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A Prospective Evaluation of Circulating Tumor Cells and Cell

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A Prospective Evaluation of Circulating Tumor Cells and Cell Published OnlineFirst June 8, 2016; DOI: 10.1158/1078-0432.CCR-16-0909 Cancer Therapy: Clinical Clinical Cancer Research A Prospective Evaluation of Circulating Tumor Cells and Cell-Free DNA in EGFR-Mutant Non– Small Cell Lung Cancer Patients Treated with Erlotinib on a Phase II Trial Masahiko Yanagita1,2, Amanda J. Redig1,2,3, Cloud P. Paweletz1,4, Suzanne E. Dahlberg5,6, Allison O'Connell5, Nora Feeney5, Myriam Taibi1, David Boucher7, Geoffrey R. Oxnard1,2,3, Bruce E. Johnson1,2,3, Daniel B. Costa2,7, David M. Jackman1,2,3, and Pasi A. Janne€ 1,2,3,4 Abstract Purpose: Genotype-directed therapy is the standard of care for 35 of 44 patients (80%), with paired CTC/cfDNA analysis advanced non–small cell lung cancer (NSCLC), but obtaining in 41 of 44 samples at baseline and 36 of 44 samples at tumor tissue for genotyping remains a challenge. Circulating progression. T790M was identified in 23 of 35 (66%) tissue tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis for noninvasive evaluation. This prospective trial evaluated CTCs at progression identified MET amplification in 3 samples in and cfDNA in EGFR-mutant NSCLC patients treated with erloti- which tissue analysis could not be performed. cfDNA anal- nib until progression. ysis identified T790M in 2 samples in which rebiopsy Experimental Design: EGFR-mutant NSCLC patients were was not possible. At diagnosis, high levels of cfDNA but enrolled in a phase II trial of erlotinib. Blood was collected at not high levels of CTCs correlated with progression-free baseline, every 2 months on study, and at disease progression. survival. Plasma genotyping was performed by droplet digital PCR for Conclusions: cfDNA and CTCs are complementary, nonin- EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, vasive assays for evaluation of acquired resistance to first-line enumerated, and analyzed by immunofluorescence for CD45 and EGFR TKIs and may expand the number of patients in whom pan-cytokeratin and EGFR and MET FISH were also performed. actionable genetic information can be obtained at acquired Rebiopsy was performed at disease progression. resistance. Serial cfDNA monitoring may offer greater clinical Results: Sixty patients were enrolled; 44 patients discon- utility than serial monitoring of CTCs. Clin Cancer Res; 1–11. tinued therapy for disease progression. Rebiopsy occurred in Ó2016 AACR. Introduction efficacy of tyrosine kinase inhibitors (TKI) targeting sensitizing mutations in EGFR (2–4) or rearrangements in ALK or ROS1 Tumor genotyping has emerged as the standard of care for (5, 6), acquired resistance is inevitable (7, 8). The most common patients with newly diagnosed non–small cell lung cancer mechanism of acquired resistance to first-line EGFR TKIs is the (NSCLC) because of the efficacy of targeted therapies for patients EGFR T790M mutation (7, 8), and in November 2015, osimerti- whose tumors are ultimately found to have activating mutations nib became the first investigational third-generation EGFR TKI to in specific oncogenic drivers (1). However, despite the initial receive FDA approval for patients with an acquired T790M muta- tion at the time of disease progression (9). Notably, the clinical use of these third-generation inhibitors requires repeat tumor 1Department of Medical Oncology, Dana-Farber Cancer Institute, Bos- ton, Massachusetts. 2Harvard Medical School, Boston, Massachusetts. biopsy at the time of disease progression to identify mechanisms 3Department of Medicine, Brigham and Women's Hospital, Boston, of acquired resistance. Biopsy of an advanced solid tumor can be 4 Massachusetts. Belfer Center for Applied Cancer Sciences, Dana- associated with increased patient morbidity; thus, the parallel Farber Cancer Institute, Boston, Massachusetts. 5Department of Bio- statistics and Computational Biology, Dana-Farber Cancer Institute, development of advanced technologies for noninvasive liquid Boston, Massachusetts. 