EGFR-mutant Lung Adenocarcinoma Research Articles

The prognostic value of tumor mutation burden in EGFR-mutant advanced lung adenocarcinoma, an analysis based on cBioPortal data base.

Tumor mutation burden (TMB) is novel biomarker of promising predict value in prediction of immune checkpoint inhibitors (ICPis) in non-small cell lung cancer (NSCLC). However, the distribution of TMB in epidermal growth factor receptor (EGFR)-mutant advanced lung adenocarcinoma (LUAD) patients and the impact on overall survival (OS) time are not well demonstrated. Information regarding gene mutations and patients' survival time in advanced LUAD was downloaded from The Cancer Genome Atlas (TCGA) database. The diversity of TMB in different EGFR-mutant types was observed and the predicted value of TMB for OS as well as other co-mutations were analyzed. The diversity of TMB was also observed in another Chinese cohort of advanced LUAD patients. The median TMB values of EGFR wild-type, other types of EGFR mutations, exon 19 deletions and L858R were 6.12, 5.66, 3.77 and 4.72, differences between wild-type and EGFR sensitive mutations (exon 19 deletion or L858R) were significant (P<0.001 and P<0.01). OS time of high TMB group was inferior to that of the low TMB group (24.03 months vs. not reached, P=0.0020). TMB and TP53 together will make more accurate prediction of OS in EGFR-mutant advanced LUAD patients. Distribution of TMB in another Chinese cohort had the same trend. In advanced LUAD patients, TMB was lower in patients with EGFR-mutant group than EGFR wild group. TMB was a negative prognostic biomarker of OS in EGFR-mutant LUAD patients, especially when TP53 was mutated together.

Effects of different brain surveillance strategies on outcomes for patients with EGFR-mutant metastatic lung adenocarcinoma under targeted therapy

ObjectivesBrain metastasis (BM) is common in patients with epidermal growth factor receptor (EGFR)-mutant lung cancer. However, the brain surveillance strategy during treatment in advanced lung cancer patients varies, and the impact on clinical outcome is unclear. Here we aimed to evaluate the effect of different brain surveillance strategies on the clinical characteristics and treatment outcome in patients with EGFR-mutant lung adenocarcinoma treated with first-line EGFR tyrosine kinase inhibitors (EGFR-TKIs). Materials and methodsThis is a retrospective observational study conducted in a medical center in an area with high prevalence of EGFR mutation. Patients with initially diagnosed stage IV EGFR-mutant lung adenocarcinoma were included. Patients undergoing regular brain magnetic resonance imaging (MRI) every 3-6 months were categorized in the regular follow-up (RFU) group, and the rest were categorized in the liberal follow-up (LFU) group. Clinical outcomes were collected and analyzed. ResultsA total of 310 patients were included, and 43.5% initially had brain metastases. Patients in the LFU group were significantly older than those in the RFU group (median age: 67 vs 62, p < 0.001). The overall survival and time-to-treatment failure of patients with initial EGFR-TKIs treatment showed no statistical difference between the two groups. However, the intracranial progression free survival was significantly shorter in the RFU group than in the LFU group (p = 0.009). The risk of mortality was similar in the LFU and RFU groups. There was no difference in the intracranial progression patterns and cause of death between the two groups. ConclusionsFor EGFR-mutant lung adenocarcinoma patients who used EGFR-TKIs as the frontline therapy, regular or liberal brain MRI follow-up showed no significant impact on the outcome, irrespective of initial brain metastasis.

P2.09-09 EGFR Is Highly Mutated in Lung Adenocarcinoma Patients with History of Breast Cancer

EGFR gene mutation has been reported to be frequent in the patients with specific clinical features such as female, adenocarcinoma, and East-Asian ethnicity. The mutation rate in lung adenocarcinoma is approximately 50% in Japan. Currently, the molecular mechanism which cause EGFR mutation has not been clarified. If the EGFR mutation in lung adenocarcinoma correlates with specific type of malignancy in the other organ, it could be a clue to find a mechanism which promote carcinogenesis of lung adenocarcinoma. In the current study, we focused on breast cancer. Patients with lung adenocarcinoma who underwent pulmonary resection in our hospital from January 2011 to December 2018 were analyzed. We retrospectively reviewed clinical information such as past illness, radiological findings, pathological diagnosis, and EGFR mutation status. Correlation was tested by chi-square test and p value of less than 0.05 was regarded as statistically significant. A total of 21 patients of lung adenocarcinoma had history of treatment for breast cancer. All patients were female. Among them, EGFR mutation was detected in 20 patients (95%). One patient who were negative for EGFR mutation had history of not only breast cancer but also cervical cancer of uterus and gastrointestinal stromal tumor, and developed angiosarcoma of the skin. Detected EGFR mutation types in 20 patients were as follows; deletion in exon 19 for 9 patients, L858R for 7 patients G719X for two patients, and L861Q for one patient. One patient showed multiple mutation (G719X and L861). In the same period, among 203 lung adenocarcinoma patients without other organ malignancy, 115 showed EGFR mutation (56.7%). There was significant difference in EGFR mutation rate between breast cancer group and no malignancy group (p=0.00058). Patients of lung adenocarcinoma with history of breast cancer showed extremely high positive rate for EGFR mutation in Japan, suggesting underlying common oncogenic molecular mechanism between lung adenocarcinoma and breast cancer. Elucidation of the mechanism may contribute to the diagnosis and treatment of carcinogenesis of breast cancer and lung cancer.

