Acta Neuropathologica
Acta Neuropathol. (Berl.) 46, 65-68 (1979)
~ Springer-Verlag '1979
DNA Content of Meningiomas P. Frederiksen 1, E. Reske-Nielsen 1, and P. Bichel 2 i Department of Neuropathology, Aarhus University School of Medicine, DK-8000 Aarhus, Denmark 2 Institute of Cancer Research (sponsored by the Danish Cancer Society), Radiumstationen, DK-8000 Aarhus, Denmark
Summary. Flow cytometric D N A analysis was carried out in 35 meningiomas. Thirty-three had diploid stem lines while two were aneuploid having a triploid D N A mode. Both aneuploid meningiomas were grossly huge nodular tumours and one had malignant histologic features. Flow cytometry in meningiomas is a rapid means of determining the D N A modes and adds objective information to microscopy. Appropriate cases may be selected for further cytogenetic studies.
Material and Methods
Fresh tissue was obtained by surgery from 35 consecutive patients suffering from meningiomasand prepared for flow cytometric DNA analysis as previously described (Frederiksen et al., 1978). Cytological smears were prepared from identical areas and stained according to May-Griinewald-Giemsa.Formalin-fixed paraffin sections were stained with haematoxylin-eosin, van GiesonHansen, and Gomori's reticulum method. The histological classification of meningiomas as adapted by Russel and Rubinstein (1977) was used. Results
Key words: D N A - Flow cytometry - Brain tumour - Meningioma
In this study the single cell D N A content was measured in 35 meningiomas derived from various intracranial and spinal sites as recorded in Table 1. Thirty-three meningiomas had diploid model D N A values (stem lines) while two had aneuploid (heteroploid) D N A stern lines. The 33 meningiomas characterized by a diploid stem line could further be subdivided in three types based on the D N A findings: 14 had solely diploid cel!s, 12 had additional smaller amounts of tetraploid cells (not exceeding 10 %) and 7 had elevated amounts of tetraploid cells (from 1 0 - 2 5 %). The histological types and the D N A contents are summarized in Table 2 and the D N A histograms are illustrated in Figs. ] -- 5. The two aneuploid meningiomas were characterized by triploid D N A stem lines. One patient (42-yearold female) had a 70 g nodular and circumscribed convexity meningioma of mixed syncytial and fibroblastic type (Fig. 4) and the other (51-year-old female) had a 112 g recurrent nodular falcine malignant meningioma invading the left hemisphere (Fig. 5).
The nuclear D N A content is closely correlated to the chromosome number. Cytophotometric studies of D N A have been consistent with proportionality between modal D N A and chromosome number, although a small discrepancy in the D N A / c h r o m o s o m e ratio can be noted often in the direction of more D N A per chromosome (Atkin et al., 1966; Kraemer et al., 1972). In a previous flow-cytometric study of D N A distributions in tumours of the brain we observed hyperploid D N A values in most malignant tumours whereas the majority of benign tumours and all reactive lesions had diploid modal values (Frederiksen et al., 1978). Meningiomas are considered benign mesenchymal tumours amenable to treatment, but some meningiomas are prone to infiltrate locally and recur. This study of the single cell D N A content was undertaken in an attempt to add further information on the intrinsic nature of the meningiomas.
Discussion
Offprint requests to: P. Frederiksen, M. D. (address see above)
In this study the usual sites and histological types of meningiomas were found. The predominant diploid
0001-6322/79/0046/0065/$1. O0
66
Acta Neuropathol. (Berl.) 46 (1979)
3
2N
4N
Figs. 1--5. DNA histograms, each based on the analysis of 12,000-50,000 cell nuclei. Ordinate indicates cells per chanel in relative units. The abscissa indicates the relative fluorescence intensity. 2N = diploid, 3 N = triploid, 4N = tetraploid cell nuclei. Microphotographs are • 120, H.-E. Fig. 1. Meningioma (No. 22449) of transitional cell type. DNA histogram showing solely diploid cells Fig.2. Meningioma (No. 23451) of syncytial type. DNA histogram showing diploid cells and an additional small fraction of tetraploid cells and no S-face nuclei Fig.3. Meningioma (No. 23859) of fibroblastic type. DNA histogram showing a major diploid peak and an additional smaller tetraploid peak (25 ~ 4N cells) 2
N 4N
Table 1. DNA ploidy and site of 35 meningiomas
Diploid stem line Aneuploid stem line
Basal
Convexity
Falcine
Ventricular
Spinal
Total
8
11 1
7 I
1
6
33 2
D N A values o b s e r v e d correlate well with c y t o g e n e t i c studies on m e n i n g i o m a s by Singer a n d Z a n g (1970) a n d by M a r k (1970). M a r k (1973) s t a t e d t h a t o u t o f 105 m e n i n g i o m a s analysed, 97 h a d d i p l o i d - h y p o d i p l o i d stem lines.
