S2552
Radiobiology - Microenvironment
ESTRO 2026
Material/Methods: PLATO (ISRCTN88455282) is a UK multicentre trial platform testing CRT dose optimisation in patients with localised ASCC. Sections of baseline formalin- fixed, paraffin embedded (FFPE) biopsies from 358 participants in the ACT 3 (58), ACT 4 (111) and ACT 5 (189) studies were stained for p16 and graded for tumour infiltrating lymphocytes (TIL)[3]. Invasive ASCC was confirmed by histopathology. Tumour sections were macro-dissected and sequencing libraries made from the resultant DNA. Sequence libraries were pooled to 150 samples per HiSeq2000 flow cell. Sequence reads were aligned to human and HPV genomes, to enable HPV genotyping. DNA sequences were aligned against microbial genomes using GATK PathSeq, before being filtered against a list of putative known gastro-intestinal taxa generated from the Genomics England 100,000 genomes project. Analysis after filtering was performed on genus-level relative abundances.Alpha diversity was calculated using the vegan package in R with Shannon index as the primary measure, and differences assessed using the Kruskal- Wallis test. Beta diversity was assessed by multivariable PERMANOVA using the adonis2 function based on Bray-Curtis dissimilarities.6-month response was available for 263 patients who underwent chemoradiotherapy (ACT 4 and ACT 5 only). Results: Multivariable PERMONOVA testing highlighted significant differences associated with treating centre (p=0.001) and sequencing batch (p=0.001) as expected, due to contamination. After adjustment for these confounders, small but significant differences in beta diversity were seen for tumour site (anal canal vs anal margin, p=0.004), age (p=0.003), sex (p=0.001), HPV status (p=0.005) and TILs score (p=0.012). No significant differences were seen by HIV status, tumour stage or p16 status. Alpha diversity was higher in HPV positive tumours than HPV negative tumours (median shannon 3.36 vs 2.96, p=0.009). There was no significant difference in alpha diversity or beta diversity between those with or without a complete response at 6-months.
Digital Poster 3402 Investigating the intratumoural microbiota in anal squamous cell carcinoma, a translational sub- study of the PLATO (ISRCTN88455282) trial platform. Hamish Sinclair 1,2 , Henry Wood 3 , Susan Richman 3 , Daniel Bottomley 3 , Joanne Copeland 4 , Joanne Webster 4 , Alexandra Smith 4 , Lindy Berkman 5 , Rebecca Muirhead 6,7 , Andrew Renehan 8,9 , Richard Adams 10 , Mark Harrison 11 , Sheila Rao 12 , Vicky Goh 13 , Maria Hawkins 14 , David Sebag-Montefiore 15 , Duncan C Gilbert 1 1 Sussex Cancer Centre, University Hospitals Sussex NHS Foundation Trust, Brighton, United Kingdom. 2 Clinical and experimental medicine, Brighton and Sussex Medical School, Falmer, United Kingdom. 3 Division of Pathology and Data Analystics, Leeds Insitutute of Medical Research, Leeds, United Kingdom. 4 Leeds Cancer Research UK Clinical Trials Unit, Leeds Institute of Clinical Trials, Leeds, United Kingdom. 5 Patient and Public Involvement Trial Representativerepresentative, (PPI), London, United Kingdom. 6 Department of Oncology, Oxford University Hospitals NHS Trust, Oxford, United Kingdom. 7 Department of Oncology, University of Oxford, Oxford, United Kingdom. 8 Colorectal and Peritoneal Oncology Centre, The Christie NHS Foundation Trust, Manchester, United Kingdom. 9 Division of Cancer Sciences, University of Manchester, Manchester, United Kingdom. 10 Centre for Trials Research, Cardiff University Heath Park, Cardiff, United Kingdom. 11 Mount Vernon Centre for Cancer Treatment, Mount Vernon Hospital, London, United Kingdom. 12 The Royal Marsden Hospital, Institute of Cancer Research, London, United Kingdom. 13 13.School of Biomedical Engineering & Imaging Sciences, Kings College, London, United Kingdom. 14 Department of Medical Physics & Biomedical Engineering, University College London, London, United Kingdom. 15 Leeds Institute of Medical Research at St James's, St James's University Hospital, Leeds, United Kingdom Purpose/Objective: Anal squamous cell carcinomas (ASCC) are strongly associated with human papilloma virus (HPV) infection; HPV-negative tumours have a worse prognosis for reasons poorly understood. Chemoradiotherapy (CRT) is standard of care for localised tumours. Emerging evidence suggests the intratumoural microbiota is an important component of the tumour microenvironment and may influence response to CRT [1,2]. We sought to investigate the intratumoural microbiota in ASCC and assess microbial differences between clinicopathological variables.
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