Polymorphisms in transcription factor binding sites and enhancer regions and pancreatic ductal adenocarcinoma risk.

Genome-wide association studies (GWAS) are a powerful tool for detecting variants associated with complex traits and can help risk stratification and prevention strategies against pancreatic ductal adenocarcinoma (PDAC). However, the strict significance threshold commonly used makes it likely that many true risk loci are missed. Functional annotation of GWAS polymorphisms is a proven strategy to identify additional risk loci. We aimed to investigate single-nucleotide polymorphisms (SNP) in regulatory regions [transcription factor binding sites (TFBSs) and enhancers] that could change the expression profile of multiple genes they act upon and thereby modify PDAC risk. We analyzed a total of 12,636 PDAC cases and 43,443 controls from PanScan/PanC4 and the East Asian GWAS (discovery populations), and the PANDoRA consortium (replication population). We identified four associations that reached study-wide statistical significance in the overall meta-analysis: rs2472632(A) (enhancer variant, OR 1.10, 95%CI 1.06,1.13, p = 5.5 × 10-8), rs17358295(G) (enhancer variant, OR 1.16, 95%CI 1.10,1.22, p = 6.1 × 10-7), rs2232079(T) (TFBS variant, OR 0.88, 95%CI 0.83,0.93, p = 6.4 × 10-6) and rs10025845(A) (TFBS variant, OR 1.88, 95%CI 1.50,1.12, p = 1.32 × 10-5). The SNP with the most significant association, rs2472632, is located in an enhancer predicted to target the coiled-coil domain containing 34 oncogene. Our results provide new insights into genetic risk factors for PDAC by a focused analysis of polymorphisms in regulatory regions and demonstrating the usefulness of functional prioritization to identify loci associated with PDAC risk.

Mucosal Microbiota from Colorectal Cancer, Adenoma and Normal Epithelium Reveals the Imprint of Fusobacterium nucleatum in Cancerogenesis.

An increasing amount of evidence suggests the emerging role of the gut microbiota in the development of colorectal cancer (CRC). This study aimed to elucidate the architecture of microbial communities within normal and neoplastic colonic mucosa. METHODS: Microbiota were analyzed by NGS and by an ensemble of metagenomics analysis tools in a total of 69 tissues from 9 patients with synchronous colorectal neoplasia and adenomas (27 specimens: 9 from normal tissues, 9 adenomas, and 9 tumours), 16 patients with only colonic adenomas (32 specimens: 16 from normal tissues and 16 adenomas), and from healthy subjects (10 specimens of normal mucosa). RESULTS: Weak differences were observed in alpha and beta metrics among the synchronous tissues from CRC and controls. Through pairwise differential abundance analyses of sample groups, an increasing trend of Rikenellaceae, Pseudomonas and Fusobacterium, and decreasing trends of Staphylococcus, Actinobacillus and Gemmiger were observed in CRC, while Staphylococcus and Bifidobacterium were decreased in patients with only adenomas. At RT-qPCR analysis, Fusobacterium nucleatum was significantly enriched in all the tissues of subjects with synchronous colorectal neoplasia. CONCLUSION: Our findings provide a comprehensive view of the human mucosa-associated gut microbiota, emphasizing global microbial diversity mostly in synchronous lesions and proving the constant presence of Fusobacterium nucleatum, with its ability to drive carcinogenesis.

Gut virome-colonising Orthohepadnavirus genus is associated with ulcerative colitis pathogenesis and induces intestinal inflammation in vivo.

