Introduction • Emerging evidence suggests that circulating tumor DNA (ctDNA) could be used to monitor treatment response. • The advent of sensitive genomic techniques such as droplet digital polymerase chain reaction (ddPCR) and tagged- amplicon sequencing (TAmSeq) has enabled detection and enumeration of somatic genetic aberrations in blood samples. • Previous studies have mainly used plasma samples for ctDNA detection, but other biofluids such as saliva could provide alternative sources for ctDNA assessment. • Here, we aimed to assess whether longitudinal mutant ctDNA monitoring using blood and saliva samples could reflect response and resistance to osimertinib and tumor burden measured by three-dimensional volumetric measurement in the setting of a prospective clinical trial of local ablative therapy (LAT) for oligoprogressive, EGFR-mutant NSCLC (NCT02759835). Methods • Schema of Clinical Trial • Patients with no prior EGFR-TKI treatment or patients with T790M-positive NSCLC after EGFR-TKI treatment receive osimertinib. Upon progression, patients with ≤ 5 progressing sites undergo local ablative therapy and resume osimertinib. • Sample collection: Blood and saliva samples were collected at baseline (Day 0), cycle 1 day 7, and the first day of each cycle thereafter. ctDNA testing • Exon 19 deletion, L858R, and T790M were detected using ddPCR EGFR-mutation detection assays • For next generation sequencing, we used an amplicon- based sequencing platform, InvisionFirst ® -Lung, which analyzes somatic changes within 36 cancer genes. Dynamic ctDNA changes reflect treatment response and tumor burden Longitudinal circulating tumor DNA analysis in blood and saliva predicts response to osimertinib and disease progression in EGFR-mutant non-small-cell lung cancer Chul Kim 1 , Liqiang Xi 2 , Constance M. Cultraro 1 , Fang Wei 3 , Gregory Jones 4 Jordan Cheng 3 , Ahmad Shafiei 5 , Trinh Hoc-Tran Pham 2 , Nitin Roper 1 , Elizabeth Akoth 1 , Vikram Misra 1 , Nina Monkash 1 , Charles Strom 6 , Michael Tu 6 , Wei Liao 6 , David Chia 7 , Clive Morris 4 , Seth M. Steinberg 8 , Mohammadhadi Bagheri 5 , David T.W. Wong 3 , Mark Raffeld 2 , Udayan Guha 1 ([email protected] ) 1 Thoracic and GI Malignancies Branch, CCR, NCI, NIH, Bethesda, MD. 2 Laboratory of Pathology, CCR, NCI, NIH, Bethesda, MD. 3 School of Dentistry, University of California, Los Angeles, Los Angeles, CA. 4 Inivata, Cambridge, UK. 5 Radiology and Imaging Sciences, Clinical Center, NIH, Bethesda, MD. 6 EZLife Bio Inc., Woodland Hills, CA. 7 Department of Pathology, and Laboratory Medicine, UCLA, Los Angeles, CA. 8 Biostatistics and Data Management Section, NCI, NIH, Bethesda, MD. • ctDNA in salivary samples were analyzed to detect EGFR mutations using EFIRM liquid biopsy (eLB) assay (EZLife Bio, Woodland Hills, CA). Volumetric measurement • All target lesions were identified at each tumor assessment CT. We also measured all other soft tissue lesions. All of these lesions were manually segmented and the volume was measured using Carestream PACS. Results • We analyzed 387 plasma samples from 20 patients by ddPCR, 126 plasma samples from 16 patients by NGS and 298 saliva samples from 18 patients by eLB. Table 1. Patient characteristics (n=20) • Among 14 patients who progressed, ctDNA progression (increased AFs two consecutive times by ddPCR) predated RECIST progression by a median of 82 days (range: 28-216 days) in 9 patients. • Of 5 patients without ctDNA progression by ddPCR, 1 patient had an increase in EGFR mutation-level by eLB and another patient had an increase in PTEN AF by NGS. Patients with no RECIST progression do not have ctDNA rise. Baseline detection of plasma ctDNA and association between baseline mutant EGFR plasma ctDNA levels and tumor burden Baseline mutant ctDNA level as well as its early clearance predicts progression-free survival while tumor volume measurements by volumetric CT did not EGFR, ERBB2, MET amplifications, PI3K alterations, and EGFR C797S mutation were key osimertinib resistance mechanisms identified by InvisionFirst-Lung Conclusions Serial assessment of mutant ctDNA in plasma and saliva predicts tumor response and resistance to osimertinib. Baseline ctDNA level and early mutant ctDNA clearance is associated with PFS. γ=0.96, p<0.001 γ=0.42, p<0.001 γ=0.45, p<0.001 γ=0.48, p=0.034 γ=0.51, p=0.044 ddPCR NGS eLB LAT001 LAT002 LAT003 LAT006 LAT007 LAT0013 LAT0014 LAT0015 LAT0010 LAT0017 ddPCR NGS eLB LAT009 LAT008 LAT0016 LAT001 Baseline images Images at 8 months follow-up