Objective
The objective of the study was to evaluate the efficacy of intravaginal application of 5% 5-fluorouracil (5-FU) for the treatment of cervical intraepithelial neoplasia (CIN) 2 in women.
Study Design
Women aged 18-29 years with CIN 2 were recruited for this randomized controlled trial of observation vs treatment with intravaginal 5-FU. Women in the observation group returned in 6 months for a Papanicolaou smear, colposcopy, and a human papillomavirus (HPV) deoxyribonucleic acid test. Women in the 5-FU group were treated with intravaginal 5-FU once every 2 weeks for a total of 16 weeks and were similarly evaluated at 6 months. All women who had a baseline visit were included in the intention-to-treat analysis. Values of P < .05 were considered statistically significant.
Results
Between August 2010 and June 2013, 60 women were randomized and had a baseline visit for intervention (n = 31) vs observation (n = 29). Of women who had cervical biopsy results at 6 months, regression of disease was demonstrated in 93% of women in the 5-FU group (26 of 28) and 56% of women in the observation group (15 of 27). Under the intention-to-treat analysis, a relative risk for cervical disease regression of 1.62 (95% confidence interval [CI], 1.10–2.56) was found between the 5-FU and observation arms ( P = .01). When the cervical biopsy, Papanicolaou smear, and HPV results were combined for the 6 month follow-up visit, 50% of the 5-FU group (14 of 28) had a documented normal biopsy, normal Papanicolaou smear, and negative HPV test compared with 22% in the observation group (6 of 27) (relative risk, 2.25; 95% confidence interval, 1.05–5.09; P < .05). There were no moderate or severe side effects in the intervention group.
Conclusion
Topical 5-FU appears to be an effective medical therapy for CIN 2 in young women. 5-FU is readily available and may be considered as an off-label treatment option for young women with CIN 2 who are interested in the treatment of disease but want to avoid excisional procedures.
Historically, most women with cervical intraepithelial neoplasia (CIN) 2 and 3 underwent excisional therapy or ablation of the cervical transformation zone. However, 5-26% of women have disease recurrence, even with negative surgical margins. Additionally, excisional treatment procedures have been associated with increased risk of premature delivery as well as anxiety, pain, bleeding, and health care expenditure. Because nearly half of CIN 2 lesions regress in young women, current guidelines endorse close observation of young women as a preferable management strategy for CIN 2 and acceptable management strategy for CIN 3. Although expectant management is appealing, approximately one-third of CIN 2 cases will persist and the remaining may progress to CIN 3 on follow-up. There are no medical therapies recommended to promote the clearance of human papillomavirus (HPV) or cervical dysplasia.
Topical 5-fluorouracil (5-FU) is used for the treatment of skin cancers and lesions caused by HPV, including genital warts, vulvar intraepithelial neoplasia and vaginal intraepithelial neoplasia. The 5-FU treatment of genital disease is an off-label use of the medication because it has not been approved by the Food and Drug Administration or recommended by the American College of Obstetricians and Gynecologists for this use.
Initial treatment regimens were associated with severe side effects such as pain and chronic ulceration. These side effects were likely a dose-related response because standard treatments require multiple daily applications; studies limiting topical 5-FU to less frequent application or diluted doses have reported favorable side effect profiles.
The objective of this study was to assess the efficacy, safety, and acceptability of intravaginal 5-FU as a primary treatment for CIN 2 in young women.
Materials and Methods
This was a prospective nonblinded, randomized trial of intravaginal 5% 5-FU vs standard-of-care observation in young women with CIN 2 (no placebo). The primary outcome was regression of disease 6 months after the diagnosis of CIN 2. Secondary outcomes included high-risk HPV status at 6 months, 12 month pathological findings, and safety and acceptability data.
This study was approved by the University of North Carolina (UNC) Institutional Review Board. All participants underwent written informed consent procedures.
