Association of Human Papillomavirus Genotype 16 Lineages With Anal Cancer Histologies Among African Americans

The incidence and mortality from anal squamous cell carcinoma (SCC) have increased over the last several decades. African American men show the greatest increase and have the highest incidence of anal SCC.^1,2 Nearly 90% of cases have been attributed to human papillomavirus (HPV),³ of which more than 200 genotypes have been reported. Of these, only 13 HPV types cause virtually all HPV-associated anogenital and oropharyngeal cancers. HPV type 16 (HPV16) is the most carcinogenic and is uniquely prevalent worldwide.⁴ HPV16 consists of 4 lineages (A, B, C, D) and 16 sublineages (A1–4, B1–4, C1–4, D1–4).⁵ Differences in carcinogenic potential have been assigned to these sub-lineages.^6,7 However, little is known about their association to anal cancer. Here, we analyzed clinical features, risk factors, and HPV16 sublineages in African Americans with anal lesions.

Methods

Study Population

This retrospective study was approved by Howard University Institutional Review Board under the protocol IRB-06-MED-39. We reviewed medical records of 386 African Americans with anal lesions (January 2007 to December 2016). Demographic, clinical, and pathologic data (HPV, human immunodeficiency virus [HIV], hepatitis C virus, diabetes mellitus, hypertension, and body mass index) were collected.

DNA Extraction, Library Preparation, and HPV16 Variants Determination

Tissue blocks from 72 patients were used for DNA extraction. Specific cores within the blocks were used: 9 normal tissues, 46 condylomas (CAs), 31 high-grade dysplasias, 1 carcinoma in situ, and 24 SCC (111 samples). A custom ThermoFisher Scientific multiplex polymerase chain reaction primer panel was used to amplify the entire 7906-base pair HPV16 genome as previously described,⁸ which then was sequenced on the Ion Torrent S5 Sequencer at the National Cancer Institute Cancer Genomics Research Laboratory. Trimmed sequencing reads were aligned to the HPV16 reference sequence. Mapped sequences were genotyped using Torrent Variant Caller version 5.0.3.⁷ HPV16 variant lineage assignment was based on the maximum likelihood tree topology constructed using RAxML MPI v7.2.8.27.⁷

Statistical Analyses

Continuous variables were presented with median values, and statistical analyses were performed using χ² and Student t tests. For HPV16 sublineage associations, a logistic regression model was used to obtain the odds ratios and 95% confidence intervals for SCC risk using different comparisons with normal and precancerous lesions. The most prevalent sublineage, A1, was used as a reference. SPSS version 21.0 and R version 3.1.2 were used, and all statistical tests were 2-sided. P < .05 was considered significant.

Results

Study Population Characteristics

Of all patients, 289 (75%) were men (median age, 44 years). CA, high-grade squamous intraepithelial lesion, SCC, and adenocarcinoma frequencies were 202 (52%), 30 (8%), 31 (8%), and 8 (2%), respectively. HPV (72%) and HIV (43%) were common infections in this population. Patients with CAs were significantly younger (median age, 36 years) than other patients with anal lesions (median age, 50 years; P < .001). Men had significantly more CAs than women (P < .001). Fifty-two percent of patients with CAs had a co-infection with HIV. Hypertension was statistically more prevalent in patients without CAs. Sixty-three percent and 77% of the high-grade squamous intraepithelial lesion and SCC patients, respectively, had HIV infection, significantly higher than patients without these lesions. SCC patients (median age, 54 years) were significantly older than non-SCC patients (median age, 42 years; P < .001).

HPV16 Sublineage Associations With SCC

The HPV16 sublineage distribution illustrates the predominance of the A1 sublineage (Figure 1A). We assessed each HPV16 sublineage and a combination of sublineages for associations with SCC compared with sublineage A1 as a reference. Patients with A4, B, C, or D sublineages combined had a significantly increased SCC risk compared with all other lesions (odds ratio, 4.2; 95% confidence interval, 1.01–17.4; P = .048) (Figure 1B). The combination of A4, B, and D sublineages, without the C sublineage, had a higher increased SCC risk (odds ratio, 10.5; 95% confidence interval, 1.8–62.4; P = .009). Interestingly, the HPV16 B sublineage alone was similarly associated with increased SCC risk (odds ratio, 10.5; 95% confidence interval, 0.96– 115.3; P = .054).

Figure 1.

(A) HPV16 sublineage distribution among condyloma, HSIL and SCC anal lesions. One sample per patient. (B) HPV16 variant lineage associations with anal squamous cell carcinoma.

Discussion

We showed that most patients with CAs are young men with both HPV and HIV infections. HIV was also a risk factor for high-grade dysplasia and progression to carcinoma. Interestingly, HPV16 sublineage B was associated with higher SCC risk.

HPV16 is uniquely carcinogenic at the anus.³ At the cervix, HPV16 sublineages are associated with different risks of precancer and cancer; these sublineage risks vary by a woman’s race or ethnicity,⁷ and this has also been observed for HPV35. Here we evaluated HPV16 lineage and sublineage associations with anal SCC in African American patients and showed that the non-A1 (or non-European) lineages and sublineages were significantly associated with SCC. Although based on small numbers, HPV16 lineage B (aka African-1 lineage), was the non-A1 lineage most associated with an increased risk of SCC. In contrast, this B lineage was associated with less cervical precancer and cancer in primarily European women.⁷ It is possible that the B lineage has a different carcinogenic potential by anatomic site (ie, anus vs cervix); however, it is more likely that the HPV16 lineage risk varies based on the infected population and ethnicity. The B lineage has been observed at a higher proportion in individuals of African ancestry, and previous data suggest that if an individual’s race or ethnicity matches that of the origin of the infecting HPV16 virus, there is an increased risk of cervical progression⁷; therefore, this is also possible for anal SCC, and the B lineage may have a stronger carcinogenic potential in this African American population. Interestingly, we did not observe any SCC with HPV16 lineage C, which is the other African lineage (ie, African-2 lineage).

Although current HPV vaccines will likely prevent most HPV-associated cancers, there are some important considerations for African Americans. First, the inclusion of HPV genotypes that are prevalent in African Americans (ie, HPV35) needs to be included in future HPV vaccine versions. Second, an investigation into HIV’s role in anal cancer for this population is warranted. Finally, sexual behavior education that specifically targets younger individuals may be beneficial to reduce anal cancer. In conclusion, our data show that most African American patients with anal lesions are young men with HPV and HIV co-infections, and HPV16 lineage B was associated with a high SCC risk.

Abbreviations used in this paper:

CA

condyloma

HIV

human immunodeficiency virus

HPV

human papillomavirus

HPV16

human papillomavirus type 16

SCC

squamous cell carcinoma