Mutational signatures and increased retrotransposon insertions in xeroderma pigmentosum variant skin tumors.

Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells are deficient in the translesion synthesis (TLS) DNA polymerase eta, responsible for bypassing different types of DNA lesions. From the exome sequencing of 11 skin tumors of a genetic XP-V patients' cluster, classical mutational signatures related to sunlight exposure, such as C>T transitions targeted to pyrimidine dimers, were identified. However, basal cell carcinomas also showed distinct C>A mutation spectra reflecting a mutational signature possibly related to sunlight-induced oxidative stress. Moreover, four samples carry different mutational signatures, with C>A mutations associated with tobacco chewing or smoking usage. Thus, XP-V patients should be warned of the risk of these habits. Surprisingly, higher levels of retrotransposon somatic insertions were also detected when the tumors were compared with non-XP skin tumors, revealing other possible causes for XP-V tumors and novel functions for the TLS polymerase eta in suppressing retrotransposition. Finally, the expected high mutation burden found in most of these tumors renders these XP patients good candidates for checkpoint blockade immunotherapy.

Skin fibrosis associated with keloid, scleroderma and Jorge Lobo's disease (lacaziosis): An immuno-histochemical study.

Fibrosis is a common pathophysiological response of many tissues and organs subjected to chronic injury. Despite the diverse aetiology of keloid, lacaziosis and localized scleroderma, the process of fibrosis is present in the pathogenesis of all of these three entities beyond other individual clinical and histological distinct characteristics. Fibrosis was studied in 20 samples each of these three chronic cutaneous inflammatory diseases. An immunohistochemical study was carried out to explore the presence of α-smooth muscle actin (α-SMA) and vimentin cytoskeleton antigens, CD31, CD34, Ki67, p16; CD105, CD163, CD206 and FOXP3 antigens; and the central fibrotic cytokine TGF-ß. Higher expression of vimentin in comparison to α-SMA in all three lesion types was found. CD31- and CD34-positive blood vessel endothelial cells were observed throughout the reticular dermis. Ki67 expression was low and almost absent in scleroderma. p16-positive levels were higher than ki67 and observed in reticular dermis of keloidal collagen in keloids, in collagen bundles in scleroderma and in the external layers of the granulomas in lacaziosis. The presence of α-actin positive cells and rarely CD34 positive cells, observed primarily in keloids, may be related to higher p16 antigen expression, a measure of cell senescence. Low FOXP3 expression was observed in all lesion types. CD105-positive cells were mainly found in perivascular tissue in close contact with the adventitia in keloids and scleroderma, while, in lacaziosis, these cells were chiefly observed in conjunction with collagen deposition in the external granuloma layer. We did not find high involvement of CD163 or CD206-positive cells in the fibrotic process. TGF-ß was notable only in keloid and lacaziosis lesions. In conclusion, we have suggested vimentin to be the main myofibroblast general marker of the fibrotic process in all three studied diseases, while endothelial-to-mesenchymal transition (EndoMT) and mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role.

Perivascular clusters of Th2 cells and M2 macrophages in allergic contact dermatitis to methylchloroisothiazolinone and methylisothiazolinone.

BACKGROUND: Methylisothiazolinone (MI) and Methylchloroisothiazolinone (MCI) are among the most common skin sensitizers, yet the immunological events that occur during MCI/MI allergic contact dermatitis (ACD) are still poorly understood. OBJECTIVES: To analyse dendrocytes, macrophage subtypes and T cells in skin during the elicitation phase of MCI/MI ACD. METHODS: Thirteen patients with positive patch test reactions to MCI/MI (ACD group) and 11 individuals with negative patch test results were selected. Skin biopsies were only performed at 48 hours of patch testing. Immunohistochemistry was conducted to assess T cells, dendrocytes (Factor XIIIa), M1 (p-Stat1, CD68) and M2 (c-Maf, CD163) macrophages. Transcriptional analyses were performed for cytokines and related factors, and further compared to atopic dermatitis samples (n=4). Immunofluorescence assays addressed T cells location, along with IL-4 or IL-13, within the skin. RESULTS: MCI/MI elicited dermal dendrocytes and macrophages, pronouncedly the M2 subtype. T cells, majorly CD4+ T cells, accumulated in the perivascular areas. Similarly, abundant IL-4 protein was detected in these areas. There was an upregulation of IL-4 and IL-13 mRNA expression, a mild increase in IFNG mRNA levels and a down-regulation of RORC in the ACD group. Immunofluorescence revealed dermal clusters of T cells co-localized with IL-4. CONCLUSIONS: M2 macrophages and Th2 cells participate in the immunopathogenesis of MCI/MI ACD. Dermal dendrocytes and M2 macrophages may assist the formation of CD4+ T cells perivascular clusters. These findings render a mechanistic insight into the MCI/MI reaction. Further analysis at different timepoints of patch testing is required to fully comprehend this ACD kinetics.

