Association between antithrombotic agents use and hepatocellular carcinoma risk: a two-sample Mendelian randomization analysis

Association between antithrombotic agents use and hepatocellular carcinoma risk: a two-sample Mendelian randomization analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Association between antithrombotic agents use and hepatocellular carcinoma risk: a two-sample Mendelian randomization analysis Fengyi Yang, Ouyang Li, Benjian Gao, Zhuo Chen, Bo Li, Jiaqi He, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4608895/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Oct, 2024 Read the published version in Journal of Cancer Research and Clinical Oncology → Version 1 posted 7 You are reading this latest preprint version Abstract Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide. Multiple observational studies demonstrated a negative correlation between the use of antithrombotic agents and the risk of HCC. However, the precise causal relationship between these factors remains uncertain. Therefore, our study used a two-sample Mendelian randomization (MR) analysis to assess the causal link between these two factors. Method: The summary statistics of single nucleotide polymorphisms (SNPs) associated with the use of antithrombotic agents were acquired from a genome-wide association study (GWAS) performed on individuals of European descent, as well as from the GWAS on the UK Biobank. A two-sample MR analysis was performed using the inverse variance weighting (IVW), the weighted median estimate, the MR-Egger regression, and the weighted-mode estimate. The robustness of the primary findings was assessed by sensitivity analysis. Results: Ten SNPs associated with the use of antithrombotic agents were selected as instrumental variables. The MR analysis performed using the four methods mentioned above revealed a negative correlation between the use of antithrombotic agents and HCC. The other methods also produced similar results. No heterogeneity and horizontal pleiotropy were found. Conclusion: Our findings suggested an inverse association of antithrombotic agents with the risk of HCC. antithrombotic agents hepatocellular carcinoma Mendelian randomization study causal relationship Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Hepatocellular carcinoma (HCC) accounts for the vast majority of liver cancer cases and fatalities. It has become a growing disease burden on a global scale in recent decades (McGlynn, Petrick, and El-Serag, 2021 ; Singal, Lampertico, and Nahon, 2020 ; Fitzmaurice et al. 2019.). The World Health Organization estimates an annual rise in the mortality rate of liver cancer patients, exceeding one million by 2030 (Villanueva, 2019 ). Hepatitis B infections are at present the main risk factors for HCC. Moreover, the presence of cirrhosis, metabolic disorders, and autoimmune diseases as well as genetic predisposition are factors increasing the risk of HCC (Shetty and Kellarai, 2022 ). Surgical resection represents the established therapeutic approach for HCC patients with good liver function and no obvious vascular invasion or distant metastasis. Although the rate of HCC relapse or development of new tumors has declined due to the development of locoregional and systemic therapies in these years, the rate is over 75% (Siegel et al. 2021 ; Heimbach et al. 2018 ; Marrero et al. 2018 ). HCC is associated with a significant pain and a substantial economic burden. Hence, it is of the utmost importance to identify as yet unknown causes and apply preventive measures to reduce the growing incidence rate. Aspirin, warfarin, and heparin are common antithrombotic agents. Aspirin, whose active principle is acetylsalicylic acid, is a commonly used medication against inflammatory and antiplatelet diseases (DiNicolantonio Jj Fau - O'Keefe, O'Keefe Jh Fau - Lavie, and Lavie, 2012). So far, no primary prevention trials have been conducted to evaluate the use of Aspirin on HCC risk, but basic and clinical research demonstrates preventive or therapeutic properties against cancer (Drew and Chan, 2021 ). HCC is classified as an inflammation-associated malignancy involving a series of interconnected processes, including hepatocyte injury, inflammation, necrosis, and subsequent regeneration. These processes lead to a state of chronic inflammation, fibrosis, cirrhosis, and genomic instability, contributing to the development and progression of HCC (Kumar, Zhao X Fau - Wang, and Wang, 2011). Platelets play a pivotal role throughout the necro-inflammatory process in the liver by stimulating the increase of inflammatory and immune cells, increasing risk of liver damage and cancer, and promoting the development of a fibrogenic microenvironment (Iannacone et al. 2005 ; Maini and Schurich, 2012 ; Pavlovic et al. 2019 ). Therefore, the use of antithrombotic agents has been associated with a decreased risk of HCC. Rebecca W. Zeng et al. used a random effects model pooling multivariable-adjusted hazard ratios for HCC using the Medline and Embase databases, revealing that the use of aspirin is associated with the risk of HCC, although its degree of significance is not yet clear (Zeng et al. 2023 ). Although many observational studies showed an inverse association between the use of antithrombotic agents and HCC risk, few have directly assessed the association between antithrombotic agents and HCC. Therefore, a randomized controlled trial would be the ideal study design to identify the effect of the use of antithrombotic agents on HCC risk. Significant advances were performed during the last decade on large-scale genome-wide association studies (GWASs) and on the powerful statistical tool mendelian randomization (MR). These progresses provide valuable opportunities to comprehensively and cost-effectively evaluate the causal relationship among various phenotypes (Wu et al. 2019 ). MR is a scientific approach that uses genetic variations as instrumental variables (IVs) to assess whether the observed association between risk factors and outcomes indicates a causal relationship (Burgess, Daniel, et al. 2015 ). MR analysis overcomes the influence of confounding factors, including behavioral and environmental factors (Burgess, Butterworth A Fau - Malarstig, et al. 2012; Smith and Ebrahim, 2004 ). Furthermore, it provides reliable evidence on causal relationships between risk factors and diseases, simultaneously guiding the performance of clinical trials and the development of pharmaceutical interventions (Davies, Holmes, and Davey Smith, 2012; Burgess, Butterworth A Fau - Malarstig, et al. 2018). Therefore, our study make use of two-sample MR to evaluate the causal relationship between the use of antithrombotic agents and HCC risk. The latest drug GWAS data that cover a wide range of populations were used, and accessing the dependability of MR results was evaluated. Our result might provide new evidence being in favor of the causal relationships between the use of antithrombotic agents and HCC risk, providing potential direction for future drug trials. 