Germline-focused analysis of tumour-detected variants in 49,264 cancer patients: ESMO Precision Medicine Working Group recommendations.

BACKGROUND: The European Society for Medical Oncology Precision Medicine Working Group (ESMO PMWG) was reconvened to update its 2018/19 recommendations on follow-up of putative germline variants detected on tumour-only sequencing, which were based on an analysis of 17 152 cancers. METHODS: We analysed an expanded dataset including 49 264 paired tumour-normal samples. We applied filters to tumour-detected variants based on variant allele frequency, predicted pathogenicity and population variant frequency. For 58 cancer-susceptibility genes, we then examined the proportion of filtered tumour-detected variants of true germline origin [germline conversion rate (GCR)]. We conducted subanalyses based on the age of cancer diagnosis, specific tumour types and 'on-tumour' status (established tumour-gene association). RESULTS: Analysis of 45 472 nonhypermutated solid malignancy tumour samples yielded 21 351 filtered tumour-detected variants of which 3515 were of true germline origin. 3.1% of true germline pathogenic variants were absent from the filtered tumour-detected variants. For genes such as BRCA1, BRCA2 and PALB2, the GCR in filtered tumour-detected variants was >80%; conversely for TP53, APC and STK11 this GCR was <2%. CONCLUSION: Strategic germline-focused analysis can prioritise a subset of tumour-detected variants for which germline follow-up will produce the highest yield of most actionable true germline variants. We present updated recommendations around germline follow-up of tumour-only sequencing including (i) revision to 5% for the minimum per-gene GCR, (ii) inclusion of actionable intermediate penetrance genes ATM and CHEK2, (iii) definition of a set of seven 'most actionable' cancer-susceptibility genes (BRCA1, BRCA2, PALB2, MLH1, MSH2, MSH6 and RET) in which germline follow-up is recommended regardless of tumour type.

Analysis of rare disruptive germline mutations in 2135 enriched BRCA-negative breast cancers excludes additional high-impact susceptibility genes.

BACKGROUND: Breast cancer has a significant heritable basis, of which ∼60% remains unexplained. Testing for BRCA1/BRCA2 offers useful discrimination of breast cancer risk within families, and identification of additional breast cancer susceptibility genes could offer clinical utility. PATIENTS AND METHODS: We included 2135 invasive breast cancer cases recruited via the Breast and Ovarian Cancer Susceptibility study, a retrospective UK study of familial breast cancer. ELIGIBILITY CRITERIA: female, BRCA-negative, white European ethnicity, and one of: (i) breast cancer family history, (ii) bilateral disease, (iii) young age of onset (<30 years), and (iv) concomitant ovarian cancer. We undertook exome sequencing of cases and carried out gene-level burden testing of rare damaging variants against those from 51 377 ethnicity-matched population controls from gnomAD. RESULTS: 159/2135 (7.4%) cases had a qualifying variant in an established breast cancer susceptibility gene, with minimal evidence of signal in other cancer susceptibility genes. Known breast cancer susceptibility genes PALB2, CHEK2, and ATM were the only genes to retain statistical significance after correcting for multiple testing. Due to the enrichment of hereditary cases in the series, we had good power (>80%) to detect a gene of BRCA1-like risk [odds ratio (OR) = 10.6] down to a population minor allele frequency of 4.6 × 10-5 (1 in 10 799, less than one-tenth that of BRCA1)and of PALB2-like risk (OR = 5.0) down to a population minor allele frequency of 2.8 × 10-4 (1 in 1779, less than half that of PALB2). Power was lower for identification of novel moderate penetrance genes (OR = 2-3) like CHEK2 and ATM. CONCLUSIONS: This is the largest case-control whole-exome analysis of enriched breast cancer published to date. Whilst additional breast cancer susceptibility genes likely exist, those of high penetrance are likely to be of very low mutational frequency. Contention exists regarding the clinical utility of such genes.

Clinical likelihood ratios and balanced accuracy for 44 in silico tools against multiple large-scale functional assays of cancer susceptibility genes.

