Immune Activation following Irbesartan Treatment in a Colorectal Cancer Patient: A Case Study.

Colorectal cancers are one of the most prevalent tumour types worldwide and, despite the emergence of targeted and biologic therapies, have among the highest mortality rates. The Personalized OncoGenomics (POG) program at BC Cancer performs whole genome and transcriptome analysis (WGTA) to identify specific alterations in an individual's cancer that may be most effectively targeted. Informed using WGTA, a patient with advanced mismatch repair-deficient colorectal cancer was treated with the antihypertensive drug irbesartan and experienced a profound and durable response. We describe the subsequent relapse of this patient and potential mechanisms of response using WGTA and multiplex immunohistochemistry (m-IHC) profiling of biopsies before and after treatment from the same metastatic site of the L3 spine. We did not observe marked differences in the genomic landscape before and after treatment. Analyses revealed an increase in immune signalling and infiltrating immune cells, particularly CD8+ T cells, in the relapsed tumour. These results indicate that the observed anti-tumour response to irbesartan may have been due to an activated immune response. Determining whether there may be other cancer contexts in which irbesartan may be similarly valuable will require additional studies.

Whole-genome and transcriptome analysis enhances precision cancer treatment options.

BACKGROUND: Recent advances are enabling delivery of precision genomic medicine to cancer clinics. While the majority of approaches profile panels of selected genes or hotspot regions, comprehensive data provided by whole-genome and transcriptome sequencing and analysis (WGTA) present an opportunity to align a much larger proportion of patients to therapies. PATIENTS AND METHODS: Samples from 570 patients with advanced or metastatic cancer of diverse types enrolled in the Personalized OncoGenomics (POG) program underwent WGTA. DNA-based data, including mutations, copy number and mutation signatures, were combined with RNA-based data, including gene expression and fusions, to generate comprehensive WGTA profiles. A multidisciplinary molecular tumour board used WGTA profiles to identify and prioritize clinically actionable alterations and inform therapy. Patient responses to WGTA-informed therapies were collected. RESULTS: Clinically actionable targets were identified for 83% of patients, of which 37% of patients received WGTA-informed treatments. RNA expression data were particularly informative, contributing to 67% of WGTA-informed treatments; 25% of treatments were informed by RNA expression alone. Of a total 248 WGTA-informed treatments, 46% resulted in clinical benefit. RNA expression data were comparable to DNA-based mutation and copy number data in aligning to clinically beneficial treatments. Genome signatures also guided therapeutics including platinum, poly-ADP ribose polymerase inhibitors and immunotherapies. Patients accessed WGTA-informed treatments through clinical trials (19%), off-label use (35%) and as standard therapies (46%) including those which would not otherwise have been the next choice of therapy, demonstrating the utility of genomic information to direct use of chemotherapies as well as targeted therapies. CONCLUSIONS: Integrating RNA expression and genome data illuminated treatment options that resulted in 46% of treated patients experiencing positive clinical benefit, supporting the use of comprehensive WGTA profiling in clinical cancer care.

Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer.

BACKGROUND: NRG1 fusion-positive lung cancers have emerged as potentially actionable events in lung cancer, but clinical support is currently limited and no evidence of efficacy of this approach in cancers beyond lung has been shown. PATIENTS AND METHODS: Here, we describe two patients with advanced cancers refractory to standard therapies. Patient 1 had lung adenocarcinoma and patient 2 cholangiocarcinoma. Whole-genome and transcriptome sequencing were carried out for these cases with select findings validated by fluorescence in situ hybridization. RESULTS: Both tumors were found to be positive for NRG1 gene fusions. In patient 1, an SDC4-NRG1 gene fusion was detected, similar gene fusions having been described in lung cancers previously. In patient 2, a novel ATP1B1-NRG1 gene fusion was detected. Cholangiocarcinoma is not a disease type in which NRG1 fusions had been described previously. Integrative genome analysis was used to assess the potential functional significance of the detected genomic events including the gene fusions, prioritizing therapeutic strategies targeting the HER-family of growth factor receptors. Both patients were treated with the pan HER-family kinase inhibitor afatinib and both displayed significant and durable response to treatment. Upon progression sites of disease were sequenced. The lack of obvious genomic events to describe the disease progression indicated that broad transcriptomic or epigenetic mechanisms could be attributed to the lack of prolonged response to afatinib. CONCLUSION: These observations lend further support to the use of pan HER-tyrosine kinase inhibitors for the treatment of NRG1 fusion-positive in both cancers of lung and hepatocellular origin and indicate more broadly that cancers found to be NRG1 fusion-positive may benefit from such a clinical approach regardless of their site of origin. CLINICAL TRIAL INFORMATION: Personalized Oncogenomics (POG) Program of British Columbia: Utilization of Genomic Analysis to Better Understand Tumour Heterogeneity and Evolution (NCT02155621).

