A chlorin e6 derivative-mediated photodynamic therapy for patients with cervical and vaginal low-grade squamous intraepithelial lesions: a retrospective analysis

Cervical and vaginal low-grade squamous intraepithelial lesions (LSIL) are associated with persistent human papillomavirus infections, and require an efficacious non-invasive treatment such as photodynamic therapy due to anatomical characteristics. In this study, we evaluated the clinical efficacy and safety of a chlorin e6 derivative named STBF-mediated photodynamic therapy (STBF-PDT) for patients with cervical and vaginal LSIL. A total of 18 patients received STBF-PDT for 3 to 6 times and followed up for 6 to 12 months; among them, 6 had cervical and vaginal LSIL and 1 had vaginal stump LSIL after hysterectomy for cervical cancer. Results showed the complete remission (CR) rates were 72.22% (13/18), and the HPV remission rates and HPV clearance rates were 88.89% (16/18) and 83.33% (15/18), respectively at 1-month follow-up. The CR reached 88.89


| INTRODUCTION
Cervical cancer is one of the most common malignant tumors that harms women's health in worldwide. Persistent high-risk human papillomaviruses (HR-HPV) infection (more than 1 year) may cause the cervical intraepithelial neoplasia (CIN) and even cervical cancer [1][2][3]. Prospective studies have shown that more than 90% of new HPV infections would regress in 6 to 18 months and more persistent infection is a prerequisite for progression to CIN [4]. Approximately 70% of cervical cancer cases contain HR-HPV, with HPV-16 and HPV-18 being the most prevalent [5]. Since 2012, as per the Lower Anogenital Squamous Terminology (LAST) consensus, the terminology CIN was replaced with squamous intraepithelial lesions (SIL) [6]. That is CIN I and p16-negative CIN II were redefined as low-grade SIL (LSIL), and p16-positive CIN II and CIN III as high-grade SIL (HSIL). Vaginal intraepithelial neoplasia (VAIN) is rare and accounts for approximately 1% to 4% of female genital tract cancer. Studies have shown that about 4.4% of patients with CIN had multicentric intraepithelial neoplasia in lower genital tracts, which can be related to high resistance to HPV infection [7]. VAIN is usually detected during cervical cancer screening, and multi-site lesions are usually caused by persistent HPV infections of the whole lower genital tract [8]. The WHO classification graded VAIN lesions as vaginal LSIL and vaginal HSIL.
Due to the special anatomical structure and physiological function requirements, the treatment of cervical and vaginal lesions is a challenge for clinicians. Current therapeutic regimes include surgery, such as loop electrosurgical excision procedure (LEEP), ablative treatment, such as cryotherapy, laser, and thermal coagulation, but most of the treatments are traumatic and may cause pain, adhesion, bleeding, scar, and other adverse reactions [9]. Photodynamic therapy (PDT) is a promising, targeted, and non-invasive approach that combines photosensitizer and illumination. The photosensitizer can produce reactive oxygen species (ROS) after irradiation with a specific light source to make a cytotoxic, lesion-ablating photodynamic reaction [10]. 5-Aminolevulinic acid (5-ALA), a precursor of the photosensitizer protoporphyrin IX (PpIX), is a prevalently applied photosensitizer in China for patients with condyloma acuminatum, CIN, and VAIN. After incubation for up to 3 to 4 hours, the 5-ALA is absorbed, transformed, and then activated by irradiation, and the effective rate for CIN was 83.3% to 90%, which varies among studies [11,12]. However, the 5-ALA-mediated photodynamic therapy (ALA-PDT) was accompanied by unbearable adverse reactions, whose rate was 74.6% as reported [13]. The main adverse reactions are pain, local discomfort, burning sensation, increased vaginal discharge, and urinary retention [14].
STBF is a derivative of chlorin e6 (Ce6), belonging to chlorophyll photosensitizers, which is extracted from spirulina and can be used as a photosensitizer for photodynamic therapy with high efficiency in generating singlet oxygen. Ce6 is a promising photosensitizer, but the hydrophobic characteristics limit its applications. At present, Ce6 is mostly wrapped in nanoparticles and delivered to the target site for therapeutic or diagnostic uses, and several chlorophyll photosensitizers have been used for tumor imaging and PDT with high safety [15][16][17]. STBF is hydrophilic and photosensitive, so there is no need for a long-time incubation and intracellular transformation when in clinical use. The excitation wavelengths of STBF range from 630 to 670 nm. It is reported that the method of simultaneous spectral fluorescence diagnostics and photodynamic therapy using intravenous administration of chlorin e6 has been established, and successfully treated for 52 cervical neoplasms patients [18]. However, there are no studies that evaluate the efficacy of topically Ce6-mediated PDT for HPV infectious diseases. The objective of this study was to assess the efficacy and safety of STBF-mediated photodynamic therapy (STBF-PDT) for cervical and vaginal LSIL. Patients were included if they met all of the following inclusion criteria: (1) Age 18 to 75 years; (2) All the included cases were evaluated by colposcopy and colposcopy-guided biopsy at the clinic; cervical or vaginal LSIL was initially histologically diagnosed by two independent pathologists; (3) Patients with cervical HR-HPV infection; Patients with or without vaginal LSIL; (4) Patients were explained about the study program and its potential risks before the start of the study and signed the informed consent. Patients were excluded for pregnancy, invasive cervical cancers, and invasive vaginal cancer. Patient characteristics include age, duration of HR-HPV infection, HPV types, history of previous cervical or vaginal diseases, and history of previous treatments.

