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EGFR Mutation Testing (Lung Cancer)

Cobas® EGFR Mutation Test v2 for In Vitro Diagnostic use (Roche).

The Cobas® EGFR Mutation Test v2 in conjunction with the Cobas® 4800 System provides the option of using FFPE tissue or plasma.

The Cobas® EGFR Mutation Test v2 is a real-time PCR test for the qualitative detection and identification of mutations in exons 18, 19, 20 and 21 of the EGFR gene using a DNA input of 50 ng. The test can detect 42 EGFR mutations including G719X (G719A, G719C and G719S) in exon 18; deletions and complex mutations in exon 19; S768I, T790M and insertions in exon 20; L858R and L861Q in exon 21.

In formalin-fixed, paraffin-embedded tumor samples, the presence of mutations is detected with an analytical specificity of at least 95% with a mutation-specific detection limit of at least 5% mutant level in a background of wild-type genomic DNA using the standard input of 50ng per reaction well.

In plasma, the presence of mutations is detected with an analytical specificity of at least 95% with a mutation-specific limit of detection of at least 1% mutant level in a background of wild-type genomic DNA using the standard input of 25μL of DNA stock per reaction well.

The Cobas® EGFR Mutation Test v2 is based on two major processes: (1) manual specimen preparation to obtain DNA from FFPET or plasma; (2) PCR amplification and detection of target DNA using complementary primer pairs and oligonucleotide probes labeled with fluorescent dyes. Mutation detection is achieved through PCR analysis with the Cobas z 480 analyzer (1, 2, 3).

Clinical Implication

Lung cancer is a leading cause of cancer death worldwide. Patients with non-small cell lung cancer (NSCLC) often present with advanced disease and treatment benefit with standard chemotherapy is modest. The introduction of therapies that target the epidermal growth factor receptor (EGFR) pathway in NSCLC, such as the tyrosin kinase inhibitors erlotinib (Tarceva), osimertinib (Tagrisso) and gefitinib (Iressa) has begun to improve outcomes in patients with advanced NSCLC. Especially those NSCLC cases harboring activating mutations in the EGFR gene show response to anti-EGFR tyrosin kinase inhibitors. The highest response rates have been shown for exon 19 deletions, G719A/C, L858R and L861Q (4). Therefore, it is important to identify EGFR mutations in NSCLC.

Treatment with first- or second-generation epidermal growth factor (EGFR)-tyrosine kinase inhibitors (TKIs) is effective for non-small cell lung cancer (NSCLC) patients harboring a sensitizing EGFR mutation. However, often resistance is acquired after 9–14 months. The most common mechanism of resistance to first- and second-generation EGFR-TKIs in the first-line setting is the EGFR T790M mutation, which accounts for approximately 60% of cases. Third-generation EGFR-TKIs targeting EGFR T790M mutation (e.g. Osimertinib), are reported to be highly active against T790M-positive NSCLC. To detect the T790M mutation in patients progressing during EGFR-TKI treatment, tumor re-biopsy is necessary. As re-biopsies with invasive procedures (bronchoscopy or needle biopsy) are often infeasible in standard care of NSCLC patients, circulating tumor DNA (ctDNA) detected in plasma is recognized as a noninvasive biomarker for the molecular analysis of NSCLC. The cobas® EGFR Mutation Test (Roche Diagnostics K.K., Switzerland.) is a companion diagnostic test for the detection of EGFR mutations in plasma specimens and has been approved to identify such patients with NSCLC. T790M monitoring in plasma ctDNA of patients receiving EGFR-TKIs could yield valuable clinical information (5).

Specimen Requirements

  1. FFPE
    • Formalin-fixed, paraffin-embedded NSCLC tissue specimens with a fixation time of 6‑48 hours
  2. Plasma
    • Whole blood or plasma derived from (K2) EDTA anti-coagulated peripheral whole blood


  1. FFPE
    • One representative paraffin block is preferred. Alternatively, 3 unstained tissue sections are requested. Of interest, if additional ALK FISH analysis is required (confirmation of ALK IHC positive case), two 3 µm thick unstained sections are required for ALK FISH analysis.
  2. Plasma
    • 8ml of whole blood or 2ml of plasma is required

Storage and Shipment Instructions

  1. FFPE
    • Maintain and ship FFPE specimens at ambient temperature.
  2. Plasma
    • Maintain and ship whole blood specimens at ambient temperature.
    • Maintain plasma specimens at -70°C or colder and ship and dry ice.


  1. FFPE
    • Insufficient tumor content may not allow the detection of EGFR mutations (< 10%); tumor content is evaluated prior to analysis and macrodissection is performed. Fixatives other than formalin or prolonged fixation time may give rise to inadequate results.
  2. Plasma
    • Samples tested at high concentrations (>105 copies/mL) may generate false results.
  3. General
    • The Cobas® EGFR Mutation Test v2 shows cross-reactivity (results of “Mutation Detected”) to the exon 19 L747S mutation, a rare acquired mutation that may confer resistance to TKI treatment.
    • Detection of a mutation is dependent on the number of copies present in the specimen and may be affected by sample integrity, amount of isolated DNA, and the presence of interfering substances. The presence of PCR inhibitors may cause false negative or invalid results.

Special Requirements


Turn-Around Time

Five to 7 business days for plasma and slides and paraffin blocks, respectively.


  1. Newton CR, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989; 17(7): 2503-16.
  2. Lopez-Rios F et al. Comparison of molecular testing methods for the detection of EGFR mutations in formalin-fixed, paraffin-embedded tissue specimens of non-small cell lung cancer. J. Clin. Pathol. 2013; 66: 381-385.
  3. Pirker R et al. Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop. J Thorac Oncol. 2010 Oct;5(10):1706-13.
  4. da Cunha Santos G et al. EGFR mutations and Lung Cancer. Annu Rev Pathol. 2011;6:49-69.
  5. Usui K et al. Plasma ctDNA monitoring during epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor treatment in patients with EGFR-mutant non-small cell lung cancer. Jpn J Clin Oncol. 2019 Feb 27.

Updated on 24 May 2019