1259P - The role of tumour neoantigens in the differential response to immunotherapy (IO) in EGFR and BRAF mutated lung cancers: Quantity or quality?

BackgroundLung cancer patients with activating EGFR mutations tend to respond poorly to IO, but this is not true for all activating mutation driven lung cancers. Recent data show that patients with BRAF mutations tend to respond better to IO. Our study estimated the tumor neoantigen burden in these tumors, and the likelihood of their presentation to cytotoxic T cells by estimating their binding affinity to major histocompatibility complex (MHC). MethodsWhole exome data for 68 patients with EGFR mutant lung adenocarcinoma and 33 with BRAF mutations were extracted from the Cancer Genome Atlas. MAFTOOLs was used to determine the tumor mutational load. Nonamers were estimated by simulating possible nonamer neoantigens from MAF files. The neoantigen (MT) and wildtype (WT) binding affinities to MHC-I were calculated using the NetMHC 4.0 server. The differential binding affinity of the neoantigens in comparison to wildtype was calculated as mean differential agretopicity index (DAI) = WT - MT. Patient survival was estimated by the Kaplan-Meier method. ResultsPatients with mutated BRAF had higher median mutational burden (445, range 165-776) than those with EGFR mutations (90.5, range 60.5-219.5, p=0.001). The median number of simulated neoantigens was higher in the BRAF (9536.5, range 3839.5-14626.0) vs the EGFR group (1895.5, range 1148.5-4766.5, p<0.001). Mean DAI for BRAF and EGFR patients were 1046 and 1033, respectively, p=0.86. In the BRAF mutant group, patients with DAI < 1,000 had significantly better 5-year survival (80% vs 40%, p=0.022) compared to patients with DAI > 1,000. No such survival benefit was identified in the EGFR mutant group (56% vs 58%, p=0.81). ConclusionsBRAF mutant lung cancers are characterized by higher neoantigen burden, but their neoantigen differential binding affinity to MHC-I complex was not different from EGFR mutant lung cancers. Our analysis suggests that the improved response to IO in the BRAF mutant population is due to increased quantity in neoantigen burden and not a qualitative difference in MHC-I binding. Further, DAI had a prognostic impact in the BRAF population suggesting that it may be a measure of tumor neoantigen immunogenicity. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureJ. Subramanian: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Research grant / Funding (institution): Paradigm; Speaker Bureau / Expert testimony: Lilly; Research grant / Funding (institution): Biocept. All other authors have declared no conflicts of interest.

P2.14-13 Elevated Expression of Eukaryotic Translation Initiation Factor 3 Subunit C Contributes to EGFR-TKI Resistance in Lung Adenocarcinoma

The treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, in patients with EGFR-mutant lung adenocarcinoma causes tumor regression and improved prognosis. However, drug resistance and tumor relapse eventually occur, and the molecular mechanisms remain to be fully revealed. In this study, we elucidated the novel mechanism of EGFR-TKI resistance and clarified the clinical significance using erlotinib-resistant lung adenocarcinoma cell lines and patient specimens. The erlotinib-resistant cell line, PC9/ER was obtained from PC9 cell line by exposing to increasing concentrations of erlotinib for 6 months. The sensitivity of erlotinib was evaluated by the MTT assay. In order to identify erlotinib-resistance related factors, we performed a proteomics analysis, followed by western blotting. To evaluate the roles of the candidate factor, we performed gene knockdown analysis using siRNAs. The autophagy activity was assessed by the amount of LC3B-II protein or visualization of autophagosomes using LC3B-RFP expression vector. Immunohistochemical staining was performed on tumor biopsies obtained from lung adenocarcinoma patients with activating mutation in EGFR, and we examined the correlation between the staining intensity and clinical EGFR-TKI resistance. Initially, we performed the sequencing analysis of EGFR exons 18 to 21, which cover the tyrosine kinase domain, and found that there was no difference between PC9 and PC9/ER cells. The proteomics analysis revealed ribosomal and translation-related proteins were abundantly identified in PC9/ER cells, compared with PC9 cells. Among them, we found that eukaryotic translation initiation factor 3 subunit C (eIF3c) was overexpressed in PC9/ER cells. The knockdown of eIF3c expression with siRNA improved the sensitivity of EGFR-TKI in PC9/ER cells. Additionally, we observed that LC3B-II increased in PC9/ER cells, but decreased by the knockdown of eIF3c. Consistently, the overexpression of eIF3c upregulated the number of autophagosomes. Moreover, the inhibition of autophagy with chloroquine attenuated erlotinib-resistance in PC9/ER cells. Finally, the frequency of eIF3c-positive samples was higher in biopsy specimens from the patients’ refractory to EGFR-TKI therapy than in those from the patients prior to the treatment with EGFR-TKI. Furthermore, patients with eIF3c-positive tumors had shorter progression-free survival in EGFR-TKI treatment than eIF3c-negative patients. The increased eIF3c expression conferred the resistance to EGFR-TKI in lung adenocarcinoma cells via enhancement of autophagy. Moreover, the patients with increased eIF3c expression in biopsy samples showed poor prognosis. The inhibition of eIF3c could be a new therapeutic strategy for overcoming EGFR-TKI resistance in lung adenocarcinoma patients.

1417P - Analysis of circulating tumour DNA in paired plasma and sputum samples of EGFR-mutated NSCLC patients

BackgroundLiquid biopsy is a minimally invasive strategy to detect genetic alterations in non-small cell lung cancer (NSCLC) patients. In particular, it is clinically useful for the detection of the EGFR T790M resistance mutation in advanced EGFR-mutated NSCLC patients who had progressed under treatment with an EGFR-TKI. While plasma analysis is commonly performed, sputum could be another potential source for circulating tumor DNA (ctDNA) in NSCLC, which has not yet been entirely evaluated. MethodsPaired plasma and sputum samples from 23 patients with EGFR-mutated lung adenocarcinoma were analyzed for EGFR exon 19 deletions (del19), L858R, and T790M mutations by droplet digital PCR (ddPCR). All patients had histologically confirmed lung adenocarcinoma with EGFR mutations in their initial tissue biopsy at diagnosis. ResultsTissue genotyping at diagnosis revealed 18 patients with EGFR del19 and 5 patients with L858R mutations. Among the 18 EGFR del19-positive patients, 8 showed concordant test results in plasma and sputum (2 positive, 6 negative), 6 had del19-positive plasma samples but del19-negative sputum samples, 4 were del19 negative in plasma and del19 positive in sputum. Among 5 L8585R-positive patients, 4 had concordant test results in plasma and sputum (3 positive, 1 negative), one patient was L858R positive in the plasma sample but L858R negative in sputum. In 10 out of 23 patients the T790M mutation was assessed in paired samples, 9 showed concordant test results in plasma and sputum (3 positive, 6 negative), one patient was T790M positive only in the sputum sample. ConclusionsOur results show a high concordance between plasma and sputum for EGFR L858R and T790M mutation testing. A lower concordance was observed for EGFR del19. Thus liquid biopsy from sputum is a promising strategy that should be further evaluated. Clinical trial identificationGS1-EK4/479-2017, 20.07.2017 EK-18-172-0918, 08.11.2018. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

P2.03-05 PHLPP1 Expression Through AKT and ERK Dual Signaling Pathways May Slow Down the Resistance to TKI in EGFR-Mutated Lung Adenocarcinoma

