Increased chromosomal instability in peripheral lymphocytes and risk of human gliomas

Randa El-Zein1, Melissa L. Bondy1, Li-E Wang1, Mariza de Andrade1, Alice J. Sigurdson1, Janet M. Bruner2, Athanassios P. Kyritsis3, Victor A. Levin3 and Qingyi Wei1,4

1 Departments of Epidemiology,
2 Pathology and
3 Neuro-Oncology, The University of Texas, M.D.Anderson Cancer Center, Houston, TX 77030, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Brain tumors exhibit considerable chromosome instability (CIN), suggesting that genetic susceptibility may contribute to brain tumorigenesis. To test this hypothesis, in this pilot study, we examined for CIN in short-term lymphocyte cultures from 25 adult glioma patients and 28 age-, sex- and ethnicity-matched healthy controls (all Caucasian). We evaluated CIN by a multicolor fluorescence in situ hybridization assay using two probes: a classic satellite probe for a large heterochromatin breakage-prone region of chromosome 1 and an alpha satellite probe for a smaller region adjacent to the heterochromatin probe. Our results showed a significant increase in the mean number of spontaneous breaks per 1000 cells in glioma patients (mean ± SD, 2.4 ± 0.8) compared with controls (1.4 ± 0.9; P < 0.001). By using the median number of breaks per 1000 cells in the controls as the cutoff value, we observed a crude odds ratio (OR) of 8.5 [95% confidence interval (CI) = 2.05–34.9, P < 0.001] for spontaneous breaks and brain tumor risk. After adjustment for age, sex and smoking status, the adjusted OR was 15.3 (95% CI, 2.71–87.8). A significant increase in cells with chromosome 1 aneuploidy (in the form of hyperdiploidy) (P < 0.001) was also observed in the glioma cases, with an adjusted OR of 6.6 (95% CI = 1.5–30, P < 0.05). These findings suggest that CIN can be detected in the peripheral blood lymphocytes of brain tumor patients and may be a marker for identifying individuals at risk.

Abbreviations: CI, confidence interval; CIN, chromosomal instability; FISH, fluorescencein situ hybridization; OR, odds ratio; PBD, phosphate-buffered detergent; SSC, standard saline citrate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Malignant tumors of the central nervous system constitute ~2.3% of all human cancers and ~17 400 new primary malignant brain tumors will have been diagnosed in the United States in 1998 (1), of which 90% would have been malignant gliomas and nearly 80% of the affected patients die within the first year after diagnosis (2). Only modest progress has been made in diagnosing and treating malignant gliomas and improving survival after diagnosis (3). The most generally accepted model of carcinogenesis postulates that cancer develops through accumulation of genetic alterations that allow the cells to escape normal growth-regulatory mechanisms (4).

A number of chromosomal loci have been reported to play a role in brain tumorigenesis because of the numerous gains and losses in those loci. For example, Bigner et al. (5), reported gain of chromosome 7 and loss of chromosome 10 in malignant gliomas as well as structural abnormalities involving chromosomes 1, 6p, 9p and 19q; Bello et al. (6) reported the involvement of chromosome 1 in oligodendrogliomas and meningiomas; and Magnani et al. (7) demonstrated the involvement of chromosomes 1, 7, 10 and 19 in anaplastic gliomas and glioblastomas. Loss of heterozygosity for loci on chromosome 17p (8) and 11p15 (9) have also been reported.

There is little published data on chromosomal alterations in the peripheral blood lymphocytes in brain tumor patients. Such alterations should provide information about pre-malignant changes that lead to tumor development. In a previous case-control study of glioma, we demonstrated that the cases had less efficient DNA repair capacity than controls, measured as an increased chromosome sensitivity to {gamma}-irradiation in stimulated peripheral blood lymphocytes. This finding was shown to be an independent risk factor for the development of gliomas (10). To extend our previous work, we conducted this pilot study to investigate whether individuals with gliomas had increased chromosomal instability (CIN) in their peripheral blood lymphocytes that accounted for their susceptibility to cancer.

