MD Santos Silva

Isabel Dos

PhD Bianca L De Stavola

MSc Costanza Pizzi

MD Tom W Meade

cancer risks: Individual participant analysis of data from three long-term cohorts

Keywords: coagulation, inflammation, fibrinogen, cancer, smoking coagulation, inflammation, fibrinogen, cancer, smoking

Background. Basic and clinical research support the hypothesis that activation of the coagulation and inflammation pathways may affect cancer onset, but there is limited epidemiological data to support this.

Methods. We examined a large range of haemostatic and inflammation markers, including fibrinogen, in 19 303 male participants from three English cohorts followed for up to 30 years. After excluding the first three years of follow-up, 2908 incident cancers were accrued. Competing risk models were fitted to estimate rate ratios (RRs) for cancer incidence, adjusting for age and other confounders.

Results. Baseline white blood cell (WBC) count and circulating levels of fibrinogen, C-reactive protein (CRP), factor VIIa and prothrombin fragment F1.2 were positively associated with risk of smokingrelated cancers, particularly lung cancer. The magnitude of these associations was highest for persistently raised fibrinogen levels. There was, however, substantial confounding by smoking with risk being fully (WBC, CRP and VIIa) or partially (fibrinogen) removed after adjustment. The pooled RR (95% CI) per one standard deviation increase in fibrinogen levels before and after adjustment for smoking habits were 1.23 (1.12, 1.36) and 1.12 (1.05, 1.20), respectively. The fibrinogen associations were present only among current smokers at entry. The effect of smoking on smoking-related cancers was partly mediated by fibrinogen levels.

Conclusions. Our results are consistent with elevated circulating levels of fibrinogen and F1.2 being predictors of risk of smoking-related cancers. Further research is necessary to clarify whether elevated levels of fibrinogen and F1.2 are causally relevant or simply correlates of the smoking-cancer association.

Introduction

Circulating levels of inflammation markers have been linked to the pathogenesis of several cancers [START_REF] Erlinger | WBC count and the risk of cancer mortality in a national sample of US adults: results from the Second National Health and Nutrition Examination Survey Mortality Study[END_REF][START_REF] Jee | White blood cell count and risk for all-cause, cardiovascular, and cancer mortality in a cohort of Koreans[END_REF][START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort[END_REF] and there is growing interest on the possible role of circulating levels of haemostatic variables. Cancer is diagnosed more often after an episode of venous thromboembolism. [START_REF] Baron | Venous thromboembolism and cancer[END_REF][START_REF] Sørensen | The risk of a diagnosis of cancer after primary deep venous thrombosis or pulmonary embolism[END_REF] Two relatively small follow-up studies have reported positive associations between circulating levels of fibrinogen and subsequent cancer mortality. [START_REF] Everett | Smoking, fibrinogen and cancer mortality[END_REF][START_REF] Yano | Plasma fibrinogen as a predictor of total and cause-specific mortality in elderly Japanese-American men[END_REF] The role of haemostatic variables on the pathogenesis of cancer is also supported by studies showing that anticoagulation may have an anti-tumour effect in addition to its antithrombotic effect.

Aspirin and other non-steroidal anti-inflammatory drugs reduce the risk of colorectal cancer [START_REF] Flossmann | on behalf of the British Doctors Aspirin Trial and the UK-TIA Aspirin Trial. Effect of aspirin on long-term risk of colorectal cancer: consistent evidence from randomised and observational studies[END_REF] and warfarin use has been found to be associated with a decreased risk of urogenital cancers, [START_REF] Schulman | Incidence of cancer after prophylaxis with warfarin against recurrent venous thromboembolism[END_REF] mainly prostate cancer. [START_REF] Tagalakis | Use of warfarin and risk of urogenital cancer: a populationbased, nested case-control study[END_REF] The aim of the present study is to examine the role of a large range of coagulation and inflammation markers on cancer incidence in three English cohorts comprising almost 20 000 male participants followed for up to 30 years.

Methods

Study subjects

We have access to data from three long-term cohort studies: the first and second Northwick Park Heart Study (NPHS-I and NPHS-II) and the Thrombosis Prevention Trial (TPT). They were coordinated by the same research group, using generally similar methods for obtaining and processing blood samples.

