360

Loss of Material from Chromosome Arm 1p during Malignant Progression of Meningioma Revealed by Fluorescent In Situ Hybridization Shinsuke Ishino, M.D.1,2 Naoya Hashimoto, M.D.1,2 Shinji Fushiki, M.D.3 Kousei Date, M.D.1 Toshiki Mori, M.D.1,2 Masahito Fujimoto, M.D.4 Yoshio Nakagawa, M.D.2 Satoshi Ueda, M.D.2 Tatsuo Abe, M.D.1 Johji Inazawa, M.D.1 1

Department of Hygiene, Kyoto Prefectural University of Medicine, Kyoto, Japan.

2

Department of Neurosurgery, Kyoto Prefectural University of Medicine, Kyoto, Japan.

3

Department of Dynamic Pathology, Research Institute for Neurological Diseases and Geriatrics, Kyoto Prefectural University of Medicine, Kyoto, Japan.

4

Department of Neurosurgery, Saiseikai Shigaken Hospital, Shiga, Japan.

BACKGROUND. Atypical and anaplastic meningiomas tend to recur and to invade adjacent brain, bone, and skin. They also can metastasize to extracranial organs such as the lung, liver, or bone, causing death. Recent reports have indicated that allelic deletion of chromosome 1p is associated with malignant progression of meningiomas. METHODS. Cytogenetic analysis of 37 meningiomas was performed using doubletarget fluorescent in situ hybridization (FISH) and focusing on chromosome arm 1p. The meningioma series included 17 benign meningiomas, 11 atypical meningiomas, and 9 anaplastic meningiomas. FISH was performed with pericentromeric (1q12) and subtelomeric (1p36) DNA probes to cell nuclei prepared from surgically extirpated tumor samples. RESULTS. A high incidence of deletion of at least part of 1p was observed in 60.0% of atypical and 85.7% of anaplastic meningiomas. Furthermore, statistically significant differences were found with respect to these data between benign versus atypical/anaplastic meningiomas. In four cases both primary and recurrent tumors from the same patient also were investigated for allelic status. CONCLUSIONS. The results of the current study support the existence of tumor suppressor gene(s) on 1p associated with malignant progression of meningioma, and suggest that detection of the allelic status of chromosome 1p by FISH may assist physicians in predicting the clinical prognosis of patients affected by this type of brain tumor. Cancer 1998;83:360 – 6. © 1998 American Cancer Society.

KEYWORDS: meningioma, malignant progression, double-target fluorescent in situ hybridization, chromosome arm 1p. Supported by a Grant-in-Aid from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan. The authors thank Dr. Daisuke Ichikawa for his helpful advice. Current address for Dr. Inazawa: Human Genome Center, Institute of Medical Science, University of Tokyo, Japan. Address for reprints: Johji Inazawa, M.D., Laboratory of Genome Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan. Received September 12, 1997; revision received December 22, 1997; accepted December 22, 1997. © 1998 American Cancer Society

M

eningiomas are common intracranial tumors that arise from cells of the meninges and account for 15–25% of all primary intracranial tumors.1 However, because new diagnostic techniques make it possible to identify asymptomatic cases of meningioma, the percentage is beginning to increase. Although the majority of these tumors are histologically benign, some meningiomas show signs of malignant histology such as marked vascularity, loss of organoid structure, mitotic figures, nuclear polymorphism, prominent nucleoli, focal necrosis, or infiltration to the adjacent brain. The World Health Organization (WHO) classifies meningiomas into three histologic grades: benign (Grade 1), atypical (Grade 2), and anaplastic (Grade 3) in accordance with clinical prognosis.2 Atypical and anaplastic meningiomas tend to recur, and to invade adjacent brain, bone, and skin. They also can metastasize to extracranial organs such as the lung, liver, or bone, resulting in death.3–5 Allelic losses of chromosome 1 frequently are documented in tu-

1p Deletions in Malignant Meningiomas/Ishino et al.

