Clinical significance of MUC-4 and associated proteins in Pancreatic and Periampullary Cancers.

OBJECTIVE: This study primarily aimed to assess the expression of MUC-4 in patients with Pancreatic Ductal Adenocarcinoma (PDAC) as compared with controls and assess its clinical relevance. METHODS: Serum MUC-4 levels and MUC-4 gene expression in snap-frozen tissue were analyzed through Surface Plasmon Resonance (SPR) and quantitative PCR respectively. Tumor tissues and control tissues were analyzed for MUC-4 and other mucins through IHC. RESULT: MUC-4 expression in tumor tissue was found to be significantly elevated in PDAC patients as compared to chronic pancreatitis (CP) tissues and normal pancreatic tissues. Periampullary carcinoma and cholangiocarcinoma tissue also showed increased expression of MUC-4 and other mucins. CONCLUSION: Differential expression of MUC-4 in pancreatic tumor tissues can help to differentiate PDAC from benign conditions.

MicroRNA Signatures for Pancreatic Cancer and Chronic Pancreatitis: Expression Profiling by NGS.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease due to the lack of early detection. Because chronic pancreatitis (CP) patients are a high-risk group for pancreatic cancer, this study aimed to assess the differential miRNA profile in pancreatic tissue of patients with CP and pancreatic cancer. METHODS: MiRNAs were isolated from formalin-fixed paraffin-embedded pancreatic tissue of 22 PDAC patients, 18 CP patients, and 10 normal pancreatic tissues from autopsy (C) cases and processed for next-generation sequencing. Known and novel miRNAs were identified and analyzed for differential miRNA expression, target prediction, and pathway enrichment between groups. RESULTS: Among the miRNAs identified, 166 known and 17 novel miRNAs were found exclusively in PDAC tissues, while 106 known and 10 novel miRNAs were found specifically in CP tissues. The pathways targeted by PDAC-specific miRNAs and differentially expressed miRNAs between PDAC versus CP tissues and PDAC versus control tissues were the proteoglycans pathway, Hippo signaling pathway, adherens junction, and transforming growth factor-ß signaling pathway. CONCLUSIONS: This study resulted in a set of exclusive and differentially expressed miRNAs in PDAC and CP can be assessed for their diagnostic value. In addition, studying the role of miRNA-target gene interactions in carcinogenesis may open new therapeutic avenues.

Elucidating the Role of miRNA-326 Modulating Hedgehog Signaling in Pancreatic Carcinoma.

BACKGROUND AND AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies worldwide characterized by poor prognosis. MicroRNAs (miRNAs) function as the key regulators in carcinogenesis and may act as noninvasive biomarkers in various malignancies including PDAC. The present study aimed to elucidate the role of miR-326, a known modulator of hedgehog (Hh) pathway in PDAC. MATERIALS AND METHODS: miR-326 circulating levels were assessed in 105 PDAC patients, 31 with chronic pancreatitis (CP) and 36 healthy controls by quantitative Polymerase chain reaction. The expression of miR-326 and smoothened (SMO) was checked in surgical PDAC tissue. SMO protein expression was analyzed by immunohistochemistry in different groups. Finally, the role of miR-326 as a modulator of Hh pathway was assessed in vitro. RESULTS: Our results demonstrate that miR-326 is downregulated in both blood and tissue of PDAC patients as compared with controls. In contrast, the target gene/protein expression of SMO is upregulated in PDAC. Moreover, the tumor stromal expression of SMO was found to be clinically associated with lymph-node metastasis and vascular encasement in PDAC. Overexpression of miR-326 in Panc1 cell line was found to induce downregulation of SMO suggesting the tumor suppressor role of miR-326 in PDAC. CONCLUSIONS: Taken together, miR-326 acts as a tumor suppressor in PDAC by modulating Hh pathway. It may be a promising target for the development of efficient drug therapies for the treatment of PDAC.

Clinical significance of Notch pathway-associated microRNA-107 in pancreatic ductal adenocarcinoma.

Background & aim: MicroRNAs associated with the Notch pathway play a critical role in the progression of pancreatic carcinoma. Our aim was to study the clinical significance of miR-107 and NOTCH2 in pancreatic ductal adenocarcinoma (PDAC). Methods: The circulating miR-107 levels in PDAC and controls were determined by qPCR. NOTCH2 protein (target) expression in tissue of PDAC, periampullary carcinoma, chronic pancreatitis and normal pancreatic tissue was assessed by immunohistochemistry. Results: The circulating miR-107 levels were found to be significantly reduced in PDAC as compared with controls. Additionally, NOTCH2 protein expression was higher in PDAC tissue as compared with controls and was clinically associated with metastasis. Conclusion: Our findings demonstrate the utility of circulating miR-107 as a potential differentiating marker in PDAC.

Analysis and forecasting of road traffic mortalities in Azad Jammu and Kashmir.

