Although there is good evidence that taking more biopsies improves the detection of cervical intraepithelial neoplasia (CIN) 2 and CIN 3,1,2 the advantages of increased sensitivity must be weighed against potential adverse effects. One possible consequence of multiple biopsies is the increased risk of acquiring new human papillomavirus (HPV) infections. Microtrauma to the epithelium is required for infectious HPV virions to reach the target epithelial cells in the basal layer.3 Cervical biopsies disrupt the epithelium and therefore potentially expose the basal layer to infectious HPV virions. Because multiple biopsies lead to a more extensive disruption of the cervical epithelium, it is conceivable that there might be a higher risk of subsequent infection with HPV, the causal factor for cancer, and its immediate precursor lesions, CIN 3 and CIN 2.4
To estimate whether multiple biopsies increased the risk of acquiring new HPV infections, we used data from the Atypical Squamous Cells of Undetermined Significance (ASCUS) and Low-Grade Squamous Intraepithelial Lesion (LSIL) Triage Study (ALTS). Clinicians were asked to take biopsies when colposcopic lesions were visible; in the majority of women, a single biopsy was taken, but in some according to clinician preference, two or more biopsies were obtained. For this analysis, we included women who had colposcopy and were not found to have CIN 2 or a more severe diagnosis at enrollment. The purpose of this analysis was to examine the effect of the number of biopsies taken at enrollment with incident HPV infections detected at the next 6-month ALTS visit.
MATERIALS AND METHODS
The ALTS was a multi-site, randomized trial comparing three management strategies for women referred for ASCUS (n=3,488) or LSIL (n=1,572) conventional cytology.5–9 We refer readers to other references for details on randomization, examination procedures, patient management, laboratory and pathology methods, and treatment protocols.5–9 The National Cancer Institute and local institutional review boards approved the study, and all participants provided written, informed consent.
Residual PreservCyt (Hologic, Bedford, MA) specimens, after being used for liquid-based cytology, were tested by Hybrid Capture 2 (hc2; Qiagen, Gaithersburg, MD), a pooled probe, signal amplification DNA test that targets a group of 13 high-risk HPV genotypes.9
Cervical specimens in spectrum transport medium (STM; Qiagen, Valencia, CA) were retrospectively tested for at least 27 HPV genotypes, and 38 HPV genotypes for most specimens, using an L1-based polymeraze chain reaction assay that uses a primer set designated PGMY09/11 on the spectrum transport medium specimen as previously described.10–12 For this analysis, the same HPV genotypes detected by hc2 were considered high-risk HPV.13,14
We restricted our analysis to women who 1) had undergone colposcopy but were not diagnosed and treated by loop electrosurgical excision procedure for CIN 2 or more severe at enrollment and 2) with test results only from the laboratory that tested for 38 HPV genotypes (approximately half of the enrollment specimens and all the follow-up specimens) (n=988) for reasons as previously described.15
We evaluated the impact of the number of biopsies taken at enrollment, categorized as zero, one, or two or more, on the subsequent acquisition of HPV. We compared the clinical and epidemiologic data on women who underwent different numbers of biopsies using Kruskal Wallis to test for statistically significant differences (P<.05) for continuous variables and Pearson χ2 for categorical variables. Our analysis considered each woman’s possible acquisition of each of 38 HPV genotypes under the assumption that the natural history of HPV genotypes is independent of whether other HPV genotypes are present.15–17 We excluded the observation for presence or absence during follow-up of HPV genotypes that were positive at enrollment because a subsequent positive could be considered a persisting infection, not a newly acquired infection.
We considered the crude, average 6-month acquisition of categories of HPV genotypes: all HPV genotypes and high-risk HPV genotypes. For each category of HPV, we calculated the average 6-month acquisition for all women, for women who tested HPV positive (with another type) at baseline, and for women who tested HPV negative (for all types) at baseline. Because each woman could acquire more than one HPV genotype, we used generalized estimating equations18 to test for trends (Wald test) for HPV acquisition across biopsy categories. We repeated the analysis stratified on baseline hc2 test results (n=898, 91%; 598 hc2 positive and 300 hc2 negative) to ensure that our findings were clinically relevant, since hc2, a U.S. Food and Drug Administration–approved test, is used in cervical cancer screening. A weighted stratified analysis approach was also used15; however, as there were only negligible differences between the crude and weighted approaches (data not shown), only the crude 6-month incidence is presented.
Finally, logistic regression using generalized estimating equations was used to calculate the odds ratio (OR) and 95% confidence interval (CI) as a measure of association for the acquisition of HPV with biopsy category. The model was adjusted for whether the women reported having a new sexual partner(s) in the 6-month follow-up, baseline HPV status (negative, positive), age group (18–22, 23–29, and 30–75 years old with few older than 40). Study arm, study center, and race were not associated with HPV acquisition in the model and therefore were not included (data not shown).
