Validation of a ctDNA-Based comprehensive genomic profiling panel (IST80 Plus) for advanced non-small cell lung cancer

Circulating tumor DNA (ctDNA) profiling can complement tissue testing in advanced non-small-cell lung cancer (NSCLC), particularly when tissue is unavailable or unable to capture tumor heterogeneity. We evaluated the analytical and clinical performance of Illumina Solid Tumor Panel 80 (IST80 Plus), a focused hybrid-capture next-generation sequencing assay for plasma genomic profiling. Validation included Seraseq-ctDNA reference materials at 2.5% and 0.5% allele frequencies, six external quality-assessment samples, supernatants from three fusion-positive cell lines, and eighteen plasma samples from patients with previously characterized NSCLC alterations. Sensitivity was assessed for small variants, gene fusions, and copy-number variants (CNVs). Quantitative agreement with reference measurements was evaluated using Pearson and Spearman correlations and Bland-Altman analysis. Robustness was explored by downsampling sequencing reads. Among 158 expected variants, 135 were within the panel's reportable range. Overall sensitivity was 95.56% (129/135), comprising 99.06% for small variants (105/106), 88.88% for fusions (16/18), and 72.73% for CNVs (8/11). For 85 detected small variants with available reference allele frequencies, Pearson's r was 0.99 and Spearman's rho was 0.82. Bland-Altman analysis showed minimal bias (0.04%), with 95% limits of agreement from -0.97% to 1.05%. Sensitivity was 98.41% for variants below 1% allele frequency and 100% for variants at or above 1%. All cell-line fusions were detected, as were three of four expected fusions in clinical samples. Following downsampling 50%, 70%, and 90% of the original reads, 81.18%, 85.88%, and 90.59% of 85 reference small variants remained detectable, respectively. Performance was strongest for small variants, whereas lower sensitivity for CNVs was noted. IST80 Plus showed high sensitivity and quantitative accuracy for small variants and reliable fusion detection in ctDNA. Its focused design may support molecular profiling of advanced NSCLC when sample availability and sequencing efficiency are limiting.