Deep whole-genome ctDNA chronology of treatment-resistant prostate cancer.

Circulating tumour DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring1. However, owing to past emphasis on targeted and low-resolution profiling approaches, our understanding of the distinct populations that comprise bulk ctDNA is incomplete2-12. Here we perform deep whole-genome sequencing of serial plasma and synchronous metastases in patients with aggressive prostate cancer. We comprehensively assess all classes of genomic alterations and show that ctDNA contains multiple dominant populations, the evolutionary histories of which frequently indicate whole-genome doubling and shifts in mutational processes. Although tissue and ctDNA showed concordant clonally expanded cancer driver alterations, most individual metastases contributed only a minor share of total ctDNA. By comparing serial ctDNA before and after clinical progression on potent inhibitors of the androgen receptor (AR) pathway, we reveal population restructuring converging solely on AR augmentation as the dominant genomic driver of acquired treatment resistance. Finally, we leverage nucleosome footprints in ctDNA to infer mRNA expression in synchronously biopsied metastases, including treatment-induced changes in AR transcription factor signalling activity. Our results provide insights into cancer biology and show that liquid biopsy can be used as a tool for comprehensive multi-omic discovery.

Empagliflozin in Patients with Chronic Kidney Disease.

BACKGROUND: The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. METHODS: We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m2 of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m2 with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to <10 ml per minute per 1.73 m2, a sustained decrease in eGFR of ≥40% from baseline, or death from renal causes) or death from cardiovascular causes. RESULTS: A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P<0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P = 0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. CONCLUSIONS: Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo. (Funded by Boehringer Ingelheim and others; EMPA-KIDNEY ClinicalTrials.gov number, NCT03594110; EudraCT number, 2017-002971-24.).

Flt3 agonist enhances immunogenicity of arenavirus vector-based simian immunodeficiency virus vaccine in macaques.

Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens are capable of inducing efficacious humoral and cellular immune responses in nonhuman primates. Several studies have evaluated the use of immune modulators to further enhance vaccine-induced T-cell responses. The hematopoietic growth factor Flt3L drives the expansion of various bone marrow progenitor populations, and administration of Flt3L was shown to promote expansion of dendritic cell populations in spleen and blood, which are targets of arenaviral vectors. Therefore, we evaluated the potential of Flt3 signaling to enhance the immunogenicity of arenaviral vaccines encoding SIV immunogens (SIVSME543 Gag, Env, and Pol) in rhesus macaques, with a rhesus-specific engineered Flt3L-Fc fusion protein. In healthy animals, administration of Flt3L-Fc led to a 10- to 100-fold increase in type 1 dendritic cells 7 days after dosing, with no antidrug antibody (ADA) generation after repeated dosing. We observed that administration of Flt3L-Fc fusion protein 7 days before arenaviral vaccine increased the frequency and activation of innate immune cells and enhanced T-cell activation with no treatment-related adverse events. Flt3L-Fc administration induced early innate immune activation, leading to a significant enhancement in magnitude, breadth, and polyfunctionality of vaccine-induced T-cell responses. The Flt3L-Fc enhancement in vaccine immunogenicity was comparable to a combination with αCTLA-4 and supports the use of safe and effective variants of Flt3L to augment therapeutic vaccine-induced T-cell responses.IMPORTANCEInduction of a robust human immunodeficiency virus (HIV)-specific CD4+ and CD8+ T-cell response through therapeutic vaccination is considered essential for HIV cure. Arenaviral vaccine vectors encoding simian immunodeficiency virus (SIV) immunogens have demonstrated strong immunogenicity and efficacy in nonhuman primates. Here, we demonstrate that the immunogenicity of arenaviral vectors encoding SIV immunogens can be enhanced by administration of Flt3L-Fc fusion protein 7 days before vaccination. Flt3L-Fc-mediated increase in dendritic cells led to robust improvements in vaccine-induced T- and B-cell responses compared with vaccine alone, and Flt3L-Fc dosing was not associated with any treatment-related adverse events. Importantly, immune modulation by either Flt3L-Fc or αCTLA-4 led to comparable enhancement in vaccine response. These results indicate that the addition of Flt3L-Fc fusion protein before vaccine administration can significantly enhance vaccine immunogenicity. Thus, safe and effective Flt3L variants could be utilized as part of a combination therapy for HIV cure.

