IJC International Journal of Cancer

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1 IJC International Journal of Cancer Gamma glutamyltransferase, alanine aminotransferase and risk of cancer: Systematic review and meta-analysis Setor K. Kunutsor 1, Tanefa A. Apekey 2, Mieke Van Hemelrijck 3, Giliola Calori 4 and Gianluca Perseghin 5 1 Department of Public Health and Primary Care, University of Cambridge, Cambridge, United Kingdom 2 Leeds Trinity University, Brownberrie Lane, Horsforth, Leeds, United Kingdom 3 Division of Cancer Studies, Cancer Unit, King s College London, School of Medicine, London, United Kingdom 4 Division of Metabolic and Cardiovascular Sciences, H. San Raffaele Scientific Institute, Milan, Italy 5 Department of Biomedical Sciences for Health, Universita degli Studi di Milano & Metabolic Medicine, Policlinico di Monza, Italy The prospective evidence for the associations of gamma glutamyltransferase (GGT) and alanine aminotransferase (ALT) with risk of cancer in the general population is uncertain. We conducted a systematic review and meta-analysis of published prospective observational studies evaluating the associations of baseline levels of GGT and ALT with risk of overall (incidence and/or mortality) and site-specific cancers. Relevant studies were identified in a literature search of MEDLINE, EMBASE, Web of Science, reference lists of relevant studies to April 2014 and contact with investigators. Study specific relative risks (RRs) were meta-analyzed using random effects models. Fourteen cohort studies with data on 1.79 million participants and 57,534 cancer outcomes were included. Comparing top versus bottom thirds of baseline circulating GGT levels, pooled RRs (95% confidence intervals) were 1.32 ( ) for overall cancer, 1.09 ( ) for cancers of the breast and female genital organs, 1.09 ( ) for cancers of male genital organs, 1.94 ( ) for cancers of digestive organs and 1.33 ( ) for cancers of respiratory and intrathoracic organs. For ALT, corresponding RRs for overall cancer were 0.96 ( ) and 1.65 ( ) in European and Asian populations, respectively. There was an increased risk of cancers of the digestive organs 2.44 ( ). The pooled RR for overall cancer per 5 U/L increment in GGT levels was 1.04 ( ). Available observational data indicate a positive log-linear association of GGT levels with overall cancer risk. The positive association was generally evident for site-specific cancers. There are geographical variations in the association of ALT and overall cancer. Gamma glutamyltransferase (GGT), a sensitive but nonspecific marker of liver dysfunction, plays a primary role in the extracellular catabolism of glutathione (the major antioxidant in mammalian cells) and has commonly been used as a biologic marker of excessive alcohol intake. 1 Alanine aminotransferase (ALT), a more specific marker of liver dysfunction, catalyses the transfer of amino groups to generate products in gluconeogenesis and amino acid metabolism. 2 Key words: gamma glutamyltransferase, alanine aminotransferase, cancer, meta-analysis Abbreviations: ALT: alanine aminotransferase; CVD: cardiovascular disease; CI: confidence interval; GGT: gamma glutamyltransferase; GLST: generalized least-squares trend estimation; NOS: Newcastle Ottawa Scale; RR: relative risk Additional Supporting Information may be found in the online version of this article. DOI: /ijc History: Received 23 Apr 2014; Accepted 9 July 2014; Online 19 July 2014 Correspondence to: Setor K. Kunutsor, Department of Public Health and Primary Care, University of Cambridge, Cambridge CB1 8RN, United Kingdom, Tel.: , Fax: , skk31@cantab.net In addition to their physiological functions, a growing body of evidence indicates that serum levels of GGT and ALT may be associated with a wide range of disease outcomes. Several prospective studies have demonstrated associations between GGT or ALT and risk of type 2 diabetes mellitus, 3,4 cardiovascular disease, 5,6 vascular and nonvascular mortality, 7,8 and all-cause mortality outcomes. 9 Emerging evidence indicates that increasing levels of GGT and ALT may each be linked to cancer risk. A number of prospective studies have been published reporting on the associations between baseline levels of these enzymes and risk of cancer, 8,10 14 but their results have been inconsistent, particularly for ALT. Whereas some studies have observed positive associations of circulating levels of these enzymes with risk of cancer, 8,10,11 others have shown inverse associations, 10 with some studies showing no associations at all. 12,13 While some of these studies have reported log-linear associations, 11,14 others have failed to evaluate nonlinearity, 12,15 leaving great uncertainty regarding the nature of the associations. Long et al. 16 have recently conducted a review on available prospective epidemiological data on the association between GGT and cancer risk and suggested a positive association. There were however some limitations to the review. First, they only reported on cancer mortality outcomes; incident cancer outcomes were not

