Circulating high sensitivity C reactive protein concentrations and risk of lung cancer: nested case-control study within Lung Cancer Cohort Consortium

Abstract Objectives To conduct a comprehensive analysis of prospectively measured circulating high sensitivity C reactive protein (hsCRP) concentration and risk of lung cancer overall, by smoking status (never, former, and current smokers), and histological sub-type. Design Nested case-control study. Setting 20 population based cohort studies in Asia, Europe, Australia, and the United States. Participants 5299 patients with incident lung cancer, with individually incidence density matched controls. Exposure Circulating hsCRP concentrations in prediagnostic serum or plasma samples. Main outcome measure Incident lung cancer diagnosis. Results A positive association between circulating hsCRP concentration and the risk of lung cancer for current (odds ratio associated with a doubling in hsCRP concentration 1.09, 95% confidence interval 1.05 to 1.13) and former smokers (1.09, 1.04 to 1.14) was observed, but not for never smokers (P<0.01 for interaction). This association was strong and consistent across all histological subtypes, except for adenocarcinoma, which was not strongly associated with hsCRP concentration regardless of smoking status (odds ratio for adenocarcinoma overall 0.97, 95% confidence interval 0.94 to 1.01). The association between circulating hsCRP concentration and the risk of lung cancer was strongest in the first two years of follow-up for former and current smokers. Including hsCRP concentration in a risk model, in addition to smoking based variables, did not improve risk discrimination overall, but slightly improved discrimination for cancers diagnosed in the first two years of follow-up. Conclusions Former and current smokers with higher circulating hsCRP concentrations had a higher risk of lung cancer overall. Circulating hsCRP concentration was not associated with the risk of lung adenocarcinoma. Circulating hsCRP concentration could be a prediagnostic marker of lung cancer rather than a causal risk factor.


Study population
We invited all prospective cohort studies that in 2009 were members in the US National Cancer Institute (NCI) Cohort Consortium to participate in the study. Additional inclusion criteria included the occurrence of at least 200 incident lung cancer cases with baseline questionnaire data and either plasma or serum samples available. Twenty-one cohorts fulfilled those criteria and accepted to participate, but one cohort failed to send biospecimens and were not included, leaving the 20 cohorts described below.

1) US cohorts The Women's Health Initiative (WHI)
WHI is a long-term health study of 161,808 post-menopausal women aged 50 to 79 years at In the present study lung cancer cases occurring during the follow-up of WHI cohorts since enrolment (1993-1998) among non-smoking women, were matched 1-1 to corresponding non-smoking lung cancer free controls, for serum and DNA analyte comparisons.

The Southern Community Cohort Study (SCCS)
The Southern Community Cohort Study (SCCS) 3 is a prospective cohort of African and non-African Americans which during 2002-2009 enrolled approximately 86,000 residents aged 40-79 years across 12 southern states. Recruitment occurred mainly at community health centers, institutions providing basic health services primarily to the medically uninsured, so that the cohort includes many adults of lower income and educational status. Each study participant completed a detailed baseline questionnaire, and nearly 90% provided a biologic specimen (approximately 45% a blood sample and 45% buccal cells). Follow-up of the cohort is conducted by linkage to national mortality registers and to state cancer registries.

Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO)
The PLCO study, a randomized trial aimed at evaluating the efficacy of screening in reducing cancer mortality, recruited approximately 155,000 men and women age 55 to 74 years from 1992 to 2001 4 . Screening for lung cancer among participants in the intervention arm included a chest x-ray at baseline followed by either three annual x-rays (for current or former smokers at enrollment) or two annual x-rays (for never smokers); participants in the control arm received routine health care. Screening-arm participants provided data on sociodemographic factors, smoking behavior, anthropometric characteristics, medical history, and family history of cancer, as well as blood samples annually for the first 6 years of the study (baseline [T0] and T1 through T5). Lung cancers were ascertained through annual questionnaires mailed to the participants, and positive reports were followed up by abstracting medical records or death certificates. Follow-up in the trial as of July 2009 was 96.7%.
We conducted a nested case-control study within the screening arm of the PLCO trial. As of December 31, 2004, 898 lung cancers were diagnosed among the 77,464 participants.
Patients were excluded because of missing baseline questionnaire, previous history of any cancer, diagnosis of multiple cancers during follow-up, missing smoking information at baseline, missing consent for utilization of biologic specimens for etiologic studies, or unavailability/insufficient quantity of serum or DNA specimens. Hemolyzed vials were excluded. Participants for this nested case-control study were sampled from the intervention arm.
Controls were individuals free of cancer at the time of a case's lung cancer diagnosis.
Controls were individually matched to lung cancer patients on sex, date of birth +/-1 year with a possible relaxation to 5 years, race, study year of blood draw, date of blood draw +/-1 month (with a possible relaxation to 3/6 months), time of blood draw (6AM-9AM, 9AM-12PM, other), smoking categories (smoking status at enrollment, never, former, or current smoker; cumulative amount of smoking (0 to 29, 30 to 39, 40 to 49, and 50+ pack-years), with additional matching for time since quitting (< 15 years and ≥ 15 years) for former smokers, cigarettes per day smoked, and number of days in the study.

The New York University Women's Health Study (NYUWHS)
The New York University Women's Health Study (NYUWHS) is a prospective cohort study of At the time of enrolment, data on demographics, anthropometric measures, medical history, reproductive and lifestyle variables were collected through self-administered questionnaires after written informed consent was obtained. Incident lung cancer cases were identified through active follow-up of the cohort conducted with questionnaires mailed approximately every two to four years and record linkages with state tumor registries in New York, New Jersey, and Florida, as well as record linkage with the National Death Index (NDI). Medical records were obtained to verify reported cancer outcomes.

The American Cancer Society Cancer Prevention Study-II (CPS-II) Nutrition Cohort
The CPS-II Nutrition Cohort is a prospective study of cancer incidence and mortality among 86,404 men and 97,786 women. The CPS-II Nutrition Cohort, which is described in detail Incident lung cancer cases were identified through self-report on a follow-up questionnaire, linkage with state cancer registries, or death certificates. Self-reported cancers were verified through medical records.

The Campaign Against Cancer and Stroke (CLUE I) and the Campaign Against Cancer and Heart Disease (CLUE II).
The CLUE studies include two large cohorts of volunteers from Washington County,

The Multiethnic Cohort (MEC)
The MEC includes over 215,000 men and women aged 45-75 years at recruitment from five different racial/ethnic groups (African Americans, Japanese Americans, Native Hawaiians, Latinos and European Americans) in Hawaii and California 6 . The cohort was assembled in 1993-1996 by mailing a self-administered, 26-page questionnaire to obtain extensive information on demographics, medical and reproductive histories, medication use, family history of various cancers, physical activity and diet. Identification of incident cancer cases is by regular linkage with the Hawaii, Los Angeles County and California SEER registries.
From 1995 to 2001, blood collection was conducted from incident cases with breast, prostate, or colorectal cancers, as well as a random sample of cohort participants to serve as controls in genomic nested case-control studies (participation rate 72% and 63%, respectively). In addition, from 2001 to 2006, blood was also collected prospectively, without regard for cancer diagnosis, from willing cohort participants. Approximately 67,000 gave a blood sample (participation rate 43%). All incident lung cancer cases diagnosed before 2010 with a pre-diagnostic blood sample were considered for inclusion in this study. Each case was matched to a control based on study site, sex, age, race/ethnicity, smoking status, hours of fasting, and date and time of blood draw.

Women's Health Study (WHS)
The WHS was a randomized trial of low-dose aspirin, vitamin E, and beta-carotene in the primary prevention of cardiovascular disease and cancer beginning in 1992 among 39,876 female US health professionals aged ≥45 years 7 . Information on major clinical, lifestyle, and dietary factors was collected via self-reports on baseline questionnaires. Women also provided baseline bloods. During more than two decades of follow-up, WHS participants reporting new cases of cancer on annual follow-up questionnaires were confirmed by medical record review by the WHS Endpoints Committee. Reports of cancer were confirmed on the basis of pathology or cytology reports or, rarely, strong clinical and radiologic or laboratory marker evidence when a pathology or cytology review was not conducted. Only confirmed cases of lung cancer were included in the present analyses, which were matched with eligible controls.

