Evaluating and comparing immunostaining and computational methods for spatial profiling of drug response in patient-derived explants.

Patient-derived explants (PDEs) represent the direct culture of fragments of freshly-resected tumour tissue under conditions that retain the original architecture of the tumour. PDEs have advantages over other preclinical cancer models as platforms for predicting patient-relevant drug responses in that they preserve the tumour microenvironment and tumour heterogeneity. At endpoint, PDEs may either be processed for generation of histological sections or homogenised and processed for 'omic' evaluation of biomarker expression. A significant advantage of spatial profiling is the ability to co-register drug responses with tumour pathology, tumour heterogeneity and changes in the tumour microenvironment. Spatial profiling of PDEs relies on the utilisation of robust immunostaining approaches for validated biomarkers and incorporation of appropriate image analysis methods to quantitatively and qualitatively monitor changes in biomarker expression in response to anti-cancer drugs. Automation of immunostaining and image analysis would provide a significant advantage for the drug discovery pipeline and therefore, here, we have sought to optimise digital pathology approaches. We compare three image analysis software platforms (QuPath, ImmunoRatio and VisioPharm) for evaluating Ki67 as a marker for proliferation, cleaved PARP (cPARP) as a marker for apoptosis and pan-cytokeratin (CK) as a marker for tumour areas and find that all three generate comparable data to the views of a histomorphometrist. We also show that Virtual Double Staining of sequential sections by immunohistochemistry results in imperfect section alignment such that CK-stained tumour areas are over-estimated. Finally, we demonstrate that multi-immunofluorescence combined with digital image analysis is a superior method for monitoring multiple biomarkers simultaneously in tumour and stromal areas in PDEs.

Development of a patient-derived explant model for prediction of drug responses in endometrial cancer.

OBJECTIVE: To undertake a pilot study to develop a novel Patient-Derived-Explant (PDE) model system for use in endometrial cancer (EC) that is capable of monitoring differential drug responses in a pre-clinical setting. METHODS: Fresh tumour was obtained post-hysterectomy from 27 patients with EC. Tumours were cut into 1-3 mm3 explants that were cultured at the air-liquid interface for 16-24 h in culture media. Explants were cultured in different media conditions to optimise viability. Explants were also treated with carboplatin/paclitaxel or pembrolizumab for 24 h and processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed followed by image analysis and quantitation of biomarker expression. RESULTS: EC samples are amenable to PDE culture with preserved histological architecture and PDE viability for up to 48 h, with the addition of autologous serum in culture media facilitating EC-PDE viability. Our PDE platform provides evidence of differential drug-response to conventional chemotherapeutics and immune checkpoint inhibition, and these responses can be assessed in the context of a preserved tumour microenvironment. CONCLUSIONS: Our PDE platform represents a rapid, low-cost pre-clinical model which can be easily integrated into drug development pipelines. PDE culture preserves original tumour architecture and enables evaluation of spatial relationships in the tumour microenvironment. PDE culture has the potential for personalised drug-testing in a pre-clinical setting which is increasingly important in an era of personalised medicine in the treatment of EC.

Exploration of Matrix Effects in Laser Ablation Inductively Coupled Plasma Mass Spectrometry Imaging of Cisplatin-Treated Tumors.

The use of a low aerosol dispersion ablation chamber within a laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS) setup allows for high-resolution, high-speed imaging of the distribution of elements within a sample. Here we show how this enhanced capability creates new analytical problems and solutions. We report the distribution of platinum at the cellular level in non-small cell lung cancer (NSCLC) explant models after treatment with clinically relevant doses of cisplatin. This revealed for the first time a correlation between the platinum signal and the presence of carbon deposits within lung tissue. We show how complementary ion beam analysis techniques, particle-induced X-ray emission (PIXE) and elastic backscattering spectrometry (EBS), can be used to explore potential matrix effects in LA-ICP-MS data. For these samples, it was confirmed that the enhancement was unlikely to have resulted from a matrix effect alone. Thus, the presence of carbon deposits within tissue has potential implications for the effective distribution of the cisplatin drug.

Correction to: Association of invasion-promoting tenascin-C additional domains with breast cancers in young women.

After the publication of this work [1] an error was noticed in Fig. 6 (b). In the MCF-7/Vector columns, the same image was used accidentally for the 0 h and 24 h time points. Both images were taken from the 0 h time point.

5-Hydroxymethylcytosine is an independent predictor of survival in malignant melanoma.

