Non-Invasive Molecular Testing for Osimertinib Acquired Resistance in T790M Positive Advanced Non-Small Cell Lung Cancer

Non-Invasive Molecular Testing for Osimertinib Acquired Resistance in T790M Positive Advanced Non-Small Cell Lung Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Non-Invasive Molecular Testing for Osimertinib Acquired Resistance in T790M Positive Advanced Non-Small Cell Lung Cancer Piyakarn Watcharenwong, Narumol Trachu, Nareenart Iemwimangsa, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7683102/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Introduction: Data concerning the acquired resistance mechanisms to osimertinib in epidermal growth factor receptor (EGFR ) T790M -positive advanced non-small cell lung cancer (NSCLC) are limited, pwithin the Asian population. This study utilized Non-invasive Molecular Testing (NIMT) to characterize these resistance mechanisms and evaluate their clinical impact. Methods Advanced NSCLC patients with EGFR T790M positivity, treated with osimertinib after failure on first- or second-generation EGFR-TKIs at Ramathibodi Hospital (January 2016–December 2019), were prospectively enrolled. Plasma samples, collected at baseline and disease progression, were analyzed via Next-Generation Sequencing (NGS). Clinical outcomes were subsequently correlated with the molecular findings. Results Fifty patients were included in the analysis. A significant proportion (38%, n = 19) of patients exhibited more than one resistance mechanism. The most common resistance mechanism observed was T790M -loss (50%), followed by PIK3CA alteration (14%), EGFR C797S (10%), and MET alteration (6%). The median overall survival (OS) was 16.4 months, and the time to treatment failure (TTF) for osimertinib was 9.3 months. Patients experiencing T790M -loss trended toward a shorter TTF compared to those with T790M maintenance (6.0 vs 10.1 months, P = 0.21). Patients with T790M-maintained status combined with C797S exhibited shorter OS compared to those with T790M -maintained status without C797S (11.0 vs 15.2 months). Furthermore, patients with T790M -loss accompanied by other co-alterations demonstrated a shorter TTF than those with T790M -loss alone (4.1 vs 10.6 months, P = 0.07). A significant correlation was found between T790M -loss and the presence of clinical brain metastasis prior to osimertinib use (P = 0.04). Additionally, the development of brain progression after osimertinib was significantly correlated with the presence of a BRAF mutation (P = 0.01). Conclusion Acquired resistance to osimertinib is diverse. The presence of T790M -loss suggests a poorer TTF, and OS is compromised in patients developing EGFR C797S or T790M -loss with co-mutations. Molecular profiling at progression is crucial for guiding subsequent therapy. Osimertinib mechanism resistance cfDNA EGFR-T790M NSCLC liquid biopsy NGS Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Non-small cell lung cancer (NSCLC) accounts for approximately 80%–85% of all global lung cancer cases and is associated with a high mortality rate [ 1 ]. In Thailand, NSCLC is also the most common cancer and the second most common cause of cancer-related death besides biliary tract cancer [ 2 ]. NSCLC harbor epidermal growth factor receptor ( EGFR) mutations in approximately 10%–20% of all lung cancer cases in Europe and North America [ 3 ], and in up to 50% of Asian patients [ 4 ]. Although first- or second-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) are initially effective in most non-small cell lung cancer (NSCLC) patients with common sensitizing EGFR mutations ( Exon 19 deletion and Exon 21 L858R ), nearly all patients eventually develop acquired resistance to these drugs, typically leading to disease progression within 9 to 14 months of starting therapy. [ 5 – 8 ]. The T790M mutation is the most frequent mechanism of acquired resistance to first- and second-generation EGFR-TKIs, being identified in approximately 50% of patients experiencing treatment failure. [ 9 , 10 ]. Osimertinib is a third-generation TKI that targets the EGFR [ 11 ] with significant efficacy against non-small cell lung cancer (NSCLC) harboring both common sensitizing EGFR mutations (Exon 19 deletion or L858R ) and the acquired resistance mutation, EGFR T790M. In the randomized Phase III AURA trial, osimertinib was shown to significantly improve progression-free survival (PFS) and the objective response rate (ORR) when compared to platinum-pemetrexed chemotherapy in patients with T790M-positive NSCLC who had progressed on prior first- or second-generation EGFR-TKIs [ 12 ]. In the first-line treatment of sensitizing EGFR-mutant NSCLC, the Phase III FLAURA trial demonstrated that osimertinib provided a significant improvement in both progression-free survival (PFS) and overall survival (OS) when compared to the first-generation EGFR-TKIs, gefitinib or erlotinib [ 13 , 14 ]. Although the use of osimertinib for the treatment of EGFR -positive NSCLC is increasing, there are limited data about the resistance mechanisms to this agent, in both upfront and sequential treatment. Previous reports have demonstrated that other EGFR mutations, including T790M loss, C797S, L718X, and L792X mutations, MET amplification, HER2 amplification, and small cell transformation, are also possible drivers of resistance [ 15 – 17 ]. Tumor biopsy remains the established standard of care for cancer diagnosis and is the principal method used to perform molecular testing that guides the selection of appropriate precision therapies. However, liquid biopsy is emerging as a valuable tool for molecular testing, offering a less invasive method to explore tumor heterogeneity and improve both cancer detection and disease monitoring. [ 18 ]. Indeed, several previous studies of liquid biopsy and cfDNA testing have demonstrated the use of cfDNA to detect resistant mechanisms in lung cancer with a good sensitivity and specificity [ 19 – 21 ]. A comprehensive understanding of resistant mechanisms to osimertinib is necessary to develop strategies to overcome osimertinib resistance. In this study, we analyzed the mechanisms of acquired resistance to osimertinib using non-invasive molecular testing (NIMT) in a cohort of 50 patients with advanced EGFR -mutant NSCLC who progressed on osimertinib in Ramathibodi hospital, Mahidol University, Bangkok, Thailand. We evaluated the molecular data, and the clinical and described features that were associated with survival outcomes. Methods Patients and study design A prospective study was performed in EGFR-T790M + ve advanced-NSCLC patients who received osimertinib after 1st/2nd-generation EGFR-TKI treatment from January 2016 to December 2019 at Ramathibodi Hospital. The patients in study fully understood and were informed of this study and had signed the Informed-Consent Form (ICF). Paired plasma samples were collected at baseline and at progression by Resist 1.1 criteria or discontinuation of osimertinib. Patients who had incomplete blood sampling or inadequate cfDNA were excluded. Clinical data were collected from Electronic Medical Record (EMR). The Human Research Ethics Committee of Ramathibodi Hospital, Mahidol University approved this study (IRB no.: EC600169). Identifies the institutional and licensing committee that approved the experiments, including any relevant details. Blood sample collection Patients provided written informed consent after being informed of the study and the procedure. Plasma samples were collected before osimertinib treatment and following disease progression or treatment discontinuation (10 ml/ time point in an EDTA blood collection tube). The EDTA blood was centrifuged within 1 hour of collection at 2000 x g for 15 minutes until the plasma was separated. A pipette was used to transfer the plasma into 1.5 ml microtubes, and the plasma was frozen at -70°C to -80°C. The paired plasma samples of each patient were analyzed by Next-Generation Sequencing (NGS; ThermoFischer Pancancer 52 genes) to explore the resistance mechanisms. The medical records of the patients who completed blood sampling at baseline and at progression were reviewed. The clinical characteristics were collected, and those that were associated with the mechanisms of resistance to osimertinib were analyzed. Statistical analysis and outcomes The primary endpoint of this study was the mechanisms of resistance after using osimertinib in T790M-positive advanced-NSCLC patients. The secondary endpoints were overall survival (OS), time to treatment failure (TTF), and clinical correlation to the osimertinib acquired-resistant mechanisms. Time to treatment failure (TTF) was defined as the time from initiation of osimertinib until discontinuation of treatment due to PD or toxicity. Overall survival (OS) defined as the time from initiation of osimertinib until death from any cause. The patients’ baseline characteristics and mechanisms of resistance to osimertinib were reported using descriptive statistics. Patients who were still alive at the last follow-up were censored for OS analysis. The Kaplan-Meier method was used to estimate the TTF, and OS. Hazard ratios and 95% confidence intervals (95% CI) were assessed with a Cox proportional-hazards model. In a part of clinical relevance of the resistance mechanism, statistical significance for categorical data was assessed using the χ2 or Fisher's exact test. For continuous data, the Student's t-test or Wilcoxon rank-sum test was employed, as appropriate for the data distribution. Clinical differences were quantified using the odds ratio (OR), along with its corresponding 95% Confidence Interval (CI) and P-value. Genomic profiling The Ion Torrent Oncomine Pan-Cancer Cell-Free Assay analyzes cell-free total nucleic acid (cfTNA) to detect four main types of somatic alterations across 52 unique cancer-related genes (ThermoFisher Pancancer): https://www.thermofisher.com/order/catalog/product/A37664 