Tuesday, January 31, 2017
Competition Funded PhD Project (European/UK Students Only)
This project aims to address one of the critical research gaps in the prevention, detection and treatment of breast cancer and will aids the development of screening approaches. The key objectives are to test the predicted molecular markers for their suitability for minimally invasive early detection and stratification of breast cancers, and to devise immunoassays for their detection. We aim to develop multiplex affinity assays for serum profiling and simpler lateral flow immunochromatographic assays, akin Clearblue pregnancy tests, for Point-of-Care (PoC) urine analyses. New assays will allow mass screening, improve the time to diagnosis, improve clinical management, will lead to better health outcomes and should help to reduce the number of deaths from breast cancer (currently 12,000 each year).
Clinical breast cancer diagnostics and routine monitoring rely on mammography and ultrasound scanning, whilst home examination is still limited to Self Breast Examination. No suitable molecular tools exist for the early detection of breast cancer, no kits are available for routine breast health monitoring and there exist no affordable means of routine mass screening.
Past studies reported that ca. 700 proteins are expressed at elevated levels in breast cancer tissues (Varnum et.al. 2003, Celis et.al. 2004, Canelle et.al. 2006, Gromov et.al. 2013, Misek&Kim 2011, Anderson et.al. 2011, Kulasingam&Diamandis 2007, Lam et.al. 2014, Aarøe et.al. 2010, Allinen et.al. 2004, Lacroix M. 2006, Alexander et.al. 2004, Choong et.al. 2010). These and other similar studies were often performed on surgically removed tumours and most of the reported proteins were intracellular and showed little correlation between breast tissues and serum or urine levels. Furthermore, only very few of these are present in serum or urine at detectable levels. Many of these proteins will be unfolded and partially degraded by the time they appear in urine.
We have compared protein concentrations and documented gene expression changes for candidate proteins and narrowed down the list of suitable protein markers to 187 (where both genes and proteins are unregulated). Of these potential markers 52 genes are the most abundant and upregulated between 10 and 200 fold in breast cancer) and the relevant are amenable to detection in serum and urine at protein or peptide levels with suitably designed antibodies. These cancer associated proteins present ideal targets for affinity based detection.
We have earlier developed Affinity Peptidomics approach to protein profiling (Soloviev & Finch. 2005, Soloviev et.al. 2007, Soloviev et al. 2008, Zhang et.al. 2010, Soloviev 2010, Zhang et.al. 2016). This technique uses anti-peptide antibodies to detect peptide targets in naturally occurring or experimentally generated complex peptide pools (including e.g. crude proteolysis digests of proteomes). Anti-peptide antibodies may not capture intact proteins, but such antibodies are highly suitable for assaying peptides. Affinity Peptidomics approach alleviates common problems of protein instability and degradation, especially when using urine, relaxes the stringent preservation requirements commonly associated with protein samples and is ideally suited for the analysis of the urine peptidome. We have our own algorithm for immunogenic peptide selection; which consistently outperforms old antigenicity prediction tools. We are therefore capable of testing secretome and degradome captured from serum or urine rather than transcriptional profiles of cancer cells. Therefore such profiling can be performed as part of routine diagnostic even before the discovery of cancer, or at earlier stages as well as during and following therapy. Serum and urine markers may also be indicative of secondary tumours and distant metastases missed by traditional screenings.
The student will select immunogenic peptides from proteins predicted to have predictive and prognostic value in breast cancer. These will be used to generate polyclonal anti-peptide antibodies. Following their validation, these will be used to establish test platforms suitable for quantitative or qualitative affinity peptidomics profiling of serum and urine. The use of anti-peptide antibodies following proteolytic digestion of samples alleviates multiple problems of many existing protein affinity assays, i.e. protein sample stability, protein degradation, heterogeneity of physical properties of proteins to name just a few. Validated peptide targets are also most suitable for the generation of molecularly imprinted polymers (MIPs, also referred to as "plastic antibodies") and their performance will be tested using lateral flow assay format. Robust nature of MIPs make them especially suitable for use in aggressive media or under denaturing conditions incompatible with traditional antibodies.
Applicants should already have or be expected to obtain a First or upper Second Class degree in a relevant discipline. This studentship is fully funded for three years. It covers tuition fees at the UK/EU rate and includes a stipend at the standard Research Council rate (currently £16,296 per annum). Funding is available for UK and EU students.
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Soloviev et.al. Protein profiling for forensic and biometric applications. In Molecular Forensics. Ed. Rapley. 2007 John Wiley & Sons, Ltd
Soloviev M, Shaw C and Andren P (Editors). Peptidomics: Methods and applications. 2008 by John Wiley & Sons, Inc.
Zhang et.al. Affinity Peptidomics: Peptide Selection and Affinity Capture on Hydrogels and Microarrays. In Peptidomics: Methods and protocols. Methods in Molecular Biology. 2010 Humana Press, USA. pp. 313-344
Soloviev (Editor). Peptidomics. Methods and Protocols. Series: Methods in Molecular Biology, Vol. 615. 1st Edition., 2010, XII, 380 p. 112 illus.
Zhang et.al. Peptides and Anti-peptide Antibodies for Small and Medium Scale Peptide and Anti-peptide Affinity Microarrays: Antigenic Peptide Selection, Immobilization, and Processing. 2016 In : Methods in Molecular Biology. 1352, p. 51-66
NHS (2013) https://www.gov.uk/guidance/nhs-population-screening-explained
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Czulak, A. Guerreiro, K. Metran, F. Canfarotta, A. Goddard, R. H. Cowan, A. W. Trochimczuk, S. Piletsky. et. al. Formation of target-specific binding sites in enzymes. Solid-phase molecular imprinting of HRP – Nanoscale, 2016, 8 (21)
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