**1. Introduction**

Anal cancer is one of the six cancers shown to have a human papillomavirus (HPV) aetiology [1]. Most HPV-positive anal cancers are caused by HPV type 16 (HPV 16), and in a recent population-based assessment in Scotland, in cases diagnosed between 2009–2018, HPV 16 was detected in 93.3% of the HPV positive cases [2], higher than the amount of HPV 16 attributable to cervical cancer [3].

Additionally, as with other HPV-driven cancers, anal cancer incidence is increasing worldwide, including in the USA and Europe [4–7].

HPVs are formally classified as "types" based on the nucleotide sequence of the open reading frame (ORF) coding for the major capsid protein: L1 [8]. HPV types differ by more than 10% of their primary sequence compared to their most closely related type [8]. Phenotypic differences in HPV types with respect to disease risk and tissue tropism are wellestablished, and this knowledge has informed the development of effective vaccines and HPV-based cervical screening assays. However, below the level of HPV type exist lineages (with 2–10% variation) and sub-lineages (0.5% to 2% variation) [9], and the implications of this level of variation on clinical outcomes of infection is less established.

**Citation:** Guerendiain, D.; Mühr, L.S.A.; Grigorescu, R.; Holden, M.T.G.; Cuschieri, K. Mapping HPV 16 Sub-Lineages in Anal Cancer and Implications for Disease Outcomes. *Diagnostics* **2022**, *12*, 3222. https://doi.org/10.3390/ diagnostics12123222

Academic Editors: Fabio Bottari and Anna Daniela Iacobone

Received: 31 October 2022 Accepted: 9 December 2022 Published: 19 December 2022

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For HPV 16, four lineages have been identified (lineages A, B, C and D), as well as 16 sub-lineages: A, including A1–A3 (previously named European) and A4 (Asian) sub-lineages; B, including B1 (African-1, Afr1a) and B2 (African-1, Afr1b), B3 and B4 sublineages; C1 (African-2, Afr2a), C2, C3 and C4; and D, including D1 (North American, NA1), D2 (Asian-American, AA2), D3 (Asian-American, AA1) and D4 sub-lineages [9].

Although some investigators have assessed the global distribution of sub-lineages, the majority have focused on cervical cancers rather than other HPV-driven cancers. In 2013, Cornet et al. looked at the HPV lineages in cervical cancers and showed that European sub-lineages (A1–A3) were the most common in all regions of the world, except in sub-Saharan Africa and East Asia, whereas the African sub-lineages dominated in the northern sub-Saharan region of Africa, and the Asian variant in East Asia [10]. Nicolás-Párraga et al. (2016) found similar results, with A1–3 present in 95.65% of the cases in Europe, 78.26% in Central/South America (D in 21.73%) and 80% in Asia (12% A4 and 7.69% D) [11].

In terms of HPV 16 sub-lineages present in the anus, data is relatively sparce. Volpini et al. (2017) investigated the HPV 16 variants in anal samples collated in Brazil, finding that 70.8% were classified as A1–3 sub-lineages and 29.2% as "other" [12]. A recent systematic review, performed by Ferreira et al. (2021) of genetic variants of HPV-16 in men, found HPV 16 lineages vary according to anatomical and geographical regions, but they found that European samples had a high prevalence (86.59%) of HPV 16 lineage A [13].

In the context of cervical disease, evidence suggests that sub-lineages and variants may be independently associated with poor clinical outcomes. Mirabello et al. (2015) [14] revealed a higher risk of disease associated with B/C/D lineages as a group compared to the A lineage. Clifford et al. (2019). also found an increased cervical cancer risk for A3, A4 and D-(sub-) lineages vs the A1 sub-lineage. A more recent study by Lang Kuhs et al. (2022) looked into the genetic variation of HPV 16 and its association with clinical outcomes in HPV 16-positive oropharyngeal cancer patients. They investigated different high-risk single nucleotide polymorphisms (SNPs) and found that those with one or more high-risk SNPs had a median survival time of 3.96 years compared to 18.67 years for those with no high-risk SNPs. Most of these SNPs were common to the D2 sub-lineage, which have also been associated with higher risk of cancer in the cervix [14]. However equivalent studies on anal cancer are rare.

We recently identified that the viral load of HPV16 in anal cancer may be informative for prognosis [2]. Now, due to the information published on the association of HPV 16 sub-lineages and cancer risk yet the comparative absence of data in the anal context, we aimed to better understand the pattern and dominance of HPV 16 sub-lineages in a population-based cohort of anal cancer and to determine whether significant associations with sub-lineage and demographic or clinical variables existed). Data obtained from the cancer cohort was contextualized and compared to variant profile in anal samples obtained from an asymptomatic population.
