del Pino, Marta MD; Torne, Aureli MD; Alonso, Immaculada MD; Mula, Raquel MD; Masoller, Narcís MD; Fuste, Victoria MD; Ordi, Jaume MD
The Pap test has reduced the incidence of cervical cancer among women. However, although repeated examinations over a lifetime improve the sensitivity of this test, overwhelming evidence have shown that cytology has a low sensitivity, ranging from 50% to 60%.1 Molecular techniques of high-risk human papillomavirus (HPV) detection have increased the sensitivity of cervical cancer screening to 95% to 97%.2
One of the main limitations of cytology is that it detects a high number of high-risk HPV-related cell abnormalities with a low potential of progression, such as low-grade squamous intraepithelial lesions and atypical squamous cells of unknown significance. The incorporation of high-risk HPV testing has resulted in the detection of a number of women with high-risk HPV infection and normal cytology.3 In most of these women, both high-risk HPV infection and the cytologic abnormalities regress, and only a minority have cervical intraepithelial neoplasia (CIN) grade 2 or 3.
Colposcopy plays an essential role in the evaluation of women with cytologic abnormalities or high-risk HPV-positive test results. Nevertheless, it has been shown that some high-grade lesions are missed in the initial colposcopy.4 However, several studies have shown that colposcopy examination with directed biopsy in individuals with atypical squamous cells of unknown significance and low-grade squamous intraepithelial lesions detects CIN 2 or 3 in 5% to 20%.1,5,6 Finally, there are limited data on whether colposcopy can provide any information on the risk of progression in these individuals.
The aim of this study was to evaluate the risk of progression to CIN 2 or 3 in women with high-risk HPV infection and borderline (atypical squamous cells of unknown significance), mild (low-grade squamous intraepithelial lesions), or no cytologic abnormalities, and to determine the accuracy of colposcopy to predict the risk of progression.
PARTICIPANTS AND METHODS
The present study was conducted at the referral Colposcopy Unit of the Hospital Clinic of Barcelona. From January 2000 to July 2008, women from six different family practice centers with abnormal cytology or HPV infection diagnosed within the previous 6 months were referred to the colposcopy clinic. Most women with an HPV-positive test and negative cytology were included after 2006, when the HPV test was recommended in Spain as an adjunct to cytology for HPV screening in women older than age 30 years. We recruited all women fulfilling the following criteria: positive high-risk HPV test results and Pap test with normal, atypical squamous cells of unknown significance or low-grade squamous intraepithelial lesions results.
According to our study protocol, on the first visit all women attending the unit routinely underwent a complete study including clinical history, Pap test, high-risk HPV detection using Hybrid Capture-2, and digital colposcopy. Colposcopy examinations were routinely conducted by three faculty colposcopists with experience in lower genital tract disease. All women with abnormal cytology (atypical squamous cells of unknown significance or higher) or positive result of HPV testing with an abnormal transformation zone underwent a colposcopy-directed biopsy, endocervical curettage, or both if the transformation zone was not visible or was only partially visible. Neither biopsy nor endocervical curettage was performed in women with normal colposcopy results and a visible transformation zone.
A total of 982 women were eligible for the study. The following were considered exclusion criteria: 1) histologic diagnosis of CIN 2 or 3 or carcinoma in the colposcopy-directed biopsy, endocervical curettage, or both; 2) previous history of cervical cancer; 3) history of abnormal biopsy results, abnormal Pap test results, or high-risk HPV-positive results between 6 months and 3 years before enrollment; and 4) previous treatment for CIN 2 or 3 performed within the previous 3 years. None of the women had previously received the HPV vaccine. A total of 568 women were initially included in the study but 103 individuals were lost during follow-up. Therefore, 465 individuals were finally included in the analysis. Figure 1 shows the flow chart of the individuals initially screened as well as those finally enrolled and monitored in the study. Individuals lost to follow-up did not differ from the population included and the results were not affected by the exclusion of this group. In 218 (46.9%) individuals, a biopsy was performed at the first visit. Individuals with CIN 1 biopsy results were classified as having low-grade squamous intraepithelial lesions regardless of the cytology result (negative, atypical squamous cells of unknown significance, or low-grade squamous intraepithelial lesions). Individuals with a negative biopsy result were classified according to their cytology. Thus, after the first visit, the individuals were grouped as having low-grade squamous intraepithelial lesions or atypical squamous cells of unknown significance or as negative according to their cytology or histology results.
