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Clinical Cancer Research Vol. 8, 1117-1124, May 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Prognostic Impact of TP53 Mutations and P53 Protein Overexpression in Supratentorial WHO Grade II Astrocytomas and Oligoastrocytomas1

Aurelia Peraud2, Friedrich W. Kreth, Otmar D. Wiestler, Paul Kleihues and Hans-Jürgen Reulen

Department of Neurosurgery, Klinikum Grosshadern, Ludwig-Maximilians-University, Munich, Germany 81377 [A. P., F. W. K., H.-J. R.]; Department of Neuropathology and Brain Tumor Reference Center, University of Bonn, Medical Center, Germany 53105 [O. D. W.]; and International Agency for Research on Cancer, Lyon, France 69372 [P. K.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Screening for TP53 Mutations...
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The purpose of this study was to analyze retrospectivelytiming, frequency, and prognostic impact of TP53 mutations and P53 protein accumulation in supratentorial WHO grade II astrocytomas and oligoastrocytomas of adult patients.

Experimental Design: We included 159 consecutively treated patients (1991–1998), and each tumor was screened for TP53 mutations and P53 protein overexpression. Prognostic evaluation was performed with the multivariate proportional hazards model.

Results: TP53 mutations (P53 protein overexpression) were detected in 49% (47%) of all tumors with a preference for the gemistocytic subtype (P < 0.05). The TP53 status of the primary tumor was predictive for the status of the recurrent tumor, and tumor recurrence/progression was associated with an increase of P53 immunopositive cells in 68% of the investigated tumors. Univariately, gemistocytic subtype and presence of TP53 mutation (but not P53 accumulation) were unfavorable predictors for progression-free survival (P < 0.05); multivariately, only the gemistocytic subtype remained unfavorable influence (P = 0.04). No overall prognostic impact of the TP53 status on survival and time to malignancy was observed (P < 0.05). Five nongemistocytic tumors with a codon 175 TP53 mutation exhibited a significantly worse prognosis as compared with those with any other mutational sites (5-year progression-free survival: 0%; 5-year malignant transformation: 100%; P < 0.001).

Conclusions: TP53 mutations are frequent and early events in the pathogenesis of WHO grade II astrocytomas/oligoastrocytomas, and most of the univariately detected overall prognostic impact of the TP53 status must be related to the influence of the gemistocytic subtype. In nongemistocytic astrocytomas, a hot spot codon 175 TP53 mutation indicates a worse prognosis in terms of time to progression and malignancy.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Screening for TP53 Mutations...
 RESULTS
 DISCUSSION
 REFERENCES
 
WHO grade II astrocytomas and oligoastrocytomas have an intrinsic tendency to progress to a more malignant phenotype, i.e., anaplastic astrocytoma (WHO grade III) and glioblastoma multiforme (WHO grade IV). Whereas some tumors progress rapidly, others remain unchanged for extended periods of time. The overall prognosis is strongly determined by pretreatment patient- and tumor-related factors. Young patients (<40 years) with small tumors volumes (<20 ml), no/minor clinical deficits, and no contrast enhancement in the computed tomography/MR3 tomography scan experience the most favorable outcome (5-year survival rates in the range of 70–80%; Refs. 1 , 2 ). Survival time decreases dramatically in patients presenting with two or three unfavorable prognostic factors. Currently available treatment strategies such as tumor resection, external beam radiation, and radiosurgery are only partially effective, and treatment responses are not uniform. A major impediment to effective treatment is incomplete knowledge of the molecular signals that underlie tumor progression. On the basis of available preliminary retrospective studies, TP53 mutations have been considered the most frequent and earliest genetic alteration in patients with WHO grade II astrocytomas/oligoastrocytomas (3, 4, 5) . However, the prognostic relevance of this finding is still debated. Small and heterogeneous study groups, different techniques for assessing the TP53 mutational status, nonstandardized treatment strategies, and the use of different histological classification systems, are major obstacles for proper evaluation of these retrospective data (3, 4, 5, 6, 7, 8, 9, 10, 11) . The present retrospective study (1991–1998) addressed these issues in the following manner: (a) a large and homogeneous cohort of adult patients with supratentorial WHO grade II astrocytomas and oligoastrocytomas was evaluated; (b) the histology of each tumor was reviewed by the National Brain Tumor Reference Center according to the criteria of the WHO 2000 (12) ; (c) standardized treatment strategies were applied (microsurgical tumor resection was the treatment of choice); (d) tumors were screened for mutations of the TP53 gene and overexpression of the P53 protein; and (e) the prognostic impact of both TP53 mutations and P53 protein accumulation was analyzed with regard to length of survival, progression-free survival, postrecurrence survival, and the risk of malignant transformation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Screening for TP53 Mutations...
 RESULTS
 DISCUSSION
 REFERENCES
 
