SnoRNA profiling in colorectal cancer and assessment of non-invasive biomarker capacity by ddPCR in fecal samples.

Small nucleolar RNAs (snoRNAs) have been identified dysregulated in several pathologies, and these alterations can be detected in tissues and in circulation. The main aim of this study was to analyze the whole snoRNome in advanced colorectal neoplasms and to identify new potential non-invasive snoRNA-based biomarkers in fecal samples by different analytical approaches. SNORA51, SNORD15B, SNORA54, SNORD12B, SNORD12C, SNORD72, SNORD89, and several members of SNORD115 and SNORD116 clusters were consistently deregulated in both tissue sets. After technical validation, SNORA51 and SNORD15B were detected in FIT+ samples. SNORA51 was significantly upregulated in FIT+ samples from CRC patients compared to healthy controls. This upregulation, together with the fecal hemoglobin concentration, was sufficient to identify, among FIT+ individuals, patients with CRC (AUC = 0.86) and individuals with advanced adenomas (AUC = 0.68). These findings portray snoRNAs as an alternative source of candidates for further studies and SNORA51 appears as a potential non-invasive biomarker for CRC detection.

Modulation of faecal miRNAs highlights the preventive effects of a Mediterranean low-inflammatory dietary intervention.

BACKGROUND: Dietary interventions have been proposed as therapeutic approaches for several diseases, including cancer. A low-inflammatory Mediterranean dietary intervention, conducted as a pilot study in subjects with Familial Adenomatous Polyposis (FAP), reduced markers of local and systemic inflammation. We aim to determine whether this diet may modulate faecal microRNA (miRNA) and gene expression in the gut. METHODS: Changes in the faecal miRNome were evaluated by small RNA sequencing at baseline (T0), after the three-month intervention (T1), and after an additional three months (T2). Changes in the transcriptome of healthy rectal mucosa and adenomas were evaluated by RNA sequencing at T0 and T2. The identification of validated miRNA-gene interactions and functional analysis of miRNA targets were performed using in silico approaches. RESULTS: Twenty-seven subjects were included in this study. It was observed that the diet modulated 29 faecal miRNAs (p < 0.01; |log2 Fold Change|>1), and this modulation persisted for three months after the intervention. Levels of miR-3612-3p and miR-941 correlated with the adherence to the diet, miR-3670 and miR-4252-5p with faecal calprotectin, and miR-3670 and miR-6867 with serum calprotectin. Seventy genes were differentially expressed between adenoma and normal tissue, and most were different before the dietary intervention but reached similar levels after the diet. Functional enrichment analysis identified the proinflammatory ERK1/2, cell cycle regulation, and nutrient response pathways as commonly regulated by the modulated miRNAs and genes. CONCLUSIONS: Faecal miRNAs modulated by the dietary intervention target genes that participate in inflammation. Changes in levels of miRNAs and genes with oncogenic and tumour suppressor functions further support the potential cancer-preventive effect of the low-inflammatory Mediterranean diet. CLINICAL TRIAL NUMBER REGISTRATION: NCT04552405, Registered in ClinicalTrials.gov.

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.

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.

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.

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.

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.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-12.

We are delighted to share with you our twelfth Journal Club and highlight some of the most interesting papers published recently [...].

Gluten-free diet affects fecal small non-coding RNA profiles and microbiome composition in celiac disease supporting a host-gut microbiota crosstalk.

Current treatment for celiac disease (CD) is adhering to a gluten-free diet (GFD), although its long-term molecular effects are still undescribed. New molecular features detectable in stool may improve and facilitate noninvasive clinical management of CD. For this purpose, fecal small non-coding RNAs (sncRNAs) and gut microbiome profiles were concomitantly explored in CD subjects in relation to strict (or not) GFD adherence over time. In this observational study, we performed small RNA and shotgun metagenomic sequencing in stool from 63 treated CD (tCD) and 3 untreated subjects as well as 66 sex- and age-matched healthy controls. tCD included 51 individuals on strict GFD and with negative transglutaminase (TG) serology (tCD-TG-) and 12 symptomatic with not strict/short-time of GFD adherence and positive TG serology (tCD-TG+). Samples from additional 40 healthy adult individuals and a cohort of 19 untreated pediatric CD subjects and 19 sex/age matched controls were analyzed to further test the outcomes. Several miRNA and microbial profiles were altered in tCD subjects (adj. p < .05). Findings were validated in the external group of adult controls. In tCD-TG-, GFD duration correlated with five miRNA levels (p < .05): for miR-4533-3p and miR-2681-3p, the longer the diet adherence, the less the expression differed from controls. tCD-TG+ and untreated pediatric CD patients showed a similar miRNA dysregulation. Immune-response, trans-membrane transport and cell death pathways were enriched in targets of identified miRNAs. Bifidobacterium longum, Ruminococcus bicirculans, and Haemophilus parainfluenzae abundances shifted (adj. p < .05) with a progressive reduction of denitrification pathways with GFD length. Integrative analysis highlighted 121 miRNA-bacterial relationships (adj. p < .05). Specific molecular patterns in stool characterize CD subjects, reflecting either the long-term GFD effects or the gut inflammatory status, in case of a not strict/short-time adherence. Our findings suggest novel host-microbial interplays and could help the discovery of biomarkers for GFD monitoring over time.

