In Vitro assessment of intermittent Afatinib treatment effect in non-small cell lung cancer cell lines

Approximately 85% of all lung cancers consist of non-small cell lung cancer (NSCLC) that harbours an epidermal growth factor receptor (EGFR) mutation and is treated with afatinib. Unfortunately, resistance emerges in most patients, making treatment ineffective. Reports have suggested intermittent treatment has the potential to delay the emergence of resistance as compared to continuous treatment. However, it is known that continuous treatment is the standard of care and has superior treatment efficacy compared to intermittent therapy. In this study, the effect of afatinib through intermittent versus continuous treatments in NSCLC was investigated in vitro. The growth inhibition of afatinib in HCC827 (sensitive cells; EGFR exon 19 deletion), H1975 (resistant cells; EGFR T790M), and co-cultured cell lines with varying resistance percentages to mimic heterogeneous tumour subpopulations was observed. Co-cultures of 0.1% and 0.5% resistance were treated with afatinib at 100 picomolar (pM) and 500 pM. Continuous treatment was treated for 96 hours, while intermittent treatment was treated for 24 hours, followed by 72- hour treatment-free period. The efficacy for both continuous and intermittent treatments were assessed at the endpoint of 192 hours with a cell counting assay. Intermittent treatment showed a higher cell count than continuous treatment, with resistant percentages of 0.1% and 0.5% at 100 pM and 500 pM. The pM concentration in the experimental setup were below the plasma threshold of what being experienced in clinical patients. Thus, the pharmacokinetic area under the curve data of clinical trial patients were converted to nanomolar (nM) concentrations to mimic an in-patient scenario. Similar experimental plan from previous setup were remodelled with an increased concentration range from pM to nM for both continuous and intermittent treatments. The higher concentration in the experimental plans resulted in both continuous and intermittent treatments showed approximately 90% reduction in viable cell count at 96 hours, followed by increased in viable cell count at 192 hours for both 0.1% and 0.5% resistance. It was hypothesized that the initial reduction at 96 hours were due to high concentration that killed majority of sensitive cells, while the increased in viable cell count at 192 hours were caused by the resistant cells driving the co-culture growth. In order to monitor and quantify the cellular growth of resistant cells in co-culture for both treatment plans, fluorescent dyes that have been designed to freely pass through cell membranes were selectively tagged onto the resistant cells in 0.1% resistance co-culture in both treatment setups for 96 hours. Resistant cells showed higher cell count and fluorescence read in continuous as compared to intermittent treatment. Based on the fluorescence cell count for resistant cells, intermittent treatment was shown to cause 50.7% resistance growth inhibition as compared to continuous treatment. These results indicated intermittent treatment resulted higher cell count than continuous treatment, the fluorescent count and read showed lower resistant cells as compared to continuous treatment. Collectively, intermittent treatment was proven to be effective in vitro to suppress the emergence of de novo resistant cells in co-cultures of 0.1% and 0.5% resistance percentages in NSCLC cell lines.

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