J. Endocrinol. Invest. 15: 31-37,1992
Epidermal growth factor receptor in human brain tumors A. Di Carlo*, A. Mariano*, P.E. Macchia*, M.C. Moroni**, L. Beguinot**, and V. Macchia* *Centro di Endocrinologia ed Oncologia Sperimentale del CNR e Dipartimento di Biologia e Patologia Cellulare e Molecolare, II Facolta di Medicina e Chirurgia, Universita di Napoli, Napoli, Italy and **Institute of Microbiology, University of Copenhagen, Denmark ABSTRACT. The expression of epidermal growth factor receptor (EGF-R) was examined in 27 primary human brain tumors (7 glioblastomas, 10 astrocytomas, 5 oligodendrogliomas, 1 schwannoma, 1 ganglioneuroma, 1 medulloblastoma, 1 ependimoma, 1 histiocitic lymphoma), in 6 brain metastases from lung carcinomas and in 20 meningiomas. Peritumoral tissues histologically normal excised surgically along with a large tumor were used as control. All plasma membranes from brain tissues tested showed specific EGF binding. The EGF re-
ceptor is expressed at low levels in the control human brain and at very high levels in 60% of the total intracranial tumors studied. When the various histological types of tumors were analyzed, the higher percentage of positive tumors was found with the meningiomas (85%) and the glioblastomas (71 %), while the lower percentage of positivity was found with the oligodendrogliomas (40%) and the astrocytomas (30%). A good correlation between binding and total amount of EGF-R protein detected by Western Blot was also observed.
INTRODUCTION
MATERIALS AND METHODS Tissue
The epidermal growth factor (EGF) receptor (EGFR) is a protoncogene (1, 2) and its overexpression in vitro gives rise to a fully transformed phenotype in the presence of the ligand (3-5). A potential role of EGF-R has been proposed in the development of human tumors based on the findings of amplification of the gene and/or increased numbers of EGFR in primary neoplasms of ectodermal origin (6, 7). Furthermore, recent studies suggest that patients with breast or bladder carcinomas with a high epidermal growth factor receptor content have poor prognosis (8, 9). However, so far there are only a few reports concerning EGF-R content in intracranial tumors (10-15) and it is not yet established whether the measurement of EGF-R may provide useful information on the malignancy of these tumors. In order to clarify these points we have studied the content of EGF receptor in human brain tissue obtained from patients with different types of malignant lesion by a radioligand assay in all samples and by Western Blot in few cases.
Pathological brain tissues were obtained in the operating room from patients undergoing surgery for neoplasms. Diagnosis of tumor was made by usual clinical and laboratory criteria and confirmed by histopathological findings according to Zulch (16). The patient's age varied between 27 and 76 years. Fifty-three malignant tumors: 20 meningiomas, 7 glioblastomas, 10 astrocytomas, 5 oligodendrogliomas and 6 brain metastases were examined. Furthermore, also five rare cases of human brain tumors were processed: 1 schwannoma, 1 ganglioneuroma, 1 medulloblastoma, 1 ependimoma and 1 histiocitic lymphoma. Three peritumoral tissues histologically normal excised surgically along with a large tumor (oligodendroglioma or astrocytoma) were used as control tissue. Freshly collected tissues were cut into two parts: the first was fixed in formalin for histological diagnosis and the second rapidly frozen and stored at -20 C to be anaIyzed for EGF receptor content.
Materials EGF was obtained from Bethesda Research Laboratories (Bethesda, MD, USA); 1251-EGF (specific activity 160 IlCi/llg) was purchased from the Radiochemical Center at Amersham; Aprotinin, PMSF, BSA and Hepes were from Sigma Chemical Co (St Louis, MO, USA); Biorad protein assay from Bio-Rad Laboratories. All other reagents were available from commercial sources.
Key-words: EGF receptor, brain tumors. Correspondence. Pro!. Vincenzo Macchia, Dipartimento di Biologia e Patologia Cellulare e Molecolare. II Facolta di Medicina e Chlrurgia, Via Sergio Pansini 5. 80131 Napoli, Italy. Received April
29, 1991, accepted October 21, 1991
31
A Di Car/o, A Mariano, PE Macchia, et at.
Homogenate preparation Approximately 500 mg of brain tissues were washed, minced in 0, 154M NaCI on ice and then homogenized with 5 strokes in a teflon-glass homogenizer in 2 ml of buffer A (10mM Tris HC1 pH 7A, 50mM NaCI, 1% Aprotinin, 1% PM SF and 5mM EOT A), The homogenate was centrifuged for 10 min at 1,500xg, the supernatant was further centrifuged for 30 min at 105,000xg and the crude membrane pellets were resuspended in ice cold buffer A using a glass-to-glass homogenizer and aliquots were stored at -80 C, Membrane proteins were determined after solubilization of appropriate aliquots in 1N NaOH by the Bradford procedure (17),
studied the effects of time, temperature, pH, 125 1_ EGF and protein plasma membrane concentrations (data not shown and Fig, 1, upper panel), The optimal binding of 1251-EGF was obtained using 1,2nM of 1251-EGF and 100 Ilg of plasma membranes at
50
1 ~
40
J!
0
0
E
'"cz
Binding of 1251-EGF to plasma membrane
:> 0
In a standard assay aliquots of crude plasma membranes (50-100 Ilg protein) were incubated in a final vol 200 III of buffer B (20mM Hepes buffer pH 7A containing 0,1% BSA) with 1 ,2nM of 125 1_ EGF (about 500,000 cpm) at 25 C for 120 min, Parallel samples containing 1 Ilg of unlabelled EGF were performed in order to calculate the nonspecific binding and subtracted from each value, At the end of the incubation, reactions were stopped by addition of 1 ml of buffer B containing 4 mg/ml BSA and centrifuged at 14,000xg for 10 min at 4 C, Pellets were washed twice with 1 ml of cold buffer B, solubilized with 0,5 ml of 1N NaOH at 37 C for 20 min and the radioactivity measured in a gammacounter, Membranes prepared from placental tissue were used in each experiment as positive control, All determinations were performed in duplicate, The content of EGF receptor was expressed as fmol of 1251-EGF bound/mg membrane protein,
ID
... "'" -
PROTEINS (I'glr.m.)
ea. ~ 0
z
:> 0
Immunoblotting Crude membrane from tissues (100-150 Ilg) and from KB (human carcinoma cell line) and A431 (human vulval epidermoid carcinoma cell line) cells extracts (50 Ilg) were solubilized 1xLaemmli Buffer (50 Ill) and run on an 8,5% SOS-polyacrylamide gel, The separated proteins were transferred to nitrocellulose and blotted either with a rabbit polyclonal antibody (Ab) against the EGF-R (Ab 2913) (18) or with control normal rabbit IgG, Antibodies were detected by a secondary biotynilated Ab and the Vecta-Stain Kit, as recommended by the manufacturer (Vector Laboratories", Burlingamm, KA, USA),
ID
...
