAP220, an Upstream Component of the Rb Pathway, in Oral Carcinogenesis

Few genes have been implicated in the development of oral cancer. In our study, we identified a novel gene in the Rb pathway that is frequently altered and overexpressed in oral tumors. Significantly, the alteration is also associated with early oral premalignant lesions (OPLs). This region was identified through a genomewide scan using randomly amplified polymorphic DNA (RAPD) PCR of 40 microdissected oral squamous cell carcinomas (SCCs). Recurrent gain of a approximately 400 bp signal was observed in multiple patients. This gain was localized to 13q14.11, a region frequently altered in multiple cancer types. Through microsatellite analysis, a 1.9 Mbp minimal region of alteration (MRA) was defined between D13S263 and D13S1227. Allelic imbalance (AI) in the MRA was present in only 28% of low-grade dysplasia, but strikingly increased with progression to 64% in high-grade dysplasia, plateauing at 61% in tumors, thus implicating this alteration in the early stages of disease development. Of the 3 genes residing within the MRA, Receptor Activator of NK-kappa-B Ligand (RANKL) and Diacylglycerol Kinase (DGKH) showed no change in expression levels in tumors compared to normal tissue. In contrast, 12 of 16 tumors showed significant overexpression of A-Kinase Anchoring Protein 220 (AKAP220). Since AKAP220 plays a role in regulating the Rb pathway, its dysregulation may contribute significantly to alterations in cell cycle regulation that facilitate progression of OPLs.

Array CGH Technologies and Their Applications to Cancer Genomes

Cancer is a disease characterized by genomic instability. Comparative genomic hybridization (CGH) is a technique designed for detecting segmental genomic alterations. Recent advances in array-based CGH technology have enabled examination of chromosomal regions in unprecedented detail, revolutionizing our understanding of tumour genomes. A number of array-based technologies have been developed, aiming to improve the resolution of CGH, enabling researchers to refine and define regions in the genome that may be causal to cancer, and facilitating gene discovery at a rapid rate. This article reviews the various array CGH platforms and their use in the study of cancer genomes. In addition, the need for high-resolution analysis is discussed as well as the importance of studying early-stage disease to discover genetic alterations that may be causal to cancer progression and aetiology.

Chromosome 5p Aberrations Are Early Events in Lung Cancer: Implication of Glial Cell Line-derived Neurotrophic Factor in Disease Progression

Lung cancer is the most widely diagnosed malignancy in the world. Understanding early-stage disease will give insight into its pathogenesis. Despite the fact that pre-invasive lesions are challenging to isolate, and often yield insufficient DNA for the analysis of multiple loci, genomic profiling of such lesions will lead to the discovery of causal genetic alterations, which may be otherwise masked by the gross instability associated with tumors. In this study, we report the identification of multiple early genetic events on chromosome 5p in lung cancer progression. Using a high-resolution 5p-specific genomic array, which contains a tiling path of DNA segments for comparative genomic hybridization, nine novel minimal regions of loss and gain were discovered in bronchial carcinoma in situ (CIS) specimens. Within these regions we identified two candidate genes novel to lung cancer. The 0.27 Mbp region at 5p15.2 contains a single gene, Triple Functional Domain, which we determined to be differentially expressed in tumors. The 0.34 Mbp region at 5p13.2 contains Glial Cell Line-Derived Neurotrophic Factor (GDNF), which is a ligand for the RET oncogene product and is normally expressed during lung development (but absent in adult lung tissue). Our data showed not only that GDNF is overexpressed at the transcript level in squamous non-small-cell lung carcinoma, but also that the GDNF protein is present in early-stage lesions. Reactivation of the fetal lung expressed GDNF in early lesions and its amplification in CIS suggests an early role in tumorigenesis. These results highlight the value of examining the genomes of pre-invasive stages of cancer at tiling resolution.

High-resolution Array CGH Increases Heterogeneity Tolerance in the Analysis of Clinical Samples

Recent advances in array comparative genomic hybridization (array CGH) technology are revolutionizing our understanding of tumor genomes. Marker-based arrays enable rapid survey at megabase intervals, while tiling path arrays examine the entire genome in unprecedented detail. Tumor biopsies are typically small and contain infiltrating stromal cells, requiring tedious microdissection. Tissue heterogeneity is a major barrier to high-throughput profiling of tumor genomes and is also an important consideration for the introduction of array CGH to clinical settings. We propose that increasing array resolution will enhance detection sensitivity in mixed tissues and as a result significantly reduce microdissection requirements. In this study, we first simulated normal cell contamination to determine the heterogeneity tolerance of array CGH and then validated this detection sensitivity model on cancer specimens using the newly developed submegabase resolution tiling-set (SMRT) array, which spans the human genome with 32,433 overlapping BAC clones.

Chromosome-wide and Promoter-specific Analyses Identify Sites of Differential DNA Methylation in Normal and Transformed Human Cells

Cytosine methylation is required for mammalian development and is often perturbed in human cancer. To determine how this epigenetic modification is distributed in the genomes of primary and transformed cells, we used an immunocapturing approach followed by DNA microarray analysis to generate methylation profiles of all human chromosomes at 80-kb resolution and for a large set of CpG islands. In primary cells we identified broad genomic regions of differential methylation with higher levels in gene-rich neighborhoods. Female and male cells had indistinguishable profiles for autosomes but differences on the X chromosome. The inactive X chromosome (Xi) was hypermethylated at only a subset of gene-rich regions and, unexpectedly, overall hypomethylated relative to its active counterpart. The chromosomal methylation profile of transformed cells was similar to that of primary cells. Nevertheless, we detected large genomic segments with hypomethylation in the transformed cell residing in gene-poor areas. Furthermore, analysis of 6,000 CpG islands showed that only a small set of promoters was methylated differentially, suggesting that aberrant methylation of CpG island promoters in malignancy might be less frequent than previously hypothesized.

Multiple Microalterations Detected at High Frequency in Oral Cancer

The development of array comparative genomic hybridization (array CGH) at tiling-path resolution has enabled the detection of gene-sized segmental DNA copy number gains and losses. Here, we present the first application of whole genome tiling-path array CGH to archival clinical specimens for the detailed analysis of oral squamous cell carcinomas (OSCC). We describe the genomes of 20 OSCCs as well as a selection of matched normal DNA in unprecedented detail. Examination of their whole genome profiles enabled the identification of alterations ranging in size from whole-arm, segmental, to gene size alterations. Tiling-path resolution enabled the detection of many more alterations within each tumor than previously reported, many of which include narrow alterations found to be frequent events among the 20 OSCCs. We report the presence of several novel frequent submegabase alterations, such as the 0.58 Mb gain at 5p15.2 containing triple functional domain (TRIO), detected in 45% of cases. We also report the first coamplification of two gene clusters, by fine-mapping the precise base pair boundaries of the high-level amplification at 11q22.2-22.3 containing both matrix metalloproteinase and baculoviral IAP repeat-containing protein 2 (BIRC) gene clusters. These results show the large improvement in detection sensitivity and resolution compared with genome interval marker arrays and the utility of tiling resolution array CGH for the detection of both submegabase and single copy gains and losses in cancer gene discovery.

Toluidine Blue Staining Identifies High-risk Primary Oral Premalignant Lesions with Poor Outcome

There is a pressing need for the development of visual aids that will facilitate the detection of oral premalignant lesions (OPLs) with a high-risk of progression. Preliminary data suggest that toluidine blue stain may be preferentially retained by OPLs with high-risk molecular clones. In this study, we monitored OPLs from 100 patients without any history of oral cancer for an average of 44 months in order to evaluate the association of toluidine blue status with clinicopathologic risk factors, molecular patterns (microsatellite analysis on seven chromosome arms: 3p, 9p, 4q, 8p, 11q, 13q, and 17p) and outcome. Toluidine blue-positive staining correlated with clinicopathologic risk factors and high-risk molecular risk patterns. Significantly, a >6-fold elevation in cancer risk was observed for toluidine blue-positive lesions, with positive retention of the dye present in 12 of the 15 lesions that later progressed to cancer (P = 0.0008). This association of toluidine blue status with risk factors and outcome was evident even when the analysis was restricted to OPLs with low-grade or no dysplasia. Our results suggest the potential use of toluidine blue in identifying high-risk OPLs.

A Stepwise Framework for the Normalization of Array CGH Data

In two-channel competitive genomic hybridization microarray experiments, the ratio of the two fluorescent signal intensities at each spot on the microarray is commonly used to infer the relative amounts of the test and reference sample DNA levels. This ratio may be influenced by systematic measurement effects from non-biological sources that can introduce biases in the estimated ratios. These biases should be removed before drawing conclusions about the relative levels of DNA. The performance of existing gene expression microarray normalization strategies has not been evaluated for removing systematic biases encountered in array-based comparative genomic hybridization (CGH), which aims to detect single copy gains and losses typically in samples with heterogeneous cell populations resulting in only slight shifts in signal ratios. The purpose of this work is to establish a framework for correcting the systematic sources of variation in high density CGH array images, while maintaining the true biological variations.

Gain of a Region on 7p22.3, Containing MAD1L1, is the Most Frequent Event in Small-cell Lung Cancer Cell Lines

Small-cell lung cancer (SCLC) is a highly aggressive lung neoplasm, which accounts for 20% of yearly lung cancer cases. The lack of knowledge of the progenitor cell type for SCLC precludes the definition of a normal gene expression profile and has hampered the identification of gene expression changes, while the low resolution of conventional genomic screens such as comparative genomic hybridization (CGH) and loss of heterozygosity analysis limit our ability to fine-map genetic alterations. The recent advent of whole genome tiling path array CGH enables profiling of segmental DNA copy number gains and losses at a resolution 100 times that of conventional methods. Here we report the analysis of 14 SCLC cell lines and six matched normal B-lymphocyte lines. We detected 7p22.3 copy number gain in 13 of the 14 SCLC lines and 0 of the 6 matched normal lines. In 4 of the 14 cell lines, this gain is present as a 350 kbp gene specific copy number gain centered at MAD1L1 (the human homologue of the yeast gene MAD1). Fluorescence in situ hybridization validated the array CGH finding. Intriguingly, MAD1L1 has been implicated to have tumor-suppressing functions. Our data suggest a more complex role for this gene, as MAD1L1 is the most frequent copy number gain in SCLC cell lines.

