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ABSTRACT: Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC). As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1) and its matched lymphocyte cell line (AGLCL) to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1), resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA), in a strand-directional manner. We also captured small RNA (<30 bp). We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA). We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.
Neoplasia (New York, N.Y.) 11/2012; 14(11):1075-86. · 5.48 Impact Factor
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Caroline Conway,
Rebecca Chalkley,
Alec High,
Kenneth Maclennan,
Stefano Berri,
Preetha Chengot,
Melissa Alsop,
Philip Egan,
Joanne Morgan,
Graham R Taylor,
John Chester,
Mehmet Sen, Pamela Rabbitts,
Henry M Wood
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ABSTRACT: Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.
The Journal of molecular diagnostics: JMD 03/2012; 14(2):104-11. · 3.48 Impact Factor
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ABSTRACT: Comparison of read depths from next-generation sequencing between cancer and normal cells makes the estimation of copy number alteration (CNA) possible, even at very low coverage. However, estimating CNA from patients' tumour samples poses considerable challenges due to infiltration with normal cells and aneuploid cancer genomes. Here we provide a method that corrects contamination with normal cells and adjusts for genomes of different sizes so that the actual copy number of each region can be estimated.
The procedure consists of several steps. First, we identify the multi-modality of the distribution of smoothed ratios. Then we use the estimates of the mean (modes) to identify underlying ploidy and the contamination level, and finally we perform the correction. The results indicate that the method works properly to estimate genomic regions with gains and losses in a range of simulated data as well as in two datasets from lung cancer patients. It also proves a powerful tool when analysing publicly available data from two cell lines (HCC1143 and COLO829).
An R package, called CNAnorm, is available at http://www.precancer.leeds.ac.uk/cnanorm or from Bioconductor.
a.gusnanto@leeds.ac.uk
Supplementary data are available at Bioinformatics online.
Bioinformatics 01/2012; 28(1):40-7. · 5.47 Impact Factor
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Ornella Belvedere,
Stefano Berri,
Rebecca Chalkley,
Caroline Conway,
Fabio Barbone,
Federica Pisa,
Kenneth MacLennan,
Catherine Daly,
Melissa Alsop,
Joanne Morgan,
Jessica Menis,
Peter Tcherveniakov,
Kostas Papagiannopoulos, Pamela Rabbitts,
Henry M Wood
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ABSTRACT: Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.
Genomics 10/2011; 99(1):18-24. · 3.02 Impact Factor
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ABSTRACT: Histologically distinct pre-invasive lesions have been identified as precursors, or putative precursors, of lung cancer; the
extent to which pre-invasive disease precedes invasive disease is, however, still unknown. In the context of lung cancer screening,
understanding the biology behind pre-invasive disease is potentially of importance as it may lead to the identification of
specific and selective markers of pre-invasive lesions at high risk to develop into invasive lung cancer, useful for both
the development of screening tools and also for the development of effective targeted strategies for chemoprevention and early
treatment of such disease. In this chapter we focus on current knowledge and future directions of research on pulmonary pre-invasive
lesions.
12/2010: pages 271-295;
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Henry M Wood,
Ornella Belvedere,
Caroline Conway,
Catherine Daly,
Rebecca Chalkley,
Melissa Bickerdike,
Claire McKinley,
Phil Egan,
Lisa Ross,
Bruce Hayward,
Joanne Morgan,
Leslie Davidson,
Ken MacLennan,
Thian K Ong,
Kostas Papagiannopoulos,
Ian Cook,
David J Adams,
Graham R Taylor, Pamela Rabbitts
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ABSTRACT: The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.
Nucleic Acids Research 08/2010; 38(14):e151. · 8.03 Impact Factor
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William Andrews,
Melissa Barber,
Luis R Hernadez-Miranda,
Jian Xian,
Sonja Rakic,
Vasi Sundaresan,
Terence H Rabbitts,
Richard Pannell, Pamela Rabbitts,
Hannah Thompson,
Lynda Erskine,
Fujio Murakami,
John G Parnavelas
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ABSTRACT: Cortical interneurons in rodents are generated in the ventral telencephalon and migrate tangentially into the cortex. This process requires the coordinated action of many intrinsic and extrinsic factors. Here we show that Robo1 and Robo2 receptor proteins are dynamically expressed throughout the period of corticogenesis and colocalize with interneuronal markers, suggesting that they play a role in the migration of these cells. Analysis of Robo mutants showed a marked increase in the number of interneurons in the cortices of Robo1(-/-), but not Robo2(-/-), animals throughout the period of corticogenesis and in adulthood; this excess number of interneurons was observed in all layers of the developing cortex. Using BrdU incorporation in dissociated cell cultures and phosphohistone-3 labeling in vivo, we demonstrated that the increased number of interneurons in Robo1(-/-) mice is, at least in part, due to increased proliferation. Interestingly, a similar increase in proliferation was observed in Slit1(-/-)/Slit2(-/-) mutant mice, suggesting that cell division is influenced by Slit-Robo signaling mechanisms. Morphometric analysis of migrating interneurons in Robo1(-/-), Robo2(-/-) and Slit1(-/-)/Slit2(-/-), but not in Slit1(-/-) mice, showed a differential increase in neuronal process length and branching suggesting that Slit-Robo signaling also plays an important role in the morphological differentiation of these neurons.
