We all know NGS analysis can be difficult to navigate. Not only is the human genome complex, but technical errors can occur throughout the entire workflow – from sample preparation, through sequencing and during analysis.
Thankfully, these technical errors can be mitigated by using high-quality reference material. Using the best standards for your assay will help both calibrate your NGS measurements and evaluate your diagnostic performance.
With advances in affordable technologies, there is now a multitude of reference types to choose from including patient material, cell line-derived standards and synthetic spike-ins. In this blog, we shine the spotlight on cell line-derived standards and the distinct advantages these have when used for NGS oncology assays.
7 advantages of cell-line derived reference material for NGS oncology assays
- Representative of full genomic complexity Reference Standards derived from cancer cell lines offer an incredibly close representation of the patient tumor sample. They contain mutations of interest in the context of the complete genome, which makes them an ideal reference for both calibrating and validating your oncology assay. Cell line-derived reference standards are classed as Commutable – i.e. they perform similarly to a patient genome sample during sequencing and analysis. This also means they can contain thousands of mutations, including SNPs, insertions, deletions and CNVs.
- Reproducible source of material Cell lines can be easily maintained, under the right conditions, allowing cell line-derived reference materials to be reproduced again and again to high quality standards. This provides a limitless source of consistently high quality reference material that is reliable over time. In comparison, real FFPE patient material is often of limited quantity and variable quality throughout the block, leaving the lab at a loss when the material either runs out, or its genetic profile changes between sections.
- Long shelf life and easy storage Cell-line derived reference material can be stored for up to 48 months at 4°C. Compared to other standard types, this is significantly low maintenance and avoids DNA damage caused by repeated freeze/thaw cycles. One area where this is a considerable advantage over real patient material is in the growing field of liquid biopsy. Real blood and extracted plasma samples from patients have limited storage time due to degradation of the DNA by enzymes naturally found within the human vascular system. Some cell-line derived standards are available in synthetic plasma, which is stable over time and free from contaminating analytes and enzymatic degradation.
- Defined mutation allele frequency In oncology, driver mutations can be present at different allele frequencies (AF) depending on what percentage of the patients’ cells contain that mutation. If the mutation is present in the patient’s germline DNA (most likely having been inherited from one or both parents, or possibly created as a novel mutation during meiosis or early zygogenesis), it will be present at either 50 or 100% AF. Alternatively, if the mutation was created during a random mutagenic event within a somatic cell at any point during the patient’s lifetime, it will be present at a much lower AF (typically under 10% AF). The challenge for NGS technology developers is to improve sequencing sensitivity to enable the detection of these low frequency somatic oncogenic mutations. This allows new cancers, or new resistance mutations that evolve during monitoring of an existing tumor, to be detected and treated as early as possible. The limit of detection (LOD) of current clinical NGS assays is around 5% AF, but this can vary depending on the assay. In fact, best clinical practice dictates that the LOD needs to be calculated using a reference standard during the introduction and validation of every new gene panel before patient testing. Cell line-derived reference standards are produced to contain mutations at a broad range of AFs to challenge labs to correctly identify their accurate LOD.
- Copy number variations (CNVs) In addition to mutation AF, copy number is an increasingly important criteria to assess in clinical oncology. Many cancers are distinguished by having an abnormal number of copies of key oncogenes, most likely caused by genome instability and largescale chromosomal rearrangements in neoplasms. Sequencing technology developers are improving their capabilities in this area, and many NGS gene panels are now able to detect CNVs in a number of actionable genes. To validate the accuracy and guide necessary optimization of these challenging new techniques, cell line-derived reference standards are provided with known copy number information. This allows users to test the accuracy of their CNV assay in a controlled way that is not always possible with real patient material as the CNV status is often undetermined in real tissue.
- Different formats of reference standards to suit your sample. Cell-line derived reference standards can be developed into a variety of formats, to suit your sample and assay. These include:
- Genomic DNA (gDNA)
- Formalin-Fixed Paraffin-Embedded (FFPE) DNA
- Cell-free DNA (cfDNA)
- Formalin compromised DNA (fcDNA)