Air pollution, deprivation and health: understanding relationships to add value to local air quality management policy and practice in Wales, UK.

Background: Air pollution exposure reduces life expectancy. Air pollution, deprivation and poor-health status combinations can create increased and disproportionate disease burdens. Problems and solutions are rarely considered in a broad public health context, but doing so can add value to air quality management efforts by reducing air pollution risks, impacts and inequalities. Methods: An ecological study assessed small-area associations between air pollution (nitrogen dioxide and particulate matter), deprivation status and health outcomes in Wales, UK. Results: Air pollution concentrations were highest in 'most' deprived areas. When considered separately, deprivation-health associations were stronger than air pollution-health associations. Considered simultaneously, air pollution added to deprivation-health associations; interactions between air pollution and deprivation modified and strengthened associations with all-cause and respiratory disease mortality, especially in 'most' deprived areas where most-vulnerable people lived and where health needs were greatest. Conclusion: There is a need to reduce air pollution-related risks for all. However, it is also the case that greater health gains can result from considering local air pollution problems and solutions in the context of wider health-determinants and acting on a better understanding of relationships. Informed and co-ordinated air pollution mitigation and public health action in high deprivation and pollution areas can reduce risks and inequalities. To achieve this, greater public health integration and collaboration in local air quality management policy and practice is needed.

Response to angiotensin blockade with irbesartan in a patient with metastatic colorectal cancer.

BACKGROUND: A patient suffering from metastatic colorectal cancer, treatment-related toxicity and resistance to standard chemotherapy and radiation was assessed as part of a personalized oncogenomics initiative to derive potential alternative therapeutic strategies. PATIENTS AND METHODS: Whole-genome and transcriptome sequencing was used to interrogate a metastatic tumor refractory to standard treatments of a patient with mismatch repair-deficient metastatic colorectal cancer. RESULTS: Integrative genomic analysis indicated overexpression of the AP-1 transcriptional complex suggesting experimental therapeutic rationales, including blockade of the renin-angiotensin system. This led to the repurposing of the angiotensin II receptor antagonist, irbesartan, as an anticancer therapy, resulting in the patient experiencing a dramatic and durable response. CONCLUSIONS: This case highlights the utility of comprehensive integrative genomic profiling and bioinformatics analysis to provide hypothetical rationales for personalized treatment options.

Personalized oncogenomics in the management of gastrointestinal carcinomas-early experiences from a pilot study.

BACKGROUND: Gastrointestinal carcinomas are genomically complex cancers that are lethal in the metastatic setting. Whole-genome and transcriptome sequencing allow for the simultaneous characterization of multiple oncogenic pathways. METHODS: We report 3 cases of metastatic gastrointestinal carcinoma in patients enrolled in the Personalized Onco-Genomics program at the BC Cancer Agency. Real-time genomic profiling was combined with clinical expertise to diagnose a carcinoma of unknown primary, to explore treatment response to bevacizumab in a colorectal cancer, and to characterize an appendiceal adenocarcinoma. RESULTS: In the first case, genomic profiling revealed an IDH1 somatic mutation, supporting the diagnosis of cholangiocarcinoma in a malignancy of unknown origin, and further guided therapy by identifying epidermal growth factor receptor amplification. In the second case, a BRAF V600E mutation and wild-type KRAS profile justified the use of targeted therapies to treat a colonic adenocarcinoma. The third case was an appendiceal adenocarcinoma defined by a p53 inactivation; Ras/raf/mek, Akt/mtor, Wnt, and notch pathway activation; and overexpression of ret, erbb2 (her2), erbb3, met, and cell cycle regulators. SUMMARY: We show that whole-genome and transcriptome sequencing can be achieved within clinically effective timelines, yielding clinically useful and actionable information.

Identification and analysis of murine pancreatic islet enhancers.

