Cotinine Conundrum--A Step Forward but Questions Remain

Abstract

There is widespread agreement that self-reported smoking quantity, though a convenient and simple measure, is an imprecise assessment of exposure to tobacco smoke and that measuring serum cotinine levels in cigarette smokers provides a more objective estimate of current smoking behavior. Initial genome-wide association studies (1–3) suggested that single-nucleotide polymorphisms (SNPs) spanning the chromosome 15q25 region encoding the a5, a3, and b4 nicotinic acetylcholine receptor (nAChR) subunit gene cluster, CHRNA5-CHRNA3-CHRNB4 (CHRNA5-A3-B4), were associated with both smoking intensity and lung cancer risk. Subsequent analyses robustly associated these SNPs with heavy smoking, nicotine dependence, craving, and related endophenotypes (4–7). The association with lung cancer, though statistically robust and initially not altered by adjustment for smoking, increasingly appears to be mediated through smoking. However, there is still uncertainty regarding a direct effect of the variants on lung cancer risk or if the risk for lung cancer is mediated solely through the genetic risk to smoking. In this issue of the Journal, Munafo et al. (8) provide convincing evidence that genetic variation at chromosome 15q25 locus influences cotinine levels more strongly than smoking quantity (self-reported cigarettes per day). Two single-nucleotide variants in this region were studied for their association with serum cotinine level and smoking intensity—rs16969968, which has a functional effect on nicotine signaling mediated by CHRNA5, and rs1051730, which is strongly correlated with rs16969968. Their data from 2932 smokers replicate and extend those reported in 2009 by Keskitalo et al. (9) in a smaller sample size. Both of these studies showed a much stronger association between variants in the CHRNA5-A3-B4 gene cluster with cotinine than with reported cigarette per day use. In an interesting and valuable application of their results to a published case–control study of cotinine levels and lung cancer risk, Munafo et al. (8) estimated that the per allele increase in cotinine level indicated a 31% increased risk of lung cancer per risk allele of rs16969968, an effect size that is very similar to the effect sizes of the GWAS odds ratios for lung cancer risk for these risk alleles. Therefore, the authors (8) conclude that the association of these variants with lung cancer risk is mediated largely, if not exclusively, through their effect on increasing tobacco exposure. It is true that the association of the chromosome 15 region and lung cancer is not seen in nonsmokers (10). Yet, a direct association of this locus with lung cancer risk, independent of its role in nicotine dependence, is still disputed. There are compelling data that nicotine and its derived carcinogenic nitrosamines can contribute directly to lung cancer risk through binding to nAChRs, which then activate proliferation, apoptosis, angiogenesis, and tumor invasion pathways, as well as phosphorylation of the AKT pathway (11). Lam et al. (12) reported different nAChR subunit gene expression patterns in non–small cell lung cancer from never and ever smokers and demonstrated that nicotine exposure in human bronchial epithelial cells resulted in reversible differences in nAChR subunit gene expression. These data all seem to implicate nicotinic receptor activity in bronchial carcinogenesis. It could be argued that the decision regarding what measure of tobacco exposure to use depends on the outcome phenotype of interest. For accurate classification of current smoking intensity, serum cotinine levels might be the measurement of choice as an objective marker of recent exposure because they remain relatively stable over time in frozen serum samples. Nevertheless, variability in the measurement and the biological limitations of cotinine as a biomarker (short half-life, poorer performance of serum cotinine than urine cotinine as a dosimeter of recent smoking), as well as cost, must be factored into widespread use of this biomarker. For nicotine dependence, there are a variety of available validated measures, including the Fagerstrom Test of Nicotine Dependence (FTND) or the Nicotine Dependence Syndrome Scale, that estimate dependence quite accurately. Chen et al. (13) have shown that none of the more comprehensive measures of smoking behaviors yielded stronger genetic associations with the chromosome 15q25 locus variants than did cigarettes per day. However, other regions of the genome may not have this same relationship between cigarettes per day and nicotine dependence measures.