"'" -
50 40
30 20
'0~\-::'2.,-L-!;2-2;;l,;~~4,5.--f.7.B,-L-~'B;;-----;!.--==.:1~~,.i:J5!!::::=~ootiB>25 1,6
3,5
9.5
EGF (nM)
Fig. 1 - Upper panel. Binding of 1251-EGF to human brain tumors p/asma membranes as a function of protein concentration. Experimental conditions are identical to those described in the text. ;]---0 medulloblastoma, .---. meningioma n. 18, ,---, placenta, A---A brain metastases n. 1; ,:)---0 glioblastoma 17. 4. (Ilg/rm = Ilg of total protein/reaction mixture) Lower panel. Effect of unlabelled EGF on the binding of 1251-EGF to human brain plasma membranes. Experimental conditions are identical to those described in the text except that each reaction mixture contained 39 Ilg of protein brain metastases (no 1) plasma membrcii1es A---A, 40 Ilg of protein glioblastoma (n. 4) plasma membrares O---J, 311lg of protein placenta plasma membranes ,---- 194 pg of protein meningioma plasma membranes .---.
RESULTS Binding of 1251-EGF to tumor brain plasma membrane In order to determine optimal assay conditions we
32
EGF receptor in brain tumors
total number of cases analyzed is too low to allow proper statistical analysis. As shown in Table 1, in meningiomas the specific binding for EGF ranged between 4.5-142 fmol/mg protein, with a mean value of 44 ± 6.9 fmol/mg protein. In grade I and II astrocytomas EGF binding was much lower, similar to the normal tissues (1.0-14.0 fmol/mg protein, mean 5.7 ± 1 A), whereas in the one case of grade III astrocytoma EGF binding was very high (210 fmol/mg protein) (Table 2). In oligodendrogliomas, EGF binding ranged between 3.6-30 fmol/mg protein (mean 13.3 ± 5.3), independently of the grade of the tumor (Table 2). In glioblastomas, specific binding ranged between 4.6-496 fmol/mg protein. In this type of tumors the amount of EGF-R expressed appears to be highly variable; this variability can probably be attributed to cell type diversity (Table 3). We also studied one case each of rare types of brain tumors. The schwannoma was the only one found slightly positive for EGF binding (12 fmol/mg protein) while the others (ependymoma, medulloblastoma, ganglioneuroma and histiocitic lymphoma) bound very low amounts of EGF (Table 3). High levels of EGF-R expression were also observed in the four cases examined of brain metastases from primary lung epidermoid carcinoma. As shown in Table 4 the specific binding varied between 93 to 280 fmol/mg protein in these specimens, while
Table 1 - EGF Binding in primary meningiomas' Sample (no.)
Histology
Age
Sex
125 1 EGF bound fmol/mg protein
M. angioblastic
74
F
22.0
2
53
F
23.2
3
37
F
66.6
4
39
M
84.0
5
51
M
45.2
60
F
64.5
65
F
84
8
67
F
44.5
9
53
F
142.0
10
6
M. transitional
7
68
F
64
11
56
F
38.5
12
63
M
4.5
13
68
M
37.7
14
57
F
38.8
15
69
F
464
16
70
F
24.6
17
84
F
86.0
18
37
F
54.8
19
M. sarcomatus
55
M
27.9
20
M. syncytial
41
F
124
1Experimental
Table 2 - EGF Binding in primary neuroepithelial tumors!.
conditions are reported in the Materials and Methods.
Sample (no.)
pH 7A for 2 h at 25 C. Under these conditions nonspecific binding was 1-5% of the total bound EGF. All plasma membranes from brain tissues tested showed specific EGF binding In the three control normal nervous tissues studied EGF binding was respectively 3.6, 1.0, and 7.2 fmol/mg protein. According to the data of Nicholson (19), we considered that a tumor expressed higher level of EGFR when the specific binding was> 0.25% (equivalent to 10 fmol/mg protein). This number agrees well with the binding observed in our few cases of normal nervous tissue available. Using this cut-off value as a measure of total amount of EGF receptor, we found that the receptor is expressed at higher levels in 60% of the total intracranial tumors studied. When the various hystological types of tumors were analyzed, the percentage of positive tumors was found with the meningiomas (85%) and the glioblastomas (71 %) while the lower percentage of positivity was found with the oligodendrogliomas (40%) and the astrocytomas (30%). However, the
Histology
Grade Age
Astrocytoma 2 3
Sex
125 1 EGF bound fmol/mg protein
42
F
1.0
6
M
1.0
5
F
10.6
4
11
45
F
14.0
5
11
46
F
7.0
48
F
2.0
6 7
11
56
M
9.5
8
11
43
M
3.9
9
11
26
M
2.1
10
I11
51
F
2100
11
36
M
3.6
12
Oligodendroglioma
38
M
20.5
13
35
M
30.0
14
11
31
F
6.7
15
I11
35
M
5.9
'Experimental conditions are reported in the Materials and MeHlOds.
33
A oi Carlo, A Mariano, PE Macchia, et al.
to the Kd value measured in the control tissue. i.e., placenta (2.5nM).
Table 3 - EGF binding in various primary brain tumorsl. Histology
Age
Sex
Glioblastoma multiforme
34
M
4.6
46
F
12.7
Sample (no.)