OBJECTIVE: To analyse road traffic accident mortalities in a geographical region. Method: The retrospective study was conducted in Azad Jammu and Kashmir based on secondary data from 2004 to 2017 collected from the police department. Duncan's multiple range test was used to assess the trends in road traffic accident fatalities with respect to districts and divisions. Different goodness-of-fit criteria were used to compare the performance of different regression models to analyse road traffic accident mortalities with respect to vehicle ownership. The parsimonious time series model was used to forecast the future trends of road traffic accident mortalities. R 3.6.0 software was used for data analysis. RESULTS: There were 5263 major road traffic accidents during the period studied, causing 2317 deaths and 12963 injuries. The number of mortalities in Mirpur division was 923(39.8%), in Muzaffarabad 794(34.3%), and inss Poonch 600(25.9%). The rates of road traffic accident mortalities per 100,000 population increased up to year 2010 and dropped slowly afterwards (Figure 1C). Some disparities were noted among different districts and divisions with respect to road traffic accident mortalities. Based on different goodness-of-fit criteria, the Smeed's model was found to be the most efficient model to analyse the trends of road traffic accident mortalities with respect to vehicle ownership (Table 1). The forecast for road traffic accident mortalities exhibited some fluctuations in the start and a uniform trend afterwards (Figure 6). CONCLUSIONS: Disparities in road traffic accident fatalities across different districts and divisions of Azad Jammu and Kashmir were observed. Though the rate of road traffic accident mortality was seen to be decreasing since 2010, the situation is for behind compared to the global Sustainable Development Goals.

Genetic polymorphisms in phase II metabolizing enzymes in alcoholic and idiopathic chronic pancreatitis: Indian scenario.

AIM: To study polymorphisms in glutathione-S-transferases (GST-T1, GST-M1, GST-P1) and uridine-5'-diphosphate-glucuronosyl-transferases (UGT1A7) genes and the risk of developing chronic pancreatitis (CP) associated with these polymorphisms. METHODS: This study included 49 alcoholic and 51 idiopathic chronic pancreatitis patients, 50 alcohol addicts and 50 healthy controls. Polymorphism(s) in GST-T1 and GST-M1 genes were assessed by multiplex polymerase chain reaction (PCR), while PCR-radiofrequency lesioning (RFLP) was employed to assess the same in GST-P1 and UGT1A7 genes. The differences in polymorphism frequency between groups and the risk of developing pancreatitis were assessed by the odds ratio. RESULTS: Strong association of the null genotype of GST-T1 with CP susceptibility was observed. Alcoholics with the Val allele of GST-P1 have higher chances of having pancreatitis. Idiopathic pancreatitis patients with higher age at the onset of pain were found to have the null genotype of GST-M1. CONCLUSION: Alcoholics with the null genotype of the GST-T1 gene and the Valine allele of the GST-P1 gene are at a higher risk of developing CP. Thus, genotyping of these genes may serve as an important screening tool for the identification of high-risk groups among alcoholics.

Association of inflammatory markers with the disease & mutation status in pancreatic cancer.

Background & objectives: Inflammation has been studied to be an important contributory factor to carcinogenesis through pro-inflammatory markers such as interleukin (IL)-6 and C-reactive protein (CRP). Furthermore, K-ras mutation is an important genetic alteration in the pathogenesis of pancreatic cancer. This study aimed to compare these inflammatory markers in pancreatic ductal adenocarcinoma (PDAC) with the diseased and healthy controls (HCs) and to check for any association between IL-6 and CRP serum levels with the disease status, survival and K-ras mutation status of PDAC patients. Methods: The study included 135 PDAC, 25 chronic pancreatitis (CP) patients and 25 HCs. The serum levels of IL-6 and CRP were detected by enzyme-linked immunosorbent assay and K-ras mutations were detected by polymerase chain reaction-restriction fragment length polymorphism technique. Results: The serum levels of both these markers were elevated in PDAC cases than that in HCs. High IL-6 levels and higher CRP levels were found to be associated with locally advanced disease, lymphatic invasion, metastasis and advanced stage of the PDAC. In patients with unresectable PDAC, higher IL-6 levels were found to be associated with the presence of K-ras mutations. Interpretation & conclusions: Higher IL-6 and CRP levels in patients with advanced PDAC suggest an important role of these inflammatory markers in tumour progression. Furthermore, the association of mutations in the K-ras gene with serum IL-6 indicates cross-talks that may contribute to the progression of the PDAC.

Panel of serum miRNAs as potential non-invasive biomarkers for pancreatic ductal adenocarcinoma.

