P H Rabbitts

University of Leeds, Leeds, England, United Kingdom

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Publications (85)422.83 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The etiology of oral verrucous carcinoma is unknown, and human papillomavirus 'involvement' remains contentious. The uncertainty can be attributed to varied detection procedures and difficulties in defining 'gold-standard' histologic criteria for diagnosing 'verrucous' lesions. Their paucity also hampers investigation. We aimed to analyze oral verrucous lesions for human papillomavirus (HPV) subtype genomes.
    Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 07/2014; 118(1):117-125.e1. · 1.50 Impact Factor
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    ABSTRACT: The catalogue of tumour-specific somatic mutations is growing rapidly owing to the advent of next-generation sequencing. Identifying those mutations responsible for the development and progression of the disease, so-called driver mutations, will increase our understanding of carcinogenesis and provide candidates for targeted therapeutics. The phenotypic consequence(s) of driver mutations cause them to be selected for within the tumour environment, such that many approaches aimed at distinguishing drivers are based on finding significantly somatically mutated genes. Currently, these methods are designed to analyse, or be specifically applied to, non-synonymous mutations: those that alter an encoded protein. However, growing evidence suggests the involvement of non-coding transcripts in carcinogenesis, mutations in which may also be disease-driving.We wished to test the hypothesis that common DNA variation rates within humans can be used as a baseline from which to score the rate of somatic mutations, irrespective of coding capacity. We preliminarily tested this by applying it to a dataset of 159,498 somatic mutations and using the results to rank genes. This resulted in significant enrichment of known cancer genes, indicating that the approach has merit. As additional data from cancer sequencing studies is made publicly available, this approach can be refined and applied to specific cancer subtypes. We named this preliminary version of our approach PRISMAD (Polymorphism Rates Indicate Somatic Mutations As Drivers) and have made it publicly accessible, with scripts, via a link at www.precancer.leeds.ac.uk/software-and-datasets. © 2014 Wiley Periodicals, Inc.
    International Journal of Cancer 05/2014; · 6.20 Impact Factor
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    ABSTRACT: Purpose The aetiology of oral verrucous carcinoma is unknown whilst human papillomavirus ‘involvement’ remains contentious. Dubiety can be attributed to varied detection procedures and difficulties in defining ‘gold-standard’ histological criteria for diagnosing ‘verrucous’ lesions. Their paucity also hampers investigation. Design We aimed to analyse oral verrucous lesions for HPV subtype genomes using ‘next generation sequencing’ for the detection of papilloma virus sequences, identifying subtypes and computing viral loads. We identified a total of 78 oral verrucous cases [62 carcinomas and 16 hyperplasias]. DNA was extracted from all and sequenced at a coverage between 2.5 and 13%. Findings An HPV-16 sequence was detected in one carcinoma and one hyperplasia, and an HPV-2 sequence was detected in one carcinoma out of the 78 cases, with viral loads of 2.24, 8.16 and 0.33 viral genomes per cell respectively. Practical implications Our results indicate no conclusive human papilloma virus involvement in oral verrucous carcinoma or hyperplasia.
    Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 04/2014; · 1.50 Impact Factor
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    ABSTRACT: Current high throughput sequencing has greatly transformed genome sequence analysis. In the context of very low- coverage sequencing (<0.1X), performing 'binning' or 'windowing' on mapped short sequences ('reads') is critical to extract genomic information of interest for further evaluation, such as copy number alterations analysis. If the window size is too small, many windows will exhibit zero counts and almost no pattern can be observed. In contrast, if the window size is too wide, the patterns or genomic features will be 'smoothed out'. Our objective is to identify an optimal window size in between the two extremes. We assume the reads density to be a step function. Given this model, we propose a data-based estimation of optimal window size based on Akaike's information criterion (AIC) and cross-validation (CV) log-likelihood. By plotting the AIC and CV log likelihood curve as a function of window size, we are able to estimate the optimal window size that minimises AIC or maximise CV log-likelihood. The proposed methods are of general purpose and we illustrate their application using low-coverage next-generation sequence datasets from real tumour samples and simulated datasets. An R package to estimate optimal window size is available in http://www1.maths.leeds.ac.uk/~arief/R/win/ CONTACT: a.gusnanto@leeds.ac.uk.
    Bioinformatics 03/2014; · 5.47 Impact Factor
  • Journal of Investigative Dermatology 01/2014; · 6.19 Impact Factor
  • Neeraj Sethi, Henry Wood, Pamela Rabbitts
    Oral Oncology. 01/2014;
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    ABSTRACT: MicroRNAs are a class of non-coding RNA which regulate gene expression. Their discovery in humans in 2000 has led to an explosion in research in this area in terms of their role as a biomarker, therapeutic target as well as trying to elucidate their function. This review aims to summarise the function of microRNAs as well as to examine how dysregulation at any step in their biogenesis or functional pathway can play a role in the development of cancer. We review which microRNAs are implicated as oncogenic or tumour suppressor in head and neck cancer as well as the data available on the use of microRNAs as diagnostic and prognostic marker. We also discuss routes for future research.
