A Combined DNA/RNA-based Next-Generation Sequencing Platform to Improve the Classification of Pancreatic Cysts and Early Detection of Pancreatic Cancer Arising From Pancreatic Cysts.

OBJECTIVE: We report the development and validation of a combined DNA/RNA next-generation sequencing (NGS) platform to improve the evaluation of pancreatic cysts. BACKGROUND AND AIMS: Despite a multidisciplinary approach, pancreatic cyst classification, such as a cystic precursor neoplasm, and the detection of high-grade dysplasia and early adenocarcinoma (advanced neoplasia) can be challenging. NGS of preoperative pancreatic cyst fluid improves the clinical evaluation of pancreatic cysts, but the recent identification of novel genomic alterations necessitates the creation of a comprehensive panel and the development of a genomic classifier to integrate the complex molecular results. METHODS: An updated and unique 74-gene DNA/RNA-targeted NGS panel (PancreaSeq Genomic Classifier) was created to evaluate 5 classes of genomic alterations to include gene mutations (e.g., KRAS, GNAS, etc.), gene fusions and gene expression. Further, CEA mRNA ( CEACAM5 ) was integrated into the assay using RT-qPCR. Separate multi-institutional cohorts for training (n=108) and validation (n=77) were tested, and diagnostic performance was compared to clinical, imaging, cytopathologic, and guideline data. RESULTS: Upon creation of a genomic classifier system, PancreaSeq GC yielded a 95% sensitivity and 100% specificity for a cystic precursor neoplasm, and the sensitivity and specificity for advanced neoplasia were 82% and 100%, respectively. Associated symptoms, cyst size, duct dilatation, a mural nodule, increasing cyst size, and malignant cytopathology had lower sensitivities (41-59%) and lower specificities (56-96%) for advanced neoplasia. This test also increased the sensitivity of current pancreatic cyst guidelines (IAP/Fukuoka and AGA) by >10% and maintained their inherent specificity. CONCLUSIONS: PancreaSeq GC was not only accurate in predicting pancreatic cyst type and advanced neoplasia but also improved the sensitivity of current pancreatic cyst guidelines.

A primer-free method that selects high-affinity single-stranded DNA aptamers using thermostable RNA ligase.

This article describes a method for selecting single-stranded DNA (ssDNA) molecules that bind with high-affinity aptamers to specific target proteins. This SELEX (systematic evolution of ligands by exponential enrichment) method is similar to other "primer-free" approaches where the random sequence ssDNA starting pool has no fixed sequences at the 5' and 3' termini. Therefore, there are no predetermined sequences that could bias selection. Like other SELEX methods, repeated cycles (typically 5-15) of selection and then amplification and reselection are used. The method differs from other primer-free approaches in that the key step for regenerating new material for subsequent rounds is ligation of the selected ssDNA to a defined sequence oligonucleotide using thermostable RNA ligase. Under specific conditions, this ligase ligated 30-nt random sequence ssDNA (5'-N(30)-3') to a specified 20-nt ssDNA with approximately 50% efficiency. Efficiency was improved to approximately 90% by the addition of a single T residue to the 3' end (5'-N(29)T-3'). High efficiency in this step is critical, especially early in the procedure because any selected material that is not ligated is lost. In this study, human immunodeficiency virus reverse transcriptase was used as the target protein, but the method could be applied to essentially any protein.

DNA aptamers to human immunodeficiency virus reverse transcriptase selected by a primer-free SELEX method: characterization and comparison with other aptamers.

A 30-nucleotide DNA aptamer (5'-AGGAAGGCTTTAGGTCTGAGATCTCGGAAT-3', denoted PF1) selected for high affinity to human immunodeficiency virus reverse transcriptase (HIV RT) using a primer-free SELEX (systematic evolution of ligands by exponential enrichment) method was characterized to determine features promoting tight binding. PF1's equilibrium dissociation constant for RT was ∼80 nM, over 10-fold lower than a random 30-mer. Changing the 2 terminal diguanosine repeats (underlined above) to diadenosine or dithymidine modestly decreased binding. Any changes to the 2 central diguanosines dramatically decreased binding. Binding was highly sensitive to length, with any truncations that deleted part of the 4 diguanosine motifs resulting in a 6-fold or more decrease in affinity. Even a construct with all the diguanosine motifs but lacking the 5' terminal A and 3 nucleotides at the 3' end showed ∼3-fold binding decrease. Changes to the nucleotides between the diguanosines, even those that did not alter PF1's low secondary structure (free energy of folding ΔG=-0.61 kcal/mol), dramatically decreased binding, suggesting sequence specificity. Despite the diguanosine motifs, circular dichroism (CD) spectra indicated that PF1 did not form a G-quartet. PF1 inhibited HIV RT synthesis with a half-maximal inhibitory value (IC(50)) of ∼60 nM. Larger, more structured RT DNA aptamers based on the HIV polypurine tract and those that formed G-quartets (denoted S4 and R1T) were more potent inhibitors, with IC(50) values of ∼4 and ∼1 nM, respectively. An RNA pseudoknot aptamer (denoted 1.1) showed an IC(50) near 4 nM. Competition binding assays with PF1 and several previously characterized RT aptamers indicated that they all bound at or near the primer-template pocket. These other more structured and typically larger aptamers bound more tightly than PF1 to RT based on filter binding assays. Results indicate that PF1 represents a new class of RT aptamers that are relatively small and have very low secondary structure, attributes that could be advantageous for further development as HIV inhibitors.