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Background: The DNA released into the bloodstream by malignant tumours· called circulating tumour DNA (ctDNA), is often a small fraction of total cell-free DNA shed predominantly by hematopoietic cells and is therefore challenging to detect. Understanding the biological properties of ctDNA is key to the investigation of its clinical relevance as a non-invasive marker for cancer detection and monitoring. Methods: We selected 40 plasma DNA samples of pancreatic cancer cases previously reported to carry a KRAS mutation at the ‘hotspot’ codon 12 and re-screened the cell-free DNA using a 4-size amplicons strategy (57 bp, 79 bp, 167 bp and 218 bp) combined with ultra-deep sequencing in order to investigate whether amplicon lengths could impact on the capacity of detection of ctDNA, which in turn could provide inference of ctDNA and non-malignant cell-free DNA size distribution. Findings: Higher KRAS amplicon size (167 bp and 218 bp) was associated with lower detectable cell-free DNA mutant allelic fractions (p

Human papillomavirus (HPV) causes oropharyngeal squamous cell carcinoma (OPSCC), although strongly divergent results have been reported regarding the prevalence of HPV16 in different countries, whether this represents important differences in etiology remains unclear. Applying rigorous protocols for sample processing, we centrally evaluated 1420 head and neck tumors (533 oropharynx, 395 oral cavity and 482 larynx) from studies conducted in the US, Europe and Brazil for mucosal HPV DNA and p16INK4a expression to evaluate regional heterogeneity in the proportion of HPV16-associated OPSCC and other head and neck cancer, and to assess covariates associated with the risk of HPV16-positive OPSCC. While majority of OPSCC in the US (60%) were HPV16-positive, this proportion was 31% in Europe and only 4% in Brazil (p

The emergence of Next-Generation Sequencing (NGS) has revolutionized the way of reaching a genome sequence, with the promise of potentially providing a comprehensive characterization of DNA variations. Nevertheless, detecting somatic mutations is still a difficult problem, in particular when trying to identify low abundance mutations such as subclonal mutations, tumour-derived alterations in body fluids or somatic mutations from histological normal tissue. The main challenge is to precisely distinguish between sequencing artefacts and true mutations, particularly when the latter are so rare they reach similar abundance levels as artefacts. Here, we present needlestack, a highly sensitive variant caller, which directly learns from the data the level of systematic sequencing errors to accurately call mutations. Needlestack is based on the idea that the sequencing error rate can be dynamically estimated from analyzing multiple samples together. We show that the sequencing error rate varies across alterations, illustrating the need to precisely estimate it. We evaluate the performance of needlestack for various types of variations, and we show that needlestack is robust among positions and outperforms existing state-of-the-art method for low abundance mutations. Needlestack, along with its source code is freely available on the GitHub plateform: https://github.com/IARCbioinfo/needlestack.

The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series.

Background: Detecting pre-clinical bladder cancer (BC) using urinary biomarkers may provide a valuable opportunity for screening and management. Telomerase reverse transcriptase (TERT) promoter mutations detectable in urine have emerged as promising BC biomarkers. Methods: We performed a nested case-control study within the population-based prospective Golestan Cohort Study (50,045 participants, followed up to 14 years) and assessed TERT promoter mutations in baseline urine samples from 38 asymptomatic individuals who subsequently developed primary BC and 152 matched controls using a Next-Generation Sequencing-based single-plex assay (UroMuTERT) and droplet digital PCR assays. Findings: Results were obtained for 30 cases and 101 controls. TERT promoter mutations were detected in 14 pre-clinical cases (sensitivity 46·67%) and none of the controls (specificity 100·00%). At an estimated BC cumulative incidence of 0·09% in the cohort, the positive and negative predictive values were 100·00% and 99·95% respectively. The mutant allelic fractions decreased with the time interval from urine collection until BC diagnosis (p = 0·033) but the mutations were detectable up to 10 years prior to clinical diagnosis. Interpretation: Our results provide the first evidence from a population-based prospective cohort study of the potential of urinary TERT promoter mutations as promising non-invasive biomarkers for early detection of BC. Further studies should validate this finding and assess their clinical utility in other longitudinal cohorts. Funding: French Cancer League, World Cancer Research Fund International, Cancer Research UK, Tehran University of Medical Sciences, the International Agency for Research on Cancer, and the U.S. National Cancer Institute. Keywords: Early detection, TERT promoter mutations, Urinary biomarker, Bladder cancer, Prospective cohort

