Evaluation of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) Samples from Advanced Non-Small Cell Lung Cancer for Whole Genome, Whole Exome and Comprehensive Panel Sequencing.

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is often the only source of tumor tissue from patients with advanced, inoperable lung cancer. EBUS-TBNA aspirates are used for the diagnosis, staging, and genomic testing to inform therapy options. Here we extracted DNA and RNA from 220 EBUS-TBNA aspirates to evaluate their suitability for whole genome (WGS), whole exome (WES), and comprehensive panel sequencing. For a subset of 40 cases, the same nucleic acid extraction was sequenced using WGS, WES, and the TruSight Oncology 500 assay. Genomic features were compared between sequencing platforms and compared with those reported by clinical testing. A total of 204 aspirates (92.7%) had sufficient DNA (100 ng) for comprehensive panel sequencing, and 109 aspirates (49.5%) had sufficient material for WGS. Comprehensive sequencing platforms detected all seven clinically reported tier 1 actionable mutations, an additional three (7%) tier 1 mutations, six (15%) tier 2-3 mutations, and biomarkers of potential immunotherapy benefit (tumor mutation burden and microsatellite instability). As expected, WGS was more suited for the detection and discovery of emerging novel biomarkers of treatment response. WGS could be performed in half of all EBUS-TBNA aspirates, which points to the enormous potential of EBUS-TBNA as source material for large, well-curated discovery-based studies for novel and more effective predictors of treatment response. Comprehensive panel sequencing is possible in the vast majority of fresh EBUS-TBNA aspirates and enhances the detection of actionable mutations over current clinical testing.

Computational immunogenomic approaches to predict response to cancer immunotherapies.

Cancer immunogenomics is an emerging field that bridges genomics and immunology. The establishment of large-scale genomic collaborative efforts along with the development of new single-cell transcriptomic techniques and multi-omics approaches have enabled characterization of the mutational and transcriptional profiles of many cancer types and helped to identify clinically actionable alterations as well as predictive and prognostic biomarkers. Researchers have developed computational approaches and machine learning algorithms to accurately obtain clinically useful information from genomic and transcriptomic sequencing data from bulk tissue or single cells and explore tumours and their microenvironment. The rapid growth in sequencing and computational approaches has resulted in the unmet need to understand their true potential and limitations in enabling improvements in the management of patients with cancer who are receiving immunotherapies. In this Review, we describe the computational approaches currently available to analyse bulk tissue and single-cell sequencing data from cancer, stromal and immune cells, as well as how best to select the most appropriate tool to address various clinical questions and, ultimately, improve patient outcomes.

The clinical utility and costs of whole-genome sequencing to detect cancer susceptibility variants-a multi-site prospective cohort study.

BACKGROUND: Many families and individuals do not meet criteria for a known hereditary cancer syndrome but display unusual clusters of cancers. These families may carry pathogenic variants in cancer predisposition genes and be at higher risk for developing cancer. METHODS: This multi-centre prospective study recruited 195 cancer-affected participants suspected to have a hereditary cancer syndrome for whom previous clinical targeted genetic testing was either not informative or not available. To identify pathogenic disease-causing variants explaining participant presentation, germline whole-genome sequencing (WGS) and a comprehensive cancer virtual gene panel analysis were undertaken. RESULTS: Pathogenic variants consistent with the presenting cancer(s) were identified in 5.1% (10/195) of participants and pathogenic variants considered secondary findings with potential risk management implications were identified in another 9.7% (19/195) of participants. Health economic analysis estimated the marginal cost per case with an actionable variant was significantly lower for upfront WGS with virtual panel ($8744AUD) compared to standard testing followed by WGS ($24,894AUD). Financial analysis suggests that national adoption of diagnostic WGS testing would require a ninefold increase in government annual expenditure compared to conventional testing. CONCLUSIONS: These findings make a case for replacing conventional testing with WGS to deliver clinically important benefits for cancer patients and families. The uptake of such an approach will depend on the perspectives of different payers on affordability.

