Heterogeneity Underlies the Emergence of EGFRT790 Wild-Type Clones Following Treatment of T790M-Positive Cancers with a Third-Generation EGFR Inhibitor

Abstract

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.

Figure 1. Results of rociletinib-resistant biopsies

Panel A. Representative images from a CT of the chest showing (I) thickening of the left pleura prior to the start of rociletinib, (II) resolution of the left pleural thickening due to disease response on day 44 of rociletinib and (III) progressive thickening of the left pleural at rociletinib progression (day 372.) Arrows depict the site of biopsies obtained at baseline and at progression. Panel B. Pie-chart showing distribution of findings on rociletinib-resistant biopsies. 6 of 13 biopsies had “lost” T790M and 2 of these underwent transformation to small cell lung cancer. 7 remained T790M-positive, and 3 of these developed EGFR amplification. Panel C. Rociletinib-resistant cell line MGH700 (derived from the rociletinib-resistant biopsy of patient 10) demonstrates in vitro cross-resistance to all tested EGFR inhibitors. MGH700 cells were treated with the indicated EGFR TKIs over a range of doses for 72 hours. CellTiter Glo was used to measure cell viability. Experiments were carried out in quadruplicate and the error bars depict standard error of the mean. Panel D. Western blot showing that treatment of the MGH700 cell line with 1^st, 2^nd or 3^rd generation EGFR TKIs leads to inhibition of EGFR phosphorylation, but fails to suppress the AKT/mTOR pathway. Lysates from MGH700 cells treated with DMSO or 1μM of Gefitinib, Afatinib, or Rociletinib for 6 hours were probed with the indicated antibodies. Panel E. Pre and post-rociletinib biopsies of patient #11. Histologic analyses illustrate that the pre-rociletinib biopsy shows moderately differentiated adenocarcinoma with an acinar pattern. The post-rociletinib biopsy (11b) exhibits nests of tumor cells with a high nuclear to cytoplasmic ratio and no gland formation. Immunohistochemical studies reveal the tumor cells to be negative for chromogranin and synaptophysin pre-rociletinib and positive for both neuroendocrine markers in the post-rociletinib liver biopsy. The results, in conjunction with the morphology, are consistent with the diagnosis of small cell carcinoma in the rociletinib-resistant specimen.

Figure 2. Intratumoral T790M heterogeneity

Panel A. A conceptual model showing that tumors with T790M “positive” resistance to erlotinib, gefitinib or afatinib may be heterogeneous, consisting of both T790M-positive (blue) and T790 wild-type (white) populations. In some cases of rociletinib resistance, T790 wild-type cells may be the dominant driver of tumor growth and may lead to a predominantly T790 wild-type tumor (top), while in other cases the T790M-positive cells may make up the majority of the population (bottom.) Panel B. (I) Two representative clones out of eight isolated from cell line MGH176, derived from an afatinib-resistant pleural effusion, demonstrate heterogeneity of T790M. Brightfield images of the clones are shown on the left and nucleotide sequences on the right. Both clones are positive for the EGFR exon 19 deletion mutation. The nucleotide sequence of clone 2 reveals the presence of T790M (*), while the mutation is absent in clone 6. (II) Summary of the single-cell clone analyses showing that overall 5 of 8 clones analyzed are T790M positive and 3 of 8 are T790 wild-type (see also supplemental figure S3.)

Figure 3. Plasma and Tissue EGFR allele analyses

Panel A. 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 biopsy and the maximum reduction in tumor volume (maximal percent change in the sum of the longest diameter of RECIST target lesions.) Patients with highest allelic fractions of T790M have the most significant reductions in tumor volume, while those with low (or absent) T790M were least likely to respond. The Spearman correlation coefficient (R) for this relationship is −0.596 (p=0.0017.) Panels B–D. Longitudinal quantitative analyses of EGFR mutations are shown with the relative copies of T790M and activating mutation per milliliter of plasma depicted by solid and dashed red lines, respectively. The corresponding radiographic response is depicted with a solid blue line, measured as the sum of the longest diameters of the target lesions as per the RECIST method. Arrows indicate the timing of rociletinib-resistant biopsies. ctDNA analysis of patient 7 (panel B) shows an increase in both Del19 and T790M when radiographic progression develops. In contrast, patients 10 (panel C) and 11 (panel D) demonstrate increasing levels of the activating mutation at the time of radiographic progression while T790M remains suppressed.