2 Kunutsor et al What s new? Emerging evidence indicates that the level of gamma glutamyltransferase (GGT) and the level of alanine aminotransferase (ALT) two markers of liver dysfunction may each be linked to cancer risk. So far, the prospective evidence on their associations has been inconsistent. However, in the review presented here, GGT level was found to be positively and log-linearly associated with overall cancer risk. The positive association was consistent for site-specific cancers. As for ALT, there were geographical variations in its association with overall cancer, and ALT was also found to be associated with an increased risk of cancers of digestive organs. assessed. Second, a pooled analysis was not performed because of the different risk comparisons reported or the different study designs (e.g., prospective versus retrospective cohort studies), therefore a reliable magnitude of the association could not be estimated. Third, the nature of the dose response relationship was not addressed, therefore it is not clear if there is a clear linear relationship to the association across the whole range of GGT levels or there is a threshold effect. To help characterize and quantify more reliably the nature and magnitude of the associations and provide robust observational evidence on the role of GGT or ALT in the development of cancer, we performed a first systematic review and meta-analysis of published prospective evidence on the associations of baseline levels of GGT and ALT, each with the risk of overall and site-specific cancers in the general population. Reported risk estimates from eligible studies were standardized to a consistent comparison (top versus bottom thirds of baseline levels of GGT and ALT) before pooling and the dose response relationship between GGT levels and overall cancer risk was examined using restricted cubic splines. Materials and Methods Data sources and search strategy We conducted this review using a predefined protocol and in accordance with PRISMA and MOOSE guidelines 17,18 (Supporting Information Appendices 1 2). Two independent authors (T.A.A. and G.C.) in duplication, searched MED- LINE, EMBASE and Web of Science up to April We sought prospective (cohort or nested case control ) population-based studies that had reported on associations of baseline circulating levels of GGT or ALT with overall cancer (defined as incidence of or mortality due to cancer at any site) or site-specific cancer incidence or mortality, in which fatal outcomes were registered according to the primary cause, on the basis of coding from the International Classification of Diseases (ICD) or according to study defined classifications. The computer-based searches combined free and MeSH search terms and combination of key words related to the exposures (e.g., gamma glutamyltransferase and alanine aminotransferase ) and outcomes (e.g., cancer and neoplasm ). There were no restrictions on language or the publication date. Reference lists of retrieved articles were manually scanned for all relevant additional studies and review articles. We restricted the search to studies of humans. Further details on the search strategy are presented in Supporting Information Appendix 3. Eligibility criteria Observational cohort studies were included if they had at least one year of follow-up, assessed associations of GGT or ALT with cancer risk in adults (over 18 years), with samples measured at baseline, and reported recruitment of participants representative of or from approximately general populations (i.e., they did not select participants on the basis of confirmed pre-existing disease conditions such as cancer, chronic kidney disease, liver disease or excluded participants with marked elevations in levels of GGT or ALT at baseline). Retrospective studies were not included. Data extraction and quality assessment The data extraction and quality assessment were conducted by two independent reviewers (T.A.A. and G.C.). A standardized predesigned data collection form was used for data extraction. Data were abstracted, where available, on study, publication date, geographical location, population source, time of baseline survey, sample population, study design, sample source (plasma/serum), nature of sample (fresh or frozen and storage temperature), assay type and source, case definition (Supporting Information Appendix 4), sample size, numbers of cancer outcomes, mean age, duration of followup and degree of adjustment for potential confounders [defined as 1 when relative risks (RRs) were adjusted for age and/or sex; 11 further adjustment for potential risk factors such as smoking status, body mass index, blood pressure, lipids or physical activity and 111 additional adjustment for alcohol consumption]. Each article was assessed using the inclusion criteria above and any disagreement regarding eligibility of an article was discussed, and agreement reached by consensus with a third reviewer (S.K.K.). We contacted authors who had conducted univariate or multivariate analyses with any of the liver enzymes as a covariate, but had not reported estimates for cancer outcomes and studies with missing information, to conduct reanalyses of data and provide the required data. Study quality was assessed based on the nine-star Newcastle Ottawa Scale (NOS) 19 using three predefined domains namely: selection of participants (population representativeness), comparability

