Humoral Response to HPV16 Proteins in Persons with Anal High-Grade Squamous Intraepithelial Lesion or Anal Cancer

Human papillomavirus (HPV) is a ubiquitous sexually transmitted virus that infects more than 80% of sexually active persons by adulthood. HPV-associated cancers, however, develop in only a small proportion of infected persons (1, 2). Globally, 35,000 anal cancer cases per year are attributable to HPV, with highest frequency among women, men who have sex with men (MSM), and people living with human immunodeficiency virus (HIV) or immunosuppression, including solid organ transplant recipients (3). In the United States, the incidence of anal cancer has increased by 2.2% each year over the past 10 years, with an estimated 8,300 cases and 1,280 deaths in 2019 (4). Although 13 HPV types have been declared oncogenic in cervical cancer, HPV16 is responsible for most HPV attributable cancers including up to 84% of anal cancers (3, 5, 6).

Persistent oncogenic anal HPV infection and anal high-grade squamous intraepithelial lesions (HSIL) are precursors to anal cancer (2). However, the prevalence of anal HPV infection is common and thus, molecular detection of HPV in the anus has a poor positive predictive value for HSIL (7). Detection of anal HSIL requires high-resolution anoscopy (HRA) with biopsy of potential lesions, a labor intensive and uncomfortable procedure. Treatment of anal HSIL can be challenging, recurrences are frequent, and up to 30% may regress without treatment (8–10). As such, identification of serologic biomarkers for anal HSIL may help focus secondary prevention on persons at highest risk for anal cancer.

In natural infection, early (E1, E2, E4, E6, and E7) and late (L1 and L2) HPV genes are expressed during the virus life cycle (11–13). The early HPV proteins may reflect virus-driven epithelial cell division in productive infection, and if so, they may be important biomarkers. The most promising candidates, based on our understanding of etiology, are antibodies to HPV E6 and E7 oncoproteins, as they are associated with the tumor suppressors, p53 and pRb, and implicated in the induction of malignant epithelial transformation (14). In most cancers, the HPV viral genome becomes integrated, which results in dysregulation of E6 and E7 proteins and continued oncogenic progression (15). Thus, seropositivity to HPV16 E6 has been linked to the risk of HPV16- driven genital, cervical, and oropharyngeal cancers (16–18). Similarly, studies indicate that antibody to HPV16 E6 oncoprotein has been associated with increased risk of anal cancer, but HPV antibodies to early proteins are not helpful predictors of anal HSIL (19–22). Using samples from a well-characterized, population-based case–control study (6) conducted in Seattle, Washington, we aimed to gain insights into serologic predictors of anal HSIL and cancer.

The study and ethics board review were conducted at the Fred Hutchinson Cancer Research Center (Seattle, Washington). The epidemiologic results have been published previously, and the methods are briefly summarized here (6). Cases were women and men ages 18–74 years with histologically confirmed anal HSIL or cancer diagnosed between 1989 and 1998. HSIL was referred as carcinoma in situ during the time span of this study. The case participants were identified from the Cancer Surveillance System and resided in King, Pierce, and Snohomish counties in Washington. Of 485 cases ascertained by the cancer registry, 306 (63%) were interviewed, and of those, 183 participants (60%) donated blood for serologic evaluation at the time of posttreatment interview.

Controls were frequency matched to the age distribution of the cases in 5-year age intervals and assigned a reference year on the basis of the distribution of diagnosis years of cases for all anogenital HPV cancer sites by sex. Controls were selected from the same geographic area as cases using random digit dialing, separately for male and female controls (23, 24). Among controls, 1,700 (70%) agreed to be interviewed; of those, 1,524 (89%) provided blood samples, and a subset of sex-specific cases and controls were selected for inclusion in this study (n = 830).

Extensive in-person interviews were administered and serum was collected. The interview focused on demographic characteristics, sexual behavior, and smoking history. None of the participants were immunized against HPV.

Serum samples were tested for the presence of antibodies to HPV16 L1, E1, E2, E4, E6, and E7 at the German Cancer Research Center (Heidelberg, Germany) using a Luminex Multiplex Serology Assay (25). Briefly, HPV antigens were expressed in E. coli as fusion proteins with N-terminal glutathione S-transferase and affinity purified on glutathione-coated fluorescence beads. Subsequently, antigen-loaded beads were incubated with sera at dilution 1:100. Primary serum antibodies bound to the antigens were quantified in a Luminex 200 Flow Cytometer as median fluorescence intensity (MFI). Antibody levels were dichotomized as seropositive or seronegative on the basis of previously defined cutoffs in MFI: L1, 422; E1, 200; E2, 679; E4, 876; E6, 484; and E7, 548.

