Mutagenesis, Vol. 17, No. 2, 171-175,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
REVIEW |
Measurement and analysis of the chemotherapy-induced genetic instability in pediatric cancer patients
Laboratory and Department of Pediatrics, 1 Department of Toxicology and 2 Department of Genetics, University of Navarra, E-31080 Pamplona, Spain
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Abstract |
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Bleomycin sensitivity has been proven to be a useful biomarker for environmental carcinogenesis and tumor genetic instability. We have previously reported a significant increase in the chromosomal aberrations induced by chemotherapy regimens. This study aimed to test whether there is an inherent increased genetic instability in cancer patients at diagnosis, to determine the increase and time course of the chemotherapy-induced instability and to test whether bleomycin sensitivity can be used as a predictor of tumor evolution or relapse. The analysis included 99 pediatric cancer patients with four different tumor types (Ewing's sarcoma, osteosarcoma, lymphoma and CNS tumors) and 25 controls. Blood samples (n = 171) were obtained before and at the end of treatment, during clinical remission and at relapse and bleomycin tests on lymphocyte cultures were performed. We detected a significant increase (P = 0.004) in mutagen sensitivity in patients at the end of treatment compared with untreated patients, regardless of the tumor type. In both the longitudinal and cross-sectional analyses maximal and similar values of mutagen sensitivity were found in patients during treatment (1.84 ± 0.82) and at relapse (1.78 ± 0.52); minimum and similar values were found in controls (0.93 ± 0.23), untreated patients (1.15 ± 0.65) and in those who had fulfilled the chemotherapy protocols for at least 2 years before their sample was collected (1.09 ± 0.53). From this preliminary data we can conclude that cytostatic drugs induce a transient increase in chromosomal instability in pediatric cancer patients that can be monitored by bleomycin-induced sensitivity tests and that the genetic instability indices should be further investigated as predictors of relapse.
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Introduction |
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The study of carcinogenesis in humans over the past few years has potentiated the search for biomarkers that allow the detection of groups that are at risk of developing cancer (Spitz and Bondy, 1993
; Olden, 1994). One of these markers is measurement of mutagen sensitivity, through the quantification of chromosome breaks induced by the radiomimetic agent bleomycin, in peripheral blood lymphocyte cultures (Hsu, 1983).
Several case–control studies have demonstrated that mutagen sensitivity is an independent risk factor for development of different tumors, such as those of the upper aerodigestive tract (Spitz et al., 1989), head and neck (Cloos et al., 1996; Wei et al., 1996), lung (Spitz et al., 1995) and liver (Wu et al., 1998). It has even been described as an indicator of tumor progression in precancerous lesions of the larynx (Gallo et al., 1996) and as a predictor of recurrence and development of secondary tumors following malignancies of the upper aerodigestive tract (Spitz et al., 1994, 1998).
On the other hand, it is well known that some cytostatic drugs are DNA-damaging agents. In fact, an increase in both spontaneous chromosome breakage and sister chromatid exchange after chemotherapy treatment has been reported by several authors (Gebhart et al., 1980; Aronson et al., 1982; Carbonell et al., 1996). In a previous pilot study of 80 pediatric patients (127 samples) we demonstrated that the transient increase in chromosomal aberrations induced by anti-tumor regimens seems to be non-random, which suggests the existence of increased chromosome fragility in specific genomic locations (López de Mesa et al., 2000). It is also accepted that a percentage of cancer patients develop secondary tumors after being treated with mutagenic agents such as cytostatic drugs (Smith et al., 1996). Therefore, it is very important to have a simple method to measure and evaluate the persistence of the genetic damage induced by such treatments.
The aims of this study were to evaluate chromosomal instability through the analysis of bleomycin tests performed before, during and after anti-tumor treatments in pediatric cancer patients with four different tumor types, in order to test the following hypotheses: (i) if there is increased genetic instability prior to the treatment; (ii) if there is an increase in genetic instability after administration of the chemotherapy treatment and to evaluate its time course throughout the treatment and afterwards; (iii) to test the possibility of using this simple test as a predictor of tumor evolution, complete remission or relapse.
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Materials and methods |
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Patients and samples
Peripheral blood samples were obtained from 99 pediatric cancer patients [35 osteosarcomas, 26 Ewing's sarcomas, 15 lymphomas and 23 central nervous system (CNS) tumors; mean age 12.92 ± 5.03; 58 males and 41 females] and 25 healthy controls (mean age 14.06 ± 4.70; 15 males and 10 females). The study was approved by the Human Ethics Committee of the University Clinic of Navarra and informed consent was obtained from the individuals included in the study or from their parents. None of the individuals included in the analysis belonged to cancer-prone families or suffered from cancer predisposition syndromes.
