A partial epithelial-mesenchymal transition signature for highly aggressive colorectal cancer cells that survive under nutrient restriction.

Partial epithelial-mesenchymal transition (p-EMT) has recently been identified as a hybrid state consisting of cells with both epithelial and mesenchymal characteristics and is associated with the migration, metastasis, and chemoresistance of cancer cells. Here, we describe the induction of p-EMT in starved colorectal cancer (CRC) cells and identify a p-EMT gene signature that can predict prognosis. Functional characterisation of starvation-induced p-EMT in HCT116, DLD1, and HT29 cells showed changes in proliferation, morphology, and drug sensitivity, supported by in vivo studies using the chorioallantoic membrane model. An EMT-specific quantitative polymerase chain reaction (qPCR) array was used to screen for deregulated genes, leading to the establishment of an in silico gene signature that was correlated with poor disease-free survival in CRC patients along with the CRC consensus molecular subtype CMS4. Among the significantly deregulated p-EMT genes, a triple-gene signature consisting of SERPINE1, SOX10, and epidermal growth factor receptor (EGFR) was identified. Starvation-induced p-EMT was characterised by increased migratory potential and chemoresistance, as well as E-cadherin processing and internalisation. Both gene signature and E-cadherin alterations could be reversed by the proteasomal inhibitor MG132. Spatially resolving EGFR expression with high-resolution immunofluorescence imaging identified a proliferation stop in starved CRC cells caused by EGFR internalisation. In conclusion, we have gained insight into a previously undiscovered EMT mechanism that may become relevant when tumour cells are under nutrient stress, as seen in early stages of metastasis. Targeting this process of tumour cell dissemination might help to prevent EMT and overcome drug resistance. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Small noncoding RNAs and sperm nuclear basic proteins reflect the environmental impact on germ cells.

BACKGROUND: Molecular techniques can complement conventional spermiogram analyses to provide new information on the fertilizing potential of spermatozoa and to identify early alterations due to environmental pollution. METHODS: Here, we present a multilevel molecular profiling by small RNA sequencing and sperm nuclear basic protein analysis of male germ cells from 33 healthy young subjects residing in low and high-polluted areas. RESULTS: Although sperm motility and sperm concentration were comparable between samples from the two sites, those from the high-pollution area had a higher concentration of immature/immune cells, a lower protamine/histone ratio, a reduced ability of sperm nuclear basic proteins to protect DNA from oxidative damage, and an altered copper/zinc ratio in sperm. Sperm levels of 32 microRNAs involved in intraflagellar transport, oxidative stress response, and spermatogenesis were different between the two areas. In parallel, a decrease of Piwi-interacting RNA levels was observed in samples from the high-polluted area. CONCLUSIONS: This comprehensive analysis provides new insights into pollution-driven epigenetic alterations in sperm not detectable by spermiogram.

A Fecal MicroRNA Signature by Small RNA Sequencing Accurately Distinguishes Colorectal Cancers: Results From a Multicenter Study.

BACKGROUND & AIMS: Fecal tests currently used for colorectal cancer (CRC) screening show limited accuracy in detecting early tumors or precancerous lesions. In this respect, we comprehensively evaluated stool microRNA (miRNA) profiles as biomarkers for noninvasive CRC diagnosis. METHODS: A total of 1273 small RNA sequencing experiments were performed in multiple biospecimens. In a cross-sectional study, miRNA profiles were investigated in fecal samples from an Italian and a Czech cohort (155 CRCs, 87 adenomas, 96 other intestinal diseases, 141 colonoscopy-negative controls). A predictive miRNA signature for cancer detection was defined by a machine learning strategy and tested in additional fecal samples from 141 CRC patients and 80 healthy volunteers. miRNA profiles were compared with those of 132 tumors/adenomas paired with adjacent mucosa, 210 plasma extracellular vesicle samples, and 185 fecal immunochemical test leftover samples. RESULTS: Twenty-five miRNAs showed altered levels in the stool of CRC patients in both cohorts (adjusted P < .05). A 5-miRNA signature, including miR-149-3p, miR-607-5p, miR-1246, miR-4488, and miR-6777-5p, distinguished patients from control individuals (area under the curve [AUC], 0.86; 95% confidence interval [CI], 0.79-0.94) and was validated in an independent cohort (AUC, 0.96; 95% CI, 0.92-1.00). The signature classified control individuals from patients with low-/high-stage tumors and advanced adenomas (AUC, 0.82; 95% CI, 0.71-0.97). Tissue miRNA profiles mirrored those of stool samples, and fecal profiles of different gastrointestinal diseases highlighted miRNAs specifically dysregulated in CRC. miRNA profiles in fecal immunochemical test leftover samples showed good correlation with those of stool collected in preservative buffer, and their alterations could be detected in adenoma or CRC patients. CONCLUSIONS: Our comprehensive fecal miRNome analysis identified a signature accurately discriminating cancer aimed at improving noninvasive diagnosis and screening strategies.