2
125 1 EGF bound fmol/mg protein
3
63
F
41.2
4
76
M
496.0
5
73
M
12.0
6
68
M
4.6
69
M
264.0
8
Schwannoma
43
M
12.0
9
Ependymoma
4
M
7.5
7
10
Histiocitic lymphoma
48
M
8.3
11
Ganglioneur.oma
36
M
4.3
12
Medulloblastoma
4
F
1.6
Western Blot Some of the tumors that we have analyzed showed very high levels of EGF binding. In order to directly look at the receptor protein we performed Western blotting using an anti-EGF-R polyclonal Ab (Ab 2913) (18). Both in the metastases (no. 1) (Fig. 2 lane 5) and in glioblastoma (no. 4) (Fig. 2 lane 4) the receptor was present as a main 175 Kd band comigrating with the normal receptor expressed in KB cells (Fig. 2 lane 2) and a 150 Kd band that represents a degradation product. In agreement with the binding data, the glioblastoma no. 4 expressed very high levels of receptor. Expression of very high amounts of receptor is usually due to amplification of the gene. We have then analyzed by Southern blotting the receptor gene copy number in the same tumors. Only in the glioblastoma we found
'Experimental conditions are reported In the Materials and Methods
binding was found essentially normal in two brain metastases derived from lung adenocarcinoma. To ensure that the EGF-R was really elevated in the lung metastases we have used normal lung tissue as control where the specific binding was found to be 6.1 ± 3.4 fmol/mg protein (data not shown). To ensure that the binding was performed under linear conditions and to determine the Kd value for EGF in the brain tumors, in prototypes of meningiomas, glioblastomas, medulloblastomas and metastases, the 125 1_ EGF binding was performed as a function of plasma membrane protein concentration (Fig. 1, upper panel) and of EGF (Fig. 1, lower panel). Analysis of Scatchard plots were compatible with the presence of a single class of binding sites (1), with an apparent Kd value of 1.6nM for the glioblastoma, 3.9nM for the meningioma and 7nM in the metastases. The values were substantially similar
Kd - 200 ~
EGF-R
- 92
-68
-45 Table 4 - EGF binding in brain metastases from lung cancer
I.
Primary tumor
Age
Sex
125 1EGF bound fmol/mg protein
Epidermoid Ca
60
M
280.0
2
47
M
120.0
3
41
M
121.0
Sample (no.)
4 5 6
Adenocarcinoma
50
M
93.0
59
M
110
63
M
7.7
2
3
4
5
6
Fig. 2 - Immunoblotting of EGF-R in human primary tumors. Lysates prepared from A431 (lane 1) and KB (lane 2) cells (30 and 60 f,lg of total proteins) or from crude membrane fractions of primary human tumors (120-140 fig of proteins), glioblastoma no. 4 (lane 4), bram metastases no. 1 (lane 5), primary lung epidermoid carcinoma (lane 6) or control brain tissue (lane 3) were separated on an 8.5% SOS polyacrylamide gels. blotted on nitrocellulose and developed with an anti-EGF-R polyc/onal Ab and the Vecta Stain system as described in the materials section. Molecular weight markers are shown on the right The arrow indicates the EGF-receptor.
, Experimerltal conditions are reported in tile Materials and Metilods.
34
EGF receptor in brain tumors
amplification of gene from 5 to 10 copies, while in the metastases and in meningioma the gene was not amplified (data not shown). A good correlation between binding and total amount of EGF-R protein detected by Western was also observed in other tumors analyzed (data not shown), however, the small amounts of pathological material available was not enough to routinely perform Western and Southern analysis as suggested by Slamon (20).
ligand produced from the same or the nearby tissues. Clearly the normal receptor even if expressed at very high levels requires stimulation by its own ligand to induce transformation (3, 5). In addition, several human tumor cell lines with high expression of the EGFR show relatively high levels of the alpha transforming growth factor (TGF) mRNA (26). It would be interesting to routinely compare EGF-R expression and alpha TGF production in the same tumor tissue to determine whether, in vivo an autocrine stimulation really exists or the ligand is produced only by the surrounding tissue. Furthermore a variability in the value of receptor numbers in the glioblastomas and in few cases of meningiomas was observed. Whereas such variability was not observed in the astrocytomas, in which the highest value of EGF-R was observed in the grade Ill, and in the oligodendrogliomas. However the variability in the first two types of tumors is not due to the presence of contaminating cells since from each part of tissue on which we performed the EGFR analysis, a small piece was removed and histopathological studies were repeated in order to be sure of the morfological characteristics of the analyzed tissues. The very high content of EGF receptors that we have detected in few brain tumors could be a secondary event due to a later stage of the tumor or to a more aggressive form. However no correlation between size of tumor and EGF-R was found. In an other group of tumors we found (27) that EGFR does not correlate with the staging of the tumors but with the grade of differentiation. For brain tumors there is no staging, therefore we could not made any correlation between staging and EGF binding. Another organ in which the EGF receptor seems to play a role in tumorigenesis is the lung (28). Interestingly, in our study, all four brain metastases from primary lung squamous carcinomas expressed very high levels of EGF-R while the two from adenocarcinomas were negative. Our preliminary results with primary tumors of the lung show relatively high levels of EGF-R in squamous carcinomas and normal or lower levels in adenocarcinomas (Fig. 2 lane 6); however the amounts are never so high as in metastatic brain tumors (27). In conclusion it is possible that also in brain tumors the levels of EGF receptor could be used as biological indicator for a bad prognosis or for anticipating a relapse of the tumor. However, for this purpose a larger number of cases has to be analyzed with proper statistical analysis and with an extensive follow-up of the patients.
