Whole-genome sequencing reveals clinically relevant insights into the aetiology of familial breast cancers.

BACKGROUND: Whole-genome sequencing (WGS) is a powerful method for revealing the diversity and complexity of the somatic mutation burden of tumours. Here, we investigated the utility of tumour and matched germline WGS for understanding aetiology and treatment opportunities for high-risk individuals with familial breast cancer. PATIENTS AND METHODS: We carried out WGS on 78 paired germline and tumour DNA samples from individuals carrying pathogenic variants in BRCA1 (n = 26) or BRCA2 (n = 22) or from non-carriers (non-BRCA1/2; n = 30). RESULTS: Matched germline/tumour WGS and somatic mutational signature analysis revealed patients with unreported, dual pathogenic germline variants in cancer risk genes (BRCA1/BRCA2; BRCA1/MUTYH). The strategy identified that 100% of tumours from BRCA1 carriers and 91% of tumours from BRCA2 carriers exhibited biallelic inactivation of the respective gene, together with somatic mutational signatures suggestive of a functional deficiency in homologous recombination. A set of non-BRCA1/2 tumours also had somatic signatures indicative of BRCA-deficiency, including tumours with BRCA1 promoter methylation, and tumours from carriers of a PALB2 pathogenic germline variant and a BRCA2 variant of uncertain significance. A subset of 13 non-BRCA1/2 tumours from early onset cases were BRCA-proficient, yet displayed complex clustered structural rearrangements associated with the amplification of oncogenes and pathogenic germline variants in TP53, ATM and CHEK2. CONCLUSIONS: Our study highlights the role that WGS of matched germline/tumour DNA and the somatic mutational signatures can play in the discovery of pathogenic germline variants and for providing supporting evidence for variant pathogenicity. WGS-derived signatures were more robust than germline status and other genomic predictors of homologous recombination deficiency, thus impacting the selection of platinum-based or PARP inhibitor therapy. In this first examination of non-BRCA1/2 tumours by WGS, we illustrate the considerable heterogeneity of these tumour genomes and highlight that complex genomic rearrangements may drive tumourigenesis in a subset of cases.

The provision of seven day multidisciplinary staffing in Scottish acute medical units: a cross-sectional study.

BACKGROUND: Acute medical units (AMUs) are a central component of the admission pathway for the majority of medical patients presenting to hospital in the United Kingdom and other international settings. Detail on multidisciplinary staffing provision on weekdays and weekends is lacking. Equity of staffing across 7 days is a strategic priority for national health services in the United Kingdom. AIM: To evaluate weekday compared with weekend multidisciplinary staffing in a national set of AMUs. DESIGN: Cross-sectional survey. METHODS: Twenty-nine Scottish AMUs were identified and all were included in the study population. Data were collected by semi-structured interviews with nursing, pharmacy, therapy, non-consultant medical and consultant staff. Staffing was quantified in staff hours. A correction factor of 0.5 was applied to non-dedicated staff. The percentage of weekend/weekday staffing was calculated for each unit and the mean of these percentages was calculated to give a summary measure for each professional group. RESULTS: As a percentage of weekday staffing levels, weekend staffing across the units was 93.8% for nursing staff; 2.2% for pharmacy staff; 13.1% for therapy staff; 69.6% for non-consultant staff and 65.0% for consultant staff. CONCLUSIONS: There is a contrast between weekday and weekend staffing on the AMU, with reductions at weekends in total staff hours, the proportion of dedicated vs. undedicated staff and the seniority of nursing staff. The weekday/weekend difference was far more pronounced for allied healthcare professional staff than any other group. These findings have potential implications for patient outcomes, quality of care, hospital flow and workforce planning.

How is it best to deliver care in acute medical units? A systematic review.

The majority of medical patients presenting to hospital in the UK are cared for in acute medical units (AMUs). Such units are also increasingly present internationally. Care delivery varies across units: this review aims to examine the evidence for how best to deliver AMU care.Six electronic databases and grey literature were searched. Inclusion criteria comprised interventions applied to undifferentiated patients in AMU settings. All studies were quality assessed. A narrative approach was undertaken.Nine studies, all conducted in the UK or Ireland, evaluated 1.3 million episodes, 3617 patients and 49 staff. There was single study evidence for beneficial effects of: enhanced pharmacy care, a dedicated occupational therapy service, an all-inclusive consultant work pattern, a rapid-access medical clinic and formalized handovers. Two studies found increased consultant presence was associated with reduced mortality; one of these studies found an association with a reduction in 28-day readmissions; and the other found an association with an increased proportion of patients discharged on the day they were admitted. Three studies provide evidence of the beneficial effects of multiple interventions developed from local service reviews.Overall, the quality of the evidence was limited. This review has identified operationally relevant evidence that increased consultant presence is associated with improved outcomes of care; has highlighted the potential to improve outcomes locally through service reviews; and has demonstrated an important knowledge gap of how best to deliver AMU care. These findings have importance given the challenges acute services currently face.

