Plum-blossom needle tapping enhances the efficacy of ALA photodynamic therapy for facial actinic keratosis in Chinese population: a randomized, multicenter, prospective, and observer-blind study.

BACKGROUND: Photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) is a reliable treatment for actinic keratosis (AK), but its effect needs to be enhanced in thick lesions. Plum-blossom needle is a traditional Chinese cost-effective instrument for enhancing the transdermal delivery of ALA. However, whether it could improve the efficacy of AK treatment has not yet been investigated. OBJECTIVE: To compare the efficacy and safety of plum-blossom needle-assisted PDT in facial AK in the Chinese population. METHODS: In this multicenter, prospective study, a total of 142 patients with AKs (grades I-III) were randomized into the plum-blossom needle-assisted PDT group (P-PDT) and control PDT group (C-PDT). In the P-PDT group, each AK lesion was tapped vertically by a plum-blossom needle before the application of 10% ALA cream. In the C-PDT group, each lesion was only wiped with regular saline before ALA cream incubation. Then, 3 hours later, all the lesions were irradiated with light-emitting diode (LED) at a wavelength of 630 nm. PDT was performed once every 2 weeks until all lesion patients achieved complete remission or completed six sessions. The efficacy (lesion response) and safety (pain scale and adverse events) in both groups were evaluated before each treatment and at every follow-up visit at 3-month intervals until 12 months. RESULTS: In the P-PDT and C-PDT groups, the clearance rates for all AK lesions after the first treatment were 57.9% and 48.0%, respectively (P < 0.05). For grade I AK lesions, the clearance rates were 56.5% and 50.4%, respectively (P = 0.34). For grade II AK lesions, the clearance rates were 58.0% and 48.9%, respectively (P = 0.1). For grade III AK lesions, the clearance rates were 59.0% and 44.2%, respectively (P < 0.05). Moreover, grade III AK lesions in the P-PDT group required fewer treatment sessions (P < 0.05). There was no significant difference in the pain score between the two groups (P = 0.752). CONCLUSION: Plum-blossom needle tapping may enhance the efficacy of ALA-PDT by facilitating ALA delivery in the treatment of AK.

Single-cell transcriptomic analysis reveals the critical molecular pattern of UV-induced cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most common nonmelanoma skin cancer characterized by high invasiveness, heterogeneity, and mainly occurs in the ultraviolet (UV)-exposed regions of the skin, but its pathogenesis is still unclear. Here, we generated single-cell transcriptome profiles for 350 cells from six primary UV-induced cSCCs, together with matched adjacent skin samples, and three healthy control skin tissues by single-cell RNA-sequencing technology based on Smart-seq2 strategy. A series of bioinformatics analyses and in vitro experiments were used to decipher and validate the critical molecular pattern of cSCC. Results showed that cSCC cells and normal keratinocytes were significantly distinct in gene expression and chromosomal copy number variation. Furthermore, cSCC cells exhibited 18 hallmark pathways of cancer by gene set enrichment analysis. Differential expression analysis demonstrated that many members belonging to S100 gene family, SPRR gene family, and FABP5 were significantly upregulated in cSCC cells. Further experiments confirmed their upregulation and showed that S100A9 or FABP5 knockdown in cSCC cells inhibited their proliferation and migration through NF-κB pathway. Taken together, our data provide a valuable resource for deciphering the molecular pattern in UV-induced cSCC at a single-cell level and suggest that S100A9 and FABP5 may provide novel targets for therapeutic intervention of cSCC in the future.

5-Aminolevulinic acid photodynamic therapy for early-stage lip squamous cell carcinoma.