High Resolution Analysis of Non-small Cell Lung Cancer Cell Lines by Whole Genome Tiling Path Array CGH

Chromosomal regions harboring tumor suppressors and oncogenes are often deleted or amplified. Array comparative genomic hybridization detects segmental DNA copy number alterations in tumor DNA relative to a normal control. The recent development of a bacterial artificial chromosome array, which spans the human genome in a tiling path manner with >32,000 clones, has facilitated whole genome profiling at an unprecedented resolution. Using this technology, we comprehensively describe and compare the genomes of 28 commonly used non-small cell lung carcinoma (NSCLC) cell models, derived from 18 adenocarcinomas (AC), 9 squamous cell carcinomas and 1 large cell carcinoma. Analysis at such resolution not only provided a detailed genomic alteration template for each of these model cell lines, but revealed novel regions of frequent duplication and deletion. Significantly, a detailed analysis of chromosome 7 identified 6 distinct regions of alterations across this chromosome, implicating the presence of multiple novel oncogene loci on this chromosome. As well, a comparison between the squamous and AC cells revealed alterations common to both subtypes, such as the loss of 3p and gain of 5p, in addition to multiple hotspots more frequently associated with only 1 subtype. Interestingly, chromosome 3q, which is known to be amplified in both subtypes, showed 2 distinct regions of alteration, 1 frequently altered in squamous and 1 more frequently altered in AC. In summary, our data demonstrate the unique information generated by high resolution analysis of NSCLC genomes and uncover the presence of genetic alterations prevalent in the different NSCLC subtypes.

Microarray-based CGH of Sporadic and Syndrome-related Pheochromocytomas Using a 0.1-0.2 Mb Bacterial Artificial Chromosome Array Spanning Chromosome Arm 1p

Pheochromocytomas (PCC) are relatively rare neuroendocrine tumors, mainly of the adrenal medulla. They arise sporadically or occur secondary to inherited cancer syndromes, such as multiple endocrine neoplasia type II (MEN2), von Hippel-Lindau disease (VHL), or neurofibromatosis type I (NF1). Loss of 1p is the most frequently encountered genetic alteration, especially in MEN2-related and sporadic PCC. Previous studies have revealed three regions of common somatic loss on chromosome arm 1p, using chromosome-based comparative genomic hybridization (CGH) and LOH analysis. To investigate these chromosomal aberrations with a higher resolution and sensitivity, we performed microarray-based CGH with 13 sporadic and 11 syndrome-related (10 MEN2A-related and 1 NF1-related) tumors. The array consisted of 642 overlapping bacterial artificial chromosome (BAC) clones mapped to 1p11.2-p36.33. Chromosomal deletions on 1p were detected in 18 of 24 cases (75%). Among 9 tumors with partial 1p loss, the deleted region was restricted to 1cen-1p32.3 in six cases (25%), indicating a region of genetic instability. The consensus regions of deletion in this study involved 1cen-1p21.1, 1p21.3-1p31.3, and 1p34.3-1p36.33. In conclusion, these data strongly suggest that chromosome arm 1p is the site for multiple tumor suppressor genes, although the potential candidate genes CDKN2C and PTPRF/LAR are not included in these regions.

Recent Advances in Array Comparative Genomic Hybridization Technologies and Their Applications in Human Genetics

Array comparative genomic hybridization (array CGH) is a method used to detect segmental DNA copy number alterations. Recently, advances in this technology have enabled high-resolution examination for identifying genetic alterations and copy number variations on a genome-wide scale. This review describes the current genomic array platforms and CGH methodologies, highlights their applications for studying cancer genetics, constitutional disease and human variation, and discusses visualization and analytical software programs for computational interpretation of array CGH data.

Epigenomics: Mapping the Methylome

DNA methylation is integral to normal development and disease processes. However, the genomic distribution of methylated sequences--the methylome--is poorly understood. We have recently developed a platform technology for rapid assessment of methylation status throughout the human genome in a high-resolution, high-throughput manner. This is achieved by coupling a methylated DNA immunoprecipitation (MeDIP) method for isolating methyl cytosine rich fragments with array-based comparative genomic hybridization (array CGH). Using a combination of whole genome tiling path BAC arrays and CpG island microarrays, DNA methylation profiles are obtained simultaneously at both genome-wide and locus-specific levels. A comparison between male and female DNA using MeDIP-array CGH revealed unexpected hypomethylation of the inactive x-chromosome in gene-poor regions. Furthermore, comparisons between cancer and noncancer cell types yielded differential methylation patterns that link genetic and epigenetic instability offering a new approach to decipher misregulation in cancer. Finally, we provide new data showing epigenomic instability in lung cancer cells with concurrent regions of genetic and epigenetic alterations harboring known oncogenes.

Comprehensive Copy Number Profiles of Breast Cancer Cell Model Genomes

Breast cancer is the most commonly diagnosed cancer in women worldwide and consequently has been extensively investigated in terms of histopathology, immunochemistry and familial history. Advances in genome-wide approaches have contributed to molecular classification with respect to genomic changes and their subsequent effects on gene expression. Cell lines have provided a renewable resource that is readily used as model systems for breast cancer cell biology. A thorough characterization of their genomes to identify regions of segmental DNA loss (potential tumor-suppressor-containing loci) and gain (potential oncogenic loci) would greatly facilitate the interpretation of biological data derived from such cells. In this study we characterized the genomes of seven of the most commonly used breast cancer model cell lines at unprecedented resolution using a newly developed whole-genome tiling path genomic DNA array.

Identification of Novel Lung Genes in Bronchial Epithelium by Serial Analysis of Gene Expression

A description of the transcriptome of human bronchial epithelium should provide a basis for studying lung diseases, including cancer. We have deduced global gene expression profiles of bronchial epithelium and lung parenchyma, based on a vast dataset of nearly two million sequence tags from 21 serial analysis of gene expression (SAGE) libraries from individuals with a history of smoking. Our analysis suggests that the transcriptome of the bronchial epithelium is distinct from that of lung parenchyma and other tissue types. Moreover, our analysis has identified novel bronchial-enriched genes such as MS4A8B, and has demonstrated the use of SAGE for the discovery of novel transcript variants. Significantly, gene expression associated with ciliogenesis is evident in bronchial epithelium, and includes the expression of transcripts specifying axonemal proteins DNAI2, SPAG6, ASP, and FOXJ1 transcription factor. Moreover, expression of potential regulators of ciliogenesis such as MDAC1, NYD-SP29, ARMC3, and ARMC4 were also identified. This study represents a comprehensive delineation of the bronchial and parenchyma transcriptomes, identifying more than 20,000 known and hypothetical genes expressed in the human lung, and constitutes one of the largest human SAGE studies reported to date.

Integrating Copy Number Polymorphisms into Array CGH Analysis Using a Robust HMM

Array comparative genomic hybridization (aCGH) is a pervasive technique used to identify chromosomal aberrations in human diseases, including cancer. Aberrations are defined as regions of increased or decreased DNA copy number, relative to a normal sample. Accurately identifying the locations of these aberrations has many important medical applications. Unfortunately, the observed copy number changes are often corrupted by various sources of noise, making the boundaries hard to detect. One popular current technique uses hidden Markov models (HMMs) to divide the signal into regions of constant copy number called segments; a subsequent classification phase labels each segment as a gain, a loss or neutral. Unfortunately, standard HMMs are sensitive to outliers, causing over-segmentation, where segments erroneously span very short regions.

Genomic Alterations in Lobular Neoplasia: a Microarray Comparative Genomic Hybridization Signature for Early Neoplastic Proliferationin the Breast

The identification of genomic alterations occurring in neoplastic lesions provides insight into both lesion occurrence and disease progression. In this study, we used microarray comparative genomic hybridization (CGH) to investigate genetic changes in atypical lobular hyperplasia (ALH) and lobular carcinoma in situ (LCIS), as the presence of these lobular neoplastic lesions is an indicator of risk in the development of invasive breast cancer. DNA was extracted from microdissected archival breast tissue containing ALH or LCIS, lacking adjacent invasive carcinoma, and subjected to whole-genome tiling path microarray-CGH using the submegabase resolution tiling set (SMRT)-array platform. Twelve ALH and 13 LCIS lesions were examined. Copy number alterations were identified using statistical criteria and validated with Real-Time PCR and fluorescence in situ hybridization. From statistical analysis, a greater number of alterations were observed in ALH compared to LCIS. Alterations common to ALH include gain at 2p11.2 and loss at 7p11-p11.1 and 22q11.1. Alterations common to LCIS include gain at 20q13.13 and loss at 19q13.2-q13.31. In both ALH and LCIS, we observed loss of 16q21-q23.1, an altered region previously identified in lobular neoplasia and invasive carcinoma. The validation of select alterations reinforces the genomic signature. This study represents the first whole-genome investigation of lobular neoplastic breast lesions using clinical archival specimens. The identified genomic signature includes copy number alterations not previously identified for lobular neoplasia. This genomic signature, common to ALH and LCIS, suggests a role for the acquisition of novel genomic alterations in the aberrant cellular proliferation that defines lobular neoplasia.

Large Fragment Bst DNA Polymerase for Whole Genome Amplification of DNA from Formalin-fixed Paraffin-embedded Tissues

Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold.

SIGMA: a System for Integrative Genomic Microarray Analysis of Cancer Genomes

The prevalence of high resolution profiling of genomes has created a need for the integrative analysis of information generated from multiple methodologies and platforms. Although the majority of data in the public domain are gene expression profiles, and expression analysis software are available, the increase of array CGH studies has enabled integration of high throughput genomic and gene expression datasets. However, tools for direct mining and analysis of array CGH data are limited. Hence, there is a great need for analytical and display software tailored to cross platform integrative analysis of cancer genomes.