3 1164 GGT, ALT and cancer risk Figure 1. Selection of studies included in the meta-analysis. ALT, alanine aminotransferase; GGT, gamma glutamyltransferase. (adjustment for confounders) and ascertainment of outcomes of interest. The NOS assigns a maximum of four points for selection, two points for comparability and three points for outcome. Nine points on the NOS reflects the highest study quality. A score of 5 indicated adequate quality for inclusion in the review. Statistical analysis The RR with 95% confidence intervals (CIs) was used as the common measure of association across studies. To enable a consistent approach to the meta-analysis and enhance interpretation of the findings, reported study-specific risk estimates (per-unit or standard deviation change, quintiles, quartiles or other groupings) were transformed to involve comparisons between the top third and bottom third of each study population s baseline distribution of GGT or ALT levels, using standard statistical methods 20,21 which have been described in detail in Supporting Information Appendix 5. Briefly, log risk estimates were transformed assuming a normal distribution (or that a transformation of the explanatory variable for which the risk ratio is based was normally distributed), with the comparison between top and bottom thirds being equivalent to 2.18 times the log risk ratio for a 1 standard deviation increase (or equivalently, as 2.18/2.54 times the log risk ratio for a comparison of extreme quarters and as 2.18/2.80 times the log risk ratio for a comparison of extreme quintiles). Standard errors of the log risk estimates were calculated using published confidence limits and were standardized in the same way. When studies published more than one estimate of the association according to subgroups (e.g., by sex), a within-study summary estimate was obtained using a fixed effect analysis. When reported risk estimates could not be transformed, we obtained the standardized estimates through correspondence with the study authors. Summary RRs were pooled using random effects models to minimize the effect of between-study heterogeneity. 22 We also performed a dose response association of circulating levels of GGT with risk of overall cancer. A 2-step generalized least-squares trend estimation (GLST) analysis as described by Greenland and Orsini, 21,23 was used to compute study-specific slopes (linear trends) from the correlated natural logs of the RRs across categories of GGT. Only studies that reported the number of cases, person-years of follow-up or noncases and the RRs with the variance estimates for at least three quantitative GGT categories were included. Potential nonlinear dose response relationships were examined by modeling levels of GGT using restricted cubic splines. Statistical heterogeneity across studies was quantified using standard chi-square tests and the I 2 statistic. 24 Studylevel characteristics including geographical location, sex differences, average duration of follow-up, number of outcomes, type of cancer endpoint (incidence or mortality), degree of adjustment and study quality were prespecified as characteristics for assessment of heterogeneity, which was conducted using stratified analysis and random effects meta-regression. 25 In analysis specified post hoc, stratified analysis was conducted to examine the difference in pooled RRs by baseline average age of participants. Sensitivity analyses were performed to assess the influence of each individual study by omitting one study at a time and calculating pooled estimates for the remainder of the studies. We assessed the potential for small study effects such as publication bias through formal tests, namely Begg s funnel plots 26 and Egger s regression symmetry test 27 and by comparing pooled results from studies involving 1,000 cancer outcomes with those from smaller studies. The overall summary estimates for the funnel plots were based on fixed effects models rather than randomeffects models as this is the preferred method; this is because a random-effects estimate gives greater relative weight to smaller studies and, therefore, will be more affected if publication bias is present. 28 We adjusted for publication bias using the Duval and Tweedie s nonparametric trim-and-fill method. 29 All analyses were conducted using Stata version 12 (Stata Corp, College Station, TX). Results Study identification and selection Our initial search identified 5,671 potentially relevant citations. After screening based on titles and abstracts, 79 articles remained for further evaluation. Following detailed assessments, 64 articles were excluded. The remaining 15 articles based on 14 unique prospective cohort studies met our inclusion criteria and were included in the meta-analysis (Fig. 1; Supporting Information Appendix 6). In aggregate, the included studies comprised 1,790,734 nonoverlapping participants and 57,534 cancer outcomes. A single study contributed over 70% of data (including up to 545,460 participants