Sensitivity of tropical montane birds to anthropogenic disturbance and management strategies for their conservation in agricultural landscapes.

Tropical montane bird communities are hypothesized to be highly sensitive to anthropogenic disturbance because species are adapted to a narrow range of environmental conditions and display high rates of endemism. We assessed avian sensitivity at regional and continental scales for a global epicenter of montane bird biodiversity, the tropical Andes. Using data from an intensive field study of cloud forest bird communities across 7 landscapes undergoing agricultural conversion in northern Peru (1800-3100 m, 2016-2017) and a pan-Andean synthesis of forest bird sensitivity, we developed management strategies for maintaining avian biodiversity in tropical countrysides and examined how environmental specialization predicts species-specific sensitivity to disturbance. In Peru, bird communities occupying countryside habitats contained 29-93% fewer species compared with those in forests and were compositionally distinct due to high levels of species turnover. Fragments of mature forest acted as reservoirs for forest bird diversity, especially when large or surrounded by mixed successional vegetation. In high-intensity agricultural plots, an addition of 10 silvopasture trees or 10% more fencerows per hectare increased species richness by 18-20%. Insectivores and frugivores were most sensitive to disturbance: abundance of 40-70% of species declined in early successional vegetation and silvopasture. These results were supported by our synthesis of 816 montane bird species studied across the Andes. At least 25% of the species declined due to all forms of disturbance, and the percentage rose to 60% in agricultural landscapes. The most sensitive species were those with narrow elevational ranges and small global range sizes, insectivores and carnivores, and species with specialized trophic niches. We recommend protecting forest fragments, especially large ones, and increasing connectivity through the maintenance of early successional vegetation and silvopastoral trees that increase avian diversity in pastures. We provide lists of species-specific sensitivities to anthropogenic disturbance to inform conservation status assessments of Andean birds.

Sensibilidad de aves montanas a perturbaciones antropogénicas y estrategias de manejo para su conservación en paisajes agrícolas Resumen Se ha hipotetizado que las comunidades de aves tropicales montanas son sumamente sensibles a la perturbación antropogénica porque las especies están adaptadas a una reducida gama de condiciones ambientales y tienen altas tasas de endemismo. Evaluamos la sensibilidad aviar a escalas regional y continental para un epicentro global de biodiversidad de aves montanas, los Andes tropicales. Utilizando datos de un estudio intensivo de campo de comunidades de aves de bosques nublados en 7 paisajes bajo conversión agrícola en el norte de Perú (1800 - 3100 m, 2016-2017) y una síntesis pan-Andina de sensibilidad de aves de bosque, desarrollamos estrategias de manejo para el mantenimiento de la biodiversidad de aves en campiñas tropicales y examinamos cómo la especialización ambiental predice la sensibilidad de cada especie a la perturbación. Las comunidades de aves ocupando hábitats campestres tropicales en Perú contenían 29 - 93% menos especies en comparación con las de bosques y tuvieron una composición distinta debido a los altos niveles de recambio de especies. Los fragmentos de bosque maduro fungieron como reservorios para la diversidad de aves de bosque, especialmente cuando eran extensos y estaban rodeados por vegetación secundaria mixta. En las parcelas con actividad agrícola intensiva, la adición de 10 árboles silvopastoriles o 10% más de cercos por hectárea incrementó la riqueza de especies en 18 - 20%. Las insectívoras y frugívoras fueron más sensibles a la perturbación: la abundancia de 40 - 70% de especies declinó en la vegetación secundaria temprana y en la silvopastura. Estos resultados fueron sustentados por nuestra síntesis de 816 especies de aves montanas estudiadas en los Andes. Por lo menos 25% de estas especies declinaron debido a todas las formas de perturbación, y el porcentaje incrementó a 60% en paisajes agrícolas. Las especies más sensibles fueron aquellas con rangos altitudinales estrechos y extensiones de distribución pequeñas, las insectívoras y carnívoras y las especies con nichos tróficos especializados. Recomendamos la protección de fragmentos de bosque, especialmente los extensos, y el incremento de la conectividad mediante el mantenimiento de vegetación secundaria temprana y árboles silvopastoriles que incrementan la diversidad de aves en los pastizales. Proporcionamos listas de la sensibilidad de cada especie a la perturbación antropogénica para contribuir a las evaluaciones del estatus de conservación de aves Andinas.

