Journal: Molecular Diagnosis & Therapy

Article Title: Dynamic ctDNA Mutational Complexity in Patients with Melanoma Receiving Immunotherapy

doi: 10.1007/s40291-023-00651-4

Figure Lengend Snippet: Detection of co-occurring BRAF and NRAS gene mutations in the ctDNA from a single patient. Analysis of the fifth plasma sample from patient MEL0035 is shown. a An Integrated Genome Browser (IGV) image of NGS sequence reads (grey bars) shows three concurrent mutations in this sample: NRAS Q61R (c.182 T > C, 25,050 depth, 0.2% VAF), BRAF V600K (c.1798 AC > TT, 47,133 depth, 6.5% VAF) and BRAF V600E (c.1799A > T, 47,089 depth, 4.6% VAF). The sequencing results are supported by concordant ddPCR analysis ( b ) and UltraSEEK MassArray results ( c )

Article Snippet: For each sample assessed, PCR amplification of up to 10 ng of cfDNA sample was analysed with the UltraSEEK MassARRAY melanoma V2 panel (Agena Biosciences [ ]), following the manufacturer’s instructions.

Techniques: Clinical Proteomics, Sequencing

Journal: Cancers

Article Title: Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling

doi: 10.3390/cancers12061588

Figure Lengend Snippet: Experimental setup to assess commercially available reference material and mutation analysis kits. ( A ) Multicenter evaluation of the Seraseq ctDNA Complete reference material using three NGS panels (a custom SureSelect design [Agilent], the AVENIO Targeted kit [Roche], and the QIAact Lung UMI Panel [QIAGEN]), two digital droplet PCR assays (ddPCR) (one ISO15189 certified [ISO] and one research use only [RUO], and the MassARRAY (UltraSeek lung panel [Agena]). Intra-run precision was evaluated by testing the VAF1% reference material in triplicate in a single complete mutational analysis workflow. Inter-run reproducibility was assessed by testing the sample in three separate mutational analysis workflows. ( B ) Using the Seraseq ctDNA reference material v2, five commercially available mutation analysis assays targeting clinically relevant genes were tested with respect to sensitivity and specificity. ( C ) Assessment of Diagnostic LeukApheresis (DLA) for inter-assay comparison at a single site (MUG) and for inter-laboratory comparisons at two sites using the AVENIO Expanded kit (MUG and UMCG). A subset of variants was validated using ddPCR.

Article Snippet: In addition, we evaluated two droplet digital PCR (ddPCR) assays (one research use only assay performed at Bayer and one ISO15189 validated assay performed at the University Medical Center Groningen—UMCG) and the UltraSeek lung panel (MassARRAY performed at Agena Bioscience).

Techniques: Mutagenesis, Diagnostic Assay, Inter Assay, Comparison

Journal: Cancers

Article Title: Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling

doi: 10.3390/cancers12061588

Figure Lengend Snippet: Inter- and intra-run variability of three qPCR-based and three NGS assays calculated from five clinically relevant mutations in three replicates. The use of the SeraCare Seraseq ctDNA Complete reference material (VAF1%) was evaluated using two digital droplet PCR assays (ddPCR) (one ISO15189 certified [ISO (UMCG)] and one research use only [RUO (Bayer)], the MassARRAY (UltraSeek lung panel) and three NGS panels (a custom SureSelect design, the AVENIO Targeted kit, and the QIAact Lung UMI Panel). Five clinically relevant mutations, i.e., BRAF V600E, EGFR T790M, EGFR L858R, KRAS G12C and KRAS G12D were taken into account. ( A ) Plotted are the coefficients of variations (CV) of observed VAFs of all tested assays. Shown are inter- and intra-run variability (three replicates each) as well as the calculated random error (shot noise) based on the availability of mutant input molecules. ( B ) Shown are VAFs for various input masses of DNA calculated from three NGS panels (20 ng), the MassARRAY (15 ng), and two ddPCR assays (8, 4, and 2 ng), demonstrating that the amount of input DNA (i.e., available molecules) clearly affects the variability of a test. Plotted are the respective VAFs as well as an inverse binomial distribution based on the trials (how many copies of DNA were analyzed), the likelihood of an event happening (0.01 for 1% VAF), and the location along the cumulative distribution function (colored line, e.g., 2.5th percentile would be 0.025). For example, when sampling a 4-ng input DNA, one can expect to observe a 1% variant between VAFs of 0.44% and 1.57% with a likelihood of 95%, or between 0.35% and 1.85% with a likelihood of and 99%.

Article Snippet: In addition, we evaluated two droplet digital PCR (ddPCR) assays (one research use only assay performed at Bayer and one ISO15189 validated assay performed at the University Medical Center Groningen—UMCG) and the UltraSeek lung panel (MassARRAY performed at Agena Bioscience).

Techniques: Mutagenesis, Sampling, Variant Assay

Journal: Cancers

Article Title: Technical Evaluation of Commercial Mutation Analysis Platforms and Reference Materials for Liquid Biopsy Profiling

doi: 10.3390/cancers12061588

Figure Lengend Snippet: Participating Cancer-ID sites and performed mutation analysis assays.

Article Snippet: In addition, we evaluated two droplet digital PCR (ddPCR) assays (one research use only assay performed at Bayer and one ISO15189 validated assay performed at the University Medical Center Groningen—UMCG) and the UltraSeek lung panel (MassARRAY performed at Agena Bioscience).

Techniques: Mutagenesis, Sequencing