Materials and Methods
The clinical trial was conducted according to European Medicines Agency and Food and Drug Administration guidelines, and in accordance with the International Conference on Harmonization Guideline for Good Clinical Practice and the Declaration of Helsinki. The study is registered in the Clinicaltrials.gov (National Institutes of Health) database ( NCT01256424 ). Ethical approval was obtained from all clinical sites and all subjects provided written informed consent before inclusion.
This was a phase 2b, prospective, randomized, double-blind, placebo-controlled, multicenter study conducted at 23 centers in the United States, Germany, Czech Republic, Slovak Republic, and Norway. A total of 262 adult women (age ≥18 years) with satisfactory colposcopy examination including visibility of the entire transformation zone with the entire lesion margin, average-sized uterine cervix (approximately 27-mm diameter), and colposcopically identified CIN 1 or CIN 2 based on local pathology read of biopsy were included. Main patient exclusion criteria were inadequate colposcopy, lesion(s) extending to the vaginal vault, suspicion of endocervical disease on colposcopy, previous treatment of CIN or invasive disease, significant vaginal infection or bleeding, porphyria, known drug sensitivity, pregnancy, or breast-feeding.
The expected proportions of patients with complete or partial response were set to 20% for placebo and 50% for HAL PDT. A 2-sided significance level of 5% and statistical power of 80% were used to calculate the sample sizes.
Eligible subjects were randomized equally to 4 arms: HAL 5%, 1%, 0.2%; and placebo ointments. Randomization was stratified by site in a block size of 4. Drug and illumination were administered by the intravaginal device ( Figure 1 ). The gynecologist spread the ointment evenly in the device cavity before positioning it on the cervix, thereafter the patient was free to return to normal daily activities. The device supported targeted delivery of HAL ointment to the portio for 5 hours, enabling selective enrichment of photoactive porphyrins in the diseased area in subjects who received active drug, followed by automatic photoactivation (illumination) for another 4.6 hours. The device was designed and programmed to deliver red light (629 nm) with a total dose of 100 J/cm 2 exclusively to the portio of the ectocervix in the active treatment groups, with no light in the placebo ointment group.
The primary study endpoint was patient response at 3 months with assessment of histology and cytology regression and clearance of oncogenic HPV. Subjects with abnormal histology or cytology at 3 months received a second treatment followed by a new biopsy 3 months after retreatment. Secondary endpoints were patient response (cytology and oncogenic HPV) 6 months after treatment and safety assessments. Subjects with disease progression or a need for immediate treatment were offered standard-of-care treatment at the discretion of the investigator. Colposcopic-directed biopsies were obtained from all suspicious areas at baseline and 3-month follow-up. In normal-appearing epithelium at colposcopy follow-up, biopsies were obtained from the most severe baseline lesion areas. For efficacy analyses all baseline and follow-up biopsies were reviewed by a central expert gynecology pathologist (DCL Laboratories, Urbana, IL) blinded to other study data.
Oncogenic HPV DNA was performed using an in-house polymerase chain reaction method, genotyping high-risk HPV DNA 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 (NorChip, Klokkarstua, Norway). Only the oncogenic HPV genotypes present at baseline were evaluated for clearance.
Subjects with baseline CIN 1 were a priori determined to have a clinical response at 3 months’ follow-up if: (i) histology and cytology were both normal; or (ii) histology and/or cytology showed low grade (CIN 1/atypical squamous cells of undetermined significance/low-grade squamous intraepithelial lesions) and the baseline HPV infection was cleared. At 6 and 9 months, subjects with baseline CIN 1 had a clinical response if: (i) cytology was negative; or (ii) cytology was atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesions and baseline HPV infection was cleared. Subjects with baseline CIN 2 were regarded as having response at 3 months’ follow-up if histology and cytology were normal or low grade. At 6 and 9 months, subjects with CIN 2 at baseline were responders if cytology was normal or low grade. According to clinical guidelines, patients underwent LEEP or colposcopy follow-up including cytology and HPV testing when indicated. Safety was evaluated by recording adverse events, vital signs, blood biochemistry, and concomitant medication use.
Statistical analyses were performed on the total CIN 1/2 population and on the CIN 1 and CIN 2 subsets. Each of the active treatment groups were compared with the placebo group. Summary statistics and data analysis were performed using software (SAS; SAS Institute Inc, Cary, NC). The efficacy endpoint analyses were based on the Fisher exact test. Safety data were summarized by treatment group. In patients who were withdrawn during follow-up, data from the previous follow-up were used for efficacy assessment (last observation carried forward).
Results
All randomized patients who received at least 1 treatment were included in the safety evaluations. A total of 262 patients were enrolled and treated. The median age was 27 years (range, 18–60), and 258 subjects (98%) were Caucasian, the remaining subjects were sub-Sahara African. Mean body mass index was 23.2 (range, 16–54). Age, race, and body mass index characteristics were similar across the treatment groups. After central pathology review of screening biopsies, 118 subjects were classified as having CIN 1 and 89 subjects with CIN 2 ( Table 1 ). The remaining subjects were normal or reclassified as having CIN 3, and were not included in the efficacy analyses. Oncogenic HPV infections were observed in 49% of subjects with CIN 1 and 83% of subjects with CIN 2.
