Abstract 5043: Achieving sensitivity of 0.1% variant allele frequency (VAF) in plasma and urine with the nRichDX revolution sample prep system evaluated on the the MassARRAY system
Authors: Jafari Nafiseh , Saidian Mayer , Saenz Jason , Lee Lauren , Dunnigan Andrew , Hernandez Carlos , Mendoza Jessica , Hernandez Francisco , Irwin Darryl 
Published: March 2024

Introduction: Achieving high sensitivity in detecting cancer variants is essential for accurate diagnosis and monitoring of diseases. In this study, we explore the effectiveness of the nRichDX Revolution Sample Prep System in conjunction with the MassARRAY System for enhancing sensitivity and achieving 0.1% VAF in plasma and urine samples. The ability to detect such low-frequency variants is significant in clinical applications.

Methods: Whole blood from healthy donors was collected in K2EDTA tubes. Plasma aliquots were extracted from 5mL (6) and 20mL (6) inputs using the Revolution Max20 cfDNA Extraction Kit. Replicate samples were extracted using the QIAamp Circulating Nucleic Acid kit from QIAGEN. Urine from healthy donors was extracted from 10mL (3) and 20mL (3) inputs using the Revolution Max20 cfDNA Extraction kit. (12) Replicate samples were extracted using a MagMAX cfDNA Isolation kit from Thermo Fisher Scientific. All the samples were spiked with a cfDNA standard containing a KRAS p.G12V mutation at 0.1% VAF. The quantity and quality of extracted cfDNA were determined using the LiquidIQ Pro Panel on the MassARRAY System, which measures amplifiable cfDNA copies. Recovery and frequency of the spiked ctDNA standard were performed with the UltraSEEK LungPanel v2.0, capable of detecting variants as low as 0.1% VAF.

Results: The spiked KRAS mutation was recovered consistently by all nRichDX 5mL plasma samples with an accepted mutation frequency and high mutation significance z-score (z-score&gt;10). However, 3/6 of the QIAGEN 5mL plasma samples did not detect the mutation. The spiked KRAS mutation was recovered consistently by all nRichDX 10 and 20mL urine samples with an accepted mutation frequency and high mutation significance z-score. However, all the MagMAX 10 and 20mL urine samples did not pass the QC (5) or detect the mutation (1). The amplifiable copies of cfDNA recovered from nRichDX 20mL plasma samples are significantly higher than Qiagen (p &lt;0.0001). The amplifiable copies of cfDNA recovered from nRichDX 10 and 20mL urine samples are significantly higher compared to MagMAX (p &lt;0.0063.)

Conclusion: The evaluation of the nRichDX Revolution Sample Prep System on the MassARRAY System has demonstrated its effectiveness in achieving 0.1% VAF detection in plasma and urine samples. The nRichDX System extracts cfDNA and ctDNA with consistently high yields. Two sample T-Test analysis shows that the nRichDX System performed significantly better than the QIAGEN and Thermo Fisher Scientific extraction kits. The results demonstrate the capability of the nRichDX system to recover more amplifiable copies with consistent cfDNA quality and ctDNA frequency with high z-scores. This advancement in sensitivity enhances our ability to detect rare cancer variants, leading to the potential for better patient outcomes in precision medicine.

Citation Format: Nafiseh Jafari, Mayer Saidian, Jason Saenz, Lauren Lee, Andrew Dunnigan, Carlos Hernandez, Jessica Mendoza, Francisco Hernandez, Darryl Irwin. Achieving sensitivity of 0.1% variant allele frequency (VAF) in plasma and urine with the nRichDX revolution sample prep system evaluated on the the MassARRAY system [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(6_Suppl):Abstract nr 5043.