PURPOSE: Where multiple in silico tools are concordant, the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) framework affords supporting evidence toward pathogenicity or benignity, equivalent to a likelihood ratio of ~2. However, limited availability of "clinical truth sets" and prior use in tool training limits their utility for evaluation of tool performance. METHODS: We created a truth set of 9,436 missense variants classified as deleterious or tolerated in clinically validated high-throughput functional assays for BRCA1, BRCA2, MSH2, PTEN, and TP53 to evaluate predictive performance for 44 recommended/commonly used in silico tools. RESULTS: Over two-thirds of the tool-threshold combinations examined had specificity of <50%, thus substantially overcalling deleteriousness. REVEL scores of 0.8-1.0 had a Positive Likelihood Ratio (PLR) of 6.74 (5.24-8.82) compared to scores <0.7 and scores of 0-0.4 had a Negative Likelihood Ratio (NLR) of 34.3 (31.5-37.3) compared to scores of >0.7. For Meta-SNP, the equivalent PLR = 42.9 (14.4-406) and NLR = 19.4 (15.6-24.9). CONCLUSION: Against these clinically validated "functional truth sets," there was wide variation in the predictive performance of commonly used in silico tools. Overall, REVEL and Meta-SNP had best balanced accuracy and might potentially be used at stronger evidence weighting than current ACMG/AMP prescription, in particular for predictions of benignity.

Collateral damage: the impact on outcomes from cancer surgery of the COVID-19 pandemic.

BACKGROUND: Cancer diagnostics and surgery have been disrupted by the response of health care services to the coronavirus disease 2019 (COVID-19) pandemic. Progression of cancers during delay will impact on patients' long-term survival. PATIENTS AND METHODS: We generated per-day hazard ratios of cancer progression from observational studies and applied these to age-specific, stage-specific cancer survival for England 2013-2017. We modelled per-patient delay of 3 and 6 months and periods of disruption of 1 and 2 years. Using health care resource costing, we contextualise attributable lives saved and life-years gained (LYGs) from cancer surgery to equivalent volumes of COVID-19 hospitalisations. RESULTS: Per year, 94 912 resections for major cancers result in 80 406 long-term survivors and 1 717 051 LYGs. Per-patient delay of 3/6 months would cause attributable death of 4755/10 760 of these individuals with loss of 92 214/208 275 life-years, respectively. For cancer surgery, average LYGs per patient are 18.1 under standard conditions and 17.1/15.9 with a delay of 3/6 months (an average loss of 0.97/2.19 LYGs per patient), respectively. Taking into account health care resource units (HCRUs), surgery results on average per patient in 2.25 resource-adjusted life-years gained (RALYGs) under standard conditions and 2.12/1.97 RALYGs following delay of 3/6 months. For 94 912 hospital COVID-19 admissions, there are 482 022 LYGs requiring 1 052 949 HCRUs. Hospitalisation of community-acquired COVID-19 patients yields on average per patient 5.08 LYG and 0.46 RALYGs. CONCLUSIONS: Modest delays in surgery for cancer incur significant impact on survival. Delay of 3/6 months in surgery for incident cancers would mitigate 19%/43% of LYGs, respectively, by hospitalisation of an equivalent volume of admissions for community-acquired COVID-19. This rises to 26%/59%, respectively, when considering RALYGs. To avoid a downstream public health crisis of avoidable cancer deaths, cancer diagnostic and surgical pathways must be maintained at normal throughput, with rapid attention to any backlog already accrued.

Germline-focussed analysis of tumour-only sequencing: recommendations from the ESMO Precision Medicine Working Group.

It is increasingly common in oncology practice to perform tumour sequencing using large cancer panels. For pathogenic sequence variants in cancer susceptibility genes identified on tumour-only sequencing, it is often unclear whether they are of somatic or constitutional (germline) origin. There is wide-spread disparity regarding both the extent to which systematic 'germline-focussed analysis' is carried out upon tumour sequencing data and for which variants follow-up analysis of a germline sample is carried out. Here we present analyses of paired sequencing data from 17 152 cancer samples, in which 1494 pathogenic sequence variants were identified across 65 cancer susceptibility genes. From these analyses, the European Society of Medical Oncology Precision Medicine Working Group Germline Subgroup has generated (i) recommendations regarding germline-focussed analyses of tumour-only sequencing data, (ii) indications for germline follow-up testing and (iii) guidance on patient information-giving and consent.

