Pamela Rabbitts - Academia.edu (original) (raw)

Papers by Pamela Rabbitts

The Journal of Pathology, 2015

The study of the relationships between pre-cancer and cancer and identification of early driver m... more 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.

International Journal of Cancer, 2015

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous c... more 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 (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN 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.

Bioinformatics (Oxford, England), Jan 5, 2015

The role of personalised medicine and target treatment in the clinical management of cancer patie... more The role of personalised 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. 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. ...

Thorax, 2007

The natural history of bronchial preinvasive lesions and the risk of developing lung cancer in pa... more The natural history of bronchial preinvasive lesions and the risk of developing lung cancer in patients with these lesions are not clear. Previous studies have treated severe dysplasia and carcinoma in situ (CIS) on the assumption that most will progress to invasive carcinoma. To define the natural history of preinvasive lesions and assess lung cancer risk in patients with these lesions. Most preinvasive lesions will not progress to invasive carcinoma but patients with these lesions will be at high risk. A cohort of patients with preinvasive lesions underwent fluorescence bronchoscopy every 4-12 months and computed tomography of the chest annually. The main end point was the development of invasive carcinoma. 22 patients with 53 lesions were followed up for 12-85 months. 11 cancers were diagnosed in 9 patients. Of the 36 high-grade lesions (severe dysplasia and CIS), 6 progressed to invasive cancers. 5 separate cancers developed at remote sites in patients with high-grade lesions. A...

European Journal of Cancer, 2014

MicroRNAs are a class of non-coding RNA which regulate gene expression. Their discovery in humans... more 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.

Journal of Investigative Dermatology, 2014

Arabia, for supporting this scholarship. The funders were not involved in study design, sample co... more Arabia, for supporting this scholarship. The funders were not involved in study design, sample collection and data analysis, decision to publish, or manuscript preparation.

The Journal of Molecular Diagnostics, 2012

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a p... more 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 previ-

Journal of Oral Pathology & Medicine, 2014

Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiologi... more 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(INK4a) 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(INK4a) . 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(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) 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(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

International Journal of Cancer, 1994

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and f... more Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p2 1-22. We examined 3 I paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p2 I. Allele loss was found in I9 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p2 i -22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehydefixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues. o 1994 Wiley-Liss, Inc.

Genomics, 2012

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal ab... more 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.

Genes, Chromosomes and Cancer, 2005

Carcinomas are believed to develop by incremental steps of increasingly abnormal morphology drive... more Carcinomas are believed to develop by incremental steps of increasingly abnormal morphology driven by accumulating somatic genetic changes. This process is often difficult to study, as the early stages are undetectable. We used fluorescence bronchoscopy, which enhances detection of preinvasive bronchial lesions, and have obtained sequential biopsies of carcinoma in situ (CIS) from a patient with no detectable tumor and from a squamous cell carcinoma that developed 19 months after presentation at the site of one of the previous CIS lesions. Biopsies of preinvasive CIS, which follow-up showed had different pathologic outcomes, and tumor were microdissected to obtain enriched cell populations and DNA prepared from them. Molecular characteristics of these biopsies were compared by loss of heterozygosity analysis, TP53 mutation analysis, and comparative genomic hybridization. Although all lesions examined had the same TP53 mutation and almost identical allelotypes, differences were observed. Loss in 5q21 and amplification of 3q25-26 were associated with the lesion that progressed and the subsequent carcinoma. Allele loss at 4p16 was detected in the tumor but not in any of the CIS lesions, suggesting it was a late event associated with tumor invasion. Amplification at 4q12 was specifically observed in the tumor and in the CIS at the site of eventual tumor formation. Although these findings may be unique to this one patient, the successful demonstration of sequential genetic changes raises the possibility that this approach, unencumbered by interpatient variability between lesions, will greatly facilitate the identification of molecular events driving the invasive process V V C 2005 Wiley-Liss, Inc.