    European Journal of Cancer. 01/2014;
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    ABSTRACT: Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK) (4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK) (4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK) (4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK) (4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK) (4a) positivity and was unable to confirm a prognostic ability for this biomarker.
    Journal of Oral Pathology and Medicine 12/2013; · 2.06 Impact Factor
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    ABSTRACT: Background:Prognostic biomarkers aim to improve on the current inadequate method of histological assessment to identify patients with oral epithelial dysplasia at greatest risk of malignant transformation. We aimed to assess the prognostic ability of six protein biomarkers linked to the epidermal growth factor receptor (EGFR) pathway, including three tetraspanins, in a large multicentre oral dysplasia cohort.Methods:One hundred and forty-eight cases with varying degrees of epithelial dysplasia underwent immunohistochemical assessment for CD9, CD151, CD82, EGFR, Her-2, and COX-2. Scoring was performed independently by two observers. Univariate analyses using both logistic and Cox regression models and a multivariate regression were performed.Results:Malignant progression was significantly greater in those cases with decreased expression of CD9 (P=0.02), and increased expression of CD151 (P=0.02), EGFR (P=0.04), and COX-2 (P=0.003). Histological grade (P=0.0002) and morphology (P=0.03) were also prognostic, whereas smoking and alcohol were not. The optimal combination by backward-variable selection was of histological grade (hazard ratio (HR) 1.64; 95% CI 1.12, 2.40), COX-2 overexpression (HR 1.12; 1.02, 1.24) and CD9 underexpression (HR 0.88; 0.80, 0.97). CD82 and Her-2 demonstrated no prognostic ability.Conclusion:This is the first study of the expression and prognostic potential of the tetraspanins in oral dysplasia. A combination of certain biomarkers with clinical factors appeared to improve the accuracy of determining the risk of malignancy in individuals with oral dysplasia. These findings may also offer potential new therapeutic approaches for this condition.British Journal of Cancer advance online publication, 7 November 2013; doi:10.1038/bjc.2013.600 www.bjcancer.com.
    British Journal of Cancer 11/2013; · 5.08 Impact Factor
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    ABSTRACT: Current methods for resolving genetically distinct subclones in tumour samples require somatic mutations to be clustered by allelic frequencies, which are determined by applying a variant calling program to next-generation sequencing data. Such programs were developed to accurately distinguish true polymorphisms and somatic mutations from the artifactual non-reference alleles introduced during library preparation and sequencing. However, numerous variant callers exist with no clear indication of the best-performer for subclonal analysis, in which the accuracy of the assigned variant frequency is as important as correctly indicating whether the variant is present or not. Furthermore, sequencing depth (the number of times that a genomic position is sequenced) affects the ability to detect low-allelic fraction variants and accurately assign their allele frequencies. We created two synthetic sequencing datasets, and sequenced real KRAS amplicons, with variants spiked in at specific ratios, in order to assess which caller performs best in terms of both variant detection and assignment of allelic frequencies. We also assessed the sequencing depths required to detect low-allelic fraction variants. We found that VarScan2 performed best overall with sequencing depths of 100x, 250x, 500x and 1000x required to accurately identify variants present at 10%, 5%, 2.5% and 1% respectively. This article is protected by copyright. All rights reserved.
    Human Mutation 06/2013; · 5.21 Impact Factor
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    ABSTRACT: Array Comparative Genomic Hybridisation (aCGH) profiling is currently the gold standard for genetic diagnosis of copy number. Next Generation Sequencing technologies provide an alternative and adaptable method of detecting copy number by comparing the number of sequence reads in non-overlapping windows between patient and control samples. Detection of copy number using the BlueGnome 8x60K oligonucleotide aCGH platform was compared with low resolution Next Generation Sequencing using the Illumina GAIIx on 39 patients referred to the Leeds Clinical Cytogenetics Laboratory with developmental delay and/or learning difficulties. Sensitivity and workflow of the two platforms were compared. Customised copy number algorithms assessed sequence counts and detected changes in copy number. Imbalances detected on both platforms were compared. Of the thirty-nine patients analysed, all eleven imbalances detected by array CGH and confirmed by FISH or Q-PCR were also detected by CNV-seq. In addition, CNV-seq reported one purported pathogenic copy number variant that was not detected by array CGH. Non-pathogenic, unconfirmed copy number calls were detected by both platforms; however few were concordant between the two. CNV-seq offers an alternative to array CGH for copy number analysis with resolution and future costs comparable to conventional array CGH platforms and with less stringent sample requirements.
    Genomics 04/2013; · 3.01 Impact Factor
