II. MATERIALS AND METHODS
4. Statistical analysis
Comparisons of frequency distributions were analyzed using Chi-squared and Fisher's exact tests. Mantel-Haenszel method was used to analyze ordinal data.
Unconditional multiple logistic regression was used to calculate odds ratios (OR) and corresponding 95% confidence intervals (CIs) to assess the associations between cervical HPV DNA status, serologic HPV 16/18 antibody status and the disease severity of cervical neoplasia or selected characteristics. The mean cervical HPV DNA titer and serum anti-HPV16/18 antibody titer was analyzed using ANOVA test after log-transformation of the variables and Bonferroni corrections were used to adjust for multiple comparisons. As measures of prognostic outcome for cervical cancer, Kaplan-Meier survival curves were compared using the log-rank test for disease-free survival and the multivariate Cox proportional hazards model was used to adjust for potential confounding factors.
Statistical analyses were performed using the SPSS PASW Statistics 18.0 (SPSS Inc, Chicago, IL, USA). All statistical tests were two-sided and considered to be statistically significant at P < 0.05.
9 III. RESULTS
1. Evaluation of cervical HPV infection and serologic HPV detection with epidemiologic risk factors.
Epidemiological characteristics according to disease severity and the association with cervical HPV DNA and serum anti-HPV 16/18 antibody status are presented in Table 1 and 2. There was a significant increase of cervical HPV DNA positivity in women who were single, in women who had first sexual intercourse at age before 20 years old and in women who had more than 5 sexual partners. Age factor revealed a significant increase of cervical HPV positivity under 30 years old than among age 30 to 49 years old and also showed an increasing tendency than age over 50 years old. Parity status revealed a significant increase of cervical HPV positivity in nulliparous women and then showed a decreasing tendency with increased parity. Considering the serum anti-HPV 16/18 antibody status, women who were single had significantly increased seropositivity to both HPV 16 and 18. Seropositivity to HPV 18 was significantly increased in women who had first sexual intercourse at age before 20 years and in women with a previous history of STD.
Seropositivity to HPV 16 and 18 both showed a decreasing tendency to age factor and parity status. There were no significant difference in cervical HPV positivity and HPV 16/18 seropositivity associated with smoking status and oral contraceptive uses. Overall, the results involving sexual behavior of which younger women, with more lifetime partners, with an early sexual debut, who were single and who had a history of STD had an increasing risk of cervical HPV positivity and HPV seropositivity.
10
Table 1. Epidemiologic characteristics according to disease severity
Characteristics Normal
Age at first sexual intercourse 0.001
< 20 74 (9.1) 1(7.1) 32(27.1) 11 (10.5)
> 20 737 (90.9) 13(92.9) 86(72.9) 94 (89.5)
Total 811 (100) 14(100) 118(100) 105 (100)
Lifetime number of sexual partners 0.577
< 5 781 (96.7) 19(100) 84(97.7) 34 (97.1)
Oral contraception use < 0.001
No 729 (85.9) 31(100) 100(95.2) 117 (99.2)
Yes 120 (41.1) 0(0) 5(4.8) 1 (0.8)
Total 849 (100) 31(100) 105(100) 118 (100)
Parity (Numbers of delivery) < 0.001
None 211 (27.4) 8(13.1) 77(33.2) 14 (8.3)
Data are presented as number (%).
11
Table 2. Comparison of epidemiologic risk factors with cervical HPV infection and serologic HPV detection
Age at first sexual intercourse
< 20 40.6 1 20.3 1 9.3 1
Lifetime number of sexual partners
< 5 21.2 1 13.1 1 3.2 1
12
2. Comparison of cervical HPV infection and serologic HPV detection according to disease severity
The mean cervical HPV DNA titers and serum anti-HPV 16/18 antibody titers were analyzed according to disease severity and are presented in Table 3. Cervical HPV DNA titer were significantly increased in patients with cervical lesions compared to normal cytology (P <0.001) and although cervical HPV DNA titer was increased in cervical cancer compared to CIN 1 (P = 0.049), overall there was no significant difference according to the severity of cervical neoplasia (Figure 1A). Serum anti-HPV 16 antibody titer was significantly increased in CIN 2/3 and cervical cancer compared with CIN 1 and normal cytology (P <0.001), but there was no significant difference between CIN 1 and normal cytology and between CIN 2/3 and cervical cancer. There was no significant difference in the serum anti-HPV 18 antibody titer according to the disease severity (Figure 1B).
Table 3. Cervical HPV DNA titer and serum anti-HPV 16/18 antibody titer according to disease severity Variables were analysed after log-transformation.
Std, Standard deviation.
13
Figure 1. Comparison of HPV titers according to disease severity. A) Cervical HPV DNA titer. B) Serum anti-HPV 16/18 antibody titer. Bar represents the mean titer and standard error are presented as error bars (I). Bonferroni corrections were used to adjust for multiple comparisons. NS, not significant. Ab, antibody.
