Skin cancer precursor immunotherapy for squamous cell carcinoma prevention

Abstract

Background: Topical calcipotriol plus 5-fluorouracil (5-FU) combination is an effective immunotherapy against actinic keratosis (AK), which is a precursor to squamous cell carcinoma (SCC). However, the long-term effectiveness of calcipotriol plus 5-FU treatment for SCC prevention is unknown.

Methods: We performed a blinded prospective cohort study on participants of a randomized double-blind clinical trial in which a 4-day course of topical calcipotriol plus 5-FU combination was compared to Vaseline plus 5-FU (control) for AK treatment. SCC and basal cell carcinoma (BCC) incidences were assessed at 1, 2, and 3 years after trial. Tissues were analyzed for calcipotriol plus 5-FU-induced T cell immunity in the skin.

Results: Calcipotriol plus 5-FU-induced tissue-resident memory T (Trm) cell formation in face and scalp skin associated with significantly higher erythema scores compared with control (P < 0.01). Importantly, more participants in the test cohort remained SCC-free over the more than 1,500-day follow-up period (P = 0.0765), and significantly fewer developed SCC on the treated face and scalp within 3 years (2 of 30 [7%] versus 11 of 40 [28%] in control group, hazard ratio 0.215 [95% CI: 0.048-0.972], P = 0.032). Accordingly, significantly more epidermal Trm cells persisted in the calcipotriol plus 5-FU-treated face and scalp skin compared with control (P = 0.0028). There was no significant difference in BCC incidence between the treatment groups.

Conclusion: A short course of calcipotriol plus 5-FU treatment on the face and scalp is associated with induction of robust T cell immunity and Trm formation against AKs and significantly lowers the risk of SCC development within 3 years of treatment.

Funding: This research was supported by internal academic funds and by grants from the Burroughs Wellcome Fund, Sidney Kimmel Foundation, Cancer Research Institute, and NIH.

Keywords: Adaptive immunity; Cancer immunotherapy; Dermatology; Oncology; Skin cancer.

Figure 1. CONSORT diagram of clinical trial followed by prospective cohort study.

Flow chart illustrates the number of clinical trial participants randomized to each treatment group who were included in analyses at 1, 2, and 3 years after treatment. Participants were excluded if they had fewer than 365 days of follow-up after the clinical trial or were immunosuppressed due to a medical condition or therapy during the follow-up period.

Figure 2. Immune activation, erythema, and Trm induction by calcipotriol plus 5-FU treatment.

(A and B) The inflammation grades of AK biopsies are compared with the (A) erythema extent and (B) erythema intensity on the biopsied anatomical sites 1 day after completion of a 4-day treatment course. Note that the data include all participants in the prospective cohort study who contributed an AK biopsy during the clinical trial. P values were calculated by Fisher’s exact test. (C) Stacked bar chart compares the distribution of participants’ erythema extent in each treatment group for each treated anatomical site. **P < 0.001, ***P < 0.0001 by Fisher’s exact test. ns, not significant. (D) Stacked bar chart compares the distribution of participants’ erythema intensity in each treatment group for each treated anatomical site. *P < 0.01, **P < 0.001, ***P < 0.0001 by Fisher’s exact test. ns, not significant. (E) Representative histologic images show AKs that were biopsied on the face and scalp 1 day after treatment with calcipotriol plus 5-FU (C+F) or Vaseline plus 5-FU (V+F). Tissue samples were stained with H&E and with antibodies against CD3 plus CD4, CD69, or CD103 to detect induction of CD4⁺ T cell immunity and CD69⁺CD103⁺ Trm cells in the lesional skin. Erythema intensity associated with the site of the biopsies: face C+F, 3; face V+F, 0; scalp C+F, 2; scalp V+F, 1. Scale bars: 100 μm. (F) CD4⁺ T, CD8⁺ T, CD103⁺CD4⁺ Trm, and CD103⁺CD8⁺ Trm cells are quantified across 24 high-power field (hpf) images of 3 test (C+F) AKs and 20 hpf images of 3 control (V+F) AKs biopsied 1 day after treatment completion. **P < 0.001, ***P < 0.0001 by Wilcoxon’s rank-sum test.

Figure 3. Proportion of SCC-free participants on the treated face and scalp over time and the accompanying Trm cell persistence in the skin.

(A) Kaplan-Meier analysis shows that at any given time over more than 1,500 days of follow-up, a higher proportion of participants in the calcipotriol plus 5-FU treatment arm remained free of squamous cell carcinoma (SCC) on the treated face and scalp compared with controls. This trend approaches statistical significance. (B) Representative immunostaining images depict epidermal CD4⁺ and CD8⁺ Trm cells in the perilesional skin regions of AK-SCC spectrum biopsies obtained from the face and scalp of the participants after trial (arrows point to epidermal CD4⁺ Trm cells). Scale bar: 100 μm. (C) Bar graphs show the number of epidermal CD4⁺ and CD8⁺ Trm cells in the perilesional normal skin from 22 participants, counted blindly and averaged across 10 random hpf per sample. Note that every AK-SCC spectrum biopsy that was obtained from the face and scalp of the participants during the >3-year period after trial completion was included in this analysis. P values were calculated by Wilcoxon’s rank-sum test.