Obstetrics & Gynecology:
Clinical Significance of Serum Anti-Human Papillomavirus 16 and 18 Antibodies in Cervical Neoplasia
Chay, Doo Byung MD, PhD; Cho, Hanbyoul MD; Kim, Bo Wook MD; Kang, Eun Suk MD, PhD; Song, Eunseop MD, PhD; Kim, Jae-Hoon MD, PhD
Departments of Obstetrics and Gynecology, Yongin Severance Hospital, Yonsei University College of Medicine, Yongin, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korean Cancer Center Hospital, Korean Institute of Radiological and Medical Sciences, Seoul, and Inha University Hospital, Inha University College of Medicine, Incheon, and the Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea.
Corresponding author: Jae-Hoon Kim, MD, PhD, Department of Obstetrics and Gynecology, Gangnam Severance Hospital, Yonsei University College of Medicine, 146-92 Dogok-Dong, Gangnam-Gu, Seoul 135-720, Korea; e-mail: firstname.lastname@example.org.
Supported by the intramural research fund (code#2008-E00306-00) from the Korea Centers for Disease Control and Prevention.
Presented at the 17th International Meeting of the European Society of Gynecologic Oncology, September 11–14, 2011, Milan, Italy (poster abstract), and at the Society of Gynecologic Oncology's 43rd Annual Meeting on Women's Cancer, March 24–27, 2012, Austin, Texas (poster abstract).
Financial Disclosure The authors did not report any potential conflicts of interest.
OBJECTIVE: To estimate the clinical significance of serum anti-human papillomavirus (HPV) antibodies and high-risk cervical HPV DNA in cervical neoplasia.
METHODS: The study population comprised patients who were histopathologically diagnosed with cervical intraepithelial neoplasia (CIN) 1 (n=64), CIN 2 and 3 (n=241), cervical cancer (n=170), and normal control participants (n=975). Cervical HPV DNA tests were performed through nucleic acid hybridization assay tests, and serum anti-HPV 16 and 18 antibodies were measured by competitive immunoassay. The associations of HPV DNA and anti-HPV antibodies were evaluated with demographic characteristics and compared according to the levels of disease severity. Anti-HPV antibodies were also investigated with clinicopathologic parameters, including survival data.
RESULTS: Among various demographic characteristics, factors involving sexual behavior had a higher tendency of HPV DNA positivity and HPV seropositivity. Human papillomavirus DNA mean titer and positivity were both increased in patients with cervical neoplasia compared with those with normal control participants, but there was no statistical difference among types of cervical neoplasia. Serum anti-HPV 16 antibodies were also able to differentiate cervical neoplasia from a normal control participant and furthermore distinguished CIN 1 from CIN 2 and 3 (odd ratio 2.87 [1.43–5.78], P=.002). In cervical cancer, HPV 16 seropositivity was associated with prolonged disease-free survival according to the univariable analysis (hazard ratio=0.12 [0.01–0.94], P=.044).
CONCLUSION: Serum anti-HPV 16 antibodies can distinguish cervical neoplasia from a normal control and has the advantage of identifying high-grade CIN. Moreover, in cervical cancer, HPV 16 seropositivity may be associated with a more favorable prognosis.
LEVEL OF EVIDENCE: II
Human papillomavirus (HPV) infection is a common sexually transmitted disease (STD) and more than 130 HPV genotypes have been identified. Risk factors associated with cervical cancer include persistent high-risk HPV infection, and approximately 70% of cervical cancers are associated with HPV types 16 and 18.1,2 Other risk factors such as multiparity, cigarette smoking, oral contraceptive use, and coinfection with another STD are known to be associated with cervical carcinogenesis.3,4 Furthermore, the probability of acquiring a HPV infection has been strongly related to the age of sexual debut and the number of associated sexual partners.5
A cervical HPV DNA test along with cytology has been clinically applied in the primary screening for the triage of equivocal cervical cytology results and in the follow-up of patients treated for high-grade cervical intraepithelial neoplasia (CIN).6,7 However, the HPV DNA test only shows that there are currently HPV antigens, whereas serum anti-HPV antibodies can provide information on cumulative exposure. Development of detectable antibodies does not always occur after natural HPV infection and antibody persistence may vary. Considering the natural history of HPV infection, spontaneous remission occurs in 30% of the patients within 3–6 months and approximately 54–69% of HPV DNA–positive women are seroconverted within 6–18 months.8–10
Previously, serologic studies on anti-HPV antibodies have focused mainly on developing optimal vaccination strategies for the general population and serologic studies against cervical neoplasia have been relatively less reported.11–14 However, in the area of postprophylactic vaccination, serologic assessments of anti-HPV antibodies in patients with cervical neoplasia may provide important basic data in the development of therapeutic vaccination.
