Objective To determine the expression of Notch pathway receptors (Notch1 and Notch4) and ligands (Jagged1 and Dll4) in cutaneous malignant melanoma (CMM) tissues,and to preliminarily explore the role of the Notch signaling pathway in the pathogenesis of CMM.Methods Immunohistochemical study was performed to determine the expression pattern and intensity of Notch1,Notch4,Jagged1 and Dll4 in 40 paraffin-embedded CMM specimens and 15 paraffin-embedded pigmented nevus specimens.Statistical analysis was carried out by chi-square test and Spearman rank correlation analysis with the SPSS 21.0 software.Results Notchl was detected in 31 (77.5％) of 40 CMM specimens,as well as in 3 of 15 pigmented nevus specimens,and the positive rates significantly differed between the two groups (x2 =15.281,P ＜ 0.001).However,no significant difference in the expression intensity of Notch1 was observed between 18 in situ melanoma tissues and 22 invasive melanoma tissues (x2 =0.631,P =0.427).In addition,the positive rates of Notch4,Jagged1 and Dll4 were also significantly higher in the CMM group than those in the pigmented nevus group (all P ＜ 0.05),and the expression intensity of Notch4,Jagged1 and Dll4 significantly differed between in situ and invasive melanoma tissues (all P ＜ 0.05).In CMM tissues,the expression of Notch1 was positively correlated with that of Jagged1 (rs =0.350,P =0.027) and Dll4 (rs =0.562,P ＜ 0.001),while the expression of Jaggedl was negatively correlated with that of Dl14 (rs =-0.734,P ＜ 0.001).Conclusion Abnormality of the Notch signaling pathway may be involved in the pathogenesis of melanoma,but further researches are still needed to elucidate the detailed mechanism.

ObjectiveTo understand the clinical and histopathologic diagnostic criteria for aneurysmal fibrous histiocytoma(AFH).MethodsThe clinical and histopathological features of 5 patients with AFH were retrospectively reviewed.ResultsThere were 3 males and 2 females in these patients.All the tumors clinically manifested as dark erythematous or brown nodules.Three cases had a recent history of rapid growth.The lesions were located on the limbs(n =3),or chest and lower mandible(n =2).Histopathological examination of skin biopsies showed typical features of dermatofibroma,accompanied by many irregular cleftlikeorcavernousblood-filledspaceswithnumeroushemosiderinpigmentsinallofthesecases.Immunohistochemically,the tumor cells were immunoreactive to vimentin and CD68 but negative for CD34 or CD31.Conclusions In view of a history of recent rapid growth,the presence of hemorrhagic pseudocysts and high vascularity,AFH should be differentiated from angiosarcoma and angiomatoid fibrous histiocytoma.

Objective To detect the expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in cutaneous malignant melanoma (CMM) tissue and to assess its relationship with melanoma proliferation,invasion and metastasis.Methods Western blot was conducted to measure the protein expression of TIMP-4 in five fresh lesional and paratumoral tissue specimens of CMM and three fresh tissue specimens of nevi.Immunohistochemistry was carried out to quantify the expression of TIMP-4,Ki-67,matrix metalloproteinase-2 (MMP-2),vascular endothelial growth factor (VEGF) and CD63 in paraffin-embedded tissue samples from 43 cases of CMM and 51 cases of nevi.The degree of malignancy of melanoma was evaluated in these lesions.Results Western blot analysis showed that the expression of TIMP-4 was significantly higher in 4 of 5 CMM tissue specimens than in corresponding paratumoral tissue specimens and nevus tissue specimens.Immunohistochemistry revealed that the expression rate of TIMP-4 was 86.04％ (37/43) in melanoma tissue,compared to 19.6％ (10/51) in nevus tissue (x2 =31.55,P ＜ 0.05).The expression of TIMP-4 increased sequentially from in situ melanoma to invasive and metastatic melanoma (rs =0.309,P ＜ 0.05).As far as CMM was concerned,the TIMP-4 expression was uncorrelated with any of the known prognostic variables including clinical stage,Clark level,Breslow depth,presence of ulcer,and Ki-67 expression (all P ＞ 0.05),but positively correlated with the expressions of VEGF (rs =0.345,P ＜ 0.05) and CD63 (rs =0.555,P ＜ 0.01).The median expression level of TIMP-4 was significantly higher in MMP-2-positive than in MMP-2-negative melanoma tissue samples (3 vs.0,P ＜ 0.01).Conclusions TIMP-4 protein is highly expressed in CMM tissue,which may be closely associated with the initiation and progression of CMM,especially with the metastasis of and angiogenesis in CMM.

