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Abstract Background Male breast cancer (MBC) is an uncommon and relatively uncharacterised disease accounting for BRCA2 mutations. Here we describe clinicopathological features and genomic BRCA1 and BRCA2 mutation status in a large cohort of familial MBCs. Methods Cases (n=60) included 3 BRCA1 and 25 BRCA2 mutation carries, and 32 non-BRCA1/2 (BRCAX) carriers with strong family histories of breast cancer. The cohort was examined with respect to mutation status, clinicopathological parameters including TNM staging, grade, histological subtype and intrinsic phenotype. Results Compared to the general population, MBC incidence was higher in all subgroups. In contrast to female breast cancer (FBC) there was greater representation of BRCA2 tumours (41.7% vs 8.3%, p=0.0008) and underrepresentation of BRCA1 tumours (5.0% vs 14.4%, p=0.0001). There was no correlation between mutation status and age of onset, disease specific survival (DSS) or other clincopathological factors. Comparison with sporadic MBC studies showed similar clinicopathological features. Prognostic variables affecting DSS included primary tumour size (p=0.003, HR:4.26 95%CI 1.63-11.11), age (p=0.002, HR:4.09 95%CI 1.65-10.12), lymphovascular (p=0.019, HR:3.25 95%CI 1.21-8.74) and perineural invasion (p=0.027, HR:2.82 95%CI 1.13-7.06). Unlike familial FBC, the histological subtypes seen in familial MBC were more similar to those seen in sporadic MBC with 46 (76.7%) pure invasive ductal carcinoma of no special type (IDC-NST), 2 (3.3%) invasive lobular carcinomas and 4 (6.7%) invasive papillary carcinoma. A further 8 (13.3%) IDC-NST had foci of micropapillary differentiation, with a strong trend for co-occurrence in BRCA2 carriers (p=0.058). Most tumours were of the luminal phenotype (89.7%), with infrequent HER2 (8.6%) and basal (1.7%) phenotype tumours seen. Conclusion MBC in BRCA1/2 carriers and BRCAX families is different to females. Unlike FBC, a clear BRCA1 phenotype is not seen but a possible BRCA2 phenotype of micropapillary histological subtype is suggested. Comparison with sporadic MBCs shows this to be a high-risk population making further recruitment and investigation of this cohort of value in further understanding these uncommon tumours.

Abstract Background Detection of mutations by DNA sequencing can be facilitated by scanning methods to identify amplicons which may have mutations. Current scanning methods used for the detection of germline sequence variants are laborious as they require post-PCR manipulation. High resolution melting (HRM) is a cost-effective rapid screening strategy, which readily detects heterozygous variants by melting curve analysis of PCR products. It is well suited to screening genes such as BRCA1 and BRCA2 as germline pathogenic mutations in these genes are always heterozygous. Methods Assays for the analysis of all coding regions and intron-exon boundaries of BRCA1 and BRCA2 were designed, and optimised. A final set of 94 assays which ran under identical amplification conditions were chosen for BRCA1 (36) and BRCA2 (58). Significant attention was placed on primer design to enable reproducible detection of mutations within the amplicon while minimising unnecessary detection of polymorphisms. Deoxyinosine residues were incorporated into primers that overlay intronic polymorphisms. Multiple 384 well plates were used to facilitate high throughput. Results 169 BRCA1 and 239 BRCA2 known sequence variants were used to test the amplicons. We also performed an extensive blinded validation of the protocol with 384 separate patient DNAs. All heterozygous variants were detected with the optimised assays. Conclusions This is the first HRM approach to screen the entire coding region of the BRCA1 and BRCA2 genes using one set of reaction conditions in a multi plate 384 well format using specifically designed primers. The parallel screening of a relatively large number of samples enables better detection of sequence variants. HRM has the advantages of decreasing the necessary sequencing by more than 90%. This markedly reduced cost of sequencing will result in BRCA1 and BRCA2 mutation testing becoming accessible to individuals who currently do not undergo mutation testing because of the significant costs involved.