BACKGROUND: Squamous cell carcinoma (SCC) of the lip removed by surgery may cause lip dysfunction and scar. ALA-PDT (5-Aminolevulinic acid photodynamic therapy) is a minimally invasive treatment for superficial SCC in situ. However, few studies reported the use of topical ALA-PDT to manage lip SCC. METHODS: Between 2015 and 2017, 6 patients with Tis to T2 lip squamous cell carcinoma without evidence of lymph node spread, were treated with topical ALA-PDT at Shanghai Skin Disease Hospital. Clinical responses and side effect were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST) at 1 month, 6 months, 12 months and 24 months after ALA-PDT. RESULTS: All of the 6 patients achieved complete response (CR) by 2-7 sessions of ALA-PDT. There was no relapse during 24 months follow up for 4 patients. Two out of 6 patients relapsed at 10 months and 20 months post PDT but achieved CR again by 1-3 more sessions of PDT. There was no functional or aesthetic problem. CONCLUSION: Topical 5-Aminolevulinic acid photodynamic could be considered a potential alternative therapeutic option for early-stage lip squamous cell carcinoma without lymph node spread.

Comparison of efficacy, adverse effects and costs between 20 % ALA-PDT and 10 % ALA-PDT for the treatment of actinic keratosis in Chinese patients.

BACKGROUND: 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is a reliable treatment for actinic keratosis (AK), though the concentration of 20 % is applied in most ALA-PDT for the treatment of AK, it was found that 10 % ALA could achieve similar outcome during clinical practice. However, there are limited number of reports comparing the efficacy and the safety of ALA-PDT with different ALA doses when treating AK. OBJECTIVES: This study was designed to compare the efficacy, the adverse effects and the costs of 10 % and 20 % ALA-PDT for AK treatment. METHODS: We reviewed the records of patients who had received ALA-PDT in our department between January 1, 2012, and April 30, 2017, the efficacy, the adverse effects were recorded, and the costs were analyzed. Follow-up data were collected until April 30, 2018. RESULTS: A total of 59 AK patients' records were analyzed for the efficacy, the adverse events, the recurrence and the costs. All the AK lesions were tapped with plum-blossom needle as a pre-treatment before receiving ALA-PDT. There was no statistical difference between 10 % ALA-PDT and 20 % ALA-PDT in terms of efficacy and adverse events. In the 10 % ALA group, there was one recurrence out of 34 patients and in the 20 % group there were none. The average treatment cost of each lesion was significantly lower in 10 % ALA group than in 20 % ALA group. CONCLUSIONS: 10 % ALA-PDT has similar efficacy and adverse effects as 20 % ALA-PDT with lower costs. Taken together, this supports the use of 10 % ALA-PDT in clinical practice.

The effect of C-X-C motif chemokine ligand 13 in cutaneous squamous cell carcinoma treated with aminolevulinic acid-photodynamic therapy.

BACKGROUND: Active recruitment of inflammatory cells into tumors may be vital for antitumor immunity in cutaneous squamous cell carcinoma (cSCC) after photodynamic therapy. Chemokines play important roles in inflammatory cell recruitment. Moreover, C-X-C motif chemokine ligand 13 (CXCL13) is thought to be a pivotal chemokine involved in inflammatory response and antitumor effect. Here, we examined the roles of CXCL13 in the response of cSCC to ALA-PDT. METHODS: Microarray analysis was used to select the chemokines involved in cSCC treated with ALA-PDT. The expression and transcriptional activity of CXCL13 were assessed by immunohistochemistry and quantitative real-time polymerase chain reaction. Western blotting was used to detect C-X-C motif chemokine receptor 5 (CXCR5) expression. The role of CXCL13 in ALA-PDT efficacy was assessed in vivo. RESULTS: Microarray analysis of total 63 chemokines and their receptors showed that the expression of 21 chemokines and 13 receptors were up-regulated in cSCC after ALA-PDT; in particular, CXCL13 was significantly upregulated. Immunohistochemistry showed that cancer-associated fibroblasts (CAFs) may be the main source of CXCL13 upregulation in the cSCC microenvironment after ALA-PDT. The efficacy of ALA-PDT in the treatment of cSCC was significantly reduced after CXCL13 inhibition. CONCLUSION: CXCL13 plays important roles in the antitumor effect of ALA-PDT for cSCC and may originate mainly from CAFs in the cSCC microenvironment.

