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Full NameMr John Burke
Organisation:Royal College of Surgeons in Ireland
- genetics, genomics and molecular biology
- epidemiology/population health research
- Clinical Trials
There are over 600 cases of rectal cancer diagnosed annually in Ireland. The treatment and survival of this condition has improved significantly through centralization, multidisciplinary team care and neoadjuvant chemoradiotherapy (CRT). The mucinous subtype of rectal cancer comprises approximately 14% of rectal cancer cases, and responds poorly to neoadjuvant CRT, with a 68% reduced likelihood of achieving tumour down-staging in response to neoadjuvant CRT with an associated adverse overall survival. Thus, this cohort of patients represents a group in urgent need of investigation.
Our research group is examining how the mutational burden in mucinous adenocarcinoma differs to non-mucinous adenocarcinoma and the mechanism via which this plays a role in the resistance to chemoradiotherapy we observe. Using a wide variety of techniques, our current program of research is determining the genetic architecture which leads to the development of mucinous rectal adenocarcinoma, exploring the intracellular signalling mechanisms that lead these tumours to be resistant and examining alternative chemotherapeutic regimens which may be more successful in achieving a response to treatment.
Using in-vitro techniques, candidate genes from our whole exome sequencing study will be knocked down using CRISPR/CAS9 genome editing technology. The knockdown will be confirmed using PCR and Western blot and the effect of this knockdown on the expression of genes such as MUC1, MUC2, MUC5AC, and MUC6 using Western blot will be determined. Synchronously, the effect on cellular apoptosis will be determined by crystal violet assay, clonogenic survival assay and flow cytometry with propidium iodide staining. The individual mucin glycoproteins induced by gene modulation will then be over-expressed using plasmid transfection to determine their effect on cellular apoptosis. Lastly, these cells over-expressing mucin glycoproteins will be treated with 5-FU, oxaliplatin, irinotecan, cetuximab and radiotherapy to determine relative in-vitro chemo- and radio-sensitivity.
A stable transfected knockdown cell line of a candidate gene from Phase 1 will be utilized in an in-vivo murine radiochemotherapy model to confirm the importance of this gene in radioresistance in rectal cancer.
The prognostic relevance of protein expression of candidate genes from our whole exome sequencing study will be assessed in a tumor micro-array to determine the association of genes of interest with radiochemotherapy response, clinicopathological variables and survival outcomes.