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Background: Despite the worldwide progress in cancer diagnostics, more sensitive diagnostic biomarkers are needed. The methylome has been extensively investigated in the last decades, but a low-cost, bisulfite-free detection method for multiplex analysis is still lacking., Methods: We developed a methylation detection technique called IMPRESS, which combines methylation-sensitive restriction enzymes and single-molecule Molecular Inversion Probes. We used this technique for the development of a multi-cancer detection assay for eight of the most lethal cancer types worldwide. We selected 1791 CpG sites that can distinguish tumor from normal tissue based on DNA methylation. These sites were analysed with IMPRESS in 35 blood, 111 tumor and 114 normal samples. Finally, a classifier model was built., Results: We present the successful development of IMPRESS and validated it with ddPCR. The final classifier model discriminating tumor from normal samples was built with 358 CpG target sites and reached a sensitivity of 0.95 and a specificity of 0.91. Moreover, we provide data that highlight IMPRESS's potential for liquid biopsies., Conclusions: We successfully created an innovative DNA methylation detection technique. By combining this method with a new multi-cancer biomarker panel, we developed a sensitive and specific multi-cancer assay, with potential use in liquid biopsies., (© 2024. The Author(s).)

DNA methylation biomarkers have emerged as promising tools for cancer detection. Common methylation patterns across tumor types allow multi-cancer detection. Droplet digital PCR (ddPCR) has gained considerable attention for methylation detection. However, multi-cancer detection using multiple targets in ddPCR has never been performed before. Therefore, we developed a multiplex ddPCR assay for multi-cancer detection. Based on previous data analyses using The Cancer Genome Atlas (TCGA), we selected differentially methylated targets for eight frequent tumor types (lung, breast, colorectal, prostate, pancreatic, head and neck, liver, and esophageal cancer). Three targets were validated using ddPCR in 103 tumor and 109 normal adjacent fresh frozen samples. Two distinct ddPCR assays were successfully developed. Output data from both assays is combined to obtain a read-out from the three targets together. Our overall ddPCR assay has a cross-validated area under the curve (cvAUC) of 0.948. Performance between distinct cancer types varies, with sensitivities ranging from 53.8% to 100% and specificities ranging from 80% to 100%. Compared to previously published single-target parameters, we show that combining targets can drastically increase sensitivity and specificity, while lowering DNA input. In conclusion, we are the first to report a multi-cancer methylation ddPCR assay, which allows for highly accurate tumor predictions., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction: Interval cancer (IC) is an important quality indicator in colorectal cancer (CRC) screening. Previously, we found that fecal immunochemical test (FIT) ICs are more common in women, older age, right-sided tumors, and advanced stage. Here, we extended our existing stage IV patient cohort with clinicopathological and molecular characteristics, to identify factors associated with FIT-IC., Methods: Logistic regression models were fit to identify variables associated with the odds of having a stage IV FIT-IC. Multivariate models were corrected for gender, age, and location., Results: A total of 292 screen-detected (SD) CRCs and 215 FIT-IC CRCs were included. FIT-IC CRC had 5 fold higher odds to be a neuroendocrine (NET) tumor and 2.5 fold higher odds to have lymphovascular invasion. Interestingly, some variables lost significance upon accounting for location. Thus, tumor location is a critical covariate that should always be included when evaluating factors related to FIT-IC., Conclusions: We identified NETs and lymphovascular invasion as factors associated with increased odds of having a stage IV FIT-IC. Moreover, we highlight the importance of tumor location as a covariate in evaluating FIT-IC related factors. More research across all stages is needed to clarify how these insights might help to optimize the Flemish CRC screening program., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Neefs, Tran, Ferrari, Janssens, Van Herck, Op de Beeck, Van Camp, Peeters, Fransen, Hoeck and Van Hal.)