CCL8 enhances sensitivity of cutaneous squamous cell carcinoma to photodynamic therapy by recruiting M1 macrophages.

BACKGROUND: Antitumor immunity induced by photodynamic therapy (PDT) is believed to depend on the degree of local and systemic inflammation. The recruitment of leukocytes, in particular by the chemokine CCL8, to the sites of tissue damage has been strongly associated with the initiation of inflammatory reactions. OBJECTIVE: To evaluate whether and how CCL8 enhances the immune response against tumors in 5-aminolevulinic acid (ALA)-mediated PDT. METHODS: In this study, we investigated the effect of ALA-PDT-induced CCL8 expression on the recruitment and polarization of macrophages using immunohistochemistry, western blot and Transwell cell migration assay. We evaluated CCL8 expression following ALA-PDT in vitro and in vivo by using RT-PCR, western blot, and ELISA in clinical cutaneous squamous cell carcinoma (cSCC) samples, a mouse model of cSCC, tumor cells, and macrophages. The effect of the combination of ALA-PDT with CCL8 treatment on anti-tumor immunity was tested in the mouse model. RESULTS: We found that ALA-PDT enhanced CCL8 expression, increased the number of macrophages in tumor, and stimulated their M1 pro-inflammatory phenotype characterized by high expression levels of CD16 and CD80, low expression level of CD163, and absence of CD206 expression. Furthermore, CCL8 enhanced the effect of ALA-PDT on cSCC in mice, such a combination of CCL8 and ALA-PDT had a stronger positive effect in the treatment of mouse cSCC than PDT alone and suppressed tumor volume regrowth. CONCLUSION: ALA-PDT induces CCL8 expression and recruits M1 macrophages, thus suppressing tumor growth.

Conventional versus daylight photodynamic therapy for actinic keratosis: A randomized and prospective study in China.

BACKGROUND: Conventional photodynamic therapy (C-PDT) is an effective treatment for actinic keratoses (AKs) in Asia, with some unmet needs. Severe PDT-associated pain results in low treatment willingness. Daylight photodynamic therapy (DL-PDT) is a more simple and tolerable treatment that has the same efficacy as C-PDT in Europe. However, few studies have been conducted with Asian patients. OBJECTIVE: To evaluate the efficacy and safety of DL-PDT vs. C-PDT for treating AK patients in China. METHODS: This randomized and prospective study was conducted in Shanghai, China. Sixty patients with AKs (grades I-III) were randomized into two groups (DL-PDT and C-PDT). PDT was performed once every two weeks for a total of three times. Patients were evaluated before each treatment (baseline and two weeks after the first and second treatment) and at one month after the third treatment. Endpoints included efficacy (lesion response) and safety (pain scale and adverse events). RESULTS: A total of 55 patients completed the study. At the first month after 3 sessions of PDT, the overall lesion clearance rate of DL-PDT (95.5%) was similar to that of C-PDT (96.8%). However, in some particular parts (eyebrow and sideburns), C-PDT resulted in higher rates of cured lesions than DL-PDT. Additionally, patients undergoing DL-PDT had nearly no pain, showing significantly lower pain scores than those undergoing C-PDT (1.7 ± 0.9 for DL-PDT vs. 5.2 ± 1.7 for C-PDT). Moreover, fewer subjects undergoing DL-PDT had related adverse events than those undergoing C-PDT (36.7% vs. 63.3%). CONCLUSION: DL-PDT was effective, better tolerated and nearly painless compared with C-PDT in AK patients in China.

Therapeutic effect of Imiquimod enhanced ALA-PDT on cutaneous squamous cell carcinoma.