Correlates of chronic hepatitis B virus infection in the general adult population of China: Systematic review and meta-analysis.

Chronic infection with hepatitis B virus (HBV) is a significant public health issue in China. Understanding factors associated with chronic HBV is important to enable targeted screening and education and to improve early diagnosis and prevention of disease progression. This systematic review and meta-analysis aimed to identify and describe correlates of chronic HBV among Chinese adults. Searches were conducted in MEDLINE, EMBASE and grey literature up to 25 June 2020. Eligible papers included observational studies in adults of the general population in China that reported factors associated with chronic HBV, measured by Hepatitis B surface antigen (HBsAg). Meta-analysis was performed using fixed-effect models of HBsAg prevalence among factors, and of adjusted odds ratios (ORs) for chronic HBV associated with each factor. Overall 39 articles were included, covering 22 factors, including a range of sociodemographic, behavioural and medical factors. In meta-analysis of eligible studies, a range of factors were significantly associated with higher HBsAg prevalence, including middle age, male sex, being married, rural residence, lower education, smoking, having a HBsAg positive household contact, family history of HBV, history of surgery or blood transfusion. The adjusted ORs varied, from 1.11 (95% CI 1.05-1.18) for smoking to 5.13 (95% CI 4.99-5.26) for having a HBsAg positive household contact. In Chinese adults, a range of factors are associated with chronic HBV infection, which may help inform targeted screening in the general population.

Spatial profiling of gastric cancer patient-matched primary and locoregional metastases reveals principles of tumour dissemination.

OBJECTIVE: Endoscopic mucosal biopsies of primary gastric cancers (GCs) are used to guide diagnosis, biomarker testing and treatment. Spatial intratumoural heterogeneity (ITH) may influence biopsy-derived information. We aimed to study ITH of primary GCs and matched lymph node metastasis (LNmet). DESIGN: GC resection samples were annotated to identify primary tumour superficial (PTsup), primary tumour deep (PTdeep) and LNmet subregions. For each subregion, we determined (1) transcriptomic profiles (NanoString 'PanCancer Progression Panel', 770 genes); (2) next-generation sequencing (NGS, 225 gastrointestinal cancer-related genes); (3) DNA copy number profiles by multiplex ligation-dependent probe amplification (MLPA, 16 genes); and (4) histomorphological phenotypes. RESULTS: NanoString profiling of 64 GCs revealed no differences between PTsup1 and PTsup2, while 43% of genes were differentially expressed between PTsup versus PTdeep and 38% in PTsup versus LNmet. Only 16% of genes were differently expressed between PTdeep and LNmet. Several genes with therapeutic potential (eg IGF1, PIK3CD and TGFB1) were overexpressed in LNmet and PTdeep compared with PTsup. NGS data revealed orthogonal support of NanoString results with 40% mutations present in PTdeep and/or LNmet, but not in PTsup. Conversely, only 6% of mutations were present in PTsup and were absent in PTdeep and LNmet. MLPA demonstrated significant ITH between subregions and progressive genomic changes from PTsup to PTdeep/LNmet. CONCLUSION: In GC, regional lymph node metastases are likely to originate from deeper subregions of the primary tumour. Future clinical trials of novel targeted therapies must consider assessment of deeper subregions of the primary tumour and/or metastases as several therapeutically relevant genes are only mutated, overexpressed or amplified in these regions.

Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome.

N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype.

PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.

Exome sequencing as a tool for Mendelian disease gene discovery.

Exome sequencing - the targeted sequencing of the subset of the human genome that is protein coding - is a powerful and cost-effective new tool for dissecting the genetic basis of diseases and traits that have proved to be intractable to conventional gene-discovery strategies. Over the past 2 years, experimental and analytical approaches relating to exome sequencing have established a rich framework for discovering the genes underlying unsolved Mendelian disorders. Additionally, exome sequencing is being adapted to explore the extent to which rare alleles explain the heritability of complex diseases and health-related traits. These advances also set the stage for applying exome and whole-genome sequencing to facilitate clinical diagnosis and personalized disease-risk profiling.