NPHS-I was the first systematic epidemiological investigation of the contribution of the haemostatic system to the thrombotic component of coronary heart disease (CHD). [START_REF] Meade | Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study[END_REF] It recruited 2167 white men aged over 18 years (80% of those eligible) between 1972 and 1978 by invitation to factory and office workers in three North West London occupational groups. NPHS-II extended the aims of NPHS-I by including several new assays for clotting factor activity and indices of thrombin production and activity, [START_REF] Cooper | Comparison of novel haemostatic risk factors and conventional risk factors for prediction of coronary heart disease[END_REF] none of which were available when NPHS-I was conducted. It began in 1989 and recruited 3052 men aged 50-61 years (76% of those eligible) who were registered in nine general practices in England and Scotland and had no history of cardiovascular disease or cancer. Of these, over 700 were resident in Scotland and were excluded from this analysis because of incomplete follow-up. TPT, a double-blind factorial randomised controlled trial of low-intensity anticoagulation with warfarin and lowdose aspirin initiated in 1989, [START_REF]Thrombosis prevention trial: randomised trial of low-intensity oral anticoagulation with warfarin and low-dose aspirin in the primary prevention of ischaemic heart disease in men at increased risk. The Medical Research Council's General Practice Research Framework[END_REF][START_REF] Rudnicka | Associations of plasma fibrinogen and factor VII clotting activity with coronary heart disease and stroke: prospective cohort study from the screening phase of the Thrombosis Prevention Trial[END_REF] recruited men aged 45-69 years and at high risk of CHD but without a history of clinical episodes. Our analysis includes baseline data from the 5057 men at high CHD risk who did not take part in the treatment phase of the trial ("high risk" group) and a sample of 12 159 men found to be at lower risk during the trial's screening phase ("low risk group"). The 5499 men at "high risk" enrolled into the trial were excluded from this analysis because treatment could have affected cancer incidence. Together, these three studies collected data on a large range of inflammation and haemostatic variables (Table 1) based on non-fasting blood samples taken at entry (or at screening in TPT). Fibrinogen was measured by clot weight in NPHS-I and the von Clauss thrombin clotting time method in NPHS-II and TPT. Fibrinolytic activity (FA) was assessed by the time to dissolution of clot (expressed as 100 time in hours). Prothrombin fragment F1.2 was measured by double-antibody radioimmunoassay and fibrinopeptide A (FPA) by commercial radioimmunoassay (Byk-Sangtee) as indices of thrombin production and activity, respectively. Factors VII and VIII were measured as described in Meade et al. [START_REF] Meade | Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study[END_REF] and VIIa, XIIa, Protein S, and C-reactive protein (CRP) as described in Cooper et al. [START_REF] Cooper | Comparison of novel haemostatic risk factors and conventional risk factors for prediction of coronary heart disease[END_REF] Haematological variables were measured by automated Coulter counts and serum cholesterol by an automated procedure (Sigma).

Blood pressure measurements were recorded by a random-zero sphygmomanometer and anthropometric variables measured using standard methods. Data on several potential confounders and effect modifiers were collected by questionnaire at the time of entry (or screening in TPT) (Table 1).

Social class was ascertained according to the occupation held at the time of recruitment. [START_REF]Office of Population Census and Surveys (OPCS)[END_REF] Entry smoking status was categorised as never-/ex-/current smoker in NPHS-I and NPHS-II, and as current (yes/no) in TPT. Life-time amount smoked up to the time of entry was estimated as pack-years (i.e. number of cigarettes, or equivalent units for pipes and cigars, per day multiplied by number of years smoked divided by 20) in NPHS-I and NPHS-II. Current alcohol intake at entry was defined as consumption of at least one alcoholic drink in the week prior to entry/screening, with weekly amount for NPHS-I and NPHS-II estimated from number of drinks per week, and expressed as numbers of equivalent glasses of wine.

Follow-up variables

In NPHS-I, all haemostatic variables were re-measured in 65% of participants six years after entry, on average, while in NPHS-II fibrinogen and factors VIIc and XIIa were measured five times at yearly intervals but, to ensure comparability with NPHS-I, only the 5-year follow-up measures were included in these analyses. These repeat measurements were used to define single-occasion (i.e. either entry or follow-up) and persistently (i.e. both entry and follow-up) raised (>4 g/L) fibrinogen levels. A cut-point of 3.5 g/L was also used. Follow-up questionnaire data on smoking status and quantity, available in both NPHS-I and NPHS-II, were used to define cumulative amount smoked up to the follow-up visit.

