Randomized trial of calcipotriol combined with 5-fluorouracil for skin cancer precursor immunotherapy

Abstract

Background: Actinic keratosis is a precursor to cutaneous squamous cell carcinoma. Long treatment durations and severe side effects have limited the efficacy of current actinic keratosis treatments. Thymic stromal lymphopoietin (TSLP) is an epithelium-derived cytokine that induces a robust antitumor immunity in barrier-defective skin. Here, we investigated the efficacy of calcipotriol, a topical TSLP inducer, in combination with 5-fluorouracil (5-FU) as an immunotherapy for actinic keratosis.

Methods: The mechanism of calcipotriol action against skin carcinogenesis was examined in genetically engineered mouse models. The efficacy and safety of 0.005% calcipotriol ointment combined with 5% 5-FU cream were compared with Vaseline plus 5-FU for the field treatment of actinic keratosis in a randomized, double-blind clinical trial involving 131 participants. The assigned treatment was self-applied to the entirety of the qualified anatomical sites (face, scalp, and upper extremities) twice daily for 4 consecutive days. The percentage of reduction in the number of actinic keratoses (primary outcome), local skin reactions, and immune activation parameters were assessed.

Results: Calcipotriol suppressed skin cancer development in mice in a TSLP-dependent manner. Four-day application of calcipotriol plus 5-FU versus Vaseline plus 5-FU led to an 87.8% versus 26.3% mean reduction in the number of actinic keratoses in participants (P < 0.0001). Importantly, calcipotriol plus 5-FU treatment induced TSLP, HLA class II, and natural killer cell group 2D (NKG2D) ligand expression in the lesional keratinocytes associated with a marked CD4+ T cell infiltration, which peaked on days 10-11 after treatment, without pain, crusting, or ulceration.

Conclusion: Our findings demonstrate the synergistic effects of calcipotriol and 5-FU treatment in optimally activating a CD4+ T cell-mediated immunity against actinic keratoses and, potentially, cancers of the skin and other organs.

Trial registration: ClinicalTrials.gov NCT02019355.

Funding: Not applicable (investigator-initiated clinical trial).

Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1. Mechanistic studies on the TSLP-dependent antitumor effect of calcipotriol against skin carcinogenesis.

(A and B) WT (TSLPR+/–) and TSLPR–/– sex-matched littermates were treated with the standard DMBA-TPA skin carcinogenesis protocol. Animals received calcipotriol (80 nmol) or EtOH (carrier only) on the back skin 3 times per week during the 15-week TPA treatment period (n ≥ 19 for each group). **P < 0.01, by log-rank test (A) and *P < 0.05 versus the WT plus EtOH group, by Student’s t test (B). (C) Scheme used to investigate the role of transient TSLP induction by calcipotriol in skin cancer development. Age- and sex-matched WT mice were treated on their back skin with the standard DMBA-TPA protocol. At the first sign of tumor development (week 5), the animals were randomized into 2 groups and received calcipotriol (80 nmol) or EtOH in their ears for 3 consecutive days. Thereafter, the animals continued to receive TPA biweekly and were analyzed at 15 weeks (n ≥ 4 for each group). (D) Serum TSLP levels after 3 days of topical treatment with calcipotriol versus EtOH. *P < 0.05, by Student’s t test. (E) Number of tumors developed on each mouse after calcipotriol/EtOH treatment. *P < 0.05, by Student’s t test. (F) Representative pictures of the tumor-bearing mice and average weight of the 7 largest tumors in each group. Scale bar: 1 cm.

Figure 2. CONSORT diagram of the clinical trial.

Flow chart shows the number of patients who were screened, randomized into the treatment groups, completed the study, and included in the final analysis.

Figure 3. Reduction in the number of actinic keratoses by week 8 after treatment.

Box-and-whisker plots demonstrate twice-daily application of calcipotriol plus 5-FU versus Vaseline plus 5-FU for 4 days efficacy in eliminating actinic keratoses (AKs) on the face, scalp, RUE, and LUE. P values were determined by Student’s t test. The percentage of reduction in the number of actinic keratoses for the participant who was excluded from the analysis due to immunosuppression is shown as a red circle on the face plot.

Figure 4. Photographs and clinical evaluation of skin reactions.

(A) Representative photographs of the 4 anatomical sites treated with calcipotriol plus 5-FU versus Vaseline plus 5-FU. Photographs are of 8 participants before (day 0) and after treatment (day 5 and week 8). (B) Pie charts demonstrate the percentage of distribution of the participants’ erythema scores (defined in Supplemental Table 2) for the 4 treated anatomical sites in the calcipotriol plus 5-FU and Vaseline plus 5-FU groups immediately after treatment (day 5). P values were determined by Fisher’s exact test.

Figure 5. Therapy-induced histological changes in actinic keratosis.

(A) Histological images of the actinic keratoses after treatment (day 5) demonstrate the magnitude of actinic keratosis inflammation, including CD4+ and CD8+ T cell infiltration into the lesions in the calcipotriol plus 5-FU versus Vaseline plus 5-FU groups. Note the degree of dermal and epidermal immune infiltrate and epidermal spongiosis and dyskeratosis present in the H&E-stained images. Tissue staining was performed on adjacent sections, and representative images of actinic keratoses on all 4 anatomical sites are shown. (B) Pie charts show the percentage of distribution of the immune-mediated actinic keratosis rejection grades (defined in Supplemental Table 4) in the calcipotriol plus 5-FU (n = 21) and Vaseline plus 5-FU (n = 20) groups after treatment (day 5). The significant difference between the 2 treatment groups was independent of the anatomical site of the actinic keratoses. P value was determined by type III test of means in a mixed random effects ANOVA.

Figure 6. Induction of TSLP, HLA class II, and NKG2D ligand in response to calcipotriol plus 5-FU combination therapy.

(A) Representative images of actinic keratoses stained with H&E, TSLP and keratin 14 (K14), and HLA DP/DQ/DR (class II) and CD4, before and after treatment. K14 staining marks the proliferative keratinocytes of actinic keratoses. Black arrow points to the site’s lymphocytic aggregate at the epidermal-dermal junction, and red arrows point to lesional keratinocytes that expressed HLA class II in the calcipotriol plus 5-FU–treated actinic keratoses. (B) Graphs demonstrate the expression of the cellular stress genes relative to GAPDH in actinic keratoses before and after treatment with calcipotriol plus 5-FU (C+F, n = 11) or Vaseline plus 5-FU (V+F, n = 10). P values are shown in each graph and were determined by paired Student’s t test. Note that the gene expression analysis was performed on actinic keratosis biopsies that were obtained before and after treatment from the same anatomical site on the face or scalp from each participant. Scale bars: 100 μm.