Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

The FANCM:p.Arg658* truncating variant is associated with risk of triple-negative breast cancer

Abstract: Breast cancer is a common disease partially caused by genetic risk factors. Germline pathogenic variants in DNA repair genes BRCA1, BRCA2, PALB2, ATM, and CHEK2 are associated with breast cancer risk. FANCM, which encodes for a DNA translocase, has been proposed as a breast cancer predisposition gene, with greater effects for the ER-negative and triple-negative breast cancer (TNBC) subtypes. We tested the three recurrent protein-truncating variants FANCM:p.Arg658*, p.Gln1701*, and p.Arg1931* for association with breast cancer risk in 67,112 cases, 53,766 controls, and 26,662 carriers of pathogenic variants of BRCA1 or BRCA2. These three variants were also studied functionally by measuring survival and chromosome fragility in FANCM−/− patient-derived immortalized fibroblasts treated with diepoxybutane or olaparib. We observed that FANCM:p.Arg658* was associated with increased risk of ER-negative disease and TNBC (OR = 2.44, P = 0.034 and OR = 3.79; P = 0.009, respectively). In a country-restricted analysis, we confirmed the associations detected for FANCM:p.Arg658* and found that also FANCM:p.Arg1931* was associated with ER-negative breast cancer risk (OR = 1.96; P = 0.006). The functional results indicated that all three variants were deleterious affecting cell survival and chromosome stability with FANCM:p.Arg658* causing more severe phenotypes. In conclusion, we confirmed that the two rare FANCM deleterious variants p.Arg658* and p.Arg1931* are risk factors for ER-negative and TNBC subtypes. Overall our data suggest that the effect of truncating variants on breast cancer risk may depend on their position in the gene. Cell sensitivity to olaparib exposure, identifies a possible therapeutic option to treat FANCM-associated tumors

CYP3A7*1C allele: linking premenopausal oestrone and progesterone levels with risk of hormone receptor-positive breast cancers

Funder: Breast Cancer Now (BCN); doi: https://doi.org/10.13039/100009794Funder: Cancer Research UK (CRUK); doi: https://doi.org/10.13039/501100000289Funder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265Funder: U.S. Department of Health & Human Services | National Institutes of Health (NIH)Funder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199; Grant(s): HEALTH-F2-2009-223175, HEALTH-F2-2009-223175Funder: Genome Canada (Génome Canada); doi: https://doi.org/10.13039/100008762Funder: Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada); doi: https://doi.org/10.13039/501100000024Funder: Quebec Breast cancer Foundation Genome QuebecFunder: U.S. Department of Health & Human Services | NIH | U.S. National Library of Medicine (NLM); doi: https://doi.org/10.13039/100000092Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))Funder: European Union’s Horizon 2020Funder: Deutsche Krebshilfe (German Cancer Aid); doi: https://doi.org/10.13039/501100005972Funder: BCAST - European Union’s Horizon 2020Funder: Breast Cancer Now; doi: https://doi.org/10.13039/501100007913Abstract: Background: Epidemiological studies provide strong evidence for a role of endogenous sex hormones in the aetiology of breast cancer. The aim of this analysis was to identify genetic variants that are associated with urinary sex-hormone levels and breast cancer risk. Methods: We carried out a genome-wide association study of urinary oestrone-3-glucuronide and pregnanediol-3-glucuronide levels in 560 premenopausal women, with additional analysis of progesterone levels in 298 premenopausal women. To test for the association with breast cancer risk, we carried out follow-up genotyping in 90,916 cases and 89,893 controls from the Breast Cancer Association Consortium. All women were of European ancestry. Results: For pregnanediol-3-glucuronide, there were no genome-wide significant associations; for oestrone-3-glucuronide, we identified a single peak mapping to the CYP3A locus, annotated by rs45446698. The minor rs45446698-C allele was associated with lower oestrone-3-glucuronide (−49.2%, 95% CI −56.1% to −41.1%, P = 3.1 × 10–18); in follow-up analyses, rs45446698-C was also associated with lower progesterone (−26.7%, 95% CI −39.4% to −11.6%, P = 0.001) and reduced risk of oestrogen and progesterone receptor-positive breast cancer (OR = 0.86, 95% CI 0.82–0.91, P = 6.9 × 10–8). Conclusions: The CYP3A7*1C allele is associated with reduced risk of hormone receptor-positive breast cancer possibly mediated via an effect on the metabolism of endogenous sex hormones in premenopausal women

