[Show abstract][Hide abstract]ABSTRACT: Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low coverage copy number sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. Copy number results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.
[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.
Full-text available · Article · Oct 2015 · Oncotarget
[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. Methods: 120 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. Results: 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. Conclusion: 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.
[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 available · 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 available · Article · Jun 2015 · The Journal of Pathology
[Show abstract][Hide abstract]ABSTRACT: Motivation: The role of personalized medicine and target treatment in the clinical management of cancer patients has become increasingly important in recent years. This has made the task of precise histological substratification of cancers crucial. Increasingly, genomic data are being seen as a valuable classifier. Specifically, copy number alteration (CNA) profiles generated by next-generation sequencing (NGS) can become a determinant for tumours subtyping. The principle purpose of this study is to devise a model with good prediction capability for the tumours histological subtypes as a function of both the patients covariates and their genome-wide CNA profiles from NGS data. Results: We investigate a logistic regression for modelling tumour histological subtypes as a function of the patients’ covariates and their CNA profiles, in a mixed model framework. The covariates, such as age and gender, are considered as fixed predictors and the genome-wide CNA profiles are considered as random predictors. We illustrate the application of this model in lung and oral cancer datasets, and the results indicate that the tumour histological subtypes can be modelled with a good fit. Our cross-validation indicates that the logistic regression exhibits the best prediction relative to other classification methods we considered in this study. The model also exhibits the best agreement in the prediction between smooth-segmented and circular binary-segmented CNA profiles.
[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 available · Article · Jan 2015 · International Journal of Cancer
[Show abstract][Hide abstract]ABSTRACT: The applications of information on copy number changes in cancer have been twofold. Recognizing that regions of copy number gain signalled the location of oncogenes and that, similarly, copy number loss signalled the location of tumour suppressor genes, has resulted in screening of the minimally defined regions for candidate genes involved in tumourigenesis. Once candidates emerged, other evidence of their role in tumours was sought, by functional assays for example, and a huge literature built up describing these gene classes. Even without knowledge of how the genes acted in the development of tumours, the second application has been to correlate the chromosomal abnormalities with various clinical parameters, again resulting in many thousands of publications, although to date the translation of laboratory observations into clinical practice is still not widespread.
[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.
[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.
Full-text available · Article · Jul 2014 · Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology
[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.
Full-text available · Article · Mar 2014 · Bioinformatics
[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 available · Article · Dec 2013 · Journal of Oral Pathology and Medicine