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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.

Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma