- Email: [email protected]
Immunocytochemical evidence of progesterone receptors in human meningiomas
172
Surg Neurol 1989;31:172-6
Immunocytochemical Evidence of Progesterone Receptors in Human Meningiomas Ernst R. Waelti, Ph.D., and Thomas-Marc Markwalder, M.D. Institute of Pathology and Department of Neurosurgery, University of Bern, Bern, Switzerland
Waelti ER, Markwalder TM. Immunocytochemical evidence of progesterone receptors in human meningiomas. Surg Neurol 1989;31:172-6.
The presence of progesterone receptors in meningioma tissue is demonstrated by use of highly specific monoclonal antibodies against the rabbit progesterone receptors which cross-react with human progesterone receptors in breast cancer cells, thus giving evidence of the existence of genuine progesterone receptors in human meningiomas. K E Y WORDS: Meningioma; Progesterone receptor; Sex steroids; Neurochemistry
Remarkably high titers of a non-estrogen-regulated [3,11,14,16,26] high-affinity/limited-capacity progesterone receptor (PR) have been detected in over 70% of meningioma cytosols [ 1,3,4,9,11-16,19,21,24]. The recent development of highly specific monoclonal antibodies against the rabbit PR which cross-reacted with human cytosolic PR [8,18] and the subsequently described immunocytochemical method for PR detection in human breast tumors [17] offer the opportunity for specific localization of PR in meningiomas. In the following report we demonstrate an immunohistochemical evaluation of PR in several meningiomas and a preliminary correlation between PR immunostaining and the concentration of cytosolic PR determined by a steroid-binding assay. Materials and Methods
Materials Mouse anti-PR antibodies were purchased from Dianova GmbH, Hamburg, FRG, and Transbio, Paris, France and are now available through an agreement with
the Institut National de la Sant6 et de la Recherche Mddicale [INSERM], Paris. Biotinylated sheep antimouse immunoglobulin (Ig) was obtained from Amersham UK. Avidin-biotinylated peroxidase complexes (ABComplex) were purchased from Dakopatts, Denmark. All other chemicals were of analytical grade.
Tissue Preparation Tissue samples from surgically removed meningiomas and breast carcinomas were rapidly frozen in liquid nitrogen and stored at -70°C in airtight capsules until sectioning for histochemical receptor localization. Two tissue blocks were sampled, one for immunocytochemical assay and one for PR-binding assay.
Immunostaining Procedure The cryostat sections were mounted on chrome-gelatincoated glass slides and air-dried in a exsiccator at room temperature. After rehydration in a 1% NaCl solution for 5 minutes, the sections were fixed for 10 minutes in acetone, air dried, and then incubated successively with monoclonal mouse anti-PR antibodies (1:25 dilution) for 1 hour at room temperature, biotinylated sheep anti-mouse (1 : 200 dilution) for 30 minutes, and performed avidin-biotinylated peroxidase complexes for 30 minutes. Each incubation was followed by a 30-minute washing in 1% NaC1 solution. Finally, the sections were incubated for 6 minutes with the 3-3' diaminobenzidine tetrahydrochloride (DAB) solution (1.05 g of citric acid monohydrat, 1.7 g of imidazole in 50 mL of water, pH 7.0, containing 0.2 mg of DAB per milliliter and 0.01% HzO2) and after a 5-minute wash counterstained with hematoxylin. Control slides consisted of sections of meningiomas in which the monoclonal antibody was replaced by normal mouse immunoglobulin.
Biochemical Determination of ProgesteroneReceptors Address reprint requests to: Ernst R. Waelti, Ph.D., Department of Pathology, University of Bern, CH-3010 Bern, Switzerland. Received March 21, 1988; accepted September 12, 1988.
© 1989 by Elsevier Science Publishing Co., Inc.
Cytosolic PR determination was measured by dextrancoated charcoal assay as previously described [27]. 0090-3019/89/$3.50
Immunostaining of Meningioma Progesterone Receptors
Surg Neurol 1989;31:172-6
173
P jte s 0
i
Figure 1. (A) Immunostaining of meningioma tissue with a very high PR content of 464 fmol/mg protein. Note the strong nuclear staining of the greatest part of the tumor cells. (B) Immunohistochemical localization of PR of an infiltrating breast carcinoma using the same monoclonal antibody (x340).