mors of the central nervous system.6 –9 The existence of certain tumor suppressor gene(s) on this chromosome has been suggested by studies involving neuroblastomas6 and medulloblastomas,7 and recent reports have indicated that allelic deletion of chromosome 1p is associated with malignant progression of meningiomas.10,11 We recently reported frequent deletions of chromosome arm 1p in a series of oligodendroglial tumors using double-target fluorescent in situ hybridization (FISH).8 That research revealed a strikingly high incidence of deletions in the 1p36 region in pure oligodendroglial tumors, and supported the feasibility of FISH for detecting allelic deletions in chromosomes. In the current study we examined meningiomas for losses of chromosome arm 1p, using double-target FISH. Atypical (Grade 2) and anaplastic (Grade 3) meningiomas displayed much higher frequencies of 1p36 deletions than did benign (Grade 1) tumors. Moreover, in cases in which we could follow recurrent tumors that showed malignant changes, we found that the allelic loss of chromosome arm 1p in meningiomas was related closely to the malignant progression of tumors that initially had been diagnosed as benign by histologic examination.

MATERIALS AND METHODS Tumor Samples A total of 37 tumor samples was obtained from 31 patients who had undergone surgery at the hospital of Kyoto Prefectural University of Medicine (KPUM) or other hospitals between 1979 and 1996 (Table 1). Thirty-three of these samples were paraffin embedded and 4 were fresh material. Because of the rarity of the malignant phenotype, we located archival samples through older records available at KPUM. Benign meningiomas were selected from recent patient records. All samples were classified according to the WHO classification of tumors of the central nervous system.2 Histopathologic evaluations were performed independently by two neuropathologists (S.F. and Y.N.) who confirmed that all the specimens were comprised of at least 80% tumor cells. Four patients had undergone surgery for meningioma at least twice; we investigated all recurrent tumors to compare their histologic changes with our genetic analysis (Table 2). None of the patients had received any preoperative irradiation or chemotherapy except for Patient no. 208 (Table 1), who had undergone irradiation for maxillary sinus sarcoma 27 years previously.

361

Tumor Cell Processing To obtain a suspension of single cells from paraffin embedded tissues, 100 mm sections were cut, dewaxed in xylene for 3 hours, immersed in ethanol for 1 hour, and dehydrated through ethanol series. After incubation at 37 °C overnight in 1X phosphate-buffered saline (PBS), the tissues were minced with scissors in 1X PBS and incubated at 37 °C for 3– 4 hours in a solution containing 200 mg/mL proteinase K. The tissues were disaggregated mechanically with a homogenizer (Iuchi-Shoeido, Tokyo, Japan). The homogenized solution was filtered through a 100 mm nylon mesh, and the cell suspensions were smeared on a slide coated with poly-L-lysine using a centrifugal smear machine (Autosmear, Sakura Seiki Co, Tokyo, Japan). With regard to the fresh materials, surgically extirpated tumor samples were soaked immediately in 0.9% saline, preserved at 4 °C and prepared within 6 hours. A suspension of single cells was obtained by aspirating the tumor with an 21-gauge needle. The cells then were soaked in 0.075M KC1 solution for 15 minutes to tear cell membranes and expose naked nuclei. After centrifugation at 3000 revolutions per minute for 5 minutes, the upper layer was exchanged with methanol/acetic acid (3:1) solution and dripped over glass slides under steam.

FISH For detection of deletions of chromosome arm 1p, we used the repetitive DNA probe pUC1.77,12 which is specific for the pericentromeric region (1q12), and the cosmid probe CI1-5335,13 which is specific for the subtelomeric region (1p36). pUC1.77 was labeled with biotin (bio)-16-dUTP (Boehringer-Mannheim Biochemicals, Indianapolis, IN) and CI1-5335 was labeled with digoxigenin (dig)-11-dUTP (Boehringer Mannheim) by nick translation. Bio- and dig-labeled probe solutions were mixed in a ratio of 7:2 (volume/volume) (pUC1.77 and CI1-5335) and 1.0 mL of Cot-1 DNA (5 mg/mL; GIBCO BRL, Gaithersburg, MD) was added to 9 mL of the mixed probe solution. Doubletarget FISH then was performed as previously described.8,14

Scoring of Interphase Nuclei Under a Nikon FXA epifluorescence microscope (Nikon, Tokyo, Japan) interphase nuclei were screened through a UV-2A filter (Nikon, Tokyo; exciter, 400–440 nanometers [nm]; barrier, 470 nm); only intact nuclei, no torn or overlapping ones, were evaluated. Signals of both probes were visualized simultaneously through a double or triple band-pass filter (Omega Optical, Brattleboro, VT).