Fecal miRNA profiles in colorectal cancers with mucinous morphology.

Diagnostic performance of molecular markers in surrogate tissues like stool may be affected by colorectal cancer (CRC) morphological heterogeneity. The mucinous histotype represents a subgroup of CRC with a peculiar molecular program and unfavorable disease progression. However, the percentage of mucinous morphology necessary to define this subtype is still a matter of debate. In this study, we investigated whether stool miRNA profiles of CRC patients differ in patients with mucinous histopathological subtypes compared to non-mucinous cancers. In this respect, we also explored how the stool miRNA signature reported in our previous multicentric study (Pardini et al., Gastroenterology 2023) behave in this histotype. Small-RNA sequencing was performed in fecal and tissue samples of an Italian cohort (n=172), including 27 CRC with mucinous morphology (mucinous cancers with >50% mucinous morphology and those with mucinous component >5% but <50%), 58 non-mucinous CRC, and 87 colonoscopy-negative controls. Results were compared with fecal miRNA profiles of a cohort from the Czech Republic (n=98). Most of the differentially expressed (DE) stool miRNAs (n=324) were in common between CRC with mucinous morphology and non-mucinous histopathological subtypes in comparison with healthy controls. Interestingly, the altered levels of 25 fecal miRNAs previously identified distinguishing CRC cases from controls in both cohorts were also confirmed after stratification for mucinous morphology. Forty-nine miRNAs were DE exclusively in CRC with mucinous morphology and 61 in non-mucinous CRC. Mucinous cancers and those with mucinous component showed fairly similar profiles that were comparable in the Czech cohort. Among the stool DE miRNAs observed in CRC with mucinous morphology, 20 were also altered in the comparison between tumor and adjacent mucosa tissue. This study highlights miRNAs specifically altered in CRC with mucinous morphology. Nevertheless, the performance of our stool miRNA signature in accurately distinguishing CRC cases from controls was not significantly affected by this histological subtype. This aspect further supports the use of stool miRNAs for noninvasive diagnosis and screening strategies.

Profiling small RNAs in fecal immunochemical tests: is it possible?

Fecal microRNAs represent promising molecules with potential clinical interest as non-invasive diagnostic and prognostic biomarkers. Colorectal cancer (CRC) screening based on the fecal immunochemical test (FIT) is an effective tool for prevention of cancer development. However, due to the poor sensitivity of FIT especially for premalignant lesions, there is a need for implementation of complementary tests. Improving the identification of individuals who would benefit from further investigation with colonoscopy using molecular analysis, such as miRNA profiling of FIT samples, would be ideal due to their widespread use. In the present study, we assessed the feasibility of applying small RNA sequencing to measure human miRNAs in FIT leftover buffer in samples from two European screening populations. We showed robust detection of miRNAs with profiles similar to those obtained from specimens sampled using the established protocol of RNA stabilizing buffers, or in long-term archived samples. Detected miRNAs exhibited differential abundances for CRC, advanced adenoma, and control samples that were consistent for FIT and RNA-stabilizing buffers. Interestingly, the sequencing data also allowed for concomitant evaluation of small RNA-based microbial profiles. We demonstrated that it is possible to explore the human miRNome in FIT leftover samples across populations and envision that the analysis of small RNA biomarkers can complement the FIT in large scale screening settings.

Variability of the Human Serum Metabolome over 3 Months in the EXPOsOMICS Personal Exposure Monitoring Study.

Liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) and untargeted metabolomics are increasingly used in exposome studies to study the interactions between nongenetic factors and the blood metabolome. To reliably and efficiently link detected compounds to exposures and health phenotypes in such studies, it is important to understand the variability in metabolome measures. We assessed the within- and between-subject variability of untargeted LC-HRMS measurements in 298 nonfasting human serum samples collected on two occasions from 157 subjects. Samples were collected ca. 107 (IQR: 34) days apart as part of the multicenter EXPOsOMICS Personal Exposure Monitoring study. In total, 4294 metabolic features were detected, and 184 unique compounds could be identified with high confidence. The median intraclass correlation coefficient (ICC) across all metabolic features was 0.51 (IQR: 0.29) and 0.64 (IQR: 0.25) for the 184 uniquely identified compounds. For this group, the median ICC marginally changed (0.63) when we included common confounders (age, sex, and body mass index) in the regression model. When grouping compounds by compound class, the ICC was largest among glycerophospholipids (median ICC 0.70) and steroids (0.67), and lowest for amino acids (0.61) and the O-acylcarnitine class (0.44). ICCs varied substantially within chemical classes. Our results suggest that the metabolome as measured with untargeted LC-HRMS is fairly stable (ICC > 0.5) over 100 days for more than half of the features monitored in our study, to reflect average levels across this time period. Variance across the metabolome will result in differential measurement error across the metabolome, which needs to be considered in the interpretation of metabolome results.

Stool microRNA profiles reflect different dietary and gut microbiome patterns in healthy individuals.

OBJECTIVES: MicroRNA (miRNA) profiles have been evaluated in several biospecimens in relation to common diseases for which diet may have a considerable impact. We aimed at characterising how specific diets are associated with the miRNome in stool of vegans, vegetarians and omnivores and how this is reflected in the gut microbial composition, as this is still poorly explored. DESIGN: We performed small RNA and shotgun metagenomic sequencing in faecal samples and dietary recording from 120 healthy volunteers, equally distributed for the different diets and matched for sex and age. RESULTS: We found 49 miRNAs differentially expressed among vegans, vegetarians and omnivores (adj. p <0.05) and confirmed trends of expression levels of such miRNAs in vegans and vegetarians compared with an independent cohort of 45 omnivores. Two miRNAs related to lipid metabolism, miR-636 and miR-4739, were inversely correlated to the non-omnivorous diet duration, independently of subject age. Seventeen miRNAs correlated (|rho|>0.22, adj. p <0.05) with the estimated intake of nutrients, particularly animal proteins, phosphorus and, interestingly, lipids. In omnivores, higher Prevotella and Roseburia and lower Bacteroides abundances than in vegans and vegetarians were observed. Lipid metabolism-related miR-425-3p and miR-638 expression levels were associated with increased abundances of microbial species, such as Roseburia sp. CAG 182 and Akkermansia muciniphila, specific of different diets. An integrated analysis identified 25 miRNAs, 25 taxa and 7 dietary nutrients that clearly discriminated (area under the receiver operating characteristic curve=0.89) the three diets. CONCLUSION: Stool miRNA profiles are associated with specific diets and support the role of lipids as a driver of epigenetic changes and host-microbial molecular interactions in the gut.

Combined miRNA and SERS urine liquid biopsy for the point-of-care diagnosis and molecular stratification of bladder cancer.

BACKGROUND: Bladder cancer (BC) has the highest per-patient cost of all cancer types. Hence, we aim to develop a non-invasive, point-of-care tool for the diagnostic and molecular stratification of patients with BC based on combined microRNAs (miRNAs) and surface-enhanced Raman spectroscopy (SERS) profiling of urine. METHODS: Next-generation sequencing of the whole miRNome and SERS profiling were performed on urine samples collected from 15 patients with BC and 16 control subjects (CTRLs). A retrospective cohort (BC = 66 and CTRL = 50) and RT-qPCR were used to confirm the selected differently expressed miRNAs. Diagnostic accuracy was assessed using machine learning algorithms (logistic regression, naïve Bayes, and random forest), which were trained to discriminate between BC and CTRL, using as input either miRNAs, SERS, or both. The molecular stratification of BC based on miRNA and SERS profiling was performed to discriminate between high-grade and low-grade tumors and between luminal and basal types. RESULTS: Combining SERS data with three differentially expressed miRNAs (miR-34a-5p, miR-205-3p, miR-210-3p) yielded an Area Under the Curve (AUC) of 0.92 ± 0.06 in discriminating between BC and CTRL, an accuracy which was superior either to miRNAs (AUC = 0.84 ± 0.03) or SERS data (AUC = 0.84 ± 0.05) individually. When evaluating the classification accuracy for luminal and basal BC, the combination of miRNAs and SERS profiling averaged an AUC of 0.95 ± 0.03 across the three machine learning algorithms, again better than miRNA (AUC = 0.89 ± 0.04) or SERS (AUC = 0.92 ± 0.05) individually, although SERS alone performed better in terms of classification accuracy. CONCLUSION: miRNA profiling synergizes with SERS profiling for point-of-care diagnostic and molecular stratification of BC. By combining the two liquid biopsy methods, a clinically relevant tool that can aid BC patients is envisaged.

