Longitudinal Circulating Tumor DNA Analysis in Blood and Saliva for Prediction of Response to Osimertinib and Disease Progression in EGFR-Mutant Lung Adenocarcinoma

Chul Kim, Liqiang Xi, Constance M Cultraro, Fang Wei, Gregory Jones, Jordan Cheng, Ahmad Shafiei, Trinh Hoc-Tran Pham, Nitin Roper, Elizabeth Akoth, Azam Ghafoor, Vikram Misra, Nina Monkash, Charles Strom, Michael Tu, Wei Liao, David Chia, Clive Morris, Seth M Steinberg, Hadi Bagheri, David T W Wong, Mark Raffeld, Udayan Guha, Chul Kim, Liqiang Xi, Constance M Cultraro, Fang Wei, Gregory Jones, Jordan Cheng, Ahmad Shafiei, Trinh Hoc-Tran Pham, Nitin Roper, Elizabeth Akoth, Azam Ghafoor, Vikram Misra, Nina Monkash, Charles Strom, Michael Tu, Wei Liao, David Chia, Clive Morris, Seth M Steinberg, Hadi Bagheri, David T W Wong, Mark Raffeld, Udayan Guha

Abstract

Background: We assessed whether serial ctDNA monitoring of plasma and saliva predicts response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma. Three ctDNA technologies-blood-based droplet-digital PCR (ddPCR), next-generation sequencing (NGS), and saliva-based EFIRM liquid biopsy (eLB)-were employed to investigate their complementary roles. Methods: Plasma and saliva samples were collected from patients enrolled in a prospective clinical trial of osimertinib and local ablative therapy upon progression (NCT02759835). Plasma was analyzed by ddPCR and NGS. Saliva was analyzed by eLB. Results: A total of 25 patients were included. We analyzed 534 samples by ddPCR (n = 25), 256 samples by NGS (n = 24) and 371 samples by eLB (n = 22). Among 20 patients who progressed, ctDNA progression predated RECIST 1.1 progression by a median of 118 days (range: 61-272 days) in 11 (55%) patients. Of nine patients without ctDNA progression by ddPCR, two patients had an increase in mutant EGFR by eLB and two patients were found to have ctDNA progression by NGS. Levels of ctDNA measured by ddPCR and NGS at early time points, but not volumetric tumor burden, were associated with PFS. EGFR/ERBB2/MET/KRAS amplifications, EGFR C797S, PIK3CA E545K, PTEN V9del, and CTNNB1 S45P were key resistance mechanisms identified by NGS. Conclusion: Serial assessment of ctDNA in plasma and saliva predicts response and resistance to osimertinib, with each assay having supplementary roles.

Keywords: EGFR; NSCLC; ctDNA; osimertinib.

Conflict of interest statement

Chul Kim receives research funding from his current institution (Georgetown University), AstraZeneca, Novartis, Debiopharm, Genentech, Karyopharm, Regeneron, Janssen, BMS, and Mirati. Chul Kim also served as a consultant for Novartis, Janssen, and PierianDx Oncology. Clive Morris and Greg Jones are employees of Inivata. Charles Strom, Michael Tu, and Wei Liao are employees of EZLife Bio Inc. David T.W. Wong receives research grant funding from NIH, Colgate-Palmolive, and Hirshberg Foundation. Udayan Guha has a clinical trial agreement (CTA) with AstraZeneca for the current study and receives research funding from AstraZeneca, Esanex, and Aurigene. David T.W. Wong has equity in Liquid Diagnostics. The other authors have no conflicts of interest to report.