OBJECTIVES: Ulcerative colitis (UC) is a chronic inflammatory disorder of unknown aetiology. Gut virome dysbiosis is fundamental in UC progression, although its role in the early phases of the disease is far from fully understood. Therefore, we sought to investigate the role of a virome-associated protein encoded by the Orthohepadnavirus genus, the hepatitis B virus X protein (HBx), in UC aetiopathogenesis. DESIGN: HBx positivity of UC patient-derived blood and gut mucosa was assessed by RT-PCR and Sanger sequencing and correlated with clinical characteristics by multivariate analysis. Transcriptomics was performed on HBx-overexpressing endoscopic biopsies from healthy donors.C57BL/6 mice underwent intramucosal injections of liposome-conjugated HBx-encoding plasmids or the control, with or without antibiotic treatment. Multidimensional flow cytometry analysis was performed on colonic samples from HBx-treated and control animals. Transepithelial electrical resistance measurement, proliferation assay, chromatin immunoprecipitation assay with sequencing and RNA-sequencing were performed on in vitro models of the gut barrier. HBx-silencing experiments were performed in vitro and in vivo. RESULTS: HBx was detected in about 45% of patients with UC and found to induce colonic inflammation in mice, while its silencing reverted the colitis phenotype in vivo. HBx acted as a transcriptional regulator in epithelial cells, provoking barrier leakage and altering both innate and adaptive mucosal immunity ex vivo and in vivo. CONCLUSION: This study described HBx as a contributor to the UC pathogenesis and provides a new perspective on the virome as a target for tailored treatments.

Evaluation of inherited germline mutations in cancer susceptibility genes among pancreatic cancer patients: a single-center study.

BACKGROUND: Germline mutations in cancer susceptibility genes were identified in pancreatic cancer (PanC) patients with a sporadic disease and in those unselected for family cancer history. METHODS: With the aim to determine the prevalence of germline predisposition genes mutations in PanC, and to evaluate whether they were associated with the presence of PanC, we profiled a custom AmpliSeq panel of 27 cancer susceptibility genes in 47 PanC patients and 51 control subjects by using the Ion Torrent PGM system. RESULTS: Multigene panel testing identified a total of 31 variants in 27 PanC (57.4%), including variants with pathogenic/likely pathogenic effect, those of uncertain significance, and variants whose clinical significance remains currently undefined. Five patients carried more than one variant in the same gene or in different genes. Eight patients (17.0%) had at least one pathogenic/likely pathogenic variant in four main genes: CFTR (10.6%), BRCA2 (8.5%), ATM and CHEK2 (2.1%). Pathogenic/likely pathogenic mutation were identified in patients with positive PanC family history (20%) or in patients without first-degree relatives affected by PanC (13.6%). All the BRCA2 mutation carriers were unselected PanC patients. The presence of mutations in BRCA2 was significantly associated with an increased occurrence of PanC and with positive family history for endometrial cancer (p = 0.018). CONCLUSIONS: This study confirmed the potential remarkable contribution of BRCA2 in assessing the presence of PanC. Overall our findings supported the recommendation of offering the germline testing to all the PanC patients with the intent to reduce the number of underdiagnosed carriers of mutations in predisposition genes, and not to preclude their relatives from the opportunity to benefit from surveillance programs.

Association between a polymorphic variant in the CDKN2B-AS1/ANRIL gene and pancreatic cancer risk.

Genes carrying high-penetrance germline mutations may also be associated with cancer susceptibility through common low-penetrance genetic variants. To increase the knowledge on genetic pancreatic ductal adenocarcinoma (PDAC) aetiology, the common genetic variability of PDAC familial genes was analysed in our study. We conducted a multiphase study analysing 7745 single nucleotide polymorphisms (SNPs) from 29 genes reported to harbour a high-penetrance PDAC-associated mutation in at least one published study. To assess the effect of the SNPs on PDAC risk, a total of 14 666 PDAC cases and 221 897 controls across five different studies were analysed. The T allele of the rs1412832 polymorphism, that is situated in the CDKN2B-AS1/ANRIL, showed a genome-wide significant association with increased risk of developing PDAC (OR = 1.11, 95% CI = 1.07-1.15, P = 5.25 × 10-9 ). CDKN2B-AS1/ANRIL is a long noncoding RNA, situated in 9p21.3, and regulates many target genes, among which CDKN2A (p16) that frequently shows deleterious somatic and germline mutations and deregulation in PDAC. Our results strongly support the role of the genetic variability of the 9p21.3 region in PDAC aetiopathogenesis and highlight the importance of secondary analysis as a tool for discovering new risk loci in complex human diseases.

A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application.

Background: Enterobius vermicularis (E. vermicularis) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Materials and methods: Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis. Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Results: Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. Conclusion: The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.