Several samples were obtained from the patients at different times before, during or after chemotherapy treatment and at relapse. For different reasons, samples at all times were not available from all of the patients. A cross-sectional study was performed with all the samples obtained (n = 171) from all of the patients included in the analysis (n = 99) and the 25 healthy controls.
The 25 controls analyzed were collected on five different dates (five controls each day) and were cultured as the same culture batch for the same day but as five different batches on the five dates of sample collection (Table I). From these data it can be deduced that the mean instability values of the five control groups are very similar (P = 0.929), even when the five measurements within each group are different due to inter-individual variability in susceptibility to bleomycin. The homogeneity in the means of samples collected on different dates and cultured as different batches allows us, from our point of view, to rule out variability induced by the culture medium.
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A longitudinal study was also performed with those patients (n = 46) for whom two samples at consecutive stages were available: 14 patients before and at the end of chemotherapy, 13 patients at the end of treatment and at 2 years in clinical remission, 16 patients followed until they had been in clinical remission for >2 years and three patients before and after relapse had occurred.
Bleomycin tests
Cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI 1640 medium supplemented with 16% fetal bovine serum (ICN Biomedicals, Irvine, UK), gentamycin sulfate (final concentration 0.048 mg/ml) (ICN Biomedicals) and L-glutamine (final concentration 2 mM) (Gibco BRL Life Technologies, Paisley, UK). Lymphocytes were stimulated with phytohemagglutinin (Seromed, Biochrom, Berlin, Germany) and incubated at 37°C. On day 3 of incubation the cultures were treated for 5 h with 0.03 U/ml bleomycin (Almirall Prodesfarma SA, Barcelona, Spain), according to the protocol described by Hsu et al. (1989).
During the last hour the cells were treated with 0.04 µg/ml colcemid (Gibco BRL Life Technologies) to arrest them in metaphase. At 72 h incubation the cells were harvested by centrifugation at 1000 r.p.m. for 10 min. Cells were washed once in RPMI 1640 medium and treated with 0.075 M KCl at 37°C for 10 min. The cells were then centrifuged and a methanol/acetic acid (3:1) solution was slowly added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative solution and dropped onto clean slides. Finally, the slides were stained with 10% Giemsa (Merck, Darmstadt, Germany) for 5 min.
Breaks were scored in 50 metaphases per sample and the number of breaks per cell (b/c index) was averaged. The scoring criteria of Hsu et al. (1989) were used. Only chromatid breaks or exchanges were recorded; chromatid gaps or attenuated regions were disregarded.
Statistical analyses
Cross-sectional study
The mean b/c of the groups (tumors and stages) were compared in a two-way interaction analysis of variance (ANOVA), followed by the Tamhane test (Tamhane, 1979) to compare the different groups with each of the others. All of the data are reported as means ± SD. Differences in mean b/c between controls and untreated patients were tested by Student's t-test and the results are reported as mean difference and 95% confidence intervals.
Longitudinal study The differences in mean b/c between consecutive stages were analyzed by t-test for paired samples. All the quantitative variables fulfilled the Shapiro–Wilk or Kolmogorov–Smirnov normality criteria. All of the probability (P) values were two-tailed and values lower than 0.05 were considered statistically significant. Statistical analyses were performed with the Statistical Package for the Social Sciences v.9.0 (SPSS, Chicago, IL).
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Results |
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We studied the genetic instability in 99 pediatric cancer patients with four different tumor types who have received chemotherapy and/or radiotherapy treatment (Table II
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Blood samples were obtained at different times: (i) at diagnosis, before any treatment was administered; (ii) in the last week of the chemotherapy treatment; (iii) at remission, between 6 and 24 months after the end of treatment; (iv) at remission, 2 or more years after the end of treatment; (v) at relapse. The bleomycin test was performed with a total of 171 samples obtained from pediatric cancer patients and 25 from children without tumors, considered as controls. The breaks were scored in 50 metaphases per sample and the mean b/c were calculated. The cross-sectional study showed that the four tumor types analyzed had similar patterns of instability throughout the stages considered (Figure 1
). A two-way ANOVA which permitted us to analyze the variables tumor type and stage independently, due to a lack of interaction, was applied. The ANOVA confirmed that there were no differences in the b/c indices among the four tumor groups (P = 0.182), nevertheless, the six different stages being considered showed highly significant differences in their b/c indices (P < 0.001) (Figure 2).
