Abstract 1975: Development and validation of detection of ESR1 mutations in circulating tumor DNA using PNA-LNA mediated PCR clamping

Background: The occurrence of ESR1 mutations is rare in estrogen receptor (ER) positive primary breast cancer patients; however these mutations are frequently reported in ER positive recurrent breast cancer patients pretreated with endocrine therapy. ESR1 mutation is an essential mechanism of resistance to endocrine therapy and predictive biomarker as well as a potential therapeutic target. The measurement of ESR1 mutations in circulating tumor DNA (ctDNA) has been studied as a non-invasive method to quickly assess endocrine therapy resistant metastatic breast cancer patients. As a simple, inexpensive, low-invasive assay, we developed a novel detection system of ESR1 mutations using peptide nucleic acid-locked nucleic acid mediated PCR clamping (PNA-LNA PCR clamping) assay. Methods: We developed a novel detection system to screen mutations at hot spot codons (E380, L536, Y537 and D538) in ESR1. Major mutations, E380Q, Y537S and D538G, were detected using PNA-LNA PCR clamping assay, and the other mutations were detected by directly sequencing amplified product of the clamp PCR. To evaluate the developed system, we used available plasma samples from ER positive breast cancer patients diagnosed at national cancer center Japan. The patient samples were screened for 9 mutations at hotspots (E380Q, L536H, L536Q, L536P, L536R, Y537S, Y537N, Y537C, and D538G) in ESR1 by an NGS panel for plasma (ctDNA), Oncomine Pan-Cancer Cell-Free Assay, and then the results were compared with the data acquired by the developed system. Results: Cell free DNA was obtained from 68 patients with ER positive breast cancer. ESR1 mutations at hotspots were found in 8.8% (6/68) of the samples by ctDNA NGS. The detected mutations were as follows: 2 samples at E380Q, 1 L536H, 3 at Y537S, 2 at Y537N, 1 at Y537C, and 5 at D538G. The average number of mutations per sample was 2.33. The concordance rate between the developed system and ctDNA NGS was calculated using results from these 6 positive samples, together with 4 negative samples, at the 9 mutations totaling 90 sites. Among the 14 hotspot mutations detected by the NGS, 12 were also detected by the developed system, giving the sensitivity of 85.7%. At 76 negative sites judged by the NGS, 74 were also judged as negative by the developed system, giving specificity of 97.4%. Combining these numbers together, the calculated concordance rate was 95.6% (86/90). Conclusion: This system using PNA-LNA mediated PCR clamping assay is a simple, ine