We hypothesize that innate immunity pathways, inflammation biomarkers and body mass index (BMI) are associated with lung cancer survival. The specific aims designed to test this central hypothesis are: C.2.1 To examine serum levels and polymorphisms in the innate immunity genes MBL2 and MASP2 and their relationship with lung cancer survival. Using the National Cancer Institute - Maryland (NCI-MD) case-control study, we recently found that functional variants in the innate immunity gene MBL2 were associated with lung cancer survival 1. As these are functional SNPs that influence expression of MBL-2 protein, we hypothesize that circulating levels of MBL-2 will also be associated with survival. Moreover, we hypothesize that SNPs and levels of the MBL-2 interacting protein, MASP-2, are also associated with lung cancer survival. To achieve this aim, the SNPs MBL2_01, MBL2_02, MBL2_03, MBL2_11, MBL2_12, MASP2_36, MASP2_42 and MASP2_43 will be tested for their association with lung cancer survival. As these SNPs were previously analyzed in a lung cancer study nested within PLCO, we will request survival data only for this analysis. Levels of serum MBL-2 and MASP-2 were assayed as part of our previous submission to PLCO (a study focused on lung cancer risk); therefore to assess the relationship between MBL-2 and MASP-2 serum levels with survival we will not require additional serum samples. Polymorphisms (individual and haplotypes) will be assessed for their association with lung cancer survival. Interactions between serum levels of MBL-2 and MASP-2 will also be tested with the functional polymorphisms previously listed. Moreover, as MBL-2 is a key activator of MASP-2 in the complement cascade, we will also investigate the potential for an interaction between increased levels of MBL-2 and MASP-2. C.2.2 To measure inflammatory markers in serum and their relationship with lung cancer survival. We recently found that increased levels of IL-6 and IL-8—measured both at (NCI-MD case-control study) and before (PLCO) diagnosis—were associated with increased risk of lung cancer incidence 1. We also found that increased levels of circulating IL-6, measured at the time of diagnosis, were associated with poor survival among lung cancer patients within the NCI-MD case-control study 2. Therefore, we propose to validate the association between IL-6 and survival in Caucasians within PLCO. Moreover, we propose to determine whether functional SNPs known to influence cytokine levels modulate the association between these inflammatory markers and lung cancer survival. Specifically, we will assess the SNPs IL-1B_03, IL-8_01 and TNFa _02. As the circulating inflammatory cytokines (IL-1B, IL-6, IL-8 and TNFa ) and SNPs listed above were measured in PLCO as part of our previously submitted proposal, this aim will only request survival data. C.2.3 To examine the relationship between body-mass-index (BMI) in relation to lung cancer risk and survival. C.2.3.1: Examine the association between BMI at age 20 years, age 50 years and age at study entry with lung cancer risk, stage at diagnosis, histology and survival among participants in the PLCO study. Data from our analyses in the NCI-MD lung cancer case-control study have demonstrated that there are significant associations between BMI at baseline and BMI 10 years prior to diagnosis in relation to the outcomes of lung cancer risk, stage and survival (manuscript in preparation). In addition, we found an association between BMI and histological sub-type of lung cancer. Specifically, we found an increased risk of lung adenocarcinoma with increased BMI prior to diagnosis. In contrast, prior to diagnosis increased BMI was associated with a reduced risk of lung squamous cell carcinoma. In order to validate our findings in a prospective cohort, we propose using the PLCO data to conduct a similar analysis of lung cancer risk, stage, histology and survival to assess the exposures BMI. C.2.3.2: Determine how changes in BMI between early, middle adulthood and baseline affect the association with lung cancer risk, survival, stage at diagnosis and histology. While our previous aim looked at static measures of BMI over the life-course and its association with lung cancer, it is also possible that changes in BMI over time could also influence lung carcinogenesis. Therefore, we will investigate whether changes in BMI between early adulthood (age 20), middle adulthood (age 50) and age at study entry affect the relationship between BMI and lung cancer risk, stage, histology and survival. C.2.3.3: Assess the relationship between BMI with selected immunity SNPs and inflammatory marker levels from Specific Aims C.2.1 and C.2.2. Little is known about inflammatory pathways that may be altered as a result of changes in BMI and their effect on lung cancer risk and survival. In addition, how genetic variations within inflammatory pathways modulate these effects are also largely unknown. To address this question, we propose using the SNPs and inflammatory markers identified in Aim C.2.1 and C.2.2 in combination with our results from Aim C.2.3 to illuminate the possible immunological factors associated with BMI and lung cancer. C.3 SIGNIFICANCE OF RESEARCH: An estimated 226,160 new cases of lung cancer may be diagnosed in 2012, accounting for approximately 15% of all cancer diagnoses 3. A number of environmental risk factors for lung cancer have been identified, however cigarette smoking is responsible for >80% of the lung cancer burden 4,5. More than 90% of lung cancer patients are smokers, but the fact that only 10% of smokers develop lung cancer suggests that genetic and acquired host factors play a role in the natural history of the disease. Epidemiologic studies