P24.07 An Ultra-Sensitive Protocol for ctDNA Mutation Detection With Application in Lung Cancers

Abstract

To establish an ultra-high sensitivity protocol for ctDNA mutation detection in blood samples, i.e., PEAC followed by NGS (PEAC+NGS), to achieve the desirable results of targeted panel sequencing and the sensitivity of digital PCR. The PEAC+NGS protocol was proposed to achieve high concordance between the matched blood and tissue samples from patients with early-stage lung cancer. The cfDNA extracted from blood samples was enriched by PEAC technique, and the obtained products were amplified by a second round PCR process to add the adapter sequences used for NGS sequencing. The PEAC+NGS protocol was applied to the blood samples of 46 lung cancer patients, including 36 patients in stage I and II, and 10 patients in stage III and IV. The testing results were used to evaluate the performance of PEAC+NGS in blood ctDNA testing and its consistency with the testing results of matched tissues. The PEAC+NGS protocol can simultaneously detect over 40 variant forms of five genes, i.e., EGFR, KRAS, NRAS, BRAF, and PIK3CA, which are closely associated with lung cancer. With a sequencing depth of over 10,000×, PEAC+NGS can achieve the ability to detect ctDNA variant at 0.01% allele frequencies, and 100% detection specificity. In 36 blood samples from patients with stage I and II lung cancer, 9 cases were detected as positive for mutations and 27 cases were negative by PEAC+NGS. The 9 blood samples with positive ctDNA variants also had positive results in matched tissue samples, while 11 of the 27 negative cases had positive testing results in matched tissues. The concordance between tissue and blood samples of stage I and II patients was 55.6%. Accordingly, for the 10 patients in stage III and IV, two blood samples detected as negative by PEAC+NGS also had negative results in the tissue; while among the remaining 8 patients that were tested positive in tissues, only one was failed to be detected by PEAC+NGS on the blood sample. The concordance between the blood and tissue samples was thus 90% for stage III and IV patients. We established an ultra-sensitive protocol for ctDNA mutation detection in blood samples, PEAC+NGS, which achieved 55.6% concordance in matched blood and tissue samples from patients with stage I and II lung cancer, and 90% concordance in stage III and IV lung cancer patients. These results demonstrate the great value of PEAC+NGS in clinical ctDNA testing.