Supplementary Glioma Gene Mutations from A Hypermutation Phenotype and Somatic MSH6 Mutations in Recurrent Human Malignant Gliomas after Alkylator Chemotherapy

Supplementary Information from A Hypermutation Phenotype and Somatic MSH6 Mutations in Recurrent Human Malignant Gliomas after Alkylator Chemotherapy

A number of methods are in common use for calculating 95th percentiles of enterococcal sampling results from recreational waters. However all are liable to give a poor indication of infection risk as calculated from the formula used in the 2003 WHO Guidelines for safe recreational water environments and similar risk based guidelines. Their limitations are particularly evident where the underlying microbial population deviates from the reference distributions used in these guidelines (lognormal, with a log10 standard distribution of ∼0.81). A Microsoft® Excel template (the EnteroTester) has been developed for generating workbooks that: estimate the infection risk (according to the formula used in the above guidelines) for any given enterococcal distribution, and calculate a 95th percentile standardised to that of the reference distribution with the same risk. Standardised enterococcal 95th percentiles allow direct comparison between different recreational waters in terms of their infection risk. They overcome the common problem of misclassifying recreational waters to the wrong microbial water quality assessment category. The template works for data sets containing eight observations or more, and can accommodate up to 80% of the results being below the limit of detection. It produces a narrower upper confidence band than the classic parametric method, thus requiring fewer samples than recommended in the above guidelines. The template also generates trigger levels for alerting recreational water managers to likely changes in the underlying distribution, and the need to consider whether further sampling or investigation is required. It has been widely trialled in Australia, demonstrating its practical application in managing recreational waters.

Protecting recreational water quality where 'whole-of-body contact' activities occur is important from a public health and economic perspective. Numerous studies have demonstrated that infectious illnesses occur when swimming in faecally polluted waters. With the release of the 2008 Australian recreational water guidelines, the Western Australian (WA) Department of Health conducted a formal evaluation to highlight the advantages of applying the microbial risk management framework to 27 swimming beaches in the Swan and Canning Rivers in Perth, WA. This involved a two-phase approach: (i) calculation of 95th percentiles using historical enterococci data; and (ii) undertaking sanitary inspections. The outcomes were combined to assign provisional risk classifications for each site. The classifications are used to promote informed choices as a risk management strategy. The study indicates that the majority of swimming beaches in the Swan-Canning Rivers are classified as 'very good' to 'good' and are considered safe for swimming. The remaining sites were classified as 'poor', which is likely to be attributed to environmental influences. Information from the study was communicated to the public via a series of press releases and the Healthy Swimming website. The guidelines provide a sound approach to managing recreational water quality issues, but some limitations were identified.

This paper presents an overview of the chemical results of the Premiers Collaborative Research Project: Characterising Secondary Treated Wastewater for Drinking Purposes Following Reverse Osmosis Treatment. The chemical groups analysed include a broad range of analytes with different physical and chemical characteristics and toxic effects. Their potential human health impacts at the concentrations observed in the post RO water were evaluated using a screening health risk assessment methodology. The results confirm that reverse osmosis is effective in controlling chemical hazards and reliably producing recycled water suitable for augmenting, through indirect potable reuse, public drinking water supplies. Specific health recommendations, detailed in the technical report, were developed for the monitoring of the Water Corporation's three-year groundwater replenishment trial.

The growing scarcity of potable water supplies is among the most important issues facing many cities, in particular those using single sources of water that are climate dependent. Consequently, urban centers are looking to alternative sources of water supply that can supplement variable rainfall and meet the demands of population growth. A diversified portfolio of water sources is required to ensure public health, as well as social, economical and environmental sustainability. One of the options considered is the augmentation of drinking water supplies with advanced treated recycled water. This paper aims to provide a state of the art review of water recycling for drinking purposes with emphasis on membrane treatment processes. An overview of significant indirect potable reuse projects is presented followed by a description of the epidemiological and toxicological studies evaluating any potential human health impacts. Finally, a summary of key operational measures to protect human health and the areas that require further research are discussed.

An assessment of potential health impacts of dioxin and dioxin-like compounds in recycled water for indirect potable reuse was conducted. Toxic equivalency factors (TEFs) for 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDFs) and dioxin-like polychlorinated biphenyls (PCBs) congeners have been developed by the World Health Organization to simplify the risk assessment of complex mixtures. Samples of secondary treated wastewater in Perth, Australia were examined pre-and post-tertiary treatment in one full-scale and one pilot water reclamation plant. Risk quotients (RQs) were estimated by expressing the middle-bound toxic equivalent (TEQ) and the upper-bound TEQ concentration in each sampling point as a function of the estimated health target value. The results indicate that reverse osmosis (RO) is able to reduce the concentration of PCDD, PCDF and dioxin-like PCBs and produce water of high quality (RQ after RO=0.15). No increased human health risk from dioxin and dioxin-like compounds is anticipated if highly treated recycled water is used to augment drinking water supplies in Perth. Recommendations for a verification monitoring program are offered.

