Analytical and Biological Factors Associated with Oncogenic Variant Yield in Tumor-Agnostic Plasma ctDNA Profiling

Background/Objectives: Tumor-agnostic plasma ctDNA profiling is used when tumor tissue is unavailable, insufficient, or unrepresentative. We investigated analytical and biological factors associated with oncogenic variant yield. Methods: In the prospective EXMASTER study, plasma cfDNA was analyzed using a 265-gene targeted panel. Technical performance was benchmarked using synthetic reference materials and tumor-informed tissue–plasma comparison. Tumor-agnostic analysis used predefined filtering and oncogenic classification. Results: Forty patients were included; 34 underwent baseline tumor-agnostic plasma profiling. All expected variants were detected in synthetic reference material at the tested nominal allele fractions, including 0.005 (0.5%). In six selected patients, 32 of 33 evaluable tissue-derived variants within the panel footprint were detected in plasma (97.0% variant-level concordance). Tumor-agnostic profiling retained 603 plasma variants, including 74 oncogenic or likely oncogenic variants. Thirty-one of 34 patients (91.2%) had at least one retained plasma variant, whereas 27 of 34 (79.4%) had at least one oncogenic or likely oncogenic variant. No statistically supported association was observed between tissue-derived MATH score and oncogenic variant yield. The correlation between total retained plasma variant burden and oncogenic variant count was considered descriptive because these measures are mathematically dependent. Increasing filtering stringency reduced the retained plasma call set while preserving most tissue-concordant variants. Conclusions: In this exploratory, tumor-heterogeneous cohort, analytical filtering strongly shaped the retained plasma variant set, whereas no statistically supported association was observed between tissue-derived MATH score and oncogenic variant yield. These findings support assay-aware interpretation of plasma sequencing as complementary rather than interchangeable with tissue profiling.