Somatic DNA Variants: Hotspot single nucleotide variants (SNVs) and short insertions/deletions (indels) in 41 genes, including KRAS , EGFR , BRAF , and TP53 . Gene Fusions: Fusions involving 12 genes ( ALK , RET , ROS1 , NTRK1/3 , etc.). Splice Variants: Detection of MET exon 14 skipping. Copy Number Variants (CNVs): Amplifications in 12 genes ( EGFR , ERBB2 , MET , MYC , etc.). The study began with the isolation of cell-free total nucleic acid (cfTNA) from cell-free biological samples using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit, followed by quantification with the Qubit 2.0 Fluorometer and the Qubit dsDNA HS Assay Kit.To prepare for sequencing, amplicon libraries targeting 52 cancer-associated genes were generated using the Oncomine™ Pan-Cancer Cell-Free Assay reagents. Four barcoded libraries were quantified, pooled, and then loaded onto an Ion 540 chip. Automated template preparation and sequencing were performed using the Ion Chef™ and the Ion GeneStudio S5 System, respectively.Finally, raw reads were processed by the Ion Reporter Software. Mutation calls were validated only if they were reported via the Oncomine™ TagSeq Pan-Cancer Liquid Biopsy - w2.0 - Single Sample workflow and met all defined ThermoFisher quality control (QC) criteria. Results Patients and treatment A total of 110 patients were eligible for participation in the current study from January 2016 to December 2019. Only 50 patients (33 females and 17 males) met the study criteria (Consort diagram shown in Supplement Fig. 1 ), and the median age was 63.5 years old. Almost all patients had an Eastern Cooperative Oncology Group (ECOG) score of 0 to 1, and the most common co-morbidities were hypertension and other diseases. The majority of patients were never smokers (82%), and adenocarcinoma was the most common histopathology (98%). The most common pre-existing sensitizing mutation was exon 19 deletion (60%), followed by exon 21 L858R (32%). In total, 90% of patients received previous 1st generation EGFR-TKI, while only 10% of patients received 2nd generation EGFR-TKI. Most of the patients used osimertinib as 2nd line treatment (68%), of whom, 30% were still alive at the time of analysis. The descriptive patient data are shown in Table 1 . Table 1 Baseline patient characteristics Characteristic Number % Age (year) Median (IQR) 63.5 (59.5–67.5) Sex Male 17 34 Female 33 66 ECOG 0 9 18 1 40 80 2 1 2 Comorbid No 29 58 T2DM 4 8 Hypertension 8 16 Dyslipidemia 5 10 Other* 8 16 Smoking status Never smoker 42 84 Ex-smoker 5 10 Current smoker 3 6 Smoking (pack/year) 0 42 84 1–10 7 14 11–20 1 2 Histology Adenocarcinoma 49 98 Adenocarcinoma mixed squamous cell carcinoma 1 2 Stage IVa 38 76 IVb 12 24 EGFR Exon 19 deletion 30 60 Exon 21 L858R 16 32 Del 19 with de novo T790M 2 4 Exon 21 L858R with de novo T790M 2 4 Method of T790M detected Plasma (by ddPCR) 50 100 Both tissue and plasma 20 40 Type of previous line of treatment Chemotherapy 15 30 EGFR-TKI 1st Gen EGFR-TKI 45 90 2nd Gen EGFR-TKI 5 10 Line of osimertinib treatment 2nd line 32 64 3rd line 10 20 ≥ 3rd line 7 14 Metastatic site at baseline Lung 38 39.4 Bone 18 19.1 Pleura 10 9.6 CNS 8 8.5 Adrenal 7 7.5 Metastatic site at osimertinib progression Lung 32 35.6 Pleura 13 14.4 LN 11 12.2 Liver 11 12.2 bone 8 8.9 Lymphangitis 6 6.7 CNS 3 3.3 *1 Vulvular heart disease, 1 Asthma, 1 Psoriasis, 1 Gout, 1 SLE, 1 BPH, 1 Ischemic heart disease, 1 Epilepsy Acquired mechanisms of resistance The 100 paired-plasma samples from 50 patients were analyzed for resistant mechanisms by NGS (ThermoFisher Pancancer 52 genes). In total, 19 patients (38%) developed more than one resistant alteration, while T790M cfDNA was detected in 32 (64%) patients by NGS. T790M-loss was the most common mutation, in 16 of 32 patients (50%), followed by PIK3CA mutation (E545K, E542K, H1047R) (14%), EGFR C797S/HER2/FGFR2 mutation (10% each) and other acquired EGFR mutation (L792F, P848L, P848Q)/ BRAF/RET/KIT mutation (8% of each mutation), MET mutation (6%), MET exon 14 skipping (4%), MET amplification (2%). EGFR C797S was only found in T790M-maintained patients. One patient (2%) was diagnosed small cell transformation by tissue biopsy, the NGS shown PIK3CA mutation (E542K) and TP53 mutation (R248Q, R306*). The results of mutational profiling are summarized in Fig. 1 and Supplement Table 1 . Efficacy of osimertinib and clinical relevance to the resistant mechanisms The median time to treatment failure (mTTF) was 9.3 months (mo) (IQR, 4.7–13.6), and the median overall survival was (mOS) was 16.4 mo (IQR, 10.3–29.3) (Supplement Fig. 3 ). The response rates of osimertinib treatment were 52% PR, 34% SD, and 14% PD, and the clinical benefit rate was 86%. The patients with T790M-loss tended to have a shorter TTF than the patients with T790M-maintained (6.0 vs 10.1 mo, hazard ratio [HR], 0.63; 95%CI, 0.3–1.3; P = 0.21), but the mOS was similar in both group 15.1 vs 15.2 mo (HR, 1.39; 95%CI, 0.60–3.28, P = 0.13) (Fig. 2 ). Univariate analysis of the clinical and T790M status revealed that CNS metastasis at baseline was significantly associated with T790M-loss. Moreover, patients with T790M-loss tended to be younger (Table 2 ) than T790M-maintained patients. Table 2 Univariate analysis of clinical and T790M status in T790M-positive advanced-NSCLC T790M -maintain n = 16 (%) T790M -loss n = 16 (%) Univariate P-value Age (yr.) (Median ± IQR) 65.81 57.81 0.05 Sex Male 6 (38) 6 (38) 1.00 Female 10 (62) 10 (62) Smoking status Never smoker 12 (75) 14 (88) 0.79 Ex-smoker 3 (19) 1 (6) Current smoker 1 (6) 1 (6) Smoking (pack/yr.) 0 13 (81) 14 (88) 0.60 1–10 3 (19) 1 (6) 11–20 0 1 (6) Histology Adenocarcinoma 16 (100) 15 (94) 1.00 Adenocarcinoma mixed squamous cell carcinoma 0 1(6) Stage IVa 12 (75) 12 (75) 1.00 IVb 4 (25) 4 (25) EGFR Exon 19 deletion 8 (50) 10 (63) 0.17 Exon 21 L858R 8 (50) 4 (25) Del 19 with de novo T790M 0 1 (6) Exon 21 L858R with de novo T790M 0 1 (6) Type of previous line of treatment EGFR-TKI 0.85 1st Gen EGFR-TKI 9 (56) 11 (69) 2nd Gen EGFR-TKI 1 (6) 1 (6) CMT and TKI 6 (38) 4 (25) Line of osimertinib treatment 2nd line 10 (63) 12 (75) 0.86 3rd line 2 (13) 2 (13) ≥ 3rd line 2 (13) 4 (25) Metastatic site at baseline Lung 11 (69) 12 (75) 1.00 Bone 9 (56) 5 (31) 0.15 Pleura 4 (25) 2 (13) 0.65 CNS 0 5 (31) 0.04 Adrenal 2 (13) 2 (13) 1.00 Metastatic site at the time progression Lung 12 (67) 20 (63) 1.00 Pleura 3 (17) 10 (31) 0.33 LN 4 (22) 7 (22) 1.00 Liver 4 (22) 7 (22) 1.00 bone 1 (6) 7 (22) 0.23 Lymphangitis 3 (9) 3 (17) 0.65 CNS 2 (11) 1 (6) 0.29 Patients who developed T790M-maintained with C797S mutation tended to have a shorter TTF and OS compared to patients with T790M-maintained without C797S (mTTF 9.3 vs 11.8 mo [HR, 0.89; 95%CI, 0.24–3.09; P = 0.86], and mOS 11.0 vs 15.2 mo [HR, 1.44; 95%CI, 0.30–6.97; P = 0.65]) (Fig. 3 ). Furthermore, patients with T790M-loss together with other co-mutations had a shorter TTF than patients with T790M-loss without other co-mutations (4.1 vs. 10.6 mo; HR, 2.8; 95%CI, 0.9–8.7; P = 0.06). Multiple logistic regression was performed using adjusted sex and line of osimertinib treatment, the result was demonstrated in the same trend (adjusted HR, 5.38; P = 0.07) (Fig. 4 ). Results of the Cox regression analyses for assessment of the clinical association with the mechanisms of resistance showed that brain metastasis prior to use of osimertinib was significantly related to T790M-loss (P = 0.04). In addition, uncommon site metastasis (for example, pericardium or kidney) had a significant association with EGFR C797S mutation (OR, 21.0; 95%CI, 2.4–178.2; P < 0.01) (Supplement Table 3), while brain progression after osimertinib treatment was related to BRAF mutation (P = 0.01) (Supplement Table 4). Nearly half of the patients who progressed on osimertinib were treated with chemotherapy followed by no other treatment, chemotherapy plus EGFR-TKI, and continued osimertinib beyond progression with radiotherapy (Supplement Table 5). The median time after stopping osimertinib to death was 6.5 mo (IQR, 1.65–16.96). At the end of the study, 15 patients (30%) were still alive and 35 patients (70%) had died. Discussion Several of the osimertinib acquired resistance mechanisms in T790M -positive advanced-NSCLC in Thailand are different from those outlined in previous reports [ 16 , 17 , 22 – 24 ]. T790M -loss (50%) and other EGFR aberrancy ( EGFR C797S [10%], P848X [4%], L792F [2%]) were common mechanisms to other studies. [ 15 , 17 , 25 – 28 ] However, there was a high prevalence of PIK3CA mutation (14%), HER2 mutation (10%), and BRAF mutation (8%), and a low prevalence of MET amplification (2%) in the Thai population compared to those reported in other studies. This might be due to the heterogeneity of cancer, the modality of molecular testing technique, and ethnicity differences. In our study, patients mostly used osimertinib as second-line or later-line treatment after 1st or 2nd generation EGFR-TKIs, and the median time to progression in our study was similar to that outlined in the AURA 3 study [ 12 ] (9.3 vs 10.1 mo). In contrast, the mOS was shorter (16.5 vs 26.8 mo) in our study compared to the AURA 3, probably because of 17 patients (34%) in our study used osimertinib in later-lines treatment which affects the OS outcome [ 29 ]. Both the ORR and disease control rate were also lower in our population compared to in the AURA 3 study. Furthermore, 60% of patients in the current study underwent detection of T790M by liquid biopsy, and only 40% of patients underwent both tissue and liquid biopsy. The T790M- loss tended to have worse TTF than T790M -maintained patients; this association was consistent across previously published reports [ 25 , 26 , 28 , 30 ]. This may be explained by intratumoral heterogeneity before treatment with osimertinib. Indeed, the previous report found that there were multiple concomitant