A Novel C-C Chemoattractant Cytokine (Chemokine) Receptor 6 (CCR6) Antagonist (PF-07054894) Distinguishes between Homologous Chemokine Receptors, Increases Basal Circulating CCR6+ T Cells, and Ameliorates Interleukin-23-Induced Skin Inflammation.

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.

Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs.

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies.

Biodistribution and Tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han Rats and Cynomolgus Macaques Reveal Different Toxicologic Profiles.

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Temporal trends in the incidence rates of keratinocyte carcinomas from 1978 to 2018 in Tasmania, Australia: a population-based study.

OBJECTIVES: We described incidence trends of keratinocyte carcinomas (KCs)-namely basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)-in the Australian state of Tasmania. METHODS: We identified histologically confirmed KCs within the Tasmanian Cancer Registry (TCR) and conducted assessments to ensure data quality. Age-standardised incidence rates were calculated for first (1985-2018) and annual KCs (1978-2018). Average annual percentage changes were computed using Joinpoint regression models. RESULTS: The TCR is a reliable source of KC data. A total of 83,536 people were registered with a KC between 1978 and 2018. Age-standardised incidence rates of first KCs increased on average by 3% per annum for BCCs and 4% per annum for SCCs, reaching 363/100,000 and 249/100,000 in 2018, respectively. Age-standardised incidence rates of annual KCs increased on average by 5% per annum for BCCs and 6% per annum for SCCs, up to 891/100,000 and 514/100,000 in 2018, respectively. This increase was steeper for females than males and highest during the late 1980s and early 1990s. A change in trend around 2014 suggested that incidence rates have started to decline. CONCLUSION: While the incidence of KCs in Tasmania increased substantially over 41 years, rates have recently plateaued and started to decline. The findings may reflect changes in sun exposure behaviours due to awareness campaigns, but high incidence rates in 2018 indicate that KCs still pose a substantial burden to this population.

Cardiac procedures in ST-segment-elevation myocardial infarction - the influence of age, geography and Aboriginality.

BACKGROUND: Timely restoration of bloodflow acute ST-segment elevation myocardial infarction (STEMI) reduces myocardial damage and improves prognosis. The objective of this study was describe the association of demographic factors with hospitalisation rates for STEMI and time to angiography, Percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Graft (CABG) in New South Wales (NSW) and the Australian Capital Territory (ACT), Australia. METHODS: This was an observational cohort study using linked population health data. We used linked records of NSW and the ACT hospitalisations and the Australian Government Medicare Benefits Schedule (MBS) for persons aged 35 and over hospitalised with STEMI in the period 1 July 2010 to 30 June 2014. Survival analysis was used to determine the time between STEMI admission and angiography, PCI and CABG, with a competing risk of death without cardiac procedure. RESULTS: Of 13,117 STEMI hospitalisations, 71% were among males; 55% were 65-plus years; 64% lived in major cities, and 2.6% were Aboriginal people. STEMI hospitalisation occurred at a younger age in males than females. Angiography and PCI rates decreased with age: angiography 69% vs 42% and PCI 60% vs 34% on day 0 for ages 35-44 and 75-plus respectively. Lower angiography and PCI rates and higher CABG rates were observed outside major cities. Aboriginal people with STEMI were younger and more likely to live outside a major city. Angiography, PCI and CABG rates were similar for Aboriginal and non-Aboriginal people of the same age and remoteness area. CONCLUSIONS: There is a need to improve access to definitive revascularisation for STEMI among appropriately selected older patients and in regional areas. Aboriginal people with STEMI, as a population, are disproportionately affected by access to definitive revascularisation outside major cities. Improving access to timely definitive revascularisation in regional areas may assist in closing the gap in cardiovascular outcomes between Aboriginal and non-Aboriginal people.

Genetic associations with clozapine-induced myocarditis in patients with schizophrenia.

