Highly sensitive genotyping assays can detect mutations in cell-free DNA (cfDNA) from cancer patients, reflecting the biology of each patient’s cancer. Because circulating tumor DNA comprises a small, variable fraction of DNA circulating in the blood, sensitive parallel multiplexing tests are required to determine mutation profiles. We prospectively examined the clinical utility of ultra-deep sequencing analysis of cfDNA from 126 non-small cell lung cancer (NSCLC) patients using the Ion AmpliSeq Cancer Hotspot Panel v2 (ICP) and validated these findings with droplet digital polymerase chain reaction (ddPCR). ICP results were compared with tumor tissue genotyping (TTG) results and clinical outcomes. A total of 853 variants were detected, with a median of four variants per patient. Overall concordance of ICP and TTG analyses was 90% for EGFR exon 19 deletion and 88% for the L858R mutation. Of 34 patients with a well-defined EGFR activating mutation defined based on the results of ICP and TTG, 31 (81.6%) showed long-term disease control with EGFR TKI treatment. Of 56 patients treated with an EGFR tyrosine kinase inhibitor (TKI), the presence of the de novo T790M mutation was confirmed in 28 (50%). Presence of this de novo mutation did not have a negative effect on EGFR TKI treatment. Ultra-deep sequencing analysis of cfDNA using ICP combined with confirmatory ddPCR was effective at defining driver genetic changes in NSCLC patients. Comprehensive analysis of tumor DNA and cfDNA can increase the specificity of molecular diagnosis, which could translate into tailored treatment.

Mutations in the EGFR gene downstream signaling pathways may cause receptor-independent pathway activation, making tumors unresponsive to EGFR inhibitors. However, the clinical significance of RAS, PIK3CA or PTEN mutations in NSCLC is unclear. In this study, patients who were initially diagnosed with NSCLC or experienced recurrence after surgical resection were enrolled, and blood samples was collected. Ultra-deep sequencing analysis of cfDNA using Ion AmpliSeq Cancer Hotspot Panel v2 with Proton platforms was conducted. RAS/PIK3CA/PTEN mutations were frequently detected in cfDNA in stage IV NSCLC (58.1%), and a high proportion of the patients (47.8%) with mutations had bone metastases at diagnosis. The frequency of RAS/PIK3CA/PTEN mutations in patients with activating EGFR mutation was 61.7%. The median PFS for EGFR-TKIs was 15.1 months in patients without RAS/PIK3CA/PTEN mutations, and 19.9 months in patients with mutations (p = 0.549). For patients with activating EGFR mutations, the overall survival was longer in patients without RAS/PIK3CA/PTEN mutations (53.8 months vs. 27.4 months). For the multivariate analysis, RAS/PIK3CA/PTEN mutations were independent predictors of poor prognosis in patients with activating EGFR mutations. In conclusion, RAS, PIK3CA and PTEN mutations do not hamper EGFR-TKI treatment outcome; however, they predict a poor OS when activating EGFR mutations coexist.

Purpose: This study investigates the effect of pregnant women s growth mindset on the stress of pregnancy ' through ego-resilience.Methods: The subjects included 551 pregnant women in Seoul and Gyeonggi area. Data were collected using a structured questionnaire; statistical, frequency, correlation, and structural equation analyses were performed using SPSS21 and M-plus 7.0.Results: First, pregnant women s growth mindset had a direct effect on pregnancy stress. Second, the mediating ' pathway that influenced pregnancy stress through the ego-resilience of growth mindset was significant. Conclusion: These results suggest that it is important to devise and practice a method to improve the growth mindset of pregnant women, enhance ego-resilience, and reduce the negative effects of stress by reducing pregnancy stress.

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 >0.1% and p<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 (<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 (<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

Non-small cell lung cancers (NSCLC) are characterized by a unique pattern of genetic driver mutations, and some of mutations may be used to predict prognosis and targeted treatment such as EGFR TKIs. Cell free (cfDNA) present in the blood stream shows much potential as a useful cancer maker for early 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 no standardized methods to identify mutations in cfDNA. In this study, we examined the viability of PGM and Proton platforms. Ion AmpliSeq Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze cfDNA of 125 serum samples from NSCLC patients. The on target was 88% and mean depth was 643x using PGM platform. And, the on target was 92% and mean depth was 22,868x in Proton platform. To validate the results of two NGS platforms, we analyzed EGFR status by sanger sequencing in available 100 tumor tissues. EGFR mutations were identified in 34 (34%) by sanger sequencing. EGFR mutations were identified in 32 (25.6%) and 28 (22.4%) by PGM and Proton platform. Interestingly, out of 34 mutations of tumor tissue, EGFR mutations were matched to 11 and 26 in PGM and Proton platform, respectively. These results showed concordance of 76.5% between the tDNA (sanger sequencing) and cfDNA (Proton). In addition, KRAS (codon 12 and 13) mutations were 4 (3.2%) and 18 (14.4%), respectively. In our study, we demonstrated that Proton platform of high depth is a useful assay to identify somatic mutations of cfDNA in NSCLCs. [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, Nak-Jung Kwon, Won-Chul Lee, Hae Mi Kim, Won Jin Jang, Yun Ji Choi, Kyung Hwa Park, Yeul Hong Kim. Comparison and evaluation of somatic mutation using PGM and proton platform in cell free DNA of non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3614.