Abstract Background: The oral MEK 1/2 inhibitor selumetinib (50-mg po, BID) has shown remarkable response rates in an ongoing phase-II trial (NCT02407405) in adult patients (pts) with neurofibromatosis type-1 (NF1) (cutaneous neurofibroma (cNF) or inoperable plexiform neurofibromas (PN). In this study, we are evaluating the pharmacodynamics (PD) of selumetinib-induced modulation of key phosphorylation sites on MEK1, MEK2, ERK1, and ERK2 using a new, validated, fit-for-purpose multiplex immunoassay of tumor biopsy extracts that distinguishes specific isoforms. Methods: Core needle tumor biopsies scheduled at baseline and pre-cycle 3 of selumetinib treatment were obtained from patients with a confirmed diagnosis of cNF or PN, snap-frozen in liquid nitrogen within 2 min at point of collection, and stored at -80°C. Additional dermal biopsies were collected (6 of 17 pts) to assess clinical suitability of the proposed PD analysis. Total cell lysates of biopsies were prepared in accordance with the NCI SOP that has been validated to preserve phospho-biomarker integrity (PMID 27001313). Samples were analyzed in a single batch on a Luminex platform, and the ratio of phosphorylated to total protein calculated individually for MEK1, MEK 2, ERK1, and ERK2 and compared by pair-wise analysis. Results: The current PD evaluation resulted from analysis of 24 paired biopsies (17 pts; 6 cNF, 18 PN) representing a range in time interval between previous dose and biopsy procedure (43 min - to 28 hrs; median 7.6, 95% CI 3.25 - 13.98 hrs). Selumetinib treatment decreased the median pERK1 ratio by 59% (95% CI: -30 to -77%; p<0.05) and the pERK2 ratio by 58% (95% CI: -27 to -67%; p<0.05). Biomarker variability was higher in cNF biopsies than PN core biopsies. The pERK1 and pERK2 ratios correlated moderately with elapsed time between previous dose and biopsy procedure (p < 0.05) but did not correlate with baseline MEK1 or MEK2 ratios. Selumetinib treatment decreased pERK1 and pERK2 ratios in 15/17 pts, and achieved >50% suppression in 12/17 pts. The last dose maintained pERK1/2 suppression (>50% decrease in their ratio values) for >8 hrs in 5 pts and >20 hrs in 2 pts. Conclusions: Our PD study provides the first evidence of engagement and control of selumetinib's molecular target in NF1. Importantly, molecular target response was demonstrable in 14 of 15 patients who responded clinically to this treatment regimen, a finding enabled by a validated high-performance assay for phosphoproteins that are key PD biomarkers of selumetinib mechanism of action and that may be useful in assessing molecular drug action of other members of this drug class. Funded by NCI Contract No HHSN261200800001E. Citation Format: William Herrick, Geraldine O'Sullivan Coyne, Andrea Gross, Eva Dombi, Jeevan Govindharajulu, Casey Kilpatrick, Jennifer Desanto, Amanda Marie Carbonell, Joanne Derdak, Dominique Corinne Pichard, Cecilia Maura Tibery, Bradford J. Wood, Lawrence Doyle, James H. Doroshow, Alice Chen, Ralph E. Parchment, Brigitte C. Widemann, Apurva K. Srivastava. Intra-tumoral pharmacodynamics of selumetinib in serial biopsies from patients with neurofibroma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 805.