Christodoulos P Pipinikas

Extensive transduction of nonrepetitive DNA mediated by L1 retrotransposition in cancer genomes

Introduction The human genome is peppered with mobile repetitive elements called long interspersed nuclear element–1 (L1) retrotransposons. Propagating through RNA and cDNA intermediates, these molecular parasites copy and insert themselves throughout the genome, with potentially disruptive effects on neighboring genes or regulatory sequences. In the germ line, unique sequence downstream of L1 elements can also be retrotransposed if transcription continues beyond the repeat, a process known as 3′ transduction. There has been growing interest in retrotransposition and 3′ transduction as a possible source of somatic mutations during tumorigenesis. The activity of individual L1 elements fluctuates during tumor evolution. In a lung tumor, hundreds of 3′ transductions arose from a small number of active L1 source elements (colored circles on outer rim of circle). As the tumor evolved from the preinvasive common ancestor to invasive cancer, individual elements exhibited variable activity over time. Rationale To explore whether 3′ transductions are frequent in cancer, we developed a bioinformatic algorithm for identifying somatically acquired retrotranspositions in cancer genomes. We applied our algorithm to 290 cancer samples from 244 patients across 12 tumor types. The unique downstream sequence mobilized with 3′ transductions effectively fingerprints the L1 source element, providing insights into the activity of individual L1 loci across the genome. Results Across the 290 samples, we identified 2756 somatic L1 retrotranspositions. Tumors from 53% of patients had at least one such event, with colorectal and lung cancers being most frequently affected (93% and 75% of patients, respectively). Somatic 3′ transductions comprised 24% of events, half of which represented mobilizations of unique sequence alone, without any accompanying L1 sequence. Overall, 95% of 3′ transductions identified derived from only 72 germline L1 source elements, with as few as four loci accounting for 50% of events. In a given sample, the same source element could generate 50 or more somatic transductions, scattered extensively across the genome. About 5% of somatic transductions arose from L1 source elements that were themselves somatic retrotranspositions. In three of the cases in which we sequenced more than one sample from a patient’s tumor, we were able to place 3′ transductions on the phylogenetic tree. We found that the activity of individual source elements fluctuated during tumor evolution, with different subclones exhibiting much variability in which elements were “on” and which were “off.” The ability to identify the individual L1 source elements active in a given tumor enabled us to study the promoter methylation of those elements specifically. We found that 3′ transduction activity in a patient’s tumor was always associated with hypomethylation of that element. Overall, 2.3% of transductions distributed exons or entire genes to other sites in the genome, and many more mobilized deoxyribonuclease I (DNAse-I) hypersensitive sites or transcription factor binding sites identified by the ENCODE project. Occasionally, somatic L1 insertions inserted near coding sequence and redistributed these exons elsewhere in the genome. However, we found no general effects of retrotranspositions on transcription levels of genes at the insertion points and no evidence for aberrant RNA species resulting from somatically acquired transposable elements. Indeed, as with germline retrotranspositions, somatic insertions exhibited a strong enrichment in heterochromatic, gene-poor regions of the genome. Conclusion Somatic 3′ transduction occurs frequently in human tumors, and in some cases transduction events can scatter exons, genes, and regulatory elements widely across the genome. Dissemination of these sequences appears to be due to a small number of highly active L1 elements, whose activity can wax and wane during tumor evolution. The majority of the retrotransposition events are likely to be harmless “passenger” mutations. Hitchhiking through the tumor genome Retrotransposons are DNA repeat sequences that are constantly on the move. By poaching certain cellular enzymes, they copy and insert themselves at new sites in the genome. Sometimes they carry along adjacent DNA sequences, a process called 3′ transduction. Tubio et al. found that 3′ transduction is a common event in human tumors. Because this process can scatter genes and regulatory sequences across the genome, it may represent yet another mechanism by which tumor cells acquire new mutations that help them survive and grow. Science, this issue p. 10.1126/science.1251343 Tumor genomes are peppered with mobile repeat sequences that carry along adjacent DNA when they insert into new genomic sites. Long interspersed nuclear element–1 (L1) retrotransposons are mobile repetitive elements that are abundant in the human genome. L1 elements propagate through RNA intermediates. In the germ line, neighboring, nonrepetitive sequences are occasionally mobilized by the L1 machinery, a process called 3′ transduction. Because 3′ transductions are potentially mutagenic, we explored the extent to which they occur somatically during tumorigenesis. Studying cancer genomes from 244 patients, we found that tumors from 53% of the patients had somatic retrotranspositions, of which 24% were 3′ transductions. Fingerprinting of donor L1s revealed that a handful of source L1 elements in a tumor can spawn from tens to hundreds of 3′ transductions, which can themselves seed further retrotranspositions. The activity of individual L1 elements fluctuated during tumor evolution and correlated with L1 promoter hypomethylation. The 3′ transductions disseminated genes, exons, and regulatory elements to new locations, most often to heterochromatic regions of the genome.