The epidermal growth factor receptor (EGFR) kinase inhibitors are effective treatments for lung cancers with EGFR activating mutations, but the magnitude of tumor regression varies and drug resistance is unavoidable. Multiple mechanisms of resistance to EGFR-TKIs have been identified, including the occurrence of secondary mutations in the EGFR gene, MET amplification, acquired BRAF rearrangements and activation of bypass pathways. Central to these mechanisms of resistance is the re-activation of AKT and ERK signaling, which enables escape of tumor cells from EGFR inhibitor treatment. However, the mechanisms of reactivation of PI3K-AKT and ERK/MAPK pathway are unclear. Pleckstrin homology (PH) domain leucine-rich repeat protein phosphatase (PHLPP) acts as tumor suppressors in various types of human cancer by suppressing cell survival pathways and promoting apoptosis through inhibiting AKT and ERK pathway activation. Here, we hypothesize that PHLPP is a key regulator of EGFR-TKI resistance in lung cancer and a potential treatment target for overcoming resistance to EGFR-TKI treatment. A transcriptomic of PHLPP1 in non-small lung cancer cell according to gefitinib sensitivity obtained from Gene Expression Omnibus (GEO) database under accession number GSE4342 were analysis. The lentivirus-mediated delivery of shRNA was used to generated stable knockdown of PHLPP1 expression lung cancer cells, and retrovirus-mediated delivery was used to generated stable overexpression of PHLPP1 lung cancer cells. Western blotting, real-time PCR (RT-PCR) and immunofluorescence were used to determined PHLPP expression in vitro. PHLPP1 expression in clinical sample was determined by Immunohistochemical (IHC) staining. MTT assay was conducted to determine the cell proliferation. Xenografts bearing PHLPP overexpression and control were evaluated EGFR-TKI induced tumor regression. PHLPP1 gene expression was higher in gefitinib-sensitive NSCLC cell lines than gefitinib-resistant NSCLC cell lines from a GEO public database. In vitro, EGFR mutated NSCLC cell line HCC827 continuously exposing to gefitinib exhibited dramatically reduced expression of PHLPP1 and increased phosphorylation AKT and ERK. Knockdown of PHLPP1 decreased cell death induced by the EGFR-TKI in EGFR-mutant lung cancer cells, overexpression of PHLPP1 enhanced gefitinib-induced apoptosis in gefitinib-resistance EGFR-mutant lung cancer cells. In xenograft model, overexpression of PHLPP showed significantly more tumor regression after gefitinib treatment at 1-week time point compared to control group. In patients, PHLPP1 were highly expressed in tumors with EGFR common mutations pre- and post-development of resistance to EGFR TKIs. PHLPP1 expression were down regulated in the post-relapse tumor samples compared to that of pre-treatment, and patients with higher PHLPP1 expression in pre-treatment had significantly longer progression-free survival (PFS). PHLPP loss may be a key molecule contributing to the resistance of EGFR-TKI by activating PI3K-AKT and ERK/MAPK signaling pathway. PHLPP may serve as a potential predictor of EGFR-TKI treatment response in these patients. Up-regulating PHLPP expression may prevent or /and delay the emergence of EGFR-TKI resistance. Further study is warranted to prove PHLPP as an effective strategy.

P1.14-44 Positive PD-L1 Expression Is Associated with Unfavorable Clinical Outcome in EGFR-Mutated Lung Adenocarcinomas Treated with EGFR-TKIs

Clinical implication of programmed death ligand 1 (PD-L1) expression in oncogene-addicted non-small cell lung cancer treated with tyrosine kinase inhibitors (TKIs) is largely unknown. The objective of this study was to determine the frequency of PD-L1 expression and the clinical outcome according to PD-L1 expression in lung adenocarcinomas harboring EGFR mutation and treated with EGFR-TKIs. We retrospectively evaluated PD-L1 expression using 22C3 pharmDx assay in lung adenocarcinoma patients with EGFR mutations at two referral hospitals between January 2017 and June 2018. Samples were obtained from surgically resected tumors, small biopsy, or cytologic cell blocks. Of all 71 patients analyzed, 44 (58.7%) had PD-L1 tumor proportion score (TPS) of < 1%, 23 (30.7%) had PD-L1 TPS of 1%-49%, and 8 (10.7%) had PD-L1 TPS of ≥ 50%. Of the 37 patients treated with first-line EGFR-TKIs, PD-L1 TPS ≥ 1% was associated with a significantly decreased response rate, compared with PD-L1 TPS < 1% (45.7% vs. 67.3%, p=0.005). Furthermore, PD-L1 TPS ≥ 1% was associated with a significantly shorter median progression free survival, compared with PD-L1 TPS < 1% (9.3 months vs. 14.2 months, p = 0.024). Multivariate analysis showed that PD-L1 TPS ≥1% is independently associated with shorter PFS (hazard ratio 1.32, p=0.012). PD-L1 was positive in approximately 40% of patients with EGFR-mutated lung adenocarcinoma. Positive PD-L1 expression may be associated with unfavorable clinical outcome to EGFR-TKIs among those patients group.