This study estimates the clinical significance and prognostic value of serum anti-HPV antibodies and HPV DNA according to the levels of disease severity.
MATERIALS AND METHODS
The study participants comprised 975 normal control participants, 64 CIN 1, 241 CIN 2 and 3, and 170 patients with cervical cancer who visited Gangnam Severance Hospital from July 2002 to December 2010. Normal control participants were cytology-negative and HPV DNA-negative women as well as colposcopically confirmed normal for those women who were cytology-negative and HPV DNA-positive. All patients with cervical neoplasia were histologically confirmed and the cervical cancer was clinically staged according to the International Federation of Gynecology and Obstetrics (FIGO) staging system. Biological samples such as vaginal swab specimen and venous blood samples were collected from patients after informed consent at the time of the diagnosis before treatment. The study protocol was approved by the institutional review board of Gangnam Severance Hospital. Sociodemographic information, histopathologic features, and clinical data were obtained through a retrospective review of the medical records. Demographic characteristics involving cervical pathogenesis were evaluated in association with the serum anti-HPV 16 and 18 antibodies and cervical HPV DNA.
The cervical HPV DNA test was performed using the Hybrid Capture II assay. To confirm the presence of high-risk type HPV DNA (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68), cells from the uterine cervix were collected using a female swab specimen collection kit (Dacron swab). Cells were placed in a liquid hybridization assay collection kit and then preserved at –20°C for further analysis. The HPV DNA test was performed according to the manufacturer's instructions. The light reaction unit was measured using a luminometer. Solutions containing 10 pg HPV 16 DNA/milliliter served as positive controls for high-risk HPV. Relative light unit:cutoff ratios were calculated as the ratio of the specimen luminescence to the positive control luminescence. A positive cutoff value was set at 1 pg HPV DNA/milliliter in each specimen.
Blood samples were centrifuged at 1,500×g for 10 minutes for serum separation and were stored in prelabeled tubes at –70°C before processing. Frozen samples were shipped to the Merck and Co, Inc testing laboratory where samples were thawed and tested for specific neutralizing antibodies to high-risk HPV subtypes 16 and 18 using a competitive Luminex immunoassay. Procedures and analysis for antibodies against HPV types 16 and type18 virus-like particles were done as described by Dias et al in 2005. Seropositivity for HPV was defined as having anti-HPV antibody titers of at least 20 milli-Merck units/milliliter for HPV type 16 and of at least 24 milli-Merck units/milliliter for HPV type 18.15
Frequency distributions were compared using χ2 and Fisher's exact tests. Mantel-Haenszel method was used to test the association of disease severity and demographic characteristic.16 Unconditional multiple logistic regressions were used to calculate odds ratios (ORs) to estimate the associations between HPV DNA or serum anti-HPV antibodies and the disease severity or characteristics of cervical neoplasia. The mean cervical HPV DNA titer and serum anti-HPV antibody titer was analyzed using the analysis of variance test after log transformation of the variables and the P value was adjusted by means of the Bonferroni correction for multiple testing. Kaplan-Meier survival curves were compared using the log-rank test for disease-free survival and the Cox regression analysis was used to adjust for the potential confounding factors. Statistical analyses were performed using SPSS PASW Statistics 18.0. All statistical tests were two-sided and considered to be statistically significant at P<.05.
Demographic characteristics according to the severity of disease and association with HPV DNA positivity and HPV seropositivity are presented in Tables 1 and 2. The following factors were found to be associated with a significant increase in HPV DNA positivity: being single, sexual debut before the age of 20 years old, and having had more than five sexual partners. Women younger than 30 years of age have had a significant increase in HPV DNA positivity, more so than those within the age groups of 30–49 years and 50 years and older. The parity status revealed that there was a significant increase in HPV DNA positivity in nulliparous women. However, as parity increased, the tendency for HPV DNA positivity decreased. Regarding anti-HPV antibodies, both HPV 16 and HPV 18 seropositivity significantly increased in women who were single. A significant increase in HPV 18 seropositivity could also be found in women who had sexual debut before the age of 20 years and in women with a history of STD. Seropositivity to HPV 16 and HPV 18 depended on age and parity status. No significant difference was found in HPV DNA positivity and HPV seropositivity in association with smoking status and oral contraceptive use. Overall, factors involving sexual behavior contributed to a higher tendency for HPV DNA positivity and HPV seropositivity than other factors.
