Epidermal growth factor receptor: An independent predictor of survival in astrocytic tumors given definitive irradiation

l

Clinical Original Contribution EPIDERMAL GROWTH FACTOR RECEPTOR: AN INDEPENDENT PREDICTOR OF SURVIVAL IN ASTRUCYTIC TUMORS GIVEN DEFINITIVE IRRADIATION AN ZHU,

M.D.,* JAMES SHAEFFER, PH.D.,* SUSAN LESLIE, B.S.,* PAUL KOLM, AND ANAS M. EL-MAHDI, M.D., Sc.D., F.A.C.R.*

PH.D. ’

*Department of Radiation Oncology & Biophysics, ‘Office of Biostatistics, Eastern Virginia Medical School. Norfolk, VA owth factor receptor (EGFR)

protein was

Results: The percentage of tumor cells which were EGFR positive related to reduced survival by Cox in both univariate @ = 0. vity was the only 1 of 11 cl recurrence-free survival by either univariate (p grow-& facIor receptor protein expression was n Courkion: Epiderrtlsl growth factor receptor . mdicator for overail survival and recurrence-free survival for irradiated patients with astrocytic $omas. Astrocytic survival.

tumors, Epidermal

growth

factor receptor, Immunohistochemistry,

Survival, Recurrence-free

The prognosis of patients with astrocytic tumors remains extremely poor. The main reason for such an outcome is the aggressive feature of the tumor or high recurrence frequency, which, to some degree, depends on the grade of malignancy (7, 10, 14- 16), histopathologic features (10, 17), age at diagnosis (7, 10, 13-18, 20, 21), and therapeutic intervention (7, 10, 13, 14, 17, 20, 21). Clinical outcome cannot be predicted in individual patients. Epidermal growth factor receptor (EGFR) is the product of v-e&B, oncogene (l), which is located in chromosome 7 (23). The EGFR gene is the most commonly affected oncogene abnormality in astrocytomas (11, 23). Epidermal growth factor receptor gene amplification was reported to stimulate progression of malignant grade (2), appeared largely restricted to glioblastoma multiforme (23), and occurred more commonly in older patients (11).

Recently, a high incidence of EGFR protein expression was reported in malignant gliomas, and it was suggested that the expression of EGFR protein in highly proliferative malignant gliomas is compatible with the role of EGFR in the induction of the malignancy ( 12). Some studies showed that the incidence of EGFR protein expression in astrocytoma is related to histologic Grade 4 (glioblastoma multiforme), and may provide a useful tool to distinguish anaplastic astrocytoma and glioblastoma multiforme (1). The proliferative ability of astrocytoma cells determined by Ki-67 was positively influenced by expression of EGFR protein, which suggested that EGFR might be involved in controlling tumor proliferation (9). Currently, the association between EGFR protein expression and patient prognosis has not yet been well evaluated. It was reported in a study of a small group of patients with astrocytic tumors that EGFR positive patients had poor survival (9), but EGF%‘s influence on

Reprint requests to: An Zhu, M.D., Department of Radiation Oncology & Biophysics, Eastern Virginia Medical School, P.O. Box 1980, Norfolk, VA 23501. Acknowledgements-The authors express their appreciation to Therese Babb, R.N., and Claudia Brigham, B.S., for help with

obtaining clinical information, to Myra Frost for preparing the manuscript, and to Mary Lou Beckett for photographic assistance. The study was supported in part by a grant from the Sentara Endowment Fund. Accepted for publication 20 October 1995.

INTRODUCTION

809

Volume 34, Number 4,

810

tumor recurrence has not been examhed. whether or not EGFR infiuences prognosis ~nde~~deu~y of other known prognostic factors is unclear. In different studies (1, 9, 12), there was lack of agreement concerning the incidence of EGFR protein expression in astrocytic tumors. We designed this study to explore the association of EGFR protein expression and (a) patient survival, (b) grade of malignancy, (c) histologic features, (d) age at diagnosis, and (e) sex. Because previous studies indirectly imply that EGFB might influence prognosis in some way, the ~tenti~ relations~p of EGFB to survival was the particular concern of this study.