Women (aged 18-29 years) presenting to the UNC’s Women’s Hospital Clinics with satisfactory colposcopic examinations, a biopsy-confirmed diagnosis of CIN 2, and in whom follow-up observation with cytology and colposcopy every 6 months was planned were approached for study enrollment. Women who were non-English speaking, human immunodeficiency virus (HIV) infected, immunosuppressed, pregnant, planning pregnancy, or breast-feeding during the study time period or were unwilling to use condoms and another form of birth control during the treatment time period were excluded from the study.
Dual contraception (condoms plus 1 of the following: oral, intravaginal, injectable, implantable, or intrauterine conception) was required for the 5-FU group because of its potential teratogenic effects demonstrated in intravenous administration. Women were counseled regarding teratogenic risk during the consent process.
Order of randomization was generated based on a simple randomization table with a 1:1 allocation ratio, and assignments were placed into sequentially numbered opaque envelopes. After verbal interest was reported by a potential participant on the phone, women were randomized to an observation or treatment group (5-FU) by study staff. Participants in the observation group were given the option to have their written consent forms mailed and completion of a background survey by phone. They were scheduled for appointments in 6 months from biopsy date and received standard phone and written appointment reminders from the health care facility in addition to reminders from study staff. Reminders consisted of phone calls, texting, or e-mailing, depending on the preference of the participant.
The 5-FU group presented to the study site for written consent procedures and received written and verbal instructions for insertion of 2 g of 5-FU via vaginal applicators every 2 weeks for a total of 8 doses. This dosing schedule was based on its reported safety, tolerability, and efficacy in a prior trial of intravaginal 5-FU for the prevention of recurrence of CIN after excisional procedure in HIV-infected women. However, because our study’s patient population was not potentially immunocompromised, we chose a shorter course of 16 weeks as per other treatment studies for HPV-related diseases.
The 5-FU participants were instructed to insert 2 g of topical 5% 5-FU cream (Efudex; Valeant Pharmaceuticals International, Quebec, Canada) at night with a vaginal applicator, which could be twisted onto the study tube for the removal of 5-FU cream. After the medication was inserted into the vagina proximal to the cervix, participants placed a tampon per vagina overnight to keep the cream at the cervix. Participants were instructed to remove the tampon in the morning, shower, and frequently hand wash and change panty liners over the next 2 days to avoid irritation from the cream.
Women were supplied home pregnancy tests for use prior to each application of cream. Condoms or abstinence from sexual activity 48 hours after use of the cream was also recommended to diminish any potential irritation to the participant’s sexual partner. All supplies were provided to the participants. Use of study drug was delayed if a participant was having her menstrual cycle.
Participants in the 5-FU group returned for a safety and acceptability visit at the study site after 8-16 weeks of use of intravaginal 5-FU to complete a survey and pelvic examination. A single unblinded coinvestigator assessed adherence and genital symptoms and performed a colposcopic examination of vaginal and cervical tissue as per the National Institutes of Health Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events: Female Genital Grading Table for Use in Microbicide Studies ( Table 3 for a listing of criterion.).
The interview included an acceptability questionnaire in which participants were surveyed regarding emotions felt while using the cream and side effects to their sexual partner; logistical concerns surrounding use of the cream were scored on a 5 point Likert scale. Prior to the 6 month visit, all participants received hospital-based and study-based reminders similar to the observation group described above.
At the 6 month visit, colposcopically guided cervical biopsies were performed at the site previously biopsied and diagnosed with CIN 2 on original histology 6 months prior for both intervention and control arms. In all participants, additional biopsies were obtained if there was clinical concern for other areas of dysplasia. We completed similar procedures at the 12 month visit. If the participant had normal results at the 6 month visit and had a normal colposcopy, no biopsies were taken at the 12 month visit unless abnormal colposcopic findings were noted. Women who had treatment procedures (because of a CIN diagnosis or personal preference because of persistent CIN 2) during the study were withdrawn from the study. The colposcopist, a board-certified obstetrician-gynecologist and the director of the colposcopy clinic, was not blinded to the study group of the participants.