The median time of the colposcopic evaluation after enrollment among women who did not undergo immediate colposcopy (ie, those in the HPV and conservative management randomization arms) was 56 days for woman with zero biopsies, 49 days for 1 biopsy, and 52 days for two or more biopsies taken at colposcopy after enrollment (P=.2, Kruskal Wallis). The median time of the 6-month follow-up visit after enrollment was 188 days for woman with zero biopsies, 185 days for woman with one biopsy, and 189 days for woman with two or more biopsies taken at colposcopy after enrollment (P=.2, Kruskal Wallis).
Table 1 describes the study population and the subpopulations of women with zero, one, and two biopsies taken at colposcopy after enrollment. Not surprisingly, despite exclusion of women with diagnoses at enrollment of CIN 2 or more severe, the number of biopsies taken was related to risk factors for CIN 2 or more severe: 1) more likely to test positive for the highest risk HPV types (P<.001); 2) more likely to have worse cytology (P<.001); and 3) more likely to have colposcopic impression of high grade (P<.001).
The 6-month HPV acquisition is shown in Table 2. There was no statistical difference (Ptrend>.05) by number of biopsies taken at enrollment in the 6-month HPV acquisition for the average acquisition of any HPV genotype and any high-risk HPV genotype (or any non–high-risk HPV, data not shown) for all women, enrollment HPV-positive women, and enrollment HPV-negative women. There was no observed effect of the number of biopsies on 6-month HPV acquisition by age group (18–22, 23–29, and 30–75 years), although not surprisingly, older women were less likely to acquire new infections than younger women. Stratification using baseline hc2 results yielded similar findings (data not shown).
Although the trend for number of biopsies and 6-month acquisition was not significant, HPV-negative women who had two or more biopsies taken (2.65%) were more likely to acquire high risk HPV than enrollment HPV-negative women who had zero biopsies taken (1.32%, P=.06) and one biopsy taken (1.31%, P=.04). Notably, the 6-month acquisition of high-risk HPV in HPV-negative women who had two or more biopsies taken was similar to the HPV-positive women with zero (2.65%), one (2.22%), or two or more (2.59%) biopsies taken.
Using a logistic regression model that adjusted for the baseline HPV status (Table 3), number of biopsies was unrelated to the risk of acquiring HPV: zero biopsies (OR 1.0, 95% CI 0.84–1.4) and two or more biopsies (OR 1.0, 95% CI 0.75–1.3) (compared with one biopsy). Acquisition of HPV was associated with having a new sexual partner(s) during follow-up (OR 1.8, 95% CI 1.3–2.4). Acquisition of HPV was negatively associated with being aged 30–75 years at enrollment (compared with 18–22 years) (OR 0.47, 95% CI 0.35–0.63).
The risk of developing CIN 3 over the next 2 years in this subgroup did not differ by number of biopsies taken at enrollment (P=.3):4.5% for zero biopsies, 7.4% for one biopsy, and 6.7% for two or more biopsies. The likelihood of 6-month clearance of HPV genotypes detected at enrollment did not differ by number of biopsies taken at enrollment (P=.3): 70.8% for zero biopsies, 66.9% for one biopsy, and 68.8% for two or more biopsies.
In our analysis of number of biopsies and the acquisition of HPV, we acknowledge that there was an unavoidable bias in the ALTS design that also influenced how many biopsies were taken. Women who were in the immediate colposcopy arm returned for their enrollment visit sooner than women enrolled in the HPV triage and conservative management arm. Therefore, the colposcopists were aware that some women were in the immediate colposcopy arm. Despite the fact that at the time colposcopy was considered the gold standard for disease detection, the colposcopists took more biopsies when there was cause, eg, testing HPV positive (HPV arm) or having high-grade squamous intraepithelial lesion cytology (HPV or conservative management arm) (Table 1). Notably, HPV status was unrelated to the number of biopsies taken at enrollment after controlling for study arm and colposcopic impression (data not shown), and HPV status (and number of biopsies) was unrelated to whether women acquired a new infection after controlling for whether or not women had a new sexual partner during the 6-month follow-up.
We recognize two other limitations for this analysis. First, we had insufficient sample size to evaluate the acquisition of individual HPV genotypes. It is plausible, although unlikely, that the impact of multiple biopsies is differential by HPV genotype. Second, women were referred into ALTS for an ASCUS or LSIL Pap test. Therefore, these results may not be generalized to women with high-grade squamous intraepithelial lesion and other rare, more severe cytologic interpretations, although it is unclear how the severity of the lesion might influence HPV acquisition after biopsy.
We emphasize that ALTS was not designed specifically to address this hypothesis. We therefore conducted a simple post hoc calculation of power for the crude 6-month acquisition (uncorrected for the contribution of multi-HPV genotype acquisition by the same woman). For 6,447 possible infections in women with two or more biopsies and 18,766 possible infections in women with one biopsy, there was 90% or 80% power (alpha=0.05) to detect a difference in 6-month acquisition of 0.66% or 0.56%, respectively. The 6-month acquisition in women with two or more biopsies was assumed to be 1.74% under the null hypothesis and 2.40% (38% increase, 90% power) and 2.30% (32% increase, 80% power) under the alternative hypothesis.