362

CANCER July 15, 1998 / Volume 83 / Number 2

TABLE 1 Summary of FISH Results in 34 Meningioma Cases Patient no.

Age (yrs)

Gender

Pathologya

Recur.

FISH CI1-5335/pUC 1.77 (%)

101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 201 202 203 204 205 206 207 208 209 210 301 302 303 304 305 306 307

68 64 39 63 42 54 55 54 37 51 60 53 59 59 58 81 64 71 40 54 59 75 39 70 55 54 82 57 57 39 58 64 41 54

F F M F F F F M M M F F F F F F M F M F M M M F F F F M M M F F M F

Meningothelial, Gr1 Meningothelial, Gr1 Angiomatous, Gr1 Meningothelial, Gr1 Fibroblastic, Gr1 Meningothelial, Gr1 Meningothelial, Gr1 Meningothelial, Gr1 Meningothelial, Gr1 Meningothelial, Gr1 Fibroblastic, Gr1 Psammomatous, Gr1 Transitional, Gr1 Meningothelial, Gr1 Meningothelial, Gr1 Fibroblastic, Gr1 Meningothelial, Gr1 Atypical, Gr2 Atypical, Gr2 Atypical, Gr2 Atypical, Gr2 Arypical, Gr2 Atypical, Gr2 Atypical, Gr2 Atypical, Gr2 Atypical, Gr2 Atypical, Gr2 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3

N N N N N N N N N N N N N N R1 N N R1 R2 N N N R1 N N N N R1 R1 N R1 N N N

1/2 2/2 2/4 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 2/2 1/2 2/2 1/2 2/2 1/2 2/2 1/1 2/2 2/2 1/2 1/2 1/2 2/2 2/2 1/2 1/2 1/2

(32.2) (82.0) (59.4) (87.7) (76.4) (67.8) (91.5) (86.9) (73.4) (94.4) (94.8) (89.1) (92.0) (80.8) (85.7) (76.1) (92.9) (42.6) (78.4) (52.2) (81.5) (78.2) (42.5) (70.1) (78.0) (73.8) (53.3) (46.7) (54.8) (66.6) (35.2) (42.8) (50.3) (70.5)

2/2 1/2 2/2 1/2 2/3 2/4 1/2 2/3 1/2 1/1 4/4 1/2 1/2 4/4 2/1 1/2 1/2 2/2 1/2 2/2 1/2 2/2 1/3 1/2 1/2 2/3 2/2 1/3 2/2 1/1 2/4 2/2 2/2 1/1

(30.3) (8.7) (11.9) (8.6) (5.5) (12.7) (2.3) (3.4) (13.7) (2.4) (2.6) (5.9) (2.7) (13.5) (5.5) (12.7) (3.3) (26.1) (11.2) (32.7) (4.4) (19.0) (22.3) (11.8) (16.5) (4.2) (38.8) (19.3) (34.8) (13.1) (21.0) (35.2) (35.9) (11.6)

% of deletionsb 2/3 1/1 2/5 2/1 1/2 1/2 2/1 1/2 1/3 1/2 1/1 2/1 2/1 1/2 1/1 4/4 1/1 1/1 2/1 0/2 4/4 2/4 1/4 2/2 1/1 3/3 2/4 2/2 1/1 1/2 2/3 2/4 2/4 2/2

(9.2) (2.7) (5.8) (1.0) (3.6) (8.5) (1.7) (1.7) (3.6) (1.6) (1.3) (4.0) (2.7) (1.9) (3.3) (5.6) (1.6) (9.6) (4.3) (8.0) (4.4) (1.4) (14.9) (11.8) (2.2) (4.2) (3.3) (14.8) (9.5) (6.1) (19.3) (8.3) (9.2) (8.9)

61.8 15.3 84.5 9.6 14.5 28.8 2.8 9.1 17.3 4.8 2.6 6.9 2.7 1.9 8.8 18.3 5.5 71.3 17.2 62.8 7.4 81.0 56.8 84.6 20.9 13.7 59.8 84.4 65.1 23.7 57.1 58.6 63.4 89.0

