Comparison of Biomarker Assays for EGFR: Implications for Precision Medicine in Patients with Glioblastoma

Abstract

Purpose: Patients with glioblastoma (GBM) have a poor prognosis and are in desperate need of better therapies. As therapeutic decisions are increasingly guided by biomarkers, and EGFR abnormalities are common in GBM, thus representing a potential therapeutic target, we systematically evaluated methods of assessing EGFR amplification by multiple assays. Specifically, we evaluated correlation among fluorescence in situ hybridization (FISH), a standard assay for detecting EGFR amplification, with other methods. Experimental Design: Formalin-fixed, paraffin-embedded tumor samples were used for all assays. EGFR amplification was detected using FISH (N ¼ 206) and whole-exome sequencing (WES, N ¼ 74). EGFR mRNA expression was measured using reverse transcription-polymerase chain reaction (RT-PCR, N ¼ 206) and transcriptome profiling (RNAseq, N ¼ 64). EGFR protein expression was determined by immunohistochemistry (IHC, N ¼ 34). Significant correlations among various methods were determined using Cohen's kappa (k ¼ 0.61-0.80 defines substantial agreement) or R 2 statistics. Results: EGFR mRNA expression levels by RNA sequencing (RNAseq) and RT-PCR were highly correlated with EGFR amplification assessed by FISH (k ¼ 0.702). High concordance was also observed when comparing FISH to WES (k ¼ 0.739). RNA expression was superior to protein expression in delineating EGFR amplification. Conclusions: Methods for assessing EGFR mRNA expression (RT-PCR, RNAseq) and copy number (WES), but not protein expression (IHC), can be used as surrogates for EGFR amplification (FISH) in GBM. Collectively, our results provide enhanced understanding of available screening options for patients, which may help guide EGFR-targeted therapeutic approaches.

Identification of New Genetic Clusters in Glioblastoma Multiforme: EGFR Status and ADD3 Losses Influence Prognosis

“…The first cluster (C1) offers a low rate of SCNAs compared with the rest. Cluster 2 (C2) and cluster 3 (C3) share a high rate of SCNAs in MTAP, CDKN2A, MSH6 and EGFR, in agreement with previous descriptions [16], and suggesting that GBs from both clusters display alterations in DNA metabolic processes (by STRING). However, the second cluster (C2) concentrates the co-existence of EGFR alterations through EGFRvIII or EGFR amplification and the highest rate of SCNAs.…”

“…STRING analysis correlates the SCNA statistically associated with amplified-EGFR GBs with NOTCH3 activation and negative regulation of cell-matrix adhesion. Crespo et al, using high-density (500K) single-nucleotide polymorphism arrays, achieved similar relevance for the axis established with MTAP [16]. In concordance with that work, our group of patients that displayed EGFRvIII, which is the one with the worst outcome, also showed the impairment of cell-matrix adhesion processes.…”

“…In addition, the identification of differential targets among GB IDH-wt genetic subgroups could lead to reach better approaches to GB management. EGFR amplification is a hallmark of glioma pathogenesis [4,15,16]. In the last decade, many molecules have shown promising results on pre-clinical or phase 1/2 trials targeting EGFR.…”

“…In the last decade, many molecules have shown promising results on pre-clinical or phase 1/2 trials targeting EGFR. However, they tend to fail, as recently happened with ABT-414 conjugated with ABBV-321 [16,17]. It is also disappointing that while TKI therapies have been successfully expanded to multiple types of cancer, none of them have improved the lifespan of GB patients [18].…”

Heterogeneous EGFR, CDK4, MDM4, and PDGFRA Gene Expression Profiles in Primary GBM: No Association with Patient Survival

“…In recent years, accumulated evidence suggested that amplification of the EGFR and other genes might play a critical role in the oncogenesis and clinical behavior of GBM [22,26,27]. Despite this, with the exception of a recent study [28], no clear association has been reported in the literature between the GEP and the genetic alterations of individual genes in GBM [29]. In order to investigate the potential association between overexpression of EGFR, CDK4, MDM4, and PDGFRA, subsequent analysis of gene amplification was performed at the DNA level.…”