In a recent publication, Lapitz et al. reported differences in serum levels that predict the development of cholangiocarcinoma (CCA) in patients with primary sclerosing cholangitis prior to clinical manifestation. We examined whether these biomarkers also predict the risk of gallbladder cancer (GBC) in European prospective plasma samples from 24 GBC cases and 90 control individuals. After logarithmic transformation and quantile normalisation of individual protein levels measured with a timsTOF PRO mass spectrometer, we fitted univariate logistic regression models and applied backward model selection to identify the optimal model for GBC risk prediction. CRP and MASP2, previously reported markers of CCA, were found to be predictive for GBC risk as well (p value < 0.05), and complemented by age at blood sampling, provided an area under the receiver operating characteristic curve of 0.80 (95% confidence interval 0.69-0.92) when combined in a prediction model for GBC risk. We further examined the mRNA expression of CRP and MASP2 in serum samples from 82 GBC cases and 79 control subjects from Chile. CRP mRNA levels were elevated in Chilean GBC cases, but MASP2 showed an opposite trend. While there are considerable differences in the design of the discovery study and ours, the finding that circulating levels of CRP and MASP2 are associated with the risk of both CCA and GBC in Europeans highlights the need for further research into potential shared mechanisms and strategies for the prevention of these two aggressive biliary tumours.

ABSTRACT Background and Aims Highly aggressive hepatobiliary tumours include gallbladder cancer (GBC), hepatocellular carcinoma (HCC), intrahepatic and extrahepatic cholangiocarcinoma (iCCA, eCCA) and ampulla of Vater cancer (AoV). We aimed to identify plasma biomarkers for the early diagnosis of hepatobiliary cancer by leveraging the metabolomic signatures of established clinical risk factors. Method Based on 273,190 participants from the UK Biobank, we (1) identified metabolites associated with gallstone-related conditions (e.g. cholecystitis), primary sclerosing cholangitis (PSC) and metabolic liver diseases (e.g. cirrhosis), and (2) evaluated the relationship between the identified metabolites and the risk of GBC, HCC, iCCA, eCCA and AoV. Findings were validated in an independent group of 227,809 participants from the UK Biobank. We also derived metabolomic scores summarizing the three risk-factor signatures and evaluated their ability to stratify cancer risk. Results We identified 27 metabolites associated with gallstone-related conditions, 11 with PSC, and 34 with metabolic liver diseases, some of which showed associations with inconsistent directions across risk factors, suggesting distinct pathogenic processes. Several metabolites were associated with cancer risk in both the discovery and validation datasets, independently of established risk factors, predominantly for HCC (16 signals) and for iCCA (4), with one for GBC and none for eCCA and AoV. Metabolomic scores clearly distinguished individuals at high risk for HCC and iCCA. Conclusion The preselection of plasma metabolites associated with established risk factors facilitated the subsequent identification and validation of biomarkers for early cancer detection. The identified metabolites suggest specific pathogenic pathways for each type of hepatobiliary cancer. Wider replication is urgently needed to advance toward clinical implementation. What you need to know BACKGROUND AND CONTEXT Clinical risk factors for hepatobiliary cancers often progress silently, making early identification of high-risk individuals difficult and highlighting the need for biological markers detectable before clinical diagnosis. NEW FINDINGS Risk-factor–based serum metabolomic profiling identified circulating metabolites that predict specific hepatobiliary cancers years before diagnosis, with strongest and most consistent signals for hepatocellular and intrahepatic cholangiocarcinoma. LIMITATIONS Clinical risk factors were assumed to be frequently underdiagnosed in UK Biobank, and event numbers were relatively small for some cancers, which may have reduced power and attenuated associations for less common endpoints. CLINICAL RESEARCH RELEVANCE This study shows that serum metabolic profiles can identify individuals at increased risk for hepatobiliary cancers long before symptoms appear, particularly for hepatocellular and intrahepatic cholangiocarcinoma. These findings support the development of precision risk-stratification strategies that may ultimately enable earlier surveillance. BASIC RESEARCH RELEVANCE By first identifying metabolites linked to specific liver and biliary clinical conditions, the study clarifies which metabolites are indirectly associated with hepatobiliary cancers through known disease pathways. Testing these metabolites again while adjusting for diagnoses of those conditions then reveals which ones also show direct, pathway-independent associations with individual hepatobiliary cancers, providing clearer insight into cancer-specific metabolic mechanisms.