| Colposcopy-guided photodynamic therapy
Before treatment, colposcopy was performed using a Leisegang 1D LED colposcope at a focal distance of 300 mm. The entire area of the abnormal epithelium including the cervix and vagina was colposcopically inspected after applying 3% acetic acid followed by iodine solution. Suspicious lesions biopsy was performed by colposcopy experts on white acetate epithelial areas and iodine test-negative area. Menopausal women without contraindications to estrogen use were treated with tibolone 2.5 mg orally for 1 week before colposcopy to ensure accuracy.
In this study, we used a new photosensitizer STBF (Guilin Xingda optoelectronic Medical Instrument Co., Ltd., Guangxi, China), the active component of which is the C67H95N11O11(Molar mass 1230.54 g/mol). In a dark room, freshly prepared 5 mg STBF powder was dissolved in 5 mL sodium bicarbonate solution to guarantee the stability and so 1 mg/mL STBF solution was ready. A thin cotton pad was soaked in the solution and covered the cervix and vaginal surface, especially the suspicious lesions, so the cotton sliver was inserted into the cervical canal to cover the cervical canal in 1 to 2 cm deep and the adjacent mucosa for 5 mm border. The external cervix was occluded with food-grade cling film and covered with thick gauze to avid light for 1 hour. The lesions were then illuminated using a red light (Diode laser, Biolitec AG, Vienna, Austria) at a wavelength of 630 nm. The illumination included two stages and in order to cover the cervical surface, and a bare fiber with a circular light spot was used in a light dose of 100 to 150 J/cm 2 . Furthermore, to ensure adequate illumination for the whole cervical canal and vaginal fornix, a 6cm columnar dispersion optical fiber whose distal end inserted into a disposable cannula is needed. The prepared optical fiber was inserted into the cervical canal at a depth of 1 to 2 cm in a light dose of 300 to 350 J/cm 2 . After irradiation, 33.3% trichloroacetic acid was applied to the suspicious lesions. All patients received STBF-PDT for 3 times with 1 to 2-week intervals, and underwent assessment 1 month after the third treatment. Patients with HPV infection and colposcopy results improved, but those with no complete remission would receive 2 to 3 times of consolidation treatments or were followed up according to their wishes.

| Follow-up and assessment
The postoperative assessment involved a cytological examination and HPV detection at first month, third month, and sixth month post-treatment. All cases continued to be followed up every year. Efficacy was evaluated by HPV detection and cytological examination first, and targeted tissue biopsy was performed by colposcopy experts. When patient's cervical squamous column junction was insufficiently visible, endocervical curettage (ECC) should be done to avoid the possibility of missed diagnosis of lesions in the cervical canal. Women without evidence of abnormal pathology were considered to demonstrate complete remission (CR). The CR rate was defined as the number of CR/ total follow-up *100%. HPV remission rates were defined as the number of HPV improvement/ total follow-up *100%. HPV clearance rates were defined as the number of HPV infection complete elimination/ total follow-up *100%. At each followup visit, the patients were interviewed and examined. Symptoms and adverse events related were recorded to evaluate the safety and tolerability of STBF-PDT.

| Statistical analysis
Data were analyzed using SPSS 20.0. For the quantitative variable, the t-test was used to compare group differences. For categorical variables, the Pearson χ 2 test was used for group comparisons. The significance level was set at P < 0.05.  Table 1 and Table 2.

| Factors affecting the efficacy of STBF-PDT
As shown in Table 3, patients vary in age, duration, and other characteristics. We tried to identify the correlative factors affecting CR by analyzing the relationships between efficacy and various factors, including age, HPV subtype, duration, treatment histories, lesion range, lesion features (adhesion or scar formation), and patient compliance (regular treatment as schedule and healthy living habits). The results showed no statistical difference due to the small number of cases shown in Table 4. It is indicated that a large sample of cases are needed for further analysis. third-month follow-up. It is exciting that 94.44% of cases achieved HPV infection remission at 6-month follow-up after 3 to 6 times of treatments, demonstrating the ability to eliminate HPV infections.

| Adverse reactions
Topical PDT has no systemic adverse reactions generally and does not destroy the normal anatomical structures and functions. Possible adverse reactions during treatment and follow-up were recorded. The former include pain, burning sensation, heaviness, defecated desire, and bleeding, and the latter include slight discomfort in the lower abdomen, increased vaginal secretion, adhesions, scar, and infection, which are shown in Table 5. There is no patient who experienced pain during the procedure and one patient felt a mild burning sensation during illumination and remit after treatment. One patient felt a slight discomfort in the lower abdomen while seven patients complained of increased vaginal secretions, both of which were bearable and relieved 3 to 5 days post-treatment. There were no other adverse reactions observed. Figure 1.

