This study demonstrates that total NNAL in serum is significantly associated with lung cancer risk in samples from the PLCO study, even after controlling for intensity and duration of smoking. The analysis was controlled simultaneously for both duration (years of smoking) and intensity (cotinine levels) of smoking and so the estimated effect of NNAL is unlikely to be due to residual confounding from smoking patterns. Further, we simultaneously controlled for both the tobacco carcinogen biomarkers to avoid mutual confounding. Based on our regression, which assumes a linear relation between concentration and log odds, for a unit standard deviation increase in NNAL of 40 fmol per ml of serum, the odds for lung cancer increased 1.57 times (95% confidence interval:,1.08, 2.28). As suggested by , the effect of other increases in NNAL can be predicted from the estimated relationship between total NNAL and risk. For example, the odds increase about 1.7 times (95% CI: 1.1, 2.8) from the 25th to 75th percentiles of total NNAL (57.3 to 108 fmol/ml), and about 4.2 times (95% CI: 1.3, 13.7) from the 5th to 95th percentiles (23.2 to 147.0 fmol/ml). This suggests that, among long-term heavy smokers, the total serum NNAL level is a major determinant of lung cancer risk conferred by cigarette smoking.
Age, the only other factor statistically significantly associated with increased lung cancer risk in the multiple logistic regression, was previously well established. Although the unadjusted two-way association of smoking duration with lung cancer risk was statistically significant, when controlled for the biomarkers and other factors it was not. This may be due to the relatively small sample size here and should not be interpreted to indicate that it is not an important risk factor. Note that the distributions of biomarkers observed in this study are similar to those measured in other studies.
To our knowledge, this is the first study to demonstrate a relation between a specific quantifiable tobacco-related lung carcinogen biomarker – total NNAL – and lung cancer in smokers. Several studies, including one prospective study, have examined the relation between lung cancer and “bulky DNA adducts,” “aromatic DNA adducts” or “PAH-DNA adducts,” in white blood cells of smokers using immunoassay or 32
P-postlabelling; the results generally demonstrate increased risk with increased adduct levels.(16
) However, these methods are non-specific to a particular carcinogen, and it is not known with certainty which cigarette-smoke carcinogens actually give rise to the measured adducts, although it is likely that PAHs are responsible, in part.
Our finding that total NNAL is related to lung cancer in smokers is biologically plausible. NNK has long been suspected to be a human lung carcinogen. NNAL and its glucuronides are metabolites of NNK in humans and do not come from any other sources; urinary total NNAL is related to NNK dose and correlates with total serum NNAL.(3
) NNK itself is extensively metabolized and cannot be detected in human urine. NNK causes lung cancer, predominantly adenocarcinoma, in all laboratory animal species tested, including rats, mice, hamsters, and ferrets, and it does so independently of the route of administration.(22
) It is particularly effective in rats, in which a total dose of only 6 mg/kg caused a significant incidence of lung tumors.(24
) The carcinogenic activity of NNAL is similar to that of NNK.(22
) Based on these data and associated mechanistic evidence from laboratory animals and humans, the International Agency for Research on Cancer has concluded that NNK and the related tobacco-specific nitrosamine N′
-nitrosonornicotine are carcinogenic to humans,(25
) and the present results certainly support that evaluation by directly estimating the effect in humans.
The measures of smoking duration (years of smoking) and intensity (serum cotinine) used are imperfect. One might hypothesize that NNAL is merely sweeping up residual effects of smoking duration and intensity through its correlation with other carcinogens, a hypothesis which an observational study cannot refute deductively. However, the fact that cotinine, for which NNAL may be a proxy, has too small a relationship with risk in this modestly sized study to be statistically significant, and inclusion of total NNAL reduces the otherwise statistically significant effect of duration, for which it is not a proxy, argues against this interpretation. Further, this hypothesis would require that NNAL, controlled for smoking duration and intensity, is highly associated with some other set of unmeasured carcinogens that do cause lung cancer, while the carcinogen with which it is most highly associated (NNK) does not cause lung cancer. This could happen if some strong physical or biological association between NNK and these putative carcinogens existed, for example, if they were products of the same biological process. But since total NNAL is most highly correlated with intake of NNK, a known lung carcinogen in many species of mammals, and much less highly correlated with other carcinogen intake (e.g., PAHs), we find this interpretation less plausible and find our results as highly confirmatory of the importance of NNK in lung cancer carcinogenesis among smokers.
