Interphase cytogenetics of brain tumors

GENES, CHROMOSOMES & CANCER 3:101-107 (1991)

Interphase Cytogenetics of Brain Tumors Edo P.J. Arnoldus, lnge A. Noordermeer, A.C. Boudewijn Peters, Joan H.C. Voormolen, Gerard T.A.M. Bob, Anton K. Raap, and Mels van der Ploeg

Departments of Neurology (E.P.J.A., I.A.N., A.C.B.P.), Neurosurgery (J.H.C.V.), Neuropathology (G.T.A.M.B.), and Cytochemistry and Cytometry (E.P.J.A., I.A.N.. A.K.R., M.v.d.P.), Leiden University, Leiden, The Netherlands

The development and application of a procedure for interphase cytogenetics on brain tumor material is described. Nuclei isolated from freshly removed brain tumor tissue were investigated for chromosomal aberrations by nonradioactive in situ hybridization with a panel of chromosome-specific probes. The panel consisted of nine satellite DNA probes specific for the centromeric regions of chromosomes I, 6,7, 10, I I, 17, 18, X, and Y. For each probe, the number of hybridization signals per cell was determined in 200 nuclei. It was inferred from the hybridization results that in I I gliomas (seven astrocytomas grade 11-IV, three oligodendrogliomas, and one ependymoma) the numerical aberrations were gains of chromosomes I (once), 7 (twice), 10 (once), I I (twice), and X (twice); losses of chromosomes I (once), 10 (twice), 17 (twice), and Y (once); and complete tetraploidy (once). Among the I8 investigated meningiomas monosomy I8 and trisomy I7 were observed once and twice, respectively. An additional hybridization with a cosmid probe for the BCR gene on 22q I I indicated monosomy 22q in I I meningiomas. These results show the value of interphase cytogenetics for the analysis of solid tumors for which it is relatively difficult to obtain sufficient metaphases of good quality for conventional cytogenetics.

INTRODUCTION

During the last few years a number of reports have been published regarding the cytogenetic fea- tures of brain tumors. Metaphase spreads of gliomas often show double minutes, a gain of chromosome 7, and losses of chromosomes 10 and 22 and sex chromosomes (Bigner et al., 1986, 1988, 1990; Rey et al., 1987a,b; Jenkins e t al., 1989). In the majority of meningiomas, the only aberration observed in metaphase preparations is usually monosomy 22 (Zang and Singer, 1967; Mark, 1973; Zang, 1982; A1 Saadi et al., 1987; Casalone et al., 1987, 1990; Maltby et al., 1988). So far, cytogenetic changes of these solid tumors have been studied mainly by karyotyping. Tissue culture of tumor material is notoriously difficult (Teyssier, 1989): Not only can cultures fail completely but they can also lead to either selective growth or spontaneous transformation of the karyotype. In addition, the yield of good quality metaphases is usually small. Therefore, approaches for the study of cytogenetic changes in solid tumors, that make culturing of the tumor material unnecessary, are urgently needed.

Taking into consideration that chromosomal aberrations may change the normal DNA content of cells, measurement of that content by DNA flow cytometry is one of the possible choices. However, the smallest changes in nuclear DNA content de- tectable are on the order of a few percent (Corne- lisse and Tanke, 1990). A dramatic genetic change, such as the loss of a chromosome 22 (0.86% of the genome; Mendelsohn et al., 1973) in meningio- 0 1991 WILEY-LISS. INC.

mas, will actually fall below the detection thresh- old (Raap et al., 1987). Furthermore, even when an abnormal DNA content would be found, it is impossible to detect the exact structural cytogenetic changes that have occurred.

The in situ hybridization technique with chromosome-specific DNA probes seems to provide a better alternative (Raap et al., 1990). An important advantage of this approach is that it can be applied to interphase nuclei as well as metaphase chromosomes. Interphase cytogenetics (Cremer et al., 1986) not only overcomes the necessity for culturing, but provides the possibility to increase the number of cells to be studied. In this way, the method can be very useful to identify tumor sub- populations with specific chromosome aberrations. In cytogenetic studies of solid tumors, interphase cytogenetics has been shown to be a reliable procedure in the study of bladder carcinomas (Hop- man et al., 1988, 1989), breast tumors (Devilee et al., 1988b), testicular tumors (Walt et al., 1989), and gastric tumors (Van Dekken et al., 1990).

For the interpretation of interphase cytogenetics results, one should realize that information is gen- erated only for those chromosome segments to which the probes hybridize. Three signals in an interphase nucleus after hybridization with a probe specific for the centromeric region may reflect the presence of three complete copies of that chromo-

Received October 1 , 1990; accepted November 18, 1990. Address reprint requests to Edo P.J. Arnoldus, Department of

Cytochemistry and Cytometry, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.

