Evaluation of nipple aspirate fluid as a diagnostic tool for early detection of breast cancer

YesThere has been tremendous progress in detection of breast cancer in postmenopausal women, resulting in two-thirds of women surviving more than 20 years after treatment. However, breast cancer remains the leading cause of cancerrelated deaths in premenopausal women. Breast cancer is increasing in younger women due to changes in life-style as well as those at high risk as carriers of mutations in high-penetrance genes. Premenopausal women with breast cancer are more likely to be diagnosed with aggressive tumours and therefore have a lower survival rate. Mammography plays an important role in detecting breast cancer in postmenopausal women, but is considerably less sensitive in younger women. Imaging techniques, such as contrast-enhanced MRI improve sensitivity, but as with all imaging approaches, cannot differentiate between benign and malignant growths. Hence, current well-established detection methods are falling short of providing adequate safety, convenience, sensitivity and specificity for premenopausal women on a global level, necessitating the exploration of new methods. In order to detect and prevent the disease in high risk women as early as possible, methods that require more frequent monitoring need to be developed. The emergence of “omics” strategies over the last 20 years, enabling the characterisation and understanding of breast cancer at the molecular level, are providing the potential for long term, longitudinal monitoring of the disease. Tissue and serum biomarkers for breast cancer stratification, diagnosis and predictive outcome have emerged, but have not successfully translated into clinical screening for early detection of the disease. The use of breast-specific liquid biopsies, such as nipple aspirate fluid (NAF), a natural secretion produced by breast epithelial cells, can be collected non-invasively for biomarker profiling. As we move towards an age of active surveillance, home-based liquid biopsy collection kits are increasingly being applied and these could provide a paradigm shift where NAF biomarker profiling is used for routine breast health monitoring. The current status of established and newly emerging imaging techniques for early detection of breast cancer and the potential for alternative biomarker screening of liquid biopsies, particularly those applied to high-risk, premenopausal women, will be reviewed.Proteomics research was supported by Yorkshire Cancer Research projects, BPP047 and B381PA, and co-funded by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation projects ΥΓΕΙΑ/ΒΙΟΣ/0311(ΒΙΕ/07) and NEKYP/0311/17

Patterns of cancer cell sphere formation in primary cultures of human oral tongue squamous cell carcinoma and neck nodes

YesRecently a sub-population of cells with stem cell characteristics, reported to be associated with initiation, growth, spread and recurrence, has been identified in several solid tumors including oral tongue squamous cell carcinoma (OTSCC). The aim of our pilot study was to isolate CD44+ cancer stem cells from primary cultures of OTSCC and neck node Level I (node-I) biopsies, grow cell spheres and observe their characteristics in primary cultures. Parallel cultures of hyperplastic lesions of tongue (non-cancer) were set up as a control. Immunohistochemistry was used to detect CD44/CD24 expression and magnetic activated cell sorting to isolate CD44+ cell populations followed by primary cell culturing. Both OTSCC and node-I biopsies produced floating spheres in suspension, however those grown in hyperplastic and node-I primary cultures did not exhibit self-renewal properties. Lymph node metastatic OTSCC, express higher CD44/CD24 levels, produce cancer cell spheres in larger number and rapidly (24 hours) compared to node negative OTSCC (1 week) and non-cancer specimens (3 weeks). In addition, metastatic OTSCC have the capacity for proliferation for up to three generations in primary culture. This in vitro system will be used to study cancer stem cell behavior, therapeutic drug screening and optimization of radiation dose for elimination of resistant cancer cells.SKMCH&RC, Yorkshire Cancer Researc

High-Throughput Proteomic Profiling of Nipple Aspirate Fluid from Breast Cancer Patients Compared with Non-Cancer Controls: A Step Closer to Clinical Feasibility.

BACKGROUND: Early detection of breast cancer (BC) is critical for increasing survival rates. However, current imaging approaches can provide ambiguous results, requiring invasive tissue biopsy for a definitive diagnosis. Multi-dimensional mass spectrometric analysis has highlighted the invaluable potential of nipple aspirate fluid (NAF) as a non-invasive source of early detection biomarkers, by identifying a multitude of proteins representative of the changing breast microenvironment. However, technical challenges with biomarker validation in large cohorts remain due to low sample throughput, impeding progress towards clinical utility. Rather, by employing a high-throughput method, that is more practicable for clinical utility, perturbations of the most abundant NAF proteins in BC patients compared with non-cancer (NC) controls could be monitored and validated in larger groups. METHOD: We characterized matched NAF pairs from BC (n = 9) and NC (n = 4) volunteers, using a rapid one dimensional liquid chromatography-mass spectrometry (1D LC-MS/MS) approach. RESULTS: Overall, 198 proteins were relatively quantified, of which 40 were significantly differentiated in BC samples, compared with NC (p ≤ 0.05), with 26 upregulated and 14 downregulated. An imbalance in immune response and proteins regulating cell growth, maintenance and communication were identified. CONCLUSIONS: Our findings show 1D LC-MS/MS can quantify changes reflected in the NAF proteome associated with breast cancer development