Outcome ascertainment

Site-specific cancer incidence, cause-specific mortality and loss-to-follow-up for the three cohorts were ascertained through the England & Wales National Health Services Central Register (NHSCR) with independent assessment from general practices, hospital records, and coroners. Events were coded according to the International Classification of Diseases (ICD), [START_REF]Manual of the International Statistical Classification of Diseases, Injuries and Causes of Death, eight, ninth and tenth revisions[END_REF] with appropriate bridging across ICD revisions.

Statistical analysis

For each study, follow-up time was calculated from the beginning of the fourth year since entry up to the occurrence of the earliest event of: cancer incidence, death, emigration, 90 th birthday, or 30 June 2006.

The first three follow-up years were excluded to minimise the potential for any effects of incipient cancer on the exposures of interest.

Analyses were carried out separately by study (and for TPT also separately by risk group) and then jointly, if appropriate. Circulating levels of fibrinogen and other haemostatic and inflammation variables, when available, were related to incidence of cancer. Analyses were based on the competing risks model proposed by Fine & Gray 17 because of the expected correlations among times to specificcancers as well as with time to cardiovascular death. The resulting estimates -referred to as competing rate ratios (RRs) -represent the effects of the explanatory variables on (a transformation of) the cumulative incidence function (CIF). As age was the time-scale of analysis all estimated RRs are ageadjusted. Further adjustment for smoking and other potential confounders was performed.

Each exposure variable was examined on both a continuous scale, in terms of effect per one standard deviation (SD) increase, and a categorical scale, in study-specific fourths of the overall distribution, with RRs computed relative to the lowest fourth (reference). Departure from linearity was assessed by including quadratic terms. Effect modification was tested by including appropriate interaction terms. When assessing the potential modifying effect of smoking, interaction terms were defined both for smoking status and cumulative amount smoked, when available. P-values, including P-values of test for linearity and for effect modification, were examined via likelihood ratio tests. [START_REF] Clayton | Statistical Models in Epidemiology[END_REF] The proportionality of effects was assessed via Schoenfeld's residuals. [START_REF] Latouche | Misspecified regression model for the subdistribution hazard of a competing risk[END_REF] When no evidence of between-study heterogeneity was found, RRs were pooled to produce summary estimates by fitting a random effects model to the studyspecific RRs. [START_REF] Skrondal | Generalized Latent Variable Modelling: Multilevel, Longitudinal, and Structural Equation Models[END_REF] To allow for regression dilution effect in the age-adjusted estimated RRs for fibrinogen, the method of moments was used to correct the estimated RRs. [START_REF] Carroll | Measurement Error in Nonlinear Models[END_REF]

Results

Baseline characteristics

After exclusions due to missed linkage to the NHSCR and to either death, cancer or migration in the first three years of follow-up, the analyses concern 19 303 men (Table 1). Participants in NPHS-I were, on average, eight years younger at entry than those in NPHS-II; they were also more likely to have been

F o r R e v i e w O n l y
6 current smokers but otherwise -given the age differences -were similar. Participants in TPT were of similar ages to those in NPHS-II, with the high risk group having a higher percentage of current smokers at entry as well as elevated average values for all the clinical, biochemical and haemostatic variables examined (Table 1). A total of 2908 cancers were accrued (Table 2) during a follow-up time ranging from 3.0 to 34.2 years (median: 25.6 years in NPHS-I, 11.7 in NPHS-II, and 12.4 in TPT overall). Smokingrelated cancers accounted for about 40% of all malignancies in NHPS-I and TPT-high risk but for only about 29% in the other studies.

(Table 1 here)

Fibrinogen levels and cancer incidence

Continuous fibrinogen levels were positively associated with overall (all-sites combined) cancer risk in each study (Table 2). Categorical fibrinogen also showed positive associations with all-sites cancer risk, with linear trends across the categories found in NPHS-II and TPT low risk, the two cohorts for which the magnitude of the effect on the continuous scale was strongest (Table 2). These results were mainly due to the association of fibrinogen with smoking-related cancers (pooled age-adjusted RR (95% CI) of smoking-related cancers per one SD increase in fibrinogen levels: 1.23 (1.12, 1.36); Figure 1). Although the magnitude of the fibrinogen effect was stronger for lung cancer (Table 2), but with evidence of between-study heterogeneity (P=0.006; Figure 2), it was also present for all the other smoking-related cancers except lung cancer (pooled age-adjusted RR (95% CI): 1.10 (0.97, 1.26)). Fibrinogen levels were not associated with risk of non-smoking related cancers overall, nor with risk of digestive, genital or haematological cancers when analysed separately (Table 2; Supplementary Figures 123).