Genetic variations in microRNA-binding sites of solute carrier transporter genes as predictors of clinical outcome in colorectal cancer.

One of the principal mechanisms of chemotherapy resistance in highly frequent solid tumors, such as colorectal cancer (CRC), is the decreased activity of drug transport into tumor cells due to low expression of important membrane proteins, such as solute carrier (SLC) transporters. Sequence complementarity is a major determinant for target gene recognition by microRNAs (miRNAs). Single-nucleotide polymorphisms (SNPs) in target sequences transcribed into messenger RNA may therefore alter miRNA binding to these regions by either creating a new site or destroying an existing one. miRSNPs may explain the modulation of expression levels in association with increased/decreased susceptibility to common diseases as well as in chemoresistance and the consequent inter-individual variability in drug response. In the present study, we investigated whether miRSNPs in SLC transporter genes may modulate CRC susceptibility and patient's survival. Using an in silico approach for functional predictions, we analyzed 26 miRSNPs in 9 SLC genes in a cohort of 1368 CRC cases and 698 controls from the Czech Republic. After correcting for multiple tests, we found several miRSNPs significantly associated with patient's survival. SNPs in SLCO3A1, SLC22A2 and SLC22A3 genes were defined as prognostic factors in the classification and regression tree analysis. In contrast, we did not observe any significant association between miRSNPs and CRC risk. To the best of our knowledge, this is the first study investigating miRSNPs potentially affecting miRNA binding to SLC transporter genes and their impact on CRC susceptibility or patient's prognosis.

Metabolic perturbations prior to hepatocellular carcinoma diagnosis: Findings from a prospective observational cohort study.

Hepatocellular carcinoma (HCC) development entails changes in liver metabolism. Current knowledge on metabolic perturbations in HCC is derived mostly from case-control designs, with sparse information from prospective cohorts. Our objective was to apply comprehensive metabolite profiling to detect metabolites whose serum concentrations are associated with HCC development, using biological samples from within the prospective European Prospective Investigation into Cancer and Nutrition (EPIC) cohort (>520 000 participants), where we identified 129 HCC cases matched 1:1 to controls. We conducted high-resolution untargeted liquid chromatography-mass spectrometry-based metabolomics on serum samples collected at recruitment prior to cancer diagnosis. Multivariable conditional logistic regression was applied controlling for dietary habits, alcohol consumption, smoking, body size, hepatitis infection and liver dysfunction. Corrections for multiple comparisons were applied. Of 9206 molecular features detected, 220 discriminated HCC cases from controls. Detailed feature annotation revealed 92 metabolites associated with HCC risk, of which 14 were unambiguously identified using pure reference standards. Positive HCC-risk associations were observed for N1-acetylspermidine, isatin, p-hydroxyphenyllactic acid, tyrosine, sphingosine, l,l-cyclo(leucylprolyl), glycochenodeoxycholic acid, glycocholic acid and 7-methylguanine. Inverse risk associations were observed for retinol, dehydroepiandrosterone sulfate, glycerophosphocholine, γ-carboxyethyl hydroxychroman and creatine. Discernible differences for these metabolites were observed between cases and controls up to 10 years prior to diagnosis. Our observations highlight the diversity of metabolic perturbations involved in HCC development and replicate previous observations (metabolism of bile acids, amino acids and phospholipids) made in Asian and Scandinavian populations. These findings emphasize the role of metabolic pathways associated with steroid metabolism and immunity and specific dietary and environmental exposures in HCC development.

Mutational landscape of plasma cell-free DNA identifies molecular features associated with therapeutic response in patients with colon cancer. A pilot study.

