Cervical intraepithelial neoplasia (CIN) refers to a precancerous condition which progresses to cervical cancer, the third most prevalent cancer worldwide [1]. Human papillomavirus (HPV) is one of the leading causes of CIN and cervical cancer as proven by epidemiological and molecular pathologic evidence [2].

CIN can be histologically classified into CIN 1, CIN 2, and CIN 3 [3]. The current treatment for CIN 2 and 3 involves mostly an excision, and of the different types of excision, conization is preferred, which is reported to cause a small number of short-term complications [4, 5]. However, conization can also lead to long-term complications such as preterm birth and perinatal mortality [6]. Moreover, a recent meta-analysis has reported that the loop electrosurgical excisional procedure (LEEP) is associated with preterm birth [7].

CIN commonly occurs among women of childbearing age [8]. However, there are currently no HPV therapeutic vaccines for the management of HPV-positive patients or medical therapy for CIN. Research on less invasive and safe methods of treatment for patients with CIN 2-3 who wish to become pregnant is currently needed.

There has been some research on imiquimod for the conservative treatment of CIN [9-11]. Imiquimod is a therapeutic agent for genital warts, which develop as a result of HPV 6 and 11 infections, and has been used clinically since being approved in 1997 [12]. Some studies have reported imiquimod to be effective for treating HPV-related vulvar intraepithelial neoplasia [13, 14].

Imiquimod is a topical immune response modifier that has agonist effects on toll-like 7 receptors [15]. It induces the expression of cytokines including interferon-alpha, tumor necrosis factor-alpha, and interleukin 1, 6, and 8 by dendritic cells, thereby activating natural killer cells, which attack the HPV-infected and carcinogenic cells. Aside from mediating this innate immune response, imiquimod also induces the maturation of Langerhans cells and moves them into the lymph nodes, where it gives rise to an adaptive immune response including a Th1 response. Through these mechanisms, imiquimod plays both antiviral and antitumor roles [12, 16, 17].

In the present study, the authors evaluated the therapeutic effects and complications of imiquimod directly applied by a gynecologic oncologist to the cervix of women with high-grade CIN (HG-CIN, CIN 2 and 3) who refused to undergo excision therapy.

This study was retrospective approved by the Institutional Review Board of the Asan Medical Center (Seoul, Korea). The authors analyzed the medical records of patients who underwent imiquimod therapy after being histologically diagnosed with CIN 2 or 3. Patients who had previously been treated for CIN were excluded from the study. A total of 55 patients underwent imiquimod therapy or were followed up after treatment between June 2014 and May 2017. After excluding one patient who became pregnant, 54 patients were included in this study. Of the 54 patients, all patients were diagnosed with CIN 2 or 3 based on a colposcopic punch biopsy at a different hospital or in at the present center. All patients refused to undergo excisional therapy and consented to use imiquimod after being informed about the off-label use of imiquimod. They were also informed about its complications. All patients provided their informed consent.

The patients visited the present center once or twice per week up to at least eight or more visits during the treatment period. The same gynecologic oncologist applied imiquimod cream 12.5 mg to the cervix of all the patients after colposcopy during every treatment session. If any abnormal or unsatisfactory findings were detected during colposcopy, a punch biopsy and Pap test were performed. An HPV test was performed for follow-up at our center, using real-time PCR.

Follow-up test took place after one, three, and six months after the treatment. During the follow-up tests, colposcopy and a Pap test were performed, and a punch biopsy was performed if colposcopy results turned out abnormal or unsatisfactory. A patient’s response to treatment was classified as cytologic or histologic regression, complete remission, and persistent state. Regression was defined as when the patient showed evidence of low-grade squamous intraepithelial lesion (LSIL), atypical squamous cells of undetermined significance (ASC-US), reactive cellular change (RCC), or negative results in a Pap test and CIN 1, negative or cervicitis in the case of a punch biopsy. Complete remission was defined as when the patient showed evidence of RCC or negative result in a Pap test and negative or cervicitis in the case of a punch biopsy. HPV clearance was defined as when the patient had a negative result in an HPV test performed at our hospital.

A chi-square test and a logistic regression test were used to study the association between the variables and the response. To compare patient responses between the HPV 16-or 18-positive groups, and non-HPV 16 and 18 groups, a chi-square test was performed. A p-value of < 0.05 in a two-sided test was regarded as statistically significant. Statistical analyses were performed using SPSS version 21.0.

A total of 55 patients were enrolled in the imiquimod study at the present center. After two treatment sessions with imiquimod, one of these patients was excluded from the study since she was pregnant. She showed no complications during the pregnancy period and gave a natural birth after a full term of pregnancy. Table 1 summarizes the characteristics of the 54 patients who continued in the study. The median age was 30 (range 21-42) years, and the median follow-up period was 13.4 (range, 2.6-32.1) months. Twenty-three (42.6%) patients had CIN 2, and 31 (57.4%) had CIN 3. Only two patients were HPV-negative, and all the remaining were HPV-positive. Twenty (37%) patients were positive for HPV 16 or 18, and 29 (53.7%) patients were positive for other high-risk HPV infections. Three (5.6%) patients were positive for unknown types of HPV.

The median length of the treatment period was two (range 1-3.7) months. The median number of imiquimod therapy sessions was eight (range 8-12). Fifty-two patients underwent eight treatment sessions, and two patients underwent 12 treatment sessions as they requested others.

Table 2 summarizes the treatment responses in all the patients. The patients’ responses to treatment were evaluated after one month later after the treatment. The regression was observed in 45 (83.3%) patients and the complete remission was observed in 32 (59.3%) patients. The HPV clearance rate was 33.3%.