Inclusion Parameter.
Adult patients with a supratentorial WHO grade II astrocytoma or oligoastrocytoma were eligible for this study. All of the tumor specimens were reviewed by the National Brain Tumor Reference Center in Bonn (O. D. W.) as well as by the founder of the WHO classification of brain tumors (P. K.), and evaluated for tumor grade as well as histological subtype (fibrillary, protoplasmic, gemistocytic astrocytomas, and oligoastrocytoma). Oligoastrocytomas are composed of a conspicuous mixture of two distinct neoplastic cell types morphologically resembling the tumor cells in oligodendroglioma and astrocytoma WHO grade II. The fraction of one component should be >=30% (12) . The recently revised WHO classification and grading guidelines were strictly applied. Tumors with signs of marked anaplasia and a proliferation index >5% were excluded. Finally, 159 patients (129 patients with a de novo tumor and 30 patients with a tumor recurrence), who were consecutively treated at the Neurosurgical Department of the Ludwig-Maximilians-University, Munich (January 1991 to December 1998), were included in the present study (demographic data are presented in Table 1Citation ).


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  		Table 1 Demographic data

 
Treatment Protocol.
Microsurgical tumor resection was the treatment of choice. In 25 patients with surgically not accessible tumors, BX was used to establish a histopathological diagnosis. EOR was determined according to the intraoperative impression of the surgeon and/or the description in the surgical records. Five patients with large residual tumors after microsurgical treatment and 7 patients, who underwent BX, were treated with additional radiation therapy. Eight patients with eloquently located small and circumscribed tumors received interstitial Iodine-125 radiosurgery. Generally, radiation therapy was applied as second line treatment in the case of progressive tumor growth or malignant transformation.

Patient Evaluation.
Clinical and neuroradiologic examination (MR) was performed at 6-month intervals and at the time of the last follow-up (April 1999) in the outpatient clinic of the department. In 102 patients a detailed questionnaire was sent out to achieve last follow-up informations. After STR any tumor volume increase >25% in the MR was classified as tumor progression. Any tumor regrowth after GTR was classified as tumor recurrence. Criteria for malignant transformation of the tumor were: (a) tumor histology classified as grade III/IV after surgery/BX; or (b) multilocular appearance or contrast enhancement of an initially hypointense (nonenhancing) tumor in combination with rapid tumor growth.


   Screening for TP53 Mutations and P53 Protein Overexpression
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Screening for TP53 Mutations...
RESULTS
 DISCUSSION
 REFERENCES
 
SSCP Analysis and Direct DNA Sequencing for TP53 Mutations.
Serial paraffin sections, 4 µm thick, were cut from the tissue blocks successive to the sections for immunohistochemistry. Typical tumor areas were identified and marked on H&E stained slides, and copied on the adjacent unstained sections. The marked areas were scraped from the slides and transferred to the bottom of sterile 1.5-ml tubes. The DNA was extracted by a xylene-ethanol-aceton procedure and a final digestion with proteinase K, as described elsewhere (13 , 14) . Prescreening for mutations by PCR-SSCP analysis in exons 5–8 of the TP53 gene was performed as described previously (14) . In short, PCR was carried out with 2 µl of DNA solution, 3 pmol of each primer, 50 µM of deoxynucleotide triphosphates, 1 µCi of [{alpha}-33P]dCTP (ICN, Costa Mesa, CA; specific activity, 3000 Ci/mmol), 10 mM Tris (pH 8.8), 50 mM KCl, and 1 mM MgCl2 and 0.2 units Taq polymerase (Sigma, Deisenhofen, Germany) in a final volume of 10 µl. The PCR mixture underwent 40 cycles of denaturation (94°C) for 50 s, annealing (60°C for exons 5B, 6, 7, and 8, and 55°C for exon 5A) for 60 s, and extension (72°C) for 60 s using a Robocycler 96 gradient temperature cycler (Stratagene GmbH, Heidelberg, Germany). Two µl 0.2 M NaOH and 9 µl stop solution (USB, Cleveland, OH) were added to the 1.5 µl PCR reaction mixture. Samples were heated at 99°C for 5 min and 4 µl were immediately loaded onto a 6% polyacrylamide nondenaturing gel containing 6% glycerol. Gels were run at 40W for 3 h with fan cooling at room temperature, dried at 80°C, and autoradiographed for 24–48 h.