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For an in-depth analysis of these differences in b/c indices depending on stage, the Tamhane multiple comparison test was performed and the following results were obtained (Table III
): (i) there are no significant differences (P = 0.936) between the b/c indices of samples from patients before treatment (1.15 ± 0.65) and those from the controls (0.93 ± 0.23); (ii) there is a significantly higher b/c index in treated patients (1.84 ± 0.82) compared with the control group (P < 0.001), with patients before treatment (P = 0.008) and with patients 2 years after the end of treatment (1.09 ± 0.53, P < 0.001); (iii) there is a high b/c index in samples obtained from patients with remissions of <2 years (1.45 ± 0.53) and in samples obtained from patients with relapsed tumors (1.78 ± 0.52) that do not significantly differ from the treated patients. In brief, maximal and similar values of mutagen sensitivity were found in patients during treatment and at relapse; on the other hand, minimum and similar values were found in controls, untreated patients and in those who had fulfilled the chemotherapy protocols at least 2 years before their sample was collected.
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In 43 patients samples from two consecutive stages were available and a longitudinal study could be performed. As shown in Table IV
, the results are very similar to those obtained in the cross-sectional study. The t-test for paired samples shows no significant differences between the b/c index of the untreated patients and that of the controls and a significant increase in the mean b/c value in patients at the end of the treatment compared with the mean before treatment (P = 0.004). There was a non-statistically significant lowering of the b/c index in samples obtained from patients that were in clinical remission of <2 years compared with their instability at the end of treatment; this instability in `short-term' remission was significantly higher than that of the controls. There was also a decrease in the b/c values obtained before and after the second year of clinical remission in the same children (P = 0.060). Three patients in the longitudinal study presented a clinical relapse and showed a significant increase in their b/c indices compared with values at clinical remission, nevertheless, due to the low number of relapsed patients, they were not included in the statistical analyses (Table IV).
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Discussion |
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Bleomycin-induced sensitivity has been proven to be an indirect indicator of several defects in DNA repair, mostly of those induced by environmental mutagens (Wei et al., 1996
; Miller et al., 1998; Gu et al., 1999) as well as in syndromes characterized by high chromosomal instability (Bloom syndrome, Fanconi anemia, ataxia telangiectasia, etc.) (Hsu et al., 1989). Given that cytostatic drugs act as exogenous mutagens, the bleomycin test might be considered a good indicator of genomic damage induced by such treatments at the chromosomal level.
The bleomycin sensitivity test has been widely used in environmental carcinogenesis studies once the reproducibility and sensitivity of the assay among healthy controls had been extensively proven (Hsu et al., 1989). This cytogenetic technique is considered to be cost effective as well as relatively easy to perform, nevertheless, it is crucial to control the reproducibility of the assay in each laboratory, particularly when the methodology is to be used with clinical samples. Certainly, a good understanding of the sources of variability, such as the use of different culture medium batches, is required. In this study the reproducibility and reliability of the results can be assessed by the coefficient of variation in the control group, which is <30% (standard deviation/mean = 0.2384/0.9364 = 25.4%), and also by the fact that the controls cultured as different batches showed a similar degree of variation in their mean instability values (see Material and methods). In addition, this level of variation is in good agreement with historical control values in our laboratory.
There are several reports in the literature that describe an increase in chromosomal aberrations and/or sister chromatid exchanges during chemotherapy treatments and for a variable period of time afterwards. This increase was associated with a higher risk of secondary tumor development (Gebhart et al., 1980; Aronson et al., 1982, Carbonell et al., 1996). A non-significant increase in the b/c index in patients with upper aerodigestive tract cancer subjected to radiotherapy has also been described (Spitz et al., 1997, 1998). In contrast, there are no studies on bleomycin-induced instability in patients undergoing chemotherapy treatments.
We were unable to complete a longitudinal analysis with all of the patients included in the study because some of them attended our center after diagnosis or due to the low number of cells in some of the samples. Nevertheless, in the patients that were followed and included in the longitudinal analysis, the difference between two consecutive samples may be attributable to the effect of chemotherapy, and a significant increase in chromosomal instability was detected among them. We could also observe a progressive decrease in instability throughout the months following the end of treatment, which was more noticeable after the second year of clinical remission.