Cancer Genetics and Cytogenetics, 1995

Human chromosomal region 3pl2-p23 is proposed to harbor at least three tumor suppressorgenes invo... more Human chromosomal region 3pl2-p23 is proposed to harbor at least three tumor suppressorgenes involved in the development oflung cancer, renal cell carcinoma, and other neoplasias. In order to identify one ofthese genes we defined sequence tagged sites (STSs) specific for 3p13-~24.2 by analyzing a chromosome 3~14 microdissection library. ST% were used for isolating yeast artificial chromosome (YAC) clones from the Centre d'Etude du Polymorphisme Humain (CEPH) YAC libraries. Thirty-eight YACs were assembled into a contig approximately 2.5 Mb in size spanning the t(3;8) and t(3;6) translocation breakpoints associated with hereditary renal cell carcinoma and hematologic malignancies, respectively. Chromosomal localization and chimeric status of 126 YACs was analyzed by fluorescence in situ hybridization (FISH). The order of 17 YACs determined by double-color FJSH was in agreement with the STS-based arrangement of the YAC-contig. INTRODUCITON Chromosomal aberrations of the short arm of human chromosome 3 are frequently observed in different types of tumors, including renal cell carcinoma (RCC) [l, 21, smallcell (SCLC) and non-small cell lung cancer (NSCLC) [3-51, head and neck carcinoma [6], breast cancer [fl , ovarian cancer [a], cervix carcinoma [9], and bladder cancer [lo]. In a hereditary RCC the chromosome 3 breakpoint of a disease-related constitutional translocation, t(3;8), was mapped to 3~14.2 [ll].

Bioinformatics, 2014

Current high-throughput sequencing has greatly transformed genome sequence analysis. In the conte... more Current high-throughput sequencing has greatly transformed genome sequence analysis. In the context of very low-coverage sequencing (<0.1×), performing 'binning' or 'windowing' on mapped short sequences ('reads') is critical to extract genomic information of interest for further evaluation, such as copy-number alteration 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 minimizes AIC or maximizes 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 at http://www1.maths.leeds.ac.uk/∼arief/R/win/.

Bioinformatics, 2012

Comparison of read depths from next-generation sequencing between cancer and normal cells makes t... more 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.

Cancer Detection <html_ent glyph="@amp;" ascii="&"/> Prevention, 1999

The production of metastases depends on changes in a large number of genes. It is also connected ... more The production of metastases depends on changes in a large number of genes. It is also connected with the interaction of tumor cells with the environment. It has been reported that primary tumor clone domination is also an important factor in metastasizing, and in many neoplasms dominating clones are the metastatic forerunners. Up to now it is unknown whether domination of a given clone in a primary renal cell carcinoma is a crucial factor in forming metastases or rather presence or absence of specific genes imposes the major advantage in the metastatic process. To study the presence or absence of the duplication and mitotic nondisjunction event as one of the phenomenon in the creation of metastases, as well as possible derivation of metastatic cells from the minor subclone of primary tumor, we examined three metastatic renal clear-cell carcinomas in which by comparative genomic hybridization we detected the loss of 3p in the primary tumor and two copies of 3p in the corresponding metastasis. Loss of heterozygosity analyses using markers for 3p25 (D3S1038), 3p21.1 (D3S1295), and 3p14.2 (D3S1481) proved heterozygosity of at least two 3p loci in all metastatic tumors, which indicates the absence of mitotic nondisjunction event as a cause of occurrence of two copies of 3p in metastases. Our results suggest that in some of the clear-cell renal carcinomas metastatic cells may derive from minor subclones of primary tumors.

The Journal of Pathology, 2015

The study of the relationships between pre-cancer and cancer and identification of early driver m... more 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.

International Journal of Cancer, 2015

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous c... more 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 (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN 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.

Bioinformatics (Oxford, England), Jan 5, 2015

The role of personalised medicine and target treatment in the clinical management of cancer patie... more The role of personalised 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. 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. ...