4 Kunutsor et al Table 1. Summary characteristics of included studies Gamma glutamyltransferase Eligible studies No. of unique studies 10 9 Median (IQR) follow-up (years) Participants 12.5 ( ) Alanine aminotransferase 12.3 ( ) Total no. of participants 840,876 1,514,177 Total no. of cancers 52,852 44,397 Median (IQR) age (years) 47 (44 60) 55 (44 63) Location Europe 8 (806,058) 4 (551,127) North America 1 (14,950) 1 (14,950) Asia-Pacific 2 (19,868) 4 (948,100) Baseline enzyme level Median (IQR) pooled level (IU/L) Type of cancer no. of studies (no. of events) ( ) 23.8 ( ) Overall cancer 10 (52,724) 7 (41,996) Breast and female 4 (11,022) genital organs Breast cancer 2 (167) Male genital organs 3 (8,562) Urinary organs Digestive organs 6 (10,574) 3 (2,679) Esophageal cancer 1 (61) 1 (2,379) Liver cancer 3 (481) 2 (300) Respiratory and 4 (4,427) intrathoracic organs Nervous system 3 (2,522) Lymphoid, hematopoietic 3 (3,300) and related tissue Bone, connective tissue and soft tissues, skin 3 (4,314) Values are number of studies (number of participants) unless stated otherwise. 1 Are not unique studies or events. Abbreviation: IQR 5 interquartile range. and 37,809 cases) to the present review. 10 Table 1 provides a breakdown of overall and site-specific cancer outcomes for GGT and ALT. Study characteristics and study quality Supporting Information etable 1 provides details of the eligible studies that evaluated the associations of baseline circulating levels of GGT or ALT with risk of overall cancer or sitespecific cancer outcomes and their quality assessment scores. All included studies were prospective cohort studies carried out in United States, Europe (Guernsey, Scotland, Netherlands, Germany, Italy, Sweden, Finland and Austria) and Asia (Korea, Japan and Taiwan). Duration of follow-up for cancer outcomes ranged from 2 to 20 years, with a median follow-up of 12 years. All included studies were judged to be of high quality (quality score: 7 9). GGT and cancer risk Circulating GGT levels in relation to subsequent risk of overall cancer was reported in 10 cohort studies, involving 780,553 participants and 52,724 cancer events recorded during an average follow-up period ranging from 2 to 20 years (Supporting Information etable 1). The pooled RR (95% CI) for overall cancer outcomes in a comparison of individuals in the top thirds versus those in the bottom thirds of the population distribution of baseline GGT level, adjusted for several potential risk factors was 1.32 ( ) (Fig. 2; Supporting Information efig. 1). Exclusion of any single study at a time from the meta-analysis had minimal effect on the pooled RRs, which ranged from 1.28 ( ) to 1.35 ( ). The combined RR excluding the single largest study was 1.34 ( ), very similar to the main finding (Supporting Information efig. 1). There was substantial heterogeneity between studies (I 2 > 70%), which was partly explained by sex (p for meta-regression ; Fig. 3). A strong association was observed in men 1.60 ( ) compared to a null association in women 1.09 ( ). In further exploration of heterogeneity, this was substantially reduced and no longer significant, when we restricted the analysis to studies of the highest quality. Among the remaining studies, the pooled RR for overall cancer was 1.37 ( ), similar to the overall combined RR. The positive association was consistently observed across several subgroups. The Egger s test for publication bias was significant (p ), consistent with observed funnel plot asymmetry (Supporting Information efig. 3), suggesting that studies with less striking results were less likely to have been reported. Despite the concern that small studies with null results often tend not to be published, we found no clear evidence of such selective reporting when studies were grouped by size in meta-regression analysis (Fig. 4). Duval and Tweedie s trim-and-fill method resulted in no additional imputed studies. There were significant positive associations for site-specific cancers such as breast cancer, cancers of male genital organs, cancers of digestive organs and liver cancer (Fig. 2). ALT and cancer risk Seven cohort studies reported on the associations of circulating ALT levels with risk of overall cancer outcomes, including a total of 712,969 participants and 41,996 events (Table 1; Supporting Information etable 1). The pooled RR (95% CI) for overall cancer in a comparison of extreme thirds of baseline level of ALT was 1.14 ( ), with substantial heterogeneity (I 2 > 70%) between the studies (Fig. 4; Supporting Information efig. 2). Exclusion of any single study at a time from the meta-analysis did not appreciably alter the