The mean HPV DNA titer was 0.37 relative light unit:cutoff ratio in normal control participant, 3.61 relative light unit:cutoff ratio in CIN 1, 4.10 relative light unit:cutoff ratio in CIN 2 and 3, 4.50 relative light unit:cutoff ratio in cervical cancer. Serum anti-HPV 16 and 18 antibody titers were 2.58/2.37 milli-Merck units/milliliter in normal control participants, 2.74/2.36 milli-Merck units/milliliter in CIN 1, 3.22/2.42 milli-Merck units/milliliter in CIN 2 and 3, and 3.18/2.38 milli-Merck units/milliliter in cervical cancer, respectively (Fig. 1). Human papillomavirus DNA titer was significantly increased in the histologically proven cases of cervical neoplasia in comparison with the normal control participant (P<.001) and although HPV DNA titer was increased in cervical cancer compared with CIN 1 (P=.049), there was no significant difference according to the severity of cervical neoplasia overall (Fig. 1A). Serum anti-HPV 16 antibody titer was significantly increased in CIN 2 and 3 and cervical cancer compared with those of CIN 1 and the normal control participants (P<.001), but there was no significant difference between CIN 1 and the normal control participants and between CIN 2 and 3 and cervical cancer. There was no significant difference in the serum anti-HPV 18 antibody titer according to the severity of disease (Fig. 1B). Cervical HPV positivity was 13.8% in the normal control participants, 84.3% in CIN 1, 92.3% in CIN 2 and 3, and 87.3% in cervical cancer. Seropositivity to HPV 16 and 18 was 8.9/3.3% in the normal control participants, 17.2/3.1% in CIN 1, 37.3/4.5% in CIN 2 and 3, and 28.2/2.4% in cervical cancer (data not shown). Human papillomavirus DNA positivity and HPV 16 seropositivity were significantly higher in the histologically proven cases of cervical neoplasia in comparison with the normal control participants (P<.001). Although HPV DNA positivity showed no significant difference in cervical neoplasia according to the severity of disease, HPV 16 seropositivity was significantly increased in CIN 2 and 3 compared with CIN 1 (OR 2.87 [1.43–5.78], P=.002). However, no significant differences between CIN 2 and 3 and cervical cancer were seen (Table 3).
Associations of serum anti-HPV 16 and 18 antibodies and prognostic parameters of cervical cancer are shown in Table 4. Patients with FIGO stage 1 cervical cancer had significantly higher HPV 16 seropositivity than those with FIGO stage 2 to 4 cervical cancer. Seropositivity to HPV 16 was also significantly higher in squamous cell carcinoma than those of other cell types. In the univariable analysis, prolonged disease-free survival was found to be significantly associated with seropositivity to HPV 16, advanced stage, tumor size, and lymph node status. However, according to the multivariable analysis, only advanced stage and lymph node status were significantly correlated with survival (Table 5). Kaplan-Meier survival estimates showed that seropositivity to HPV 16 was significantly associated with better disease-free survival (P=.017) (Fig. 2A) and seropositivity to HPV 18 showed a trend (P=.523, not significant) for better disease-free survival (Fig. 2B).
