Inserm U1245, Normandie Univ, UNIROUEN, IRON group, Normandy Centre for Genomic and Personalized Medicine, Rouen University Hospital, F-76031, Rouen, France.
Department of Medical Oncology, Cancer Centre Henri Becquerel, Rue d'Amiens, F-76038, Rouen, France.
Acta Neuropathol Commun. 2020 Apr 17;8(1):52. doi: 10.1186/s40478-020-00917-6.
Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2-7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.
表皮生长因子受体（EGFR）扩增和 EGFR 变体 III（EGFRvIII，外显子 2-7 的缺失）对胶质母细胞瘤具有临床意义。目的是开发一种基于数字 PCR（dPCR）的方法，使用锁核酸（LNA）水解探针，同时检测 EGFR 扩增和 EGFRvIII 变体。共纳入 62 例患者。探索性队列（n=19）用于使用 EGFR 基因内的三个选定扩增子开发 dPCR 检测，针对内含子 1（EGFR1）、外显子 3 和内含子 3 的连接（EGFR2）和内含子 22（EGFR3）。通过比较 EGFR1、EGFR2 和 EGFR3 扩增子的相对定量与参考基因的扩增子的液滴，估计 EGFR 的拷贝数。通过比较 EGFR2 扩增子的拷贝数与 EGFR1 或 EGFR3 扩增子的拷贝数来确定 EGFRvIII。dPCR 结果与荧光原位杂交（FISH）和下一代测序（用于扩增）进行比较；并与基于 RT-PCR 的方法比较 EGFRvIII。然后在验证队列（n=43）中测试 dPCR 检测。在探索性队列中，8/19 例 EGFR 扩增和 5/19 例 EGFRvIII 阳性肿瘤被鉴定。与 FISH 相比，EGFR3 dPCR 检测到所有 EGFR 扩增肿瘤（8/8，100%），与 NGS 估计的拷贝数具有最高的一致性。使用 EGFR2 和 EGFR3 探针之间的拷贝数差异为 10.8 的绝对差异，用于检测 EGFRvIII 的 RT-PCR 和 dPCR 之间的一致性也为 100%。在验证队列中，与 FISH（19/19）相比，使用 EGFR3 探针的 dPCR 对 EGFR 扩增检测的敏感性和特异性均为 100%。dPCR 在 8 例 EGFR 扩增患者中检测到 EGFRvIII，并通过 RT-PCR 确认。与 FISH 相比，EGFR2/EGFR3 dPCR 检测的成本值估计为一半。这些结果表明，dPCR 允许同时检测胶质母细胞瘤的 EGFR 扩增和 EGFRvIII。
