Enhancing the implementation of provider-to-provider telehealth in rural and remote areas: A mixed methods study protocol.

Background: Virtual healthcare solutions are proposed as a way to combat the inequity of access to healthcare in rural and remote areas, and to better support the front-line providers who work in these areas. Rural provider-to-provider telehealth (RPPT) connects rural and remote clinicians to a 'hub' of healthcare specialists who can increase access to emergency and specialised healthcare via an integrated model. Reported benefits for the place-based provider include enhanced knowledge, expanded professional development opportunities, improved scope of practice, and increased confidence in treating more complex cases. These reported benefits could have implications for supporting and futureproofing our health workforce in terms of productivity, burnout, recruitment, and retention. Methods: The research uses an explanatory sequential mixed methods approach across multiple phases to evaluate the current implementation of Western Australia Country Health Service's (WACHS) Command Centre (CC) services and explore factors associated with their differential use. The primary population of interest and participants in this study are the place-based providers in country Western Australia (WA). Patient data constitutes the secondary population, informing the access and reach of CC services into country WA. Data collection will include service data, an online survey, and semi-structured interviews with the primary population. The data will be interpreted to inform evidence-based strategies and recommendations to improve the implementation and sustainment of RPPT. Discussion: Innovative and sustained workforce models and solutions are needed globally. Virtual healthcare, including provider-to-provider models, demonstrate potential, especially in rural and remote areas, designed to increase access to specialised expertise for patients and to support the local workforce. This research will generate new data around behaviour, perceptions, and value from the WACHS rural and remote workforce about provider-to-provider telehealth, to explore the implementation and investigate strategies for the long-term sustainment of RPPT services.

Public preference on sharing health data to inform research, health policy and clinical practice in Australia: A stated preference experiment.

OBJECTIVE: To investigate public willingness to share sensitive health information for research, health policy and clinical practice. METHODS: A total of 1,003 Australian respondents answered an online, attribute-driven, survey in which participants were asked to accept or reject hypothetical choice sets based on a willingness to share their health data for research and frontline-medical support as part of an integrated health system. The survey consisted of 5 attributes: Stakeholder access for analysis (Analysing group); Type of information collected; Purpose of data collection; Information governance; and Anticipated benefit; the results of which were analysed using logistic regression. RESULTS: When asked about their preference for sharing their health data, respondents had no preference between data collection for the purposes of clinical practice, health policy or research, with a slight preference for having government organisations manage, govern and curate the integrated datasets from which the analysis was being conducted. The least preferred option was for personal health records to be integrated with insurance records or for their data collected by privately owned corporate organisations. Individuals preferred their data to be analysed by a public healthcare provider or government staff and expressed a dislike for any private company involvement. CONCLUSIONS: The findings from this study suggest that Australian consumers prefer to share their health data when there is government oversight, and have concerns about sharing their anonymised health data for clinical practice, health policy or research purposes unless clarity is provided pertaining to its intended purpose, limitations of use and restrictions to access. Similar findings have been observed in the limited set of existing international studies utilising a stated preference approach. Evident from this study, and supported by national and international research, is that the establishment and preservation of a social license for data linkage in health research will require routine public engagement as a result of continuously evolving technological advancements and fluctuating risk tolerance. Without more work to understand and address stakeholder concerns, consumers risk being reluctant to participate in data-sharing and linkage programmes.

Australian general practitioner perceptions to sharing clinical data for secondary use: a mixed method approach.

OBJECTIVE: The potential for data collected in general practice to be linked and used to address health system challenges of maintaining quality care, accessibility and safety, including pandemic support, has led to an increased interest in public acceptability of data sharing, however practitioners have rarely been asked to share their opinions on the topic. This paper attempts to gain an understanding of general practitioner's perceptions on sharing routinely collected data for the purposes of healthcare planning and research. It also compares findings with data sharing perceptions in an international context.  MATERIALS AND METHODS: A mixed methods approach combining an initial online survey followed by face-to-face interviews (before and during COVID-19), designed to identify the barriers and facilitators to sharing data, were conducted on a cross sectional convenience sample of general practitioners across Western Australia (WA). RESULTS: Eighty online surveys and ten face-to-face interviews with general practitioners were conducted from November 2020 - May 2021. Although respondents overwhelmingly identified the importance of population health research, their willingness to participate in data sharing programs was determined by a perception of trust associated with the organisation collecting and analysing shared data; a clearly defined purpose and process of collected data; including a governance structure providing confidence in the data sharing initiative simultaneously enabling a process of data sovereignty and autonomy. DISCUSSION: Results indicate strong agreement around the importance of sharing patient's medical data for population and health research and planning. Concerns pertaining to lack of trust, governance and secondary use of data continue to be a setback to data sharing with implications for primary care business models being raised. CONCLUSION: To further increase general practitioner's confidence in sharing their clinical data, efforts should be directed towards implementing a robust data governance structure with an emphasis on transparency and representative stakeholder inclusion as well as identifying the role of government and government funded organisations, as well as building trust with the entities collecting and analysing the data.

