RESUMO
BACKGROUND: Early diagnosis of pancreatic ductal adenocarcinoma (PDAC) has been a longstanding challenge. The prognosis of patients with PDAC depends on the stage at diagnosis. It is necessary to identify biomarkers for the detection and differentiation of pancreatic tumors and optimize PDAC sample preparation procedures for DNA and RNA analysis. Most molecular studies are done using paraffin-embedded blocks; however, the integrity of DNA and RNA is often compromised in this format. Moreover, RNA isolated from human pancreatic tissue samples is generally of low quality, in part, because of the high concentration of endogenous pancreatic RNAse activity present. AIM: To assess the potential of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to obtain specimens from pancreatic neoplasms for subsequent RNA molecular profiling, including next-generation sequencing (NGS). METHODS: Thirty-four EUS-FNA samples were included in this study: PDAC (n = 15), chronic pancreatitis (n = 5), pancreatic cysts (n = 14), mucinous cysts (mucinous cystic neoplasia/intraductal papillary mucinous neoplasia) n = 7, serous cystic neoplasms n = 5, and pseudocysts n = 2. Cyst material consisted of cyst fluid and cyst wall samples obtained by through-the-needle biopsy (TTNB). Samples were stored at -80 °C until analysis. RNA purity (A260/230, A260/280 ratios), concentration, and integrity (RIN) were assessed. Real-time polymerase chain reaction was conducted on all samples, and small RNA libraries were prepared from solid mass samples. RESULTS: RNA was successfully extracted from 29/34 (85%) EUS-FNA samples: 100% pancreatic adenocarcinoma samples, 100% chronic pancreatitis samples, 70% pancreatic fluid cyst samples, and 50% TTNB samples. The relative expression of GAPDH and HPRT were obtained for all successfully extracted RNA samples (n = 29) including low-quality RNA specimens. Low concentration and nonoptimal RIN values (no less than 3) of RNA extracted from EUS-FNA samples did not prevent NGS library preparation. The suitability of cyst fluid samples for RNA profiling varied. The quality of RNA extracted from mucinous cyst fluid had a median RIN of 7.7 (5.0-8.2), which was compatible with that from solid neoplasms [6.2 (0-7.8)], whereas the quality of the RNA extracted from all fluids of serous cystic neoplasms and TTNB samples had a RIN of 0. CONCLUSION: The results demonstrate the high potential of EUS-FNA material for RNA profiling of various pancreatic lesions, including low-quality RNA specimens.
RESUMO
Background: Necrotizing enterocolitis (NEC) is a common gastrointestinal emergency among neonates which is characterized by acute intestinal inflammation and necrosis. The main risk factors for NEC are prematurity, low birth weight, and some preexisting health conditions such as congenital heart defects (CHDs). Investigation of the potential genetic predisposition to NEC is a promising approach that might provide new insights into its pathogenesis. One of the most important proteins that play a significant role in the pathogenesis of NEC is Toll-like receptor 4 (TLR4) which recognizes lipopolysaccharide found in Gram-negative bacteria. In intestinal epithelial cells, a protein encoded by the SIGIRR gene is a major inhibitor of TLR4 signaling. A few SIGIRR variants, including rare p.Y168X and p.S80Y, have already been identified in preterm infants with NEC, but their pathogenic significance remains unclear. This study aimed to investigate the spectrum of SIGIRR genetic variants in term newborns with CHD and to assess their potential association with NEC. Methods and Results: A total of 93 term newborns with critical CHD were enrolled in this study, 33 of them developed NEC. SIGIRR genetic variants were determined by Sanger sequencing of all exons. In total, eight SIGIRR genetic variants were identified, two of which were found only in newborns with NEC (P = 0.12). The rare missense p.S80Y (rs117739035) variant in exon 4 was found in two infants with NEC stage IIA. Two infants with NEC stage III and stage IB carried a novel duplication c. 102_121dup (rs552367848) variant in exon 10 that has not been previously associated with any clinical phenotype. Conclusions: The presence of both variants only in neonates who developed NEC, together with earlier published data, may suggest their potential contribution to the risk of developing NEC in term infants with CHD and allow planning larger cohort studies to clarify their relevance.
