The immunological milieu of oropharyngeal squamous cell carcinoma (OPC) according to HPV and smoking status and its influence on prognosis.
Authors: Guimaraes de Sousa Luana , Mota Siqueira Juliana , McGrail Daniel , Marques-Piubelli Mario , Lazar Neto Felippe , Ferri Borgogno Sammy , Daumas Nunes Fabio , Matos Leandro , Kowalski Luiz Paulo , Hoff Camilla Oliveira , Dai Hui , Mitani Yoshitsugu , Li Kaiyi , Williams Michelle D. , Lin Shiaw-Yih , El-Naggar Adel K. , Sikora Andrew G. , Burks Jared , Gillison Maura L. , Ferrarotto Renata 
Published: June 2024

3147 Background: OPC exhibits distinct epidemiological, genetic, and clinical characteristics, significantly influenced by the presence of HPV. Despite multidisciplinary therapy, approximately 40% of OPC patients (pts) experience recurrence, and those who achieve cure often endure late and morbid sequelae of treatment. In this study, we comprehensively explored the composition and functional status of the tumor immune microenvironment (TIME) in a cohort of OPC pts treated with curative intent and investigated TIME differences based on HPV infection, smoking status, and clinical outcomes. Methods: Two tissue microarrays with baseline OPC tissues were stained with a previously validated panel containing 33 metal-tagged markers. Imaging mass cytometry was performed with the Fluidigm Helios CyTOF instrument, and analysis performed with Visiopharm software. HPV status was determined by p16 IHC and/or DNA testing. Comparison of cell types and markers densities among subgroups (HPV positive vs negative) were analyzed with Wilcoxon signed-rank test and its association with clinical outcomes (recurrence vs no-recurrence) with log-rank and Cox Proportional Hazards. Multiple comparisons were corrected by Benjamini Hochberg procedure. Results: A total of 99 samples from 61 pts from a single institution were included in this study. The majority of pts were male (N=47, 77%) and heavy smokers (N=55, 90%); 72% were HPV-negative. The median age was 61 years (range, 39 – 87 years), and 52% of cases were diagnosed with stages III-IVB (AJCC 8th edition). Most pts (57%) was treated with surgery (±RT). Thirty-three pts (54%) experienced recurrent disease. Smoking, stage III-IVB, and HPV-negative tumors were associated with higher recurrence rates (p <0.01). Regarding the OPC TIME, the most prevalent cell populations in the stroma were fibroblasts (22%), followed by T-cells (17%) and endothelial cells (10%). The stroma of HPV+ tumors exhibited higher densities of memory T cells (FDR < 0.01), B cells (FDR = 0.02), and T cells (FDR = 0.02) compared to HPV negative counterparts, which displayed an increased density of neutrophils (FDR = 0.04). The density of cancer stem cells was directly associated with smoking history (FDR<0.001), and an increased density of intra-tumoral B-cells was found in non-smokers (FDR=0.01). Higher intra-tumoral neutrophil density was linked to recurrent disease (FDR < 0.001). Conversely, higher densities of memory CD8+ T cells and non-M2 macrophages in the TIME were associated with a lower chance of recurrence (FDR < 0.1). Conclusions: The intricate analysis of the OPC TIME revealed significant differences according to HPV and smoking status and highlighted the prognostic role of neutrophils, memory CD8+ T cells, and non-M2 macrophages. These findings may guide the development of personalized therapeutic strategies for OPC.