Evaluation of HealthPathways: an appraisal of usage, experiences and opinions of healthcare professionals in Australia and New Zealand.

Objectives HealthPathways, pioneered in Canterbury, New Zealand, in 2008, is a web-based tool designed to promote health care integration and patient management in primary care and to reduce fragmentation in the delivery of health services. This cross-sectional study evaluated the utilisation and perceptions of this tool among health professionals in Australia and New Zealand. Methods A cross-sectional survey was administered online through Research Electronic Data Capture (REDCap) to general practitioners (GPs), practice nurses and managers, nurse practitioners, specialist and community nurses, hospital clinicians, nurses, managers, and allied health professionals between April and September 2018. The frequency of HealthPathways use in the previous month was modelled as an ordered response using an ordered logistic regression model after adjusting for the possible effects of sex, age, years in clinical practice, location and time spent in practice. Results Health professionals perceived HealthPathways to be useful in primary care management and referral, as well as in the prereferral treatment of patients. GPs in New Zealand, New South Wales and Victoria were 73%, 47% and 27% more likely to have used HealthPathways ≥10 times in the previous month respectively. Conclusion The results suggest that HealthPathways is having a positive effect on healthcare systems in New Zealand and Australia. However, differences in uptake suggests the need for focused implementation, integration into eReferral software and expanding the tool to medical students, registrars, allied health professionals and potentially patients to encourage behavioural change. What is known about the topic? Early evaluations suggest that HealthPathways is a useful tool for health professionals, although uptake and utilisation may be limited. However, there is no comparative evidence regarding uptake and implementation of the tool. What does the paper add? This study is among the first to provide a comparative narrative of the literature assessing the implementation and uptake of HealthPathways across Australia and New Zealand. It is also among the first to compare the perceptions of allied health professionals in the use of HealthPathways across Australia and New Zealand. What are the implications for practitioners? The results of this study suggest the need for focused implementation, integration into eReferral software and expanding the tool to medical students, registrars, allied health professionals and potentially patients to encourage behavioural change.

Exploring the costs and effectiveness of the Drug and Alcohol Withdrawal Network: a home-based alcohol and other drug withdrawal service.

The Drug and Alcohol Withdrawal Network (DAWN) is a home-based withdrawal service based in Perth, Western Australia. Literature on outcomes, costs and client attitudes towards this type of home-based detoxification in Australia is sparse. Therefore, this study assessed these factors for clients enrolled over a 5-year period (July 2011-June 2016). Client experience was explored through semi-structured interviews with 10 clients. Over the study period, 1800 clients (54% male, mean age 38 years) were assessed, and there were 2045 episodes of care. Although most first-episode clients (52%) listed alcohol as the primary drug of concern, the proportion listing methamphetamine increased from 4% in 2011-12 to 23% in 2015-16. In 94% (n=639) of withdrawal detoxification episodes with completed surveys, clients used their 'drug of primary concern' most days or more often at baseline; this had reduced to 23% (n=149) at the conclusion of detoxification. Five-year direct costs were A$4.8million. Clients valued the person-centred holistic approach to care, including linking with other health providers. Barriers included low awareness of the program and difficulties finding an appropriate support person. Further exploration of cost-effectiveness would substantiate the apparently lower per client cost, assuming medical suitability for both programs, for home-based relative to inpatient withdrawal.

Investigating articulated heavy-vehicle crashes in Western Australia using a spatial approach.