BACKGROUND:Chagas disease, caused by the parasite Trypanosoma cruzi (T. cruzi), affects around 6-7 million people in Latin America and hundreds of thousands in the United States and Europe. A main complication of chronic Chagas disease is cardiomyopathy, possibly manifesting as arrhythmias, heart failure, or sudden cardiac death. Understanding the link between T. cruzi infection and cardiomyopathy is essential for early diagnosis and adequate treatment. METHODS:We sequenced small RNAs in serum samples from 228 Chagas patients recruited in Chile, Bolivia and Italy. After bioinformatic processing of sequencing data to quantify serum miRNA expression, robust logistic regression was applied to identify miRNAs differentially expressed in Chagas patients with abnormalities in electrocardiography (ECG), bundle-branch block on ECG, and high Kuschnir scores. We also investigated the association between genotype-based miRNA expression and the risk of abnormal ECG findings. FINDINGS:As reported, the risk of abnormal ECG findings was higher in male patients and increased with age. Three miRNAs showed lower serum expression levels in patients with abnormal ECG: miRNA-101-3p, miRNA-576-3p and miRNA-629-5p (p < 0.0002), especially in patients with high Kuschnir scores. The expression of miRNA-629-5p was negatively correlated with the CCL5 expression (p = 3.7×10-8), a chemokine frequently reported in Chagas disease. Gene enrichment analyses indicated involvement of cytokine signalling in Chagas cardiomyopathy. INTERPRETATION:The findings demonstrate the potential of circulating miRNAs as diagnostic biomarkers for Chagas cardiomyopathy. The associations found with disease severity and immune response may help to improve our knowledge of complications' development in Chagas disease.

The European–Latin American Research Consortium towards Eradication of Preventable Gallbladder Cancer (EULAT Eradicate GBC) is identifying geographical, environmental, lifestyle, genetic and molecular risk factors for gallbladder cancer. Collaborative research is essential to improve prevention of this aggressive tumour, especially in high-incidence, low-income and middle-income regions.

Gallbladder cancer (GBC) is an aggressive disease with limited treatment options but high prevention potential. GBC tumours take 10-20 years to develop, a timeframe that holds potential for early detection. MicroRNAs (miRNAs) play a central role in abnormal cell processes, and circulating miRNAs may constitute valuable biomarkers of early disease. We used microarray data to pre-select differentially expressed miRNAs in formalin-fixed paraffin-embedded (FFPE) gallbladder tissue samples (GBC n = 40, normal n = 8). We then applied small-RNA sequencing to screen for miRNA expression differences in serum samples from three European prospective cohorts (n = 37 GBC case-control pairs), and validated the most promising candidates in three independent cohorts (n = 36 GBC case- control pairs). Statistical analyses included robust linear regression, pathway and meta-analysis, and examination of expression correlation between miRNAs and target genes. MiR-4533 and miR-671-5p were overexpressed in GBC tissue and serum samples, and meta-analysis confirmed the overexpression of miR-4533 in GBC serum samples from the prospective cohorts (p-value = 4.1×10-4), especially in individuals of female sex, under 63.5 years, or with a BMI below 26.2 kg/m2. Pathway and correlation analyses revealed that miR-4533 targets SIPA1L2 in the Rap1 signalling pathway, and SIPA1L2 was downregulated in GBC serum samples. Our study highlights the advantage of integrating small-RNA sequencing results from different types of samples and independent datasets, and the need for international research collaborations to identify and validate biomarkers for secondary prevention of rare tumours such as GBC. The function of miR-4533 and its interaction with SIPA1L2 in GBC development need to be further investigated.