Ferroptosis is a cell death pathway that can be promoted by peroxidizable polyunsaturated fatty acids in cancer cells. Here, we investigated the mechanisms underlying the toxicity of punicic acid (PunA), an isomer of conjugated linolenic acids (CLnAs) bearing three conjugated double bonds highly prone to peroxidation, on prostate cancer (PCa) cells. PunA induced ferroptosis in PCa cells and triggered massive lipidome remodeling, more strongly in PC3 androgen-negative cells than in androgen-positive cells. The greater sensitivity of androgen-negative cells to PunA was associated with lower expression of glutathione peroxidase 4 (GPX4). We then identified the phospholipase PLA2G7 as a PunA-induced ferroptosis suppressor in PCa cells. Overexpressing PLA2G7 decreased lipid peroxidation levels, suggesting that PLA2G7 hydrolyzes hydroperoxide-containing phospholipids, thus preventing ferroptosis. Importantly, overexpressing both PLA2G7 and GPX4 strongly prevented PunA-induced ferroptosis in androgen-negative PCa cells. This study shows that PLA2G7 acts complementary to GPX4 to protect PCa cells from CLnA-induced ferroptosis., Competing Interests: The authors declare that they have no conflict of interest with the content of this article., (© 2024 The Author(s).)

Methylmercury (MeHg) is a pervasive environmental contaminant in aquatic ecosystems that can reach elevated concentrations in fish of high trophic levels, such as salmonids. The present study aims at investigating the individual and combined impacts of dietary MeHg and fatty acids on lipid metabolism in juvenile rainbow trout (Oncorhynchus mykiss) with a focus on two key organs, adipose tissue and liver. MeHg and fatty acids are both known to act on energy homeostasis although little is known about their interplay on lipid metabolism in fish. Fish were fed diets enriched in linoleic acid (LA, 18:2 n-6), α-linolenic acid (ALA, 18:3 n-3), eicosapentaenoic acid (EPA, 20:5 n-3) or docosahexaenoic acid (DHA, 22:6 n-3) for ten weeks, with the addition of MeHg to the diets during the last six weeks (0, 2.4 or 5.5 mg MeHg/kg dry matter). LA and ALA are polyunsaturated fatty acids (PUFA) typical of plant-derived oils whereas EPA and DHA are n-3 long chain PUFA largely found in fish oil, all used in feed formulation in aquaculture. The results showed that the LA-enriched diet induced a higher whole-body lipid content compared to the three other diets. On the contrary, the addition of MeHg led to a significant reduction of the whole-body lipid content, regardless of the diet. Interestingly, the adipocytes were larger both in presence of LA, compared to EPA and DHA, or MeHg, indicating a lipogenic effect of these two compounds. No effect was, however, observed on lipid accumulation per gram of adipose tissue. The fatty acid composition of adipose tissue and liver was significantly modified by the dietary lipids, reflecting both the fatty acid composition of the diets and the high bioconversion capacity of the rainbow trout. Exposure to MeHg selectively led to a release of n-6 PUFA from the hepatic membranes of fish fed the LA-enriched diet, showing a disruption of the pathways using n-6 PUFA. This study highlights the significant impact of MeHg exposure and dietary fatty acids on lipid metabolism in fish. Further investigation is needed to elucidate the underlying mechanisms and to explore the potential involvement of other organs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Biomarker discovery in colorectal cancer has mostly focused on methylation patterns in normal and colorectal tumor tissue, but adenomas remain understudied. Therefore, we performed the first epigenome-wide study to profile methylation of all three tissue types combined and to identify discriminatory biomarkers., Results: Public methylation array data (Illumina EPIC and 450K) were collected from a total of 1 892 colorectal samples. Pairwise differential methylation analyses between tissue types were performed for both array types to "double evidence" differentially methylated probes (DE DMPs). Subsequently, the identified DMPs were filtered on methylation level and used to build a binary logistic regression prediction model. Focusing on the clinically most interesting group (adenoma vs carcinoma), we identified 13 DE DMPs that could effectively discriminate between them (AUC = 0.996). We validated this model in an in-house experimental methylation dataset of 13 adenomas and 9 carcinomas. It reached a sensitivity and specificity of 96% and 95%, respectively, with an overall accuracy of 96%. Our findings raise the possibility that the 13 DE DMPs identified in this study can be used as molecular biomarkers in the clinic., Conclusions: Our analyses show that methylation biomarkers have the potential to discriminate between normal, precursor and carcinoma tissues of the colorectum. More importantly, we highlight the power of the methylome as a source of markers for discriminating between colorectal adenomas and carcinomas, which currently remains an unmet clinical need., (© 2023. The Author(s).)