| DISCUSSION
PDT has been confirmed as a safe and effective treatment option for condyloma acuminatum, actinic keratosis, and other dermatologic conditions, and the most commonly used photosensitizer is 5-ALA. Hillemanns, Peter et al. [19] reviewed 93 VIN patients treated with either CO 2 laser vaporization, PDT, excision, or vulvectomy between 1991 and 2001, and found the recurrence rates between the CO 2 laser, PDT, and surgical excision did not differ significantly. A previous study revealed that the remission rate of CIN1 treated with ALA-PDT was 57%, and the clearance rate of ALA-PDT combined with CO 2 laser for vaginal LSIL was 85% [20]. In a retrospective study, 285 cervical LSIL patients associated with HR-HPV infection were treated with ALA-PDT, 84.88% of whom achieved CR and 64.34% got HR-HPV cleared [21]. Ran R et al. [22] enrolled 33 patients with cervical LSIL to receive ALA-PDT, and the histology remission rate 1 mo after six sessions treatment was 69.57%. The CR rate in this pilot study was 72.22% and 83.33% at the first-and the third-month follow-up, which was similar to previously reported studies of ALA-PDT. Half of the patients were over 50 y old, and they have been treated with surgery and repeated laser treatments. Seven cases had adhesion and scar formation, which may lead to insufficient illumination and affect the effect, although there is no statistical difference.
The HPV remission rate and HPV clearance rate were 88.89% and 83.33% at the first-month follow-up, respectively. The HPV clearance rates is similar to previous studies [23], and those results indicated that PDT can help in clearing HPV infections and patients could benefit from it. In addition, several patients could achieve CR after adding the treatment times, suggesting that the treatment parameters of STBF-PDT need further optimization, including the light dose, treatment interval, frequency, and so on. A direct comparison of efficacy between STBF-PDT and ALA-PDT may require further large-sample, multicenter studies.
PDT is a non-invasive technique during which photosensitizers can selectively accumulate in warty lesions, subclinical lesions, and latent HPV-infected cells, and produce ROS to exert a photodynamic effect, killing diseased cells [24]. In this study, the new photosensitizer named STBF is a derivative of Ce6 with high singlet oxygen generation efficiency and can be quickly eliminated from the human body, making it a promising photosensitizer [25]. STBF is the hydrophilic modification of Ce6 and makes it good biosafety. In addition, STBF solution was applied topically during treatment, and strong red fluorescence can be observed within a short incubation time due to its photosensitivity and there was no need for intracellular transformation. In this study, the cervical and vaginal lesions were incubated with STBF solution for 1 h, and obvious fluorescence could be observed under the Wood's lamp. Compared with 5-ALA, the latter requires absorption and transformation, so the incubation time is longer, even up to 3 to 4 h, which prolongs the hospitalization time. In addition, we found that the absorption of STBF in mucous appeared better than that of skin in practice, which may indicate the application prospect of this photosensitizer in oral, genital, and bladder diseases. However, the characteristics of this photosensitizer and the specific mechanisms still need further research.
There were no obvious adverse effects observed during and after STBF-PDT, and several patients complained of burning sensation, slight discomfort in the lower abdomen, and increased vaginal secretions, and got relieved soon. In particular, none of the patients complained of pain during illumination and quit treatments. When treated with ALA-PDT, some patients complained of unbearable adverse reactions and had to interrupt the treatment. Pain is the main adverse reaction of ALA-PDT, which usually reaches a peak within a few minutes after the start of irradiation and affects the patient's treatment experience [10]. It has been reported that 24.1% patients feel mild-to-moderate pain during ALA-PDT [14]. Besides, other side effects such as burning sensation during treatment, urinary retention, and keeping in a fixed position for a long time could affect patient's satisfaction and compliance.
Multicentric lesions are considered a risk factor for recurrence [26]; therefore, for patients with LSIL in both the cervix and vagina, comprehensive treatment is needed. Although CO 2 laser vaporization is established as the standard procedure for the conservative treatment of VAIN, it is difficult to cover multipoint lesions, while PDT can be extensive and repeated. On the other hand, the laser may cause serious side effects, such as bleeding, adhesion, and "dead angle" [27]. The safety and tolerability of STBF-PDT were demonstrated in this study.

| CONCLUSION
In conclusion, STBF-PDT is a novel and noninvasive therapy for cervical and vaginal LSIL. Particularly, it not only has high efficacy on cervical and vaginal LSIL but is also safe and tolerable with lower adverse reaction rates. This method has many unique advantages and promises a wide scope of application. However, prospective studies with more cases and longer follow-up times are necessary for further clinical applications.

FUNDING INFORMATION
This work was supported by the general project of Science and Technology Commission of Shanghai Municipality (21Y11905000).

CONFLICT OF INTEREST
All authors have read and agreed to the published version of the manuscript. The authors confirm no financial interests in any business or commercial entity that are related to the manuscript.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.