Tumour biopsy tissue is the most common sample type used for mutation analysis.1,2 However, because it may be difficult to obtain biopsy samples and in some cases there may be insufficient tumour sample, the use of surrogate samples has been explored.2–5
Circulating tumour DNA (ctDNA) isolated from blood (or plasma) samples provides an alternative to tumour biopsy samples for evaluating epidermal growth factor receptor (EGFR) mutation status at primary diagnosis.6 In many patients with advanced non-small cell lung cancer (NSCLC), ctDNA can be found in the blood.6
At primary diagnosis of advanced NSCLC, EGFR mutation test results help physicians to decide if a patient is likely to benefit from treatment with an EGFR-TKI. EGFR mutation test results should therefore be reported clearly and include the mutation type to allow the physician to make a treatment decision based on the evidence available.
When testing for EGFR mutations, it is recommended that a tumour sample be used to confirm the presence of EGFR mutations prior to treatment. However, ctDNA obtained from blood (plasma) may be used if a tumour sample is not available or if the test is inconclusive.7
When considering the use of IRESSA treatment, if a tumour sample is not evaluable, then ctDNA obtained from a blood plasma sample may be used as per IRESSA (gefitinib) EU SmPC. Regulatory conditions may differ from country to country. Please refer to your locally approved licence and take into consideration when viewing information in this resource.
EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer
IRESSA (gefitinib) EU Summary of Product Characteristics. Available at http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/001016/WC500036358.pdf
Based on the results of a Phase IV open-label, efficacy, safety and tolerability study, IRESSA Follow‐Up Measure (IFUM),8 the IRESSA EU label was updated in 2014. This made IRESSA (gefitinib) the first EGFR‐TKI to allow the use of ctDNA obtained from a blood (or plasma) sample to assess the EGFR mutation status at diagnosis in patients where a tumour sample is not an option.7
DNA extraction methods optimised for ctDNA, together with robust and sensitive EGFR mutation testing methods are essential to maximise the likelihood of successful analysis.9 New methods and procedures may further improve EGFR mutation detection rates in the future.
For ctDNA testing, due to limited stability, samples should be processed promptly following blood draw, however, stabilisation collection tubes provide an alternative when this is not possible (e.g. Streck cell-free DNA blood collection tubes, PAXgene Blood DNA Tubes).10–12
Please note: heparin collection tubes must not be used as this can interfere with downstream PCR applications.13
Evidence supporting the use of ctDNA (plasma) samples for EGFR mutation testing, and a summary of baseline mutation status for tumour tissue biopsy and plasma-derived ctDNA samples in all screened patients in IFUM who were evaluable for both samples is shown in the figure below.14
ctDNA, circulating tumour DNA; EGFR, epidermal growth factor receptor; M+, mutation positive; M–, mutation negative*;
PPV, positive predictive value
Tumour biopsy and matched plasma-derived ctNDA samples. ctDNA extracted using Qiagen QIAamp® Circulating Nucleic Acid Kit and EGFR mutations detected in singleton using Qiagen Therascreen® EGFR RCQ PCT Kit
Concordance: rate of agreement in positive or negative EGFR mutation status between matched biopsy and plasma (ctDNA) samples
Sensitivity: proportion of EGFR mutation-positive biopsy samples with a matched EGFR mutation-positive plasma (ctDNA) sample
Specificity: proportion of EGFR mutation-negative biopsy samples with a matched EGFR mutation-negative plasma (ctDNA) sample
PPV: proportion of EGFR mutation-positive plasma (ctDNA) samples with a matched EGFR mutation-positive biopsy sample
Adapted from Douillard J-Y et al. J Thorac Oncol 2014:9:1345–1353
These data are consistent with the pre-planned exploratory Japanese subgroup analysis in IPASS (IRESSA Pan-ASia Study), in which sensitivity was 43.1% and specificity was 100%.15 (In IPASS, ctDNA derived from serum was used for EGFR mutation analysis using the EGFR Mutation Test Kit (DxS, Manchester, UK) [N=86]).15
The ASSESS study investigated the use of ctDNA isolated from blood (or plasma) samples for EGFR mutation testing in a real-world setting. Patients were enrolled from Europe and Japan. The primary endpoint was the level of concordance between the EGFR mutation status obtained from tissue/cytologic and from blood (plasma) samples.16
An improvement in positive predictive value and sensitivity was observed when the samples were tested with more sensitive methods, including the Roche cobas EGFR Mutation Test (Roche Molecular Diagnostics, CA, US).16
The concordance between the tissue/cytologic samples and blood (plasma) samples (89%) suggests that ctDNA is a viable option for real-world EGFR mutation analysis if sensitive DNA extraction and analysis methods are used, and when tumour samples are unavailable.16 However, it should be noted that this is lower than the concordance rate reported for IFUM (94.3%; sensitivity 65.7%, specificity 99.8%, positive predictive value 98.6%, and negative predictive value 93.8%).14
PPV, positive predictive value; n, number of samples; N, total number of samples; NPV, negative predictive value
Reck et al. J Thorac Oncol. 2016 Oct;11(10):1682-9
The study demonstrated that further improvements in ctDNA testing, including assay sensitivity, validation and quality, are needed to reduce the rate of false negative results.16
The IGNITE study was a non-interventional, real-world evidence, diagnostic study, which investigated the EGFR mutation frequency in patients with advanced NSCLC of adenocarcinoma (ADC) and non-ADC histology.17
The primary endpoint was EGFR mutation frequency by histology (tested locally). The secondary endpoints were EGFR mutation status concordance between matched tissue/cytology and ctDNA isolated from blood (or plasma) samples (China, Taiwan, South Korea and Russia only); correlation between mutation status and demographic/disease status; treatment decisions; and EGFR mutation testing practices.17
The frequency of EGFR mutations in the IGNITE study was consistent with published findings showing a higher frequency of EGFR mutations in Asian populations than Caucasian populations.19-21
ADC, adenocarcinoma; n, number of samples; N, total number of samples
Han et al. Ann Oncol 2015;26 (Supplement 1): i29–i44
The concordance between the tissue/cytologic samples and ctDNA isolated from blood (plasma) samples in Asia-Pacific and Russia (table below),18 suggests that ctDNA could be viable option for real-world EGFR mutation analysis when tumour samples are not evaluable.
PPV, positive predictive value; N, total number of participants analysed; NPV, negative predictive value
Clinicaltrial.gov. Asia Pacific and Russia Diagnostic Study for EGFR Testing (IGNITE). NCT01788163. Last updated July 29, 2016.
The findings from the study support the use of EGFR mutation testing in all patients (ADC and non-ADC histology).17
When comparing the findings from the ASSESS, IGNITE and IFUM studies, the data suggest that ctDNA isolated from blood (or plasma) may be a feasible and suitable alternative for EGFR mutation testing when a tissue/cytology sample is unavailable, and a robust, sensitive method validated for ctDNA extraction and mutation detection are used.14,16-18
The IRESSA (gefitinib) EU SmPC states that, when considering the use of treatment with gefitinib, ctDNA obtained from blood (plasma) may be used if a tumour sample is not evaluable.7
For further information on EGFR mutation testing, please visit www.EGFR‐mutation.com