Analysis of markers of cell division cycle, apoptosis and autophagy flux in glioblastoma
Thesis or dissertation
- © 2012 Jawad Yousaf. All rights reserved. No part of this publication may be reproduced without the written permission of the copyright holder.
Glioblastomas are the most aggressive and most common type of primary brain neoplasms and are associated with poor prognosis despite advances in surgical and oncological treatments. Currently available treatments include surgical resection, fractionated external beam radiotherapy and chemotherapy. This study aimed to investigate markers of cell division cycle, apoptosis and autophagy flux in an attempt to identify biomarkers with prognostic and/or predictive significance.
The cell cycle markers studied included: Mcm2, expressed throughout the cell cycle; Cyclin A, an S-phase cyclin; Geminin, a protein that prevents re-initiation; and Phosphohistone H3 (PHH3), a marker of mitosis. Apoptotic markers included two anti-apoptotic proteins, Bcl-2 and Bcl-xl; a pro-apoptotic protein, Bak; and a final executioner caspase, caspase 3. Markers of autophagy flux included LC3B, a ubiquitin like protein that form part of the core autophagy machinery; and p62, a mammalian autophagy receptor that binds ubiquitinated proteins.
A total of 66 patients were recruited to the study between 2007 and 2009. Data were collected on patient demographics, pre-operative Karnofsky score, surgical and adjuvant treatment and survival. A tissue micro-array, constructed using glioblastoma tissue was immunohistochemically-stained using antibodies against a panel of markers against the molecules described above. A semiquantitative labelling index (LI) was calculated for cell cycle and apoptotic markers using an average of 18 high power fields (hpf) in three replicate cores. Staining scores were calculated for markers of autophagy flux on the basis of cytoplasmic staining intensity (1-3) and percentage of cells with nuclear staining (1<50%, 2>50%).
Cell cycle marker LI were calculated from a cohort of 66 patients, who were further subdivided into two groups: Group 1 (n=50) underwent surgery and radiotherapy with 24 patients receiving temozolomide; and Group 2 (n=16) received surgical treatment only. In group 1, a LI, higher than the median value for Geminin and Cyclin A correlated with prolonged survival when tumours received adjuvant treatment (Kaplan Meier test, p=0.0046 and p =0.0063 respectively). In group 1, Mcm2 and PHH3 LI did not correlate significantly with survival. There was no relationship between patient survival and LI for any marker in group 2. A reduction in the LI of Mcm2, Geminin and Cyclin A was observed following administration of adjuvant treatment in three patients with recurrent glioblastoma.
Apoptotic marker LI were calculated in 28 patients, due to limited tissue availability; values below the median for Bak expression conferred a survival advantage in these patients by Kaplan Meier analysis (p = 0.0039).
LC3b and p62 staining scores were calculated in 45 patients and correlated significantly with each other. Whilst no significant correlation was observed between LC3b staining score and patient survival, p62 staining above the median conferred a survival disadvantage (Kaplan Meier analysis, p =0.017).
Geminin and Cyclin A, each showed potential as independent prognostic markers in glioblastomas receiving adjuvant treatment. This may reflect the fact that geminin and cyclin A both estimate proliferating cell sub-populations sensitive to radiotherapy/chemotherapy. The addition of these markers could therefore contribute valuable prognostic information if added to the glioblastoma diagnostic panel. The association of high Bak expression with survival advantage suggests a possible, as yet unknown, role of this pro-apoptotic protein in glioblastoma oncogenesis. The association of high p62 expression with decreased survival confirms the important role of autophagy flux in glioblastoma resistance to treatment and suggests a target for future research and targeted therapy.
- Postgraduate Medical Institute, The University of Hull
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