Computational Methods for the Analysis of Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a technique for assaying the copy number status of cancer genomes. The widespread use of this technology has lead to a rapid accumulation of high throughput data, which in turn has prompted the development of computational strategies for the analysis of array CGH data. Here we explain the principles behind array image processing, data visualization and genomic profile analysis, review currently available software packages, and raise considerations for future software development.

Cytogenetically Balanced Translocations Are Associated with Focal Copy Number Alterations

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated.

Whole Genome Tiling Path Array CGH Analysis of Segmental Copy Number Alterations in Cervical Cancer Cell Lines

Cervical cancer is the second most common malignancy in women worldwide, with high risk subtypes of human papillomavirus (HPV) constituting the major etiological agent. However, only a small percentage of women infected by the virus develop disease, suggesting that additional host genetic alterations are necessary for disease progression. In this study we examined the genomes of a panel of commonly used model cervical cancer cell lines using a recently developed whole genome tiling path array for CGH analysis. Detailed analysis of genomic profiles enabled the detection of many novel aberrations, which may have been missed by conventional cytogenetic methods. In total, 27 minimal regions of recurrent copy number alteration were identified that are potentially involved in tumorigenesis. Interestingly, fine mapping of the 3q gain, which is associated with the progression of precursor lesions to invasive cervical cancer, identified a minimal region of alteration harboring genes distinct from previous candidates. Novel regions of gene amplification, including the coamplification of both the Birc and MMP gene clusters on 11q22, were also evident. Lastly, characterization of genomic structure at sites of HPV integration identified the copy number gain of host cellular sequences between the viral-host genomic boundaries in both SiHa and SW756, suggesting a direct role for HPV integration in the development of genetic abnormalities that initiate cervical cancer. This work represents the highest resolution look at a cervical cancer genome to date and offers definitive characterization of the alteration status of these cancer cell lines.

A Comprehensive Analysis of Common Copy-number Variations in the Human Genome

Segmental copy-number variations (CNVs) in the human genome are associated with developmental disorders and susceptibility to diseases. More importantly, CNVs may represent a major genetic component of our phenotypic diversity. In this study, using a whole-genome array comparative genomic hybridization assay, we identified 3,654 autosomal segmental CNVs, 800 of which appeared at a frequency of at least 3%. Of these frequent CNVs, 77% are novel. In the 95 individuals analyzed, the two most diverse genomes differed by at least 9 Mb in size or varied by at least 266 loci in content. Approximately 68% of the 800 polymorphic regions overlap with genes, which may reflect human diversity in senses (smell, hearing, taste, and sight), rhesus phenotype, metabolism, and disease susceptibility. Intriguingly, 14 polymorphic regions harbor 21 of the known human microRNAs, raising the possibility of the contribution of microRNAs to phenotypic diversity in humans. This in-depth survey of CNVs across the human genome provides a valuable baseline for studies involving human genetics.

Resolving the Resolution of Array CGH

Many recent technologies have been designed to supplant conventional metaphase CGH technology with the goal of refining the description of segmental copy number status throughout the genome. However, the emergence of new technologies has led to confusion as to how to describe adequately the capabilities of each array platform. The design of a CGH array can incorporate a uniform or a highly variable element distribution. This can lead to bias in the reporting of average or median resolutions, making it difficult to provide a fair comparison of platforms. In this report, we propose a new definition of resolution for array CGH technology, termed "functional resolution," that incorporates the uniformity of element spacing on the array, as well as the sensitivity of each platform to single-copy alterations. Calculation of these metrics is automated through the development of a Java-based application, "ResCalc," which is applicable to any array CGH platform.

Heritable Skewed X-chromosome Inactivation Leads to Haemophilia A Expression in Heterozygous Females

Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentage-activated normal X-chromosome was found in HA heterozygous females (R(2)=0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (>80:20 or <20:80) (P<0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (P<0.025), with more females having higher degrees of skewing. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, and X-inactive-specific transcript mutations, are not consistent with our results. This study shows that FVIII activity in HA heterozygous females can be directly related to XCI skewing, and that low FVIII activity in females in this family is due to unfavourable XCI skewing. Further, the findings suggest that these XCI ratios are genetically influenced, consistent with a novel heritable human X controlling element (XCE) functioning similarly to the mouse Xce.

Skewed X-chromosome Inactivation is Associated with Primary but Not Secondary Ovarian Failure

Premature ovarian failure (POF) is the occurrence of menopause before the age of 40, and may present with either primary or secondary amenorrhea. Numerous cases of POF in women with X-chromosome deletions or translocations have been reported; thus, it is possible that smaller rearrangements undetectable by conventional cytogenetics may contribute to POF in some patients. In females with an abnormal X chromosome, cells with inactivation of the normal X may be selected against, causing skewed X-chromosome inactivation (XCI). We therefore assessed XCI by methylation sensitive restriction digestion and PCR amplification at the androgen receptor (AR) locus, in 4 primary and 55 secondary POF patients and 109 control women. In samples heterozygous at AR and therefore informative for the skewing assay, the frequency of skewed XCI among the women with secondary amenorrhea was identical to that in control women, with 4 out of 48 (8.3%) secondary ovarian failure patients and 8 out of 97 (8.2%) control women having > or =90% skewing. Notably, all three primary amenorrhea patients that were informative at AR had skewed XCI > or =90% (P = 0.001 vs. control women; Fisher's exact test). To investigate whether X-chromosome copy number alterations were responsible, DNA from selected patients with skewed XCI was examined by high resolution DNA microarray, however no potential regions of DNA addition or deletion were confirmed by FISH or PCR. X-chromosome abnormalities undetectable by array, or reduced follicular pool due to an early trisomic rescue event, may explain the skewed XCI observed in POF patients presenting with primary amenorrhea.

Whole-genome Analysis and HLA Genotyping of Enteropathy-type T-cell Lymphoma Reveals 2 Distinct Lymphoma Subtypes

Enteropathy-type T-cell lymphoma (ETL) is an aggressive extranodal T-cell non-Hodgkin lymphoma assumed to arise in the setting of celiac disease.

Comprehensive Serial Analysis of Gene Expression of the Cervical Transcriptome

More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE).

Modeling Recurrent DNA Copy Number Alterations in Array CGH Data

Recurrent DNA copy number alterations (CNA) measured with array comparative genomic hybridization (aCGH) reveal important molecular features of human genetics and disease. Studying aCGH profiles from a phenotypic group of individuals can determine important recurrent CNA patterns that suggest a strong correlation to the phenotype. Computational approaches to detecting recurrent CNAs from a set of aCGH experiments have typically relied on discretizing the noisy log ratios and subsequently inferring patterns. We demonstrate that this can have the effect of filtering out important signals present in the raw data. In this article we develop statistical models that jointly infer CNA patterns and the discrete labels by borrowing statistical strength across samples.

Genetic Changes in the Evolution of Multidrug Resistance for Cultured Human Ovarian Cancer Cells

The multidrug resistant (MDR) phenotype is often attributed to the activity of ATP-binding cassette (ABC) transporters such as P-glycoprotein (ABCB1). Previous work has suggested that modulation of MDR may not necessarily be a single gene trait. To identify factors that contribute to the emergence of MDR, we undertook integrative genomics analysis of the ovarian carcinoma cell line SKOV3 and a series of MDR derivatives of this line (SKVCRs). As resistance increased, comparative analysis of gene expression showed conspicuous activation of a network of genes in addition to ABCB1. Functional annotation and pathway analysis revealed that many of these genes were associated with the extracellular matrix and had previously been implicated in tumor invasion and cell proliferation. Further investigation by whole genome tiling-path array CGH suggested that changes in gene dosage were key to the activation of several of these overexpressed genes. Remarkably, alignment of whole genome profiles for SKVCR lines revealed the emergence and decline of specific segmental DNA alterations. The most prominent alteration was a novel amplicon residing at 16p13 that encompassed the ABC transporter genes ABCC1 and ABCC6. Loss of this amplicon in highly resistant SKVCR lines coincided with the emergence of a different amplicon at 7q21.12, which harbors ABCB1. Integrative analysis suggests that multiple genes are activated during escalation of drug resistance, including a succession of ABC transporter genes and genes that may act synergistically with ABCB1. These results suggest that evolution of the MDR phenotype is a dynamic, multi-genic process in the genomes of cancer cells.

Effect of Active Smoking on the Human Bronchial Epithelium Transcriptome

Lung cancer is the most common cause of cancer-related deaths. Tobacco smoke exposure is the strongest aetiological factor associated with lung cancer. In this study, using serial analysis of gene expression (SAGE), we comprehensively examined the effect of active smoking by comparing the transcriptomes of clinical specimens obtained from current, former and never smokers, and identified genes showing both reversible and irreversible expression changes upon smoking cessation.

Frequent Occurrence of Deletions in Primary Mediastinal B-cell Lymphoma

Primary mediastinal B-cell lymphoma (PMBCL) is a distinct subtype of diffuse large B-cell lymphoma. PMBCL has been previously studied with a variety of genomic techniques resulting in frequent detection of chromosomal gains; however, chromosomal losses have been rarely reported. This finding contrasts many other types of lymphoma, in which deletions are common. We hypothesize that segmental losses do exist but may have escaped detection by methods used in the previous studies. Using array comparative genomic hybridization to a tiling-resolution microarray encompassing the entire human genome, PMBCL samples were analyzed for genomic copy number alterations. An almost equal number of gains and losses of chromosomal material were detected throughout the genome (216 vs. 193, respectively). A selection of these DNA copy number alterations were confirmed by quantitative real-time PCR. Recurrent gains were detected at all previously reported regions of gain, including 9p seen in approximately 70% of cases. Recurrent chromosomal losses were observed at 1p, 3p, 4q, 6q, 7p, and 17p, with a novel event at 1p13.1-p13.2 representing the most frequent at 42% of cases analyzed. We conclude that consistent losses are present in the PMBCL genome. Given the similar frequency of losses to that of segmental gains of DNA, they are likely to play an important role in the pathogenesis of PMBCL.