genomic alterations, including clonal and subclonal co-alterations in lung cancer, and T790M positive and wild-type cell clones coexisting in the tumors. Osimertinib was a potent TKI against the EGFR T790M mutation, and may almost suppress this clone, causing an increase in pre-existing T790M wild-type clones with or without extra EGFR- independent resistance mechanisms [ 31 ]. Moreover, this phenomenon might be consistent in patients with loss of T790M but who carry other co-mutations which tended to have worse TTF and OS than the patients who had T790M -loss alone. In our study, T790M -maintained or loss was not translated to OS benefit; this was probably because of the subsequent treatment after progression. The current mainstay treatment after osimertinib failure is chemotherapy with or without immunotherapy; however, we still need to explore novel treatment for this clinical setting [ 32 ]. In addition, we first reported the clinical correlations to T790M- loss in late middle-aged patients (median age, 57.8 years old), who were younger than the T790M -maintained patient group (median age, 65 years old). Brain metastasis was a significant clinical characteristic found in T790M-loss patients at baseline before treatment with osimertinib. The C797S mutation was only seen in T790M -maintained patients [ 33 ]. This concomitant mutation tended to have poorer clinical outcomes, including TTF and OS; this result was consistent with a previous report from the Dana-Farber Cancer Institute, Boston, Massachusetts [ 25 ]. The point mutation of PIK3CA in exon 9 ( E545K, E542K ) and exon 20 (H1047R) was found to be similar to that outlined in the previous study, but it did not correlate to TTF and OS in our study [ 34 ]. The BRAF mutation was also found to be higher in our study (8%) compared to previous studies [ 17 ]. Furthermore, the prevalence of non-V600 mutation (6%) in our study was higher than the V600 mutation (2%); the non-V600 mutations were N581S and F595L , both of which were reported as potential resistant mechanisms because of their locations in the kinase domain [ 35 ]. Besides the resistant mutations, we first demonstrated the significant correlation of BRAF mutation with the patients who had brain progression after osimertinib treatment failure. Our study had several limitations. Firstly, we used non-invasive molecular testing to identify the resistant mechanism, and this may have low sensitivity to detect cfDNA at a low level in some resistant mechanisms.[ 20 ] Secondly, we did not routinely perform repeat or follow-up plasma cfDNA, so we were unable to determine the onset or dynamic change of each resistant mechanism at follow-up. Finally, there was low statistical power to detect the association between clinical characteristics and mechanisms of resistance; probably because of the small sample size. The strength of the study is that we investigated the association between the mechanism of acquired resistance of osimertinib and the clinical relevance using a non-invasive molecular technique that is feasible to use in routine clinical practice compared to sequential tissue biopsies. Further analysis of the molecular resistant mechanisms to osimertinib with a larger cohort and monitoring the dynamic change of molecular profile will be a challenge. In conclusion, there were heterogeneous mechanisms of acquired resistance to osimertinib in T790M -positive NSCLC, and patients with T790M- loss tended to have worse TTF compared to T790M -maintained patients. C797S mutation and T790M- loss with other co-mutations are the most important factors that affect the survival outcomes in this group of patients. Novel treatment, guided by resistant gene profiling after failure of T790M treatment, should be explored to improve survival of EGFR -positive patients. In conclusion, this study demonstrates a heterogeneity of acquired resistance mechanisms to osimertinib in T790M -positive NSCLC. A significant finding is that patients experiencing T790M -loss tend to exhibit a shorter time to treatment failure (TTF) compared to those who maintain the T790M mutation. The most critical determinants of survival outcomes in this patient group are the emergence of the C797S mutation and T790M -loss concurrent with other co-mutations. Therefore, future research must focus on exploring novel treatment strategies guided by a comprehensive resistance gene profiling following the failure of T790M -targeted therapy to ultimately improve the survival of EGFR -positive patients. Abbreviations EGFR epidermal growth factor receptor NSCLC advanced non-small cell lung cancer NIMT non-invasive molecular testing TTF time to treatment failure ORR objective response rate mPFS median progression free survival mOS median overall survival cfDNA cell-free DNA ECOG Eastern Cooperative Oncology Group EMR Electronic Medical Record Declarations Acknowledgement This research was funded by Thailand Center of Excellence for Life Sciences (TCELS) and Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Division of Medical Oncology, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Center for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Authors’ contribution: Piyakarn Watcharenwong: Conceptualization, methodology, formal analysis, investigation, data curation, writing-original draft, visualization. Narumol Trachu: Software, validation, investigation, resources, project administration Writing-review & editing. Nareenart Iemwimangsa: Software, formal analysis, investigation, resources, data curation. Dittapol Munthum: software, formal analysis, data curation Nanamon Monnamo: Resources., Angkana Charoenyingwattana : Project administration, Chayanin Nitiwarangkul: Writing-review & editing, resources. Wichana Chamroonrat: Writing-review & editing, resources. Phichai Chansriwong: Writing-review & editing, resources. Touch Ativitavas: Writing-review & editing, resources. Thitiya Dejthevaporn: Writing-review & editing, resources. Wasun Chantratita: Software, supervision, funding acquisition, writing-review & editing. Thanyanan Reungwetwattana: Conceptualization, methodology, formal analysis, investigation, resources, data curation, writing-review & editing, visualization, supervision, funding acquisition. Funding: This research was funded by Thailand Center of Excellence for Life Sciences (TCELS) and Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand Data availability: The raw data (VCF files) relevant to our submission are now accessible as a reference in EMBL-EBI under project ID: PRJEB98151 on EMBL-EBI. https://www.ebi.ac.uk/eva/?eva-study=PRJEB98151 Ethics approval and consent to participate: The Human Research Ethics Committee of Ramathibodi Hospital, Mahidol University, Bangkok, Thailand with the IRB number EC600169 approved this study. 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Loss of T790M mutation is associated with early progression to osimertinib in Chinese patients with advanced NSCLC who are harboring EGFR T790M. Lung Cancer. 2019;128:33–9. Mehlman C, et al. Resistance mechanisms to osimertinib in EGFR-mutated advanced non-small-cell lung cancer: A multicentric retrospective French study. Lung Cancer. 2019;137:149–56. Zhao Z, et al. The Status of the EGFR T790M Mutation is associated with the Clinical Benefits of Osimertinib Treatment in Non-small Cell Lung Cancer Patients: A Meta-Analysis. J Cancer. 2020;11(11):3106–13. Wu YL et al. Overall survival (OS) from the AURA3 phase III study: Osimertinib vs platinum-pemetrexed (plt-pem) in patients (pts) with EGFR T790M advanced non-small cell lung cancer (NSCLC) and progression on a prior EGFR-tyrosine kinase inhibitor (TKI). Ann Oncol, 2019. 30. Hong S, et al. Concomitant Genetic Alterations With Response to Treatment and Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients With EGFR-Mutant Advanced Non-Small Cell Lung Cancer. JAMA Oncol. 2018;4(5):739–42. Blakely CM, et al. Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. Nat Genet. 2017;49(12):1693–704. Mu Y, et al. Clinical Modality of Resistance and Subsequent Management of Patients with Advanced Non-small Cell Lung Cancer Failing Treatment with Osimertinib. Target Oncol. 2019;14(3):335–42. Yang Z, et al. Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients. Clin Cancer Res. 2018;24(13):3097–107. Eng J, et al. Impact of Concurrent PIK3CA Mutations on Response to EGFR Tyrosine Kinase Inhibition in EGFR-Mutant Lung Cancers and on Prognosis in Oncogene-Driven Lung Adenocarcinomas. J Thorac Oncol. 2015;10(12):1713–9. Sheikine Y, et al. BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations. Jco Precision Oncol. 2018;2:1–15. Additional Declarations No competing interests reported. Supplementary Files SupplementFigures.pdf Supplement Figure 1. Patient flowchart Supplement Figure 2. Comparison of mutations by NIMT pre and post-osimertinib Supplement Figure 3. Kaplan-Meier estimates of mTTF and mOS in patients with EGFR T790M– positive NSCLC Supplement table 1. Mechanisms of resistance to osimertinib in T790M+ve NSCLC (N=50) Supplement Table 2. Time to detect T790M compare from the option of treatments Supplement Table 3. Association between type of mutation and metastatic organ before treatment with osimertinib (P-value) Supplement Table 4. Association between types of mutation and metastatic organs after treatment with osimertinib (P-value) Supplement Table 5. Subsequence treatment after progression on osimertinib SupplementTables.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 10 Nov, 2025 Reviewers agreed at journal 05 Nov, 2025 Reviewers invited by journal 05 Nov, 2025 Editor assigned by journal 27 Oct, 2025 Editor invited by journal 06 Oct, 2025 Submission checks completed at journal 06 Oct, 2025 First submitted to journal 06 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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T790M-loss was the most common mutation, in 16 of 32 patients (50%), followed by PIK3CA mutation (E545K, E542K, H1047R) (14%), EGFR C797S/HER2/FGFR2 mutation (10% each) and other acquired EGFR mutation (L792F, P848L, P848Q)/ BRAF/RET/KIT mutation (8% of each mutation), MET mutation (6%), MET exon 14 skipping (4%), MET amplification (2%).