BACKGROUND: Recent developments in Western Australia's economy including widespread traffic congestion as well as road safety issues are increasingly becoming prominent. Previous studies relied on traditional statistical methods to investigate patterns and characteristics of motor vehicle crashes. Although useful, statistical analysis alone is incapable of providing a spatial context and is therefore unable to associate existing crash characteristics with a spatial distribution. AIMS: To identify concentrations or "hotspots" of articulated heavy vehicle crashes in WA between the years 2001-2013, by using a spatial analysis approach. METHODS: Spatial modelling and spatio-temporal analytical methods such as Emerging Hotspots were used to identify emerging hotspots on specific roads in Western Australia using the Integrated Road Information System (IRIS). RESULTS: The results suggest that the majority of articulated heavy vehicles crashes occurred in the vicinity or within the Perth metropolitan area. Based on spatial-temporal trend analyses, our findings highlight some regions that are emerging as areas of interest. DISCUSSION: This study was one of the first attempts to adopt a spatial analysis approach in studying heavy-vehicle crashes in Western Australia. Applying spatial methodologies to road safety data has the potential of obtaining previously undiscovered insights, which can be extended further, and provide future avenues to research in this field.

Impact of geography on the control of type 2 diabetes mellitus: a review of geocoded clinical data from general practice.

OBJECTIVE: To review the clinical data for people with diabetes mellitus with reference to their location and clinical care in a general practice in Australia. MATERIALS AND METHODS: Patient data were extracted from a general practice in Western Australia. Iterative data-cleansing steps were taken. Data were grouped into Statistical Area level 1 (SA1), designated as the smallest geographical area associated with the Census of Population and Housing. The data were analysed to identify if SA1s with people aged 70 years and older, and with relatively high glycosylated haemoglobin (HbA1c) were significantly clustered, and whether this was associated with their medical consultation rate and treatment. The analysis included Cluster and Outlier Analysis using Moran's I test. RESULTS: The overall median age of the population was 70 years with more males than females, 53% and 47%, respectively. Older people (>70 years) with relatively high HbA1c comprised 9.3% of all people with diabetes in the sample, and were clustered around two 'hotspot' locations. These 111 patients do not attend the practice more or less often than people with diabetes living elsewhere in the practice (p=0.098). There was some evidence that they were more likely to be recorded as having consulted with regard to other chronic diseases. The average number of prescribed medicines over a 13-month time period, per person in the hotspots, was 4.6 compared with 5.1 in other locations (p=0.26). Their prescribed therapy was deemed to be consistent with the management of people with diabetes in other locations with reference to the relevant diabetes guidelines. CONCLUSIONS: Older patients with relatively high HbA1c are clustered in two locations within the practice area. Their hyperglycaemia and ongoing cardiovascular risk indicates causes other than therapeutic inertia. The causes may be related to the social determinants of health, which are influenced by geography.

The Australian primary healthcare experiment: a national survey of Medicare Locals.

OBJECTIVE: The objectives of this study are to evaluate the development and implementation of Medicare Locals as new primary care organisations and consider the implications of these findings for the wider challenge of strengthening primary healthcare in Australia and internationally. DESIGN: National survey of Medicare Locals which involved the use of content analysis and a descriptive survey tool. SETTING: 61 Medicare Locals in Australia. PARTICIPANTS: The survey was distributed electronically to all 61 Medicare Local Chief Executive Officers (CEOs) between October and December 2013. MAIN OUTCOME MEASURES: The research was interested in exploring the following areas; the form and function of Medicare Locals; the confidence and capacity of Medicare Locals to perform against their objectives around population planning and system integration; their ability to engage relevant stakeholder groups; and the barriers and facilitators to reform. RESULTS: A total of 43 (70%) of Medicare Locals completed the survey with representation from six of the eight Australian states and Territories. Results suggest differences in the form and function of the Medicare Local organisations and considerable diversity in the implementation of Medicare Local organisations across Australia. This diversity and lack of guidance from government impacted on the overall success of the reform. Other barriers to reform included difficulties in stakeholder relationships and limited incentives (financial and other) to drive and influence change. CONCLUSIONS: Findings from this study produce important insights for primary care reform in Australia; and internationally it adds to the growing body of knowledge around primary care reform.

HealthPathways: creating a pathway for health systems reform.