FISH: fluorescent in situ hybridization; recur.: recurrence; F: female; M: male; Gr1: World Health Organization Grade 1; N: not a recurrence (primary tumor); R1: first recurrence; Gr2: World Health Organization Grade 2; R2: second recurrence; Gr3: World Health Organization Grade 3. a Histopathologic diagnosis. b % of deletions shows summed percentage. Includes all counted nuclei in any population considered to have deletions of 1p36; see also Hashimoto et al.8

Hybridization signals were counted in 200 interphase nuclei, and the numbers of pericentromeric signals and subtelomeric signals were recorded for each nucleus. The percentage of deletion was defined as the fraction of all nuclei scored having more pericentromeric signals than 1p36 signals and included all nuclei having only one pericentromeric signal and one or no 1p36 specific signals.8

Statistical Analysis of FISH Results To compare the FISH results with histological grading, the Mann–Whitney U test was performed on the FISH results between each group of WHO grades. In this analysis we included the 17 Grade 1 meningiomas

(Nos. 101–117), 10 Grade 2 meningiomas (Nos. 201– 210), and 7 Grade 3 meningiomas (Nos. 301–307) (Table 1). The recurrent tumor samples without histologic upgrading (Table 2) were excluded.

RESULTS Although we used samples prepared in the 1970s or 1980s, hybridization signals were discernible (Fig. 1). When judging whether a brain tumor sample had deletion of chromosome arm 1p using double-target FISH, we set a cutoff value of 35.1% that resulted from control hybridizations previously performed under the same conditions in the same laboratory.8 Control hybridizations on normal brain tissues had revealed

1p Deletions in Malignant Meningiomas/Ishino et al.

363

TABLE 2 Recurrent Meningiomas in the Same Patients

Pathology

% of deletions

Date of recur.

Location

Simpson surgical grade

F F F M M F F

Atypical, Gr2 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3 Anaplastic, Gr3 Meningothelial, Gr1 Atypical, Gr2

62.8 57.1 65.2 63.4 79.9 61.8 71.3

1st op 4 yrs 1 mo 6 yrs 1st op 7 mos 1st op 2 yrs, 6 mos

Rt. convexity Rt. falx & convexity Rt. falx Lt. parasagittal Rt. parasagittal Lt. falcotentorial Lt. falcotentorial

I II I IV II IV IV

M M M

Meningothelial, Gr1 Atypical, Gr2 Atypical, Gr2

17.3 56.8 67.2

1st op 2 yrs, 1 mo 3 yrs, 9 mos

Lt. falx Rt. parasagittal Rt. parasagittal

II III III

Patient no.

Age (yrs)

Gender

203 304

54 58 60 41 42 68 71 37 39 40

306 101 201 109 206

Particular findings Skull invasion Brain infiltration Postoperative irradiation SSS occlusion, skin invasion SSS occlusion Transverse sinus occlusion Straight, transverse, SSS occlusion SSS occlusion SSS occlusion

Outcome

Alive at 10 yrs, 10 mos Alive at 6 mos Died 5 yrs, 1 mo

Alive at 3 yrs, 10 mos

Recur: recurrence; F: female; M: male; Gr2: World Health Organization Grade 2; Gr3: World Health Organization Grade 3; Gr1: World Health Organization Grade 1; Op: operation; Rt: right; Lt: left; SSS: superior sagittal sinus.

FIGURE 1. Enhanced computed tomography. (a) Left falcotentorial meningioma (Case 1). (b) Left parasagittal meningioma (Case 2). (c) Right parasagittal meningioma (Case 2).

that the average number of nuclei with deletions according to our criteria was 27% (standard deviation [SD] 5 2.7%) in populations of normal cells. On the basis of a mean of 13 SD, we could judge as a significant population any preparation in which . 35.1% of signal patterns indicated deletion. Table 1 summarizes the results, including the percentages of nuclei with 1p36 deletions in the cell populations from each tumor that showed the three most common signal patterns. We detected 1p36 deletions in 6 samples of 7 anaplastic meningiomas (Grade 3) (85.7%); 6 of 10 atypical meningiomas (Grade 2) (60.0%) had lost 1p36; whereas only 2 of 17 benign meningiomas (Grade 1) (11.8%) were considered to have losses. The percentages of deletions were analyzed for each combination of two of the three histologic groups using the Mann–Whitney U test. A significant difference was noted between benign versus

atypical (P 5 0.0078) and benign versus anaplastic (P 5 0.0017), but the difference between atypical versus anaplastic was not significant (P 5 0.2416). We had access to samples from four patients whose tumors had recurred. The clinical profiles of these four patients, together with the FISH results, are shown in Table 2. Eight of the ten samples from these patients showed a malignant phenotype such as invasion to adjacent tissues and/or metastasis to other organs.