Early-stage diagnosis of pancreatic ductal adenocarcinoma (PDAC) is difficult due to non-specific symptoms. Circulating miRNAs in body fluids have been emerging as potential non-invasive biomarkers for diagnosis of many cancers. Thus, this study aimed to assess a panel of miRNAs for their ability to differentiate PDAC from chronic pancreatitis (CP), a benign inflammatory condition of the pancreas. Next-generation sequencing was performed to identify miRNAs present in 60 FFPE tissue samples (27 PDAC, 23 CP and 10 normal pancreatic tissues). Four up-regulated miRNAs (miR-215-5p, miR-122-5p, miR-192-5p, and miR-181a-2-3p) and four down-regulated miRNAs (miR-30b-5p, miR-216b-5p, miR-320b, and miR-214-5p) in PDAC compared to CP were selected based on next-generation sequencing results. The levels of these 8 differentially expressed miRNAs were measured by qRT-PCR in 125 serum samples (50 PDAC, 50 CP, and 25 healthy controls (HC)). The results showed significant upregulation of miR-215-5p, miR-122-5p, and miR-192-5p in PDAC serum samples. In contrast, levels of miR-30b-5p and miR-320b were significantly lower in PDAC as compared to CP and HC. ROC analysis showed that these 5 miRNAs can distinguish PDAC from both CP and HC. Hence, this panel can serve as a non-invasive biomarker for the early detection of PDAC.

Clinical significance of promoter methylation status of tumor suppressor genes in circulating DNA of pancreatic cancer patients.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive cancer. There are various sub-cellular events (both genetic and epigenetic) that get dysregulated leading to tumorigenesis. Methylation in promoters of tumor suppressor genes is one of these epigenetic phenomena contributing to the pathogenesis of cancer. Genes analyzed for promoter methylation status in this study namely SPARC (Secreted Protein Acidic and Rich in Cysteine, UCHL1 (ubiquitin carboxy-terminal hydrolase L1), NPTX2 (neuronal pentraxin 2), PENK (proenkephalin) had been studied in pancreatic cancer, but there is a need to check methylation in these genes as circulatory non-invasive markers. This study analyzed the absolute quantification of methylation levels of SPARC, UCHL1, PENK, and NPTX2 genes promoters in PDAC patients as well as in chronic pancreatitis (CP) patients and healthy subjects (HC) and evaluated its clinical significance in PDAC. MATERIALS AND METHODS: The study included 65 PDAC patients, 25 CP patients, and 25 healthy controls. DNA was extracted from their plasma samples and subsequently given bisulfite treatment. Absolute quantization of methylated and unmethylated copies of gene promoters of all the four genes was performed using real-time PCR (SYBR green) by the standard curve method. Methylation levels were expressed as methylation index (MI) for each gene in each patient. MI was calculated from absolute copy numbers as follows: MI-methylated copy number/methylated copy number + unmethylated copy number). These indices were used to compare gene methylation levels within different groups and to correlate with clinicopathological features and survival of pancreatic cancer patients. An appropriate statistical analysis was applied. RESULTS: Methylation indices for all the four genes in PDAC cases were found to be significantly higher as compared to that in healthy individuals. SPARC MI values were found to differentiate early-stage PDAC patients from CP patients. PDAC patients with the metastasized disease and stage IV disease were found to have high MI for the SPARC gene as well as for the NPTX2 gene, while a higher UCHL1 methylation index was found to correlate with an advanced stage of the disease. Higher MI values for SPARC and NPTX2 genes were found to associate with poor survival in patients with PDAC. CONCLUSION: Methylation load in the form of MI for each of the four genes assessed in plasma may emerge as a non-invasive biomarker to differentiate pancreatic cancer from healthy individuals. But only SPARC and NPTX2 hypermethylation were able to distinguish pancreatic cancer from chronic pancreatitis. Association of aberrant methylation in SPARC and NPTX2 gene with metastasis and poor survival of patients suggest the role of methylation in these genes as prognostic markers.

Progression of Chronic Pancreatitis to Pancreatic Cancer: Is There a Role of Gene Mutations as a Screening Tool?

OBJECTIVES: Early detection of pancreatic ductal adenocarcinoma still remains a challenge. Patients with chronic pancreatitis (CP) have a markedly increased risk of pancreatic cancer. Mutations in oncogenes and/or tumor suppressor genes play a role in development of pancreatic ductal adenocarcinoma. This study assessed mutations in KRAS and p53 gene in blood as a screening tool for malignant transformation in CP patients. METHODS: This was a cohort, single-center study including 294 CP patients. DNA was isolated from plasma of CP patients, and KRAS mutations were identified using polymerase chain reaction-restriction fragment length polymorphism. Patients with positive KRAS mutation were screened for malignancy using positron emission tomography or endoscopic ultrasound. Mutations in p53 gene were analyzed by sequencing. Tissue samples from CP and pancreatic cancer patients were also tested for mutations in KRAS and p53 genes. RESULTS: The plasma samples of 64 CP patients were positive for KRAS mutation, and 4 had mutation in p53 gene also. No patient positive for KRAS mutation and/or p53 mutation was found to have malignant transformation. CONCLUSION: Detection of KRAS or p53 mutation in plasma is not an effective screening tool for pancreatic cancer because accumulation of multiple mutations is required for malignant transformation in the pancreas.