Claudin expression profile in flat wart and cutaneous squamous cell carcinoma in epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) is a genodermatosis related to human beta-papillomavirus (beta-HPV), with a high risk of cutaneous squamous cell carcinoma (cSCC). Claudins are transmembrane proteins expressed in epithelia and may be altered during carcinogenesis. For a better understanding of the role of beta-HPV in cutaneous carcinogenesis, this claudin expression study was conducted on lesions of patients with and without EV. In this study, claudins-1, -2, -3, -4, -5, -7 and -11 expressions were analyzed by applying the immunohistochemistry technique, in samples of 108 normal skin, 39 flat warts and 174 cSCC. The cSCC samples were organized in tissue microarrays. We found that claudin-1 and claudin-3 focal expressions were associated with cSCC (p < 0.001), and claudin-2 focal or negative expression with flat wart (p < 0.001), in EV and NEV (non-EV) groups. For claudin-5, EV group showed a lower chance of focal and negative expression (p < 0.001), and its negative expression was associated with flat wart (p < 0.001) and lower mean age (p < 0.001). Claudins-4, -7 and -11 showed a diffuse expression in almost all studied samples. Our findings suggest that claudin-5 increased expression observed on normal skin, flat wart and cSCC showed association with EV. Claudin-1 and -3 down expression were also observed, but they could not be related to beta-HPV infection.

Immune privilege disruption in folliculotropic mycosis fungoides: investigation of major histocompatibility complex antigen expression.

BACKGROUND: Folliculotropic mycosis fungoides (FMF) is a cutaneous T-cell lymphoma mainly affecting the hair follicle, which seems to represent a place of immune privilege phenomenon. OBJECTIVES: To explore a possible role of immune privilege (IP) in FMF analyzing the major histocompatibility complex (MHC) expression. METHODS: Immunohistochemistry for HLA-G and MHC-II was performed to formalin-fixed paraffin-embedded cutaneous skin biopsies of FMF patients (n = 43), conventional mycosis fungoides (CMF; n = 13), alopecia areata (AA; n = 13), and normal scalp skin (NS; n = 12). RESULTS: HLA-G expression was lower in FMF (34%: 14/41) and CMF (18%: 2/11) groups compared to alopecia areata (92%:11/12) and normal scalp skin group (100%: 12/12). MHC-II expression in hair follicle was greater in the FMF group (18/42: 43%) compared to AA (0%) and NS (0%). HLA-G and MHC-II expression in cellular infiltrate had no difference among FMF and CMF groups and was different compared to the AA group. CONCLUSIONS: Our data support the hypothesis of disruption of immune privilege based on the lower expression of HLA-G and higher expression of MHC-II in the follicular epithelium in mycosis fungoides compared to alopecia areata and normal scalp skin. The lack of difference between FMF and CMF groups did not support the role of these molecules as a driver of folliculotropism. The expression of MHC molecules seems to be different between neoplastic and inflammatory infiltrates. The definitive significance of expression of the MHC molecules remains unclear, and more studies are necessary to fully understand the role of these molecules in cutaneous lymphomas.

Histoid leprosy: clinical and histopathological analysis of patients in follow-up in University Clinical Hospital of endemic country.