The 8q24 region hosts miRNAs altered in biospecimens of colorectal and bladder cancer patients.

BACKGROUND: The 8q24 locus is enriched in cancer-associated polymorphisms and, despite containing relatively few protein-coding genes, it hosts the MYC oncogene and other genetic elements connected to tumorigenesis, including microRNAs (miRNAs). Research on miRNAs may provide insights into the transcriptomic regulation of this multiple cancer-associated region. MATERIAL AND METHODS: We profiled all miRNAs located in the 8q24 region in 120 colorectal cancer (CRC) patients and 80 controls. miRNA profiling was performed on cancer/non-malignant adjacent mucosa, stool, and plasma extracellular vesicles (EVs), and the results validated with The Cancer Genome Atlas (TCGA) data. To verify if the 8q24-annotated miRNAs altered in CRC were dysregulated in other cancers and biofluids, we evaluated their levels in bladder cancer (BC) cases from the TCGA dataset and in urine and plasma EVs from a set of BC cases and healthy controls. RESULTS: Among the detected mature miRNAs in the region, 12 were altered between CRC and adjacent mucosa (adj. p < 0.05). Five and four miRNAs were confirmed as dysregulated in the CRC and BC TCGA dataset, respectively. A co-expression analysis of tumor/adjacent tissue data from the CRC group revealed a correlation between the dysregulated miRNAs and CRC-related genes (PVT1 and MYC) annotated in 8q24 region. miR-30d-5p and miR-151a-3p, altered in CRC tissue, were also dysregulated in stool of CRC patients and urine of BC cases, respectively. Functional enrichment of dysregulated miRNA target genes highlighted terms related to TP53-mediated cell cycle control. CONCLUSIONS: Altered expression of 8q24-annotated miRNAs may be relevant for the initiation and/or progression of cancer.

Integrative genomic and transcriptomic analyses illuminate the ontology of HER2-low breast carcinomas.

BACKGROUND: The "HER2-low" nomenclature identifies breast carcinomas (BCs) displaying a HER2 score of 1+/2+ in immunohistochemistry and lacking ERBB2 amplification. Whether HER2-low BCs (HLBCs) constitute a distinct entity is debated. METHODS: We performed DNA and RNA high-throughput analysis on 99 HLBC samples (n = 34 cases with HER2 score 1+/HLBC-1, n = 15 cases with HER2 score 2+ and ERBB2 not amplified/HLBC-2N, and n = 50 cases with score 2+ and ERBB2 copy number in the equivocal range/HLBC-2E). We compared the mutation rates with data from 1317 samples in the Memorial Sloan-Kettering Cancer Center (MSKCC) BC cohort and gene expression data with those from an internal cohort of HER2-negative and HER2-positive BCs. RESULTS: The most represented mutations affected PIK3CA (31/99, 31%), GATA3 (18/99, 18%), TP53 (17/99, 17%), and ERBB2 (8/99, 8%, private to HLBC-2E). Tumor mutational burden was significantly higher in HLBC-1 compared to HLBC-2E/N (P = 0.04). Comparison of mutation spectra revealed that HLBCs were different from both HER2-negative and HER2-positive BCs, with HLBC-1 resembling more HER2-negative tumors and HLBC-2 mutationally related to HER2-addicted tumors. Potentially actionable alterations (annotated by using OncoKB/ESCAT classes) affected 52 patients. Intra-group gene expression revealed overlapping features between HLBC-1 and control HER2-negative BCs, whereas the HLBC-2E tumors showed the highest diversity overall. The RNA-based class discovery analysis unveiled four subsets of tumors with (i) lymphocyte activation, (ii) unique enrichment in HER2-related features, (iii) stromal remodeling alterations, and (iv) actionability of PIK3CA mutations (LAURA classification). CONCLUSIONS: HLBCs harbor distinct genomic features when compared with HER2-positive and HER2-negative BCs; however, differences across IHC classes were also unveiled thus dissecting the full picture of heterogeneity across HER2-low disease. The HLBC-2E category harbors most distinctive features, whereas HLBC-1 seems superimposable to HER2-negative disease. Further studies are needed to ascertain whether the four genomic-driver classes of the LAURA classification hold prognostic and/or predictive implications.