Human immunodeficiency virus (HIV) antibodies were evaluated in all women with the ADVIA Centaur HIV 1/0/2 Assay. All HIV-positive individuals had been receiving antiretroviral therapy since the diagnosis of HIV infection. CD4 levels and plasma viral load (Versant HIV-1 RNA 1.0 kinetic PCR) were determined at every follow-up visit. The present study protocol was approved by the ethical review board of the Hospital Clinic, Barcelona.
Cytology samples were obtained with an Ayre spatula and a cytobrush, and the result was reported according to the Bethesda 2001 criteria.7 All cytologic samples were examined by three pathologists with special training in cytology. Cervical specimens for high-risk HPV testing were collected using the Digene cervical sampler kit following the manufacturer's protocol. Digital colposcopy was performed using a colposcope Olympus Evis Exera II CV-180. First, the colposcopist applied 5% acetic acid and let it remain for 2 minutes. The acetic acid was reapplied to the cervix using cotton balls repeatedly over 1 to 2 minutes to detect “fast fader” lesions. Colposcopy findings were described following the criteria of the International Federation for Cervical Pathology and Colposcopy (Barcelona 2002). Normal colposcopic findings included original squamous epithelium, columnar epithelium, and the transformation zone. Minor changes (suggestive of low-grade disease) included a smooth surface with irregular outer border, slight acetowhite change, slow to appear, and quick to disappear; mild, speckled iodine partial positivity; and fine punctuation or fine regular mosaic. Finally, major changes (suggestive of high-grade disease) included: smooth surface with a sharp outer border; dense acetowhite change appearing early and being slow to resolve; iodine negativity in a previously densely white epithelium; coarse punctuation or wide irregular mosaic; and dense acetowhite change within columnar epithelium The size of the lesion was recorded as quadrants of the cervix involved.
The follow-up visits were scheduled every 6 months. Cytology, high-risk HPV testing, and colposcopy, with colposcopy-directed biopsy or endocervical curettage if indicated, were performed on each visit. Indications of colposcopy-directed biopsy or endocervical curettage during follow-up were high-grade squamous intraepithelial lesions result of the Pap test or significant worsening in the colposcopy pattern or an increase in lesion size. All individuals with persistence of high-risk HPV test results or abnormal cytology were monitored for a minimum period of 12 months. Thus, each individual had at least two colposcopy examinations, two Pap tests, and two tests for high-risk HPV.
Regression was defined as one or more negative Pap test result, with a negative high-risk HPV test, and, if available, a negative histologic result. Persistent cytologic or histologic abnormalities (atypical squamous cells of unknown significance or low-grade squamous intraepithelial lesions or CIN 1), positive high-risk HPV test result, or both were considered as persistent disease. Progression was defined as a histologic diagnosis of CIN 2 or 3. All individuals with CIN 2 or 3 were treated using the loop electrosurgical excision procedure (loop electrosurgical excision procedure). Individuals with CIN 2 or 3 or who were negative for both cytology and the high-risk HPV tests were excluded from the follow-up protocol.
The data were analyzed with SPSS software. The results are presented as absolute numbers and percentages or median and range. The χ2 or Fisher exact tests were used for comparisons between categorical variables. All the analyses were repeated after stratifying by age (younger than 30 years old compared with 30 years old or older), viral load (1–10, 10–100, more than 100 relative light units), previous cervical lesion, and HIV status to evaluate the influence of these factors. Logistic regression analyses were performed to determine the risk factors for progression to CIN 2 or 3 with independent variables such as age, smoking status, HIV status, previous cervical lesion, initial diagnoses, initial viral load, and initial colposcopy findings. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were evaluated.
The risk of progression and regression in each of the three groups (normal cytology, atypical squamous cells of unknown significance, and low-grade squamous intraepithelial lesions) for each time interval was computed by dividing the number of cases diagnosed in that interval by the number of women at risk during that interval. Using the Kaplan-Meier method, we calculated cumulative incidence rates with 95% CI for each interval up to 90.9 months. Progression and regression curves were compared using the log-rank test. A P value of 0.05 was considered statistically significant.
The individuals were grouped into three diagnostic categories: negative (92 women, 19.8%), atypical squamous cells of unknown significance (67 women, 14.4%), and low-grade squamous intraepithelial lesions or CIN 1 (306 women, 65.8%). Table 1 shows the demographic characteristics of the three groups.
The median follow-up period was 19.5 months (range 12–90.9). Forty-three individuals (9.3%) had progression to CIN 2 or 3 during follow-up, 161 (34.6%) had spontaneous regression, and 261 (56.1%) persisted with abnormal cytology, a high-risk HPV–positive test result, or both. Of the women with persistent lesions at the end of the follow-up, 145 (55.6%) had abnormal cytology and a positive high-risk HPV test result, 96 (36.8%) had persistent high-risk HPV–positive test results with negative cytologic results, and 20 (7.7%) showed a negative test result for high-risk HPV and persistence of atypical squamous cells of unknown significance or low-grade squamous intraepithelial lesions in the cytology.