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/f9b869c407e66d80215d0e46.jpg"},{"id":96247708,"identity":"b3f0c33e-687c-496c-8878-e8fff8d34205","added_by":"auto","created_at":"2025-11-19 07:27:41","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan–Meier estimates of OS \u0026amp; TTF According to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT790M\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e status.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patients with T790M-loss tended to have a shorter TTF than the patients with T790M-maintained (6.0 vs 10.1 mo, hazard ratio [HR], 0.63; 95%CI, 0.3–1.3; P = 0.21(A). The mOS was similar in both group 15.1 vs 15.2 mo (HR, 1.39; 95%CI, 0.60–3.28, P = 0.13) (B).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/ebfe6ad1710e1f09ab205d88.jpg"},{"id":96076726,"identity":"6210c00a-ee64-4d95-a1ce-3b74c27a675b","added_by":"auto","created_at":"2025-11-17 10:45:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaplan–Meier estimates of OS and TTF by \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eT790M\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u0026amp; \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eC797S\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients who developed T790M-maintained with C797S mutation tended to have a shorter TTF and OS compared to patients with T790M-maintained without C797S (mTTF 9.3 vs 11.8 mo [HR, 0.89; 95%CI, 0.24–3.09; P = 0.86] (A.), and mOS 11.0 vs 15.2 mo [HR, 1.44; 95%CI, 0.30–6.97; P = 0.65] (B.))\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/33637e054668bab0a413b440.jpg"},{"id":96076728,"identity":"4208932b-a423-4c3c-913e-8afec35687bb","added_by":"auto","created_at":"2025-11-17 10:45:39","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":175221,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsimertinib TTF by Candidate Resistance Mechanisms and T790M status\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTTF (mo)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe patients with T790M-loss together with other co-mutations had a shorter TTF than patients who had only T790M-loss (4.1 vs. 10.6 mo; HR, 2.8; 95%CI, 0.9–8.7; P = 0.06). Multiple logistic regression was performed using adjusted sex and line of osimertinib treatment, the result was demonstrated in the same trend (adjusted HR, 5.38; P = 0.07).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/3c32585a85093239472921c8.jpg"},{"id":96256118,"identity":"85dc8442-d747-4962-af8b-1a216babf44b","added_by":"auto","created_at":"2025-11-19 07:49:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1822370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/db139904-d5e3-4493-abc5-acc18298f93e.pdf"},{"id":96076724,"identity":"0c2d31fe-da46-45e5-9aff-2776aa1af426","added_by":"auto","created_at":"2025-11-17 10:45:39","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":111553,"visible":true,"origin":"","legend":"\u003cp\u003eSupplement Figure 1. Patient flowchart\u003c/p\u003e\n\u003cp\u003eSupplement Figure 2. Comparison of mutations by NIMT pre and post-osimertinib\u003c/p\u003e\n\u003cp\u003eSupplement Figure 3. Kaplan-Meier estimates of mTTF and mOS in patients with EGFR T790M–\u003c/p\u003e\n\u003cp\u003epositive NSCLC\u003c/p\u003e\n\u003cp\u003eSupplement table 1. Mechanisms of resistance to osimertinib in T790M+ve NSCLC (N=50)\u003c/p\u003e\n\u003cp\u003eSupplement Table 2. Time to detect \u003cem\u003eT790M\u003c/em\u003e compare from the option of treatments\u003c/p\u003e\n\u003cp\u003eSupplement Table 3. Association between type of mutation and metastatic organ before treatment\u003c/p\u003e\n\u003cp\u003ewith osimertinib (P-value)\u003c/p\u003e\n\u003cp\u003eSupplement Table 4. Association between types of mutation and metastatic organs after treatment\u003c/p\u003e\n\u003cp\u003ewith osimertinib (P-value)\u003c/p\u003e\n\u003cp\u003eSupplement Table 5. Subsequence treatment after progression on osimertinib\u003c/p\u003e","description":"","filename":"SupplementFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/67448c15ceb8b38f58c3e790.pdf"},{"id":96249083,"identity":"eff78db5-846d-4a30-9c97-ab4a0f9a8f09","added_by":"auto","created_at":"2025-11-19 07:30:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":123001,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementTables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7683102/v1/10061898ceb0dd4fc7f874b5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Non-Invasive Molecular Testing for Osimertinib Acquired Resistance in T790M Positive Advanced Non-Small Cell Lung Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNon-small cell lung cancer (NSCLC) accounts for approximately 80%\u0026ndash;85% of all global lung cancer cases and is associated with a high mortality rate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Thailand, NSCLC is also the most common cancer and the second most common cause of cancer-related death besides biliary tract cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NSCLC harbor epidermal growth factor receptor (\u003cem\u003eEGFR)\u003c/em\u003e mutations in approximately 10%\u0026ndash;20% of all lung cancer cases in Europe and North America [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and in up to 50% of Asian patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough first- or second-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) are initially effective in most non-small cell lung cancer (NSCLC) patients with common sensitizing EGFR mutations (\u003cem\u003eExon 19 deletion and Exon 21 L858R\u003c/em\u003e), nearly all patients eventually develop acquired resistance to these drugs, typically leading to disease progression within 9 to 14 months of starting therapy. [\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The \u003cem\u003eT790M\u003c/em\u003e mutation is the most frequent mechanism of acquired resistance to first- and second-generation EGFR-TKIs, being identified in approximately 50% of patients experiencing treatment failure. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOsimertinib is a third-generation TKI that targets the \u003cem\u003eEGFR\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] with significant efficacy against non-small cell lung cancer (NSCLC) harboring both common sensitizing EGFR mutations (Exon 19 deletion or \u003cem\u003eL858R\u003c/em\u003e) and the acquired resistance mutation, \u003cem\u003eEGFR T790M.\u003c/em\u003e In the randomized Phase III AURA trial, osimertinib was shown to significantly improve progression-free survival (PFS) and the objective response rate (ORR) when compared to platinum-pemetrexed chemotherapy in patients with T790M-positive NSCLC who had progressed on prior first- or second-generation EGFR-TKIs [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the first-line treatment of sensitizing EGFR-mutant NSCLC, the Phase III FLAURA trial demonstrated that osimertinib provided a significant improvement in both progression-free survival (PFS) and overall survival (OS) when compared to the first-generation EGFR-TKIs, gefitinib or erlotinib [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although the use of osimertinib for the treatment of \u003cem\u003eEGFR\u003c/em\u003e-positive NSCLC is increasing, there are limited data about the resistance mechanisms to this agent, in both upfront and sequential treatment. Previous reports have demonstrated that other \u003cem\u003eEGFR\u003c/em\u003e mutations, including \u003cem\u003eT790M loss, C797S, L718X, and L792X\u003c/em\u003e mutations, \u003cem\u003eMET\u003c/em\u003e amplification, \u003cem\u003eHER2\u003c/em\u003e amplification, and small cell transformation, are also possible drivers of resistance [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTumor biopsy remains the established standard of care for cancer diagnosis and is the principal method used to perform molecular testing that guides the selection of appropriate precision therapies. However, liquid biopsy is emerging as a valuable tool for molecular testing, offering a less invasive method to explore tumor heterogeneity and improve both cancer detection and disease monitoring. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Indeed, several previous studies of liquid biopsy and cfDNA testing have demonstrated the use of cfDNA to detect resistant mechanisms in lung cancer with a good sensitivity and specificity [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA comprehensive understanding of resistant mechanisms to osimertinib is necessary to develop strategies to overcome osimertinib resistance. In this study, we analyzed the mechanisms of acquired resistance to osimertinib using non-invasive molecular testing (NIMT) in a cohort of 50 patients with advanced \u003cem\u003eEGFR\u003c/em\u003e-mutant NSCLC who progressed on osimertinib in Ramathibodi hospital, Mahidol University, Bangkok, Thailand. We evaluated the molecular data, and the clinical and described features that were associated with survival outcomes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePatients and study design\u003c/h2\u003e\u003cp\u003eA prospective study was performed in EGFR-T790M\u0026thinsp;+\u0026thinsp;ve advanced-NSCLC patients who received osimertinib after 1st/2nd-generation EGFR-TKI treatment from January 2016 to December 2019 at Ramathibodi Hospital. The patients in study fully understood and were informed of this study and had signed the Informed-Consent Form (ICF). Paired plasma samples were collected at baseline and at progression by Resist 1.1 criteria or discontinuation of osimertinib. Patients who had incomplete blood sampling or inadequate cfDNA were excluded. Clinical data were collected from Electronic Medical Record (EMR). The Human Research Ethics Committee of Ramathibodi Hospital, Mahidol University approved this study (IRB no.: EC600169). Identifies the institutional and licensing committee that approved the experiments, including any relevant details.