Objective To measure the expression of hypoxia-inducible factor (HIF)-1α in acral malignant melanoma (MM) tissue and to investigate its relationship with the stem cell factor (SCF)/c-kit pathway.Methods Immunohistochemical staining was performed to measure the expression of HIF-1α in tissue specimens from lesions of 93 patients with acral MM,21 with non-acral MM,39 with acral melanocytic nevi,and from the normal acral skin of 15 healthy human controls.Meanwhile,the expression of c-kit was detected by immunohistochemical staining in the 93 acral MM tissue specimens.Statistical comparisons were carried out by chi-square test and Mann-Whitney U test.The relationship of HIF-1α expression with c-kit expression as well as tumor progression and staging was assessed by Spearman correlation analysis.Results Immunohistochemistry showed that the expression rate of HIF-1α was 87.10％ (81/93) in acral MM specimens,90.48％ (19/21) in non-acral MM specimens,15.38％ (6/39) in acral melanocytic nevus specimens,but 0 (0/15) in the normal acral skin specimens.The expression of HIF-1α was significantly higher in acral MM lesions than in normal acral skin and acral melanocytic nevus lesions (both P ＜ 0.01),and significantly different between acral MM and non-acral MM lesions (P ＜ 0.01).Moreover,HIF-1α expression was positively correlated with Clark level and Breslow depth of melanoma (rs =0.442,0.368,respectively,both P ＜ 0.01),with the progression of acral MM (from in situ to aggressive and metastatic MM) (rs =0.420,P ＜ 0.01),and with the expression of c-kit (rs =0.307,P ＜ 0.01).Conclusions HIF-1α is highly expressed in acral MM,positively correlated with the staging,progression and aggression of MM,and co-expressed with c-kit in acral MM tissue,suggesting that both HIF-1α and c-kit take part in the pathogenesis of acral MM.

Objective To investigate the in vitro anti-proliferation effect of a histone deacetylase inhibitor,chidamide,on a cutaneous malignant melanoma cell line,A375.Methods Cultured A375 cells were treated with different concentrations of chidamide(5,10,50,100,500 μmol/L)and aichostatin A (TSA)(0.1,0.25,0.5,1.0 μmol/L),respectively,for various durations(24,48,72,96,120 hours).Subsequently,cell proliferation,apoptosis and cell cycle were detected by MTT assay,annexin Vfluorescein isothiocyanate and propidium iodide double staining,and DNA ploid analysis,respectively.Results The proliferation of A375 cells was inhibited in a dose-dependent manner by chidamide of 5-500μmol/L and TSA of 0.1-1 μmol/L,and in a time-dependent manner from 0 to 120 hours after the beginning of trealment with ehidamide of 5-500μmol/L and TSA of 0.25-1μmol/L.The 48-hour 50% growth inhibition concentration(IC50)of ehidamide and TSA on A375 cells was about 250 μmol/L and 0.7μmol/L,respectively.After 48-hour treatment,the apoptosis mte was 80.27%±3.06%,79.53%±5.70%,83.13%±6.90%in A375 cells treated with chidamide of 62.5,125,250 μmol/L,respectively,16.27%±2.46%,28.83%±2.55%,83.40%±8.65%in those treated with TSA of 0.175,0.35,0.7 μmol/L,respectively,10.43%±0.96%in ontreated cells;a statistical increase was noticed in chidamide-treated cells and TSA-treated cells vs.untreated cells(all P＜0.001).A positive correlation was observed between the apoptosis rate and concentrations of TSA(r=0.955,P=0.000).Cell cycle analysis indicated that treatment with chidamide induced cell cycle arrest in G0/G1 phase,with the cell proportion in G0/G1 phase being 76.30%±6.06%,82.79%±0.74%,88.91%±5.29%in A375 cells treated with chidamide of 62.5,125,250μmol/L,respectively,versus 38.73%±3.36%in untreated cells.While after 48-hour treatment with TSA of 0.35 and 0.7 μmol/L,the proportion of cells in G2/M phases was 25.15%±2.71%and 58.71%±3.45%,respectively,compared to 15.73%±0.23%in untreated cells(P＜0.01).Conclusion Chidamide and TSA could induce cell cycle arrest and apoptosis,as well as inhibit the growth of A375 ceils in vitro.