Adherence to Gluten-Free Diet Restores Alpha Diversity in Celiac People but the Microbiome Composition Is Different to Healthy People.

Celiac disease (CD) is an autoimmune disease with the destruction of small intestinal villi, which occurs in genetically predisposed individuals. At the present moment, a gluten-free diet (GFD) is the only way to restore the functionality of gut mucosa. However, there is an open debate on the effects of long-term supplementation through a GFD, because some authors report an unbalance in microbial taxa composition. METHODS: For microbiome analysis, fecal specimens were collected from 46 CD individuals in GFD for at least 2 years and 30 specimens from the healthy controls (HC). Data were analyzed using an ensemble of software packages: QIIME2, Coda-lasso, Clr-lasso, Selbal, PICRUSt2, ALDEx2, dissimilarity-overlap analysis, and dysbiosis detection tests. RESULTS: The adherence to GFD restored the alpha biodiversity of the gut microbiota in celiac people but microbial composition at beta diversity resulted as different to HC. The microbial composition of the CD subjects was decreased in a number of taxa, namely Bifidobacterium longum and several belonging to Lachnospiraceae family, whereas Bacteroides genus was found to be more abundant. Predicted metabolic pathways among the CD bacterial communities revealed an important role in tetrapyrrole biosynthesis. CONCLUSIONS: CD patients in GFD had a non-dysbiotic microbial composition for the crude alpha diversity metrics. We found significant differences in beta diversity, in certain taxon, and pathways between subjects with inactive CD in GFD and controls. Collectively, our data may suggest the development of new GFD products by modulating the gut microbiota through diet, supplements of vitamins, and the addition of specific prebiotics.

Healthy and pro-inflammatory gut ecology plays a crucial role in the digestion and tolerance of a novel Gluten Friendly™ bread in celiac subjects: a randomized, double blind, placebo control in vivo study.

Gluten Friendly™ (GF) is a new gluten achieved through a physicochemical process applied to wheat kernels. The goal of this research was to assess the in vivo effects of Gluten Friendly™ bread on celiac gut mucosa and microbiota. In a double-blind placebo-controlled intervention study, 48 celiac disease (CD) patients were randomized into 3 groups to eat 100 g of bread daily, containing different doses (0; 3 g; 6 g) of GF for 12 weeks. The small-bowel morphology (VH/CrD), intraepithelial densities of CD3+, celiac serology, MUC2, CB1, gut permeability, proinflammatory cytokines, gluten in stools, symptoms, and gut microbial composition were assessed. All 48 CD subjects experienced no symptoms. K-means analysis evidenced celiac subjects clustering around unknown parameters independent of GF dosage: K1 35%; K2 30%; K3 35%. VH/CrD significantly decreased in K1 and K2. VH/CrD did not correlate with IEL increase in K2. 33-mer was not detected in 47% and 73% of patients in both K1 and K2, respectively. VH/CrD and IEL did not change significantly and strongly correlated with the absence of 33-mer in K3. Inflammation and VH/CrD decrease are strongly related with the presence of proinflammatory species at the baseline. A boost in probiotic, butyrate-producing genera, is strongly related with GF tolerance at the end of the trial. Our research suggests that a healthy and proinflammatory ecology could play a crucial role in the digestion and tolerance of the new gluten molecule in celiac subjects. However, GF can be completely digested by gut microbiota of CD subjects and shapes it toward gut homeostasis by boosting healthy butyrate-producing populations. The clinical trial registry number is NCT03137862 (https://clinicaltrials.gov).

Germline Alterations in Patients With IBD-associated Colorectal Cancer.

