EGFR mutation testing of tumor sample DNA from patients with nonsquamous advanced NSCLC is advocated by clinical guidelines (1, 2) and working groups (3, 4) and is required to confirm whether patients should receive EGFR tyrosine kinase inhibitor (TKI) treatment. However, tissue samples are not always available or evaluable for diagnosis and mutation testing, leaving some patients ineligible for EGFR testing and targeted therapies. To overcome these issues, cell-free circulating tumor DNA (ctDNA) isolated from patient liquid biopsy may be a non-invasive option to obtain the necessary genetic follow-up data.
The IFUM study (5) has already shown the feasibility of using ctDNA isolated from plasma to determine EGFR mutation status in a small cohort of Caucasian NSCLC patients. However, the accuracy, suitability and feasibility of ctDNA for mutation analysis in clinical practice must be confirmed outside controlled clinical trials. ctDNA analysis is technically challenging, and is not yet widely implemented in local diagnostic labs. In order to enable wider access to ctDNA mutation testing, and to provide more patients the opportunity to receive precise, targeted therapies, a group of lung cancer experts across the world performed the ASSESS study, the largest real patient study for plasma as a surrogate for tissue for EGFR testing (6). In this study over 1300 patients were enrolled across 56 centers throughout Europe and Japan. The ASSESS study was designed to evaluate how local practices for tissue and plasma sample testing compare with well-controlled clinical testing, as performed in a single expert laboratory. Using real-world data, results from this large, non-interventional diagnostic study confirm that ctDNA is a feasible patient sample for EGFR mutation analysis, with an overall mutation status concordance of 89% between 1162 matched tissue and plasma samples. The ASSESS study confirms findings from the IFUM study, which indicate that EGFR mutations detected in ctDNA can be trusted, and that the results can be used to determine whether TKI therapy is likely to benefit the patient.
By far, the main method for EGFR testing in the European centers of the ASSESS study was QIAGEN’s therascreen EGFR assays. The therascreen_EGFR_RGQ_PCR_Kit† was used for almost 34% of all tissue tests and the therascreen_EGFR_Plasma_RGQ_PCR_Kit†† was used for nearly 50% of all plasma tests. Other testing methods used in these centers were mainly a variety of lab developed tests (LDTs) involving DNA sequencing, fragment length analysis, pyrosequencing or PNA-LNA PCR Clamp technologies. Other commercial kits were tested in the European centers as well, however less frequently. The ASSESS study reports substantial differences in results obtained among the different mutation testing methodologies used, and show that commercial tests perform better than LDTs for EGFR testing from plasma by delivering better positive predictive values (PPV) and sensitivity. Among commercial tests used the QIAGEN and Roche tests had the best analytical sensitivities, which were very comparable (~75%) and the best analytical specificities (~100%). However, the Roche study arm was poorly powered, with only 23 patients tested in both tissue and plasma compared to 138 patients using the QIAGEN assays for both specimen types.
An initial concern was the potentially false-positive results obtained from plasma samples. The authors determined this to be rather an artifact caused by false-negative results from tumor samples, which means the positive plasma test result from tests such as QIAGEN’s therascreen EGFR Plasma test was correct, but the initial tissue assessment was incorrect. Erroneous tissue testing can occur due to spatial and temporal tumor heterogeneity (10), inexperience or use of methods with low sensitivity, such as Sanger sequencing or LDTs. The authors conclude that mutation testing of both tumor and plasma samples should be conducted using robust/sensitive methods to ensure that patients receive appropriate treatments that target the specific molecular characteristics of their disease. Due to low amounts of ctDNA in plasma, comprising less than 1% of total circulating free DNA (cfDNA) from both normal and tumor cells (7), clinically appropriate DNA extraction and mutation analysis methodologies are fundamental to the feasibility of this method for patient EGFR mutation detection. Standardized and robust pre-analytical methods for blood and plasma collection are also necessary for successful mutation detection from ctDNA (8).
To support using liquid biopsy for clinical research, QIAGEN offers an entire workflow_of_solutions to enable robust cfDNA isolation from plasma and biomarker mutation detection and analysis from ctDNA samples, and the portfolio is growing rapidly. QIAGEN recently launched their PAXgene_Blood_ccfDNA_Tubes** which contain a unique non-crosslinking reagent that ensures cfDNA levels are preserved during collection and transport. The PAXgene ccfDNA Tubes contribute to a fully integrated, complete and standardized system, covering pre-analytical workflow steps from blood collection, stabilization, transport, storage and isolation of high quality cfDNA. The QIAamp_Circulating_Nucleic_Acid_Kit * – considered a gold standard sample technology for the processing of of cfDNA (9) – provided the basis for ctDNA sample preparation for EGFR plasma testing in the ASSESS study, both using QIAGEN’s therascreen kit, and using LDT methods. QIAGEN is in the process of finalizing development of a fully automated IVD kit for extraction of cfDNA on the QIAsymphony, which will make sample preparation even quicker and easier. This kit incorporates significant improvements in extraction chemistry to enhance processing of large sample volumes (e.g., 4 ml plasma) and to increase throughput on the QIAsymphony SP instrument. This improved cfDNA extraction kit is planned to launch in Q1 2017. In June, QIAGEN introduced a liquid_biopsy_workflow for the QIAact_Tumor_Panels** on the GeneReader system, which enables detection of a broad range of biomarkers in clinical research to add valuable insights about the progression of the most common cancers. For research labs focusing on new biomarker discovery using Illumina or Ion Torrent sequencers, the QIAseq_cfDNA_All-in-One_Kit * uniquely combines cfDNA extraction from plasma with NGS library preparation in a way that avoids sample loss while ensuring optimal sample conversion at every step. The cfDNA extraction step is based on the same technology as the gold standard QIAamp Circulating Nucleic Acid Kit.
QIAGEN also offers resources to support labs developing liquid biopsy techniques. Watch this webinar on EGFR testing from liquid biopsy, hosted by Dr. Marc G. Denis, from Nantes University Hospital (France). Check out an interview with Dr. Nicola Normanno, one of the principal investigators from the ASSESS study, where he discusses liquid biopsy and the need for robust, sensitive methods and multiplex technologies to make testing from liquid biopsy more widely implemented. The Normanno lab uses QIAGEN solutions for both research and clinical application. See Dr. Normanno and his lab in action by watching this short video, and listen to his vision for the development of precision medicine.
QIAGEN continues to engage with thought leaders around the world to ensure strategies for oncology solutions meet the evolving market need for cancer research and molecular diagnostics. The therascreen_EGFR_Plasma_RGQ_PCR_Kit used in the ASSESS study, and the array of products QIAGEN offers for liquid biopsy, are just a few examples of QIAGEN’s leadership and innovation. Read more about QIAGEN’s recent advances in liquid biopsy technologies, and the broad range of oncology research and diagnostic solutions available in the US and throughout Europe.
† The therascreen EGFR RGQ PCR Kit is approved for IVD use in Europe, US, China, Japan and many others.
†† The therascreen EGFR Plasma RGQ PCR Kit is available for IVD use in Europe.
* The QIAamp Circulating Nucleic Acid Kit and the QIAseq cfDNA All-in-One Kit are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
** The PAXgene Blood ccfDNA Tubes and the GeneRead QIAact Panel are for research use only (RUO).
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- 2. NICE. EGFR-TK mutation testing in adults with locally advanced or metastatic non-small-cell lung. Link. Accessed July 8, 2016.
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- 9. PR newswire press release, Jan 12, 2015. QIAGEN Announces First-ever Regulatory Registration of a Lung Cancer Companion Diagnostic Based on Liquid Biopsies. Link
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