Objective To measure the expression of CC chemokine ligand 18(CCL18)in cutaneous malignant melanoma (CMM) tissues, and to explore its clinical significance, as well as relationship with vascular endothelial growth factor (VEGF) and Ki67 antigen expressions. Methods Immunohistochemistry was performed to measure CCL18, VEGF and Ki67 expressions in 58 paraffin?embedded CMM tissue specimens, as well as CCL18 expression in 20 paraffin?embedded pigmented nevus specimens, and immunofluorescence assay to confirm the expression of CCL18 in fresh CMM tissue specimens. Correlations of CCL18 expression with CMM clinicopathologic features, VEGF and Ki67 expressions were analyzed. Results CCL18 was detected in 49 (84.48%) of 58 paraffin?embedded CMM specimens, but in none of the 20 paraffin?embedded pigmented nevus specimens, with a significant difference in the positive rate of CCL18 between the CMM group and pigmented nevus group(χ2=45.46, P<0.01). The expression of CCL18 in paraffin?embedded CMM tissues was positively correlated with Clark′s level and Breslow thickness of CMM (rs = 0.609, 0.644 respectively, both P < 0.01), and was significantly different between ulcerated and non?ulcerated CMM(P<0.05), as well as between patients with and without lymphatic metastasis(P<0.05). However, there were no significant differences in the expression of CCL18 among patients of different age, gender, or between acral and non?acral CMM(all P>0.05). In addition, the expression of CCL18 in CMM tissues was positively correlated with that of VEGF(rs = 0.727, P < 0.05), but unrelated to that of Ki67(P > 0.05). Immunofluorescence assay showed CCL18 expression in the cytoplasm of tumor cells in CMM tissues. Conclusion CCL18 is highly expressed in CMM tissues, and may be involved in tumor invasion and metastasis.

Objective To measure histidine triad nucleotide?binding protein 1(HINT1)protein expression and gene promoter methylation, and to analyze the relationship between HINT1 gene promoter methylation and clinical pathological features of melanoma. Methods Fifty?six patients with melanoma and 51 patients with nevus were enrolled as subjects and controls, respectively. Methylation?specific PCR (MSP) was performed to measure the methylation of HINT1 gene promoter in lesional and paratumoral tissue specimens from the patients with melanoma, as well as in lesional specimens from the patients with nevus. Immunohistochemistry was carried out to quantify the expression of HINT1 protein in these tissue specimens. Results MSP showed that the methylation rate of HINT1 gene promoter was significantly higher in melanoma tissues than in paratumoral and nevus tissues(76.8%[43/56]vs. 33.9%[19/56]and 35.3%[18/51], χ2 = 20.810 and 18.749, respectively, both P < 0.05), but was insignificantly different between paratumoral and nevus tissues(χ2=0.022, P>0.05). Immunohistochemistry revealed that the expression rate of HINT1 was 21.4%(12/56)in melanoma tissues, compared to 82.4%(42/51)in nevus tissues(χ2 = 39.633, P <0.01). There was a significant difference in the methylation rate of HINT1 promoter between HINT1?positive and ?negative melanoma tissues(6/12 vs. 37/44[84.1%], P<0.05), and between Clark levelⅠ-ⅡandⅢ-Ⅴmelanoma tissues(59.1%[13/22]vs. 88.2%[30/34],χ2=6.365,P=0.012). Conclusions HINT1 protein is lowly expressed in melanoma, which may be associated with high methylation of its gene promoter. Moreover, the high methylation ofHINT1 gene promoter may be involved in the initiation and progression of melanoma.

Objective To analyze clinicopathologic features of sebaceoma. Methods Clinical, pathologic and immunohistochemical findings from 31 cases of sebaceoma were retrospectively analyzed. The clinicopathologic features of sebaceoma were investigated. Results There were 9 males and 22 females. The patients′ age was 53.90 ± 15.40 years, and the clinical course was 9.41 ± 13.75 years. Sebaceoma predominantly affected the face. The common lesion of sebaceoma was red, yellowish?red, skin?colored or slight brown papules, with no subjective symptoms in most cases. Histopathologically, neoplasms had symmetric structures, and were located in the dermis. Epidermal involvements were found in 9 cases. The neoplasm cells were mainly composed of basaloid cells, a few mature sebocytes and some transition cells. The proportion of mature sebocyts was less than 1%in 26 cases, less than 20%in 2 cases, and 20%-40%in 3 cases. Mitoses were occasionally found in 5 cases. One patient was complicated by eccrine poroma. Varying amounts of ducts were found in all the patients. Immunohistochemical staining showed that epithelial membrane antigen was expressed on ducts and mature sebocytes in all the patients, while epithelial antigen was undetected in any of the patients. Carcinoembryonic antigen, androgen receptor and D2?40 were found in 20, 24 and 28 patients with sebaceoma, respectively. Conclusions The diagnosis of sebaceoma mainly depends on histopathological examination. Combined immunohistochemical detection of epithelial membrane antigen, androgen receptor and D2?40 is beneficial to its differential diagnosis.

