In this multi stage analysis, using independent sets of cases and controls for discovery and replication, we were able to successfully replicate 6 SNPs in the inflammation pathways in former smokers and 5 different SNPs in current smokers that were statistically significant in both groups with almost identical risk estimates in the discovery and replication phases. In a subsequent meta-analysis from three additional external studies, two of these variants achieved statistical significance. These were rs1544669 in BCL2L14 in former smokers and rs2235330 in IL2RB in current smokers.
Inflammation is a physiological response to cellular and tissue damage. Appropriate response to this damage is tightly regulated through a balance between pro-inflammatory and anti-inflammatory cytokines and signaling molecules (25
). It is suggested, in fact, that the tumor microenvironment, and especially its inflammatory component may be a critical element of carcinogenesis (26
). Since common variations in a single gene contribute only modestly to risk, it seems logical that rather than focusing on a few SNPs and/or genes with the strongest evidence of disease association, one considers multiple variants in interacting or related genes in the same pathway to improve the power to detect causal pathways and disease mechanisms (27
). Loza et al. (6
) constructed a comprehensive inflammation pathway gene list and functionally defined subpathways that formed the basis for our own analysis.
The replicated SNP in current smokers was in the Interleukin-2 receptor subunit beta (IL2RB)
gene, a cytokine signaling gene. IL-2 exerts both stimulatory and regulatory functions in the immune system and is a member of the cytokine family that is central to immune homeostasis (28
). IL-2 binds to its receptor complex, IL-2R alpha, beta, and gamma chains, and exerts its effect via second messengers, mainly tyrosine kinases, which ultimately are involved in T cell-mediated immune responses. Local blockade of the beta-chain of the IL-2R restored an immunosuppressive cytokine milieu that ameliorated both inflammation and airway hyperresponsiveness in experimental allergic asthma (29
). Interleukin-2 (IL-2) is the major growth factor for activated T-lymphocytes and stimulates clonal expansion and maturation of these lymphocytes.
In former smokers, we replicated a SNP in the BCL2L14
gene, located on chromosome 12, and that encodes Apoptosis facilitator Bcl-2-like protein 14
. Loss of heterozygosity of the short arm of chromosome 12 is a frequent event in both hematological malignancies and solid tumors. (30
, also known as Bcl-G
, is a proapoptotic member of the Bcl-2 family which regulates cell death (31
). This gene has been shown to be a transcriptional target of TP53 (32
) and is a candidate tumor suppressor. Association of this gene with lung cancer has not been previously reported. Since apoptosis plays an essential role in protecting against cellular carcinogenesis, such as one due to oxidative damage from cigarette smoke, it is biologically plausible that this cell death regulator might influence the pathogenesis of lung cancer through the TP53 pathway. It is not surprising that different findings were observed in current versus former smokers. For example, current and former smokers differ with respect to the role that COX-2 plays in maintaining bronchial epithelial proliferation (33
), and differences between these two groups with respect to bronchial epithelial biology have been reported (34
). Different biology is also suggested by the observation that active smokers have faired poorly in large-scale chemoprevention trials, whereas former smokers have exhibited no effect or favorable trends (33
There are a few published, generally small, candidate gene studies on polymorphisms in inflammation-related genes that have been linked with increased lung cancer risk, but with limited replication of the study results, as reviewed by Engels (36
). For example, Hart et al (37
) studied 11 SNPs in nine genes in 882 subjects and reported risk by combination of adverse genotypes, but there was no replication phase. Vogel et al (38
) included 7 inflammation pathway SNPS in their case-cohort study that included 428 cases. Carriers of a variant allele of IL10 and IL1B were at increased risk, although the latter was statistically significant only in current smokers. We (39
) have previously published case control data on over 1000 cases and a similar number of controls from the study base and reported a significant association with a SNP in ILIB
. In the International Lung Cancer Consortium (ILCCO), we conducted a coordinated genotyping study of 10 common variants including this IL1B variant in 4588 cases and 6453 controls but found no association with this IL1B
), nor was this specific IL1B
variant included in our discovery analysis.
Our discovery and replication data are derived from a retrospective case-control analysis and therefore we are unable to effectively evaluate any meaningful association with inflammatory marker levels since pre-diagnostic sera are not available. However, an analysis of pre-diagnostic C reactive protein levels in the PLCO data showed a significant association with subsequent lung cancer risk (41
). Another limitation of our study was the lack of genotyping for all of the SNPs included in our discovery study so that we could not validate risk score predictions. Further studies that independently evaluate the risk scores we developed are needed.
There are clear advantages to this pathway-based approach. Since we restricted our analysis to a specific pathway, we have reduced to some extent the issue of false positive reporting, and increased the power of our analyses. Our genes are classified by functional subpathways and thus we were able to evaluate associations in a more biologically driven manner. We cannot exclude the possibility that, despite our three stage analytic approach, the findings represent false positives. However, the relatively large sample sizes with dual discovery and replication populations, detailed epidemiologic information, and comprehensive query of genes and SNPs ensure robust power and greater genetic coverage to detect true-positive findings. Follow-up analysis in African American lung cancer cases and controls is ongoing to confirm and extend these findings.