2.1.1. Anal Cancer Cohort, Collection and Annotation

For the present work, we used the same anal cancer (*n* = 150) sample set as described in detail in Guerendiain et al. (2022) [2]. Briefly, nucleic acid extract associated with archived formalin-fixed, paraffin-embedded tissue was genotyped using the Seegene Anyplex II 28 (Seoul, Korea), followed by storage at −80 ◦C. Anal cancer biopsy samples were taken between 2009 and 2018 as part of the management of patients with anal disease from 3 of the 14 territorial health boards in Scotland (NHS Lothian, NHS Borders and NHS Fife).

HPV typing was performed at the Scottish HPV Reference Laboratory, Edinburgh, UK. One 10 µm section per sample was obtained and incubated in Seegene Universal Lysis Buffer (LB) at 65 ◦C overnight. DNA extraction was performed using the Microlab Nimbus IVD (Hamilton, Reno, USA) with the StarMAg Universal cartridge Kit (Seegene), following manufacturers' instructions. Mastermix was prepared with the Nimbus and PCR on the CFX Real-Time PCR instrument (Biorad, CA, USA).

As described in Guerendiain et al. (2022), clinico-demographic information was obtained in January 2020, specifically the patient's age, sex, stage of cancer (using the American Joint Committee on Cancer (AJCC) TNM system) [15], response to treatment, date of diagnosis and vital (dead/alive) status. Age and stage of cancer were considered at the time of diagnosis. Vital status information and date of death data was censored in July 2020.

Cases categorized according to the various clinical and demographic variables are summarized in Table 1. Age was stratified in 4 different groups: <50, 50–59, 60–69 and ≥0. Response to treatment was organized in 3 groups: yes, no or unknown, following the ESMO guidelines for anal cancer [16]. Cancer stage was aggregated in 5 groups: I, II, III, IV and unknown, following the AJCC system effective January 2018 [15].


**Table 1.** Anal cancer cohort: clinical characteristics and demographics with valid NGS analysis.