Correlation of Smoking-Associated DNA Methylation Changes in Buccal Cells With DNA Methylation Changes in Epithelial Cancer

IMPORTANCE The utility of buccal cells as an epithelial source tissue for epigenome-wide association studies (EWASs) remains to be demonstrated. Given the direct exposure of buccal cells to potent carcinogens such as smoke, epigenetic changes in these cells may provide insights into the development of smoke-related cancers. OBJECTIVE To perform an EWAS in buccal and blood cells to assess the relative effect of smoking on the DNA methylation (DNAme) patterns in these cell types and to test whether these DNAme changes are also seen in epithelial cancer. DESIGN, SETTING, AND PARTICIPANTS In 2013, we measured DNAme at more than 480,000 CpG sites in buccal samples provided in 1999 by 790 women (all aged 53 years in 1999) from the United Kingdom Medical Research Council National Survey of Health and Development. This included matched blood samples from 152 women. We constructed a DNAme-based smoking index and tested its sensitivity and specificity to discriminate normal from cancer tissue in more than 5000 samples. MAIN OUTCOMES AND MEASURES CpG sites whose DNAme level correlates with smoking pack-years, and construction of an associated sample-specific smoking index, which measures the mean deviation of DNAme at smoking-associated CpG sites from a normal reference. RESULTS In a discovery set of 400 women, we identified 1501 smoking-associated CpG sites at a genome-wide significance level of P < 10-7, which were validated in an independent set of 390 women. This represented a 40-fold increase of differentially methylated sites in buccal cells compared with matched blood samples. Hypermethylated sites were enriched for bivalently marked genes and binding sites of transcription factors implicated in DNA repair and chromatin architecture (P < 10-10). A smoking index constructed from the DNAme changes in buccal cells was able to discriminate normal tissue from cancer tissue with a mean receiver operating characteristic area under the curve of 0.99 (range, 0.99-1.00) for lung cancers and of 0.91 (range, 0.71-1.00) for 13 other organs. The corresponding area under the curve of a smoking signature derived from blood cells was lower than that derived from buccal cells in 14 of 15 cancer types (Wilcoxon signed rank test, P = .001). CONCLUSIONS AND RELEVANCE These data point toward buccal cells as being a more appropriate source of tissue than blood to conduct EWASs for smoking-related epithelial cancers.

Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors

Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to “neutral drift” of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease. DOI: http://dx.doi.org/10.7554/eLife.00966.001

Prognostic Impact of Novel Molecular Subtypes of Small Intestinal Neuroendocrine Tumor

Purpose: Small intestinal neuroendocrine tumors (SINET) are the commonest malignancy of the small intestine; however, underlying pathogenic mechanisms remain poorly characterized. Whole-genome and -exome sequencing has demonstrated that SINETs are mutationally quiet, with the most frequent known mutation in the cyclin-dependent kinase inhibitor 1B gene (CDKN1B) occurring in only ∼8% of tumors, suggesting that alternative mechanisms may drive tumorigenesis. The aim of this study is to perform genome-wide molecular profiling of SINETs in order to identify pathogenic drivers based on molecular profiling. This study represents the largest unbiased integrated genomic, epigenomic, and transcriptomic analysis undertaken in this tumor type. Experimental Design: Here, we present data from integrated molecular analysis of SINETs (n = 97), including whole-exome or targeted CDKN1B sequencing (n = 29), HumanMethylation450 BeadChip (Illumina) array profiling (n = 69), methylated DNA immunoprecipitation sequencing (n = 16), copy-number variance analysis (n = 47), and Whole-Genome DASL (Illumina) expression array profiling (n = 43). Results: Based on molecular profiling, SINETs can be classified into three groups, which demonstrate significantly different progression-free survival after resection of primary tumor (not reached at 10 years vs. 56 months vs. 21 months, P = 0.04). Epimutations were found at a recurrence rate of up to 85%, and 21 epigenetically dysregulated genes were identified, including CDX1 (86%), CELSR3 (84%), FBP1 (84%), and GIPR (74%). Conclusions: This is the first comprehensive integrated molecular analysis of SINETs. We have demonstrated that these tumors are highly epigenetically dysregulated. Furthermore, we have identified novel molecular subtypes with significant impact on progression-free survival. Clin Cancer Res; 22(1); 250–8. ©2015 AACR.