Illustrative Cases Case 1 (tumor samples 101/201) A 68-year-old female with headache and memory disturbance was admitted in December 1986. A left falcotentorial meningioma (Fig. 1a) was removed partially (Simpson’s surgical Grade IV15). Although the pathologic diagnosis was a benign meningioma

364

CANCER July 15, 1998 / Volume 83 / Number 2

FIGURE 2. Histology of meningiomas, showing a progression from World Health Organization Grade 1 to Grade 2. (A) Patient no. 101 (Case 1). Prominent whorl formation is noted without recognizable atypia. (B) Patient no. 201 (Case 1). Cellularity is increased with large nuclei. Note mitotic figure (arrowhead). (C) Patient no. 109. (Case 2). The tumor is comprised of interlacing bundles of long, narrow, spindle cells. (D) Patient no. 206. (Case 2). The tumor cells have large irregular-shaped nuclei, with frequent mitoses (arrowheads) (A–D: magnification 3 120).

(Grade 1) (Fig. 2A), a sample from the primary tumor (No. 101) showed deletions of 1p36 in nearly 62% of the cells; the deletion pattern is shown in Figure 3a. This tumor recurred 2.5 years after the first surgery. The sample from the recurrent tumor (No. 201) was diagnosed pathologically as an atypical meningioma (Grade 2) (Fig. 2B) and the 1p36 deletions had increased to 71%.

Case 2 (tumor samples 109/206) A 37-year-old male with headache, right hemihypesthesia, and dysarthric speech was admitted in April 1992. A large left falx meningioma (Simpson’s surgical Grade II) (Fig. 1b) was removed completely. Histologic examination of the tumor detected a benign meningioma (Grade 1) (Fig. 2C). Follow-up at the usual intervals by computed tomography scan revealed no recurrence until rapid growth of a contralateral (right side)

tumor (Fig. 1c) caused acute left hemiparesis in June 1994. The histologic findings in the tumor removed in the second surgery indicated malignancy (Grade 2) (Fig. 2D). A retrospective FISH study (Fig. 3b) using a paraffin embedded sample obtained 2 years previously (No. 109) revealed deletions of 1p36 in only 17.3% of the cells in the primary tumor. The sample from the recurrent tumor (No. 206) showed the summed percentage of populations with 1p36 deletions to be 56.8% (Fig. 3c). After the third surgery, histologic examination of the right parasagittal tumor still indicated a Grade 2 atypical meningioma, although the 1p36 deletions had increased to 67.2%.

DISCUSSION Conventional cytogenetic analysis of meningiomas often reveals the frequent entire or partial losses of chromosome 22.16 Extensive studies of loss of het-

1p Deletions in Malignant Meningiomas/Ishino et al.

365

FIGURE 3. Results of double-target fluorescent in situ hybridization using probes specific for 1q12 (yellow) and 1p36 (red) to interphase nuclei prepared from meningioma samples. Representative patterns of hybridization signals are seen in (a) Patient no. 101, (b) Patient no. 109, and (c) Patient no. 206 sample. The numbers of signals for 1p36 (red) and 1q12 (green) could be detected unambiguously as (a) 1/2, (b) 2/2, and (c) 1/3. The patterns in panels a and c indicate that the 1p36 band is missing from at least one chromosome 1 in these nuclei, whereas the patterns in panel b show that neither homologue of chromosome 1 has lost 1p36. In the sample from Patient no. 101 (Case 1), despite the diagnosis of World Health Organization Grade 1 meningioma, the majority of cells were believed to show the deletion of this region as indicated in panel a. In the sample from Patient no. 109 (Case 2), which was diagnosed as benign meningioma, 73.4% of cells exhibited the pattern of 2/2, indicating the retention of both alleles as shown in panel b. However, in the recurrent sample from Patient no. 206 (atypical meningioma), 22.3% of cells were of the 1/3 pattern as shown in panel c. Bottom panels of each show DAPI-stained nuclei of panels a, b, and c, respectively.

