EGFR Abnormalities in Cancer

Dysregulation of EGFR has a causal role in the development and maintenance of certain human carcinomas.3

Amplification, Overexpression, Mutation

In cancer, the EGFR gene is often amplified, overexpressed, or mutated, resulting in abnormal signaling and malignant cellular behaviors.3

  • EGFR-directed therapies have improved outcomes in a number of tumor types.4
    • Sustained responses to EGFR-positive tumors may be limited due to the complexity of EGFR signaling.5,6
    • EGFR-directed tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) are associated with serious adverse events, most notably skin rash, diarrhea, hepatotoxicity, and interstitial lung disease.7,8


Amplification of the EGFR gene is among the most common genetic alterations in glioblastoma (GBM), occurring in approximately 50% of GBM tumors.3,9 EGFR amplification status is stable from GBM diagnosis to recurrence at a rate of 84% to 93% when treated with the current standard of care.10,11

  • The prognostic value of EGFR amplification in GBM is unclear.12-14 However, lower-grade gliomas with IDH wild type (IDH-WT) display prognostic heterogeneity, and EGFR amplification is one marker associated with a worse prognosis.15,16

EGFRvIII is another common mutation in GBM, occurring almost excusively in EGFR-amplified GBM. Approximately 25% of all GBMs harbor both EGFR amplification and the EGFRvIII mutation.3,17-19


The most commonly used diagnostic tools for clinical evaluation of EGFR gene amplification are fluorescence in situ hybridization (FISH) or colorimetric in situ hybridization (CISH).20

Fluorescence In Situ Hybridization (FISH)

Fluorescence-tagged, sequence-specific probes (single-stranded DNA or RNA) are hybridized to complementary DNA or RNA sequences in cells or tissues.21-24

  • Is specific, simple, reliable, and quantitative25
  • Concurrent use of a second probe, commonly to a centromeric region of a chromosome, allows for differentiation of a specific gene amplification from polysomy of a chromosome22
  • Fluorescence allows for more accurate image analysis, quantification of copy number, and multiplexing to resolve structural variants and copy number abnormalities20,23,26
  • Short turnaround time (ie, days) compared to some other technologies (ie, weeks)27
  • Requires technical expertise and specialized instruments for analysis22; immediate image capture may be necessary as fluorescence fades over time28

Colorimetric In Situ Hybridization (CISH) & Dual In Situ Hybridization (DISH)

Colorimetric in situ hybridization, also known as chromogenic in situ hybridization (CISH), is similar to FISH, but probes are labeled with a chromogen instead of fluorescence.

Dual in situ hybridization (DISH) is a form of CISH that utilizes 2 differentially labeled probes that are cohybridized and visualized on the same slide.

  • Semiquantitatively detects presence of gene amplification (>10 copies per cell, 3-10 copies per cell, or absence of amplification)20
  • Evaluates histologic, morphologic, and molecular alterations simultaneously with a bright field standard light microscope20,29-31
  • Can detect structural gene variants20
  • Hybridization signals do not fade over time29
  • Time-efficient32
  • Multiplexing is limited33

In addition to FISH and CISH/DISH, a number of other methods are available for the clinical evaluation of EGFR copy number, directly or indirectly.

Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS) technology allows high-throughput sequencing of millions of DNA templates in a single reaction with multiple patient samples. Applications include sequencing of the whole genome or of targeted protein-coding regions.22

Whole-genome sequencing20,22,34

  • Extensive data on genomic alterations, including single nucleotide variants, insertions and deletions, complex structural arrangements, and copy number changes in a single run
  • Comprehensive view of unique genomic alterations in cancer tissue compared to matched surrounding normal tissue
  • Multiplexing allows simultaneous sequencing of large sample numbers in a single run

Targeted NGS/exome sequencing20,22,34

  • Sequences/regions of interest or clinical significance are selectively amplified before NGS
  • Can provide highly confident data on mutations and sequence variants; is less sensitive to copy number and structural variants
  • Allows sequencing of several hundred to thousands of targeted regions simultaneously in a single run
  • High multiplexing capacity provides fast turnaround time

Potential drawbacks include34:

  • Requires specialized bioinformatics analysis
  • Longer turnaround time compared to ISH and IHC
  • Cell and tissue morphology and molecular alterations cannot be evaluated simultaneously
  • May require macrodissection of sample to enrich tumor content

Polymerase Chain Reaction (PCR)

PCR allows the rapid amplification of a specific DNA fragment from a complex pool of DNA.35 Double-stranded DNA is denatured, sequence-specific primers are annealed to the single DNA strands, and the primers are elongated by DNA polymerase.35,36

  • Standard PCR on genomic DNA provides qualitative information regarding the presence or absence of a DNA sequence.
  • Reverse Transcription PCR (RT-PCR) uses RNA as the template. Total RNA is reverse-transcribed into complementary DNA (cDNA), which is then amplified in a standard PCR or quantitative PCR (qPCR) reaction. In qPCR, fluorescent labeling enables the collection of data through each cycle of the PCR reaction. The amount of DNA or RNA in a biological sample can be quantified.
  • Anchored multiplex PCR (AMP) combined with NGS can be effective in detecting gene rearrangements, single nucleotide variants, insertions, deletions, and copy number changes.37
  • Potential drawbacks include35-37:
    • Cell and tissue morphology and molecular alterations cannot be evaluated simultaneously
    • RT-PCR detects mRNA expression as a surrogate for amplification

Immunohistochemistry (IHC) & Immunofluorescence (IF)

IHC and IF semiquantitatively measure protein levels only, and do not measure EGFR gene amplification. Antibodies are bound to a specific antigen on a protein of interest in fixed cells or tissues and then visualized.38,39

  • IHC: antibodies are linked with a chromogen and visualized with a bright-field standard light microscope
  • IF: antibodies are linked to fluorescence moieties; specialized instruments are necessary for fluorescence analysis
  • Wild-type and mutant protein expression can be measured in the same cell20
  • Short turnaround (ie, days)20
  • Evaluates cell and tissue morphology and protein expression levels simultaneously

Potential drawbacks include38,39:

  • Measures protein expression only, not gene amplification specifically
  • Is only semiquantitative
  • May have false-positives
    and -negatives
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