Among various research conducted on HPV infection, studies on serologic response have not delivered practical contribution to the clinicians. The reasons are an officially recognized serologic test remains unavailable because international standardization for HPV antigen has only been recently introduced and the fact that HPV infection does not often induce a systemic infection that leads to the production of antibodies detectable using serologic test. Such low immunity is the result of HPV infections restricted to the intraepithelial layer, absence of a bloodborne phase, lack of proinflammatory cytokine release, and antigen-presenting cells inactivation.17–19 As a result of the mentioned disadvantages, the serologic HPV assay has less been used compared with cervical HPV DNA detection, yet HPV DNA detection has its limitation in representing only the current infection because most of the HPV infection regresses and clears away naturally and current HPV infection does not always imply progression into cervical lesions. On the other hand, serum anti-HPV antibodies, which include cumulative HPV exposures and immune clearance, have the advantage of providing more comprehensive information on HPV infection.3,8–10
This study investigates the association of clinical aspects involved in the sequential step of cervical carcinogenesis with cervical HPV DNA and serum anti-HPV 16 and 18 antibodies, respectively. Foremost, we evaluated the relationship between HPV infection status and previously known demographic characteristics involved in cervical carcinogenesis. Similar to previous studies, HPV DNA positivity and HPV seropositivity were related to younger age, single marital status, a higher number of lifetime partners, an early sexual debut, and a history of STD,20,21 yet, on the contrary to past findings, the seropositivity decreased as the number of full-term deliveries increased in this study, and this can be explained by the recent changes in the Korean sexual culture wherein younger women are preferring liberalism and either nulliparity or single parity.9,22,23
In addition, although HPV DNA can only distinguish cervical neoplasia from normal cytology, serum anti-HPV 16 antibodies were capable of further distinguishing CIN 1 from CIN 2 and 3. Distinguishing CIN 1 from CIN 2 and 3 is important because, although CIN 1 pathologically undergoes viral replication with no cell proliferation and shows a high spontaneous regression, CIN 2 and 3 undergoes cell proliferation and thus clinically requires surgical intervention as a result of the risk of progression.3,10,24 The results of anti-HPV 16 antibodies in this study suggest the possibility of using serologic HPV assay as an adjuvant tool in identifying women at risk of developing a high-grade CIN.
According to our findings on invasive cervical cancer, HPV 16 seropositivity was associated with squamous cell carcinoma as expected because more than 70% of the squamous cell carcinoma worldwide is contributed by the HPV type 16.3,10,25 In addition, HPV 16 seropositivity was higher in FIGO stage 1 than in FIGO stage 2–4. Considering that the HPV 16 seropositivity was also high in CIN 2 and 3 compared with CIN 1 and together with the aforementioned result, HPV may be participating in the early stages of carcinogenesis.
Survival analysis revealed that seropositivity to HPV 16 was associated with prolonged disease-free survival in the univariable analysis, and this may suggest a potential association of anti-HPV antibodies with prognosis. Explanations on such possible association between HPV 16 seropositivity and prognosis have been suggested in several previous studies. Skiba et al reported that HPV 16 seropositivity was correlated with prolonged disease-free and overall survival, particularly in the patients with early FIGO stage 1 and 2 according to the univariable analysis. They state that seronegative patients exhibited a decrease in HPV-specific immune competence, allowing the virus to escape the immune system and consequently reducing the immunosuppression of HPV-induced tumor.26 Moreover, Heim et al previously reported that HPV capsid protein antibodies can indicate malignant lesions with persistent episomal HPV DNA and that these lesions with episomal forms are found to be associated with better disease-free survival than those with only integrated DNA. This is because malignant transformation accompanied by the expressions of oncoprotein is increased when episomal HPV DNA is integrated into the chromosomal DNA. Thus, remaining episomal HPV DNA in lesions implied that both the HPV DNA integration and malignant transformation are yet to be complete. Our findings together with previous studies suggest that cancer progresses more rapidly as a result of either the evasion of immunosurveillance or the decrease in the patient's immunocompetence in advanced stages.27
There are several limitations in this study. First is its cross-sectional nature representing a single time point during the disease process. Another is the hybrid capture II test that detects 13 high-risk types of HPV at a time and provides an overall status with a lack of HPV DNA type-specific information. The sensitivity of the hybrid capture II tests in detecting lesions of CIN 2 or more has been reported with a wide variation (66–100%) with the overall sensitivity of 90%.28,29 This could be the reason for the 87.3% HPV DNA positivity among cervical cancer in this study. Furthermore, there is limitation for the sample size of recurrence in this study causing low statistical power. The event rate was 12.9% in this study, and an event rate of 40% is needed to establish an adequate statistical power.30
In conclusion, among various demographic characteristics, sexual behavior factors were associated with higher tendency of cervical and serologic HPV positivity. Our results revealed that although HPV DNA is limited in distinguishing cervical neoplasia from normal control participants, serologic HPV assay using anti-HPV 16 antibodies is capable of distinguishing CIN 1 from CIN 2 and 3. Furthermore, our results show that serum anti-HPV 16 antibodies may also have the possibility of presenting a favorable prognostic value in cervical cancer.
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