5 1166 GGT, ALT and cancer risk Figure 2. Associations of gamma glutamyltransferase with overall and site-specific cancers. The summary estimates presented were calculated using random effects models; Size of data markers are proportional to the inverse of the variance of the relative ratio; CI, confidence interval (bars); GGT, gamma glutamyltransferase; RR, relative risk. Forest plot for individual studies of overall cancer are provided in Supporting Information. Figure 3. Associations of gamma glutamyltransferase and alanine aminotransferase levels with risk of overall cancer, grouped according to several study characteristics. The summary estimates presented were calculated using random effects models; CI, confidence interval (bars); ALT, alanine aminotransferase; GGT, gamma glutamyltransferase; RR, relative risk; *, p value for meta-regression. pooled RRs. The combined RR excluding the single largest study was 1.18 ( ), also very similar to the main finding. The inconsistency across the studies was to a large part explained by geographical location (p for metaregression < ; Fig. 3). The association was inverse in European populations 0.96 ( ) and positive in Asian populations 1.65 ( ), respectively. Of the four European studies, only the single largest study reported a

6 Kunutsor et al Figure 4. Associations of alanine aminotransferase with overall and site-specific cancers. The summary estimates presented were calculated using random effects models; Size of data markers are proportional to the inverse of the variance of the relative ratio; CI, confidence interval (bars); ALT, alanine aminotransferase; RR, relative risk. Forest plot for individual studies of overall cancer are provided in Supporting Information. nonlinear relation between GGT levels and overall cancer risk (p for nonlinearity50.57). The pooled RR per 5 U/L increment in GGT levels was 1.04 ( ). Figure 5. Dose response relation between levels of gamma glutamyltransferase and relative risks of overall cancer for pooled results of six studies providing relevant data. Adjusted relative risks and 95% confidence intervals (CIs; dashed lines) are reported. Data were modeled with restricted cubic splines with three knots in random-effects dose response models. The median value (5.5 U/L) of the lowest reference range was used to estimate all relative risks. The vertical axes are on log scales. significant inverse association for overall cancer. The Egger s test for publication bias was not significant (p ), consistent with observed funnel plot symmetry (Supporting Information efig. 3). Summary estimates were 2.44 ( ) for cancers of the digestive organs (three studies) and 3.27 ( ) for liver cancers (two studies). Dose response analyses Figure 5 shows the dose response relationship of GGT levels with risk of overall cancer outcomes for pooled results of six studies providing relevant data. There was no evidence of a Discussion The findings of this review indicate a significant positive association between circulating levels of GGT and risk of overall cancer incidence or mortality, consistent with a log-linear dose response relationship. Our pooled findings of a log-linear association of GGT levels with risk of overall cancer are consistent with findings from two previous published large rigorously conducted studies on the topic. 11,14 The positive association was generally consistent for site-specific cancers. There was no strong evidence of any association of ALT with overall cancer, however, stratified analysis by geographical location showed an inverse association in European populations and a positive association in Asian populations. Pooled analysis of three studies indicated a strong positive association of ALT levels with cancers of the digestive organs. Though the evidence indicates that GGT might be involved in tumor biology, the underlying pathways remain unclear. It is uncertain if GGT has a direct etiological role in carcinogenesis or may just be a marker of an underlying etiology. Several potential mechanisms for the positive association between GGT and cancer have been postulated. GGT plays a major role in glutathione metabolism, the major thiol antioxidant in the body, thereby playing an important role in cellular defence and protection of cells against further oxidative stress. However, GGT is also known to mediate oxidative stress, as it is a source of reactive oxygen species (ROS) during glutathione metabolism. Low levels of pro-oxidants (ROS) may modulate a range of biological responses involved in cellular growth, proliferation and apoptosis and immune

7 1168 GGT, ALT and cancer risk system functions. 30,31 The persistent production of ROS, resulting from GGT-mediated metabolism may thus contribute to tumor progression and invasion. GGT activity is increased in malignant lesions and has been considered to confer rapid turnover and survival advantages for neoplastic cells. 32 Elevated GGT levels are associated with hyperglycemia, which can lead to the overproduction of ROS, which has been implicated as the underlying link for the increased risk of cancer in patients with diabetes. 33 This is also supported by epidemiological data on the stronger associations between GGT and cancers observed for groups with elevated glucose levels compared to those with lower levels. 