Relationships between MRI fat distributions and sleep apnea and obesity hypoventilation syndrome in very obese patients.

PURPOSE: Obesity is associated with both obstructive sleep apnea (OSA) and obesity hypoventilation. Differences in adipose tissue distribution are thought to underlie the development of both OSA and hypoventilation. We explored the relationships between the distribution of upper airway, neck, chest, abdominal and muscle fat in very obese individuals. METHODS: We conducted a cross-sectional cohort study of individuals presenting to a tertiary sleep clinic or for assessment for bariatric surgery. Individuals underwent magnetic resonance (MR) imaging of their upper airway, neck, chest, abdomen and thighs; respiratory polygraphy; 1 week of autotitrating CPAP; and morning arterial blood gas to determine carbon dioxide partial pressure and base excess. RESULTS: Fifty-three individuals were included, with mean age of 51.6 ± 8.4 years and mean BMI of 44.3 ± 7.9 kg/m2; there were 27 males (51%). Soft palate, tongue and lateral wall volumes were significantly associated with the AHI in univariable analyses (p < 0.001). Gender was a significant confounder in these associations. No significant associations were found between MRI measures of adiposity and hypoventilation. CONCLUSIONS: In very obese individuals, our results indicate that increased volumes of upper airway structures are associated with increased severity of OSA, as previously reported in less obese individuals. Increasingly large upper airway structures that reduce pharyngeal lumen size are likely to lead to OSA by increasing the collapsibility of the upper airway. However, we did not show any significant association between regional fat distribution and propensity for hypoventilation, in this population.

Implications of polygenic risk for personalised colorectal cancer screening.

BACKGROUND: We modelled the utility of applying a personalised screening approach for colorectal cancer (CRC) when compared with standard age-based screening. In this personalised screening approach, eligibility is determined by absolute risk which is calculated from age and polygenic risk score (PRS), where the PRS is relative risk attributable to common genetic variation. In contrast, eligibility in age-based screening is determined only by age. DESIGN: We calculated absolute risks of CRC from UK population age structure, incidence and mortality rate data, and a PRS distribution which we derived for the 37 known CRC susceptibility variants. We compared the number of CRC cases potentially detectable by personalised and age-based screening. Using Genome-Wide Complex Trait Analysis to calculate the heritability attributable to common variation, we repeated the analysis assuming all common CRC risk variants were known. RESULTS: Based on the known CRC variants, individuals with a PRS in the top 1% have a 2.9-fold increased CRC risk over the population median. Compared with age-based screening (aged 60: 10-year absolute risk 1.96% in men, 1.19% in women, as per the UK NHS National Bowel Screening Programme), personalised screening of individuals aged 55-69 at the same risk would lead to 16% fewer men and 17% fewer women being eligible for screening with 10% and 8%, respectively, fewer screen-detected cases. If all susceptibility variants were known, individuals with a PRS in the top 1% would have an estimated 7.7-fold increased risk. Personalised screening would then result in 26% fewer men and women being eligible for screening with 7% and 5% fewer screen-detected cases. CONCLUSION: Personalised screening using PRS has the potential to optimise population screening for CRC and to define those likely to maximally benefit from chemoprevention. There are however significant technical and operational details to be addressed before any such programme is introduced.

Ecological speciation in sympatric palms: 1. Gene expression, selection and pleiotropy.

Ecological speciation requires divergent selection, reproductive isolation and a genetic mechanism to link the two. We examined the role of gene expression and coding sequence evolution in this process using two species of Howea palms that have diverged sympatrically on Lord Howe Island, Australia. These palms are associated with distinct soil types and have displaced flowering times, representing an ideal candidate for ecological speciation. We generated large amounts of RNA-Seq data from multiple individuals and tissue types collected on the island from each of the two species. We found that differentially expressed loci as well as those with divergent coding sequences between Howea species were associated with known ecological and phenotypic differences, including response to salinity, drought, pH and flowering time. From these loci, we identified potential 'ecological speciation genes' and further validate their effect on flowering time by knocking out orthologous loci in a model plant species. Finally, we put forward six plausible ecological speciation loci, providing support for the hypothesis that pleiotropy could help to overcome the antagonism between selection and recombination during speciation with gene flow.

