EGFR mutation testing: collecting the sample

Determining the EGFR mutation status of a tumour is key to choosing first-line therapy for patients with stage IIIb/IV NSCLC.

• In order for a lab to carry out a successful analysis, a sufficient quantity of tumour material is required 

Tumour sample Eberhard 2008 

• At diagnosis, a sample is collected for histological evaluation. This can also be used for mutation analysis
• Tissue from a tumour biopsy is the most commonly used and preferred sample type for mutation analysis
• Biopsy tissue is usually fixed with formalin and embedded in paraffin for testing. Frozen tumour yields higher quality DNA but is rarely available

Other tissues Kimura and Fujiwara 2006, Kimura 2006, Oshita 2006, Molina-Vila 2008

• Due to difficulty in obtaining biopsy samples and lack of tumour sample in some cases, the use of surrogate samples (non-tumour) is being explored.
  These include serum or plasma samples and cytology, which include bronchial alveolar lavage, bronchial scrapings and fine needle aspirates (FNAs)
• Further work is required before these can be used in routine clinical practice

Amount of tissue

• For accurate mutation analysis there must be sufficient DNA to analyse
• 5–10 5µm sections of the tumour biopsy should provide adequate DNA
• In some cases, successful analyses may be obtained from less tissue

Sending the biopsy to the lab

• At diagnosis, a needle core biopsy or fine needle aspirate (FNA) will be obtained. The sample is transported to a pathology laboratory where it is processed, embedded in a paraffin block, sectioned, stained and analysed by a pathologist
• The pathologist will provide a report confirming the underlying histology (small cell vs large cell vs non-small cell lung cancer; squamous vs adenocarcinoma)
• Once histological diagnosis and stage of disease are established, additional tests and therapeutic options are considered, including EGFR mutation analysis

Tips for obtaining the biopsy
Where possible the following is recommended

• Clarify in advance how best to obtain the tissue (for example, endoscopically, percutaneously)
• Determine how the tissue should be sent to the pathologist
• Take 4–5 biopsies per tumour
• Biopsy different areas of the tumour
• Biopsy the metastases

Tips for the pathologist responsible for sending the biopsy for mutation analysis Eberhard 2008 

• Check with your molecular pathology lab which sample they require for mutation analysis
• Check if your molecular pathology lab would like the hematoxylin and eosin (H&E) stained sections in order for them to be able to perform macrodissection. If so send H&E stained sections with every block/sections for testing. All histological sections should be returned to you following the EGFR mutation analysis
• Send either one formalin-fixed paraffin embedded (FFPE) block or all FFPE blocks depending on the requirements

Next: EGFR exon mutation analysis