Cell-free DNA (cfDNA) has recently been used as a non-invasive diagnostic tool for detecting tumour-specific mutations. cfDNA may also be used for monitoring disease progression and treatment response, but so far researchers focused on one or few genes only. A genomic profile may provide better information on patient prognosis compared to single specific mutations. In this hypothesis-generating study, we profiled by whole exome sequencing serial plasma samples from 10 colon cancer (CC) patients collected before and after 5-fluorouracil-based therapy, and one year after diagnosis to determine alterations associated with treatment response. In parallel, genome profiling was also performed in patients' corresponding tumour tissue to ascertain the molecular landscape of resistant tumours. The mutation concordance between cfDNA and tumour tissue DNA was higher in more advanced tumour stages than in the early stages of the disease. In non-responders, a specific mutation profile was observed in tumour tissues (TPSD1 p.Ala92Thr, CPAMD8 p.Arg341Gln, OBP2A p.ArgTyr123CysHis). A pathogenic APC mutation (p.Ser1315Ter) was detected only in cfDNA of one poor responder one year after the diagnosis and after therapy termination. Another poor responder presented a likely pathogenic TP53 mutation (p.Arg110Pro) in cfDNA of all plasma samplings and in tumour tissue. In conclusion, cfDNA could be used for genetic characterisation of CC patients and might be clinically useful for non-invasive therapy response monitoring.

Genetic variations in 3'UTRs of SMUG1 and NEIL2 genes modulate breast cancer risk, survival and therapy response.

Breast cancer (BC) is the most frequent malignancy in women accounting for approximately 2 million new cases worldwide annually. Several genetic, epigenetic and environmental factors are known to be involved in BC development and progression, including alterations in post-transcriptional gene regulation mediated by microRNAs (miRNAs). Single nucleotide polymorphisms (SNPs) located in miRNA binding sites (miRSNPs) in 3'-untranslated regions of target genes may affect miRNA-binding affinity and consequently modulate gene expression. We have previously reported a significant association of miRSNPs in the SMUG1 and NEIL2 genes with overall survival in colorectal cancer patients. SMUG1 and NEIL2 are DNA glycosylases involved in base excision DNA repair. Assuming that certain genetic traits are common for solid tumours, we have investigated wherever variations in SMUG1 and NEIL2 genes display an association with BC risk, prognosis, and therapy response in a group of 673 BC patients and 675 healthy female controls. Patients with TC genotype of NEIL2 rs6997097 and receiving only hormonal therapy displayed markedly shorter overall survival (HR = 4.15, 95% CI = 1.7-10.16, P = 0.002) and disease-free survival (HR = 2.56, 95% CI = 1.5-5.7, P = 0.02). Our results suggest that regulation of base excision repair glycosylases operated by miRNAs may modulate the prognosis of hormonally treated BC.

Short-term personal and outdoor exposure to ultrafine and fine particulate air pollution in association with blood pressure and lung function in healthy adults.

Studies reporting on associations between short-term exposure to outdoor fine (PM2.5), and ultrafine particles (UFP) and blood pressure and lung function have been inconsistent. Few studies have characterized exposure by personal monitoring, which especially for UFP may have resulted in substantial exposure measurement error. We investigated the association between 24-h average personal UFP, PM2.5, and soot exposure and dose and the health parameters blood pressure and lung function. We further assessed the short-term associations between outdoor concentrations measured at a central monitoring site and near the residences and these health outcomes. We performed three 24-h personal exposure measurements for UFP, PM2.5, and soot in 132 healthy adults from Basel (Switzerland), Amsterdam and Utrecht (the Netherlands), and Turin (Italy). Monitoring of each subject was conducted in different seasons in a one-year study period. Subject's activity levels and associated ventilation rates were measured using actigraphy to calculate the inhaled dose. After each 24-h monitoring session, blood pressure and lung function were measured. Contemporaneously with personal measurements, UFP, PM2.5 and soot were measured outdoor at the subject's residential address and at a central site in the research area. Associations between short-term personal and outdoor exposure and dose to UFP, PM2.5, and soot and health outcomes were tested using linear mixed effect models. The 24-h mean personal, residential and central site outdoor UFP exposures were not associated with blood pressure or lung function. UFP mean exposures in the 2-h prior to the health test was also not associated with blood pressure and lung function. Personal, central site and residential PM2.5 exposure were positively associated with systolic blood pressure (about 1.4 mmHg increase per Interquartile range). Personal soot exposure and dose were positively associated with diastolic blood pressure (1.2 and 0.9 mmHg increase per Interquartile range). No consistent associations between PM2.5 or soot exposure and lung function were observed. Short-term personal, residential outdoor or central site exposure to UFP was not associated with blood pressure or lung function. Short-term personal PM2.5 and soot exposures were associated with blood pressure, but not lung function.