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    ABSTRACT: Chromosomal translocations and other abnormalities are central to the initiation of cancer in all cell types. Understanding the mechanism is therefore important to evaluate the evolution of cancer from the cancer initiating events to overt disease. Recent work has concentrated on model systems to develop an understanding of the molecular mechanisms of translocations but naturally occurring events are more ideal case studies since biological selection is absent from model systems. In solid tumours, nonreciprocal translocations are most commonly found, and accordingly we have investigated the recurrent nonreciprocal t(3;5) chromosomal translocations in renal carcinoma to better understand the mechanism of these naturally occurring translocations in cancer. Unexpectedly, the junctions of these translocations can be associated with site-specific, intrachromosomal inversion involving at least two double strand breaks (DSB) in cis and rejoining by nonhomologous end joining or micro-homology end joining. However, these translocations are not necessarily associated with transcribed regions questioning accessibility per se in controlling these events. In addition, intrachromosomal deletions also occur. We conclude these naturally occurring, nonreciprocal t(3;5) chromosomal translocations occur after complex and multiple unresolved intrachromosomal DSBs leading to aberrant joining with concurrent interstitial inversion and that clonal selection of cells is the critical element in cancer development emerging from a plethora of DSBs that may not always be pathogenic. © 2013 Wiley Periodicals, Inc.
    Genes Chromosomes and Cancer 01/2013; · 3.55 Impact Factor
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    ABSTRACT: Squamous cell carcinoma (SCC) of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding) of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.
    PLoS ONE 01/2013; 8(11):e78823. · 3.53 Impact Factor
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    ABSTRACT: Metagenomics, the study of microbial genomes within diverse environments, is a rapidly developing field. The identification of microbial sequences within a host organism enables the study of human intestinal, respiratory, and skin microbiota, and has allowed the identification of novel viruses in diseases such as Merkel cell carcinoma. There are few publicly available tools for metagenomic high throughput sequence analysis. We present Integrated Metagenomic Sequence Analysis (IMSA), a flexible, fast, and robust computational analysis pipeline that is available for public use. IMSA takes input sequence from high throughput datasets and uses a user-defined host database to filter out host sequence. IMSA then aligns the filtered reads to a user-defined universal database to characterize exogenous reads within the host background. IMSA assigns a score to each node of the taxonomy based on read frequency, and can output this as a taxonomy report suitable for cluster analysis or as a taxonomy map (TaxMap). IMSA also outputs the specific sequence reads assigned to a taxon of interest for downstream analysis. We demonstrate the use of IMSA to detect pathogens and normal flora within sequence data from a primary human cervical cancer carrying HPV16, a primary human cutaneous squamous cell carcinoma carrying HPV 16, the CaSki cell line carrying HPV16, and the HeLa cell line carrying HPV18.
    PLoS ONE 01/2013; 8(5):e64546. · 3.53 Impact Factor
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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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    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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    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. · 3.01 Impact Factor
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    ABSTRACT: The term 'field cancerization' is used to describe an epithelial surface that has a propensity to develop cancerous lesions, and in the case of the aerodigestive tract this is often as a result of chronic exposure to carcinogens in cigarette smoke 1, 2. The clinical endpoint is the development of multiple tumours, either simultaneously or sequentially in the same epithelial surface. The mechanisms underlying this process remain unclear; one possible explanation is that the epithelium is colonized by a clonal population of cells that are at increased risk of progression to cancer. We now address this possibility in a short case series, using individual genomic events as molecular biomarkers of clonality. In squamous lung cancer the most common genomic aberration is 3q amplification. We use a digital PCR technique to assess the clonal relationships between multiple biopsies in a longitudinal bronchoscopic study, using amplicon boundaries as markers of clonality. We demonstrate that clonality can readily be defined by these analyses and confirm that field cancerization occurs at a pre-invasive stage and that pre-invasive lesions and subsequent cancers are clonally related. We show that while the amplicon boundaries can be shared between different biopsies, the degree of 3q amplification and the internal structure of the 3q amplicon varies from lesion to lesion. Finally, in this small cohort, the degree of 3q amplification corresponds to clinical progression.
    The Journal of Pathology 06/2011; 224(2):153-9. · 7.59 Impact Factor

Publication Stats

1k Citations
422.83 Total Impact Points

Institutions

  • 2007–2014
    • University of Leeds
      • • Leeds Institute of Molecular Medicine (LIMM)
      • • Department of Statistics
      Leeds, England, United Kingdom
    • University College London
      • Division of Medicine
      London, ENG, United Kingdom
  • 1994–2010
    • University of Cambridge
      • • Centre for Family Research
      • • Department of Oncology
      Cambridge, ENG, United Kingdom
  • 2008
    • Saint James School Of Medicine
      Park Ridge, Illinois, United States
  • 2002
    • The University of Edinburgh
      • Credit Research Centre
      Edinburgh, SCT, United Kingdom
  • 1988–1999
    • Medical Research Council (UK)
      • MRC Laboratory of Molecular Biology
      Londinium, England, United Kingdom
    • Ludwig Institute for Cancer Research
      La Jolla, California, United States
  • 1987–1988
    • Cancer Research UK Cambridge Institute
      Cambridge, England, United Kingdom