14
Cervical HPV positivity and HPV 16/18 seropositivity in women with normal cytology, patients with CIN 1, CIN 2/3 and cervical cancer are presented in Table 4. Cervical HPV positivity was 13.8% in normal cytology, 84.3% in CIN 1, 92.3% in CIN 2/3 and 87.3% in cervical cancer. Seropositivity to HPV 16 was 8.9% in normal cytology, 17.2% in CIN 1, 37.3% in CIN 2/3 and 28.2% in cervical cancer. While Cervical HPV positivity and seropositivity to HPV 16 were significantly different among cervical neoplasia (P <0.001) there was no significant difference with seropositivity to HPV 18 (P = 0.658).
Table 4. Cervical HPV positivity and HPV 16/18 seropositivity according to disease severity
Characteristics Normal
cytology CIN 1 CIN 2/3 Cervical
cancer P value
HPV DNA < 0.001
Negative 785 (86.2) 8(15.7) 13(7.7) 10 (12.7)
Positive 126 (13.8) 43(84.3) 155(92.3) 69 (87.3)
Total 911 (100) 51(100) 168(100) 79 (100)
HPV 16 Ab < 0.001
Negative 888 (91.1) 53(82.8) 151(62.7) 122 (71.8)
Positive 87 (8.9) 11(17.2) 90(37.3) 48 (28.2)
Total 975 (100) 64(100) 241(100) 170 (100)
HPV18 Ab 0.658
Negative 943 (96.7) 62(96.9) 230(95.5) 166 (97.6)
Positive 32 (3.3) 2(3.1) 11(4.5) 4 (2.4)
Total 975 (100) 64(100) 241(100) 170 (100)
Data are presented as number (%).
Cut off value : HPV 16 Ab ≥ 20 mMU/ml , HPV 18 Ab ≥ 24 mMU/ml.
15
Table 5. Comparison of cervical HPV positivity and HPV 16/18 seropositivity according to disease severity
In the analysis according to disease severity, cervical HPV positivity and HPV 16 seropositivity was significantly higher in the presence of cervical lesions compared to normal cytology (P <0.001). Cervical HPV positivity showed no significance between the severity of cervical neoplasia, whereas HPV 16 seropositivity was significantly increased in CIN 2/3 compared with CIN 1 (P = 0.002) but showed no significant differences between CIN 2/3 and cervical cancer (Table 5). The concordance between cervical HPV positivity and seropositivity to HPV 16 was 11.9% in normal cytology, 16.3% in CIN 1, 35.5% in CIN 2/3 and 57.5% in cervical cancer. For seropositivity to HPV 18, the concordance with cervical HPV positivity was 4.0% in normal cytology, 4.6% in CIN 1, 4.5% in CIN 2/3 and 2.9% in cervical cancer (Table 6).
16
Table 6. Concordance of serologic HPV detection with cervical HPV infection according to disease severity
Characteristics Neutralizing antibody
HPV 16 Ab - HPV 16 Ab + P-value HPV 18 Ab -
HPV 18 Ab + P-value
Normal cytology 0.236 0.190
HPV DNA – 719 (91.6) 66 (8.4) 761 (96.9) 24(3.1)
Cervical cancer 0.233 1.000
HPV DNA – 9 (90.0) 1 (10.0) 10 (100.0) 0 (0.0)
HPV DNA + 50 (72.5) 19 (57.5) 67 (97.1) 2 (2.9)
Data are presented as number (%). P values were analyzed by Fisher’s test.
3. Evaluation of serologic HPV detection as a prognostic parameter in cervical cancer patients
Correlation of serologic HPV detection and prognostic parameters of cervical cancer are shown in Table 7. Patients with Stage 1 cervical cancer had significantly higher HPV 16 seropositivity compared to stage 2 to 4 cervical cancer patient. Seropositivity to HPV 16 was also significantly higher in Squamous Cell Carcinoma (SCC) type compared to other cell types.