Model-based integration analysis revealed presence of novel prognostic miRNA targets and important cancer driver genes in triple-negative breast cancers

YesBackground: miRNAs (microRNAs) play a key role in triple-negative breast cancer (TNBC) progression, and its heterogeneity at the expression, pathological and clinical levels. Stratification of breast cancer subtypes on the basis of genomics and transcriptomics profiling, along with the known biomarkers’ receptor status, has revealed the existence of subgroups known to have diverse clinical outcomes. Recently, several studies have analysed expression profiles of matched mRNA and miRNA to investigate the underlying heterogeneity of TNBC and the potential role of miRNA as a biomarker within cancers. However, the miRNA-mRNA regulatory network within TNBC has yet to be understood. Results and Findings: We performed model-based integrated analysis of miRNA and mRNA expression profiles on breast cancer, primarily focusing on triple-negative, to identify subtype-specific signatures involved in oncogenic pathways and their potential role in patient survival outcome. Using univariate and multivariate Cox analysis, we identified 25 unique miRNAs associated with the prognosis of overall survival (OS) and distant metastases-free survival (DMFS) with “risky” and “protective” outcomes. The association of these prognostic miRNAs with subtype-specific mRNA genes was established to investigate their potential regulatory role in the canonical pathways using anti-correlation analysis. The analysis showed that miRNAs contribute to the positive regulation of known breast cancer driver genes as well as the activation of respective oncogenic pathway during disease formation. Further analysis on the “risk associated” miRNAs group revealed significant regulation of critical pathways such as cell growth, voltage-gated ion channel function, ion transport and cell-to-cell signalling. Conclusion: The study findings provide new insights into the potential role of miRNAs in TNBC disease progression through the activation of key oncogenic pathways. The results showed previously unreported subtype-specific prognostic miRNAs associated with clinical outcome that may be used for further clinical evaluation.EPSRC (EP/R043787/1)

The emotional well-being of young people: a review of the literature.

Suicide is increasingly described by governments and policy-makers as a global public health problem. Between 1950 and 1995 global suicide rates have increased by 60%. In recent years concerns have been expressed in Scotland and the UK about rising suicide rates amongst children and young people and the accumulation of increasing evidence that the adoption of negative coping strategies is contributing to rising levels of deliberate self harm (DSH).This literature review was in part used and incorporated into chapter two of the final report, The emotional wellbeing of young people: final report of phase one of a 'Choose Life' research project in Aberdeenshire (March 2004-March 2007), which can be found at http://hdl.handle.net/10059/439

A proteomic investigation to discover candidate proteins involved in novel mechanisms of 5-fluorouracil resistance in colorectal cancer

YesOne of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC.Sadr ul-Shaheed and the University of Bradford Proteomics Facility were supported by Yorkshire Cancer Research, UK (Cancer Medicine Discovery II, grant B381PA)

Stem Cell Organoids in Primary Cultures of Human Non-Malignant and Malignant Colon

YesA sub-population of cells named cancer stem cells (CSCs) that initiate and promote tumour growth have been demonstrated to exist in several malignancies including colon carcinoma. The objective of our pilot study was to isolate CD133+CD26+CD44+ CSCs from patient colon tumours, culture spheres or organoids and observe their proliferation in primary cultures. Parallel cultures of non-cancer controls from colon normal lining and nonadenomatous polyps were set up. Magnetic activated cell sorting was used to isolate CD133+CD26+CD44+ cell populations followed by primary cell culturing under stem cell culture conditions. Number, cells/organoid and daughter generations of organoids were calculated using phase contrast microscope. Trypan blue exclusion method was used to test the viability of the cells. Both colon tumour and colon non-adenomatous polyp formed floating organoids in suspension; however non-adenomatous polyp cultures did not show self-renewal properties for more than 1 passage. Normal colon singlecell suspension did not create organoids. Metastatic colon tumours rapidly produce cancer cell organoids in less than 24 hours in larger numbers compared to non-metastatic colon tumours (1-3 weeks). Metastatic colon tumour organoids have the ability for proliferation for upto five daughter generations in primary culture compared to three generations for those grown from non-metastatic tumours. This in vitro CSC organoid model will help study colon cancer biology, in particular providing a valuable source of primary cell-derived tissue for studying personalized molecular profiling using ‘omics strategies to direct therapeutic intervention

