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Background & Methods In this study, a novel restriction enzyme (RE) digestion-based droplet digital polymerase chain reaction (ddPCR) assay was designed for cg005575921 within the AHRR gene body and compared with matching results obtained by bisulfite conversion (BIS) ddPCR and Illumina DNA methylation array. Results The RE ddPCR cg05575921 assay appeared concordant with BIS ddPCR ( r 2  = 0.94, P  < 0.0001) and, when compared with the Illumina array, had significantly better smoking status classification performance for current versus never smoked (AUC 0.96 versus 0.93, P  < 0.04) and current versus ex-smoker (AUC 0.88 versus 0.83, P  < 0.04) comparisons. Conclusions The RE ddPCR cg05575921 assay accurately predicts smoking status and could be a useful component of ‘precision-medicine’ chronic disease risk screening tools.

Background Endometrial cancer (EC) is the most common gynaecological malignancy globally, with rising incidence and notable disparities in outcomes. In New Zealand, EC rates have increased significantly, particularly among Māori and Pacific women, who face higher risks of advanced disease and poorer outcomes. Microbial dysbiosis has been implicated in EC pathogenesis, but characterising the uterine microbiome is challenging due to low microbial biomass and high contamination risk. Aims This study aimed to pilot a protocol that could inform the preparation of a larger cohort trial. Short‐read Illumina MiSeq and long‐read Oxford Nanopore Technologies (ONT) 16S rRNA gene sequencing were investigated to profile the uterine microbiome in people with EC. Methods and Results Uterine and vaginal swabs were analysed to assess platform performance in terms of DNA recovery, sequencing success, diversity metrics, and taxonomic resolution. The impact of sample freezing or immediate lysis prior to DNA extraction was also evaluated. ONT sequencing provided enhanced species‐level resolution and improved detection of low‐abundance taxa but showed variable performance in low‐yield samples. Freezing prior to cell DNA extraction modestly increased bacterial 16S copy numbers and improved community consistency. Contamination was a problem across both platforms, particularly in low‐biomass samples, but can be minimised during data analysis. Conclusion This study provides practical guidance for sequencing platform selection and sample handling in uterine microbiome research. Our findings support future efforts to elucidate microbial contributions to EC pathogenesis and highlight the importance of rigorous contamination control. Importantly, this is the first presentation of a New Zealand cohort and contributes valuable data from an underrepresented population and informs future research in diverse clinical settings.

The nucleic acid-binding protein YB-1, a member of the cold-shock domain protein family, has been implicated in the progression of breast cancer and is associated with poor patient survival. YB-1 has sequence similarity to LIN28, another cold-shock protein family member, which has a role in the regulation of small noncoding RNAs (sncRNAs) including microRNAs (miRNAs). Therefore, to investigate whether there is an association between YB-1 and sncRNAs in breast cancer, we investigated whether sncRNAs were bound by YB-1 in two breast cancer cell lines (luminal A-like and basal cell-like), and whether the abundance of sncRNAs and mRNAs changed in response to experimental reduction of YB-1 expression. RNA-immunoprecipitation with an anti-YB-1 antibody showed that several sncRNAs are bound by YB-1. Some of these were bound by YB-1 in both breast cancer cell lines; others were cell-line specific. The small RNAs bound by YB-1 were derived from various sncRNA families including miRNAs such as let-7 and miR-320, transfer RNAs, ribosomal RNAs and small nucleolar RNAs (snoRNA). Reducing YB-1 expression altered the abundance of a number of transcripts encoding miRNA biogenesis and processing proteins but did not alter the abundance of mature or precursor miRNAs. YB-1 binds to specific miRNAs, snoRNAs and tRNA-derived fragments and appears to regulate the expression of miRNA biogenesis and processing machinery. We propose that some of the oncogenic effects of YB-1 in breast cancer may be mediated through its interactions with sncRNAs.

TP53, the most commonly-mutated gene in cancer, undergoes complex alternative splicing. Different TP53 transcripts play different biological roles, both in normal function and in the progression of diseases such as cancer. The study of TP53’s alternative RNA splice forms and their use as clinical biomarkers has been hampered by limited specificity and quantitative accuracy of current methods. TP53 RNA splice variants differ at both 5’ and 3’ ends, but because they have a common central region of 618 bp, the individual TP53 transcripts are impossible to specifically detect and precisely quantitate using standard PCR-based methods or short-read RNA sequencing. Therefore, we devised multiplex probe-based long amplicon droplet digital PCR (ddPCR) assays, which for the first time allow precise end-to-end quantitation of the seven major TP53 transcripts, with amplicons ranging from 0.85 to 1.85 kb. Multiple modifications to standard ddPCR assay procedures were required to enable specific co-amplification of these long transcripts and to overcome issues with secondary structure. Using these assays, we show that several TP53 transcripts are co-expressed in breast cancers, and illustrate the potential for this method to identify novel TP53 transcripts in tumour cells. This capability will facilitate a new level of biological and clinical understanding of the alternatively-spliced TP53 isoforms.

