Robust Detection of EGFR Copy Number Changes and EGFR Variant III: Technical Aspects and Relevance for Glioma Diagnostics

Abstract

Epidermal growth factor receptor (EGFR) is commonly affected in cancer, generally in the form of an increase in DNA copy number and/or as mutation variants [e.g., EGFR variant III (EGFRvIII), an in-frame deletion of exons 2–7]. While detection of EGFR aberrations can be expected to be relevant for glioma patients, such analysis has not yet been implemented in a routine setting, also because feasible and robust assays were lacking. We evaluated multiplex ligation-dependent probe amplification (MLPA) for detection of EGFR amplification and EGFRvIII in DNA of a spectrum of 216 diffuse gliomas. EGFRvIII detection was verified at the protein level by immunohistochemistry and at the RNA level using the conventionally used endpoint RT-PCR as well as a newly developed quantitative RT-PCR. Compared to these techniques, the DNA-based MLPA assay for EGFR/EGFRvIII analysis tested showed 100% sensitivity and specificity. We conclude that MLPA is a robust assay for detection of EGFR/EGFRvIII aberrations. While the exact diagnostic, prognostic and predictive value of such EGFR testing remains to be seen, MLPA has great potential as it can reliably and relatively easily be performed on routinely processed (formalin-fixed, paraffin-embedded) tumor tissue in combination with testing for other relevant glioma markers.

Assessment and prognostic significance of the epidermal growth factor receptor vIII mutation in glioblastoma patients treated with concurrent and adjuvant temozolomide radiochemotherapy

“…34 Some of these studies evaluated different methods for EGFRvIII detection in malignant gliomas, with variable results. For example, MLPA-based detection of EGFRvIII was reported to demonstrate the same sensitivity and specificity as analyses by RT-PCR or immunohistochemistry.…”

Significance of complete 1p/19q co-deletion, IDH1 mutation and MGMT promoter methylation in gliomas: use with caution

“…25 MGMT promoter hypermethylation was assessed with MS-MLPA assay ME-011 in 433 tumors, and IDH1 mutation analysis was performed in 442 tumors by direct sequencing as described previously. [26][27][28][29] In addition, in 440 cases MLPA assay P105 (lot nos. 0306, 0407 or 1008) was used to detect copy number changes in the genes CDKN2A, PTEN and EGFR.…”

“…0306, 0407 or 1008) was used to detect copy number changes in the genes CDKN2A, PTEN and EGFR. 28 Owing to limited amount of DNA of some samples it was not possible to asses all molecular markers on all samples.…”

AMG 595, an Anti-EGFRvIII Antibody–Drug Conjugate, Induces Potent Antitumor Activity against EGFRvIII-Expressing Glioblastoma

“…EGFR expression is associated with a malignant phenotype in multiple cancers, including colon cancer (6), head and neck squamous cell carcinoma (7), and GBM (8). Amplification of EGFR is observed in approximately 40% to 70% of GBM patients (9,10). EGFR gene alterations are observed in tumors with amplified EGFR (11).…”