Background: Aristolochic acid (AA) is a nephrotoxicant associated with AA nephropathy (AAN) and upper urothelial tract cancer (UUTC). Whole-genome sequences of 14 Romanian cases of renal cell carcinoma (RCC) recently exhibited mutational signatures consistent with AA exposure, although RCC had not been previously linked with AAN and AA exposure was previously reported only in localised rural areas. Methods: We performed mass spectrometric measurements of the aristolactam (AL) DNA adduct 7-(deoxyadenosin-N6-yl) aristolactam I (dA-AL-I) in nontumour renal tissues of the 14 Romanian RCC cases and 15 cases from 3 other countries. Results: We detected dA-AL-I in the 14 Romanian cases at levels ranging from 0.7 to 27 adducts per 108 DNA bases, in line with levels reported in Asian and Balkan populations exposed through herbal remedies or food contamination. The 15 cases from other countries were negative. Interpretation: Although the source of exposure is uncertain and likely different in AAN regions than elsewhere, our results demonstrate that AA exposure in Romania exists outside localised AAN regions and provide further evidence implicating AA in RCC.

// Sandra Perdomo 1, 2, * , Patrice H. Avogbe 1, * , Matthieu Foll 1 , Behnoush Abedi-Ardekani 1 , Violeta Lescher Facciolla 3 , Devasena Anantharaman 1,9 , Priscilia Chopard 1 , Florence Le Calvez-Kelm 1 , Marta Vilensky 4 , Jerry Polesel 5 , Ivana Holcatova 6 , Lorenzo Simonato 7 , Cristina Canova 7 , Pagona Lagiou 8 , James D. McKay 1 and Paul Brennan 1 1 International Agency for Research on Cancer (IARC), Lyon 69372, France 2 Institute of Nutrition, Genetics and Metabolism Research, Faculty of Medicine, Universidad El Bosque, Bogota 110121, Colombia 3 Departamento de Saude Coletiva, Faculdade de Ciencias Medicas da Santa Casa de Sao Paulo, Sao Paulo CEP01221-020, Brazil 4 Instituto Angel Roffo, Buenos Aires C1417DTB, Argentina 5 CRO Aviano National Cancer Institute, Aviano 33081, Italy 6 Charles University, 1st Faculty of Medicine, Prague 116 36, Czech Republic 7 Laboratory of Public Health and Population Studies, University of Padova, Padova 35122, Italy 8 Department of Hygiene, Epidemiology and Medical Statistics, School of Medicine, National and Kapodistrian University of Athens, Athens 11527, Greece 9 Current/present address: Rajiv Gandhi Centre for Biotechnology, Trivandrum 695010, India * These authors have contributed equally to this work Correspondence to: Paul Brennan, email: BrennanP@iarc.fr Keywords: ctDNA, head and neck, cancer, mutation, detection Abbreviations: cfDNA: cell free DNA; ctDNA: circulating tumor DNA; HNSCC: head and neck squamous cell carcinoma; AF: allelic frequency Received: April 27, 2017 Accepted: July 12, 2017 Published: August 07, 2017 ABSTRACT The use of non-invasive biomarkers such as circulating tumor DNA (ctDNA) in head and neck tumors may be of relevance in early diagnosis and eventually improved outcome. We evaluated two different approaches from two case series in Europe and South America including (i) targeted screening of ctDNA mutations, and (ii) detection of TP53 mutations in plasma and oral rinses without previous knowledge of mutational status in tumor samples. Targeted sequencing in 5 genes identified ctDNA mutations in plasma among 42% of HNSCC cases, 67% of who were early stage cases. No association was found between ctDNA mutation detection and overall survival. Sequencing of the entire coding region of the TP53 gene resulted in identification of TP53 mutations in 76% of tumor cases. However, concordance of mutation detection was low between tumor, oral rinses (11%) and plasma (2,7%) samples. Identification of 5 pathogenic TP53 mutations in oral rinses from 3 non-cancer controls gives additional evidence of mutation occurrence in individuals without a diagnosed cancer and presents an additional challenge for the development of ctDNA diagnostic assays.