Those samples that showed a mobility shift in the SSCP analysis were additionally analyzed by direct DNA sequencing. After PCR amplification as described above, 10 µl PCR products were digested with 2 units of shrimp alkaline phosphatase and 10 units of exonuclease I at 37°C for 15 min. After inactivation of these enzymes at 80°C for 15 min, sequencing primer (9 pmol) and 2 µl of 5X sequenase buffer [200 mM Tris-HCl (pH 7.5), 100 mM MgCl2, and 250 mM NaCl] were added. Template-primer mixture was heated at 100°C for 3 min and then placed in ice-cold water. Subsequently, 0.1 M DTT, 3 units Sequenase version 2.0 (USB), and 0.5 µCi of [{alpha}-33P]dCTP were added to samples, which were then divided into four tubes each containing termination mixture. Samples were incubated at 37°C for 10 min and mixed with 5 µl stop solution (USB). After being heated at 80°C for 3 min, samples were loaded onto a 6% polyacrylamide/7 M urea gel. Gels were dried at 80°C and autoradiographed for 12–48 h. Primer sequences for PCR and DNA sequencing were described previously (14) .

P53 Immunohistochemistry.
Tumor samples for standard histopathological evaluation and immunohistochemistry were fixed in buffered formalin, processed through graded alcohols and xylene, embedded in paraffin, and sectioned at 5 µm. Immunohistochemistry for P53 was performed as described previously (11) . For this purpose the monoclonal antibody IgG2b mouse antihuman P53 M 7001 (Dako, Glostrup, Denmark) was used. The primary antibody reacts with the wild-type as well with the mutated P53 protein and recognizes the epitope at the amino-end of the human P53 protein. The antibody was applied to formalin-fixed, paraffin-embedded sections after dilution 1:100 in PBS. The sections were boiled for 15 min in 10 mM sodium citrate buffer (1.8 mM citric acid, and 8.2 mM sodium citrate) in a steam pot. Incubation was carried out overnight at 4°C after blocking of nonspecific binding with PBS and Brij twice for 10 min. The reaction was visualized using the biotinylated monoclonal antimouse antibody E 0354 (Dako), streptavidin-horseradish peroxidase (Dako), and diaminobenzidine substrate solution. Sections were counterstained with hematoxylin. Formalin-fixed, paraffin-embedded glioblastoma sections with strong immunoreactivity for P53 protein were used as positive controls. Sections without the primary antibody served as negative control. Only the nuclear staining was considered as positive result, and only sections without background staining were used for analysis. Results of immunohistochemistry were recorded as -, negative; +, isolated positive cells; ++, clusters of positive cells; and +++, majority of cells positive.

Statistical Analyses.
Reference points for this study were the date of the first surgical procedure. The date of the last follow-up examination was April 1999. End points used were death, date of tumor recurrence/tumor progression, and date of malignant transformation. Progression-free survival was defined as time to tumor progression after STR or time to tumor recurrence after GTR. Postrecurrence survival was the time interval between tumor recurrence/tumor progression and death/date of the last follow-up. Survival time, progression-free survival, postrecurrence survival, and rate of malignant transformation were analyzed with the Kaplan-Meier method (15) . Prognostic factors were obtained from the proportional hazards model (16) . The correlation between prognostic factors was analyzed using {chi}2 statistics. The prognostic importance of each covariate was first tested univariately; additional multivariate analysis was performed whenever possible (sufficient number of events). In the case of low number of events a few alternative models with subsets of variables were tested. Comparison of alternative models was performed by computing the maximized likelihood. The BEST model contained only variables that were significantly associated with a chosen end point after adjustment for the effects of the other variables of the model; it was validated by a final check, i.e., no term in the model should be added or omitted without significantly changing the maximized likelihood. The following variables were tested: age (>= 40 years versus < 40 years), histological subtype (gemistocytic versus others), sex, Karnofsky Performance Scale (>= 70 versus < 70), EOR (GTR versus STR and BX), radiation therapy (yes versus no), TP53 mutational status (TP53 mutation versus no TP53 mutation), and mutations in hot spot codons.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Screening for TP53 Mutations...
 RESULTS
DISCUSSION
 REFERENCES
 