Results As described by the documentation of the producer, the antibodies were raised against purified rabbit PR. They were selected among 70 monoclonals for giving optimal immunocytochemical or immunoblot reactions with either rabbit and guinea pig (mPRII) or human (mPRI) receptors; mPRI is an IgG1 and will detect the intact receptor. The use ofmPRI for the immunocytochemical study of PR in human tissues, especially breast cancer, was described in detail by Perrot-Applanat et al [17], who showed by immunoblotting analysis that mPRI indeed bound to human PR in the cytosol of the PR containing T47D human breast cancer cells (4 pmol of PR per milligram of protein). We tested the monoclonal antibodies in T47D monolayer cultures by an immunocytochemical assay and found an intense staining of the cell nuclei (results not shown).
The results of immunostaining in five cases in meningiomas with monoclonal antibodies against PR are summarized in Figures 1 and 2 and Table 1. Concentrations of PR in cytosolic extracts varied from 464 to 4 fmol/mg protein. As demonstrated in Figure 1, A and B, the monoclonal antibody reacting with PR of human breast cancer cells also stained PR of meningioma cells. This finding suggests that the well-characterized, high-affinity PR in human mammary carcinomas may be identical to PR identified in meningiomas by use of the same monoclonal antibody. In the case depicted by Figure 1 A (PR content of 464 fmol/mg protein) essentially all tumor cells were stained strongly. The staining of varying intensity was predominantly located in the nucleus. Nuclear and specific cytoplasmic staining was only observed in case F.D. Figure 2, A - D illustrates different patterns of staining distribution obtained in this study: tumor staining ranged from a clear-cut predominance of strongly stained cells as seen in Figure 2 A (151 fmol of PR per milligram of protein) and Figure 2 B (110 fmol of PR per milligram of protein) or only a few stained
Figure 2. Representative immunostaining of two cases of meningiomas with high PR content (A, l 51 fmol/mg protein; B, I 10 fmol/mg protein), one case with a lower content (C, 55 fmol/mg proteinL and a negative case (D, 4 fmol/mg protein) where all of the cell nuclei were stained blue. Each large filled arrow indicates a PR-positive nucleus with strong immunostaining which appears in black: each open arrow marks a PR-negative nuclear staining which appears in gray. Cytoplasmic staining (small arrows~ is visible in C. In all instances slides were counterstained with hematoxylin (×340).
Immunostaining of Meningioma Progesterone Receptors
Table
1.
Surg Neurol 1989;31:172-6
175
ProgesteroneReceptor Status and Intensity of Progesterone Receptor Staining in Meningiomas
Case identification no. R.J.L. Sch. A. F.G. F.D. U.H. Sch.M.
Cytosolic PR binding (fmol/mg protein)
Staining Nuclear
Cytoplasmic
464 154 110 55 31 4
+ + + + + 0
0 0 0 + 0 0
Staining intensity" lll-IV I 11 II-lll 11 1 --
Histologic type Meningothelial Meningothelial Meningothelial Fibroblastic Meningothelial Transitional
" 1, weak staining; IV, intense staining. Very weak cytoplasmic staining was interpreted as nonspecific background staining.
cells per field (Figure 2 C, 55 fmol of PR per milligram of protein and case U.H., 31 fmol of PR per milligram of protein, photograph not shown) to a complete absence of positive cells in Figure 2 D (4 fmol of PR per milligram protein). Comparing Figures 1 A and 2 D, for example, the difference between a PR-positive and a PR-negative meningioma tissue is eye-catching. For better discrimination of positive and negative staining characteristics, positive nuclei are marked with a filled arrow and negative nuclei with an open arrow in Figure 2, A and B. A relative intense staining was observed in cytoplasm of meningioma cells of case F.D. (55 fmol of PR per milligram of protein) as demonstrated in Figure 2 C (small arrows indicate cytoplasmic staining, whereas the big arrow indicates a positive nucleus). Although the number of meningiomas studied is not sufficient to allow a thorough statistical analysis and it is difficult to establish clear criteria for the detection of qualitative differences in staining intensity since such differences relate to a certain degree to the observer, there appears to exist a relationship among the staining intensity, percentage of positive tumor cells, and the quantitative PR value obtained by steroid-binding assay (Table 1).