J 6 r g e n s h a u s (1978) studied the n u c l e a r D N A content o f 14 m e n i n g i o m a s by m e a n s o f feulgen c y t o p h o tometry. In c o n t r a s t to o u r findings h y p e r d i p l o i d a n d t r i p l o i d D N A stem lines p r e d o m i n a t e d in the m a t e r i a l o f J6rgenshaus. H o w e v e r , it is i m p o s s i b l e in o u r study
P. Frederiksenet al.: DNA Content of Meningiomas
67
9 3N
6N
I 3N
6N
Fig.4. Aneuploid meningioma (No. 23534). Mixed fibroblastic and syncytial type. DNA histogram showing two distinct peaks : diploid cells and triploid cells plus a "shoulder" in the 4 - 6 N area
Fig.5. Aneuploid meningioma (No. 23989). Malignant meningiolna with mitotic figures and (not shown) focal necroses. DNA histogram showing two large peaks: diploid cells and triploid cells plus additional smaller peaks in the 4N and 6N area
Table 2. Histologic type and DNA content in 35 meningiomas. 2 N
sequent mitotic division (Adler et al., 1977). Nevertheless, the possibility of a certain amount of clumping cannot be excluded although the purity of the nuclear suspension was examined under the microscope. The two meningiomas having aneuploid stern lines were huge nodular tumours. One had malignant histological features (Fig. 5) while the other did not show significant malignant changes on microscopy (Fig. 4). Aneuploid meningiomas were reported in cytogenetic studies by Zankl et al. (1971) and by Mark (1973) but their cases did not have histologic malignant features. Histological malignancy versus clinical malignancy was discussed by Zfilch (1974) and it is well-known that histological features in meningiomas can be uncertain considering prognostic statements. In the meningioma series by Skullerud and L6ken (1974) and Jellinger and Slowik (1975) parameters of primary prognostic importance were incomplete surgical remowd and falcine location while histological details were of minor importance except for a high degree of cellularity and increased mitotic rates. The findings of aneuploidy in otherwise benign meningiomas cannot presently be evaluated. A developing step in carcinogenesis could be considered. If that be so meningiomas cover a spectrum extending from entirely benign tumours to malignant tumours. In
indicates all counted cell nuclei in the diploid DNA area. 2 N (4 N) indicates an additional small fraction of less than 10 ~ tetraploid cells. 2 N 4 N indicates an additional significant fraction oftetraploid cells (10-25 %). 2 N 3 N indicates an additional significant fraction of triploid cells Histologic type
Diploid stem line 2N
2N
Aneuploid stem line 2N
2N
(4N) 4N
3N
Transitional Syncytial Fibroblastic Mixed Malignant
12 8 5 9 1
8 2 2 2 0
4 4 1 3 0
0 2 2 3 0
0 0 0 1 1
Total
35
14
12
7
2
to distinguish between diploid and near diploid cells, which may explain some of the differences. The findings in the present material of an elevated amount of tetraploid cells in a substantial number of cases can hardly be due }o premitotic G 2 cells but may rather be parallel to the frequent findings of tetraploid cell nuclei in, e.g., liver and heart muscle, possibly caused by endoreduplication of DNA without sub-
68
this context flow cytometric DNA analysis adds objective information to microscopy. Furthermore, this method is a rapid means of determining the DNA modes and in this way it should be possible to select appropriate cases of meningiomas for further chromosome studies. Acknowledgements.We wish to thank Mrs. B. Kirkegaard, Mrs. H. V. S~brensen, Mrs. B. Fastrup, Mrs. B. Olson, and Mrs. V. Rosleff for excellent technical and secretarial assistance.