ARNOLDUS ET AL 102

some. However, a centromeric sequence present in, e.g., a marker chromosome or a dicentric chromosome can also cause the third signal. Therefore, one must be careful with the interpretation of the interphase cytogenetics results. A combination of GTG-banding and in situ hybridization, as described by Smit e t al. (1990), can improve the characterization of complex karyotypes, although such an approach has its own limitations.

In this report, the development and application of a reliable procedure for sensitive interphase cytogenetics on brain tumors is described. The im- plications of this technique for the study of karyo- typic changes in brain tumors are discussed.

MATERIALS AND METHODS Tumor Material

Fresh tumor tissue of seven astrocytomas (grade 11-IV, according to WHO classification; Zulch, 1979), three oligodendrogliomas, one papillary ependymoma, and 18 meningiomas were collected in culture medium during neurosurgery. The size of the specimen varied from 0.02 to 1.0 cm3. At the neuropathological examination of this material, special care was given to prevent sampling errors for the in situ hybridization studies.

T h e glioma group included eight males and three females, with a mean age of 43 years. In the meningioma group, there were seven males and 1 1 females, with a mean age of 56 years. The clinico- pathological data of the 29 patients are summarized in Tables 2 and 3.

Preparation of Nuclei Suspensions

The tumor tissue was mechanically disaggre- gated with a Potter-Elvehjem homogenizer (with a

TABLE I , Hybridization Results on Tumor A4*

Observer A Observer B (spotdnucleus) (spotdnucleus)

Chromosome I 2 3 I 2 3

I 6 7

10 I I 17 18 X Y

7 5 I

79 4 8

12 99 99

90 3 3 9 5 2 95 0 4 96 0 28 71 I 30 69 21 0 78 22 0 96 0 7 93 0 92 0 10 88 2 88 0 7 92 I

I 0 9 8 2 0 I 0 99 I 0

* For nine centromere-specific probes, we present the percentages of nuclei with one, two, or three pots as determined by two independent observers (number of nuclei counted = 200).

TABLE 2. Summary of the Chromosomal Aberrations Observed in I I Gliomas by Interphase Cytogenetics Using a

Panel of Nine Centromere-Specific Probes

Histology and Aberration

No. Sex (years) grade (% nuclei) Age

A l M 33 A2 M 42 A3 M 33 A4 M 56 A5 M 62 A6 M 36 A7 M 52 E l F 52

01 F 29 0 2 M 39 0 3 F 43

Astrocytoma II Astrocytoma II Normal Astrocytoma ll-Ill Tetraploid (34) Astrocytoma 111 Astrocytoma 111 - 10 (82) Astrocytoma IV Astrocytoma IV Papi I lary

- I7 ? (28)

- 10 (79), +7 (70)

+/+ +7 (I 2/25) -Y (loo), +X (80) +/+ + I (27/47)

ependymoma 111 +I++ 10 (30/40)

+/++X (16169) + I I (49)

Oligodendroglioma Normal Oligodendroglioma + I I (72) Oligodendroglioma - I (36), - 17 ? (26)

TABLE 3. Summary of the Chromosomal Aberrations Observed in 18 Meningiomas by lnterphase Cytogenetics Using a Panel of Nine Centromere-Specific Probes Plus a

Cosmid Probe for BCR on 22q I I

Age No. Sex (years) Type’ Aberration (% nuclei)

MI M2 M3 M4 M5 M6 M7 M8 M9 MI0 MI I T I T2 T3 T4 F I HI H2

M 30 F 41 F 54

M 60 F 67 F 67

M 68 F 68

M 69 M 76 F 83 F 43

M 43 F 51 F 57 F 41

M 35 F 49

men. men. men. men. men. men. men. men. men. men. men. tran. tran. tran. tran. fibr. hem. hem.

+ I7 (35)

Normal

Normal

Normal

+ I7 (81) Normal

- I8 (59) Normal Normal

-22 (50)

-22 (43) -22 (55)

-22 (39)

-22 (80) -22 (62) -22 (86)

-22 (58)

-22 (68) -22 (46)

-22 (49)

a men., meningothelid; tran.. transitional; fibr.. fibrous hem., hemangio- pericytic.

piston+ylindrical vessel distance of 200 pm). The suspension was fixed in 1% acid-free formaldehyde (Merck No. 3999, Darmstadt, Germany), 0.9% NaCl, 10 mM MgSO,. After centrifuging for 10 min at 400g, the pellet was dissolved in lysis buffer (0.15 M NH,Cl, 10 mM KHCO,, 10 mM EDTA, pH 7.4) to lyse the remaining erythrocytes. The