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eBlood sample collection\u003c/h3\u003e\n\u003cp\u003e Patients provided written informed consent after being informed of the study and the procedure. Plasma samples were collected before osimertinib treatment and following disease progression or treatment discontinuation (10 ml/ time point in an EDTA blood collection tube). The EDTA blood was centrifuged within 1 hour of collection at 2000 x g for 15 minutes until the plasma was separated. A pipette was used to transfer the plasma into 1.5 ml microtubes, and the plasma was frozen at -70\u0026deg;C to -80\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe paired plasma samples of each patient were analyzed by Next-Generation Sequencing (NGS; ThermoFischer Pancancer 52 genes) to explore the resistance mechanisms.\u003c/p\u003e\u003cp\u003e The medical records of the patients who completed blood sampling at baseline and at progression were reviewed. The clinical characteristics were collected, and those that were associated with the mechanisms of resistance to osimertinib were analyzed.\u003c/p\u003e\n\u003ch3\u003eStatistical analysis and outcomes\u003c/h3\u003e\n\u003cp\u003eThe primary endpoint of this study was the mechanisms of resistance after using osimertinib in T790M-positive advanced-NSCLC patients.\u003c/p\u003e\u003cp\u003eThe secondary endpoints were overall survival (OS), time to treatment failure (TTF), and clinical correlation to the osimertinib acquired-resistant mechanisms. Time to treatment failure (TTF) was defined as the time from initiation of osimertinib until discontinuation of treatment due to PD or toxicity. Overall survival (OS) defined as the time from initiation of osimertinib until death from any cause.\u003c/p\u003e\u003cp\u003eThe patients\u0026rsquo; baseline characteristics and mechanisms of resistance to osimertinib were reported using descriptive statistics. Patients who were still alive at the last follow-up were censored for OS analysis. The Kaplan-Meier method was used to estimate the TTF, and OS. Hazard ratios and 95% confidence intervals (95% CI) were assessed with a Cox proportional-hazards model.\u003c/p\u003e\u003cp\u003eIn a part of clinical relevance of the resistance mechanism, statistical significance for categorical data was assessed using the χ2 or Fisher's exact test. For continuous data, the Student's t-test or Wilcoxon rank-sum test was employed, as appropriate for the data distribution. Clinical differences were quantified using the odds ratio (OR), along with its corresponding 95% Confidence Interval (CI) and P-value.\u003c/p\u003e\n\u003ch3\u003eGenomic profiling\u003c/h3\u003e\n\u003cp\u003eThe Ion Torrent Oncomine Pan-Cancer Cell-Free Assay analyzes cell-free total nucleic acid (cfTNA) to detect four main types of somatic alterations across 52 unique cancer-related genes (ThermoFisher Pancancer): \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.thermofisher.com/order/catalog/product/A37664\u003c/span\u003e\u003cspan address=\"https://www.thermofisher.com/order/catalog/product/A37664\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003cp\u003eSomatic DNA Variants: Hotspot single nucleotide variants (SNVs) and short insertions/deletions (indels) in 41 genes, including \u003cem\u003eKRAS\u003c/em\u003e, \u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eBRAF\u003c/em\u003e, and \u003cem\u003eTP53\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eGene Fusions: Fusions involving 12 genes (\u003cem\u003eALK\u003c/em\u003e, \u003cem\u003eRET\u003c/em\u003e, \u003cem\u003eROS1\u003c/em\u003e, \u003cem\u003eNTRK1/3\u003c/em\u003e, etc.).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSplice Variants: Detection of MET exon 14 skipping.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eCopy Number Variants (CNVs): Amplifications in 12 genes (\u003cem\u003eEGFR\u003c/em\u003e, \u003cem\u003eERBB2\u003c/em\u003e, \u003cem\u003eMET\u003c/em\u003e, \u003cem\u003eMYC\u003c/em\u003e, etc.).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eThe study began with the isolation of cell-free total nucleic acid (cfTNA) from cell-free biological samples using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit, followed by quantification with the Qubit 2.0 Fluorometer and the Qubit dsDNA HS Assay Kit.To prepare for sequencing, amplicon libraries targeting 52 cancer-associated genes were generated using the Oncomine\u0026trade; Pan-Cancer Cell-Free Assay reagents. Four barcoded libraries were quantified, pooled, and then loaded onto an Ion 540 chip. Automated template preparation and sequencing were performed using the Ion Chef\u0026trade; and the Ion GeneStudio S5 System, respectively.Finally, raw reads were processed by the Ion Reporter Software. Mutation calls were validated only if they were reported via the Oncomine\u0026trade; TagSeq Pan-Cancer Liquid Biopsy - w2.0 - Single Sample workflow and met all defined ThermoFisher quality control (QC) criteria.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePatients and treatment\u003c/h2\u003e\u003cp\u003eA total of 110 patients were eligible for participation in the current study from January 2016 to December 2019. Only 50 patients (33 females and 17 males) met the study criteria (Consort diagram shown in Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and the median age was 63.5 years old. Almost all patients had an Eastern Cooperative Oncology Group (ECOG) score of 0 to 1, and the most common co-morbidities were hypertension and other diseases. The majority of patients were never smokers (82%), and adenocarcinoma was the most common histopathology (98%). The most common pre-existing sensitizing mutation was exon 19 deletion (60%), followed by exon 21 L858R (32%). In total, 90% of patients received previous 1st generation EGFR-TKI, while only 10% of patients received 2nd generation EGFR-TKI. Most of the patients used osimertinib as 2nd line treatment (68%), of whom, 30% were still alive at the time of analysis. The descriptive patient data are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline patient characteristics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCharacteristic\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (year)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMedian (IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e63.5 (59.5\u0026ndash;67.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eECOG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eComorbid\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eT2DM\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHypertension\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDyslipidemia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOther*\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eSmoking status\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNever smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEx-smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCurrent smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eSmoking (pack/year)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e84\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eHistology\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdenocarcinoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdenocarcinoma mixed squamous cell carcinoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eStage\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIVa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIVb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eEGFR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExon 19 deletion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExon 21 L858R\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDel 19 with de novo T790M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eExon 21 L858R with de novo T790M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eMethod of T790M detected\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePlasma (by ddPCR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBoth tissue and plasma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eType of previous line of treatment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eChemotherapy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEGFR-TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1st Gen EGFR-TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2nd Gen EGFR-TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eLine of osimertinib treatment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2nd line\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3rd line\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;3rd line\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eMetastatic site at baseline\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLung\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePleura\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdrenal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003e\u003cb\u003eMetastatic site at osimertinib progression\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLung\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePleura\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLiver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ebone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLymphangitis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e*1 