Int. J. Cancer (Pred. Oncol.): 79, 116–120 (1998) r 1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer DNA PLOIDY AND CELL-CYCLE ANALYSIS IN INTRACRANIAL MENINGIOMAS AND HEMANGIOPERICYTOMAS: A STUDY WITH HIGH-RESOLUTION DNA FLOW CYTOMETRY Anton ZELLNER1, Jürgen MEIXENSBERGER2*, Wolfgang ROGGENDORF3, Michael JANKA2, Holger HOEHN4 and Klaus ROOSEN2 of Neurology, University of Würzburg, Würzburg, Germany 2Department of Neurosurgery, University of Würzburg, Würzburg, Germany 3Department of Pathology, University of Würzburg, Würzburg, Germany 4Department of Genetics, University of Würzburg, Würzburg, Germany 1Department Although various DNA flow-cytometric studies have been performed on meningiomas, the role of DNA ploidy and the S-phase fraction (SPF) in predicting biological tumor behavior remains unresolved. Discrepant results in earlier studies might be due to different preparing, staining and measuring techniques; different quality standards; and lack of sophisticated computer software. In this study, high-resolution DNA flow cytometry using the DNA-specific dye DAPI (48, 68diamidino-2-phenylindol) was performed on stored frozen tissue from 128 microsurgically resected meningiomas and 7 hemangiopericytomas, including 17 recurrent meningiomas and 4 recurrent hemangiopericytomas. The computer software Multicycle 2.5 was used to determine the ploidy level and to perform cell-cycle analysis. DNA aneuploidy and SPF were significantly higher in atypical, anaplastic and recurrent meningiomas and correlated well with histopathological features such as focal necrosis, infiltration of dura mater and mitotic activity. Among 128 meningiomas, 42 had additional DNA aneuploid stem lines. No association between hypo- and hyperploidy and either histological subtype or clinical outcome was found. In 7 hemangiopericytomas, SPF was significantly higher compared to the benign meningioma group, while only 1 tumor was aneuploid. In all 42 DNA aneuploid tumors, cell-cycle analysis was performed separately for the euploid and aneuploid stem lines. The proliferation parameters (SPF, G2/M phase) were significantly higher in the DNA aneuploid stem lines. DNA ploidy and SPF are thus useful indicators of different biological behavior within identical histological subgroups in meningiomas. Int. J. Cancer (Pred. Oncol.) 79:116–120, 1998. r 1998 Wiley-Liss, Inc. Despite sophisticated histological grading systems based on morphological and immunohistochemical parameters, it is not always possible to predict the individual proliferative potential and tendency of recurrence among tumors within the same histological subtype; this is particularly true for low- and middle-grade tumors (Coons et al., 1994; Ironside et al., 1987; May et al., 1989). Thus, even among meningiomas that are histologically classified as benign and completely resected by microsurgery, the number of tumor relapses varies between 5% and 20%. Flow-cytometric parameters, such as DNA ploidy and S-phase fraction (SPF), were significantly associated with tumor behavior in many tumor entities, such as breast cancer, non-small cell lung cancer, ovarian carcinoma and colorectal carcinoma (Coon et al., 1987; Giaretti and Santi, 1990; Merkel and McGuire, 1990; Seckinger et al., 1989). Although many studies have been performed on meningiomas and neuro-epithelial tumors, the role of DNA ploidy and SPF in these tumor groups remains unresolved (Rabson, 1994). Discrepant results might be due to technical reasons (e.g., sample preparation, staining) or variable definitions (e.g., definition of DNA aneuploidy and DNA index). In addition, many studies do not incorporate internal cell standards or achieve only poor coefficients of variation (CV) in their measurements (Dressler, 1990; Hiddemann et al., 1984; McCarthy and Fetterhoff, 1989; Shapiro, 1989). Finally, in earlier studies, sophisticated computer software was not available to perform cell-cycle analysis simultaneously in different stem lines of the same tumor sample. The present study aimed at avoiding these problems by using high-resolution DNA histograms the sophisticated Multicycle 2.5 software. Our prospective study seeks to answer the following questions: 1. Do flow-cytometric DNA parameters such as ploidy and SPF have prognostic relevance in predicting an aggressive tumor behavior? 