Replication data on fibrinogen from NPHS-II, consisting of 267 independent repeat measures taken 6-weeks apart, were used to correct the age-adjusted estimates, leading to increased RRs by 15% for smoking-related cancers (corrected pooled age-adjusted RRs (approximate 95% CI) per one SD increase in fibrinogen levels: 1.40 (1.19, 1.63)) and by about 24% for the study-specific RRs for lung cancer.

(Table 2 here)

Confounding by smoking and other factors

Smoking was associated with higher entry levels of fibrinogen in each study and, among current smokers, mean fibrinogen levels increased progressively with increasing amount smoked in both NPHS-I and NPHS-II (e.g. mean (SD) fibrinogen levels for NPHS-I never-smokers, ex-smokers of 1-24 and ≥25 packyears, and current smokers of 1-24 and ≥25 pack-years: 2.68 (0.57), 2.82 (0.63), 3.06 (0.64), 2.94 (0.71) and 3.20 (0.64)) (Supplementary Table 1). For smoking-related cancers there was evidence of substantial confounding of the effect of fibrinogen by smoking, with the magnitude of the pooled RRs per one SD increase in levels declining from 1.23 (95% CI: 1.12, 1.36) to 1.12 (1.05, 1.20) (Figure 1). Similar reductions in effects were found for lung cancer (Supplementary Table 2; Figure 2). Further adjustment for other potential confounders only moderately reduced these estimates (Supplementary Table 2; Figures 12). Equivalent analyses for non-smoking related cancers overall (Supplementary Table 2) and separately for digestive, genital and haematological cancers (Supplementary Figures 123) showed no association with fibrinogen levels.

The association between fibrinogen and risk of smoking-related cancers was further examined in the separate strata of current-/ex-and never-smokers at entry in NPHS-I and NPHS-II. As the RRs for these two studies were similar estimates were derived from the combined data. The positive association between fibrinogen levels and risk of smoking-related cancers was present only among current-smokers (Table 3).

(Table 3 here)

Single-occasion versus persistently raised fibrinogen levels

Similar positive associations with risk of smoking-related cancers were observed when analysing followup rather than baseline fibrinogen levels in the subsets of 1507 NPHS-I and 1446 NHPS-II participants with follow-up measurements taken 5-6 years after entry (combined age-smoking-adjusted RR (95% CI) for smoking-related and lung cancers per one SD increase in fibrinogen levels: 1.37 (1.19, 1.58) and 1.62 (1.33, 1.96), respectively). Persistently raised fibrinogen levels were strongly associated with increased risk of both smoking-related cancers and lung cancer (combined age-smoking-adjusted RR (95% CI):

2.61 (1.40, 4.86) and 2.99 (1.27, 7.08), respectively; Supplementary Table 3). In contrast, single-occasion high fibrinogen level was only moderately associated with these outcomes. Among those with persistently raised levels, 61% were current smokers and 30% were ex-smokers; the corresponding figures among those with single-occasion raised levels were 36% and 34%, respectively. Similar results were obtained when 3.5 g/L was used as the cut-point (Supplementary Table 3).

Fibrinogen as a mediator of the effect of smoking on lung cancer incidence

To assess whether fibrinogen levels could mediate the association between smoking and lung cancer we compared the effect of smoking before and after adjustment for fibrinogen levels. In NPHS-I and NPHS-II combined, the age-adjusted rates of lung cancer were about 15-times (combined age-adjusted RR=14.5; 95% CI: 5.3, 10.3) higher in current smokers and 4-times higher among ex-smokers (combined ageadjusted RR=3.5; 95% CI: 1.2, 10.3) relative to never-smokers. Further adjustment for baseline In the subset of 2953 NPHS-I and NPHS-II participants with follow-up data the reduction in the magnitude of the smoking effects was slightly more marked on simultaneous adjustment for both baseline and follow-up fibrinogen levels. Relative to never-smokers the combined age-adjusted RR (95% CI) for lung cancer was 19.0 (4.6, 78.2) for current-smokers and 5.1 (1.2, 22.4) for ex-smokers, relatively to never smokers, with the magnitude of these associations declining respectively to 14.2 (3.4, 58.9) and 4.6

(1.0, 20.1), with further simultaneous adjustment for baseline and follow-up fibrinogen levels. These results would be consistent with fibrinogen being a possible mediator of the effect of smoking on lung cancer incidence.