DISCUSSION Several lines of evidence support the hypothesis that an increased level of EGF receptor represents an important event in the development of human tumors. In human tumor cell lines (21-23) and in primary tumors (6-15) amplification and overexpression of the normal EGF receptor have been reported. A431 cells, which contain more than 106 receptor/cell, possess all the features of transformed cells while clones selected to express a lower number of EGF-R molecules display a reduced tumoringenicity. In addition, overexpression of the full length human EGF receptor cDNA confers a fully transformed phenotype to normal mouse fibroblasts in the presence of EGF (3, 5). Malignancies of the central nervous system are the main category of tumors in which the EGF-R is found elevated and often amplified (10-15). In the present study most of the plasma membranes from brain tumors indeed show a specific binding which is higher than the normal counterpart. However the majority of the tumors show only a moderate increase in their levels of EGF receptor (2-10 fold), while in few cases (5-10% of the total) a major increase in the amounts of EGF-R expressed can be detected. In addition, most of the glioblastomas that we have studied, although showing high levels of the receptor, do not show a consistent amplification of the gene, as previously reported (12). Only in one case we have detected gene amplification and this is associated with extremely high levels of EGF-R. Since in most of the glioblastoma that we have analyzed we do not find amplification of the gene, we have not detected altered forms of the EGF-R due to rearrangement of the gene as the one recently reported in gliomas (24, 25). This truncated form, which is a rare event (25), would not be detected by the 1251-EGF screening procedure since is lacking part of the binding domain, but would be detected by Western blotting since the antibody used recognizes the intracellular domain (18). The number of tumors (53) studied, however, is too small to allow general conclusions. It is possible that even low levels of receptors are able to initiate the tumor growth if appropriately stimulated by the
ACKNOWLEDGMENTS We would like to thank Dr. Ira Pastan for helpful discussions. L. Beguinot is supported by grants from the Danish Cancer Society
35
A. Di Car/a, A. Mariano, PE. Macchia, et ai,
Expression of epidermal growth factors receptors in human brain tumors. Cancer Res. 44, 753, 1984. 11. Libermann TA, Nusbaum H,R., Razon N., Kris R, Lax I, Soreq H., Whittle N, Waterfield MD, Ullrich A, Schlessinger J, Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin, Nature 313.144,1985.
and Danish Biotechnology Center, M,C. Moroni is a recipient of an EEC Fellowship (Biotechnology Action Program),
REFERENCES 1, Carpenter G, Receptors for epidermal growth factor and other polypeptide mitogens, Annu, Rev, Biochem. 56,881,1987, 2, Downward J" Yarden y" Mayes E" Scrace G" Totty N, Stockwell p" Ullrich A, Schlessinger J" Waterfield MD. Close similarity of epidermal growth factor receptor and v-erbB oncogene protein sequences, Nature 307' 521, 1984, 3, Velu T.J" Beguinot L" Wass WC" Willingham M,C, Merlino GT, Pastan I" Lowy OR Epidermal growth factor dependent transformation by a human EGF receptor proto-oncogene, Science 238.1408,1987. 4. Velu T.J., Beguinot L, Wass W.C., Zhang K" Pastan I, Lowy D,R. Retrovirus expressing different levels of the normal epidermal growth factor receptor: biological properties and new biossay. J. Cell. Biochem. 39: 153,1989. 5. Di Fiore P,P" Pierce J,H., Fleming T.P., Hazan R., Ullrich A, King C,R., Schlessinger J, Aaronson SA Overexpression of the human EGF receptors confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 51.' 1063,1987, 6. Hendler F., Ozanne B. Human squamous cell lung cancers express increased epidermal growth factors receptors. J. Clin. Invest 74,647,1984. 7. Ozanne B., Shum A, Richards CS, Cassells D., Grossman 0, Trent J" Gusterson B., Hendler F, Evidence for an increase of EGF receptors in epidermoid malignancies. In: Cancer Cells, Growth Factors and Transformatio Cold Spring Harbor, NY, 1985, vol. Ill, p. 41, 8. Sainsbury J,R,C., Farndom J.R" Needham G,K., Malcolm AJ., Harris AL. Epidermal growth factor receptor receptor status as predictor of early recurrence of and death from breast cancer, Lancet 1:1398,1987. 9. Neal DE, Marsh C, Bennett M.K" Abel PD., Hall R.R, Sainsbury J.R.C" Harris AL Epidermal growth factor receptors in human bladder cancer: comparison of invasive and superficial tumors, Lancet 1: 366,1985. 10. Libermann TA, Razon N, Bartal AD., Yarden Y., Schlessinger J, Soreq H.
12. Wong AJ., Bigner S,H" Bigner DD" Kinzler KW, Hamilton S,R., Vogelstein B. Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc, Natl. Acad, Sci. USA 84: 6899,1987. 13, Filmus J., Pollak M,N, Cairncross J,G., Buick RN Amplified, overexpressed and rearranged epidermal growth factor receptor gene in a human astrocytoma cell line, Biochem, Biophys, Res, Commun, 131.' 207,1985. 14, Weisman AS., Raguet SS., Kelly PA Characterization of the epidermal growth factor receptor in human meningioma. Cancer Res, 47' 2172, 1987. 15, Yamazaki H., Fukui Y., Ueyama Y, Tamaoki N, Kawamoto T., Taniguchi S., Shibuya M. Amplification of the structurally and functionally altered epidermal growth factor receptor gene (cerbB) in human brain tumors, Mol. Cell. BioI. 8, 1816, 1988, 16. Zulch K,J. Principles of the new World Health Organization (WHO) classification of the brain tumors, Neuroradiology 19: 59, 1980, 17. Bradford M.M, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72: 248, 1976, 18, Beguinot L" Werth D., Ito S., Richert N, Willingham M.C., Pastan I. Functional studies on the EGF receptor with an antibody that recognized the intracellular portion of the receptor. J. Bioi. Chem, 261.1801,1986, 19. Nicholson S, Sainsbury J.R.C, Needham G K., Chambers P, Farndom J.R, Harris AL, Quantitative assays of epidermal growth factor receptor in human breast cancer: cut-off points of clinical relevance. I~, J, Cancer 4236, 1988. 20. Slamon D,J" Clark G,M., Wong SG, Levin WJ, Ullrich A, McGuire WL, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235,' 177, 1987.
