EGFR Mutation Testing of circulating free tumour DNA (ctdna)

Transcription

1 EGFR Mutation Testing of circulating free tumour DNA (ctdna) This booklet describes EGFR mutation testing in circulating-free tumour DNA (ctdna). ctdna may be considered for EGFR mutation testing, where no tumour sample is available EGFR mutations identified in ctdna are highly predictive of EGFR mutation positive tumours. However it is not always possible to detect EGFR mutations using this sample type1, 3, 17 Robust DNA extraction and sensitive EGFR mutation testing methods are essential to maximise the likelihood of successful analysis EGFR mutation testing should be performed on a tumour sample where available AstraZeneca 2015 FOR HEALTHCARE PROFESSIONALS ONLY ATLAS ID: 724, Date of preparation 27/4/15. Expiry Date 27/4/16

2 CONTENTS PAGE What is circulating free tumour DNA (ctdna)? 3 The use of ctdna for EGFR mutation testing When is it appropriate to use ctdna? 4 Positive predictive value of ctdna versus false negative rate 5 Prediction of response to EGFR-TKI treatment 6 Sample processing methods Sample types 8 Pre-analytical processing 9 DNA extraction methods 10 EGFR mutation testing methods 11 Summary 14 Further Information 15 References 16 AstraZeneca 2015 FOR HEALTHCARE PROFESSIONALS ONLY

3 Circulating free tumour DNA is present in the blood of many patients with advanced NSCLC Circulating free tumour DNA (ctdna) released from tumour cells is present in the blood of many, but not all, patients with advanced non-small cell lung cancer (NSCLC) 1, 4. Several studies have demonstrated the utility of ctdna in the advanced NSCLC setting. EGFR mutations have been detected using ctdna isolated from blood plasma/serum 1, 2, 3, 5, 17, and EGFR mutations present in ctdna have been shown to predict response to EGFR-TKI treatment 1, 5, 17. Proposed mechanisms by which tumour cells release DNA into the bloodstream4, 6, 7 Necrosis or apoptosis of the tumour cells Lysis of circulating tumour cells Spontaneous active secretion of DNA by the tumour Healthy cells Neoplastic cells Neoplastic cells undergoing necrosis/lysis Neoplastic cells undergoing apoptosis DNA 3

4 Where tumour samples are not available, ctdna may be used for EGFR mutation testing Tumour samples (e.g. biopsy, resection, cytology) which are used for the diagnosis of advanced NSCLC are the preferred sample type for EGFR mutation testing. These samples are collected directly from, or local to, the primary tumour or metastatic sites, and typically contain sufficient tumour material to obtain an accurate EGFR mutation result. However not all patients are able to provide an adequate sample and re-biopsy is not always possible. In the IFUM study, EGFR mutation tumour status was unable to be determined in 19% of eligible patients due to reasons including; absence of biopsy, low tumour content, poor sample quality, insufficient quantity, poor / inappropriate fixation, or no DNA 17. In these instances, ctdna provides an alternative sampling method for EGFR mutation testing. Analysing ctdna samples from patients where tumour or cytology samples are not available could increase patient access to EGFR mutation testing, allowing them to be treated appropriately based on their EGFR mutation status (i) Number of patients tested for EGFR mutations Number of patients tested for EGFR mutations????? Tumour or cytology sample analysed Tumour, cytology or ctdna sample analysed Evaluable sample: mutation detected Evaluable sample: mutation not detected? Non-evaluable sample / No sample available (i) Figure for illustrative purposes only, EGFR mutation frequencies may vary depending on patient population 4

