Gary M. Clifford

Human papillomavirus infection and vaccination among young females in rural Uganda

Abstract Cervical cancer is the most common cancer in Uganda. In 2015, a national human papillomavirus (HPV) vaccination program was initiated, targeting girls aged 10 years. To provide a pre‐vaccination baseline to monitor HPV vaccine effectiveness, first‐void urine (FVU) samples were collected from females aged 16–21 years in the General Population Cohort (GPC) in South‐East Uganda, between 2019 and 2023. HPV vaccination status was obtained from questionnaires and vaccination cards. FVU samples were tested for 28 HPV types using Allplex HPV28. Among 1009 participants, 28 type prevalence was 33%, and was higher among females reporting sexual intercourse (aPR = 3.7, 95%CI 2.8–4.8) and HIV infection (PR = 1.4, 95%CI 1.1–1.8). HPV16/18 prevalence was 4.8% overall, and lower in 146 vaccinated (1.4%) than 783 unvaccinated (5.6%) females (aPR = 0.4, 95%CI 0.1–1.4). No decrease was observed in other high‐risk (aPR = 1.5, 95%CIs 1.0–2.2) or low‐risk (aPR = 1.4, 95%CIs 1.0–2.1) types which were more prevalent in vaccinated females. Among vaccinated 16–21 year‐olds, 30.8% ( n  = 45) received one, 44.5% ( n  = 65) two, and 14.3% ( n  = 21) three doses. Vaccination status was also obtained from 1121 younger girls aged 10–15 years from the same GPC population, among whom 42.8% ( n  = 480) were vaccinated, 47.1% ( n  = 226) with one, 44.2% ( n  = 212) two, and 6.7% ( n  = 32) three doses. In conclusion, we report high HPV prevalence in young women in Uganda and see first impacts of vaccination on HPV16/18 infection. This population, shown to have suboptimal HPV vaccine coverage and heterogeneity in doses received, can serve as a robust baseline for future evaluations of HPV vaccine effectiveness.

Whole‐genome sequencing of 1,083 HPV45 cases and controls identifies genetic variants associated with glandular cervical lesions

AbstractHuman papillomavirus type 45 (HPV45) causes ~6% of all cervical cancers and an even greater proportion of adenocarcinomas, the latter of which are challenging to detect using current cervical cancer screening. Little is known about how HPV45 genetic variation is related to the risk of cervical precancer/cancer. To investigate this, we whole‐genome sequenced a total of 1,083 HPV45‐positive samples from two large studies. We evaluated associations of HPV45 genetic variation (sublineages, subclades, and SNPs) with histology‐specific precancer/cancer risk using logistic regression and evaluated risk modification by self‐reported race/ethnicity. Compared to the common A1 sublineage, A2 and B1 were associated with increased precancer/cancer (A2, OR = 3.9, 95% CI = 1.9–8.5; B1, OR = 2.7, 95% CI = 1.3–5.8; B2, OR = 3.3, 95% CI = 1.6–7.3), and most strongly with the glandular precancers/cancers (AIS/ADC; A2, OR = 6.9, 95% CI = 1.0–184; B1, OR = 6.2, 95% CI = 1.1–159). The A2 sublineage was most prevalent in women in East Asia and women who self‐reported as Asian/Pacific Islander (PI) in the U.S.; East Asian and Asian/PI women had the greatest precancer/cancer risk associated with A2 infections (OR = 5.8, 95% CI = 1.3–37.4) compared to all other sublineages among these women. We further evaluated precancer/cancer risk associations for 262 individual HPV45 SNPs and identified four SNPs significantly associated with only glandular precancers/cancers after correction for multiple tests (ORs ranged 7.8–20.7). One of these SNPs was a nonsynonymous variant in both overlapping viral E2/E4 ORFs. In summary, we show that HPV45 genetic variation influences the risk of precancer/cancer, specifically glandular precancer/cancer. Further studies of these genetic variants may improve our understanding of glandular lesions.

