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[Cancer Research 66, 6473-6476, July 1, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Association of Mutant TP53 with Alternative Lengthening of Telomeres and Favorable Prognosis in Glioma

Yu-Jen Chen1, Vicky Hakin-Smith3, Mario Teo3, George E. Xinarianos3, David A. Jellinek4, Thomas Carroll4, David McDowell2, Martin R. MacFarlane5, Ronald Boet5, Bruce C. Baguley6, Antony W. Braithwaite1,7, Roger R. Reddel7 and Janice A. Royds1

1 Department of Pathology, University of Otago; 2 Dunedin Public Hospital, Dunedin, New Zealand; 3 Institute for Cancer Studies, Division of Genomic Medicine, University of Sheffield; 4 Royal Hallamshire Hospital, Sheffield, United Kingdom; 5 Christchurch Hospital, Christchurch, New Zealand; 6 Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand; and 7 Children's Medical Research Institute, Sydney, New South Wales, Australia

Requests for reprints: Janice A. Royds, Department of Pathology, Dunedin School of Medicine, University of Otago, P.O. Box 913, Dunedin, New Zealand. Phone: 64-3-4797471; Fax: 64-3-4797136; E-mail: Janice.royds{at}stonebow.otago.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular basis for alternative lengthening of telomeres (ALT), a prognostic marker for glioma patients, remains unknown. We examined TP53 status in relation to telomere maintenance mechanism (TMM) in 108 patients with glioblastoma multiforme and two patients with anaplastic astrocytoma from New Zealand and United Kingdom. Tumor samples were analyzed with respect to telomerase activity, telomere length, and ALT-associated promyelocytic leukemia nuclear bodies to determine their TMM. TP53 mutation was analyzed by direct sequencing of coding exons 2 to 11. We found an association between TP53 mutation and ALT mechanism and between wild-type TP53 and telomerase and absence of a known TMM (P < 0.0001). We suggest that TP53 deficiency plays a permissive role in the activation of ALT. (Cancer Res 2006; 66(13): 6473-6)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The behavior of gliomas and hence the survival of patients with these tumors are determined by the molecular nature of the malignant cells. We have shown that, for patients with glioblastoma multiforme, telomere maintenance mechanisms (TMM) correlate with patient outcome (1). Patients whose tumors have an alternative lengthening of telomeres (ALT) mechanism (2) have a prolonged survival, whereas telomerase confers a poor survival. Interestingly, a significant fraction of glioblastoma multiformes has no known mechanism for telomere maintenance. Germline mutations in the TP53 tumor suppressor gene are associated with increased risk of gliomas (3), and somatic mutations are found in a significant proportion of sporadic gliomas (4). The effect of these mutations on survival is equivocal (5–9), and the relationship of TP53 status with TMM has not been studied previously. We correlated TP53 status of gliomas with telomere maintenance and patient outcome. We show here for the first time that mutant TP53 correlates strongly with the ALT mechanism and good prognosis, whereas, for patients with telomerase-positive tumors, mutant TP53, although rare, confers a worse prognosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient population. We obtained tissue samples from 110 patients recruited from Neurosurgical Units at Dunedin, Christchurch, Auckland (from year 2002-2005), Hull, and Sheffield (from year 1997-2002). The study was approved by institutional ethics committees, and all patients provided informed consent. The inclusion and exclusion criteria were the same as published previously (1). The histologic diagnoses were made by consultant neuropathologists at each center. All patients received surgical debulking followed by 60 Gy of fractionated radiotherapy using megavoltage X-ray. Patient age and survival were calculated from the time of first surgery. Patients were followed up until date of death or until Nov 10, 2005. Ninety-seven patients were included in the survival analysis (6 patients were excluded because the length of their follow-up was less than the reported median survival of 8 months; ref. 1; 7 patients were lost during follow-up.). This number of patients was considered appropriate for the survival analysis, as it was more than thrice greater than the minimum number of 30 events (10 events per degree of freedom; 3 degrees of freedom) required for the Cox proportional hazard regression model used in the study (10). The follow-up period for the six survivors in the survival analysis was 1,872, 900, 510, 450, 330, and 330 days. The median follow-up time for all patients was 243 days.

Telomere maintenance mechanism analysis. To determine the TMM of each sample, we measured telomerase activity and telomere length from frozen tissues using TeloTAGGG Telomerase PCR ELISA Plus kit (Roche Applied Science, Penzberg, Germany) and TeloTAGGG Telomere Length Assay kit (Roche Applied Science, Mannheim, Germany) as described previously (1). ALT status was confirmed for samples with long telomeres by staining for ALT-associated promyelocytic leukemia bodies (APB) on paraffin sections as described previously (11).