To detect background instability in these patients, we measured the level of CIN at hyper-breakable regions in the genome. There is increasing evidence that the human heterochromatin regions, particularly those of chromosomes 1, 9, 16 and Y, are frequently involved in stable chromosome rearrangements (11,12). Smith and Grosovsky (13) and Grosovsky et al. (14) reported that breakage affecting the centromeric and pericentromeric heterochromatin regions of human chromosomes can lead to mutations and chromosomal rearrangements and increase genomic instability. Previous studies with human lymphocytes have shown an elevated frequency of breakage in the heterochromatin regions 1q12 and 9q12 after exposure to a variety of environmental clastogens (1517). In this study, we used a multicolor fluorescence in situ hybridization (FISH) assay to detect background CIN in the peripheral blood lymphocytes of 25 glioma patients and 28 matched controls. The probes used hybridized to a small centromeric region and a large pericentromeric region on chromosome 1 (1cen–q12) and allowed the detection of both structural and numerical aberrations simultaneously.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
The cases were patients with histopathologically confirmed, previously untreated malignant gliomas, of whom 15 (60%) had glioblastoma multiforme, six (24%) had astrocytomas and four (16%) had oligodendrogliomas. These patients were registered at The University of Texas M.D.Anderson Cancer Center between 1994 and 1997. They were also participants in an ongoing family study of glioma described previously (10). The study subjects were recruited sequentially with no age, sex, ethnic or stage restrictions. Those patients who received chemotherapy or radiotherapy prior to blood sampling were excluded. The controls were blood donors who either came to the M.D. Anderson Blood Bank to donate blood or participated in an off-site employee blood drive. The controls were matched with the cases by age (±5 years), sex and ethnicity. Information on medical and family history of cancer, smoking habits and occupational history was obtained through an interviewer-administered questionnaire as well as from review of the patients' hospital records.

FISH analysis
For the FISH experiments, blood cultures were established by a standard procedure (18). Briefly, 1 ml of fresh blood from each study subject was inoculated into a T-25 culture flask with 9 ml of RPMI 1640 that contained 12.5 µg/ml phytohemagglutinin (Murex Diagnosis, Norcross, GA). At 1 h 30 min before harvesting, the cultures were treated with colcemid (0.04 µg/ml) to arrest the cells in mitosis. The cultures were then harvested at 72 h after initiation, and slides were prepared by using Oncor's and Boehringer Mannheim's protocols for human satellite III DNA probe and alpha satellite chromosome 1 probe, respectively. These two different chromosome 1-specific DNA probes were a human satellite III DNA probe labeled with fluorescein that hybridizes with the breakage prone pericentric region of chromosome 1 (Boehringer Mannheim, Indianapolis, IN), and an alpha satellite chromosome 1-specific probe labeled with digoxigenin that targets a smaller, less breakage-prone centromeric region (Oncor, Gaithersburg, MD). The slides were aged in 2x standard saline citrate (SSC) for 30 min at 37°C, washed in an ethanol series (70, 85 and 100%) at room temperature for 2 min each time and left to dry at room temperature. The hybridization solution consisted of 1.5 µl of each probe and 30 µl of hybridization mix (Hybrisol, Oncor). This probe mix was heated in water at 70–72°C for 5 min to denature the probe DNA and then put immediately into 4°C ice-bath. The target DNA was denatured by immersing the slides in 70% formamide and 2x SSC at 72°C for 3 min then passing them through a dehydration series of 70, 85 and 100% cold ethanol for 2 min each time. The slides were then placed on a 42°C slide warmer, 30 µl of hybridization mix was added to each warm slide and a coverslip was sealed onto the slide with rubber cement (Oncor). The slides were hybridized overnight in a humidified chamber at 37°C. The post-hybridization washes consisted of three washes in 60% formamide and 2x SSC for 5 min each time at 43°C and one wash in 2x SSC for 5 min at 37°C. After a 1x phosphate-buffer detergent (PBD) rinse for 15 min, the hybridized alpha probe was detected with 60 µl of anti-digoxigenin rhodamine antibody, and the slides were covered with coverslips and incubated for 30 min at 37°C. The slides were then rinsed three times in 1x PBD at room temperature for 2 min each time. For signal amplification, each slide was treated with 60 µl of rabbit anti-sheep antibody solution, covered with a coverslip, incubated for 30 min at 37°C and rinsed three times in 1x PBD at room temperature for 2 min each time. Then, the slides were each treated with 60 µl of rhodamine sheep anti-rabbit antibody solution by the procedures described above and counterstained with 60 µl of 4,6-diamidino-2-phenylindole.

Cytogenetic evaluation
All scoring was performed from coded, randomly ordered slides without an identification of subjects by using a Nikon Phot-Lab2 microscope with a fluorescence attachment equipped with a triple-band-pass filter for the multicolor FISH studies. A thousand interphase nuclei randomly selected from each sample of cases and controls blinded to the examiner (R.E.Z.) were analyzed. As indicated by two different chromosome 1-specific DNA probes, a normal cell had the interphase nuclei with two labeled FISH signals, each containing a large green spot adjacent to a small red spot (corresponding to the classic and alpha probes, respectively (Figure 1AGo). In contrast, an abnormal cell had one labeled signal containing a large green spot adjacent to a red spot and another signal with two widely separated green signals (Figure 1BGo, as shown by the arrow), indicating a break in the classic probe. Hyperdiploidy in chromosome 1 is indicated if the cells had more than two sets of the tandem labels (Figure 1CGo). Both a break and hyperdiploidy of chromosome 1 were considered CIN in this study.