EP1.01-40 Outcome of EGFR-Mutated and Non-Mutated Lung Adenocarcinoma Receiving Standard Therapy - A Nepalese Cohort

EP1.01-40 Outcome of EGFR-Mutated and Non-Mutated Lung Adenocarcinoma Receiving Standard Therapy - A Nepalese Cohort

P1.14-39 Acquired ALK Rearrangement in EGFR-Mutant Lung Adenocarcinoma Treated with EGFR TKIs

P1.14-39 Acquired ALK Rearrangement in EGFR-Mutant Lung Adenocarcinoma Treated with EGFR TKIs

EP1.01-13 A Phase 2 Trial Assessing Osimertinib Activity Against Leptomeningeal Carcinomatosis in EGFR-Mutant Lung Cancer

Leptomeningeal carcinomatosis (LMC) occurs in 10 % of EGFR-mutant lung adenocarcinoma (LA). Retrospective studies have suggeted a clinical benefit of osimertinib in treating LMC in patients with EGFR-mutant LA. We conducted a phase 2 prospective trial to demonstrate the clinical efficacy, activity, and LMC-specific mechanisms of resitance to osimertinib in EGFR-mutant LA patients. We performed gadalidium-enhanced brain MRIs at the time of enrollment, followed by scheduled brain imaging once every six weeks. Clinical neurological exams were performed every four weeks. Adverse Events (AEs) were assessed by NCI-CTCAE Ver. 4. Cerebral spinal fluid (CSF) and plasma was obtained on day 1 and 21 for all patients. The CSF osimertinib penetration rate was measured by liquid-chromatography mass spectrometry (LC-MSMS) and compared to plasma osimertinib levels. Targeted droplet digital PCR (ddPCR) was used to detect copy number changes of Met and the EGFR C797S mutation in patients CSF-DNA and plasma cell free (cf) DNA. We enrolled six pre EGFR-TKI treated EGFR mutant LA with LMC. We observed a LMC-specific progression free survival of 3.7 months and overall survival of 6.2 months or longer with osimertinib. Four and three of six patients showed neurological and radiological responses, respectively. No patients had any AEs greater than grade 3 and all patients continued osimertinib beyond PD. The osimertinib CSF penetration rate was 1.2±0.5%. Met copy number was normal in plasma, but increased in the CSF of 2/6 patients prior to progression. Met copy number was confirmed in 1/6 patients at the time of progression. An aquired EGFR C797S mutation was observed in 1/6 patients prior to progression.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Osimertinib 80 mg is efficacious in the treatment of LMC in EGFR mutated LA. CSF-specific MET copy number gain and EGFR C797S mutations arise prior to progression in EGFR-mutant LA patients treated with osimeritinib.

P2.16-26 Opportunity of WBRT in EGFR-Mutated Lung Adenocarcinoma Patients with Multiple Brain Metastases: A Retrospective Study Based on DS-GPA

P2.16-26 Opportunity of WBRT in EGFR-Mutated Lung Adenocarcinoma Patients with Multiple Brain Metastases: A Retrospective Study Based on DS-GPA

P1.01-57 Association of Initial PD-L1 Expression with T790M-Acquired Resistance in Advanced EGFR-Mutant Lung Adenocarcinoma Patients

The primary objective was to investigate the association between initial programmed cell death-ligand 1 (PD-L1) expression levels and the frequency of T790M-acquired resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase Inhibitor (TKI) in patients with advanced EGFR-mutant lung adenocarcinoma. We examined patients with advanced EGFR-mutant lung adenocarcinoma whose initial PD-L1 expression levels and T790M-acquired mutation status after EGFR-TKI failure (gefitinib, erlotinib or afatinib) could be evaluated. We retrospectively investigated the patients’ characteristics and survival, as well as the association between initial PD-L1 expression levels and frequency of T790M-acquired resistance Overall, 100 patients were enrolled. Nine (9%) patients had an initial PD-L1 tumor proportion score (TPS) of ≥50%, 19(19%) had PD-L1 TPS of 1%-49%, and 72 (72%) had PD-L1 TPS of <1%. T790M-acquired mutation (T790M+) was identified in 57 (57%) patients. Initial PD-L1 expression levels were not associated with the frequency of T790M-acquired mutations in patients (p=0.822). Positive PD-L1 expression (PD-L1+) was associated with lower OS compared with negative PD-L1 expression (PD-L1-) (median overall survival [OS], 40.3 vs 74.3 months, p=0.0053). Furthermore, T790M+ was associated with longer OS compared with T790M- in total and PD-L1- population (total: median OS, 74.3 vs 41.3 months, p=0.0154, PD-L1-: median OS, 82.0 vs 41.2 months, p=0.00412), but not in PD-L1+ population (median OS, 36.2 vs 46.2 months, p=0.792). Among 57 patients with T790M-aquired mutation, 49 received osimertinib treatment. The estimated median progression-free survival rate of osimertinib was 13.2 months in PD-L1- patients (n=37) and 6.9 months in PD-L1+ patients (n=12) (p=0.224). There was no association between initial PD-L1 expression levels and the frequency of T790M-aquired mutations in patients. However, PD-L1+ expression levels in patients with treatment-naïve advanced EGFR-mutant lung adenocarcinoma predicted poorer outcomes. Intriguingly, T790M-aquired mutations were related to longer OS in PD-L1- patients, but not in PD-L1+. Thus, for patients with T790M-acquired mutant and initial PD-L1 positive lung adenocarcinoma in whom the use of osimertinib is indicated, other treatment options should be considered without hesitation if the treatment effects are not as expected.

