Ex vivo evaluation of cytotoxicity and melanocyte viability after A-101 hydrogen peroxide topical solution 40% or cryosurgery treatment in seborrheic keratosis lesions

To the Editor: Seborrheic keratosis (SK) is one of the most common types of dermatologic lesions and is, therefore, the most common skin tumor seen by dermatologists in everyday practice.1Coyne J.D. Classification of the seborrheic keratosis.Int J Surg Pathol. 2016; 24: 51-52Crossref PubMed Scopus (4) Google Scholar Currently, cryosurgery is one of the most commonly employed and successful techniques to physically resolve an SK.2Farhangian M.E. Snyder A. Huang K.E. Doerfler L. Huang W.W. Feldman S.R. Cutaneous cryosurgery in the United States.J Dermatolog Treat. 2016; 27: 91-94PubMed Google Scholar However, all patients are susceptible to some degree of blistering, infection, scarring, recurrence, and pigmentary changes after treatment, but darker-skinned individuals are particularly vulnerable to the last 3 adverse events.3Andrews M.D. Cryosurgery for common skin conditions.Am Fam Physician. 2004; 69: 2365-2372PubMed Google Scholar To help predict the potential toxicologic impact of A-101, a recently Food and Drug Administration–approved topical 40% hydrogen peroxide solution for the treatment of SK,4Draelos Z.D. Kempers S. Smith S.R. et al.Safety and efficacy of A-101 hydrogen peroxide topical solution in adults with seborrheic keratosis: results from the phase 3, randomized, double-blind, vehicle-controlled, parallel-group study.Skin J Cutan Med. 2017; 1: s119Crossref Google Scholar compared with the gold standard cryosurgery, we evaluated skin architecture, metabolic activity, and cytotoxicity with a particular emphasis on melanocytes, using a validated ex vivo human reconstituted full thickness model derived from Fitzpatrick V skin (MRHE).5Yoon T.J. Lei T.C. Yamaguchi Y. Batzer J. Wolber R. Hearing V.J. Reconstituted 3-dimensional human skin of various ethnic origins as an in vitro model for studies of pigmentation.Anal Biochem. 2003; 318: 260-269Crossref PubMed Scopus (60) Google Scholar MRHEs were treated with cryosurgery (5-second and 10-second freeze cycles) or A-101 (1 μL and 2 μL) and analyzed by MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] assay, Fontana-Masson, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling), and S100 immunohistochemical staining to determine skin architecture, metabolic activity, and cytotoxicity. Histologic evaluation of untreated and A-101 vehicle–treated tissues revealed a normal stratum corneum, with spinous and basal cell layers overlying a well-organized dermis with scattered fibroblasts. MRHEs treated with cryosurgery for 5 s or 10 s both demonstrated overall thinning of the epidermis, with more pyknotic cells noted in the deeper spinous layer of the 10-second specimens. In both groups, dermal-epidermal junction separation was noted, though more prominent in the 10-second group. In both A-101–treated groups, acanthosis of the epidermis and mild pallor was noted, though not to the extent of the cryosurgery-treated specimens, without epidermal clefting (data not shown). To quantify the degree of epidermal damage incurred by treatment with cryosurgery or A-101, TUNEL staining was utilized to identify apoptotic cells. Cryosurgery for 5 s and 10 s resulted in 16.4 ± 0.6424 TUNEL-positive cells/high-power field (HPF) and 20.6 ± 0.6424 TUNEL-positive cells/HPF, higher values compared with the 2 doses of A-101 used, 8.65 ± 0.4122 TUNEL-positive cells/HPF and 12.4 ± 0.3728 TUNEL-positive cells/HPF for 1 μL and 2 μL, respectively (Fig 1, A). Cellular metabolic activity was assessed using MTT assay, showing reduced metabolic activity indicative of reduced viability in cryosurgery-treated MRHE samples compared with A-101–treated groups (Fig 1, B). Within HPFs, untreated and vehicle-treated MRHEs were found to have 2.5 ± 0.1987 melanocytes and 2.0 ± 0.5000 melanocytes, respectively, based on S100 staining (Fig 1, C). After cryosurgery for 5 s and 10 s, the number of melanocytes were 0.45 ± 0.1535 cells and 0.2 ± 0.0918 cells, respectively, and after A-101 treatment for 1 μL and 2 μL, 1.95 ± 0.1535 cells and 1.95 ± 0.1535 cells, respectively. Together, we show that cryosurgery is more cytotoxic than A-101, with special consideration of melanocyte survival. Our data suggests that A-101 might be a less caustic option for SK removal that reduces the risk of posttreatment pigmentary alterations. However, to best appreciate these findings, a clinical study that examines the risk for hypopigmentation or hyperpigmentation in darker skin types after A-101 treatment is warranted and is ongoing (ClinicalTrials.gov Identifier: NCT03224598).