Background
Anal squamous cell carcinomas (ASCC) are largely human papillomavirus (HPV) driven cancers. Overexpression of p16, a surrogate marker for HPV infection, is associated with improved overall survival (OS). However, immune checkpoint blockade (ICB) has shown limited efficacy in improving outcomes for ASCC patients irrespective of p16 status. Here we assessed, characterised the tumour microenvironment in p16 positive versus p16 negative, and primary treatment naïve versus recurrent, ASCC.
Methods
Custom T cell centric OPAL multiplex immunohistochemistry (n=39) and NanoString PanCancer transcriptomics profiling (n=12) were used to assess the immune microenvironment. Digital cytometry was utilised to quantitate cell abundance using the nSolver computational platform. Baseline patient, tumour characteristics, and survival data were correlated to the immune data.
Results
In total, 22 primary ASCC and 17 relapsed tumours were included. Primary ASCC had an overall higher abundance of CD8+, CD4+, double negative (DN), and FOXP3+ T cells in the intratumoural compartments compared to relapsed tumours. Primary and p16 positive tumours had a statistically significant higher density of CD4+ (p=0.027 and p=0.044) and FOXP3+ PD-1 Treg cells (p=0.039 and p=0.049) in the intratumoural sections. Spatial analyses demonstrated immune localisation within the 100mm intratumoural border in primary and p16 positive tumours except for PD-1 expressing CD8+ and DN T cell subsets. Low CD4+ (Hazard Ratio (HR) 3.6, 95% confidence interval (CI) 1.39-9.31, p=0.012), DN (HR 3.3, 95% CI 1.22-8.90, p=0.010), and Treg (HR 2.9, 95% CI 1.11-7.53, p=0.028) intratumoural infiltration were associated with inferior OS. Primary ASCC tumours were enriched in genes promoting T, B cell activation, cytotoxicity, and immune cell localisation in the tumours. In contrast, recurrent tumours were enriched for macrophage and neutrophil infiltration. In p16 negative tumours, myeloid leucocyte activation with a neutrophil predominant cell composition was identified.
Conclusion
Treatment naïve and p16 positive ASCC are characterised by a pro-inflammatory anti-tumour microenvironment with recruitment of T, B cells, differentiation, activation pathways and upregulation of cytokine signalling. Recurrent and p16 negative tumours are enriched in myeloid signature with a macrophage and neutrophil population. Future studies should focus on stratifying these immune subgroups and consider instituting ICB early in primary/p16 positive tumours.