erozygosity (LOH) also have shown frequent allelic losses on the long arm of this chromosome, pinpointing the tumor suppressor locus in the vicinity of the NF-2 gene.17–19 Although mutations of the NF-2 gene have been observed in up to 59% of sporadic meningiomas,20 it remains controversial whether another tumor suppressor exists distal to NF-2 on 22q. After chromosome 22 anomalies, aberrations of the short arm of chromosome 1 are among the most frequent alterations detected by cytogenetic analysis of meningiomas.16,21 Together with the fact that LOH on chromosome arm 1p is observed in a variety of tumor types,6,8,9,16 these findings prompted us to investigate the allelic status of chromosome arm 1p in meningiomas as well as other brain tumors we observe in daily clinical practice. The FISH technique has provided new insights into interphase cytogenetics,8 and now is applicable to clinical practice22 because it need not be time-consuming and can be applied to various clinical materials including paraffin embedded tumor samples or biopsy samples. An additional advantage of FISH is its consistently informative nature, even in individuals who are homozygous at the locus of interest; this feature results in a high efficiency for detecting allelic loss. We previously confirmed that FISH can detect loss of genetic information as reliably as LOH studies by directly comparing FISH results and LOH investigations.8 Recently at least two groups of researchers inde-

pendently reported that the frequency of LOH on chromosome arm 1p tends to increase with tumor grade in patients with meningiomas.10,11 Bello et al.10 observed LOH on 1p in 12 of 17 Grade 2 and 3 meningiomas (71%) with 5 highly polymorphic markers. In a subsequent report, LOH on 1p was observed in 6 of 15 Grade 2 meningiomas (40%) and 7 of 10 Grade 3 meningiomas (70%).11 Our data show a slightly higher frequency of deletions (60.0% in Grade 2 and 85.7% in Grade 3) but are consistent with their results. These findings as well as cytogenetic data16 strongly indicate the existence of one or more tumor suppressor genes on chromosome arm 1p, which may contribute to progression of Grade 1 meningioma to Grade 2. However, this speculation merely is based on the observation that 1p deletions are found more frequently in histologically malignant meningiomas than in histologically benign meningiomas. It would be important to determine how the benign meningioma progresses to malignancy in individual cases. Therefore we investigated 1p deletions in both primary and recurrent tumors from four patients. In two of the recurrent cases the histologically benign meningioma showed malignant changes in the recurrent tumor. In Case 1, the histologically benign meningioma showed deletions of 1p as early as the first surgery; the recurrent tumor showed malignant histology. In Case 2, the histologically benign meningioma did not show 1p deletions, but when malignant

366

CANCER July 15, 1998 / Volume 83 / Number 2

changes in histology were evident 1p deletions had occurred in the recurrent tumor. The change in FISH results corresponded to the histopathologic upgrading from Grade 1 meningioma to Grade 2. Together with the fact that the recurrent tumors showed histologic malignancy, the results can be interpreted to mean that the intrinsic tumor malignancy was detectable by means of FISH before the pathologic changes occurred. It would be reasonable to assume that genetic changes would precede the morphologic changes. We suggest that the allelic status of chromosome arm 1p detected by FISH may be a useful predictor of malignant progression in individual meningiomas. From this point of view, Patient 103, whose tumor sample showed a high percentage of 1p36 deletions (84.5%) in spite of its pathologically benign appearance, should be followed carefully.

11.

12.

13.

14.

15.

REFERENCES 1.

