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.

中文翻译：

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使用基于数字PCR的方法在胶质母细胞瘤中同时检测EGFR扩增和EGFRvIII变异。

表皮生长因子受体（EGFR）扩增和EGFR变体III（EGFRvIII，外显子2-7缺失）在胶质母细胞瘤中具有临床意义。目的是开发一种基于数字PCR（dPCR）的方法，该方法使用基于锁定核酸（LNA）的水解探针，从而可以同时检测EGFR扩增和EGFRvIII变体。包括62例患者。探索性队列（n = 19）用于开发dPCR分析，使用EGFR基因内的三个选定扩增子，靶向内含子1（EGFR1），外显子3和内含子3（EGFR2）和内含子22（EGFR3）。EGFR的拷贝数通过与参考基因的液滴相比对EGFR1，EGFR2和EGFR3扩增子液滴的相对定量来估计。通过比较EGFR2扩增子与EGFR1或EGFR3扩增子的拷贝数来鉴定EGFRvIII。将dPCR结果与荧光原位杂交（FISH）和下一代测序进行比较；以及基于RT-PCR的EGFRvIII方法。然后在验证队列中对dPCR分析进行了测试（n = 43）。在探索性队列中总共鉴定出8/19 EGFR扩增和5/19 EGFRvIII阳性肿瘤。与FISH相比，EGFR3 dPCR检测可检测到所有EGFR扩增的肿瘤（8 / 8，100％），并且与NGS估计的拷贝数具有最高的一致性。使用EGFR2和EGFR3探针之间拷贝数的绝对差为10.8，RT-PCR与dPCR之间的一致性也达到100％。在验证队列中，与FISH（19/19）相比，使用EGFR3探针进行dPCR的敏感性和特异性为EGFR扩增检测的100％。通过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分析的成本价值估计为一半。这些结果表明，dPCR可以同时检测胶质母细胞瘤的EGFR扩增和EGFRvIII。