Abstract Background RNA extracted from formalin-fixed paraffin-embedded (FFPE) samples is chemically modified and degraded, which compromises its use in gene expression studies. Most of the current approaches for RNA quality assessment are not suitable for FFPE derived RNA. Results We have developed a single-tube multiplex endpoint RT-PCR assay specifically designed to evaluate RNA extracted from FFPE tissues for mRNA integrity and performance in reverse transcription - quantitative real-time PCR (RT-qPCR) assays. This single-tube quality control (QC) assay minimises the amount of RNA used in quality control. mRNA integrity and the suitability of RNA for RT-PCR is evaluated by the multiplex endpoint RT-PCR assay using the TBP gene mRNA as the target sequence. The RT-PCR amplicon sizes, 92, 161, 252 and 300 bp, cover a range of amplicon sizes suitable for a wide range of RT-qPCR assays. The QC assay was used to evaluate RNA prepared by two different protocols for extracting total RNA from needle microdissected FFPE breast tumour samples. The amplification products were analysed by gel electrophoresis where the spectrum of amplicon sizes indicated the level of RNA degradation and thus the suitability of the RNA for PCR. The ability of the multiplex endpoint RT-PCR QC assay to identify FFPE samples with an adequate RNA quality was validated by examining the Cq values of an RT-qPCR assay with an 87 bp amplicon. Conclusions The multiplex endpoint RT-PCR assay is well suited for the determination of the quality of FFPE derived RNAs, to identify which RT-PCR assays they are suitable for, and is also applicable to assess non-FFPE RNA for gene expression studies. Furthermore, the assay can also be used for the evaluation of RNA extraction protocols from FFPE samples.

Abstract Background Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes that are also known to act as tumour suppressor genes. Increased succinate or fumarate levels as a consequence of SDH and FH deficiency inhibit hypoxia inducible factor-1α (HIF-1α) prolyl hydroxylases leading to sustained HIF-1α expression in tumours. Since HIF-1α is frequently expressed in breast carcinomas, DNA methylation at the promoter regions of the SDHA, SDHB, SDHC and SDHD and FH genes was evaluated as a possible mechanism in silencing of SDH and FH expression in breast carcinomas. Findings No DNA methylation was identified in the promoter regions of the SDHA, SDHB, SDHC, SDHD and FH genes in 72 breast carcinomas and 10 breast cancer cell lines using methylation-sensitive high resolution melting which detects both homogeneous and heterogeneous methylation. Conclusion These results show that inactivation via DNA methylation of the promoter CpG islands of SDH and FH is unlikely to play a major role in sporadic breast carcinomas.

Abstract Background Epithelial growth factor receptor (EGFR) and KRAS mutation status have been reported as predictive markers of tumour response to EGFR inhibitors. High resolution melting (HRM) analysis is an attractive screening method for the detection of both known and unknown mutations as it is rapid to set up and inexpensive to operate. However, up to now it has not been fully validated for clinical samples when formalin-fixed paraffin-embedded (FFPE) sections are the only material available for analysis as is often the case. Methods We developed HRM assays, optimised for the analysis of FFPE tissues, to detect somatic mutations in EGFR exons 18 to 21. We performed HRM analysis for EGFR and KRAS on DNA isolated from a panel of 200 non-small cell lung cancer (NSCLC) samples derived from FFPE tissues. Results All 73 samples that harboured EGFR mutations previously identified by sequencing were correctly identified by HRM, giving 100% sensitivity with 90% specificity. Twenty five samples were positive by HRM for KRAS exon 2 mutations. Sequencing of these 25 samples confirmed the presence of codon 12 or 13 mutations. EGFR and KRAS mutations were mutually exclusive. Conclusion This is the first extensive validation of HRM on FFPE samples using the detection of EGFR exons 18 to 21 mutations and KRAS exon 2 mutations. Our results demonstrate the utility of HRM analysis for the detection of somatic EGFR and KRAS mutations in clinical samples and for screening of samples prior to sequencing. We estimate that by using HRM as a screening method, the number of sequencing reactions needed for EGFR and KRAS mutation detection can be reduced by up to 80% and thus result in substantial time and cost savings.

Abstract Background A recurrent somatic mutation, E17K, in the pleckstrin homology domain of the AKT1 gene, has been recently described in breast, colorectal, and ovarian cancers. AKT1 is a pivotal mediator of signalling pathways involved in cell survival, proliferation and growth. The E17K mutation stimulates downstream signalling and exhibits transforming activity in vitro and in vivo. Findings We developed a sensitive high resolution melting (HRM) assay to detect the E17K mutation from formalin-fixed paraffin-embedded tumours. We screened 219 non-small cell lung cancer biopsies for the mutation using HRM analysis. Four samples were identified as HRM positive. Subsequent sequencing of those samples confirmed the E17K mutation in one of the cases. A rare single nucleotide polymorphism was detected in each of the remaining three samples. The E17K was found in one of the 14 squamous cell carcinomas. No mutations were found in 141 adenocarcinomas and 39 large cell carcinomas. Conclusion The AKT1 E17K mutation is very rare in lung cancer and might be associated with tumorigenesis in squamous cell carcinoma. HRM represents a rapid cost-effective and robust screening of low frequency mutations such as AKT1 mutations in clinical samples.