Clozapine is the most effective antipsychotic drug for schizophrenia, yet it can cause life-threatening adverse drug reactions, including myocarditis. The aim of this study was to determine whether schizophrenia patients with clozapine-induced myocarditis have a genetic predisposition compared with clozapine-tolerant controls. We measured different types of genetic variation, including genome-wide single-nucleotide polymorphisms (SNPs), coding variants that alter protein expression, and variable forms of human leucocyte antigen (HLA) genes, alongside traditional clinical risk factors in 42 cases and 67 controls. We calculated a polygenic risk score (PRS) based on variation at 96 different genetic sites, to estimate the genetic liability to clozapine-induced myocarditis. Our genome-wide association analysis identified four SNPs suggestive of increased myocarditis risk (P < 1 × 10-6), with odds ratios ranging 5.5-13.7. The SNP with the lowest P value was rs74675399 (chr19p13.3, P = 1.21 × 10-7; OR = 6.36), located in the GNA15 gene, previously associated with heart failure. The HLA-C*07:01 allele was identified as potentially predisposing to clozapine-induced myocarditis (OR = 2.89, 95% CI: 1.11-7.53), consistent with a previous report of association of the same allele with clozapine-induced agranulocytosis. Another seven HLA alleles, including HLA-B*07:02 (OR = 0.25, 95% CI: 0.05-1.2) were found to be putatively protective. Long-read DNA sequencing provided increased resolution of HLA typing and validated the HLA associations. The PRS explained 66% of liability (P value = 9.7 × 10-5). Combining clinical and genetic factors together increased the proportion of variability accounted for (r2 0.73, P = 9.8 × 10-9). However, due to the limited sample size, individual genetic associations were not statistically significant after correction for multiple testing. We report novel candidate genetic associations with clozapine-induced myocarditis, which may have potential clinical utility, but larger cohorts are required for replication.

A comparison of immunoglobulin IGHV, IGHD and IGHJ genes in wild-derived and classical inbred mouse strains.

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflects differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ) and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ and PWD/PhJ) and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the international ImMunoGeneTics information system. By contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. By contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand interstrain variation in models of antibody-mediated disease.

Single-cell profiling of peanut-responsive T cells in patients with peanut allergy reveals heterogeneous effector TH2 subsets.

BACKGROUND: The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood. OBJECTIVES: Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of subjects with and without peanut allergy (PA). METHODS: We obtained samples from patients with PA, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (Consortium of Food Allergy Research [CoFAR] 6). Subjects were confirmed as having PA, or if they passed a 1-g peanut challenge, they were termed high-threshold subjects. Healthy control (HC) subjects were also recruited. Peanut-responsive T cells were identified based on CD154 expression after 6 to 18 hours of stimulation with peanut extract. Cells were analyzed by using flow cytometry and single-cell RNA sequencing. RESULTS: Patients with PA had tissue- and follicle-homing peanut-responsive CD4+ T cells with a heterogeneous pattern of TH2 differentiation, whereas control subjects had undetectable T-cell responses to peanut. The PA group had a delayed and IL-2-dependent upregulation of CD154 on cells expressing regulatory T (Treg) cell markers, which was absent in HC or high-threshold subjects. Depletion of Treg cells enhanced cytokine production in HC subjects and patients with PA in vitro, but cytokines associated with highly differentiated TH2 cells were more resistant to Treg cell suppression in patients with PA. Analysis of gene expression by means of single-cell RNA sequencing identified T cells with highly correlated expression of IL4, IL5, IL9, IL13, and the IL-25 receptor IL17RB. CONCLUSIONS: These results demonstrate the presence of highly differentiated TH2 cells producing TH2-associated cytokines with functions beyond IgE class-switching in patients with PA. A multifunctional TH2 response was more evident than a Treg cell deficit among peanut-responsive T cells.

Identification of a novel RASD1 somatic mutation in a USP8-mutated corticotroph adenoma.

Cushing's disease (CD) is caused by pituitary corticotroph adenomas that secrete excess adrenocorticotropic hormone (ACTH). In these tumors, somatic mutations in the gene USP8 have been identified as recurrent and pathogenic and are the sole known molecular driver for CD. Although other somatic mutations were reported in these studies, their contribution to the pathogenesis of CD remains unexplored. No molecular drivers have been established for a large proportion of CD cases and tumor heterogeneity has not yet been investigated using genomics methods. Also, even in USP8-mutant tumors, a possibility may exist of additional contributing mutations, following a paradigm from other neoplasm types where multiple somatic alterations contribute to neoplastic transformation. The current study utilizes whole-exome discovery sequencing on the Illumina platform, followed by targeted amplicon-validation sequencing on the Pacific Biosciences platform, to interrogate the somatic mutation landscape in a corticotroph adenoma resected from a CD patient. In this USP8-mutated tumor, we identified an interesting somatic mutation in the gene RASD1, which is a component of the corticotropin-releasing hormone receptor signaling system. This finding may provide insight into a novel mechanism involving loss of feedback control to the corticotropin-releasing hormone receptor and subsequent deregulation of ACTH production in corticotroph tumors.