INTERPHASE CYTOGENETICS OF BRAIN TUMORS 103

cells were centrifuged again, after which the pellet was resuspended in 0.5% (w/v) pepsin (Serva No. 31855, Heidelberg, Germany) in 0.9% NaCl, 10 mM MgSO, (pH adjusted to 2.0 with 1 M HCl). The resulting suspension was filtered through a nylon filter of 80 pm to remove remaining clumps of cells. Finally, the suspension of nuclei was fixed several times in cold 70% ethanol and stored at - 20°C. Shortly before the in situ hybridization ex- periment, the nuclei were centrifuged onto glass slides with a bucket centrifuge as previously described (Arnoldus et al., 1989).

Probes and Labeling

In this study nine probes specific for the (per;)- centromeric regions of chromosomes 1 (pUC1.77; Cooke and Hindley, 1979), 6 (p308; Jabs et al., 1984), 7 (p7tl; Waye et al., 1987b), 10 (DlOZl; Devilee et al., 1988a), 11 (pLCllA, Waye et al., 1987a), 17 (p17H8; Waye and Willard 1986), 18 (L1.84; Devilee e t al., 1986), X (pBamX5; Willard et al., 1983), and Y (DYZ3; Cooke et al., 1982) were used. These probes, which recognize highly repetitive satellite DNA sequences clustered around the centromere, were selected on basis of their relevance for brain tumors and their good ap- plicability.

For the detection of chromosome 22 aberrations in meningiomas, the cosmid probe Cos-BCR-19-1 was used, because we found that the probe for the chromosome 22 centromeric region (D2223; Metz- dorf et al., 1988) is not specific enough for this purpose. This cosmid probe which contains a 34 kb fragment of the BCR gene on 22qll (Hermans et al., 1989) was combined with the cosmid clone Cos-ABL-18, which contains a 40 kb fragment of the human ABL gene in the q34 band of chromosome 9 (Heisterkamp et al., 1983), as a control for hybridization efficiency in a double hybridization.

T h e probes were labeled by standard nick- translation with either biotin-1 1-dUTP (Sigma, St. Louis, MO) or digoxigenin-1 1-dUTP (Boehringer, Mannheim, Germany). The labeled DNA was ethanol-precipitated with salmon sperm DNA and yeast RNA as a carrier. The precipitate was dissolved in a mixture of 60% deionized formamide (v/v), 2x SSC ( l x SSC = 0.15 M NaCl, 0.015 M sodium citrate) to a probe concentration of 1 ng/pl of pUC1.77 and 2.5 ng/pl of the other satellite DNA probes.

In the double cosmid in situ hybridization experiments the hybridization mixtures contained 20 ng of each labeled cosmid probe plus a 500-fold excess of human genomic DNA as a competitor,

dissolved in 10 pl of 50% formamide, 10% dextran sulphate, and Z X SSC.

In Situ Hybridization (ISH)

Before hybridization, cells were treated with pepsin for 10 min at 37°C (0.01% pepsin [Serva 318551 in 0.01 M HCl), and postfixed for 10 min in 1% acid-free formaldehyde in PBS. For the satellite DNA probes 5 p1 of the hybridization mixtures were added under a coverslip of 18 X 18 mmz, and probe and target DNA were denatured simulta- neously for 10 min at 80°C. For the double cosmid hybridization the slides and the hybridization mixture were denatured separately. Then 10 pl of the hybridization mixture was applied and sealed under a 18 X 18 mm2 coverslip with a rubber solution. In situ hybridization was allowed to take place overnight at 37°C in a moist chamber with 60% formamide and 2X SSC.

lrnrnunocytochernical Detection

After hybridization, the slides that were hybrid- ized with the satellite DNA probes were washed in 60% formamide, 2X SSC at 37°C. T h e hybrids were detected by three consecutive immunocyto- chemical steps of 30 min at 37°C in 4X SSC, 5% nonfat dry milk. The first and third layer contained 5 pg avidin-D-FITC/ml (Vector Laboratories, Bur- lingame, CA). T h e second layer consisted of biotinylated goat antiavidin (Vector), 1: 100. Finally, the cells were dehydrated and embedded in a glycerol mixture with propidium iodide for counterstaining and the antifade reagent DABCO (Sigma).