Performance of visual inspection, partial genotyping, and their combination for the triage of women living with HIV who are screen positive for human papillomavirus: Results from the AIMA‐CC ANRS 12375 multicentric screening study

AbstractThe WHO recommends the use of human papillomavirus (HPV) testing for primary cervical cancer (CC) screening because of its high sensitivity. However, triage is desirable to correctly identify HPV+ women who have high‐grade lesions (CIN2+) and require treatment. The ANRS‐12375 study was conducted in Côte d'Ivoire, Burkina Faso and Cambodia to assess the performance, feasibility and benefits of different triage options for detecting CIN2+ lesions: partial (HPV16 and HPV16/18/45) and extended genotyping, visual inspection (VIA) alone and VIA combined with partial genotyping. VIA was performed by gynecologists. The sensitivity, specificity, and diagnostic likelihood ratio (DLR) of each triage option for detecting CIN2+ lesions with histology as a reference standard were calculated. Of the 2253 women living with HIV (WLHIV) included, 932 (41%) were HPV+. A CIN2+ lesion was identified in 105 (13%) of the 777 participants with histopathology results. The sensitivity of VIA as a triage test for CIN2+ patients was 89%, while that for extended genotyping was 89%, that for HPV16/18/45 partial genotyping was 51%, and that for HPV16 partial genotyping was 36%. The specificities for these tests were 45%, 29%, 72%., and 85%, respectively. Combining VIA and/or partial genotyping positivity slightly increased the sensitivity (94%) at the cost of lower specificity (28%). There was significant intersite heterogeneity (p = .04). Among the three triage tests with a sensitivity ≥85%, the VIA had the highest specificity and positive likelihood ratio (p < .001). VIA and extended genotyping, whether independent or combined, are good triage options with high sensitivity for identifying WLHIV needing treatment for CIN2+.

Human papillomavirus genotypes in cervical and other HPV‐related anogenital cancer in Rwanda, according to HIV status

The study aim was to describe human papillomavirus (HPV)‐attributable cancer burden in Rwanda, according to anogenital cancer site, HPV type, age and HIV status. Tissue specimens of cervical, vulvar, vaginal, penile and anal cancer diagnosed in 2012–2018 were retrieved from three cancer referral hospitals and tested for high‐risk (HR) HPV DNA. Cervical cancer represented the majority of cases (598 of 738), of which 96.0% were HR‐HPV positive. HPV‐attributable fractions in other cancer sites varied from 53.1% in 81 penile, through 76.7% in 30 vulvar, 83.3% in 24 vaginal, up to 100% in 5 anal cases. HPV16 was the predominant HR‐HPV type in cervical cancer (55.0%), followed by HPV18 (16.6%) and HPV45 (13.4%). HPV16 also predominated in other cancer sites (60–80% of HR‐HPV‐attributable fraction). For cervical cancer, type‐specific prevalence varied significantly by histology (higher alpha‐9 type prevalence in 509 squamous cell carcinoma vs. higher alpha‐7 type prevalence in 80 adenocarcinoma), but not between 501 HIV‐negative and 97 HIV‐positive cases. With respect to types targeted, and/or cross‐protected, by HPV vaccines, HPV16/18 accounted for 73%, HPV31/33/45/52/58 for an additional 22% and other HR‐HPV types for 5%, of HPV‐attributable cancer burden, with no significant difference by HIV status nor age. These data highlight the preventive potential of the ongoing national HPV vaccination program in Rwanda, and in sub‐Saharan Africa as a whole. Importantly for this region, the impact of HIV on the distribution of causal HPV types was relatively minor, confirming type‐specific relevance of HPV vaccines, irrespective of HIV status.

Antibodies against high‐risk human papillomavirus proteins as markers for noncervical HPV‐related cancers in a Black South African population, according to HIV status

AbstractHuman papillomavirus (HPV) proteins may elicit antibody responses in the process toward HPV‐related malignancy. However, HPV seroepidemiology in noncervical HPV‐related cancers remains poorly understood, particularly in populations with a high prevalence of human immunodeficiency virus (HIV). Using a glutathione S‐transferase‐based multiplex serology assay, antibodies against E6, E7 and L1 proteins of HPV16 and HPV18 were measured in sera of 535 cases of noncervical HPV‐related cancers (anal (n = 104), vulval (n = 211), vaginal (n = 49), penile (n = 37) and oropharyngeal (n = 134)) and 6651 non‐infection‐related cancer controls, from the Johannesburg Cancer Study that recruited Black South African with newly diagnosed cancer between 1995 and 2016. Logistic and Poisson regression models were used to calculate adjusted odds ratios (aOR) and prevalence ratios (aPR) and 95% confidence intervals (CI) in cases versus controls. HPV16 E6 was more strongly associated with noncervical HPV‐related cancers than HPV16 L1 or E7, or HPV18 proteins: anal (females (HPV16 E6 aOR = 11.50;95%CI:6.0–22.2), males (aOR = 10.12;95%CI:4.9–20.8), vulval (aOR = 11.69;95%CI:7.9–17.2), vaginal (aOR = 10.26;95%CI:5.0–21), penile (aOR = 18.95;95%CI:8.9–40), and oropharyngeal (females (aOR = 8.95;95%CI:2.9–27.5), males (aOR = 3.49;95%CI:1.8–7.0)) cancers. HPV16‐E6 seropositivity ranged from 24.0% to 35.1% in anal, vulval, vaginal and penile cancer but was significantly lower (11.2%) in oropharyngeal cancer. After adjustment for HIV, prevalence of which increased from 22.2% in 1995–2005 to 54.1% in 2010–2016, HPV16 E6 seropositivity increased by period of diagnosis (aPR for 2010–2016 vs. 1995–2006 = 1.84;95%CI:1.1–3.0). Assuming HPV16 E6 seroprevalence reflects HPV attributable fraction, the proportion of certain noncervical‐HPV‐related cancers caused by HPV is increasing over time in South Africa. This is expected to be driven by the increasing influence of HIV.