Force protection in contingency operations: an evaluation of temperature monitoring in Sierra Leone.

OBJECTIVES: The deployment of the UK-led Joint Inter-Agency Taskforce to Sierra Leone in September 2014 brought the era of contingency operations into focus. Daily health screening of such deployed personnel forms a key element of medical force protection. We have performed a service evaluation of an existing screening programme and detail the comparison of the two thermometers used in this role. METHODS: Data from the existing screening programme were used to inform a sample size required to enable statistically and clinically significant differences to be detected between the two interchangeably used thermometer systems in use. A prospective service evaluation on these devices was then carried out over a 10-day period and the data analysed by parametric tools. 10 personnel had their temperature recorded by both devices at the same time by a single operator every day. RESULTS: For the screened population, a mean temperature of 36.55°C and SD of 0.32°C was revealed. Powered to 80% with a two-tailed α of 0.05, the evaluation of the two thermometers revealed no significant difference between recordings taken with either device (p=0.115). The low SD meant that a pyrexial patient (>37.5°C) would require a recording over 3 SD from the population mean. DISCUSSION: Evaluations of medical force protection will carry increasing consequence as the UK deploy on short notice operations to regions of considerable endemic threat. Presence of pyrexia is a key early indicator of illness affecting deployed personnel, and two different thermometer types are provided for this function. We have shown for the first time with statistical and clinical significance that the two thermometers used in contingency force protection are interchangeable. The narrow variance is reassuring and confirms that the chosen trigger (>37.5°C) would warrant further investigation in the pyrexial patient.

Cardiovascular event rates in the MOSAIC trial: 2-year follow-up data.

The Multicentre Obstructive Sleep Apnoea Intervention Cardiovascular (MOSAIC) trial investigated the effect of continuous positive airway pressure (CPAP) on both sleepiness and predicted cardiovascular risk over 6 months in minimally symptomatic patients with obstructive sleep apnoea. Although there was clear benefit in terms of Epworth Sleepiness Score, there was no improvement in blood pressure and predicted vascular risk score. In order to calculate the required size of future trials, with real vascular events as the endpoint, the rate of such events in this population is needed. 188 patients from the original trial were followed for 2 years. The overall number of new vascular events over the 2 years was 25, and all-cause mortality was 4. There was a weak statistically significant reduction in vascular events in the CPAP group (p=0.049). Large-scale randomised trials are needed to determine if CPAP causes a real reduction in vascular events in minimally symptomatic patients. Based on our figures, future trials of CPAP versus no treatment would need to randomise approximately 2540 patients to not miss a real reduction in vascular events and over 6000 for mortality.

Pea aphid biotype performance on diverse Medicago host genotypes indicates highly specific virulence and resistance functions.

Aphid-plant interactions depend on genotypes of both organisms, which determine the two-way molecular exchange that leads to compatible or incompatible outcomes. The underlying genes are mostly unknown, making it difficult to predict likelihood of aphid success or host resistance, and hampering crop genetic improvement. Here we screened eight pea aphid clonal genotypes collected from diverse legume hosts, on a species-wide panel of Medicago truncatula (Mt) genotypes. Aphid virulence was measured by survival, fecundity and growth rate, together with scores for chlorosis and necrosis as host response indicators. Outcomes were highly dependent on the specific aphid-host genotype combinations. Only one Mt line was fully resistant against all clones. Aphid-induced host chlorosis and necrosis varied greatly, but correlated with resistance only in a few combinations. Bi-clustering analysis indicated that all aphid clones could be distinguished by their performance profiles across the host genotypes tested, with each clone being genetically differentiated and potentially representing a distinct biotype. Clones originating from Medicago sativa ranged from highly virulent to almost completely avirulent on both Medicago species, indicating that some were well adapted, whereas others were most likely migrants. Comparisons of closely related pairs of Australian Mt genotypes differing in aphid resistance revealed no enhanced resistance to European pea aphid clones. Based on the extensive variation in pea aphid adaptation even on unfamiliar hosts, most likely reflecting multiple biotype-specific gene-for-gene interactions, we conclude that robust defences require an arsenal of appropriate resistance genes.

