Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling

Abstract

Non-small cell lung cancers (NSCLCs) that harbor mutations within the epidermal growth factor receptor (EGFR) gene are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. Unfortunately, all patients treated with these drugs will acquire resistance, most commonly as a result of a secondary mutation within EGFR (T790M). Because both drugs were developed to target wild-type EGFR, we hypothesized that current dosing schedules were not optimized for mutant EGFR or to prevent resistance. To investigate this further, we developed isogenic TKI-sensitive and TKI-resistant pairs of cell lines that mimic the behavior of human tumors. We determined that the drug-sensitive and drug-resistant EGFR-mutant cells exhibited differential growth kinetics, with the drug-resistant cells showing slower growth. We incorporated these data into evolutionary mathematical cancer models with constraints derived from clinical data sets. This modeling predicted alternative therapeutic strategies that could prolong the clinical benefit of TKIs against EGFR-mutant NSCLCs by delaying the development of resistance.

Fig. 1

Derivation and characterization of TKI-resistant cells. (A and B) PC-9 erlotinib-resistant cells (PC-9/ER) (panel A) and PC-9 BIBW-2992–resistant cells (PC-9/BR) (panel B) were derived after ~120 days of culture with increasing concentrations of drug. Growth inhibition assays show that these cells are resistant to respective TKIs compared to the parental cells. (C) Direct dideoxynucleotide sequencing chromatograms from EGFR exon 20 (ex20) show the presence of the T790M mutation (*ACG→ATG) in the PC-9/ER and PC-9/BR cells but not in parental cells. F, forward; R, reverse directions.

Fig. 2

Growth characteristics of TKI-sensitive and TKI-resistant cells. (A) Polyclonal PC-9/BR cells cultured without BIBW-2992 for 8 and 16 passages (P-8 and P-16, respectively) regained intermediate and complete sensitivity to BIBW-2992, respectively. (B) Sequencing of EGFR exon 20 (ex20) showed a decrease in the T790M allele that correlates with restored TKI sensitivity. Genomic DNA was extracted from cells after 9 and 17 passages (P-9 and P-17, respectively) without drug (*ACG→ATG). (C) Parental and late-passage resistant cells (P-29) show decreased phosphorylation of EGFR and its downstream targets in the presence of BIBW-2992, whereas signaling in the PC-9/BR cells remained intact. Cells were treated with vehicle (DMSO) or BIBW-2992 (B) for 3 hours. (D) T790M-containing PC-9/BR cells proliferated more slowly than parental cells over 72 hours in the absence of inhibitor. Graphs represent the average of triplicate wells ± SD. *P < 0.01. (E) [³H]Thymidine incorporation confirms the slower proliferation rate of the PC-9/BR cells compared to parental cells. Cells were treated with DMSO, erlotinib (E), or BIBW-2992 (B) for 24 hours. Data are expressed as counts per million (CPM) relative to each other. (F) Cell counts for PC-9 parental cells, BR (polyclonal), BR late-passage (P-25), and T790M-containing BR clones (1, 2, 4, 5, 6, 8) show that the clones grow more slowly than parental and P-25 cells. (G) PC-9 parental and PC-9/BR cells (clones 1 and 4) were injected subcutaneously into nude mice, and tumor growth in the absence of drug was monitored over time. The slower growth pattern of T790M-harboring PC-9/BR clones 1 and 4 is maintained in vivo. Data are average tumor volumes (n = 3 per group) ± SEM. (H) At the onset of acquired resistance, an EGFR-mutant tumor (blue) develops T790M in a small proportion of cells (red) after exposure to erlotinib (E; left). Upon withdrawal of drug, previously growth-arrested TKI-sensitive cells repopulate the tumor. Alternatively (right), all cells contain some level of T790M at progression. Upon discontinuation of the inhibitor, all cells revert back to parental genotype.