Multi-omic features of oesophageal adenocarcinoma in patients treated with preoperative neoadjuvant therapy.

Oesophageal adenocarcinoma is a poor prognosis cancer and the molecular features underpinning response to treatment remain unclear. We investigate whole genome, transcriptomic and methylation data from 115 oesophageal adenocarcinoma patients mostly from the DOCTOR phase II clinical trial (Australian New Zealand Clinical Trials Registry-ACTRN12609000665235), with exploratory analysis pre-specified in the study protocol of the trial. We report genomic features associated with poorer overall survival, such as the APOBEC mutational and RS3-like rearrangement signatures. We also show that positron emission tomography non-responders have more sub-clonal genomic copy number alterations. Transcriptomic analysis categorises patients into four immune clusters correlated with survival. The immune suppressed cluster is associated with worse survival, enriched with myeloid-derived cells, and an epithelial-mesenchymal transition signature. The immune hot cluster is associated with better survival, enriched with lymphocytes, myeloid-derived cells, and an immune signature including CCL5, CD8A, and NKG7. The immune clusters highlight patients who may respond to immunotherapy and thus may guide future clinical trials.

Network structure of avian mixed-species flocks decays with elevation and latitude across the Andes.

Birds in mixed-species flocks benefit from greater foraging efficiency and reduced predation, but also face costs related to competition and activity matching. Because this cost-benefit trade-off is context-dependent (e.g. abiotic conditions and habitat quality), the structure of flocks is expected to vary along elevational, latitudinal and disturbance gradients. Specifically, we predicted that the connectivity and cohesion of flocking networks would (i) decline towards tropical latitudes and lower elevations, where competition and activity matching costs are higher, and (ii) increase with lower forest cover and greater human disturbance. We analysed the structure of 84 flock networks across the Andes and assessed the effect of elevation, latitude, forest cover and human disturbance on network characteristics. We found that Andean flocks are overall open-membership systems (unstructured), though the extent of network structure varied across gradients. Elevation was the main predictor of structure, with more connected and less modular flocks upslope. As expected, flocks in areas with higher forest cover were less cohesive, with better defined flock subtypes. Flocks also varied across latitude and disturbance gradients as predicted, but effect sizes were small. Our findings indicate that the unstructured nature of Andean flocks might arise as a strategy to cope with harsh environmental conditions. This article is part of the theme issue 'Mixed-species groups and aggregations: shaping ecological and behavioural patterns and processes'.

Wet and dry extremes reduce arthropod biomass independently of leaf phenology in the wet tropics.

Warming temperatures are increasing rainfall extremes, yet arthropod responses to climatic fluctuations remain poorly understood. Here, we used spatiotemporal variation in tropical montane climate as a natural experiment to compare the importance of biotic versus abiotic drivers in regulating arthropod biomass. We combined intensive field data on arthropods, leaf phenology and in situ weather across a 1700-3100 m elevation and rainfall gradient, along with desiccation-resistance experiments and multi-decadal modelling. We found limited support for biotic drivers with weak increases in some herbivorous taxa on shrubs with new leaves, but no landscape-scale effects of leaf phenology, which tracked light and cloud cover. Instead, rainfall explained extensive interannual variability with maximum biomass at intermediate rainfall (130 mm month-1 ) as both 3 months of high and low rainfall reduced arthropods by half. Based on 50 years of regional rainfall, our dynamic arthropod model predicted shifts in the timing of biomass maxima within cloud forests before plant communities transition to seasonally deciduous dry forests (mean annual rainfall 1000-2500 mm vs. <800 mm). Rainfall magnitude was the primary driver, but during high solar insolation, the 'drying power of air' (VPDmax ) reduced biomass within days contributing to drought related to the El Niño-Southern Oscillation (ENSO). Highlighting risks from drought, experiments demonstrated community-wide susceptibility to desiccation except for some caterpillars in which melanin-based coloration appeared to reduce the effects of evaporative drying. Overall, we provide multiple lines of evidence that several months of heavy rain or drought reduce arthropod biomass independently of deep-rooted plants with the potential to destabilize insectivore food webs.