Physicians' Health Study (PHS)
The PHS I began in 1982 as a randomized trial of aspirin and beta-carotene for the primary prevention of heart disease and cancer among 22,071 male, Caucasian physicians initially aged 40 to 84 years 8 , followed by the PHS II trial beginning in 1997 to evaluate betacarotene, vitamin C, vitamin E, and a daily multivitamin on the prevention of cancer, CVD, and other endpoints. The PHS II included 14,641 men, with 7,641 participants from the PHS I plus 7,000 new physicians, for a total of 29,071 PHS participants 9 . A wide range of demographic, clinical, and lifestyle factors were assessed via baseline questionnaires, along with baseline bloods. PHS participants reported major clinical endpoints, including cancer, yearly in a mailed questionnaire and postcards every six months. Self-reported, incident lung cancer cases were confirmed through medical record review by the PHS Endpoints Committee in included in the present analyses.

The Nurses' Health Study (NHS)
The Nurses' Health Study (NHS) 10  The follow-up rate has been greater than 90%. The institutional review board at the Brigham and Women's Hospital approved the study. As approved by the committee, return of the questionnaires was considered to imply informed consent. Cases of lung cancer were selfreported by the participants or identified on their death certificates and were subsequently confirmed by medical records.

Health Professionnals Follow-up Study (HPFS)
The Health Professionals Follow-up Study (HPFS) 17 is an ongoing cohort study of 51,529 U.S. male professionals who were aged 40 to 75 years at baseline in 1986. Questionnaires have been mailed to participants every 2 years since baseline to collect updated information on demographics, lifestyle factors, mwedical history, and disease outcomes. The follow-up rate has been greater than 90%. The institutional review board at the Harvard T.H. Chan School Public Health approved this study. As approved by the committee, return of the questionnaires was considered to imply informed consent. A semi-quantitative food frequency questionnaire (FFQ) was administered to obtain information on usual dietary intake over the previous year. The FFQ was first administered in 1986 and was repeated every 4 years thereafter. The reproducibility and validity of the FFQ have been estabished 14 18 . For each food item, the questionnaire specified a common serving size and queried respondents on average intake during the previous year; responses in 9 categories ranged from almost never to 6 or more per day. Cases of lung cancer were self-reported by the participants or identified on their death certificates and were subsequently confirmed by medical records.
We would like to thank the participants and staff of the Nurses' Health Study and the Health

2) European/Australian cohorts The Melbourne Collaborative Cohort Study (MCCS)
The MCCS is a prospective cohort study of 41,514 participants (17,

The Malmö Diet and Cancer Study (MDCS)
The Malmö Diet and Cancer Study (MDCS) is a population-based prospective cohort study that between 1991 and 1996 recruited men and women aged 44 to 74 years of age living in Malmö, Sweden 20 . The main goal of the MDCS is to study the impact of diet on cancer incidence and mortality. It consists of a baseline examination including dietary assessment, a self-administered questionnaire, anthropometric measurements and collection of blood samples.

The Northern Sweden Health and Disease Study Cohort (NSHDS)
The Northern Sweden Health and Disease Study (NSHDS) encompasses several prospective cohorts, the current study involving study participants from the Västerbotten Intervention Project (VIP), a sub-cohort within NSHDS 21 . VIP is an ongoing prospective cohort and intervention study intended for health promotion of the general population of the Västerbotten County in northern Sweden. VIP was initiated in 1985 and all residents in the Västerbotten County were invited to participate by attending a health check-up at 40, 50 and 60 years of age. Participants were asked to complete a self-administered questionnaire including various demographic factors such as education, smoking habits, physical activity and diet. In addition, height and weight were measured and participants were asked to donate a fasting blood sample for future research. Incident lung cancer cases were identified through linkage with the regional cancer registry.

The Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC)
The ATBC Study was a randomized, double-blind, placebo-controlled, primary cancer prevention trial testing daily supplementation with α-tocopherol (50 mg/day) or β-carotene (20 mg/day), or both 22  For the current study, study participants were selected randomly from the different arms of the ATBC study.
Comprehensive data on life style, health status, symptoms, diseases and anthropometrics have been collected through questionnaires, interviews and clinical examinations, and in HUNT2 and HUNT3 biological material as blood and urine additionally were collected and stored. Incident cancer cases were identifiead via linkage to the Norwegian Cancer Registry.
The HUNT Study is a collaboration between HUNT Research Centre (Faculty of Medicine and Health Sciences, NTNU, Norwegian University of Science and Technology, Nord-Trøndelag County Council, Central Norway Health Authority, and the Norwegian Institute of Public Health.

3) Asian cohorts The Shanghai Men's Health Study (SMHS) and the Shanghai Women's Health Study (SWHS)
The SMHS and SWHS are population-based cohort studies conducted in eight communities of urban Shanghai. Their designs and methods have been described elsewhere 23 24 .

The Singapore Chinese Health Study (SCHS)
The design of the SCHS study has been described 25 26 . Briefly, the cohort was drawn from permanent residents or citizens of Singapore who resided in government-built housing estates (86% of the Singapore population reside in such facilities). The eligible age range for cohort enrolment was 45-74 years. We restricted study subjects to the two major dialect groups of Chinese in Singapore: the Hokkiens and the Cantonese, who originated from The cohort has been passively followed for death and cancer occurrence through regular record linkage with the population-based Singapore Cancer Registry and the Singapore Registry of Births and Deaths. Migration out of Singapore, especially among housing estate residents, is negligible. As of latest update, only 55 individuals from this cohort were known to be lost to follow-up due to migration and other reason.
A nested case-control study of incident lung cancer cases within the Singapore Chinese Health Study was established. Briefly, 422 lung cancer cases were identified among cohort participants with available prediagnostic plasma samples as of 12/31/2011. For each case, one control subject was randomly selected from all eligible cohort members who were alive and free of cancer on the date of cancer diagnosis of the index case. The control subject was individually matched to the index case by gender, dialect group (Hokkien, Cantonese), age at enrolment (±3 years), date of baseline interview (±2 year), date of biospecimen collection (±6 months), and smoking status (current, former, and never smokers). For current smokers, cases and controls were further matched by number of cigarettes per day (<15, ≥15 cigarettes/day). For former smokers, cases and controls were further matched by years since quitting smoking (<10, ≥10 years). One plasma aliquot per subject was retrieved from the biorepository and all plasma samples were sent to the laboratory (B-vital) for measurements.

The Shanghai Cohort Study (SCS)
The SCS study is a residential cohort of 18,244 men in Shanghai, China, assembled during 1986-89 when subjects were between the ages of 45 and 64 years. Approximately 80% of eligible men participated in the study. At the time of recruitment, each cohort subject was interviewed in-person by a trained nurse interviewer using a structured questionnaire that included background information, history of tobacco and alcohol use, current diet, and medical history 27 28 .
At the completion of the interview, the nurse collected a 10 ml blood and a single void urine specimen from the study participant. Blood and urine samples were kept in insulated boxes with ice (0-2 o C). The serum was separated from blood specimen within 3-4 hours after collection. Two sets of serum (2 ml and 1 ml, respectively) and two sets of urine samples (10 ml each) per subject have been stored at -80 o C. A nested case-control study of incident lung cancer cases within the Shanghai Cohort Study was established. Briefly, 516 lung cancer cases were identified among cohort participants with available serum samples as of 12/31/2006. For each case, we randomly selected one control subject from all cohort members who were free of cancer and alive at the time of cancer diagnosis of the index case. Controls were matched to the index case by age at enrolment (±2 years), date of biospecimen collection (±1 month) and neighbourhood of residence at recruitment, and smoking status (current, former and never smokers) as established previously for other studies. For former smokers, cases and controls were further matched by years since quitting smoking (<10 vs ≥10 years). One serum vial per subject was retrieved from biorepository and all serum samples were sent to the laboratory (B-vital) for measurements.