For Your Reference

Though next-generation sequencing (NGS) technologies have revolutionized cancer genomics by enabling comprehensive analysis of tumor genomes, the complexity of data interpretation poses a challenge for clinical practice. Clinicians, pharmaceutical companies, and companion diagnostics providers must navigate a vast array of genomic alterations, each with varying clinical significance. This work could include assessment of single genes (such as BRAF, EGFR or ALK), a composite genetic signature (such as mismatch repair or homologous recombination deficiency), or a comprehensive genome profiling (1).

What’s more, such molecular assays lack value without validation. With this in mind, we look at recent innovations in molecular reference standards for both solid and liquid samples in oncology.

The role of reference standards
 

Reference standards are well-established reference materials containing known variants across the entire genome. They serve as benchmarks for validating laboratory workflows and calibrating computational tools used for variant calling from patient samples. Scientists are able to compare identified variants in the reference standard with the known truth set. In this way, laboratories can assess their workflows’ sensitivity, specificity, and reproducibility, ensuring reliable detection and interpretation of patient mutations. 

In 2013, the Next-generation Sequencing: Standardization of Clinical Testing (Nex-StoCT) workgroup recognized the use of reference standards as a key element of the implementation of NGS workflows (2). This conclusion directly led to the creation of “genome in a bottle” sample sets that are used by multiple laboratories around the world. 

In oncology, no such reference standards exist. Recognizing this gap, a consortium led by the Medical Device Innovation Consortium (MDIC) started the Somatic Reference Samples (SRS) Initiative. This collaborative initiative is designed to elevate the accuracy of NGS-based oncology diagnostics. One of its primary goals is to establish publicly available cell-line derived reference samples and a global genomic data resource library. These resources are expected to be instrumental across the entire life cycle of NGS-based diagnostics.

The impact of the SRS Initiative extends far beyond the development of reference samples. It’s about transforming the entire ecosystem of NGS-based cancer diagnostics. From accelerating diagnostic development and regulatory approvals to enhancing reimbursement decisions and supporting precision medicine, SRSs are poised to make a profound difference in the lives of cancer patients.  

A pilot project has started to create an initial set of 10 reference samples together with validated data sets. Work is ongoing by Revvity Mimix to engineer clinically relevant cancer variants individually into a well-characterized cell line HG002 (PGP/GIAB) background to be made commercially available in an FFPE format.

Liquid biopsy and minimal residual disease testing
 

With the introduction of liquid biopsy techniques, circulating tumor DNA (ctDNA) has recently gained popularity and has proved transformative in cancer diagnosis and treatment decisions. New NGS-based assays for liquid biopsy, especially those targeting minimal or molecular residual disease (MRD), require rigorous validation using appropriate reference materials. Like standard DNA assays, MRD testing requires the detection of very low quantities of tumor-derived ctDNA fragments – often orders of magnitude lower than typical detection limits. 

For precise evaluation of assay performance including accuracy, analytical sensitivity, specificity, robustness and limit of detection of an assay, especially in the context of MRD detection, the development and use of “commutable” reference materials mimicking circulating tumor DNA (ctDNA) in real-world patient samples with extremely low variant allele frequencies are essential.  

Precision medicine
 

The field of personalized medicine in oncology has witnessed significant growth, fueled by the increased use of NGS and molecular assays for cancer detection, research, and diagnosis. The growing number of instrument platforms, assays, and targeted drugs have made the field of oncology companion diagnostics a hotbed of innovation. All these innovations drive the need for accurate and well-qualified quality controls and reference materials for assay development and implementation.

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