2. Can these parameters identify different prognostic groups within histopathologically graded and defined tumor subgroups? 3. Is there a significant difference in the proliferative activity between DNA diploid and DNA aneuploid tumor stem lines? MATERIAL AND METHODS We studied 135 samples from complete microsurgically resected tumors that were frozen in liquid nitrogen immediately after surgery. Our protocol was approved by the local ethics committee. Tissue samples were stored at 280°C for an average of 6 months prior to flow-cytometric analysis. Table I summarizes essential data and histological grading (WHO) of the tumor material. Immediately after thawing, tumor samples were minced and prepared as single-cell-nuclei suspensions using citric acid and Tween 20 (Sigma, Deisenhofen, Germany) as detergent according to the modified protocol of Otto (1990). Cell nuclei were stained with the AT-specific dye DAPI (48, 68-diamidino-2-phenylindol) (Sigma). Chicken erythrocytes and calf thymocytes were treated like tumor tissue and were used as internal DNA standards. In about 40 tumors, we prepared and measured an average of 3 samples within different areas of the same tumor in order to evaluate tissue heterogeneity. All flow-cytometric measurements were performed on a PAS II flow cytometer (Partec, Reinach, Switzerland) using a mercury arc lamp as light source. More than 400 measurements with an average of 19,500 signals (cell nuclei) counted in each sample were evaluated. Mean values for nuclear aggregation (clumping) were 6.14 6 3.54% and for debris 7.54 6 8.19%. The resulting DNA histograms (Fig. 1) were evaluated with the computer software Multicycle 2.5 (Phoenix Flow Systems, Seattle, WA; Rabinovitch). Histological classification was performed according to the WHO classification for meningiomas (Kleihues et al., 1991). Simultaneously, a qualitative and semi-quantitative morphological investigation concentrated on the number of mitoses, infiltrative growth, tightness and pleomorphism of cells, as well as tumor necrosis. *Correspondence to: Department of Neurosurgery, Universitätsklinik Würzburg, Josef-Schneiderstrasse 11, D-97080 Würzburg, Germany. Fax: 011-49-931-2012635. E-mail: [email protected] Received 8 August 1997; Revised 17 October 1997 DNA FLOW CYTOMETRY IN MENINGIOMAS AND HEMANGIOPERICYTOMAS TABLE I – CLINICAL DATA AND HISTOLOGICAL GRADING (WHO) ON THE TUMOR MATERIAL Histology Meningioma subtype Meningothelial Transitional Fibroblastic Psammomatous Microcystic Secretory Myxomatous Atypical Anaplastic Hemangioblastic Hemangiopericytomas Total case number Cases (n) WHO grade Age (mean, years) Sex ratio (m:f) Recurrent tumors (n) 39 36 22 2 3 2 1 17 5 1 7 135 1 1 1 1 1 1 1 2 3 2 2 57 61 61 60 44 58 42 61 63 52 52 11:28 10:26 1:21 1:1 1:2 0:2 1:0 3:14 3:2 0:1 2:5 5 3 0 0 0 0 0 7 2 0 4 21 TABLE II – DISTRIBUTION OF ANEUPLOID TUMORS AND PERCENTAGES OF THE ANEUPLOID POPULATION (% ANTOT) IN ANEUPLOID TUMORS Histology Cases Aneuploid (n) tumors (n) Meningioma subtype Meningothelial 39 Transitional 36 Fibroblastic 22 Psammomatous 2 Microcystic 3 Secretory 2 Myxomatous 1 Atypical 17 Anaplastic 5 Hemangioblastic 1 Hemangiopericytomas 7 Total case number 135 11 9 0 0 3 0 1 12 5 1 1 43 % Antot (mean 6 SD) Recurrent tumors (n) 53.55 (622.17) 65.04 (611.75) — — 70.45 (62.95) — 51.2 67.93 (617.63) 77.42 (610.81) 54.3 Not evaluable 63.97 (618.43) 5 3 0 0 0 0 0 7 2 0 4 21 117 S phase SPF indicates how many cells of the entire population are replicating DNA at a given time point. SPF is computed by the polynomial S-phase fitting method described by Dean and Jett (1980). DNA ploidy level The relation of the tumor stem cell peak to the reference peaks (chicken and calf thymocytes) is defined as the DNA index (DI) and relates to the DNA-specific fluorescence emitted by a given cell population. By definition, the DI for the diploid G0/G1 peak is set at 1, the DI for the G2/M peak is 2 and additional aneuploid stem lines have a DI ,1 (hypodiploid) or .1 (hyperdiploid). In our series, the existence of additional DNA aneuploid stem lines was accepted only when 2 clearly visible and distinct (G0/G1 ) peaks, which could be reproduced in at least 2 independent measurements, were demonstrated in the histograms. Identification and calculation of tetraploid stem lines is more difficult because the tetraploid G0/G1 peak occupies the same position in the histogram as the G2/M peak of the diploid stem line. In our study, tetraploidy was considered when the G2/M phase of the diploid stem line exceeded 10% of all measured cell nuclei and a corresponding G2/M phase of the presumptive tetraploid stem line was evident. Coeffıcient of variation The CV is used to describe the width of the peaks in the DNA histograms. It is a normalized standard deviation defined as CV 5 100 times SD divided by the mean of the peak. Statistical analysis Results are given as mean 6 SD for the different groups. Statistical analysis was performed using the x2 test, t-test, Wilcoxon test and variance analysis, as appropriate. RESULTS FIGURE 1 – DNA histogram of a DNA euploid meningioma with internal standard (chicken erythrocytes) evaluated by the computer software Multicycle 2.5. Tumor necrosis was assessed only in pre-operatively nonembolized tumor tissue. DNA classification The program Multicycle calculates 3 cell-cycle compartments (G0/G1, S, G2/GM) separately for different stem lines within a tumor sample. As shown in Table I, 128 cases of intracranial meningiomas and 7 hemangiopericytomas were included in our study. Among these, 21 recurrent tumors could be evaluated (17 meningiomas and 4 hemangiopericytomas). The average CV for all diploid G0/G1 peaks was 2.01 6 0.58% and for all aneuploid peaks 1.86 6 0.46%. A tumor sample was considered unusable when the CV in repeated measurements exceeded 3.5%. This was the case in about 20 tumor samples, which were not considered in our series; 43 of the 135 tumors had an additional DNA aneuploid stem line, which was hypodiploid in 18 cases (41.9%) and hyperdiploid in 19 cases (44.2%). Four tumors (9.3%) had a tetraploid stem line. In 2 tumors, either the cell-cycle parameters (case in Fig. 2b) or the DI of the aneuploid stem line (the one aneuploid hemangiopericytoma) could not be accurately determined by the program Multicycle (one case in Fig. 2b) and, therefore, were not considered in statistical analyses. No association between hypo- and hyperdiploidy and either histological subtype or tumor recurrence was noted. Concerning the peak position of the aneuploid stem lines, there appeared to be a tendency of the aneuploid G0/G1 peaks to be more distant from the diploid stem line (DI 5 1) along with an increasing malignant potential or WHO grade (Figs. 3, 4). Table II lists the distribution of aneuploid tumors among the histological subgroups and the relative percentages of the aneuploid tumor fraction (% Antot). All of the 5 anaplastic meningiomas (WHO 3) and 12 of the 17 (70.6%) atypical meningiomas (WHO 2) exhibited DNA aneuploid stem lines, while of the benign (WHO 1) meningiomas