COX-2 expression is predictive for early relapse and aromatase inhibitor resistance in patients with ductal carcinoma in situ of the breast, and is a target for treatment.

BACKGROUND: Stratification of patients for treatment of ductal carcinoma in situ (DCIS) is suboptimal, with high systemic overtreatment rates. METHODS: A training set of 95 tumours from women with pure DCIS were immunostained for proteins involved in cell survival, hypoxia, growth factor and hormone signalling. A generalised linear regression with regularisation and variable selection was applied to a multiple covariate Cox survival analysis with recurrence-free survival 10-fold cross-validation and leave-one-out iterative approach were used to build and test the model that was validated using an independent cohort of 58 patients with pure DCIS. The clinical role of a COX-2-targeting agent was then tested in a proof-of-concept neoadjuvant randomised trial in ER-positive DCIS treated with exemestane 25 mg day(-1)± celecoxib 800 mg day(-1). RESULTS: The COX-2 expression was an independent prognostic factor for early relapse in the training (HR 37.47 (95% CI: 5.56-252.74) P=0.0001) and independent validation cohort (HR 3.9 (95% CI: 1.8-8.3) P=0.002). There was no significant interaction with other clinicopathological variables. A statistically significant reduction of Ki-67 expression after treatment with exemestane ± celecoxib was observed (P<0.02) with greater reduction in the combination arm (P<0.004). Concomitant reduction in COX-2 expression was statistically significant in the exemestane and celecoxib arm (P<0.03) only. CONCLUSIONS: In patients with DCIS, COX-2 may predict recurrence, aiding clinical decision making. A combination of an aromatase inhibitor and celecoxib has significant biological effect and may be integrated into treatment of COX2-positive DCIS at high risk of recurrence.

Effect of trilostane and mitotane on aldosterone secretory reserve in dogs with pituitary-dependent hyperadrenocorticism.

BACKGROUND: Maximal aldosterone secretion in healthy dogs occurs 30 minutes postadrenocorticotropin (ACTH; 5 µg/kg IV) stimulation. The effect of trilostane and mitotane on aldosterone at that time is unknown. OBJECTIVES: To assess the effect of trilostane and mitotane in dogs with pituitary-dependent hyperadrenocorticism on aldosterone secretory reserve. To determine if aldosterone concentration correlates with electrolyte concentrations. ANIMALS: Serum collected from 79 client-owned dogs and 33 stored samples. METHODS: Client-owned dogs had ACTH stimulation tests with cortisol concentrations measured at 0 and 60 minutes and aldosterone concentrations measured at 0, 30, and 60 minutes. Stored samples had aldosterone concentrations measured at 0 and 60 minutes. Ten historical clinically healthy controls were included. All had basal sodium and potassium concentrations measured. RESULTS: The aldosterone concentrations in the mitotane- and trilostane-treated dogs at 30 and 60 minutes post-ACTH were significantly lower than in clinically healthy dogs; no significant difference was detected in aldosterone concentration between 30 and 60 minutes in treated dogs. However, a significantly higher percentage of dogs had decreased aldosterone secretory reserve detected at 30 minutes than at 60 minutes. At 30 minutes, decreased secretory reserve was detected in 49% and 78% of trilostane- and mitotane-treated dogs, respectively. No correlation was detected between aldosterone and serum electrolyte concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: Decreased aldosterone secretory reserve is common in trilostane- and mitotane-treated dogs; it cannot be predicted by measurement of serum electrolyte concentrations. Aldosterone concentration at 30 minutes post-ACTH stimulation identifies more dogs with decreased aldosterone secretory reserve than conventional testing at 60 minutes.

Mutations in EGFR, BRAF and RAS are rare in triple-negative and basal-like breast cancers from Caucasian women.