Research biopsies in kidney transplantation: an evaluation of surgical techniques and optimal tissue mass allowing molecular and histological analyses

Background: Research biopsies have great potential to advance scientific knowledge by helping to establish predictors of favourable or unfavourable outcomes in kidney transplantation. We evaluated punch and core biopsies of different sizes to determine the optimal size for clinical use. Methods: A total of 54 punch biopsies and 18 core needle biopsies were retrieved by three transplant surgeons. Each surgeon obtained three separate 2 mm, 3 mm and 4 mm punch biopsy samples and three 23 mm (length) core needle biopsies from two pig kidneys. Results: 4 mm punch biopsies yielded the greatest amount of protein (2.11 ± 0.41 mg) with good reproducibility between surgeons and biopsy types (Coefficient of Variation ∼ 22.13%). All surgeons found 2 mm biopsies technically challenging to obtain and sample processing was difficult due to the sample size. Shotgun proteomics identified 3853 gene products with no significant difference in the quantitative proteome of 2 mm and 3 mm punch biopsies. However, the expression of 158 Kidney enriched genes, was higher in bigger and deeper 4 mm punch and core needle biopsies compared to 2 mm biopsy. Only 80% of 2 mm biopsies demonstrated the presence of glomeruli, whereas glomeruli were present in 100% of all other biopsy sizes. Conclusions: The 2 mm punch biopsy has been shown to be challenging to use and frequently provides inadequate tissue for histology and proteomics while 3 mm research biopsies were the smallest size that were technically obtainable with adequate tissue for molecular studies

Heterochromatic sequences in a Drosophila whole-genome shotgun assembly

BACKGROUND: Most eukaryotic genomes include a substantial repeat-rich fraction termed heterochromatin, which is concentrated in centric and telomeric regions. The repetitive nature of heterochromatic sequence makes it difficult to assemble and analyze. To better understand the heterochromatic component of the Drosophila melanogaster genome, we characterized and annotated portions of a whole-genome shotgun sequence assembly. RESULTS: WGS3, an improved whole-genome shotgun assembly, includes 20.7 Mb of draft-quality sequence not represented in the Release 3 sequence spanning the euchromatin. We annotated this sequence using the methods employed in the re-annotation of the Release 3 euchromatic sequence. This analysis predicted 297 protein-coding genes and six non-protein-coding genes, including known heterochromatic genes, and regions of similarity to known transposable elements. Bacterial artificial chromosome (BAC)-based fluorescence in situ hybridization analysis was used to correlate the genomic sequence with the cytogenetic map in order to refine the genomic definition of the centric heterochromatin; on the basis of our cytological definition, the annotated Release 3 euchromatic sequence extends into the centric heterochromatin on each chromosome arm. CONCLUSIONS: Whole-genome shotgun assembly produced a reliable draft-quality sequence of a significant part of the Drosophila heterochromatin. Annotation of this sequence defined the intron-exon structures of 30 known protein-coding genes and 267 protein-coding gene models. The cytogenetic mapping suggests that an additional 150 predicted genes are located in heterochromatin at the base of the Release 3 euchromatic sequence. Our analysis suggests strategies for improving the sequence and annotation of the heterochromatic portions of the Drosophila and other complex genomes

Systems Analysis of miRNA Biomarkers to Inform Drug Safety

microRNAs (miRNAs or miRs) are short non-coding RNA molecules which have been shown to be dysregulated and released into the extracellular milieu as a result of many drug and non-drug-induced pathologies in different organ systems. Consequently, circulating miRs have been proposed as useful biomarkers of many disease states, including drug-induced tissue injury. miRs have shown potential to support or even replace the existing traditional biomarkers of drug-induced toxicity in terms of sensitivity and specificity, and there is some evidence for their improved diagnostic and prognostic value. However, several pre-analytical and analytical challenges, mainly associated with assay standardization, require solutions before circulating miRs can be successfully translated into the clinic. This review will consider the value and potential for the use of circulating miRs in drug-safety assessment and describe a systems approach to the analysis of the miRNAome in the discovery setting, as well as highlighting standardization issues that at this stage prevent their clinical use as biomarkers. Highlighting these challenges will hopefully drive future research into finding appropriate solutions, and eventually circulating miRs may be translated to the clinic where their undoubted biomarker potential can be used to benefit patients in rapid, easy to use, point-of-care test systems