Colorectal cancer (CRC) is one of the leading cancer-related causes of death in the world. Since the 70s, many countries have adopted different CRC screening programs, which has resulted in a decrease in mortality. However, current screening test options still present downsides. The commercialized stool-based tests present high false-positive rates and low sensitivity, which negatively affects the detection of early stage carcinogenesis. The gold standard colonoscopy has low uptake due to its invasiveness and the perception of discomfort and embarrassment that the procedure may bring. In this review, we collected and described the latest data about alternative CRC screening techniques that can overcome these disadvantages. Web of Science and PubMed were employed as search engines for studies reporting on CRC screening tests and future perspectives. The searches generated 555 articles, of which 93 titles were selected. Finally, a total of 50 studies, describing 14 different CRC alternative tests, were included. Among the investigated techniques, the main feature that could have an impact on CRC screening perception and uptake was the ease of sample collection. Urine, exhaled breath, and blood-based tests promise to achieve good diagnostic performance (sensitivity of 63-100%, 90-95%, and 47-97%, respectively) while minimizing stress and discomfort for the patient.

The present study aimed at investigating interactive effects between dietary lipids and both short- and long-term exposures to a low, environmentally realistic, cadmium (Cd) concentration. Juvenile rainbow trout were fed four isolipidic diets (31.7 g/kg) enriched in either linoleic acid (LA, 18:2n-6), alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3). From the 4th week of this 10-week experiment, the lipid level of the diet was increased (120.0 g/kg) and half of the fish fed each diet were aqueously exposed to Cd (0.3 μg/L) while the other half were not exposed to Cd (control). Fish were sampled and their liver was harvested for fatty acid profile, hepatic Cd and calcium concentrations, total glutathione level and gene expression assessment, either (i) after 4 weeks of feeding and 24 h of Cd contamination (day 29) (short-term Cd exposure) or (ii) after 10 weeks of feeding and 6 weeks of Cd contamination (day 70) (long-term Cd exposure). We found that both dietary lipids and Cd exposure influenced fatty acid homeostasis and metabolism. The hepatic fatty acid profile mostly reflected that of the diet (e.g. n-3/n-6 ratio) with some differences, including selective retention of specific long chain polyunsaturated fatty acids (LC-PUFAs) like DHA and active biotransformation of dietary LA and ALA into LC-PUFAs. Cd effects on hepatic fatty acid profiles were influenced by the duration of the exposure and the nutritional status of the fish. The effects of diet and Cd exposure on the fatty acid profiles were only sparsely explained by variation of the expression pattern of genes involved in fatty acid metabolism. The biological responses to Cd were also influenced by dietary lipids. Fish fed the ALA-enriched diet seemed to be the least affected by the Cd exposure, as they showed a higher detoxifying ability against Cd with an early upregulation of protective metallothionein a (MTa) and apoptosis regulator BCL2-Like1 (BCLx) genes, an increased long-term phospholipid synthesis and turnover and fatty acid bioconversion efficiency, as well as a lower long-term accumulation of Cd in their liver. In contrast, fish fed the EPA-enriched diet seemed to be the most sensitive to a long-term Cd exposure, with an impaired growth performance and a decreased antioxidant capacity (lower glutathione level). Our results highlight that low, environmentally realistic aqueous concentrations of Cd can affect biological response in fish and that these effects are influenced by the dietary fatty acid composition., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Methylmercury (MeHg) is a ubiquitous contaminant largely found in aquatic environments, especially in species at high trophic level such as salmonids. The aim of this study was to evaluate the effects of MeHg on adipocyte differentiation and lipid metabolism in rainbow trout. Primary cultured preadipocytes were exposed to increasing concentrations of MeHg during six days with or without a hormonal cocktail. Main results showed a dose-dependent intracellular accumulation of neutral lipids with a preferential uptake of n-3 polyunsaturated fatty acids. Interestingly, this accumulation occurred after a fairly low uptake of MeHg by preadipocytes and was maintained after the cellular exposure to MeHg. In membrane phospholipids, arachidonic acid (20:4 n-6) was