Latin Americans have a rich genetic make-up that translates into heterogeneous fractions of the autosomal genome in runs of homozygosity (FROH) and heterogeneous types and proportions of indigenous American ancestry. While autozygosity has been linked to several human diseases, very little is known about the relationship between inbreeding, genetic ancestry, and cancer risk in Latin Americans. Chile has one of the highest incidences of gallbladder cancer (GBC) in the world, and we investigated the association between inbreeding, GBC, gallstone disease (GSD), and body mass index (BMI) in 4029 genetically admixed Chileans. We calculated individual FROH above 1.5 Mb and weighted polygenic risk scores for GSD, and applied multiple logistic regression to assess the association between homozygosity and GBC risk. We found that homozygosity was due to a heterogeneous mixture of genetic drift and consanguinity in the study population. Although we found no association between homozygosity and overall GBC risk, we detected interactions of FROH with sex, age, and genetic risk of GSD that affected GBC risk. Specifically, the increase in GBC risk per 1% FROH was 19% in men (p-value = 0.002), 30% in those under 60 years of age (p-value = 0.001), and 12% in those with a genetic risk of GSD above the median (p-value = 0.01). The present study highlighted the complex interplay between inbreeding, genetic ancestry, and genetic risk of GSD in the development of GBC. The applied methodology and our findings underscored the importance of considering the population-specific genetic architecture, along with sex- and age-specific effects, when investigating the genetic basis of complex traits in Latin Americans.

List of software and databases used for the analysis and functional follow up

Supplementary table and figure legends

Since 2006, Chile has been implementing a gallbladder cancer (GBC) prevention program based on prophylactic cholecystectomy for gallstone patients aged 35 to 49 years. The effectiveness of this prevention program has not yet been comprehensively evaluated. We conducted a retrospective study of 473 Chilean GBC patients and 2137 population-based controls to develop and internally validate three GBC risk prediction models. The Baseline Model accounted for gallstones while adjusting for sex and birth year. Enhanced Model I also included the non-genetic risk factors: body mass index, educational level, Mapuche surnames, number of children and family history of GBC. Enhanced Model II further included Mapuche ancestry and the genotype for rs17209837. Multiple Cox regression was applied to assess the predictive performance, quantified by the area under the precision-recall curve (AUC-PRC) and the number of cholecystectomies needed (NCN) to prevent one case of GBC at age 70 years. The AUC-PRC for the Baseline Model (0.44%, 95%CI 0.42-0.46) increased by 0.22 (95%CI 0.15-0.29) when non-genetic factors were included, and by 0.25 (95%CI 0.20-0.30) when incorporating non-genetic and genetic factors. The overall NCN for Chileans with gallstones (115, 95%CI 104-131) decreased to 92 (95%CI 60-128) for Chileans with a higher risk than the median according to Enhanced Model I, and to 80 (95%CI 59-110) according to Enhanced Model II. In conclusion, age, sex and gallstones are strong risk factors for GBC, but consideration of other non-genetic factors and individual genotype data improves risk prediction and may optimize allocation of financial resources and surgical capacity.

A strong association between the proportion of indigenous South American Mapuche ancestry and the risk of gallbladder cancer (GBC) has been reported in observational studies. Chileans show the highest incidence of GBC worldwide, and the Mapuche are the largest indigenous people in Chile. We set out to assess the confounding-free effect of the individual proportion of Mapuche ancestry on GBC risk and to investigate the mediating effects of gallstone disease and body mass index (BMI) on this association. Genetic markers of Mapuche ancestry were selected based on the informativeness for assignment measure, and then used as instrumental variables in two-sample Mendelian randomization analyses and complementary sensitivity analyses. Results suggested a putatively causal effect of Mapuche ancestry on GBC risk (inverse variance-weighted (IVW) risk increase of 0.8% per 1% increase in Mapuche ancestry proportion, 95% CI 0.4% to 1.2%, p = 6.7 × 10−5) and also on gallstone disease (3.6% IVW risk increase, 95% CI 3.1% to 4.0%), pointing to a mediating effect of gallstones on the association between Mapuche ancestry and GBC. In contrast, the proportion of Mapuche ancestry showed a negative effect on BMI (IVW estimate −0.006 kg/m2, 95% CI −0.009 to −0.003). The results presented here may have significant implications for GBC prevention and are important for future admixture mapping studies. Given that the association between the individual proportion of Mapuche ancestry and GBC risk previously noted in observational studies appears to be free of confounding, primary and secondary prevention strategies that consider genetic ancestry could be particularly efficient.