Inefficiencies in the co-ordination and integration of primary and secondary care services in Australia, have led to increases in waiting times, unnecessary presentations to emergency departments and issues around poor discharge of patients. HealthPathways is a program developed in Canterbury, New Zealand, that builds relationships between General Practitioners and Specialists and uses information technology so that efficiency is maximised and the right patient is given the right care at the right time. Healthpathways is being implemented by a number of Medicare Locals across Australia however, little is known about the impact HealthPathways may have in Australia. This article provides a short description of HealthPathways and considers what it may offer in the Australian context and some of the barriers and facilitators to implementation.

The expression level of small non-coding RNAs derived from the first exon of protein-coding genes is predictive of cancer status.

Small non-coding RNAs (smRNAs) are known to be significantly enriched near the transcriptional start sites of genes. However, the functional relevance of these smRNAs remains unclear, and they have not been associated with human disease. Within the cancer genome atlas project (TCGA), we have generated small RNA datasets for many tumor types. In prior cancer studies, these RNAs have been regarded as transcriptional "noise," due to their apparent chaotic distribution. In contrast, we demonstrate their striking potential to distinguish efficiently between cancer and normal tissues and classify patients with cancer to subgroups of distinct survival outcomes. This potential to predict cancer status is restricted to a subset of these smRNAs, which is encoded within the first exon of genes, highly enriched within CpG islands and negatively correlated with DNA methylation levels. Thus, our data show that genome-wide changes in the expression levels of small non-coding RNAs within first exons are associated with cancer.

Barnacle: detecting and characterizing tandem duplications and fusions in transcriptome assemblies.

BACKGROUND: Chimeric transcripts, including partial and internal tandem duplications (PTDs, ITDs) and gene fusions, are important in the detection, prognosis, and treatment of human cancers. RESULTS: We describe Barnacle, a production-grade analysis tool that detects such chimeras in de novo assemblies of RNA-seq data, and supports prioritizing them for review and validation by reporting the relative coverage of co-occurring chimeric and wild-type transcripts. We demonstrate applications in large-scale disease studies, by identifying PTDs in MLL, ITDs in FLT3, and reciprocal fusions between PML and RARA, in two deeply sequenced acute myeloid leukemia (AML) RNA-seq datasets. CONCLUSIONS: Our analyses of real and simulated data sets show that, with appropriate filter settings, Barnacle makes highly specific predictions for three types of chimeric transcripts that are important in a range of cancers: PTDs, ITDs, and fusions. High specificity makes manual review and validation efficient, which is necessary in large-scale disease studies. Characterizing an extended range of chimera types will help generate insights into progression, treatment, and outcomes for complex diseases.

Complete genomic landscape of a recurring sporadic parathyroid carcinoma.

Parathyroid carcinoma is a rare endocrine malignancy with an estimated incidence of less than 1 per million population. Excessive secretion of parathyroid hormone, extremely high serum calcium level, and the deleterious effects of hypercalcaemia are the clinical manifestations of the disease. Up to 60% of patients develop multiple disease recurrences and although long-term survival is possible with palliative surgery, permanent remission is rarely achieved. Molecular drivers of sporadic parathyroid carcinoma have remained largely unknown. Previous studies, mostly based on familial cases of the disease, suggested potential roles for the tumour suppressor MEN1 and proto-oncogene RET in benign parathyroid tumourigenesis, while the tumour suppressor HRPT2 and proto-oncogene CCND1 may also act as drivers in parathyroid cancer. Here, we report the complete genomic analysis of a sporadic and recurring parathyroid carcinoma. Mutational landscapes of the primary and recurrent tumour specimens were analysed using high-throughput sequencing technologies. Such molecular profiling allowed for identification of somatic mutations never previously identified in this malignancy. These included single nucleotide point mutations in well-characterized cancer genes such as mTOR, MLL2, CDKN2C, and PIK3CA. Comparison of acquired mutations in patient-matched primary and recurrent tumours revealed loss of PIK3CA activating mutation during the evolution of the tumour from the primary to the recurrence. Structural variations leading to gene fusions and regions of copy loss and gain were identified at a single-base resolution. Loss of the short arm of chromosome 1, along with somatic missense and truncating mutations in CDKN2C and THRAP3, respectively, provides new evidence for the potential role of these genes as tumour suppressors in parathyroid cancer. The key somatic mutations identified in this study can serve as novel diagnostic markers as well as therapeutic targets.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

The clonal and mutational evolution spectrum of primary triple-negative breast cancers.