For the double cosmid hybridization, the slides were washed in 50% formamide, 2x SSC at 45"C, followed by washes in 0.1 X SSC at 60°C. T h e im- munocytochemical detection consisted of four steps of 30 min at 37°C. T h e first layer contained 5 p.g avidin-D-FITC/ml in 4X SSC, 5% nonfat dry milk. The second layer consisted of biotinylated goat antiavidin, 1: 100, plus mouse antidigoxigenin (a generous gift from Boehringer, Tutzing, Ger- many), 1:1,000. T h e third layer was avidin-FITC plus rabbit antimouse-TRITC, 1: 1,000; the fourth layer was goat antirabbit-TRITC, 1: 1,000. T h e last three layers were diluted in 0.1 M Tris HCl (pH 7.5), 0.15 M NaCl, and 0.5% Boehringer blocking reagent. Finally, the cells were dehydrated and embedded in a glycerol mixture with DAPI for counterstaining and DABCO.

Evaluation and Interpretation of In Situ Hybridization Signals

The evaluation of the in situ hybridization sig-

104 ARNOLDUS ET AL.

nals was performed using similar criteria as previously described (Hopman et al., 1988; Arnoldus et al., 1989). In contrast to the method of Hopman et al., nuclei without any signals were also counted. For all probes used, the number of signals per nucleus after hybridization was counted in 200 nuclei. As can be seen in Table 1, the interobserver vari- ability was low.

For the sake of simplicity, we have assumed that the actual copy number of a certain chromosome was identical to the number of hybridization signals obtained with its specific centromeric probe. Thus two clear spots per nucleus represent the normal two copies of the chromosome. Nuclei with only one signal may reflect a monosomy for that specific chromosome. T o rule out the possibility that the in situ hybridization partly failed in these nuclei, double hybridizations with another probe were performed in those cases (Fig. lC,D). However, it is not uncommon to have 1040% nuclei with only one signal in normal tissue (Devilee et al., 1988b; Nederlof et al., 1989). In this study, therefore, one signal per nucleus was only interpreted as an indi- cation of a monosomy when it was present in more than 20% of the nuclei. More than two signals usually give less interpretation problems and in this case a value of more than 7.5% of nuclei with three signals was used to indicate a subpopulation with a trisomy.

RESULTS

Results of the experiments with the panel of satellite DNA probes on glioma interphase nuclei are summarized in Table 2. Only two of the ten investigated gliomas (one astrocytoma grade I1 and one oligodendroglioma) did not show any abnormalities with this panel of probes. The observed aberrations in the seven astrocytomas 11-IV were: - 10 (twice), - 17 (once) and - Y (once); +/+ + 7 (twice), and + X (once) and complete tetraploidy (once). T h e papillary ependymoma displayed: +/+ + 1, +/+ + 10, + 11, and +/+ +X. The observed aberrations in the three oligodendrogliomas were: - 1 (once), - 17 (once), and + 11 (once). For chromosome 17, control experiments on normal brain tissue revealed that the centromeres are often paired in astrocytes, resulting in only one, slightly larger, spot per nucleus (Arnoldus et al., 1990a). This complicates the interpretation of the apparent loss of a chromosome 17 (indicated by a question mark in Table 2) observed in 28% and 26% of the nuclei of tumors A1 and 0 3 , respectively.

The results of in situ hybridization on meningi-

omas are summarized in Table 3. With the panel of probes specific for the centromeric regions only three changes were found in 18 meningiomas: - 18 (once) and +17 (twice). T h e hybridization with the cosmid probes for BCR (22qll) and ABL (9q34) revealed more aberrations. Eleven of 18 meningiomas showed one signal for 22qll and two signals for 9q34 in 39-86% of the nuclei, implicating a loss of 22q (Fig. lE,F). The majority of the remaining nuclei showed two signals for both probes.

DISCUSSION

The first important step in a protocol for interphase cytogenetics is the preparation of a suspension of intact nuclei from freshly collected (brain) tumor tissue that can be stored until use. Since the clinical samples are often small, it is important to obtain high nuclear yields. Mechanical disaggrega- tion with a Potter-Elvehjem homogenizer gave a much higher yield than simple cutting with razor blades; even the smallest clinical specimen (0.02 cm3) thus rendered sufficient nuclei for 16 hybridizations. Pepsin was then used to disaggregate the remaining clumps of cells into single nuclei. Fi- nally, the nuclei were fixed with 70% ethanol, after which the suspensions could be kept at - 20°C for months without loss of in situ hybridization efficiency.