Discussion We have demonstrated immunohistochemically the presence of high-affinity PR in meningiomas by using a monoclonal antibody reacting with PR in human breast cancer cells. Since this monoclonal antibody recognizes the same antigenic determinant on PR molecules in meningiomas as on the high-affinity PR in breast cancer tissue, we must conclude that these receptors are not merely progesterone-binding proteins but indeed genuine high-affinity PR. Moreover, Perrot-Applanat et al [17] submitted cytosols from human breast cancer T47-D cells containing PR to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and analyzed the monoclonal antibody concerned by immunoblotting: a single immunogenic protein with molecular weight of 110,000, identical to the rabbit PR, was
found. Our immunohistochemical results also confirm the data of previous reports obtained by Scatchard plot analysis [1,5,6,19,20,23], which demonstrate that a large proportion of human meningiomas are rich in PR. However, the results of our study are in contrast to the views of Schwartz et al [22] who concluded that neither the estrogen- nor progesterone-binding proteins found in meningioma cytosols are likely to be receptors. Nevertheless, they also reported high levels of progesterone binding in the majority of their meningiomas. On the other hand, the existence of estrogen receptors in human meningiomas is still a matter of debate [7], although the virtual absence of ER seems to have been sufficiently documented. Moreover, the search for ER in meningiomas tissue sections with monoclonal antibodies against the human ER failed [2]. Monoclonal antibodies against PR used a immunocytochemical assay offer new criteria for positivity of the meningioma tissue. Low levels of apparent PR content determined by the dextran-coated charcoal method may be verified by these antibodies, since there is a relationship among the intensity of staining, the number of positive tumor cells, and the PR content measured by radioligand assay. Our finding suggests that immunohistochemistry in tissue sections of meningiomas with monoclonal antibodies against PR may be of predictive value concerning the possibility of hormonal manipulation and the clinical behavior of these tumors. It may be a further explanation for the observed effects of medroxyprogesterone acetate on the growth fractions of meningioma cells in vivo immunostained by the monoclonal antibody Ki-67 specific for cycling cells [10] and on the growth of meningioma cells in culture [25].
References 1. Blaauw G, Blankenstein MA, Lamberts WJ. Sex steroid receptors in h u m a n meningiomas. Acta N e u r o c h i r 1986;79:42-7. 2. Blankenstein MA, Berns PMJJ, Blaauw G, Mulder E, Thijssen J H H . Search for estrogen receptors in h u m a n meningioma tissue sections with a monoclonal antibody against the h u m a n estrogen receptor. Cancer Res (Suppl) 1986;46:4268s-70s.
176
Surg N e u r o l 1989;31:172-6
3. Blankenstein MA, Blaauw G, Lamberts WJ, Mulder E. Presence of progesterone receptors and absence of estrogen receptors in human intracranial meningioma cytosols. EurJ Cancer Clin Oncol 1983;19:365-70. 4. Cahill DW, Bashirelahi N, Solomon LW, Dalton T, Salcman M, Ducker TB. Estrogen and progesterone receptors in meningiomas J Neurosurg 1984;60:985-93. 5. Glick RP, Molteni A, Fors EM. Hormone binding in brain tumors. Neurosurgery 1983;13:513-9. 6. Hayward E, Whitwell H, Paul KS, et al. Steroid receptors in human meningioma. Clin Neuropharmacol 1984;7:351-6. 7. Lesch KP, Schott W, Gross S. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: Evidence for multiple estrogen binding sites. Surg Neurol 1987;28:181-8. 8. Logeat F, Vu Hai MT, Fournier A, Legrain P, Buttin G, Milgrom E. Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors. Proc Natl Acad Sci USA 1983;80:6456-9. 9. Magdenelat H, Pertuiset BF, Poisson M, Martin PM, PhilipponJ, Pertuiset B, Buge A. Progestin and oestrogen receptors in meningiomas: biochemical characterization, clinical and pathological correlations in 42 cases. Acta Neurochir 1982;64:199213.