Nine patients were in the cytologic or histologic persistent state. Six patients immediately underwent LEEP, and three patients refused to undergo excisional therapy. Of these three patients, one patient wished to continue the imiquimod therapy but had to undergo LEEP after four additional imiquimod treatment sessions, since she was found to be still in the persistent state for follow-up tests. Two patients were lost to follow-up after they refused excisional therapy. Of the 45 patients who entered regression after the imiquimod therapy, five (11.1%) patients showed signs of recurrence in the follow-up tests during six months of the follow-up period.

Table 3 compares the rates of complete remission, regression, and HPV clearance between the HPV 16-or 18-positive group, and the non-HPV 16 and 18 group. The rate of HPV clearance was significantly higher in the non-HPV 16 and 18 group.

All 43 patients underwent a Pap test only after completing four treatment sessions during eight or 12 sessions of the total treatment. The proportion of patients who showed regression in an interim test was significantly associated with the proportion of patients who showed regression after the treatment period (p-value < 0.001).

Table 4 summarizes the complications that arose during the treatment period. Over half of all the patients experienced no complications. Headache was the most common complication, followed by fever and febrile sense.

In young women, especially nulliparous, it is not simple to decide on an excisional therapy for high-grade CIN. Excisional therapy including conization and LEEP can have long-term complications such as preterm birth and perinatal mortality [6, 7]. Moreover, a recent study suggests that large loop excision of the transformation zone (LLETZ) can lead to subfertility [18]. Margot et al. compared the patients’ preferences for imiquimod vs. LLETZ, for the treatment of high-grade CIN, and found that the desire for a future pregnancy is a major factor influencing the preference for imiquimod treatment [19].

The present authors retrospectively analyzed 54 patients who were treated with imiquimod for high-grade CIN. This study showed that direct application of imiquimod over the cervix by a gynecologic oncologist was effective and safe for treating CIN 2 and 3. The cytologic or histologic regression rate after the imiquimod therapy was 83.5%, and the cytologic or histologic complete remission rate was 59.3%. The present data are consistent with the findings of Grimm et al., who conducted a randomized controlled trial to compare the effectiveness of imiquimod and placebo in the treatment of CIN 2 and 3 [11]. In their study, the histologic regression rate after imiquimod therapy was 73%, and the complete histologic remission rate was 47%. They demonstrated that topical imiquimod could effectively treat CIN, and claimed it to be a novel discovery [11].

In the present study, 12.5 mg of imiquimod was applied to the cervix by a gynecologic oncologist at a median frequency of once per week up to a median of 8 total sessions for a median period of two months. In the study by Grimm et al., 6.25 mg of imiquimod was administered as a self-applied vaginal suppository for 16 weeks, one suppository per week in the first two weeks, two suppositories per week for the subsequent two weeks, and three suppositories per week starting at the 5^th week [11]. Despite the smaller therapeutic imiquimod total dose used and the shorter treatment duration of this study, the response rates were higher than those found by Grimm et al., possibly because of the direct application of imiquimod on the cervix.

During the six months of follow-up period after imiquimod treatment, the recurrence rate was 11.1%. All patients who had a recurrence underwent LEEP. According to a review by Witte et al., three studies have been conducted on imiquimod as a potential therapeutic agent for CIN, and one of these has reported on the cytologic or histologic recurrence rate [20]. Pachman et al. conducted a randomized clinical trial comparing patients who underwent standard excisional or ablation treatment for CIN with and without an earlier imiquimod therapy. In this study, the rate of two-year dysplasia recurrence was 14% in both the groups [20].

Of the 54 patients in this study, two patients were diagnosed with an invasive lesion. These two patients had no children. One patient showed HSIL after the treatment and underwent LEEP accordingly, after which she was diagnosed with invasive squamous cell carcinoma FIGO (International Federation of Gynecology and Obstetrics) stage IA1, without lymphovascular space invasion. The patient underwent no additional treatment and was disease-free 15 months later. The other patient underwent LEEP after showing HSIL in six months after the imiquimod therapy. The patient was diagnosed with superficially invasive squamous cell carcinoma with a depth and width of 0.1 mm and margin negative, and became disease-free six months after the diagnosis. They are currently attempting to become pregnant. In the randomized controlled trial by Grimm et al., only three patients in the placebo group of a total 59 patients included in the study were diagnosed with micro-invasive cervical cancer. Grimm et al. explained that their results most likely reflected the underdiagnosed occult cancer in the cervical canal [11].

In the present study, the HPV clearance rate was 33.3%, and it differed significantly between the HPV 16 or 18 positive group and the non-HPV 16 and 18 groups. The clearance rate was considerably lower in the HPV 16 or 18 positive group. In the study by Pachman et al., the group treated with imiquimod showed an HPV clearance rate of 42% [9], while in the study by Grimme et al. it was 60% [11].

Although the present study used a small sample population, it demonstrates an association between the interim response and the regression rate. In other words, early responders showed a higher regression rate. In the next prospective study, the authors may plan their study designs in consideration of this fact.

In this study, 55.6% of the patients did not experience any complications from the topical application of imiquimod, indicating that it is both tolerable and safe, For the other 44.4%, complications were tolerable and did not require the patient to cease the treatment. Three studies have been conducted on the use of imiquimod in the treatment of CIN. These studies reported that complications commonly occurred during the imiquimod therapy, but were all tolerable [20].

A limitation of this study is retrospective nature with a small number of cases. However, the sample size of high-grade CIN is the largest among previous studies [10, 11]. This study may serve as a stepping stone for future studies regarding imiquimod therapy for CIN.

In conclusion, this study showed that direct application of imiquimod to the cervix was a safe, effective, and feasible method of treatment for high-grade CIN in patients who desired to become pregnant. It could be an alternative option for them. A prospective study with a larger cohort is needed to confirm these findings.