Afatinib demonstrates remarkable activity against HER2-amplified uterine serous endometrial cancer in vitro and in vivo

C L Schwab, S Bellone, D P English, D M Roque, S Lopez, E Cocco, R Nicoletti, I Bortolomai, E Bonazzoli, E Ratner, D-A Silasi, M Azodi, P E Schwartz, T J Rutherford, A D Santin, C L Schwab, S Bellone, D P English, D M Roque, S Lopez, E Cocco, R Nicoletti, I Bortolomai, E Bonazzoli, E Ratner, D-A Silasi, M Azodi, P E Schwartz, T J Rutherford, A D Santin

Abstract

Background: Uterine serous carcinomas (USCs) are an aggressive form of uterine cancer that may rely on HER2/neu amplification as a driver of proliferation. The objective of this paper is to assess the sensitivity of USC cell lines with and without HER2/neu gene amplification to afatinib, an irreversible ErbB tyrosine kinase inhibitor, and to test the efficacy of afatinib in the treatment of HER2-amplified USC xenografts.

Methods: Eight of fifteen primary USC cell lines (four with HER2 amplification and four without) demonstrating similar in vitro growth rates were treated with scalar concentrations of afatinib. Effects on cell growth, signalling and cell cycle distribution were determined by flow cytometry assays. Mice harbouring xenografts of HER2/neu-amplified USC were treated with afatinib by gavage to determine the effect on tumour growth and overall survival.

Results: Primary chemotherapy-resistant USC cell lines harbouring HER2/neu gene amplification were exquisitely sensitive to afatinib exposure (mean ± s.e.m. IC50=0.0056 ± 0.0006 μM) and significantly more sensitive than HER2/neu-non-amplified USC cell lines (mean ± s.e.m. IC50=0.563 ± 0.092 μM, P<0.0001). Afatinib exposure resulted in abrogation of cell survival, inhibition of HER2/neu autophosphorylation and S6 transcription factor phosphorylation in HER2/neu overexpressing USC and inhibited the growth of HER2-amplified tumour xenografts improving overall survival (P=0.0017).

Conclusions: Afatinib may be highly effective against HER2/neu-amplified chemotherapy-resistant USC. The investigation of afatinib in patients harbouring HER2/neu-amplified USC is warranted.

Figures

Figure 1
Figure 1
Comparison of mean IC50 values between HER2-amplified and -non-amplified cell lines. (A) Comparison of mean IC50 values for each HER2-amplified and -non-amplified cell line run in triplicate. (B) Comparison of the overall mean IC50 values for HER2-amplified and -non-amplified cell lines each represented in triplicate.
Figure 2
Figure 2
Comparison of mean fluorescence intensity (MFI) of ErbB1 in HER2-amplified vs -non-amplified cell lines. The figure depicts a comparison of the MFI of ErbB1 by direct staining as detected using flow cytometry between HER2-amplified and -non-amplified cell lines.
Figure 3
Figure 3
Representative changes in S6 phosphorylation with afatinib treatment HER2-amplified vs -non-amplified cell lines. Representative changes in phosphorylation of the transcription factor S6 after treatment with scalar amounts of afatinib in (A) HER2-amplified (ARK2) and (B) -non-amplified (ARK11) cell lines 8 h after treatment.
Figure 4
Figure 4
Comparison of mean fluorescence intensity (MFI) for phosphorylated HER2 and phosphorylated S6. Comparison of the MFI of phosphorylated HER2 (A) and phosphorylated S6 at 10 h (B) in HER2-amplified cell line ARK 2 after treatment with afatinib at a concentration of 5 nM (IC50), 50 nM (human serum concentration after 24 h of 40 mg of afatinib by mouth), and 175 nM (human serum concentration after 28 days of 40 mg of afatinib by mouth daily).
Figure 5
Figure 5
Comparison of mean tumour volume and overall survival in mice treated with afatinib vs vehicle. (A) Comparison of mean tumour size between vehicle and afatinib-treated mice harbouring xenografts of ARK2. (B) Comparison of overall survival in mice harbouring ARK2 xenografts that were treated with afatinib daily or with vehicle.

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