Over the past decade, liquid biopsy-based testing, especially using next-generation sequencing (NGS) to detect circulating tumor DNA (ctDNA) in plasma, has been increasingly adopted in clinical practice for cancer screening, diagnosis, therapy selection, and treatment response monitoring. This is due to the advantages it offers over tissue biopsy: less invasive nature, lower cost, real-time information on the state of the tumor and, in some cases, the ability to overcome the issue of tumor heterogeneity.1 However, only two NGS-based plasma ctDNA tests have been approved by the U.S. Food and Drug Administration (FDA) to date.2 These tests can identify single nucleotide variants (SNV), small insertions/deletions (indels), and structural alterations in cancer-related genes, as well as assessing tumor mutation burden (TMB) and microsatellite instability (MSI).2 The test results may have an immediate impact on the patient’s disease management for early cancer detection, staging, early relapse detection, real-time monitoring of therapeutic efficacy, and detection of therapeutic targets and resistance mechanisms. However, to become a part of routine clinical care, NGS-based plasma ctDNA assays must be thoroughly validated to demonstrate their analytical validity, clinical validity, and clinical utility as required by the FDA’s guidance for Medical Devices.3
Recently, the Association for Molecular Pathology (AMP) and the College of American Pathologists (CAP) jointly published recommendations to facilitate assessment of the analytical and clinical performance of plasma ctDNA assays.4 Among the seven recommendations for clinical laboratory testing of ctDNA, emphasis was put on validation of the assay performance characteristics. These include sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and concordance, not only as a whole of the assay but also for each variant or at least each variant class, including SNV, indel, copy number variation (CNV), SV, or mutational signature (MSI, TMB, or homologous recombination deficiency). Similarly, the validation of limit of detection (LoD) of the ctDNA assays should be performed for each variant or variant class. It was also emphasized that appropriate controls should be included to remove potential sources of assay interference and sources of result interpretation error, such as variants from clonal hematopoiesis of indeterminate potential (CHIP) and the presence of germline variants. Moreover, laboratories were recommended to define and describe orthogonal method confirmations as well as comparison to tissue-based specimens.
To carry out a successful validation, laboratories need to have a thoroughly laid-out plan and prepare all needed components, such as reagents, instruments, software, operators, locations, and most importantly validation samples, in advance. When preparing validation samples, clinical specimens are the recommended first choice, but the following considerations should be kept in mind.
When putting together all required tests recommended by the AMP/CAP guidelines plus necessary replicates recommended by BloodPAC,4,5 a large number of clinical specimens will be required in order to accumulate enough variants of each variant class for validating their individual sensitivity and specificity. The amount of ctDNA needed to accomplish even one aspect of the validation will be huge and unachievable with a single patient specimen, in which ctDNA accounts for only 0.1–10% of the total circulating cell-free DNA (cfDNA), with normal plasma cfDNA levels ranging from 10–100 ng/ml.6 Therefore, pooled clinical specimens may be needed to provide enough ctDNA for all aspects of the validation.
Another challenge of ctDNA assay validation is that the allele frequencies (AFs) of variants in the clinical specimens may not be at the same level. Therefore, not all variants present are informative, especially for validation of LoD, which requires assessing variants with AFs around the targeted LoD. Again, accumulating enough variants of each variant class at a similar AF for LoD validation requires a large number of clinical specimens.
Concordance studies of plasma ctDNA with tumor tissue biopsy have a few inherent limitations, which may not be resolved using the selected sample types.
The above challenges clearly demonstrate how critical appropriate samples are in ctDNA assay validation. While all other components of the validation are relatively easy to assemble, sourcing samples takes most of the time and effort. Thus far, the lack of appropriate types or numbers of clinical specimens has been the major limitation hindering ctDNA assay validation. These limitations inherent in clinical specimens can be mitigated with well characterized contrived samples. It is anticipated that ideal contrived samples with the following characteristics will greatly facilitate the validation of the NGS-based plasma ctDNA assays.
Contrived samples or reference materials have been recommended for use in NGS-based plasma ctDNA assay validation guidelines from AMP/CAP and BloodPAC.4,5 While ideal contrived samples or reference materials mimicking the patient samples and fulfilling all the requirements of ctDNA assay validation are yet to be created, a few applicable ones are already commercially available depending on your validation goals. At LGC Clinical Diagnostics, we have created several ctDNA reference materials (with more under development) to target different cancer types, including pan-cancer, solid tumor, lymphoma, and myeloid malignancies, or different study purposes, such as ctDNA mutation profiling, minimal residual disease monitoring, TMB scoring, and methylation screening. The newest version of ctDNA reference material, to be released soon, has 93 variants, including 43 SNVs, 22 indels, 12 CNVs, 10 SVs, and 6 MSI biomarkers.
You can learn more about those ctDNA reference materials, download the poster.
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