BACKGROUND: Histoid leprosy (HL) is a rare form of lepromatous leprosy, characterized by hyperchromic indurated nodules above normal skin. Its main histopathological aspect is spindle cells. Because it may simulate other aspects, such as dermatofibroma and neurofibroma, histoid leprosy poses itself as a diagnostic challenge. METHODS: This is a retrospective study with all patients having been selected from the leprosy clinic of the Hospital das Clínicas da Universidade de São Paulo from 2006 to 2016. RESULTS: There were 12 patients in this study, eight in the histoid group and four in the lepromatous leprosy group. The prevalence of HL was 1.12% in all leprosy subjects. All individuals from HL group were "de novo" cases, and the histopathological analysis of skin lesions presented spindle cells generating a storiform pattern. Immunohistochemistry for CD68, vimentin, and anti-BCG were positive in all 12 cases. Factor XIIIa was visualized only in the papillary dermis, and S100 protein was negative in all biopsies. Smooth-muscle actin was present in 62.5% of the HL samples. CONCLUSION: The prevalence of HL was similar to previous reports. However, all histoid patients were "de novo" cases, differing from published studies. Fusocellular macrophage transformation could be explained by the differences in cytoskeleton proteins expressed in histoid lesions in comparison to other leprosy variants, with emphasis on vimentin and smooth muscle actin.

High density of M2-macrophages in acral lentiginous melanoma compared to superficial spreading melanoma.

AIMS: Acral lentiginous melanoma (ALM) is the most common type of melanoma in people with darker skin phototypes. There is some evidence that the aetiology, pathogenesis, risk factors and natural history of ALM differ from those of superficial spreading melanoma (SSM). ALM behaves more aggressively than SSM, but the biological explanation for these differences remains unknown. The presence of one subtype of macrophages, termed M2-macrophage (M2-M), has been found to be related to local progression, metastasis and poor prognosis in several neoplasms. The aim of this study was to compare the density of M2-Ms in ALMs versus SSMs, and to examine whether or not the density of M2-Ms is associated with histopathological features predictive of adverse prognosis in cutaneous melanoma (CM), as well as development of metastasis. METHODS AND RESULTS: Sixty-seven ALMs and 67 SSMs cases were analysed. The tumours were classified according to thickness, ulceration, mitosis and metastasis. M2-M quantity was evaluated using immunohistochemistry with anti-CD163 and anti-CD206 antibodies. M2-Ms were increased in ALM compared with SSM, and were related to the histopathological features predictive of adverse prognosis in CM, such as thickness > 1.0 mm, ulceration and mitotic activity, and the development of metastasis. CONCLUSIONS: Our study is the first, to our knowledge, to demonstrate the increased presence of M2-Ms in ALM compared with SSM. Our findings suggest that the increased M2-Ms in ALM are associated with the main histopathological features predictive of adverse prognosis in CM, as well as the presence of metastasis, and that these cells can be related to the aggressive behaviour seen in ALMs.

Inate immunity in rosacea. Langerhans cells, plasmacytoid dentritic cells, Toll-like receptors and inducible oxide nitric synthase (iNOS) expression in skin specimens: case-control study.

Rosacea is a chronic inflammatory condition with predominant facial involvement. Because of that, many patients sense that rosacea affects quality of life. The etiology of rosacea remains unknown. Recent studies have suggested that aberrant innate immunity is central to this disease. The aim of this study was to examine the presence of Langerhans cells, plasmacytoid dentritic cells (PDC), the expression of Toll-like receptors (TLR) and inducible oxide nitric synthase (iNOS) in skin of patients with rosacea, to highlight the participation of innate immunity in its pathogenesis. 28 biopsy specimens were taken from patients with clinical and histopathological findings of rosacea. Immunohistochemical demonstration of Langerhans cells (anti-CD1a antibody), PDC (anti-CD 123 antibody), TLR2, TLR4 and iNOS was performed in skin samples and compared with normal skin controls. The expression of Langerhans cells was lower in rosacea group than in control group. PDC were found in skin samples of rosacea as isolated cells and forming small clusters. Expression of TLR2, TLR4 and iNOS was higher in rosacea samples than in normal skin controls. This research demonstrates early and late stage components of innate immunity in specimens of rosacea ratifying the existence of an altered innate immunity in its pathogenesis.