El aumento de las temperaturas está incrementando los extremos de precipitación, pero las respuestas de los artrópodos a las fluctuaciones climáticas siguen siendo poco conocidas. Aquí, utilizamos la variación espaciotemporal en el clima montano tropical como un experimento natural para comparar la importancia de los factores bióticos versus abióticos en la regulación de la biomasa de artrópodos. Combinamos datos de campo intensivos de artrópodos, fenología de las hojas y clima in situ a lo largo de un gradiente altitudinal de 1700 a 3100 m y un gradiente de precipitación, junto con experimentos de resistencia a la desecación y modelos multi-decenales. Encontramos evidencia limitada para los factores bióticos con aumentos débiles en algunos taxones de herbívoros en arbustos con hojas nuevas, pero no hubo efectos a escala de paisaje en la fenología de la hoja, que rastreaba la luz y la cubierta de nubes. En cambio, las precipitaciones explicaron la amplia variabilidad interanual con una biomasa máxima en precipitaciones intermedias (130 mm mes−1 ), ya que los tres meses de precipitaciones altas y bajas redujeron los artrópodos a la mitad. Basándose en 50 años de precipitación regional, nuestro modelo dinámico de artrópodos predijo cambios en el momento de los máximos de biomasa dentro del bosque nuboso antes de que las comunidades de plantas hicieran la transición al bosque seco estacional caducifolio (precipitación media anual 1000-2500 mm vs. <800 mm). La magnitud de las lluvias fue el principal factor, pero durante la alta insolación solar, el "poder de secado del aire" (VPDmax ) redujo la biomasa en cuestión de días, lo que contribuyó a la sequía relacionada con El Niño-Southern Oscillation (ENSO). Destacando los riesgos de la sequía, los experimentos demostraron la susceptibilidad de toda la comunidad a la desecación, excepto en el caso de algunas orugas en las que la coloración a base de melanina parece reducir los efectos de la desecación por evaporación. En resumen, proporcionamos múltiples líneas de evidencia de que varios meses de fuertes lluvias o sequías reducen la biomasa de artrópodos independientemente de las plantas de raíces profundas con el potencial de desestabilizar las redes alimentarias de los insectívoros.

Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes.

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes. SIGNIFICANCE: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711.

Analysis of hereditary cancer gene variant classifications from ClinVar indicates a need for regular reassessment of clinical assertions.

The clinical classification of variants may change with new information, however, there is limited guidance on how often significant changes in variant classification occur. We used ClinVar to examine how variant classification changes over time. We developed a custom parser and accessed variant data from ClinVar between January 2015 and July 2021. The ClinVar-assigned "aggregate" classification of variants in 121 hereditary cancer genes was harmonized across releases to align to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology terms. Aggregate classification categories were grouped as: benign/likely benign (B/LB); likely pathogenic/pathogenic (LP/P); variant of uncertain significance (VUS); conflicting interpretations of pathogenicity (Conflicting); or Other. We profiled changes in aggregate variant classification between consecutive semi-annual ClinVar releases. The proportion of variants that changed aggregate classification between semi-annual ClinVar releases ranged from 0.6% to 6.4%. The most frequent changes were "VUS to conflicting," "other to LP/P," and "B/LB to Conflicting." A limited number of variants changed aggregate classification from "LP/P to B/LB," or vice versa. Our analysis indicates need for regular reassessment of clinical variant interpretations. The parser developed for this project will facilitate extraction of relevant interpretation data from ClinVar.