of familial aggregation of lung cancer provide indirect evidence for a genetic role in predisposition to lung cancer 6. Both smoking and non-smoking relatives of lung cancer patients have an increased risk of lung cancer. Moreover, lung cancer patients with a first-degree relative with lung cancer have, on average, poorer outcomes than those without such a history 7,8. Thus, susceptibility to lung cancer, and an individual's outcome, may be in part due to inter-individual genetic variation in the form of genetic polymorphisms, or common allelic variants 9. In addition to the second highest incidence rate, lung cancer has the highest mortality rate of any cancer; 160,340 deaths are expected in 2012 3. Although the 1-year survival rate for lung cancer has increased in recent years, due largely to improvements in surgical techniques and targeted, combination therapies, the 5-year survival rate is still less than 17%. It is notable that this rate increases to ~50% when cancer is diagnosed at an early stage; however, the majority of lung cancers are diagnosed at a late stage 3. Despite downward trends of lung cancer mortality in men, death from lung cancer continues to rise in women and remains by far the leading cause of cancer-related death in men and women. Epidemiological studies show that chronic inflammation is associated with lung cancer. For example, several triggers of chronic inflammation, such as asthma, chronic interstitial lung fibrosis and COPD increase cancer risk 6,10-13. Moreover, environmental exposures including tobacco smoke and occupational exposures that cause inflammation in the respiratory tract and lung parenchyma are important risk factors for lung cancer. Inflammatory cells may participate in lung carcinogenesis by generating reactive oxygen and nitrogen species, by secreting growth stimulatory cytokines, and by contributing to the formation of DNA adducts. In addition, the tumorigenic potential of an inflammatory environment may be augmented in cases where the genetic background interacts with inflammatory markers. Conversely, the immune system may also provide protection against malignancies, and innate immunity pathways may regulate survival and migration of tumor cells 12,14. Identifying biomarkers that quantify such signals and inform on both diagnosis and prognosis could prove extremely useful in the management of lung cancer. Previous work has defined the association between chronic inflammation and lung cancer risk. However, few studies have examined the association between chronic inflammation and lung cancer survival. Some have found that patients with a systemic inflammatory response had an increased risk of lung cancer mortality 15-17. Moreover, there is accumulating evidence that inflammation can modulate response to therapy 18-20. As evidence for a relationship between inflammation and cancer survival mounts, there is a possibility that inflammation could be a possible target for the treatment of cancer. Phase I and phase II clinical trials, with the recently developed monoclonal antibody against IL-6, are further evidence of this possibility 21. However, the precise mediators of this relationship have not been extensively studied. Inflammation is also thought to be one of the key mediators in the relationship between obesity and cancer. The prevalence of obesity in the U.S. has more than doubled over the past 50 years. Combined, individuals who are either overweight (32.7%) or obese (34.3%) represent approximately two thirds of the adult U.S. population. The health risks associated with being overweight or obese include diabetes, cardiovascular disease and cancer. Moreover, the heightening obesity epidemic comes with a hefty economic burden also; current annual estimates are approximately $147 billion 22. The National Cancer Institute estimates that 5.5% of all new cancers are due to obesity, and deaths from cancer attributed to obesity are 14% in men and 20% in women. Chronic inflammation is one mechanism that has been proposed to explain how obesity alters cancer risk. BMI has been associated with pro-inflammatory cytokines IL-6 and TNFa in lung cancer 23, though the role of the obesity-associated cytokine leptin in lung cancer risk and survival remains controversial 24,25. Interestingly, several studies show an inverse relationship between lung cancer and BMI 22,26-30 that is independent of the residual confounding effects from cigarette smoking 31. Lung cancer survival is also affected by BMI both at baseline and prior to diagnosis 30,32-35, however, few epidemiological studies have addressed the question of how BMI several years before diagnosis or changes over time in BMI alters the etiology of lung cancer. Thus, in line with the objectives of the PLCO, we will investigate the potential ability of inflammatory cytokines to predict populations at higher risk of lung cancer mortality, so that those at risk can be targeted for more rigorous screening and treatment. As pre-diagnostic levels of IL-6 and IL-8 are associated with an increased risk of lung cancer, we will investigate the tangible possibility that these biomarkers are also associated with lung cancer survival. Our analyses will follow an integrated approach with serum biomarkers and genetic markers. We will also explore the relationship between BMI with lung cancer risk, stage, histology and survival. Upon completion of this study, our findings regarding BMI and its association with lung cancer risk, histology, stage and survival will identify some of the most important lung cancer outcomes associated with BMI. Our approach will also address how chronic obesity and/or changes in weight prior to cancer diagnosis impact these outcomes. Preliminary data