BACKGROUND: Patients with inflammatory bowel diseases (IBD), both ulcerative colitis (UC) and Crohn's disease (CD), are at risk of developing a colorectal cancer (CRC). No information is available on the contribution of patients' genetic background to CRC occurrence. This study investigates germline alterations in patients with IBD-associated CRC. METHODS: We profiled a panel of 39 genes potentially involved in cancer predisposition and searched for germline variants in IBD patients with CRC or high-grade dysplasia. RESULTS: After clinical exclusion of genetic cancer syndromes, 25 IBD patients (4 CD and 21 UC) with CRC or high-grade dysplasia were studied. After excluding variants with low likelihood of pathogenicity (classes 1 or 2 according the International Agency for Research on Cancer [IARC]), the panel identified pathogenic variants, likely pathogenic, or variants with unknown significance in 18 patients (72%). Six patients (24%) carried pathogenic or likely variants (IARC class 5 or 4). Of the identified variants, 4 encompassed the APC region, 3 the MLH1 gene, and the remaining ones the MSH2, MSH3, monoallelic MUTYH, EPCAM, BRCA1, CHEK2, POLD1, POLE, CDKN2A, and PDGFRA genes. Four patients carried at least 2 variants in different genes. Duration of IBD was significantly shorter in carriers of 4 or 5 IARC variants (7 years; range 0-21; P = .002) and in those with variants with unknown significance (12 years; range 0-22; P = .005) compared with patients without or with only benign variations (23.5 years; range 15-34). CONCLUSIONS: In silico analysis and sequence-based testing of germline DNA from IBD patients with CRC or high-grade dysplasia detected 24% of variants positioned in pathogenic classes. In patients with type 3, 4, and 5 variants, the onset of high-grade dysplasia or CRC was significantly earlier than in patients with benign or unidentified variants. The screening for these genes could identify IBD patients requiring a more intensive endoscopic surveillance for earlier detection of dysplastic changes.

Downexpression of miR-200c-3p Contributes to Achalasia Disease by Targeting the PRKG1 Gene.

Achalasia is an esophageal smooth muscle motility disorder with unknown pathogenesis. Taking into account our previous results on the downexpression of miR-200c-3p in tissues of patients with achalasia correlated with an increased expression of PRKG1, SULF1, and SYDE1 genes, our aim was to explore the unknown biological interaction between these genes and human miR-200c-3p and if this relation could unravel their functional role in the etiology of achalasia. To search for putative miR-200c-3p binding sites in the 3'-UTR of PRKG1, SULF1 and SYDE1, a bioinformatics tool was used. To test whether PRKG1, SULF1, and SYDE1 are targeted by miR-200c-3p, a dual-luciferase reporter assay and quantitative PCR on HEK293 and fibroblast cell lines were performed. To explore the biological correlation between PRKG1 and miR-200c-3p, an immunoblot analysis was carried out. The overexpression of miR-200c-3p reduced the luciferase activity in cells transfected with a luciferase reporter containing a fragment of the 3'-UTR regions of PRKG1, SULF1, and SYDE1 which included the miR-200c-3p seed sequence. The deletion of the miR-200c-3p seed sequence from the 3'-UTR fragments abrogated this reduction. A negative correlation between miR-200c-3p and PRKG1, SULF1, and SYDE1 expression levels was observed. Finally, a reduction of the endogenous level of PRKG1 in cells overexpressing miR-200c-3p was detected. Our study provides, for the first time, functional evidence about the PRKG1 gene as a direct target and SULF1 and SYDE1 as potential indirect substrates of miR-200c-3p and suggests the involvement of NO/cGMP/PKG signaling in the pathogenesis of achalasia.

Microbiome Analysis of Mucosal Ileoanal Pouch in Ulcerative Colitis Patients Revealed Impairment of the Pouches Immunometabolites.