AIMS/HYPOTHESIS: The paucity of information on the epigenetic barriers that are blocking reprogramming protocols, and on what makes a beta cell unique, has hampered efforts to develop novel beta cell sources. Here, we aimed to identify enhancers in pancreatic islets, to understand their developmental ontologies, and to identify enhancers unique to islets to increase our understanding of islet-specific gene expression. METHODS: We combined H3K4me1-based nucleosome predictions with pancreatic and duodenal homeobox 1 (PDX1), neurogenic differentiation 1 (NEUROD1), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MAFA) and forkhead box A2 (FOXA2) occupancy data to identify enhancers in mouse islets. RESULTS: We identified 22,223 putative enhancer loci in in vivo mouse islets. Our validation experiments suggest that nearly half of these loci are active in regulating islet gene expression, with the remaining regions probably poised for activity. We showed that these loci have at least nine developmental ontologies, and that islet enhancers predominately acquire H3K4me1 during differentiation. We next discriminated 1,799 enhancers unique to islets and showed that these islet-specific enhancers have reduced association with annotated genes, and identified a subset that are instead associated with novel islet-specific long non-coding RNAs (lncRNAs). CONCLUSIONS/INTERPRETATIONS: Our results indicate that genes with islet-specific expression and function tend to have enhancers devoid of histone methylation marks or, less often, that are bivalent or repressed, in embryonic stem cells and liver. Further, we identify a subset of enhancers unique to islets that are associated with novel islet-specific genes and lncRNAs. We anticipate that these data will facilitate the development of novel sources of functional beta cell mass.

Phase diffusion in graphene-based Josephson junctions.

We report on graphene-based Josephson junctions with contacts made from lead. The high transition temperature of this superconductor allows us to observe the supercurrent branch at temperatures up to ∼2 K, at which point we can detect a small, but nonzero, resistance. We attribute this resistance to the phase diffusion mechanism, which has not been yet identified in graphene. By measuring the resistance as a function of temperature and gate voltage, we can further characterize the nature of the electromagnetic environment and dissipation in our samples.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans.

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia.

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite 'E' (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 µm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite 'E'. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5'-AGTGCGGTGGTATCGACAAG) and EFCF6 (3'-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

The impact of deprivation on youth violence: a comparison of cities and their feeder towns.

BACKGROUND AND AIMS: 66,000 children and adolescents are treated at emergency departments (EDs) in England and Wales each year for assault injury. The aim of this study was to compare adolescent assault injury rates in cities and towns and determine how assault injury varies with deprivation and gender. METHODS: The study was set in three cities in Wales, UK, and their surrounding towns. Subjects were 11-17 year olds treated for assault injury at one of seven EDs from 1 October 2005 to 30 September 2006. Area of residence (electoral divisions, EDivs) was identified from patient postcode. EDivs were aggregated into deprivation fifths for males and females and cities and towns. Assault injury rates, rate ratios and 95% CIs were calculated. RESULTS: 1472 children and adolescents of 11-17 years old were treated for assault injury. Male city assault injury rates were 14.2/1000 11-17 year olds; and 13.1 in towns. Female city assault injury rates were 6.0; and 5.6 in towns. In the most deprived city areas males had assault injury rates 2.6 times (95% CI 1.85 to 3.59) that of the most affluent, compared with 2.0 times in towns (95% CI 1.39 to 2.86). For females, the most deprived city areas had assault injury rates 5.3 times that of the most affluent (95% CI 2.93 to 9.41), compared with 2.8 times in towns (95% CI 1.47 to 5.28). CONCLUSIONS: Injury in youth violence increased with increasing deprivation in cities and their feeder towns. This was true for boys and girls, though rates for boys were consistently higher. This link between assault injury and deprivation was stronger for girls in cities than in feeder towns. Strategies to prevent youth violence need to include improved safeguarding arrangements for girls living in the most deprived city areas.

Small DNA deletions creating avirulence in Streptococcus pyogenes.

The M protein is the antigen on the surface of group A streptococci that allows these bacteria to resist phagocytosis. DNA encoding the M12 protein was cloned into Escherichia coli and used as an isotopically labeled hybridization probe to compare genomic DNA's isolated from M+ and M- isogenic cultures in an effort to elucidate the genetic basis of this variation. DNA's from two spontaneous, independent M- variants contained small (approximately 50 base pairs) deletions which were mapped to identical restriction fragments within or adjacent to the M protein coding sequence. Taken together with the pleiotropic nature of these deletions, this suggests that they define a regulatory switch.