Association of HPV35 with cervical carcinogenesis among women of African ancestry: Evidence of viral‐host interaction with implications for disease intervention

AbstractHPV35 has been found in only ∼2% of invasive cervical cancers (ICC) worldwide but up to 10% in Sub‐Saharan Africa, warranting further investigation and consideration of impact on preventive strategies. We studied HPV35 and ethnicity, in relation to the known steps in cervical carcinogenesis, using multiple large epidemiologic studies in the U.S. and internationally. Combining five U.S. studies, we measured HPV35 positivity and, in Northern California, observed HPV35 type‐specific population prevalence and estimated 5‐year risk of developing precancer when HPV35‐positive. HPV35 genetic variation was examined for differences in carcinogenicity in 1053 HPV35+ cervical specimens from a U.S. cohort and an international collection. African‐American women had more HPV35 (12.1% vs 5.1%, P < .001) and more HPV35‐associated precancers (7.4% vs 2.1%, P < .001) compared to other ethnicities. Precancer risks after HPV35 infection did not vary by ethnicity (global P = .52). The HPV35 A2 sublineage showed an increased association with precancer/cancer in African‐Americans (OR = 5.6 vs A1, 95% CI = 1.3‐24.8) and A2 was more prevalent among ICC in Africa than other world regions (41.9% vs 10.4%, P < .01). Our analyses support a strong link between HPV35 and cervical carcinogenesis in women of African ancestry. Current HPV vaccines cover the majority of cervical precancer/cancer across all ethnic groups; additional analyses are required to determine whether the addition of HPV35 to the already highly effective nine‐valent HPV vaccine would provide better protection for women in Africa or of African ancestry.

FAM19A4/miR124‐2 methylation in invasive cervical cancer: A retrospective cross‐sectional worldwide study

Widespread adoption of primary human papillomavirus (HPV)‐based screening has encouraged the search for a triage test which retains high sensitivity for the detection of cervical cancer and precancer, but increases specificity to avoid overtreatment. Methylation analysis of FAM19A4 and miR124‐2 genes has shown promise for the triage of high‐risk (hr) HPV‐positive women. In our study, we assessed the consistency of FAM19A4/miR124‐2 methylation analysis in the detection of cervical cancer in a series of 519 invasive cervical carcinomas (n = 314 cervical scrapes, n = 205 tissue specimens) from over 25 countries, using a quantitative methylation‐specific PCR (qMSP)‐based assay (QIAsure Methylation Test®). Positivity rates stratified per histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region were calculated. In total, 510 of the 519 cervical carcinomas (98.3%; 95% CI: 96.7–99.2) tested FAM19A4/miR124‐2 methylation‐positive. Test positivity was consistent across the different subgroups based on cervical cancer histotype, FIGO stage, hrHPV status, hrHPV genotype, sample type and geographical region. In conclusion, FAM19A4/miR124‐2 methylation analysis detects nearly all cervical carcinomas, including rare histotypes and hrHPV‐negative carcinomas. These results indicate that a negative FAM19A4/miR124‐2 methylation assay result is likely to rule out the presence of cervical cancer.