| Variable, n (%) | HAL 5% (n = 65) | HAL 1% (n = 67) | HAL 0.2% (n = 62) | Placebo (n = 68) | Total a (n = 262) |
|---|---|---|---|---|---|
| CIN 1 | 34 (52) | 27 (40) | 26 (43) | 31 (46) | 118 (45) |
| Oncogenic HPV DNA+ | 19 (56) | 12 (44) | 12 (46) | 15 (48) | 58 (49) |
| HPV 16/18 b | 7 (37) | 3 (25) | 4 (33) | 3 (20) | 17 (29) |
| Oncogenic HPV DNA- | 15 (44) | 15 (57) | 14 (54) | 16 (52) | 60 (51) |
| CIN 2 | 19 (29) | 30 (45) | 19 (31) | 21 (31) | 89 (34) |
| Oncogenic HPV DNA+ | 13 (68) | 26 (87) | 17 (89) | 18 (86) | 74 (83) |
| HPV 16/18 b | 6 (46) | 15 (58) | 7 (41) | 6 (33) | 33 (46) |
| Oncogenic HPV DNA- | 6 (32) | 4 (13) | 2 (11) | 3 921) | 15 (20) |
| Normal, CIN 3, or missing | 12 (18) | 10 (15) | 17 (2) | 16 (24) | 55 (21) |
a Of 262 patients included, based on local pathology, 207 subjects were verified as having CIN 1 or CIN 2 based on central read and included in main statistical analysis. 55 of 262 subjects were diagnosed with CIN 3, normal, or missing histology after central read
b Percentage of high-risk HPV 16/18 that is oncogenic DNA positive.
The majority of patients (88%) completed the study. Of 31 patients who discontinued, 13 withdrew consent, 8 were lost to follow-up, and 10 were withdrawn: 3 by the gynecologist, 1 patient due to noncompliance, and 6 due to other causes (eg pregnancy and progression to CIN 3). The number of withdrawals was similar across treatment groups.
In all, 136 patients (52%) received a retreatment, due to initial noncomplete response. A lower number of retreatments in the low-dose and placebo groups were due to patient withdrawal in these groups.
Efficacy
There were 262 patients in the intention-to-treat set, which was used for all statistical analyses.
In the CIN 1 and combined CIN 1/2 populations there were no statistically significant differences in the number of responders in the HAL dose groups compared to placebo at any time point.
In subjects with CIN 2, HAL 5% showed a significantly higher response of 95% (18/19 patients) compared to 57% (12/21 patients) in the placebo group ( P = .009), with a clear dose response ( Figure 2 and Table 2 ). Of the 18 responders in the HAL 5% group, 4 had normal histology and cytology. In the placebo group, no patients had normal histology and cytology at 3 months after last treatment. Six months after last treatment a sustained response of 95% (18/19 patients) was observed in the HAL 5% group, still significantly higher compared to the placebo effect of 62% (13/21 patients) ( P = .021) ( Figure 2 ). HAL 1% and HAL 0.2% were not significantly different from placebo at any time points.
| Variable | HAL 5% | HAL 1% | HAL 0.2% | Placebo |
|---|---|---|---|---|
| Patients with CIN 2, n | 19 | 29 | 19 | 21 |
| 3 mo after last treatment | ||||
| Responders, n (%) | 18 (95) | 20 (69) | 12 (63) | 12 (57) |
| P value | .009 | .551 | .755 | |
| 6 mo after last treatment | ||||
| Responders, n (%) | 18 (95) | 23 (79) | 15 (79) | 13 (62) |
| P value | .021 | .213 | .311 | |
Retreatment of 2 subjects in the HAL 5% group with remaining high-grade disease (CIN 2+) after first treatment demonstrated histological regression and HPV 16/18 clearance in both patients. Retreatment of 5 subjects with remaining low-grade disease (histology or cytology) demonstrated lesion clearance in 1 patient.
The proportion of CIN 2 subjects with oncogenic HPV clearance 3 months after last treatment was 62% (8/13 subjects) in the HAL 5% group and 28% (5/18 subjects) in the placebo group ( P = .079). At 6 months after last treatment, this increased to 77% (10/13 patients) in the HAL 5% group and 39% (7/18 patients) in the placebo group ( P = .067) ( Figure 3 ).
Among subjects with CIN 2, the clearance of high-risk oncogenic HPV 16/18 in the HAL 5% group demonstrated a sustainable 83% response (5/6 patients) at both 3 and 6 months after last treatment compared to 0% (0/6 patients) and 33% (2/6 patients), respectively, after 3 and 6 months in the placebo group ( Figure 4 ).
As HPV clearance is an important goal of effective treatment, a post hoc analysis was conducted in subjects with CIN 2 at 6 months’ follow-up after last treatment defining complete response as absence of CIN 2+ and HPV. Using 3-month histology results at 6 months together with 6-month HPV results, there was again a clear dose-related response of 84% (16/19 patients) in the HAL 5% group, 48% (14/29 patients) in the HAL 1% group, 42% (8/19 patients) in the HAL 0.2% group, and 38% (8/21 patients) in the placebo group.
Safety
A total of 261 adverse events were reported by 125 patients. Mainly local and self-limiting adverse reactions were reported, with vaginal discharge, local discomfort, and spotting being the most common. A higher proportion of subjects in the HAL 5% group reported adverse reactions (54%) compared with the other groups (31-34%) ( Table 3 ).
| Variable, n (%) | HAL 5% (n = 65) | HAL 1% (n = 67) | HAL 0.2% (n = 62) | Placebo (n = 68) |
|---|---|---|---|---|
| Patients with at least 1 | ||||
| Adverse reaction a | 35 (54) | 21 (31) | 21 (34) | 22 (32) |
| Discharge | 21 (32) | 12 (18) | 9 (15) | 8 (12) |
| Local discomfort b | 24 (37) | 12 (18) | 9 (15) | 10 (15) |
| Hemorrhage (spotting) | 5 (8) | 8 (12) | 5 (8) | 4 (6) |
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