Vulvular heart disease, 1 Asthma, 1 Psoriasis, 1 Gout, 1 SLE, 1 BPH, 1 Ischemic heart disease, 1 Epilepsy\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAcquired mechanisms of resistance\u003c/h3\u003e\n\u003cp\u003eThe 100 paired-plasma samples from 50 patients were analyzed for resistant mechanisms by NGS (ThermoFisher Pancancer 52 genes). In total, 19 patients (38%) developed more than one resistant alteration, while T790M cfDNA was detected in 32 (64%) patients by NGS. T790M-loss was the most common mutation, in 16 of 32 patients (50%), followed by PIK3CA mutation (E545K, E542K, H1047R) (14%), EGFR C797S/HER2/FGFR2 mutation (10% each) and other acquired EGFR mutation (L792F, P848L, P848Q)/ BRAF/RET/KIT mutation (8% of each mutation), MET mutation (6%), MET exon 14 skipping (4%), MET amplification (2%). EGFR C797S was only found in T790M-maintained patients. One patient (2%) was diagnosed small cell transformation by tissue biopsy, the NGS shown PIK3CA mutation (E542K) and TP53 mutation (R248Q, R306*). The results of mutational profiling are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplement Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eEfficacy of osimertinib and clinical relevance to the resistant mechanisms\u003c/h3\u003e\n\u003cp\u003eThe median time to treatment failure (mTTF) was 9.3 months (mo) (IQR, 4.7\u0026ndash;13.6), and the median overall survival was (mOS) was 16.4 mo (IQR, 10.3\u0026ndash;29.3) (Supplement Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The response rates of osimertinib treatment were 52% PR, 34% SD, and 14% PD, and the clinical benefit rate was 86%.\u003c/p\u003e\u003cp\u003eThe patients with T790M-loss tended to have a shorter TTF than the patients with T790M-maintained (6.0 vs 10.1 mo, hazard ratio [HR], 0.63; 95%CI, 0.3\u0026ndash;1.3; P\u0026thinsp;=\u0026thinsp;0.21), but the mOS was similar in both group 15.1 vs 15.2 mo (HR, 1.39; 95%CI, 0.60\u0026ndash;3.28, P\u0026thinsp;=\u0026thinsp;0.13) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eUnivariate analysis of the clinical and T790M status revealed that CNS metastasis at baseline was significantly associated with T790M-loss. Moreover, patients with T790M-loss tended to be younger (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) than T790M-maintained patients.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eUnivariate analysis of clinical and T790M status in T790M-positive advanced-NSCLC\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eT790M\u003c/em\u003e-maintain\u003c/p\u003e\u003cp\u003en\u0026thinsp;=\u0026thinsp;16 (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eT790M\u003c/em\u003e-loss\u003c/p\u003e\u003cp\u003en\u0026thinsp;=\u0026thinsp;16 (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUnivariate\u003c/p\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eAge (yr.)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e(Median\u0026thinsp;\u0026plusmn;\u0026thinsp;IQR)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e65.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.05\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 (38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6 (38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (62)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 (62)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eSmoking status\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNever smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14 (88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.79\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eEx-smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (19)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCurrent smoker\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eSmoking (pack/yr.)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13 (81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14 (88)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (19)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eHistology\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdenocarcinoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e16 (100)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15 (94)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdenocarcinoma mixed squamous cell carcinoma\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1(6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cb\u003eStage\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIVa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12 (75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIVb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eEGFR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eExon 19 deletion\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (50)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 (63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eExon 21 L858R\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 (50)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eDel 19 with de novo T790M\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eExon 21 L858R with de novo T790M\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e\u003cb\u003eType of previous line of treatment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eEGFR-TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e0.85\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1st Gen EGFR-TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9 (56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e11 (69)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2nd Gen EGFR-TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCMT and TKI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6 (38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eLine of osimertinib treatment\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2nd line\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10 (63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12 (75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3rd line\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (13)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026ge;\u0026thinsp;3rd line\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (25)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eMetastatic site at baseline\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLung\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (69)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12 (75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9 (56)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 (31)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePleura\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5 (31)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.04\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAdrenal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2 (13)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003e\u003cb\u003eMetastatic site at the time progression\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLung\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20 (63)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePleura\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (17)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10 (31)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7 (22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLiver\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4 (22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7 (22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ebone\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7 (22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLymphangitis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 (9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (17)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCNS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2 (11)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 (6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ePatients who developed T790M-maintained with C797S mutation tended to have a shorter TTF and OS compared to patients with T790M-maintained without C797S (mTTF 9.3 vs 11.8 mo [HR, 0.89; 95%CI, 0.24\u0026ndash;3.09; P\u0026thinsp;=\u0026thinsp;0.86], and mOS 11.0 vs 15.2 mo [HR, 1.44; 95%CI, 0.30\u0026ndash;6.97; P\u0026thinsp;=\u0026thinsp;0.65]) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, patients with T790M-loss together with other co-mutations had a shorter TTF than patients with T790M-loss without other co-mutations (4.1 vs. 10.6 mo; HR, 2.8; 95%CI, 0.9\u0026ndash;8.7; P\u0026thinsp;=\u0026thinsp;0.06). Multiple logistic regression was performed using adjusted sex and line of osimertinib treatment, the result was demonstrated in the same trend (adjusted HR, 5.38; P\u0026thinsp;=\u0026thinsp;0.07) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eResults of the Cox regression analyses for assessment of the clinical association with the mechanisms of resistance showed that brain metastasis prior to use of osimertinib was significantly related to T790M-loss (P\u0026thinsp;=\u0026thinsp;0.04). In addition, uncommon site metastasis (for example, pericardium or kidney) had a significant association with EGFR C797S mutation (OR, 21.0; 95%CI, 2.4\u0026ndash;178.2; P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Supplement Table\u0026nbsp;3), while brain progression after osimertinib treatment was related to BRAF mutation (P\u0026thinsp;=\u0026thinsp;0.01) (Supplement Table\u0026nbsp;4).\u003c/p\u003e\u003cp\u003eNearly half of the patients who progressed on osimertinib were treated with chemotherapy followed by no other treatment, chemotherapy plus EGFR-TKI, and continued osimertinib beyond progression with radiotherapy (Supplement Table\u0026nbsp;5). The median time after stopping osimertinib to death was 6.5 mo (IQR, 1.65\u0026ndash;16.96). At the end of the study, 15 patients (30%) were still alive and 35 patients (70%) had died.