only 11 of 39 (28.9%) meningothelial, 9 of 36 (25%) transitional and 0 of 22 fibroblastic tumors were DNA aneuploid. This difference (WHO 1 vs. WHO 2/3) was highly significant ( p , 0.001). In contrast with these results, only 1 of 7 (14.2%) hemangiopericytomas (WHO 2) showed a DNA aneuploid cell line. One 118 ZELLNER ET AL. A FIGURE 4 – Distribution of DNA aneuploid stem lines (according to the DNA indices of the corresponding G0/G1 peaks) in 16 atypic and anaplastic (WHO 2/3) meningiomas. A DNA index of 1 refers to the position of the diploid G0/G1 peak. B FIGURE 5 – Among DNA aneuploid meningiomas (n 5 43, gray and black columns), the SPF, G2/M phase and PI of the aneuploid stem line were significantly higher compared to the diploid stem line, ***p , 0.0001. As a comparison, the mean values of DNA euploid meningiomas (n 5 92) are indicated by the white columns. FIGURE 2 – DNA histograms of 2 different tumor probes of the same atypical recurrent meningioma depicting local tumor heterogeneity. While in probe 1 (a) only 1 additional hypoploid stem line could be detected, probe 2 (b) showed 2 additional hypoploid stem lines. The exact positions of the peaks were determined in histograms with internal DNA standards (not shown). Cell-cycle parameters in this case (b) were not calculated accurately by Multicycle. FIGURE 3 – Distribution of DNA aneuploid stem lines (according to the DNA indexes of the corresponding G0/G1 peaks) in 24 benign (WHO 1) meningiomas. A DNA index of 1 refers to the position of the diploid G0/G1 peak. recurrent, atypical meningioma had 2 DNA hypoploid stem lines (Fig. 2a,b). Tumors with aneuploid stem lines showed a significantly higher rate of histomorphological evidence for focal necrosis ( p , 0.01), infiltration of dura mater ( p , 0.05) and a higher mitotic activity ( p , 0.001). No significant correlation between the percentages of the aneuploid tumor fractions (% Antot) and parameters of proliferation (SPF, proliferation index [PI]) was observed, although there was an unequivocal tendency toward increased % Antot fraction in tumors with higher rates of focal necrosis, infiltration of dura mater and high mitotic activity. Among the 21 recurrent tumors, 11 had additional aneuploid stem lines (52.3%), while only 32 of the 114 primary tumors (28.1%) were DNA aneuploid. This difference was significant ( p , 0.05). Cell-cycle analysis The SPF of all DNA diploid tumors (n 5 92) was compared between the histological subtypes. The atypical meningiomas (n 5 5, WHO 2) and the hemangiopericytomas (n 5 6, WHO 2) showed a significantly higher ( p , 0.001) mean SPF of 2.1 6 1.4% and 1.6 6 1.6% compared to the meningothelial (0.7 6 0.6%, n 5 28), transitional (0.6 6 0.6%, n 5 27) and fibroblastic (0.7 6 0.6%, n 5 22) subgroups. Among the 43 tumors with aneuploid stem lines the SPF, G2M-phase and the PI (SPF 1 G2/M-phase) were calculated separately for the diploid stem line and the DNA aneuploid stem line fractions. All parameters were significantly ( p , 0.001) higher in the aneuploid stem lines (Fig. 5). Additionally, in contrast with diploid tumors, no significant difference in the SPF between histological subgroups was evident. In accordance with the ploidy evaluation results, significantly higher levels of SPF were noted in meningiomas with higher mitotic rates ( p , 0.05), whereas tumors infiltrating the cortex or exhibiting focal necrosis had no significantly different SPF levels. The 21 recurrent tumors had significantly higher SPF levels than the 114 primary tumors ( p , 0.05). DNA FLOW CYTOMETRY IN MENINGIOMAS AND HEMANGIOPERICYTOMAS DISCUSSION Quality criteria Although various previous DNA flow-cytometric studies have been performed on meningiomas (Ironside et al., 1987; May et al., 1989; Ahyai and Spaar, 1987; Appley et