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.

Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers.

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co-deletion.

Whole-genome sequencing and social-network analysis of a tuberculosis outbreak.

BACKGROUND: An outbreak of tuberculosis occurred over a 3-year period in a medium-size community in British Columbia, Canada. The results of mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) genotyping suggested the outbreak was clonal. Traditional contact tracing did not identify a source. We used whole-genome sequencing and social-network analysis in an effort to describe the outbreak dynamics at a higher resolution. METHODS: We sequenced the complete genomes of 32 Mycobacterium tuberculosis outbreak isolates and 4 historical isolates (from the same region but sampled before the outbreak) with matching genotypes, using short-read sequencing. Epidemiologic and genomic data were overlaid on a social network constructed by means of interviews with patients to determine the origins and transmission dynamics of the outbreak. RESULTS: Whole-genome data revealed two genetically distinct lineages of M. tuberculosis with identical MIRU-VNTR genotypes, suggesting two concomitant outbreaks. Integration of social-network and phylogenetic analyses revealed several transmission events, including those involving "superspreaders." Both lineages descended from a common ancestor and had been detected in the community before the outbreak, suggesting a social, rather than genetic, trigger. Further epidemiologic investigation revealed that the onset of the outbreak coincided with a recorded increase in crack cocaine use in the community. CONCLUSIONS: Through integration of large-scale bacterial whole-genome sequencing and social-network analysis, we show that a socioenvironmental factor--most likely increased crack cocaine use--triggered the simultaneous expansion of two extant lineages of M. tuberculosis that was sustained by key members of a high-risk social network. Genotyping and contact tracing alone did not capture the true dynamics of the outbreak. (Funded by Genome British Columbia and others.).

De novo assembly and analysis of RNA-seq data.

We describe Trans-ABySS, a de novo short-read transcriptome assembly and analysis pipeline that addresses variation in local read densities by assembling read substrings with varying stringencies and then merging the resulting contigs before analysis. Analyzing 7.4 gigabases of 50-base-pair paired-end Illumina reads from an adult mouse liver poly(A) RNA library, we identified known, new and alternative structures in expressed transcripts, and achieved high sensitivity and specificity relative to reference-based assembly methods.

Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors.

BACKGROUND: Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment. RESULTS: In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways. CONCLUSIONS: We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.

System-level analysis of neuroblastoma tumor-initiating cells implicates AURKB as a novel drug target for neuroblastoma.

PURPOSE: Neuroblastoma (NB) is an aggressive tumor of the developing peripheral nervous system that remains difficult to cure in the advanced stages. The poor prognosis for high-risk NB patients is associated with common disease recurrences that fail to respond to available therapies. NB tumor-initiating cells (TICs), isolated from metastases and primary tumors, may escape treatment and contribute to tumor relapse. New therapies that target the TICs may therefore prevent or treat tumor recurrences. EXPERIMENTAL DESIGN: We undertook a system-level characterization of NB TICs to identify potential drug targets against recurrent NB. We used next-generation RNA sequencing and/or human exon arrays to profile the transcriptomes of 11 NB TIC lines from six NB patients, revealing genes that are highly expressed in the TICs compared with normal neural crest-like cells and unrelated cancer tissues. We used gel-free two-dimensional liquid chromatography coupled to shotgun tandem mass spectrometry to confirm the presence of proteins corresponding to the most abundant TIC-enriched transcripts, thereby providing validation to the gene expression result. RESULTS: Our study revealed that genes in the BRCA1 signaling pathway are frequently misexpressed in NB TICs and implicated Aurora B kinase as a potential drug target for NB therapy. Treatment with a selective AURKB inhibitor was cytotoxic to NB TICs but not to the normal neural crest-like cells. CONCLUSION: This work provides the first high-resolution system-level analysis of the transcriptomes of 11 primary human NB TICs and identifies a set of candidate NB TIC-enriched transcripts for further development as therapeutic targets.

Conserved role of intragenic DNA methylation in regulating alternative promoters.

Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.