Single-cell nanobiopsy enables multigenerational longitudinal transcriptomics of cancer cells.

Single-cell RNA sequencing has revolutionized our understanding of cellular heterogeneity, but routine methods require cell lysis and fail to probe the dynamic trajectories responsible for cellular state transitions, which can only be inferred. Here, we present a nanobiopsy platform that enables the injection of exogenous molecules and multigenerational longitudinal cytoplasmic sampling from a single cell and its progeny. The technique is based on scanning ion conductance microscopy (SICM) and, as a proof of concept, was applied to longitudinally profile the transcriptome of single glioblastoma (GBM) brain tumor cells in vitro over 72 hours. The GBM cells were biopsied before and after exposure to chemotherapy and radiotherapy, and our results suggest that treatment either induces or selects for more transcriptionally stable cells. We envision the nanobiopsy will contribute to transforming standard single-cell transcriptomics from a static analysis into a dynamic assay.

Variation in care and outcomes for people after hip fracture with and without cognitive impairment; results from the Australian and New Zealand Hip Fracture Registry.

BACKGROUND: People with dementia have poorer outcomes after hip fracture and this may be due in part to variation in care. We aimed to compare care and outcomes for people with and without cognitive impairment after hip fracture. METHODS: Retrospective cohort study using Australian and New Zealand Hip Fracture Registry data for people ≥50 years of age who underwent hip fracture surgery (n = 49,063). Cognitive impairment or known dementia and cognitively healthy groups were defined using preadmission cognitive status. Descriptive statistics and multivariable mixed effects models were used to compare groups. RESULTS: In general, cognitively impaired people had worse care and outcomes compared to cognitively healthy older people. A lower proportion of the cognitively impaired group had timely pain assessment (≤30 min of presentation: 61% vs 68%; p < 0.0001), were given the opportunity to mobilise (89% vs 93%; p < 0.0001) and achieved day-1 mobility (34% vs 58%; p < 0.0001) than the cognitively healthy group. A higher proportion of the cognitively impaired group had delayed pain management (>30 mins of presentation: 26% vs 20%; p < 0.0001), were malnourished (27% vs 15%; p < 0.0001), had delirium (44% vs 13%; p < 0.0001) and developed a new pressure injury (4% vs 3%; p < 0.0001) than the cognitively healthy group. Fewer of the cognitively impaired group received rehabilitation (35% vs 64%; p < 0.0001), particularly patients from RACFs (16% vs 39%; p < 0.0001) and were prescribed bone protection medication on discharge (24% vs 27%; p < 0.0001). Significantly more of the cognitively impaired group had a new transfer to residential care (46% vs 11% from private residence; p < 0.0001) and died at 30-days (7% vs 3% from private residence; 15% vs 10% from RACF; both p < 0.0001). In multivariable models adjusting for covariates with facility as the random effect, the cognitively impaired group had a greater odds of being malnourished, not achieving day-1 walking, having delirium in the week after surgery, dying within 30 days, and in those from private residences, having a new transfer to a residential care facility than the cognitively healthy group. CONCLUSIONS: We have identified several aspects of care that could be improved for patients with cognitive impairment - management of pain, mobility, nutrition and bone health, as well as delirium assessment, prevention and management strategies and access to rehabilitation. Further research is needed to determine whether improvements in care will reduce hospital complications and improve outcomes for people with dementia after hip fracture.

Improving hip fracture care: A five-year review of the early contributors to the Australian and New Zealand Hip Fracture Registry.

