Humoral immunity in colorectal cancer : evaluation of the anti-p53 and anti-hTERT auto-antibody responses
Thesis or dissertation
- © 2010 Aravind Suppiah. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Carcinogenesis is a multi-factorial and multi-aetiological process involving suppressions, alterations and re-activation of key biomolecular markers. Some of these changes are recognised by the humoral system and are known as Tumour Associated Antigens (TAA) against which the humoral system is able to mount an auto-antibody response. Cancer cells are subject to two key mortality barriers (M1 & M2) as described in the "2-hit" hypothesis which are overcome by p53 dysfunction (M1) and hTERT re-expression (M2). These two molecular events generate TAA which are recognised by the humoral immune system with a corresponding auto-antibody response. The aim of this thesis was to investigate the significance of the humoral anti-p53 auto-antibody and anti-hTERT auto-antibody responses in colorectal cancer (CRC). This was performed by evaluating all published literature (1979-2009) on anti-p53 auto-antibody response and its association with p53 mutation to provide the largest cumulative sample size to date spanning 30 years. A critical review was performed of all anti-p53 auto-antibody studies in CRC, followed by an investigation into the long-term prognostic significance (minimum 5 years follow-up) of anti-p53 auto-antibody in CRC. The second aim of this thesis was to optimise a method of detecting anti-hTERT in CRC patients and correlate this with anti-p53 auto-antibody in order to investigate the significance of a joint humoral response against the two key TAA responsible for CRC immortality.
The overall prevalence of anti-p53 auto-antibody was 18.4% (3292/17,859) in all cancers and 2.2% (88/3,946) in normal/benign disease controls. The anti-p53 autoantibody presence in all published cancers reports was plotted against the reported p53 mutational rates in individual cancers and showed partial correlation (R2=0.5, correlation=0.7) between anti-p53 auto-antibody presence and p53 mutation. Anti-p53 was present in 21.5% (786/3,653) in all CRC only studies, and 19.9% (479/2,409) in CRC studies using ELISA. Anti-p53 was not associated with clinico-pathological factors or prognosis in majority of the studies. Only 4 studies associate anti-53 autoantibody with adverse clinico-pathological parameters, mostly in selective groups. The weaknesses of these studies are discussed. This association leads to anti-p53 association with adverse prognosis but only in selective analysis. The prognostic significance is observed in univariate analysis but lost in multivariate analysis when stronger traditional prognostic factors are incorporated.
This thesis initially compared serum with plasma anti-p53 auto-antibody titres and excluded plasma titres from further analysis due to the potential contamination by non-specific binding leading to falsely elevated levels (17-73% variation) of anti-p53 auto-antibody. Serum anti-p53 auto-antibody was present in 21.7% (20/92) CRC patients and 0% (0/20) controls. There was no association with age (p=0.750), sex (p-0.468), Dukes' / TNM stage (p=1.000), T- (p=0.900), N- (p=0.912), M-stage (p=0.632), location (p=0.175), differentiation (p=0.117) or mucinous component (p=0.699). The median follow-up was 97 months with median DPS and OS of 73 months and 62 months respectively. Dukes' / TNM stage, T-, N-, M-stage were prognostic indicators in univariate DFS and OS analysis. Only Dukes'/TNM stage remained an independent prognostic indicators in multivariate analysis (p=0.001). Anti-p53 auto-antibody did not display prognostic significance in univariate or multivariate analysis of OS or DFS.
Anti-hTERT auto-antibody has only been reported once in the literature, using molecular recombination to develop hTERT antigen. This thesis optimisation processes aimed develop a method of detecting anti-hTERT using less restrictive technology, and further development of a WB or ELISA to allow mass detection of serum anti-hTERT. The first step aimed to isolate hTERT using a streptavidin immune-affinity column with biotinylated anti-hTERT to capture hTERT from cancer cell lysates. This was unsuccessful and further attempts were made at identifying hTERT using Western blot (WB). Five different anti-hTERT antibodies and a multitude of WB conditions were trialled in duplicate (>100 WB), each with multiple ECL exposures. hTERT was not identified. The reason for this was narrowed down in the final experiments to the lack of specificity of the primary antibodies available. The raising of a sufficiently specific anti-hTERT antibody is required to isolate hTERT antigen.
Early CRC detection is vital in improving outcomes. The humoral response to TAA incarcinogenesis could enable earlier identification of CRC and impact prognosis.
- Postgraduate Medical Institute, The University of Hull
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