In Vivo Evaluation of Melasma Pathologic Features and Treatment Response by 2-Photon Microscopy

Importance: Melasma, a complex and clinically challenging facial pigmentary disorder, lacks in vivo pathologic tools for definitive diagnosis and accurate efficacy assessment. Objective: To use 2-photon microscopy (TPM) for noninvasive pathologic detection of melasma and therapeutic response monitoring to hydroquinone cream, 2%. Design, Setting, and Participants: This single-center, evaluator-blinded, observational study consisting of 2 sequential phases was conducted at the Dermatology Outpatient Clinic of China-Japan Friendship Hospital from January to September 2024. The study population included patients aged 18 to 60 years with clinically diagnosed melasma who were not treated for the condition in the past 3 months, without other facial dermatoses, free of malignant disease history, and not pregnant and nonlactating. Exposure and Intervention: In the first phase, participants underwent noninvasive TPM 3-dimensional imaging of lesional and perilesional areas. In the second phase, all participants were treated with hydroquinone cream, 2% twice daily for 12 weeks. Main Outcomes and Measures: In phase 1, the main outcome was TPM pathologic features. In phase 2, the main measures were modified Melasma Area and Severity Index (mMASI) scores, dermoscopic scores, and TPM indexes at baseline and weeks 4, 8, and 12. Results: The TPM images of 120 lesional and perilesional areas from 60 patients (57 female individuals, 3 male individuals; median [IQR] age, 39.5 [37.0-45.8] years) were analyzed. Compared with the perilesions, melasma lesions showed increased melanin across all epidermal layers (stratum corneum: median [IQR], 1 [0-2] vs 0 [0-0]; stratum granulosum: median [IQR], 1 [1-2] vs 0 [0-0]; superficial stratum spinosum: median [IQR], 2 [1-3] vs 1 [1-1]; deep stratum spinosum: median [IQR], 3 [2-3] vs 2 [1-2]; stratum basale: median [IQR], 3 [3-4] vs 2 [2-3]) in a mottled distribution, reduced viable epidermal thickness (median [IQR], 43.5 [39.0-48.0] µm vs 48.0 [43.9-54.0] µm; median difference, -4.50; 95% CI, -6.58 to -3.70), flattened rete ridges (94.17%; 95% CI, 88.45%-97.15% vs 69.17%; 95% CI, 60.42%-76.73%), increased activated (median [IQR], 2 [1-2] vs 1 [1-1]; median difference, 1; 95% CI, 0.70-1.00) and pendulous (median [IQR], 0 [0-1] vs 0 [0-0]) melanocytes at the dermal-epidermal junction (DEJ), and more severe solar elastosis (median [IQR], 2 [2-3] vs 2 [1-2]) (all P<.001). Among them, 53 patients (88.3%) completed 12 weeks of hydroquinone treatment, the mean mMASI scores and dermoscopic pigmentary scores decreased by 29.81% (95% CI, 22.75%-36.72%; P < .001) and 36.16% (95% CI, 30.07%-42.25%; P < .001), respectively, after treatment. TPM showed significant decreases in melanin content of each epidermal layer (stratum corneum: median [IQR], 1 [1-2] vs 1 [0-1]; stratum granulosum: median [IQR], 2 [1-2] vs 1 [0-1]; superficial stratum spinosum: median [IQR], 2 [2-3] vs 1 [1-2]; deep stratum spinosum: median [IQR], 3 [3-3] vs 2 [2-3]; and stratum basale: median [IQR], 3 [3-4] vs 2 [2-3]; all P < .001) and activated melanocytes at the DEJ (median [IQR], 2 [2-2] vs 1 [1-2]; P < .001) after treatment, but no statistical changes in pendulous melanocytes or solar elastosis during treatment. Conclusions and Relevance: This observational study found that TPM may offer a new paradigm in pigmentary disorder management by enabling in vivo detection of pathologic features and assessment of cellular-level treatment responses.