The role of other inflammatory and haemostatic factors

WBC count, available only in NPHS-I, and continuous CRP and VIIa, available only in NPHS-II, were associated with incidence of smoking-related cancers and, with the exception of VIIa, lung cancer.

However, each of these associations was fully confounded by smoking (Table 4). Prothrombin fragment F1.2, only available in NPHS-II, was associated with risk of smoking-related cancers and lung cancer even after controlling for smoking (Table 4). Analyses restricted to never-smokers were hampered by small number of cases among never-smokers. No cancer associations were found with any other haemostatic or inflammation variable (Supplementary Table 4).

Discussion

The present analysis revealed positive associations between circulating levels of fibrinogen, CRP, VIIa and prothrombin fragment F1.2, and also of WBC, with subsequent risk of smoking-related cancers.

Measurement error may have underestimated the magnitude of these associations, possibly by about 20% for fibrinogen. Smoking was, however, a strong confounder and, with the exception of F1.2, these associations were either considerably attenuated (fibrinogen) or removed (WBC, CRP and VIIa) after adjustment for this variable. Furthermore, no associations with fibrinogen were observed among neversmokers suggesting that smoking accounted for most of the excess risks at these cancer sites. Cigarette smoking is an important determinant of levels of fibrinogen, and other inflammation markers, and it is also a strong risk factor for many cancers. So, while we cannot exclude the possibility of a true independent association between levels of inflammatory and haemostatic variables and subsequent incidence of smoking-related cancers, residual confounding by smoking is a likely explanation for any remaining effects. Nevertheless, activation of the inflammation and coagulation cascades may still be important in the pathogenesis of smoking-related cancers, with inflammation and haemostatic factors being markers of susceptibility to the effects of smoking and/or markers of the biological effects of this exposure on cells in the target tissues. Our findings, particularly those based on both baseline and followup fibrinogen levels, are consistent with fibrinogen being a potential mediator of the smoking effect on the incidence of lung cancer. In contrast, the association of F1.2 with smoking-related cancer incidence was not reduced by adjustment for smoking, suggesting a thrombogenic effect via activation of the coagulation pathway, but this finding was based on data from only one study.

Previous epidemiological studies have found associations between fibrinogen levels and cancer mortality, [START_REF] Everett | Smoking, fibrinogen and cancer mortality[END_REF][START_REF] Yano | Plasma fibrinogen as a predictor of total and cause-specific mortality in elderly Japanese-American men[END_REF] mostly as in the present study, with smoking-related cancers. [START_REF] Everett | Smoking, fibrinogen and cancer mortality[END_REF] A recent meta-analysis, focused on cardiovascular diseases, reported positive associations between fibrinogen levels and mortality from smoking-related cancers among all participants and among never-smokers only. [START_REF]Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis[END_REF] Others have examined the role of WBC count on cancer mortality, with one reporting a positive association 1 and another no association. [START_REF] Jee | White blood cell count and risk for all-cause, cardiovascular, and cancer mortality in a cohort of Koreans[END_REF] Fewer studies have examined risks in relation to cancer incidence. A US prospective study found positive associations with circulating levels of inflammation markers such as interleukin-6, CRP and tumour necrosis factor-α, but interestingly these associations were stronger for cancer mortality than for cancer incidence. [START_REF] Il'yasova | Circulating levels of inflammatory markers and cancer risk in the health aging and body composition cohort[END_REF] Similarly to our findings, a recent meta-analysis [START_REF] Heikkilä | Associations of circulating C-reactive protein and interleukin-6 with cancer risk: findings from two prospective cohorts and a meta-analysis[END_REF] found an association between CRP levels and cancer incidence, essentially driven by a positive CRP -lung cancer association. Miller et al. [START_REF] Miller | Increased evidence of neoplasia of the digestive tract in men with persistent activation of the coagulation pathway[END_REF] found that persistent activation of the haemostatic pathway was associated with an elevated risk of digestive cancers. However, many of the cancers arose within a short time of recruitment into the study so that reverse causality was a real possibility. We were unable to confirm the finding in the present analysis although there was evidence in NPHS-II of a positive association between F1.2 and smoking-related cancers. Laboratory-based research has considered pathways through which activation of the coagulation cascade might be associated with cancer, but it is unclear whether the proposed mechanisms (e.g. stimulation of tumour growth and angiogenesis; [START_REF] Caine | The Hypercoagulable state of malignancy: pathogenesis and current debate[END_REF][START_REF] Dotsenko | Thrombosis and cancer[END_REF] promotion of metastatic potential by impeding natural killer cell elimination of tumour cells; [START_REF] Palumbo | Fibrinogen is an important determinant of metastatic potential of circulating tumour cells[END_REF][START_REF] Palumbo | Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumour cells[END_REF] smoking-induced damage with oxidation and nitration of DNA and plasma proteins such as fibrinogen [START_REF] Pignatelli | Nitrated and oxidized plasma proteins in smokers and lung cancer patients[END_REF] ) are a cause or a consequence of the disease.

