Development of Serum Proteomics

It is thought that peptides and/or proteins that are present in the blood reflect the status of the tissue that they surround and as such blood presents an attractive target for the identification of novel biomarkers that could provide prognostic, diagnostic or surrogate markers of response to a particular therapy. The advantages of using blood, as opposed to other more invasive and expensive methods to gain clinically relevant knowledge about disease status are many, and include the fact that it is thought that multiple blood sampling will enable the dynamics of a disease status or treatment outcome to be more closely monitored, with greater clinical benefit. Recent advances in mass spectrometry technology and direct peptide labelling approaches have led us, in tandem with Professor Tony Whetton’s laboratory within MCRC, to set up a proteomics initiative in our laboratory that aims to look for novel circulating biomarkers. Using tandem MS/MS and iTRAQ labelling we hope to gain deeper penetration and greater depth of coverage into the blood proteome and thus generate novel fingerprints and/or discover new biomarkers that will enable us to move closer towards earlier diagnosis and better treatment regimens for cancer therapy. With this aim we have optimised a workflow that we think will translate best to a high throughput method for screening many patient samples. We have also invested in bioinformatics within CEP and work collaboratively with the Informatics Group led by Dr Crispin Miller at PICR. The first clinical samples are being collected for analysis.

Circulating Tumour Cells

In 2007 CEP obtained CRUK funding for the Veridex CellSearch System that allows enumeration and evaluation of circulating tumour cells (CTCs). CEP is asking whether CTC numbers can predict disease relapse and whether, if sufficiently pure populations can be isolated, their biology can inform on optimal drug treatment strategies. With the firm aim of molecular characterisation additional technologies are being evaluated including the ISET filtration device (acquired 2008) isolating cells on the basis of size. Downstream applications will include analysis by immunohistochemistry, FISH and gene expression. Our portfolio of CTC trials spans a range of tumour types including NSCLC, CRC, pancreatic cancer and melanoma.  Initial studies have focused on SCLC and with the approaching clinical evaluation of Bcl-2 inhibitors at DCU, we are also examining the levels of molecular determinants of drug resistance to such agents in CTCs, data that can be correlated with our panel of serological biomarkers of cell death. Our serological alliance with AstraZeneca has expanded our portfolio for CTC evaluation even further. We will explore the effects of novel agents on CTC numbers and assess the feasibility of linking targeted therapy with molecular determinants from CTCs.

Q Dot Immunohistochemistry

The Pharmacodynamic evaluation of new drugs that target defined biological processes is based on detecting changes in drug-target molecules and/or in downstream effectors. For instance, proof that a novel VEGF inhibitor inhibits angiogenesis will require methods to prove the mechanism (e.g. staining VEGF receptor expression and phosphorylation status to determine % activated receptors) and other methods to prove the principle (e.g. staining CD31 (total blood vessels) and CD105 (proliferating blood vessels)) to determine the expression ratio between the two as an indicator of therapy response. Combined Q-dots to discriminate resting and proliferating endothelial cells and identify apoptotic molecular events could, with method optimisation and validation, demonstrate directly the effects of an anti-angiogenic drug on the fate of the key cells that mediate angiogenesis.  Traditionally, it has been very difficult to apply these methodologies to the same tissue sections and thus cell-cell comparisons have been hard to achieve. Although double staining techniques exist they are challenging to conduct and to analyse. Q-dots are nanometer scale, semiconductor crystals that fluoresce over a range of wavelengths tuneable dependent upon crystal diameter. The development of Q-dots now facilitates measurement of multiple antibodies (and mRNA probes for in situ hybridisation) simultaneously on the same section of tissue. These sections can then be evaluated through spectral image analysis and a cell by cell assessment of antigen expression and modulation can then be conducted. In addition to the quantitative advantage, the Q-dot approach has additional advantages of high stability and enhanced brightness of the output signal. Respectively, these advantages permit accurate re-evaluation of slides with time (e.g. through a trial period of 19 months) and an increased resolution particularly useful for low abundance targets in clinical specimens. Q-dot labelled staining reagents (antibodies and RNA probes) therefore have enormous potential in clinical trials. However, these assays need to be comprehensively validated before they can be used in the clinical trial setting. This research is currently underway in collaboration with Dr Richard Byers, University of Manchester and Prof Andrew Hughes, Discovery Medicine, AstraZeneca.