Table 1. Characteristics of the patient population Number of patients t%) Category Sex Age

Prior surgery

Tumor size METHODS

AND MATERIALS Tumor location*

Between 1976 and 1989, 123 patients with astrocytic tumors were given definitive radiation therapy at this institution. All patients were treated with modified wholebrain irradiation. For those patients having prior surgery, a safety rn~~n of 2-3 cm around the shack defect was used. For those patients having biopsy only, the computerized t~ograp~c (CT) image was used to define the volume irradiated. In general, patients with cerebral lesions had their whole brain irradiated, with the exception of infratentorial structures. For those patients with tumors in the posterior fossa, most of the cranium was include in the treatment volume, excluding the frontal lobe. Radiation treatments were administered as 60 Gy in 30 fractions at 2 Gy per fraction, 5 times per week. A 4 MV linear accelerator was used to treat all patients. Clinical info~ation was obtained for all 123 patients, which included age at diagnosis, sex, overall survival, and recurrence-free survival. Survival times were computed from the date of diagnosis. The criteria for tumor recurrence were radiographic, not histopathologic, and thus, were subject to interpretive error. In those patients treated before the advent of CT, ~gio~s were used to help define disease progression. After the advent of CT, these examinations were performed routinely at 6-month intervals. Clinical deterioration was not observed in the presence of stable imaging. Depending on the site of the lesion and other factors related to the patient’s condition, selected patients were reopened for a “second look” and surgical debulking was performed. Histologic grading Formalin-fixed, paraffin-embedded blocks were available for 71 of the 123 patients. Table 1 lists the clinical characteristics for the patient population, both for the entire (n = 123) irradiated population as well as those (n = 71) for whom archival paraffin blocks could be obtained. ~istopathologic evaluation and grading of the malignancy ’ Sigma Chemical Co., St. Lo&s, MU. ’ DAK0 Corp., Carpinteria, CA.

19%

Variable

n = 71

n = 123

Fern&e Male 40 r50 Biopsy Partial Total Not spec%ed --c5cm ~5 cm Not specified Parieti Front& Temporal Occipital Cerebellar Corpus callosum

29 (41) 42 (59) 25 (35) 46 (65) 1 Of 51 (72) 16 (23) 3 (41 45 (63) 21 (30) 5 (7) 38 (54) 30 (42) 27 (38)

50 (41) 13 (59) 42 (34) 81 t6Ei) 1m 96 (78) 22 (18) 4 (3) 18 (63) 36 (29) 9 (7) 58 (47) 50 (41) 40 (33) 16 (13) 4 (3) 1 (11

6 (8)

0 (01 0 to‘,

* Some patients had multiple sites of tumor ovation.

was done according to the three-tiered system used by the National Brain Tumor Study Group (3). This analysis revealed 55 cases of glioblastoma multiforme (GBM), 14 ~apl~tic as~~~orna (APA), and 2 ~~~ytorn~ CAST). Immunohistochemistry and assessment of EGFR protein expressitln The monoclonal mouse antibody F4’ and the DAK@labeled s~ep~vi~n biotin kit were-used for immunohist~ chemical st~n~g. After being dep~~z~, the 5 pm sections were covered with 0.2% trypsin,’ incubated 5 min, and rinsed with phosphate-buffered saline (PBS). All operations were done at room temperature. Three percent hydrogen peroxide was applied to the specimens, which were incubated 5 min, and rinsed in PBS. The specimens were covered with blocking serum and incubated 30 min. Excess serum was tapped off the specimens, to which were applied mouse anti-EGFR at a 1:3000 dilution. Specimens were then incubated at 4°C overnight. After rinsing in PBS, sections were incubated with link antibody for 30 min, rinsed again, and incuba~ with diluted s~ep~vi~n for 30 min. Substrate solution was applied to the specimens, which were monitored under a microscope until satisfactory staining was achieved in positive control sections. Slides were lightly counterstained with hematoxylin and coverslipped. Epidermal growth factor receptor protein positive cells were examined and quantified. Because of regional heterogeneity of EGFR staining, tumor cells were evaluated from all available fields. Endothelial cells and areas of tumor necrosis were excluded from