References Adler, C. P., Hartz, A., Sandritter, W. : Form and structure of cell nuclei in growing and hypertrophied human hearts. Beitr. Pathol. 161, 3 4 2 - 362 (1977) Aktin, N. B., Mattinson, G., Baker, M. C.: A comparison of the DNA content and chromosome number of fifty human tumours. Br. J. Cancer 20, 8 4 - 101 (1966) Frederiksen, P., Reske-Nielsen, E., Bichel, P. : Flow cytometry in tumours of the brain. Acta Neuropathol. (Berl.) 41, 179-183 (1978)
Acta Neuropathol. (Berl.) 46 (1979) Jellinger, K., Slowik, F. : Histological subtypes and prognostic problems in meningiomas. J. Neurol. 208, 279-298 (1975) J6rgenshaus, W.: Zytophotometrische Untersuchungen fiber die DNS-Verteilung in menschlichen Meningomen. Inaug.-Diss., Univ. K61n 1978 Kraemer, P. M., Deaven, L. L., Crissman, H. A., Van Dilla, M. A. : DNA constancy dispite variability in chromosome number. In: Advances in cell and molecular biology. Vol. 2, pp. 47-108. New York, London: Academic Press 1972 Mark, J.: Chromosomal patterns in human meningiomas. Eur. J. Cancer 6, 489-498 (1970) Mark, J. : The fluorescence karyotypes of three human meningiomas with hyperdiploid-hypotriploid stemlines. Acta Neuropathol. (Berl.) 25, 4 6 - 5 3 (1973) Russell, D. S., Rubinstein, L. J. : Pathology of tumours of the nervous system. 4th ed., pp. 65-91. London: Arnold 1977 Skullerud, K., L6ken, A. C. : The prognosis in meningiomas. Acta Neuropathol. (Berl.) 29, 337-344 (1974) Singer, H., Zang, K. D. : Cytologische und cytogenetische Untersuchungen an Hirntumoren. Humangenetik 9, 172-184 (1970) Zankl, H., Singer, H., Zang, K. D. : Cytological and cytogenetical studies on brain tumours. Humangenetik 11, 253-257 (1971) Zfilch, K. J.: Atlas of gross neurosurgical pathology, pp. 31-34. Berlin, Heidelberg, New York: Springer 1975 Received October 3, 1978/Accepted November 23, 1978
Acta Neuropathol. (Berl.) 46, 65-68 (1979)
~ Springer-Verlag '1979
DNA Content of Meningiomas P. Frederiksen 1, E. Reske-Nielsen 1, and P. Bichel 2 i Department of Neuropathology, Aarhus University School of Medicine, DK-8000 Aarhus, Denmark 2 Institute of Cancer Research (sponsored by the Danish Cancer Society), Radiumstationen, DK-8000 Aarhus, Denmark
Summary. Flow cytometric D N A analysis was carried out in 35 meningiomas. Thirty-three had diploid stem lines while two were aneuploid having a triploid D N A mode. Both aneuploid meningiomas were grossly huge nodular tumours and one had malignant histologic features. Flow cytometry in meningiomas is a rapid means of determining the D N A modes and adds objective information to microscopy. Appropriate cases may be selected for further cytogenetic studies.
Material and Methods
Fresh tissue was obtained by surgery from 35 consecutive patients suffering from meningiomasand prepared for flow cytometric DNA analysis as previously described (Frederiksen et al., 1978). Cytological smears were prepared from identical areas and stained according to May-Griinewald-Giemsa.Formalin-fixed paraffin sections were stained with haematoxylin-eosin, van GiesonHansen, and Gomori's reticulum method. The histological classification of meningiomas as adapted by Russel and Rubinstein (1977) was used. Results
Key words: D N A - Flow cytometry - Brain tumour - Meningioma
In this study the single cell D N A content was measured in 35 meningiomas derived from various intracranial and spinal sites as recorded in Table 1. Thirty-three meningiomas had diploid model D N A values (stem lines) while two had aneuploid (heteroploid) D N A stern lines. The 33 meningiomas characterized by a diploid stem line could further be subdivided in three types based on the D N A findings: 14 had solely diploid cel!s, 12 had additional smaller amounts of tetraploid cells (not exceeding 10 %) and 7 had elevated amounts of tetraploid cells (from 1 0 - 2 5 %). The histological types and the D N A contents are summarized in Table 2 and the D N A histograms are illustrated in Figs. ] -- 5. The two aneuploid meningiomas were characterized by triploid D N A stem lines. One patient (42-yearold female) had a 70 g nodular and circumscribed convexity meningioma of mixed syncytial and fibroblastic type (Fig. 4) and the other (51-year-old female) had a 112 g recurrent nodular falcine malignant meningioma invading the left hemisphere (Fig. 5).