Waelti and Markwalder
16.
17.
18.
19.
20.
21.
22.
10. Markwalder TM, Gerber HA, Waelti E, Schaffner T, Markwalder RV. Hormonotherapy of meningiomas with medroxyprogesterone acetate: Immunohistochemical demonstration of the effect of MPA on growth fractions ofmeningioma cells using the monoclonal antibody Ki-67. Surg Neurol 1988;30:97-101.
24.
11. Markwalder TM, Markwalder RV, Zava DT. Estrogen and progestin receptors in meningiomas: clinicopathological correlations. Clin Neuropharmacol 1984;7:368-74.
25.
12. Markwalder TM, Waelti E, K6nig MP. Endocrine manipulation of meningiomas with medroxyprogesterone acetate: Effect of MPA on receptor status of meningioma cytosols. Surg Neurol 1987;28:3-9.
26.
23.
13. Markwalder TM, Zava DT. Sex steroid hormones and meningioma cell growth (letter). J Neurosurg 1986;64:341-2. 14. Markwalder TM, Zava DT, Goldhirsch A, Markwalder RV. Estrogen and progesterone receptors in meningiomas in relation to clinical and pathological features. Surg Neurol 1983 ;20:42-7. 15. Martuza RL, MacLaughlin DT, Ojeman RG. Specific estradiol
27.
binding in schwannomas, meningiomas and neurofibromas. Neurosurgery 1981 ;9:665-71. Martuza RL, Miller DC, MacLaughlin DT. Estrogen and progestin binding by cytosolic and nuclear fractions of human meningiomas. J Neurosurg 1985;62:750-6. Perrot-Applanat M, Groyer-Picard MT, Lorenzo F, Jolivet A, Vu MT, Pallud C, Spyratos F, Milgrom E. Immunocytochemical study with monoclonal antibodies to progesterone receptor in human breast tumors. Cancer Res 1987;47:2652-61. Perrot-Applanat M, Logeat M, Groyer-Picard MT, Milgrom E. Immunocytochemical study of mammalian progesterone receptor monoclonal antibodies. Endocrinology 1985;116:1473-84. Poisson M, Magdenelat H, Foncin JF, Bleibel JM, Philippon J, Pertuiset B, Buge A. R&epteurs d'oestrog~nes et de progesterone dans les m~ningiomes. Rev Neurol (Paris) 1980;136:193203. Schnegg JF, Gomez F, Le Marchand-B~raud T, Tribolet N. Presence of sex steroid hormone receptors in meningioma tissue. Surg Neurol 1981;15:415-8. Schoenberg BS, Christine BW, Whisnant JP. Nervous system neoplasms and primary malignancies of other sites: the unique association between meningiomas and breast cancer. Neurology 1975;25:705-12. Schwartz MR, Randolph RL, Cech DA, Rose JE, Panko WB. Steroid hormone binding macromolecules in meningiomas: Failure to meet criteria of specific receptors. Cancer 1984;53:922-7. Tilzer LL, Plapp FV, Evans JP, Stone D, Alward K. Steroid receptor proteins in human meningioma. Cancer 1982;49:633-6. Vaquero J, Marcos ML, Martinez R, Bravo G. Estrogen-and progesterone receptor proteins in intracranial tumors. Surg Neurol 1983;19:11-3. Waelti E, Markwalder TM. Endocrine manipulation of meningiomas with medroxyprogesterone acetate: effect of MPA on growth of primary meningioma cells in monolayer tissue culture. Surg Neurol 1989;31:96-100. Zava DT, Markwalder TM, Markwalder RV. Biological expression of steroid hormone receptors in primary meningioma cells in monolayer culture. Clin Neuropharmacol 1984;7:382-8. Zava DT, Wyler-von Ballmoos A, Goldhirsch A, Roos W, Takahashi A, Eppenberger U, Arrenbrecht S, Martz G, Losa G, Gomez F, Guelpa C. A quality control study to assess the interlaboratory variability of routine estrogen and progesterone receptor assays. Eur Cancer Clin Oncol 1982;18:713-21.