This series comprised 159 patients (129 patients with de novo tumors) with an average age of 37.4 ± 12.3 years and a median follow-up period of 49.1 ± 36.6 months for the survivors. The most important clinical data are presented in Table 1Citation . Eighty-four patients experienced tumor progression/tumor recurrence; forty-five of these patients underwent a second and 9 a third microsurgical procedure. Radiation therapy alone or in combination with surgical retreatment was applied in 47 patients. At the time of the last follow-up, 36 patients had died. The overall 5-year survival, progression-free survival, and malignant transformation rates were 80.5%, 48.8%, and 26.1%, respectively (Fig. 1)Citation . The overall median postrecurrence survival was 26 months.



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  		Fig. 1. Graphs showing cumulative progression-free survival, survival (A), and rate of malignant transformation (B) in WHO grade II astrocytomas and oligoastrocytomas (n, number of events).

 
TP53 Mutation and P53 Protein Overexpression.
TP53 mutations were present in 49% of the whole cohort (73 of 149 patients) and in 43.4% of those with de novo tumors (53 of 122 patients). There were 64 tumors with 1, 7 with 2, and 2 with 3 mutations (total number of mutations: 84). Thirty-six mutations were found in hot spot codons (codon 175, 5 mutations; Table 2Citation ; codon 248, 4 mutations; and codon 273, 27 mutations). The rates of hot spot codon mutations were similar in tumors with single (32 of 64, 50%) and multiple TP53 mutations (6 of 9, 66.7%; 4 of them in codon 273; Table 3Citation ). The prevalence of TP53 mutations in nongemistocytic astrocytomas and oligoastrocytomas was comparable (P = 0.12). The gemistocytic subtype was significantly more often associated with a mutated TP53 gene (7 of 9 tumors with TP53 mutations; P < 0.05); Four hot spot mutations (3 mutations in codon 273 and 1 mutation in codon 248) and 1 double mutation could be detected in this small subpopulation. Of 84 mutations, there were three deletions with frameshift and 81 missense mutations with amino acid exchange. CpG sites were involved in 64.3% (54 of 84 mutations). Development of new TP53 mutations was not observed at the time of tumor progression/tumor recurrence (84 tumors) or malignant transformation (39 tumors) in any of the investigated tumors, i.e., the TP53 status of the primary tumor was always predictive of the TP53 status in the recurrent/progressive/malignant anaplastic tumor.


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  		Table 2 Cases with TP53 mutations in the hot spot codon 175

 

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  		Table 3 Cases with multiple TP53 mutations

 
Immunostaining revealed P53 protein overexpression in 47%. Tumors with TP53 mutations showed an accumulation of the respective gene product in 91.8%. Six tumors with TP53 mutations (all point mutations with amino acid exchange) showed negative immunoreactivity for P53, whereas in 2 cases positive immunostaining for P53 was present without evidence for a TP53 mutation. The amount of P53 immunopositive cells increased from the first to the recurrent or second recurrent tumor in 15 of 22 immunopositive tumors investigated.

Prognostic Factors and Length of Survival.
The prognostic impact of variables was tested in several alternative models, which did not contain more than four variables (because of the low number of events). The only unfavorable predictor for length of survival was increasing age (>=40 years) after univariate and multivariate analysis (P < 0.05; risk ratio, 2.3; 95% confidence interval, 1.1–4.9). Nonsignificant variables were KPS, sex, seizures at the time of presentation, histological subtype, TP53 mutational status, and treatment related factors (EOR and radiation therapy). No prognostic factor could be identified for postrecurrence survival. The same prognostic pattern was obtained for 129 patients with de novo tumors (data not shown).

