Mutagenesis Advance Access originally published online on July 26, 2006
Mutagenesis 2006 21(4):261-264; doi:10.1093/mutage/gel030
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Reliability of mutagen sensitivity assay: an inter-laboratory comparison
University of New Mexico, School of Medicine, Department of Internal Medicine and New Mexico Cancer Research and Treatment Center Albuquerque, NM, USA 1 The University of Texas M. D. Anderson Cancer Center Houston, TX, USA 2 Memorial Sloan-Kettering Cancer Center New York, NY, USA 3 Mount Sinai School of Medicine New York, NY, USA
Mutagen sensitivity is regarded as a genetic susceptibility phenotype for various cancers; it is cytogenetically based and probably involves a number of genes from different DNA repair pathways. This assay has been used in a number of laboratories in the field of epidemiology, where it has been investigated and appears to be a useful susceptibility biomarker for epidemiological studies assessing cancer risks at the population level. One concern about phenotypic assays, such as the mutagen sensitivity assay, has been that there could be wide variation in results depending on the timing of the assay (within individual variation), the individual performing the assay (within observer variation) and the laboratory where the assay has been performed (inter-laboratory variation). We conducted an inter-laboratory comparison study between the Memorial Sloan-Kettering Cancer Center and M. D. Anderson, in which we assessed all these concerns. We did not find any significant variation in any of the assays. The correlation was high for all tests. The good concordance rate between laboratories supports the continued use of the mutagen sensitivity assay by different laboratories, and demonstrates its potential to identify at-risk subgroups among normal individuals and cancer patients alike.
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The search for ways to measure cancer susceptibility has expanded geometrically as understanding of the human genome has evolved. Focus on genetic variation in the form of disease-associated mutations and polymorphisms have produced much data but few new understandings as yet. Genotype–phenotype correlations are currently being investigated in many laboratories in order to find potentially useful genotypes for association studies of disease among the general population. Although decrements in DNA repair capacity are probably involved in the carcinogenic process, to date there is no widely accepted consensus as to which measures of DNA repair, either phenotypic or genetic, might be useful to help predict individual susceptibility to carcinogenesis.
The bleomycin-induced mutagen sensitivity assay is an in vitro measure of DNA repair capacity developed by Hsu (1
). This assay is an indirect measure of both DNA damage and DNA repair expressed as ‘breaks per cell’ (b/c) in short-term cultured lymphocytes. It is a relatively simple test in which a higher number of bleomycin-induced chromatid breaks indicates higher ‘mutagen sensitivity’ and lower DNA repair. For example, if the number of ‘breaks per cell’ is greater than the median of the controls, usually 
0.8, the examined subject is considered ‘mutagen sensitive’ (2). Mutagen sensitivity is regarded as a genetic susceptibility phenotype for various cancers; it is cytogenetically based and likely involves a number of genes from different DNA repair pathways. However, the specific genes accounting for the traits are still unknown (3).
Hsu (1) developed a functional or phenotypic assay, (the bleomycin-induced the mutagen sensitivity assay), for measuring ‘overall unrepaired DNA’, which has been found to discriminate between individuals with cancer and healthy controls (2). This assay has also been found to distinguish between individuals with cancer who will develop second malignancies and those who will not (4–6), as well as those with a family history of cancer and those without (7–12). It also became clear that mutagen sensitivity assay measured cellular responses based on environmental exposure and individual susceptibility (13). In the past 15 years, the mutagen sensitivity assay has been used in a number of laboratories in the field of epidemiology, where it has been investigated and appears to be a useful susceptibility biomarker for epidemiological studies assessing cancer risks at the population level (14). Large prospective studies have been conducted that demonstrate that chromosomal aberrations can predict cancer occurrence among a cancer-free cohort 15 years later (15,16); however, in these studies only baseline chromosome breaks were counted, no chemically induced mutagen treatments were applied as in this study.
One concern about phenotypic assays, such as the mutagen sensitivity assay, has been of the typical wide variation in results that may depend on the timing of the assay (within individual variation), the individual performing the assay (within observer variation) and the laboratory where the assay has been performed (inter-laboratory variation). As the mutagen sensitivity assay is somewhat time-consuming and expensive to conduct, we wanted to evaluate its reliability before continuing its use in epidemiological studies and going further to develop a higher throughput assay. Therefore, we measured the variability of the mutagen sensitivity assay in these contexts.