Surg Neurol 1989;31:172-6
Immunocytochemical Evidence of Progesterone Receptors in Human Meningiomas Ernst R. Waelti, Ph.D., and Thomas-Marc Markwalder, M.D. Institute of Pathology and Department of Neurosurgery, University of Bern, Bern, Switzerland
Waelti ER, Markwalder TM. Immunocytochemical evidence of progesterone receptors in human meningiomas. Surg Neurol 1989;31:172-6.
The presence of progesterone receptors in meningioma tissue is demonstrated by use of highly specific monoclonal antibodies against the rabbit progesterone receptors which cross-react with human progesterone receptors in breast cancer cells, thus giving evidence of the existence of genuine progesterone receptors in human meningiomas. K E Y WORDS: Meningioma; Progesterone receptor; Sex steroids; Neurochemistry
Remarkably high titers of a non-estrogen-regulated [3,11,14,16,26] high-affinity/limited-capacity progesterone receptor (PR) have been detected in over 70% of meningioma cytosols [ 1,3,4,9,11-16,19,21,24]. The recent development of highly specific monoclonal antibodies against the rabbit PR which cross-reacted with human cytosolic PR [8,18] and the subsequently described immunocytochemical method for PR detection in human breast tumors [17] offer the opportunity for specific localization of PR in meningiomas. In the following report we demonstrate an immunohistochemical evaluation of PR in several meningiomas and a preliminary correlation between PR immunostaining and the concentration of cytosolic PR determined by a steroid-binding assay. Materials and Methods
Materials Mouse anti-PR antibodies were purchased from Dianova GmbH, Hamburg, FRG, and Transbio, Paris, France and are now available through an agreement with
the Institut National de la Sant6 et de la Recherche Mddicale [INSERM], Paris. Biotinylated sheep antimouse immunoglobulin (Ig) was obtained from Amersham UK. Avidin-biotinylated peroxidase complexes (ABComplex) were purchased from Dakopatts, Denmark. All other chemicals were of analytical grade.
Tissue Preparation Tissue samples from surgically removed meningiomas and breast carcinomas were rapidly frozen in liquid nitrogen and stored at -70°C in airtight capsules until sectioning for histochemical receptor localization. Two tissue blocks were sampled, one for immunocytochemical assay and one for PR-binding assay.
Immunostaining Procedure The cryostat sections were mounted on chrome-gelatincoated glass slides and air-dried in a exsiccator at room temperature. After rehydration in a 1% NaCl solution for 5 minutes, the sections were fixed for 10 minutes in acetone, air dried, and then incubated successively with monoclonal mouse anti-PR antibodies (1:25 dilution) for 1 hour at room temperature, biotinylated sheep anti-mouse (1 : 200 dilution) for 30 minutes, and performed avidin-biotinylated peroxidase complexes for 30 minutes. Each incubation was followed by a 30-minute washing in 1% NaC1 solution. Finally, the sections were incubated for 6 minutes with the 3-3' diaminobenzidine tetrahydrochloride (DAB) solution (1.05 g of citric acid monohydrat, 1.7 g of imidazole in 50 mL of water, pH 7.0, containing 0.2 mg of DAB per milliliter and 0.01% HzO2) and after a 5-minute wash counterstained with hematoxylin. Control slides consisted of sections of meningiomas in which the monoclonal antibody was replaced by normal mouse immunoglobulin.
Biochemical Determination of ProgesteroneReceptors Address reprint requests to: Ernst R. Waelti, Ph.D., Department of Pathology, University of Bern, CH-3010 Bern, Switzerland. Received March 21, 1988; accepted September 12, 1988.
© 1989 by Elsevier Science Publishing Co., Inc.
Cytosolic PR determination was measured by dextrancoated charcoal assay as previously described [27]. 0090-3019/89/$3.50
Immunostaining of Meningioma Progesterone Receptors
Surg Neurol 1989;31:172-6
173
P jte s 0
i
Figure 1. (A) Immunostaining of meningioma tissue with a very high PR content of 464 fmol/mg protein. Note the strong nuclear staining of the greatest part of the tumor cells. (B) Immunohistochemical localization of PR of an infiltrating breast carcinoma using the same monoclonal antibody (x340).