Genomics and Premalignant Breast Lesions: Clues to the Development and Progression of Lobular Breast Cancer

Advances in genomic technology have improved our understanding of the genetic events that parallel breast cancer development. Because almost all mammary carcinomas develop in the terminal duct lobular units of the breast, understanding the events involved in mammary gland development make it possible to recognize those events that, when altered, contribute to breast neoplasia. In this review we focus on lobular carcinomas, discussing the pathology, development, and progression of premalignant lobular lesions from a genomic point of view. We highlight studies utilizing genomic approaches and describe how these investigations have furthered our understanding of the complexity of premalignant breast lesions.

Public Databases and Software for the Pathway Analysis of Cancer Genomes

The study of pathway disruption is key to understanding cancer biology. Advances in high throughput technologies have led to the rapid accumulation of genomic data. The explosion in available data has generated opportunities for investigation of concerted changes that disrupt biological functions, this in turns created a need for computational tools for pathway analysis. In this review, we discuss approaches to the analysis of genomic data and describe the publicly available resources for studying biological pathways.

Using LongSAGE to Detect Biomarkers of Cervical Cancer Potentially Amenable to Optical Contrast Agent Labelling

Sixteen longSAGE libraries from four different clinical stages of cervical intraepithelial neoplasia have enabled us to identify novel cell-surface biomarkers indicative of CIN stage. By comparing gene expression profiles of cervical tissue at early and advanced stages of CIN, several genes are identified to be novel genetic markers. We present fifty-six cell-surface gene products differentially expressed during progression of CIN. These cell surface proteins are being examined to establish their capacity for optical contrast agent binding. Contrast agent visualization will allow real-time assessment of the physiological state of the disease process bringing vast benefit to cancer care. The data discussed in this publication have been submitted to NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE6252.

Sub-megabase Resolution Tiling (SMRT) Array-based Comparative Genomic Hybridization Profiling Reveals Novel Gains and Losses of Chromosomal Regions in Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma Cell Lines

Hodgkin lymphoma (HL) and Anaplastic Large Cell Lymphoma (ALCL), are forms of malignant lymphoma defined by unique morphologic, immunophenotypic, genotypic, and clinical characteristics, but both overexpress CD30. We used sub-megabase resolution tiling (SMRT) array-based comparative genomic hybridization to screen HL-derived cell lines (KMH2 and L428) and ALCL cell lines (DEL and SR-786) in order to identify disease-associated gene copy number gains and losses.

Up Regulation in Gene Expression of Chromatin Remodelling Factors in Cervical Intraepithelial Neoplasia

The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development.

Inverted Duplication with Terminal Deletion of 5p and No Cat-like Cry

We report on a 6-year-old boy referred for cytogenetics study. A few non-specific features were observed in the newborn: hypotonia, failure to thrive, seizures, pre-auricular skin tags. Cat-like cry was not identified. No remarkable facial dysmorphism, gastrointestinal, respiratory or cardiac abnormalities were identified. At age 4 years, speech and motor skill delays were apparent. Karyotyping and FISH analysis revealed a de novo rearranged chromosome 5p, with subtelomeric deletion of 5p and a duplication of the cri-du-chat critical region. Array CGH using sub-megabase resolution tiling-set (SMRT) array followed by FISH analysis with labeled BACs showed a deletion of 5pter to 5p15.31 (0-6.9 Mb) and an inverted duplication of the greater part of 5p15.31 to the distal end of 5p14.3 (6.9-19.9 Mb). Although very rare, inverted duplications with terminal deletion (inv dup del) have been reported at different chromosomal ends. Our finding adds a second patient of inv dup del 5p to this growing list, and the potential causative mechanisms for this rearrangement are discussed. Review of the mapping information of cri-du-chat patients and the comparison with a previously reported patient suggested that the critical region for cat-like cry is located within a 0.6 Mb region.

Epigenetics of Cancer Progression

Alteration in epigenetic regulation of gene expression is a frequent event in human cancer. CpG island hypermethylation and downregulation is observed for many genes involved in a diverse range of functions and pathways that become deregulated in cancer. Paradoxically, global hypomethylation is a hallmark of almost all human cancers. Methylation profiles can be used as molecular markers to distinguish subtypes of cancers and potentially as predictors of disease outcome and treatment response. The role of epigenetics in diagnosis and treatment is likely to increase as mechanisms leading to the transcriptional silencing of genes involved in human cancers are revealed. Drugs that inhibit methylation are used both as a research tool to assess reactivation of genes silenced in cancer by hypermethylation and in the treatment of some hematological malignancies. Multidimensional analysis, evaluating genetic and epigenetic alterations on a global and locus-specific scale in human cancer, is imperative to understand mechanisms driving changes in gene dosage, and as a means towards identifying pathways driving cancer initiation and progression.

Comment Re: a Comparison of DNA Copy Number Profiling Platforms

MD-SeeGH: a Platform for Integrative Analysis of Multi-dimensional Genomic Data

Recent advances in global genomic profiling methodologies have enabled multi-dimensional characterization of biological systems. Complete analysis of these genomic profiles require an in depth look at parallel profiles of segmental DNA copy number status, DNA methylation state, single nucleotide polymorphisms, as well as gene expression profiles. Due to the differences in data types it is difficult to conduct parallel analysis of multiple datasets from diverse platforms.

Genomic Markers for Malignant Progression in Pulmonary Adenocarcinoma with Bronchioloalveolar Features

Bronchioloalveolar carcinoma (BAC), a subtype of lung adenocarcinoma (ADC) without stromal, vascular, or pleural invasion, is considered an in situ tumor with a 100% survival rate. However, the histological criteria for invasion remain controversial. BAC-like areas may accompany otherwise invasive adenocarcinoma, referred to as mixed type adenocarcinoma with BAC features (AWBF). AWBF are considered to evolve from BAC, representing a paradigm for malignant progression in ADC. However, the supporting molecular evidence remains forthcoming. Here, we have studied the genomic changes of BAC and AWBF by array comparative genomic hybridization (CGH). We used submegabase-resolution tiling set array CGH to compare the genomic profiles of 14 BAC or BAC with focal area suspicious for invasion with those of 15 AWBF. Threshold-filtering and frequency-scoring analysis found that genomic profiles of noninvasive and focally invasive BAC are indistinguishable and show fewer aberrations than tumor cells in BAC-like areas of AWBF. These aberrations occurred mainly at the subtelomeric chromosomal regions. Increased genomic alterations were noted between BAC-like and invasive areas of AWBF. We identified 113 genes that best differentiated BAC from AWBF and were considered candidate marker genes for tumor invasion and progression. Correlative gene expression analyses demonstrated a high percentage of them to be poor prognosis markers in early stage ADC. Quantitative PCR also validated the amplification and overexpression of PDCD6 and TERT on chromosome 5p and the prognostic significance of PDCD6 in early stage ADC patients. We identified candidate genes that may be responsible for and are potential markers for malignant progression in AWBF.

High-resolution Whole Genome Tiling Path Array CGH Analysis of CD34+ Cells from Patients with Low-risk Myelodysplastic Syndromes Reveals Cryptic Copy Number Alterations and Predicts Overall and Leukemia-free Survival

Myelodysplastic syndromes (MDSs) pose an important diagnostic and treatment challenge because of the genetic heterogeneity and poorly understood biology of the disease. To investigate initiating genomic alterations and the potential prognostic significance of cryptic genomic changes in low-risk MDS, we performed whole genome tiling path array comparative genomic hybridization (aCGH) on CD34(+) cells from 44 patients with an International Prognostic Scoring System score less than or equal to 1.0. Clonal copy number differences were detected in cells from 36 of 44 patients. In contrast, cells from only 16 of the 44 patients displayed karyotypic abnormalities. Although most patients had normal karyotype, aCGH identified 21 recurring copy number alterations. Examples of frequent cryptic alterations included gains at 11q24.2-qter, 17q11.2, and 17q12 and losses at 2q33.1-q33.2, 5q13.1-q13.2, and 10q21.3. Maintenance of genomic integrity defined as less than 3 Mb total disruption of the genome correlated with better overall survival (P = .002) and was less frequently associated with transformation to acute myeloid leukemia (P = .033). This study suggests a potential role for the use of aCGH in the clinical workup of MDS patients.

Characterization of Genes Differentially Expressed Within Macrophages by Virulent and Attenuated Mycobacterium Tuberculosis Identifies Candidate Genes Involved in Intracellular Growth

To identify genes involved in the intracellular survival of Mycobacterium tuberculosis we compared the transcriptomes of virulent (H37Rv) and attenuated (H37Ra) strains during their interaction with murine bone-marrow-derived macrophages. Expression profiling was accomplished via the bacterial artificial chromosome fingerprint array (BACFA) technique. Genes identified with BACFA, and confirmed via qPCR to be upregulated in the attenuated H37Ra at 168 h post-infection, were frdB, frdC and frdD. Genes upregulated in the virulent H37Rv were pks2, aceE and Rv1571. Further qPCR analysis of these genes at 4 and 96 h post-infection revealed that the frd operon (encoding the fumarate reductase enzyme complex) is expressed at higher levels in the virulent H37Rv at earlier time points while the expression of aceE and pks2 is higher in the virulent strain throughout the course of infection. Assessment of frd transcripts in oxygen-limited cultures of M. tuberculosis H37Ra and H37Rv showed that the attenuated strain displayed a lag in frdA and frdB expression at the onset of microaerophilic culture, when compared to microaerophilic cultures of H37Rv and aerated cultures of H37Ra. Lastly, treatment of intracellular bacteria with a putative inhibitor of fumarate reductase resulted in a significant reduction of bacterial growth.

Evolving Strategies for Global Gene Expression Analysis of Cancer

The advent of high throughput gene expression profiling, from microarrays to sequence based assays has yielded vast insight into the biology of tumors. New technologies are constantly being unveiled which promise to generate more accurate maps of tumor gene deregulation, and demand the development of new strategies in data analysis. This review details the challenges faced in profiling tumor transcriptomes, and highlights the emerging strategies to utilize global profiling approaches to advance our understanding of causal genetic and epigenetic events and their impact on gene expression and tumor phenotype and behavior, through high throughput profiling, and integration of multiple dimensions of genomic data.