Processed pseudogenes acquired somatically during cancer development

Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5′ truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context.

Cell migration leads to spatially distinct but clonally related airway cancer precursors

Background Squamous cell carcinoma of the lung is a common cancer with 95% mortality at 5 years. These cancers arise from preinvasive lesions, which have a natural history of development progressing through increasing severity of dysplasia to carcinoma in situ (CIS), and in some cases, ending in transformation to invasive carcinoma. Synchronous preinvasive lesions identified at autopsy have been previously shown to be clonally related. Methods Using autofluorescence bronchoscopy that allows visual observation of preinvasive lesions within the upper airways, together with molecular profiling of biopsies using gene sequencing and loss-of-heterozygosity analysis from both preinvasive lesions and from intervening normal tissue, we have monitored individual lesions longitudinally and documented their visual, histological and molecular relationship. Results We demonstrate that rather than forming a contiguous field of abnormal tissue, clonal CIS lesions can develop at multiple anatomically discrete sites over time. Further, we demonstrate that patients with CIS in the trachea have invariably had previous lesions that have migrated proximally, and in one case, into the other lung over a period of 12 years. Conclusions Molecular information from these unique biopsies provides for the first time evidence that field cancerisation of the upper airways can occur through cell migration rather than via local contiguous cellular expansion as previously thought. Our findings urge a clinical strategy of ablating high-grade premalignant airway lesions with subsequent attentive surveillance for recurrence in the bronchial tree.

Epigenetic dysregulation and poorer prognosis in DAXX-deficient pancreatic neuroendocrine tumours

Exome sequencing of sporadic pancreatic neuroendocrine tumours (PNETs) has identified mutually exclusive mutations in the chromatin regulators α- thalassaemia/mental retardation X-linked (ATRX) and death associated protein 6 (DAXX) genes in 43% of cases (18% and 23% of cases respectively in 68 cases studied) (Elsasser et al. 2011; Jiao et al. 2011). ATRX and DAXX are chromatin remodelers; their loss leads to alternative lengthening of telomeres (ALT) and chromosomal instability (CIN) (Heaphy et al. 2011). ALT is a telomerase independent mechanism for maintenance of telomere stabilisation. Although it was initially reported that ATRX/DAXX mutant tumours had superior 10-year survival and outcome (Jiao et al. 2011), a recent larger study on 243 tumours has demonstrated that ATRX and DAXX loss and associated ALT in PNETs correlates with CIN, advanced tumour stage, development of metastases and poorer progression free (PFS) and overall survival (OS) (Marinoni et al. 2014) ...