5 EGFR mutations identified in ctdna are highly predictive of EGFR mutation positive tumours, however false negative results are often observed An extremely high specificity and concordance rate are observed between matched EGFR mutation positive ctdna and EGFR mutation positive tumour biopsy samples 1, 3, 17. However, the proportion of mutant tumour DNA within the circulation may be low, or not present at all 3, 8. Limitations in DNA extraction and EGFR mutation testing methods may also impact detection rates in ctdna 9. Therefore it is not always possible to detect EGFR mutations using this sample type 1, 3, 17. Percentage of EGFR mutation positive tumours identified from matched ctdna samples (sensitivity) 43.1 % 65.7 % IPASS 1 (i) Tumour mutation status IFUM 17 (ii) Tumour mutation status M+ M- Total M+ M- Total ctdna mutation status M M Total ctdna mutation status M M Total Positive predictive value of ctdna = 100% Specificity = 100% Concordance = 66.3 % Positive predictive value of ctdna = 98.6 % Specificity = 99.8% Concordance = 94.3% (i) Tumour biopsy and matched serum-derived ctdna samples. CtDNA extracted using Qiagen QIAamp DNA Blood Kit and EGFR mutations detected using DxS EGFR Mutation Test Kit. False negative rate 56.9 % 1 (ii) Tumour biopsy and matched plasma-derived ctdna samples. CtDNA extracted using Qiagen QIAamp Circulating Nucleic Acid Kit and EGFR mutations detected in singleton using Qiagen Therascreen EGFR RGQ PCR Kit. False negative rate 34.3 % 17 5

6 EGFR mutation positive ctdna predicts response to EGFR-TKI treatment (IPASS) In 2012, Goto et al compared the outcomes of 194 Japanese patients with NSCLC, randomised to gefitinib or carboplatin/paclitaxel, whose ctdna EGFR mutation status had been assessed (IPASS) 1. A significantly longer progression free survival (PFS) and higher objective response rate (ORR) were observed in the ctdna EGFR mutation-positive patients when treated with gefitinib, compared to carboplatin/paclitaxel 1 : PFS: [HR], 0.29; 95 % [CI], ; p < ORR: [OR], 1.71; 95 % [CI], ; 75.0 % versus 63.6 %; p = 0.40 Probability of progression-free survival n Events Gefitnib (62.5%) Carboplatin / paclitaxel (86.4%) HR = 0.29 (95% CI, ) p < Kaplan-Meier curve of PFS in ctdna (serum-derived) EGFR mutationpositive patients in the Japanese subset of IPASS 1. Hazard ratio (HR) <1 indicates a difference in favour of gefitinib; CI, confidence interval; OR, odds ratio Time (months) Matched tumour and ctdna results were available for 86 patients. Fewer patients were EGFR mutation positive when assessed using ctdna (23.7 %) versus tumour tissue derived DNA (61.5 %). ctdna results identified no false positives, but a high rate of false negatives were observed when compared to tumour samples (56.9 %) 1. 6

7 EGFR mutation positive ctdna predicts response to EGFR-TKI treatment (IFUM) In 2014, a study by Douillard et al assessed 106 NSCLC patients with EGFR mutation positive tumours, to determine the efficacy of gefitinib in Caucasian population (IFUM) 17. EGFR mutation status was also assessed in duplicate ctdna samples. 69 patients whose tumour were EGFR mutation positive were also found to harbour EGFR mutation positive ctdna 17. When treated with gefitinib, a similar objective response rate was observed in the tumour and ctdna positive subgroup, compared with the overall tumour positive population 18. This finding in Caucasian patients is consistent with the IPASS data in Japanese patients. Objective response rate in patients with EGFR mutation positive ctdna treated with gefitinib in IFUM17, Objective response rate (%) % 76.9% 0 Tumour M+ ctdna M+ ORR in Tumour biopsy and matched plasma-derived ctdna samples. CtDNA extracted using Qiagen QIAamp Circulating Nucleic Acid Kit and EGFR mutations detected in duplicate using Qiagen Therascreen EGFR RGQ PCR Kit 17 All ctdna M+ samples are also tumour M+, with the exception of a single sample which was M+ by ctdna but M- by tumour This study also observed an extremely high concordance rate in duplicate ctdna samples (96.9%), indicating that plasma-derived ctdna can reliably be used to perform EGFR mutation testing using a single sample when tumour tissue is unavailable or exhausted. 7

8 Both plasma and serum can be used to isolate ctdna suitable for EGFR mutation testing Plasma is superior to serum for EGFR testing 10, however either may be used providing appropriate DNA extraction and EGFR mutation detection methods are used. Higher detection levels of EGFR mutations have been observed in plasma when compared to matched serum samples 10 As a guide, approximately 5 ml of plasma/serum is obtained from every 10 ml of blood collected. Successful analysis can be achieved using 1-2 ml of plasma Recommended procedures to obtain plasma and serum from blood Plasma Serum Collect blood (i) 5ml 2ml EDTA 5ml 2ml No anticoagulant Minimum volume Minimum volume Process to plasma / serum As soon as possible As soon as possible Immediately freeze -70 C / -80 C -70 C / -80 C (i) Heparin collection tubes must not be used, as the heparin interferes with downstream PCR applications 8