al., 1990; Assietti et al., 1990; Butti et al., 1989; Crone et al., 1988; Cruz-Sanchez et al., 1993; Kawamoto et al., 1989; Nishizaki et al., 1993; Spaar et al., 1987), rather conflicting results have been reported concerning the number of DNA aneuploid stem lines and the distribution of the respective cell-cycle phases. The reasons for such discrepancies might be different methods of cell preparation and differences in instrumentation and flow cytometry, including histogram evaluation. Another explanation might be the smaller sample numbers or differences in definition of DNA aneuploidy. Quality criteria for flow-cytometric measurements set in earlier studies (Dressler, 1990; Hiddemann et al., 1984; McCarthy and Fetterhoff, 1989; Shapiro, 1989) often have been ignored. In addition to parameters such as the number of counted cells, amount of debris and cell aggregation (clumping), CV is one of the most important criteria in evaluating flow-cytometric DNA histograms (Dressler, 1990; McCarthy and Fetterhoff, 1989). Lower CVs imply higher precision and a greater chance to identify DNA aneuploid stem lines within tumor tissue. The CV of such measurements, therefore, should not exceed 3% (Seckinger et al., 1989; Shapiro, 1989). In our study, the average CV for all diploid G0/G1 peaks was 2.01 6 0.58% and for all aneuploid peaks 1.86 6 0.46%. Moreover, compared to earlier investigations, our series of 128 meningiomas comprises a very large and homogeneous group. The role of DNA aneuploidy as an indicator of an aggressive biological tumor behavior is unresolved. Butti et al. (1989) and Spaar et al. (1987) described a significantly higher rate of aneuploid stem lines in malignant meningiomas. Crone et al. (1988) and Ironside et al. (1987) have shown an increase in SPF and PI in DNA aneuploid meningiomas. Cruz-Sanchez et al. (1993) observed that DNA aneuploidy was significantly associated with tumor recurrence. However, Mathiesen et al. (1989) and Akachi et al. (1991) found no increased DNA aneuploidy in malignant meningiomas and Nishizaki et al. (1990) showed that evaluating tumor prognosis based on the presence of aneuploid stem lines yields very uncertain estimates. Concerning proliferation parameters like SPF and PI, many authors agree on their significance in indicating an aggressive biological behavior (Ironside et al., 1987; May et al., 1989; Akachi et al., 1991; Butti et al., 1989; Crone et al., 1988; Cruz-Sanchez et al., 1993; Finn et al., 1994) in malignant meningiomas. In our series, only 24 of 105 (22.9%) benign (WHO 1) meningiomas were DNA aneuploid, while 12 of 17 (64%) atypical tumors and all malignant meningiomas (n 5 5) had additional aneuploid stem lines. SPF was significantly higher ( p , 0.001) in atypical meningiomas compared to benign tumors. In atypical meningiomas (WHO 2), an intermediate position between benign and malignant tumors with respect to DNA ploidy was demonstrated here. Both DNA aneuploidy and SPF were significantly increased in recurrent tumors (n 5 21) compared to the primary tumor group (n 5 114). In the fibroblastic meningioma group (n 5 22), no aneuploid stem line and no tumor recurrence was found. During a post-operative follow-up period ranging from 8 to 54 months (median 25 months), we observed 8 recurring tumors among 6 different patients in our series. In every case, the primary tumor had an aneuploid stem line, whereas none of the 92 primary DNA euploid tumors showed evidence of recurrence. Evaluation of tissue heterogeneity of 35 tumors with an average of 3 different samples/tumor tested revealed no variation of DI for both diploid and aneuploid stem lines. Except in 1 case (Fig. 2a,b), no different aneuploid stem lines were detected in different areas of the same tumor. However, the percentages of the aneuploid