Prevalence of HPV 16 and HPV 18 lineages in Galicia, Spain

Abstract

Genetic variants of human papillomavirus types 16 and 18 (HPV16/18) could differ in their cancer risk. We studied the prevalence and association with high-grade cervical lesions of different HPV16/18 variant lineages in a case-control study including 217 cases (cervical intraepithelial neoplasia grade 2 or grade 3 or worse: CIN2 or CIN3+) and 116 controls (no CIN2 or CIN3+ in two-year follow-up). HPV lineages were determined by sequencing the long control region (LCR) and the E6 gene. Phylogenetic analysis of HPV16 confirmed that isolates clustered into previously described lineages: A (260, 87.5%), B (4, 1.3%), C (8, 2.7%), and D (25, 8.4%). Lineage D/lineage A strains were, respectively, detected in 4/82 control patients, 19/126 CIN3+ cases (OR = 3.1, 95%CI: 1.0-12.9, p = 0.04), 6/1 glandular high-grade lesions (OR = 123, 95%CI: 9.7-5713.6, p<0.0001), and 4/5 invasive lesions (OR = 16.4, 95%CI: 2.2-113.7, p = 0.002). HPV18 clustered in lineages A (32, 88.9%) and B (4, 11.1%). Lineage B/lineage A strains were respectively detected in 1/23 control patients and 2/5 CIN3+ cases (OR = 9.2, 95%CI: 0.4-565.4, p = 0.12). In conclusion, lineages A of HPV16/18 were predominant in Spain. Lineage D of HPV16 was associated with increased risk for CIN3+, glandular high-grade lesions, and invasive lesions compared with lineage A. Lineage B of HPV18 may be associated with increased risk for CIN3+ compared with lineage A, but the association was not significant. Large well-designed studies are needed before the application of HPV lineage detection in clinical settings.

Figure 1. Study population.

CIN2: Cervical intraepithelial neoplasia grade 2. CIN3+ includes CIN grade 3-carcinoma in situ, invasive squamous cell carcinoma, adenocarcinoma in situ, and adenocarcinoma. Controls for the analysis were women without progression to CIN2 or CIN3+ during the two-year follow-up of the prospective study of HPV16/18-positive women. Cases were women with histological diagnosis of HPV16/18-positive CIN2 or CIN3+ at enrolment or during the two-year follow-up of the prospective study. Enrolment of the prospective study: from 2009 to 2010. Additional cases were HPV16-positive CIN2 or CIN3+ cases collected from 2006 to 2008 and 2011 to 2012.

Figure 2. Nucleotide sequence variations of LCR/E6 among HPV16 isolates.

Position number refers to the HPV 16 prototype sequence previously described .

Figure 3. Phylogenetic tree of the HPV16 isolates based on LCR/E6 sequences.

Phylogenetic analysis confirmed the presence of the four lineages : A, B, C and D. A maximum likelihood (ML) tree was inferred from an alignment of ten reference sequences and fifteen study sequences of HPV16 LCR-E6 using RAxML HPC v8 . Highly related sequences (<0.4 differences) from the study were not included in this figure. Reference sequences were denominated as lineage|strain.

Figure 4. Nucleotide sequence variations of LCR/E6 among HPV18 isolates.

Position number refers to the HPV18 prototype sequence previously described .

Figure 5. Phylogenetic tree of the HPV18 isolates based on LCR/E6 sequences.

Phylogenetic analysis confirmed the presence of two lineages : A and B. A maximum likelihood (ML) tree was inferred from an alignment of nine reference sequences and seven study sequences of HPV18 LCR-E6 using RAxML HPC v8 . Highly related sequences (<0.4 differences) from the study were not included in this figure. Reference sequences were denominated as lineage|strain.

Figure 6. Nucleotide sequence variations of L1 among HPV16 isolates.

Position number refers to the HPV 16 prototype sequence previously described. Asterisk indicates amino acid substitution.