Objective This paper describes the 4-year journey of Hunter and New England HealthPathways - a password-protected web-based portal designed to provide localised evidence-informed clinical and referral information to support general practice at the point of care. Methods A process evaluation was conducted in 2013, with a case study comparison performed in 2014 to assess impact of HealthPathways on patient referral and access to specialist care, followed by a review in 2016 of utilisation of the online portal to assess whether healthcare providers continued to access HealthPathways. Results Increased utilisation was correlated with an increase in the number of pathways published online. Clinical leadership and the process of developing pathways built relationships between primary care and specialist teams. Case studies indicated that a comprehensive approach to pathway implementation accompanied by service redesign resulted in higher pathway use and improved access to specialist care. Senior management support and a formal partnership between major health care providers led to strong governance of HealthPathways and the delivery of other integrated care initiatives. There was significant growth in utilisation over the 4 years, increasing to an average of 6679 sessions per month in 2016 and more general practices reported use of HealthPathways. Conclusions HealthPathways is a vehicle for building strong foundations to support system change and integrated care. The critical elements for acceptability, growth and sustainability are the strong relationships between primary care and specialist clinicians, as well as formal partnerships that are built from the processes used to develop HealthPathways. What is known about the topic? HealthPathways and similar web-based evidence-informed guidelines aimed at improving system integration are increasing in Australia. There are few published papers that describe approaches to inform the ongoing implementation of such programs. What does this paper add? This paper describes iterative methodology for evaluating complex programs, such as HealthPathways, that identifies the critical factors required to build sustainable models of integrated care. What are the implications for practitioners? The 4-year experience of Hunter and New England HealthPathways provides an approach to improve the implementation, sustainability and spread of similar programs and associated integrated care initiatives.

This study sought to understand the unique types of social support salient to mental health service-users from the perspective of case managers. The sample consisted of case managers working in county mental health agencies in the southwest and west coast. Data was gathered from three focus groups and analyzed using NVivo 10 and Consensual Qualitative Research. Six themes were described including relational support, consistency support, validation and affirmation support, social connection support, day-to-day living support and vocational support. While the social support domains described in this study share conceptual underpinnings with traditional conceptualizations of support, our findings reveal unique types of support from the perspective of case managers. Findings from this study offer an important perspective—case managers—to the extant body of research investigating the meaning of social support for people with lived mental health experiences. Of particular interest is the finding that relational support, affirmative and validation support, and consistency support are salient case manager functions.

Custom-compounded subcutaneous implants are being used widely in Australia for gender-affirming hormone therapy. However, there is no published literature regarding their use for this purpose. Electronic medical records were audited for consecutive clients who received oestradiol implants April 2019-November 2022 in gender clinics held within Hunter New England Health District in New South Wales, Australia. Serum oestradiol levels were analysed for implant doses 50-200mg, and predicted oestradiol level was modelled following 100mg implant insertion. An electronic consumer survey was sent to a convenience sample of implant recipients. A total of 38 clients received 88 implants, with 100mg oestradiol implants being the most frequently used (68%). The median interval between insertion procedures was 270 (IQR 186-399) days. The median serum oestradiol levels following implant insertion, for all implants combined, were within the target range of 250-600pmol/L at 1-, 3-, 6-, 9- and 12-month time points. Following insertion of a 100mg implant, the estimated time to reach a predicted serum oestradiol of ≤250pmol/L was 4months after an initial implant, and 13months after subsequent implants. Seventeen consumer surveys were received from 28 invitations. All respondents had previous experience of oral and/or transdermal oestradiol use. Oestradiol implants were preferred due to ease of use, perceived effectiveness, and the belief that other methods were less safe or associated with intolerance and side effects. Oestradiol implants are effective in achieving target serum oestradiol levels over a sustained period. Further research with larger cohorts could identify the optimal dosage regimen.

Background Circulating tumour DNA (ctDNA) analysis promises to improve the clinical care of people with cancer, address health inequities and guide translational research. This observational cohort study used ctDNA to follow 29 patients with advanced-stage cutaneous melanoma through multiple cycles of immunotherapy. Method A melanoma-specific ctDNA next-generation sequencing (NGS) panel, droplet digital polymerase chain reaction (ddPCR) and mass spectrometry analysis were used to identify ctDNA mutations in longitudinal blood plasma samples from Aotearoa New Zealand (NZ) patients receiving immunotherapy for melanoma. These technologies were used in conjunction to identify the breadth and complexity of tumour genomic information that ctDNA analysis can reliably report. Results During the course of immunotherapy treatment, a high level of dynamic mutational complexity was identified in blood plasma, including multiple BRAF mutations in the same patient, clinically relevant BRAF mutations emerging through therapy and co-occurring sub-clonal BRAF and NRAS mutations. The technical validity of this ctDNA analysis was supported by high sample analysis–reanalysis concordance, as well as concordance between different ctDNA measurement technologies. In addition, we observed > 90% concordance in the detection of ctDNA when using cell-stabilising collection tubes followed by 7-day delayed processing, compared with standard EDTA blood collection protocols with rapid processing. We also found that the undetectability of ctDNA at a proportion of treatment cycles was associated with durable clinical benefit (DCB). Conclusion We found that multiple ctDNA processing and analysis methods consistently identified complex longitudinal patterns of clinically relevant mutations, adding support for expanded clinical trials of this technology in a variety of oncology settings.