Robert D. Burk

Case-Control Study of Cervicovaginal β/γ–Human Papillomavirus Infection in Women With Human Immunodeficiency Virus and Its Relation to Incident Cervical Precancer

Abstract We studied cervicovaginal β/γ–human papillomavirus (HPV) and their relationship to cervical precancer in women with human immunodeficiency virus; having previously reported strong positive associations of β/γ-HPV with incident head and neck cancer in the general population. Case patients (n = 124) had cervical intraepithelial neoplasia (CIN) 3 or 2. Controls (n = 247) were individually matched 2:1 to case patients. Unexpectedly, multivariate analyses found strong inverse associations between β/γ-HPV and CIN-2/3 (odds ratio, 0.19 [95% confidence interval, .04–.86]; P = .03; Ptrend < .01]). This is, to our knowledge, the first study of β/γ-HPV and cervical precancer. If confirmed, a strong inverse (protective) association would be of potential clinical and biologic relevance.

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.

Somatic mutations in 3929 HPV positive cervical cells associated with infection outcome and HPV type

AbstractInvasive cervical cancers (ICC), caused by HPV infections, have a heterogeneous molecular landscape. We investigate the detection, timing, and HPV type specificity of somatic mutations in 3929 HPV-positive exfoliated cervical cell samples from individuals undergoing cervical screening in the U.S. using deep targeted sequencing in ICC cases, precancers, and HPV-positive controls. We discover a subset of hotspot mutations rare in controls (2.6%) but significantly more prevalent in precancers, particularly glandular precancer lesions (10.2%), and cancers (25.7%), supporting their involvement in ICC carcinogenesis. Hotspot mutations differ by HPV type, and HPV18/45-positive ICC are more likely to have multiple hotspot mutations compared to HPV16-positive ICC. The proportion of cells containing hotspot mutations is higher (i.e., higher variant allele fraction) in ICC and mutations are detectable up to 6 years prior to cancer diagnosis. Our findings demonstrate the feasibility of using exfoliated cervical cells for detection of somatic mutations as potential diagnostic biomarkers.

Genetic and Epigenetic Variations of HPV52 in Cervical Precancer

The goal of this study was to identify human papillomavirus (HPV) type 52 genetic and epigenetic changes associated with high-grade cervical precancer and cancer. Patients were selected from the HPV Persistence and Progression (PaP) cohort, a cervical cancer screening program at Kaiser Permanente Northern California (KPNC). We performed a nested case-control study of 89 HPV52-positive women, including 50 cases with predominantly cervical intraepithelial neoplasia grade 3 (CIN3) and 39 controls without evidence of abnormalities. We conducted methylation analyses using Illumina sequencing and viral whole genome Sanger sequencing. Of the 24 CpG sites examined, increased methylation at CpG site 5615 in HPV52 L1 region was the most significantly associated with CIN3, with a difference in median methylation of 17.9% (odds ratio (OR) = 4.8, 95% confidence interval (CI) = 1.9–11.8) and an area under the curve of 0.73 (AUC; 95% CI = 0.62–0.83). Complete genomic sequencing of HPV52 isolates revealed associations between SNPs present in sublineage C2 and a higher risk of CIN3, with ORs ranging from 2.8 to 3.3. This study identified genetic and epigenetic HPV52 variants associated with high risk for cervical precancer, improving the potential for early diagnosis of cervical neoplasia caused by HPV52.

The D2 and D3 Sublineages of Human Papilloma Virus 16–Positive Cervical Cancer in Guatemala Differ in Integration Rate and Age of Diagnosis

Abstract Human papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence. Significance: This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

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.

Epidemiological evidence that common HPV types may be common because of their ability to evade immune surveillance: Results from the Women's Interagency HIV study

Infection by human papillomavirus (HPV) type 16, the most oncogenic HPV type, was found to be the least affected by HIV‐status and CD4 count of any of the approximately 13 oncogenic HPV types. This relative independence from host immune status has been interpreted as evidence that HPV16 may have an innate ability to avoid the effects of immunosurveillance. However, the impact of immune status on other individual HPV types has not been carefully assessed. We studied type‐specific HPV infection in a cohort of 2,470 HIV‐positive (HIV[+]) and 895 HIV‐negative (HIV[−]) women. Semi‐annually collected cervicovaginal lavages were tested for >40 HPV types. HPV type‐specific prevalence ratios (PRs), incidence and clearance hazard ratios (HRs), were calculated by contrasting HPV types detected in HIV[+] women with CD4 < 200 to HIV[−] women. HPV71 and HPV16 prevalence had the weakest associations with HIV‐status/CD4 count of any HPV, according to PRs. No correlations between PRs and HPV phylogeny or oncogenicity were observed. Instead, higher HPV type‐specific prevalence in HIV[−] women correlated with lower PRs (ρ = −0.59; p = 0.0001). An alternative (quadratic model) statistical approach (PHIV+ = a*PHIV− + b*PHIV−2; R2 = 0.894) found similar associations (p = 0.0005). In summary, the most prevalent HPV types in HIV[−] women were the types most independent from host immune status. These results suggest that common HPV types in HIV[−] women may have a greater ability to avoid immune surveillance than other types, which may help explain why they are common.

HPV73 a nonvaccine type causes cervical cancer

HPV73 is classified as possibly oncogenic. It is neither routinely evaluated in HPV screening, nor covered by any of the prophylactic vaccines. We sought to investigate the carcinogenic characteristics of HPV73. Molecular studies were performed on eight cervix cancer biopsy specimens containing HPV73 from a cross‐sectional cancer cohort of 590 women referred to the National Cancer Institute in Rio de Janeiro, Brazil. Transcriptional activity of HPV73 was evaluated by detection of spliced transcripts of E6/E6* and E1^E4 in cDNA created from RNA isolated from fresh tissue. Disruption of viral E1 and E2 genes in the tumor DNA was assessed by overlapping PCR amplification. Evaluation of viral integration was performed using a customized capture panel and next‐generation sequencing, and an in‐house bioinformatic pipeline. HPV73 E6/E6* transcripts were found in 7/7 specimens with available RNA, and three also had HPV73 E1^E4 transcripts. Disruption of E1 and E2 genes was observed in 4/8 specimens. Integration of HPV73 sequences into the cancer cell genomes was identified in all cervix cancer tissues. These results provide evidence that HPV73 is an oncogenic virus that can cause invasive cervix cancer. With current molecular screening and HPV vaccination, not all cervix cancers will be prevented.

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.