Fig. 3

Reconstitution experiments to study T790M-mediated resistance. (A) T790M-containing PC-9 cells (BR, clone 1) were spiked into parental PC-9 cells at various proportions. The increased proportion of the T790M allele (*ACG→ATG) is evident from representative direct sequencing chromatograms of EGFR exon 20. (B) Mixed populations of cells were treated with increasing concentrations of erlotinib for 72 hours, at which point growth inhibition was measured. (C) Cell populations with varying proportions of T790M-containing cells were grown in the presence of DMSO or erlotinib (1 μM) to mimic various states of a TKI-resistant heterogeneous solid tumor. Total cell number was determined after 72 hours and graphed as the percent growth compared to parental cells (0%) ± SD. *P < 0.05; **P < 0.01.

Fig. 4

Indolent progression of T790M-harboring patient tumors. (A) A subset of patients with EGFR-mutant tumors (n = 114) treated with first-line gefitinib (7) displayed prolonged responses to treatment. The average time on gefitinib before progression was 0.9 years. (B) Serial computed tomography scans from a patient with an EGFR-mutant tumor (ex 19 del) [images from (11)]. (C) Serial bidimensional measurements taken from the time of best response for the patient in panel B illustrate the slow rate of progression in this lesion. (D) Patients receiving first-line erlotinib as part of a phase II trial. Four of 14 patients (28%; patients B, C, D, and H) were continued on treatment with single-agent TKI (erlotinib or gefitinib) for >6 months after RECIST progression. Asterisk denotes the presence of T790M.

Fig. 5

Evolutionary cancer modeling predictions to delay the development of resistance. (A) PC-9 cell birth rate at erlotinib concentrations of 0, 1, 3, 10, and 20 μM. (B) PC-9 cell death rate as a function of increasing erlotinib concentration. (C) PC-9/ER cell birth rate in the presence of erlotinib (0, 1, 3, 10, and 20 μM). (D) PC-9/ER cell death rates as a function of increasing erlotinib concentration. (E) Probability of preexisting T790M-harboring cells in a population of 3 million cells initiating from one cell harboring just a drug-sensitive EGFR mutation that grew in the absence of drug for a range of mutation rates (10⁻⁴ to 10⁻⁸ per cell division). (F) Expected number of resistant cells present in the population, both averaged over all cases and averaged only over the subset of cases, where at least one resistant cell is present. (G) An initial population of 750,000 cells, 10% of which harbor T790M, treated with continuous low-dose erlotinib (1 and 3 μM) selects for the emergence of T790M-harboring cells (green and black lines). The addition of one or two high-dose erlotinib “pulses” (20 μM) followed by 1 μM for the remaining days of a 7-day cycle decreases the expected number of resistant cells (red and blue lines). (H) Analogous results as in panel (G) starting with an initial population with 25% T790M-harboring cells.

Fig. 6

Effect of pulsed high-dose TKI treatment on the number of T790M-harboring cells. (A) Using a T790M-harboring clone (PC-9/BR, c1), we mixed cell populations to have 0, 25, or 100% resistant cells. The baseline panel shows forward sequence tracings from exon 20 of EGFR (the underlined codon encodes T790). Cell populations were then treated with erlotinib at the indicated doses for 7 days. (B) Chromatograms display exon 20 forward sequences of EGFR from PC-9 cells treated with different drugs (*ACG→ATG).

Fig. 7

Effect of continuation of TKI therapy with chemotherapy on heterogeneous TKI-resistant tumors. (A) Schematic outline of treatment options for patients with EGFR-mutant disease. (B) PC-9/BR c1–resistant cells were diluted in parental cells at various concentrations (see Fig. 3) and treated with chemotherapy (cisplatin, 500 nM) or chemotherapy plus erlotinib (3 μM). In all cases, the TKI-chemotherapy combination was more efficacious at inhibiting cell growth. (C) Athymic nude mice with established tumors (50:50 mixture of PC-9 parental and BR c1 cells) were administered vehicle, cisplatin (4 mg/kg), erlotinib (12.5 mg/kg), or cisplatin plus erlotinib. At the start of treatment, T790M-containing cells made up ~25% of the population, as measured by direct sequencing (bottom right). Tumor volumes were graphed as averages (n = 5 per group) ± SEM. mpk, mg/kg. * indicates residue of interest.