10 Additionally, GGT has been implicated as a marker of exposure to certain carcinogens, which include persistent environmental pollutants such as lead, dioxins or organochlorine pesticides. 34,35 Our analyses also showed GGT to be associated with site-specific cancers such as those of the digestive (including liver cancers) and genital organs, which are well known to be influenced by lifestyle and environmental factors (such as diet, smoking, alcohol consumption and pollutants), 36,37 and which also influence levels of GGT. 34,35,38 A particularly strong association was demonstrated for liver cancer, but the pooled estimate was imprecise (wide CIs) because of the available number of studies with small sample sizes. The GGT isoenzyme, GGTII, has been considered as a useful tumor marker for the diagnosis of small hepatocellular carcinoma, 39 and it has been speculated that an increased risk of diabetes might mediate the association of GGT and liver cancer. Alcohol consumption, which has a direct role in carcinogenesis, has been particularly linked to the positive association between GGT and these site-specific cancers, with suggestions that GGT may be a marker of alcohol-related cancers. 40 Despite the uncertain mechanisms underlying the association between GGT and cancer, our findings of a graded increase in overall cancer with increasing GGT levels, suggest the possibility of a causal relationship, but establishing this requires robust evidence from clinical trials. Several interventions are available that alter levels of GGT, however, these also influence levels of several hepatic and lipid factors. 41 In the absence of clinical trials however, Mendelian randomization (MR) studies of genetic variants specifically related to GGT levels may provide another route to assess whether GGT is directly causal in cancer. 42 A substantial proportion of the variance of GGT is under strong genetic regulation, with its heritability estimate reported to be as high as 77%. 43 There is evidence to suggest that the GGT1 locus, which is the main protein-coding gene for GGT, may be specific for GGT levels and therefore variants within this locus may be valid instrumental variables for MR studies. 44 Conen et al. 45 have recently used the rs variant of the GGT1 gene as an instrumental variable in a MR study and found evidence for a causal effect of GGT on fasting insulin. There were geographical variations in the association of ALT levels with overall cancer a positive association in Asians and an inverse association in European populations. The reasons for these variations are unclear, but we have observed similar findings in our recent reviews of ALT with all-cause mortality and cardiovascular disease outcomes. 9,46 Given that the liver is involved in hormone synthesis, it is expected that poor liver function would be associated with an increased risk of hormonally modulated cancers. There is a possibility that ALT may simply be a marker of an underlying etiology. Our findings also suggest that ALT is strongly associated with the development of cancers of the digestive organs (which may be largely driven by cancers of the liver). The exact mechanisms underlying the association remain unclear, but factors such as alcohol consumption, smoking, obesity, diet and insulin resistance have been postulated to be linked to this association. 47 Further studies are needed to dissect the biological mechanisms underlying these observed associations. Our findings are relevant and may have implications. They underscore a potentially deleterious role of increasing levels of these enzymes, particularly GGT, on future risk of cancer in general populations. Results from individual studies and metaanalyses 41,48,49 have found evidence to support a substantial lowering effect of variety of interventions (including lifestylerelated factors such as weight loss, 49 physical activity and dietary factors 48 ) on circulating levels of GGT and ALT. Carefully designed randomized controlled trials are warranted to investigate the impact of these interventions on risk of cancer. In the absence of such studies, individuals with elevated levels of these enzymes may need further clinical evaluation and close monitoring. The finding of an inverse association of ALT and overall cancer outcomes in European populations may need to be replicated in further studies. The strengths and potential limitations of this review and meta-analysis deserve consideration. This is the first metaanalysis of the prospective associations of GGT and ALT with cancer outcomes and had enhanced power to examine the associations in greater detail. Our meta-analysis included studies that recruited participants from approximately general populations, had long follow-up durations and sensitivity analyses yielded similar results when studies with less than 15 years follow-up durations were excluded (subgroup analyses), therefore minimising reverse causation and selection biases. We employed standardized risk estimates comparing extreme thirds of baseline distribution of GGT and ALT levels each, from contributing studies to allow a consistent combination of estimates across studies. Dose response relationships were also assessed using GLST analyses. Our review was limited by the moderate amount of data on several site-specific cancer outcomes. We were unable to examine fully the impact of adjustment for potential confounding factors such as alcohol consumption, diet, physical activity and genetic factors, because the present review was based on variably adjusted data reported by the eligible studies. Majority of studies included in the review, however, adjusted for some of these confounders, most notably alcohol consumption, and grouping the studies by degree of adjustment did

