Molecular prognostic markers in renal cell carcinoma
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Date
27/11/2015Author
Laird, Alexander
Metadata
Abstract
Renal cell carcinoma (RCC) is the most deadly of urological malignancies. While
metastatic disease affects one third of patients at diagnosis, a further third of patients
who undergo extirpative surgery with curative intent subsequently develop metastatic
disease. Inconsistency in the clinical course ensures predicting subsequent metastasis
is notoriously difficult, despite the routine use of prognostic clinico-pathological
parameters in risk stratification. With greater understanding of pathways involved in
disease pathogenesis, a number of biomarkers have been proposed to be of
prognostic significance; however there are currently no molecular prognostic
markers in clinical use. Genetic intra-tumoural heterogeneity (genetic ITH) has been
described in clear cell RCC (ccRCC) and may limit the clinical translation of
biomarkers. There has been no assessment of ITH at other molecular levels. The aim
of this work was to define and compare proteomic, transcriptomic and DNA
methylation ITH in ccRCC, and identify potential prognostic biomarkers.
Using reverse phase protein arrays to study protein expression in multiple spatially
separate regions of primary and metastatic ccRCC, proteomic ITH was demonstrated
for the first time. Interestingly there was no significant difference in proteomic ITH
in metastatic ccRCC tumour deposits compared to primary tumours. However, on
analysis of differential protein expression between primary and metastatic ccRCC
tissue using a tissue microarray and automated analysis of immunofluorescence,
there was significantly greater expression of Ki67, p53, VEGFR1, SLUG and SNAIL
in the metastases compared to the primary tumours. Subsequent profiling of gene
expression and DNA methylation in multiple areas of the same primary tumours
confirmed transcriptomic and methylomic ITH. On comparison of this multimolecular
ITH, significantly greater proteomic ITH was seen compared to gene
expression and DNA promoter methylation heterogeneity.
Recent evidence suggests DNA methylation may be prognostically important in RCC
and given the lower methylomic ITH in ccRCC, the identification of prognostic
DNA methylation changes in ccRCC were pursued using the Infinium
HumanMethylation450K Beadchip. Following development of an analysis pipeline,
identification and validation of prognostic differentially methylated regions (DMR)
was performed on an experimental cohort and published dataset respectively. Five
DMRs, which were associated with disease recurrence in ccRCC, were identified.
NEFM gene promoter methylation was the only DMR associated with cancer
specific survival, independent of TNM stage and nuclear grade on multivariate
analysis, which was confirmed on a third independent published dataset.
This thesis therefore demonstrates multi-molecular ITH in ccRCC for the first time.
Despite this, NEFM promoter methylation may be a useful independent prognostic
marker of cancer specific survival.