Thorax, 2007

The natural history of bronchial preinvasive lesions and the risk of developing lung cancer in pa... more The natural history of bronchial preinvasive lesions and the risk of developing lung cancer in patients with these lesions are not clear. Previous studies have treated severe dysplasia and carcinoma in situ (CIS) on the assumption that most will progress to invasive carcinoma. To define the natural history of preinvasive lesions and assess lung cancer risk in patients with these lesions. Most preinvasive lesions will not progress to invasive carcinoma but patients with these lesions will be at high risk. A cohort of patients with preinvasive lesions underwent fluorescence bronchoscopy every 4-12 months and computed tomography of the chest annually. The main end point was the development of invasive carcinoma. 22 patients with 53 lesions were followed up for 12-85 months. 11 cancers were diagnosed in 9 patients. Of the 36 high-grade lesions (severe dysplasia and CIS), 6 progressed to invasive cancers. 5 separate cancers developed at remote sites in patients with high-grade lesions. A...

European Journal of Cancer, 2014

MicroRNAs are a class of non-coding RNA which regulate gene expression. Their discovery in humans... more 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.

Journal of Investigative Dermatology, 2014

Arabia, for supporting this scholarship. The funders were not involved in study design, sample co... more Arabia, for supporting this scholarship. The funders were not involved in study design, sample collection and data analysis, decision to publish, or manuscript preparation.

The Journal of Molecular Diagnostics, 2012

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a p... more 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 previ-

Journal of Oral Pathology & Medicine, 2014

Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiologi... more 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(INK4a) 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(INK4a) . 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(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) 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(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

International Journal of Cancer, 1994

Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and f... more Loss of heterozygosity (LOH) has been extensively studied on the short arm of chromosome 3, and functional proofs have been obtained defining a tumor-suppressor locus at 3p2 1-22. We examined 3 I paraffin-embedded cervical cancer samples for LOH, using 5 PCR-primer pairs, located around 3p2 I. Allele loss was found in I9 out of the 27 informative samples (70%) while 13 out of 23 informative samples (56%) had LOH located at 3p2 i -22. More of the human papillomavirus (HPV)-positive samples had LOH compared to the HPV-negative samples, giving only a weak association between loss of allele and HPV integration. Modifications of the DNA in the formaldehydefixed samples were detected, and further studies will be required to clarify how such artifacts may affect restriction fragment length polymorphism (RFLP) studies on fixed tissues. o 1994 Wiley-Liss, Inc.

Genomics, 2012

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal ab... more 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.

Genes, Chromosomes and Cancer, 2005

Carcinomas are believed to develop by incremental steps of increasingly abnormal morphology drive... more Carcinomas are believed to develop by incremental steps of increasingly abnormal morphology driven by accumulating somatic genetic changes. This process is often difficult to study, as the early stages are undetectable. We used fluorescence bronchoscopy, which enhances detection of preinvasive bronchial lesions, and have obtained sequential biopsies of carcinoma in situ (CIS) from a patient with no detectable tumor and from a squamous cell carcinoma that developed 19 months after presentation at the site of one of the previous CIS lesions. Biopsies of preinvasive CIS, which follow-up showed had different pathologic outcomes, and tumor were microdissected to obtain enriched cell populations and DNA prepared from them. Molecular characteristics of these biopsies were compared by loss of heterozygosity analysis, TP53 mutation analysis, and comparative genomic hybridization. Although all lesions examined had the same TP53 mutation and almost identical allelotypes, differences were observed. Loss in 5q21 and amplification of 3q25-26 were associated with the lesion that progressed and the subsequent carcinoma. Allele loss at 4p16 was detected in the tumor but not in any of the CIS lesions, suggesting it was a late event associated with tumor invasion. Amplification at 4q12 was specifically observed in the tumor and in the CIS at the site of eventual tumor formation. Although these findings may be unique to this one patient, the successful demonstration of sequential genetic changes raises the possibility that this approach, unencumbered by interpatient variability between lesions, will greatly facilitate the identification of molecular events driving the invasive process V V C 2005 Wiley-Liss, Inc.