8 Kunutsor et al not alter the magnitude of the associations. The statistical methods used to standardize the risk estimates require assumptions such as approximate normality of the exposure variable or its transformation, which are commonly not perfectly fulfilled; despite the limitations, these methods provide approximate risk estimates and have proved useful for quantitative reviews of this nature. 20 There was a potential that the single study contributing over 70% of data to the present review may have largely driven the present results. However, sensitivity testing excluding this large study in all analyses yielded comparable results, indicating the robustness of our findings. Cancer outcomes in our review were defined as a mix of incident and mortality cases (Supporting Information Appendix 4). Given that cancer mortality cases are prone to disease misclassification due to both misdiagnoses and confounding effects of deaths from other causes, 50 this may have introduced biases in our results. However, stratified analysis by cancer endpoints (cancer incidence or deaths) showed consistent associations. It was not possible to correct the estimates for within-individual variation in levels of GGT or ALT, because the included studies lacked serial assessments of circulating levels of these exposures in the same individuals. Visual inspection of plots and formal tests demonstrated statistical evidence of publication bias for studies of GGT, though there was no clear evidence of such selective reporting when studies were grouped by size. Despite the lack of strong evidence of publication bias for studies of ALT, it is not possible to discount completely the influence of selective reporting; since tests for publication bias have low statistical power, especially when the number of studies is limited. There was substantial heterogeneity among the contributing prospective studies, however, we systematically explored and identified the possible sources of heterogeneity using stratified analyses, meta-regression and sensitivity analyses. More detailed analyses under a broader range of circumstances and exploration of potential sources of heterogeneity will require collaborative pooling of individual participant data from prospective studies. In conclusion, available observational evidence suggest a positive association of baseline circulating levels of GGT with overall cancer risk, consistent with a log-linear dose response relationship. The positive association was generally evident for site-specific cancers. There are geographical variations in the association of ALT and overall cancer risk and an increased risk of cancers of digestive organs with increasing ALT levels. Studies are needed to determine whether the association of GGT with cancer represents a causal relationship and if lowering levels of these enzymes may be beneficial in the prevention of cancer. Acknowledgements We thank Ruben Hernaez, PhD, Department of Medicine, John Hopkins School of Medicine, 2024 East Monument Street, Suite 2 600, Baltimore, MD 21287, for readily providing data on request. References 1. Whitfield JB. Gamma glutamyl transferase. Crit Rev Clin Lab Sci 2001;38: Wroblewski F, Ladue JS. Serum glutamic pyruvic transaminase in cardiac with hepatic disease. Proc Soc Exp Biol Med 1956;91: Fraser A, Harris R, Sattar N, et al. Alanine aminotransferase, gamma-glutamyltransferase, and incident diabetes: the British Women s Heart and Health Study and meta-analysis. Diabetes Care 2009;32: Kunutsor SK, Apekey TA, Walley J. Liver Aminotransferases and Risk of Incident Type 2 Diabetes: a Systematic Review and Meta-Analysis. Am J Epidemiol doi: /aje/kws Fraser A, Harris R, Sattar N, et al. Gamma-glutamyltransferase is associated with incident vascular events independently of alcohol intake: analysis of the British Women s Heart and Health Study and Meta-Analysis. Arterioscler Thromb Vasc Biol 2007;27: Schindhelm RK, Dekker JM, Nijpels G, et al. Alanine aminotransferase predicts coronary heart disease events: a 10-year follow-up of the Hoorn Study. Atherosclerosis 2007;191: Ruttmann E, Brant LJ, Concin H, et al. Gammaglutamyltransferase as a risk factor for cardiovascular disease mortality: an epidemiological investigation in a cohort of 163,944 Austrian adults. Circulation 2005;112: Kim HC, Nam CM, Jee SH, et al. Normal serum aminotransferase concentration and risk of mortality from liver diseases: prospective cohort study. 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