Functional response traits and altered ecological niches drive the disassembly of cloud forest bird communities in tropical montane countrysides.

Anthropogenic disturbance contributes to global change by reshaping the ecological niche space available to biological communities. Quantifying the range of functional response traits required for species persistence is central towards understanding the mechanisms underlying community disassembly in disturbed landscapes. We used intensive field surveys of cloud forest bird communities across seven replicate landscapes undergoing agricultural conversion in the Peruvian Andes to examine how a suite of 16 functional response traits related to morphology, diet, foraging behaviour and environmental niche breadth predict (1) species-specific abundance changes in countryside habitats compared to forest and (2) differential changes to the ecological niche space occupied by communities. Our analyses relied on (1) hierarchical distance sampling models to examine the functional predictors of abundance change across the agricultural land use gradient while accounting for imperfect detection and (2) n-dimensional hypervolumes to quantify the expansion and contraction of ecological niche space in countryside habitats. Key traits related to increased abundance in early successional and mixed-intensity agricultural areas included (1) morphological adaptations to dense understorey habitats, (2) plant-based diets (flowers, fruit and seeds) and (3) broad elevational range limits and habitat breadth. Species occupying mixed and high-intensity agricultural land use regimes had mean elevational range limits 20%-60% wider than species found within forests. Collectively, ecological niche space expanded within agricultural habitats for traits related to diet and environmental niche breadth, while contracting for foraging and dispersal traits. Such changes were driven by species with unique functional trait combinations. Our results reveal the dynamic changes to ecological niche space that underly community structure in disturbed landscapes and highlight how increased niche breadth can ameliorate disturbance sensitivity for generalist species. We emphasize that functional traits can be used to predict changes in community structure across disturbance gradients, allowing insights into specific mechanisms underlying community disassembly beyond emergent patterns of functional diversity. By identifying key functional trait groups that align with different countryside habitats, we demonstrate how conservation practitioners can contribute to the retention of avian functional diversity in agricultural landscapes throughout the world.

La perturbación antropogénica contribuye al cambio global al remodelar el espacio de nicho ecológico disponible para las comunidades biológicas. Cuantificar la gama de rasgos de respuesta funcional requeridos para la persistencia de las especies es fundamental para comprender los mecanismos que subyacen al desensamble de la comunidad en los paisajes perturbados. Utilizamos muestreos de campo intensivos de las comunidades de aves del bosque nublado en siete paisajes replicados convertidos a uso agrícola en los Andes peruanos para examinar cómo un conjunto de 16 rasgos de respuesta funcional relacionados con la morfología, dieta, comportamiento de forrajeo, y la amplitud del nicho ambiental predicen (1) cambios en las abundancias de especies específicas en paisajes agrícolas ("countrysides") en comparación con el bosque y (2) cambios diferenciales en el espacio del nicho ecológico ocupado por las comunidades. Nuestros análisis se basaron en (1) modelos jerárquicos de muestreo por la distancia para examinar los predictores funcionales del cambio de abundancia a través del gradiente de uso de suelo para agricultura teniendo en cuenta la detección imperfecta, y (2) "n-dimensional hypervolumes" para cuantificar la expansión y contracción del espacio de nicho ecológico en los hábitats agrícolas. Los rasgos clave relacionados con el aumento de la abundancia en áreas agrícolas de sucesión temprana y de intensidad mixta incluyeron (1) adaptaciones morfológicas a hábitats de sotobosque denso, (2) dietas basadas en plantas (flores, frutas y semillas), y (3) amplios límites de rango de elevación y amplitud de hábitat. Las especies que ocupan regímenes de suelo agrícola mixto y de alta intensidad tenían límites de rango de elevación promedio 20%-60% más amplios que las especies que se encuentran en los bosques. En conjunto, el espacio del nicho ecológico se expandió dentro de los hábitats agrícolas para los rasgos relacionadas con la dieta y la amplitud del nicho ambiental, mientras que se contrajo para los rasgos de forrajeo y dispersión. Dichos cambios fueron impulsados por especies con combinaciones de rasgos funcionales únicos. Nuestros resultados revelan los cambios dinámicos en el espacio del nicho ecológico que subyacen a la estructura de la comunidad en los paisajes perturbados y destacan cómo una mayor amplitud del nicho puede mejorar la sensibilidad a las perturbaciones para las especies generalistas. Enfatizamos que los rasgos funcionales pueden utilizarse para predecir los cambios en la estructura de la comunidad a través de gradientes de perturbación, lo que permite comprender los mecanismos específicos que subyacen al desensamble de la comunidad más allá de los patrones emergentes de diversidad funcional. Al identificar grupos de rasgos funcionales claves que se alinean con diferentes hábitats agrícolas como en "countrysides" demostramos cómo los profesionales de la conservación pueden contribuir a la retención de la diversidad funcional de las aves en los paisajes agrícolas en todo el mundo.