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by hotspot mutations in the KRAS gene (codons 12, 13 or 61) in 85-90% of cases. Codon 12 KRAS mutations have been detected in pancreatic juice, blood and stool samples from pancreatic cancer cases and represent promising biomarkers for early detection. However, the proportion of tumor-derived KRAS mutations in cell-free DNA fragments (cfDNA) has shown large variations, probably because of the heterogeneity in biosamples and assays tested. Deep sequencing technologies (NGS) allow the identification of low-abundance somatic variants, but have not previously been applied to the detection of KRAS hotspot mutations in cfDNA of PDAC cases. Moreover, variant calling methods have rarely been tested against cancer-free individuals so the proportion of false positives is unknown. We investigated whether deep sequencing of KRAS mutations at codons 12, 13 and 61 in plasma samples could represent a robust assay to distinguish pancreatic cancer from chronic pancreatitis and healthy controls. Methods: We developed an Ion Torrent-based NGS KRAS assay (partial exons 2 and 3, totalling 208bp) to screen cfDNA from plasma samples of 461 PDAC cases, 154 individuals with chronic pancreatitis and 421 healthy controls. cfDNA extraction (>4ng) and sequencing (>1000x coverage on average, and absence of systematic high sequencing error rates on the 208bp) performed well on 431 (93%) PDAC cases, 138 (90%) chronic pancreatitis, and 388 (95%) controls. We fit a robust negative-binomial regression to estimate the distribution of the sequencing errors at each DNA bp position and identified outlying samples, which were considered as KRAS positive when q-value Results: Sequencing of the serial dilutions of KRAS p.G12V mutated DNA indicated a detection threshold at a minor allele frequency of 0.2%. KRAS mutations in cfDNA were detected in 83 (19.3%) PDAC cases (73 on codon 12; 8 on codon 61; 1 on codon 13; and 1 multiple codons, i.e., similar in proportions as reported in tumor tissue from the International Cancer Genomic Consortium); 3 (2.2%) chronic pancreatitis (all on codon 12); and 8 (2.1%) healthy controls (4 on codon 12 and 4 on codon 61). Stage was significantly associated with the proportion of detected mutations in cancer cases (chi-squared p = 0.0005): the proportions of cases with detectable KRAS mutations in plasma were 7.9%, 14.9%, and 31.1% for local, regional, and advanced stages, respectively. Conclusions: The NGS-based KRAS mutation screening is a sensitive approach to detect low allelic fraction in plasma cfDNA, although its utility for early detection is still limited. However, it has the capacity to identify specific KRAS mutations, which could be useful in a panel of other non-invasive biomarkers. Citation Format: Florence Le Calvez-Kelm, Matthieu Foll, Magdalena B. Wozniak, Geoffroy Durand, Priscilia Chopard, Maroulio Pertesi, Tiffany Delhomme, Ivana Holcatova, Lenka Foretova, Vladimir Janout, Eleonora Fabianova, Maxime P. Vallée, Paul Brennan, James D. McKay, Graham Byrnes, Ghislaine Scélo. NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3137.

Background The US National Lung Cancer Screening Trial (NLST) demonstrated in 2011 that screening with computed tomography (CT) scans could reduce lung cancer mortality by 20%, but with important financial costs and high number of false positives. The identification of novel biomarkers is a need to obtain the maximum benefit from CT screening. Given its economical and minimally invasive nature, screening for somatic mutations in circulating tumor DNA (ctDNA) using next-generation sequencing may complement existing screening tools. However, for application in early detection, variant detection must also be done agnostically, i.e. without prior knowledge from tumour tissue of the mutations expected and unfortunately, most currently available variant callers are not adapted for this task. Methods We performed multiplex PCR on circulating free DNA (cfDNA) extracted from the plasma of 35 lung squamous cell carcinoma (SCC) and 64 small-cell carcinoma (SCLC) patients. We additionally included 133 hospital controls to evaluate the specificity of ctDNA. We applied (>10,000X) Ion torrent targeted sequencing on the full-coding region of TP53 since this gene is known to be mutated in more than 70% and 90% of SCC and SCLC, respectively. Each amplification, library preparation, and sequencing was performed in duplicate to control for amplification and sequencing errors. Detecting mutations on ctDNA raises important statistical and bioinformatics challenges as it represents only a small fraction of cfDNA. We therefore developed and applied a method based on the idea that a data-derived model of sequencing errors has the potential to improve our ability to detect low-allelic fraction (AF) somatic variants. Results We detected TP53 non-synonymous coding mutations with AFs between 0.04% and 85% (median 1.7%) in 8 (23%) SCC patients, 28 (44%) SCLC patients, and 8 (6%) controls. We estimated odds ratios of 4.6 (p = 0.006) for SCC and 12.0 (p = 6.7×10-10) for SCLC. Observations in controls are surprising, but in this instance there was no information regarding a subsequent cancer diagnosis. Conclusion We show that it is possible to detect ctDNA in the cfDNA of lung cancer patients. Since only patients with early stage (I-IIA) SCC tumours were included, these results support the potential utility of the approach for early detection. Nevertheless, if such mutations are found prior to diagnosis has not been explored in a prospective study design with pre-diagnostic plasma samples and individuals without a cancer diagnosis through a follow-up period. Citation Format: Patrice H. Avogbe, Tiffany Delhomme, Noémie Leblay, Florence Le Calvez-Kelm, Priscilia Chopard, Valérie Gaborieau, Ghislaine Scelo, Behnoush Abedi-Ardekani, David Zaridze, Anush Mukeria, Graham Byrnes, Paul Brennan, Lynnette Fernandez-Cuesta, Matthieu Foll, James D. McKay. NGS-based screening for TP53 mutations in circulating cell-free DNA: A first step towards early detection of lung cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3156.