The nuclear D N A content is closely correlated to the chromosome number. Cytophotometric studies of D N A have been consistent with proportionality between modal D N A and chromosome number, although a small discrepancy in the D N A / c h r o m o s o m e ratio can be noted often in the direction of more D N A per chromosome (Atkin et al., 1966; Kraemer et al., 1972). In a previous flow-cytometric study of D N A distributions in tumours of the brain we observed hyperploid D N A values in most malignant tumours whereas the majority of benign tumours and all reactive lesions had diploid modal values (Frederiksen et al., 1978). Meningiomas are considered benign mesenchymal tumours amenable to treatment, but some meningiomas are prone to infiltrate locally and recur. This study of the single cell D N A content was undertaken in an attempt to add further information on the intrinsic nature of the meningiomas.
Discussion
Offprint requests to: P. Frederiksen, M. D. (address see above)
In this study the usual sites and histological types of meningiomas were found. The predominant diploid
0001-6322/79/0046/0065/$1. O0
66
Acta Neuropathol. (Berl.) 46 (1979)
3
2N
4N
Figs. 1--5. DNA histograms, each based on the analysis of 12,000-50,000 cell nuclei. Ordinate indicates cells per chanel in relative units. The abscissa indicates the relative fluorescence intensity. 2N = diploid, 3 N = triploid, 4N = tetraploid cell nuclei. Microphotographs are • 120, H.-E. Fig. 1. Meningioma (No. 22449) of transitional cell type. DNA histogram showing solely diploid cells Fig.2. Meningioma (No. 23451) of syncytial type. DNA histogram showing diploid cells and an additional small fraction of tetraploid cells and no S-face nuclei Fig.3. Meningioma (No. 23859) of fibroblastic type. DNA histogram showing a major diploid peak and an additional smaller tetraploid peak (25 ~ 4N cells) 2
N 4N
Table 1. DNA ploidy and site of 35 meningiomas
Diploid stem line Aneuploid stem line
Basal
Convexity
Falcine
Ventricular
Spinal
Total
8
11 1
7 I
1
6
33 2
D N A values o b s e r v e d correlate well with c y t o g e n e t i c studies on m e n i n g i o m a s by Singer a n d Z a n g (1970) a n d by M a r k (1970). M a r k (1973) s t a t e d t h a t o u t o f 105 m e n i n g i o m a s analysed, 97 h a d d i p l o i d - h y p o d i p l o i d stem lines.
J 6 r g e n s h a u s (1978) studied the n u c l e a r D N A content o f 14 m e n i n g i o m a s by m e a n s o f feulgen c y t o p h o tometry. In c o n t r a s t to o u r findings h y p e r d i p l o i d a n d t r i p l o i d D N A stem lines p r e d o m i n a t e d in the m a t e r i a l o f J6rgenshaus. H o w e v e r , it is i m p o s s i b l e in o u r study
P. Frederiksenet al.: DNA Content of Meningiomas
67
9 3N
6N
I 3N
6N
Fig.4. Aneuploid meningioma (No. 23534). Mixed fibroblastic and syncytial type. DNA histogram showing two distinct peaks : diploid cells and triploid cells plus a "shoulder" in the 4 - 6 N area
Fig.5. Aneuploid meningioma (No. 23989). Malignant meningiolna with mitotic figures and (not shown) focal necroses. DNA histogram showing two large peaks: diploid cells and triploid cells plus additional smaller peaks in the 4N and 6N area
Table 2. Histologic type and DNA content in 35 meningiomas. 2 N
sequent mitotic division (Adler et al., 1977). Nevertheless, the possibility of a certain amount of clumping cannot be excluded although the purity of the nuclear suspension was examined under the microscope. The two meningiomas having aneuploid stern lines were huge nodular tumours. One had malignant histological features (Fig. 5) while the other did not show significant malignant changes on microscopy (Fig. 4). Aneuploid meningiomas were reported in cytogenetic studies by Zankl et al. (1971) and by Mark (1973) but their cases did not have histologic malignant features. Histological malignancy versus clinical malignancy was discussed