Exploring the feasibility and utility of exome-scale tumour sequencing in a clinical setting.

BACKGROUND: Technology has progressed from single gene panel to large-scale genomic sequencing. This is raising expectations from clinicians and patients alike. The utility and performance of this technology in a clinical setting needs to be evaluated. AIM: This pilot study investigated the feasibility of using exome-scale sequencing (ESS) to identify molecular drivers within cancers in real-time for Precision Oncology in the clinic. METHODS: Between March 2014 and March 2015, the Victorian Comprehensive Cancer Centre Alliance explored the feasibility and utility of ESS in a pilot study. DNA extracted from the tumour specimens underwent both ESS and targeted 'hotspot' sequencing (TS). Blood was taken for germline analysis. A multi-disciplinary molecular tumour board determined the clinical relevance of identified mutations; in particular, whether they were 'actionable' and/or 'druggable'. RESULTS: Of 23 patients screened, 15 (65%) met the tissue requirements for genomic analysis. TS and ESS were successful in all cases. ESS identified pathogenic somatic variants in 73% (11/15 cases) versus 53% (8/15 cases) using TS. Clinically focused ESS identified 63 variants, consisting of 30 somatic variants (including all 13 identified by TS) and 33 germline variants. Overall, there were 48 unique variants. ESS had a clinical impact in 53% (8/15 cases); 47% (7/15 cases) were referred to the familial cancer clinic, and 'druggable' targets were identified in 53% (8/15 cases). CONCLUSION: ESS of tumour DNA impacted clinical decision-making in 53%, with 20% more pathogenic variants identified through ESS than TS. The identification of germline variants in 47% was an unexpected finding.

Preparing for genomic medicine: a real world demonstration of health system change.

Organisations and governments seeking to implement genomics into clinical practice face numerous challenges across multiple, diverse aspects of the health care system. It is not sufficient to tackle any one aspect in isolation: to create a system that supports genomic medicine, they must be addressed simultaneously. The growing body of global knowledge can guide decision-making, but each jurisdiction or organisation needs a model for genomic (or personalised) medicine that is tailored to its unique context, its priorities and the funds available. Poor decisions could greatly reduce the benefits that could potentially arise from genomic medicine. Demonstration projects enable models to be tested, providing valuable evidence and experience for subsequent implementation. Here, we present the Melbourne Genomics Health Alliance demonstration project as an exemplar of a collaborative, holistic approach to phased implementation of genomics across multiple autonomous institutions. The approach and lessons learned may assist others in determining how best to integrate genomics into their healthcare system.

RAS Mutations as Predictive Biomarkers in Clinical Management of Metastatic Colorectal Cancer.

The use of anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapies in patients with metastatic colorectal cancer is guided by the presence of activating point mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS and NRAS genes in the primary tumor. Although these mutations have been incorporated into the prescribing information for both cetuximab and panitumumab, highlighted in the National Comprehensive Cancer Network Guidelines, and routinely tested, a number of controversial issues and unanswered questions related to these mutations and their clinical significance remain. In the present review, we explored the contradictory data related to the prognostic value of KRAS mutations, the reported frequent discordance of KRAS mutations, and the reported nonequivalence of some of these mutations. We also considered the issues related to incorporating additional mutations into the already accredited and approved assays and the challenges created by changing an assay's analytical and clinical limits of detection. We also discuss the lack of biologic data supporting the pathogenicity of newly described clinically actionable mutations and explore the uncertainty regarding the clinical significance of low-frequency mutations, highlighting the importance of correcting allele frequencies for tumor purity. We also considered the importance of distinguishing the significance of low-frequency RAS mutations in tumors previously not treated or treated with anti-EGFR therapies and explore new technologies capable of detecting emerging polyclonal RAS mutations that appear to confer drug resistance.

Scabrosin esters and derivatives: chemical derivatization studies and biological evaluation.

Several derivatives of the natural scabrosin esters were synthesized in order to elucidate the structural features present, which are responsible for the biological activities. The studies demonstrate that full anti-proliferative activities of the scabrosin esters, both the carboskeleton core as well as the ability to form the dithiol and/or the disulfide linkage of the epidithiopiperazine-2,5-dione are required. The presence of the epoxide rings on the scabrosin esters do not contribute to the observed biological activities.

Evidence that the lichen-derived scabrosin esters target mitochondrial ATP synthase in P388D1 cells.

Scabrosin esters (SEs), which have been recently isolated from the lichen Xanthoparmelia scabrosa, belong to the epipolythiodioxopiperazine (ETP) class of secondary metabolites characterized by possession of a reactive disulfide bond. Colony forming assays show that these toxins are active against human tumor cell lines at nanomolar concentrations. Other members of the ETP class of toxins such as gliotoxin have been shown to induce apoptosis in cells, although the cellular target(s) of the ETP toxins is currently unknown. ETP toxins have been shown to inhibit a variety of enzymes via interaction with sensitive cysteine residues. Here we show that the typical scabrosin ester acetate butyrate induces early mitochondrial membrane hyperpolarization assessed by JC-1 staining accompanied by apoptotic cell death. The toxin lowers ATP in intact cells and inhibits the rate of ATP synthesis in permeabilzed cells. Comparison with the effects of the known ATP synthase inhibitor oligomycin B is consistent with ATP synthase as an early target in scabrosin ester-induced cell death.