Domenica Gioffreda

Thiopurines are employed in inflammatory bowel diseases (IBDs; Crohn’s disease, CD; ulcerative colitis, UC) to induce remission, prevent relapse, and reduce the steroid dosage, although they can sometimes be ineffective and present side effects. Genetic variations in the TPMT and NUDT15 genes are well recognized to influence the therapeutic response, despite notable regional differences in their frequencies across various ethnic populations. Herein, the risk haplotypes TPMT*3A, *3B, *3C, and the variant NUDT15*3 were examined in a retrospective cohort of 383 Italian IBD patients who received azathioprine or 6-mercaptopurine. TPMT and NUDT15 genotyping was performed by Sanger sequencing and TaqMan allelic discrimination, respectively. Allelic and genotype frequencies and genotype–phenotype correlations in non-responder and intolerant patients were assessed in comparison to responders. In total, 17% of patients did not respond to treatment, while 20% experienced adverse events, with leukopenia found in 13% of patients. TPMT haplotypes were found in 3.1% of patients, and 1.6% had the NUDT15*3 variant. CD patients with leukopenia had a higher frequency of the TPMT risk haplotype (40% vs. 4%, p = 0.024). Although additional validation through larger prospective studies or meta-analyses is needed, our findings support the importance of TPMT gene-variant assessment for forecasting azathioprine-related leukopenia in Italian IBD patients.

Pancreatic ductal adenocarcinoma (PDAC) is associated with intratumoral microbiota changes. However, defining the normal pancreatic microbial composition remains a challenge. Herein, we tested the hypothesis that the microbial profiling of normal pancreatic tissue from healthy organ donors (HC) could help in determining the signature of microbiota in PDAC. Matched pairs of tumor and normal tissues from PDAC patients (n = 32) and normal pancreatic tissues from HC (n = 17) were analyzed by 16S rRNA gene sequencing. Dissimilarities in all the beta metrics emerged in both normal samples and tumor samples, compared to HC (Bray–Curtis dissimilarity and Jaccard distance: p = 0.002; weighted UniFrac distances: p = 0.42 and p = 0.012, respectively; unweighted UniFrac distance: p = 0.009); a trend toward a lower Faith’s phylogenetic distance was found at the tumor level vs. HC (p = 0.08). Within PDAC, a lower Faith’s phylogenetic distance (p = 0.003) and a significant unweighted UniFrac distance (p = 0.024) were observed in tumor samples vs. normal samples. We noted the presence of a decreased abundance of bacteria with potential beneficial effects (Jeotgalicoccus) and anticancer activity (Acinetobacter_guillouiae) in PDAC vs. HC; bacteria involved in immune homeostasis and suppression of tumor progression (Streptococcus_salivarius, Sphingomonas) were reduced, and those implicated in tumor initiation and development (Methylobacterium-Methylorubrum, g_Delftia) were enhanced in tumor samples vs. normal samples. Metagenomic functions involved in fatty acid synthesis were reduced in normal samples compared to HC, while peptidoglycan biosynthesis IV and L-rhamnose degradation were more abundant in tumor samples vs. normal samples. Future prospective studies on larger populations, also including patients in advanced tumor stages and considering all potential existing confounding factors, as well as further functional investigations, are needed to prove the role of microbiota-mediated pathogenicity in PDAC.

Diagnosis of biliopancreatic cancers by the available serum tumor markers, imaging, and histopathological tissue specimen examination remains a challenge. Circulating cell-free DNA derived from matched pairs of secretin-stimulated duodenal fluid (DF) and plasma from 10 patients with biliopancreatic diseases and 8 control subjects was analyzed using AmpliSeq™ HD technology for Ion Torrent Next-Generation Sequencing to evaluate the potential of liquid biopsy with DF in biliopancreatic cancers. The median cfDNA concentration was greater in DF-derived than in plasma-derived samples. A total of 13 variants were detected: 11 vs. 1 were exclusive for DF relative to the plasma source, and 1 was shared between the two body fluids. According to the four-tier systems, 10 clinical tier-I–II (76.9%), 1 tier–III (7.7%), and 2 tier–IV (15.4%) variants were identified. Notably, the 11 tier-I-III variants were exclusively found in DF-derived cfDNA from five patients with biliopancreatic cancers, and were detected in seven genes (KRAS, TP53, BRAF, CDKN2A, RNF43, GNAS, and PIK3CA); 82% of the tier-I-III variants had a low abundance, with a VAF < 6%. The mutational profiling of DF seems to be a reliable and promising tool for identifying cancer-associated alterations in malignant cancers of the biliopancreatic tract.

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.