Impact of ischemia on sample quality of human pancreatic tissues.

BACKGROUND: Successful clinical evaluation of human tumors relies on proper handling of tissue samples to maximally preserve the cellular and metabolic states in vivo. Pancreatic samples are particularly sensitive to sample mishandling due to the abundance of digestive enzymes. We study how the duration of ischemia, in vivo and ex vivo, both of which are unavoidable lagging periods following surgical dissection, significantly impact the utility of pancreatic samples. METHODS: We systematically characterize a wide range of tissue integrity features, including histological patterns, cellular structures, DNA/RNA quality and activity of major signaling pathways in normal pancreases and pancreatic ductal adenocarcinoma (PDAC) tumor tissues from 41 patients with different ischemia. RESULTS: We reveal that tissues experiencing longer periods of ischemia exhibit significant deterioration and could potentially mislead disease diagnosis and preclinical research. Based on these analyses, we propose an optimal procedure that balances better clinical practice and high tissue sample quality. CONCLUSIONS: Our work provides a guideline for pancreatic sample handling and could have wide implications in clinical diagnosis and translational research.

Gut resistome profiling reveals high diversity and fluctuations in pancreatic cancer cohorts.

Background: Pancreatic cancer is one of the deadliest cancer, with a 5-year overall survival rate of 11%. Unfortunately, most patients are diagnosed with advanced stage by the time they present with symptoms. In the past decade, microbiome studies have explored the association of pancreatic cancer with the human oral and gut microbiomes. However, the gut microbial antibiotic resistance genes profiling of pancreatic cancer patients was never reported compared to that of the healthy cohort. Results: In this study, we addressed the gut microbial antibiotic resistance genes profile using the metagenomic data from two online public pancreatic cancer cohorts. We found a high degree of data concordance between the two cohorts, which can therefore be used for cross-sectional comparisons. Meanwhile, we used two strategies to predict antibiotic resistance genes and compared the advantages and disadvantages of these two approaches. We also constructed microbe-antibiotic resistance gene networks and found that most of the hub nodes in the networks were antibiotic resistance genes. Conclusions: In summary, we describe the panorama of antibiotic resistance genes in the gut microbes of patients with pancreatic cancer. We hope that our study will provide new perspectives on treatment options for the disease.

Convergence between germline and somatic mutations in pancreatic neuroendocrine tumors.

Objectives: The pancreatic neuroendocrine tumors (PanNETs) are a group of clinically heterogeneous neoplasms. Although previous studies illustrated the somatic mutation pattern for PanNETs, the germline mutation pattern is still unclear. Here, we comprehensively screened the underlying germline mutations in a cohort of multiple endocrine neoplasia type 1 (MEN1)-related and sporadic PanNETs to reveal the characteristics of germline mutation in PanNET patients. Methods: Patients diagnosed with PanNETs by biopsy or surgical pathology were enrolled in this study. Peripheral blood samples were used for genomic DNA purification and subsequent sequencing. The following sequencing techniques were used and compared for validation: (1) targeted gene capture with a customized panel; (2) whole exome sequencing data from previous study. Results: A total of 184 PanNET patients were enrolled, including 20 MEN1-related and 164 sporadic cases. In this study, MEN1 mutation rate in MEN1-related PanNETs was 60% (12/20), of which 50% were novel mutation sites. For sporadic PanNETs, the overall germline mutation rate was very low. Besides the rare MEN1 mutation, previously unreported germline variant in DAXX was found in one non-functional PanNET. Conclusions: This study revealed distinctive germline mutation rates between MEN1-related and sporadic PanNETs. The novel MEN1 mutations contribute to revealing the spectrum of MEN1 mutations in PanNETs. The newly discovered germline variant of DAXX in sporadic PanNET implies a tendency of convergence between germline and somatic mutation genes.

Effects of ex vivo ischemia time and delayed processing on quality of specimens in tissue biobank.

The RNA quality of tissue biobank is crucial for translational research; however, the effects of the ex vivo ischemia time on RNA integrity and expression of genes related to hypoxia, stress, apoptosis and autophagy remains elusive. A total of 18 carcinoma tissues were stored at room temperature for 15 min, 30 min, 1, 2, 4, 8 and 24 h. The integrity and purity of isolated RNA were analyzed. Furthermore, the gene expression of mTOR, hypoxia­inducible factor 1α, phosphatidylinositol 4,5­bisphosphate 3­kinase catalytic subunit ß isoform (PI3KCB), threonine kinase 1 (AKT1), NF­κB, protein kinase AMP­activated catalytic subunit α1 (AMPKα1), caspase 8 (CASP8), unc­51 like autophagy activating kinase 1 and Fas cell surface death receptor were analyzed using reverse transcription­quantitative PCR. The results demonstrated that RNA integrity numbers (RINs) remained stable in carcinoma tissues following ex vivo ischemia for 2 h at room temperature and that degradation began at 4 h (P<0.001). Additionally, the expression of PI3KCB, AKT1, AMPKα1 and CASP8 decreased at time points 8­24 h following ex vivo ischemia and delayed processing (P<0.001). In conclusion, >2 h of ex vivo ischemia and delayed processing induced RNA degradation and RIN, and the gene expressions of PI3KCB, AKT, AMPKα1 and CASP8 may be considered as markers to evaluate tissue quality at the gene expression level, providing a method for the standard processing and assessment of tissue specimen.

Circular RNA expression and association with the clinicopathological characteristics in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Circular RNAs (circRNAs) are a novel class of RNAs, with higher stability and tissue specificity, which may be of value as novel clinical markers. High­throughput RNA sequencing was used to profile the expression of circRNAs in 5 pairs of cancer and normal tissues, and reverse transcription­quantitative PCR (RT­qPCR) analysis was employed to verify the results of the RNA sequencing in 45 cases of PTC. The dysregulated circRNA expression and clinicopathological characteristics were assessed and the potential roles of circRNAs in the cellular miRNA and mRNA network were predicted using bioinformatics analysis. The results demonstrated that, compared with normal tissues, a total of 53 circRNAs were dysregulated in tumour tissues, and 8 circRNAs were validated at the mRNA level (P<0.001 and P<0.01). Among those, the expression of chr5:161330882­161336769­ (P=0.015), chr9:22046750­22097364+ (P=0.041) and chr8:18765448­18804898­ (P=0.036) were obviously associated with the BRAFV600E mutation, chr12:129699809­129700698­ was associated with capsular invasion (P=0.025) and chr5:38523418­38530666­ was associated with pT stage (P=0.037) and lymph node metastasis (P=0.002). Therefore, some dysregulated circRNAs were found to be associated with BRAFV600E mutation, capsular invasion, advanced pT stage and lymph node metastasis of PTC, indicating that circRNAs may be involved in tumourigenesis and cancer progression, and they may be putative biomarkers for the diagnosis and evaluation of progression of PTC.