Scientific background: Human papillomavirus (HPV) is an important etiologic factor in oropharyngeal squamous cell carcinoma. It has been established that HPV-positive oropharyngeal squamous cell carcinoma respond better to concomitant radiochemotherapy with platinum based agents than HPV-negative oropharyngeal squamous cell carcinoma. This initiated the idea of de-intensification of the treatment in patients with HPV-positive tumors. The prerequisite is to maintain the curability rate achieved with standard dose radiotherapy (fractionated irradiation with total dose of 70 Gy) and platinum-based chemotherapy, whereas the aim is to reduce the treatment-related side effects. According to the research done so far, better results of HPV-positive oropharyngeal squamous cell carcinoma treatment are the consequence of higher sensitivity of HPV-positive tumors to radio(chemo)therapy. Molecular mechanisms of the higher sensitivity of HPV-positive oropharyngeal squamous cell carcinoma to radiotherapy are not entirely clear, but are probably based on impaired DNA damage repair. Preclinical research on the effects of radiotherapy on HPV-positive tumor models is scarce and preclinical research on the response of HPV-positive oropharyngeal squamous cell carcinoma tumor models to concomitant radiochemotherapy, which could lead to better treatment planning, is even scarcer.
Aim and hypotheses: The aim of this study was to determine the possible decrease in irradiation dose in HPV-positive tumors without a negative influence on the antitumor effect. Our hypotheses were that the radiosensitivity of HPV-positive oropharyngeal squamous cell carcinoma in combination with cisplatin in vitro is higher than that of HPV-negative cells; that the radiosensitivity of HPV-positive tumor cells is higher because of impaired DNA damage repair mechanisms and lastly, that in HPV-positive tumor models the same level of cures, as in HPV-negative, can be reached with a lower irradiation dose in combination with cisplatin.
Methods: In the in vitro part, we determined the response of HPV-positive and HPV-negative cell line to radiotherapy, cisplatin and the combined treatment using clonogenic assay. We evaluated the difference in the levels of DNA damage and kinetics of DNA repair in HPV-positive and HPV-negative tumor cells in vitro, using genotoxicologic studies. We evaluated the levels of single and double-strand DNA breaks (using comet assay) and the formation of foci of phosphorylated histones γH2AX (which are located on the sites of double-strand DNA breaks) after irradiation, after cisplatin therapy and after the combined treatment. We also monitored the presence of bystander effect after the irradiation or combined treatment in HPV-positive and HPV-negative tumor cells. Additionally, we analyzed the cell morphology after the combined treatment and evaluated the number of apoptotic and mitotic cells. We also determined the changes in the cell cycle progression after different therapies using flow cytometry. The same human tumor cell lines as in vitro were used for the induction of tumors on immunodeficient laboratory mice (SCID). We evaluated the response of both tumor models, first to radiotherapy and then to the combined treatment, using tumor growth delay assay. In the end, we determined the dose modifying factor of the irradiation dose in HPV-positive tumors after the combined treatment.
Results: Both in vitro and in vivo results showed that HPV-positive cells and tumors are more sensitive to irradiation than HPV-negative cells and tumors. The treatment with low doses of cisplatin alone resulted in a comparable response in both cell lines. Potentiation of irradiation with cisplatin was also present in both cell lines. In the in vivo study, the addition of cisplatin to irradiation resulted in better potentiation in HPV-negative tumors, however, the overall response to the combined treatment was better in HPV-positive tumors. The dose modifying factor, which was evaluated by the comparison of the effectiveness of irradiation and the combined treatment according to the HPV-status of the tumor, was 1.3 for irradiation alone and 1.2 for the combined treatment. This means that HPV-positive tumors responded 30% better to radiotherapy and 20% better to the combined treatment than HPV-negative tumors. Furthermore, HPV-positive cells exhibited higher levels of double-strand DNA breaks and slower DNA damage repair after irradiation alone or in combination with cisplatin (compared to HPV-negative cells). After the combined treatment, morphologic analysis showed higher levels of apoptotic HPV-positive cells and lower levels of mitotic HPV-positive cells compared to HPV-negative cells. The analysis of the cell cycle showed arrest in G2/M phase of HPV-positive cells. Finally, bystander effect was observed only in HPV-positive cells after the combined treatment.
Conclusions: The results of the study confirmed that HPV-positive cells and tumors are more sensitive to radiotherapy and that addition of cisplatin leads to an additive effect irrespective of the HPV-status. The results led to the suggestion that the irradiation dose in the concomitant treatment of HPV-positive oropharyngeal squamous cell carcinoma could be decreased for 20% in order to achieve the same level of effectiveness as in HPV-negative tumors treated with standard doses of radiochemotherapy. The results of the in vitro study showed that higher sensitivity of HPV-positive oropharyngeal squamous cell carcinoma to the described treatment is the consequence of higher levels of DNA damage, slower DNA damage repair, cell cycle arrest in G2/M phase and higher levels of apoptosis.
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