17
Table 7. Correlation of serologic HPV detection and prognostic parameters in cervical cancer
Characteristics HPV 16 Ab - HPV 16 Ab + P-value HPV 18 Ab - HPV 18 Ab + P-value
Age 0.098 0.958
<30 2 (66.7) 1 (33.3) 3 (100) 0 (0)
30-49 57 (64.8) 31 (35.5) 86 (97.7) 2 (2.3)
≥ 50 63 (79.7) 16 (20.3) 77 (97.5) 2 (2.5)
Stage 0.041 1.000
1 65 (65.7) 34 (34.3) 96 (97.0) 3 (3.0)
2-4 46 (82.1) 10 (17.9) 55 (98.2) 1 (1.8)
Grade 0.565 1.000
1-2 35 (74.5) 12 (25.5) 45 (95.7) 2 (4.3)
3 14 (66.7) 7 (33.3) 20 (85.2) 1 (4.8)
Cell type 0.046 1.000
SCC type 95 (68.3) 44 (31.7) 135 (97.1) 4 (2.9)
Others 27 (87.1) 4 (12.9) 31 (100) 0 (0)
Tumor size 0.769 0.575
< 4 cm 80 (71.4) 32 (28.6) 108 (96.4) 4 (3.6)
≥ 4 cm 31 (73.8) 11 (26.2) 42 (100) 0 (0)
LN mets 0.300 1.000
Negative 66 (68.8) 30 (31.3) 93 (96.9) 3 (3.1)
Positive 18 (81.8) 4 (22) 2 (100) 0 (0)
Data are presented as number (%).
Cut off value: HPV 16 Ab > 20 mMU/ml, HPV 18 Ab > 24 mMU/ml.
In the univariate survival analysis, seropositivity to HPV 16, FIGO stage, tumor size and lymph-node status were found to be significantly associated with
18
longer disease-free survival. However among the significant parameters, only advanced stage 2 or more patients and lymph node metastasis status were found to be significantly correlated with survival and other factors showed no significant differences in the multivariate analysis (Table 8). Kaplan-Meier survival estimates revealed that seropositivity to HPV 16 was significantly associated with better disease-specific survival (P = .017) (Figure 2A) and seropositivity to HPV 18 showed a trend (P = .523, not significant) for better disease-free survival (Figure 2B).
Table 8. Multivariate cox proportional hazards analysis on disease free survival of cervical cancer
Disease-Free Survival
Univariate Multivariate
Age NS
HPV 16 Ab positive 0.12 [0.01-0.94], 0.044 NS
HPV 18 Ab positive NS
Stage
1 1 1
2-4 10.02 [3.37-29.81], < 0.001 8.14 [2.01-32.94], 0.003 Grade
1-2 1
3 NS
Cell type NS
Tumor size (> 4 cm) 2.98 [1.26-7.04], 0.013 NS
LN mets 6.25 [2.09-18.69], 0.001 3.63 [1.14-11.52],0.029
NS, not significant.
Cut off value: HPV 16 Ab ≥ 20 mMU/ml , HPV 18 Ab ≥ 24 mMU/ml.
19
Figure 2. Kaplan-Meier analysis of disease free survival. A) Disease free survival according to HPV 16 seropositivity. B) Disease free survival according to HPV 18.
20 IV. DISCUSSION
This study investigated serum anti-HPV 16/18 antibody and cervical HPV DNA status in women with cervical neoplasia, revealing the association according to
the disease severity,
with epidemiological risk factors in cervical carcinogenesis and with prognostic factors of cervical cancer. The overall findings in this study showed that cervical HPV DNA indicated current infection status and distinguished cervical neoplasia with normal cytology, while serum antibodies to HPV 16/18, revealing cumulative status of sustained HPV exposure including past infections, distinguished CIN 1 with advanced cervical neoplasia (more than CIN 2). The
findings were more positively associated with seropositivity to HPV 16 and this may be due to the lower power for the HPV-18 analysis since the seropositivity to HPV 18 was low in patients with CIN 1 (3.1%), CIN 2/3 (4.5%) and cervical cancer (2.4%) and the mean titers were low compared to HPV 16. The limitations of this study was mainly due to the cross sectional nature representing a single time point during the disease process and a lack of cervical HPV DNA type-specific information since HCII test detects 13 high-risk types of HPV at a time and provides the overall current status of HR-HPV infection.Regarding the association with epidemiological risk factors in cervical carcinogenesis the results involving sexual behavior of which younger women, with more lifetime partners, with an early sexual debut, who were single and who had a history of STD had an increasing risk of cervical HPV DNA positivity and HPV16/18 seropositivity. On the other hand the results on parity, which has been recognized as a risk factor for cervical carcinogenesis decreased with the number of full term delivery in this study. Hormonal influences and long maintenance of the transformation zone on the exocervix is suggested for the explanation of the risk in multiparity, and the lack of this influence in this study can be presumed, but still a feasible explanation is needed for the
21 decreased risk with parity.22,23
The main interest of this study was the evaluation of serum anti-HPV 16/18 antibody and cervical HPV DNA status according to disease severity since it has been reported in many studies that there was a strong association between HPV infection and advanced cervical neopasia.24-26 Persistent detection of cervical HPV DNA was an evident predictor of cervical neoplasia, and serum HPV antibodies were associated with persistent DNA detection indicating life time exposure to HPV.27,28 In accordance with the findings previously reported, cervical HPV positivity was 13.8% in normal cytology, 84.3% in CIN 1, 92.3%