HPV testing was performed at the 6 and 12 month visits using the Digene Hybrid Capture 2 high risk HPV DNA test at the Department of Pathology, Yale University (New Haven, CT). A single pathologist coinvestigator, board-certified in cytopathology with a focused subspecialty practice in gynecological surgical and cytopathology, was blinded to our randomization scheme and reviewed all cytology and pathologic specimens for entry criteria and for follow-up cytology and histology. Additional p16 immunohistochemistry staining was conducted if clinically indicated.
Sample size and statistical analysis
Sample size calculations were based on the primary outcome of the regression of disease. Based on published literature, we assumed that 50% of CIN 2 lesions would regress with observation alone, and a clinically significant difference was estimated at 30%. The total sample size required for the study was 89 women (45 in each arm, α = 0.05, β = 0.80 and 2-sided Student t test).
The differences between the treatment and observation groups were assessed for all categorical baseline variables and outcomes using 2-sided Fisher exact tests of association. Continuous baseline variables were assessed using exact Wilcoxon rank sum tests. Histological biopsy results at the 6 month visit were separated into a dichotomous variable indicating regression of disease vs persistence or progression of disease.
The association between the regression of disease (regression or persistence/progression) and group status (5-FU and observation) was determined using a 2-sided Fisher exact test of association. Additionally, relative risk (RR) with exact confidence interval (CI) was calculated to aid in understanding the direction of the association. The analyses were completed using intention-to-treat (ITT) methodology and by imputing histological biopsy results for the women with missing 6 month measurements. Imputed values were found using the last observation carried forward method (ie, women without results were designated to still have their baseline result [CIN 2, CIN 3]). Furthermore, a sensitivity analysis was conducted by removing the women with missing 6 month measurements and repeating the analysis to ensure the imputation method was conservative. Values of P < .05 were considered statistically significant. Other variables assessed were histological results at 12 months after diagnosis, cervical cytology, HPV presence, and genotype. All analyses were completed using SAS 9.3 (SAS Institute, Cary, NC) or StatXact version 9 (Cytel, Cambridge, MA).
Results
Between Aug. 1, 2010, and June 1, 2012, 93 women who met inclusion criteria were approached for enrollment ( Figure ). Twenty-six women declined and another 7 were subsequently excluded because of protocol violations prior to the baseline visit. Sixty women were randomized and underwent a baseline enrollment survey. Baseline characteristics of both groups are described in Table 1 .
| Demographics | 5-FU | Observation | P value | ||
|---|---|---|---|---|---|
| n | % | n | % | ||
| Race | |||||
| White | 24 | 77.4 | 18 | 62.1 | .26 |
| African American | 6 | 19.4 | 7 | 24.1 | |
| Other a | 1 | 3.2 | 4 | 13.8 | |
| Ethnicity | |||||
| Hispanic | 3 | 9.7 | 4 | 13.8 | .70 |
| Non-Hispanic | 28 | 90.3 | 25 | 86.2 | |
| Age, y | |||||
| 19-20 | 2 | 6.5 | 1 | 3.5 | .28 |
| 21-25 | 17 | 54.8 | 22 | 75.9 | |
| 26-30 | 12 | 38.7 | 6 | 20.7 | |
