Elsevier

Lung Cancer

Volume 127, January 2019, Pages 146-152
Lung Cancer

Efficacy of afatinib or osimertinib plus cetuximab combination therapy for non-small-cell lung cancer with EGFR exon 20 insertion mutations

https://doi.org/10.1016/j.lungcan.2018.11.039Get rights and content

Highlights

  • Ba/F3 cells with EGFR exon 20 insertion mutations were used in vitro and in vivo.

  • Cetuximab can increase the effect of afatinib or osimertinib against these mutations.

  • These combination treatments were well tolerable in vivo.

Abstract

Objectives

Epidermal growth factor receptor (EGFR) mutation-positive lung cancer accounts for a significant subgroup of non-small cell lung cancers (NSCLC). Approximately 4–10% of EGFR mutations in NSCLC are EGFR exon 20 insertion mutations, which are reportedly associated with resistance to EGFR tyrosine kinase inhibitor (EGFR-TKI) treatment. NSCLC patients carrying these mutations are rarely treated with EGFR-TKIs. The purpose of this study was to evaluate the efficacy of afatinib or osimertinib plus cetuximab combination therapy in experimental NSCLC models with EGFR exon 20 insertion mutations.

Materials and methods

The EGFR mutations examined in this study were A763_Y764insFQEA, Y764_V765insHH, A767_V769dupASV, and D770_N771insNPG. Ba/F3 cells constitutively expressing wild type or mutated EGFR were used to determine the efficacy of afatinib or osimertinib plus cetuximab combination therapy in vitro. To determine the efficacy of the combination therapy in vivo, female BALB/c-nu mice were injected subcutaneously with 1 million Ba/F3 cells carrying EGFR A767_V769dupASV or Y764_V765insHH.

Results

We observed a mild but significant (P < 0.05) additive effect of the combination therapy against several EGFR exon 20 insertion mutations in vitro. Regarding EGFR A767_V769dupASV and EGFR Y764_V765insHH, cetuximab and afatinib single treatment did not induce significant inhibition of tumor formation; however, afatinib plus cetuximab combination treatment induced significant (P < 0.05) tumor growth inhibition without significant body weight loss or skin rash.

Conclusion

The combination therapy induced a more potent inhibitory effect against several EGFR exon 20 insertion mutations than either therapy alone. Cetuximab can potentially increase the efficacy of afatinib or osimertinib in NSCLC with EGFR exon 20 insertion mutations.

Introduction

Lung cancer is a leading cause of cancer-related deaths worldwide [1]. However, in recent decades, the elucidation of key molecular mechanisms contributed to the improvement of prognosis in patients with lung cancer, especially in those with non-small cell lung cancer (NSCLC) [2]. In 2004, an association between somatic mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) gene and the response to EGFR tyrosine kinase inhibitor (TKI) treatment were reported by several groups [3,4]. Among the EGFR mutations found in NSCLC, in-frame deletions near the LREA motif in exon 19 (exon 19 deletions) and the L858R point mutation in exon 21 are classic mutations, accounting for approximately 80–90% of EGFR mutations [5]. These mutations destabilize the inactive conformation of EGFR and induce its constitutive activation [[6], [7], [8]]. Activated EGFR transduces downstream signaling, including the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase/protein kinase B (AKT) pathways [5,9]. After this discovery, multiple clinical trials demonstrated the efficacy of EGFR-TKIs in NSCLC patients carrying classic EGFR mutations [[10], [11], [12], [13]]. Multiple EGFR-TKIs, including gefitinib, erlotinib (first-generation), afatinib, dacomitinib (second-generation), and osimertinib (third-generation), are in clinical use for the treatment of NSCLC patients with these EGFR mutations [[14], [15], [16]]. However, the EGFR exon 20 insertion mutations are reportedly associated with resistance to EGFR-TKIs [17,18]. One exception is A763_Y764insFQEA, which we identified as an EGFR-TKI sensitive EGFR exon 20 insertion mutation [18]. EGFR exon 20 insertion mutations account for approximately 4–10% of all EGFR mutations associated with NSCLC [19]. Afatinib had a response rate of 8.3% in EGFR exon 20 insertion mutation-positive NSCLC, indicating the limited efficacy of EGFR-TKIs against this subgroup of NSCLC [19]. To improve the prognosis in NSCLC patients harboring EGFR exon 20 insertion mutations, the development of an effective therapy is urgently needed. Our study findings show that cetuximab combination therapy would be a potential strategy for the treatment of these patients.

Section snippets

Reagents

Erlotinib and afatinib were purchased from LC Laboratories (Woburn, MA, USA). Osimertinib was purchased from Selleck Chemicals (Houston, TX, USA). Cetuximab was purchased from Keio University Hospital (Tokyo, Japan). Total EGFR antibody (#2232), total AKT antibody (#9272), phospho-AKT (S473; D9E) antibody (#4060), total p44/42 MAPK antibody (#9102S), and phospho-p44/42 MAPK (T202/204) antibody (#9101S) were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-EGFR (Y1068)

Efficacy of afatinib or osimertinib plus cetuximab combination therapy on EGFR exon 20 insertion mutations

To evaluate the efficacy of cetuximab on EGFR exon 20 insertion mutations, we performed the MTS cell proliferation assay with cetuximab in a panel of Ba/F3 cells transduced with EGFR exon 20 insertion mutations (A767_V769dupASV, A764_V765insHH, D770_N771insNPG, and A763_Y764 in. FQEA). Except for EGFR A763_Y764insFQEA, an EGFR-TKI sensitive mutation, cetuximab did not induce an efficient inhibition of Ba/F3 cell proliferation (Fig. 1). These data indicated that cetuximab monotherapy has no

Discussion

In this study, we evaluated the in vitro and in vivo efficacy of afatinib or osimertinib plus cetuximab combination therapy on EGFR exon 20 insertion mutations. An earlier report indicated a limited efficacy of 1st- and 2nd-generation EGFR-TKIs in NSCLC with EGFR exon 20 insertion mutations [19]. Therefore, NSCLC patients carrying these mutations cannot be effectively treated with EGFR-TKIs. Recently, we reported preclinical evidence for the potential efficacy of osimertinib on EGFR exon 20

Funding

This work was supported in part by the Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to K.S. (15K09229), T.B. (15H04833), H.Y. (17K09667) and H.T. (18K08184). This work was supported in part by Takeda Science Foundation to H. Yasuda and H. Terai.

Conflicts of interest statement

There are no potential conflicts of interest to disclose.

Acknowledgements

We would like to thank Professor Tomoko Betsuyaku for financial assistance and technical advice. We are grateful to Ms. Mikiko Shibuya for her excellent technical assistance. We are also grateful to the Collaborative Research Resources at the Keio University School of Medicine for assistance with cell sorting.

References (26)

  • S.V. Sharma et al.

    Epidermal growth factor receptor mutations in lung cancer

    Nat. Rev. Cancer

    (2007)
  • D.B. Costa et al.

    BIM mediates EGFR tyrosine kinase inhibitor-induced apoptosis in lung cancers with oncogenic EGFR mutations

    PLoS Med.

    (2007)
  • T.S. Mok et al.

    Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma

    N. Engl. J. Med.

    (2009)
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