Background Head and neck carcinomas (HNC) are the world’s sixth most common cancer. Most of HNCs are associated with tobacco and other environmental factors but a growing part of oropharyngeal tumors are caused by persistent infection of human papillomavirus (HPV). Patients with HPV positive cancers have a better prognosis with fewer recurrences. This may be caused by different anti-tumor immune response and immune profile of patients. Multispectral fluorescent immunohistochemistry (fIHC) is a powerful tool for a detailed analysis of the tumor microenvironment. This method allows to access the phenotype and calculate cells in tumor parenchyma and stroma of the tumor since in comparison to flow cytometry, an architecture of the tissue remains preserved. fIHC is uniquely suited to study interaction of immune and cancer cells in situ.

Materials and Methods Number of 97 formalin fixed paraffine-embedded slides of the human HNC tissue with known etiology were examined using 4 different panels of 5 antibodies each. These panels include antibodies suitable for phenotyping of immune cells (CD3e, CD4, CD8, FOXP3) or their functional description (PD1, CTLA4, ICOS, CCR4). Additionally, antibodies against Ki67, VEGF and cell cytokeratin were used. Slides were stained using Opal™ 7-Color Fluorescent IHC Kit (Akoya Biosciences). The quantity of immune cells was evaluated in stroma and tumor compartment using InForm™ 2.4.6. software (Akoya Biosciences). For all patients the demographic and clinical data were available and these patients were followed for up to 18 years.

Results Our results have shown significantly higher abundance of Th and Tc in both compartments of HPV+ samples. Besides HPV etiology Th and Tc in the tumor microenvironment predict independently better survival of patients. We did not observed difference in number of Tregs (characterized as a CD3+CD4+FOXP3+ cells) in tumors of different etiology, but we detected higher number of ICOS+Tregs in stroma of HPV- tumors. We also quantified the subpopulations of Th and Tc cells expressing regulatory receptors PD1 and CTLA4. PD1 showed significantly higher expression on Th and Tc both in tumor and stroma of HPV+ tumors, but CTLA4 expression was significantly higher only on Th located in stroma of HPV- tumors. Moreover, we detected significantly higher VEGF expression in both compartments and higher proliferating activity of tumor cells in HPV- tumors.

Conclusions Detailed analyses of the tumor infiltrating lymphocytes allows for selection of prognostic markers in HNC of different etiology. Our results may also help to understand the better prognosis of HPV+ patients. More detailed survival analyses with inclusion of other clinical and demographic data will be presented.

Disclosure Information B. Pokrývková: None. M. Grega: None. J. Klozar: None. O. Vencalek: None. J. Nunvar: None. R. Tachezy: None.