| Median age, y b | 24 | (19–29) | 23 | (20–29) | .32 |
| Marital status | |||||
| Married | 6 | 19.4 | 5 | 17.2 | .65 |
| Divorced/separated | 2 | 6.5 | 4 | 13.8 | |
| Never married | 23 | 74.2 | 20 | 69.0 | |
| Education | |||||
| High school or less | 8 | 25.8 | 8 | 27.6 | 1.00 |
| More than high school | 23 | 74.2 | 21 | 72.4 | |
| Health insurance (public or private) | 18 | 58.1 | 10 | 34.5 | .08 |
| Sexual history | |||||
| Current contraception use | 28 | 90.3 | 24 | 82.8 | .47 |
| Ever condom use | 18 | 58.1 | 13 | 44.8 | .44 |
| Age at first intercourse, y | |||||
| 12-15 | 10 | 32.3 | 12 | 41.4 | .79 |
| 16-20 | 20 | 64.5 | 16 | 55.2 | |
| 21-25 | 1 | 3.2 | 1 | 3.5 | |
| Number of sexual partners over lifetime | |||||
| 1-5 | 10 | 32.3 | 15 | 51.7 | .31 |
| 6-10 | 12 | 38.7 | 9 | 31.0 | |
| >10 | 9 | 29.0 | 5 | 17.2 | |
| Number of sexual partners in last year | |||||
| 1 | 23 | 74.2 | 19 | 65.5 | .58 |
| ≥2 | 8 | 25.1 | 10 | 34.5 | |
| Medical history | |||||
| Current tobacco use | 7 | 22.6 | 8 | 27.6 | .77 |
| Past tobacco use | 14 | 45.2 | 10 | 34.5 | .44 |
| HPV vaccine (1-3 doses) | 10 | 32.3 | 6 | 20.7 | .39 |
| Ever had an STD | 5 | 16.1 | 6 | 20.7 | .74 |
| Nulliparous | 23 | 74.2 | 15 | 51.7 | .11 |
| First abnormal Papanicolaou smear | 13 | 41.9 | 13 | 44.8 | 1.00 |
| Prior history CIN 2 or CIN 3 c | 6 | 19.4 | 1 | 3.5 | .10 |
| Prior history cryotherapy or excision | 4 | 12.9 | 1 | 3.5 | .35 |
a Asian (n = 1), American Indian or Alaskan Native (n = 1), unknown (n = 3)
b Age in years is represented by the median in the n column and the range in the % column
c One participant in the observation group unsure of a prior history of CIN 2 or 3.
There were no statistically significant differences between the 5-FU and observation groups. The median age of participants was 24 and 23 years in the 5-FU and observation groups, respectively ( P = .32). There were no differences between the groups in regard to the following CIN- or HPV-related parameters: tobacco use, first abnormal Papanicolaou smear, a prior history of CIN 2 or 3, or a prior history of cryotherapy or loop electrosurgical excision procedure. Two women in the 5-FU group and 1 woman in the observation group also were noted to have findings consistent with both CIN 2 and 3. All histological, cytological, and HPV results at each measured time point are described in Table 2 .
| Variable | Baseline (n = 60) | 6 months (n = 56) | ||||||
|---|---|---|---|---|---|---|---|---|
| 5-FU | Control | 5-FU | Control | |||||
| n | % | n | % | n | % | n | % | |
| Pathology | ||||||||
| No biopsy | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 1 | 3.6 |
| Normal | 0 | 0.0 | 0 | 0.0 | 24 | 85.7 | 12 | 42.9 |
| CIN 1 | 0 | 0.0 | 0 | 0.0 | 2 | 7.1 | 3 | 10.7 |
| CIN 2 | 29 | 93.6 | 28 | 96.6 | 1 | 3.6 | 8 | 28.6 |
| CIN 3 | 2 | 6.5 | 1 | 3.5 | 1 | 3.6 | 4 | 14.3 |
| Papanicolaou smear | ||||||||
| Normal | 0 | 0.0 | 0 | 0.0 | 16 | 57.1 | 7 | 25.0 |
| ASCUS | 9 | 29.0 | 4 | 13.8 | 7 | 25.0 | 4 | 14.3 |
| ASCH | 8 | 25.8 | 2 | 6.9 | 1 | 3.6 | 2 | 7.1 |
| LSIL | 8 | 25.8 | 16 | 55.2 | 2 | 7.1 | 6 | 21.4 |
| HSIL | 6 | 19.4 | 7 | 24.1 | 2 | 7.1 | 8 | 28.6 |
| Unknown | 0 | 0.0 | 0 | 0.0 | 0 | 0.0 | 1 | 3.6 |
| HPV result | ||||||||
| Yes | NT | NT | 12 | 42.9 | 20 | 71.4 | ||
| No | NT | NT | 15 | 53.6 | 7 | 25.0 | ||
| Unknown | NT | NT | 1 | 3.6 | 1 | 3.6 | ||
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