2. Material and Methods 2.1 Data sources Our workflow diagram is shown in Fig. 1 . This is an MR study investigating the causal relationship between the use of antithrombotic agents and the risk of HCC. A publicly available summary statistics datasets were obtained from the FinnGen and UK Biobank studies. The exposure variable involved the performance of GWAS meta-analysis on antithrombotic agents among individuals of European descent (n = 153,639), while the outcome variable was based on the GWAS data from the UK Biobank (n = 475,638). The cancer outcomes were classified according to the data from the national cancer registry, electronic health records, hospital episode statistics, death certificates and verified self-reported information. The International Classification of Disease version 10 (ICD-10) code C22.0 was used to identify HCC. 2.2 Filter instrumental variables IVs are selected based on predetermined criteria. There are three conditions that MR study need to satisfy. First, the IV is cosely associated with the exposure. Second,no confounding factors could influence the outcome, only exposure could modulate the influence of IVs on the outcome. Third, IVs is not relevant to the outcome (Slob and Burgess, 2020 ). Single nucleotide polymorphisms (SNPs) showing a significant association with the use of antithrombotic agents at the conventional GWAS threshold were extracted to avoid a too small number of SNPs (P < 5×10 − 8 ) (Sanna et al. 2019 ). Linkage disequilibrium (LD) shows that the population exhibits a notable deviation from random expectation with regards to the relatively high frequency of inheritance of two genes located at distinct loci (Sanna et al. 2019 ). We have implemented a number of steps for strict quality control. We selected SNPs with genome-wide significant using a clumping function in PLINK(r 2 < 0.001, kb = 10,000). Meanwhile, no LD was found in summary statistics. The F statistic of the IVs was calculated with the formula below and those with F less than 10 showed that genetic variants were considered insufficient and excluded (Cui et al. 2021 ). F = r 2 (N-2) / (1-r 2 ) in which N is the sample size, r 2 is the variance explained by IVs (Kurilshikov et al. 2021 ) The PhenoScanner was used to assess whether the SNPs were related to HCC confounders such as hepatitis B, cirrhosis, obesity, and non-alcoholic fatty liver disease according to the HCC guidelines(Kamat et al. 2019 ; Vogel et al. 2022 ). The remaining SNPs were used for MR analysis after the removal of the confounding factors. 2.3 MR analysis The inverse variance-weighted (IVW) approach was used as the principal analysis method, while three other methods, such as MR-Egger regression, weighted median analysis, and weighted mode, were used as secondary references. The IVW is a robust approach assuming that all genetic variants are valid IVs, specifically only one specific pathway of exposure (antithrombotic agents) acts on the target outcome (HCC) (Burgess, Butterworth A Fau - Thompson, and Thompson, 2013). Next, the IVW method was used to examine the association between the use of antithrombotic agents and the risk of HCC. IVW with a multiplicative random-effect method provides a more precise estimation and confidence interval (CI) than the fixed-effect IVW method in case of heterogeneity (Burgess, Davey Smith, et al. 2023). The fixed-effect IVW method was used as the primary approach in the absence of heterogeneity, while the multiplicative random-effects IVW method was used in the presence of heterogeneity. Additional sensitivity analysis was performed only when the IVW results yielded meaningful findings. Egger regression distinguishes itself from IVW by adding an intercept term to detect horizontal pleiotropy, also known as the MR-Egger intercept test (Hemani et al. 2018 ; Cho et al. 2020 ). On the other hand, the weighted median method assigns higher importance to SNPs with larger beta values, contributing more to the estimation of result.31 The advantage of the weighted median method is that only half of the valid SNPs provide unbiased causal estimates (Walker et al. 2019 ). The IVW results were trustworthy when no heterogeneity and pleiotropy were present (Bowden, Davey Smith, and Burgess, 2015 ). 2.4 Sensitivity analysis The global test was initially performed using the Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) approach to assess the horizontal pleiotropy. Any outliers (i.e., SNPs with P < 0.05) indicating the presence of horizontal pleiotropy were subsequently removed. Furthermore, MR-Egger regression was used to evaluate the potential bias resulting from horizontal pleiotropy. The absence of horizontal pleiotropy was indicated when the intercept approached 0 (not significant P values) (Burgess and Thompson, 2017 ). Next, Cochran's Q statistics were also performed to confirm the heterogeneity among the selected IVs (Bowden and Holmes, 2019 ), and the influence of a single outlier on the overall findings was evaluated by conducting a leave-one-out analysis, whereby individual SNPs were sequentially excluded and the remaining SNP effects were recalculated. This method can effectively reduce the pleiotropy or heterogeneity of SNPs. 2.5 Statistical analysis Analyses were carried out in R version 4.0.1 using the “Two-Sample MR” package. P < 0.05 was considered statistically significant. 3. Results A total of 29 SNPs were included in this study. The positive results of these SNPs were evaluated using PhenoScanner and 19 SNPs were found associated with the confounders mentioned in the paragraph 2.2. The F-statistics of all SNPs were > 10. The MR analysis revealed a negative correlation between the use of antithrombotic agents and HCC (Table 1 ). The infuence of SNPs sizes on the exposure (antithrombotic agents) and outcome (HCC) was demonstrated by the scatter plots (Fig. 2 ). The Steiger’s Z test direction was true (P < 0.05), showing that SNPs acted on the exposure first, and next on the outcome. Table 1 MR analysis for antithrombotic agents use on HCC. Method Number of SNPs OR 95% Confidence Interval P Inverse variance weighted 10 0.444 0.279, 0.707 0.001 MR Egger 10 0.259 0.061, 1.084 0.102 Weighted median 10 0.462 0.257, 0.831 0.010 Weighted mode 10 0.693 0.237, 2.024 0.520 Abbreviations: HCC, hepatocellular carcinoma; MR, Mendelian randomization; SNPs, single nucleotide polymorphisms; OR, odds ratio. Cochran’s Q-test results showed that no significant results after the use of antithrombotic agents, indicating that all of the IVs were no heterogeneous [IVW, Q (df) 12.339 (9) P = 0.1949; MR–Egger, Q (df) 11.454 (8), P = 0.1773]. The funnel plot indicated evidence of symmetrical distribution, indicating the lack of directional horizontal pleiotropy (Fig. 3 ), suggesting that a potential bias was less likely to influence the causal association. The MR–Egger regression for IVs confirmed the absence of horizontal pleiotropy (intercept β = 0.0319, P = 0.454). The findings of the leave-one-out analysis were firm in the evaluation of the outcomes of this MR analysis, as shown in Fig. 4 ; SNPs had no fundamental effect on the results regardless of which