The effect of total NNAL estimated in this study appears modest compared to the effect of smoking overall because it is the estimated difference in risk for individuals with a given smoking history, age, sex, and family history of lung cancer. Therefore, the reported effect is due only to differences in total NNAL levels. These differences could arise from differing concentrations of NNK in the cigarettes smoked or from biological variability in the absorption and metabolism of NNK. In any case, the NNAL effect cannot be compared to the effect of smoking vs. not smoking. Rather, this increased risk is a modifier of the known, large risk of smoking and thus, importantly, supports the hypothesis that NNK and NNAL are human lung carcinogens. Of further note, NNK and NNAL require metabolic activation to exert their carcinogenic effects.(22
) Thus, the carcinogenic effects of NNK are related to dose plus the extent of metabolic activation. In this study we measured only dose; higher levels of metabolic activation (e.g., due to inherited differences or inducers/inhibitors) would potentially increase risk. We did not measure metabolites of NNK beyond total NNAL, but in future research inclusion of such biomarkers – or their ratios – may allow us to estimate the effects of activation and detoxification and to control for additional heterogeneity in risk among smokers. Such future studies may result in even stronger predictive relations between measured biomarker levels and lung cancer risk.
We did not observe associations of either PheT with lung cancer risk in this study. PheT has been proposed as a biomarker of PAH dose plus metabolic activation.(7
) PheT levels correlate with those of 1-hydroxypyrene, a well established biomarker of PAH uptake. But PheT, the product of the PAH diol epoxide metabolic activation pathway, is also a measure of metabolic activation. The lack of an association of PheT with lung cancer in this study was somewhat surprising in view of the strong evidence linking PAH to lung cancer etiology in smokers.(4
) However, unlike NNAL, PheT is not tobacco-specific, and diet is another more variable source of PAH exposure which may attenuate the observed effect of PAH from cigarette smoking. Also, note that the rather high variance of PheT measurements relative to mean levels makes the power for a given proportional change smaller than that for NNAL. This finding motivates further studies to evaluate PheT as a marker of risk for lung cancer. Markers of tobacco-specific PAH exposure in general may be intractable due to the many environmental sources for PAH; nonetheless, finding biomarkers of PAH metabolic activation would be valuable to understanding the role of tobacco smoke in lung cancer.
In this study, we observed no association between serum cotinine levels and lung cancer risk, although serum cotinine correlated with serum total NNAL and smoking intensity (data not shown), consistent with previous studies of urinary cotinine and total NNAL.(3
) We included cotinine in the regression as a more accurate proxy for intensity of smoking exposure rather than using categories of reported number of cigarettes per day. Boffetta and co-workers found that serum cotinine was a predictor of lung cancer risk in a study of smokers which was far larger than ours;(27
) however, they did not measure or control for total NNAL exposure. Our results suggest that total NNAL, metabolically formed from the carcinogen NNK, is a better risk marker than cotinine, a metabolite of the non-carcinogen nicotine.
Our study has certain limitations. Sample size was modest, but obviously adequate to detect the association for which it was designed. A larger study would, of course, have had more power to investigate the strength of the association of this and other biomarkers with lung cancer and within histologic subtypes. A larger study would also allow us to more closely examine the mathematical nature of the relation of NNAL to smoking patterns and to the other biomarkers and allow for more precise estimates of the relations of the biomarkers to risk. We hope these results will motivate such expanded research.
Each biological sample in this study represents only a single time point – the initial screening visit. Although no split-sample testing was done in this study due to the small amount of sample available, a previous publication estimated the within-day and between-day relative standard deviations for the NNAL laboratory assay to be 7.7% and 11.7%, respectively. However, we have shown, in a recently completed longitudinal study (unpublished), that the average coefficient of variation of serum NNAL levels over a one-year period in 50 smokers sampled every 2 months was about 30%. Thus, most of the variability within a subject comes from biological variability rather than assay reliability. This variability would serve to attenuate the estimated effect of the NNAL, suggesting that the true effect might likely be even larger than the estimated effect.
The results of this study have potentially profound implications for lung cancer etiology, because they support the hypothesis that NNK is a lung carcinogen in smokers. By focusing on NNK and the pathways related to its activation and detoxification, we may be able to develop preventive approaches. Previous studies using total NNAL to measure NNK uptake in smokers who used different products or reduced their smoking, and in non-smokers exposed to secondhand tobacco smoke demonstrate great variability in levels of exposure to NNK.(3
) Now those data can also be seen to reflect potential variability in lung cancer risk and have implications for future studies that use total NNAL as a biomarker. Furthermore, lung cancer chemoprevention approaches based on NNK in rodent models now gain more salience to humans.(34
) Ultimately, as further research refines its estimated effect and delineates how it interacts with other biological measures, total NNAL may become part of an algorithm that includes metabolic activation and DNA repair measures to predict lung cancer susceptibility in people who choose to smoke cigarettes. In long-term smokers, such an algorithm could be used in cessation programs or surveillance activities. In newer smokers, better risk information could better motivate early cessation efforts. The assay is limited in that it can only be done on current smokers, and so could not help those considering smoking initiation or those who have already given it up.
In summary, we report here for the first time that total NNAL, a biomarker of uptake of the tobacco-specific lung carcinogen NNK, is related to the risk of lung cancer in smokers. These results are consistent with previous carcinogenicity and mechanistic studies that point to NNK as an important cause of lung cancer in smokers.