P2.03-03 Landscape of Gene Fusions in Lung Adenocarcinoma Patients with Minimal Cigarette Exposure Identified on Malignant Pleural Effusions

Gene fusions in lung adenocarcinoma (LuAD) involving tyrosine kinase receptors such as ROS1, ALK or RET are recurrent oncogenic drivers (∼10%), enriched in light or never-smokers. Some of them represent emerging and predictive biomarkers for targeted therapies. Here we report the fusions detected in a cohort of metastatic LuAD patients with low tobacco exposure (never or former-smokers). Patient-derived cancer cell lines (PDC) were successfully established from malignant pleural effusions from 11 patients diagnosed with LuAD. We assessed the genetic and molecular profile by whole-exome sequencing (WES) and RNA sequencing (RNA-seq) in each cell line. Patients’ characteristics: median age, 58 (39-86); 9 were female. Eight of eleven were never-smokers and three, former-smokers. Seven patients were treatment naïve when pleural effusion samples were collected. A cytological examination of pleural fluid was performed by a lung pathologist and all samples were positive for malignant cells. Known driver mutations in lung primary tumours included one ALK translocation detected by FISH and three EGFR Del19 mutations by targeted sequencing. The three EGFR-mutant LuAD patients progressed to first or second-generation EGFR-TKI and we were able to stablish paired PDC after progressing to tyrosine kinase inhibitors (TKI) in two of them. We identified an acquired FGFR3-TACC3 fusion in one paired PDC after gefitinib progression (T790M-negative), that led to overexpression of FGFR3 concurrent with an enrichment of squamous cell lineage transcripts (e.g. TP63, SOX2) and MDM2 amplification. Among EGFR wild type (wt) patients, two RET rearrangements, CCDC6-RET and KIF5B-RET, and one EML4-ALK fusion -also detected in the primary tumour- were identified in PDC models. In addition, in two of the samples we discovered novel gene fusions that will be described in detail, involving proteins that are not kinases, and thus, their potential role in cancer is still unknown. In this cohort enriched with never-smoking LuAD patients presenting pleural effusions at diagnosis, the presence of known driver fusions during the disease’s course detected by RNA-Seq was 36% (4/11), including a FGFR3-TACC3 fusion as an acquired resistance mechanism to EGFR-TKI. Further study is ongoing in our PDC models to test the functional role of these fusions in order to facilitate precision medicine.

ES11.01 Lung Adenocarcinoma to Squamous Cell Carcinoma Transdifferentiation and Drug Resistance

Lung cancer is notorious for high heterogeneity and strong plasticity, which might contribute to the development of drug resistance. Lineage transition from lung adenocarcinoma (ADC) to squamous cell carcinoma (SCC), as implicated by clinical observation of mixed ADC and SCC pathologies in adenosquamous cell carcinoma (Ad-SCC), reflects strong cancer plasticity and potentially links to drug resistance. Using Genetically Engineered Murine Model (GEMM), we have provided in vivo evidence in supporting the ADC to SCC transdifferentiation (AST): Lkb1-deficient mouse lung ADC transdifferentiates into SCC progressively via pathologically mixed Ad-SCC. Mechanistically, we find that down-regulation of reactive oxygen species (ROS) level through N-acetyl cysteine (NAC) treatment or NRF2 expression inhibits this transition, highlighting the functional importance of ROS in regulating cancer plasticity. Pentose phosphate pathway deregulation and impaired fatty acid oxidation collectively contribute to the redox imbalance and functionally affect the AST process. Importantly, similar tumor and redox heterogeneity are also found in human LKB1-inactivated lung cancer. In preclinical trials toward metabolic stress, Lkb1-inactivated ADC can develop drug resistance through squamous transdifferentiation. Recent observations in clinic further suggest that such pathological transition might be responsible for resistance to tyrosine kinase inhibitor (TKI) therapy and chemotherapy in relapsed EGFR-mutant lung ADC patients. These findings demonstrate that lung cancer plasticity potentially affects therapeutic response and precision medicine through histological transition.