Bacterial Artificial Chromosome Fingerprint Arrays for the Differentiation of Transcriptomic Differences in Mycobacteria

Although microarray technology has become more widespread as a discovery tool for bacterial pathogenesis, it remains a method available only to laboratories with access to expensive equipment and costly analysis software. Mycobacterium tuberculosis, the causative agent for tuberculosis (TB), afflicts one-third of the global population, and kills between 2 and 3 million people per year. While the majority of cases of TB occur in developing areas of the world, facilities in these regions may not be able to support microarray analysis. Additionally, a major limitation of microarrays is that only genes on the array are being assayed. With acquired virulence and drug resistance in microbes, a method less dependent on a predetermined list of gene targets is advantageous. We present a method of expression analysis based on bacterial artificial chromosomes (BACs) that can be applied with standard laboratory equipment and free analysis software. This technique, bacterial artificial chromosome fingerprint arrays (BACFA), was developed and utilised to identify expression differences between intracellular strains of M. tuberculosis, one virulent (H37Rv) and one attenuated (H37Ra). Southern blots of restriction-enzyme digested BAC fragments were sequentially hybridised with strain-specific cDNA probes to generate expression profiles that were used to isolate expression differences in broth grown and intracellular bacteria. Repeat comparisons of intracellular profiles via BACFA identified genomic regions differentially expressed by the two strains. Quantitative real-time PCR was used to assess the genes located in these fragments in order to confirm or deny the differential regulation of genes. In total, we identified six genes that were differentially regulated between strains inside the host cell (pks2, aceE, Rv1571, and frdBCD). We report that BACFA is an effective technique in the expression analysis of bacteria and can be considered complementary to the high-throughput analysis offered by microarrays.

PIK3CA Mutations and Copy Number Gains in Human Lung Cancers

We investigated the frequency and function of mutations and increased copy number of the PIK3CA gene in lung cancers. PIK3CA mutations are one of the most common gene changes present in human cancers. We analyzed the mutational status of exons 9 and 20 and gene copy number of PIK3CA using 86 non-small cell lung cancer (NSCLC) cell lines, 43 small cell lung cancer (SCLC) cell lines, 3 extrapulmonary small cell cancer (ExPuSC) cell lines, and 691 resected NSCLC tumors and studied the relationship between PIK3CA alterations and mutational status of epidermal growth factor receptor (EGFR) signaling pathway genes (EGFR, KRAS, HER2, and BRAF). We also determined PIK3CA expression and activity and correlated the findings with effects on cell growth. We identified mutations in 4.7% of NSCLC cell lines and 1.6% of tumors of all major histologic types. Mutations in cell lines of small cell origin were limited to two ExPuSC cell lines. PIK3CA copy number gains were more frequent in squamous cell carcinoma (33.1%) than in adenocarcinoma (6.2%) or SCLC lines (4.7%). Mutational status of PIK3CA was not mutually exclusive to EGFR or KRAS. PIK3CA alterations were associated with increased phosphatidylinositol 3-kinase activity and phosphorylated Akt expression. RNA interference-mediated knockdown of PIK3CA inhibited colony formation of cell lines with PIK3CA mutations or gains but was not effective in PIK3CA wild-type cells. PIK3CA mutations or gains are present in a subset of lung cancers and are of functional importance.

SIGMA2: a System for the Integrative Genomic Multi-dimensional Analysis of Cancer Genomes, Epigenomes, and Transcriptomes

High throughput microarray technologies have afforded the investigation of genomes, epigenomes, and transcriptomes at unprecedented resolution. However, software packages to handle, analyze, and visualize data from these multiple 'omics disciplines have not been adequately developed.

Integrative Genomic and Gene Expression Analysis of Chromosome 7 Identified Novel Oncogene Loci in Non-small Cell Lung Cancer

Lung cancer accounts for over a quarter of cancer deaths, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of cases. Several genome studies have been undertaken in both cell models of NSCLC and clinical samples to identify alterations underlying disease behaviour, and many have identified recurring aberrations of chromosome 7. The presence of recurring chromosome 7 alterations that do not span the well-studied oncogenes EGFR (at 7p11.2) and MET (at 7q31.2) has raised the hypothesis of additional genes on this chromosome that contribute to tumourigenesis. In this study, we demonstrated that multiple loci on chromosome 7 are indeed amplified in NSCLC, and through integrative analysis of gene dosage alterations and parallel gene expression changes, we identified new lung cancer oncogene candidates, including FTSJ2, NUDT1, TAF6, and POLR2J. Activation of these key genes was confirmed in panels of clinical lung tumour tissue as compared with matched normal lung tissue. The detection of gene activation in multiple cohorts of samples strongly supports the presence of key genes involved in lung cancer that are distinct from the EGFR and MET loci on chromosome 7.

Multiple Aberrations of Chromosome 3p Detected in Oral Premalignant Lesions

The study of oral premalignant lesions (OPL) is crucial to the identification of initiating genetic events in oral cancer. However, these lesions are minute in size, making it a challenge to recover sufficient DNA from microdissected cells for comprehensive genomic analysis. As a step toward identifying genetic aberrations associated with oral cancer progression, we used tiling-path array comparative genomic hybridization to compare alterations on chromosome 3p for 71 OPLs against 23 oral squamous cell carcinomas. 3p was chosen because although it is frequently altered in oral cancers and has been associated with progression risk, its alteration status has only been evaluated at a small number of loci in OPLs. We identified six recurrent losses in this region that were shared between high-grade dysplasias and oral squamous cell carcinomas, including a 2.89-Mbp deletion spanning the FHIT gene (previously implicated in oral cancer progression). When the alteration status for these six regions was examined in 24 low-grade dysplasias with known progression outcome, we observed that they occurred at a significantly higher frequency in low-grade dysplasias that later progressed to later-stage disease (P < 0.003). Moreover, parallel analysis of all profiled tissues showed that the extent of overall genomic alteration at 3p increased with histologic stage. This first high-resolution analysis of chromosome arm 3p in OPLs represents a significant step toward predicting progression risk in early preinvasive disease and provides a keen example of how genomic instability escalates with progression to invasive cancer.

Disruption of the Non-canonical WNT Pathway in Lung Squamous Cell Carcinoma

Disruptions of beta-catenin and the canonical Wnt pathway are well documented in cancer. However, little is known of the non-canonical branch of the Wnt pathway. In this study, we investigate the transcript level patterns of genes in the Wnt pathway in squamous cell lung cancer using reverse-transcriptase (RT)-PCR. It was found that over half of the samples examined exhibited dysregulated gene expression of multiple components of the non-canonical branch of the WNT pathway. In the cases where beta catenin (CTNNB1) was not over-expressed, we identified strong relationships of expression between wingless-type MMTV integration site family member 5A (WNT5A)/ frizzled homolog 2 (FZD2), frizzled homolog 3 (FZD3) / dishevelled 2 (DVL2), and low density lipoprotein receptor-related protein 5 (LRP5)/ secreted frizzled-related protein 4 (SFRP4). This is one of the first studies to demonstrate expression of genes in the non-canonical pathway in normal lung tissue and its disruption in lung squamous cell carcinoma. These findings suggest that the non-canonical pathway may have a more prominent role in lung cancer than previously reported.

MET Gene Amplification or EGFR Mutation Activate MET in Lung Cancers Untreated with EGFR Tyrosine Kinase Inhibitors

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real-time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in-situ hybridization. Total and phospho-MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High-level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR-TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho-MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho-MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA-mediated knockdown of EGFR abolished phospho-MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho-MET expression in 2 cell lines with amplified MET gene was not down-regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC.

Array-comparative Genomic Hybridization in Sporadic Benign Pheochromocytomas

Pheochromocytomas (PCC) are catecholamine-producing tumors arising from the adrenal medulla that occur either sporadically or in the context of hereditary cancer syndromes, such as multiple endocrine neoplasia type 2 (MEN2), von Hippel-Lindau disease (VHL), neurofibromatosis type 1, and the PCC-paraganglioma syndrome. Conventional comparative genomic hybridization studies have shown loss of 1p and 3q in the majority of sporadic and MEN2-related PCC, and 3p and 11p loss in VHL-related PCC. The development of a submegabase tiling resolution array enabled us to perform a genome-wide high-resolution analysis of 36 sporadic benign PCC. The results show that there are two distinct patterns of abnormalities in these sporadic PCC, one consisting of loss of 1p with or without concomitant 3q loss in 20/36 cases (56%), the other characterized by loss of 3p with or without concomitant 11p loss in 11/36 (31%). In addition, we found loss of chromosome 22q at high frequency (35%), as well as the novel finding of high frequency chromosome 21q loss (21%). We conclude that there appear to be two subgroups of benign sporadic PCC, one of which has a pattern of chromosomal abnormalities that is comparable with PCC from patients with MEN2 and the other that is comparable with the PCC that arise in patients with VHL disease. In addition, genes on 21q and 22q might play a more important role in PCC pathogenesis than had been assumed thus far.

Defining Genomic Alteration Boundaries for a Combined Small Cell and Non-small Cell Lung Carcinoma

In the rare case of a male patient presenting with a combined small cell lung carcinoma (SCLC), large cell neuroendocrine carcinoma and adenocarcinoma, we used whole genome analysis by tiling-path array comparative genomic hybridization to evaluate the clonal relationship between nodules. In two areas of SCLC distinguishable by divergent neuroendocrine marker expression (CD56 and chromogranin-A), the presence of identical genomic breakpoints and rearrangements indicated a common origin, with the presence of additional distinct genomic alterations in these two components indicating diverging clonal evolution. The absence of shared genome alteration features for the adenocarcinoma and large cell neuroendocrine carcinoma components suggested that these tumors evolved independently from the SCLC. Taken together, the array comparative genomic hybridization data demonstrate the development and evolution of three independent primary lung cancers in close proximity to each other to form a combined carcinoma. Application of whole genome analysis shows the potential utility of high resolution molecular tools in resolving the origin and delineating the clonal relationships of a tumor that contains heterogeneous histologic components leading to an ambiguous histogenesis.

Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines

Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.