Results As described by the documentation of the producer, the antibodies were raised against purified rabbit PR. They were selected among 70 monoclonals for giving optimal immunocytochemical or immunoblot reactions with either rabbit and guinea pig (mPRII) or human (mPRI) receptors; mPRI is an IgG1 and will detect the intact receptor. The use ofmPRI for the immunocytochemical study of PR in human tissues, especially breast cancer, was described in detail by Perrot-Applanat et al [17], who showed by immunoblotting analysis that mPRI indeed bound to human PR in the cytosol of the PR containing T47D human breast cancer cells (4 pmol of PR per milligram of protein). We tested the monoclonal antibodies in T47D monolayer cultures by an immunocytochemical assay and found an intense staining of the cell nuclei (results not shown).
The results of immunostaining in five cases in meningiomas with monoclonal antibodies against PR are summarized in Figures 1 and 2 and Table 1. Concentrations of PR in cytosolic extracts varied from 464 to 4 fmol/mg protein. As demonstrated in Figure 1, A and B, the monoclonal antibody reacting with PR of human breast cancer cells also stained PR of meningioma cells. This finding suggests that the well-characterized, high-affinity PR in human mammary carcinomas may be identical to PR identified in meningiomas by use of the same monoclonal antibody. In the case depicted by Figure 1 A (PR content of 464 fmol/mg protein) essentially all tumor cells were stained strongly. The staining of varying intensity was predominantly located in the nucleus. Nuclear and specific cytoplasmic staining was only observed in case F.D. Figure 2, A - D illustrates different patterns of staining distribution obtained in this study: tumor staining ranged from a clear-cut predominance of strongly stained cells as seen in Figure 2 A (151 fmol of PR per milligram of protein) and Figure 2 B (110 fmol of PR per milligram of protein) or only a few stained
Figure 2. Representative immunostaining of two cases of meningiomas with high PR content (A, l 51 fmol/mg protein; B, I 10 fmol/mg protein), one case with a lower content (C, 55 fmol/mg proteinL and a negative case (D, 4 fmol/mg protein) where all of the cell nuclei were stained blue. Each large filled arrow indicates a PR-positive nucleus with strong immunostaining which appears in black: each open arrow marks a PR-negative nuclear staining which appears in gray. Cytoplasmic staining (small arrows~ is visible in C. In all instances slides were counterstained with hematoxylin (×340).
Immunostaining of Meningioma Progesterone Receptors
Table
1.
Surg Neurol 1989;31:172-6
175
ProgesteroneReceptor Status and Intensity of Progesterone Receptor Staining in Meningiomas
Case identification no. R.J.L. Sch. A. F.G. F.D. U.H. Sch.M.
Cytosolic PR binding (fmol/mg protein)
Staining Nuclear
Cytoplasmic
464 154 110 55 31 4
+ + + + + 0
0 0 0 + 0 0
Staining intensity" lll-IV I 11 II-lll 11 1 --
Histologic type Meningothelial Meningothelial Meningothelial Fibroblastic Meningothelial Transitional
" 1, weak staining; IV, intense staining. Very weak cytoplasmic staining was interpreted as nonspecific background staining.
cells per field (Figure 2 C, 55 fmol of PR per milligram of protein and case U.H., 31 fmol of PR per milligram of protein, photograph not shown) to a complete absence of positive cells in Figure 2 D (4 fmol of PR per milligram protein). Comparing Figures 1 A and 2 D, for example, the difference between a PR-positive and a PR-negative meningioma tissue is eye-catching. For better discrimination of positive and negative staining characteristics, positive nuclei are marked with a filled arrow and negative nuclei with an open arrow in Figure 2, A and B. A relative intense staining was observed in cytoplasm of meningioma cells of case F.D. (55 fmol of PR per milligram of protein) as demonstrated in Figure 2 C (small arrows indicate cytoplasmic staining, whereas the big arrow indicates a positive nucleus). Although the number of meningiomas studied is not sufficient to allow a thorough statistical analysis and it is difficult to establish clear criteria for the detection of qualitative differences in staining intensity since such differences relate to a certain degree to the observer, there appears to exist a relationship among the staining intensity, percentage of positive tumor cells, and the quantitative PR value obtained by steroid-binding assay (Table 1).