The pathogenesis of ulcerative colitis (UC) is unknown, although genetic loci and altered gut microbiota have been implicated. Up to a third of patients with moderate to severe UC require proctocolectomy with ileal pouch ano-anastomosis (IPAA). We aimed to explore the mucosal microbiota of UC patients who underwent IPAA. METHODS: For microbiome analysis, mucosal specimens were collected from 34 IPAA individuals. Endoscopic and histological examinations of IPAA were normal in 21 cases, while pouchitis was in 13 patients. 19 specimens from the healthy control (10 from colonic and 9 from ileum) were also analyzed. Data were analyzed using an ensemble of software packages: QIIME2, coda-lasso, clr-lasso, PICRUSt2, and ALDEx2. RESULTS: IPAA specimens had significantly lower bacterial diversity as compared to normal. The microbial composition of the normal pouch was also decreased also when compared to pouchitis. Faecalibacterium prausnitzii, Gemmiger formicilis, Blautia obeum, Ruminococcus torques, Dorea formicigenerans, and an unknown species from Roseburia were the most uncommon in pouch/pouchitis, while an unknown species from Enterobacteriaceae was over-represented. Propionibacterium acnes and Enterobacteriaceae were the species most abundant in the pouchitis and in the normal pouch, respectively. Predicted metabolic pathways among the IPAA bacterial communities revealed an important role of immunometabolites such as SCFA, butyrate, and amino acids. CONCLUSIONS: Our findings showed specific bacterial signature hallmarks of dysbiosis and could represent bacterial biomarkers in IPAA patients useful to develop novel treatments in the future by modulating the gut microbiota through the administration of probiotic immunometabolites-producing bacterial strains and the addition of specific prebiotics and the faecal microbiota transplantation.

Circulating levels of cytokines, chemokines and growth factors in patients with achalasia.

Idiopathic achalasia is a disease that is characterized by the absence of peristalsis and incomplete relaxation of the lower esophageal sphincter, which is accompanied by dysphagia, regurgitation, chest pain and weight loss. The role of inflammatory infiltrates in the pathogenesis of achalasia remains controversial, although the infiltrating cell profile in the tissue has been previously characterized histologically and immunohistochemically. The present study aimed to evaluate the serum levels of 27 protein biomarkers to determine their association with achalasia and the clinical disease characteristics. The cytokine, chemokine and growth factor serum profiles of 68 patients with achalasia and 39 healthy individuals were explored using the 27-Bio-Plex Pro Human Cytokine assay. Reductions in the levels of inflammatory mediators IL-1ß, IL-2, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interferon-γ, monocyte chemoattractant protein-1, macrophage inflammatory protein-1 (MIP-1)α and MIP-1ß, regulated upon activation normal T cell expressed and presumably secreted, TNF-α and VEGF were detected in the serum samples of patients with achalasia compared with those in the control group (P<0.05). However, significant associations between the expression in the levels of inflammatory factors and clinical characteristics of the patients were not found (P>0.05). These results suggest that achalasia is a disease that has a local but not a systemic inflammatory pattern. Further studies are required to improve the current understanding of the mechanism underlying this disease.

Impact of the COVID-19 outbreak and the serum prevalence of SARS-CoV-2 antibodies in patients with inflammatory bowel disease treated with biologic drugs.

BACKGROUND: Patients receiving biologic therapies are at risk for viral infections. This study investigated the impact of the SARS-CoV-2 infection and the serum prevalence of SARS-CoV-2 antibodies in patients with inflammatory bowel disease (IBD) treated with biologic drugs. METHODS: Information on demography, co-morbidities, clinical data regarding IBD, symptoms suggestive of the SARS-CoV-2 infection, close contacts with SARS-CoV-2 positive patients, hospitalization, and therapies administered for COVID-19 was collected for all patients who were being treated with biologic drugs. All patients underwent SARS-CoV-2 antibody testing. RESULTS: Two hundred and fifty-nine patients (27 children) with a mean age of 42.2 ±â€¯16.7 years (range 9 - 88) and a mean duration of disease of 13.4 ±â€¯10 years (range 0.2 - 49) were enrolled. One hundred four patients (40.2%) had ulcerative colitis, and 155 (59.8%) had Crohn's disease. About the therapy: 62 patients were receiving infliximab, 89 adalimumab, 20 golimumab, 57 vedolizumab, 27 ustekinumab, 1 thalidomide, and 3 an experimental compound. The mean Charlson Comorbidity Index was 2. Thirty-two patients (12.3%) reported respiratory symptoms, and 2 of them were hospitalized (0.77%). Two patients resulted positive for IgG against SARS-CoV-2 (0.77%). CONCLUSIONS: In patients with IBD, treatment with biologic drug does not represent a risk factor for the SARS-CoV-2 infection.

False-positive results of SARS-CoV-2 IgM/IgG antibody tests in sera stored before the 2020 pandemic in Italy.