The committee of brain tumor registry of Japan. General rules for clinical and pathological studies on brain tumors. Tokyo: Kanehara & Company, LTD., 1995:9 –11. 2. Kleihues P, Burger PC, Scheithauer BW. Histological typing of tumors of the central nervous system. Berlin: SpringerVerlag, Inc., 1993:33– 42. 3. Mahmood A, Caccamo DV, Tomecek FJ, Malik GM. Atypical and malignant meningiomas: a clinicopathological review. Neurosurgery 1993;33:955– 63. 4. Ja¨a¨skela¨inen J, Haltia M, Servo A. Atypical and anaplastic meningiomas: radiology, surgery, radiotherapy, and outcome. Surg Neurol 1986;25:233– 42. 5. Maier H, Ofner D, Hittmair A, Kitz HK, Budka H. Classic, atypical, and anaplastic meningioma. J Neurosurg 1992;77: 616 –23. 6. Brodeur GM, Fong CT. Molecular biology and genetics of human neuroblastoma. Cancer Genet Cytogenet 1989;41:153–74. 7. Bigner SH, Mark J, Friedman HS, Biegel JA, Bigner DD. Structural chromosomal abnormalities in human medulloblastoma. Cancer Genet Cytogenet 1988;30:91–101. 8. Hashimoto N, Ichikawa D, Arakawa Y, Date K, Ueda S, Nakagawa Y, et al. Frequent deletions of material from chromosome arm 1p in oligodendroglial tumors revealed by double-target fluorescence in situ hybridization and microsatellite analysis. Genes Chromosom Cancer 1995;14:295–300. 9. Bello MJ, Leone PE, Nebreda P, de Campos JM, Kusak HE, Vaquero J, et al. Allelic status of chromosome 1 in neoplasms of the nervous system. Cancer Genet Cytogenet 1995; 83:160 – 4. 10. Bello MJ, Campos JM, Kusak E, Vaquero J, Sarasa JL, Pestan ˜ a A, et al. Allelic loss at 1p is associated with tumor

16.

17.

18.

19.

20.

21.

22.

progression of meningiomas. Gene Chromosome Cancer 1994;9:296 – 8. Simon M, von Deimling A, Larson JJ, Wellenreuther R, Kaskel P. Allelic losses on chromosomes 14, 10, and 1 in atypical and malignant meningiomas: a genetic model of meningioma progression. Cancer Res 1995;55:4696 –701. Cooke HJ, Hindley J. Cloning of human satellite 3 DNA: different components are on different chromosomes. Nucleic Acids Res 1979;6:3177–97. Ariyama T, Inazawa J, Ezaki T, Nakamura Y, Horii A, Abe T. High-resolution cytogenetic mapping of the short arm of chromosome 1 with newly isolated 411 cosmid makers by fluorescence in situ hybridization: the precise order of 18 markers on 1p36.1 on prophase chromosomes and ‘‘stretched’’ DNAs. Genomics 1995;25:114–23. Inazawa J, Ariyama T, Abe T. Physical ordering of three polymorphic DNA markers spanning the regions containing a tumor suppressor gene of renal cell carcinoma by threecolor fluorescence in situ hybridization. Jpn J Cancer Res 1992;83:1248 –52. Simpson D. The recurrence of intracranial meningiomas after surgical treatment. J Neurol Neurosurg Psychiatry 1957; 20:22–39. Mittelman F, Kaneko Y, Berger R. Report of the committee on chromosome changes in neoplasia. In: Cuticchia AJ, Chipperfield MA, Foster, PA, editors. Human gene mapping 1995: a compendium. Baltimore: Johns Hopkins University Press, 1996:1332–50. Dumanski JP, Rouleau GA, Nordenskjold M, Collins VP. Molecular genetic analysis of chromosome 22 in 81 cases of meningioma. Cancer Res 1990;50:5863–7. Ruttledge MH, Xie YG, Han FY, Peyrard M, Collins VP, Nordenskjold M, et al. Deletions of chromosome 22 in sporadic meningioma. Genes Chromosom Cancer 1994;10: 122–30. Akagi K, Kurahashi H, Arita N, Hayakawa T, Monden M, Mori T, et al. Deletion mapping of the long arm of chromosome 22 in human meningiomas. Int J Cancer 1995;60:178 – 82. Ruttledge MH, Sarrazin J, Rangaratham S, Phelan CM, Twist E, Merel P, et al. Evidence for the complete inactivation of the NF2 gene in the majority of sporadic meningiomas. Nat Genet 1994;6:180 – 4. Jimene´z-Lara AM, Rey JA, Bello MJ, de Campos JM, Vaquero J, Kusak ME, et al. Cytogenetics of human meningiomas: an analysis of 125 cases [abstract]. Cancer Genet Cytogenet 1992;63:174. Ichikawa D, Hashimoto N, Hoshima M, Yamaguchi T, Sawai K, Nakamura Y, et al. Analysis of numerical aberrations in specific chromosomes by fluorescence in situ hybridization (FISH) as a diagnostic tool in breast cancer. Cancer 1996;77: 2064 –9.