SNP was removed.This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn. 4. Discussion This MR study intended to elucidate the prospective causal relationship between the use of antithrombotic agents and the risk of HCC in the European population. Our results demonstrated an impressive inverse association between the two, confirmed by the subsequent sensitivity analysis. The awareness of this association has grown in recent years. Nonetheless, observational studies are unable to establish the correct direction of causality. Our study is the first confirming the causal relationship between these two factors using MR analysis. MR methods are builded on the principle of Mendel's laws of inheritance. The prediction of antithrombotic agent responsiveness based on genetic variation is determined at birth, and does not influence the progression or treatment of the disease. As a result, causal inferences are protected from biases arising from confounding and reverse causality. The establishment of a causal relationship between the use of antithrombotic agents and HCC is scarce. Numerous studies currently indicate a 41% diminution in the risk of HCC and a 45% decrease in the risk of mortality due to chronic liver disease when aspirin is used compared to when it is not (Sahasrabuddhe et al. 2012 ). Furthermore, The Liver Cancer Pooling Project demonstrated a 32% reduction in the risk of HCC associated to the use of aspirin, while no significant association was observed using ibuprofen (Petrick et al. 2015 ). Moreover, Petrick, J.L., et al found that the decrease in HCC incidence is more pronounced among individuals who regularly and daily use aspirin and at low doses. Importantly, this reduction in HCC risk remains consistent regardless of the duration of the treatment (Petrick et al. 2015 ). TThe pooled analysis of the data from the Nurses' Health Study and the Health Professionals Follow-up Study demonstrated a significant 49% in the reduction of HCC incidence among both male and female participants who regularly use aspirin (Simon et al. 2018 ). The National Cohort Study of Korean Adults discovered a dose-dependent 13% reduction in the risk of HCC associated with the use of aspirin compared to when it is not (Hwang et al. 2018 ). These retrospective studies performed in the general population are subjected to various limitations, including the non-randomized assignment of the treatment. However, a study that used Taiwan's Longitudinal Health Insurance database noted an approximate 50% decrease in the risk of HCC among both males and females who carry HCV and use aspirin (Liao et al. 2020 ). A retrospective cohort study performed among Korean patients suffering from alcoholic cirrhosis demonstrated a significant 87% reduction in HCC risk associated with the use of low-dose aspirin compared to when it is not (Shin et al. 2020 ). Similarly, an independent correlation between the use of lower dose aspirin and a 29% decrease in the risk of HCC was found in a retrospective population-based cohort study performed in Taiwan among patients with hepatitis B virus infection (Lee et al. 2019 ). Our results are consistent with all these previous research indicating the association between the use of antithrombotic agents and the decreased risk of HCC. Antithrombotic agents inhibit the progression of HCC by cyclooxygenase-dependent and independent mechanisms. These mechanisms include the control of inflammation or cell proliferation, leading apoptosis or autophagy, the reduction of fibrosis, and the repression of platelet function (Huang et al. 2018 ; Shi et al. 2020 ; Wang et al. 2019 ). However, no statistically relevant association between the use of aspirin and a reduction in the risk of HCC among patients with underlying cirrhosis was found in a multivariable stratified analysis (Lee et al. 2019 ). Chiu et al. reported a lack of significant impact of aspirin on the decreased risk of HCC in a mass case study (Chiu et al. 2010). Other studies reported conflicting helpful influences of aspirin on the decreased of HCC risk in hepatitis B virus patients (Tsan et al. 2012 ; Kim et al. 2013). A meta-analysis considering 12 studies also indicated a lack of a significant impact of aspirin on the recurrence of HCC (Pang et al. 2017 ). Therefore, the involvement of antithrombotic agents in the risk of HCC continues to be a controversial topic. Our finding was relied on a greater robust study design different from the above previous research. This discrepancy can be attributed to the inherent constraints of observational studies, which fail to establish a causal relationship, as well as the use of small sample sizes, which may not provide adequate evidence to draw a definitive conclusion. Our study presents numerous strengths. The 10 SNPs stringently selected in Europeans were unattached and strictly associated with the use of antithrombotic agents, avoiding the influence of LD on the causal estimate. The use of MR analysis reduced the likelihood of bias from confounding factors and reversed the causation in comparison to traditional observational studies. Consequently, the SNPs used in this investigation and the matching research results are significantly valid and dependable. Our results supplied more compelling evidence to support the causal relationship between the use of antithrombotic agents and the decreased risk of HCC. However, our study is not without limitations. The GWAS data associated to antithrombotic agents and HCC predominantly originates from individuals of European descent, thereby lacking representation from other ethnic populations. Therefore, the results of our MR analysis should be exercised with caution when generalizing to other ethnic groups, since its applicability might be limited to populations of European ancestry. Moreover, it is important to acknowledge the inherent limitations of the two-sample MR analysis since it provides estimates of hypothetical causal associations despite this analysis serves as a important device for examining the causal relationship between antithrombotic agents and HCC. Accordingly, further analyses are essential to confirm these results. 5. Conclusion The use of antithrombotic agents results in a negative association with the risk of HCC. Nevertheless, further comprehensive investigations are needed due to the limitations of this study to deepen the relationship between antithrombotic agents and HCC risk, including beneficiary populations and underlying mechanisms to improve the rational guidance for HCC treatment. Declarations Author Contributions F.Y., O.L., J.H. and X.Y. contributed to study, concept,and design. F.Y., O.L., B.G., and Z.C. contributed to the acquisition of data. F.Y. and C.Z. performed the Mendelian randomization analysis, visualized the data and wrote the first draft of the manuscript. F.Y., O.L., B.G., Z.C., J.H. and X.Y. revised the manuscript. F.Y., O.L., B.G., Z.C., J.H. and X.Y. contributed equally to this work. Acknowledgments We would like to thank MogoEdit (https://www.mogoedit.com) for its English editing during the preparation of this manuscript. Funding ： This work was supported by the Key Research and Development Project of the Science & Technology Department of Sichuan Province (Nos.2023YFQ0101), Sichuan Science and Technology Program (Nos.2022YFS0625) and Luzhou Science and Technology and Talent Work Bureau (Nos.2022-SYF-55). Conflict of interest All authors have no conflicts of interest. 