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EGF receptor in brain tumors
21. Kamata N., Chida K, Rikimaru K, Horikoshi M., Enomoto S., Kuroki T. Growth-inhibitory effects of epidermal growth factor and over expression of its receptors on human squamous cell carcinomas in culture. Cancer Res. 46.' 1648, 1986. 22. Yao M., Shuin T., Misaki H., Kubota Y. Enhanced expression of c-myc and epidermal growth factor receptor (C-erbB-1) genes in primary human renal cancer. Cancer Res. 48. 6753, 1988.
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26.
23. Yamamoto 1., Kamata N., Kawano H, Shimizu S, Kuroki T., Toyoshima K., Rikimaru K., Nomura N., Ishizaki R., Pastan I., Gamou S., Shimizu N. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res. 46: 414, 1986.
27.
24. Humphrey P.A, Wong AJ., Vogelstein B., Zalutsky MH, Fuller GN, Archer G.E., Friedman H.S., Kwatra MM, Bigner S.H, Bigner 0.0. Anti-synthetic peptide antibody reacting at the fu-
28.
37
sion junction of deletion-mutant epidermal growth factor receptors gene in human glioblastoma. Proc. Natl. Acad. Sci. USA 87' 4207, 1990. Sugawa N., Ekstrand AJ, James CD, Collins V.P. Identical splicing of aberrant EGF-R transcripts from amplified rearranged genes in human glioblastomas. Proc. Natl. Acad. Sci. USA 87 8602,1990. Derynck R., Goeddel DV, Ullrich A, Gutterman J.U., Williams RD, Bringman T.S., Berger WH Synthesis of messenger RNAs for transforming growth factors alfa and beta and the epidermal growth factor receptor by human tumors. Cancer Res. 47: 707,1987. Elia S, Cecere C, Ferrante G., Di Carlo A, Mariano A, Macchia V. Epidermal growth factor receptor (EGFr) in human endothoracic neoplasms. Che~9~131S, 1989. Veale D., Kerr N., Gibson G.J., Harris AL. Characterization of epidermal growth factor receptor in primary human non-small cell lung cancer. Cancer Res. 4~ 1313,1989.
Epidermal growth factor receptor in human brain tumors A. Di Carlo*, A. Mariano*, P.E. Macchia*, M.C. Moroni**, L. Beguinot**, and V. Macchia* *Centro di Endocrinologia ed Oncologia Sperimentale del CNR e Dipartimento di Biologia e Patologia Cellulare e Molecolare, II Facolta di Medicina e Chirurgia, Universita di Napoli, Napoli, Italy and **Institute of Microbiology, University of Copenhagen, Denmark ABSTRACT. The expression of epidermal growth factor receptor (EGF-R) was examined in 27 primary human brain tumors (7 glioblastomas, 10 astrocytomas, 5 oligodendrogliomas, 1 schwannoma, 1 ganglioneuroma, 1 medulloblastoma, 1 ependimoma, 1 histiocitic lymphoma), in 6 brain metastases from lung carcinomas and in 20 meningiomas. Peritumoral tissues histologically normal excised surgically along with a large tumor were used as control. All plasma membranes from brain tissues tested showed specific EGF binding. The EGF re-
ceptor is expressed at low levels in the control human brain and at very high levels in 60% of the total intracranial tumors studied. When the various histological types of tumors were analyzed, the higher percentage of positive tumors was found with the meningiomas (85%) and the glioblastomas (71 %), while the lower percentage of positivity was found with the oligodendrogliomas (40%) and the astrocytomas (30%). A good correlation between binding and total amount of EGF-R protein detected by Western Blot was also observed.
INTRODUCTION
MATERIALS AND METHODS Tissue
The epidermal growth factor (EGF) receptor (EGFR) is a protoncogene (1, 2) and its overexpression in vitro gives rise to a fully transformed phenotype in the presence of the ligand (3-5). A potential role of EGF-R has been proposed in the development of human tumors based on the findings of amplification of the gene and/or increased numbers of EGFR in primary neoplasms of ectodermal origin (6, 7). Furthermore, recent studies suggest that patients with breast or bladder carcinomas with a high epidermal growth factor receptor content have poor prognosis (8, 9). However, so far there are only a few reports concerning EGF-R content in intracranial tumors (10-15) and it is not yet established whether the measurement of EGF-R may provide useful information on the malignancy of these tumors. In order to clarify these points we have studied the content of EGF receptor in human brain tissue obtained from patients with different types of malignant lesion by a radioligand assay in all samples and by Western Blot in few cases.
Pathological brain tissues were obtained in the operating room from patients undergoing surgery for neoplasms. Diagnosis of tumor was made by usual clinical and laboratory criteria and confirmed by histopathological findings according to Zulch (16). The patient's age varied between 27 and 76 years. Fifty-three malignant tumors: 20 meningiomas, 7 glioblastomas, 10 astrocytomas, 5 oligodendrogliomas and 6 brain metastases were examined. Furthermore, also five rare cases of human brain tumors were processed: 1 schwannoma, 1 ganglioneuroma, 1 medulloblastoma, 1 ependimoma and 1 histiocitic lymphoma. Three peritumoral tissues histologically normal excised surgically along with a large tumor (oligodendroglioma or astrocytoma) were used as control tissue. Freshly collected tissues were cut into two parts: the first was fixed in formalin for histological diagnosis and the second rapidly frozen and stored at -20 C to be anaIyzed for EGF receptor content.
Materials EGF was obtained from Bethesda Research Laboratories (Bethesda, MD, USA); 1251-EGF (specific activity 160 IlCi/llg) was purchased from the Radiochemical Center at Amersham; Aprotinin, PMSF, BSA and Hepes were from Sigma Chemical Co (St Louis, MO, USA); Biorad protein assay from Bio-Rad Laboratories. All other reagents were available from commercial sources.
Key-words: EGF receptor, brain tumors. Correspondence. Pro!. Vincenzo Macchia, Dipartimento di Biologia e Patologia Cellulare e Molecolare. II Facolta di Medicina e Chlrurgia, Via Sergio Pansini 5. 80131 Napoli, Italy. Received April
29, 1991, accepted October 21, 1991
31
A Di Car/o, A Mariano, PE Macchia, et at.
