Inflammatory pathways confer resistance to chemoradiotherapy in anal squamous cell carcinoma

Acantholytic squamous cell carcinoma (ASCC) is associated with immunosuppression and infection with human papillomavirus (HPV). Response to standard chemoradiotherapy (CRT) varies considerably. A comprehensive molecular characterization of CRT resistance is lacking, and little is known about the interplay between tumor immune contexture, host immunity, and immunosuppressive and/or immune activating effects of CRT. Patients with histologically confirmed, localized ASCC, treated between 1998 and 2019 at three different sites (Frankfurt, Berlin, Tübingen) of the German Cancer Consortium (DKTK) were included. All patients received 5-FU/MMC-based CRT. Patient cohorts for molecular analysis were defined based on availability and quality of samples, including baseline formalin fixed paraffin embedded biopsies for immunohistochemistry (n=130), baseline RNA sequencing (n=98), peripheral blood immune profiling by sequential multiparametric flow cytometry (n=47), and serum cytokine multiplex assays measurement (n=35). Tumor response, freedom from locoregional failure (FFLF), and freedom from distant metastasis (FFDM) were correlated with clinicopathologic and molecular findings. Baseline FFPE tumor samples were processed for total RNA isolation, library preparation and raw counts generation. Total RNA was isolated from FFPE samples using Qiagen RNeasy FFPE Kit (Qiagen) and depleted from ribosomal RNA (Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA)) to prepare sequencing libraries (NEBNext Ultra RNA Library Prep Kit for Illumina). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). Samples were sequenced using a 2 × 150 paired end configuration (HiSeq 2x150 PE HO) and quality guarantee ≥80% of bases ≥Q3.