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Am J Gastroenterol. 106(5):894-898. doi:10.1038/ajg.2010.475 Tsan YT, Lee CH, Wang JD, Chen PC (2012). Statins and the risk of hepatocellular carcinoma in patients with hepatitis B virus infection [published correction appears in J Clin Oncol. 2013 Aug 20;31(24):3049]. J Clin Oncol. 30(6):623-630. doi:10.1200/JCO.2011.36.0917 Kim G, Jang SY, Han E, et al (2017). Effect of statin on hepatocellular carcinoma in patients with type 2 diabetes: A nationwide nested case-control study. Int J Cancer. 2017;140(4):798-806. doi:10.1002/ijc.30506. Pang Q, Jin H, Qu K, et al (2017). The effects of nonsteroidal anti-inflammatory drugs in the incident and recurrent risk of hepatocellular carcinoma: a meta-analysis. Onco Targets Ther. 10:4645-4656. Published 2017 Sep 20. doi:10.2147/OTT.S143154 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 22 Oct, 2024 Read the published version in Journal of Cancer Research and Clinical Oncology → Version 1 posted Editorial decision: Revision requested 07 Jul, 2024 Reviews received at journal 05 Jul, 2024 Reviewers agreed at journal 01 Jul, 2024 Reviewers invited by journal 24 Jun, 2024 Editor assigned by journal 20 Jun, 2024 Submission checks completed at journal 20 Jun, 2024 First submitted to journal 19 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4608895","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323843979,"identity":"8ce62da8-99b9-40c9-872a-1a7b62a108c1","order_by":0,"name":"Fengyi Yang","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fengyi","middleName":"","lastName":"Yang","suffix":""},{"id":323843980,"identity":"385ccc6f-4bdb-4bca-afcb-2f40421080e2","order_by":1,"name":"Ouyang Li","email":"","orcid":"","institution":"Huadong Hospital Affiliated to Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Ouyang","middleName":"","lastName":"Li","suffix":""},{"id":323843981,"identity":"0f43deb7-0f9b-4060-af7f-401ba364df6d","order_by":2,"name":"Benjian Gao","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Benjian","middleName":"","lastName":"Gao","suffix":""},{"id":323843982,"identity":"159e2112-9873-403f-9380-156161cdec2f","order_by":3,"name":"Zhuo Chen","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuo","middleName":"","lastName":"Chen","suffix":""},{"id":323843983,"identity":"96807383-a657-4e62-8123-52281624eb14","order_by":4,"name":"Bo Li","email":"","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Li","suffix":""},{"id":323843984,"identity":"7ce1f10f-6837-43ef-917a-6d24732907e7","order_by":5,"name":"Jiaqi He","email":"","orcid":"","institution":"Huadong Hospital Affiliated to Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jiaqi","middleName":"","lastName":"He","suffix":""},{"id":323843985,"identity":"12f33372-fbd8-4a5f-b4da-9e54931920be","order_by":6,"name":"Xiaoli Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACeYYDiQ8+GNgw87M3EKnFsPHAY8MZFWnskj0HiLXm8MFn0jxnDvEbzEggUgdj2+E0Cd62A9IGko833mCosYkmqIWd51iyhWTbHWNz6bRiC4ZjabkNBG2ZcSbxhmHbs2TL2TlmEowNhwlrYbj//oNEYtvh+g03zxCr5cCBJIkDZw4zG9zgIVKLYcOBZMOGijRmyR6gXxKI8QsoKh//AUfl4Y03PtTYEOEwJGAgkUCKcogWUnWMglEwCkbByAAAivJHibkwrKYAAAAASUVORK5CYII=","orcid":"","institution":"The Affiliated Hospital of Southwest Medical University","correspondingAuthor":true,"prefix":"","firstName":"Xiaoli","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-06-20 03:39:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4608895/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4608895/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00432-024-05960-7","type":"published","date":"2024-10-22T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60617315,"identity":"d8012b14-0b83-4e61-be6d-85e489830524","added_by":"auto","created_at":"2024-07-18 20:29:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":59390,"visible":true,"origin":"","legend":"\u003cp\u003eWorkflow of the MR analysis.Assumption 1 represents that instrumental variables (IVs) are strongly associated with exposure and only affected the outcome through the exposure; Assumption 2 represents that IVs are not associated with confounders; Assumption 3 represents that IVs are not directly associated with the outcome.MR, Mendelian randomization\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4608895/v1/174da884c5b0fb750436d8a8.png"},{"id":60617318,"identity":"4e962a64-0530-4247-b176-eca4b84c9717","added_by":"auto","created_at":"2024-07-18 20:29:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":34395,"visible":true,"origin":"","legend":"\u003cp\u003eScatter plots of the MR analysis. Each point represents a single SNP, and the line on each point represents 95% CI. The slope of each line represents the MR effect for each method. CI, confidence interval; MR, Mendelian randomization; SNP, single nucleotide polymorphism.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4608895/v1/f23339f235ea29d0f95b40ee.png"},{"id":60618619,"identity":"4b15aa82-9b34-4b75-9f04-33bd31a7c2bd","added_by":"auto","created_at":"2024-07-18 20:37:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14300,"visible":true,"origin":"","legend":"\u003cp\u003eFunnel plot of the MR analysis. The vertical blue and dark blue lines represent the causal effects estimated using the inverse variance weighting and MR–Egger methods, respectively. IV, instrumental variable; MR, Mendelian randomization; SE, standard error.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4608895/v1/f0beb57b6462d049fb7af50a.png"},{"id":60617316,"identity":"b88b477d-4d84-4fa2-a6e5-d908eb3d122b","added_by":"auto","created_at":"2024-07-18 20:29:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16516,"visible":true,"origin":"","legend":"\u003cp\u003eLeave-one-out results. Each black point represents result of the IVW MR method applied to estimate the causal effect of antithrombotic agents use on the reduced risk of HCC excluding particular SNP from the analysis. Each red point depicts the IVW estimate using all SNPs.No matter which SNP was removed, it did not have a fundamental effect on the results.