Cancer Genetics and Cytogenetics, 1995

Human chromosomal region 3pl2-p23 is proposed to harbor at least three tumor suppressorgenes invo... more Human chromosomal region 3pl2-p23 is proposed to harbor at least three tumor suppressorgenes involved in the development oflung cancer, renal cell carcinoma, and other neoplasias. In order to identify one ofthese genes we defined sequence tagged sites (STSs) specific for 3p13-~24.2 by analyzing a chromosome 3~14 microdissection library. ST% were used for isolating yeast artificial chromosome (YAC) clones from the Centre d'Etude du Polymorphisme Humain (CEPH) YAC libraries. Thirty-eight YACs were assembled into a contig approximately 2.5 Mb in size spanning the t(3;8) and t(3;6) translocation breakpoints associated with hereditary renal cell carcinoma and hematologic malignancies, respectively. Chromosomal localization and chimeric status of 126 YACs was analyzed by fluorescence in situ hybridization (FISH). The order of 17 YACs determined by double-color FJSH was in agreement with the STS-based arrangement of the YAC-contig. INTRODUCITON Chromosomal aberrations of the short arm of human chromosome 3 are frequently observed in different types of tumors, including renal cell carcinoma (RCC) [l, 21, smallcell (SCLC) and non-small cell lung cancer (NSCLC) [3-51, head and neck carcinoma [6], breast cancer [fl , ovarian cancer [a], cervix carcinoma [9], and bladder cancer [lo]. In a hereditary RCC the chromosome 3 breakpoint of a disease-related constitutional translocation, t(3;8), was mapped to 3~14.2 [ll].

Bioinformatics, 2014

Current high-throughput sequencing has greatly transformed genome sequence analysis. In the conte... more Current high-throughput sequencing has greatly transformed genome sequence analysis. In the context of very low-coverage sequencing (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.1×), performing &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;binning&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; or &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;windowing&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; on mapped short sequences (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;reads&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;) is critical to extract genomic information of interest for further evaluation, such as copy-number alteration 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 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;smoothed out&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;. 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;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 minimizes AIC or maximizes 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 at http://www1.maths.leeds.ac.uk/∼arief/R/win/.

Bioinformatics, 2012

Comparison of read depths from next-generation sequencing between cancer and normal cells makes t... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; 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.

Cancer Detection <html_ent glyph="@amp;" ascii="&"/> Prevention, 1999

The production of metastases depends on changes in a large number of genes. It is also connected ... more The production of metastases depends on changes in a large number of genes. It is also connected with the interaction of tumor cells with the environment. It has been reported that primary tumor clone domination is also an important factor in metastasizing, and in many neoplasms dominating clones are the metastatic forerunners. Up to now it is unknown whether domination of a given clone in a primary renal cell carcinoma is a crucial factor in forming metastases or rather presence or absence of specific genes imposes the major advantage in the metastatic process. To study the presence or absence of the duplication and mitotic nondisjunction event as one of the phenomenon in the creation of metastases, as well as possible derivation of metastatic cells from the minor subclone of primary tumor, we examined three metastatic renal clear-cell carcinomas in which by comparative genomic hybridization we detected the loss of 3p in the primary tumor and two copies of 3p in the corresponding metastasis. Loss of heterozygosity analyses using markers for 3p25 (D3S1038), 3p21.1 (D3S1295), and 3p14.2 (D3S1481) proved heterozygosity of at least two 3p loci in all metastatic tumors, which indicates the absence of mitotic nondisjunction event as a cause of occurrence of two copies of 3p in metastases. Our results suggest that in some of the clear-cell renal carcinomas metastatic cells may derive from minor subclones of primary tumors.