Discussion We have demonstrated immunohistochemically the presence of high-affinity PR in meningiomas by using a monoclonal antibody reacting with PR in human breast cancer cells. Since this monoclonal antibody recognizes the same antigenic determinant on PR molecules in meningiomas as on the high-affinity PR in breast cancer tissue, we must conclude that these receptors are not merely progesterone-binding proteins but indeed genuine high-affinity PR. Moreover, Perrot-Applanat et al [17] submitted cytosols from human breast cancer T47-D cells containing PR to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate and analyzed the monoclonal antibody concerned by immunoblotting: a single immunogenic protein with molecular weight of 110,000, identical to the rabbit PR, was
found. Our immunohistochemical results also confirm the data of previous reports obtained by Scatchard plot analysis [1,5,6,19,20,23], which demonstrate that a large proportion of human meningiomas are rich in PR. However, the results of our study are in contrast to the views of Schwartz et al [22] who concluded that neither the estrogen- nor progesterone-binding proteins found in meningioma cytosols are likely to be receptors. Nevertheless, they also reported high levels of progesterone binding in the majority of their meningiomas. On the other hand, the existence of estrogen receptors in human meningiomas is still a matter of debate [7], although the virtual absence of ER seems to have been sufficiently documented. Moreover, the search for ER in meningiomas tissue sections with monoclonal antibodies against the human ER failed [2]. Monoclonal antibodies against PR used a immunocytochemical assay offer new criteria for positivity of the meningioma tissue. Low levels of apparent PR content determined by the dextran-coated charcoal method may be verified by these antibodies, since there is a relationship among the intensity of staining, the number of positive tumor cells, and the PR content measured by radioligand assay. Our finding suggests that immunohistochemistry in tissue sections of meningiomas with monoclonal antibodies against PR may be of predictive value concerning the possibility of hormonal manipulation and the clinical behavior of these tumors. It may be a further explanation for the observed effects of medroxyprogesterone acetate on the growth fractions of meningioma cells in vivo immunostained by the monoclonal antibody Ki-67 specific for cycling cells [10] and on the growth of meningioma cells in culture [25].
References 1. Blaauw G, Blankenstein MA, Lamberts WJ. Sex steroid receptors in h u m a n meningiomas. Acta N e u r o c h i r 1986;79:42-7. 2. Blankenstein MA, Berns PMJJ, Blaauw G, Mulder E, Thijssen J H H . Search for estrogen receptors in h u m a n meningioma tissue sections with a monoclonal antibody against the h u m a n estrogen receptor. Cancer Res (Suppl) 1986;46:4268s-70s.
176
Surg N e u r o l 1989;31:172-6
3. Blankenstein MA, Blaauw G, Lamberts WJ, Mulder E. Presence of progesterone receptors and absence of estrogen receptors in human intracranial meningioma cytosols. EurJ Cancer Clin Oncol 1983;19:365-70. 4. Cahill DW, Bashirelahi N, Solomon LW, Dalton T, Salcman M, Ducker TB. Estrogen and progesterone receptors in meningiomas J Neurosurg 1984;60:985-93. 5. Glick RP, Molteni A, Fors EM. Hormone binding in brain tumors. Neurosurgery 1983;13:513-9. 6. Hayward E, Whitwell H, Paul KS, et al. Steroid receptors in human meningioma. Clin Neuropharmacol 1984;7:351-6. 7. Lesch KP, Schott W, Gross S. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: Evidence for multiple estrogen binding sites. Surg Neurol 1987;28:181-8. 8. Logeat F, Vu Hai MT, Fournier A, Legrain P, Buttin G, Milgrom E. Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors. Proc Natl Acad Sci USA 1983;80:6456-9. 9. Magdenelat H, Pertuiset BF, Poisson M, Martin PM, PhilipponJ, Pertuiset B, Buge A. Progestin and oestrogen receptors in meningiomas: biochemical characterization, clinical and pathological correlations in 42 cases. Acta Neurochir 1982;64:199213.