Objective:To investigate clinicopathological features and prognosis of transformed mycosis fungoides (TMF) .Methods:A retrospective analysis was performed on clinicopathological data collected from 24 patients with TMF, as well as on flow cytometry results of 16 peripheral blood samples obtained from 11 of the 24 patients, who visited Hospital of Dermatology, Chinese Academy of Medical Sciences between 2014 and 2020.Results:Among the 24 patients, 11 were males and 13 were females. Their average age at diagnosis of TMF was 50.0 years (range: 18 - 77 years), and patients with early-stage TMF (9 cases) and tumor-stage TMF (15 cases) were aged 44.8 and 52.6 years on average, respectively. The average time interval from diagnosis of MF to large cell transformation was 3.7 years, and 8 patients were diagnosed with TMF at the initial visit. Histopathologically, large cells infiltrated in a diffuse pattern in 20 cases, as well as in a multifocal pattern in 4, and the proportion of large cells in 7 cases was greater than 75%. Immunohistochemically, 18 patients showed positive staining for CD30, and the proportion of CD30-positive large cells was greater than 75% in 9; negative staining for CD30 was observed in 6. Flow cytometry of 16 peripheral blood samples showed the presence of cell subsets expressing clonal T cell receptor (TCR) -vβ in 2 of 4 patients with early-stage TMF and 10 of 12 with tumor-stage TMF, and tumor cells with higher forward scatter than normal lymphocytes were detected in 16 samples. During the follow-up, among the patients with early-stage TMF, 3 progressed to tumor-stage TMF 3.3 years on average after large cell transformation, 1 progressed to erythrodermic MF in stage IIIA, and the other 4 still showed an indolent course; among the patients with tumor-stage TMF, 1 progressed to stage-IV TMF, and 5 died 3.3 (1.5 - 6) years after large cell transformation.Conclusion:Large cell transformation may occur in patients with MF in any stage, some patients have poor prognosis, so close follow-up is needed for patients with TMF.

Objective:To investigate the expression of epigenetic inhibitor polycomb group proteins such as enhancer of zeste homolog 1/2 (EZH1/EZH2), embryonic ectoderm development protein (EED) and suppressor of zeste 12 (SUZ12) in common cutaneous T-cell lymphomas and lymphoproliferative disorders (CTCL/LPD) .Methods:Totally, 93 paraffin-embedded skin samples of CTCL/LPD and 8 of lichen planus were collected from Hospital for Skin Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College between 2012 and 2019, and subjected to immunohistochemical staining to determine the protein expression of EZH2, EED, SUZ12 and EZH1. Statistical analysis was carried out with SPSS 25.0 software by using chi-square test and Spearman correlation analysis.Results:The 93 cases of CTCL/LPD included 44 cases of mycosis fungoides (MF), 17 natural killer/T cell lymphoma (NK/TCL), 8 primary cutaneous anaplastic large cell lymphoma (PC-ALCL), 8 lymphomatoid papulosis (LyP), 8 hydroa vacciniforme-like lymphoproliferative disorder (HV-like LPD) and 8 cases of subcutaneous panniculitis-like T cell lymphoma (SPTCL). Among the 93 CTCL/LPD cases, 83 (89.2%) were positive for EZH2, 81 (87.1%) for EED, 78 (83.9%) for SUZ12 and 37 (39.8%) for EZH1; among the 8 cases of lichen planus, 1 was positive for EZH2, all were positive for EZH1, and all were negative for EED and SUZ12. The expression of EZH2, EED, SUZ12 and EZH1 in lichen planus samples significantly differed from all the CTCL/LPD samples ( χ2 = 41.75, 39.74, 39.36, 32.83, respectively, all P < 0.001), and from MF, NK/TCL, PC-ALCL, LyP, HV-like LPD and SPTCL samples separately (α = 0.008 3, all P < 0.001). Meanwhile, the score of EZH2 expression was negatively correlated with that of EZH1 expression in the MF, NK/TCL, PC-ALCL, LyP, HV-like LPD and SPTCL tissues ( rs = -0.60, -0.68, -0.89, -0.74, -0.93, -0.80, respectively, all P < 0.05) . Conclusion:Polycomb group proteins EZH2, EED, SUZ12 and EZH1 are abnormally expressed in CTCL/LPD lesions.