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Pathological and immunological analysis of RDEB-SCC for the development of novel biomarkers (Marshall 4)

Completed
Project lead Dr John Marshall
Organisation Queen Mary University of London, Centre for Cell Biology and Cutaneous Research, London, UK
Project budget EUR 119,228.00
Start date / Duration 01. Apr 2010 / 24 months
Funder(s) / Co-Funder(s) DEBRA Austria, MSAP/EBEP Recommended
Research area Skin cancer & fibrosis

Project details

Short lay summary

For many years the EB research community has been unable to determine why squamous cell carcinoma in RDEB patients have a metastatic propensity (and kill patients) compared with non-EB SCC which is rarely metastatic. Since development of SCC in RDEB is usually associated with chronic wound healing this suggests that this host response provides a permissive environment for cancer to develop and spread. Whereas others have concentrated on the transformed keratinocytes in SCC we shall use a non-candidate approach to examine the stroma of RDEB SCC. Thus to identify biomarkers associated with the EB-SCC we will 1) use expression proteomic analysis of stromal isolated using PALM laser dissection 2) incubate EB-SCC tissues with scFv antibody phage libraries and identify those stromal proteins differentially expressed in the EB-SCC tumour stroma versus normal skin or non-EB SCC 3) for the first time we shall undertake a systematic histopathological analysis of EB-SCC versus non-EB SCC archival tissues that will direct immunopathology analysis of the tumour-associated environment (stromal fibroblasts, EMT, angiogenesis, protease activity as inflammatory infiltrate). These data, which will be collated as a database for the EB community, should identify factors associated specifically with EB-SCC. 

 

Strategic relevance

All cancers develop by talking to the normal cells that surround them. In some instances, they send signals to this ‘microenvironment’ which can result in some of these completely normal cells changing their behaviour in such a way that it actually helps the cancers to grow faster and spread more easily. So modern cancer therapy must consider stopping both the cancer cells AND those normal cells that produce factors that help the cancer cells. The skin cancers that develop in EB patients, especially those with RDEB, tend to develop more frequently and then spread more easily than similar types of skin cancer that develop in people without EB. Colleagues have found however that the actual skin cancer cells themselves in people with EB and without EB are extremely similar. Thus we considered that the reason the cancers in EB patients do worse is that the response of their microenvironment is different. We believe that the proteins produced in the microenvironment are different in skin cancers from EB patients compared with non-EB patients and that one or more of these proteins may help the EB cancers to spread more easily. Therefore we have use 2 techniques to study this possibility.

What did this project achieve?

Firstly we have used special microscopy techniques to collect the area around the skin cancers (the so called microenvironment) in EB patients and then, using a complex technique called Mass Spectrometry, examined which proteins are present, and then compared this list with the proteins we found in non-EB skin cancers and normal skin. To date we have found over 100 proteins whose presence varies significantly. We are now checking is samples from skin cancers from EB patients to confirm whether our results are true for all patients or only some. These studies will result in additional experiments to discover how these new proteins affect skin cancer development.

In a second project we have developed a technique where we can take many millions of small virus like particles (called bacteriophage) that each have on their surface a different mini-antibody (called single chain Fv antibodies or scFv) and place them on top of slices of cancers taken from people with EB. These antibodies can stick the bacteriophage to proteins in the microenvironment. We then collect only those bacteriophage that have stuck to the microenvironment, use special techniques to increase their number and then repeat the binding process.  This allows us to enrich the ‘library’ of bacteriophage to include only those that bind to the microenvironment. We can then use the specificity of the mini-antibody to identify which protein they were sticking to. We hope to use this method to show, not only that there are many different proteins in the microenvironment of skin cancers of EB patients, but that we have a mini-antibody that binds to it. We hope that these mini-antibodies might then be developed into targeting agents for future therapies that are against the microenvironment of these cancers.


Keywords

dystrophic EB
DEB
SCC
Marshall 4
Marshall
Queen Mary University
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