Minimum Altered Regions in Early Prostate Cancer Progression Identified by High Resolution Whole Genome Tiling Path BAC Array Comparative Hybridization

Carcinoma of the prostate (CaP) is a serious health problem. The altered molecular mechanisms that lead to this disease are poorly understood.

High-resolution Array CGH Identifies Novel Regions of Genomic Alteration in Intermediate-risk Prostate Cancer

Approximately one-third of prostate cancer patients present with intermediate risk disease. Interestingly, while this risk group is clinically well defined, it demonstrates the most significant heterogeneity in PSA-based biochemical outcome. Further, the majority of candidate genes associated with prostate cancer progression have been identified using cell lines, xenograft models, and high-risk androgen-independent or metastatic patient samples. We used a global high-resolution array comparative genomic hybridization (CGH) assay to characterize copy number alterations (CNAs) in intermediate risk prostate cancer. Herein, we show this risk group contains a number of alterations previously associated with high-risk disease: (1) deletions at 21q22.2 (TMPRSS2:ERG), 16q22-24 (containing CDH1), 13q14.2 (RB1), 10q23.31 (PTEN), 8p21 (NKX3.1); and, (2) amplification at 8q21.3-24.3 (containing c-MYC). In addition, we identified six novel microdeletions at high frequency: 1q42.12-q42.3 (33.3%), 5q12.3-13.3 (21%), 20q13.32-13.33 (29.2%), 22q11.21 (25%), 22q12.1 (29.2%), and 22q13.31 (33.3%). Further, we show there is little concordance between CNAs from these clinical samples and those found in commonly used prostate cancer cell models. These unexpected findings suggest that the intermediate-risk category is a crucial cohort warranting further study to determine if a unique molecular fingerprint can predict aggressive versus indolent phenotypes.

Comparative Genomic Hybridization on BAC Arrays

Alterations in genomic DNA are a key feature of many constitutional disorders and cancer. The discovery of the underlying regions of gene dosage has thus been essential in dissecting complex disease phenotypes and identifying targets for therapeutic intervention and diagnostic testing. The development of array comparative genomic hybridization (aCGH) using bacterial artificial chromosomes (BACs) as hybridization targets has facilitated the discovery and fine mapping of novel genomic alterations allowing rapid identification of target genes. In BAC aCGH, DNA samples are first labeled with fluorescent dyes through a random priming reaction with 100-400 ng of genomic DNA. This probe is then co-hybridized to an array consisting of BAC clones, either tiling the genome (approximately 50 kbp resolution) or spaced at intervals (e.g., 1 Mbp resolution). The resulting arrays are then imaged and the signal at each locus is compared between a reference and test sample to determine the copy number status. The DNA samples to be analyzed may be derived from either fresh, frozen, or formalin-fixed paraffin-embedded material, and sample requirements are currently significantly lower than those for oligonucleotide platforms due to the high probe-binding capacity of BAC clone targets (approximately 150 kbp) compared to oligonucleotides (25-80 bp). In this chapter, we describe in detail the technical procedure required to perform copy number analysis of genomes with BAC aCGH.

Methylation Analysis by DNA Immunoprecipitation (MeDIP)

Alteration in epigenetic regulation of gene expression is a common event in human cancer and developmental disease. CpG island hypermethylation and consequent gene silencing is observed for many genes involved in a diverse range of functions and pathways that become deregulated in the disease state. Comparative profiling of the methylome is therefore useful in disease gene discovery. The ability to identify epigenetic alterations on a global scale is imperative to understanding the patterns of gene silencing that parallel disease progression. Methylated DNA immunoprecipitation (MeDIP) is a technique that isolates methylated DNA fragments by immunoprecipitating with 5'-methylcytosine-specific antibodies. The enriched methylated DNA can then be analyzed in a locus-specific manner using PCR assay or in a genome-wide fashion by comparative genomic hybridization against a sample without MeDIP enrichment. This article describes the detailed protocol for MeDIP and hybridization of MeDIP DNA to a whole-genome tiling path BAC array.

Multiple Pathways in the FGF Signaling Network Are Frequently Deregulated by Gene Amplification in Oral Dysplasias

Genetic alteration in oral premalignant lesions (OPLs), the precursors of oral squamous cell carcinomas (OSCCs), may represent key changes in disease initiation and development. We ask if DNA amplification occurs at this early stage of cancer development and which oncogenic pathways are disrupted in OPLs. Here, we evaluated 50 high-grade dysplasias and low-grade dysplasias that later progressed to cancer for gene dosage aberrations using tiling-path DNA microarrays. Early occurrences of DNA amplification and homozygous deletion were frequently detected, with 40% (20/50) of these early lesions exhibiting such features. Expression for 88 genes in 7 recurrent amplicons were evaluated in 5 independent head and neck cancer datasets, with 40 candidates found to be overexpressed relative to normal tissues. These genes were significantly enriched in the canonical ERK/MAPK, FGF, p53, PTEN and PI3K/AKT signaling pathways (p = 8.95 x 10(-3) to 3.18 x 10(-2)). These identified pathways share interactions in one signaling network, and amplification-mediated deregulation of this network was found in 30.0% of these preinvasive lesions. No such alterations were found in 14 low-grade dysplasias that did not progress, whereas 43.5% (10/23) of OSCCs were found to have altered genes within the pathways with DNA amplification. Multitarget FISH showed that amplification of EGFR and CCND1 can coexist in single cells of an oral dysplasia, suggesting the dependence on multiple oncogenes for OPL progression. Taken together, these findings identify a critical biological network that is frequently disrupted in high-risk OPLs, with different specific genes disrupted in different individuals.

Genomic Imbalances in Precancerous Tissues Signal Oral Cancer Risk

Oral cancer develops through a series of histopathological stages: through mild (low grade), moderate, and severe (high grade) dysplasia to carcinoma in situ and then invasive disease. Early detection of those oral premalignant lesions (OPLs) that will develop into invasive tumors is necessary to improve the poor prognosis of oral cancer. Because no tools exist for delineating progression risk in low grade oral lesions, we cannot determine which of these cases require aggressive intervention. We undertook whole genome analysis by tiling-path array comparative genomic hybridization for a rare panel of early and late stage OPLs (n = 62), all of which had extensive longitudinal follow up (>10 years). Genome profiles for oral squamous cell carcinomas (n = 24) were generated for comparison. Parallel analysis of genome alterations and clinical parameters was performed to identify features associated with disease progression. Genome alterations in low grade dysplasias progressing to invasive disease more closely resembled those observed for later stage disease than they did those observed for non-progressing low grade dysplasias. This was despite the histopathological similarity between progressing and non-progressing cases. Strikingly, unbiased computational analysis of genomic alteration data correctly classified nearly all progressing low grade dysplasia cases. Our data demonstrate that high resolution genomic analysis can be used to evaluate progression risk in low grade OPLs, a marked improvement over present histopathological approaches which cannot delineate progression risk. Taken together, our data suggest that whole genome technologies could be used in management strategies for patients presenting with precancerous oral lesions.

Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells

Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.

Methylation Analysis by DNA Immunoprecipitation

DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)-based methods to evaluate methylation status of DNA have been instrumental in such genome-wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome-wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed.

Integrating the Multiple Dimensions of Genomic and Epigenomic Landscapes of Cancer

Advances in high-throughput, genome-wide profiling technologies have allowed for an unprecedented view of the cancer genome landscape. Specifically, high-density microarrays and sequencing-based strategies have been widely utilized to identify genetic (such as gene dosage, allelic status, and mutations in gene sequence) and epigenetic (such as DNA methylation, histone modification, and microRNA) aberrations in cancer. Although the application of these profiling technologies in unidimensional analyses has been instrumental in cancer gene discovery, genes affected by low-frequency events are often overlooked. The integrative approach of analyzing parallel dimensions has enabled the identification of (a) genes that are often disrupted by multiple mechanisms but at low frequencies by any one mechanism and (b) pathways that are often disrupted at multiple components but at low frequencies at individual components. These benefits of using an integrative approach illustrate the concept that the whole is greater than the sum of its parts. As efforts have now turned toward parallel and integrative multidimensional approaches for studying the cancer genome landscape in hopes of obtaining a more insightful understanding of the key genes and pathways driving cancer cells, this review describes key findings disseminating from such high-throughput, integrative analyses, including contributions to our understanding of causative genetic events in cancer cell biology.

Transcriptome Profiles of Carcinoma-in-situ and Invasive Non-small Cell Lung Cancer As Revealed by SAGE

Non-small cell lung cancer (NSCLC) presents as a progressive disease spanning precancerous, preinvasive, locally invasive, and metastatic lesions. Identification of biological pathways reflective of these progressive stages, and aberrantly expressed genes associated with these pathways, would conceivably enhance therapeutic approaches to this devastating disease.

Patient-derived First Generation Xenografts of Non-small Cell Lung Cancers: Promising Tools for Predicting Drug Responses for Personalized Chemotherapy

Current chemotherapeutic regimens have only modest benefit for non-small cell lung cancer (NSCLC) patients. Cumulative toxicities/drug resistance limit chemotherapy given after the first-line regimen. For personalized chemotherapy, clinically relevant NSCLC models are needed for quickly predicting the most effective regimens for therapy with curative intent. In this study, first generation subrenal capsule xenografts of primary NSCLCs were examined for (a) determining responses to conventional chemotherapeutic regimens and (b) selecting regimens most effective for individual patients.

Copy Number Variations in the Human Genome and Strategies for Analysis

The structure and sequence of the genome is immensely variable in the human population. Segmental copy number variants (CNVs) contribute to the extensive phenotypic diversity among humans and have been shown to associate with disease susceptibility. In this article, we provide a detailed review of human genetic variations and the experimental approaches used to discover, catalog, and genotype CNVs.

An Integrative Multi-dimensional Genetic and Epigenetic Strategy to Identify Aberrant Genes and Pathways in Cancer

Genomics has substantially changed our approach to cancer research. Gene expression profiling, for example, has been utilized to delineate subtypes of cancer, and facilitated derivation of predictive and prognostic signatures. The emergence of technologies for the high resolution and genome-wide description of genetic and epigenetic features has enabled the identification of a multitude of causal DNA events in tumors. This has afforded the potential for large scale integration of genome and transcriptome data generated from a variety of technology platforms to acquire a better understanding of cancer.