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Fig. 1. (A) Three normal cells, where each interphase nucleus had two labeled signals, each signal containing a large green spot (the classic probe) and an adjacent red spot (the alpha probe). (B) One labeled signal containing a large green spot adjacent to a small red spot and an abnormal signal with two widely separated green signals, indicating a break in the classic probe (arrow). (C) Aneuploid cell containing three signals (trisomy).

 
Statistical analysis
We calculated odd ratios (ORs) and 95% confidence intervals (CIs) as estimates of relative risk to determine the association between CIN and the risk of development of brain tumors. Non-parametric bootstrap samplings were generated to assess the sensitivity of the cutoff point and the validity of the results. We used the SAS (Version 6, Statistical Analysis System, Cary, NC) and S-plus (Version 4, Mathsoft, Seattle, WA) statistical software programs to analyze and plot the data. The chi-square test was used to test distribution differences in the frequency of CIN between cases and controls, using a probability level of 0.05 as the criterion for significance. Because the cases and the controls were frequency matched only, the OR adjusted for age and sex was also calculated.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subject characteristics
The study included 25 cases and 28 controls. The characteristics of the study population are presented in Table IGo. The mean age was 49.4 years (SD ± 11.8) for the cases and 48.5 years (SD ± 10.8) for the controls. All subjects were Caucasian, and there were 14 male and 11 female cases, and 17 male and 11 female controls. There were more smokers in controls (54%) than in cases (36%). However, the differences were not statistically significant between cases and controls (P > 0.05).


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Table I. Distribution of glioma cases and controls and OR of selected variables
 
Chromosome aberration frequencies
Our results indicated that the cases had a significantly higher frequency of total breaks (2.4 ± 0.8 per 1000 cells) than did the controls (1.4 ± 0.9; P < 0.001). The range of the number of breaks/1000 cells was 0–3 in the controls (median = 1) and 1–4 in the cases (median = 2) (Figure 2Go). We confirmed by bootstrap resampling techniques that the median number of breaks/1000 of the controls was the optimal cutoff value, which was used for OR estimate. The risk (unadjusted OR) of glioma associated with breaks compared with the controls was 8.46 (95% CI = 2.1–34.9). When we adjusted for age, sex and smoking status by using logistic regression, the adjusted OR was 15.3 (95% CI = 2.7–87.8), which indicated that the break frequency was significantly associated with the risk of glioma. There were slight differences in the level of breaks when we stratified by age, sex or smoking status among patients, but these differences were not statistically significant (Table IIGo). For instance, the mean number of breaks in ever-smokers and never-smokers was 2.1 ± 0.6 and 2.6 ± 0.9 in cases, respectively, and 1.3 ± 0.9 and 1.5 ± 0.9 in controls, respectively (Table IIGo), suggesting the increased frequency of breaks was not induced by smoking. Further classification of the breaks into those occurring in the classic region and those occurring in the classic alpha junction indicated that >99% of the breaks occurred in the former region in all studied groups.



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Fig. 2 . Box plot showing the distribution of breaks/1000 cells in the cases and the controls. The upper and lower horizontal lines indicate the range of all values, and the boxes represent the middle 50% of values (lower 25th percentile to upper 75th percentile). The medians of both groups (2 for cases and 1 for controls) were in the 25th percentiles of the values, indicating that the distribution was skewed to low values.

 

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Table II. Frequency of CIN between glioma cases and controls by age, sex and smoking status
 
The frequency of hyperdiploidy in chromosome 1, as determined by the number of cells having more than two sets of the tandem labels (Figure 1CGo), was significantly higher in the cases (1.2 ± 1.5) than in the controls (0.3 ± 0.5; Table IIGo). Using the median hyperdiploid frequency in the controls as the cutoff point, the unadjusted OR for hyperdiploidy and glioma risk was 6.6 (95% CI = 1.5–29.8). No substantial change in the risk was observed after controlling for age, sex and smoking using logistic regression. No statistical difference was detected in the frequencies of hyperdiploidy when we stratified by age, sex or smoking status among patients (Table IIGo). In the cases, the hyperdiploidy frequency was higher (1.7 ± 1.6) in never-smokers than in ever-smokers whereas the frequency was almost identical between the control smokers (0.3 ± 0.7) and never-smokers (0.2 ± 0.3), further suggesting that CIN was not induced by smoking but could be genetically determined.