Genomic profiling reveals mutational landscape in parathyroid carcinomas.

Parathyroid carcinoma (PC) is an extremely rare malignancy lacking effective therapeutic intervention. We generated and analyzed whole-exome sequencing data from 17 patients to identify somatic and germline genetic alterations. A panel of selected genes was sequenced in a 7-tumor expansion cohort. We show that 47% (8 of 17) of the tumors harbor somatic mutations in the CDC73 tumor suppressor, with germline inactivating variants in 4 of the 8 patients. The PI3K/AKT/mTOR pathway was altered in 21% of the 24 cases, revealing a major oncogenic pathway in PC. We observed CCND1 amplification in 29% of the 17 patients, and a previously unreported recurrent mutation in putative kinase ADCK1. We identified the first sporadic PCs with somatic mutations in the Wnt canonical pathway, complementing previously described epigenetic mechanisms mediating Wnt activation. This is the largest genomic sequencing study of PC, and represents major progress toward a full molecular characterization of this rare malignancy to inform improved and individualized treatments.

Mesothelioma trends in the ACT and comparisons with the rest of Australia.

OBJECTIVES: Inhalation of asbestos fibres is the predominant cause of malignant mesothelioma. Domestic exposure to asbestos is a major community concern in the Australian Capital Territory (ACT) because of loose-fill asbestos home insulation. Little is known about how trends in mesothelioma rates in the ACT compare with those elsewhere. The objective of this study was to describe trends in mesothelioma rates in the ACT and compare them with those for the rest of Australia. METHODS: We used de-identified data from the ACT Cancer Registry (1982- 2014), and the Western Australia (WA) Cancer Registry and the Australian Cancer Database (1982-2011). We calculated crude mesothelioma rates, by 3-year periods, for the ACT and for the rest of Australia (excluding WA). We used Poisson regression to analyse mesothelioma trends from 1994 to 2011 (complete reporting period) using an indirect standardisation approach to adjust for age and sex. RESULTS: There were 140 mesothelioma cases reported to the ACT Cancer Registry between 1982 and 2014 - 81% male and 19% female. Between 1994 and 2011, age- and sex-adjusted mesothelioma rates in the ACT increased over time, on average by 12% per 3-year period (relative risk [RR] 1.12; 95% confidence interval [CI] 0.99, 1.26). Compared with the rest of Australia (excluding WA), ACT rates were, on average, lower (RR 0.84; 95% CI 0.69, 1.02), but they increased at a higher rate (RR 1.12 per 3-year period; 95% CI 0.99, 1.27). These results are strongly influenced by the higher rate of mesothelioma observed in the ACT in 2009-2011, when ACT rates became similar to those for the rest of Australia (excluding WA). CONCLUSIONS: Although mesothelioma rates may have increased more in the ACT than the rest of Australia (excluding WA) during the past two decades, there is considerable uncertainty in the trends. More information is needed regarding the health risks associated with living in a house with loose-fill asbestos insulation. This is the subject of further studies within the ACT Asbestos Health Study.

Development and clinical application of an integrative genomic approach to personalized cancer therapy.

