Introduction• Emerging evidence suggests that circulating tumor DNA(ctDNA) could be used to monitor treatment response.• The advent of sensitive genomic techniques such as dropletdigital polymerase chain reaction (ddPCR) and tagged-amplicon sequencing (TAmSeq) has enabled detection andenumeration of somatic genetic aberrations in blood samples.• Previous studies have mainly used plasma samples forctDNA detection, but other biofluids such as saliva couldprovide alternative sources for ctDNA assessment.• Here, we aimed to assess whether longitudinal mutantctDNA monitoring using blood and saliva samples could reflectresponse and resistance to osimertinib and tumor burdenmeasured by three-dimensional volumetric measurement in thesetting 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 withT790M-positive NSCLC after EGFR-TKI treatment receiveosimertinib. Upon progression, patients with ≤ 5 progressingsites undergo local ablative therapy and resume osimertinib.

• Sample collection: Blood and saliva samples were collectedat baseline (Day 0), cycle 1 day 7, and the first day of eachcycle 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, whichanalyzes 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 Kim1, Liqiang Xi2, Constance M. Cultraro1, Fang Wei3, Gregory Jones4 Jordan Cheng3, Ahmad Shafiei5, Trinh Hoc-Tran Pham2, Nitin Roper1, Elizabeth Akoth1, Vikram Misra1, Nina Monkash1, Charles Strom6, Michael Tu6, Wei Liao6, David Chia7, Clive Morris4, Seth M. Steinberg8, Mohammadhadi Bagheri5, David T.W. Wong3, Mark Raffeld2, Udayan Guha1 (udayan.guha@nih.gov)

1Thoracic and GI Malignancies Branch, CCR, NCI, NIH, Bethesda, MD. 2Laboratory of Pathology, CCR, NCI, NIH, Bethesda, MD. 3School of Dentistry, University of California, Los Angeles, Los Angeles, CA. 4Inivata, Cambridge, UK. 5Radiology and Imaging Sciences, Clinical Center, NIH, Bethesda, MD. 6EZLife Bio Inc., Woodland Hills, CA. 7Department of Pathology, and Laboratory Medicine, UCLA, Los Angeles, CA. 8Biostatistics and Data Management Section, NCI, NIH, Bethesda, MD.

• ctDNA in salivary samples were analyzed to detect EGFRmutations using EFIRM liquid biopsy (eLB) assay (EZLifeBio, Woodland Hills, CA).

Volumetric measurement• All target lesions were identified at each tumor assessment

CT. We also measured all other soft tissue lesions. All ofthese lesions were manually segmented and the volumewas measured using Carestream PACS.

Results• We analyzed 387 plasma samples from 20 patients by

ddPCR, 126 plasma samples from 16 patients by NGS and298 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) predatedRECIST progression by a median of 82 days (range: 28-216days) in 9 patients.

• Of 5 patients without ctDNA progression by ddPCR, 1patient had an increase in EGFR mutation-level by eLB andanother 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

ConclusionsSerial assessment of mutant ctDNA in plasma and salivapredicts tumor response and resistance to osimertinib.Baseline ctDNA level and early mutant ctDNA clearance isassociated 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

LAT0

01LA

T002

LAT0

03LA

T006

LAT0

07LA

T001

3LA

T001

4LA

T001

5LA

T001

0LA

T001

7

ddPCR NGS eLB

LAT0

09LA

T008

LAT0

016

LAT001Baseline images Images at 8 months follow-up