The Prognosis Of Segmentectomy And Adjuvant EGFR TKI Treatment For Early-stage Non-small-cell Lung Cancer And The Mechanisms Of 3^rd-generation TKI Resistance

BACKGROUND:The wide use of low-dose computed tomography for screening has led to a dramatic increase of early-detected lung cancer with small-size.These patients are mostly clinical node-negative,and are potential candidates for sublobar resection.It remains unclear whether the histologic subtypes of invasive adenocarcinoma impact the survival of patients undergoing sublobar resection,and whether lymphadenectomy during segmentectomy could improve survival.It has been demonstrated that adjuvant chemotherapy could improve the survival of patients after complete resection;epidermal growth factor receptor（EGFR）tyrosine kinase inhibitors（TKIs）show great advantage in advanced non-small cell lung cancer（NSCLC）patients,but its efficacy in the adjuvant treatment for operable patients is controversial in previous studies.Osimertinib（AZD9291）is a potent,irreversible 3^rd generation EGFR TKI for NSCLC patients harboring EGFR mutations,and the majority develop resistance to the treatment after 10-20 months.However,the resistant mechanisms are unknown among about 40%of the patients.PATIENTS AND METHODS:Part Ⅰ:Clinicopathologic data and outcomes of 3669 consecutive patients with stage I lung adenocarcinoma of 3 cm of smaller treated in Shanghai Chest Hospital were retrospectively analyzed,including 226 segmentectomy,472 wedge resection and 2971 lobectomy.A histology-based risk classification was proposed for stratification analysis.Survival was assessed by Kaplan-Meier method and Cox regression analysis.Part II:The clinicopathologic data of patients undergoing segmentectomy for NSCLC from July 2011 to December 2014 were retrospectively analyzed.Survival analysis was performed by Kaplan-Meier method and Cox regression analysis.Part III:The study advanced the previous meta-analysis and included a comprehensive range of relevant studies in PubMed.Disease-free survival（DFS）with hazard ratios（HRs）was calculated using random and/or fixed-effects models.Subgroup analysis and meta-regression analysis were also performed.Part IV:We used the genetically engeering mouse models the mutant EGFR,to investigate whether L718Q mutation could lead to osimertinib resistance in vivo.We continuously treated the EGFR^L858R-T790M（EGFR TL）mouse model with osimertinib （5mg/kg,P.O.,Q.D.）,to establish the acquired osimertinib-resistant mouse model and investigate the novel resistant mechanisms.RESULTS:Part Ⅰ:Compared with lobectomy,the 3-year cumulative incidence of recurrence of segmentectomy was equivalent（3.4%vs.6.9%,Gray’s test P=0.076）,and wedge resection was almost three-fold higher（18.1%,Gray’s test P<0.001）.On multivariable analysis,wedge resection was significantly associated with inferior recurrence-free survival（RFS）（hazard ratio[HR],2.98;95%confidence interval[CI],2.23-3.98）,but segmentectomy wasn’t（HR,1.21;95%CI,0.56-2.59）.In the low-risk subgroup,RFS among the three procedures were indistinguishable（log-rank P=0.73）.In the intermediate and high-risk subgroups,segmentectomy also had equivalent RFS to lobectomy（log-rank P=0.87 and 0.79,respectively）,while,wedge resection had significantly worse RFS（log-rank P<0.001 and P=0.002,respectively）,and locoregional recurrence-free survival（log-rank P=0.013 and P<0.001,respectively）.Propensity-score analysis further confirmed the equivalence of segmentectomy and lobectomy regardless of histology（log-rank P=0.37）.Part II:A total of 259patients with NSCLC were eligible for analysis.Patients with harvested lymph node（LN）≥6 had higher frequency of nodal metastasis in pathologic examination（9.4%vs.1.5%,P=0.005）.The 3-year recurrence-free survival（RFS）of patients with harvested LN≥6（92.8%）was significantly higher than that of patients with harvested LN<6（73.7%,log-rank P=0.022）.Multivariable Cox analysis indentified harvested LN≥6 as an independent predictor for improved RFS[hazard ratio（HR）,0.29;95%confidence interval（CI）,0.11-0.77;P=0.014].There was no significant difference in RFS between patients with harvested LN station≥3 and<3（log-rank P=0.34）.Part III:A total of 2223 patients in seven studies were eligible for the analysis.Adjuvant EGFR-TKIs administration was significantly associated with superior DFS（HR,0.60;95%CI,0.42-0.87）,corresponding to an absolute benefit of 3.4%at 3 years,yet with significant heterogeneity（I²=80.0%,P<0.001）.EGFR mutation rate of included patients was found to be a source of heterogeneity by meta-regression analysis（P=0.005）.In the EGFR-mutant sub-population,HR for DFS was 0.51（95%CI,0.39-0.65）,corresponding to an absolute benefit of 7.1%at 3 years.The rate of overall grade 3 or greater adverse events was 38.9%（95%CI,35.9-41.9%）.Part IV:Mice with EGFR^{L858R-T790M-L718Q}（EGFR TLL）mutant developed lung tumors after 22-25 weeks of Adeno-Cre induction,which were diagnosed with adenocarcinoma in HE-statining slides.The lung tumor burderns of EGFR TLL mice increased significantly within 6-week treatment of osimertinib（259%,93%and 228%,respectively）,while tumors of EGFR TL mice dramtically regressed after treatment（72%and 87%,respectively）.IHC statining showed that osimertinib could not inhibit the phosphorylation of EGFR,AKT and ERK in EGFR TLL tumors.Afatinib could not reverse the resistance caused by L718Q mutation.EGFR TL mice developed acquired resistance to osimertinib after treatment for 9-12 months,and subcutaneously implanted tumors in nude mice remained resistant to osimertinib.Histology examination showed that the resistant tumors were adenocarcinoma,no evidence of small cell caner transformation or epithelial to mesenchymal transition（EMT）.Sanger sequencing of EGFR gene showed that there was no loss of T790M mutation,no C797S,L718Q or other novel mutations.No significant EGFR,HER2 or MET gene amplification was detected in the resistant tumors.CONCLUSIONS:For early-stage invasive lung adenocarcinoma,segmentectomy could offer comparable oncologic outcomes with lobectomy,and wedge resection should be indicated only in lepidic predominant adenocarcinoma without high-architectural grade patterns.The number of harvest LN≥6 was independently associated with improved RFS for NSCLC patients undergoing segmentectomy,supporting the NCCN guidelines of appropriate LN sampling.The meta-analysis provided strengthened evidence of significant DFS advantage of adjuvant EGFR-TKI treatment for patients with EGFR-mutant NSCLC after complete resection.L718Q mutation is a novel resistant mechanism to osimertinib in vivo,which could not be overcome by current EGFR TKIs.Continuous treatment of osimertinib for EGFR TL mice was a feasible method to establish acquired resistant model and the resistance was not caused by currently known mechanisms,and RNA-seq might help to discover novel osimertinib resistant mechanisms.