tumor fraction (% Antot) varied up to 10% in the benign and up to 22% in the atypical and anaplastic meningiomas. The variability of the 119 G0/G1 phase and SPF was below 5% in benign and below 10% in atypical and anaplastic meningiomas. In earlier flow-cytometric investigations, comparison of cellcycle data in different DNA stem lines within the same tumor was performed rarely due to a lack of qualified computer software (Assietti et al., 1990). In our study, among 43 DNA aneuploid tumors, the diploid and aneuploid cell cycles were evaluated and compared separately with the computer software Multicycle. The SPF, G2/M phase and PI were strongly increased in the DNA aneuploid stem lines compared with the diploid stem lines (Fig. 5). Only 2 previous studies attempted to define hypo- and hyperploid cell lines. While Ironside et al. (1987) found 6 hypoploid and 10 hyperploid cell lines among 16 DNA aneuploid tumors, Cruz-Sanchez et al. (1993) detected 10 hypoploid and 7 hyperploid lines among 17 aneuploid tumors and found a slightly increased tendency of recurrence in the hyperploid tumor group. There is only sparse published evidence concerning the possible relevance of either hypo- or hyperploidy for different biological and therapeutic behaviors of intracranial tumors. Sandberg and Turc-Carel (1987) associated chromosomal hypodiploidy with an aggressive behavior in meningiomas. For cultivated glioma cell lines, Shapiro and Shapiro (1984) demonstrated that hypo- or near-diploid cell lines become more readily resistant to chemotherapy, while hyperploid cell lines are more sensitive. Similar results are known for gliomas (Kobayashi and Hoshino, 1985), neuroblastomas (Look et al., 1984), lymphoblastic leukemia (Williams et al., 1982) and bladder carcinomas (Quirke, 1986). In contrast to our results, most cytogenetic studies analyzing chromosomal patterns of intracranial tumors only occasionally provide evidence for additional hyperploid stem lines (Heim and Mitelman, 1987; Katsuyama et al., 1986; Maltby et al., 1988; Westphal et al., 1989, Zang, 1981). Likewise, the hypothesis of Poulsgard et al. (1989), that the general tendency of clonal evolution in meningiomas goes toward hypodiploidy and that this appears to be correlated with a more aggressive behavior of the tumor (Sandberg and Turc-Carel, 1987), was not confirmed in our study. A possible explanation for these discrepant results might be changes of the ploidy status of the tumor in cell culture (Kawamoto et al., 1989; Coons and Johnson, 1993). Shapiro and Shapiro (1984) described an increased instability for hyperploid glioma cell lines compared to near- or hypodiploid cell lines. It is also true for meningiomas that flow-cytometric data from cell culture tissue and fresh tissue are not strictly comparable. This issue is currently under investigation in our laboratory. The biological behavior of hemangiopericytomas is difficult to predict (Enzinger and Weiss, 1988). Although histomorphological features have been used to differentiate between benign and malignant hemangiopericytomas, it is generally assumed that many of these tumors have an uncertain malignant potential (Finn et al., 1994; Enzinger and Weiss, 1988). Finn et al. (1994) found no evidence for a DNA aneuploid stem line among 22 hemangiopericytic tumors, while high SPF and PI indicated an aggressive behavior in cases of metastasis, local recurrence following complete surgical excision or direct invasive disease. Likewise, in our series of 7 hemangiopericytomas, only 1 tumor had a DNA aneuploid stem line, while the SPF was significantly increased ( p , 0.001) compared to benign meningiomas. In agreement with Finn et al. (1994), DNA ploidy does not appear to be a useful indicator of biological behavior in hemangiopericytomas, while cell-cycle-proliferative parameters such as SPF and PI should be studied in larger series of hemangiopericytic tumors. In conclusion, DNA ploidy and SPF determined by highresolution DNA flow cytometry are useful indicators of different biological behaviors in meningiomas. In tumors with diploid and aneuploid DNA stem lines, the aneuploid line exhibited a significantly higher proliferative activity (SPF, PI). 