For the logistic model, we examined the power post hoc under a number of scenarios, while accounting for the possible within woman correlation using generalized estimating equations. For a 2% or 3% acquisition, there was greater than 80% power to detect an OR of 0.85 when OR for age ranged from 0.30 to 0.40 and for new sexual partners (yes/no) ranged from 1.5 to 1.7.
Previous studies on cervical epithelial healing after various procedures have shown that epithelial integrity is quickly reconstituted. Laser ablation completely heals within 3–4 weeks.19 The small epithelial damage induced by cervical biopsy is expected to completely heal within 1–2 weeks, women are usually recommended to refrain from sexual intercourse for at least 1 week after cervical biopsy. Thus, the 6-month window after cervical biopsy should be long enough to consider valid our study of biopsy-related HPV acquisition.
Thus, the benefit of taking multiple biopsies for more sensitive diagnosis of precancerous lesions, especially CIN 3, seems clear, provided that taking multiple biopsies is affordable and does not cause adverse effects that could include increasing the number of false-positive CIN 2 results.20 Better disease ascertainment by taking more biopsies among screen-positive women provides both patients and clinicians greater reassurance that follow-up rather than aggressive management is warranted when disease is not found. In support of taking more biopsies, we found no evidence in this study for a theoretical concern: multiple biopsies did not meaningfully increase HPV acquisition.
1. Gage JC, Hanson VW, Abbey K, Dippery S, Gardner S, Kubota J, et al. Number of cervical biopsies and sensitivity of colposcopy. Obstet Gynecol 2006;108:264–72.
2. Pretorius RG, Zhang WH, Belinson JL, Huang MN, Wu LY, Zhang X, et al. Colposcopically directed biopsy, random cervical biopsy, and endocervical curettage in the diagnosis of cervical intraepithelial neoplasia II or worse. Am J Obstet Gynecol 2004;191:430–4.
3. Doorbar J. Molecular biology of human papillomavirus infection and cervical cancer. Clin Sci (Lond) 2006;110:525–41.
4. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007;370:890–907.
5. Schiffman M, Adrianza ME. ASCUS-LSIL Triage Study. Design, methods and characteristics of trial participants. Acta Cytol 2000;44:726–42.
6. Human papillomavirus testing for triage of women with cytologic evidence of low-grade squamous intraepithelial lesions: baseline data from a randomized trial. The Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesions Triage Study (ALTS) Group. J Natl Cancer Inst 2000;92:397–402.
7. ASCUS-LSIL Triage Study (ALTS) Group. A randomized trial on the management of low-grade squamous intraepithelial lesion cytology interpretations. Am J Obstet Gynecol 2003;188:1393–400.
8. ASCUS-LSIL Triage Study (ALTS) Group. Results of a randomized trial on the management of cytology interpretations of atypical squamous cells of undetermined significance. Am J Obstet Gynecol 2003;188:1383–92.
9. Solomon D, Schiffman M, Tarone R. Comparison of three management strategies for patients with atypical squamous cells of undetermined significance: baseline results from a randomized trial. J Natl Cancer Inst 2001;93:293–9.
10. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A, et al. Improved amplification of genital human papillomaviruses. J Clin Microbiol 2000 Jan;38:357–61.
11. Schiffman M, Wheeler CM, Dasgupta A, Solomon D, Castle PE. A comparison of a prototype PCR assay and hybrid capture 2 for detection of carcinogenic human papillomavirus DNA in women with equivocal or mildly abnormal papanicolaou smears. Am J Clin Pathol 2005;124:722–32.
12. Peyton CL, Gravitt PE, Hunt WC, Hundley RS, Zhao M, Apple RJ, et al. Determinants of genital human papillomavirus detection in a US population. J Infect Dis 2001;183:1554–64.
13. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995;87:796–802.
14. Cogliano V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F. Carcinogenicity of human papillomaviruses. Lancet Oncol 2005;6:204.
15. Castle PE, Kreimer AR, Wacholder S, Wheeler CM, Koutsky LA, Rydzak G, et al. Influence of loop electrosurgical excision procedure on subsequent acquisition of new human papillomavirus infections. J Infect Dis 2009;199:1612–20.
16. Plummer M, Schiffman M, Castle PE, Maucort-Boulch D, Wheeler CM. A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion. J Infect Dis 2007;195:1582–9.
17. Liaw KL, Hildesheim A, Burk RD, Gravitt P, Wacholder S, Manos MM, et al. A prospective study of human papillomavirus (HPV) type 16 DNA detection by polymerase chain reaction and its association with acquisition and persistence of other HPV types. J Infect Dis 2001;183:8–15.
18. Zeger SL, Liang KY. Longitudinal data analysis for discrete and continuous outcomes. Biometrics 1986;42:121–30.
19. Sharp GL, Cordiner JW, Murray EL, More IA. Healing of cervical epithelium after laser ablation of cervical intraepithelial neoplasia. J Clin Pathol 1984;37:611–5.
20. Castle PE, Stoler MH, Solomon D, Schiffman M. The relationship of community biopsy-diagnosed cervical intraepithelial neoplasia grade 2 to the quality control pathology-reviewed diagnoses: An ALTS Report. Am J Clin Pathol 2007;127:805–15.