Ultra-fast conductive media for RNA electrophoretic mobility shift assays.

The use of RNA electrophoretic mobility shift assays (REMSAs) for analysis of RNA-protein interactions have been limited to lengthy assay time and qualitative assessment. To vastly improve assay efficiency, feasibility and quality of data procured from REMSAs, we combine here some of the best-known labeling and electrophoretic techniques. Nucleic acid fragments are end-labeled with fluorescent tags, as opposed to the radioactive or biotin tags. The fluorescent probes may be detected directly from the electrophoresis gel, eliminating the need for cumbersome membrane transfer and immunoblotting. Modifying the REMSA protocol to include low-molarity, lithium borate conductive media and near-infrared-labeled probes allows for a reduction assay time, quantitative comparison between experimental conditions and crisp band resolution (i.e., optimized results)

A step towards personalizing next line therapy for resected pancreatic and related cancer patients: A single institution\u27s experience

Background: There is a lack of precision medicine in pancreatic ductal adenocarcinoma (PDA) and related cancers, and outcomes for patients with this diagnosis remain poor despite decades of research investigating this disease. Therefore, it is necessary to explore novel therapeutic options for these patients who may benefit from personalized therapies. Objective: Molecular profiling of hepatopancreaticobiliary malignancies at our institution, including but not limited to PDA, was initiated to assess the feasibility of incorporating molecular profiling results into patient oncological therapy planning. Methods: All eligible patients from Thomas Jefferson University (TJU) with hepatopancreaticobiliary tumors including PDA, who agreed to molecular testing profiling, were prospectively enrolled in a registry study from December 2014 to September 2017 and their tumor samples were tested to identify molecular markers that can be used to guide therapy options in the future. Next generation sequencing (NGS) and protein expression in tumor samples were tested at CLIA-certified laboratories. Prospective clinicopathologic data were extracted from medical records and compiled in a de-identified fashion. Results: Seventy eight (78) patients were enrolled in the study, which included 65/78 patients with PDA (local and metastatic) and out of that subset, 52/65 patients had surgically resected PDA. Therapy recommendations were generated based on molecular and clinicopathologic data for all enrolled patients. NGS uncovered actionable alterations in 25/52 surgically resected PDAs (48%) which could be used to guide therapy options in the future. High expression of three proteins, TS (p = 0.005), ERCC1 (p = 0.001), and PD-1 (p = 0.04), was associated with reduced recurrence-free survival (RFS), while TP53 mutations were correlated with longer RFS (p = 0.01). Conclusions: The goal of this study was to implement a stepwise strategy to identify and profile resected PDAs at our institution. Consistent with previous studies, approximately half of patients with resected PDA harbor actionable mutations with possible targeted therapeutic implications. Ongoing studies will determine the clinical value of identifying these mutations in patients with resected PDA

Genetic Drivers of Pancreatic Cancer Are Identical Between the Primary Tumor and a Secondary Lesion in a Long-Term (>5 Years) Survivor After a Whipple Procedure

Background: A new mass in the remnant pancreas of a patient with previously resected pancreatic ductal adenocarcinoma (PDA) typically represents either a recurrence of the initial primary tumor or a second primary tumor. Recent advances in next-generation sequencing (NGS) strategies allow us to compare the genetic makeup of primary and secondary lesions. Case presentation: A 50-year-old Caucasian female presented for a surgical evaluation of a new biopsy-proven PDA at the junction of the body and tail of the pancreas. Six years prior, in 2011, the patient was found to have a T3N0M0 PDA of the pancreatic head, which was surgically resected with a classic Whipple procedure and concurrent hemicolectomy. Pathology showed pancreatic intraepithelial neoplasia grade 2 and PDA with negative surgical margins, positive perineural spread, and negative lymphovascular spread, and the patient received adjuvant chemotherapy and local radiation. In 2017, she was diagnosed with a new PDA lesion in the remaining pancreatic body far from the previous anastomosis site and was taken to surgery for a completion pancreatectomy and revision of the gastrojejunostomy. NGS was performed on both specimens. Both lesions shared identical mutations in KRAS, TP53, and CDKN2A genes. Amplifications of MYC and mutant KRAS were identified in the 2017 tumor and an ACVR1B mutation was identified in the 2011 tumor, but was not found in the 2017 tumor. Conclusions: This case demonstrates the ability to evaluate similarities between key genetic drivers from a resected primary tumor and a PDA lesion that presented in the same patient 6 years later. Histological analysis and NGS can be used to understand potential differences and similarities between lesions and may be useful in future studies as predictive markers or to provide insight into resistance mechanisms (e.g., MYC amplification)