In the cross-sectional study the analysis of interaction showed that tumor type did not condition the b/c index throughout the different stages and, therefore, both variables could be analyzed independently. The analysis showed a highly significant increase in mutagen sensitivity in treated patients compared with controls, with cancer patients before treatment and with patients in clinical remission of >2 years, which supports the data obtained in the longitudinal analysis.
The majority of bleomycin analyses published aimed at evaluating mutagen sensitivity in cancer patients compared with healthy individuals in specific types of cancer, mostly those of the upper aerodigestive tract (Berwick and Vineis, 2000).
Given the types of tumors included in this study (osteosarcomas, Ewing's sarcomas, lymphomas and CNS tumors) and the low number of patients available, the main interest of this research was not to measure genetic instability in the cancer patients before treatment, but to evaluate the chromosome damage induced by the drugs used in chemotherapy, in the short and long term. Nevertheless, the analysis of mutagen sensitivity in the group of untreated patients bearing tumors showed a non-statistically significant increase in the b/c index compared with the control group; this increase was especially marked in patients affected by Ewing's sarcoma (0.69 ± 0.38, n = 4). The low number of cases did not allow us to suggest a genetic predisposition, but we think that this preliminary observation merits further research. So far, we have not found case–control studies dealing with bleomycin-induced mutagen sensitivity in patients with bone tumors, and in those with lymphomas and CNS tumors the authors did not find a significant increase in sensitivity (Hsu et al., 1989; Strom et al., 1997). In fact, in our series, the group of CNS tumors showed a genetic instability that was statistically lower than that of any other tumor type at any stage of the study. This supports the hypothesis that the genetic susceptibility mechanism may be different in tumors primarily determined by environmental or exogenous factors and in those with a hereditary origin (Schantz et al., 1989).
The fact that bleomycin-induced instability is significantly increased in patients during treatment has two possible explanations, which do not exclude each other. First, it may be that some of the breaks recorded could be caused not by the bleomycin added to the cell cultures but by the drugs given to the patients, most of which are capable of inducing DNA damage. In this case the b/c index would reflect the effect of the genotoxic exposure. However, it could also reflect underlying genomic damage that increased as a consequence of the treatment. This genomic damage could be due either to reduced DNA repair capacity or to inhibition of apoptosis in damaged cells, which survive, leading to an apparent increase in chromosomal damage. This second hypothesis is supported by the fact that the b/c index remains relatively high in patients after several months remission and explains the high b/c index found in patients with relapsed tumors, most of whom relapsed in the months immediately following the end of treatment, when the instability index remained increased.
Several authors have reported the usefulness of b/c indices as predictors of relapse and/or secondary tumors (Spitz et al., 1994, 1998; Gallo et al., 1996), although this predictive power depends on tumor type and its pathogenesis because, for example, this association has not been found in lymphomas (Strom et al., 1997). In our samples, although the b/c index increased significantly in relapsed tumors compared with controls and with patients in clinical remission for >2 years, the sample size does not allow us to draw definitive conclusions with regard to the predictive value of recurrence or secondary tumors of this test in our series. However, we think that these preliminary results suggest that it could be, at least, a useful predictive biomarker.
We conclude from the present study that transiently increased genetic instability is a typical finding in lymphocytes of children exposed to anti-tumor regimens. Analysis of the bleomycin-induced mutagen sensitivity may be considered a useful and reasonable biomarker to monitor genomic damage induced by the chemotherapy treatments, revealing an underlying chromosomal fragility, although this should be supported with studies of DNA damage at the molecular level. The availability of biomarkers to identify groups of pediatric cancer patients with increased mutagen susceptibility would allow not only the early detection of relapse and secondary tumors in certain tumor types, but also the follow-up and use of preventive measures in these high risk patients under exposure to exogenous mutagenic agents. In fact, at present preventive measures, such as the use of vitamins and antioxidant agents, are being carefully analyzed (Trizna et al., 1991, 1992; Goodman et al., 1998), with interesting conclusions, such as the protective potential of retinoic acid (Hong et al., 1990).
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Acknowledgments |
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This investigation was supported by a grant from the Health Service of the Gobierno Foral de Navarra.
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Notes |
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3 To whom correspondence should be addressed. Tel: +34 948 425653; Fax: +34 948 425652; Email: apatigar{at}unav.es
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References |
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Received on May 23, 2001; accepted on November 21, 2001.
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