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4608895/v1/1e5660c8b316528458d63cb8.png"},{"id":67682611,"identity":"a184b3ed-7264-4c28-9bd9-1e927d6db627","added_by":"auto","created_at":"2024-10-28 16:14:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":502352,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4608895/v1/180212f5-f5f7-4819-8438-21d05bf1e5e3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Association between antithrombotic agents use and hepatocellular carcinoma risk: a two-sample Mendelian randomization analysis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) accounts for the vast majority of liver cancer cases and fatalities. It has become a growing disease burden on a global scale in recent decades (McGlynn, Petrick, and El-Serag, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singal, Lampertico, and Nahon, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fitzmaurice et al. 2019.). The World Health Organization estimates an annual rise in the mortality rate of liver cancer patients, exceeding one million by 2030 (Villanueva, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Hepatitis B infections are at present the main risk factors for HCC. Moreover, the presence of cirrhosis, metabolic disorders, and autoimmune diseases as well as genetic predisposition are factors increasing the risk of HCC (Shetty and Kellarai, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Surgical resection represents the established therapeutic approach for HCC patients with good liver function and no obvious vascular invasion or distant metastasis. Although the rate of HCC relapse or development of new tumors has declined due to the development of locoregional and systemic therapies in these years, the rate is over 75% (Siegel et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Heimbach et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Marrero et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). HCC is associated with a significant pain and a substantial economic burden. Hence, it is of the utmost importance to identify as yet unknown causes and apply preventive measures to reduce the growing incidence rate.\u003c/p\u003e \u003cp\u003eAspirin, warfarin, and heparin are common antithrombotic agents. Aspirin, whose active principle is acetylsalicylic acid, is a commonly used medication against inflammatory and antiplatelet diseases (DiNicolantonio Jj Fau - O'Keefe, O'Keefe Jh Fau - Lavie, and Lavie, 2012). So far, no primary prevention trials have been conducted to evaluate the use of Aspirin on HCC risk, but basic and clinical research demonstrates preventive or therapeutic properties against cancer (Drew and Chan, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). HCC is classified as an inflammation-associated malignancy involving a series of interconnected processes, including hepatocyte injury, inflammation, necrosis, and subsequent regeneration. These processes lead to a state of chronic inflammation, fibrosis, cirrhosis, and genomic instability, contributing to the development and progression of HCC (Kumar, Zhao X Fau - Wang, and Wang, 2011). Platelets play a pivotal role throughout the necro-inflammatory process in the liver by stimulating the increase of inflammatory and immune cells, increasing risk of liver damage and cancer, and promoting the development of a fibrogenic microenvironment (Iannacone et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Maini and Schurich, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pavlovic et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, the use of antithrombotic agents has been associated with a decreased risk of HCC. Rebecca W. Zeng et al. used a random effects model pooling multivariable-adjusted hazard ratios for HCC using the Medline and Embase databases, revealing that the use of aspirin is associated with the risk of HCC, although its degree of significance is not yet clear (Zeng et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although many observational studies showed an inverse association between the use of antithrombotic agents and HCC risk, few have directly assessed the association between antithrombotic agents and HCC. Therefore, a randomized controlled trial would be the ideal study design to identify the effect of the use of antithrombotic agents on HCC risk.\u003c/p\u003e \u003cp\u003eSignificant advances were performed during the last decade on large-scale genome-wide association studies (GWASs) and on the powerful statistical tool mendelian randomization (MR). These progresses provide valuable opportunities to comprehensively and cost-effectively evaluate the causal relationship among various phenotypes (Wu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). MR is a scientific approach that uses genetic variations as instrumental variables (IVs) to assess whether the observed association between risk factors and outcomes indicates a causal relationship (Burgess, Daniel, et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). MR analysis overcomes the influence of confounding factors, including behavioral and environmental factors (Burgess, Butterworth A Fau - Malarstig, et al. 2012; Smith and Ebrahim, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Furthermore, it provides reliable evidence on causal relationships between risk factors and diseases, simultaneously guiding the performance of clinical trials and the development of pharmaceutical interventions (Davies, Holmes, and Davey Smith, 2012; Burgess, Butterworth A Fau - Malarstig, et al. 2018).\u003c/p\u003e \u003cp\u003eTherefore, our study make use of two-sample MR to evaluate the causal relationship between the use of antithrombotic agents and HCC risk. The latest drug GWAS data that cover a wide range of populations were used, and accessing the dependability of MR results was evaluated. Our result might provide new evidence being in favor of the causal relationships between the use of antithrombotic agents and HCC risk, providing potential direction for future drug trials.\u003c/p\u003e"},{"header":"2. Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Data sources\u003c/h2\u003e \u003cp\u003eOur workflow diagram is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. This is an MR study investigating the causal relationship between the use of antithrombotic agents and the risk of HCC. A publicly available summary statistics datasets were obtained from the FinnGen and UK Biobank studies. The exposure variable involved the performance of GWAS meta-analysis on antithrombotic agents among individuals of European descent (n\u0026thinsp;=\u0026thinsp;153,639), while the outcome variable was based on the GWAS data from the UK Biobank (n\u0026thinsp;=\u0026thinsp;475,638). The cancer outcomes were classified according to the data from the national cancer registry, electronic health records, hospital episode statistics, death certificates and verified self-reported information. The International Classification of Disease version 10 (ICD-10) code C22.0 was used to identify HCC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Filter instrumental variables\u003c/h2\u003e \u003cp\u003eIVs are selected based on predetermined criteria. There are three conditions that MR study need to satisfy. First, the IV is cosely associated with the exposure. Second,no confounding factors could influence the outcome, only exposure could modulate the influence of IVs on the outcome. Third, IVs is not relevant to the outcome (Slob and Burgess, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Single nucleotide polymorphisms (SNPs) showing a significant association with the use of antithrombotic agents at the conventional GWAS threshold were extracted to avoid a too small number of SNPs (P\u0026thinsp;\u0026lt;\u0026thinsp;5\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e) (Sanna et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Linkage disequilibrium (LD) shows that the population exhibits a notable deviation from random expectation with regards to the relatively high frequency of inheritance of two genes located at distinct loci (Sanna et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We have implemented a number of steps for strict quality control. We selected SNPs with genome-wide significant using a clumping function in PLINK(r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, kb\u0026thinsp;=\u0026thinsp;10,000). Meanwhile, no LD was found in summary statistics. The F statistic of the IVs was calculated with the formula below and those with F less than 10 showed that genetic variants were considered insufficient and excluded (Cui et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). F\u0026thinsp;=\u0026thinsp;r\u003csup\u003e2\u003c/sup\u003e (N-2) / (1-r\u003csup\u003e2\u003c/sup\u003e) in which N is the sample size, r\u003csup\u003e2\u003c/sup\u003e is the variance explained by IVs (Kurilshikov et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe PhenoScanner was used to assess whether the SNPs were related to HCC confounders such as hepatitis B, cirrhosis, obesity, and non-alcoholic fatty liver disease according to the HCC guidelines(Kamat et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vogel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The remaining SNPs were used for MR analysis after the removal of the confounding factors.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 MR analysis\u003c/h2\u003e \u003cp\u003eThe inverse variance-weighted (IVW) approach was used as the principal analysis method, while three other methods, such as MR-Egger regression, weighted median analysis, and weighted mode, were used as secondary references. The IVW is a robust approach assuming that all genetic variants are valid IVs, specifically only one specific pathway of exposure (antithrombotic agents) acts on the target outcome (HCC) (Burgess, Butterworth A Fau - Thompson, and Thompson, 2013). Next, the IVW method was used to examine the association between the use of antithrombotic agents and the risk of HCC. IVW with a multiplicative random-effect method provides a more precise estimation and confidence interval (CI) than the fixed-effect IVW method in case of heterogeneity (Burgess, Davey Smith, et al. 2023). The fixed-effect IVW method was used as the primary approach in the absence of heterogeneity, while the multiplicative random-effects IVW method was used in the presence of heterogeneity. Additional sensitivity analysis was performed only when the IVW results yielded meaningful findings. Egger regression distinguishes itself from IVW by adding an intercept term to detect horizontal pleiotropy, also known as the MR-Egger intercept test (Hemani et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cho et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). On the other hand, the weighted median method assigns higher importance to SNPs with larger beta values, contributing more to the estimation of result.31 The advantage of the weighted median method is that only half of the valid SNPs provide unbiased causal estimates (Walker et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The IVW results were trustworthy when no heterogeneity and pleiotropy were present (Bowden, Davey Smith, and Burgess, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Sensitivity analysis\u003c/h2\u003e \u003cp\u003eThe global test was initially performed using the Mendelian Randomization Pleiotropy RESidual Sum and Outlier (MR-PRESSO) approach to assess the horizontal pleiotropy. Any outliers (i.e., SNPs with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) indicating the presence of horizontal pleiotropy were subsequently removed. Furthermore, MR-Egger regression was used to evaluate the potential bias resulting from horizontal pleiotropy. The absence of horizontal pleiotropy was indicated when the intercept approached 0 (not significant P values) (Burgess and Thompson, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Next, Cochran's Q statistics were also performed to confirm the heterogeneity among the selected IVs (Bowden and Holmes, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and the influence of a single outlier on the overall findings was evaluated by conducting a leave-one-out analysis, whereby individual SNPs were sequentially excluded and the remaining SNP effects were recalculated. This method can effectively reduce the pleiotropy or heterogeneity of SNPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eAnalyses were carried out in R version 4.0.1 using the \u0026ldquo;Two-Sample MR\u0026rdquo; package. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eA total of 29 SNPs were included in this study. The positive results of these SNPs were evaluated using PhenoScanner and 19 SNPs were found associated with the confounders mentioned in the paragraph 2.2. The F-statistics of all SNPs were \u0026gt;\u0026thinsp;10. The MR analysis revealed a negative correlation between the use of antithrombotic agents and HCC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The infuence of SNPs sizes on the exposure (antithrombotic agents) and outcome (HCC) was demonstrated by the scatter plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The Steiger\u0026rsquo;s Z test direction was true (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), showing that SNPs acted on the exposure first, and next on the outcome.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMR analysis for antithrombotic agents use on HCC.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethod\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of SNPs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95% Confidence Interval\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInverse variance weighted\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.279, 0.707\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMR Egger\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.061, 1.084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.102\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeighted median\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.257, 0.831\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.010\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeighted mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.237, 2.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.520\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eAbbreviations: HCC, hepatocellular carcinoma; MR, Mendelian randomization; SNPs, single nucleotide polymorphisms; OR, odds ratio.