BACKGROUND: Personalized therapy provides the best outcome of cancer care and its implementation in the clinic has been greatly facilitated by recent convergence of enormous progress in basic cancer research, rapid advancement of new tumor profiling technologies, and an expanding compendium of targeted cancer therapeutics. METHODS: We developed a personalized cancer therapy (PCT) program in a clinical setting, using an integrative genomics approach to fully characterize the complexity of each tumor. We carried out whole exome sequencing (WES) and single-nucleotide polymorphism (SNP) microarray genotyping on DNA from tumor and patient-matched normal specimens, as well as RNA sequencing (RNA-Seq) on available frozen specimens, to identify somatic (tumor-specific) mutations, copy number alterations (CNAs), gene expression changes, gene fusions, and also germline variants. To provide high sensitivity in known cancer mutation hotspots, Ion AmpliSeq Cancer Hotspot Panel v2 (CHPv2) was also employed. We integrated the resulting data with cancer knowledge bases and developed a specific workflow for each cancer type to improve interpretation of genomic data. RESULTS: We returned genomics findings to 46 patients and their physicians describing somatic alterations and predicting drug response, toxicity, and prognosis. Mean 17.3 cancer-relevant somatic mutations per patient were identified, 13.3-fold, 6.9-fold, and 4.7-fold more than could have been detected using CHPv2, Oncomine Cancer Panel (OCP), and FoundationOne, respectively. Our approach delineated the underlying genetic drivers at the pathway level and provided meaningful predictions of therapeutic efficacy and toxicity. Actionable alterations were found in 91 % of patients (mean 4.9 per patient, including somatic mutations, copy number alterations, gene expression alterations, and germline variants), a 7.5-fold, 2.0-fold, and 1.9-fold increase over what could have been uncovered by CHPv2, OCP, and FoundationOne, respectively. The findings altered the course of treatment in four cases. CONCLUSIONS: These results show that a comprehensive, integrative genomic approach as outlined above significantly enhanced genomics-based PCT strategies.

Whole-genome sequencing identifies emergence of a quinolone resistance mutation in a case of Stenotrophomonas maltophilia bacteremia.

Whole-genome sequences for Stenotrophomonas maltophilia serial isolates from a bacteremic patient before and after development of levofloxacin resistance were assembled de novo and differed by one single-nucleotide variant in smeT, a repressor for multidrug efflux operon smeDEF. Along with sequenced isolates from five contemporaneous cases, they displayed considerable diversity compared against all published complete genomes. Whole-genome sequencing and complete assembly can conclusively identify resistance mechanisms emerging in S. maltophilia strains during clinical therapy.

The relationship between basal and squamous cell skin cancer and smoking related cancers.

BACKGROUND: We compared the risk of being diagnosed with smoking-related cancers (lung, oral cavity, upper digestive and respiratory organs, bladder, kidney, anogenital cancers and myeloid leukaemia) among people with squamous cell carcinoma (SCC) or basal cell carcinoma of the skin (BCC), with risks found in the general population using data from an Australian population-based cancer registry. METHODS: People diagnosed with BCC or SCC in 1980-2003 reported to the Tasmanian Cancer Registry, Australia, were followed-up by linkage within the registry, until diagnosis of a subsequent smoking-related cancer, death, or until 31 December 2003. Risk of developing a future smoking-related cancer was assessed using age Standardised Incidence Ratios (SIR). RESULTS: People diagnosed with SCC had an increased risk of lung cancer (men: SIR = 1.89, 95% confidence interval: 1.61-2.21; women: SIR = 2.04, 1.42-2.83) and all other smoking-related cancers (men: SIR = 1.38, 1.19-1.60; women: SIR = 1.78, 1.34-2.33). Men with BCC had a significant increased risk of lung cancer (SIR = 1.26, 1.10-1.44) but not of any of the other smoking-related cancers (SIR = 1.09, 0.97-1.23). CONCLUSIONS: Individuals with a history of SCC having an increased risk of developing smoking related cancers cancer suggests smoking as a common etiology. The relationship between BCC and smoking-related cancers is less certain.

Australia's notifiable disease status, 2008: annual report of the National Notifiable Diseases Surveillance System.

In 2008, 65 communicable diseases and conditions were nationally notifiable in Australia. States and territories reported a total of 160,508 notifications of communicable diseases to the National Notifiable Diseases Surveillance System, an increase of 9% on the number of notifications in 2007. In 2008, the most frequently notified diseases were sexually transmissible infections (69,459 notifications, 43% of total notifications), vaccine preventable diseases (34,225 notifications, 21% of total notifications) and gastrointestinal diseases (27,308 notifications, 17% of total notifications). There were 18,207 notifications of bloodborne diseases; 8,876 notifications of vectorborne diseases; 1,796 notifications of other bacterial infections; 633 notifications of zoonoses and 4 notifications of quarantinable diseases.