Locuszoom plots of the regions with pleiotropic association. (A) TNP1 (2q35), (B) RUVBL1 and GATA2 (3q21), (C) PHC3 (3q26), (D) TET2 (4q24), (E) POU5F1, BAG6 and EHMT2 (6p21),(F) L3MBTL3 (6q23), (G) HDAC9 (7p21), (H) LOXL2 (8p21), (I) BRCA2 (13q12), (J) WDR61 and MORF4L1 (15q25), (K) TADA2A (17q12), (L) BABAM1 (19p13), (M) DPF1 (19q13) and (N) TGFB1 (19q13).

Venn diagrams showing the number of SNPs associated across all five cancer types, without differentiation for subtypes. A. Including MORF4L1, B. Excluding MORF4L1* *http://bioinformatics.psb.ugent.be/webtools/Venn/

Summary of candidate genes with identified risk associations, chromosomal region, previously through GWAS identified risk associations in these regions and list of additional genes located in the selected regions.

Although the method tests for associations of both directions (preventive and susceptible), here we report only the significant direction.

Genes selected for the study using GO and GeneCard databases and literature search to represent 10 epigenetic sub-pathways, specifically DNA methylation, DNA demethylation, histone acetylation, histone deacetylation, chromatin remodeling, histone methylation, histone demethylation, other histone modification, chromatin modification and histones.

Summary of the functional annotation of all significant SNPs based on FunciSNP (SNPs that were annotated as not functional are not listed).

BACKGROUND & AIMS:Gallstones (cholelithiasis) constitute a major health burden with high costs related to surgical removal of the gallbladder (cholecystectomy), generally indicated for symptomatic gallstones. The association between gallstones and cholecystectomy and kidney cancer is controversial. We comprehensively investigated this association, considering age at cholecystectomy and time from cholecystectomy to kidney cancer diagnosis, and assessed the causal effect of gallstones on kidney cancer risk by Mendelian randomization (MR).METHODS:We compared the risk of kidney cancer in cholecystectomized and noncholecystectomized patients (16.6 million in total) from the Swedish nationwide cancer, census, patient, and death registries using hazard ratios (HRs). For 2-sample and multivariable MR, we used summary statistics based on 408,567 UK Biobank participants.RESULTS:During a median follow-up of 13 years, 2627 of 627,870 cholecystectomized Swedish patients developed kidney cancer (HR, 1.17; 95% CI, 1.12-1.22). Kidney cancer risk was particularly increased in the first 6 months after cholecystectomy (HR, 3.79; 95% CI, 3.18-4.52) and in patients cholecystectomized before age 40 years (HR, 1.55; 95% CI, 1.39-1.72). MR results based on 18,417 patients with gallstones and 1788 patients with kidney cancer from the United Kingdom revealed a causal effect of gallstones on kidney cancer risk (9.6% risk increase per doubling in gallstone prevalence; 95% CI, 1.2%-18.8%).CONCLUSIONS:Both observational and causal MR estimates based on large prospective cohorts support an increased risk of kidney cancer in patients with gallstones. Our findings provide solid evidence for the compelling need to diagnostically rule out kidney cancer before and during gallbladder removal, to prioritize kidney cancer screening in patients undergoing cholecystectomy in their 30s, and to investigate the underlying mechanisms linking gallstones and kidney cancer in future studies.