The panel of 60 human cancer cell lines (the NCI-60) assembled by the National Cancer Institute for anticancer drug discovery is a widely used resource. The NCI-60 has been characterized pharmacologically and at the molecular level more extensively than any other set of cell lines. However, no systematic mutation analysis of genes causally implicated in oncogenesis has been reported. This study reports the sequence analysis of 24 known cancer genes in the NCI-60 and an assessment of 4 of the 24 genes for homozygous deletions. One hundred thirty-seven oncogenic mutations were identified in 14 (APC, BRAF, CDKN2, CTNNB1, HRAS, KRAS, NRAS, SMAD4, PIK3CA, PTEN, RB1, STK11, TP53, and VHL) of the 24 genes. All lines have at least one mutation among the cancer genes examined, with most lines (73%) having more than one. Identification of those cancer genes mutated in the NCI-60, in combination with pharmacologic and molecular profiles of the cells, will allow for more informed interpretation of anticancer agent screening and will enhance the use of the NCI-60 cell lines for molecularly targeted screens.

Abstract Malignant gliomas have a very poor prognosis. The current standard of care for these cancers consists of extended adjuvant treatment with the alkylating agent temozolomide after surgical resection and radiotherapy. Although a statistically significant increase in survival has been reported with this regimen, nearly all gliomas recur and become insensitive to further treatment with this class of agents. We sequenced 500 kb of genomic DNA corresponding to the kinase domains of 518 protein kinases in each of nine gliomas. Large numbers of somatic mutations were observed in two gliomas recurrent after alkylating agent treatment. The pattern of mutations in these cases showed strong similarity to that induced by alkylating agents in experimental systems. Further investigation revealed inactivating somatic mutations of the mismatch repair gene MSH6 in each case. We propose that inactivating somatic mutations of MSH6 confer resistance to alkylating agents in gliomas in vivo and concurrently unleash accelerated mutagenesis in resistant clones as a consequence of continued exposure to alkylating agents in the presence of defective mismatch repair. The evidence therefore suggests that when MSH6 is inactivated in gliomas, alkylating agents convert from induction of tumor cell death to promotion of neoplastic progression. These observations highlight the potential of large scale sequencing for revealing and elucidating mutagenic processes operative in individual human cancers. (Cancer Res 2006; 66(8): 3987-91)

The protein kinase gene family is the most frequently mutated in human cancer. Previous work has documented activating mutations in the KIT receptor tyrosine kinase in testicular germ-cell tumors (TGCT). To investigate further the potential role of mutated protein kinases in the development of TGCT and to characterize the prevalence and patterns of point mutations in these tumors, we have sequenced the coding exons and splice junctions of the annotated protein kinase family of 518 genes in a series of seven seminomas and six nonseminomas. Our results show a remarkably low mutation frequency, with only a single somatic point mutation, a K277E mutation in the STK10 gene, being identified in a total of more than 15 megabases of sequence analyzed. Sequencing of STK10 in an additional 40 TGCTs revealed no further mutations. Comparative genomic hybridization and LOH analysis using SNP arrays demonstrated that the 13 TGCTs mutationally screened through the 518 protein kinase genes were uniformly aneuploid with consistent chromosomal gains on 12p, 8q, 7, and X and losses on 13q, 18q, 11q, and 4q. Our results do not provide evidence for a mutated protein kinase implicated in the development of TGCT other than KIT. Moreover, they demonstrate that the general prevalence of point mutations in TGCT is low, in contrast to the high frequency of copy number changes.

We examined the coding sequence of 518 protein kinases, ∼1.3 Mb of DNA per sample, in 25 breast cancers. In many tumors, we detected no somatic mutations. But a few had numerous somatic mutations with distinctive patterns indicative of either a mutator phenotype or a past exposure.

Protein kinases are frequently mutated in human cancer and inhibitors of mutant protein kinases have proven to be effective anticancer drugs. We screened the coding sequences of 518 protein kinases (approximately 1.3 Mb of DNA per sample) for somatic mutations in 26 primary lung neoplasms and seven lung cancer cell lines. One hundred eighty-eight somatic mutations were detected in 141 genes. Of these, 35 were synonymous (silent) changes. This result indicates that most of the 188 mutations were "passenger" mutations that are not causally implicated in oncogenesis. However, an excess of approximately 40 nonsynonymous substitutions compared with that expected by chance (P = 0.07) suggests that some nonsynonymous mutations have been selected and are contributing to oncogenesis. There was considerable variation between individual lung cancers in the number of mutations observed and no mutations were found in lung carcinoids. The mutational spectra of most lung cancers were characterized by a high proportion of C:G > A:T transversions, compatible with the mutagenic effects of tobacco carcinogens. However, one neuroendocrine cancer cell line had a distinctive mutational spectrum reminiscent of UV-induced DNA damage. The results suggest that several mutated protein kinases may be contributing to lung cancer development, but that mutations in each one are infrequent.

The protein-kinase family is the most frequently mutated gene family found in human cancer and faulty kinase enzymes are being investigated as promising targets for the design of antitumour therapies. We have sequenced the gene encoding the transmembrane protein tyrosine kinase ERBB2 (also known as HER2 or Neu) from 120 primary lung tumours and identified 4% that have mutations within the kinase domain; in the adenocarcinoma subtype of lung cancer, 10% of cases had mutations. ERBB2 inhibitors, which have so far proved to be ineffective in treating lung cancer, should now be clinically re-evaluated in the specific subset of patients with lung cancer whose tumours carry ERBB2 mutations.