6Department of Biostatistics, Harvard biopsies, analysis of circulating tumor cells (CTC; ref. 10) or cell- 7 T.H. Chan School of Public Health, Boston, Massachusetts. Depart- free plasma DNA (cfDNA; ref. 11), represents potential adjunctive ment of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts. or alternative method(s) for identifying targetable mechanisms of acquired resistance in patients with progression on first-line TKIs. Note: Supplementary data for this article are available at Clinical Cancer fi Research Online (http://clincancerres.aacrjournals.org/). CTCs can be identi ed in blood samples from patients with many different types of cancer, including lung cancer (12). Ongo- M. Yanagita and A.J. Redig contributed equally to this article. ing research efforts focus not only on improving analyses of these Corresponding Author: Pasi A. Janne,€ Dana-Farber Cancer Institute, 450 Brook- circulating single cells but also on identifying prognostic and line Avenue, Boston, MA 02215. Phone: 617-632-6036; Fax: 617-582-7683; therapeutic applications within the treatment context of a specific E-mail: [email protected] cancer. In NSCLC, CTCs have been detected in the plasma at doi: 10.1158/1078-0432.CCR-16-0909 different time points throughout disease treatment and in patients Ó2016 American Association for Cancer Research. with different stages of disease (13–15). However, the prognostic www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst June 8, 2016; DOI: 10.1158/1078-0432.CCR-16-0909 Yanagita et al. EGFR-mutant NSCLC treated with erlotinib until disease progres- Translational Relevance sion. Exploratory endpoints of this study included baseline Ongoing research efforts to elucidate the mechanism(s) of assessment of CTCs and cfDNA in patients at study entry along acquired resistance to targeted therapies in patients with non– with serial evaluation of both CTCs and cfDNA throughout small cell lung cancer (NSCLC) have led to emerging new treatment and at disease progression. The goal accrual for this strategies to target acquired resistance. However, some recently study was determined based upon the primary endpoint. Specif- approved drugs, such as osimertinib for the acquired EGFR ically, a sample size of 60 patients was identified based upon T790M resistance mutation, require repeat biopsy for genomic achieving a goal of 41 evaluable patients with sufficient tissue testing. Although analyzing tumor tissue remains the gold available for mutation analysis from rebiopsy performed at the standard, noninvasive biopsy methods, including evaluation time of disease progression. A cohort of 41 patients with adequate of circulating tumor cells (CTC) or cell-free DNA (cfDNA), rebiopsy allows for an 80% confidence interval (CI) of Æ10% for represent a novel way of detecting specific oncogenic muta- an outcome (acquired T790M mutation) with an expected pro- tions or following genomic changes within a tumor over time portion of 50%. For the exploratory analyses included in this without the need for a repeat tissue biopsy. This prospective study, this represents a reasonable estimate of the true incidence of trial evaluated CTCs and cfDNA at baseline, at progression, this genetic mechanism of secondary resistance. The protocol was and throughout treatment in patients with EGFR-mutant approved by the Institutional Review Board of the Dana-Farber/ NSCLC treated with erlotinib until disease progression and Harvard Cancer Center (Boston, MA; DF/HCC). Patients were compared the outcomes with repeat tissue biopsy to more fully enrolled at two DF/HCC institutions, the Dana-Farber Cancer evaluate and compare the clinical utility of these noninvasive Institute and the Beth Israel Deaconess Medical Center (Boston, biopsy methods with tissue biopsy. MA). All patients provided written informed consent prior to initiating erlotinib therapy. Patients Eligible patients had advanced stage NSCLC with a sensitiz- significance of isolating and quantifying CTCs and/or observing ing EGFR mutation identified through testing in a CLIA- temporal changes from an individual patient remains incom- approved laboratory and no prior history of treatment with pletely understood (16, 17). Mutations or rearrangements in EGFR-targeted agents. Patients could have had up to 1 prior line EGFR or ALK have been identified in circulating cells from some of non-EGFR–directed systemic therapy. Patients were required NSCLC patients (18–20), suggesting that evaluation of CTCs to be at least 18 years of age with an Eastern Cooperative could represent a noninvasive way to follow tumor response and Oncology Group performance status of 0 to 1. Exclusion criteria assess acquired genomic changes in advanced NSCLC over time included untreated or unstable central nervous system (CNS) during treatment with targeted therapies. disease. A more detailed list of inclusion and exclusion criteria Similarly, sequencing of cfDNAhasbeenusedtoidentify can be found in Supplementary Table S1. Patient records and activating mutations and acquired resistance mutations in baseline screening exams were also evaluated to identify the EGFR (19, 21), monitor changes in cfDNA throughout treat- number of metastatic sites at study entry. Metastatic sites were ment (22), and evaluate cfDNA as a prognostic or predictive defined as pleura, extrathoracic lymph nodes, bone, skin, biomarker in NSCLC patients (23, 24). cfDNA evaluation has omentum, CNS,
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