9 Optimal pre-analytical processing steps improve ctdna isolation from plasma Blood cells begin to lyse soon after blood draw, releasing genomic DNA into plasma. The increased abundance of normal genomic DNA (gdna) dilutes the tumour derived ctdna making it more difficult to detect mutations. In addition the DNA also begins to degrade after blood collection. Delays in plasma isolation after blood draw leads to blood cell lysis and increased DNA yield at both RT and 4 C. 11 (The upper and lower limits of the boxes and the line across the boxes indicate the max and min values and the mean, respectively). The implementation of a couple of simple pre-analytical processing steps help to improve sample stability and improve mutation detection from ctdna Pre-analytical Improvement step Optimal sample collection vessel Swift processing into plasma Double centrifugation spin Details Collection in EDTA tubes is recommended. Heparin tubes should not be used. Cell free DNA blood collection tubes (Streck) may prove to be a more optimum stabilisation method 13, but these have not yet been clinically validated. Process within 2hr of blood draw Blood cells begin to lyse and release gdna into the plasma immediately after blood draw, faster preparation reduces gdna contamination. 11,19. 1 st low speed spin ( g ) for 10 mins 2 nd high (or low (i)) speed spin ( g) for 10 mins 24 The additional spin helps to remove all cell debris from the plasma 20 9 (i) Low speed 2 nd spin is better than no 2 nd spin, high speed spin is optimal, but not always possible

10 Robust DNA extraction methods are required to obtain sufficient quality DNA DNA extraction methods specifically developed for extraction of DNA from plasma should be used to maximise the yield and quality of ctdna, in order to increase assay success rate and reduce the risk of false negatives. A number of commercial kits are available for the extraction of ctdna from plasma or serum, yet ctdna yields resulting from the different methods vary. The QIAamp Circulating Nucleic Acid Kit (QIAGEN) has been shown to generate good yields of ctdna 21,22 and has been clinically validated 17. Differences in DNA yield are observed using different DNA extraction methods(i) 21 QIAamp circulating nucleic acid kit Two other commercial plasma DNA extraction kits QIAamp DNA blood mini kit (ii) Manufacturer / Method Kit Plasma / Serum Volume ml (iii) Elution Volume L Qiagen Silica-based separation membrane used in combination with spin columns or QIAvac vacuum technology QIAamp Circulating Nucleic Acid Kit12, (i) (ii) (iii) Yield of ctdna assessed by qpcr of ALUJ (grey bars) and TERT (white bars) Kit not specifically developed for extraction from serum / plasma Modifications to process may be required for larger starting volumes 10

11 Reliable, sensitive testing methods are required to maximise the likelihood of obtaining an accurate result Different EGFR mutation detection methods have varied sensitivities, and are often validated on tumour-derived DNA. To date, publications on method comparison data for use with ctdna are limited. To minimise the risk of false negative results, the use of a highly sensitive EGFR mutation detection method is strongly advised when analysing ctdna samples 9. Sensitivity of Amoy EGFR Mutations Detection Kit vs. sequencing for detection of EGFR mutations in ctdna (i) 20 Number of samples n = 13 n = 18 EGFR M- EGFR M+ n = 5 0 Amoy EGFR Kit Sequencing (i) Data generated from plasma-derived ctdna samples extracted using Qiagen QIAamp DNA Blood Kit. EGFR mutations detected using Amoy EGFR Mutations Detection Kit and sequencing. DNA sequencing is less sensitive than qpcr methods, such as the Amoy kit. Matched tumour data not available 9 Further information can be found in the Sensitive EGFR Mutation Testing brochure and at 11

12 Reliable, sensitive testing methods are required to maximise the likelihood of obtaining an accurate result The choice of testing method is often influenced by a laboratory s expertise and available equipment: labs can use commercially available kits, develop their own tests, or send samples to an external specialist testing facility. The chosen test should be optimised for the samples to be analysed. Method Advantages Commercial kits Rapid Quality controlled Well tested Often with In-Vitro Diagnostic (IVD) status Lab developed tests Can be less expensive and uses equipment that is commonly available External testing labs No internal investment required to establish a testing lab or method Choice of either commercial kits or lab developed tests 12