Comprehensive genomic and tumour immune profiling reveals potential therapeutic targets in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) has a poor overall survival with few treatment options. Whole genome sequencing (WGS) combined with the immune features of MPM offers the prospect of identifying changes that could inform future clinical trials. METHODS: We analysed somatic mutations from 229 MPM samples, including previously published data and 58 samples that had undergone WGS within this study. This was combined with RNA-seq analysis to characterize the tumour immune environment. RESULTS: The comprehensive genome analysis identified 12 driver genes, including new candidate genes. Whole genome doubling was a frequent event that correlated with shorter survival. Mutational signature analysis revealed SBS5/40 were dominant in 93% of samples, and defects in homologous recombination repair were infrequent in our cohort. The tumour immune environment contained high M2 macrophage infiltrate linked with MMP2, MMP14, TGFB1 and CCL2 expression, representing an immune suppressive environment. The expression of TGFB1 was associated with overall survival. A small subset of samples (less than 10%) had a higher proportion of CD8 T cells and a high cytolytic score, suggesting a 'hot' immune environment independent of the somatic mutations. CONCLUSIONS: We propose accounting for genomic and immune microenvironment status may influence therapeutic planning in the future.

Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma and enrichment for CRKL amplifications in acral melanoma. We modelled these changes in transgenic zebrafish models and found that CRKL-driven tumours formed predominantly in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin and limb melanocytes, when compared with body melanocytes, revealed a positional identity gene programme typified by posterior HOX13 genes. This positional gene programme synergized with CRKL to amplify insulin-like growth factor (IGF) signalling and drive tumours at acral sites. Abrogation of this CRKL-driven programme eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

Patient-derived xenograft models capture genomic heterogeneity in endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. METHODS: Here, we report whole exome or whole genome sequencing of 11 PDX models and their matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. RESULTS: PDX models were successfully generated from grade 2/3 tumors, including three uterine carcinosarcomas. The models showed similar histomorphology to the primary tumors and represented all four molecular subtypes of EC, including five mismatch-repair deficient models. The different PDX lineages showed a wide range of inter-tumor and intra-tumor heterogeneity. However, for most PDX models, one arm recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease; however, all lacked the HR deficiency genomic signature. CONCLUSIONS: EC PDX models represent the four molecular subtypes of disease and can capture intra-tumor heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi; however, deeper and more durable responses will likely require combination of PARPi with other agents.

Multiomic profiling of checkpoint inhibitor-treated melanoma: Identifying predictors of response and resistance, and markers of biological discordance.