Our hybridization results showed that chromosomal abnormalities frequently occur in gliomas. Often, however, only a fraction of the nuclei showed a chromosomal aberration. Although special care was taken by the neurosurgeons to make sure that the samples used for interphase cytogenetics consisted of tumor tissue only, it cannot be ruled out completely that they also contained some normal cells. This implicates that the percentage of nuclei with a certain aberration might be underes- timated. For instance, 79% of the nuclei of tumor A4 displayed one signal for the centromeric region of chromosome 10, whereas the remaining 21% showed the normal number of two signals. This might reflect the presence of different subpopula- tions of the tumor, but it could also be caused by normal cells in the neurosurgical sample.

Interestingly, only two of 11 gliomas (18%) showed a trisomy for chromosome 7. This is less frequent than the 50% that Rey et al. (1987b) re- ported in cultured material. Recently Heim et al. (1989) published findings of chromosomal aberrations in normal brain tissue after short-term cultures. The changes observed were - Y (in all eight males), - X (one of three females), and + 7 in three patients. In our analysis of brain tumors, we


Figure I. lnterphase cytogenetics results. A Nuclei of an oligodendroglioma show two hybridization signals (FITC. yellow as a result of the filter combination) for chromosome I centromeres; red propidium iodide counterstaining. B: Nuclei of meningioma M I I display three hybridization signals (FITC) for chromosome I7 centromeres; red propidium iodide counterstaining. C: Nuclei of attrocytoma A4 show after double hybridization only one green signal for chromosome 10 centromere (FITC) but three red signals for chromosome 7 centromeres (TRITC); blue DAPl counterstaining, triple-exposure microphotoglaph. D: Nuclei of meningioma FI display after double hybridization two red signals for chromosome 7 centromeres (TRITC) but only one green signal for chromosome I8 centromere (FITC); blue DAPl counterstaining. triple-exposure microphotograph. E Hybridization with the cosmid probes for A61 and BCR on interphase nuclei of meningioma M7. Left: blue DAPl counterstaining; middle: two red ABL signals (TRITC) in each nucleus; right: the arrows indicate the single (small) green BCR signals (FITC) in each nucleus. F: Another interphase nucleus from meningioma M7 displaying two red A61 signals but only one green 6CR signal (FITC) after a double hybridization. indicating a loss of 22q; DAPl counterstaining, triple-exposure microphotograph.

found in seven gliomas from male patients only once a loss of the Y chromosome. This discrepancy might be caused by the fact that small mosaicisms, if present, would not be classified as such using our criteria for monosomy (more than 20% of nuclei

with one spot) and trisomy (more than 7.5% of nuclei with three spots). However, in a recent study with interphase cytogenetics on samples of normal brain tissue, we found - X, - Y or + 7 in an average of only 0.3%, 0.3%, and 0.5% of the

106 ARNOLDUS ET AL.

nuclei, respectively (Arnoldus et al., 1990a). These facts suggest that the numerical changes observed by Heim et al. are induced by the culture procedure and that they do not reflect an in vivo mosa- icism.

The majority of the meningiomas (62.5%) displayed, as expected, only one signal per nucleus after hybridization with the cosmid probe for 22ql1, indicating a loss of 22q (Fig. lE,F). Some- times the nuclei with only one signal for 22qll (3946%) could be distinguished from the normal nuclei as a result of their slightly larger diameter. The three meningiomas with other changes (+ 17, - 18) did not show the different clinical behavior as suggested by A1 Saadi et al. (1987). There was no difference between the mean age of patients with chromosome anomalies (54.8 years) and the mean age of patients with normal findings (56.2 years).

The present study shows the feasibility of study- ing numerical chromosome aberrations in brain tumors by interphase cytogenetics using nonradioactive in situ hybridization. Recently it has been shown that interphase cytogenetics is also capable of detecting specific structural aberrations (Arnol- dus et al., 1990b). The number of meningiomas and gliomas studied is still too small to allow any generalizations about possible correlations between specific chromosome aberrations and the histopath- ological type and grade of the tumors. The results of the cosmid hybridizations in this study show that the sensitivity of the nonradioactive in situ hybridization on clinical material is at the single copy gene level. When small probes of direct pathoge- netic significance become available, these too can be implemented, and interphase cytogenetics will help to narrow the gap between molecular genetics and classical cytogenetics in solid tumors.

ACKNOWLEDGMENTS

T h e authors thank F.C. De Beer, H. Van Dulken, R.T.W.M. Thomeer, and M.J.H.M. Herpers from the Department of Neurosurgery of the Academic Hospital Leiden for the supply of clinical material; G.C. Grosveld from the Depart- ment of Cell Biology and Genetics of the Erasmus University, Rotterdam, for the gift of the two cosmid probes; H.F. Willard for a number of satellite DNA probes; and G.C. Beverstock from the Department of Human Genetics, Leiden Univer- sity, for critical review of the manuscript.

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Interphase cytogenetics of brain tumors