EGFR in astrocytoma0 A. ZHJ et al.

evaluation. Epidermal growth factor receptor positive cells were estimated as a percentage of stained tumor cells from the total number of cells examined. The in-lass correlation coefficient (ICC), calculated by the method of Winer (24), was used to assess intraobserver reliability of percent positive and intensity of EGFR sta&ing in a double-bled study using two observers. The KC was 0.866 for positivity, and the mean for these two observers was 0.928. For intensity, the ICC was 0.564, and the mean was 0.721. Thus, the was a high degree of agreement between the two observers for positivity, and a lesser, but adequate, reliability for EGFR intensity. Image analysis and ~s~ss~nt of h~st~~tha~og~c features Using an image analysis system4 fitted with a 100X oil immersion lens, tumor cells on ~~oxy~ and e&n slides were meas for nuclear ama and nuclear roundness factor (NRF). Nuclear roundness factor was selected as a morphometric feature to quantify the grading criterion of nuclear pl~rn~~rn. Within a given case, different tumor areas with varying degn2e.sof differentiation were often observed. For this reason, the areas with the poorest level of diffemntiation were selected for image analysis. Within these areas, individual cells were included for analysis based on nuclear integrity and nonoverlapping of nuclei. A minimum of 100 tumor nuclei were analyzed for each case. The rn~~~~c features of ceil density (number of nuclei/mm2), mitotic in&x (mitotic figures/100 tumor nuclei), necrosis, pseudopalisading cells, and giant cells were assessed on the same image analysis system fitted with a 40x objective lens. CelI density and mitotic index represent two of the histopatbologic grading criteria used in a variety of three-tiered systems for classifying astrocytic gliomas (3, 6). For each case, five different microscopic fields with the higbest mitosis and cellularity were selected for image analysis. Necmsis was estimated as the percentage of necrosis within the tumor area. For each case, the presence or absence of pseu~i~~g cells and giant cells was noted. Statistical analysis Treating EGFR as positive (> 0) or negative (0), comparisons with tumor grade, sex, pseudop~i~ding cells, and giant cells were made using chi-square. Comparisons of mean age, nuclear roundness factor, nuclear area, cell density, mitotic index, and necrosis between EGFR positive and EGFR negative were made with the Student’s ttest. Treating EGFR as a continuous variable, Pearson product-moment correlations were computed for EGFR with each of the other variables. Overall survival and recurrence-free survival were analyzed by the log rank test for EGFR positive and negative categories, and by Cox Proportional I-Iazard Regression for EGFR as a continuous variable.

’ Videoplan

III, Carl Zeiss, Inc., Thornwood,

NY.