OBJECTIVES: Aside from the outbreak of the coronavirus disease 2019 (COVID-19), serological tests are not well known for their diagnostic value. We assessed the performance of serological tests using stored sera from patients with a variety of pathologic conditions, collected before the 2020 pandemic in Italy. METHODS: Rapid lateral flow tests and Enzyme-Linked Immunosorbent Assays (ELISA) that detect Immunoglobulin M (IgM) and Immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were carried out using 1150 stored human serum samples that had been collected in 2018 and 2019. The tests were also run using samples from 15 control patients who had positive or negative oral swab test results, as assessed using real-time reverse transcription-polymerase chain reaction (rRT-PCR). The urea dissociation test was employed to rule out false-positive reactivity in the two antibody detection methods. RESULTS: The lateral flow tests revealed 21 positive samples from the stored sera: 12 for IgM, four for IgG, and five for IgM/IgG. Among the nine rRT-PCR- positive controls, six individuals presented IgG and three IgM/IgG positivity. Using the urea (6 mol/L) dissociation test, two of the twelve stored samples that had shown IgM positivity were confirmed to be positive. The ELISA test detected four IgM-positive and three IgG-positive specimens. After treatment with 4 mol/L urea, the IgM-positive samples became negative, whereas the IgG positivity persisted. All of the rRT-PCR-positive controls were found to retain IgM or IgG positivity following the urea treatment. CONCLUSIONS: Our findings highlight the limited utility of serological testing for the SARS-CoV-2 virus based on the results of specimens collected before the outbreak of the infection.

Polygenic and multifactorial scores for pancreatic ductal adenocarcinoma risk prediction.

BACKGROUND: Most cases of pancreatic ductal adenocarcinoma (PDAC) are asymptomatic in early stages, and the disease is typically diagnosed in advanced phases, resulting in very high mortality. Tools to identify individuals at high risk of developing PDAC would be useful to improve chances of early detection. OBJECTIVE: We generated a polygenic risk score (PRS) for PDAC risk prediction, combining the effect of known risk SNPs, and carried out an exploratory analysis of a multifactorial score. METHODS: We tested the associations of the individual known risk SNPs on up to 2851 PDAC cases and 4810 controls of European origin from the PANcreatic Disease ReseArch (PANDoRA) consortium. Thirty risk SNPs were included in a PRS, which was computed on the subset of subjects that had 100% call rate, consisting of 839 cases and 2040 controls in PANDoRA and 6420 cases and 4889 controls from the previously published Pancreatic Cancer Cohort Consortium I-III and Pancreatic Cancer Case-Control Consortium genome-wide association studies. Additional exploratory multifactorial scores were constructed by complementing the genetic score with smoking and diabetes. RESULTS: The scores were associated with increased PDAC risk and reached high statistical significance (OR=2.70, 95% CI 1.99 to 3.68, p=2.54×10-10 highest vs lowest quintile of the weighted PRS, and OR=14.37, 95% CI 5.57 to 37.09, p=3.64×10-8, highest vs lowest quintile of the weighted multifactorial score). CONCLUSION: We found a highly significant association between a PRS and PDAC risk, which explains more than individual SNPs and is a step forward in the direction of the construction of a tool for risk stratification in the population.

Transcriptome and Gene Fusion Analysis of Synchronous Lesions Reveals lncMRPS31P5 as a Novel Transcript Involved in Colorectal Cancer.

Fusion genes and epigenetic regulators (i.e., miRNAs and long non-coding RNAs) constitute essential pieces of the puzzle of the tumor genomic landscape, in particular in mechanisms behind the adenoma-to-carcinoma progression of colorectal cancer (CRC). In this work, we aimed to identify molecular signatures of the different steps of sporadic CRC development in eleven patients, of which synchronous samples of adenomas, tumors, and normal tissues were analyzed by RNA-Seq. At a functional level, tumors and adenomas were all characterized by increased activity of the cell cycle, cell development, cell growth, and biological proliferation functions. In contrast, organic survival and apoptosis-related functions were inhibited both in tumors and adenomas at different levels. At a molecular level, we found that three individuals shared a tumor-specific fusion named MRPS31-SUGT1, generated through an intra-chromosomal translocation on chromosome 13, whose sequence resulted in being 100% identical to the long non-coding RNA (lncRNA) MRPS31P5. Our analyses suggest that MRPS31P5 could take part to a competitive endogenous (ce)RNA network by acting as a miRNA sponge or/and as an interactor of other mRNAs, and thus it may be an important gene expression regulatory factor and could be used as a potential biomarker for the detection of early CRC events.