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeveral of the osimertinib acquired resistance mechanisms in \u003cem\u003eT790M\u003c/em\u003e-positive advanced-NSCLC in Thailand are different from those outlined in previous reports [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eT790M\u003c/em\u003e-loss (50%) and other \u003cem\u003eEGFR\u003c/em\u003e aberrancy (\u003cem\u003eEGFR C797S\u003c/em\u003e [10%], \u003cem\u003eP848X\u003c/em\u003e [4%], \u003cem\u003eL792F\u003c/em\u003e [2%]) were common mechanisms to other studies. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] However, there was a high prevalence of \u003cem\u003ePIK3CA\u003c/em\u003e mutation (14%), \u003cem\u003eHER2\u003c/em\u003e mutation (10%), and \u003cem\u003eBRAF\u003c/em\u003e mutation (8%), and a low prevalence of \u003cem\u003eMET\u003c/em\u003e amplification (2%) in the Thai population compared to those reported in other studies. This might be due to the heterogeneity of cancer, the modality of molecular testing technique, and ethnicity differences.\u003c/p\u003e\u003cp\u003eIn our study, patients mostly used osimertinib as second-line or later-line treatment after 1st or 2nd generation EGFR-TKIs, and the median time to progression in our study was similar to that outlined in the AURA 3 study [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] (9.3 vs 10.1 mo). In contrast, the mOS was shorter (16.5 vs 26.8 mo) in our study compared to the AURA 3, probably because of 17 patients (34%) in our study used osimertinib in later-lines treatment which affects the OS outcome [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Both the ORR and disease control rate were also lower in our population compared to in the AURA 3 study. Furthermore, 60% of patients in the current study underwent detection of \u003cem\u003eT790M\u003c/em\u003e by liquid biopsy, and only 40% of patients underwent both tissue and liquid biopsy.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eT790M-\u003c/em\u003eloss tended to have worse TTF than \u003cem\u003eT790M\u003c/em\u003e-maintained patients; this association was consistent across previously published reports [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This may be explained by intratumoral heterogeneity before treatment with osimertinib. Indeed, the previous report found that there were multiple concomitant genomic alterations, including clonal and subclonal co-alterations in lung cancer, and \u003cem\u003eT790M\u003c/em\u003e positive and wild-type cell clones coexisting in the tumors. Osimertinib was a potent TKI against the \u003cem\u003eEGFR T790M\u003c/em\u003e mutation, and may almost suppress this clone, causing an increase in pre-existing \u003cem\u003eT790M\u003c/em\u003e wild-type clones with or without extra \u003cem\u003eEGFR-\u003c/em\u003eindependent resistance mechanisms [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, this phenomenon might be consistent in patients with loss of \u003cem\u003eT790M\u003c/em\u003e but who carry other co-mutations which tended to have worse TTF and OS than the patients who had \u003cem\u003eT790M\u003c/em\u003e-loss alone. In our study, \u003cem\u003eT790M\u003c/em\u003e-maintained or loss was not translated to OS benefit; this was probably because of the subsequent treatment after progression. The current mainstay treatment after osimertinib failure is chemotherapy with or without immunotherapy; however, we still need to explore novel treatment for this clinical setting [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In addition, we first reported the clinical correlations to \u003cem\u003eT790M-\u003c/em\u003eloss in late middle-aged patients (median age, 57.8 years old), who were younger than the \u003cem\u003eT790M\u003c/em\u003e-maintained patient group (median age, 65 years old). Brain metastasis was a significant clinical characteristic found in T790M-loss patients at baseline before treatment with osimertinib.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eC797S\u003c/em\u003e mutation was only seen in \u003cem\u003eT790M\u003c/em\u003e-maintained patients [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This concomitant mutation tended to have poorer clinical outcomes, including TTF and OS; this result was consistent with a previous report from the Dana-Farber Cancer Institute, Boston, Massachusetts [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe point mutation of \u003cem\u003ePIK3CA\u003c/em\u003e in exon 9 (\u003cem\u003eE545K, E542K\u003c/em\u003e) and exon 20 \u003cem\u003e(H1047R)\u003c/em\u003e was found to be similar to that outlined in the previous study, but it did not correlate to TTF and OS in our study [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eBRAF\u003c/em\u003e mutation was also found to be higher in our study (8%) compared to previous studies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, the prevalence of \u003cem\u003enon-V600\u003c/em\u003e mutation (6%) in our study was higher than the \u003cem\u003eV600\u003c/em\u003e mutation (2%); the \u003cem\u003enon-V600\u003c/em\u003e mutations were \u003cem\u003eN581S\u003c/em\u003e and \u003cem\u003eF595L\u003c/em\u003e, both of which were reported as potential resistant mechanisms because of their locations in the kinase domain [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Besides the resistant mutations, we first demonstrated the significant correlation of \u003cem\u003eBRAF\u003c/em\u003e mutation with the patients who had brain progression after osimertinib treatment failure.\u003c/p\u003e\u003cp\u003eOur study had several limitations. Firstly, we used non-invasive molecular testing to identify the resistant mechanism, and this may have low sensitivity to detect cfDNA at a low level in some resistant mechanisms.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] Secondly, we did not routinely perform repeat or follow-up plasma cfDNA, so we were unable to determine the onset or dynamic change of each resistant mechanism at follow-up. Finally, there was low statistical power to detect the association between clinical characteristics and mechanisms of resistance; probably because of the small sample size. The strength of the study is that we investigated the association between the mechanism of acquired resistance of osimertinib and the clinical relevance using a non-invasive molecular technique that is feasible to use in routine clinical practice compared to sequential tissue biopsies. Further analysis of the molecular resistant mechanisms to osimertinib with a larger cohort and monitoring the dynamic change of molecular profile will be a challenge.\u003c/p\u003e\u003cp\u003eIn conclusion, there were heterogeneous mechanisms of acquired resistance to osimertinib in \u003cem\u003eT790M\u003c/em\u003e-positive NSCLC, and patients with \u003cem\u003eT790M-\u003c/em\u003eloss tended to have worse TTF compared to \u003cem\u003eT790M\u003c/em\u003e-maintained patients. \u003cem\u003eC797S\u003c/em\u003e mutation and \u003cem\u003eT790M-\u003c/em\u003eloss with other co-mutations are the most important factors that affect the survival outcomes in this group of patients. Novel treatment, guided by resistant gene profiling after failure of T790M treatment, should be explored to improve survival of \u003cem\u003eEGFR\u003c/em\u003e-positive patients.\u003c/p\u003e\u003cp\u003eIn conclusion, this study demonstrates a heterogeneity of acquired resistance mechanisms to osimertinib in \u003cem\u003eT790M\u003c/em\u003e-positive NSCLC. A significant finding is that patients experiencing \u003cem\u003eT790M\u003c/em\u003e-loss tend to exhibit a shorter time to treatment failure (TTF) compared to those who maintain the \u003cem\u003eT790M\u003c/em\u003e mutation. The most critical determinants of survival outcomes in this patient group are the emergence of the \u003cem\u003eC797S\u003c/em\u003e mutation and \u003cem\u003eT790M\u003c/em\u003e-loss concurrent with other co-mutations. Therefore, future research must focus on exploring novel treatment strategies guided by a comprehensive resistance gene profiling following the failure of \u003cem\u003eT790M\u003c/em\u003e-targeted therapy to ultimately improve the survival of \u003cem\u003eEGFR\u003c/em\u003e-positive patients.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEGFR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eepidermal growth factor receptor\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNSCLC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eadvanced non-small cell lung cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNIMT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003enon-invasive molecular testing\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTTF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003etime to treatment failure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eORR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eobjective response rate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emPFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emedian progression free survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emOS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003emedian overall survival\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ecfDNA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecell-free DNA\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eECOG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eEastern Cooperative Oncology Group\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEMR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eElectronic Medical Record\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eThis research was funded by Thailand Center of Excellence for Life Sciences (TCELS) and Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.