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Abstract The detection of mutations in the KRAS gene highly associated with cancer is one of the most widely used Sanger sequencing capillary electrophoresis (CE) applications in molecular diagnostics. In addition to their traditional role in cancer diagnostic, KRAS mutations have recently gained additional relevance as pharmacogenomics (PGx) markers for a significant number of new oncology therapeutics. KRAS mutation detection assays are used as companion diagnostics (CDx) for these cancer treatments. Analysis by Sanger Sequencing of KRAS mutations is cost effective and lends itself for use in resource limited healthcare settings. While there are currently no commercial kits for targeted sequencing of the KRAS mutation hotspots available, the online Applied Biosystems Primer Designer™ Tool (thermofisher. com) allows standardization of the PCR primer design between labs and repeat orders of the KRAS assays by using these commercially available assay ID numbers. Here we demonstrate the use of Primer Designer KRAS mutation detections assays for mutation in codon 12, 13 and 61 totaling to 8 allele variants. The data was generated on the SeqStudio Flex CE instrument using reference standard DNA as well as DNA extracted from FFPE solid tumor samples. We show accuracy of variant call (OPA 100% (64/64) ) and concordance (OPA 98. 3% (59/60) ) in a side-by-side comparison of the SeqStudio Flex with the legacy Model 3500 Genetic Analyzer instrument using a set of commercially available (Horizon Discovery) KRAS reference DNA samples. Primary solid tumor samples are processed to Fresh Frozen Paraffin Embedded samples (FFPE) for further disease marker evaluation. These samples are stable for long term at ambient temperatures and provide the opportunity to evaluate prospective biomarkers at large scale to understand their potential utility. Discovery Life Sciences has generated a large-scale biorepository of human biospecimen, including FFPE, plasma/sera, buffy coat, and PBMCs. Archival FFPE samples from oncology indications associated with KRAS mutations, such as non-small cell lung cancer and colorectal cancer, were analyzed and identified samples with KRAS mutations, demonstrating the potential diagnostic utility of these assays. Finally, cell-free (cf) DNA from double spun plasma from relevant indications was also successfully evaluated for KRAS mutations, highlighting the potential for these assays in liquid biopsy studies. The study demonstrates the robustness of the Primer Designer KRAS mutation detection sequencing assays using reference standard DNA as well as DNA derived from FFPE DNA and cfDNA from patient derived tumor samples. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Achim Karger, Ami Patel, Kaylee Tseng, Camila Ornelas, Mary Napier, Rod Beck, Kerri Collwell, Shawn Fahl, Carole Bornarth. KRAS mutation detection by Sanger sequencing using the SeqStudio Flex capillary electrophoresis instrument abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 6423.
Karger et al. (Fri,) studied this question.