The Effect of Response Bias on the Personality Inventory for DSM-5 (PID-5).

Valid self-report assessment of psychopathology relies on accurate and credible responses to test questions. There are some individuals who, in certain assessment contexts, cannot or choose not to answer in a manner typically representative of their traits or symptoms. This is referred to, most broadly, as test response bias. In this investigation, we explore the effect of response bias on the Personality Inventory for DSM-5 (PID-5; Krueger, Derringer, Markon, Watson, & Skodol, 2013 ), a self-report instrument designed to assess the pathological personality traits used to inform diagnosis of the personality disorders in Section III of DSM-5. A set of Minnesota Multiphasic Personality Inventory Restructured Form (MMPI-2-RF; Ben-Porath & Tellegen, 2008 / 2011 ) validity scales, which are used to assess and identify response bias, were employed to identify individuals who engaged in either noncredible overreporting (OR) or underreporting (UR), or who were deemed to be reporting or responding to the items in a "credible" manner-credible responding (CR). A total of 2,022 research participants (1,587 students, 435 psychiatric patients) completed the MMPI-2-RF and PID-5; following protocol screening, these participants were classified into OR, UR, or CR response groups based on MMPI-2-RF validity scale scores. Groups of students and patients in the OR group scored significantly higher on the PID-5 than those students and patients in the CR group, whereas those in the UR group scored significantly lower than those in the CR group. Although future research is needed to explore the effects of response bias on the PID-5, results from this investigation provide initial evidence suggesting that response bias influences scale elevations on this instrument.

Haploinsufficiency of SF3B4, a component of the pre-mRNA spliceosomal complex, causes Nager syndrome.

Nager syndrome, first described more than 60 years ago, is the archetype of a class of disorders called the acrofacial dysostoses, which are characterized by craniofacial and limb malformations. Despite intensive efforts, no gene for Nager syndrome has yet been identified. In an international collaboration, FORGE Canada and the National Institutes of Health Centers for Mendelian Genomics used exome sequencing as a discovery tool and found that mutations in SF3B4, a component of the U2 pre-mRNA spliceosomal complex, cause Nager syndrome. After Sanger sequencing of SF3B4 in a validation cohort, 20 of 35 (57%) families affected by Nager syndrome had 1 of 18 different mutations, nearly all of which were frameshifts. These results suggest that most cases of Nager syndrome are caused by haploinsufficiency of SF3B4. Our findings add Nager syndrome to a growing list of disorders caused by mutations in genes that encode major components of the spliceosome and also highlight the synergistic potential of international collaboration when exome sequencing is applied in the search for genes responsible for rare Mendelian phenotypes.

Targeted capture and massively parallel sequencing of 12 human exomes.

Genome-wide association studies suggest that common genetic variants explain only a modest fraction of heritable risk for common diseases, raising the question of whether rare variants account for a significant fraction of unexplained heritability. Although DNA sequencing costs have fallen markedly, they remain far from what is necessary for rare and novel variants to be routinely identified at a genome-wide scale in large cohorts. We have therefore sought to develop second-generation methods for targeted sequencing of all protein-coding regions ('exomes'), to reduce costs while enriching for discovery of highly penetrant variants. Here we report on the targeted capture and massively parallel sequencing of the exomes of 12 humans. These include eight HapMap individuals representing three populations, and four unrelated individuals with a rare dominantly inherited disorder, Freeman-Sheldon syndrome (FSS). We demonstrate the sensitive and specific identification of rare and common variants in over 300 megabases of coding sequence. Using FSS as a proof-of-concept, we show that candidate genes for Mendelian disorders can be identified by exome sequencing of a small number of unrelated, affected individuals. This strategy may be extendable to diseases with more complex genetics through larger sample sizes and appropriate weighting of non-synonymous variants by predicted functional impact.

