Lung cancer is the most common cancer worldwide, with non-small-cell lung carcinoma (NSCLC) accounting for more than 1 in 10 of all cancers diagnosed in men (1). More than 30 years ago, epidermal growth factor receptor (EGFR) was identified as being more concentrated in human lung tumors than in normal lung membrane tissue (2). EGFR gene mutations occur in a substantial number (10-50%) of NSCLC tumors (3,4). Tyrosine kinase inhibitor (TKI) treatment, such as with gefitinib (Iressa) or erlotinib (Tarceva), for certain types of EGFR mutations (e.g., exon 19 deletion, exon 21 L858R, exon 18 G719X) has been shown to be beneficial in these cases, but screening patients for mutations indicative of treatment can be challenging. Likewise, monitoring for acquired resistance to EGFR TKI therapy that is associated with a site mutation at EGFR position T790M could be beneficial to direct treatment. Tissue biopsy analysis is the standard for detecting EGRF mutations in NSCLC patients, but it is often a risky procedure (5). As many as 27–31% of patients with NSCLC may be unable to provide a biopsy sample usable for EGFR mutation analysis at diagnosis (6). Prompt identification and monitoring of tumor mutation status is critical for effective treatment and survival of these patients.
Analysis of ccfDNA (circulating cell-free DNA) including ctDNA (circulating tumor DNA) from blood samples offers advantages over tissue biopsy due to ease of sampling and the ability to sample repeatedly over days, weeks or months, thus allowing routine monitoring of cancer status. Although qPCR and dPCR has been the norm for detecting EGRF mutations, a recent article by Malapelle, U. et al. (2016) examined EGFR next-generation sequencing (NGS) mutational analysis using ccfDNA from NSCLC patients (7). During their work, they were able to identify critical workflow and validation processes for successful tumor identification and monitoring. Their recommended approach for analysis is by ultra-deep sequencing, using focused gene panels to restrict coverage to clinically relevant targets. Each read is sequenced thousands of times, and thus provides a high degree of sensitivity to allow detection of low abundant ccfDNA in blood. Critical NGS workflow points discussed in the publication along the workflow are included:
Blood collection and ccfDNA extraction
When using EDTA-containing tubes (Vacutainer) for blood collection, plasma collection using centrifugation should be performed within 1 hour of blood collection since ccfDNA has a short (15 minute) half-life. There are newer alternative blood collection tubes, and systems are available that contain formaldehyde-free preservative agents which prevent the release of genomic DNA by stabilizing nucleated blood cells and inhibiting ccfDNA degradation.
The newly launched PAXgene Blood ccfDNA Tubes from QIAGEN standardize preanalytical processing of samples, rendering them stable for transport and storage at room temperature for up to 7 days. PAXgene Blood ccfDNA Tubes can be used with either the QIAamp MinElute ccfDNA Kit (a new faster spin-column-based kit that offers advantages over the QIAamp Circulating Nucleic Acid Kit) for manual processing of samples, or as part of the QIAsymphony PAXgene Blood ccfDNA System (the system includes both tubes and the QIAsymphony PAXgene Blood ccfDNA Kit) for automated ccfDNA purification on the QIAsymphony SP Instrument. With the later, the included tubes can be processed directly as primary blood collection tubes after plasma separation on the QIAsymphony SP.
Library preparation and sequencing
Rather than performing whole exome sequencing, use of targeted panels is recommended to provide a low limit of detection (LOD) that is required for ccfDNA. Several targeted panels are commercially available that cover 15–30 kb of the exome for analysis of 20–50 genes containing key oncogenic mutational hotspots. However, these commercial panels did not provide sufficient LOD to identify somatic mutations in ccfDNA for Malapelle, U. et al.
QIAGEN’s new generation of targeted DNA panels (the QIAseq Targeted DNA panels) use Unique Molecular Indices (UMIs) introduced before library PCR amplification. This allows more accurate locus-specific interpretation of results. A key point for appropriate variant calling sensitivity in ccfDNA NGS analysis is the balance between DNA input and sequencing depth. Additional platform-specific indexes allow multiplexing of up to 384 samples per sequencing run.
The Human Lung Cancer QIAseq DNA Panel is a targeted panel to enrich for specific lung cancer genes and construct libraries for NGS analysis of 72 genes most commonly mutated in human lung cancer samples and involved in lung cancer development and progression.
Variant calling and visual inspection
Although a large number of bioinformatics tools currently exist for tissue-based analyses, our understanding of appropriate parameter selection for ccfDNA variant calling is limited. Thus, different bioinformatics pipelines can show variability in their variant calling, even when based upon the same human reference sequence (currently hg19).
As part of the Advanced Testing Solution from QIAGEN, our end-to-end workflow features Biomedical Genomics Workbench and Ingenuity Variant Analysis to combine analysis and interpretation of targeted ccfDNA data. It features optimal parameter settings across the complete workflow, to easily help identify and inspect known and new potential pathogenic and actionable cancer variants. Samples from several patients can also be compared afterwards in Ingenuity Variant Analysis to allow the identification of new upcoming pathogenic variants.
In summary, a careful validation process that includes blood collection and stabilization, ccfDNA purification, library preparation, NGS and variant calling can pave the way for routine use of ccfDNA analysis using NGS for diagnosis and to support treatment decisions for NSCLC patients.
Download our latest research poster that shows how blood collection and stabilization is critical for ccfDNA isolation and analysis of cancer biomarkers.
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- 1. Cancer Research UK. Worldwide Cancer Statistics webpage: http://www.cancerresearchuk.org/health-professional/cancer-statistics/worldwide-cancer#heading-Zero.
- 2. Veale, D., Kerr, N., Gibson, G.J., Harris A.L. (1989) Characterization of epidermal growth factor receptor in primary human non-small cell lung cancer. Cancer Res. 49, 1313–1317. Link
- 3. Markman, M. (2016) Genetics of Non-Small Cell Lung Cancer. Medscape webpage. Link
- 4. Rosell, R. et al. (2009) Screening for epidermal growth factor receptor mutations in lung cancer. N. Engl. J. Med. 361, 958–967. Link
- 5. Morgensztern, D., Politi, K., Herbst, R. (2015) EGFR mutations in non-small-cell lung cancer: Find, divide, and conquer. JAMA Oncol. 1, 146–148. Link
- 6. Normanno, N., Denis, M.G., Thress, K.S., Ratcliffe, M., Reck, M. (2017) Guide to detecting epidermal growth factor receptor (EGFR) mutations in ctDNA of patients with advanced non-small-cell lung cancer. Oncotarget 8, 12501–12516. Link
- 7. Malapelle, U. et al. (2016) Next generation sequencing techniques in liquid biopsy: focus on non-small cell lung cancer patients. Transl. Lung Cancer Res. 5, 505–510. Link