Unraveling and targeting radiotherapy response in HPV related head and neck cancers

Head and neck squamous cell carcinoma (HNSCC) is, with approximately half a million new cases every year, the sixth most common cancer worldwide and is etiologically linked to tobacco and alcohol consumption and high-risk human papillomavirus (HPV) infections. It is generally accepted that HPV related HNSCC are a distinct entity with biological, demographical as well as clinical differences. One of the interesting features of HPV related HNSCC is that they have better response rates after treatment with chemoradiotherapy (cRT). However, despite this fact all HNSCC patients are treated with the same highly toxic standard treatment cRT schedules. Treatment adaptation through de-intensification of current standard cRT treatments or through introduction of tumor specific targeted approaches can form a solution to spare patients from the long-term complications. However, for both of these approaches thorough understanding of the biology of the radiation response of HPV related HNSCC is needed. The main objective of this thesis was to further elucidate the radiobiology of human HPV related HNSCC (see specific aim 1.1) and to implement this knowledge in order to improve the radio-therapeutic index by better stratification of patients (see specific aim 1.2) and by innovative, effective combination therapies (see specific aim 1.3). Here we mainly focus on p16INK4a, which is an important regulator of the cell cycle and is currently accepted as a surrogate marker for HPV+HNSCC. However, to our knowledge no functional assessment of this protein in relation to the RT response is made. To achieve the main objective of the thesis, we mainly focus on the role of p16INK4a in radiation and DNA damage response. Below a short summary of the major findings of our research is described. 1.1nbsp; Understanding the underlying mechanism of radiation response in HPV positive head and neck cancers In this section of the project the DNA damage response and the interaction with p16INK4a was studied in vitro and the results were verified in a patient cohort treated with cRT. Briefly, we found that HPV- cells showed higher survival after treatment with radiation compared to p16INK4a/HPV+ cells. Interestingly, we found that the radiation sensitivity seen in HPV+ cells was directly related to p16INK4a overexpression. Further assessment of the function of p16INK4a in radiation response revealed that p16INK4a actively modulates DNA repair by impairing the recruitment of RAD51, a marker for homologous recombination mediated DNA repair (HRR), through down-regulation of cyclin D1 protein levels. These results clearly indicate that p16INK4a expression mediates rsquo; HRR repair and p16INK4a+ cells are characterized with genomic instability. In agreement with our in vitro results patients with high p16INK4a expression showed a better therapy response compared to patients with lower p16INK4a expression. In conclusion, these data confirmed the importance of p16INK4a expression in modulation of RT response and indicates the possibility to use p16INK4a as a marker for this response. The latter is further investigated in the second specific aim of this thesis. 1.2nbsp; Investigating the importance and localization of p16INK4a in HPV positive head and neck cancers Despite the fact that the prognostic value of HPV in HNSCC is well-described in literature, there is still a lack of consensus regarding the detection method. As direct methods of HPV detection are complex to implement in a clinical setup, p16INK4a expression is suggested as ideal surrogate marker for HPV positivity. However, lack of specificity and uniform scoring guidelines makes it difficult to reach a consensus regarding the role of p16INK4a as a marker for HPV detection. Furthermore, several papers indicate in subpopulation analysis that p16INK4a expression even in the absence of HPV seems to predict better prognosis after RT treatment. Since we demonstrated an active role of p16INK4a in RT response we hypothesized that p16INK4a is more than just surrogate marker for HPV detection, but can potentially predict RT response in HNSCC patients on its own. Moreover, because our research indicates that p16INK4a is involved in active DNA repair, which takes mainly place in the nucleus, we also have taken the localization of p16INK4a into account. Briefly, we analyzed 241 patient tumor samples (oropharynx) for p16INK4a expression and examined the predictive value of this protein in response to RT. These tumor samples were pre-treatment biopsies of HNSCC patients (oropharynx) treated with cRT. Indeed, we demonstrated that nuclear p16INK4a expressing HNSCC patients showed significant (p<0.05) better locoregional control rates (5-year 82%) compared to p16INK4a cytoplasmic positive (5-year 55%) and p16INK4a negative patients (5-year 48%). Moreover, only nuclear p16INK4a expression was a significant prognostic factor for locoregional control with a hazard ratio of 0.48 (p<0.05; 95% CI: 0.22-101). The less pronounced effect of nuclear p16INK4a expression on overall survival (p