[Show abstract][Hide abstract] ABSTRACT: Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to assess the efficacy of neoadjuvant anastrozole and fulvestrant treatment of large operable or locally advanced hormone-receptor-positive breast cancer not eligible for initial breast-conserving surgery, and to identify genomic changes occurring after treatment.
One hundred and twenty post-menopausal patients were randomised to receive 1 mg anastrozole (61 patients) or 500 mg fulvestrant (59 patients) for 6 months. Genomic DNA copy number profiles were generated for a subgroup of 20 patients before and after treatment.
A total of 108 patients were evaluable for efficacy and 118 for toxicity. The objective response rate determined by clinical palpation was 58.9% (95% CI=45.0-71.9) in the anastrozole arm and 53.8% (95% CI=39.5-67.8) in the fulvestrant arm. The breast-conserving surgery rate was 58.9% (95% CI=45.0-71.9) in the anastrozole arm and 50.0% (95% CI=35.8-64.2) in the fulvestrant arm. Pathological responses >50% occurred in 24 patients (42.9%) in the anastrozole arm and 13 (25.0%) in the fulvestrant arm. The Ki-67 score fell after treatment but there was no significant difference between the reduction in the two arms (anastrozole 16.7% (95% CI=13.3-21.0) before, 3.2% (95% CI=1.9-5.5) after, n=43; fulvestrant 17.1% (95%CI=13.1-22.5) before, 3.2% (95% CI=1.8-5.7) after, n=38) or between the reduction in Ki-67 in clinical responders and non-responders. Genomic analysis appeared to show a reduction of clonal diversity following treatment with selection of some clones with simpler copy number profiles.
Both anastrozole and fulvestrant were effective and well-tolerated, enabling breast-conserving surgery in over 50% of patients. Clonal changes consistent with clonal selection by the treatment were seen in a subgroup of patients.British Journal of Cancer advance online publication 14 July 2015; doi:10.1038/bjc.2015.247 www.bjcancer.com.
Full-text · Article · Jul 2015 · British Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognised, especially now the extent of clonal heterogeneity in fully invasive disease is being realised. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they too are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations together with copy number analysis from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas, and ultimately, the diversity of processes by which tumours are initiated, spread and metastasise. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly-related genomes. We show that oral pre-cancer exhibits considerable sub-clonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable.
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Full-text · Article · Jun 2015 · The Journal of Pathology
[Show abstract][Hide abstract] ABSTRACT: The catalogue of tumour-specific somatic mutations (SMs) 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, nonsynonymous mutations: those that alter an encoded protein. However, growing evidence suggests the involvement of noncoding 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 SMs, irrespective of coding capacity. We preliminarily tested this by applying it to a dataset of 159,498 SMs 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 are 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.
Full-text · Article · Jan 2015 · International Journal of Cancer
[Show abstract][Hide abstract] 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.
No preview · Article · Oct 2014 · European Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Objective:
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.
We used next-generation sequencing for the detection of papillomavirus 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%.
An HPV-16 sequence was detected in 1 carcinoma and 1 hyperplasia, and an HPV-2 sequence was detected in 1 carcinoma out of the 78 cases, with viral loads of 2.24, 8.16, and 0.33 viral genomes per cell, respectively.
Our results indicate no conclusive human papillomavirus involvement in oral verrucous carcinoma or hyperplasia.
[Show abstract][Hide abstract] 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.
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%.
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.
Our results indicate no conclusive human papilloma virus involvement in oral verrucous carcinoma or hyperplasia.
[Show abstract][Hide abstract] 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: email@example.com.
[Show abstract][Hide abstract] 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.
Full-text · Article · Dec 2013 · Journal of Oral Pathology and Medicine
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
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.
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.
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.
Full-text · Article · Nov 2013 · British Journal of Cancer
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.