Figures

Figure 1
Figure 1
Clinical protocol schema.
Figure 2
Figure 2
(A,B) Spearman correlation between EGFR mutation AF by ddPCR and tumor volume (A). Spearman correlation between EGFR mutation AF by NGS and tumor volume (B).
Figure 3
Figure 3
(A–C) Correlations between ddPCR, NGS, and EFIRM liquid biopsy (eLB) assays.
Figure 4
Figure 4
(A–O) Serial monitoring of ctDNA by ddPCR, NGS, and eLB for prediction of response and the detection of emergence of resistance. Solid lines represent genomic alterations and red dotted lines represent calculated tumor volume measured by volumetric CT measurement. AFs for ddPCR and NGS and current values for eLB are plotted on the left y-axis. Calculated tumor volume measured by volumetric CT measurement is plotted on the right T-axis. Treatment duration is plotted on the x-axis. The shaded gray areas indicate the duration of RECIST 1.1 response (complete or partial response). ctDNA progression, defined as increases in mutant EGFR AF by ddPCR, preceded RECIST 1.1 progression by a median of 118 days (range: 61–272 days) in 11 patients (LAT001, 002, 003, 005, 010, 013, 014, 015, 017, 023, 028). Of the 10 patients without ctDNA progression by ddPCR, 2 patients had an increase in EGFR mutation-level by eLB (LAT006, LAT026), 1 patient had an increase in the AF of PTEN Y88* by NGS (LAT007), and another patient had an increase in TP53 V157F by NGS (LAT016). The arrowhead (in black) indicates the beginning of ctDNA progression.
Figure 4
Figure 4
(A–O) Serial monitoring of ctDNA by ddPCR, NGS, and eLB for prediction of response and the detection of emergence of resistance. Solid lines represent genomic alterations and red dotted lines represent calculated tumor volume measured by volumetric CT measurement. AFs for ddPCR and NGS and current values for eLB are plotted on the left y-axis. Calculated tumor volume measured by volumetric CT measurement is plotted on the right T-axis. Treatment duration is plotted on the x-axis. The shaded gray areas indicate the duration of RECIST 1.1 response (complete or partial response). ctDNA progression, defined as increases in mutant EGFR AF by ddPCR, preceded RECIST 1.1 progression by a median of 118 days (range: 61–272 days) in 11 patients (LAT001, 002, 003, 005, 010, 013, 014, 015, 017, 023, 028). Of the 10 patients without ctDNA progression by ddPCR, 2 patients had an increase in EGFR mutation-level by eLB (LAT006, LAT026), 1 patient had an increase in the AF of PTEN Y88* by NGS (LAT007), and another patient had an increase in TP53 V157F by NGS (LAT016). The arrowhead (in black) indicates the beginning of ctDNA progression.
Figure 4
Figure 4
(A–O) Serial monitoring of ctDNA by ddPCR, NGS, and eLB for prediction of response and the detection of emergence of resistance. Solid lines represent genomic alterations and red dotted lines represent calculated tumor volume measured by volumetric CT measurement. AFs for ddPCR and NGS and current values for eLB are plotted on the left y-axis. Calculated tumor volume measured by volumetric CT measurement is plotted on the right T-axis. Treatment duration is plotted on the x-axis. The shaded gray areas indicate the duration of RECIST 1.1 response (complete or partial response). ctDNA progression, defined as increases in mutant EGFR AF by ddPCR, preceded RECIST 1.1 progression by a median of 118 days (range: 61–272 days) in 11 patients (LAT001, 002, 003, 005, 010, 013, 014, 015, 017, 023, 028). Of the 10 patients without ctDNA progression by ddPCR, 2 patients had an increase in EGFR mutation-level by eLB (LAT006, LAT026), 1 patient had an increase in the AF of PTEN Y88* by NGS (LAT007), and another patient had an increase in TP53 V157F by NGS (LAT016). The arrowhead (in black) indicates the beginning of ctDNA progression.
Figure 5
Figure 5
(A–E) Progression free survival (PFS) based on the baseline mutant EGFR ctDNA copy number, its clearance on therapy at early time periods on treatment, and baseline tumor volume assessed by volumetric CT. PFS based on mutant EGFR copy number at baseline and on day 21/28 by ddPCR (A,B), EGFR AF at baseline and day 7 by NGS (C,D), calculated tumor volume at baseline (E), and difference in tumor volume between day 42/46 vs. baseline (F).
Figure 6
Figure 6
Detection of co-occurring genomic alterations and resistance mechanisms by NGS. Each row represents genomic alterations and columns indicate each patient. Co-occurring mutations at baseline prior to exposure to osimertinib are color-coded in green and genomic alterations seen at progression are in dark purple. Germline mutations are shown in gray. Patients whose disease did not progress at the time of analysis are color-coded in blue (LAT004, LAT008, LAT019, LAT024).

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