The present analysis is based on three male cohorts and, thus, its findings may not be generalisable to women. Other limitations include inadequate power to examine separately risks in relation to individual cancers, other than lung and colon-rectum, and limited data on smoking habits for the two TPT groups.

In summary, our results show that pre-diagnostic levels of coagulation and inflammatory markers, particularly fibrinogen, are associated with risk of smoking-related cancers. The aetiological significance of these findings is unclear. Residual confounding by smoking may account for most of the remaining observed effects among smokers. Alternatively, fibrinogen may be a mediator of the smoking-cancer [START_REF] Smith | Mendelian Randomisation": can genetic epidemiology contribute to the understanding of environmental determinants of disease?[END_REF] to investigate potential associations between genetic determinants of circulating levels of fibrinogen (and other coagulation markers) and cancer risks in large-scale observational studies. Overview results from trials of fibrates, which lower fibrinogen levels by about 15%, may also provide evidence for or against an independent effect of fibrinogen on cancer onset. A recent trial of fenofibrate reported no difference in cancer risks, but based on small number of cases, between the intervention and the placebo arms. [START_REF] Field | Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial[END_REF]

Key message box

• The association between a large range of haemostatic and inflammation markers was examined in 19,303 English men followed for up to 30 years.

• Baseline circulating levels of fibrinogen were positively associated with risk of smoking-related cancers, particularly lung cancer, but only among current smokers. Adjustment for smoking habits attenuated but did not abolish the associations.

• Baseline white blood cell count and levels of C-reactive protein, factor VIIa and prothrombin fragment F1.2 were also positively associated with risk of smoking-related cancers but, with the exception of F1.2, their effects were confounded by smoking habits.

• Further research is required to clarify the role of these markers on cancer onset and establish whether they are causally relevant or simply markers of the smoking-cancer association.

F

- - - - - - - - - XII antigen (XIIa) (ng/ml) 2 - - - 1,758 1.8 1.3 Fibrinopetide A (FPA) (µmol/L) 2 - - - 2,071 1.2 0.9 - - - - - - Protrombin fragment F1.2 (µmol/L) 2 - - - 2,070 0.7 0.4 - - - - - - C-reactive protein (CRP) (mg/L) 2 - - - 1,938 2.9 5.1 -
- - - - - Protein S (% standard) - - - 1,714 111.5 20.5 - - - - -

  the smoking effects only marginally (combined age-fibrinogen-adjusted RRs (95% CI): 12.7 (4.6, 34.6) and 3.4 (1.2, 10.0), respectively).

  simply a correlate of the effects of smoking on target tissues. Assessment of the aetiological significance of high fibrinogen levels in relation to cancer risks will require strategies that can minimise potential residual biases by smoking and other variables. One possible avenue would be the use of Mendelian randomisation

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Study-specific incidence rate ratios (RR) for lung cancer associated with one standard deviation increment in fibrinogen levels, adjusting for several potential confounders

13 List of Tables and Figures TablesTable 1 .

 1 Baseline characteristics of the participants, by study

	o F o F o
	r r r
	R e v i e w R e v i e w R e v i e w
	O n O n O n
	l y l y l y

Table 2 .