Homogenate preparation Approximately 500 mg of brain tissues were washed, minced in 0, 154M NaCI on ice and then homogenized with 5 strokes in a teflon-glass homogenizer in 2 ml of buffer A (10mM Tris HC1 pH 7A, 50mM NaCI, 1% Aprotinin, 1% PM SF and 5mM EOT A), The homogenate was centrifuged for 10 min at 1,500xg, the supernatant was further centrifuged for 30 min at 105,000xg and the crude membrane pellets were resuspended in ice cold buffer A using a glass-to-glass homogenizer and aliquots were stored at -80 C, Membrane proteins were determined after solubilization of appropriate aliquots in 1N NaOH by the Bradford procedure (17),
studied the effects of time, temperature, pH, 125 1_ EGF and protein plasma membrane concentrations (data not shown and Fig, 1, upper panel), The optimal binding of 1251-EGF was obtained using 1,2nM of 1251-EGF and 100 Ilg of plasma membranes at
50
1 ~
40
J!
0
0
E
'"cz
Binding of 1251-EGF to plasma membrane
:> 0
In a standard assay aliquots of crude plasma membranes (50-100 Ilg protein) were incubated in a final vol 200 III of buffer B (20mM Hepes buffer pH 7A containing 0,1% BSA) with 1 ,2nM of 125 1_ EGF (about 500,000 cpm) at 25 C for 120 min, Parallel samples containing 1 Ilg of unlabelled EGF were performed in order to calculate the nonspecific binding and subtracted from each value, At the end of the incubation, reactions were stopped by addition of 1 ml of buffer B containing 4 mg/ml BSA and centrifuged at 14,000xg for 10 min at 4 C, Pellets were washed twice with 1 ml of cold buffer B, solubilized with 0,5 ml of 1N NaOH at 37 C for 20 min and the radioactivity measured in a gammacounter, Membranes prepared from placental tissue were used in each experiment as positive control, All determinations were performed in duplicate, The content of EGF receptor was expressed as fmol of 1251-EGF bound/mg membrane protein,
ID
... "'" -
PROTEINS (I'glr.m.)
ea. ~ 0
z
:> 0
Immunoblotting Crude membrane from tissues (100-150 Ilg) and from KB (human carcinoma cell line) and A431 (human vulval epidermoid carcinoma cell line) cells extracts (50 Ilg) were solubilized 1xLaemmli Buffer (50 Ill) and run on an 8,5% SOS-polyacrylamide gel, The separated proteins were transferred to nitrocellulose and blotted either with a rabbit polyclonal antibody (Ab) against the EGF-R (Ab 2913) (18) or with control normal rabbit IgG, Antibodies were detected by a secondary biotynilated Ab and the Vecta-Stain Kit, as recommended by the manufacturer (Vector Laboratories", Burlingamm, KA, USA),
ID
...
"'" -
50 40
30 20
'0~\-::'2.,-L-!;2-2;;l,;~~4,5.--f.7.B,-L-~'B;;-----;!.--==.:1~~,.i:J5!!::::=~ootiB>25 1,6
3,5
9.5
EGF (nM)
Fig. 1 - Upper panel. Binding of 1251-EGF to human brain tumors p/asma membranes as a function of protein concentration. Experimental conditions are identical to those described in the text. ;]---0 medulloblastoma, .---. meningioma n. 18, ,---, placenta, A---A brain metastases n. 1; ,:)---0 glioblastoma 17. 4. (Ilg/rm = Ilg of total protein/reaction mixture) Lower panel. Effect of unlabelled EGF on the binding of 1251-EGF to human brain plasma membranes. Experimental conditions are identical to those described in the text except that each reaction mixture contained 39 Ilg of protein brain metastases (no 1) plasma membrcii1es A---A, 40 Ilg of protein glioblastoma (n. 4) plasma membrares O---J, 311lg of protein placenta plasma membranes ,---- 194 pg of protein meningioma plasma membranes .---.
RESULTS Binding of 1251-EGF to tumor brain plasma membrane In order to determine optimal assay conditions we
32
EGF receptor in brain tumors
total number of cases analyzed is too low to allow proper statistical analysis. As shown in Table 1, in meningiomas the specific binding for EGF ranged between 4.5-142 fmol/mg protein, with a mean value of 44 ± 6.9 fmol/mg protein. In grade I and II astrocytomas EGF binding was much lower, similar to the normal tissues (1.0-14.0 fmol/mg protein, mean 5.7 ± 1 A), whereas in the one case of grade III astrocytoma EGF binding was very high (210 fmol/mg protein) (Table 2). In oligodendrogliomas, EGF binding ranged between 3.6-30 fmol/mg protein (mean 13.3 ± 5.3), independently of the grade of the tumor (Table 2). In glioblastomas, specific binding ranged between 4.6-496 fmol/mg protein. In this type of tumors the amount of EGF-R expressed appears to be highly variable; this variability can probably be attributed to cell type diversity (Table 3). We also studied one case each of rare types of brain tumors. The schwannoma was the only one found slightly positive for EGF binding (12 fmol/mg protein) while the others (ependymoma, medulloblastoma, ganglioneuroma and histiocitic lymphoma) bound very low amounts of EGF (Table 3). High levels of EGF-R expression were also observed in the four cases examined of brain metastases from primary lung epidermoid carcinoma. As shown in Table 4 the specific binding varied between 93 to 280 fmol/mg protein in these specimens, while
Table 1 - EGF Binding in primary meningiomas' Sample (no.)
Histology
Age
Sex
125 1 EGF bound fmol/mg protein
M. angioblastic
74
F
22.0
2
53
F
23.2
3
37
F
66.6
4
39
M
84.0
5
51
M
45.2
60
F
64.5
65
F
84
8
67
F
44.5
9
53
F
142.0
10
6
M. transitional
7
68
F
64
11
56
F
38.5
12
63
M
4.5
13
68
M
37.7
14
57
F
38.8
15
69
F
464
16
70
F
24.6
17
84
F
86.0
18
37
F
54.8
19
M. sarcomatus
55
M
27.9
20
M. syncytial
41
F
124
1Experimental
Table 2 - EGF Binding in primary neuroepithelial tumors!.
conditions are reported in the Materials and Methods.
