EGFR R521K Polymorphism Is Not a Major Determinant of Clinical Cetuximab Resistance in Head and Neck Cancer.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are among the most abundant malignancies worldwide. Patients with recurrent/metastatic disease undergo combination chemotherapy containing cetuximab, the monoclonal antibody used against the epidermal growth factor receptor (EGFR). Cetuximab augments the effect of chemotherapy; however, a significant number of patients show therapy resistance. The mechanism of resistance is yet to be unveiled, although extracellular alterations of the receptor have been reported, and their role in cetuximab failure has been proposed. AIMS: Here, we investigate possible effects of the multi-exon deletion variant (EGFRvIII), and the single nucleotide polymorphism EGFR R521K on cetuximab efficacy. RESULTS: Our results show that in HNSCC patients, the EGFRvIII allele frequency is under 1%; therefore, it cannot lead to common resistance. EGFR R521K, present in 42% of the patients, is investigated in vitro in four HNSCC cell lines (two wild-type and two heterozygous for EGFR R521K). While no direct effect is found to be related to the EGFR status, cells harboring R521K show a reduced sensitivity in ADCC experiments and in vivo xenograft experiments. However, this preclinical difference is not reflected in the progression-free or overall survival of HNSCC patients. Furthermore, NK cell and macrophage presence in tumors is not related to EGFR R521K. DISCUSSION: Our results suggest that EGFR R521K, unlike reported previously, is unable to cause cetuximab resistance in HNSCC patients; therefore, its screening before therapy selection is not justifiable.

EGFR Alterations Influence the Cetuximab Treatment Response and c-MET Tyrosine-Kinase Inhibitor Sensitivity in Experimental Head and Neck Squamous Cell Carcinomas.

Background: Anti-EGFR antibody therapy is still one of the clinical choices in head and neck squamous cell carcinoma (HNSCC) patients, but the emergence of cetuximab resistance questioned its effectiveness and reduced its applicability. Although several possible reasons of resistance against the antibody treatment and alternative therapeutic proposals have been described (EGFR alterations, activation of other signaling pathways), there is no method to predict the effectiveness of anti-EGFR antibody treatments and to suggest novel therapeutics. Our study investigated the effect of EGFR R521K alteration on efficiency of cetuximab therapy of HNSCC cell lines and tried to find alternative therapeutic approaches against the resistant cells. Methods: After genetic characterization of HNSCC cells, we chose one wild type and one R521K+ cell line for in vitro proliferation and apoptosis tests, and in vivo animal models using different therapeutic agents. Results: Although the cetuximab treatment affected EGFR signalization in both cells, it did not alter in vitro cell proliferation or apoptosis. In vivo cetuximab therapy was also ineffective on R521K harboring tumor xenografts, while blocked the tumor growth of EGFR-wild type xenografts. Interestingly, the cetuximab-resistant R521K tumors were successfully treated with c-MET tyrosine kinase inhibitor SU11274. Conclusion: Our results suggest that HNSCC cell line expressing the R521K mutant form of EGFR does not respond well to cetuximab treatment in vitro or in vivo, but hopefully might be targeted by c-MET tyrosine kinase inhibitor treatment.

[Consequences of extracellular alterations of EGFR on cetuximab therapy in HNSCC]. / Az EGFR extracelluláris módosulásainak hatása fej-nyaki daganatok cetuximabterápiájára.

Head and neck squamous cell carcinomas (HNSCC) take many lifes worldwide. Patients with recurrent/metastatic disease receive combination chemotherapy containing anti-EGFR antibody cetuximab. However, resistance often hurdles therapy. The mechanism is yet to unveil, although EGFR extracellular alterations and activity of c-Met signaling were accused. We investigated the effects of EGFR-vIII and EGFR-R521K on cetuximab efficacy in HNSCC in cellular, xenograft, and clinical setup. Furthermore, we investigated the efficacy of c-Met inhibition in HNSCC in vitro and in vivo. We showed that EGFR-vIII is very rare in HNSCC, while the common R521K polymorphism abolishes antibody-dependent cellular cytotoxicity and in vivo antitumor effect of cetuximab. This selectivity was not reflected in immunophenotype or survival data of HNSCC patients, suggesting a more complex mechanism behind. Interestingly, c-Met inhibitor SU11274 was more effective in cetuximab-resistant, EGFR R521K heterozygous cells and xenografts, raising the possible importance of simultaneous targeting of the two receptors.