OBJECTIVE: The aim of this study was to examine temporal trends (2016-2020) in hip fracture care in Australian and New Zealand (ANZ) hospitals that started providing patient-level data to the ANZ Hip Fracture Registry (ANZHFR) on/before 1 January 2016 (early contributors). METHODS: Retrospective cohort study of early contributor hospitals (n = 24) to the ANZHFR. The study cohort included patients aged ≥50 years admitted with a low trauma hip fracture between 1 January 2016 and 31 December 2020 (n = 26,937). Annual performance against 11 quality indicators and 30- and 365-day mortality were examined. RESULTS: Compared to 2016/2017, year-on-year improvements were demonstrated for preoperative cognitive assessment (2020: OR 3.57, 95% confidence interval [95% CI] 3.29-3.87) and nerve block use prior to surgery (2020: OR 4.62, 95% CI 4.17-5.11). Less consistent improvements over time from 2016/2017 were demonstrated for emergency department (ED) stay of <4 h (2017; 2020), pain assessment ≤30 min of ED presentation (2020), surgery ≤48 h (2020) and bone protection medication prescribed on discharge (2017-2020; 2020 OR 2.22, 95% CI 2.03-2.42). The odds of sustaining a hospital-acquired pressure injury increased in 2019-2020 compared to 2016. The odds of receiving an orthogeriatric model of care and being offered the opportunity to mobilise on Day 1 following surgery fluctuated. There was a reduction in 365-day mortality in 2020 compared to 2016 (OR 0.86, 95% CI 0.74-0.98), whereas 30-day mortality did not change. CONCLUSIONS: Several quality indicators improved over time in early contributor hospitals. Indicators that did not improve may be targets for future care improvement activities, including considering incentivised hip fracture care, which has previously been shown to improve care/outcomes. COVID-19 and reporting practices may have impacted the study findings.

A randomised trial of the effect of omega-3 polyunsaturated fatty acid supplements on the human intestinal microbiota.

OBJECTIVE: Omega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22). DESIGN: A randomised, open-label, cross-over trial of 8 weeks' treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week 'washout' period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry. RESULTS: Both omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or ß diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects. CONCLUSION: Omega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure. TRIAL REGISTRATION NUMBER: ISRCTN18662143.

Preoperative chemoradiation with capecitabine, irinotecan and cetuximab in rectal cancer: significance of pre-treatment and post-resection RAS mutations.

BACKGROUND: The influence of EGFR pathway mutations on cetuximab-containing rectal cancer preoperative chemoradiation (CRT) is uncertain. METHODS: In a prospective phase II trial (EXCITE), patients with magnetic resonance imaging (MRI)-defined non-metastatic rectal adenocarinoma threatening/involving the surgical resection plane received pelvic radiotherapy with concurrent capecitabine, irinotecan and cetuximab. Resection was recommended 8 weeks later. The primary endpoint was histopathologically clear (R0) resection margin. Pre-planned retrospective DNA pyrosequencing (PS) and next generation sequencing (NGS) of KRAS, NRAS, PIK3CA and BRAF was performed on the pre-treatment biopsy and resected specimen. RESULTS: Eighty-two patients were recruited and 76 underwent surgery, with R0 resection in 67 (82%, 90%CI: 73-88%) (four patients with clinical complete response declined surgery). Twenty-four patients (30%) had an excellent clinical or pathological response (ECPR). Using NGS 24 (46%) of 52 matched biopsies/resections were discrepant: ten patients (19%) gained 13 new resection mutations compared to biopsy (12 KRAS, one PIK3CA) and 18 (35%) lost 22 mutations (15 KRAS, 7 PIK3CA). Tumours only ever testing RAS wild-type had significantly greater ECPR than tumours with either biopsy or resection RAS mutations (14/29 [48%] vs 10/51 [20%], P=0.008), with a trend towards increased overall survival (HR 0.23, 95% CI 0.05-1.03, P=0.055). CONCLUSIONS: This regimen was feasible and the primary study endpoint was met. For the first time using pre-operative rectal CRT, emergence of clinically important new resection mutations is described, likely reflecting intratumoural heterogeneity manifesting either as treatment-driven selective clonal expansion or a geographical biopsy sampling miss.

Comparing mutation calls in fixed tumour samples between the affymetrix OncoScan® array and PCR based next-generation sequencing.

BACKGROUND: The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material. METHODS: We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls. RESULTS: A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5-10%) than product claims indicated. CONCLUSIONS: This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results.

Examining the potential use and long-term stability of guaiac faecal occult blood test cards for microbial DNA 16S rRNA sequencing.