released in a dose-dependent manner. At the transcriptional level, the expression of several adipocyte-specific genes (perilipin 2 and apolipoprotein Eb) as well as lipid-related genes (fatty acid synthase and fatty acid binding protein 11a) was up-regulated in preadipocytes exposed to MeHg. These results highlight for the first time the disrupting effect of MeHg in trout adipocyte metabolism, providing new insights regarding the role of environmental pollutants in adipose tissue dysfunction and related pathologies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Polyunsaturated fatty acids (PUFAs) have key biological roles in fish cells. We recently showed that the phospholipid composition of rainbow trout liver cells (RTL-W1 cell line) modulates their tolerance to an acute cadmium (Cd) challenge. Here, we investigated (i) the extent to which PUFAs and Cd impact fatty acid homeostasis and metabolism in these cells and (ii) possible mechanisms by which specific PUFAs may confer cytoprotection against Cd. First, RTL-W1 cells were cultivated for one week in growth media spiked with 50 μmol L -1 of either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), linoleic acid (LA, 18:2n-6) or arachidonic acid (AA, 20:4n-6) in order to modulate their fatty acid profile. Then, the cells were challenged with Cd (0, 50 or 100 μmol L -1 ) for 24 h prior to assaying viability, fatty acid profile, intracellular Cd content, proteomic landscape and expression levels of genes involved in fatty acid metabolism, synthesis of PUFA-derived signalling molecules and stress response. We observed that the fatty acid supply and, to a lesser extent, the exposure to Cd influenced cellular fatty acid homeostasis and metabolism. The cellular fatty acid composition of fish liver cells modulated their tolerance to an acute Cd challenge. Enrichments in ALA, EPA, and, to a lesser extent, AA conferred cytoprotection while enrichment in LA had no impact on cell viability. The present study ruled out the possibility that cytoprotection reflects a decreased Cd burden. Our results rather suggest that the PUFA-derived cytoprotection against Cd occurs through a reduction of the oxidative stress induced by Cd and a differential induction of the eicosanoid cascade, with a possible role of peroxiredoxin and glutaredoxin (antioxidant enzymes) as well as cytosolic phospholipase A2 (enzyme initiating the eicosanoid cascade)., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lipids, and their constitutive fatty acids, are key nutrients for fish health as they provide energy, maintain cell structure, are precursors of signalling molecules and act as nuclear receptor ligands. These specific roles may be of crucial importance in a context of exposure to pollutants. We recently showed that the fatty acid profile of rainbow trout liver cell phospholipids modulates sensitivity to an acute methylmercury challenge. In order to investigate mechanisms of effects, we herein tested whether specific polyunsaturated fatty acids (PUFAs) may protect cells from methylmercury through decreasing intracellular mercury accumulation and/or enhancing cellular defences (e.g. via modulation of gene expression patterns). We also investigated the inverse relationship and assessed the impact of methylmercury on cellular fatty acid metabolism. To do so, the fatty acid composition of rainbow trout liver cell phospholipids was first modified by incubating them in a medium enriched in a specific PUFA from either the n-3 family (alpha-linolenic acid, ALA; eicosapentaenoic acid, EPA) or the n-6 family (linoleic acid, LA; arachidonic acid, AA). Cells were then exposed to methylmercury (0.15 or 0.50 μM) for 24 h and sampled thereafter for assessing phospholipid fatty acid profile, intracellular total mercury burden, and expression pattern of genes involved in fatty acid metabolism, synthesis of PUFA-derived signalling molecules and stress response. We observed that cells incorporated the given PUFA and some biotransformation products in their phospholipids. Methylmercury had few impacts on this cellular phospholipid composition. None of the PUFA enrichments affected the cellular mercury burden, suggesting that the previously observed cytoprotection conferred by ALA and EPA was not linked to a global decrease in cellular accumulation of mercury. Fatty acid enrichments and methylmercury exposure both modulated gene expression patterns. Genes involved in the synthesis of PUFA-derived signalling molecules, in stress response and the orphan cytochrome P450 20A1 were identified as possible sites of interaction between fatty acids and methylmercury in rainbow trout liver cells., (Copyright © 2018. Published by Elsevier B.V.)