 2 Age-adjusted incidence rate ratios for selected cancer sites in relation to categorical and continuous fibrinogen levels at entry, by study

Table 3 .

 3 Age-adjusted incidence rate ratios for smoking-related cancers in relation to categorical and continuous fibrinogen levels at entry stratified by smoking status, NPHS-I and NPHS-II combined.

Table 4 .

 4 Incidence rate ratios (RR) for smoking-related and lung cancers in relation to categorical and continuous white blood cell (WBC) count, C-reactive protein (CRP) and prothrombin fragment F1.2, adjusting for several potential confounders

Table 1 .

 1 Baseline characteristics of the participants, by study

	NPHS-I	NPHS-II	TPT low risk	TPT high risk

Table 2 .

 2 Age-adjusted incidence rate ratios for selected cancer sites in relation to categorical and continuous fibrinogen levels at entry, by study

	-

Table 3 .

 3 Age-adjusted incidence rate ratios for smoking-related cancers in relation to categorical and continuous fibrinogen levels at entry stratified by smoking status, NPHS-I and NPHS-II combined

	F o
	r
	R e v i e w
	O n
	l y

Table 4 .

 4 Incidence rate ratios of smoking-related and lung cancer in relation to categorical and continuous white blood cell (WBC) count, C-reactive protein (CRP), VII antigen (VIIa), and F1.2 at entry, adjusting for several potential confounders

	Supplementary
	F o
	r
	R e v i e w
	O n
	l y

Table 1 .

 1 Circulating levels of coagulation/inflammation markers at entry by smoking status at entry, NPHS-I and NPHS-II

		Non-smokers		Ex-smokers			Current smokers
				by pack-years (p-yrs)		by pack-years (p-yrs)
				1-24 p-yrs	25+ p-yrs	1-24 p-yrs	25+ p-yrs
	Study (n)	Mean SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
	NPHS I (2,053)
	Fibrinogen	2.68 0.57	2.82	0.63	3.06	0.64	2.94	0.71	3.20	0.64
	WBC	6.20 1.30	6.44	1.38	6.58	1.35	7.47	2.08	8.34	1.97
	NPHS II (2,075)
	Fibrinogen	2.67 0.50	2.68	0.50	2.73	0.56	2.88	0.62	3.06	0.62
	CRP 1	2.15 3.45	2.47	3.93	3.62	4.68	3.50	7.47	5.35	9.87
	VIIa 1	123	42	131	46	132.5	45	131	46	128	43.5
	F1.2 1	0.67 0.33	0.68	0.33	0.72	0.40	0.77	0.36	0.81	0.37
	1 Median and inter-quartile range are shown

Table 3 .

 3 Age-and smoking-adjusted incidence rate ratios for selected cancer sites in relation to single-occasion and persistently high fibrinogen levels, NPHS-I and NPHS-II combined (n=2,953) Single-occasion high fibrinogen levels defined as fibrinogen levels greater than the cut-point either at entry or at follow-up; persistent high levels are defined as fibrinogen levels greater than the cut-point at both entry and follow-up.

		Fibrinogen cut-point used to define activation
		4 g/L			3.5 g/L
		Single-occasion 1	Persistent 1	Single-occasion 1	Persistent 1
	Cancer site (d) 2	RR 3 (95% CI) RR 3	(95% CI)	RR 3 (95% CI) RR 3 (95% CI)
	Smoking related (179)	1.16 0.79; 1.72 2.61 1.40; 4.86 1.23 0.89; 1.70 1.37 0.91; 2.06
	Lung cancer (76)	1.72 1.01; 2.90 2.99 1.27; 7.08 1.29 0.80; 2.09 2.15 1.26; 3.66
	Non-smoking related (358) 0.78 0.55; 1.12 1.13 0.53; 2.41 1.03 0.80; 1.33 0.72 0.47; 1.12
	Digestive (137)	0.54 0.28; 1.04 1.55 0.57; 4.25 0.86 0.55; 1.29 0.85 0.45; 1.60
	Colorectal (74)	0.72 0.31; 1.67 0.80 0.11; 5.86 0.96 0.54; 1.71 0.67 0.24; 1.87
	Genital (158)	0.64 0.35; 1.15 1.09 0.35; 3.46 0.80 0.54; 1.21 0.85 0.45; 1.60
	Haematological (37)	0.63 0.19; 2.08 1.44 0.19; 10.78 1.10 0.51; 2.36 0.57 0.13; 2.41
	CI=confidence interval; RR=rate ratio; SD=standard deviation
	1 2 d=no. of incident cancer cases

Table 4 .