Outcomes for melanoma patients vary within cancer stage. Prognostic biomarkers are potential adjuncts to provide more precise prognostic information. Simple, low-cost biomarker assays, such as those based on immunohistochemistry, have strong translational potential. 5-hydroxymethylcytosine (5 hmC) shows prognostic potential in melanoma but prior studies were small. We, therefore, analysed 5 hmC in a retrospective cohort to provide external validation of its prognostic value. Two hundred primary melanomas were evaluated for 5 hmC expression using immunohistochemistry. The primary objective was to assess the effect on overall survival while controlling for important confounders. Univariable and multivariable analyses were performed. REMARK guidelines were followed. The 5 hmC immunohistochemistry scoring showed very strong inter-observer agreement (ICC 0.88) and expression was significantly related to age, site, Breslow thickness, ulceration, mitotic rate, and stage. Kaplan-Meier analysis showed 5 hmC was associated with metastasis-free, melanoma-specific, and overall survival, P<0.0001 for each. In univariable Cox proportional hazards models, 5 hmC hazard ratios were significant and remained so in a multivariable model. A two-step cox model was created using stage and 5 hmC, as stage is the gold standard for clinical practice. The addition of 5 hmC produced significant improvement in the model and 5 hmC and stage were independent significant predictors. This is the largest study of the prognostic value of 5 hmC immunohistochemistry in melanoma. The 5 hmC scoring was easily and reproducibly performed and it was an independent predictor of metastasis-free survival, melanoma-specific survival, and overall survival. This work supports further development of 5 hmC as a prognostic biomarker and suggests that it could add more precision to American Joint Committee on Cancer staging.

The time course of endogenous erythropoietin, IL-6, and TNFα in response to acute hypoxic exposures.

Erythropoietin (EPO) rapidly decreases on return to sea level (SL) after chronic altitude exposure. Acute hypoxia may provide an additional stimulus to prevent the decline in EPO. Proinflammatory cytokines, interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα) have been shown to inhibit EPO production. Optimal normobaric hypoxic exposure has not been established; therefore, investigation of methods eliciting the greatest response in EPO to limit physiological stress is required. Eight men (age 27 ± 4 years, body mass 77.5 ± 9.0 kg, height 179 ± 6 cm) performed four passive exposures to different normobaric hypoxic severities [FiO2 : 0.209 (SL), FiO2 : ~0.135 (3600 m), FiO2 : ~0.125 (4200 m) and FiO2 : ~0.115 (4800 m)] in a hypoxic chamber for 2 h. Venous blood was drawn pre-exposure and then at 1, 2, 4, 6, and 8 h to determine EPO concentration ([EPO]), IL-6, and TNFα. During 4200 and 4800 m, [EPO] increased from 5.9 ± 1.5 to 8.1 ± 1.5 mU/mL (P = 0.009) and 6.0 ± 1.4 to 8.9 ± 2.0 mU/mL (P = 0.037), respectively, with [EPO] increase peaking at 4 h (2 h post-exposure). There were no differences in IL-6 or TNFα during or post-exposure. Increased [EPO] was found 2 h post hypoxic exposure as result of 2 h of normobaric hypoxia ≥4200 m. There was no dose-response relationship in [EPO] between simulated hypoxia severities.

microRNA-10b is a prognostic biomarker for melanoma.

Malignant melanoma is an aggressive form of skin cancer. Recently, drug therapy of advanced disease has been revolutionized by new agents. More therapeutic options, coupled with the desire to extend treatment to the adjuvant setting mean that prognostic biomarkers that can be assayed from formalin-fixed paraffin-embedded clinical would be valuable. microRNAs have potential to fill this need. We analyzed 377 microRNAs in 79 primary melanomas and 32 metastases using a split sample discovery strategy. From a discovery analysis using 40 thick primary melanomas (20 cases with metastasis and 20 controls without metastasis at 5 years), microRNA expression was measured by quantitative RT-PCR (QRT-PCR). MiR-10b emerged as a candidate prognostic microRNA. This was confirmed in an independent validation set of thick primary melanomas (20 cases with metastasis and 19 controls without metastasis at 5 years). In the combined discovery and validation cohorts (n=79), miR-10b expression showed a 3.7-fold increase in expression between cases and controls (P=0.005) and showed a trend of increasing expression between primary melanomas and their matched metastases (P<0.001). In situ hybridization showed expression was in melanoma cells and correlated with expression measured by QRT-PCR (P=0.0005). We used the combined discovery and validation samples to verify the prognostic value of additional candidate microRNAs identified from other studies, and proceeded to analyze miR-200b. We demonstrated that miR-10b and miR-200b showed independent prognostic value (P=0.002 and 0.047, respectively) in multivariable analysis alongside known clinico-pathological prognostic features (eg, Breslow thickness) using a Cox proportional hazards regression model. Furthermore, the addition of these microRNAs to the clinico-pathological features led to an improved regression model with better identification of aggressive thick melanomas. Taken together, these data suggest that miR-10b is a new prognostic microRNA for melanoma and that there could be a place for microRNA analysis in stratifying melanoma for therapy.

Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." METHODS: We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). RESULTS: Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. CONCLUSIONS: Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease.

Nationwide outbreak of multidrug-resistant Salmonella Heidelberg infections associated with ground turkey: United States, 2011.

On 23 May 2011, CDC identified a multistate cluster of Salmonella Heidelberg infections and two multidrug-resistant (MDR) isolates from ground turkey retail samples with indistinguishable pulsed-field gel electrophoresis patterns. We defined cases as isolation of outbreak strains in persons with illness onset between 27 February 2011 and 10 November 2011. Investigators collected hypothesis-generating questionnaires and shopper-card information. Food samples from homes and retail outlets were collected and cultured. We identified 136 cases of S. Heidelberg infection in 34 states. Shopper-card information, leftover ground turkey from a patient's home containing the outbreak strain and identical antimicrobial resistance profiles of clinical and retail samples pointed to plant A as the source. On 3 August, plant A recalled 36 million pounds of ground turkey. This outbreak increased consumer interest in MDR Salmonella infections acquired through United States-produced poultry and played a vital role in strengthening food safety policies related to Salmonella and raw ground poultry.

'One Health' investigation: outbreak of human Salmonella Braenderup infections traced to a mail-order hatchery - United States, 2012-2013.

Human salmonellosis linked to contact with live poultry is an increasing public health concern. In 2012, eight unrelated outbreaks of human salmonellosis linked to live poultry contact resulted in 517 illnesses. In July 2012, PulseNet, a national molecular surveillance network, reported a multistate cluster of a rare strain of Salmonella Braenderup infections which we investigated. We defined a case as infection with the outbreak strain, determined by pulsed-field gel electrophoresis, with illness onset from 25 July 2012-27 February 2013. Ill persons and mail-order hatchery (MOH) owners were interviewed using standardized questionnaires. Traceback and environmental investigations were conducted. We identified 48 cases in 24 states. Twenty-six (81%) of 32 ill persons reported live poultry contact in the week before illness; case-patients named 12 different MOHs from eight states. The investigation identified hatchery D as the ultimate poultry source. Sampling at hatchery D yielded the outbreak strain. Hatchery D improved sanitation procedures and pest control; subsequent sampling failed to yield Salmonella. This outbreak highlights the interconnectedness of humans, animals, and the environment and the importance of industry knowledge and involvement in solving complex outbreaks. Preventing these infections requires a 'One Health' approach that leverages expertise in human, animal, and environmental health.

Identification of high-risk Dukes B colorectal cancer by microRNA expression profiling: a preliminary study.

AIM: MicroRNAs (miRNAs) from tumour tissue and common gene mutations were studied to determine whether they predict the development of metastasis in patients with Dukes B colorectal cancer. METHOD: Patients who underwent curative resection for Dukes B colorectal cancer who subsequently developed distant metastatic disease at some stage in the following 5 years ('high-risk B') were compared with case-matched controls of Dukes A, Dukes B (no metastases, 'low-risk B') and Dukes C patients without any detectable metastasis at 5 years of follow-up. MiRNAs from tumour and adjacent normal tissue and common gene mutations (KRAS, BRAF, PIK3CA) in primary cancer tissue were analysed to identify prognostic tissue markers for the development of metastasis in patients with Dukes B colorectal cancer. RESULTS: Expression of miR-15b and miR-135b was significantly downregulated (P < 0.001) in 'high-risk B' tumours compared with Dukes A, 'low-risk B' and C without metastasis. No significant differences were noted for mutation status and the development of metastasis. CONCLUSION: The study suggests that the development of metastasis in Dukes B tumours may be predictable based on the miRNA expression of miR-15b and miR-135b. This requires further study on a much larger cohort.

Nationwide outbreak of Salmonella Montevideo infections associated with contaminated imported black and red pepper: warehouse membership cards provide critical clues to identify the source.