Teachers' needs for an FASD-informed resource: a qualitative interview needs assessment based on the ADAPT-ITT framework.

Background: Fetal alcohol spectrum disorders (FASD) represent a wide range of neurodevelopmental differences associated with prenatal alcohol exposure and are highly prevalent. The current study represents the initial stages in adapting the Families Moving Forward (FMF) Program, an evidence-based behavioral consultation intervention for caregivers of children with FASD, to a website for teachers. Aims: To understand teachers' needs and preferences for an FASD-informed intervention website and to assess the goodness of fit of the FMF Program to teachers and the school setting. Methods: Twenty-three teachers with experience teaching students with FASD were interviewed. Interviews were conducted via Zoom and lasted about 53 minutes on average. Data were transcribed verbatim and analyzed using qualitative content analysis in Dedoose. Results: Three overarching themes represented teachers' needs for an FASD-informed resource: teachers need evidence-based FASD information and strategies, teachers have very little extra time, and the needs of special and general education teachers vary. Teachers were positive about the concepts of the FMF Program and felt they would have good fit. Conclusions: Teachers need an evidence-based FASD-informed intervention that is easy to use, concise, and responsive to varying needs and levels of experience. Results will inform the adaptation process of the FMF Program.

Positive behavior support (PBS) with children with fetal alcohol spectrum disorders: Characterizing caregivers' use of PBS using follow-up clinical trial data.

BACKGROUND: Fetal alcohol spectrum disorders (FASD) are associated with neurodevelopmental challenges leading to difficulties with everyday life tasks. The Families Moving Forward (FMF) Program teaches caregivers to use positive behavior support (PBS), integrated with other techniques. However, it is unknown how caregivers retain and use these PBS strategies after the intervention. METHODS: About 4.5 months after completing the FMF Program, twenty-three caregivers of children with FASD aged 4-12 were interviewed about problem behaviors targeted during the FMF Program and their continued use of PBS strategies. Interviews were recorded and coded thematically by a five-coder team. Higher-level pattern codes were developed to facilitate themes across descriptive codes. RESULTS: Caregivers commonly targeted task incompletion and rule breaking, and problem behaviors were often complex or combined. Caregivers identified environmental and interpersonal triggers for problem behavior. They used many accommodations to prevent problem behaviors, most often related to task or environment simplification. Caregivers also used consequence-based strategies. CONCLUSIONS: This study is the first to characterize caregivers' use of PBS strategies for children with FASD using mixed methods. Problem behaviors such as rule breaking were more difficult to target. Caregivers found most success when using a combination of multiple different accommodations per problem behavior. WHAT THIS PAPER ADDS: This is the first study to use mixed methods to characterize how caregivers of children with fetal alcohol spectrum disorders (FASD) use positive behavior support (PBS) strategies to target problem behavior after completion of the empirically validated Families Moving Forward (FMF) Program. Among other techniques involved in the FMF Program, PBS strategies are taught to caregivers and are used to target two distinct, caregiver-identified problem behaviors. This data provides essential information about behaviors responsive to PBS supports, for children with FASD, to inform clinical intervention and research. Notably, multiple problem behaviors often occurred together, emphasizing complexity of behavior challenges in this population and the resulting need for individualized supports. This study is the first to describe commonly observed triggers (antecedents) and commonly used supports (accommodations) from the perspective of caregivers of children with FASD. Importantly, results indicate that use of a wide variety of accommodations, or antecedent-based strategies, are effective in supporting behavior in children with FASD. However, success was most common when caregivers used multiple accommodations for any given concerning behavior. Findings represent 'real-world' strategies caregivers use to support adaptive behavior in their children several months after completion of the FMF Program, suggesting these strategies are applicable to clinical practice.