Abstract Background Germline inactivating mutations in BRCA1 and BRCA2 underlie a major proportion of the inherited predisposition to breast and ovarian cancer. These mutations are usually detected by DNA sequencing. Cost-effective and rapid methods to screen for these mutations would enable the extension of mutation testing to a broader population. High resolution melting (HRM) analysis is a rapid screening methodology with very low false negative rates. We therefore evaluated the use of HRM as a mutation scanning tool using, as a proof of principle, the three recurrent BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population in addition to other mutations that occur in the same regions. Methods We designed PCR amplicons for HRM scanning of BRCA1 exons 2 and 20 (carrying the founder mutations185delAG and 5382insC respectively) and the part of the BRCA2 exon 11 carrying the 6174delT founder mutation. The analysis was performed on an HRM-enabled real time PCR machine. Results We tested DNA from the peripheral blood of 29 individuals heterozygous for known mutations. All the Ashkenazi founder mutations were readily identified. Other mutations in each region that were also readily detected included the recently identified Greek founder mutation 5331G>A in exon 20 of BRCA1. Each mutation had a reproducible melting profile. Conclusion HRM is a simple and rapid scanning method for known and unknown BRCA1 and BRCA2 germline mutations that can dramatically reduce the amount of sequencing required and reduce the turnaround time for mutation screening and testing. In some cases, such as tracking mutations through pedigrees, sequencing may only be necessary to confirm positive results. This methodology will allow for the economical screening of founder mutations not only in people of Ashkenazi Jewish ancestry but also in other populations with founder mutations such as Central and Eastern Europeans (BRCA1 5382insC) and Greek Europeans (BRCA1 5331G>A).

Abstract Background p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. Methods We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. Results One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. Conclusion HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53.

Background: Little is known about what components geriatricians routinely incorporate into outpatient comprehensive geriatric assessments (CGAs)., Aims: This study explored what components of CGAs are routinely incorporated into geriatricians' letters and assessed their consistency with the Medicare Benefits Schedule (MBS) and a recently published survey of geriatricians., Methods: We completed a manual content analysis, supplemented by qualitative thematic analysis, of 34 letters from five geriatricians, collected as part of the GOAL Trial., Results: While more than 80% of letters included each of the key clinical domains described in the Medicare Benefits Schedule and survey of geriatricians, only 62% included advanced care planning and 47% mentioned immunisations. Forty-seven percent of letters included goal setting. Few letters showed evidence of multidisciplinary working. Issues identified by the geriatrician centred around the themes of advance care planning, symptom identification and management, medical comorbidities, strategies to support quality of life and interventions to manage frailty. Patient concerns identified in the letters were cognition and mood, declining function, future planning and symptom management., Conclusions: Analysis of geriatricians' letters provides important and novel insights into usual CGA practice. The letters provide evidence of multidimensional assessments of physical, functional, social and psychological health, and most include use of standardised tools. However, less than 50% include evidence of goal setting or multidisciplinary working. The results allow consideration of how CGAs might be carried out in the outpatient setting, so that interventions focused on improving the quality and efficacy of this intervention can be implemented., (© 2024 The Author(s). Internal Medicine Journal published by John Wiley & Sons Australia, Ltd on behalf of Royal Australasian College of Physicians.)