In November 2009, we initiated a multistate investigation of Salmonella Montevideo infections with pulsed-field gel electrophoresis pattern JIXX01.0011. We identified 272 cases in 44 states with illness onset dates ranging from 1 July 2009 to 14 April 2010. To help generate hypotheses, warehouse store membership card information was collected to identify products consumed by cases. These records identified 19 ill persons who purchased company A salami products before onset of illness. A case-control study was conducted. Ready-to-eat salami consumption was significantly associated with illness (matched odds ratio 8·5, 95% confidence interval 2·1-75·9). The outbreak strain was isolated from company A salami products from an environmental sample from one manufacturing plant, and sealed containers of black and red pepper at the facility. This outbreak illustrates the importance of using membership card information to assist in identifying suspect vehicles, the potential for spices to contaminate ready-to-eat products, and preventing raw ingredient contamination of these products.

Atomistic simulation of structure and dynamics of the plastic crystal diethyl(methyl)(isobutyl)phosphonium hexafluorophosphate.

Molecular dynamics simulations have been performed to investigate the interrelations between structures, transport mechanisms, and phase transitions of an organic ionic plastic crystal material, diethyl(methyl)(isobutyl)phosphonium hexafluorophosphate ([P1,2,2,4][PF6]), in both solid and liquid phases. Examination of the temperature dependence of supercell parameters and radial distribution functions provides evidence of plastic phase transitions. Nonlinear increments of cell size within the temperature range 123-413 K are consistent with the plastic phase transitions identified from experimental analysis. The time- and temperature-dependent microstructure and dynamics have been intensively studied through analysis of trajectory files. The rotational motion and diffusion of the matrix ions are quantitatively analysed via rotational correlation functions and mean square displacements. We present new information on the evolution of molecular motions in different phases, and compare and contrast our findings with previously reported hypotheses based on nuclear magnetic resonance results. This work provides valuable information at an atomistic level to explain the experimental observations, which helps further understanding of the molecular motions underlying the plastic phase transitions.

Prognostic tissue markers in melanoma.

Prognostic tissue markers in melanoma Prognosis for patients diagnosed with cutaneous melanoma is currently based upon histopathological features alone, although tumours which are morphologically similar can behave differently. Numerous putative biomarkers have been identified in an attempt to aid prognostication for primary melanoma, using methods which include immunhistochemistry, polymerase chain reaction (PCR), array comparative genomic hybridization (CGH) and gene expression arrays. Despite this wide body of research, no biomarkers for prognosis in melanoma have been translated or are close to translation into clinical practice. In this review selected prognostic biomarkers are evaluated and the factors influencing successful biomarker translation, including phases of biomarker development and study design, are explored in an attempt to highlight the current gap between prognostic melanoma biomarker research and clinical translation.

Statins mediate anti- and pro-tumourigenic functions by remodelling the tumour microenvironment.

Anti-cancer properties of statins are controversial and possibly context dependent. Recent pathology/epidemiology studies of human lung adenocarcinoma showed reduced pro-tumourigenic macrophages associated with a shift to lower-grade tumours amongst statin users but, paradoxically, worse survival compared with that of non-users. To investigate the mechanisms involved, we have characterised mouse lung adenoma/adenocarcinoma models treated with atorvastatin. Here, we show that atorvastatin suppresses premalignant disease by inhibiting the recruitment of pro-tumourigenic macrophages to the tumour microenvironment, manifested in part by suppression of Rac-mediated CCR1 ligand secretion. However, prolonged atorvastatin treatment leads to drug resistance and progression of lung adenomas into invasive disease. Pathological progression is not driven by acquisition of additional driver mutations or immunoediting/evasion but is associated with stromal changes including the development of desmoplastic stroma containing Gr1+ myeloid cells and tertiary lymphoid structures. These findings show that any chemopreventive functions of atorvastatin in lung adenocarcinoma are overridden by stromal remodelling in the long term, thus providing mechanistic insight into the poor survival of lung adenocarcinoma patients with statin use.

The use of portable XRF as a forensic geoscience non-destructive trace evidence tool for environmental and criminal investigations.

Hand-held, portable X-Ray fluorescence instruments (pXRF) provide a means of rapid, in-situ chemical characterisation that has considerable application as a rapid trace evidence characterisation tool in forensic geoscience. This study presents both a control test study which demonstrates optimisation of the data collection process, alongside a range of individual forensic case studies, including heavy metal contamination, conflict archaeology, forensic soil characterisation, and verification of human remains, which together validate the technique and provide some comparison between field-based and laboratory-based pXRF applications. Results highlight the time-efficiency and cost-effectiveness of in-situ, field-based pXRF analyses for material characterisation when compared with other trace evidence methods. Analytical precision of various analytes during in-situ analysis was sufficient to demonstrate considerable application of field-based pXRF as a tool for rapid identification of specific areas of interest to be further investigated. Laboratory-based pXRF analyses yielded greater accuracy which could provide an efficient compromise between field-based pXRF and traditional laboratory-based analytical techniques (e.g. WD-XRF, ICP-MS). Further studies should collect more advanced datasets in more diverse locations to further validate the techniques capability to rapidly conduct geochemical surveys in a range of environments.