Paraffin-embedded tissue blocks from 139 cases were tested for HPV DNA using PCR-based methods as described previously (6, 26). Representative tumor blocks were selected from each tumor, and to deter contamination across blocks, fresh blades were used to cut each block. In addition, quality control measures included a noncancer tissue control (heart) specimen, cut after every 10th study specimen and included in HPV genotyping assays. The beta-globin gene was also amplified to ensure amplification.

We compared demographic characteristics by case–control status. We used logistic regression and calculated ORs with 95% confidence intervals (CI) to estimate the association between HPV antibodies and anal HSIL or cancer. Using a backward stepwise elimination approach with significance at P ≤ 0.05, we built multivariable logistic regression models considering potential confounders that were selected a priori. Final models were adjusted for gender, age (continuous variable), smoking status (never, former, and current smoking), and number of sex partners (1, 2–4, and ≥5 partners). In sensitivity analyses, we estimated the association between HPV antibody seropositivity and anal HSIL or cancer restricted to cases with detectable HPV16 DNA on tissue biopsy. Statistical analyses were performed in R (version 3.4.3).

The study included 67 participants with anal HSIL, 116 participants with anal cancer, and 830 controls. Among those with anal HSIL, most were men (54%) and among those with cancer, most were women (63%; Table 1). Men with HSIL were younger than women with HSIL (median 39 vs. 50 years, respectively). Most participants with HSIL were current smokers (61%) and reported five or more lifetime sex partners (54%). The median age of men and women with anal cancer was 53 and 59 years, respectively. As with HSIL, those with anal cancer tended to be current smokers (56%) and most (62%) reported five or more sex partners.

HPV16 DNA was detected in 101 of 139 (73%) of available HSIL (n = 36) or cancer (n = 65) tumor samples (Supplementary Table S1). In addition, non-HPV16 oncogenic types (HPV18, 31, and 33) were detected in 13 samples (9%). The median days of serum collection relative to the HSIL diagnosis were 356 and 338 among men and women, respectively. In contrast, the time of serum collection relative to cancer diagnosis was slightly shorter in men (280 days) and women (315 days; Supplementary Table S2).

A higher proportion of HSIL (64.2%) cases than controls (13.1%) were seropositive for L1 (Table 2). L1 and E4 seropositivities were associated with higher odds of having anal HSIL. After adjusting for potential confounders, significant associations between seropositivity to L1 and E4 antibodies and risk of HSIL remained. In addition, E6 seropositivity was significantly associated with HSIL after adjustment. In sensitivity analyses, we restricted the analysis to HSIL cases with HPV16 DNA detected in tumor tissue (n = 36); in this small subset, E4, E6, and L1 remained associated with anal HSIL (Supplementary Table S1).

A higher proportion of cancer (57.8%) cases than controls (13.1%) were seropositive for L1 (Table 2). In univariable analysis of factors associated with anal cancer versus controls, the risk of anal cancer was elevated for all HPV16 antibodies. Except for HPV 16 E7, associations between HPV16 seropositivity and anal cancer were magnified and remained significant following adjustment [L1: adjusted OR (aOR) = 12.5, 95% CI, 7.3–21.7; E1: aOR = 24.9, 95% CI, 10.3–59.9; E2: aOR = 6.3, 95% CI, 3.4–11.7; E4: aOR = 2.8, 95% CI, 1.6–4.8; and E6: aOR = 32.5, 95% CI, 14.2–74.4). When the model was restricted to anal cancer cases with confirmed HPV16 DNA in tissue, E1, E2, E4, E6, and L1 detection remained strongly associated with anal cancer (Supplementary Table S1).

This large, population-based case–control study provides important insights into HVP16 antibodies detection and risk of anal HSIL and cancer. We observed a significant association between seropositivity to HPV16 L1, E4, and E6 and anal HSIL. We also found strong associations between anal cancer and antibodies to HPV16 L1 and all early antibodies (E1, E2, E4, E6, and E7); the strongest association was observed for E6, due to low E6 seroprevalence in controls. Our findings confirm previously published studies (19, 20, 22).