Introduction: The GOAL Cluster Randomised Controlled Trial (NCT04538157) is now underway, investigating the impact of comprehensive geriatric assessment (CGA) for frail older people with chronic kidney disease (CKD). The primary outcome is the attainment of patient-identified goals at 3 months, assessed using the goal attainment scaling process. The protocol requires a dedicated process evaluation that will occur alongside the main trial, to investigate issues of implementation, mechanisms of impact and contextual factors that may influence intervention success. This process evaluation will offer novel insights into how and why CGA might be beneficial for frail older adults with CKD and provide guidance when considering how to implement this complex intervention into clinical practice., Methods and Analysis: This process evaluation protocol follows guidance from the Medical Research Council and published guidance specific for the evaluation of cluster-randomised trials. A mixed methodological approach will be taken using data collected as part of the main trial and data collected specifically for the process evaluation. Recruitment and process data will include site feasibility surveys, screening logs and site issues registers from all sites, and minutes of meetings with intervention and control sites. Redacted CGA letters will be analysed both descriptively and qualitatively. Approximately 60 semistructured interviews will be analysed with a qualitative approach using a reflexive thematic analysis, with both inductive and deductive approaches underpinned by an interpretivist perspective. Qualitative analyses will be reported according to the Consolidated criteria for Reporting Qualitative research guidelines. The Standards for Quality Improvement Reporting Excellence guidelines will also be followed., Ethics and Dissemination: Ethics approval has been granted through Metro South Human Research Ethics Committee (HREC/2020/QMS/62883). Dissemination will occur through peer-reviewed journals and feedback to trial participants will be facilitated through the central coordinating centre., Trial Registration Number: NCT04538157., Competing Interests: Competing interests: DJ has received consultancy fees, research grants, speaker’s honoraria and travel sponsorships from Baxter Healthcare and Fresenius Medical Care, consultancy fees from Astra Zeneca, Bayer and AWAK, speaker’s honoraria from ONO and Boehringer Ingelheim & Lilly, and travel sponsorships from Ono and Amgen., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Comprehensive geriatric assessment (CGA) is the gold standard model of care for older adults with frailty. However, despite a large number of published clinical trials, there remain many unanswered questions about how CGA works in different circumstances. This uncertainty stems from CGA being a deeply complex intervention that is heavily modified by context. This review describes recent and novel methodological approaches that explore the active ingredients of CGA and their interaction with context. Future research should continue to embrace broad methodologies that can help us better understand this intervention, in such a way that it can be implemented with fidelity and associated with positive outcomes for older adults., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objectives: To assess whether the Cognitive Functional Independence Measure (Cog-FIM) is correlated with the Standardised Mini-Mental State Examination (SMMSE) and Rowland Universal Dementia Assessment Scale (RUDAS) and whether there is agreement between the Cog-FIM and these two tests., Methods: Functional Independence Measure assessments were undertaken on 98 subacute patients. Forty-eight (48) patients were administered the SMMSE, and 50 were administered the RUDAS. Agreement was examined using the Bland-Altman plot., Results: Correlation was observed between the Cog-FIM and both the SMMSE and the RUDAS. The Bland-Altman analysis demonstrated agreement between the Cog-FIM and RUDAS, but not the Cog-FIM and SMMSE. The limits of agreement between the Cog-FIM and RUDAS were -13 to 13., Conclusions: The Cog-FIM is correlated with the RUDAS, but the agreement is unclear. Despite agreement of the means, the limits of agreement are large, which may suggest a clinically meaningful difference. The study should be repeated with a larger sample size., (© 2021 AJA Inc.)

Background: Frailty is prevalent in older adults with lung cancer, however the impact of frailty in this population is not well understood. The aim of this review was to evaluate the outcomes that are measured in frail older adults with lung cancer, and to determine the associations between frailty and these outcomes., Methods: A systematic online search of PubMed, EMBASE, and Cochrane databases was conducted to identify all English-language studies between January 2015 and May 2022 prospectively evaluating frailty and outcomes in older adults (median age > 65 years) with lung cancer. Studies were excluded if frailty was defined by a single domain assessment or not clearly defined. Quality was assessed using the Newcastle-Ottawa Scale., Results: Of 1891 studies screened, 16 met inclusion criteria. The median number of patients was 96 (range 26-494) and the mean age was 76.6 years. Eight different frailty assessments were used, and frailty definitions varied widely. The most frequently assessed outcomes were overall survival (n = 13,81%), treatment-related toxicity (n = 8,50%), hospitalisation (n = 5,31%), and treatment completion/discontinuation (n = 4,25%). Quality of life (n = 3,19%), function (n = 1,6%), frailty trajectory (n = 1,6%), and emergency visits (n = 1,6%) were infrequently assessed. Frailty had a strong and consistent association with mortality (Hazard Ratio range: 3.5-11.91). It was also associated with treatment-related toxicity and treatment selection. The remaining outcomes were not statistically significant., Conclusion: These data support frailty as an important predictor of mortality in older adults with lung cancer, however further research is warranted to determine the association between frailty and other meaningful endpoints for this vulnerable population., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr James Fletcher reports honorarium from Astra Zeneca. Dr Rahul Ladwa reports honorarium from Astra Zeneca, Bristol Myers Squibb and MSD, and consulting fees from Roche and Astra Zeneca. The remaining authors report no conflicts of interest., (Copyright © 2022. Published by Elsevier Ltd.)