AIMS: With a growing interest in the influence the gut microbiome has on the development of colorectal cancer (CRC), we investigated the feasibility and stability of isolating and typing microbial DNA from guaiac faecal occult blood test (gFOBt) cards. This has the future potential to screen the microbial populations present in confirmed colorectal neoplasia cases with aims to predict the presence and development of CRC. METHODS: Fresh stool samples from three healthy volunteers were applied to gFOBt cards. DNA was extracted from both the cards and fresh stool samples. A series of additional cards were prepared from one volunteer, and extracted at time points between 2 weeks and 3 years. The V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq at 2×250 bp read lengths. Data were analysed using QIIME software. RESULTS: Samples were grouped both by volunteer and by type (fresh or gFOBt), and compared a variety of ways: visual inspection of taxa, α and ß diversity, intraclass correlation. In all comparisons, samples grouped by volunteer, and not by sample type. The different time points showed no appreciable differences with increased storage time. CONCLUSIONS: This study has demonstrated that there is good concordance between microbial DNA isolated from fresh stool sample, and from the matched gFOBt card. Samples stored for up to 3 years showed no detrimental effect on measureable microbial DNA. This study has important future implications for investigating microbial influence on CRC development and other pathologies.

HER2 overexpression and amplification as a potential therapeutic target in colorectal cancer: analysis of 3256 patients enrolled in the QUASAR, FOCUS and PICCOLO colorectal cancer trials.

HER2 overexpression/amplification is linked to trastuzumab response in breast/gastric cancers. One suggested anti-EGFR resistance mechanism in colorectal cancer (CRC) is aberrant MEK-AKT pathway activation through HER2 up-regulation. We assessed HER2-amplification/overexpression in stage II-III and IV CRC patients, assessing relationships to KRAS/BRAF and outcome. Pathological material was obtained from 1914 patients in the QUASAR stage II-III trial and 1342 patients in stage IV trials (FOCUS and PICCOLO). Tissue microarrays were created for HER2 immunohistochemistry. HER2-amplification was assessed using FISH and copy number variation. KRAS/BRAF mutation status was assessed by pyrosequencing. Progression-free survival (PFS) and overall survival (OS) data were obtained for FOCUS/PICCOLO and recurrence and mortality for QUASAR; 29/1342 (2.2%) stage IV and 25/1914 (1.3%) stage II-III tumours showed HER2 protein overexpression. Of the HER2-overexpressing cases, 27/28 (96.4%) stage IV tumours and 20/24 (83.3%) stage II-III tumours demonstrated HER2 amplification by FISH; 41/47 (87.2%) also showed copy number gains. HER2-overexpression was associated with KRAS/BRAF wild-type (WT) status at all stages: in 5.2% WT versus 1.0% mutated tumours (p < 0.0001) in stage IV and 2.1% versus 0.2% in stage II-III tumours (p = 0.01), respectively. HER2 was not associated with OS or PFS. At stage II-III, there was no significant correlation between HER2 overexpression and 5FU/FA response. A higher proportion of HER2-overexpressing cases experienced recurrence, but the difference was not significant. HER2-amplification/overexpression is identifiable by immunohistochemistry, occurring infrequently in stage II-III CRC, rising in stage IV and further in KRAS/BRAF WT tumours. The value of HER2-targeted therapy in patients with HER2-amplified CRC must be tested in a clinical trial. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cross-laboratory validation of the OncoScan® FFPE Assay, a multiplex tool for whole genome tumour profiling.

BACKGROUND: Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. METHODS: In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). RESULTS: Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. CONCLUSIONS: Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.

A homeopatia e uma resposta placebo? / Is the homeopathy a placebo response?

Num experimento duplo-cego randomizado, foi testada a hipotese de que as potencias homeopaticas sao placebo. O modelo escolhido para estudo comparou, os efeitos de uma preparacao de mistura de polens de capim com placebo, em 144 pacientes com Febre do Feno ativa. A resposta era mais significativa quando os resultados eram corrigidos para a dose de polen, e quando ela estava associada a uma diminuicao de 50% da necessidade de anti-histaminicos. Nos pacientes que receberam o tratamento homeopatico foi notada, inicialmente, uma agravacao mais frequente dos sintomas, seguida de melhora. Nao houve evidencia que sustentasse a ideia de que a acao do placebo explica, completamente, as respostas clinicas para as drogas homeopaticas