The Non-Coding RNA Journal Club: Highlights on Recent Papers-11.

We are delighted to share with you our eleventh Journal Club and highlight some of the most interesting papers published recently [...].

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.

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.

Plasma concentrations of persistent organic pollutants and pancreatic cancer risk.

BACKGROUND: Findings and limitations of previous studies on persistent organic pollutants (POPs) and pancreatic cancer risk support conducting further research in prospective cohorts. METHODS: We conducted a prospective case-control study nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Participants were 513 pancreatic cancer cases and 1020 matched controls. Concentrations of 22 POPs were measured in plasma collected at baseline. RESULTS: Some associations were observed at higher concentrations of p, p'-DDT, trans-nonachlor, ß-hexachlorocyclohexane and the sum of six organochlorine pesticides and of 16 POPs. The odds ratio (OR) for the upper quartile of trans-nonachlor was 1.55 (95% confidence interval 1.06-2.26; P for trend = 0.025). Associations were stronger in the groups predefined as most valid (participants having fasted >6 h, with microscopic diagnostic confirmation, normal weight, and never smokers), and as most relevant (follow-up ≥10 years). Among participants having fasted >6 h, the ORs were relevant for 10 of 11 exposures. Higher ORs were also observed among cases with microscopic confirmation than in cases with a clinical diagnosis, and among normal-weight participants than in the rest of participants. Among participants with a follow-up ≥10 years, estimates were higher than in participants with a shorter follow-up (for trans-nonachlor: OR = 2.14, 1.01 to 4.53, P for trend = 0.035). Overall, trans-nonachlor, three PCBs and the two sums of POPs were the exposures most clearly associated with pancreatic cancer risk. CONCLUSIONS: Individually or in combination, most of the 22 POPs analysed did not or only moderately increased the risk of pancreatic cancer.

Comparison of Fecal Sample Collection Methods for Microbial Analysis Embedded within Colorectal Cancer Screening Programs.

BACKGROUND: Colorectal cancer screening programs with fecal sample collection may provide a platform for population-based gut microbiome disease research. We investigated sample collection and storage method impact on the accuracy and stability of the V3-V4 region of the 16S rRNA genes and bacterial quantity across seven different collection methods [i.e., no solution, two specimen collection cards, and four types of fecal immunochemical test (FIT) used in four countries] among 19 healthy volunteers. METHODS: Intraclass correlation coefficients (ICC) were calculated for the relative abundance of the top three phyla, the most abundant genera, alpha diversity metrics, and the first principal coordinates of the beta diversity matrices to estimate the stability of microbial profiles after storage for 7 days at room temperature, 4°C or 30°C, and after screening for the presence of occult blood in the stool. In addition, accuracy was estimated for samples frozen immediately compared to samples with no solution (i.e., the putative gold standard). RESULTS: When compared with the putative gold standard, we observed significant variation for all collection methods. However, interindividual variability was much higher than the variability introduced by the collection method. Stability ICCs were high (≥0.75) for FIT tubes that underwent colorectal cancer screening procedures. The relative abundance of Actinobacteria (0.65) was an exception and was lower for different FIT tubes stored at 30°C (range, 0.41-0.90) and room temperature (range, 0.06-0.94). CONCLUSIONS: Paper-based collection cards and different types of FIT are acceptable tools for microbiome measurements. IMPACT: Our findings inform on the utility of commonly used fecal sample collection methods for developing microbiome-focused cohorts nested within screening programs.

Faecal miRNA profiles associated with age, sex, BMI, and lifestyle habits in healthy individuals.

For their stability and detectability faecal microRNAs represent promising molecules with potential clinical interest as non-invasive diagnostic and prognostic biomarkers. However, there is no evidence on how stool miRNA profiles change according to an individual's age, sex, and body mass index (BMI) or how lifestyle habits influence the expression levels of these molecules. We explored the relationship between the stool miRNA levels and common traits (sex, age, BMI, and menopausal status) or lifestyle habits (physical activity, smoking status, coffee, and alcohol consumption) as derived by a self-reported questionnaire, using small RNA-sequencing data of samples from 335 healthy subjects. We detected 151 differentially expressed miRNAs associated with one variable and 52 associated with at least two. Differences in miR-638 levels were associated with age, sex, BMI, and smoking status. The highest number of differentially expressed miRNAs was associated with BMI (n = 92) and smoking status (n = 84), with several miRNAs shared between them. Functional enrichment analyses revealed the involvement of the miRNA target genes in pathways coherent with the analysed variables. Our findings suggest that miRNA profiles in stool may reflect common traits and lifestyle habits and should be considered in relation to disease and association studies based on faecal miRNA expression.