ctDNA testing at primary diagnosis

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

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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

EGFR mutation testing at primary diagnosis

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

EGFR mutation testing at primary diagnosis in advanced NSCLC

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

Study data supporting the use of ctDNA testing

IFUM

In the IFUM study, EGFR mutation status was assessed in tumour tissue biopsy and plasma-derived ctDNA samples, using the Therascreen EGFR RGQ PCR kit (Qiagen, Crawley, UK).14

  • Both tumour tissue biopsy and plasma-derived ctDNA and samples were evaluable for 652 patients out of 1060 screened.
  • The objective response rate in those patients who were tumour tissue biopsy and plasma-derived ctDNA mutation positive was 76.9% (95% confidence interval 65.4%, 85.5%) and in those who were tumour-only mutation positive was 59.5% (95% CI 43.5%, 73.7%).

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

Evidence supporting the use of ctDNA (plasma) samples for EGFR mutation testing
Evidence supporting the use of ctDNA (plasma) samples for EGFR mutation testing

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

ASSESS

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

  • 1288 patients were eligible.
  • 50 laboratories across Europe and Japan performed the testing.
    • In Europe, 43 academic, hospital and commercial laboratories performed the tissue/cytologic testing and five central/regional expert laboratories were used for blood (plasma) testing.
    • In Japan, two commercial laboratories performed both tissue/cytologic and blood (plasma) testing.

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

Evidence supporting the use of ctDNA (plasma) samples for EGFR mutation testing
Concordance of EGFR mutation status

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

IGNITE

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

  • 3382 patients were enrolled across 90 centres in Asia-Pacific and Russia17,18
    • 2410 patients from Asia-Pacific (includes China, Taiwan, South Korea, Thailand, Singapore, Malaysia, Indonesia and Australia)
    • 972 patients from Russia
  • 2291 and 1753 patients from Asia-Pacific were evaluable for tissue/cytology and ctDNA isolated from blood (or plasma), respectively
  • 924 and 941 patients from Russia, were evaluable for tissue/cytology and ctDNA isolated from blood (or plasma), respectively

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

EGFR mutation frequency by histology between matched tissue/cytology and ctDNA isolated from blood (or plasma) samples
EGFR mutation frequency

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.

Concordance of EGFR mutation status between matched tissue/cytology and ctDNA isolated from blood (or plasma) samples
Concordance of EGFR mutation status

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

Summary

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

References

  1. Leary AF et al. Establishing an EGFR mutation screening service for non‐small cell lung cancer – sample quality criteria and candidate histological predictors. Eur J Cancer 2012; 48: 61–67.
  2. Ellison G et al. EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples. J Clin Pathol 2013; 66: 79–89.
  3. Malapelle U et al. EGFR mutations detected on cytology samples by a centralized laboratory reliably predict response to gefitinib in non-small cell lung cancer patients. Cancer Cytopathol 2013; 121: 552–560.
  4. Yang JC-H et al. Epidermal growth factor receptor mutation analysis in previously unanalyzed histology samples and cytology samples from the phase III Iressa Pan-ASia Study (IPASS). Lung Cancer 2014; 83: 174–181.
  5. Liu X et al. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies. J Clin Pathol 2013; 66: 1065–1069.
  6. Vallée A et al. Plasma is a better source of tumor-derived circulating cell-free DNA than serum for the detection of EGFR alterations in lung tumor patients. Lung Cancer 2013; 82: 373–374.
  7. IRESSA EU Summary of Product Characteristics. Available at http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/001016/WC500036358.pdf. Accessed 03 July 2017.
  8. Douillard J-Y et al. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study. Br J Cancer 2014; 110: 55–62.
  9. Liu Y et al. A comparison of ARMS and direct sequencing for EGFR mutation analysis and tyrosine kinase inhibitors treatment prediction in body fluid samples of non-small-cell lung cancer patients. J Exp Clin Cancer Res 2011; 30: 111–118.
  10. Qin Z et al. Cell-free circulating tumor DNA in cancer. Chin J Cancer. 2016 Apr 7;35:36.
  11. Streck cell-free DNA BCT instructions for use. Available at: https://www.streck.com/resources/Cell_Stabilization/Cell-Free_DNA_BCT/01_Instructions_(IFU)/01_IFU_Cell-Free_DNA_BCT_IFU.pdf. Accessed 03 July 2017.
  12. PAXgene Blood DNA System brochure. Available at: https://www.qiagen.com/no/resources/resourcedetail?id=1cf6f25d-7cf3-4890-8445-cee3caca3466&lang=en. Accessed 03 July 2017.
  13. El Messaoudi S et al. Circulating cell free DNA: preanalytical considerations. Clin Chim Acta 2013; 424: 222–230.
  14. Douillard J-Y et al. Gefitinib treatment in EGFR mutated Caucasian NSCLC. Circulating-free tumour DNA as a surrogate for determination of EGFR status. J Thorac Oncol 2014; 9: 1345–1353.
  15. Goto K et al. Epidermal growth factor receptor mutation status in circulating free DNA in serum. J Thorac Oncol 2012; 7: 115–121.
  16. Reck et al. ctDNA Determination of EGFR Mutation Status in European and Japanese Patients with Advanced NSCLC: The ASSESS Study. J Thorac Oncol 2016 Oct;11(10):1682-9.
  17. Han B et al. Determining the prevalence of EGFR mutations in Asian and Russian patients (pts) with advanced non-small-cell lung cancer (aNSCLC) of adenocarcinoma (ADC) and non-ADC histology: ignite study. Ann Oncol. 2015;26 (Supplement 1): i29–i44.
  18. Clinicaltrial.gov. Asia Pacific and Russia Diagnostic Study for EGFR Testing (IGNITE). NCT01788163. Last updated July 29, 2016. Accessed 03 July 2017. Available from https://clinicaltrials.gov/ct2/show/study/NCT01788163?term=NCT01788163&rank=1&sect=Xdfe0156.
  19. Dearden S et al. Mutation incidence and coincidence in non small-cell lung cancer: meta-analyses by ethnicity and histology (mutMap). Ann Oncol. 2013 Sep;24(9):2371-6.
  20. Sharma S et al. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007; 7: 169–181.
  21. Shigematsu H et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005; 97: 339–346.