Different Association of Human Papillomavirus 16 Variants with Early and Late Presentation of Cervical Cancer

<div><p>The median age of cervical cancer (CC) presentation coincides with the mean age of menopause presentation (49 years) in Mexico. Here, we investigated the association between different HPV16 variants and early (≤ 49 years) or delayed (≥ 50 years) CC presentation. We conducted a case-case study that included 462 CCs, 386 squamous cell carcinomas (SCC), 63 adenocarcinomas (ACC), and 13 additional cell types. Variants were identified by PCR and DNA sequencing. The risk conferred by each variant for developing CC earlier than 50 years was analyzed using a univariate logistic regression model considering old-aged patients (≥ 50 years) and non-HPV16 cases as the reference variables. Overall, the frequency of HPV16 was 50.9%, and the only identified variants were the European A1/2 (31.2%) and the Asian-American D2 (10.8%), and D3 (8.9%). D2 was mainly associated with ≤ 49-year-old patients (15.9%); A1/2 was uniformly distributed between the two age groups (~31%), whereas D3 increased with age to a frequency of 11.8% in the older group. Only the D2 variant conferred a 3.3-fold increase in the risk of developing CC before 50 years of age (OR = 3.3, 95% CI = 1.7–6.6, p < 0.001) in relation with non-HPV16 cases. Remarkably, this risk was higher for ACC (OR = 6.0, 95% CI = 1.1–33, p < 0.05) than for SCC (OR = 2.8, 95% CI = 1.3–5.9, p < 0.01). Interestingly, when analyzing only the HPV16-positive CC, D2 increases (OR = 2.5, 95% CI = 1.2–5, p < 0.05) and D3 decreases (OR = 0.45, 95% CI 0.2–0.9, p < 0.05) the risk to develop CC before 50 years old in relation with A1/2 variant. These results indicated that D2 variant is associated with early and D3 with delayed CC presentation, whereas A1/2 variant was uniformly distributed between the two age groups.</p></div

Association between HPV16 variants and early presentation of cervical cancer.

<p>Association between HPV16 variants and early presentation of cervical cancer.</p

Nucleotide sequence changes in E6, L1 (MY), and LCR regions from A1/2, D2 and D3 HPV16 variants.

<p>The figure shows nucleotide changes in the E6, L1 (MY) and LCR regions which were amplified from 235 DNA isolates derived from cervical carcinomas positive for HPV16. The nucleotide positions at which variations were observed are written vertically. Classification of HPV16 variants were performed according to Yamada et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169315#pone.0169315.ref021" target="_blank">21</a>] and Burk et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169315#pone.0169315.ref022" target="_blank">22</a>]. The E6 panel shows 14 variant positions, all used by Yamada and all but one (183) by Burk. The LCR panel shows 21 positions, all used by Burk and Yamada. The MY panel shows 14 positions that were used by Yamada. Variant-sequence positions that do not vary in relation with the HPV16-reference sequence are marked with dashes. The number of positive CC samples (n) is shown at the right side. The HPV16R sequence (7906 bp), listed as NC_001526.4 in GenBank, was used as the reference sequence for all the alignments. The reference sequences used to classify the specific sublineages were as follows: NC_001526.4 (Sublineage A1), AF536179 (Sublineage A2), AY686579 (Sublineage D2) and AF402678 (Sublineage D3). In addition, for D variant classification we specially used the diagnostic SNPs in the LCR, positions 7507 and 7743 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169315#pone.0169315.ref023" target="_blank">23</a>], and the MY, positions 6803 and 6862 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169315#pone.0169315.ref021" target="_blank">21</a>].</p

HPV types distribution by 5-years age intervals in the whole sample of CC patients.

<p>The figure shows the frequency distribution of HPV16 single infections (blue circles), pooled frequency of HPV18, HPV45, and HPV39 single infections (green circles), and the pooled frequency of other HPV types (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109406#pone-0109406-g002" target="_blank">Figure 2</a>) plus HPV31 and all double infections (orange circles) over 5-years age intervals in the whole CC patients (n = 462).</p

Percent positivity of HPV16 variant by 5-year age intervals.

<p>The figure shows the percentage of HPV16 infections as a whole and as segregated by lineages D (D2 and D3) and A (A1/2) based on 5-year age intervals for all CC patients (n = 462). The decrease in HPV16 and D2 percent positivity with patient age to the 51–55 year-old interval were statistically significant (p < 0.05, Spearman correlation). The HPV16+ plot has already been published in a previous paper [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169315#pone.0169315.ref009" target="_blank">9</a>], but it was included as a reference for the trend of HPV16 variants.</p

Frequencies of different HPV types in CC patients according to age and FIGO staging.

<p>The figure shows the relative frequency (%) of HPV types in CC patients grouped by age in ≤40, between 41 and 55 and>55 years old. Panel A included the patients with FIGO I/II, and panel B patients with FIGO III/IV. Bars labeled as HPV16, HPV18, HPV45, HPV31 and HPV39 include only single infections. Other HPVs group include single infection of HPV types 6, 11, 26, 33, 35, 42, 51, 52, 53, 56, 58, 59, 61, 66, 68, 69, 70, 73, 82, 39-like, 51-like, 82-like and all double infections.</p

Age distribution of patients with cervical cancer (CC).

<p>The age distribution of CC patients divided into 5-year intervals according to tumor histological type (A). The frequency of patients by age, divided into three groups: <41 years, 41–55 years, and>55 years, according to FIGO staging (B). IND, undifferentiated; ASCC, adenosquamous cell carcinoma; ACC, adenocarcinoma; SCC, squamous cell carcinoma.</p

Mean age of cervical cancer patients according to HPV type (n = 462).

<p>a. S.D.  =  standard deviation.</p><p>b. Other HPVs included HPV6, HPV26, HPV39Like, HPV42, HPV51Like, HPV53, HPV61, HPV66, HPV68, HPV69, HPV70, HPV73, HPV82, HPV82Like.</p><p>The mean±S.D.(n) in the whole sample was 50.6±13.0 (462)</p><p>Mean age of cervical cancer patients according to HPV type (n = 462).</p

Frequency of single and double HPV infections in cervical cancer patients (n = 462).

a<p>Includes HPV68, HPV51Like, HPV66, HPV26, HPV39Like, HPV42, HPV61, HPV70, HPV73, HPV82, HPV82Like.</p><p>Frequency of single and double HPV infections in cervical cancer patients (n = 462).</p

Association of life-style factors and HPV types with delayed onset of cervical cancer.

a<p>Patient group ≤40 yrs was taken as reference group and odds ratios were calculated using a logistic regression model including all significant variables of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109406#pone-0109406-t003" target="_blank">table 3</a>; reference variable (OR = 1), p value and 95% confidence interval are shown.</p>b<p>Other HPVs includes single infections other than HPV16/18/45/39 and double infections.</p>c<p>Information of six patients was missed.</p>d<p>Include nulliparous (3.4% of total cases).</p>e<p>Patients that have been assisted at least once to Pap screening.</p><p>*Lower-risk factor or reference factor for CC.</p><p>Association of life-style factors and HPV types with delayed onset of cervical cancer.</p