The frequencies of breaks among the different histological types of gliomas were very similar, with a mean of 2.4 ± 0.8 for glioblastoma multiforme cases (n = 15) and 2.3 ± 0.8 for the other tumors (six astrocytomas and four oligodendrogliomas), indicating the breaks were more likely constitutional rather than tumor-related.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this pilot study, we demonstrated that CIN as measured by the FISH technique in peripheral blood lymphocytes was associated with an increased risk of developing gliomas. Individuals with a significantly higher level of background CIN had a 15-fold increased risk of gliomas. Although previous studies have demonstrated the presence of CIN in brain tumor tissues (1922), to our knowledge, this is the first study to investigate the role of background CIN in the peripheral blood lymphocytes in human gliomas. Our findings suggest that accumulated chromosomal damage in peripheral blood lymphocytes may be an important biomarker for identifying individuals at risk of developing gliomas.

Another interesting observation in this study was the detection of aneuploidy in peripheral blood lymphocytes of glioma patients. The significantly higher level of hyperdiploidy was associated with a nearly 7-fold increased risk. CIN leading to abnormal chromosome number or aneuploidy has been observed in many cancer types (23). Loss of chromosomal material has been postulated to be one of the early events in carcinogenesis. When the hypodiploid cells replicate, their daughter cells are hyperdiploid (24). Because our study was retrospective, it is possible that presence of a glioma may have caused the observed CIN in normal lymphocytes detected by our FISH method. However, recently Duesberg et al. (25) and Lengauer et al. (23) reported that aneuploidy itself was a sufficient cause of genetic instability that is independent of gene mutation in tumor cells (25). In this study, we examined a breakage-prone site in the genome (2628) of interphase lymphocytes, which were unlikely affected by glioma carcinogenesis. These breakage sites are thought to be involved in both the early and late stages of tumor development (11,12). Therefore, our findings in these lymphocytes provide some insight of genetic instability that may have occurred in glioma tumor cells. This speculation is consistent with previous reports of multiple structural and numerical aberrations of chromosomes in brain tumors of various different histological types (2,2022).

Although the probes we used in our study are non-specific for detecting CIN, a number of reports in the literature suggest that chromosome 1 is involved in brain tumors. For example, Wernicke et al. (21) detected a gain in chromosome 1p or the whole chromosome 1 in 17 of 26 brain tumors studied. Debiec-Rychter et al. (29) reported structural changes in the form of deletions, duplications and translocations of chromosome 1 that resulted in trisomy of 1p, 1q or both, in tumor tissues. Combaret et al. (30) proposed using chromosome 1 aberrations as an indicator of prognosis in other central nervous system tumors such as neuroblastomas. Our data raise the possibility that structural and numerical aberrations involving chromosome 1 detected in peripheral lymphocytes may not be random, and may be a marker for risk of brain tumorigenesis.

Smoking status (ever- or never-smokers) in either the cases or controls did not influence the frequency of CIN, possibly because of the small sample size in this pilot study, i.e. there were only nine smokers in the cases and 15 smokers in the controls; although in previous studies, we reported that smoking affected the frequency of chromosomal aberrations (17,31).

In conclusion, in this pilot study, we showed that the multicolor FISH assay for detecting CIN in peripheral blood lymphocytes may be a potential biomarker of glioma susceptibility. This approach has the advantage of using interphases of peripheral blood lymphocytes without further laboratory processing, which should make the technique applicable to many different cell types, such as exfoliated cells. However, the findings from this pilot case-control study are preliminary, addressing an association rather than establishing causality. Larger studies are needed to further test the hypothesis that CIN in peripheral blood lymphocytes correlates with the cytogenetic changes in the target tissues. If this is true, then CIN may prove to be a biomarker for identifying individuals at risk.


    Acknowledgments
 
We thank Drs R.Lotan and M.Spitz for their invaluable suggestions and constructive comments about this manuscript, Ms Yongli Guan for technical support, Ms Phyllis Adatto for patient recruitment, Dr Maureen Goode (Department of Scientific Publication) for editorial assistance, and Ms Joanne Sider and Joyce Brown for manuscript preparation. This work was supported in part by National Cancer Institute grants CA 70334 and CA 74851 (to Q.W.), CA 70917 (to M.L.B.) and CA 55261 (to V.A.L.).


    Notes
 
4 To whom correspondence should be addressed Email: qwei{at}notes.mdacc.tmc.edu Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 13, 1998; revised December 10, 1998; accepted December 23, 1998.