Contrasting Transcriptional Responses of a Virulent and an Attenuated Strain of Mycobacterium Tuberculosis Infecting Macrophages

H37Rv and H37Ra are well-described laboratory strains of Mycobacterium tuberculosis derived from the same parental strain, H37, that show dramatically different pathogenic phenotypes.

FACADE: a Fast and Sensitive Algorithm for the Segmentation and Calling of High Resolution Array CGH Data

The availability of high resolution array comparative genomic hybridization (CGH) platforms has led to increasing complexities in data analysis. Specifically, defining contiguous regions of alterations or segmentation can be computationally intensive and popular algorithms can take hours to days for the processing of arrays comprised of hundreds of thousands to millions of elements. Additionally, tumors tend to demonstrate subtle copy number alterations due to heterogeneity, ploidy and hybridization effects. Thus, there is a need for fast, sensitive array CGH segmentation and alteration calling algorithms. Here, we describe Fast Algorithm for Calling After Detection of Edges (FACADE), a highly sensitive and easy to use algorithm designed to rapidly segment and call high resolution array data.

Array Comparative Genomic Hybridization of Peripheral Blood Granulocytes of Patients with Myelodysplastic Syndrome Detects Karyotypic Abnormalities

The diagnosis of myelodysplastic syndromes (MDSs) relies largely on morphologic and karyotypic abnormalities, present in about 50% of patients with MDS. Array-based genomic platforms have identified copy number alterations in 50% to 70% of bone marrow samples of patients with MDS with a normal karyotype, suggesting a diagnostic role for these platforms. We investigated whether blood granulocytes harbor the same copy number alterations as the marrow of affected patients. Of 11 patients, 4 had cytogenetic abnormalities shown by conventional karyotyping involving chromosomes 5, 8, 11, 20, and X, and these changes were seen in the granulocytes of all 4 patients by using array comparative genomic hybridization (aCGH). Cryptic alterations were identified at a significantly higher level in marrow CD34+ cells compared with granulocytes (P < .0001). These data suggest that aCGH analysis of circulating granulocytes may be useful in detecting gross karyotypic alterations in patients with MDS when marrow examination has failed or not been done.

Integrative Genomic Analyses Identify BRF2 As a Novel Lineage-specific Oncogene in Lung Squamous Cell Carcinoma

Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes--adenocarcinoma (AC) and squamous cell carcinoma (SqCC)--respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome.

Lung Cancer Cell Lines As Tools for Biomedical Discovery and Research

Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and non-small cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel.

A Sequence-based Approach to Identify Reference Genes for Gene Expression Analysis

An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer) may not be suitable in another (e.g. breast cancer). Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate.

Copy Number Alterations at Polymorphic Loci May Be Acquired Somatically in Patients with Myelodysplastic Syndromes

Loss of genomic integrity is thought to be one of the underlying causes of myelodysplastic syndromes (MDS). However, it is unclear whether changes in copy number at loci that are common sites of copy number polymorphisms play a pathogenic role. Here we show that copy number changes in the MDS clone that occur at polymorphic loci are frequently somatic alterations rather than constitutional variants, and the extent of copy number changes at polymorphic loci is increased in CD34(+) cells of MDS patients compared to age-matched controls. This study suggests a potential pathophysiological role for copy number alterations at polymorphic loci in patients with MDS, and highlights the need for somatic control tissues for each patient studied in high-resolution genome-wide investigations.

Genome-wide Identification of Human MicroRNAs Located in Leukemia-associated Genomic Alterations

Cytogenetic alterations, such as amplifications, deletions, or translocations, contribute to myeloid malignancies. MicroRNAs (miRNAs) have emerged as critical regulators of hematopoiesis, and their aberrant expression has been associated with leukemia. Genomic regions containing sequence alterations and fragile sites in cancers are enriched with miRNAs; however, the relevant miRNAs within these regions have not been evaluated on a global basis. Here, we investigated miRNAs relevant to acute myeloid leukemia (AML) by (1) mapping miRNAs within leukemia-associated genomic alterations in human AML cell lines by high-resolution genome arrays and (2) evaluating absolute expression of these miRNAs by massively parallel small RNA sequencing. Seventy-seven percent (542 of 706) of miRNAs mapped to leukemia-associated copy-number alterations in the cell lines; however, only 18% (99 of 542) of these miRNAs are expressed above background levels. As evidence that this subset of miRNAs is relevant to leukemia, we show that loss of 2 miRNAs identified in our analysis, miR-145 and miR-146a, results in leukemia in a mouse model. Small RNA sequencing identified 28 putative novel miRNAs, 18 of which map to leukemia-associated copy-number alterations. This detailed genomic and small RNA analysis points to a subset of miRNAs that may play a role in myeloid malignancies.

Deciphering Squamous Cell Carcinoma Using Multidimensional Genomic Approaches

Squamous cell carcinomas (SqCCs) arise in a wide range of tissues including skin, lung, and oral mucosa. Although all SqCCs are epithelial in origin and share common nomenclature, these cancers differ greatly with respect to incidence, prognosis, and treatment. Current knowledge of genetic similarities and differences between SqCCs is insufficient to describe the biology of these cancers, which arise from diverse tissue origins. In this paper we provide a general overview of whole genome approaches for gene and pathway discovery and highlight the advancement of integrative genomics as a state-of-the-art technology in the study of SqCC genetics.

Comparison of Genome-wide Array Genomic Hybridization Platforms for the Detection of Copy Number Variants in Idiopathic Mental Retardation

Clinical laboratories are adopting array genomic hybridization as a standard clinical test. A number of whole genome array genomic hybridization platforms are available, but little is known about their comparative performance in a clinical context.

Cyclin E1 is Amplified and Overexpressed in Osteosarcoma

Osteosarcoma is a genetically complex malignancy, predominantly afflicting the adolescent population and associated still with relatively poor long-term outcomes. Although there has been some improvement in the understanding of osteosarcoma biology, this has not yet translated particularly well into therapeutic advances. By using a whole-genome tiling path array for comparative genomic hybridization analysis, we sought to evaluate DNA copy number changes in 22 osteosarcoma tumor samples. Regions of most frequent gains or losses generated by Genomic Identification of Significant Targets in Cancer analysis were evaluated for genes of interest. Correlation of the copy number data with preexisting expression data for these genes yielded not only targets known to be important in osteosarcoma but also novel targets, notably cyclin E1. Fluorescence in situ hybridization and immunohistochemical analysis confirmed the findings. Overexpression of cyclin E1 has potential prognostic and therapeutic implications that are discussed herein.

The Functional Role of Long Non-coding RNA in Human Carcinomas

Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently aberrantly expressed in a variety of human cancers, however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into the functional roles they may play in tumorigenesis. In this review, we highlight the emerging functional role of lncRNAs in human cancer.

MicroRNA Gene Dosage Alterations and Drug Response in Lung Cancer

Chemotherapy resistance is a key contributor to the dismal prognoses for lung cancer patients. While the majority of studies have focused on sequence mutations and expression changes in protein-coding genes, recent reports have suggested that microRNA (miRNA) expression changes also play an influential role in chemotherapy response. However, the role of genetic alterations at miRNA loci in the context of chemotherapy response has yet to be investigated. In this study, we demonstrate the application of an integrative, multidimensional approach in order to identify miRNAs that are associated with chemotherapeutic resistance and sensitivity utilizing publicly available drug response, miRNA loci copy number, miRNA expression, and mRNA expression data from independent resources. By instigating a logical stepwise strategy, we have identified specific miRNAs that are associated with resistance to several chemotherapeutic agents and provide a proof of principle demonstration of how these various databases may be exploited to derive relevant pharmacogenomic results.

Chromosome-wide DNA Methylation Analysis Predicts Human Tissue-specific X Inactivation

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E-16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset of genes predicted different XCI statuses in different tissues. Using previously published expression data the effect of transcription on gene-body methylation was investigated and while X-linked introns of highly expressed genes were more methylated than the introns of lowly expressed genes, exonic methylation did not differ based on expression level. We conclude that the XCI status predicted using methylation of X-linked promoters with CpG islands was usually the same as determined by expression analysis and that 12% of X-linked genes examined show tissue-specific XCI whereby a gene has a different XCI status in at least one of the four tissues examined.

Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components is a Key Mechanism of NF-kappaB Pathway Activation in Lung Cancer

Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-β/IKK-2), which activates NF-κB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-κB pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-κB activation in non-small cell lung cancer.

Long Non-coding RNAs Are Expressed in Oral Mucosa and Altered in Oral Premalignant Lesions

Oral epithelial dysplasias are believed to progress through a series of histopathological stages; from mild to severe dysplasia, to carcinoma in situ, and finally to invasive OSCC. Underlying this change in histopathological grade are gross chromosome alterations and changes in gene expression of both protein-coding genes and non-coding RNAs. Recent papers have described associations of aberrant expression of microRNAs, one class of non-coding RNAs, with oral cancer. However, expression profiling of long non-coding RNAs (lncRNAs) has not been reported. Long non-coding RNAs are a novel class of mRNA-like transcripts with no protein coding capacity, but with a variety of functions including roles in epigenetics and gene regulation. In recent reports, the aberrant expression of lncRNAs has been associated with human cancers, suggesting a critical role in tumorigenesis. Here, we present the first long non-coding RNA expression map for the human oral mucosa. We describe the expression of 325 long non-coding RNAs, suggesting lncRNA expression contributes significantly to the oral transcriptome. Intriguingly, ∼60% of the detected lncRNAs show aberrant expression in oral premalignant lesions. A number of these lncRNAs have been previously associated with other human cancers.

MiR-101 DNA Copy Loss is a Prominent Subtype Specific Event in Lung Cancer

MicroRNA-101 (miR-101) is frequently downregulated in cancer and exhibits antitumorigenic properties, suggesting that miR-101 is a putative tumor suppressor. miR-101 is encoded at two loci in the human genome: 1p31.3 (miR-101-1) and 9p24.1 (miR-101-2). We sought to investigate miR-101 locus-specific deletions and genomic loss in the major subtypes of lung cancer.

