Centre for Academic Surgery -

1. A low-cost, rapid and robust qPCR assay for the identification of Clostridium difficile and its toxins

Clostridium difficile (CD)–associated infection (CDI) is a substantial cause of morbidity and mortality, responsible for more deaths than all other intestinal infections combined. Furthermore, incidence and CDI related hospitalisation, and mortality is on the rise. Attributable deaths rose by 72% between 2005 and 2006 to around 6,500 people in the UK alone. Symptoms range from mild watery diarrhoea to life-threatening intestine perforation. The economic impact of CDI, largely attributable to the extended hospital stays incurred, is considerable, recently estimated to be at least $3.2 billion in the US and €3 billion a year in the EU. Current forms of diagnosis, designed to detect the organism and screen for the presence of its toxins, are inadequate as they are slow, costly and poorly standardised.  The high frequency of CDI, the likelihood of a poor clinical outcome for cases that are not treated promptly and the emergence of new more virulent CD strains, highlights the urgent need for more rapid and flexible, as well as less costly methods for detecting CDI and identifying the best course of treatment.
The high sensitivity and specificity of the real-time polymerase chain reaction (qPCR), a quantitative DNA amplification assay, has the potential to overcome these limitations; however, currently available qPCR assays lack sensitivity and are very expensive. In addition, none are specifically optimised for thorough CD characterisation and all rely on the detection of DNA probes, accounting for their high cost and complexity.
Our approach, which uses several innovative features, shows strong potential for the development of a rapid, flexible, reliable, sensitive and low-cost assay for the simultaneous detection of CD and its toxins. This would result in a significant advance in clinical efficiency, severely reducing the economic burden attributed to CD by reducing the length of hospital stays and significantly reducing morbidity and mortality. Importantly, the assay design also shows potential for the detection of any known pathogen or combination of pathogens.

2. Prognostic markers: Development of a tumour atlas in colorectal cancer

Aim: to generate clinically relevant RNA signatures from microdissected tumours for outcome and treatment evaluation in colorectal cancer.

The development of molecular analytical techniques and their application to clinical diagnostic studies has been rapid. However, the reliable interpretation of results obtained from gene expression analyses is largely influenced by the selection of appropriate study material. Colorectal cancer biopsies contain multiple cell types, are multiclonal and are characterised by distinctive histopathologies with discrete gene expression profiles. As a result, gene expression profiles from bulk biopsies obscure clinically relevant expression signatures from areas associated with poor treatment outcome, such as those characterised by vascular and lymphatic invasion and lymphocytic infiltration. A critical consequence of this heterogeneity is that such samples are unable to provide robust expression signatures capable of accurate prognostic stratification of individual colorectal cancer patients following surgery. Our solution is to use microdissection to establish gene expression profiles from specific histopathological areas that are clinically relevant and associated with poor outcome. This will allow the development of a tumour atlas associating defined histopathological areas with specific gene expression profiles and treatment outcome.

Scientific relevance: This study represents a systematic and novel approach to identifying RNA signatures that are more appropriate for predicting disease outcome than those obtained from bulk biopsies. They should define distinct gene expression features associated with high metastatic potential and poor treatment outcome in primary colorectal cancers.