Unlike most viral infections, virologically documented infection with HPV does not universally result in detectable serum antibodies. For example, in a cross-sectional study of 281 men and 93 women without history of cancer or HPV immunization in the United States, only 10% of women and 4% of men had detectable serum antibodies to HPV16 L1 using the same assay utilized for this study (27). This limits the usefulness of antibody assays to assess the frequency of current and past HPV infection; however, they may be a useful biomarker of tumorigenesis. Furthermore, the seroprevalence of natural HPV L1 antibody can differ by testing methodology, gender, sexual behavior, age, and site of the HPV disease. For instance, the HPV16 L1 seroprevalence using ELISA methods in the United States, prior to introduction of the HPV vaccine, ranged from 7.9% in men to 17.9% in women ages 12–59 years (28). In a younger cohort of Korean students ages 15–29 years, the HPV16 L1 prevalence was only 2.4% in men and 2.7% in women using multiplex HPV serology methods (17). Studies consistently have also shown a higher likelihood of HPV16 L1 antibody detection in women and MSM compared with heterosexual men, two groups at higher risk for HPV-associated malignancy (26, 29, 30). The mechanisms underlying these differences are not well understood. One potential explanation is that the humoral immune response to infection of the keratinized genital penile epithelium is lower compared with the cervix or the anal canal (31–35).

The associations between seropositivity to HPV16 antibodies and anal HSIL are somewhat inconsistent in different studies. For example, HPV16 L1 seropositivity among HIV-negative and HIV-positive gay and bisexual men from the longitudinal Australian Study of the Prevention of Anal Cancer was associated with nearly 10-fold increased risk of anal HSIL (29). The investigators also reported that an association with borderline significance was found between HPV16 E6 seropositivity and diagnosis of HSIL. In contrast, findings from the Dutch HIV and HPV in MSM Study (H2M) did not support the association between HPV16 L1 or E6 antibody detection and HSIL diagnosis (21). Using the same quantitative serology assays and cutoff values used in these studies and ours, we observed that the odds of having anal HSIL were 12 times higher in those seropositive for HPV16 L1. Furthermore, the odds of having anal HSIL were 5-fold increased in HPV16 E6- and 2-fold increased in HPV16 E4-seropositive individuals. Differences between our findings and the other studies could be explained by several limitations of this study, including use of posttreatment blood draws, undetected HIV coinfection, and a case–control rather than longitudinal study design. Furthermore, our HSIL population was diagnosed before HRA was widely used in Seattle, and men and women in this study with HSIL were either diagnosed because of symptoms, often with advanced disease, or diagnosed after perianal skin tag removal, hemorrhoidectomy, or incidental diagnosis during colonoscopy. Therefore, it is possible that we detected different associations than have been found in more consistently defined and evaluated cohorts. The association between HPV16 E6 antibody detection and anal HSIL should be further evaluated in a longitudinal study as we cannot elucidate whether the cases with anal HSIL and HPV16 E6 seropositivity could have developed anal cancer subsequently (22) or had undiagnosed genital or oropharyngeal HPV-associated cancer.

Among patients with documented HPV-driven anal cancer, several studies showed high prevalence for antibodies against HPV16 early proteins compared with controls (19, 20, 22). In our case–control study, we found an HPV16 E6 seroprevalence of 29% among persons with cancer compared with 1.4% in controls. Although HPV16 E6 antibody detection is not expected in those cases without HPV16 DNA detected in tumor, positive HPV16 E6 in such cases may be related to potential laboratory error, HSIL or cancer lesions yet to be diagnosed, or concomitant HPV16 infection at other anatomic sites. A recent study from the Swiss cohort reporting HPV16 antibody kinetics prior to anal cancer development in persons living with HIV showed an HPV16 E6 seroprevalence of 23.3% in samples obtained less than 2 years prior to cancer diagnosis. HPV16 E6 seroprevalence decreased with increased time prior to cancer (e.g., 16.7% at 2–4 years and 7.0% at 10 years or more; ref. 22). A case–control study nested in the European Prospective Investigation into Cancer and Nutrition cohort, reported HPV16 E6 seropositivity was 29.2% among persons who developed anal cancer compared with 0.6% in cancer-free controls (19). Among immunocompetent persons, antibody to HPV16 E6 protein was associated with a 75-fold increased future risk of anal cancer development and was detected a mean of 8 years before cancer diagnosis (19). Our study also found increased odds of anal cancer in persons with HPV16 E6 seropositivity compared with controls; this association was magnified when cancer cases were restricted to those with detectable HPV16 in tumor tissue. Although a clear association between HPV16 E6 antibodies and anal cancer has been demonstrated, the relatively low sensitivity of HPV serologic markers may preclude the use of antibody detection for population-level cancer prevention programs. It is possible, however, that there is a potential role for serology in high-risk populations.