The polyunsaturated fatty acid (PUFA) composition of fish tissues, which generally reflects that of the diet, affects various cellular properties such as membrane structure and fluidity, energy metabolism and susceptibility to oxidative stress. Since these cellular parameters can play an important role in the cellular response to organic and inorganic pollutants, a variation of the PUFA supply might modify the toxicity induced by such xenobiotics. In this work, we investigated whether the cellular fatty acid profile has an impact on the in vitro cell sensitivity to two environmental pollutants: methylmercury and cadmium. Firstly, the fatty acid composition of the rainbow trout liver cell line RTL-W1 was modified by enriching the growth medium with either alpha-linolenic acid (ALA, 18:3n-3), eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), linoleic acid (LA, 18:2n-6), arachidonic acid (AA, 20:4n-6) or docosapentaenoic acid (DPA, 22:5n-6). These modified cells and their control (no PUFA enrichment) were then challenged for 24h with increasing concentrations of methylmercury or cadmium. We observed that (i) the phospholipid composition of the RTL-W1 cells was profoundly modulated by changing the PUFA content of the growth medium: major modifications were a high incorporation of the supplemented PUFA in the cellular phospholipids, the appearance of direct elongation and desaturation metabolites in the cellular phospholipids as well as a change in the gross phospholipid composition (PUFA and monounsaturated fatty acid (MUFA) levels and n-3/n-6 ratio); (ii) ALA, EPA and DPA enrichment significantly protected the RTL-W1 cells against both methylmercury and cadmium; (iv) DHA enrichment significantly protected the cells against cadmium but not methylmercury; (v) AA and LA enrichment had no impact on the cell tolerance to both methylmercury and cadmium; (vi) the abundance of 20:3n-6, a metabolite of the n-6 biotransformation pathway, in phospholipids was negatively correlated to the cell tolerance to both methylmercury and cadmium. Overall, our results highlighted the importance of the fatty acid supply on the tolerance of fish liver cells to methylmercury and cadmium., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Microarray-based copy number analysis has found its way into routine clinical practice, predominantly for the diagnosis of patients with unexplained mental retardation. However, the clinical interpretation of submicroscopic copy number variants (CNVs) is complicated by the fact that many CNVs are also present in the general population. Here we introduce and discuss a workflow that can be used in routine diagnostics to assess the clinical significance of the CNVs identified. We applied this scheme to our cohort of 386 individuals with unexplained mental retardation tested using a genome-wide tiling-resolution DNA microarray and to 978 additional patients with mental retardation reported in 15 genome-wide microarray studies extracted from the literature. In our cohort of 386 patients we identified 25 clinically significant copy number losses (median size 2.6 Mb), nine copy number gains (median size 2.0 Mb), and one mosaic numerical chromosome aberration. Accordingly, the overall diagnostic yield of clinically significant CNVs was 9.1%. Taken together, our cohort and the patients described in the literature include a total of 1,364 analyses of DNA copy number in which a total of 11.2% (71.9% losses, 19.6% gains, 8.5% complex) could be identified, reflecting the overall diagnostic yield of clinically significant CNVs in individuals with unexplained mental retardation., (2008 Wiley-Liss, Inc.)

We present a 4-year-old boy with developmental delay and several into minor dysmorphic features due to an interstitial deletion of 17.5 Mb on the long arm of chromosome 13 [46,XY,del (13)(q14.3q21.33)]. The deletion was detected initially during routine cytogenetic screening and further analyzed on a genome-wide BAC array. In contrast to several previous papers reporting a short stature, our patient was tall with a 1 year advanced skeletal age. In this paper, we compare growth and clinical features of this patient with previously reported cases, with a similar interstitial deletion on the long arm of chromosome 13.