Expression of tenascin-C and its isoforms in the breast.

Tenascin-C (TNC) is an extracellular matrix glycoprotein which is frequently up-regulated in a variety of pathological conditions including chronic inflammation and cancer. TNC has been implicated in the modulation of cell migration, proliferation, invasion and angiogenesis. Multiple isoforms of TNC can be generated through the alternative splicing of nine exons located in the fibronectin type III region of the molecule. The profile of isoforms expressed differs between cancers and normal breast, with the fully truncated TNC isoform being predominant in normal and benign tissues and higher molecular weight isoforms induced predominantly in cancer. The addition of extra domains within the fibronectin type III repeat domain greatly affects TNC function with multiple exon combinations available for splicing. Exons 14 and 16 are considered to be tumour-associated and have been shown to affect breast cell line invasion and growth in vitro to a greater extent than the full-length TNC isoform. This mini review will provide a summary of the literature to date regarding the expression of TNC isoforms in the breast and also discuss more recent developments in the field regarding exon AD1.

The septins: roles in cytokinesis and other processes.

The septins are a novel family of proteins that were first recognized in yeast as proteins associated with the neck filaments. Recent work has shown that septins are also present in other fungi, insects, and vertebrates. Despite the apparent differences in modes of cytokinesis amongst species, septins appear to be essential for this process in both fungal and animal cells. The septins also appear to be involved in various other aspects of the organization of the cell surface.

Cytokinesis: an emerging unified theory for eukaryotes?

In animal and fungal cells, cytokinesis involves an actomyosin ring that forms and contracts at the division plane. Important new details have emerged concerning the composition, assembly, and dynamics of these contractile rings. In addition, recent advances suggest that targeted membrane addition is a central feature of cytokinesis in animal cells - as it is in fungi and plants - and the coordination of actomyosin ring function with targeted exocytosis at the cleavage plane is being explored. Important new information has also emerged about the spatial and temporal regulation of cytokinesis, especially in relation to the function of the spindle midzone in animal cells and the control of cytokinesis by GTPase systems.

Association of invasion-promoting tenascin-C additional domains with breast cancers in young women.

INTRODUCTION: Tenascin-C (TNC) is a large extracellular matrix glycoprotein that shows prominent stromal expression in many solid tumours. The profile of isoforms expressed differs between cancers and normal breast, with the two additional domains AD1 and AD2 considered to be tumour associated. The aim of the present study was to investigate expression of AD1 and AD2 in normal, benign and malignant breast tissue to determine their relationship with tumour characteristics and to perform in vitro functional assays to investigate the role of AD1 in tumour cell invasion and growth. METHODS: Expression of AD1 and AD2 was related to hypoxanthine phosphoribosyltransferase 1 as a housekeeping gene in breast tissue using quantitative RT-PCR, and the results were related to clinicopathological features of the tumours. Constructs overexpressing an AD1-containing isoform (TNC-14/AD1/16) were transiently transfected into breast carcinoma cell lines (MCF-7, T-47 D, ZR-75-1, MDA-MB-231 and GI-101) to assess the effect in vitro on invasion and growth. Statistical analysis was performed using a nonparametric Mann-Whitney test for comparison of clinicopathological features with levels of TNC expression and using Jonckheere-Terpstra trend analysis for association of expression with tumour grade. RESULTS: Quantitative RT-PCR detected AD1 and AD2 mRNA expression in 34.9% and 23.1% of 134 invasive breast carcinomas, respectively. AD1 mRNA was localised by in situ hybridisation to tumour epithelial cells, and more predominantly to myoepithelium around associated normal breast ducts. Although not tumour specific, AD1 and AD2 expression was significantly more frequent in carcinomas in younger women (age ≤40 years; P < 0.001) and AD1 expression was also associated with oestrogen receptor-negative and grade 3 tumours (P < 0.05). AD1 was found to be incorporated into a tumour-specific isoform, not detected in normal tissues. Overexpression of the TNC-14/AD1/16 isoform significantly enhanced tumour cell invasion (P < 0.01) and growth (P < 0.01) over base levels. CONCLUSIONS: Together these data suggest a highly significant association between AD-containing TNC isoforms and breast cancers in younger women (age ≤40 years), which may have important functional significance in vivo.