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCochran\u0026rsquo;s Q-test results showed that no significant results after the use of antithrombotic agents, indicating that all of the IVs were no heterogeneous [IVW, Q (df) 12.339 (9) P\u0026thinsp;=\u0026thinsp;0.1949; MR\u0026ndash;Egger, Q (df) 11.454 (8), P\u0026thinsp;=\u0026thinsp;0.1773]. The funnel plot indicated evidence of symmetrical distribution, indicating the lack of directional horizontal pleiotropy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), suggesting that a potential bias was less likely to influence the causal association.\u003c/p\u003e \u003cp\u003eThe MR\u0026ndash;Egger regression for IVs confirmed the absence of horizontal pleiotropy (intercept β\u0026thinsp;=\u0026thinsp;0.0319, P\u0026thinsp;=\u0026thinsp;0.454). The findings of the leave-one-out analysis were firm in the evaluation of the outcomes of this MR analysis, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; SNPs had no fundamental effect on the results regardless of which SNP was removed.This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.\u003c/p\u003e "},{"header":"4. Discussion","content":"\u003cp\u003eThis MR study intended to elucidate the prospective causal relationship between the use of antithrombotic agents and the risk of HCC in the European population. Our results demonstrated an impressive inverse association between the two, confirmed by the subsequent sensitivity analysis. The awareness of this association has grown in recent years. Nonetheless, observational studies are unable to establish the correct direction of causality. Our study is the first confirming the causal relationship between these two factors using MR analysis.\u003c/p\u003e \u003cp\u003eMR methods are builded on the principle of Mendel's laws of inheritance. The prediction of antithrombotic agent responsiveness based on genetic variation is determined at birth, and does not influence the progression or treatment of the disease. As a result, causal inferences are protected from biases arising from confounding and reverse causality. The establishment of a causal relationship between the use of antithrombotic agents and HCC is scarce. Numerous studies currently indicate a 41% diminution in the risk of HCC and a 45% decrease in the risk of mortality due to chronic liver disease when aspirin is used compared to when it is not (Sahasrabuddhe et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, The Liver Cancer Pooling Project demonstrated a 32% reduction in the risk of HCC associated to the use of aspirin, while no significant association was observed using ibuprofen (Petrick et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, Petrick, J.L., et al found that the decrease in HCC incidence is more pronounced among individuals who regularly and daily use aspirin and at low doses. Importantly, this reduction in HCC risk remains consistent regardless of the duration of the treatment (Petrick et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). TThe pooled analysis of the data from the Nurses' Health Study and the Health Professionals Follow-up Study demonstrated a significant 49% in the reduction of HCC incidence among both male and female participants who regularly use aspirin (Simon et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The National Cohort Study of Korean Adults discovered a dose-dependent 13% reduction in the risk of HCC associated with the use of aspirin compared to when it is not (Hwang et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These retrospective studies performed in the general population are subjected to various limitations, including the non-randomized assignment of the treatment. However, a study that used Taiwan's Longitudinal Health Insurance database noted an approximate 50% decrease in the risk of HCC among both males and females who carry HCV and use aspirin (Liao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A retrospective cohort study performed among Korean patients suffering from alcoholic cirrhosis demonstrated a significant 87% reduction in HCC risk associated with the use of low-dose aspirin compared to when it is not (Shin et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, an independent correlation between the use of lower dose aspirin and a 29% decrease in the risk of HCC was found in a retrospective population-based cohort study performed in Taiwan among patients with hepatitis B virus infection (Lee et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our results are consistent with all these previous research indicating the association between the use of antithrombotic agents and the decreased risk of HCC. Antithrombotic agents inhibit the progression of HCC by cyclooxygenase-dependent and independent mechanisms. These mechanisms include the control of inflammation or cell proliferation, leading apoptosis or autophagy, the reduction of fibrosis, and the repression of platelet function (Huang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, no statistically relevant association between the use of aspirin and a reduction in the risk of HCC among patients with underlying cirrhosis was found in a multivariable stratified analysis (Lee et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Chiu et al. reported a lack of significant impact of aspirin on the decreased risk of HCC in a mass case study (Chiu et al. 2010). Other studies reported conflicting helpful influences of aspirin on the decreased of HCC risk in hepatitis B virus patients (Tsan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kim et al. 2013). A meta-analysis considering 12 studies also indicated a lack of a significant impact of aspirin on the recurrence of HCC (Pang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, the involvement of antithrombotic agents in the risk of HCC continues to be a controversial topic. Our finding was relied on a greater robust study design different from the above previous research. This discrepancy can be attributed to the inherent constraints of observational studies, which fail to establish a causal relationship, as well as the use of small sample sizes, which may not provide adequate evidence to draw a definitive conclusion.\u003c/p\u003e \u003cp\u003eOur study presents numerous strengths. The 10 SNPs stringently selected in Europeans were unattached and strictly associated with the use of antithrombotic agents, avoiding the influence of LD on the causal estimate. The use of MR analysis reduced the likelihood of bias from confounding factors and reversed the causation in comparison to traditional observational studies. Consequently, the SNPs used in this investigation and the matching research results are significantly valid and dependable. Our results supplied more compelling evidence to support the causal relationship between the use of antithrombotic agents and the decreased risk of HCC.\u003c/p\u003e \u003cp\u003eHowever, our study is not without limitations. The GWAS data associated to antithrombotic agents and HCC predominantly originates from individuals of European descent, thereby lacking representation from other ethnic populations. Therefore, the results of our MR analysis should be exercised with caution when generalizing to other ethnic groups, since its applicability might be limited to populations of European ancestry. Moreover, it is important to acknowledge the inherent limitations of the two-sample MR analysis since it provides estimates of hypothetical causal associations despite this analysis serves as a important device for examining the causal relationship between antithrombotic agents and HCC. Accordingly, further analyses are essential to confirm these results.