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

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.

Meta-analysis of tumor- and T cell-intrinsic mechanisms of sensitization to checkpoint inhibition

“…Next, we considered focal (<3 Mb) ( Krijgsman et al., 2014 ) amplifications (defined as copy number ≥ 5) ( Fontanilles et al., 2020 ) and homozygous deletions (copy number = 0) in oncogenes and tumor suppressor genes respectively, to understand if these events are associated with CPI response. The most significant association was found to be significantly lower rates of CPI response in tumors with CCND1 amplification (response rate = 16.3%) compared to wild-type (26.6%) (p = 4.8 × 10 −2 ; Figure 6 A).…”

EGFR-Targeted Cellular Delivery of Therapeutic Nucleic Acids Mediated by Boron Clusters

“…B-ASOs have a dual therapeutic potential against cancer cells: anti-EGFR inhibitory and boron carrier ( 10 B) delivery for BNCT which we have described previously [ 23 , 24 , 26 , 27 , 44 ]. Importantly, the ASO sequences of these structures were designed to be effective silencers towards EGFR mRNA, as well as EGFR variant III mRNA [ 14 , 61 ]. We observed that B-ASO (80 pmol) can be freely taken up into A431 cells and mediated by FESAN-EGFR.…”

Identification of potential targets of the curcumin analog CCA-1.1 for glioblastoma treatment : integrated computational analysis and in vitro study

“…EGFRvIII undergoes a 6–273 amino acid deletion at exon 2–7, encoding the extracellular domain of EGFR 133 , and EGFRvIII can undergo dimerization via a ligand-independent activation pathway 132 . EGFRvIII differs from mutant EGFR on the extracellular domain, namely due to the deletion of certain amino acids causing slow receptor internalization, as well as a slower constitutively phosphorylation level compared to EGFR isoform 1 134 . The results of NGS showed the downregulation of EGFRvIII due to CCA-1.1 treatment.…”