in CIN 2/3 and 87.3% in cervical cancer.29-31 Cervical HPV DNA titer were significantly increased in patients with cervical neoplasia compared to normal cytology (P <0.001). Seropositivity to HPV 16 was higher in patients with CIN 1 (17.2%), CIN 2/3 (37.3%) and cervical cancer patients (28.2%) compared with normal cytology (8.9%). The trend for a high percentage of HPV16 seropositivity with advanced cervical neoplasia suggests that HPV antibodies may correlate with the inability of a patient to clear their HPV infection. Thus, continued exposure to HPV increases the likelihood of seroconversion.32,33 The findings that HPV 16 seropositivity were significantly higher with CIN 2/3 than CIN 1 and normal cytology, reveals a strong association between advanced cervical neoplasia. Also serum anti-HPV 16 antibody titers which were significantly higher with CIN 2/3 and cervical cancer compared with CIN 1 and normal cytology supports the hypothesis that serum HPV antibodies correlates with the presence of advanced cervical neoplasia and suggest that serum HPV antibody assay may be useful as an additional tool in combination with cervical HPV DNA testing for the identification of women at risk for advanced cervical neoplasia. The concordance of cervical HPV positivity and HPV 16 seropositivity was 11.9% in normal cytology, 16.3% in CIN 1, 35.5% in CIN 2/3 and 57.5% in cervical cancer but there showed no statistical significance (P
> 0.05, Fisher’s test). Concordance can be effected by several factors such as
22
lag time required for seroconversion, waning of detectable antibodies, and variable persistence of type-specific antibodies especially in women with normal cytology. Interestingly the discordance showed a tendency to decrease with disease severity and this may be suggestive, but was not statistically significant, support for the utilization of adjuvant serological assays in detecting advanced cervical lesions.
Since positive antibody responses indicates a prolonged exposure to replicating virus and antibodies to HPV16/18 are a highly type-specific markers for HPV infections associated with developing cervical cancer, the correlation of serum anti-HPV 16/18 antibody with prognostic parameters and survival were investigated in patients with cervical cancer. Among the prognostic parameters, squamous cell carcinoma (SCC) cell type and early FIGO stage1 were significantly related with higher HPV 16 seropositivity and detection of antibodies to HPV-16 was associated with a better disease-free survival in patients with cervical cancer in this study. Higher HPV 16 seropositivity in SCC cell type can be explained since HPV 16 contributes to more than 70% of squamous cell carcinoma worldwide, but the association in early FIGO stage1 and with a better survival prognosis is unclear.34 In previous reported studies, Heim et al. have proposed an explanation of the observed correlation between prognosis and HPV serostatus.35 Within benign HPV lesions, the HPV genome is episomal, whereas in malignant lesions HPV DNA integration into the host chromosome is frequently observed. In advanced CIN, a subset of lesions can be identified in which the viral genome has integrated and there is a greater risk of malignant progression.36 In invasive cancer, HPV capsid protein expression is rare, but persistent episomal HPV DNA was detected in 38% of invasive cervical carcinomas. Interestingly, tumors containing episomal DNA were associated with longer survival than tumors with only integrated DNA. The presence of antibodies directed against capsid proteins could therefore indicate the persistence of episomal HPV DNA and more prolonged expression of late
23
genes. In tumors with a lower rate of integration, therefore, the viral genome must remain extra chromosomally which would provide a mechanism for expression of the late viral genes.37,38 Thus, a relationship between the presence of capsid antibodies and the time elapsing before integration of HPV DNA in different subsets of tumors may explain the association between HPV serology and prognosis. Another similar study involving 150 cervical cancer patients by Skiba et al. reported that anti-HPV16 seropositivity correlated with prolonged, progression-free and overall survival in the FIGO stage 1 and 2 patients. They hypothesized that the seronegative patients had reduced HPV-specific immune competence, so that the virus might have been able to escape both the humoral and the cellular defense mechanisms, resulting in an impaired ability of the immune system to control the HPV-induced tumor.39,40 The results in this study also revealed a correlation with HPV 16 seropositivity and a better disease free survival supporting the above hypothesis.
V. CONCLUSION
The result of this study reveals the features of serum anti-HPV 16/18 antibody and cervical HPV DNA in women with cervical neoplasia. Among the epidemiologic correlates, sexual behavioral factors were more at risk with both cervical and serological HPV positivity. While serum anti-HPV 18 antibody demonstrated no significant difference according to disease severity, cervical HPV DNA detection and serum anti-HPV 16 antibody detection maybe useful in cervical screening as an adjuvant test revealing cervical neoplasia. Although the low prevalence of serum anti-HPV 16 antibody suggest insufficient provocation of immunogenic response in cervical neoplasia, serologic detection of anti-HPV 16 antibodies has the advantage of representing a more advanced cervical neoplasia (more than CIN 2) and also may have the possibility for a favorable prognostic value in cervical cancer.
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