Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping. We identified patients with advanced non-small cell lung cancer (NSCLC) with , or mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations. We first studied plasma ddPCR results from 58 patients with -mutant NSCLC. Two had G12X detected in cfDNA, and both were present in PBC, including one where the mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 V617F mutations derived from PBC. In addition, 108 mutations were detected in cfDNA; for 33 of the mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH. We find that most mutations, some mutations, and rare mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy..

Plasma HPV cfDNA monitoring recapitulates fluctuations in disease status. While blood-based HPV DNA monitoring does not currently have a role in managing HPV+ OPC, these data speak to their broad clinical potential in an era of precision medicine.

BackgroundBlood-based biomarkers of anti-solid tumor immune checkpoint blockade (ICB) response are lacking. We hypothesized that changes in systemic cytokine levels with the initial doses of programmed cell death protein 1 (PD-1) pathway inhibitors would correlate with clinical responses. New ultrasensitive ELISA technology enables quantitation of plasma proteins in sub-picogram-per-milliliter concentrations.MethodsWe measured plasma cytokines by ultrasensitive single-molecule array assays in patients with non-small-cell lung carcinoma before and during treatment with anti-PD-1 therapy. Association with best overall response and progression-free survival (PFS) was assessed by Kruskall-Wallis test and Kaplan-Meier plots with log-rank test, respectively.ResultsA decrease in interleukin 6 (IL-6) levels was associated with improved PFS (n=47 patients, median PFS: 11 vs 4 months, HR 0.45 (95% CI 0.23 to 0.89), p=0.04). The extent of change in IL-6 differed between best overall response categories (p=0.01) and correlated with changes in C reactive protein levels. We also explored plasma cytokine levels in relation to immune-related adverse effects and observed some correlation.ConclusionsThis study suggests the presence of a systemic, proteomic reflection of successful ICB outside the tumor microenvironment with plasma decreases in IL-6 and CRP.

Plasma genotyping represents an opportunity for convenient detection of clinically actionable mutations in advanced cancer patients, such has been well-documented in non-small cell lung cancer (NSCLC). Oncogenic gene fusions are complex variants that may be more challenging to detect by next-generation sequencing (NGS) of plasma cell-free DNA (cfDNA). Rigorous evaluation of plasma NGS assays in the detection of fusions is needed to maximize clinical utility. Materials and methods: : Additional plasma was collected from patients with advanced NSCLC and ALK, ROS1, or RET gene fusions in tissue who had undergone clinical plasma NGS using Guardant360 ™ (G360, Guardant Health). We then sequenced extracted cfDNA with a plasma NGS kit focused on known driver mutations in NSCLC (ctDx-Lung, Resolution Bioscience) with cloud-based bioinformatic analysis and blinded variant calling. Results: Of 16 patients assayed known to harbor anALK, ROS1, or RET in tumor, G360 detected fusions in 7 cases, ctDx-Lung detected fusions in 13 cases, and 3 cases were detected by neither. Of the 7 fusions detected by both assays, G360 reported lower mutant allelic fractions (AF). In cases missed by G360, tumor derived TP53 mutations were often detected confirming presence of tumor DNA. Raw sequencing data showed that inverted or out-of-frame variants were overrepresented in cases detected using ctDx-Lung but not by G360. Conclusion: Focusing on complex, clinically actionable mutations using tumor as a reference standard allows for evaluation of technical differences in plasma NGS assays that may impact clinical performance. Noting the heterogeneity of fusion sequences observed in NSCLC, we hypothesize that differences in hybrid capture techniques and bioinformatic calling may be sources of variations in sensitivity among these assays.

No abstract

Background Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. Results All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5–20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. Conclusion This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

No abstract

PURPOSE Plasma circulating tumor DNA (ctDNA) analysis is routine for genotyping of advanced non–small-cell lung cancer (NSCLC); however, early response assessment using plasma ctDNA has yet to be well characterized. MATERIALS AND METHODS Patients with advanced EGFR-mutant NSCLC across three phase I NCI osimertinib combination trials were analyzed in this study, and an institutional cohort of patients with KRAS-, EGFR-, and BRAF-mutant advanced NSCLC receiving systemic treatment was used for validation. Plasma was collected before treatment initiation and serially before each cycle of therapy, and key driver mutations in ctDNA were characterized by droplet digital polymerase chain reaction. Timing of plasma versus imaging response was compared in a separate cohort of patients with EGFR-mutant NSCLC treated with osimertinib. Across cohorts, we also studied ctDNA variability before treatment start. RESULTS In the NCI cohort, 14/16 (87.5%) patients exhibited ≥ 90% decrease in mutation abundance by the first on-treatment timepoint (20-28 days from treatment start) with minimal subsequent change. Similarly, 47/56 (83.9%) patients with any decrease in the institutional cohort demonstrated ≥ 90% decrease in mutation abundance by the first follow-up draw (7-30 days from treatment start). All 16 patients in the imaging cohort with radiographic partial response showed best plasma response within one cycle, preceding best radiographic response by a median of 24 weeks (range: 3-147 weeks). Variability in ctDNA levels before treatment start was observed. CONCLUSION Plasma ctDNA response is an early phenomenon, with the majority of change detectable within the first cycle of therapy. These kinetics may offer an opportunity for early insight into treatment effect before standard imaging timepoints.