TP53 mutation analysis. In the United Kingdom samples, exons 2 to 11 of TP53 were screened for mutations, including the intron-exon boundaries as described previously (12). For the samples from New Zealand, TP53 exons together with the intron-exon boundaries were amplified by PCR; the products were annealed with products from the control cell line, A549, and then subjected to denaturing high-performance liquid chromatography done on a Wave DNA Fragment Analysis System (Transgenomics Limited, Crewe, United Kingdom; ref. 13). Samples with dissociation curves that differed from the control were sequenced. The likely effects of the identified TP53 sequence changes were predicted using the Sorting Intolerant From Tolerant (SIFT) program and the "TP53 mutations database" from the Universal Mutation Database.8

Statistical analysis. All the assays were done blinded to the study end point. {chi}2 test and Fisher's exact test were done using GraphPad InState (version 3.05 for Macintosh, GraphPad Software, San Diego, CA). Survival was analyzed by the Kaplan-Meier method using GraphPad Prism version 4.00 for Macintosh (GraphPad Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TP53 mutations. All 110 tumors studied were high-grade gliomas, including anaplastic astrocytoma in 2 (1.8%) patients and glioblastoma multiforme in 108 (98.2%) patients. Overall, 18 of 110 (16.4%) gliomas had evidence of ALT. Thirty-three of 110 (30.0%) showed telomerase activity (Table 1 ). Fifty-nine (53.6%) tumors had no detectable TMM ("none"). TP53 mutation was detected in 28 of 110 (25.5%) patients, which is consistent with another report (14). One patient had mutations in two exons (Table 2 ). Overall, 29 mutations were detected, of which 27 (93.1%) were missense mutations, 1 (3.5%) was a nonsense mutation, and 1 (3.5%) was a splicing mutation. Nineteen of 29 (65.5%) were transition mutations. Twenty-seven (93.1%) mutations were located in the commonly mutated regions, exons 5 to 8. Outside exons 5 to 8, 1 mutation was found in exon 10, and a splicing mutation was found in intron 4. The most frequent mutation found in this study was R273H, which was detected in an ALT tumor and in three "none" tumors. Interestingly, we did not find any mutations in codons 175 or 245. Although SIFT results showed all mutations apart from R273H were deleterious, the R273H mutation has been reported to have defective activity toward promoters of various genes, including BAX, MDM2, and WAF1 (15). We have also found that the R273H mutation fails to enhance activity of adenoviral major late promoter, the principal promoter regulating expression of late virus protein in the presence of E1A (16). The average age of patients with mutant TP53 was significantly younger at diagnosis than those with wild-type (WT) TP53 (49.1 ± 3.5 and 59.4 ± 1.4 years, respectively; P = 0.0102), which agrees with Louis et al. (17). In our study, TP53 mutations were observed in 14 of 18 (77.8%) ALT tumors, which is significantly different from telomerase-positive tumors (7 of 36, 19.4%; P < 0.0001) and "none" tumors (7 of 59, 11.9%; P < 0.0001; Table 1). Mutant TP53 and ALT were more likely to occur in younger patients, whereas WT TP53, telomerase, and "none" were more likely to occur in older patients.


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  		Table 1. Clinical data for groups stratified by telomere maintenance mechanism phenotypes

 

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  		Table 2. Histologic and clinical characteristics of 28 high-grade gliomas with TP53 mutations

 
ALT confers a survival advantage over and above mutant TP53. Previous reports on the prognostic value of TP53 mutation in glioma are equivocal (5–9). In this study, TP53 mutation alone affected the overall survival of glioblastoma multiforme patients (Fig. 1A ). The median survival of glioblastoma multiforme patients carrying TP53 mutation versus those with WT TP53 was 12.2 and 7.6 months [ratio, 1.599; 95% confidence interval (95% CI), 0.9713-2.226; P = 0.0032]. The hazard ratio (HR) was 0.5202 (95% CI, 0.3323-0.8015; P = 0.004).


Figure 1
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  		Figure 1. Kaplan-Meier analysis of patient survival with different (A) TP53 status and (B) TMM and TP53 status. Labels are patients who were still alive.