Larger-scale feasibility trial of the families moving forward (FMF) connect mobile health intervention for caregivers raising children with fetal alcohol spectrum disorders.

Objective: Caregivers raising children with fetal alcohol spectrum disorders (FASD) have limited access to evidence-based supports. This single-arm feasibility trial assesses the Families Moving Forward (FMF) Connect app to determine readiness for a larger randomized controlled trial (RCT). Methods: Eligibility for this online trial included caregivers of children (ages 3-12) with FASD residing in the United States. Caregivers received FMF Connect for 12 weeks on their personal smartphones (iOS or Android). Pre- and post-assessments included child behavior, parenting and family functioning, and app quality; user experience interviews were conducted post-intervention. Usage and crashes were monitored. Study objectives assessed feasibility of the trial (recruitment, attrition, measure sensitivity), intervention (technical functionality, acceptability), and implementation (caregiver usage). Results: Recruitment strategies proved sufficient with 171 caregivers screened and 105 deemed eligible. Analyses identified a few predictive demographic and outcome variables related to attrition. Several study measures were sensitive to change. Additional trial and measurement improvements were identified. From a technological perspective, the FMF Connect app was functional; the Android prototype required more.

BRCA1 testing should be offered to individuals with triple-negative breast cancer diagnosed below 50 years.

BACKGROUND: Triple-negative (TN) tumours are the predominant breast cancer subtype in BRCA1 mutation carriers. Recently, it was proposed that all individuals below 50 years of age with TN breast cancer should be offered BRCA testing. We have evaluated the BRCA1 mutation frequency and the implications for clinical practice of undertaking genetic testing in women with TN breast cancer. METHODS: We undertook BRCA1 mutation analysis in 308 individuals with TN breast cancer, 159 individuals from unselected series of breast cancer and 149 individuals from series ascertained on the basis of young age and/or family history. RESULTS: BRCA1 mutations were present in 45 out of 308 individuals. Individuals with TN cancer <50 years had >10% likelihood of carrying a BRCA1 mutation in both the unselected (11 out of 58, 19%) and selected (26 out of 111, 23%) series. However, over a third would not have been offered testing using existing criteria. We estimate that testing all individuals with TN breast cancer <50 years would generate an extra 1200 tests annually in England. CONCLUSION: Women with TN breast cancer diagnosed below 50 years have >10% likelihood of carrying a BRCA1 mutation and are therefore eligible for testing in most centres. However, implementation may place short-term logistical and financial burdens on genetic services.

Genome-wide association studies provide new insights into the genetic basis of testicular germ-cell tumour.

Testicular germ-cell tumour (TGCT) is the most common cancer in young men, and genetic epidemiological studies suggest that the disease has a strong genetic basis. Until 2009, very little of this genetic component had been explained. Genome-wide association studies have since identified eight SNPs at six loci which together account for approximately 15% of the genetic risk of TGCT and offer novel biological insights into testicular germ-cell oncogenesis. In this review, we summarize the genetic epidemiology of TGCT, detail the contribution genome-wide association studies have made to our understanding of the genetic basis of TGCT and reflect on how future technological advances may assist in revealing the remaining genetic factors underlying TGCT susceptibility.

A challenging case of hypercapnic respiratory failure during pregnancy.

We describe a 40-year-old female who presented with progressive breathlessness and hypercapnic respiratory failure during pregnancy secondary to undiagnosed muscle-specific kinase myasthenia gravis. Her presentation was progressive and protracted, having over five contacts with healthcare professionals over nine months, many of these predating her pregnancy. Her atypical presentation for myasthenia with minimal limb weakness led to consideration of other causes of hypercapnic respiratory failure. Once diagnosed, she was treated with intravenous immunoglobulin and non-invasive ventilation. She gave birth to a pre-term infant by planned caesarean section. Her insidious presentation and the progressive nature of her breathlessness were unusual and our report highlights the predominant involvement of respiratory muscles in muscle-specific kinase myasthenia. Her pregnancy may have further delayed her diagnosis due the attribution of some symptoms to normal pregnancy. Early recognition and treatment of myasthenia gravis are important to prevent life-threatening complications.