The prognostic role of claudins in head and neck squamous cell carcinomas.

The expression of tight junction proteins is frequently altered in epithelial cancers. The loss of cell-cell adhesion associates with enhanced metastatic potential, which underlies the role of altered expression pattern of tight junction component claudins (CLDNs). Our study assessed the expression of CLDN 1, 2, 3, 4, 7, 8 and 10 in squamous cell carcinoma of the head and neck region (HNSCC) including oropahrynx, larynx, and hypopharynx in comparison to normal epithelial tissue of the same patient. The surgical samples were examined by tissue microarray and immunohistochemistry, the expression was calculated by H-score, which took account of intensity and percentage of positivity as well. Both normal and cancerous tissue proved negative for CLDN 3, 8 and 10. Normal epithelia showed mild expression of CLDN 4, but the minimal positivity disappeared in squamous cancer. In case of CLDN 1 and CLDN 7 we demonstrated significantly increased intensity in cancer, while CLDN 2 showed decreased expression compared with normal epithelium. The normal polarity and distribution of claudins were lost in HNSCC. Moreover, preserved expression of CLDN 2 (but not that of 1 and 7) was associated with better survival, which suggested a potential prognostic role of CLDN 2.

Clinical significance of genetic alterations and expression of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas.

The significance of epidermal growth factor receptor (EGFR) signaling is well studied in a number of different tumors, but limited data is available with regard to head and neck squamous cell carcinoma (HNSCC). Since anti-EGFR therapies are currently under investigation in these malignancies as well, comprehensive information about the alteration of EGFR in HNSCC is necessary to design the most appropriate therapeutic protocols. We examined retrospectively the gene copy number of EGFR by FISH and the protein expression by immunohistochemistry using different epitope-specific antibodies in paraffin-embedded primary tumors of five different regions, from 71 HNSCC patients who had not been treated with anti-EGFR therapy. In seven cases corresponding lymph node metastases were also available for comparative analyses. We also determined the mutational status of tyrosine kinase (TK) domain (exon 19 and 21) and the extracellular deletion mutation (vIII) of EGFR, the KRAS mutation at codon 12 and the presence of HPV infection. Eight of the 71 cases (11.3%) showed EGFR gene amplification (most of them localized into the hypopharyngeal region) and the increased gene copy number (amplification+polysomy) was 43.7%. Despite pronounced intratumoral heterogeneity of EGFR protein expression being found, the high EGFR expression correlated with poor prognosis. On the other hand, the phosphorylation of EGFR was associated with prolonged survival. No mutations in the TK domain of EGFR were found in any of the HNSCC patients and only two cases were KRAS mutant at codon 12. We detected vIII deletion mutation of EGFR in 21% of the samples, but there was no statistically significant correlation between the presence of vIII mutant form and patient survival. EGFR vIII mutation was, however, associated with increased gene copy number. Fourteen of 71 cases (19.7%) were HPV-positive and the incidence of infection showed a decreasing tendency from the oral cavity towards the larynx. Interestingly, in contrast to previous findings, we could not observe improved survival in HPV-positive patients compared to non-infected patients, most probably due to the fact that the majority of these HNSCC patients were smokers and alcohol consumers. In conclusion, we found that increased EGFR protein levels and gene copy numbers (not gene amplification alone) have prognostic significance in the investigated HNSCC patient population. However, the relatively high incidence of the EGFR-vIII mutant form warrants careful therapeutic decision-making when choosing between different anti-EGFR treatment options.