At the first visit, colposcopy examinations showed normal findings in 53.5% of women, minor changes in 42.8%, and major changes in 3.7%. Table 2 shows the correlation between the referral colposcopy pattern and the cytologic or histologic results at enrollment and the outcome after follow-up. Progression to CIN 2 or 3 was observed in 19 of 249 (7.6%) of the individuals with an initial normal colposcopy result, 22 of 178 (12.4%) of women with a lesion involving less than two quadrants of the cervix, and 2 of 38 (5.3%) individuals with lesions involving more than two quadrants at the initial visit (P=.170). No differences in terms of time of progression were observed depending on the initial colposcopy category (P=.510).
In the last colposcopy examination conducted at the end of the follow-up, 300 women (64.5%) showed normal colposcopy results, 138 (29.7%) had minor changes, and 27 (5.8%) had major changes. Progression to CIN 2 or 3 was identified in 8 out of 27 (29.6%) women showing major changes in the last control, in 20 out of 138 (14.5%) women with minor changes, and in 15 out of 300 (5.0%) women having a normal colposcopy result at the end of the follow-up (P<.001). Progression to CIN 2 or 3 was identified in 15 out of 306 (4.9%) women whose colposcopy pattern became normal or improved and in 27 out of 159 (16.9%) women showing worsening of the colposcopy pattern in the last follow-up control (P<.001).
Table 3 shows the outcome of individuals according to the referral cytology, the cytology or histology category at enrollment, and the high-risk HPV relative light units at the first visit. No association was observed between these parameters and outcome.
The median time of progression to CIN 2 or 3 was 19.1 months (range 6.0–63.7). No differences in terms of time of progression were observed depending on the initial cytologic result (P=.201), the initial colposcopy category (P=.510), or high-risk HPV relative light units (P=.513). Figure 2 shows the overall cumulative incidence rate of progression to CIN 2 or 3 (Fig. 2A) and regression (Fig. 2B) according to the initial cytology result. No significant differences were observed in progression or regression rates between the different groups. All the women who had progression to CIN 2 or 3 had persistent HPV infection. None of the 181 (0%; 95% CI 0%–2.1%) women who were negative for high-risk HPV during follow-up had progression to CIN 2 or 3, whereas 43 out of 284 (15.1%; 95% CI 11.4%–19.8%) women who persisted with a high-risk HPV positive test result had progression to a high-grade lesion (P<.001).
Table 4 shows the risk of progression according to age at enrollment, history of cervical lesion or treatment, and HIV result. In all the women, the previous lesion had been diagnosed more than 3 years before entry into the current study and all had several normal cytology results, negative high-risk HPV test results, or both in the 3 years before enrollment. On the univariate proportional logistic regression, age older than 30 years (OR 2.79, 95% CI 1.37–5.77) and previous cervical lesion (OR 8.82, 95% CI 4.58–16.98) were associated with an increased risk of progression to CIN 2 or 3. In the multivariate analysis, previous cervical lesion maintained the association with progression (OR 7.88, 95% CI 4.06–15.32). Smoking, HIV status, initial diagnoses, viral load, and colposcopy findings were not found to be independent risk factors for progression to CIN 2 or 3. Fifty-seven (12.3%) women were HIV-positive. CD4 cells were within normal ranges and plasma viral load was not detectable in any of the women. No differences in terms of risk of progression were detected between HIV-positive and HIV-negative individuals.
The most relevant finding of our study is that HPV-positive women with low-grade, borderline, or no cytologic or histologic abnormalities had none of the colposcopy characteristics at referral (neither classification of the colposcopy abnormality nor lesion size) that were associated with a different risk of progression (that is, colposcopy findings at referral cannot predict the risk of progression in this group of women). Recent studies have concluded that visual signs of precancer have low specificity and are neither well-reproducible nor adequate in a screening setting.8 Other studies evaluating the accuracy of colposcopy in the diagnostic setting have reported excellent performance when performed by experienced clinicians. In this study, only impairment in the colposcopy features of individuals with an initial normal colposcopy test result was a worrisome finding associated with progression.