13 Reliable, sensitive testing methods are required to maximise the likelihood of obtaining an accurate result The following sensitive testing methods have been used to analyse the EGFR mutation status of ctdna samples. Commercial Kits Manufacturer / Kit Approx. Sensitivity / Limit of Detection (i) Reference for use with ctdna Samples Qiagen therascreen EGFR Plasma RGQ PCR Kit 1 % Douillard 17 AmoyDx EGFR Mutations Detection Kit (ii) Roche Cobas EGFR Blood Test (Not yet commercially available - test in development) 1 % Liu 9 1% Weber 23 Lab Developed Tests Method Approx. Sensitivity / Limit of Detection (i) Reference for use with ctdna Samples PNA Clamp (iii) 1 % Rosell 2 Microfluidics Digital PCR 0.1 % Yung 3 BEAMing (iv) 15, % Taniguchi (i) (ii) (iii) (iv) Minimum % of mutant alleles in a wild-type background required for reliable detection (in tumour-derived DNA) Current version: EGFR 29 Mutations Detection Kit Service providers include Pangaea Biotech ( Service providers include Inostics (

14 Reliable, sensitive testing methods are required to maximise the likelihood of obtaining an accurate result Further information can be found in the Sensitive EGFR Mutation Testing brochure and at 14

15 Summary Tumour samples (e.g. biopsy, resection, cytology) are the most robust and well documented method for determining EGFR mutation status in advanced NSCLC. EGFR mutation testing in ctdna often results in a high number of false negatives: the primary tumour may be EGFR mutation positive even if the ctdna test is negative1, 2, 3,17 EGFR mutations identified in ctdna are however highly predictive of EGFR mutation positive tumours 1, 3, 17. Positive results may be used to select therapies suitable for EGFR mutation positive tumours, however atumour sample should be used where available. ctdna can be isolated from blood plasma and serum samples, however plasma is associated with higher EGFR detection 10. DNA extraction methods and pre-analytical processing steps optimised and validated for ctdna, together with reliable and sensitive EGFR mutation testing methods, are essential to maximise the likelihood of successful analysis 9,21. ctdna has been shown to be a viable alternative for EGFR mutation detection when a tumour sample is not evaluable. In 2014, the EMA approved a label update for IRESSA for use of ctdna in such situations. Further Information Commercial EGFR mutation testing kits: Lab developed EGFR mutation testing methods: Local EGFR testing labs: Diagnostic questions: diagnostics-questions@astrazeneca.com 15

16 References 1. Goto, K et al. (2012) Journal of Thoracic Oncology 7: Rosell, R et al. (2009) The New England Journal of Medicine 361: Yung, TKF et al. (2009) Clinical Cancer Research 15: Pathak, AK et al. (2006) Clinical Chemistry 52: Kimura, H et al. (2006) Clinical Cancer Research 12: Gormally, E et al. (2007) Mutat Res Fund Mol Mech Mut 635: Aung, KL et al. (2010) The HUGO Journal 4: Diehl, F et al. (2005) Proceedings of the National Academy of Sciences 102: Liu, Y et al. (2011) Journal of Experimental and Clinical Cancer Research 30: Vallée, A et al. (2013) Lung Cancer 82: Xue, X et al. (2009) Clinica Chimica Acta 404: Morgan, SR et al. (2012) Clinical Medicine Insights: Pathology 5: Norton, SE et al. (2013) Journal of Clinical Laboratory Analysis 27: Taniguchi, K et al. (2011) Clinical Cancer Research 17: Diehl, F et al. (2006) Nature Methods 3: Li, M et al. (2006) Nature Methods 3: Douillard, J-Y et al. (2014) British Journal of Cancer 110: Douillard, J-Y et al. (2013) P IASLC 15 th World Conference of Lung Cancer 19. El Messaoudi, S et al. (2013) Clinica Chimica Acta 424: Swinkels, DW et al. (2003) Clinical Chemistry : Devonshire, AS et al. (2014) Anal Bioanal Chem 406: Page, K et al. (2013) PLOS ONE 8 (10 ) e Weber, B et al (2014) BioMed Central 14: ECMC 2014 cfdna consensus meeting report ( ATLAS ID: 724, Date of preparation: 27/4/15 Date of expiry: 27/4/16 AstraZeneca 2015 FOR HEALTHCARE PROFESSIONALS ONLY