An ensemble of features based deep learning neural network for reduction of inappropriate atrial fibrillation detection in implantable cardiac monitors

Background: Multiple studies have reported on classification of raw electrocardiograms (ECGs) using convolutional neural networks (CNNs). Objective: We investigated an application-specific CNN using a custom ensemble of features designed based on characteristics of the ECG during atrial fibrillation (AF) to reduce inappropriate AF detections in implantable cardiac monitors (ICMs). Methods: An ensemble of features was developed and combined to form an input signal for the CNN. The features were based on the morphological characteristics of AF, incoherence of RR intervals, and the fact that AF begets more AF. A custom CNN model and the RESNET18 model were trained using ICM-detected AF episodes that were adjudicated to be true AF or false detections. The trained models were evaluated using a test dataset from independent patients. Results: The training and validation datasets consisted of 31,757 AF episodes (2516 patients) and 28,506 false episodes (2126 patients). The validation set (20% randomly chosen episodes of each type) had an area under the curve of 0.996 for custom CNN (0.993 for RESNET18). Thresholds were chosen to obtain a relative sensitivity and specificity of 99.2% and 92.8%, respectively (99.2% and 87.9% for RESNET18, respectively). The performance in the independent test set (4546 AF episodes from 418 patients; 5384 false episodes from 605 patients) showed an area under the curve of 0.993 (0.991 for RESNET18) and relative sensitivity and specificity of 98.7% and 91.4%, respectively, at chosen thresholds (98.9% and 88.2% for RESNET18, respectively). Conclusion: An ensemble of features-based CNNs was developed that reduced inappropriate AF detection in ICMs by over 90% while preserving sensitivity

Evaluation of a clinical score for predicting atrial fibrillation in cryptogenic stroke patients with insertable cardiac monitors: results from the CRYSTAL AF study

Background: The HAVOC score was previously developed to predict the risk of atrial fibrillation (AF) after cryptogenic stroke (CS) or transient ischemic attack (TIA). The purpose of this study was to apply the HAVOC score to patients who received insertable cardiac monitors (ICMs) in the CRYSTAL AF study. Methods: All patients from the CRYSTAL AF study who received an ICM were included. HAVOC score (one point each for peripheral vascular disease and obesity with body mass index >30, two points each for hypertension, age ⩾ 75, valvular heart disease, and coronary artery disease, 4 points for congestive heart failure) was computed for all patients. The primary endpoint was AF detection by 12 months of ICM monitoring. Results: A total of 214 patients who received ICM were included. AF was detected in 40 patients while the remaining 174 patients were AF negative. The HAVOC score was significantly higher among patients with AF [median 3.0 with interquartile range (IQR) 2–4] than those without AF [median 2.0 (IQR 0–3)], p = 0.01. AF increased significantly across the three HAVOC score groups: 11% in Group A (score 0–1), 18% in Group B (score 2–3), and 32 % in Group C (score ⩾ 4) with p = 0.02. Conclusions: The HAVOC score was shown in this post hoc analysis of CRYSTAL AF to successfully stratify AF risk post CS or TIA. The 11% AF rate in the lowest HAVOC score group highlights the significance of nontraditional contributors to AF and ischemic stroke

Lower heart rates and beta-blockers are associated with new-onset atrial fibrillation