XII

RESULTS An i~unohist~~~c~ positive faction for EGFR was present in the cytoplasm a.r#or cytoplasmic membrane of tumor cells (Fig. 1). No EGFR staining was present in tumor nuclei. The percentage of cells positive for EGFR varied from 5 to 90% in the positive cases. Forty-nine (69%) of 71 tumors had EGFR positive cells. By grade, these included 1 (50%) of 2 astrocytomas, 9 (64%) of 14 ana.plastic astrocytomas, and 39 (71%) of the 55 glioblastoma multiforme cases. The proportion of EGFR positive tumors appeared to increase with grade of malignancy; however, this relationship was not statistically significant @ = 0.7494). Neither age* sex, nor percentage of necrosis was associated with EGFR expression O;, = 0.7954, 0.9941, and 0.08, respectively). Fifty-nine of the 71 patients did not survive. Univariate analysis of the influence of EGFR protein expression on survival and recurrence-free survival was made with EGFR positivity treated in two different ways: as a continuous variable and as a dichotomous variable. Treated as a continuous variable, EGFR was a significant survival predictor. Higher percentages of tumor cells positive for EGFR were significantly related to reduced survival t’~ = 0.0424). Figure 2 plots predicted survival for selected EGFR percentages from the Cox regression model based on the 71 patients of the study. For example, the percentages of patients surviving at 12 months were predicted to be 62, 50, and 38% for patients with 0,40, and 80% EGFR positive cells, respectively. At 60 months, the corresponding percentages of surviving patients for these same EGFR groups were 24, 12, and 6%. respectively. However, when treated as a dichotomous variable, there was no significant difference between EGFR positive and negative patients with respect to survival @ = 0.1062). Epidermal grow& factor receptor was a significant predictor of recurrence-fme survival in the Cox regression model (Fig. 3). The percentage of cells positive for EGFR was associated with decreased recurrence-free survival t$ = 0.0353). The 12-month recurrence-free survival rates for the patients with 0,40, and &O% EGFR positive cells were 90, 81, and 63%, respectively. Sixty-month recurrence-free survival rates for the same groups of patients were 62, 39, and 16%, respectively. There was no significant difference (p = 0.301 I) in recurrence-f&e survival when EGFR was analyzed as ~chotomous (positive or negative). When considering the possible influence of other factors on the effect of EGFR to survival and tumor recurrence, all the clinical and histologic variables were entered into a multivariate analysis to determine whether the prognostic effect of EGFR was independent or associated with other factors. Cox proportional hazards regression was used to analyze tbe effect of age, sex, grade. tumor nuclear

1. J. Radiation Oncology 0 Biology 0 Physics

Volume 34, Number 4, 19%

Fig. 1. High-powered photomicrographs of glioblastoma multiforme specimens demonstrating intracellular patterns of epidermal growth factor receptor (EGJ?R) distribution. Photomicrographs were taken using an oil immersion lens (magnification X 1OCQ. Specimens were lightly counters&&d with hematoxylin. Some EGFR-positive cells demonstrated cytoplasmic EGFR staining with pronounced staining of the cell membrane as shown by the cell located between the three arrows (a), whereasother cells exhibited primarily cytoplasmic EGFR staining patterns, shown by the cell between two arrows (b). There were no patterns of nuclear EC.WRstaining.

area, tumor nuclear rounduess factor, cell density, mitotic index :, percentage of EGFR positive cells, percentage of necrc Isis, pseudop~isading, and giant cell on patient survival and recurrence-free survival. Although several of the vsariables had a significant influence on survival when analy rzcd separately, multiv~ate analysis indicated that only EGFR and age remained significantly and indepen-

dcntly related to survival when adjusted for the other variables (Table 2). The percentage of positive EGFR cells was the only variable that had a significant relationship to recur-rence, even when adjusted for all other variables (Tab1 .e 3). These results indicate that EGFR was an indepel predictor for both survival and recurrence-free surerival.

EGFR in astrocytoma Predicted Survival for Selected EGFR Percentages

~

!

0% 20% 40% 6QY 80%

08 $ E 2 6.z

07 I!:

-

0 6~

survival

x

-~

Grade Age Sex Nuclear area

\,

05

i! (

A. ZHU or al.

Nuclearroundness Ceil density Mitotic index EGFR Pseudopalisading cells Giant cells Necrosis

L

Fig. 2. Overall survival of patientswith astrocytic gliomaswas si~i~c~tly reduced@ = 0.0434) as &hepercentageof EGFR positive cells in the tumor increased.

Correlation of EGFR as a cou~uous variable with gmde of malignancy, tumor nuclearronndnessfactor, nuclear ama, ceil density mitotic index, percent necrosis,pseudopalisading cells, and giant cells indicated that none of these variabtbles were related to EGF’R protein expression.

p-values .---.