Agnostic Cys34-albumin adductomics and DNA methylation: Implication of N-acetylcysteine in lung carcinogenesis years before diagnosis.

Although smoking and oxidative stress are known contributors to lung carcinogenesis, their mechanisms of action remain poorly understood. To shed light into these mechanisms, we applied a novel approach using Cys34-adductomics in a lung cancer nested case-control study (n = 212). Adductomics profiles were integrated with DNA-methylation data at established smoking-related CpG sites measured in the same individuals. Our analysis identified 42 Cys34-albumin adducts, of which 2 were significantly differentially abundant in cases and controls: adduct of N-acetylcysteine (NAC, p = 4.15 × 10-3 ) and of cysteinyl-glycine (p = 7.89 × 10-3 ). Blood levels of the former were found associated to the methylation levels at 11 smoking-related CpG sites. We detect, for the first time in prospective blood samples, and irrespective of time to diagnosis, decreased levels of NAC adduct in lung cancer cases. Altogether, our results highlight the potential role of these adducts in the oxidative stress response contributing to lung carcinogenesis years before diagnosis.

DNA repair and cancer in colon and rectum: Novel players in genetic susceptibility.

Interindividual differences in DNA repair systems may play a role in modulating the individual risk of developing colorectal cancer. To better ascertain the role of DNA repair gene polymorphisms on colon and rectal cancer risk individually, we evaluated 15,419 single nucleotide polymorphisms (SNPs) within 185 DNA repair genes using GWAS data from the Colon Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), which included 8,178 colon cancer, 2,936 rectum cancer cases and 14,659 controls. Rs1800734 (in MLH1 gene) was associated with colon cancer risk (p-value = 3.5 × 10-6 ) and rs2189517 (in RAD51B) with rectal cancer risk (p-value = 5.7 × 10-6 ). The results had statistical significance close to the Bonferroni corrected p-value of 5.8 × 10-6 . Ninety-four SNPs were significantly associated with colorectal cancer risk after Binomial Sequential Goodness of Fit (BSGoF) procedure and confirmed the relevance of DNA mismatch repair (MMR) and homologous recombination pathways for colon and rectum cancer, respectively. Defects in MMR genes are known to be crucial for familial form of colorectal cancer but our findings suggest that specific genetic variations in MLH1 are important also in the individual predisposition to sporadic colon cancer. Other SNPs associated with the risk of colon cancer (e.g., rs16906252 in MGMT) were found to affect mRNA expression levels in colon transverse and therefore working as possible cis-eQTL suggesting possible mechanisms of carcinogenesis.

Consumption of Fish and Long-chain n-3 Polyunsaturated Fatty Acids Is Associated With Reduced Risk of Colorectal Cancer in a Large European Cohort.