We concurrently examine the whole genome, transcriptome, methylome, and immune cell infiltrates in baseline tumors from 77 patients with advanced cutaneous melanoma treated with anti-PD-1 with or without anti-CTLA-4. We show that high tumor mutation burden (TMB), neoantigen load, expression of IFNγ-related genes, programmed death ligand expression, low PSMB8 methylation (therefore high expression), and T cells in the tumor microenvironment are associated with response to immunotherapy. No specific mutation correlates with therapy response. A multivariable model combining the TMB and IFNγ-related gene expression robustly predicts response (89% sensitivity, 53% specificity, area under the curve [AUC], 0.84); tumors with high TMB and a high IFNγ signature show the best response to immunotherapy. This model validates in an independent cohort (80% sensitivity, 59% specificity, AUC, 0.79). Except for a JAK3 loss-of-function mutation, for patients who did not respond as predicted there is no obvious biological mechanism that clearly explained their outlier status, consistent with intratumor and intertumor heterogeneity in response to immunotherapy.

Evaluation of Crizotinib Treatment in a Patient With Unresectable GOPC-ROS1 Fusion Agminated Spitz Nevi.

IMPORTANCE: Spitz nevi are benign melanocytic neoplasms that classically present in childhood. Isolated Spitz nevi have been associated with oncogenic gene fusions in approximately 50% of cases. The rare agminated variant of Spitz nevi, thought to arise from cutaneous genetic mosaicism, is characterized by development of clusters of multiple lesions in a segmental distribution, which can complicate surgical removal. Somatic single-nucleotide variants in the HRAS oncogene have been described in agminated Spitz nevi, most of which were associated with an underlying nevus spilus. The use of targeted medical therapy for agminated Spitz nevi is not well understood. OBSERVATIONS: A girl aged 30 months presented with facial agminated Spitz nevi that recurred rapidly and extensively after surgery. Owing to the morbidity of further surgery, referral was made to a molecular tumor board. The patient's archival nevus tissue was submitted for extended immunohistochemical analysis and genetic sequencing. Strong ROS1 protein expression was identified by immunohistochemistry. Consistent with this, analysis of whole-genome sequencing data revealed GOPC-ROS1 fusions. These results indicated likely benefit from the oral tyrosine kinase inhibitor crizotinib, which was administered at a dosage of 280 mg/m2 twice daily. An excellent response was observed in all lesions within 5 weeks, with complete flattening after 20 weeks. CONCLUSIONS AND RELEVANCE: Given the response following crizotinib treatment observed in this case, the kinase fusion was believed to be functionally consequential in the patient's agminated Spitz nevi and likely the driver mutational event for growth of her nevi. The repurposing of crizotinib for GOPC-ROS1 Spitz nevi defines a new treatment option for these lesions, particularly in cases for which surgery is relatively contraindicated.

Tumor Signature Analysis Implicates Hereditary Cancer Genes in Endometrial Cancer Development.

Risk of endometrial cancer (EC) is increased ~2-fold for women with a family history of cancer, partly due to inherited pathogenic variants in mismatch repair (MMR) genes. We explored the role of additional genes as explanation for familial EC presentation by investigating germline and EC tumor sequence data from The Cancer Genome Atlas (n = 539; 308 European ancestry), and germline data from 33 suspected familial European ancestry EC patients demonstrating immunohistochemistry-detected tumor MMR proficiency. Germline variants in MMR and 26 other known/candidate EC risk genes were annotated for pathogenicity in the two EC datasets, and also for European ancestry individuals from gnomAD as a population reference set (n = 59,095). Ancestry-matched case-control comparisons of germline variant frequency and/or sequence data from suspected familial EC cases highlighted ATM, PALB2, RAD51C, MUTYH and NBN as candidates for large-scale risk association studies. Tumor mutational signature analysis identified a microsatellite-high signature for all cases with a germline pathogenic MMR gene variant. Signature analysis also indicated that germline loss-of-function variants in homologous recombination (BRCA1, PALB2, RAD51C) or base excision (NTHL1, MUTYH) repair genes can contribute to EC development in some individuals with germline variants in these genes. These findings have implications for expanded therapeutic options for EC cases.

Meta-Analysis and Systematic Review of the Genomics of Mucosal Melanoma.

