Genomic evidence of pre-invasive clonal expansion, dispersal and progression in bronchial dysplasia

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. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

The genomic road to invasion - examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.

Background: It is frequently assumed that pre-invasive lesions are simpler precursors of cancer, and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments, and early detection methodologies. Methods: We examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing. Results: Evidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One third of dysplasias showed independent copy number events. The remainder had a similar or simpler copy number pattern to the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma. Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient’s disease developed in a different way. Examining the numbers of shared mutations, and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these. Conclusions: These findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event

A computational index derived from whole-genome copy number analysis is a novel tool for prognosis in early stage lung squamous cell carcinoma.

AbstractSquamous 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

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material

Sequential screening for lung cancer in a high-risk group: randomised controlled trial: LungSEARCH: a randomised controlled trial of Surveillance using sputum and imaging for the EARly detection of lung Cancer in a High-risk group.

BACKGROUND: Low-dose computed tomography (LDCT) screening detects early-stage lung cancer and reduces mortality. We proposed a sequential approach targeted to a high-risk group as a potentially efficient screening strategy. METHODS: LungSEARCH was a national multicentre randomised trial. Current/ex-smokers with mild/moderate chronic obstructive pulmonary disease (COPD) were allocated (1:1) to have 5 years surveillance or not. Screened participants provided annual sputum samples for cytology and cytometry, and if abnormal were offered annual LDCT and autofluorescence bronchoscopy (AFB). Those with normal sputum provided annual samples. The primary end-point was the percentage of lung cancers diagnosed at stage I/II (nonsmall cell) or limited disease (small cell). RESULTS: 1568 participants were randomised during 2007-2011 from 10 UK centres. 85.2% of those screened provided an adequate baseline sputum sample. There were 42 lung cancers among 785 screened individuals and 36 lung cancers among 783 controls. 54.8% (23 out of 42) of screened individuals versus 45.2% (14 out of 31) of controls with known staging were diagnosed with early-stage disease (one-sided p=0.24). Relative risk was 1.21 (95% CI 0.75-1.95) or 0.82 (95% CI 0.52-1.31) for early-stage or advanced cancers, respectively. Overall sensitivity for sputum (in those randomised to surveillance) was low (40.5%) with a cumulative false-positive rate (FPR) of 32.8%. 55% of cancers had normal sputum results throughout. Among sputum-positive individuals who had AFB, sensitivity was 45.5% and cumulative FPR was 39.5%; the corresponding measures for those who had LDCT were 100% and 16.1%, respectively. CONCLUSIONS: Our sequential strategy, using sputum cytology/cytometry to select high-risk individuals for AFB and LDCT, did not lead to a clear stage shift and did not improve the efficiency of lung cancer screening

IMSA: Integrated metagenomic sequence analysis for identification of exogenous reads in a host genomic background

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

An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing

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

Temporal and spatial expression of two isoforms of the Dutt1/Robo1 gene in mouse development

AbstractThe mammalian homologue of the Drosophila axonal guidance receptor roundabout is expressed in a wide range of tissues. Here we show that alternative splicing of the Dutt1/Robo1 gene results in two mRNA transcripts with different signal peptides, which are differentially expressed throughout mouse embryogenesis. Since mice with a targeted deletion in the Dutt1/Robo1 gene have abnormal lung pathology, immunohistochemistry was used to identify the cellular expression pattern of Dutt1/Robo1 during lung development. Dutt1/Robo1 expression was widespread and diffuse in the lung at embryonic day 17.5 but became increasingly localised to the bronchial epithelium in newborn and adult mice