Prognostic Factors and Progression-Free Survival.
Unfavorable predictors of progression-free survival were a mutated TP53 status (P = 0.04; Fig. 2Citation ) and the gemistocytic subtype (P = 0.01) after univariate analysis (Fig. 3)Citation . The prognostic influence of P53 protein accumulation did not reach the level of significance (P = 0.07). In the BEST multivariate model, only the gemistocytic subtype remained a significant unfavorable influence (P < 0.05; risk ratio, 2.6; 95% confidence interval, 1.2–5.6). Five-year progression-free survival for patients without TP53 mutations was 58.7% (35.9% for patients with TP53 mutations) and for patients with nongemistoctic astrocytomas was 47.0% (15% for patients with gemistocytic astrocytomas). The same prognostic pattern was obtained when only 129 patients with de novo tumors were considered (data not shown).



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  		Fig. 2. Cumulative progression-free survival in tumors with and without TP53 mutations. Time to progression was significantly shorter in patients with TP53 mutations.

 


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  		Fig. 3. Cumulative progression-free survival in WHO grade II gemistocytic astrocytomas as compared with all other subtypes, indicating a worse prognosis for the gemistocytic subtype.

 
Prognostic Factors and the Risk of Malignant Transformation.
The 5-year malignant transformation rate was 24.2% for patients without TP53 mutations and 35.5% for those with a mutant TP53 status (P = 0.36). No overall prognostic influence of TP53 mutations could be observed and no other clinical patient-, tumor-, and treatment-related prognostic factor could be identified. Even although the gemistocytic subtype showed a higher rate of malignant transformation (5-year malignant transformation rate: 53.3% versus 28.7% for nongemistocytic tumors), this difference did not reach the level of significance (P = 0.24).

Prognostic Influence of Hot Spot Codon Mutations and Multiple Mutations.
A mutation at the hot spot codon 175 of the TP53 gene (5 patients) always resulted in an amino acid exchange of arginine to histidine (Table 2)Citation and affected only nongemistocytic astrocytomas of this series; it was associated with a significantly shorter progression-free survival (5-year progression free survival: 0%; P < 0.03) and a higher risk of malignant transformation (5-year malignant transformation rate: 100%; P = 0.0001) as compared with codon 248 and 273 mutations or non-hot spot mutations (Fig. 4)Citation .



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  		Fig. 4. Cumulative progression-free survival A) and rate of malignant transformation B) for different codons of the TP53 tumor suppressor gene with special emphasis of the hot spot codons 175, 248, and 273. Patients with codon 175 mutations did significantly poor in terms of progression-free survival and risk of malignant transformation.

 
Even although patients with multiple mutations (9 patients; Table 3Citation ) did worse as compared with those with one mutation, no significant difference could be detected, i.e., 5-year progression-free survival for patients with no mutation (76 patients), one mutation (64 patients), and multiple mutations were 58.7%, 37.7%, and 25.9%, and 5-year malignant transformation rates were 24.2%, 29.7%, and 66.1%, respectively.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Screening for TP53 Mutations...
 RESULTS
 DISCUSSION
REFERENCES
 
WHO grade II astrocytomas and oligoastrocytomas display a heterogeneous clinical course. Whereas some patients remain recurrence free for several years, others rapidly develop a recurrent tumor, not seldomly accompanied by a malignant transformation to a higher grade glioma (2 , 17, 18, 19, 20, 21, 22, 23) .

The analysis of correlations between the clinical course and TP53 mutations, which have been considered a genetic hallmark of tumor initiation or early tumor progression, may constitute a promising approach to foster new paradigms of risk assessment, early detection, determination of prognosis, and antineoplastic therapies in patients with WHO grade II astrocytomas/oligoastrocytomas. However, preliminary retrospective studies on this topic have led to divergent conclusions, and no prospective analysis has been performed thus far (5 , 6 , 8 , 11 , 24, 25, 26, 27, 28) . In the current report, the overall prognostic impact of both the TP53 status and P53 protein accumulation on progression-free survival could be analyzed for the first time after adjustment for the influence of all of the important patient-, tumor-, and treatment-related factors; this was possible because of the large number of events during the follow-up period (84 patients underwent tumor progression). In contrast, prognostic implications of TP53 inactivation regarding length of survival, postrecurrence survival, and the risk of malignant transformation were based on a low number of events (because of the relative short follow-up period of this study) and should be regarded more cautiously.