Additional bevacizumab in EGFR-mutant lung adenocarcinoma patients who had oligo-progression after the failure of EGFR-TKI: A single-institute retrospective study

IntroductionIn EGFR-mutant NSCLC patients with oligo-progression disease (oligo-PD) after the EGFR-TKI failure, additional local ablative therapy (LAT) including stereotactic ablative radiotherapy reportedly extends the duration of the current EGFR-TKI and prolongs survival times. In clinical practice, however, all the patients cannot receive LAT for oligo-PD. MethodsWe retrospectively evaluated the efficacy and tolerability of additional bevacizumab as an alternative to LAT for oligo-PD after the EGFR-TKI failure in previously treated lung adenocarcinoma patients (median number of previous therapies, 2 regimens). Oligo-PD was defined as a situation in which disease progression has occurred in less than 5 anatomical sites after EGFR-TKI that has achieved at least stable disease. ResultsDuring a median 29.6-month follow-up period from the initiation of EGFR-TKI, 9 patients developed oligo-PD. One patient underwent LAT, but other 8 patients did not because of a few micro-metastatic lesions (n = 2), meningitis (n = 1), no indication of pleurodesis (n = 1), patient refusal (n = 2) or oligo-PD in the LAT treated sites (n = 3). Additional bevacizumab with continuation of the current EGFR-TKI had a disease control rate of 100% and a median time of progression-free survival from additional bevacizumab until another PD was 8.8 months. The reason for the discontinuation was because of another PD (n = 6) or treatment-related adverse events (n = 3). Four patients received sequential therapy and overall survival from additional bevacizumab was 10.1 months. ConclusionsAdditional bevacizumab could be useful for EGFR-mutant adenocarcinoma patients with oligo-PD after the EGFR-TKI failure.

The influence of tumor heterogeneity on sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs

Background: Studies have shown that extensive genetic or spatial heterogeneity is present within tumors. The present study explored the influence of heterogeneous cells in the tumor microenvironment (TME) on the sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs and further investigated associated molecular mechanisms. Methods: Tumor heterogeneity was simulated using transwell co-culture technique with H1975, A549 or MRC-5 cells grown in the upper chambers and PC-9 cells cultured in the bottom chamber. Each co-culture system was grouped based on different proportions of cells in the upper and lower chambers (from 1% to 300%), and each group was subdivided into erlotinib treatment group (erlotinib+) and erlotinib non-treatment group (erlotinib−). The viability of PC-9 cells was analyzed by CCK-8; HGF, IGF-1 and TGF-α were determined by ELISA; MET amplification was detected by qRT-PCR, and the relevant proteins were detected by Western blot. Results: The viability of PC-9 cells increased with increased proportion of A549/PC-9 or MRC-5/PC-9 cells from 1% to 300%. HGF increased with increased proportion of H1975 cells from 1% to 300%. In all three co-culture systems, TGF-α production in the erlotinib+ subgroup was significantly lower than that in erlotinib- subgroup, and phosphorylated AKT protein showed an ascending tendency with the increased proportion of upper chamber cells relative to PC-9 cells from 1% to 300%. H1975 cells could induce MET amplification of PC-9 cells. MRC5 cells inhibited MET amplification. Conclusions: Tumor heterogeneity partially accounts for the resistance of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs. The possible mechanisms may be related to AKT signaling pathways activated by growth factors, which are secreted by heterogeneous cells in the TME under the pressure of EGFR-TKIs.

The influence of tumor heterogeneity on sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs.

BackgroundStudies have shown that extensive genetic or spatial heterogeneity is present within tumors. The present study explored the influence of heterogeneous cells in the tumor microenvironment (TME) on the sensitivity of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs and further investigated associated molecular mechanisms.MethodsTumor heterogeneity was simulated using transwell co-culture technique with H1975, A549 or MRC-5 cells grown in the upper chambers and PC-9 cells cultured in the bottom chamber. Each co-culture system was grouped based on different proportions of cells in the upper and lower chambers (from 1% to 300%), and each group was subdivided into erlotinib treatment group (erlotinib+) and erlotinib non-treatment group (erlotinib−). The viability of PC-9 cells was analyzed by CCK-8; HGF, IGF-1 and TGF-α were determined by ELISA; MET amplification was detected by qRT-PCR, and the relevant proteins were detected by Western blot.ResultsThe viability of PC-9 cells increased with increased proportion of A549/PC-9 or MRC-5/PC-9 cells from 1% to 300%. HGF increased with increased proportion of H1975 cells from 1% to 300%. In all three co-culture systems, TGF-α production in the erlotinib+ subgroup was significantly lower than that in erlotinib- subgroup, and phosphorylated AKT protein showed an ascending tendency with the increased proportion of upper chamber cells relative to PC-9 cells from 1% to 300%. H1975 cells could induce MET amplification of PC-9 cells. MRC5 cells inhibited MET amplification.ConclusionsTumor heterogeneity partially accounts for the resistance of EGFR-mutant lung adenocarcinoma cells to EGFR-TKIs. The possible mechanisms may be related to AKT signaling pathways activated by growth factors, which are secreted by heterogeneous cells in the TME under the pressure of EGFR-TKIs.

