Abstract Introduction: Detection of ctDNA in plasma samples permits temporal assessment of tumor mutation status during treatment. Poziotinib is an oral pan-HER TKI that has been demonstrated to be efficacious in NSCLC patients harboring HER2 exon 20 insertion mutations. We assessed serial plasma samples for changes in tumor genotype in both treatment naive and second line patients to evaluate correlation with clinical response. Methods: For NSCLC patients, HER2 exon 20 insertion mutations identified by tumor tissue based NGS were required for entry into the ZENITH20 study. Plasma samples were collected prior to treatment and at C3D1 (8 weeks post treatment 16mg poziotinib QD). The Guardant360® 74-gene liquid biopsy assay, which assessed changes in ctDNA and, subsequently, mean variant allele fraction (mVAF), was utilized for analysis of samples. The Guardant360 Response™ Molecular Response (MR) algorithm was calculated as a ratio of mVAF of oncogenic alterations at baseline compared to poziotinib treatment at C3D1. Results: 23 patients with tumor tissue confirmed NSCLC harboring HER2 exon 20 insertion mutations were studied. 22 of 23 (96%) had baseline plasma samples with detectable ctDNA. 21 of 22 samples had detectable HER2 exon 20 insertion mutations, resulting in a concordance of 95% versus tissue based NGS. The most prevalent HER2 exon 20 insertion alteration was the A775_G776ins YVMA variant, found in 50% of the baseline blood and tumor samples using both methods (100% concordance). 16 of 17 patients had both baseline and C3D1 samples, permitting assessment of temporal response. 15 of the 16 (94%) patients demonstrated a decrease in mVAF at C3D1 compared to the mVAF at baseline. 12 of 15 patients demonstrated an >50% reduction in mVAF at C3D1, with 7 of the 12 patients showing >95% reduction in mVAF at C3D1 with clinical outcomes of 5 PRs, 1 non-CR/non-PD and 1 SD. Interestingly, 3 of these patients showed complete clearance of ctDNA target HER2 exon 20 insertions at the C3D1 timepoint. Conclusions: Baseline plasma ctDNA genotyping correlated with tumor tissue based NGS in an NSCLC patient population with HER2 mutations. Poziotinib treatment resulted in mVAF reduction, which correlated with clinical response per RECIST1.1. Assessment of longitudinal changes in ctDNA during drug therapy may potentially be used to predict patient response and possibly tumor resistance. Further evaluation in larger cohorts and longer duration of treatment is required to help elucidate the impact of these findings. Citation Format: Arunthi Thiagalingam, Sribalaji Lakshmikanthan, Allysia J. Mak, Scott A. Shell, Sharon Leu, Rocky Washington, Lyndah Dreiling, Gajanan Bhat, Francois Lebel, John A. Barrett. Predictive ability of circulating tumor DNA by Guardant360 in poziotinib-treated patients with NSCLC harboring HER2 exon 20 insertion mutations abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3400.