Background: Lower heart rates (HRs) prolong diastole, which increases filling pressures and wall stress. As a result, lower HRs may be associated with higher brain natriuretic peptide (BNP) levels and incident atrial fibrillation (AF). Beta-blockers may increase the risk for AF due to suppression of resting HRs. Objective: Examine the relationships of HR, BNP, beta-blockers and new-onset AF in the REVEAL-AF and SPRINT cohort of subjects at risk for developing AF. Methods: In REVEAL-AF, 383 subjects without a history of AF and a mean CHA2DS2VASC score of 4.4 ± 1.3 received an insertable cardiac monitor and were followed up to 30 months. In SPRINT, 7595 patients without prior history of AF and a mean CHA2DS2VASC score of 2.3 ± 1.2 were followed up to 60 months. Results: The median daytime HR in the REVEAL-AF cohort was 75bpm [IQR 68–83]. Subjects with below-median HRs had 2.4-fold higher BNP levels compared to subjects with above-median HRs (median BNP [IQR]: 62 pg/dl [37−112] vs. 26 pg/dl [13–53], p < 0.001). HRs <75bpm were associated with a higher incidence of AF: 37% vs. 27%, p < 0.05. This was validated in the SPRINT cohort after adjusting for AF risk factors. Both a HR < 75bpm and beta-blocker use were associated with a higher rate of AF: 1.9 vs 0.7% (p < 0.001) and 2.5% vs. 0.6% (p < 0.001), respectively. The hazard ratio for patients on beta-blockers to develop AF was 3.72 [CI 2.32, 5.96], p < 0.001. Conclusions: Lower HRs are associated with higher BNP levels and incident AF, mimicking the hemodynamic effects of diastolic dysfunction. Suppression of resting HR by beta-blockers could explain their association with incident AF

Simulation of Daily Snapshot Rhythm Monitoring to Identify Atrial Fibrillation in Continuously Monitored Patients with Stroke Risk Factors.

BACKGROUND:New technologies are diffusing into medical practice swiftly. Hand-held devices such as smartphones can record short-duration (e.g., 1-minute) ECGs, but their effectiveness in identifying patients with paroxysmal atrial fibrillation (AF) is unknown. METHODS:We used data from the TRENDS study, which included 370 patients (mean age 71 years, 71% men, CHADS2 score≥1 point: mean 2.3 points) who had no documentation of atrial tachycardia (AT)/AF or antiarrhythmic or anticoagulant drug use at baseline. All were subsequently newly diagnosed with AT/AF by a cardiac implantable electronic device (CIED) over one year of follow-up. Using a computer simulation approach (5,000 repetitions), we estimated the detection rate for paroxysmal AT/AF via daily snapshot ECG monitoring over various periods, with the probability of detection equal to the percent AT/AF burden on each day. RESULTS:The estimated AT/AF detection rates with snapshot monitoring periods of 14, 28, 56, 112, and 365 days were 10%, 15%, 21%, 28%, and 50% respectively. The detection rate over 365 days of monitoring was higher in those with CHADS2 scores ≥2 than in those with CHADS2 scores of 1 (53% vs. 38%), and was higher in those with AT/AF burden ≥0.044 hours/day compared to those with AT/AF burden <0.044 hours/day (91% vs. 14%; both P<0.05). CONCLUSIONS:Daily snapshot ECG monitoring over 365 days detects half of patients who developed AT/AF as detected by CIED, and shorter intervals of monitoring detected fewer AT/AF patients. The detection rate was associated with individual CHADS2 score and AT/AF burden. TRIAL REGISTRATION:ClinicalTrials.gov NCT00279981

Estimated detection rates for daily snapshot monitoring classified based on low/high CHADS₂ score and low/high AT/AF burden (n = 370).

<p>Estimated detection rates for daily snapshot monitoring classified based on low/high CHADS₂ score and low/high AT/AF burden (n = 370).</p