American Association for Cancer Research
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Supplementary Figures S1 - S5 from Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor

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posted on 2023-04-03, 20:47 authored by Zofia Piotrowska, Matthew J. Niederst, Chris A. Karlovich, Heather A. Wakelee, Joel W. Neal, Mari Mino-Kenudson, Linnea Fulton, Aaron N. Hata, Elizabeth L. Lockerman, Anuj Kalsy, Subba Digumarthy, Alona Muzikansky, Mitch Raponi, Angel R. Garcia, Hillary E. Mulvey, Melissa K. Parks, Richard H. DiCecca, Dora Dias-Santagata, A. John Iafrate, Alice T. Shaw, Andrew R. Allen, Jeffrey A. Engelman, Lecia V. Sequist

Supplementary Figure S1. CONSORT diagram. Supplementary Figure S2. Histologic analysis of the pre-rociletinib biopsy from patient 12 (H&E 400x, left panel) demonstrates characteristic adenocarcinoma with an open chromatin pattern, ample cytoplasm and variable cell density. Supplementary Figure S3. Panel A. The nucleotide sequences of single-cell cloning analysis for six additional clones isolated from the afatinib-resistant cell line MGH176 are shown (see also figure 2B). Panel B. Summary of single-clone analyses performed on two additional patient-derived cell lines. Supplementary Figure S4. Longitudinal quantitative analyses of EGFR mutations in the ctDNA are shown for patients 8 (panel A), 6 (panel B) and 3 (panel C). Supplementary Figure S5. Scatterplot showing the relationship between the allelic fraction of T790M (calculated as the percent of T790M alleles/percent of EGFR activating mutation alleles as quantified by allele-specific PCR or NGS) in the pre-rociletinib tumor biopsy (y-axis) and in the pre-rociletinib plasma (as quantified by BEAMing) (x-axis).



Rociletinib is a third-generation EGFR inhibitor active in lung cancers with T790M, the gatekeeper mutation underlying most first-generation EGFR drug resistance. We biopsied patients at rociletinib progression to explore resistance mechanisms. Among 12 patients with T790M-positive cancers at rociletinib initiation, six had T790–wild-type rociletinib-resistant biopsies. Two T790–wild-type cancers underwent small cell lung cancer transformation; three T790M-positive cancers acquired EGFR amplification. We documented T790–wild-type and T790M-positive clones coexisting within a single pre-rociletinib biopsy. The pretreatment fraction of T790M-positive cells affected response to rociletinib. Longitudinal circulating tumor DNA (ctDNA) analysis revealed an increase in plasma EGFR-activating mutation, and T790M heralded rociletinib resistance in some patients, whereas in others the activating mutation increased but T790M remained suppressed. Together, these findings demonstrate the role of tumor heterogeneity when therapies targeting a singular resistance mechanism are used. To further improve outcomes, combination regimens that also target T790–wild-type clones are required.Significance: This report documents that half of T790M-positive EGFR-mutant lung cancers treated with rociletinib are T790–wild-type upon progression, suggesting that T790–wild-type clones can emerge as the dominant source of resistance. We show that tumor heterogeneity has important clinical implications and that plasma ctDNA analyses can sometimes predict emerging resistance mechanisms. Cancer Discov; 5(7); 713–22. ©2015 AACR.See related commentary by Ichihara and Lovly, p. 694.This article is highlighted in the In This Issue feature, p. 681

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