 
Previously, we have shown that patients with ALT-positive glioblastoma multiformes had a better survival than those with ALT-negative tumors (1). In this study, we found that ALT patients had better overall survival irrespective of their TP53 status (22.4 and 20.65 months for mutant TP53 and WT TP53 each) than patients with telomerase/mutant TP53, telomerase/WT TP53, none/mutant TP53, and none/WT TP53 (7, 6.7, 11, and 7.8 months, respectively; P = 0.0008; Fig. 1B). Patients with ALT-positive glioblastoma multiformes and WT TP53 did as well as those with mutant TP53 (Fig. 1B). It would be interesting to know if the TP53 pathway is disturbed by other means in the ALT/WT TP53 tumors. Moreover, ALT-negative/mutant TP53 tumor patients did less well than ALT-positive/mutant TP53 (7.6 and 22.4 months; P = 0.0010; Fig. 1B), showing that ALT seems to confer a survival advantage over and above mutant TP53. Thus, ALT per se has a positive effect on outcome and is not just a surrogate marker for TP53 mutation. There is a trend that patients with none/mutant TP53 had a better overall outcome than those with telomerase/mutant TP53, although it is not quite significant (P = 0.0687). This suggests that some of the "none" tumors are essentially different from telomerase tumors. TP53 is no longer significant for outcome after adjusting for age, gender, and TMM (P = 0.550), but TMM is significant after adjusting for age, gender, and TP53 status (P = 0.006 and 0.001 for the "none" and the telomerase groups compared with the ALT group; HR, 2.9258 and 3.7788).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mutant TP53 predisposes to ALT. Our data provide the first clinical evidence to support the notion that activation of ALT during tumorigenesis requires loss of normal TP53 function. This is supported by the observation that >95% of ALT cell lines are impaired in the p53 pathway, either through TP53 mutations or the expression of viral oncogenes (18, 19), and by in vitro studies, indicating that p53 inhibits DNA synthesis in ALT cells by a mechanism that seems to involve DNA binding and suppression of recombination (20). It therefore seems likely that TP53 deficiency plays a permissive role in the activation of the ALT recombinational mechanism, so we speculate that occurrence of TP53 mutation early in tumorigenesis predisposes to ALT. These findings may prompt similar studies in other forms of cancer for which ALT has been described (21).

We show that TP53 mutation correlates with a less aggressive type of tumor, which is more likely to occur in young patients. We have shown previously that ALT is far and away the most powerful prognostic factor yet found for glioblastoma multiforme (1). Patients with an ALT-negative phenotype have poor prognosis irrespective of TP53 status and/or young age. The prognostic implication of TP53 status, which has been controversial for gliomas, can now be explained in terms of the association between mutant TP53 and ALT and thus a more benign biology. Hence, the proportion of ALT patients in a study population has a highly significant effect on whether TP53 mutation correlates with good overall patient survival or not.

Our work shows that mutant TP53 can no longer be regarded as a universal marker of poor survival. These data also suggest that targeting mutant TP53 could form the basis of selective therapy for low-grade astrocytic lesions, which are likely to acquire an early TP53 mutation (4) and the ALT mechanism of TMM (21) and to progress to secondary glioblastoma multiforme.


   Acknowledgments
 
Grant support: Cancer Society of New Zealand and Health Research Council of New Zealand (J.A. Royds), program grant from the Cancer Council New South Wales and fellowship of the National Health and Medical Research Council of Australia (R.R. Reddel), Health Research Council of New Zealand (A.W. Braithwaite), Yorkshire Cancer Research and Internal Divisional Funds from the Division of Genomic Medicine, University of Sheffield Medical School (G.E. Xinarianos), Wolfson National Award from the Royal College of Physicians and Damai Medical and Heart Clinic, Malaysia (M. Teo), and University of Otago Prestigious Scholarship (Y-J. Chen).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Christchurch Tissue Bank for their help with collection of tumor samples; Anna Wiles, Lisa Gallagher, Patricia Fogarty, Tim Morgan, Elaine Marshall, and Shafa Al Nadaf for excellent technical work; Peter Herbison for help with statistical analysis; Graham Stevens, Wendy Telling, and David Levy for clinical data collection; and Jeremy Henson in setting up APB analysis.


   Footnotes
 
Note: Current address for M. Teo: Glasgow Royal Infirmary, Glasgow, Scotland, United Kingdom; G. Xinarianos: Roy Castle Lung Cancer Research Programme, University of Liverpool Cancer Research Centre, Liverpool, United Kingdom.

8 http://www.umd.be:2072/index.shtml. Back

Received 3/10/06. Revised 4/24/06. Accepted 5/ 5/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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