Mucosal melanoma is a rare subtype of melanoma. To date, there has been no comprehensive systematic collation and statistical analysis of the aberrations and aggregated frequency of driver events across multiple studies. Published studies using whole genome, whole exome, targeted gene panel, or individual gene sequencing were identified. Datasets from these studies were collated to summarize mutations, structural variants, and regions of copy-number alteration. Studies using next-generation sequencing were divided into the "main" cohort (n = 173; fresh-frozen samples), "validation" cohort (n = 48; formalin-fixed, paraffin-embedded samples) and a second "validation" cohort comprised 104 tumors sequenced using a targeted panel. Studies assessing mutations in BRAF, KIT, and NRAS were summarized to assess hotspot mutations. Statistical analysis of the main cohort variant data revealed KIT, NF1, BRAF, NRAS, SF3B1, and SPRED1 as significantly mutated genes. ATRX and SF3B1 mutations occurred more commonly in lower anatomy melanomas and CTNNB1 in the upper anatomy. NF1, PTEN, CDKN2A, SPRED1, ATM, CHEK2, and ARID1B were commonly affected by chromosomal copy loss, while TERT, KIT, BRAF, YAP1, CDK4, CCND1, GAB2, MDM2, SKP2, and MITF were commonly amplified. Further notable genomic alterations occurring at lower frequencies indicated commonality of signaling networks in tumorigenesis, including MAPK, PI3K, Notch, Wnt/ß-catenin, cell cycle, DNA repair, and telomere maintenance pathways. This analysis identified genomic aberrations that provide some insight to the way in which specific pathways may be disrupted. IMPLICATIONS: Our analysis has shown that mucosal melanomas have a diverse range of genomic alterations in several biological pathways. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/6/991/F1.large.jpg.

Considerations for using population frequency data in germline variant interpretation: Cancer syndrome genes as a model.

Aggregate population genomics data from large cohorts are vital for assessing germline variant pathogenicity. However, there are no specifications on how sequencing quality metrics should be considered, and whether exome-derived and genome-derived allele frequencies should be considered in isolation. Germline genome sequence data were simulated for nine read-depths to identify a minimum acceptable read-depth for detecting variants. gnomAD exome-derived and genome-derived datasets were assessed for read-depth, for six key cancer genes selected for variant curation by ClinGen expert panels. Non-Finnish European allele frequency (AF) or filter AF of coding variants in these genes, assigned into frequency bins using modified ACMG-AMP criteria, was compared between exome-derived and genome-derived datasets. A 30X read-depth achieved acceptable precision and recall for detection of substitutions, but poor recall for small insertions/deletions. Exome-derived and genome-derived datasets exhibited low read-depth for different gene exons. Individual variants were mostly assigned to non-divergent AF bins (>95%) or filter AF bins (>97%). Two major bin divergences were resolved by applying the minimal acceptable read-depth threshold. These findings show the importance of assessing read-depth separately for population datasets sourced from different short-read sequencing technologies before assigning a frequency-based ACMG-AMP classification code for variant interpretation.

Tumour gene expression signature in primary melanoma predicts long-term outcomes.

Adjuvant systemic therapies are now routinely used following resection of stage III melanoma, however accurate prognostic information is needed to better stratify patients. We use differential expression analyses of primary tumours from 204 RNA-sequenced melanomas within a large adjuvant trial, identifying a 121 metastasis-associated gene signature. This signature strongly associated with progression-free (HR = 1.63, p = 5.24 × 10-5) and overall survival (HR = 1.61, p = 1.67 × 10-4), was validated in 175 regional lymph nodes metastasis as well as two externally ascertained datasets. The machine learning classification models trained using the signature genes performed significantly better in predicting metastases than models trained with clinical covariates (pAUROC = 7.03 × 10-4), or published prognostic signatures (pAUROC < 0.05). The signature score negatively correlated with measures of immune cell infiltration (ρ = -0.75, p < 2.2 × 10-16), with a higher score representing reduced lymphocyte infiltration and a higher 5-year risk of death in stage II melanoma. Our expression signature identifies melanoma patients at higher risk of metastases and warrants further evaluation in adjuvant clinical trials.