Timing, Frequency, and Pattern of TP53 Mutations.
This study confirms that TP53 mutations are frequent and early events in the pathogenesis of astrocytomas and oligoastrocytomas with a pronounced preference for the gemistocytic subtype (11) . Even although the prevalence of TP53 mutations in astrocytomas and oligoastrocytomas was comparable, it could not be excluded that the positive rate of TP53 mutations might be falsely lowered in patients with oligoastrocytomas, because investigations on LOH for 1p/19q were not performed. The TP53 status of the primary tumor was always predictive for the TP53 status of the recurrent tumor. Mutational sites within the TP53 gene were comparable with those published previously (4 , 10 , 11) . The occurrence of 2 or more TP53 mutations has been occasionally reported, and the frequency of multiple mutations in the current series (6%) was similar to that reported by Watanabe et al. (3 of 33 patients with double mutations, 9%; Ref. 11 ). Whether multiple mutations occurred in different tumor clones and indicate an early state of genetic instability of the tumor or mutagenic conditions in the tumor environment remains unclear (4 , 11) .

TP53 Status and P53 Protein Expression.
Concordance between TP53 mutations and P53 accumulation was found in 89% of all tumors investigated. Only 6 patients showed a TP53 mutation without P53 protein accumulation, and 2 patients showed P53 protein overexpression without a TP53 mutation. Similar distributions have been described by other investigators. Discordance between the TP53 status and P53 protein accumulation has been explained by a complex formation of P53 protein with other cellular or viral oncoproteins that stabilizes P53 protein and/or post-translational modification of P53 expression (9 , 29, 30, 31, 32) . In contrast to the findings of Watanabe et al. (11) , no correlation could be found between specific mutation types (such as deletions or insertions with frameshift) and the absence of P53 protein accumulation. An increase in the fraction of P53 immunopositive cells from the primary to the recurrent tumor was observed in 68% of patients with TP53 mutations irrespective of malignant transformation (3 , 5 , 8 , 33, 34, 35) . This finding suggests, in accordance with other reports, a clonal expansion of P53 immunopositive and probably mutant tumor cells, and/or an up-regulation/stabilization of the P53 protein attributable to DNA damage for a considerable number of patients (8) .

Overall Prognostic Impact of TP53 Gene Status and P53 Protein Expression.
Given the limitations of available retrospective data it is not surprising that there is no consensus about the prognostic role of TP53 mutations and P53 protein expression in patients with WHO grade II astrocytomas and oligoastrocytomas, even when SSCP analysis and direct DNA sequencing were used. In the study of van Meyel et al. (5) , for example, patients with TP53 mutations survived twice as long as those without mutations. However, only 15 patients (those for whom paraffin embedded tissue was available) have been selected for DNA sequencing and highly different treatment strategies were applied for unknown reasons. The largest series of Ishii et al. (4) revealed a strong negative impact of TP53 mutation on both progression-free survival and the risk of malignant transformation after multivariate evaluation of 36 patients with de novo or recurrent WHO grade II astrocytomas or oligoastrocytomas. However, it was irritating that radiation therapy (applied to 8 patients for unknown reasons) gained the strongest negative impact (which was not in accordance with prospective randomized data published previously; Refs. 36 , 37 ).

In the current report no overall prognostic impact of the TP53 status could be detected for length of survival (which was inversely correlated with increasing age), postrecurrence survival, and time to malignancy. Univariately, a mutated TP53 status was an unfavorable predictor for progression-free survival. However, in the final multivariate model only the gemistocytic subtype was negatively associated with progression-free survival; thus, most of the univariately detected negative influence of mutant TP53 must be related to the influence of the gemistocytic subtype (which shows a mutant TP53 status significantly more often). Whether the poorer outcome in patients with gemistocytic astrocytomas can be attributed to the detected higher frequency of TP53 mutations and/or other/additional not yet defined factors remains unknown.

