The CBMN assay is a genotoxicity assay that provides simultaneous information on a variety of chromosomal damage endpoints that reflect chromosomal breakage, chromosome rearrangements, and gene amplification. In a recent study, we reported that lung cancer cases and matched controls had differential sensitivity to the genotoxic effects of the tobacco-specific nitrosamine NNK. The lymphocytes from patients with lung cancer were significantly more sensitive to NNK than were those from controls, with 1.6-, 3.4-, and 8.9-fold increases in micronuclei, nucleoplasmic bridges, and nuclear bud frequencies, respectively. Our results also indicated that these cytogenetic endpoints appear to be highly predictive of lung cancer status (30
). Over the past few years, the CBMN assay has been modified to measure not only chromosome breakage, chromosome loss, and nondis-junctions but also other cellular events, such as apoptosis and necrosis (8
), and is now known as the CBMN Cyt assay (11
). To our knowledge, our current report is the first case-control study to evaluate the added benefit of including these additional variables (that could be simultaneously evaluated as part of the CBMN Cyt assay) to test their effect on improving the predictive value of this assay and also to provide insight into the underlying mechanisms in lung carcinogenesis.
Our results showed that the cases had significantly higher levels of baseline micronuclei in mononucleated cells than did the controls, indicating a significantly higher level of in vivo
genetic damage and cytotoxicity. It is possible that some of the lymphocytes already harbor micronuclei before they are stimulated to divide in culture (10
). This is especially plausible in situations of increased genomic instability or chronic exposure to genotoxins. Micronuclei in mononucleated cells may also represent cells in which the replicated DNA escaped nuclear division or cells that divided but escaped cytochalasin B block. Micronuclei in mononucleated cells may also represent cells that are at a very early stage of induced cell death (either apoptosis or necrosis;ref. 10
). In our study, the cases had significantly higher levels of NNK-induced micronuclei in mononucleated cells, which may reflect an increase in the number of damaged cells that failed to divide. This possibility is supported by the cell division data showing a significantly reduced number of cells proceeding to the second and subsequent cell divisions in the cases compared with controls. Our data did not reveal any substantial differences when the frequencies of spontaneous or induced micronuclei in mononucleated cells were stratified by patient age or smoking history.
It is important to note that all the cases and controls were heavy smokers and within the same age group and were heavy smokers; therefore, the effect of age or smoking could not be evaluated. In addition, our data showed no substantial differences when the frequencies of the endpoints (spontaneous or induced) were stratified by disease stage or tumor histology, thus excluding the probability of being a disease marker.
The extent of the observed DNA damage is dependent on the extent of other cellular events, such as necrosis and/or apoptosis. Necrosis leads to the release of degradative enzymes that cause partial digestion of the DNA during the early stages (12
). Our results indicate that the lung cancer cases had a significantly higher level of spontaneous necrotic cells than did the controls (P
< 0.001). This could be due to the chronic inflammatory milieu in the cases with increased necrosis (36
). Similarly, the cases showed significantly higher levels of NNK-induced necrosis compared with controls, reflecting a higher differential sensitivity of blood lymphocytes in the cases not only to the genotoxic effect of the NNK but also to the cytotoxic effects of the chemical. This observation is supported by the significantly reduced NDCI in cases compared with controls.
The lung cancer cases in our study had a higher baseline level of apoptotic cells than did the controls. Although intriguing, this observation could potentially be explained by the increased oxidative burden in the cancer cases. Oxidative stress generates reactive oxygen species that are known to induce cellular senescence and apoptosis (37
). The tobacco carcinogen NNK has been reported to induce apoptosis, either through induction of reactive oxygen species (39
) or through a mechanism that involves β-adrenergic-mediated release of arachidonic acid (40
). Although the cancer cases had a higher frequency of apoptotic cells than did the controls, the induction of apoptosis by NNK was lower in cases than in controls, indicating the possibility of defective apoptotic machinery in the cases, thus allowing for the survival of damaged cells. NNK is considered to be a major contributor to lung carcinogenesis in smokers (41
). It has been reported that NNK induces DNA damage (29
), DNA adduct formation, increased oxidative stress (43
), and p53 and Ras mutations (29
). It has been recently reported that NNK induces phosphorylation of Bcl2, facilitating the interaction between Bcl2 and c-Myc, which in turn stabilizes c-Myc protein and enhances its global inhibition of apoptosis, thereby contributing to lung cancer development and proliferation of tumor cells (44
One of the added advantages to the application of the CBMN Cyt assay is that it could be applied to large populations, because only a small amount of blood is needed, a wide spectrum of genetic damage in cells could potentially be assessed at a relatively low cost, and it is technically not as demanding as the conventional chromosome aberrations assays (45
). To comprehensively evaluate the added benefit of using the overall data, we used FPC analysis to identify the measures that are most important for distinguishing among cases and controls and to predict case status. Our data indicated that including the MN-Mono data in the analysis improved the PPV from 94.8% [PPV derived from NNK-induced MN-BN, NPB-BN, and Buds-BN] to 98.7% [NNK-induced MN-BN, NPB-BN, Buds-BN, and MN-Mono]. Similarly, the best NPV were observed when MN-Mono data were included in the analysis: 95.6% [NPV derived from NNK-induced MN-BN, NPB-BN, Buds-BN, and MN-Mono] versus 92.6% [NNK-induced MN-BN, NPB-BN, and Buds-BN]. Unfortunately, owing to the relatively small sample sizes that led to model convergence problems, we were not able to simultaneously evaluate the discriminatory power of the FPC (including MN-Mono) and NDCI. Larger sample sizes are therefore needed to comprehensively evaluate the added effect of cellular events (such as apoptosis, necrosis, and cell proliferation) on improving the predictive values of this biomarker assay.
In summary, our study showed that the CBMN Cyt assay is an exquisitely sensitive and specific predictor of lung cancer risk. The addition to the assay of micronuclei in the mononucleated cells not only improves the PPV and the NPV but also provides an added advantage to evaluating the level of in vivo
genetic instability and delay in cell proliferation that may in turn shed light on the underlying mechanisms of the carcinogenic process. The simplicity, rapidity, and sensitivity of the CBMN test make it a valuable tool for screening and possibly for prioritizing potential cases for intensive surveillance. This assay appears to provide results that yield more accurate predictions than other phenotypic assays currently undergoing assessment in this population of lung cancer cases and controls. Lung cancer is the leading cause of cancer mortality in the United States, and there is an urgent need to improve outcome by identifying and validating markers to predict risk (1
). Prevention of even 10% of annual deaths from lung cancer would save an estimated 17,000 lives, equivalent to all the annual deaths in the United States from ovarian cancer and almost all the annual deaths from brain cancers. Clinically, the ability to identify high-risk subgroups is imperative, where such individuals might benefit from increased screening surveillance that is not appropriate for low-risk individuals. Additionally, the high-risk subgroups could be targeted for prevention trials.