BACKGROUND: Topical photodynamic therapy (PDT) is approved treatment for actinic keratosis, basal cell carcinoma and Bowen's disease. But currently it is not recommended for invasive squamous cell carcinoma (SCC) of the skin because inadequate penetration of topically applied photosensitizers lead to poor treatment response. Imiquimod (IMQ) as an immune response modifier and Toll-like receptor 7 (TLR7) agonist, is known to exhibit antitumor activity. As an adjunct therapy, it is recently seen to enhance the effect of PDT. METHOD: This is an in vivo experiment performed on 52 SCC implanted mice model. The mice were equally divided into four groups: IMQ group, IMQ + PDT group, PDT group and control group. The mice in IMQ + PDT group were treated with 3 sessions of 5% IMQ cream and ALA-PDT. Mice in IMQ group received only 5% IMQ cream. Similarly, mice in PDT group received only ALA-PDT and control mice received no treatment. The treatment efficacy was compared among these groups via tumor volume and digital photographs. In addition, immunohistochemical (IHC) markers, q PCR and detection of apoptosis were studied on 12 UV induced mice model. After successful result of this animal experiment, we performed human study on two patients with invasive cSCC on lips and foot. The patients were treated with daily application of 5% imiquimod cream and ALA-PDT at 2 weeks interval. Treatment response was assessed via clinical examination, digital photographs and dermoscopy findings. RESULTS: The study demonstrated that combination approach of IMQ + PDT has better effect than IMQ alone or PDT alone. It also showed increased expression of IL-6, IL-8, IFN-α, CXCL9, CXCL10 and TNF-α in IMQ + PDT group but at different time points following treatment (P < 0.05). IHC staining showed that the number of CD4+ cells was similar in IMQ + PDT and PDT groups but CD8+ cells was almost double in IMQ + PDT group when compared to PDT group. In addition, the number of apoptotic cell was maximum in IMQ + PDT group. Human study also delivered excellent results in both the patients with complete clearance of lesion after 3-6 sessions of treatment. CONCLUSION: PDT combined with imiquimod may have enhanced effect for the treatment of invasive cSCC. Maximum number of apoptotic cells in IMQ + PDT group can be attributed to increased number of CD8 + T cells in this group. Additional mechanism of enhanced efficacy in IMQ + PDT group may be due to increased expression of markers tested in this study.

[Analysis of KIT mutations in five patients from two Han Chinese pedigrees affected with Piebaldism].

OBJECTIVE: To screen for KIT gene mutations in two Han Chinese pedigrees affected with Piebaldism. METHODS: Clinical data of the pedigrees was collected. Genomic DNA was extracted from blood samples collected from the pedigrees and 120 unrelated healthy controls. All coding exons of the KIT gene were subjected to PCR amplification and direct sequencing. RESULTS: Two missense mutations, c.1861G>A(p.Ala621Thr) and c.1872G>A(p.Met624Ile), were identified respectively in the two pedigrees. Neither mutation was found among healthy members from the respective pedigree and the 120 unrelated healthy controls. c.1872G>A is a novel mutation. CONCLUSION: Mutations of the KIT gene may affect the structure and function of the transmembrane receptor KIT, which lead to the disease.

[Analysis of clinical and genetic features of nine patients with disseminated superfacial actinic porokeratosis].

OBJECTIVE: To analyze the clinical and genetic features of 9 ethnic Han Chinese patients with disseminated superfacial actinic porokeratosis (DSAP). METHODS: Genomic DNA was extracted from peripheral blood samples collected from the patients. PCR and direct sequencing were carried out for five patients from a family, 4 sporadic cases, and 120 healthy controls to identify potential mutations of four genes (MVK, MVD, PMVK, FDPS) involved in the mevalonate pathway as well as SLC17A9, SSH1, and SART3 genes. Pathogenecity of suspected mutations were assessed with SIFT, and Polyphen-2 scores. RESULTS: A c.746T>C mutation was identified in the family and two sporadic cases, while a c.875A>G mutation was identified in another sporadic case. No mutation was identified in the remainder genes among all patients. Scoring has suggested that the c.746T>C and c.875A>G mutations of the MVD gene are probably pathogenic. CONCLUSION: c.746 T>C and c.875A>G of the MVD gene are most common mutations. Skin rashes of the patients have a strong connection with the sunlight, albeit a significant difference among patients was discovered.

