*2.7. Assessment of Variants According to Clinic-Demographic Characteristics and Survival Analysis*

To assess the relationship between HPV sub-lineages and different factors (two or more independent variables), a univariate logistic regression analysis was performed between HPV 16 sub-lineages (HPV 16 A1-positive vs. HPV 16 non-A1-positive), age at diagnosis, collection year and health board of diagnosis. Adjustment was performed for age group (<50, 50–59, 60–69 and 70 or over), sex, response to treatment, stage of cancer and vital status (dead or alive). Comparison was performed between A1 vs. non-A1 sub-lineages due to the small number of samples identified from the different sub-lineages. The non-A1 group includes the following sub-lineages: A2, A3, A4, B1, B2, B3, B4, C1, C2, C3, C4, D1, D2, D3 and D4.

Odds ratios (OR) were calculated to quantify the strength of the association between HPV 16 sub-lineages and the demographic and clinical data. All the statistics were obtained using R-studio macOS, (version 1.2.1335) [28]. The distribution of sub-lineages in anal cancers vs. the asymptomatic population was assessed with sequences from the two groups displayed in a phylogenetic tree.

Overall survival by HPV 16 sub-lineages (HPV 16 A1-positive vs. HPV 16 non-A1 positive) was analyzed using the Kaplan-Meier method. The univariate and multivariate hazard ratios of HPV 16 sub-lineages (HPV 16 A1-positive vs. HPV 16 non-A1-positive) for all-cause death were derived using the cox proportional hazard model. A univariate and multivariate model was derived; age (<50, 50–59, 60–69, 70+), sex, stage (I, II, III, IV) and response to treatment (no, yes) were adjusted for. All the statistical analyses were performed using R-studio (version 1.2.1335) [29]. Differences in prevalence of the HPV 16 sub-lineages between the anal cancer cohort and asymptomatic control cohort are presented descriptively.