_-I

o.ooos 4.000f 0.4808 o.s419 0.0232

0.0004
0.0016 0.8524

p-values

I”

0.378 I mOO2 0.6392 0.3819 0.1393 0.7456 0.4083 0.0016 0.3738 0.6442

0.1010 0.1295 -I^_~~. __--EGFR = epidermalgrowth factor receptor.

by flow cytometry was not significantly related to either overall survival or disease-free survival in our group of irradiated astrocytic glioma patients ( 191, which prompted us to examine other possible tumor markers. This study has demonstrated percentage EGFR positivity to be significantly and independently related to overall survival in a group of patients given definitive r~~othem~y. This finding both corroborates and extends a previous study (9), which demonstrated reduced survival in patients with

DISCUSSION Historically, traditional variables for overall survival of patients with astrocytic gliomas have included patient factors, including age at diagnosis and Kamofsky performance status, and tumor factors, such as grade, size, and location. The relationship of sex to overall survival has been controversial for astrocytic gliomas, with the majority of studies demonstrating no relationship (5, 16, 20) and one recent study reporting a better outcome in male patients (4). Previously, we reported that DNA content Predicted Recurrence-Free

1.t

Table 2. Univariate and multivariate analysisof predictorsfor overall survival of patients with astrocytic tumors in a Cnx regression model -..-Predictors for Univariate Multivariate

, of----09 4

0

Survival for Selected EGFR Percentage

positive, as opposed to negative, EGFR expression; that is, EGFR was assessed as a dichotomous variable. Our study has shown that when analyzed as a continuous variable, EGFR positivity can be useful to predict outcome in EGFR positive patients. Markers for local tumor recurrence may be of even greater interest to the treating physician, because such markers may be helpful in identifying patients requiring closer follow-up or different treatment management. The results of this study have shown EGFR positivity not only was a significant predictor of local recurrence following radiotherapy, but also that it was the only one of I 1 Table 3. Univariate and multivariate analysisof predictorsfor recurrence-freesurvival of patientswith astrocytic tumorsin a Cox regressionmodel - ..-----Predictorsfor Univariate Multivariate p-values recurrence-freesurvival ---..--------- p-values Grade Age Sex

O “i -~----0

?

2

3

4

I

I

I

I

I

I

,

5

6

7

8

9

10

11

Tme(years)

Fig. 3. The recurrence-free survival rate of patients with astrocytic gliomaswas significantly @ = 0.0353) reducedas the percentageof EGF%? positive cells in the tumor increased.

Nuclear area Nuclear roundness Cell density Mitotic index EGFR Necrosis Pseudopalisading cells Giant cells

0.8650 0.2400 0.4545 0.4395 0.3607 0.3261 0.5933

0.0353 0.9037 0.6599 0.9436 ~”

EGFR = epidermalgrowth factor receptor.

0.9108 0.1054 0.4265 0.2780 0.4799 0.5 108 0.1154 0.0182 0.2398 0.6284 -._--- 0.2999