Two novel ADAR1 gene mutations in two patients with dyschromatosis symmetrical hereditaria from birth.

Dyschromatosis symmetrica hereditaria (DSH) is a rare type of pigmentary genodermatosis, which is autosomal dominantly inherited with high penetrance. The onset of DSH is typically during infancy or childhood. Cases of patients born with skin lesions have rarely been reported. Additionally, there have been few significant non­cutaneous complications reported with DSH. The present study reported two sporadic cases of patients born with DSH, confirmed by the identification of ADAR1 mutations. Additionally, comorbidity of DSH, congenital heart disease (CHD) and hemangioma disease were first reported. In the patient with isolated DSH from birth, a nonsense mutation (p.Y1192X) was identified, whereas in the second patient with DSH, CHD and hemangioma from birth, a frameshift mutation (p.Glu673ValfsX652) in ADAR1 was identified. To the best of the authors' knowledge, >120 mutations in ADAR1 have been reported to cause DSH; however, no previous studies have reported mutations in ADAR1 in DSH at birth, with CHD and hemangioma. The novel variants described in the current study add to the current knowledge of ADAR1 mutations in DSH.

[Analysis of ATP2A2 gene mutations in a pedigree and a sporadic case with Darier disease].

OBJECTIVE: To detect mutations of ATP2A2 gene in a pedigree and a sporadic case with Darier disease (DD) and explore the underlying molecular mechanism. METHODS: Clinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from blood samples of four members from the pedigree (including three patients and one healthy member), the sporadic case and 100 healthy controls. PCR was performed to amplify all coding exons of the ATP2A2 gene. And the products were directly sequenced to detect mutations. RESULTS: A missense mutation c.1484C>T (p.S495L) in exon 12 was detected in all patients of the pedigree. For the sporadic case, a novel splicing mutation c.325-2A>G was detected at the junction between intron 4 and exon 5. The same mutations were not found in the 100 healthy controls. CONCLUSION: Mutations of the ATP2A2 gene may lead to the occurrence of DD in both familial and sporadic cases with DD.

Novel mutations in one allele in a Chinese family with neurofibromatosis type 1: Including a complex insertion-deletion mutation.

Neurofibromatosis type 1 (NF1) is a hereditary disease with variable clinical manifestations. This study was performed in a Chinese three-generation family containing two members with NF1. Two novel mutations, c.853_854insTC and c.1975_1976delinsTA, were identified in the same allele in both patients by direct sequencing. By reverse transcription polymerase chain reaction, we found that the NF1 transcript contained the first mutation instead of the second mutation, suggesting a pathological role of c.853_854insTC mutation. Case reports of patients with two NF1 mutations in the same allele have not been reported. Our findings expand the known spectrum of NF1 mutations and the ongoing recognition of different mutations may give insight into the mysterious NF1 pathogenesis.

Clinical and Molecular Characterization of NF1 Patients: Single-Center Experience of 32 Patients From China.

Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, and the presence of pseudogenes.Our goal was to establish a sensitive, feasible, and comparatively economical protocol to detect NF1 mutations using blood samples.We developed a method to screen patients for mutations. Thirty-two NF1 patients from 32 unrelated families and 120 unrelated population-match controls were investigated in this study. Specific primers were designed for NF1 to avoid pseudogenes. NF1 mutations were detected by sequencing at the deoxyribonucleic acid (DNA) and complementary DNA (cDNA) levels, and multiplex ligation-dependent probe amplification (MLPA) and familial segregation analyses were used.Forty-four specific primers designed according to the NF1 structure were successfully used for polymerase chain reaction (PCR) and DNA sequencing, which was more feasible and useful than cDNA sequencing. Thirty distinct NF1 mutations were identified in 32 patients. Thirteen mutations were novel and most were frameshift mutations (33.3%). Mutations were detected at a rate of 93.8%.Our study suggests that this sensitive, feasible, and comparatively economical protocol is effective for the detection of NF1 mutations.