Background Third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as osimertinib are the last line of targeted treatment of metastatic non-small-cell lung cancer (NSCLC) EGFR-mutant harboring T790M. Different mechanisms of acquired resistance to third-generation EGFR-TKIs have been proposed. It is therefore crucial to identify new and effective strategies to overcome successive acquired mechanisms of resistance. Methods For Amplicon-seq analysis, samples from the index patient (primary and metastasis lesions at different timepoints) as well as the patient-derived orthotopic xenograft tumors corresponding to the different treatment arms were used. All samples were formalin-fixed paraffin-embedded, selected and evaluated by a pathologist. For droplet digital PCR, 20 patients diagnosed with NSCLC at baseline or progression to different lines of TKI therapies were selected. Formalin-fixed paraffin-embedded blocks corresponding to either primary tumor or metastasis specimens were used for analysis. For single-cell analysis, orthotopically grown metastases were dissected from the brain of an athymic nu/nu mouse and cryopreserved at -80°C. Results In a brain metastasis lesion from a NSCLC patient presenting an EGFR T790M mutation, we detected MET gene amplification after prolonged treatment with osimertinib. Importantly, the combination of capmatinib (c-MET inhibitor) and afatinib (ErbB-1/2/4 inhibitor) completely suppressed tumor growth in mice orthotopically injected with cells derived from this brain metastasis. In those mice treated with capmatinib or afatinib as monotherapy, we observed the emergence of KRAS G12C clones. Single-cell gene expression analyses also revealed intratumor heterogeneity, indicating the presence of a KRAS-driven subclone. We also detected low-frequent KRAS G12C alleles in patients treated with various EGFR-TKIs. Conclusion Acquired resistance to subsequent EGFR-TKI treatment lines in EGFR-mutant lung cancer patients may induce genetic plasticity. We assess the biological insights of tumor heterogeneity in an osimertinib-resistant tumor with acquired MET-amplification and propose new treatment strategies in this situation.

中文翻译：

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MET和ERBB双重抑制克服了奥希替尼耐药晚期非小细胞肺癌（NSCLC）的肿瘤内可塑性。

背景 第三代表皮生长因子受体酪氨酸激酶抑制剂 (EGFR-TKI) 如奥希替尼是靶向治疗携带 T790M 的转移性非小细胞肺癌 (NSCLC) EGFR 突变体的最后一线药物。已经提出了对第三代 EGFR-TKI 获得性耐药的不同机制。因此，确定新的有效策略来克服连续获得的耐药机制至关重要。方法 对于 Amplicon-seq 分析，使用来自指示患者（不同时间点的原发性和转移性病变）以及对应于不同治疗组的患者来源的原位异种移植肿瘤的样本。所有样品均经福尔马林固定石蜡包埋，由病理学家选择和评估。对于微滴式数字 PCR，选择了 20 名在基线时诊断为 NSCLC 或进展到不同 TKI 治疗线的患者。对应于原发性肿瘤或转移标本的福尔马林固定石蜡包埋块用于分析。对于单细胞分析，从无胸腺 nu/nu 小鼠的大脑中解剖原位生长的转移瘤，并在 -80°C 下冷冻保存。结果 在 NSCLC 患者的脑转移病灶中，呈现 EGFR T790M 突变，我们在长期使用奥希替尼治疗后检测到 MET 基因扩增。重要的是，卡马替尼（c-MET 抑制剂）和阿法替尼（ErbB-1/2/4 抑制剂）的组合完全抑制了原位注射源自这种脑转移的细胞的小鼠的肿瘤生长。在那些用卡马替尼或阿法替尼作为单一疗法治疗的小鼠中，我们观察到 KRAS G12C 克隆的出现。单细胞基因表达分析还揭示了肿瘤内异质性，表明存在 KRAS 驱动的亚克隆。我们还在接受各种 EGFR-TKI 治疗的患者中检测到低频 KRAS G12C 等位基因。结论 EGFR 突变肺癌患者对后续 EGFR-TKI 治疗线的获得性耐药可能诱导遗传可塑性。我们评估了具有获得性 MET 扩增的奥希替尼耐药肿瘤中肿瘤异质性的生物学见解，并在这种情况下提出了新的治疗策略。结论 EGFR 突变肺癌患者对后续 EGFR-TKI 治疗线的获得性耐药可能诱导遗传可塑性。我们评估了具有获得性 MET 扩增的奥希替尼耐药肿瘤中肿瘤异质性的生物学见解，并在这种情况下提出了新的治疗策略。结论 EGFR 突变肺癌患者对后续 EGFR-TKI 治疗线的获得性耐药可能诱导遗传可塑性。我们评估了具有获得性 MET 扩增的奥希替尼耐药肿瘤中肿瘤异质性的生物学见解，并在这种情况下提出了新的治疗策略。