Landmarks for dual biological therapy in inflammatory bowel disease: lesson from two case reports of vedolizumab in combination with ustekinumab.

Inflammatory bowel disease (IBD) is a chronic and disabling disorder. Severity of IBD is prominent among refractory with patients with concomitant immune-mediated disorders. Among those patients, dual biological therapy (DBT) has been suggested as an alternative approach to spare steroids and avoid surgery. However, pieces of evidence on clinical outcomes among patients receiving DBT are still limited. We present two cases of IBD patients, with dermatological comorbidity, treated with a combination of vedolizumab and ustekinumab, identifying possible landmarks to address therapeutic choice. No patient experienced adverse events in the follow-up period and both obtained complete clinical remission. DBT may be an effective approach to consider in selected patients with refractory IBD with concomitant severe immune-mediated diseases taking into account medical history of the patient, presence, and type of concomitant extraintestinal manifestations, safety profile of selected DBT, licensed therapeutic indications, and costs.

microRNA-mRNA network model in patients with achalasia.

BACKGROUND: Achalasia is a rare idiopathic disease with a complex etio-pathogenesis still unknown. This study aimed to identify microRNA (miRNA)-mRNA regulatory networks underlying achalasia. METHODS: The investigation was performed in tissue specimens from 11 patients and five controls using the microarray technology followed by an integrated bioinformatics analysis. KEY RESULTS: One hundred and six miRNAs were significantly up-regulated and 64 were down-regulated in achalasia patients. The expression of the most 10 differential expressed miRNAs (miR-122-5p, miR-133a-3p, miR-504-5p, miR-187-3p, miR-133b, miR-200c-3p, miR-375, miR-200b-5p, miR-200b-3p, and miR203a) was confirmed by droplet digital PCR in an independent cohort. The interactions between the significant miRNAs and their targets uncovered 14 miRNA-mRNA interacting pairs with experimentally predicted genes (ie, FN1, ROCK2, DPYSL2), and 35 pairs with not experimentally target genes (ie, SULF1, MRVI1, PRKG1); all genes were involved in immune cell trafficking, skeletal and muscular system development, nervous system development macro-processes. CONCLUSION & INFERENCES: The mRNA-miRNA regulatory networks described in this study provide new insights in the genetic background of the disease, suggesting further investigations in novel pathogenic mechanisms.

Droplet digital PCR quantification of miR-1290 as a circulating biomarker for pancreatic cancer.

Droplet digital PCR was used to validate miR-1290 as circulating biomarker for pancreatic cancer (PC). The diagnostic performance of miR-1290 was evaluate in 167 PC patients and 267 healthy subjects at clinical risk of developing the disease (HS). MiR-1290 plasma levels were compared to CA 19-9 determinations, and the combination of the two biomarkers was also taken into account. Plasma levels of miR-1290 were higher in PC patients compared to HS (p = 2.55 × 10-16). A similar trend was observed for CA 19-9 determinations (p = 1.03 × 10-47). ROC curve analysis revealed that miR-1290 in combination with CA 19-9 was effective for discriminating between PC patients and HS (AUC = 0.956, 95% CI = 0.933-0.979) than the two biomarkers tested alone (miR-1290: AUC = 0.734, 0.678-0.789; CA 19-9: AUC = 0.914, 0.877-0.951). The discriminating ability was higher when only PC patients with low or slightly increased CA 19-9 levels were compared with HS. MiR-1290 concentrations were not able to differentiate between PC patients with single or multiple risk factors for developing PC. Our data suggest that the absolute quantification of circulating miR-1290 levels does not allow to select patients at clinical risk of PC for entry into a surveillance program, and underline the methodological challenges still existing in utilizing circulating miRNAs as new promising biomarkers for PC.