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Materials and methods |
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Subjects
All subjects signed informed consent. All data were blinded as to subject status.
Intra-individual variation
In the Memorial Sloan-Kettering Cancer Center (MSKCC) Molecular Epidemiology Laboratory we conducted the mutagen sensitivity assay at three separate times, 1 month apart on seven individuals, healthy women with family histories of breast cancer (21 x 50 metaphases = 1050 readings).
Intra-observer variation
In the MSKCC laboratory one observer read 50 metaphase spreads from 28 cancer patients in a study of soft tissue sarcoma assessed at two separate times, 1 week apart (1400 readings). We used the same slide from each individual for each set of readings in this part of the analysis as we were addressing the issue of reader variability.
Inter-laboratory variation
To measure inter-laboratory variability in assessing mutagen sensitivity, we digitally photographed 50 metaphases from nine healthy subjects (Zeiss Spotmatic and Kodak) and saved them on a CD. These images (450 readings) were read at MSKCC and also sent to The University of Texas M. D. Anderson Cancer Center Molecular Epidemiology Laboratory of Dr Qingyi Wei for evaluation. Both laboratories thus read the same, selected metaphases for identification of chromatid breaks.
In the second phase of this inter-laboratory reliability study, the MSKCC laboratory reviewed 45 slides from 45 individuals—cancer patients and controls—and then sent these to be evaluated by the M. D. Anderson laboratory. Finally, the M. D. Anderson laboratory sent 45 slides from 45 individuals to be evaluated by the MSKCC laboratory. Each laboratory read 50 metaphases from each slide for each individual. Five slides were damaged in the exchange; so altogether 85 slides (4250 readings) were compared in the statistical analyses.
Assay
The mutagen sensitivity assay was carried out as described previously (2). From each subject, two whole-blood cultures were used for this assay. In brief, 1 ml blood was added to 9 ml media [RPMI 1640, 2 mM L-glutamine, 15% fetal calf serum (FCS), 1.5% phytohaemagglutinin, 100 U/ml penicillin and 100 µg/ml streptomycin; Sigma-Aldrich, St Louis, MO] in 25 ml cell culture flasks and incubated at 37°C, with 5% CO2 for 72 h; two for each subject. At 67 h after culture initiation, bleomycin (0.03 U/ml; Sigma-Aldrich) was added to one culture; another culture remained as an untreated control. Due to extremely low values of spontaneous chromosome breaks in all control slides, these b/c values were considered negligible and data were not recorded for this analysis. At 70 h after culture initiation, colcemid (0.04 mg/ml; Sigma-Aldrich) was added into each culture to block cells in mitosis. After 2 h, the cultures were harvested according to conventional procedures: the cells were treated with 0.06 M hypotonic potassium chloride solution for 15–20 min, fixed, washed with freshly prepared mixture of methanol and acetic acid (3:1), and air-dried on wet slides. The slides were then stained using Giemsa solution without banding and analysed for the presence of chromatid breaks or exchanges. We recognized chromatid breaks when the intervening achromatic segment was longer than or equal to the diameter of the chromatid and when the fragment was displaced (17). The frequency of breakage was expressed as breaks per cell, or b/c. For the quantification of mutagen sensitivity, 50 metaphase cells/donor were analysed, a number found necessary for acceptable reliability of the assay (18). In 1995 one of the MSKCC personnel (MB, then affiliated to MSKCC, currently with UNM) visited Dr Hsu to learn this assay. Dr Wei also learned the assay from Dr Hsu at about the same time. Each of them taught this assay to their laboratory technicians.
