Multilaboratory Assessment of a New Reference Material for Quality Assurance of Cell-Free Tumor DNA Measurements

Abstract

We conducted a multilaboratory assessment to determine the suitability of a new commercially available reference material with 40 cancer variants in a background of wild-type DNA at four different variant allele frequencies (VAFs): 2%, 0.50%, 0.125%, and 0%. The variants include single nucleotides, insertions, deletions, and two structural variations selected for their clinical importance and to challenge the performance of next-generation sequencing (NGS) methods. Fragmented DNA was formulated to simulate the size distribution of circulating wild-type and tumor DNA in a synthetic plasma matrix. DNA was extracted from these samples and characterized with different methods and multiple laboratories. The various extraction methods had differences in yield, perhaps because of differences in chemistry. Digital PCR assays were used to measure VAFs to compare results from different NGS methods. Comparable VAFs were observed across the different NGS methods. This multilaboratory assessment demonstrates that the new reference material is an appropriate tool to determine the analytical parameters of different measurement methods and to ensure their quality assurance.

Targeted Next-Generation Sequencing of Plasma Cell-Free DNA in Korean Patients with Hepatocellular Carcinoma

“…Of the 16 pathogenic variants of TP53 , 12 (75%) were missense, 13 (81%) were located in the DNA-binding domain in exons 4–8 of TP53 , and two (13%) were located at codon 245, which is one of the major hotspots of somatic TP53 pathogenic variants. Of the nine variants of CTNNB1 , four were located at known hotspots [ 18 - 21 ], two (S33Y, G34Y) were located within the β-TrCP binding domain (D32-S37), one (S45F) involved S45, an amino acid residue involved in the phosphorylation/degradation of β-catenin, and one was located in the armadillo repeat six domain (N387). Probably due to the small number of patients included in this study, we did not find a statistically significant association between clinical characteristics and the presence of TP53 and/or CTNNB1 cfDNA variants ( Table 3 ).…”

“…Seraseq ctDNA Reference Material v.2 (SeraCare Life Sciences, Milford, MA, USA) was used to validate the limit of detection. The reference material consisted of 40 cancer-relevant somatic variants spiked into a background of wild-type DNA (purified from a reference cell line, GM24385) at defined variant allele frequencies (VAFs) of 2%, 1%, 0.5%, 0.25%, 0.125%, and 0% [ 20 ]. Seraseq ctDNA was extracted and analyzed in duplicate.…”

Validation of ctDNA Quality Control Materials Through a Precompetitive Collaboration of the Foundation for the National Institutes of Health

“…All specimens were then sent to each participating laboratory and assayed as described in the Data Supplement (Fig 1). [12][13][14][15][16][17][18][19] Horizon and Thermo Fisher Scientific QCM had broader fragment distributions than observed for the LGC SeraCare material and healthy donor and lung cancer cfDNA (shown for reference). Distribution patterns from QCM were further validated by sequencing of whole genome libraries at AstraZeneca Translational Medicine Laboratory (AZ; Data Supplement).…”

Decline in circulating viral and human tumor markers after resection of head and neck carcinoma

“…For the remaining five patients (cases #7‐10 and 20) tumor markers were defined based on preresection plasma results. Analytic sensitivity and specificity of the Plasma Mutation Panel is 100% at >0.3% allele fraction as assessed in mock plasma specimens having 23 human cancer gene variants (SeraSeq; SeraCare, Milford, Massachusetts) 34,35 . Specificity is >99% for somatic mutations at >0.3% in a cohort of 20 healthy adults.…”