Phylogenomic Analysis of Human Papillomavirus Type 31 and Cervical Carcinogenesis: A Study of 2093 Viral Genomes

Human papillomavirus (HPV) type 31 (HPV31) is closely related to the most carcinogenic type, HPV16, but only accounts for 4% of cervical cancer cases worldwide. Viral genetic and epigenetic variations have been associated with carcinogenesis for other high-risk HPV types, but little is known about HPV31. We sequenced 2093 HPV31 viral whole genomes from two large studies, one from the U.S. and one international. In addition, we investigated CpG methylation in a subset of 175 samples. We evaluated the association of HPV31 lineages/sublineages, single nucleotide polymorphisms (SNPs) and viral methylation with cervical carcinogenesis. HPV31 A/B clade was >1.8-fold more associated with cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) compared to the most common C lineage. Lineage/sublineage distribution varied by race/ethnicity and geographic region. A viral genome-wide association analysis identified SNPs within the A/B clade associated with CIN3+, including H23Y (C626T) (odds ratio = 1.60, confidence intervals = 1.17–2.19) located in the pRb CR2 binding-site within the E7 oncogene. Viral CpG methylation was higher in lineage B, compared to the other lineages, and was most elevated in CIN3+. In conclusion, these data support the increased oncogenicity of the A/B lineages and suggest variation of E7 as a contributing risk factor.

Age‐specific burden of cervical cancer associated with HIV: A global analysis with a focus on sub‐Saharan Africa

AbstractHIV substantially worsens human papillomavirus (HPV) carcinogenicity and contributes to an important population excess of cervical cancer, particularly in sub‐Saharan Africa (SSA). We estimated HIV‐ and age‐stratified cervical cancer burden at a country, regional and global level in 2020. Proportions of cervical cancer (a) diagnosed in women living with HIV (WLHIV), and (b) attributable to HIV, were calculated using age‐specific estimates of HIV prevalence (UNAIDS) and relative risk. These proportions were validated against empirical data and applied to age‐specific cervical cancer incidence (GLOBOCAN 2020). HIV was most important in SSA, where 24.9% of cervical cancers were diagnosed in WLHIV, and 20.4% were attributable to HIV (vs 1.3% and 1.1%, respectively, in the rest of the world). In all world regions, contribution of HIV to cervical cancer was far higher in younger women (as seen also in empirical series). For example, in Southern Africa, where more than half of cervical cancers were diagnosed in WLHIV, the HIV‐attributable fraction decreased from 86% in women ≤34 years to only 12% in women ≥55 years. The absolute burden of HIV‐attributable cervical cancer (approximately 28 000 cases globally) also shifted toward younger women: in Southern Africa, 63% of 5341 HIV‐attributable cervical cancer occurred in women <45 years old, compared to only 17% of 6901 non‐HIV‐attributable cervical cancer. Improved quantification of cervical cancer burden by age and HIV status can inform cervical cancer prevention efforts in SSA, including prediction of the impact of WLHIV‐targeted vs general population approaches to cervical screening, and impact of HIV prevention.

Clinical performance of methylation as a biomarker for cervical carcinoma in situ and cancer diagnosis: A worldwide study

AbstractThe shift towards primary human papillomavirus (HPV)‐based screening has necessitated the search for a secondary triage test that provides sufficient sensitivity to detect high‐grade cervical intraepithelial neoplasia (CIN) and cancer, but also brings an improved specificity to avoid unnecessary clinical work and colposcopy referrals. We evaluated the performance of the previously described DNA‐methylation test (S5) in detecting CIN3 and cancers from diverse geographic settings in high‐, medium‐ and low‐income countries, using the cut‐off of 0.80 and exploratory cut‐offs of 2.62 and 3.70. Assays were performed using exfoliated cervical specimens (n = 808) and formalin‐fixed biopsies (n = 166) from women diagnosed with cytology‐negative results (n = 220), CIN3 (n = 204) and cancer stages I (n = 245), II (n = 249), III (n = 28) and IV (n = 22). Methylation increased proportionally with disease severity (Cuzick test for trend, P < .0001). S5 accurately separated women with negative‐histology from CIN3 or cancer (P < .0001). At the 0.80 cut‐off, 543/544 cancers were correctly identified as S5 positive (99.81%). At cut‐off 3.70, S5 showed a sensitivity of 95.77% with improved specificity. The S5 odds ratios of women negative for cervical disease vs CIN3+ were significantly higher than for HPV16/18 genotyping at all cut‐offs (all P < .0001). At S5 cut‐off 0.80, 96.15% of consistently high‐risk human papillomavirus (hrHPV)‐negative cancers (tested with multiple hrHPV‐genotyping assay) were positive by S5. These cancers may have been missed in current primary hrHPV‐screening programmes. The S5 test can accurately detect CIN3 and malignancy irrespective of geographic context and setting. The test can be used as a screening and triage tool. Adjustment of the S5 cut‐off can be performed considering the relative importance given to sensitivity vs specificity.