Competing Interests: Competing interests: None declared.

Objective: A pilot questionnaire was developed to investigate the cognitive tests that clinicians employ in their clinical practice and whether they perceive the Functional Independence Measure (FIM) to add clinical value., Methods: A 10-item pilot questionnaire was created and distributed to 43 health professionals of a single health service in Australia. A snowballing approach to sampling was used., Results: There were 39 respondents. Less than half of respondents thought the FIM added clinical value, and 15% stated that they would use the FIM to assess cognition on subacute wards. Ninety-seven per cent (97%) of respondents stated that they would rely more heavily on cognitive screening tests than the FIM., Conclusion: Participant responses to this survey of a single health service raise interesting questions about how useful clinicians perceive the FIM to be, beyond a costing and benchmarking role. Clinicians may prefer cognitive screening tests to the FIM, in clinical practice., (© 2021 AJA Inc.)

Common factors are nonspecific therapeutic elements common across different varieties of psychotherapy. In a recent study, 68 expert psychotherapy researchers with a variety of allegiances collectively rated biofeedback as being negatively associated with many common factors (Tschacher et al. in Clin Psychol Psychother 21(1):82-96, 2014), including the therapeutic alliance. However, it seems implausible that biofeedback could benefit so many people while being incompatible with the therapeutic alliance and other common factors. The present study investigated the experiences of biofeedback clients who participated in a brief heart rate variability biofeedback protocol in order to explore the potential roles of common factors in biofeedback. The results of this study offer preliminary evidence that many common factors-including therapeutic alliance, self-efficacy expectation, mastery experiences, provision of explanatory scheme, mindfulness, and even cognitive restructuring-may play a role in biofeedback outcomes. Future research on this topic should include mediation and moderation models investigating the role of specific common factors on outcome and process studies to help determine what clinician behaviors are most helpful. Deeper investigation of common factors in biofeedback may benefit future biofeedback research and practice and address the concerns of colleagues outside of the biofeedback community who believe that biofeedback is at odds with common factors.

Drinking water disinfection by-products (DBPs), including the ubiquitous trihalomethanes (THMs), are formed during the treatment of water with disinfectants (e.g., chlorine, chloramines) to produce and distribute potable water. Brominated THMs (Br-THMs) are activated to mutagens via glutathione S-transferase theta 1 (GSTT1); however, iodinated THMs (I-THMs) have never been evaluated for activation by GSTT1. Among the I-THMs, only triiodomethane (iodoform) has been tested previously for mutagenicity in Salmonella and was positive (in the absence of GSTT1) in three strains (TA98, TA100, and BA13), all of which have error-prone DNA repair (pKM101). We evaluated five I-THMs (chlorodiiodomethane, dichloroiodomethane, dibromoiodomethane, bromochloroiodomethane, and triiodomethane) for mutagenicity in Salmonella strain RSJ100, which expresses GSTT1, and its homologue TPT100, which does not; neither strain has pKM101. We also evaluated chlorodiiodo-, dichloroiodo-, and dibromoiodo-methanes in strain TA100 +/- rat liver S9 mix; TA100 has pKM101. None was mutagenic in any of the strains. The I-THMs were generally more cytotoxic than their brominated and chlorinated analogues but less cytotoxic than analogous trihalonitromethanes tested previously. All five I-THMs showed similar thresholds for cytotoxicity at ~2.5 μmoles/plate, possibly due to release of iodine, a well-known antimicrobial. Although none of these I-THMs was activated by GSTT1, iodoform appears to be the only I-THM that is mutagenic in Salmonella, only in strains deficient in nucleotide excision repair (uvrB) and having pKM101. Given that only iodoform is mutagenic among the I-THMs and is generally present at low concentrations in drinking water, the I-THMs likely play little role in the mutagenicity of drinking water., (© 2021 Wiley Periodicals LLC.)