TRAF6 is an Amplified Oncogene Bridging the RAS and NF-κB Pathways in Human Lung Cancer

Somatic mutations and copy number alterations (as a result of deletion or amplification of large portions of a chromosome) are major drivers of human lung cancers. Detailed analysis of lung cancer-associated chromosomal amplifications could identify novel oncogenes. By performing an integrative cytogenetic and gene expression analysis of non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) cell lines and tumors, we report here the identification of a frequently recurring amplification at chromosome 11 band p13. Within this region, only TNF receptor-associated factor 6 (TRAF6) exhibited concomitant mRNA overexpression and gene amplification in lung cancers. Inhibition of TRAF6 in human lung cancer cell lines suppressed NF-κB activation, anchorage-independent growth, and tumor formation. In these lung cancer cell lines, RAS required TRAF6 for its oncogenic capabilities. Furthermore, TRAF6 overexpression in NIH3T3 cells resulted in NF-κB activation, anchorage-independent growth, and tumor formation. Our findings show that TRAF6 is an oncogene that is important for RAS-mediated oncogenesis and provide a mechanistic explanation for the previously apparent importance of constitutive NF-κB activation in RAS-driven lung cancers.

Human Cancer Long Non-coding RNA Transcriptomes

Once thought to be a part of the 'dark matter' of the genome, long non-coding RNAs (lncRNAs) are emerging as an integral functional component of the mammalian transcriptome. LncRNAs are a novel class of mRNA-like transcripts which, despite no known protein-coding potential, demonstrate a wide range of structural and functional roles in cellular biology. However, the magnitude of the contribution of lncRNA expression to normal human tissues and cancers has not been investigated in a comprehensive manner. In this study, we compiled 272 human serial analysis of gene expression (SAGE) libraries to delineate lncRNA transcription patterns across a broad spectrum of normal human tissues and cancers. Using a novel lncRNA discovery pipeline we parsed over 24 million SAGE tags and report lncRNA expression profiles across a panel of 26 different normal human tissues and 19 human cancers. Our findings show extensive, tissue-specific lncRNA expression in normal tissues and highly aberrant lncRNA expression in human cancers. Here, we present a first generation atlas for lncRNA profiling in cancer.

Arsenic Biotransformation As a Cancer Promoting Factor by Inducing DNA Damage and Disruption of Repair Mechanisms

Chronic exposure to arsenic in drinking water poses a major global health concern. Populations exposed to high concentrations of arsenic-contaminated drinking water suffer serious health consequences, including alarming cancer incidence and death rates. Arsenic is biotransformed through sequential addition of methyl groups, acquired from s-adenosylmethionine (SAM). Metabolism of arsenic generates a variety of genotoxic and cytotoxic species, damaging DNA directly and indirectly, through the generation of reactive oxidative species and induction of DNA adducts, strand breaks and cross links, and inhibition of the DNA repair process itself. Since SAM is the methyl group donor used by DNA methyltransferases to maintain normal epigenetic patterns in all human cells, arsenic is also postulated to affect maintenance of normal DNA methylation patterns, chromatin structure, and genomic stability. The biological processes underlying the cancer promoting factors of arsenic metabolism, related to DNA damage and repair, will be discussed here.

Arsenic Exposure and the Induction of Human Cancers

Arsenic is a metalloid, that is, considered to be a human carcinogen. Millions of individuals worldwide are chronically exposed through drinking water, with consequences ranging from acute toxicities to development of malignancies, such as skin and lung cancer. Despite well-known arsenic-related health effects, the molecular mechanisms involved are not fully understood; however, the arsenic biotransformation process, which includes methylation changes, is thought to play a key role. This paper explores the relationship of arsenic exposure with cancer development and summarizes current knowledge of the potential mechanisms that may contribute to the neoplastic processes observed in arsenic exposed human populations.

Induction of Human Squamous Cell-type Carcinomas by Arsenic

Arsenic is a potent human carcinogen. Around one hundred million people worldwide have potentially been exposed to this metalloid at concentrations considered unsafe. Exposure occurs generally through drinking water from natural geological sources, making it difficult to control this contamination. Arsenic biotransformation is suspected to have a role in arsenic-related health effects ranging from acute toxicities to development of malignancies associated with chronic exposure. It has been demonstrated that arsenic exhibits preference for induction of squamous cell carcinomas in the human, especially skin and lung cancer. Interestingly, keratins emerge as a relevant factor in this arsenic-related squamous cell-type preference. Additionally, both genomic and epigenomic alterations have been associated with arsenic-driven neoplastic process. Some of these aberrations, as well as changes in other factors such as keratins, could explain the association between arsenic and squamous cell carcinomas in humans.

Allelic Loss of the Loci Containing the Androgen Synthesis Gene, StAR, is Prognostic for Relapse in Intermediate-risk Prostate Cancer

BACKGROUND: Androgen deprivation therapy (ADT) and novel agents targeting the androgen synthesis axis (e.g., abiraterone acetate) are adjuvant therapies that are currently, or may in the future be, combined with radiotherapy to reduce the chance of disease relapse. Little is known about allelic loss or gain pertaining to genes associated with the androgen synthesis axis and whether this is prognostic in patients who receive localized radiotherapy. In this hypothesis generating study, we conducted an array comparative genomic hybridization (aCGH) analysis of 33 androgen synthesis genes to identify potential prognostic factors for radiotherapy outcome. METHODS: aCGH analysis of tumor DNA prospectively derived from frozen needle biopsies of 126 men with intermediate-risk disease who underwent image-guided radiotherapy (IGRT) to a mean dose of 76.4 Gy was conducted. Statistical analyses were conducted for allelic loss or gain in genes as potential prognostic factors relative to prostate specific antigen, Gleason-score, and T-category. RESULTS: We observed that allelic losses of loci containing the genes StAR and HSD17B2 were associated with increased genetic instability (as determined by percentage genome alteration). On multivariate analyses these loci were prognostic for biochemical disease-free relapse (StAR: HR = 2.84, 95% CI: 1.44-5.61, P = 0.00269; HSD17B2: HR = 1.97, 95% CI: 1.06-3.64, P = 0.031). The results were validated in a surgical cohort of 131 intermediate-risk patients. CONCLUSIONS: Allelic losses of the loci containing StAR and HSD17B2 have significant prognostic value for intermediate-risk prostate cancer. With this hypothesis generating information future studies should test StAR and HSD17B2 losses as biomarkers of androgen response in combined modality protocols. Prostate © 2011 Wiley Periodicals, Inc.

NKX3.1 Haploinsufficiency is Prognostic for Prostate Cancer Relapse Following Surgery or Image-guided Radiotherapy

Despite the use of prostate specific antigen (PSA), Gleason-score, and T-category as prognostic factors, up to 40% of patients with intermediate-risk prostate cancer will fail radical prostatectomy or precision image-guided radiotherapy (IGRT). Additional genetic prognosticators are needed to triage these patients toward intensified combination therapy with novel targeted therapeutics. We tested the role of the NKX3.1 gene as a determinant of treatment outcome given its reported roles in tumor initiating cell (TIC) renewal, the DNA damage response, and cooperation with c-MYC during prostate cancer progression.

Copy Number Alterations of C-MYC and PTEN Are Prognostic Factors for Relapse After Prostate Cancer Radiotherapy

Despite the use of PSA, Gleason score, and T-category as prognosticators in intermediate-risk prostate cancer, 20-40% of patients will fail local therapy. In order to optimize treatment approaches for intermediate-risk patients, additional genetic prognosticators are needed. Previous reports using array comparative genomic hybridization (aCGH) in radical prostatectomy cohorts suggested a combination of allelic loss of the PTEN gene on 10q and allelic gain of the c-MYC gene on 8q were associated with metastatic disease. We tested whether copy number alterations (CNAs) in PTEN (allelic loss) and c-MYC (allelic gain) were associated with biochemical relapse following modern-era, image-guided radiotherapy (mean dose 76.4 Gy). We used aCGH analyses validated by fluorescence in-situ hybridization (FISH) of DNA was derived from frozen, pre-treatment biopsies in 126 intermediate-risk prostate cancer patients. Patients whose tumors had CNAs in both PTEN and c-MYC had significantly increased genetic instability (percent genome alteration; PGA) compared to tumors with normal PTEN and c-MYC status (p < 0.0001). We demonstrate that c-MYC gain alone, or combined c-MYC gain and PTEN loss, were increasingly prognostic for relapse on multivariable analyses (hazard ratios (HR) of 2.58/p = 0.005 and 3.21/p = 0.0004; respectively). Triaging patients by the use of CNAs within pre-treatment biopsies may allow for better use of systemic therapies to target sub-clinical metastases or locally recurrent disease and improve clinical outcomes. Cancer 2012;. © 2012 American Cancer Society.

Translating Cancer 'omics' to Improved Outcomes

The genomics era has yielded great advances in the understanding of cancer biology. At the same time, the immense complexity of the cancer genome has been revealed, as well as a striking heterogeneity at the whole-genome (or omics) level that exists between even histologically similar tumors. The vast accrual and public availability of multi-omics databases with associated clinical annotation including tumor histology, patient response, and outcome are a rich resource that has the potential to lead to rapid translation of high-throughput omics to improved overall survival. We focus on the unique advantages of a multidimensional approach to genomic analysis in this new high-throughput omics age and discuss the implications of the changing cancer demographic to translational omics research.

Integrative Genomics Identified RFC3 As an Amplified Candidate Oncogene in Esophageal Adenocarcinoma

PURPOSE: Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett's esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers.EXPERIMENTAL DESIGN: Whole genome tiling-path array CGH was used to identify significant regions of copy number (CN) alteration in 20 EACs and 10 matching BE tissues. CN and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes.RESULTS: We identified RFC3 as a novel, candidate oncogene activated by amplification in ~25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification, and was differentially expressed between non-malignant and EAC tissues. CN gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased CN, but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types.CONCLUSIONS: RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker.

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