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

Progressive epigenetic dysregulation in neuroendocrine tumour liver metastases

The incidence of small intestinal neuroendocrine tumours (SINETs) is increasing and distant metastases are present at diagnosis in 70% of cases, the liver being the commonest site of metastasis (Yao et al. 2008). Despite this, our understanding of the mechanisms underlying metastatic progression of SINETs is currently limited, and prior studies of the molecular biology of SINET liver metastases (LM) have been performed predominantly in small cohorts utilising candidate-based techniques. SINETs have a low rate of mutations compared to most cancers. The most frequently mutated gene is CDKN1B (encoding p27, a cell cycle regulator); however, mutations in this gene occur in only 8% of tumours, and there is no characteristic mutational hotspot (Francis et al. 2013). Furthermore, mutation of CDKN1B does not correlate with the expression of p27 (Crona et al. 2015). We previously identified that SINETs are epigenetically dysregulated, and a panel of candidate driver epimutation genes has been identified (Karpathakis et al. 2016). Therefore, we postulated that metastatic progression in SINETs may also be epigenetically regulated. Here, we present the findings from the largest molecular profiling study of SINET LM performed to date, integrating copy number variance (CNV), DNA methylation, and RNA expression profiling to characterise the mechanisms underlying metastatic progression. Experimental details of DNA methylation, CNV and RNA expression profiling are as previously published (Karpathakis et al. 2016). Patients provided informed consent for their tissue to be analysed in this study, which was Research Ethics Committee approved (Ref: 09/H0722/27). All cases were reviewed by two expert NET histopathologists (TVL/MN). Nucleic acids were extracted using standard methods (Qiagen, QIAamp DNA Mini kit; Roche, High Pure RNA Paraffin kit). H&E-stained sections were evaluated to ensure >80% purity of tumour specimens. Methylation profiling was performed on the HumanMethylation450 BeadChip (HM450) (Illumina). Methylation data were analysed using ChAMP pipeline (https://www.bioconductor.org/packages/release/bioc/ html/ChAMP.html). Whole-genome methylation profiling using methylated DNA immunoprecipitation sequencing (MeDIP) was performed as previously described. MeDIP data were analysed using the custom pipeline MeDUSA, v2.0 (https://www.ucl.ac.uk/cancer/research/departmentcancer-biology/medical-genomics-group/past-projects/ medusa-project). Gene expression levels were quantified using the whole genome cDNA-mediated annealing, selection and ligation (DASL) (Illumina) assay. Expression data were analysed using the ‘LIMMA’ package in R (https://bioconductor.org/packages/release/bioc/html/ limma.html). Raw data from this study has previously been deposited in GEO (Accession number: GSE73832) (Karpathakis et al. 2016). In summary, n = 90 samples underwent array-based DNA methylation analysis, n = 26 samples underwent methylation-specific immunoprecipitation followed by DNA sequencing and n = 49 underwent array-based RNA expression analysis. Of cases with relevant clinical data, 93% had received no systemic treatment prior to specimen collection (27/29 cases). The CNV profile of SINET LM (n = 20) mirrors that of primary tumours with the most frequent alteration of chr18 LOH seen in 79% of cases. A greater proportion of LM demonstrates amplification of chr20 (42%) and deletion of chr19 (35%), whereas gain of 17q is found only in LM (21%). A trend of increased incidence of CNVs was seen in LM compared to that of SINET primary tumours (SINET primary: median 78 mbp; LM: median 114 mbp, P = 0.08). Comparison of methylation profiles of SINET LM to those of primary SINETs identified 29,263 methylation

1133OMOLECULAR PROFILING OF SMALL INTESTINAL NEUROENDOCRINE TUMOURS

ABSTRACT Aim: Aberrant DNA methylation is known to play an important role in the pathogenesis of many human cancers, however little is known about its role in small intestinal neuroendocrine tumour (SI NET) development. We report the first unbiased genome wide DNA methylation analysis of a large cohort of SI NET, aiming to identify key epigenetic changes specific to SI NET which may contribute to tumorigenesis. Methods: Illumina Infinium HumanMethylation450 Array (interrogating 99% of RefSeq genes) analysis was performed on DNA extracted from macrodissected SI NET primary tumours (n = 49) and normal small intestine (SI)(n = 21). Publicly available methylation data on >600 samples from The Cancer Genome Atlas was also assessed for comparison (colorectal, pancreatic and gastric adenocarcinoma and healthy tissue). Gene expression was determined using the Illumina DASL array on RNA extracted from SI NET primary tumours (n = 32) and normal SI (n = 6). Analysis was performed using ChAMP and limma R packages. A Bonferroni adjusted significance threshold value of p Results: Comparison of SI NET with normal SI identified a total of 130,083 significant methylation variable positions, including 1841 sites hypermethylated by >30% in tumour compared to normal tissue. 626 genes were found to have significant >3fold differential expression between SI NET and normal SI. Integrated analysis identified a group of 11 candidate genes where altered methylation and expression was significant and concordant (downregulated: CDX1, FBP1, C20orf54, GATA5; upregulated: PTPRN, PCSK1, PRLHR, CELSR3, GIPR, LMX1B, SCGN). Hypermethylation of GIPR (gastic inhibitory polypeptide receptor) was most significant (SI NET median methylation 0.67 vs normal SI 0.29 p Conclusions: This study is the first comprehensive analysis of the epigenetic profile of SI NET and identifies hypermethylation of GIPR as a potential novel biomarker. Novel radioligands targeting GIPR have been developed for use as imaging tools and it is a promising target for novel therapeutic agents. Disclosure: All authors have declared no conflicts of interest.