Nonmethylated transposable elements and methylated genes in a chordate genome.

The genome of the invertebrate chordate Ciona intestinalis was found to be a stable mosaic of methylated and nonmethylated domains. Multiple copies of an apparently active long terminal repeat retrotransposon and a long interspersed element are nonmethylated and a large fraction of abundant short interspersed elements are also methylation free. Genes, by contrast, are predominantly methylated. These data are incompatible with the genome defense model, which proposes that DNA methylation in animals is primarily targeted to endogenous transposable elements. Cytosine methylation in this urochordate may be preferentially directed to genes.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Parecoxib for analgesia after craniotomy.

BACKGROUND: Pain after craniotomy is often under-treated. Opiates carry distinct disadvantages. Non-steroidal anti-inflammatory drugs have an anti-platelet action and carry a bleeding risk. Cyclo-oxygenase 2 inhibitors such as parecoxib are not associated with a bleeding risk and would be welcome analgesics if shown to be effective. METHODS: In a prospective double-blind, randomized, placebo-controlled study, we investigated the analgesic effect of a single dose of parecoxib 40 mg given at dural closure in 82 patients undergoing elective craniotomies. Remifentanil was used intraoperatively, and i.v. morphine was titrated to the requirement in the post-anaesthetic unit. On the ward, i.m. morphine 5 mg as required and regular acetaminophen was prescribed. Morphine use and visual analogue pain scores were recorded at 1, 6, 12, and 24 h after surgery. RESULTS: Parecoxib reduced pain scores at 6 h and morphine use at 6 and 12 h after operation. However, overall, it had only minimal impact on postoperative analgesia. We found a wide variability in analgesic requirements where 11% of patients required no opioids and 16% required more than 15 mg i.v. morphine 1 h after the surgery. CONCLUSIONS: We found only limited evidence to support parecoxib as an analgesic after craniotomy.

An update and lessons from whole-genome sequencing projects.

A number of prokaryotic and eukaryotic genomes are currently being sequenced. Already, the nucleotide sequences of four yeast chromosomes and of 2.2 Mb from Caenorhabditis elegans have been reported. Human genomic sequences have also been used in comparative studies with both mouse and Fugu rubripes.

The advocacy in action study a cluster randomized controlled trial to reduce pedestrian injuries in deprived communities.

BACKGROUND: Road traffic-related injury is a major global public health problem. In most countries, pedestrian injuries occur predominantly to the poorest in society. A number of evaluated interventions are effective in reducing these injuries. Very little research has been carried out into the distribution and determinants of the uptake of these interventions. Previous research has shown an association between local political influence and the distribution of traffic calming after adjustment for historical crash patterns. This led to the hypothesis that advocacy could be used to increase local politicians knowledge of pedestrian injury risk and effective interventions, ultimately resulting in improved pedestrian safety. OBJECTIVE: To design an intervention to improve the uptake of pedestrian safety measures in deprived communities. SETTING: Electoral wards in deprived areas of England and Wales with a poor record of pedestrian safety for children and older adults. METHODS: Design mixedmethods study, incorporating a cluster randomized controlled trial. Data mixture of Geographical Information Systems data collision locations, road safety interventions, telephone interviews, and questionnaires. Randomization 239 electoral wards clustered within 57 local authorities. Participants 615 politicians representing intervention and control wards. Intervention a package of tailored information including maps of pedestrian injuries was designed for intervention politicians, and a general information pack for controls. OUTCOME MEASURES: Primary outcome number of road safety interventions 25 months after randomization. Secondary outcomes politicians interest and involvement in injury prevention cost of interventions. Process evaluation use of advocacy pack, facilitators and barriers to involvement, and success.

cisRED: a database system for genome-scale computational discovery of regulatory elements.

We describe cisRED, a database for conserved regulatory elements that are identified and ranked by a genome-scale computational system (www.cisred.org). The database and high-throughput predictive pipeline are designed to address diverse target genomes in the context of rapidly evolving data resources and tools. Motifs are predicted in promoter regions using multiple discovery methods applied to sequence sets that include corresponding sequence regions from vertebrates. We estimate motif significance by applying discovery and post-processing methods to randomized sequence sets that are adaptively derived from target sequence sets, retain motifs with p-values below a threshold and identify groups of similar motifs and co-occurring motif patterns. The database offers information on atomic motifs, motif groups and patterns. It is web-accessible, and can be queried directly, downloaded or installed locally.

A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.