Abstract 6604: Analytical development and validation of a personalized hybrid capture based assay for high sensitivity monitoring minimal residual disease (MRD)

Abstract Introduction: 5-30% of patients with early-stage cancer relapse, even though most patients had undergone radical surgery. There is increasing evidence that circulating tumor DNA (ctDNA) minimal residual disease (MRD) predicts relapse following treatment for solid tumors. However, liquid biopsy analyses are challenged by the very low concentrations of ctDNA in blood samples. Existing ctDNA methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. Here, we describe a tumor-informed personalized hybrid capture based method using next-generation sequencing (NGS) to highly sensitive and specific detect MRD and predict recurrence in plasma cell-free DNA. To validate the performance of the assay, we present a technical validation study on DNA from cancer cell line material, including lung cancer cell line, reference material, Onco gDNA reference standard and wild type gDNA reference standard. Method: The performance of the assay was assessed using samples from Onco gDNA reference standard and lung cancer cell line. To estimate number of monitoring single nucleotide variants (SNVs) at input volume of 20-80ng, Onco gDNA reference standard was diluted into 8 concentrations (10, 20, 30, 40, 50, 60, 150 and 750 ppm with a variant allele frequency [VAF] range of 0.001% to 0.075%).To estimate limit of detection (LoD) of the assay at input volume of 20ng and 40ng, lung cancer cell line was diluted into 5 concentrations (10, 20, 40, 80 and 160 ppm with a variant allele frequency [VAF] range of 0.001% to 0.016%).10 samples were performed for each concentration, as well as 10 negative reference samples. Personalized panel was performed using ultradeep (100,000X) NGS with customized 55 or 60 SNVs. Result: Validation results of Onco gDNA reference standard: using 40 variants, sensitivity of input volume of 20ng and 40ng are 95% and 98% at 40 ppm, with a specificity of 100%, respectively. Using 40 variants, sensitivity of input volume of 60ng are 95% at 20 ppm, with a specificity of 100%. Using 50 variants, sensitivity of input volume of 60ng are 70% at 10 ppm, with a specificity of 100%. Validation results of lung cancer cell line: using 40 variants, sensitivity of input volume of 20ng and 40ng are 98% and 100% at 50 ppm, with a specificity of 100%, respectively. Conclusion: The assay was validated by sequentially diluting of lung cancer cell line and Onco gDNA reference standard. With input volume of 20ng, sensitivity of the assay is 95% at 40ppm when monitoring 40 variants, while with input volume of 60ng the LoD reached as low as 10 ppm with sensitivity of 70% when monitoring 50 variants. The assay demonstrates high sensitivity, while maintaining specificity above 99%. Citation Format: Xiaoqiang Wang, Lei Ye, Chao Zhang, Wanglong Deng, Yongjie Wei, Jing Chen, Xuan Tang, Xiaoxuan Wang, Chao Song, Qing Xu, Yong Ren. Analytical development and validation of a personalized hybrid capture based assay for high sensitivity monitoring minimal residual disease (MRD) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6604.