BACKGROUND & AIMS: There is an unclear association between intake of fish and long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs) and colorectal cancer (CRC). We examined the association between fish consumption, dietary and circulating levels of n-3 LC-PUFAs, and ratio of n-6:n-3 LC-PUFA with CRC using data from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. METHODS: Dietary intake of fish (total, fatty/oily, lean/white) and n-3 LC-PUFA were estimated by food frequency questionnaires given to 521,324 participants in the EPIC study; among these, 6291 individuals developed CRC (median follow up, 14.9 years). Levels of phospholipid LC-PUFA were measured by gas chromatography in plasma samples from a sub-group of 461 CRC cases and 461 matched individuals without CRC (controls). Multivariable Cox proportional hazards and conditional logistic regression models were used to calculate hazard ratios (HRs) and odds ratios (ORs), respectively, with 95% CIs. RESULTS: Total intake of fish (HR for quintile 5 vs 1, 0.88; 95% CI, 0.80-0.96; Ptrend = .005), fatty fish (HR for quintile 5 vs 1, 0.90; 95% CI, 0.82-0.98; Ptrend = .009), and lean fish (HR for quintile 5 vs 1, 0.91; 95% CI, 0.83-1.00; Ptrend = .016) were inversely associated with CRC incidence. Intake of total n-3 LC-PUFA (HR for quintile 5 vs 1, 0.86; 95% CI, 0.78-0.95; Ptrend = .010) was also associated with reduced risk of CRC, whereas dietary ratio of n-6:n-3 LC-PUFA was associated with increased risk of CRC (HR for quintile 5 vs 1, 1.31; 95% CI, 1.18-1.45; Ptrend < .001). Plasma levels of phospholipid n-3 LC-PUFA was not associated with overall CRC risk, but an inverse trend was observed for proximal compared with distal colon cancer (Pheterogeneity = .026). CONCLUSIONS: In an analysis of dietary patterns of participants in the EPIC study, we found regular consumption of fish, at recommended levels, to be associated with a lower risk of CRC, possibly through exposure to n-3 LC-PUFA. Levels of n-3 LC-PUFA in plasma were not associated with CRC risk, but there may be differences in risk at different regions of the colon.

Exosomal microRNAs and other non-coding RNAs as colorectal cancer biomarkers: a review.

The circulating human transcriptome, which includes both coding and non-coding RNA (ncRNA) molecules, represents a rich source of potential biomarkers for colorectal cancer (CRC) that has only recently been explored. In particular, the release of RNA-containing extracellular vesicles (EVs), in a multitude of different in vitro cell systems and in a variety of body fluids, has attracted wide interest. The role of RNA species in EVs is still not fully understood, but their capacity to act as a form of distant communication between cells and their higher abundance in association with cancer demonstrated their relevance. In this review, we report the evidence from both in vitro and human studies on microRNAs (miRNAs) and other ncRNA profiles analysed in EVs in relation to CRC as diagnostic, prognostic and predictive markers. The studies so far highlighted that, in exosomes, the most studied category of EVs, several miRNAs are able to accurately discriminate CRC cases from controls as well as to describe the progression of the disease and its prognosis. Most of the time, the in vitro findings support the miRNA profiles detected in human exosomes. The expression profiles measured in exosomes and other EVs differ and, interestingly, there is a variability of expression also among different subsets of exosomes according to their proteic profile. On the other hand, evidence is still limited for what concerns exosome miRNAs as early diagnostic and predictive markers of treatment. Several other ncRNAs that are carried by exosomes, mostly long ncRNAs and circular RNAs, seem also to be dysregulated in CRC. Besides various technical challenges, such as the standardisation of EVs isolation methods and the optimisation of methodologies to characterise the whole spectrum of RNA molecules in exosomes, further studies are needed in order to elucidate their relevance as CRC markers.

Expression quantitative trait loci in ABC transporters are associated with survival in 5-FU treated colorectal cancer patients.

The chemotherapeutic efficacy in colorectal cancer (CRC) is limited due to the inter-individual variability in drug response and the development of tumour resistance. ATP-binding cassette (ABC) transporters are crucial in the development of resistance by the efflux of anticancer agents from cancer cells. In this study, we identified 14 single nucleotide polymorphisms (SNPs) in 11 ABC transporter genes acting as an expression of quantitative trait loci (eQTLs), i.e. whose variation influence the expression of many downstream genes. These SNPs were genotyped in a case-control study comprising 1098 cases and 1442 healthy controls and analysed in relation to CRC development risk and patient survival. Considering a strict correction for multiple tests, we did not observe any significant association between SNPs and CRC risk. The rs3819720 polymorphism in the ABCB3/TAP2 gene was statistically significantly associated with shorter overall survival (OS) in the codominant, and dominant models [GA vs. GG, hazard ratio (HR) = 1.48; P = 0.002; AA vs. GG, HR = 1.70; P = 0.004 and GA + AA vs. GG, HR = 1.52; P = 0.0006]. Additionally, GA carriers of the same SNP displayed worse OS after receiving 5-FU based chemotherapy. The variant allele of rs3819720 polymorphism statistically significantly affected the expression of 36 downstream genes. Screening for eQTL polymorphisms in relevant genes such as ABC transporters that can regulate the expression of several other genes may help to identify the genetic background involved in the individual response to the treatment of CRC patients.