Hidaka Noriko, Iwama Eiji, Kubo Naoki, Harada Taishi, Miyawaki Kohta, Tanaka Kentaro, Okamoto Isamu, Baba Eishi, Akashi Koichi, Sasaki Hiroyuki, Nakanishi Yoichi
Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka 8128582, Japan.
Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka 8128582, Japan; Department of Comprehensive Clinical Oncology, Faculty of Medical Sciences, Kyushu University, Fukuoka 8128582, Japan.
Lung Cancer. 2017 Jun;108:75-82. doi: 10.1016/j.lungcan.2017.02.019. Epub 2017 Mar 1.
The T790M and C797S mutations of the epidermal growth factor receptor gene (EGFR) confer resistance to first- and third-generation EGFR tyrosine kinase inhibitors (TKIs), respectively, in patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR. C797S has been identified in cis or in trans with T790M in tumor specimens from patients who experienced treatment failure with first- and third-generation EGFR-TKIs. The allelic relation between T790M and activating mutations of EGFR has not been well characterized, however. We have now developed a digital polymerase chain reaction (dPCR)-based method for determination of the allelic relation between two types of EGFR mutation (T790M and either C797S or an activating mutation).
Seven clinical NSCLC specimens and two NSCLC cell lines harboring both an activating mutation and T790M were analyzed with this new method to identify the allelic relation between these EGFR mutations.
The median ratio of the number of alleles positive for both an activating mutation and T790M to the number of T790M-positive alleles was 97.1% (range, 90.0-100%). Confirmatory analysis by next-generation sequencing yielded a corresponding value of 96.7% (range, 89.1-99.5%). Our dPCR method thus reliably identifies the allelic relation between two EGFR mutations in a quantitative manner.
Almost all T790M mutations were detected in cis with activating mutations of EGFR regardless of the de novo or acquired status of T790M, with cancer cells harboring T790M and activating mutations on the same allele appearing to be selected and enriched during EGFR-TKI treatment.
表皮生长因子受体基因（EGFR）的T790M和C797S突变分别使携带EGFR激活突变的非小细胞肺癌（NSCLC）患者对第一代和第三代EGFR酪氨酸激酶抑制剂（TKIs）产生耐药性。在接受第一代和第三代EGFR-TKIs治疗失败的患者的肿瘤标本中，已鉴定出C797S与T790M呈顺式或反式。然而，T790M与EGFR激活突变之间的等位基因关系尚未得到充分表征。我们现已开发出一种基于数字聚合酶链反应（dPCR）的方法，用于确定两种EGFR突变（T790M和C797S或激活突变）之间的等位基因关系。
使用这种新方法分析了7个临床NSCLC标本和2个同时携带激活突变和T790M的NSCLC细胞系，以确定这些EGFR突变之间的等位基因关系。
激活突变和T790M均为阳性的等位基因数量与T790M阳性等位基因数量的中位数之比为97.1%（范围为90.0 - 100%）。下一代测序的验证分析得出相应值为96.7%（范围为89.1 - 99.5%）。因此，我们的dPCR方法能够以定量方式可靠地鉴定两种EGFR突变之间的等位基因关系。
几乎所有T790M突变均与EGFR激活突变呈顺式被检测到，无论T790M是从头出现还是获得性的，在EGFR-TKI治疗期间，同一等位基因上携带T790M和激活突变的癌细胞似乎被选择并富集。