Sample (no.)
pH 7A for 2 h at 25 C. Under these conditions nonspecific binding was 1-5% of the total bound EGF. All plasma membranes from brain tissues tested showed specific EGF binding In the three control normal nervous tissues studied EGF binding was respectively 3.6, 1.0, and 7.2 fmol/mg protein. According to the data of Nicholson (19), we considered that a tumor expressed higher level of EGFR when the specific binding was> 0.25% (equivalent to 10 fmol/mg protein). This number agrees well with the binding observed in our few cases of normal nervous tissue available. Using this cut-off value as a measure of total amount of EGF receptor, we found that the receptor is expressed at higher levels in 60% of the total intracranial tumors studied. When the various hystological types of tumors were analyzed, the percentage of positive tumors was found with the meningiomas (85%) and the glioblastomas (71 %) while the lower percentage of positivity was found with the oligodendrogliomas (40%) and the astrocytomas (30%). However, the
Histology
Grade Age
Astrocytoma 2 3
Sex
125 1 EGF bound fmol/mg protein
42
F
1.0
6
M
1.0
5
F
10.6
4
11
45
F
14.0
5
11
46
F
7.0
48
F
2.0
6 7
11
56
M
9.5
8
11
43
M
3.9
9
11
26
M
2.1
10
I11
51
F
2100
11
36
M
3.6
12
Oligodendroglioma
38
M
20.5
13
35
M
30.0
14
11
31
F
6.7
15
I11
35
M
5.9
'Experimental conditions are reported in the Materials and MeHlOds.
33
A oi Carlo, A Mariano, PE Macchia, et al.
to the Kd value measured in the control tissue. i.e., placenta (2.5nM).
Table 3 - EGF binding in various primary brain tumorsl. Histology
Age
Sex
Glioblastoma multiforme
34
M
4.6
46
F
12.7
Sample (no.)
2
125 1 EGF bound fmol/mg protein
3
63
F
41.2
4
76
M
496.0
5
73
M
12.0
6
68
M
4.6
69
M
264.0
8
Schwannoma
43
M
12.0
9
Ependymoma
4
M
7.5
7
10
Histiocitic lymphoma
48
M
8.3
11
Ganglioneur.oma
36
M
4.3
12
Medulloblastoma
4
F
1.6
Western Blot Some of the tumors that we have analyzed showed very high levels of EGF binding. In order to directly look at the receptor protein we performed Western blotting using an anti-EGF-R polyclonal Ab (Ab 2913) (18). Both in the metastases (no. 1) (Fig. 2 lane 5) and in glioblastoma (no. 4) (Fig. 2 lane 4) the receptor was present as a main 175 Kd band comigrating with the normal receptor expressed in KB cells (Fig. 2 lane 2) and a 150 Kd band that represents a degradation product. In agreement with the binding data, the glioblastoma no. 4 expressed very high levels of receptor. Expression of very high amounts of receptor is usually due to amplification of the gene. We have then analyzed by Southern blotting the receptor gene copy number in the same tumors. Only in the glioblastoma we found
'Experimental conditions are reported In the Materials and Methods
binding was found essentially normal in two brain metastases derived from lung adenocarcinoma. To ensure that the EGF-R was really elevated in the lung metastases we have used normal lung tissue as control where the specific binding was found to be 6.1 ± 3.4 fmol/mg protein (data not shown). To ensure that the binding was performed under linear conditions and to determine the Kd value for EGF in the brain tumors, in prototypes of meningiomas, glioblastomas, medulloblastomas and metastases, the 125 1_ EGF binding was performed as a function of plasma membrane protein concentration (Fig. 1, upper panel) and of EGF (Fig. 1, lower panel). Analysis of Scatchard plots were compatible with the presence of a single class of binding sites (1), with an apparent Kd value of 1.6nM for the glioblastoma, 3.9nM for the meningioma and 7nM in the metastases. The values were substantially similar
Kd - 200 ~
EGF-R
- 92
-68
-45 Table 4 - EGF binding in brain metastases from lung cancer
I.
Primary tumor
Age
Sex
125 1EGF bound fmol/mg protein
Epidermoid Ca
60
M
280.0
2
47
M
120.0
3
41
M
121.0
Sample (no.)
4 5 6
Adenocarcinoma
50
M
93.0
59
M
110
63
M
7.7
2
3
4
5
6
Fig. 2 - Immunoblotting of EGF-R in human primary tumors. Lysates prepared from A431 (lane 1) and KB (lane 2) cells (30 and 60 f,lg of total proteins) or from crude membrane fractions of primary human tumors (120-140 fig of proteins), glioblastoma no. 4 (lane 4), bram metastases no. 1 (lane 5), primary lung epidermoid carcinoma (lane 6) or control brain tissue (lane 3) were separated on an 8.5% SOS polyacrylamide gels. blotted on nitrocellulose and developed with an anti-EGF-R polyc/onal Ab and the Vecta Stain system as described in the materials section. Molecular weight markers are shown on the right The arrow indicates the EGF-receptor.
, Experimerltal conditions are reported in tile Materials and Metilods.
34
EGF receptor in brain tumors
amplification of gene from 5 to 10 copies, while in the metastases and in meningioma the gene was not amplified (data not shown). A good correlation between binding and total amount of EGF-R protein detected by Western was also observed in other tumors analyzed (data not shown), however, the small amounts of pathological material available was not enough to routinely perform Western and Southern analysis as suggested by Slamon (20).