The immunohistochemical approach was less sensitive. Patients with P53 protein accumulation had a shorter progression-free survival; however, the difference was not statistically significant, which was in accordance with other reports, and indicated limitations and inaccuracy of immunohistochemistry in the determination of the TP53 status (9 , 29 , 31) .

Taking into consideration shortcomings of both the Ishii study (Ref. 4 ; e.g., small sample size, poorly defined treatment strategies) and the current report (short follow-up), no definitive conclusions could be drawn at this moment about the prognostic influence of the overall TP53 status regarding survival, postrecurrence survival, and risk of malignant transformation. However, the prognostic role of the overall TP53 status on progression-free survival could be accurately described for the first time (this was possible because of the large number of events).

Prognostic Relevance of Hot Spot Codon and Multiple Mutations.
For astroglial tumors TP53 mutations are located in highly conserved regions with clusters at codon 175 (7.5%), 248 (17.5%), and 273 (7.5%; Refs. 11 , 38 ). One of the major findings of the current study was that tumors with TP53 mutations in codon 175 had a significantly shorter progression-free survival and a higher risk of malignant transformation as compared with those with mutations in other hot spot codons (such as 273 or 248) or any other mutational sites. Moreover, the unfavorable prognostic influence of codon 175 mutations exclusively concerned patients with nongemistocytic tumors, and no association with multiple mutations was observed indicating an independent prognostic role of codon 175 mutations in highly selected patients (5 patients of this series). Codon 175 point mutations with amino acid exchange of arginine to histidine (but not codon 248 and 273 mutations) result in conformation changes of the P53 protein and loss of the ability to inhibit cell proliferation as described by Ory et al. (39) . In accordance with these observations, studies on colorectal cancer have reported aggressive tumor behavior and worse outcome for patients with codon 175 mutations (40 , 41) . Results of TP53 investigations in breast cancer have suggested that direct DNA contact mutants, which are found in two-thirds of the hot spot codons 248 and 273, were associated with a better prognosis because they retain some tumor suppressor activity (42) .

The prognostic relevance of multiple mutations remains unclear, and only a few studies have reported on single cases with more than one TP53 mutation (4 , 5 , 8 , 9 , 11) . In the present series patients with multiple mutations did worse in terms of progression-free survival and the risk of malignant transformation as compared with those with a single mutation. However, the difference was not statistically significant (probably because of the low number of events). More data and longer follow-up periods will be necessary for valid estimation as to the prognostic impact of this rather rare phenomenon.

It was concluded that TP53 mutations are frequent and early events in the pathogenesis of WHO grade II astrocytomas and oligoastrocytomas with a clear preference for the gemistcytic subtype. Most of the univariately detected prognostic impact of the TP53 status must be related to the influence of gemistocytic tumors. A highly selected subpopulation of nongemistocytic tumors harboring codon 175 TP53 mutations exhibited a significantly shorter progression-free survival and a higher rate of malignant transformation. The therapeutic implications of these findings have to be defined.


   ACKNOWLEDGMENTS
 
We thank Prof. Dr. P. Mehrain, the Institute for Neuropathology, Ludwig-Maximilians-University of Munich, for providing paraffin sections of low-grade astrocytomas and oligoastrocytomas. We also thank Julia Winkler for excellent technical assistance, and Mélanie F. Daffner, Konstantinos Sarvanakis, and Magnus Meschede for help in patient data acquisition.


   FOOTNOTES
 
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.

1 Supported by the Deutsche Forschungsgemeinschaft (PE 758/1-1) and by the Deutsche Krebshilfe (to O. D. W.). Back

2 To whom requests for reprints should be addressed, at Department of Neurosurgery, Klinikum Grosshadern, Marchioninistrasse 15, 81377 Munich, Germany. Phone: 49-89-7095-1; Fax: 49-89-7095-5584; E-mail: Aurelia.Peraud{at}helios.med.uni-muenchen.de Back

3 The abbreviations used are: MR, magnetic resonance; BX, stereotactic biopsy; EOR, extent of resection; GTR, gross total resection; STR, subtotal resection; SSCP, single-strand conformational polymorphism. Back

Received 11/14/01; revised 2/20/02; accepted 2/22/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Screening for TP53 Mutations...
 RESULTS
 DISCUSSION
 REFERENCES
 

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