 4 Incidence rate ratios for selected cancer sites in relation to continuous haematological and coagulation factors adjusting for several potential confounders, by study

	Fig. 1
	Smoking-related cancers
			Rate ratio (95% CI)	P for between-study
					heterogeneity
	Age-adjusted NPHS-I NPHS-II TPT low risk TPT high risk Pooled (I 2 = 52.8%) Age & smoking adjusted NPHS-I TPT high risk Pooled (I 2 = 0.0%) Multiple-adjusted * NPHS-I F o r R e v i e w O n l y 1.21 (1.05, 1.39) 1.39 (1.18, 1.64) 1.29 (1.14, 1.46) 1.12 (1.01, 1.23) 1.23 (1.12, 1.36) 1.08 (0.93, 1.26) 1.08 (0.98, 1.20) 1.12 (1.05, 1.20) 1.11 (0.95, 1.29) 1.21 (1.05, 1.39) 1.39 (1.18, 1.64) 1.29 (1.14, 1.46) 1.12 (1.01, 1.23) 1.23 (1.12, 1.36) 1.08 (0.93, 1.26) 1.08 (0.98, 1.20) 1.12 (1.05, 1.20) 1.11 (0.95, 1.29) NPHS-II TPT low risk 1.20 (1.01, 1.44) 1.17 (1.03, 1.33) 1.20 (1.01, 1.44) 1.17 (1.03, 1.33)	0.10 0.63
	NPHS-II			1.21 (1.01, 1.45) 1.21 (1.01, 1.45)	0.32
	TPT low risk			1.18 (1.04, 1.34) 1.18 (1.04, 1.34)
	TPT high risk			1.03 (0.93, 1.15) 1.03 (0.93, 1.15)
	Pooled (I 2 = 15.3%)			1.12 (1.04, 1.20) 1.12 (1.04, 1.20)
	.75	1 1	1.5	2
		Rate Ratio

* As described in Supplementary Table

1

RR (95% CI)

Acknowledgements:

We are grateful to Professor Steve Humphries for access to the late George Miller's data on NPHS-II.

Funding: This work was supported by the Medical Research Council (NPHS-I, NPHS-II and TPT) and the British Heart Foundation (TPT). IdSS is part of the Cancer Research UK Epidemiology and Genetics Group. TM is supported by the Medical Research Council.

Conflicts of interest: None

Guarantor of the paper: Isabel dos Santos Silva

Authors' contribution: IdSS, BDS and TM conceived the study and planned the analysis; BDS and CP conducted all the statistical analyses; IdSS prepared the first draft of the manuscript; all authors contributed to the interpretation of the findings and to the final preparation of the manuscript.

Supplementary Tables and Figures (to be included in the web-version of the paper)

Supplementary Table 1. Circulating levels of coagulation/inflammation markers at entry by smoking status at entry, NPHS I and II. 2. Incidence rate ratios for selected cancer sites in relation to categorical and continuous fibrinogen at entry adjusting for several potential confounders, by study Supplementary Table 3. Age-and smoking-adjusted incidence rate ratios for selected cancer sites in relation to single-occasion and persistently high fibrinogen levels, NPHS-I and NPHS-II combined (n=2,953) Supplementary Table 4. Incidence rate ratios for selected cancer sites in relation to continuous haematological and coagulation factors at entry adjusting for several potential confounders, by study Supplementary Figure 1: Study-specific and pooled incidence rate ratios for digestive cancers associated with one standard deviation increment in fibrinogen levels at entry, adjusting for several potential confounders Supplementary Figure 2: Study-specific and pooled incidence rate ratios for genital cancers associated with one standard deviation increment in fibrinogen levels at entry, adjusting for several potential confounders Supplementary Figure 3: Study-specific and pooled incidence rate ratios for haematological cancers associated with one standard deviation increment in fibrinogen levels at entry, adjusting for several potential confounders

Supplementary Table