120 ZELLNER ET AL. REFERENCES AHYAI, A. and SPAAR, F.W., DNA and prognosis of meningiomas: a comparative cytological and fluorescence cytophotometrical study of 71 tumors. Acta Neurochir., 87, 119–141 (1987). AKACHI, K., MATSUMOTO, M., YASUE, M., NAKAMURA, N., KAMADA, M. and OHNO, T., DNA analysis of meningiomas using paraffin-embedded surgical specimens in connection with clinical recurrence. No. Shinkei. Geka., 19, 1129–1134 (1991). APPLEY, A.J., FITZGIBBONS, P.L., CHANDRASOMA, P.T., HINTON, D.R. and APUZZO, M.L., Multiparameter flow cytometric analysis of neoplasms of the central nervous system: correlation of nuclear antigen p105 and DNA content with clinical behaviour. Neurosurgery, 20, 688–694 (1990). ASSIETTI, R., BUTTI, G., MAGRASSI, L., DANOVA, M., RICCARDI, A. and GAETANI, P., Cell-kinetic characteristics of human brain tumors. Oncology, 47, 344–351 (1990). BUTTI, G., GAETANI, P., DANOVA, M., ASSIETTI, R., GIRINO, M. and RICCARDI, A., Cell kinetic studies of human intracranial tumors. J. neurosurg. Sci., 33, 47–53 (1989). COON, J.S., LANDAY, A.L. and WEINSTEIN, R.S., Advances in flow cytometry for diagnostic pathology. Lab. Invest., 57, 453 (1987). COONS, S.W. and JOHNSON, P.C., Regional heterogeneity in the DNA content of human gliomas. Cancer, 72, 3052–3060 (1993). COONS, S.W., JOHNSON, P.C. and PEARL, D.K., Prognostic significance of flow cytometry DNA analysis of human oligodendrogliomas. Neurosurgery, 34, 680–687 (1994). CRONE, K.R., CHALLA, V.R., KUTE, T.E., MOODY, D.M. and KELLY, D.L., JR., Relationship between flow cytometric features and clinical behaviour of meningiomas. Neurosurgery, 23, 720–724 (1988). CRUZ-SANCHEZ, F. F., MIQUEL, R., ROSSI, M.L., FIGOLS, J., PALACIN, A. and CORDESA, A., Clinico-pathological correlations in meningiomas: a DNA and immunohistochemical study. Histol. Histopathol., 8, 1–8 (1993). DEAN, P. and JETT, J., Mathematical analysis of DNA distributions derived from flow microfluorimetry. Cytometry, 1, 71–77 (1980). DRESSLER, L.G., Controls, standards and histogram interpretation in DNA flow cytometry. Methods Cell Biol., 33, 157–171 (1990). ENZINGER, F.M. and WEISS, S.W., Hemangiopericytoma. In: F.M. Enzinger and S.W. Weiss (eds.), Soft tissue tumors, 2nd ed., pp. 596–613, C. Mosby, St. Louis (1988). FINN, W.G., GOOLSBAY, C.L. and RAO, S., DNA flow cytometric analysis of hemangiopericytoma. Amer. J. clin. Pathol., 101, 181–185 (1994). GIARETTI, W. and SANTI, L., Tumor progression by DNA flow cytometry in human colorectal cancer. Int. J. Cancer, 45, 597–603 (1990). HEIM, S. and MITELMAN, F., Cancer cytogenetics, pp. 243–245, A.R. Liss, New York (1987). HIDDEMANN, W., SCHUMANN, J. and ANDREEF, M., Convention on nomenclature for DNA cytometry. Cancer Genet. Cytogenet., 13, 181–183 (1984). IRONSIDE, J.W., BATTERSBY, R.D.E., LAWRY, J., LOOMES, R.S., DAY, C.A. and TIMPERLEY, W.R., DNA in meningioma tissues and explant cell cultures: a flow cytometric study with clinicopathological correlates. J. Neurosurg., 66, 588–594. (1987). KATSUYAMA, J., PAPENHAUSEN, P.R., HERZ, F., GAZIVODA, P., HIRANO, A. and KOSS, L.G., Chromosome abnormalities in meningiomas. Cancer Genet. Cytogenet., 22, 63–68 (1986). KAWAMOTO, K., NUMA, Y., FUJIWARA, H., OHUCHI, M. and MATSUMURA, H., Flow cytometric study on cell kinetics of brain tumors and their cultured cells. Acta Neurochir., 97, 150–157 (1989). KLEIHUES, R., BURGER, P.C. and