Trans genomic capture and sequencing of primate exomes reveals new targets of positive selection.

Comparison of protein-coding DNA sequences from diverse primates can provide insight into these species' evolutionary history and uncover the molecular basis for their phenotypic differences. Currently, the number of available primate reference genomes limits these genome-wide comparisons. Here we use targeted capture methods designed for human to sequence the protein-coding regions, or exomes, of four non-human primate species (three Old World monkeys and one New World monkey). Despite average sequence divergence of up to 4% from the human sequence probes, we are able to capture ~96% of coding sequences. Using a combination of mapping and assembly techniques, we generated high-quality full-length coding sequences for each species. Both the number of nucleotide differences and the distribution of insertion and deletion (indel) lengths indicate that the quality of the assembled sequences is very high and exceeds that of most reference genomes. Using this expanded set of primate coding sequences, we performed a genome-wide scan for genes experiencing positive selection and identified a novel class of adaptively evolving genes involved in the conversion of epithelial cells in skin, hair, and nails to keratin. Interestingly, the genes we identify under positive selection also exhibit significantly increased allele frequency differences among human populations, suggesting that they play a role in both recent and long-term adaptation. We also identify several genes that have been lost on specific primate lineages, which illustrate the broad utility of this data set for other evolutionary analyses. These results demonstrate the power of second-generation sequencing in comparative genomics and greatly expand the repertoire of available primate coding sequences.

Exome sequencing identifies a spectrum of mutation frequencies in advanced and lethal prostate cancers.

To catalog protein-altering mutations that may drive the development of prostate cancers and their progression to metastatic disease systematically, we performed whole-exome sequencing of 23 prostate cancers derived from 16 different lethal metastatic tumors and three high-grade primary carcinomas. All tumors were propagated in mice as xenografts, designated the LuCaP series, to model phenotypic variation, such as responses to cancer-directed therapeutics. Although corresponding normal tissue was not available for most tumors, we were able to take advantage of increasingly deep catalogs of human genetic variation to remove most germline variants. On average, each tumor genome contained ~200 novel nonsynonymous variants, of which the vast majority was specific to individual carcinomas. A subset of genes was recurrently altered across tumors derived from different individuals, including TP53, DLK2, GPC6, and SDF4. Unexpectedly, three prostate cancer genomes exhibited substantially higher mutation frequencies, with 2,000-4,000 novel coding variants per exome. A comparison of castration-resistant and castration-sensitive pairs of tumor lines derived from the same prostate cancer highlights mutations in the Wnt pathway as potentially contributing to the development of castration resistance. Collectively, our results indicate that point mutations arising in coding regions of advanced prostate cancers are common but, with notable exceptions, very few genes are mutated in a substantial fraction of tumors. We also report a previously undescribed subtype of prostate cancers exhibiting "hypermutated" genomes, with potential implications for resistance to cancer therapeutics. Our results also suggest that increasingly deep catalogs of human germline variation may challenge the necessity of sequencing matched tumor-normal pairs.

Cocaine and amphetamine regulated transcript (CART) mediates sex differences in binge drinking through central taste circuits.

The neuropeptide cocaine- and amphetamine-regulated transcript (CART) has been implicated in alcohol consumption and reward behaviours, yet mechanisms mediating these effects have yet to be identified. Using a transgenic CART knockout (KO) mouse line we uncovered a sexually dimorphic effect of CART in binge drinking, with male CART KO mice increasing intake, whilst female CART KO mice decreased their alcohol intake compared to controls. Female CART KO mice show greater sensitivity to bitter solutions that can be overshadowed through addition of a sweetener, implicating taste as a factor. Further we identify that this is not driven through peripherally circulating sex hormones, but the central nucleus of the amygdala (CeA) is a locus where CART contributes to the regulation of alcohol consumption, with CeA CART neutralisation specifically reducing plain alcohol, but not sweetened alcohol consumption in female mice. These findings may have implications for the development of sex-specific treatment options for alcohol use disorders through targeting the CART system.