\u003c/li\u003e\n \u003cli\u003eDivision of Medical Oncology, Department of Medicine, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand\u003c/li\u003e\n \u003cli\u003eCenter for Medical Genomics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePiyakarn Watcharenwong:\u003c/strong\u003e Conceptualization, methodology, formal analysis, investigation, data curation, writing-original draft, visualization.\u0026nbsp;\u003cstrong\u003eNarumol Trachu:\u003c/strong\u003e Software, validation, investigation, resources, project administration Writing-review \u0026amp; editing. \u003cstrong\u003eNareenart Iemwimangsa:\u003c/strong\u003e Software, formal analysis, investigation, resources, data curation. \u003cstrong\u003eDittapol Munthum:\u003c/strong\u003e software, formal analysis, data curation \u003cstrong\u003eNanamon Monnamo:\u003c/strong\u003e Resources., \u003cstrong\u003eAngkana Charoenyingwattana\u003c/strong\u003e: Project administration, \u003cstrong\u003eChayanin Nitiwarangkul:\u003c/strong\u003e Writing-review \u0026amp; editing, resources.\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003cstrong\u003eWichana Chamroonrat:\u003c/strong\u003e Writing-review \u0026amp; editing, resources. \u003cstrong\u003ePhichai Chansriwong:\u003c/strong\u003e\u0026nbsp; Writing-review \u0026amp; editing, resources. \u003cstrong\u003eTouch Ativitavas:\u003c/strong\u003e Writing-review \u0026amp; editing, resources. \u003cstrong\u003eThitiya\u0026nbsp;Dejthevaporn:\u003c/strong\u003e Writing-review \u0026amp; editing, resources. \u003cstrong\u003eWasun Chantratita:\u003c/strong\u003e Software, supervision, funding acquisition, writing-review \u0026amp; editing. \u003cstrong\u003eThanyanan Reungwetwattana:\u003c/strong\u003e Conceptualization, methodology, formal analysis, investigation, resources, data curation, writing-review \u0026amp; editing, visualization, supervision, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by Thailand Center of Excellence for Life Sciences (TCELS) and Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The raw data (VCF files) relevant to our submission are now accessible as a reference in EMBL-EBI under project ID: PRJEB98151 on EMBL-EBI. https://www.ebi.ac.uk/eva/?eva-study=PRJEB98151\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e The Human Research Ethics Committee of Ramathibodi Hospital, Mahidol University, Bangkok, Thailand with the IRB number\u0026nbsp;EC600169\u0026nbsp;approved this study. The study adhered to the Declaration of Helsinki to this effect in the Ethics approval and consent to precipitate section or appropriate national guidelines. All methods were carried out following relevant guidelines and\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003elocal\u0026nbsp;regulations. Informed consent was obtained from all patients in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e All authors declare no competing interests to this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThe American Cancer Society. 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Transl Lung Cancer Res. 2016;5(6):695\u0026ndash;708.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTang ZH, Lu JJ. Osimertinib resistance in non-small cell lung cancer: Mechanisms and therapeutic strategies. Cancer Lett. 2018;420:242\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim SM, et al. Acquired resistance to EGFR targeted therapy in non-small cell lung cancer: Mechanisms and therapeutic strategies. Cancer Treat Rev. 2018;65:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOxnard GR, et al. Assessment of Resistance Mechanisms and Clinical Implications in Patients With EGFR T790M-Positive Lung Cancer and Acquired Resistance to Osimertinib. JAMA Oncol. 2018;4(11):1527\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhao S, et al. Loss of T790M mutation is associated with early progression to osimertinib in Chinese patients with advanced NSCLC who are harboring EGFR T790M. 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Concomitant Genetic Alterations With Response to Treatment and Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Patients With EGFR-Mutant Advanced Non-Small Cell Lung Cancer. JAMA Oncol. 2018;4(5):739\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBlakely CM, et al. Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. Nat Genet. 2017;49(12):1693\u0026ndash;704.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMu Y, et al. Clinical Modality of Resistance and Subsequent Management of Patients with Advanced Non-small Cell Lung Cancer Failing Treatment with Osimertinib. Target Oncol. 2019;14(3):335\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYang Z, et al. Investigating Novel Resistance Mechanisms to Third-Generation EGFR Tyrosine Kinase Inhibitor Osimertinib in Non-Small Cell Lung Cancer Patients. Clin Cancer Res. 2018;24(13):3097\u0026ndash;107.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEng J, et al. Impact of Concurrent PIK3CA Mutations on Response to EGFR Tyrosine Kinase Inhibition in EGFR-Mutant Lung Cancers and on Prognosis in Oncogene-Driven Lung Adenocarcinomas. J Thorac Oncol. 2015;10(12):1713\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSheikine Y, et al. BRAF in Lung Cancers: Analysis of Patient Cases Reveals Recurrent BRAF Mutations, Fusions, Kinase Duplications, and Concurrent Alterations. Jco Precision Oncol. 2018;2:1\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Osimertinib, mechanism resistance, cfDNA, EGFR-T790M, NSCLC, liquid biopsy, NGS","lastPublishedDoi":"10.21203/rs.3.rs-7683102/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7683102/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e\u003cp\u003eData concerning the acquired resistance mechanisms to osimertinib in epidermal growth factor receptor (EGFR\u003cem\u003e) T790M\u003c/em\u003e-positive advanced non-small cell lung cancer (NSCLC) are limited, pwithin the Asian population. This study utilized Non-invasive Molecular Testing (NIMT) to characterize these resistance mechanisms and evaluate their clinical impact.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eAdvanced NSCLC patients with \u003cem\u003eEGFR T790M\u003c/em\u003e positivity, treated with osimertinib after failure on first- or second-generation EGFR-TKIs at Ramathibodi Hospital (January 2016\u0026ndash;December 2019), were prospectively enrolled. Plasma samples, collected at baseline and disease progression, were analyzed via Next-Generation Sequencing (NGS). Clinical outcomes were subsequently correlated with the molecular findings.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eFifty patients were included in the analysis. A significant proportion (38%, n\u0026thinsp;=\u0026thinsp;19) of patients exhibited more than one resistance mechanism. The most common resistance mechanism observed was \u003cem\u003eT790M\u003c/em\u003e-loss (50%), followed by PIK3CA alteration (14%), \u003cem\u003eEGFR C797S\u003c/em\u003e (10%), and \u003cem\u003eMET\u003c/em\u003e alteration (6%). The median overall survival (OS) was 16.4 months, and the time to treatment failure (TTF) for osimertinib was 9.3 months. Patients experiencing \u003cem\u003eT790M\u003c/em\u003e-loss trended toward a shorter TTF compared to those with T790M maintenance (6.0 vs 10.1 months, P\u0026thinsp;=\u0026thinsp;0.21). Patients with T790M-maintained status combined with C797S exhibited shorter OS compared to those with \u003cem\u003eT790M\u003c/em\u003e-maintained status without C797S (11.0 vs 15.2 months). Furthermore, patients with \u003cem\u003eT790M\u003c/em\u003e-loss accompanied by other co-alterations demonstrated a shorter TTF than those with \u003cem\u003eT790M\u003c/em\u003e-loss alone (4.1 vs 10.6 months, P\u0026thinsp;=\u0026thinsp;0.07). A significant correlation was found between \u003cem\u003eT790M\u003c/em\u003e-loss and the presence of clinical brain metastasis prior to osimertinib use (P\u0026thinsp;=\u0026thinsp;0.04). Additionally, the development of brain progression after osimertinib was significantly correlated with the presence of a BRAF mutation (P\u0026thinsp;=\u0026thinsp;0.01).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eAcquired resistance to osimertinib is diverse. The presence of \u003cem\u003eT790M\u003c/em\u003e-loss suggests a poorer TTF, and OS is compromised in patients developing \u003cem\u003eEGFR C797S\u003c/em\u003e or \u003cem\u003eT790M\u003c/em\u003e-loss with co-mutations. Molecular profiling at progression is crucial for guiding subsequent therapy.\u003c/p\u003e","manuscriptTitle":"Non-Invasive Molecular Testing for Osimertinib Acquired Resistance in T790M Positive Advanced Non-Small Cell Lung Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-17 10:45:34","doi":"10.21203/rs.3.rs-7683102/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-11-10T23:07:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98514699348981968221724315086533099526","date":"2025-11-05T21:53:29+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-05T21:47:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-27T08:11:56+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-06T10:55:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-06T06:24:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-10-06T06:20:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2dceabd-d61d-4fb6-94f2-736eb51f70a0","owner":[],"postedDate":"November 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T10:45:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-17 10:45:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7683102","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7683102","identity":"rs-7683102","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}