Advances in precision medicine, using genetic biomarkers for molecular diagnostics, have transformed cancer diagnosis and treatment. The biological pathways that underpin cancers are notoriously complex and vary greatly across patients and tumor types. With the rapid emergence of liquid biopsy applications, tools to improve patient outcome have been identified. These include developments in early diagnosis, selection of targeted treatments, and monitoring of real-time treatment responses.
Next-generation sequencing (NGS) underpins many clinical tests aimed at detecting genetic variations; however, errors can be introduced at various stages of the workflow, from sample preparation to bioinformatic analysis. Due to an increasing focus on molecular assays for cancer diagnostics, it is more important than ever to make sure that quality reference standards, which closely represent patient samples, are used throughout the process.
Broadening the reach of cancer diagnostics
Genetic studies for cancer patients can be broadly grouped into cytogenetics and molecular genetics. Cytogenetics detects major chromosomal aberrations, whereas molecular genetics provides a deeper insight into specific mutations. Insight at the molecular level is crucial for differential diagnosis, prognosis, and disease management.
Liquid biopsy, a more recently adopted application, utilizes the analysis of cell-free DNA (cfDNA) and biomarkers in non-solid biological samples, such as blood, to identify and profile specific tumor types.1 cfDNA consists of short fragments of nucleic acids present in peripheral blood, shed by dying cells. Epigenetic and genetic information can be obtained from DNA fragments shed by cancer cells, allowing for earlier detection and diagnosis, as well as the effective measurement of therapeutic responses to inform further treatment decisions. Though tumor DNA constitutes only a small fraction of the total cfDNA population, it is proving an important biomarker as it reduces the need for recurrent, invasive, solid tumor biopsies.
Though promising, liquid biopsy is not without its challenges. Preanalytical workflows—sample collection, storage, and processing—are critical to cfDNA quality and quantity, and standardization across each stage is essential for reproducible results. For example, improper storage can result in cfDNA contamination with genomic DNA released from lysed white blood cells; specialized tubes are used to prevent lysis; however, there could be variations in sample preservation capabilities between different tubes.2 Consistent and appropriate storage conditions are therefore crucial to maintaining the integrity of cfDNA.
Variant allele frequencies (VAFs) of tumor-specific mutations are much lower in cfDNA populations compared to primary tumors, and thus accurate detection of low-level VAFs demands analytical tools with high sensitivity and specificity. Advances in NGS technologies have driven the continued success of tumor profiling by allowing for variant detection in multiple genes simultaneously, and higher scalability and throughput compared with PCR-based methods.3
Beyond preanalytical workflows, molecular assays, including NGS, involve multiple steps that are prone to both technical and biological errors. For any test to be useful, and hold clinical value, it is imperative to identify and control variabilities.
Improving diagnostics with cell line-derived controls
Despite significant progress both analytical and bioinformatic techniques to improve specificity and sensitivity in genetic diagnostics, the use of reference standards is essential to cement confidence in workflows, and to explain variabilities (Figure 1). Reference standards used in molecular diagnostics need to be homogenous, well-characterized, and mimic the properties intended for use in analytical measurements. Enter, cell line-derived controls; a renewable source that offers the complexity of the human genome, and thus commutability to patient samples.
Cell line-derived controls have grown from single gene references to highly multiplexed gene panels that contain a larger number of genes and variants implicated in cancers. These multiplexed panels are particularly useful in validating NGS large gene panel assays, where multiple variant types are analyzed.
Though patient-derived samples are a plausible source of reference material, they are non-renewable with properties that greatly depend on patient health status. Furthermore, differences in sample collection, storage, and processing may introduce variabilities of their own. Cell line-derived controls are a renewable and reproducible resource, that mimic patient samples while alleviating their limitations, and ensure consistency throughout the development and validation of diagnostic assays.
Cell line-derived controls are available in a range of formats, including cfDNA and formalin-fixed paraffin-embedded (FFPE) cell line sections, which mimic liquid biopsy and solid tumor samples, respectively. To allow researchers to validate their analysis pipeline further, reference standards are orthogonally validated by droplet digital PCR.
To ensure standardization across platforms, regulatory bodies have published guidelines for NGS-based cancer diagnostics tests that establish the validation requirements for test use in clinical and public health laboratories. Recommendations include the use of positive, negative, and sensitivity controls in the NGS workflow, and state that the reference material should mimic patient samples.4 Using standards derived from cell lines, offers researchers a comprehensive, reliable, and consistent source of reference material that complies with regulatory standards and ensures accurate diagnosis.
On the horizon
Liquid biopsy holds immense promise for diagnostics in oncology, and a breadth of other genetic diseases. Enabled by NGS technologies, researchers are now able to define signatures for specific tumors from patient samples, as well as identify the underlying molecular causations, to facilitate early diagnosis and the development of more targeted therapies. To realize its full potential for clinical use, it is crucial that effective reference standards, such as cell line-derived controls, are used to monitor assay performance and support the exciting advances in this field.
Figure 1. Using appropriate reference standards at each stage of the liquid biopsy workflow provides confidence in the analysis.
References
1. National Cancer Institute. Liquid Biopsy: Using DNA in Blood to Detect, Track, and Treat Cancer. (2017).
2. Sato, A. et al. Investigation of appropriate pre-analytical procedure for circulating free DNA from liquid biopsy. Oncotarget 9, 31904-31914 (2018).
3. Buzdin, A. et al. Editorial: Next Generation Sequencing Based Diagnostic Approaches in Clinical Oncology. Frontiers in Oncology 10, (2021).
4. New York State Department of Health. Oncology – Molecular and Cellular Tumor Markers. “Next Generation” Sequencing (NGS) guidelines for somatic genetic variant detection. (2015).