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

Maxime Fontanilles,Pascal Chambon,Doriane Richard,Olivier Langlois,Cristina Alexandru,Mathieu Viennot,Florent Marguet,Ludivine Beaussire,Florian Clatot,Kevin Cassinari,Pierre-Julien Viailly,Philippe Ruminy,Nasrin Sarafan-Vasseur,Isabelle Tennevet,Annie Laquerrière,Adrien Noel,Frédéric Di Fiore,Carole Basset

doi:10.1186/s40478-020-00917-6

Abstract

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.

Highlights

Glioblastoma is the most frequent primary brain tumor in adults, with 125,000 to 150,000 new cases per year worldwide [1]
Among the 19 epidermal growth factor receptor (EGFR)-amplified glioblastomas, EGFRvIII was identified in 8 patients by digital PCR (dPCR) EGFR2/ EGFR3 assay, and all were confirmed using LD-RT-PCR
This study shows that the specific dPCR assay using locked nucleic acid (LNA)-hydrolysis probes from Universal Probe Library® (UPL)® is a reliable and simple method to simultaneously detect an EGFR amplification and EGFRvIII variant, and this can be used in clinical practice in glioblastoma

Summary

Introduction

Glioblastoma is the most frequent primary brain tumor in adults, with 125,000 to 150,000 new cases per year worldwide [1]. Identification of EGFR amplification associated with either a telomerase reverse transcriptase promoter (TERTp) mutation or chromosomal alterations (chromosome 7 gain and chromosome 10 loss) in diffuse or anaplastic astrocytoma has led to a reclassification proposal of grade II-III 1p19q non-codeleted gliomas into glioblastoma-like tumors [14, 15]. Taken together, these data support that the detection of EGFR alterations may be considered relevant in patients treated for glioblastoma

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