Basal-like and triple-negative breast cancers usually display a high level of genomic instability and often carry TP53 mutations. Mutations in EGFR have been reported in about 10 % triple-negative tumours from Chinese women, and there is some evidence that triple-negative and basal-like tumours might carry additional mutations against which targeted therapies are available. We, therefore, sought to determine the frequency of 238 targetable mutations in 19 oncogenes (including EGFR) in a panel of basal-like and triple-negative breast cancers from Caucasian women. We used the OncoCarta panel to screen for 238 mutations across 19 common oncogenes in 107 basal-like and triple-negative breast cancers from Caucasian women. Mutations were then verified using Sanger sequencing or primer extension by iPLEX. We identified and validated 10 mutations across five genes. Most of the mutations were observed in the PIK3CA gene (18/107, 16.8 %), while mutations in KRAS, NRAS, MET and AKT1 were present in only one tumour each (1/107, 0.9 %). Among the missense substitutions in PIK3CA the point mutation resulting in the amino acid change H1047R was the most frequent (8/18, 44 %). All mutations were mutually exclusive, apart from one basal-like breast tumour which harboured mutations in both MET (p.T992I) and PIK3CA (p.H1047R). We did not identify any mutations in the EGFR gene. In conclusion, we found that with the exception of mutations in PIK3CA, these actionable oncogenic mutations on the Oncocarta panel are rare in basal-like and triple-negative breast cancers from Caucasian women. Custom panels, designed to detect mutations identified by exome sequencing of basal-like and triple-negative breast cancers, are, therefore, needed to identify women who might be eligible for targeted treatment.

Proliferation, apoptosis, and survival in high-level microsatellite instability sporadic colorectal cancer.

Sporadic colorectal cancer (CRC) characterized by high-level DNA microsatellite instability (MSI-H) has a favorable prognosis. The reason for this MSI-H survival advantage is not known. The aim of this study was to correlate proliferation, apoptosis, and prognosis in CRC stratified by MSI status. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody in a selected series of 100 sporadic colorectal cancers classified according to the level of MSI as 31 MSI-H, 29 MSI-Low (MSI-L), and 40 microsatellite stable (MSS). The Ki-67 index was significantly higher in MSI-H cancers (P < 0.0001) in which the PI was 90.1 +/- 1.2% (mean +/- SE) compared with 69.5 +/- 3.1% and 69.5 +/- 2.3% in MSI-L and MSS subgroups, respectively. There was a positive linear correlation between the apoptotic index (AI) and PI (r = 0.51; P < 0.001), with MSI-H cancers demonstrating an increased AI:PI ratio indicative of a lower index of cell production. A high PI showed a trend toward predicting improved survival within MSI-H cancers (P = 0.09) but did not predict survival in MSI-L or MSS cancers. The AI was not associated with survival in any MSI subgroup. In conclusion, this is the first study to show that sporadic MSI-H cancers are characterized by a higher AI:PI ratio and increased proliferative activity compared with MSI-L and MSS cancers, and that an elevated PI may confer a survival advantage within the MSI-H subset.

Consultant outreach, 1991 to 1998. An update and extension on its distribution in Scotland.

OBJECTIVE: To assess the extent and distribution of consultant outreach in Scotland between 1991 and 1998. DESIGN: The paper has three parts. First a description of the trends in consultants and consultant activity provides the background. This is followed by the results of an update of the 1991 survey of all health centres in Scotland and its extension to all GP premises considered suitable to hold consultant clinics. Finally, binary regression analysis of outreach is used to test the importance of total list size, distance to alternative provision and deprivation. Fourteen of the most common consultant specialties are studied. SETTING AND SUBJECTS: Scotland-wide data on consultants and consultant activity using annual data over the 1990s; and a Scotland-wide survey of 231 health centres and 312 GP premises over the period July to December 1998. RESULTS AND CONCLUSIONS: Consultant full time equivalents (ftes) increased and, with minor exceptions, consultant activity did so too. In respect of outreach, the increase was largely at GP premises and for psychiatry. For only two specialties of the fourteen studied, obstetrics and general psychiatry, could outreach be considered important. Such outreach provision as was made went where the total list size was largest and alternative provision farthest distant. The evidence that deprivation had an influence on outreach varies with specialty and is qualified.

Acinar cell apoptosis and the origin of tubular complexes in caerulein-induced pancreatitis.

The interrelationship between acinar cell apoptosis and tubular complex formation was examined in caerulein-induced pancreatitis using histology, immunohistochemistry, electron microscopy and DNA gel electrophoresis. Rats were given 8 hourly subcutaneous injections of caerulein, 24 micrograms/kg, for up to 2 days. Morphologically and biochemically typical apoptosis affected 4.6 and 8.9% of acinar cells at 1 and 2 days, respectively, resulting in removal of most acinar cells by 2 days. Consequently, pancreatic ducts, the lining cells expressing bcl-2 and therefore resistant to apoptosis, became much more closely approximated to form the basis of tubular complexes; small numbers of immunohistochemically discrete acinar cells in their lining were either pre-apoptotic resistant to it or newly formed. Proliferation of duct-like lining cells was associated with apoptosis, an increase in islet cells and acinar cell regeneration. There was evidence of duct to acinar cell differentiation but the main increase in acinar cell numbers appeared to derive from proliferation of newly formed acinar cells.

Apoptosis in the human thymus in sudden and delayed death.

We counted apoptotic thymic cortical lymphocytes in semi-thin plastic sections of 266 human autopsy thymuses. The decreased patients included in the study were all aged less than 40 yrs and all died within 72 hrs of the onset of symptoms. Based on published data on animal and human material it was anticipated that individuals dying within 3 hrs of the onset of their fatal condition--"sudden deaths"--would have low apoptotic counts and those dying 3 to 72 hrs after the onset of their fatal condition--"delayed deaths"--would have elevated counts. Results showed that the sudden death group did have low apoptotic counts (mean 0.24 +/- 0.03% Standard Error of the Mean (SEM); n = 208) and the delayed death group did have significantly higher counts (mean 17.94 +/- 3.38% SEM; n = 58; p < 0.001). However, there were a number of cases in the latter group with low counts (n = 19). All of these patients had suffered injuries or clinical conditions that have been shown by others to be capable of interfering with the production of cortisol. As elevated cortisol is a major cause of apoptosis in thymic cortical lymphocytes, any such interference might prevent elevated apoptotic counts of thymic cortical lymphocytes.

Molecular analysis of a human interferon-inducible gene family.

Three functional members of the 1-8 gene family have been isolated on a single human genomic DNA fragment of less than 18 kb. The 1-8U and 1-8D genes are extremely similar; each is contained within a less than 2-kb fragment, has in its 5'flanking region two adjacent 14-base-pair sequences showing high similarity to interferon-stimulable response elements (ISREs) and has two highly related exons. The third gene (9-27) has a similar overall structure, shows substantial similarity to the 1-8s but has only one ISRE which is 3' of two CCAAT boxes not present in the 1-8U and D genes. The cDNA corresponding to the three genes share 120 nucleotides of identical sequence and show greater than 90% identity over 70% of the coding sequence. For the 1-8U and D genes the high similarity extends into the 5' non-coding and flanking regions. The open reading frames encode polypeptides that are likely to be of very similar structure. Antiserum to a conserved peptide detects a polypeptide(s) of about 14 kDa on PAGE which separates into three components on isoelectric focussing. The 9-27 and 1-8U genes are highly interferon-inducible the 1-8D gene is much less so. These differences are mimicked by the activities of the corresponding ISREs placed 5' of a marker gene in expression constructs. They presumably reflect differences in the interaction of the ISREs with the various interferon-inducible and constitutive factors that govern the interferon response.

Differential response of the human 6-16 and 9-27 genes to alpha and gamma interferons.

9-27 mRNA is expressed to a high level in response to both alpha and gamma interferons. In contrast, 6-16 mRNA is expressed well in response to alpha but very poorly in response to gamma interferon in human cells. The factors governing these different levels of expression were investigated. For both genes the major effect of both interferons is on transcription. A transcriptional bias in the 6-16 promoter/enhancer accounts in large part for the differential response of 6-16 to the two interferons. No single DNA element appears responsible; the smaller the 5' region analysed the lower the absolute activity and the smaller the differential response to alpha and gamma interferons observed. Both the 6-16 and 9-27 mRNAs are very stable and no effect of the interferons on stability was detected. Nor was any direct evidence obtained for preferential processing of the 9-27 mRNA. Nevertheless, differentials between the transcription and accumulation of mature mRNAs, particularly for 6-16 mRNA in response to gamma interferon, suggest that post-transcriptional control(s) must additionally operate. The 9-27 5' promoter/enhancer is much less active than that from 6-16 when placed 5' of a marker gene, despite the similar response of the two genes to alpha interferon.

A single DNA response element can confer inducibility by both alpha- and gamma-interferons.

Genomic and cDNA clones corresponding to 9-27, a member of the human 1-8 gene family highly inducible by alpha- and gamma-interferons (IFNs), have been isolated and characterized. A 1.7-kilobase genomic clone contains a complete functional gene with two exons, encoding a 125-amino acid polypeptide of unknown function. The 5' flanking region of the gene contains a 13-base-pair IFN-stimulable response element (ISRE), homologous to the ISREs of the 6-16, ISG 15, and ISG 54 genes, which are predominantly inducible by IFN-alpha, beta. Analysis of constructs containing native and mutated ISREs suggests that this motif is essential for the response of 9-27 to IFN-gamma as well as IFN-alpha. Furthermore, the 9-27 (GGAAATAGAAACT) and 6-16 (GGGAAAATGAAACT) ISREs can each confer a response to both types of IFN when placed on the 5' side of a marker gene. Since the 6-16 gene does not normally respond to IFN-gamma, the context of the ISRE must determine the specificity of the response.

Health and the Groote eylandter.

Concepts of health in traditional Groote Eylandt culture are examined, and the difference between these and European concepts are described. The importance of magic as a causal factor in serious illness is noted along with the absence of any specialist medical role. The community basis of health care is highlighted, and the implications of this for the development of more adequate health care measures are discussed.