suggests that obesity, as assessed by BMI, is associated with a reduced risk of lung cancer incidence and mortality. Moreover, in the context of this proposal, we will also start to tease apart the relationship between BMI and several key pro-inflammatory pathways and their relationships with lung cancer risk and survival. Beyond stage, there are no validated biomarkers that can accurately predict the aggressiveness of lung tumors. Thus, the results from these studies have the potential to inform the use of biomarkers within screening programs. We hypothesize that innate immunity pathways, inflammation biomarkers and body mass index (BMI) are associated with lung cancer survival. The specific aims designed to test this central hypothesis are: C.2.1 To examine serum levels and polymorphisms in the innate immunity genes MBL2 and MASP2 and their relationship with lung cancer survival. Using the National Cancer Institute - Maryland (NCI-MD) case-control study, we recently found that functional variants in the innate immunity gene MBL2 were associated with lung cancer survival 1. As these are functional SNPs that influence expression of MBL-2 protein, we hypothesize that circulating levels of MBL-2 will also be associated with survival. Moreover, we hypothesize that SNPs and levels of the MBL-2 interacting protein, MASP-2, are also associated with lung cancer survival. To achieve this aim, the SNPs MBL2_01, MBL2_02, MBL2_03, MBL2_11, MBL2_12, MASP2_36, MASP2_42 and MASP2_43 will be tested for their association with lung cancer survival. As these SNPs were previously analyzed in a lung cancer study nested within PLCO, we will request survival data only for this analysis. Levels of serum MBL-2 and MASP-2 were assayed as part of our previous submission to PLCO (a study focused on lung cancer risk); therefore to assess the relationship between MBL-2 and MASP-2 serum levels with survival we will not require additional serum samples. Polymorphisms (individual and haplotypes) will be assessed for their association with lung cancer survival. Interactions between serum levels of MBL-2 and MASP-2 will also be tested with the functional polymorphisms previously listed. Moreover, as MBL-2 is a key activator of MASP-2 in the complement cascade, we will also investigate the potential for an interaction between increased levels of MBL-2 and MASP-2. C.2.2 To measure inflammatory markers in serum and their relationship with lung cancer survival. We recently found that increased levels of IL-6 and IL-8—measured both at (NCI-MD case-control study) and before (PLCO) diagnosis—were associated with increased risk of lung cancer incidence 1. We also found that increased levels of circulating IL-6, measured at the time of diagnosis, were associated with poor survival among lung cancer patients within the NCI-MD case-control study 2. Therefore, we propose to validate the association between IL-6 and survival in Caucasians within PLCO. Moreover, we propose to determine whether functional SNPs known to influence cytokine levels modulate the association between these inflammatory markers and lung cancer survival. Specifically, we will assess the SNPs IL-1B_03, IL-8_01 and TNFa _02. As the circulating inflammatory cytokines (IL-1B, IL-6, IL-8 and TNFa ) and SNPs listed above were measured in PLCO as part of our previously submitted proposal, this aim will only request survival data. C.2.3 To examine the relationship between body-mass-index (BMI) in relation to lung cancer risk and survival. C.2.3.1: Examine the association between BMI at age 20 years, age 50 years and age at study entry with lung cancer risk, stage at diagnosis, histology and survival among participants in the PLCO study. Data from our analyses in the NCI-MD lung cancer case-control study have demonstrated that there are significant associations between BMI at baseline and BMI 10 years prior to diagnosis in relation to the outcomes of lung cancer risk, stage and survival (manuscript in preparation). In addition, we found an association between BMI and histological sub-type of lung cancer. Specifically, we found an increased risk of lung adenocarcinoma with increased BMI prior to diagnosis. In contrast, prior to diagnosis increased BMI was associated with a reduced risk of lung squamous cell carcinoma. In order to validate our findings in a prospective cohort, we propose using the PLCO data to conduct a similar analysis of lung cancer risk, stage, histology and survival to assess the exposures BMI. C.2.3.2: Determine how changes in BMI between early, middle adulthood and baseline affect the association with lung cancer risk, survival, stage at diagnosis and histology. While our previous aim looked at static measures of BMI over the life-course and its association with lung cancer, it is also possible that changes in BMI over time could also influence lung carcinogenesis. Therefore, we will investigate whether changes in BMI between early adulthood (age 20), middle adulthood (age 50) and age at study entry affect the relationship between BMI and lung cancer risk, stage, histology and survival. C.2.3.3: Assess the relationship between BMI with selected immunity SNPs and inflammatory marker levels from Specific Aims C.2.1 and C.2.2. Little is known about inflammatory pathways that may be altered as a result of changes in BMI and their effect on lung cancer risk and survival. In addition, how genetic variations within inflammatory pathways modulate these effects are also largely unknown. To address this question, we propose using the SNPs and inflammatory markers identified in Aim C.2.1 and C.2.2 in combination with our results from Aim C.2.3 to illuminate the possible immunological factors associated with BMI and lung cancer.