Abstract 5680: Clinical significance of low frequency EGFR and KRAS mutations of cell free DNA using Ion AmpliSeq Cancer Hotspot Panel in lung cancer patients
Authors: Sung Jae Sook , Lee Jong Won , Kim Boyeon , Lee Saet Byeol , Park Chang Won , Kim Hae Mi , Kwon Nak-Jung , Jang Won Jin , Choi Yoon Ji , Choi Jung Yoon , Kang Eun Joo , Park Kyung Hwa , Lee Sung Yong , Kim Yeul Hong 
Published: July 2017

Cell free DNA (cfDNA) present in the blood stream shows great potential as a useful cancer marker for molecular diagnosis and cancer progression monitoring. Especially, analyzing the cfDNA with Next Generation Sequencing (NGS) technology allows high through put examination of various genes concurrently at a low cost. However, there are still debates regarding clinically meaningful variant frequency to identify mutations in cfDNA, especially with ultra-deep sequencing. In this study, we examined the clinical utility of Ion AmpliSeq Cancer Hotspot Panel v2 (ICP; Ion Torrent) with Proton platforms. ICP, covering 2800 COSMIC mutations from 50 cancer genes was used to analyze cfDNA of 125 serum samples from lung cancer patients. The percentage of on target was 92% with mean depth of 22,868x. We identified aberrations of TP53 (72%), EGFR (43%), PTEN (26%), PIK3CA (26%), BRAF (16%), KRAS (14%), KIT (10%) and RET (10%) with the cut-off criteria of variant frequency &gt;0.1% and p&lt;0.01. To validate the results, we analyzed EGFR gene status by direct sequencing in available 100 FFPE tumor tissues (tDNA). Out of 17 patients with EGFR mutations in tDNA, 9 patients showed very low frequency (&lt;0.05%) of same EGFR mutation in cfDNA. To validate the results of ICP, droplet digital PCR (ddPCR) was carried out with same cfDNA. From those 9 patients, EGFR mutations in cfDNA were detected in five patients (minimum frequency 0.01%) by ddPCR. From the patients with wild type EGFR in tDNA, EGFR exon 19 deletion or exon 21 point mutation were detected by ICP in 19 patients using cfDNA. Again, ddPCR was carried out with same cfDNA to confirm the result. EGFR mutations were confirmed in nine patients (47.4%) by cfDNA ddPCR and among the 6 patients treated with EGFR TKI, 4 patients showed response or stabilization of disease. Also, we identified 18 patients with KRAS mutations in ICP results of 125 cfDNA. The result of ddPCR was matched in 80% of patients. Interestingly, 2 patients had multiple KRAS mutations in cfDNA with ICP as well as ddPCR. In our study, we demonstrated that ICP with Proton system is a useful assay to identify somatic mutations using cfDNA in lung cancer patients. Also, we suggest that even EGFR mutation of very low frequency (&lt;0.05%) might have clinical significance in NGS analysis using cfDNA. Serial blood sample obtained during treatment in these patients will be analyzed by ICP and ddPCR. [This research was supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C0066)]

Citation Format: Jae Sook Sung, Jong Won Lee, Boyeon Kim, Saet Byeol Lee, Chang Won Park, Hae Mi Kim, Nak-Jung Kwon, Won Jin Jang, Yoon Ji Choi, Jung Yoon Choi, Eun Joo Kang, Kyung Hwa Park, Sung Yong Lee, Yeul Hong Kim. Clinical significance of low frequency EGFR and KRAS mutations of cell free DNA using Ion AmpliSeq Cancer Hotspot Panel in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5680. doi:10.1158/1538-7445.AM2017-5680