Statistical analysis
First we verified that our experimental values for breaks (b/c) satisfied the normality assumption based on Quantile–Quantile (Q–Q) plots and the Kolmogorov–Smirnov goodness-of-fit test. The overall distribution of the data is graphed in Figure 1 using scatter plots. Descriptive statistics such as mean, standard deviation (SD), correlation coefficient, coefficient of variation (CV) and 95% confidence intervals (CI) were examined for all types of variability and the intra-individual variability results are summarized in Table I. Figure 1 also shows the high positive correlation between different readings. To test the hypothesis that there were no intra-individual, intra-observer and inter-laboratory difference in b/c values, the standard paired t-test was employed. Test statistics related to correlation and paired t-test for the difference in the means of the b/c values were demonstrated in Table II.
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The distributions of b/c reading differences were centred at zero and we did not find any noteworthy patterns in variation (Figure 1). The correlations were very high and every 95% confidence interval for the reading differences included zero.
Intra-individual variation
We were interested in whether the timing of the blood collection had any influence on the number of breaks/cell of the study subjects. Intra-individual variation did not vary significantly over time. P-values of 0.34, 0.97 and 0.28 for each comparison demonstrate that there were no significant differences between readings due to intra-individual variation and that no trend over time was detected (Table I). This result is in accordance with some previous findings that have examined the specificity of the mutagen sensitivity assay and found that was not influenced by confounders, such as gender, and diet (19–21). Others emphasize that mutagen sensitivity might be considered as a complex expression of toxicological responses, therefore exposure to alcohol and smoking can be important contributors to initiate these processes (22). The low frequency of b/c values 
1 (23%) in our study population showed that subjects possibly have not been exposed to these mutagenic factors or to a lesser extent than other population reported.
Intra-observer variation
The repeated evaluation of two sets of metaphases from 28 individuals with the same technician reading the slides demonstrated no significant difference in b/c values. Table II shows the similarity of the empirical cumulative distributions from the two metaphase readings. McIntyre et al. (23) also examined reader variability of the mutagen sensitivity assay and found that 
value (Kappa statistic) was fair. However, our intra-observer variability was excellent, so training may play a difference.
Inter-laboratory variation
Two laboratories have reviewed metaphases. In one set of analyses, where researchers from each laboratory read the exact same, selected metaphase spreads, there was no significant difference in the readings (P-value = 0.39; Table II).
In a second set of analyses, one slide from each individual was independently scored by a technician from each laboratory without preselection of specific metaphases from the slide and found no statistically significant differences between readings (P-value = 0.51; Table II). Our evaluation shows that two separate laboratories have surprisingly good reproducibility of results for the mutagen sensitivity assay. As laboratory personnel of both laboratories were trained by using same technique, our future plan will be to explore inter-laboratory concordance between different settings and countries.
The application of bleomycin, a radiomimetic drug capable of inducing both single- and double-strand chromosome breaks (independently of cell cycle phases) in standard lymphocyte cultures allows investigators seeking cancer risk assessments to quantify individual overall DNA repair capacity within a relatively short time period. The mutagen sensitivity assay can also be adapted to accommodate different mutagens that offer additional information about the involvement of specific DNA damage and repair pathways. For example, using benzo[a]pyrene diol epoxide (24), gamma radiation (25) and ultraviolet light (26) that are well-known carcinogens as the test mutagens can help address the susceptibility to cancer with a specific aetiology. These make the assay potentially useful for larger scale molecular epidemiological studies. Furthermore, mutagen sensitivity may be an important susceptibility biomarker showing consistent association with cancer risk, variability among controls and good reproducibility. The good concordance rate between laboratories supports the continued use of the mutagen sensitivity assay by different laboratories, and demonstrates its potential to identify at-risk subgroups among normal individuals and cancer patients alike.
It would also be valuable to extend inter-laboratory studies to other laboratories in other countries with potentially different environmental exposures. This type of study could also add important insights into the applicability of the mutagen sensitivity assay.
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*To whom correspondence should be addressed at: University of New Mexico, School of Medicine, Department of Internal Medicine, Division of Epidemiology and Biostatistics, Molecular Epidemiology Laboratory, MSC08 4630, 1 University of New Mexico Cancer Research Facility, Room 103, Albuquerque, NM 87131-0001, USA. Tel: +1 505 272 4431 or +1 505 272 2577; Fax: +1 505 272 2570; Email: EErdei{at}salud.unm.edu
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Received on December 15, 2005; revised on January 9, 2006; accepted on June 12, 2006.
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