The signaling pathway of the epidermal growth factor receptor (EGFR) is commonly activated in HNSCC and represents a target for therapy. Among the different anti-EGFR agents, such as tyrosine kinase inhibitors and monoclonal antibodies (mAbs), the reversible inhibitor Cetuximab is the only approved for HNSCC treatment. However, treatment with these reversible tyrosine kinase inhibitors produces objective responses in only a small subset of patients. Despite an initial positive response, these patients often develop or acquire secondary resistance to the inhibitors, leading to relapse after several months. Thus, the resistance to anti-EGFR reversible inhibitors has emerged as an important clinical problem, making irreversible inhibitors studies essential for HNSCC tumors, such as clinical trials with Afatinib and Allitinib. The aim of this study is to evaluate sensitivity and resistance to anti-EGFR targeted drugs in a panel of HNSCC cell lines (HPV positive and negative), and correlate this profile with genetic alterations.
Five HNSCC HPV(+) cell lines and five HNSCC HPV(-) cell lines were used to test the treatment efficacy of Cetuximab, Afatinib and Allitinib by cell viability assays (MTS). Cells were seeded in 96 wells plates, exposed to increasing doses of each anti-EGFR inhibitors (0 - 2.5 µM) for 72 hours. For the mutation analysis, a panel of primers covering the entire coding extension of TP53, NOTCH1, P16, PTEN, PIK3CA, FBXW7, HRAS, TP63, CASP8, FAT1, MLL2, RB1, IRF6, NSD1 and EZH2 genes has been customized using AmpliSeq Custom Panel (Life Technologies). The mutational profile of these tumor-related genes was assessed by next-generation sequencing using the Ion Torrent PGM platform.
The results showed that only ten genes (TP53, NOTCH1, PTEN, HRAS, TP63, CASP8, FAT1, MLL2, RB1 and IRF6) had at least one cell line mutated. Several combinations were performed as a panel, where a positive panel was defined as at least three genes being mutated in the sample, and the result was that 80% of HPV negative cell lines were positive for the panel. Regarding to response profile, all TP53 wild type cell lines were Afatinib sensitive (p=0.033).
The “mutational positive” panel related to HPV negative cell lines corroborate with the literature, where HPV negative tumors are more likely to have these genes mutated than HPV positive tumors. Moreover, TP53 mutational status in HNSCC cell lines may predict Afatinib response, showing its feasibility as a potential biomarker in clinical setting.