814

1. J. RadiationOncology 0 Biology l Physics

variables studied that was signi~c~tly related to tumor recurrence. On the other hand, there was no relationship between EGFR and the time from recurrence until death (r = 0.11). Fifteen out of 17 (88%) of the recurrences subsequently died from their cancer, with a median time to death that was 9 months, and unrelated to EGFR. EGFR is a transmembrane glycoprotein. When EGF binds to the receptor on the cell membrane, this results in the intracellular stimulation of tyrosine kinase activity, which in tnrn, results in increased levels of DNA synthesis and cell division @, 9, 12). The increased EGFR protein expression in ~ghly proliferative as~~~orna is compatible with the role of EGFR in stimulating the growth of the malignancy (12). Our findings agree with the suggestion that EGFR expression represents a determinant of aggressive biological beha$or of @&rocytic tumors (I), and extend$,this hypothesis to the f&@er suggestionx that the aggress&@ behaiibr de&+-mined by the’ percentage’of EGm-p&i&e &ejls ultimately iiire&Iy aff&ts both local recurrenke and overall sutiival, Our’ observations are atso cons&+ w$h the reported significant re~ations~p between EGFR score (percentage positivity and stoning i~~ensi~) and the ~rcentage of ~-67-positive tumor c&si whidh. &presents the proliferative potential of the tuiIx?r, (9). ’ IXrect comparisons of EGFR positivity and grade between our study and other pnb~ished studies (1,9, 12) are difficult at best, considering the differences in material (frozen vs. paraffin), grading systems (three different four-tiered grading systems vs. our three-tiered system), possible differences in distribution of patients as percentage of as~~ytorn~ anaplastic as~~yto~~ and gtioblas-

1. Apsti, R. M.; Leuthold, M.; Gullick, W. J.; Yasargil, M. G.; Wiestler, 0. D. Expression of the epidermal growth factor receptor in astrocytic trout is s~ific~ly associated with glioblastoma muitiforme, Virchows Arch. A. Pathol. Anat. 420:321-325; 1992. 2. Bigner, S. H.; Vogelstein, B. Cytogenetics and molecular genetics of malignant gliomas and mednlXoblastomas. Brain Pathol. 1:12-18; 1990. 3. Burger, P. C.; Vogel, F. S.; Green, S. B.; Strike, T. A. G~ob~~torna rnu~~fo~e and anapIastic ~s~~~rna: Pa~olo~~ criteria and prognostic implications. Cancer 56:1106-1111; 1985. 4. Coons, S. W.; Johnson, P. C.; Pearl, D. K. Prognostic significance of flow cytometry deoxyribonucleic acid analysis of human astrocytomas. Neurosurgery 35~119-126; 1994. 5. Curran. W. J., Jr.; Scott, C. El.; Horton, J.; Nelson, J, S.; Weinstein, A. S.; Fischbach, A. J.; Chang, C. H.; Rotman, M.; Asbell, S. 0.; Krisch, R. B.; Nelson, D. F. Recursive p~tionin8 analysis in three Mayan therapy oncology group malignant glioma trials. J. Natl. Cancer Inst. 85:704710; 1993. 6. ~~~-D~~~

C.; Scheithauer, B.; O’Fallo~, J.; Kelly, P. Grading of ~~ytorn~. A simple and r~~u~~ble method. Cancer 622152-65; 1988. 7. Duncan,G. G.; GocJdman, G. B.; Ludgate, C. M.; Rheaume,

Volume 34, Number 4, 1996

toma m~tifo~e, as well as ~~erences in staining technique. Nonetheless, our overall rate of EGFR positivity (49 out of 7 I or 69%) compares favorably with the values of Jaros et aZ. (9) (32 out of 43 or 74%) and Maruno et al. (12) (16 out of 19 or 84%), but are considerably higher than those of Agosti et ak. (1) (17 out of 103 or 17%). In addition to tumor grade, we compared EGFR positivity to several quantitative m~fes~tions of grading criteria, such as NRF, necrosis, and mitotic index. When analyzed as a con~nuous variable, EGFR was not significantly related to any of the histologic grading criteria, and was only rn~gin~~y related &I = 0.08) to n~ros~s when analyzed as a dichotomous variable. In this report, OUT results agree with the general finding of Ago&i et al. (l), who, likewise, were unable to demonstrate correlation between EGFR expression and specific histologic features. It must be mentioned, however, that our study used computer-assisted morphometric analysis, whereas traditional microscopic review was used by Agosti et nl. (1) to assess histologic features. Thus, EGFR positivity appears to be a significant predictor of overall survival in patients with as&cytic gliomas. It is ~de~ndent from patient age at diagnosis. It is not si~fi~dy related to histologic grade or divide grading criteria. Perhaps more i~~t, Em is a si@ficant predictor of local failure following definitive radiation therapy in these patients. There is promise that EGFR levels may be useful in identifying a subset of astrucytic glioma patients, for instance, that may benefit from altered radiation fractionation regimens or biological response modifiers because of the high proliferative potential of tumors with high EGFR levels. This promise, however, must first be confirmed by more extensive studies.