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe use of antithrombotic agents results in a negative association with the risk of HCC. Nevertheless, further comprehensive investigations are needed due to the limitations of this study to deepen the relationship between antithrombotic agents and HCC risk, including beneficiary populations and underlying mechanisms to improve the rational guidance for HCC treatment.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.Y., O.L., J.H. and X.Y. contributed to study, concept,and design. F.Y., O.L., B.G., and Z.C. contributed to the acquisition of data. F.Y. and C.Z. performed the Mendelian randomization analysis, visualized the data and wrote the first draft of the manuscript. F.Y., O.L., B.G., Z.C., J.H. and X.Y. revised the manuscript. F.Y., O.L., B.G., Z.C., J.H. and X.Y. contributed equally to this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank MogoEdit (https://www.mogoedit.com) for its English editing during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThis work was supported by the Key Research and Development Project of the Science \u0026amp; Technology Department of Sichuan Province (Nos.2023YFQ0101), Sichuan Science and Technology Program (Nos.2022YFS0625) and Luzhou Science and Technology and Talent Work Bureau (Nos.2022-SYF-55).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are openly available in OpenGWAS and GWAS Catalog.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcGlynn KA, Petrick JL, El-Serag HB (2021). 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Q., Giovannucci, E. L., Fuchs, C. S., Meyerhardt, J. A., Corey, K. E., Chung, R. T., Zhang, X., \u0026amp; Chan, A. T. (2018). Association Between Aspirin Use and Risk of Hepatocellular Carcinoma. JAMA oncology, 4(12), 1683\u0026ndash;1690. doi:10.1001/jamaoncol.2018.4154\u003c/li\u003e\n\u003cli\u003eHwang IC, Chang J, Kim K, Park SM (2018). Aspirin Use and Risk of Hepatocellular Carcinoma in a National Cohort Study of Korean Adults. Sci Rep. 8(1):4968. doi:10.1038/s41598-018-23343-0\u003c/li\u003e\n\u003cli\u003eLiao YH, Hsu RJ, Wang TH, et al (2020). Aspirin decreases hepatocellular carcinoma risk in hepatitis C virus carriers: a nationwide cohort study. BMC Gastroenterol. 20(1):6. Published 2020 Jan 9. doi:10.1186/s12876-020-1158-y\u003c/li\u003e\n\u003cli\u003eShin S, Lee SH, Lee M, et al (2020). Aspirin and the risk of hepatocellular carcinoma development in patients with alcoholic cirrhosis. 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Published 2017 Sep 20. doi:10.2147/OTT.S143154\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-cancer-research-and-clinical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jocr","sideBox":"Learn more about [Journal of Cancer Research and Clinical Oncology](https://www.springer.com/journal/432)","snPcode":"432","submissionUrl":"https://submission.nature.com/new-submission/432/3","title":"Journal of Cancer Research and Clinical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"antithrombotic agents, hepatocellular carcinoma, Mendelian randomization study, causal relationship","lastPublishedDoi":"10.21203/rs.3.rs-4608895/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4608895/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eHepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide. Multiple observational studies demonstrated a negative correlation between the use of antithrombotic agents and the risk of HCC. However, the precise causal relationship between these factors remains uncertain. Therefore, our study used a two-sample Mendelian randomization (MR) analysis to assess the causal link between these two factors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eThe summary statistics of single nucleotide polymorphisms (SNPs) associated with the use of antithrombotic agents were acquired from a genome-wide association study (GWAS) performed on individuals of European descent, as well as from the GWAS on the UK Biobank. A two-sample MR analysis was performed using the inverse variance weighting (IVW), the weighted median estimate, the MR-Egger regression, and the weighted-mode estimate. The robustness of the primary findings was assessed by sensitivity analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Ten SNPs associated with the use of antithrombotic agents were selected as instrumental variables. The MR analysis performed using the four methods mentioned above revealed a negative correlation between the use of antithrombotic agents and HCC. The other methods also produced similar results. No heterogeneity and horizontal pleiotropy were found.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur findings suggested an inverse association of antithrombotic agents with the risk of HCC.\u003c/p\u003e","manuscriptTitle":"Association between antithrombotic agents use and hepatocellular carcinoma risk: a two-sample Mendelian randomization analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 20:29:51","doi":"10.21203/rs.3.rs-4608895/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T00:27:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-06T03:42:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217660698568264330089861444278874188324","date":"2024-07-01T06:17:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-24T13:19:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-20T13:55:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-20T13:54:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Cancer Research and Clinical Oncology","date":"2024-06-20T03:38:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-cancer-research-and-clinical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jocr","sideBox":"Learn more about [Journal of Cancer Research and Clinical Oncology](https://www.springer.com/journal/432)","snPcode":"432","submissionUrl":"https://submission.nature.com/new-submission/432/3","title":"Journal of Cancer Research and Clinical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b9923468-f7a1-4fc2-8e07-cf2453af527f","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:11:06+00:00","versionOfRecord":{"articleIdentity":"rs-4608895","link":"https://doi.org/10.1007/s00432-024-05960-7","journal":{"identity":"journal-of-cancer-research-and-clinical-oncology","isVorOnly":false,"title":"Journal of Cancer Research and Clinical Oncology"},"publishedOn":"2024-10-22 15:57:40","publishedOnDateReadable":"October 22nd, 2024"},"versionCreatedAt":"2024-07-18 20:29:51","video":"","vorDoi":"10.1007/s00432-024-05960-7","vorDoiUrl":"https://doi.org/10.1007/s00432-024-05960-7","workflowStages":[]},"version":"v1","identity":"rs-4608895","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4608895","identity":"rs-4608895","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}