by Zfilch (1974) and it is well-known that histological features in meningiomas can be uncertain considering prognostic statements. In the meningioma series by Skullerud and L6ken (1974) and Jellinger and Slowik (1975) parameters of primary prognostic importance were incomplete surgical remowd and falcine location while histological details were of minor importance except for a high degree of cellularity and increased mitotic rates. The findings of aneuploidy in otherwise benign meningiomas cannot presently be evaluated. A developing step in carcinogenesis could be considered. If that be so meningiomas cover a spectrum extending from entirely benign tumours to malignant tumours. In
indicates all counted cell nuclei in the diploid DNA area. 2 N (4 N) indicates an additional small fraction of less than 10 ~ tetraploid cells. 2 N 4 N indicates an additional significant fraction oftetraploid cells (10-25 %). 2 N 3 N indicates an additional significant fraction of triploid cells Histologic type
Diploid stem line 2N
2N
Aneuploid stem line 2N
2N
(4N) 4N
3N
Transitional Syncytial Fibroblastic Mixed Malignant
12 8 5 9 1
8 2 2 2 0
4 4 1 3 0
0 2 2 3 0
0 0 0 1 1
Total
35
14
12
7
2
to distinguish between diploid and near diploid cells, which may explain some of the differences. The findings in the present material of an elevated amount of tetraploid cells in a substantial number of cases can hardly be due }o premitotic G 2 cells but may rather be parallel to the frequent findings of tetraploid cell nuclei in, e.g., liver and heart muscle, possibly caused by endoreduplication of DNA without sub-
68
this context flow cytometric DNA analysis adds objective information to microscopy. Furthermore, this method is a rapid means of determining the DNA modes and in this way it should be possible to select appropriate cases of meningiomas for further chromosome studies. Acknowledgements.We wish to thank Mrs. B. Kirkegaard, Mrs. H. V. S~brensen, Mrs. B. Fastrup, Mrs. B. Olson, and Mrs. V. Rosleff for excellent technical and secretarial assistance.
References Adler, C. P., Hartz, A., Sandritter, W. : Form and structure of cell nuclei in growing and hypertrophied human hearts. Beitr. Pathol. 161, 3 4 2 - 362 (1977) Aktin, N. B., Mattinson, G., Baker, M. C.: A comparison of the DNA content and chromosome number of fifty human tumours. Br. J. Cancer 20, 8 4 - 101 (1966) Frederiksen, P., Reske-Nielsen, E., Bichel, P. : Flow cytometry in tumours of the brain. Acta Neuropathol. (Berl.) 41, 179-183 (1978)
Acta Neuropathol. (Berl.) 46 (1979) Jellinger, K., Slowik, F. : Histological subtypes and prognostic problems in meningiomas. J. Neurol. 208, 279-298 (1975) J6rgenshaus, W.: Zytophotometrische Untersuchungen fiber die DNS-Verteilung in menschlichen Meningomen. Inaug.-Diss., Univ. K61n 1978 Kraemer, P. M., Deaven, L. L., Crissman, H. A., Van Dilla, M. A. : DNA constancy dispite variability in chromosome number. In: Advances in cell and molecular biology. Vol. 2, pp. 47-108. New York, London: Academic Press 1972 Mark, J.: Chromosomal patterns in human meningiomas. Eur. J. Cancer 6, 489-498 (1970) Mark, J. : The fluorescence karyotypes of three human meningiomas with hyperdiploid-hypotriploid stemlines. Acta Neuropathol. (Berl.) 25, 4 6 - 5 3 (1973) Russell, D. S., Rubinstein, L. J. : Pathology of tumours of the nervous system. 4th ed., pp. 65-91. London: Arnold 1977 Skullerud, K., L6ken, A. C. : The prognosis in meningiomas. Acta Neuropathol. (Berl.) 29, 337-344 (1974) Singer, H., Zang, K. D. : Cytologische und cytogenetische Untersuchungen an Hirntumoren. Humangenetik 9, 172-184 (1970) Zankl, H., Singer, H., Zang, K. D. : Cytological and cytogenetical studies on brain tumours. Humangenetik 11, 253-257 (1971) Zfilch, K. J.: Atlas of gross neurosurgical pathology, pp. 31-34. Berlin, Heidelberg, New York: Springer 1975 Received October 3, 1978/Accepted November 23, 1978