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14, P~i~n, J. H.; Clemenceau, S. I-I.; Fat&on, F. H.; Fontin, J. F. Su~~n~~~ low-grade ~~~~ornas in adults. Nemosurgery 32554-559; 1993. 15. Salmon, I.; Dewitte, 0.; Pasteels, J. L.; Flament-Durand, J.; Brotchi, J.; Vereerstraeten, P.; Kiss, R. Prognostic scoring in adult astrocytic tumors using patient age, histopathologic grade and DNA histogram type. J. Neurosurg, 80877-883; 1994. 16. Salmon. I.; Kiss, R.; Dew&e, 0.; Gras, T.; Pasteels, J. L.; Brotchi, 3.; ~~~t~~~~~ J. ~stopa~olo~~ grading and DNA ploidy in relation to survival amoug 206 adult as~ytic

~orpatienEss.Caneer70:538-54a;

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17. Side-Wolves, M.; Modern, P.; S~rnbl~ L, G.; Anderson,If.; &xgstr6m, S.; Brun, A.; Cronqvist,S.; Hougaard,K.; Salford, L. G. A randomizedstudyof~~mo~erspy with procarbaxine,vincristine and lomostinewith and without radiation therapy for astrocytomaGrade 3 and/or 4. Cancer6822-29; 1991. 18. S&old, S. C.; Hemdon, J. E.; Burger, P. C.; Halperin, E. C.; Vick, N. A.; Cairncross,J. G.; Macdonald, D. R.; Dropcho, E. J.; Morawetz, R.; Bigner, D. D.; Mahaley, M. S. Randomizedcomparisonof diaziquoneand carmnstine in the treatment of adults with anaplasticghoma. J. Chin. Oncol. 11~77-83; 1993.

19. Shaeffer-J.; Zhu, A.; Yu, J.; Kolm, P.; El-M&d& A. M. Correlationof DNA contentand ~stolog~cgradewith survival of patientswith astrocyticgliomas.CancerMol. Biol. 1:305-310; 1994. 20. Shibamoto,Y.; Kitakabu, Y,; Takahashi,M.; Yamashita, J.; Gda, Y.; Kikuchi, H.; Abe, M. Supratentoriallow-grade astrocytoma.Cancer7219% 195; 1993. 21. Smith, D. F.; Hutton, J. L.; Foy, P. M.; Shaw, M. D, M.; Williams,I. R.; Chadwick,D. W. The prognosisof primary intracerebraltnmourspresentingwith epilepsy: The outcomeof medicalandsurgicalm~g~ent. J. Neural. Neurosurg. Psyc~a~ 54z915-9zck 1991. 22. Soffieti, R.; Chib, A,; Giordana,M. T.; Vasario,E-; Schiffer, D. Prognostic factors in we~~~ff~nti~ cerebral ~~torn~

in the ad&

~e~o~~~

~&S&-692;

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23. van Deimling, A,; Louis, D. N.; van Ammo% I(.; Petersen, I.; Hoell, T.; Chung, R, Y.; Martuza, R. L.; Schoenfeld, D. A.; Yasargil, M. G.; Wiestler, 0. D.; Seizinger, B. R. Associationof epidermalgrowth factor receptor geneamplification with lossof chromosome10 in humangliablastoma multiforme. J. Neurosurg.72295-301; 1992. 24. Winer, B. J. Statistical principlesin experimentaldesign, 2nd ed. New York: M~~aw~H~li; I97 1.