Multiregion sampling of de novo metastatic prostate cancer reveals complex polyclonality and augments clinical genotyping.

De novo metastatic prostate cancer is highly aggressive, but the paucity of routinely collected tissue has hindered genomic stratification and precision oncology. Here, we leveraged a rare study of surgical intervention in 43 de novo metastatic prostate cancers to assess somatic genotypes across 607 synchronous primary and metastatic tissue regions plus circulating tumor DNA. Intra-prostate heterogeneity was pervasive and impacted clinically relevant genes, resulting in discordant genotypes between select primary restricted regions and synchronous metastases. Additional complexity was driven by polyclonal metastatic seeding from phylogenetically related primary populations. When simulating clinical practice relying on a single tissue region, genomic heterogeneity plus variable tumor fraction across samples caused inaccurate genotyping of dominant disease; however, pooling extracted DNA from multiple biopsy cores before sequencing can rescue misassigned somatic genotypes. Our results define the relationship between synchronous treatment-sensitive primary and metastatic lesions in men with de novo metastatic prostate cancer and provide a framework for implementing genomics-guided patient management.

Prediction of plasma ctDNA fraction and prognostic implications of liquid biopsy in advanced prostate cancer.

No consensus strategies exist for prognosticating metastatic castration-resistant prostate cancer (mCRPC). Circulating tumor DNA fraction (ctDNA%) is increasingly reported by commercial and laboratory tests but its utility for risk stratification is unclear. Here, we intersect ctDNA%, treatment outcomes, and clinical characteristics across 738 plasma samples from 491 male mCRPC patients from two randomized multicentre phase II trials and a prospective province-wide blood biobanking program. ctDNA% correlates with serum and radiographic metrics of disease burden and is highest in patients with liver metastases. ctDNA% strongly predicts overall survival, progression-free survival, and treatment response independent of therapeutic context and outperformed established prognostic clinical factors. Recognizing that ctDNA-based biomarker genotyping is limited by low ctDNA% in some patients, we leverage the relationship between clinical prognostic factors and ctDNA% to develop a clinically-interpretable machine-learning tool that predicts whether a patient has sufficient ctDNA% for informative ctDNA genotyping (available online: https://www.ctDNA.org ). Our results affirm ctDNA% as an actionable tool for patient risk stratification and provide a practical framework for optimized biomarker testing.

Neuroanatomical distribution of fluorophores within adult RXFP3 Cre-tdTomato/YFP mouse brain.

Relaxin-family peptide 3 receptor (RXFP3) is activated by relaxin-3 in the brain to influence arousal and related functions, such as feeding and stress responses. Two transgenic mouse lines have recently been developed that co-express different fluorophores within RXFP3-expressing neurons: either yellow fluorescent protein (YFP; RXFP3-Cre/YFP mice) or tdTomato (RXFP3-Cre/tdTomato mice). To date, the characteristics of neurons that express RXFP3-associated fluorophores in these mice have only been investigated in the bed nucleus of the stria terminalis and the hypothalamic arcuate nucleus. To better determine the utility of these fluorophore-expressing mice for further research, we characterised the neuroanatomical distribution of fluorophores throughout the brain of these mice and compared this to the published distribution of Rxfp3 mRNA (detected by in situ hybridisation) in wildtype mice. Coronal sections of RXFP3-Cre/YFP (n = 8) and RXFP3-Cre/tdTomato (n = 8) mouse brains were imaged, and the density of fluorophore-expressing cells within various brain regions/nuclei was qualitatively assessed. Comparisons with our previously reported RXFP3 mRNA distribution revealed that of 212 brain regions that contained either fluorophore or RXFP3 mRNA, approximately half recorded densities that were within two qualitative measurements of each other (on a 9-point scale), including hippocampal dentate gyrus and amygdala subregions. However, many brain areas with likely non-authentic, false-positive, or false-negative fluorophore expression were also detected, including the cerebellum. Therefore, this study provides a guide to which brain regions should be prioritized for future study of RXFP3 in these mice, to better understand the neuroanatomy and function of this intriguing, neuronal peptide receptor.