Developmental Biology 02/2008; 313(2):648-58. · 4.07 Impact Factor
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ABSTRACT: The RASSF1A gene is a tumor suppressor gene inactivated by hypermethylation in a very wide variety of malignant tumors including prostate cancer.
In this study we have used laser capture microdissection to provide pure cell populations to investigate the methylation status of 16 CpG sites in the promoter region of this gene in prostatic intra-epithelial neoplasia, in histologically normal epithelial cells associated with these lesions and in epithelial cells from benign prostatic hyperplasia.
Unexpectedly, frequent methylation, detected by sequence analysis following bisulphite treatment, was observed in benign epithelium as well as in the lesions associated with prostatic intra-epithelial neoplasia and at high risk of cancer formation. Fifty percent or more CpG sites were methylated in 7/14 prostatic intra-epithelial neoplasms, 8/11 histologically normal epithelial cells and 8/12 specimens of benign prostatic tissue.
These observations suggest that methylation of the RASSF1A gene is present in both pre-malignant and benign epithelia and suggests quantitation is required for it to be an effective marker of early prostate cancer.
The Prostate 06/2007; 67(6):638-44. · 3.48 Impact Factor
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ABSTRACT: The DUTT1 gene is located on human chromosome 3, band p12, within a region of nested homozygous deletions in breast and lung tumors. It is therefore a candidate tumor suppressor gene in humans and is the homologue (ROBO1) of the Drosophila axonal guidance receptor gene, Roundabout. We have shown previously that mice with a targeted homozygous deletion within the Dutt1/Robo1 gene generally die at birth due to incomplete lung development: survivors die within the first year of life with epithelial bronchial hyperplasia as a common feature. Because Dutt1/Robo1 heterozygous mice develop normally, we have determined their tumor susceptibility. Mice with a targeted deletion within one Dutt1/Robo1 allele spontaneously develop lymphomas and carcinomas in their second year of life with a 3-fold increase in incidence compared with controls: invasive lung adenocarcinomas are by far the predominant carcinoma. In addition to the mutant allele, loss of heterozygosity analysis indicates that these tumors retain the structurally normal allele but with substantial methylation of the gene's promoter. Substantial reduction of Dutt1/Robo1 protein expression in tumors is observed by Western blotting and immunohistochemistry. This suggests that Dutt1/Robo1 is a classic tumor suppressor gene requiring inactivation of both alleles to elicit tumorigenesis in these mice.
Cancer Research 10/2004; 64(18):6432-7. · 7.86 Impact Factor
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ABSTRACT: The mammalian homologue of the Drosophila axonal guidance receptor roundabout is expressed in a wide range of tissues. Here we show that alternative splicing of the Dutt1/Robo1 gene results in two mRNA transcripts with different signal peptides, which are differentially expressed throughout mouse embryogenesis. Since mice with a targeted deletion in the Dutt1/Robo1 gene have abnormal lung pathology, immunohistochemistry was used to identify the cellular expression pattern of Dutt1/Robo1 during lung development. Dutt1/Robo1 expression was widespread and diffuse in the lung at embryonic day 17.5 but became increasingly localised to the bronchial epithelium in newborn and adult mice.
FEBS Letters 08/2002; 523(1-3):12-6. · 3.54 Impact Factor
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ABSTRACT: The human homologue of the Drosophila Roundabout gene DUTT1 (Deleted in U Twenty Twenty) or ROBO1 (Locus Link ID 6091), a member of the NCAM family of receptors, was recently cloned from the lung cancer tumour suppressor gene region 2 (LCTSGR2 or U2020 region) at 3p12. DUTT1 maps within a region of overlapping homozygous deletions characterized in both small cell lung cancer lines (SCLC) and in a breast cancer line. In this report we (a) defined the genomic organization of the DUTT1 gene, (b) performed mutation and expression analysis of DUTT1 in lung, breast and kidney cancers, (c) identified tumour specific promoter region methylation of DUTT1 in human cancers. The gene was found to contain 29 exons and spans at least 240 kb of genomic sequence. The 5' region contains a CpG island, and the poly(A)(+) tail has an atypical 5'-GATAAA-3' signal. We analysed DUTT1 for mutations in lung, breast and kidney cancers, no inactivating mutations were detected by PCR-SSCP. However, seven germline missense changes were found and characterized. DUTT1 expression was not detectable in one out of 18 breast tumour lines analysed by RT-PCR. Bisulfite sequencing of the promoter region of DUTT1 gene in the HTB-19 breast tumour cell line (not expressing DUTT1) showed complete hypermethylation of CpG sites within the promoter region of the DUTT1 gene (-244 to +27 relative to the translation start site). The expression of DUTT1 gene was reactivated in HTB-19 after treatment with the demethylating agent 5-aza-2'-deoxycytidine. The same region was also found to be hypermethylated in six out of 32 (19%) primary invasive breast carcinomas and eight out of 44 (18%) primary clear cell renal cell carcinomas (CC-RCC) and in one out of 26 (4%) primary NSCLC tumours. Furthermore 80% of breast and 75% of CC-RCC tumours showing DUTT1 methylation had allelic losses for 3p12 markers hence obeying Knudson's two hit hypothesis. Our findings suggest that DUTT1 warrants further analysis as a candidate for the tumour suppressor gene (TSG) at 3p12, a region defined by hemi and homozygous deletions and functional analysis.
Oncogene 06/2002; 21(19):3020-8. · 6.37 Impact Factor