Waelti and Markwalder
16.
17.
18.
19.
20.
21.
22.
10. Markwalder TM, Gerber HA, Waelti E, Schaffner T, Markwalder RV. Hormonotherapy of meningiomas with medroxyprogesterone acetate: Immunohistochemical demonstration of the effect of MPA on growth fractions ofmeningioma cells using the monoclonal antibody Ki-67. Surg Neurol 1988;30:97-101.
24.
11. Markwalder TM, Markwalder RV, Zava DT. Estrogen and progestin receptors in meningiomas: clinicopathological correlations. Clin Neuropharmacol 1984;7:368-74.
25.
12. Markwalder TM, Waelti E, K6nig MP. Endocrine manipulation of meningiomas with medroxyprogesterone acetate: Effect of MPA on receptor status of meningioma cytosols. Surg Neurol 1987;28:3-9.
26.
23.
13. Markwalder TM, Zava DT. Sex steroid hormones and meningioma cell growth (letter). J Neurosurg 1986;64:341-2. 14. Markwalder TM, Zava DT, Goldhirsch A, Markwalder RV. Estrogen and progesterone receptors in meningiomas in relation to clinical and pathological features. Surg Neurol 1983 ;20:42-7. 15. Martuza RL, MacLaughlin DT, Ojeman RG. Specific estradiol
27.
binding in schwannomas, meningiomas and neurofibromas. Neurosurgery 1981 ;9:665-71. Martuza RL, Miller DC, MacLaughlin DT. Estrogen and progestin binding by cytosolic and nuclear fractions of human meningiomas. J Neurosurg 1985;62:750-6. Perrot-Applanat M, Groyer-Picard MT, Lorenzo F, Jolivet A, Vu MT, Pallud C, Spyratos F, Milgrom E. Immunocytochemical study with monoclonal antibodies to progesterone receptor in human breast tumors. Cancer Res 1987;47:2652-61. Perrot-Applanat M, Logeat M, Groyer-Picard MT, Milgrom E. Immunocytochemical study of mammalian progesterone receptor monoclonal antibodies. Endocrinology 1985;116:1473-84. Poisson M, Magdenelat H, Foncin JF, Bleibel JM, Philippon J, Pertuiset B, Buge A. R&epteurs d'oestrog~nes et de progesterone dans les m~ningiomes. Rev Neurol (Paris) 1980;136:193203. Schnegg JF, Gomez F, Le Marchand-B~raud T, Tribolet N. Presence of sex steroid hormone receptors in meningioma tissue. Surg Neurol 1981;15:415-8. Schoenberg BS, Christine BW, Whisnant JP. Nervous system neoplasms and primary malignancies of other sites: the unique association between meningiomas and breast cancer. Neurology 1975;25:705-12. Schwartz MR, Randolph RL, Cech DA, Rose JE, Panko WB. Steroid hormone binding macromolecules in meningiomas: Failure to meet criteria of specific receptors. Cancer 1984;53:922-7. Tilzer LL, Plapp FV, Evans JP, Stone D, Alward K. Steroid receptor proteins in human meningioma. Cancer 1982;49:633-6. Vaquero J, Marcos ML, Martinez R, Bravo G. Estrogen-and progesterone receptor proteins in intracranial tumors. Surg Neurol 1983;19:11-3. Waelti E, Markwalder TM. Endocrine manipulation of meningiomas with medroxyprogesterone acetate: effect of MPA on growth of primary meningioma cells in monolayer tissue culture. Surg Neurol 1989;31:96-100. Zava DT, Markwalder TM, Markwalder RV. Biological expression of steroid hormone receptors in primary meningioma cells in monolayer culture. Clin Neuropharmacol 1984;7:382-8. Zava DT, Wyler-von Ballmoos A, Goldhirsch A, Roos W, Takahashi A, Eppenberger U, Arrenbrecht S, Martz G, Losa G, Gomez F, Guelpa C. A quality control study to assess the interlaboratory variability of routine estrogen and progesterone receptor assays. Eur Cancer Clin Oncol 1982;18:713-21.