SCHEITHAUER, B.W., Histological typing of tumors of the central nervous system, Springer-Verlag, Berlin (1991). KOBAYASHI, S. and HOSHINO, T., Clonogenicity and BCNU response of subpopulations of human glioma sorted according to DNA content. No. To. Shinkei, 37, 707–713 (1985). LOOK, A.T., HAYES, F.A., NITSCHKE, R., MCWILLIAMS, N.B. and GREEN, A.A., Cellular DNA content as a predictor of response to chemotherapy in infants with unresectable neuroblastoma. N. Engl. J. Med., 311, 231 (1984). MALTBY, E.L., IRONSIDE, J.W. and BATTERSBY, R.D.E., Cytogenetic studies in 50 meningiomas. Cancer Genet. Cytogenet., 31, 199–210 (1988). MATHIESEN, T., VON HOLST, H., ASKENSTEN, U. and COLLINS, P.V., DNA determination in the clinical management of patients with meningioma or hemangioblastoma. Brit. J. Neurosurg., 3, 575–582 (1989). MAY, P.L., BROOME, J.C., LAWRY, J., BUXTON, R.A. and BATTERSBY, R.D., The prediction of recurrence in meningiomas. A flow cytometric study of paraffin embedded archival material. J. Neurosurg., 71, 347–351 (1989). MCCARTHY, R.C. and FETTERHOFF, T.J., Issues for quality assurance in clinical flow cytometry. Arch. Pathol. Lab. Med., 113, 658–666 (1989). MERKEL, D.E. and MCGUIRE, W.L., Ploidy, proliferative activity and prognosis DNA flow cytometry of solid tumors. Cancer, 65, 1294–1205 (1990). NAGASHIMA, T., HOSHINO, T., CHO, K.G., SENEGOR, M., WALDMAN, F. and NOMURA, K., Comparison of bromodeoxyuridine labeling indices obtained from tissue sections and flow cytometry of brain tumors. J. Neurosurg., 86, 388–392 (1988). NISHIZAKI, T., OHSHITA, N. and NAGATSUGU, Y., Clinical evaluation of DNA index in human brain tumors. J. Neurooncol., 17, 9–13 (1993). NISHIZAKI, T., ORITA, T., KAJIWARA, K., IKEDA, N., OHSHITA, N., NAKYAMA, H., FURUTAMI, Y., AKIMURA, T. and KAMIRYO, T., Correlation of in vitro bromodeoxyuridine labeling index and DNA aneuploidy with survival or recurrence in brain-tumor patients. J. Neurosurg., 73, 396–400 (1990). OTTO, F., DAPI-staining of fixed cells for high-resolution flow cytometry of nuclear DNA. Methods Cell Biol., 33, 105–110 (1990). POULSGARD, L., RONNE, M. and SCHRODER, H.D., Cytogenetic studies of 19 meningiomas and their clinical significance. Anticancer Res., 9, 109–112 (1989). QUIRKE, P., Flow cytometry: methodology and applications in pathology. Arch. Pathol. Lab. Med., 113, 591–597 (1986). RABINOVITCH, P.S., Handbook ‘‘Multicycle Vers. 2.5,’’ Phoenix Flow Systems, Seattle. RABSON, A.R., Flow cytometry in the diagnosis of brain tumors. Neurosurg. Clin. N. Amer. 5, 135–146 (1994). SANDBERG, A.A. and TURC-CAREL, C., The cytogenetics of solid tumors. Relations to diagnosis, classification and pathology. Cancer, 59, 387–395 (1987). SECKINGER, D., SUGARBAKER, E. and FRANKFURT, O., DNA content in human cancer. Arch. Pathol. Lab. Med., 113, 619–626 (1989). SHAPIRO, H.M., Flow cytometry of DNA content and other indicators of proliferative activity. Arch. Pathol. Lab. Med., 113, 591–597 (1989). SHAPIRO, J.R. and SHAPIRO, W.R., Clonal tumor cell heterogeneity. Prog. exp. Tumor Res., 27, 49–66 (1984). SPAAR, F.W., AHYAI, A. and BLECH, M., DNA-fluorescence cytometry and prognosis (grading) of meningioma—a study of 104 surgically removed tumors. Neurosurg. Rev., 10, 35–39 (1987). WESTPHAL, M., HD´ ANSEL, M., KUNZMANN, R., HOLZEL, F. and HERRMANN, H.D., Spectrum of karyotypic aberrations in cultured human meningiomas. Cytogenet. Cell Genet., 52, 45–49 (1989). WILLIAMS, D.L., TSIATIS, A., BRODEUR, G.M., LOOK, A.T., MELVIN, S.L., BOWMANN, W.P., KALWINSKY, D.K., RIVERA, G. and DAHL, G.V., Prognostic importance of chromosome number in 136 untreated children with acute lymphoblastic leukemia. Blood, 60, 864 (1982). ZANG, K.D., Cytological and cytogenetical studies on human meningioma. Cancer Genet. Cytogenet., 6, 249–274 (1981).