ligand produced from the same or the nearby tissues. Clearly the normal receptor even if expressed at very high levels requires stimulation by its own ligand to induce transformation (3, 5). In addition, several human tumor cell lines with high expression of the EGFR show relatively high levels of the alpha transforming growth factor (TGF) mRNA (26). It would be interesting to routinely compare EGF-R expression and alpha TGF production in the same tumor tissue to determine whether, in vivo an autocrine stimulation really exists or the ligand is produced only by the surrounding tissue. Furthermore a variability in the value of receptor numbers in the glioblastomas and in few cases of meningiomas was observed. Whereas such variability was not observed in the astrocytomas, in which the highest value of EGF-R was observed in the grade Ill, and in the oligodendrogliomas. However the variability in the first two types of tumors is not due to the presence of contaminating cells since from each part of tissue on which we performed the EGFR analysis, a small piece was removed and histopathological studies were repeated in order to be sure of the morfological characteristics of the analyzed tissues. The very high content of EGF receptors that we have detected in few brain tumors could be a secondary event due to a later stage of the tumor or to a more aggressive form. However no correlation between size of tumor and EGF-R was found. In an other group of tumors we found (27) that EGFR does not correlate with the staging of the tumors but with the grade of differentiation. For brain tumors there is no staging, therefore we could not made any correlation between staging and EGF binding. Another organ in which the EGF receptor seems to play a role in tumorigenesis is the lung (28). Interestingly, in our study, all four brain metastases from primary lung squamous carcinomas expressed very high levels of EGF-R while the two from adenocarcinomas were negative. Our preliminary results with primary tumors of the lung show relatively high levels of EGF-R in squamous carcinomas and normal or lower levels in adenocarcinomas (Fig. 2 lane 6); however the amounts are never so high as in metastatic brain tumors (27). In conclusion it is possible that also in brain tumors the levels of EGF receptor could be used as biological indicator for a bad prognosis or for anticipating a relapse of the tumor. However, for this purpose a larger number of cases has to be analyzed with proper statistical analysis and with an extensive follow-up of the patients.
DISCUSSION Several lines of evidence support the hypothesis that an increased level of EGF receptor represents an important event in the development of human tumors. In human tumor cell lines (21-23) and in primary tumors (6-15) amplification and overexpression of the normal EGF receptor have been reported. A431 cells, which contain more than 106 receptor/cell, possess all the features of transformed cells while clones selected to express a lower number of EGF-R molecules display a reduced tumoringenicity. In addition, overexpression of the full length human EGF receptor cDNA confers a fully transformed phenotype to normal mouse fibroblasts in the presence of EGF (3, 5). Malignancies of the central nervous system are the main category of tumors in which the EGF-R is found elevated and often amplified (10-15). In the present study most of the plasma membranes from brain tumors indeed show a specific binding which is higher than the normal counterpart. However the majority of the tumors show only a moderate increase in their levels of EGF receptor (2-10 fold), while in few cases (5-10% of the total) a major increase in the amounts of EGF-R expressed can be detected. In addition, most of the glioblastomas that we have studied, although showing high levels of the receptor, do not show a consistent amplification of the gene, as previously reported (12). Only in one case we have detected gene amplification and this is associated with extremely high levels of EGF-R. Since in most of the glioblastoma that we have analyzed we do not find amplification of the gene, we have not detected altered forms of the EGF-R due to rearrangement of the gene as the one recently reported in gliomas (24, 25). This truncated form, which is a rare event (25), would not be detected by the 1251-EGF screening procedure since is lacking part of the binding domain, but would be detected by Western blotting since the antibody used recognizes the intracellular domain (18). The number of tumors (53) studied, however, is too small to allow general conclusions. It is possible that even low levels of receptors are able to initiate the tumor growth if appropriately stimulated by the
ACKNOWLEDGMENTS We would like to thank Dr. Ira Pastan for helpful discussions. L. Beguinot is supported by grants from the Danish Cancer Society
35
A. Di Car/a, A. Mariano, PE. Macchia, et ai,
Expression of epidermal growth factors receptors in human brain tumors. Cancer Res. 44, 753, 1984. 11. Libermann TA, Nusbaum H,R., Razon N., Kris R, Lax I, Soreq H., Whittle N, Waterfield MD, Ullrich A, Schlessinger J, Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin, Nature 313.144,1985.
and Danish Biotechnology Center, M,C. Moroni is a recipient of an EEC Fellowship (Biotechnology Action Program),
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12. Wong AJ., Bigner S,H" Bigner DD" Kinzler KW, Hamilton S,R., Vogelstein B. Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification. Proc, Natl. Acad, Sci. USA 84: 6899,1987. 13, Filmus J., Pollak M,N, Cairncross J,G., Buick RN Amplified, overexpressed and rearranged epidermal growth factor receptor gene in a human astrocytoma cell line, Biochem, Biophys, Res, Commun, 131.' 207,1985. 14, Weisman AS., Raguet SS., Kelly PA Characterization of the epidermal growth factor receptor in human meningioma. Cancer Res, 47' 2172, 1987. 15, Yamazaki H., Fukui Y., Ueyama Y, Tamaoki N, Kawamoto T., Taniguchi S., Shibuya M. Amplification of the structurally and functionally altered epidermal growth factor receptor gene (cerbB) in human brain tumors, Mol. Cell. BioI. 8, 1816, 1988, 16. Zulch K,J. Principles of the new World Health Organization (WHO) classification of the brain tumors, Neuroradiology 19: 59, 1980, 17. Bradford M.M, A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem, 72: 248, 1976, 18, Beguinot L" Werth D., Ito S., Richert N, Willingham M.C., Pastan I. Functional studies on the EGF receptor with an antibody that recognized the intracellular portion of the receptor. J. Bioi. Chem, 261.1801,1986, 19. Nicholson S, Sainsbury J.R.C, Needham G K., Chambers P, Farndom J.R, Harris AL, Quantitative assays of epidermal growth factor receptor in human breast cancer: cut-off points of clinical relevance. I~, J, Cancer 4236, 1988. 20. Slamon D,J" Clark G,M., Wong SG, Levin WJ, Ullrich A, McGuire WL, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235,' 177, 1987.
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EGF receptor in brain tumors
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23. Yamamoto 1., Kamata N., Kawano H, Shimizu S, Kuroki T., Toyoshima K., Rikimaru K., Nomura N., Ishizaki R., Pastan I., Gamou S., Shimizu N. High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines. Cancer Res. 46: 414, 1986.
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24. Humphrey P.A, Wong AJ., Vogelstein B., Zalutsky MH, Fuller GN, Archer G.E., Friedman H.S., Kwatra MM, Bigner S.H, Bigner 0.0. Anti-synthetic peptide antibody reacting at the fu-
28.
37
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