(1089-B) Detection of cancer mutations from Formalin fixed Paraffin Embedded tumor tissues using high throughput nucleic acid extraction and digital PCR platform

Abstract:

Introduction: Formalin-fixed paraffin-embedded (FFPE) tissues are the most frequently archived clinical tissue samples and a vital resource for investigating tumor mutations in cancer research. Over the past decades, advancements in sequencing and PCR have enabled the identification of numerous frequent somatic mutations, involved in multiple cellular pathways across various cancer types. Despite significant advancements in cancer research, the demand for high throughput processing of molecular techniques using FFPE tissues remains unmet. In this study, we conducted a comprehensive evaluation of high throughput workflow for nucleic acid extraction and dPCR analysis to detect cancer mutation in FFPE samples.

Methods: FFPE samples from breast, colon, and lung tumor tissues underwent protease treatment in Autolys tubes. The resulting lysates were utilized for nucleic acid extraction using the KingFisher Presto system, integrated with the Hamilton Nimbus automated liquid handler. The DNA and RNA extracted in the high throughput Nimbus system were compared with extractions performed on a KingFisher Flex purification instrument. Concentration and yield of DNA and RNA were measured using Nanodrop and Qubit instruments, while DNA and RNA integrity were assessed using Agilent Tapestation and Agilent Bioanalyzer. TaqMan qPCR for housekeeping genes - GAPDH and β-Actin was performed using the QuantStudio 7 system for both DNA and RNA. For tumor mutation determination, next-generation sequencing (NGS) was performed on the Ion Torrent Genexus Sequencer utilizing the Oncomine Comprehensive assay. Additionally, digital PCR assays were performed using the Applied Biosystems™ QuantStudio™ Absolute Q™ Digital PCR System with custom assays.

Results: The DNA and RNA extracted using the automated Hamilton Nimbus and Kingfisher Flex systems exhibited similar yields and concentrations, ensuring reliable nucleic acid recovery. Additionally, the DNA and RNA integrity and molecular weight distribution demonstrated consistent profiles across both platforms. furthermore, the dPCR assay successfully detected tumor mutations, highlighting the accuracy and efficacy of the experimental approach.

Conclusion: High throughput nucleic acid isolation using Hamilton Nimbus and processing in dPCR platform facilitates a streamlined workflow and minimizes user manipulation.