Macrophage subtypes in recurrent nodular basal cell carcinoma after Mohs micrographic surgery.

BACKGROUND: The macrophages associated with solid tumors are related to the progression or regression of tumors, depending on the differentiation in M1 or M2. M2 subtype promotes angiogenesis, remodeling, and tissue repair (tumor proliferation). In contrast, M1 produces toxic mediators and presents antigens, destroying microorganisms and tumor cells. The microenvironment of most aggressive forms of basal cell carcinoma (BCC) shows an increase in macrophages due to M2 phenotype compared to noninvasive forms. The treatment of nodular BCC by Mohs micrographic surgery (MMS) provides high cure rates, but relapses can occur. AIMS: To compare the total population of macrophages and their subpopulations M1 and M2 in cases of recurrent and nonrecurrent nodular BCC after excision by MMS. MATERIALS & METHODS: Histological sections obtained from paraffin blocks of nine cases of recurrent nodular BCC after MMS and 18 cases of nonrecurrent nodular BCC operated by MMS were immunostained for iNOS, CD204, CD163, and CD68. The expression of these markers was analyzed by image analysis. RESULTS: No significant differences were found between the groups in relation to the average percentage of M1 cells, M2 cells, and total cells. DISCUSSION AND CONCLUSION: A relationship was not seen between tumor-associated macrophages (TAM) and tumor recurrence.

Comparative study of p16 protein expression in squamous cell carcinomas from patients with epidermodysplasia verruciformis and patients without the disease.

Epidermodysplasia verruciformis (EV) is a rare genodermatosis associated with susceptibility to beta-human papilloma virus (HPV) infection. EV patients develop disseminated warts and non-melanoma skin cancer, mainly squamous cell carcinomas (SCC) that are locally aggressive. EV pathogenesis is not yet fully understood, but alterations in the p16 gene play a role in the pathogenesis of neoplasms caused by high-risk genital HPV. To explore its role in EV lesions, we compared p16 expression in SCC from patients with and without EV. Tissue microarray slides composed of 27 SCC from EV patients, and 35 from non-EV patients were stained with an anti-p16 antibody. Twenty (74%) EV tumors exhibited diffuse (nuclear and cytoplasmic) p16 expression, one (4%) displayed focal expression, and six (22%) displayed no p16 staining. Eleven (31%) SCC from non-EV patients presented diffuse p16 staining, 14 (40%) displayed focal expression and 10 (29%) did not express p16. The frequency of diffuse p16 expression was higher in EV tumors than in SCC from patients without EV. The frequency of diffuse p16 expression in moderately and poorly differentiated EV-SCC was similarly higher than non-EV tumors with the same degree of differentiation. The diffuse expression of p16 in EV-SCC suggests that changes in the p16 gene, probably resulting in a functionally defective protein, may be one factor determining the locally aggressive clinical behavior of SCC in young EV patients.

Langerhans Cells Express IL-17A in the Epidermis of Chromoblastomycosis Lesions.

Chromoblastomycosis (CBM) is a chronic fungal infection that affects skin and subcutaneous tissue, and little is known about the immunological aspects of such lesions. We have previously described the high expression of IL-17 in this group. Understanding the innate immune response of patients with CBM would improve the knowledge of its immunopathogenesis and contribute to the most appropriate therapies. Nineteen biopsies of verrucous form were obtained from patients with clinical and histopathological diagnosis of CBM, without treatment. This was done with a double immunostaining with conventional immunohistochemistry and immunofluorescence technique as well as confocal microscopy to detect Langerin and IL-17 expression. All of the specimens that were analyzed showed expression of Langerin in the epidermis - the same as the control group. However, only the CBM group presented cells expressing CD207 in the dermis. Interestingly, the coexpression of IL-17 and Langerin was visualized along the epidermis and dermis in 100% of the lesion group. We demonstrated for the first time in situ coexpression of IL-17 and Langerin (CD207) in epidermal cells of patients with CBM and speculated on their role as IL-17-producing cells or whether they could be a new subpopulation of dendritic cells distinct from Langerhans cells.

Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM.

Unusual remodeling of the hyalinization band in vulval lichen sclerosus by type V collagen and ECM 1 protein.

OBJECTIVES: The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus. METHODS: Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed. RESULTS: A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers of the lichen sclerosus zone of collagen remodeling confirmed the presence of densely clustered COLV. CONCLUSIONS: Increased deposition of abnormal COLV and its correlation with extracellular matrix 1 and elastic fibers suggest that COLV may be a trigger in the pathogenesis of lichen sclerosus.

Unusual remodeling of the hyalinization band in vulval lichen sclerosus by type V collagen and ECM 1 protein

OBJECTIVES: The vulva is the primary site affected in lichen sclerosus, a chronic dermatosis in women that is histologically characterized by a zone of collagen remodeling in the superior dermis. The normal physiological properties of the vulva depend on the assembly of collagen types I (COLI), III (COLIII) and V (COLV), which form heterotypic fibers, and extracellular matrix protein interactions. COLV regulates the heterotypic fiber diameter, and the preservation of its properties is important for maintaining normal tissue architecture and function. In the current work, we analyzed the expression of COLV and its relationship with COLI, COLIII, elastic fibers and extracellular matrix protein 1 in vulvar biopsies from patients with lichen sclerosus. METHODS: Skin biopsies from 21 patients with lichen sclerosus, classified according to Hewitt histological criteria, were studied and compared to clinically normal vulvar tissue (N=21). Morphology, immunohistochemistry, immunofluorescence, 3D reconstruction and morphometric analysis of COLI, COLIII, COLV deposition, elastic fibers and extracellular matrix 1 expression in a zone of collagen remodeling in the superior dermis were performed. RESULTS: A significant decrease of elastic fibers and extracellular matrix 1 protein was present in the hyalinization zone of lichen sclerosus compared to healthy controls. The non-homogeneous distribution of collagen fibers visualized under immunofluorescence in the hyalinization zone of lichen sclerosus and control skin was confirmed by histomorphometry. Lichen sclerosus dermis shows a significant increase of COLI, COLIII and COLV expression compared to the healthy controls. Significant inverse associations were found between elastic fibers and COLV and between COLV and extracellular matrix 1 expression. A direct association was found between elastic fiber content and extracellular matrix 1 expression. Tridimensional reconstruction of the heterotypic fibers ...

Analysis of microvasculature phenotype and endothelial activation markers in skin lesions of lacaziosis (Lobomycosis).

Jorge Lobo's disease is a rare mycosis characterized by chronic inflammation, which causes skin lesions in the absence of visceral dissemination. The disease occurs mainly in hot and humid climates and most cases have been registered in the Brazilian Amazon region. This study investigated possible microvascular alterations in skin lesions caused by infection with Lacazia loboi which may interfere with the clinical progression of the disease. Immunohistochemistry was used to evaluate the density of blood and lymphatic vessels, as well as expression of the cell adhesion molecules ICAM-1, VCAM-1 and E-selectin. The results showed a reduced number of blood (62.66 ± 20.30 vessels/mm(2)) and lymphatic vessels (3.55 ± 5.84 vessels/mm(2)) in Jorge Lobo's disease when compared to control skin (169.66 ± 66.38 blood vessels/mm(2) and 8 ± 2.17 lymphatic vessels/mm(2)). There were a larger number of vessels expressing ICAM-1 (27.58 ± 15.32 vessels/mm(2)) and VCAM-1 (7.55 ± 6.2 vessels/mm(2)). No difference was observed in the expression of E-selectin (4.66 ± 11 vessels/mm(2)). Taken together, the results indicate changes in the local microvasculature which may interfere with the development of an efficient cell-mediated immune response and may explain restriction of the fungus to the site of injury.