Interestingly, all individuals testing positive for high-risk HPV showed a similar risk of progression to CIN 2 or 3, independently of whether the Pap test, biopsy, or both showed negative results of atypical squamous cells of unknown significance or low-grade squamous intraepithelial lesions. Data on the risk of progression of women with normal cytology and high-risk HPV infection are limited, and there are no objective criteria to standardize the management of these women. Most reports previously published on this issue include both high-risk HPV-negative and HPV-positive women,9 whereas our study includes only high-risk HPV-positive women. The results of our study are in keeping with those from the ASC-US/Low grade squamous intraepithelial lesion Triage study, showing that individuals with high-risk HPV and atypical squamous cells of unknown significance or low-grade squamous intraepithelial lesions have a similar risk of progression.10 Our study suggests that individuals with negative cytology may also have a similar risk of progression.
No association was found between viral load measured as relative light units in Hybrid Capture-2 and risk of progression to CIN 2 or 3, similar to what has been found in other studies.11,12 However, our data confirm that persistent high-risk HPV infection is a key prognostic risk factor for development of CIN 2 or 3.9,11 In the present study, none of the women whose high-risk HPV test yielded negative results had CIN 2 or 3 develop during the follow-up. The initial relative light units in this study did not predict the risk of progression in any group, and most studies have found no association between viral load and the risk of progression.9,13
In agreement with other studies,14 we observed that women older than 30 years of age were at higher risk for progression than younger women. Although some contradictory results have been reported,9,15 our results support the current guidelines that recommend closer follow-up for women older than 30 years of age.1 No differences were found in the risk of progression in our series in HIV-positive individuals compared with HIV-negative women. In contrast, women with a history of cervical lesions had almost an eightfold higher risk of CIN 2 or 3 development than women without a previous cervical lesion. This higher risk of progression to CIN 2 or 3 was independent of the cytologic result at enrollment. Data on the long-term risk of CIN recurrence among women with previous CIN are limited. The higher risk of CIN 2 or 3 or cervical cancer development in previously treated women has been reported in several studies.16,17 In a meta-analysis, Soutter et al18 reported a 2.8-fold higher risk of cervical cancer developing in women treated for CIN compared with those with no history of lesion. Our study confirms that women with a history of cervical lesion are at higher risk for progression to CIN 2 or 3 and should be carefully observed.
There are some possible limitations of this study. Differences were frequently found between the diagnosis at referral and that at enrollment in our study. It is well-established that concordance in the diagnosis of cervical lesions (either cytologic or histologic) is approximately 50%. Many studies have reported high interobserver variability with κ values less than 0.5.19 Furthermore, different cytologic results have been reported in more than 50% of cases when cytology tests are repeated in less than 3 months.20 A second possible limitation is the low number of HPV-positive women with normal cytology included in the study. This could be partially explained by the fact that high-risk HPV testing had been recently included in the cervical cancer prevention guidelines in Spain.21 Our series also included a limited number of women with atypical squamous cells of unknown significance. Nevertheless, in contrast with other studies, our series was focused only on high-risk HPV-positive individuals, and the number of cases included in our study was similar to that of other previous reports.15,22 Considering the differences obtained in the statistical comparisons conducted between the different groups in the present study, it has a statistical power of 49%. A sample size ranging from 790 to 3,441 individuals would be necessary to provide a 80% statistical power of avoiding a type II error and a 5% chance of making a type I error. Another possible limitation is that a number of individuals underwent a biopsy, and this procedure might have altered the evolution of the lesion. However, there were no differences in the outcomes of the individuals in whom a biopsy was performed compared to the individuals with only a cytology examination. Moreover, studies on the effect of cytology and histology on the natural history of cervical lesions have shown no effect of biopsy on the short-term evolution of the disease.23 Finally, no high-risk HPV-typing data were available. One of the weaknesses of the Hybrid Capture-2 assay is that it does not allow the identification of specific high-risk HPV types. It has been shown that certain high-risk HPV types, mainly types 16 and 18, are preferentially associated with cervical neoplasia.24,25 It is currently accepted that molecular genotyping assays that detect HPV 16 and HPV 18 are clinically useful.1
In conclusion, our findings suggest that women with high-risk HPV infection having normal, borderline, or low-grade abnormalities in cytology, but no CIN 2 or 3, have a similar risk of progression to high-grade lesions and may benefit from a similar follow-up strategy. Initial colposcopy findings do not provide relevant information on the risk of progression. Follow-up of these women would be optimally undertaken with high-risk HPV testing and referral for persistence. Colposcopy during follow-up appears to predict new CIN 2 or 3 lesions, and biopsies should be performed in women with persistent high-risk HPV and persistence or impairment of colposcopic changes. Women with negative results on high-risk HPV tests appear to be at minimal risk for CIN 2 or 3 and can be monitored according to routine protocols.
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