BSCI-26. COMPARATIVE METHYLATION PROFILING OF EGFR MUTANT LUNG ADENOCARCINOMA AND PAIRED BRAIN METASTASIS

BACKGROUND: Adenocarcinomas (ADC) are the most common lung tumours and EGFR-mutant lesions have a higher risk of brain metastasis development, which confers poorer survival. Genetic and epigenetic signatures of brain metastasis have not been comprehensively identified. The aim of this study is to compare the methylome of EGFR-mutant primary lung tumor and matched brain metastasis to identify mechanisms of brain metastasis and new treatment targets. METHOD: Seven matched primary to brain metastasis tumours were profiled using the Illumina Infinium MethylationEPIC BeadChip array. Hierarchical clustering and principal component analyses (PCA) were performed using most variable CpG sites. Supervised analyses were performed between lung and brain tumour samples. Copy number variation (CNV) plots identified alterations between pairs along with Leukocytes unmethylation for purity (LUMP) score and prediction lymphocyte proportion analyses to measure immune infiltration. RESULTS: Unsupervised clustering showed that the fourteen tumours clustered according to patient with similar methylation profiles between each of seven matched pairs. On the supervised analysis using 83K significant CpG sites, the fourteen samples clustered into two groups based on tumour site being lung or brain. Of these 83K CpG sites, 2.4K were either hypermethylated or hypomethylated in all lung samples. One quarter of these 2.4K CpG sites were located in promoter regions. CNV analyses showed losses of FGFR1, C19MC, CDKN2A, PTCH1, and MYCN genes with higher deep deletions in brain versus lung primary samples. Immune infiltration measures were similar between lung and brain metastasis pairs (LUMP-score=0.64) consistent with high immune cell infiltration. CONCLUSION: In this EGFR-mutant lung adenocarcinomas and matched brain metastases, differentially methylated CpG sites and CNV alterations are identified that distinguish lung from brain samples. Further work with additional matched samples may further elucidate signatures specific to brain metastasis and aid in our understanding of the mechanisms of brain metastasis.

Combination therapy of brain radiotherapy and EGFR-TKIs is more effective than TKIs alone for EGFR-mutant lung adenocarcinoma patients with asymptomatic brain metastasis

BackgroundThe treatment strategy for brain metastasis (BM) in patients with epidermal growth factor receptor (EGFR) -mutant lung adenocarcinoma (LAC) remains controversial. In the present study, we compared the efficacy of brain radiotherapy (RT) in combination with tyrosine kinase inhibitors (TKIs) and TKIs alone for advanced LAC patients with EGFR mutations and BM.MethodsWe retrospectively studied 78 patients diagnosed with EGFR-mutant LAC who developed BM. These patients were divided into two groups: 49 patients in the combination treatment group who received brain RT in combination with EGFR-TKIs (including 23 patients with asymptomatic BM before RT); 29 patients in the TKI group who received EGFR-TKI targeted therapy alone (including 22 patients with asymptomatic BM before TKI treatment).ResultsThe median intracranial progression-free survival (iPFS) of the combination treatment group was longer than that of the TKI alone group (21.5 vs. 15 months; P = 0.036). However, there were no significant differences in median progression-free survival (PFS, 12 vs. 13 months; P = 0.242) and median overall survival (mOS, 36 vs. 23 months; P = 0.363) between the two groups. Further analysis of asymptomatic BM showed that both the median iPFS and the mOS of the combination treatment group were significantly longer than for the TKI alone group (iPFS, 21.5 vs. 14.8 months, P = 0.026; mOS, 36 vs. 23 months, P = 0.041). Cox multivariate regression analysis found no independent adverse predictors of iPFS in all patients.ConclusionsThe synchronous combination of brain RT and TKIs was superior to EGFR-TKIs alone for EGFR-mutant LAC patients with BM. The combination treatment group exhibited longer iPFS, while the PFS and OS were not significantly different between the two groups. In addition, the combination treatment could result in better iPFS and OS in those with asymptomatic BM. Therefore, addition of brain RT was useful for intracranial metastatic lesions.