Antibodies to HPV16 proteins have also been evaluated as potential biomarkers of HPV-driven cancer across genital and oropharyngeal sites. For instance, Combes and colleagues found a significant association between HPV16 L1 and early proteins and cervical cancer risk (16). In patients with oropharyngeal cancers, seropositivity to HPV16 E6 is a highly sensitive (95%) and specific (98%) marker for HPV-driven oropharyngeal cancer (36). Furthermore, the HPV Cancer Cohort Consortium suggested that HPV16 E6 seropositivity was associated with a 93-fold higher risk of HPV-associated oropharyngeal squamous cell carcinoma and the antibody was detected up to 28 years before the cancer diagnosis (18, 37, 38). Detection of antibodies to HPV16 could serve as a biomarker to facilitate the identification of patients that might benefit from a more intensive evaluation versus those that can be followed more conservatively.

A limitation of this study is that we were not able to take into account the risk of nonanal cancer, particularly oropharyngeal HPV-driven cancer among older men or cervical cancer among women. In addition, our analysis is limited by the study design, including unmeasured confounders not addressed in the analysis such as lack of ascertainment of HIV status or self-determined sexual identity (e.g., MSM and men who have sex with women). Although the HIV prevalence in the Seattle area was reported at less than 0.5%, lack of HIV status limits our ability to determine a difference in HPV16 antibody response among this expected subgroup within our cases, as it is possible that persons living with HIV, particularly MSM, were more likely to be evaluated and diagnosed with HSIL in Seattle during the period of the study (39). The study is also limited because of the unavailability of HPV DNA tests on some tumor tissues and lack of HPV mRNA testing in all tissue samples to identify HPV-driven cancer, which could affect the precision of the study estimates. Although blood samples were tested for HPV16 antibodies in a single batch to mitigate potential differences by storage time, we found that year of sample collection did not significantly alter the key associations. Finally, the participation rate for in-person interview in the original study was 63% among cases and 68% among controls. This could potentially introduce selection bias; however, it is unlikely that people with certain HPV16 antibody seropositivity will be more or less likely to participate in this study. Despite these limitations, this is one of the largest case–control studies to evaluate association of HPV16 antibodies to anal HSIL and cancer to date.

In summary, multiple diagnostic tools with various sensitivities and specificities have been evaluated for screening patients at risk for anal cancer, including anal cytology, HRA, high-risk HPV genotyping, HPV and host methylation, and HPVE6/E7 mRNA (40–45). Noninvasive screening tools are needed for detection and prognosis of anal HSIL, particularly in populations at higher risk of anal cancer. In our study, participants with anal HSIL or anal cancer had a distinct humoral immune response to multiple HPV16 antibodies. Additional research should focus on prospective replication of our findings in high-risk populations with the aim of identifying potential biomarkers and machine learning algorithms that could serve as less-invasive anal cancer screening tools in the future.

T. Waterboer reports serving on advisory boards for Merck Sharp & Dohme. A. Magaret reports grants from NIH during the conduct of the study. D.R. Doody reports grants from NIH during the conduct of the study. A. Wald reports personal fees and nonfinancial support from Merck (Data and Safety Monitoring Board participation) outside the submitted work. No potential conflicts of interest were disclosed by the other authors.

H.C. Stankiewicz Karita: Conceptualization, formal analysis, methodology, writing–original draft, writing–review and editing. T. Waterboer: Resources, supervision, methodology. A. Magaret: Conceptualization, formal analysis, supervision, methodology, writing–review and editing. D.R. Doody: Data curation, formal analysis, supervision, methodology, writing–original draft. M. Pawlita: Resources, supervision. N. Brenner: Supervision, writing–review and editing. D.A. Galloway: Resources, supervision. A. Wald: Conceptualization, formal analysis, supervision, methodology, writing–review and editing. M.M. Madeleine: Conceptualization, formal analysis, supervision, methodology, writing–review and editing.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.