Aims

Presently, three generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are approved against oncogene addicted EGFR-mutant non-small cell lung cancer (NSCLC). Patients with actionable EGFR mutations invariably develop resistance. This resistance can be intrinsic (primary) or acquired (secondary).

Materials and methods

This was a retrospective study carried out between January 2016 and April 2021 analysing 486 samples of NSCLC for primary and secondary resistance to first- (erlotinib, gefitinb), second- (afatinib) and/or third-generation (osimertinib) TKIs in EGFR-mutant NSCLCs by next generation sequencing (NGS). Tissue NGS was carried out using the Thermofischer Ion Torrent™ Oncomine™ Focus 52 gene assay; liquid biopsy NGS was carried out using the Oncomine Lung Cell-Free Total Nucleic Acid assay. All cases were previously tested for a single EGFR gene with the Therascreen® EGFR RGQ PCR kit.

Results

The results were divided into four groups: (i) group 1: primary resistance to first- and/or second-generation TKIs. This group, with 21 cases, showed EGFR exon 20 insertions, dual, complex mutations and variant of unknown significance, de novo MET gene amplification besides other mutations. (ii) Group 2: primary resistance to third-generation TKIs. This group showed two cases, with one showing dual EGFR mutation (L858R and E709A) and EGFR gene amplification. (iii) Group 3: secondary resistance to first- and second-generation TKIs. This group had 27 cases, which were previously reported negative for EGFR T790M by single gene testing. Significant findings were MET gene amplification in four cases, with one also showing MET exon 14 skipping mutation. Three cases showed small cell change and one showed loss of primary mutation. (iv) Group 4: secondary resistance to third-generation TKIs. The latter group was further subgrouped into group 4A: secondary resistance to osimertinib (third-generation TKI) when offered as second-line therapy after first- and second-generation TKIs on detection of T790M mutation. This group had 15 cases. EGFR T790M mutation was lost in 10 (10/15; 67%) cases and was retained in five cases. Patients with T790M loss experienced early resistance (6.9 months versus 12.6 months mean, P = 0.0024) compared with cases that retained T790M. Two cases gained MET amplification as the resistance mechanisms. Other mutations that were found when EGFR T790M was lost were in FGFR3, KRAS, PIK3CA, CTNNB1, BRAF genes. One case had EML4-ALK translocation. Two cases showed driver EGFR deletion 19, retained T790M and C797S mutation in Cis form. Group 4B: secondary resistance to osimertinib (when given as first-line therapy) in EGFR-mutant NSCLC. This group had three cases. The duration of osimertinib treatment ranged from 11 to 17 months. Two patients showed additional C797S mutation along with primary EGFR mutation.

Conclusion

This study shows the wide spectrum of primary and secondary EGFR resistance mechanisms to first, second and third generation of TKIs and helps us to identify newer therapeutic targets that could carry forward the initial advantage offered by EGFR TKIs.

中文翻译：

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三级中心已知表皮生长因子受体突变型非小细胞肺癌患者对各代酪氨酸激酶抑制剂原发性和继发性耐药的分子机制的审核

宗旨

目前，三代表皮生长因子受体 (EGFR) 酪氨酸激酶抑制剂 (TKI) 被批准用于治疗致癌基因成瘾的 EGFR 突变非小细胞肺癌 (NSCLC)。具有可操作的 EGFR 突变的患者总是会产生耐药性。这种阻力可以是内在的（主要的）或获得的（次要的）。

材料和方法

这是一项在 2016 年 1 月至 2021 年 4 月期间进行的回顾性研究，分析了 486 个 NSCLC 样本对 EGFR 中的第一代（厄洛替尼、吉非替尼）、第二代（阿法替尼）和/或第三代（奥希替尼）TKI 的原发性和继发性耐药性通过下一代测序(NGS)检测突变的 NSCLC 。使用 Thermofischer Ion Torrent™ Oncomine™ Focus 52 基因检测进行组织 NGS；使用 Oncomine 肺无细胞总核酸测定进行液体活检 NGS 。所有病例之前都使用 Therascreen® EGFR RGQ PCR 试剂盒对单个 EGFR 基因进行了检测。

结果

结果分为四组：(i) 第 1 组：对第一代和/或第二代 TKI 的原发性耐药。本组 21 例，显示 EGFR 外显子 20 插入，双重复杂突变和意义不明的变异，从头MET 基因扩增除了其他突变。(ii) 第 2 组：对第三代 TKI 的主要耐药性。本组病例2例，1例EGFR双重突变（L858R和E709A）和EGFR基因扩增。(iii) 第 3 组：对第一代和第二代 TKI 的继发性耐药。本组27例，既往单基因检测EGFR T790M阴性。重要发现是 4 例 MET 基因扩增，其中 1 例还显示 MET 外显子 14 跳跃突变。3例表现为小细胞改变，1例表现为原发突变丢失。(iv) 第 4 组：对第三代 TKI 的继发性耐药。后一组进一步细分为 4A 组：在第一代和第二代 TKI 检测 T790M 突变后作为二线治疗提供奥希替尼（第三代 TKI）的继发性耐药。本组15例。10 例 (10/15; 67%) EGFR T790M 突变丢失，5 例保留。T790M 丢失的患者经历了早期耐药（平均 6.9 个月对 12.6 个月，P = 0.0024) 与保留 T790M 的病例相比。两例以MET扩增为耐药机制。当 EGFR T790M 丢失时发现的其他突变位于 FGFR3 、KRAS、PIK3CA、CTNNB1、BRAF 基因中。1例存在EML4-ALK易位。两例显示驱动 EGFR 缺失 19，保留顺式形式的 T790M 和 C797S 突变。4B 组：EGFR 突变 NSCLC 对奥希替尼（当作为一线疗法给予时）的继发性耐药。本组有3例。奥希替尼治疗的持续时间为 11 至 17 个月。两名患者显示出额外的 C797S 突变以及原发性 EGFR 突变。

结论

这项研究显示了针对第一代、第二代和第三代 TKI 的广泛的初级和次级 EGFR 耐药机制，并帮助我们确定可以继承 EGFR TKI 提供的初始优势的新治疗靶点。