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ABSTRACT: Perineural infiltration refers to a neoplastic cell involvement in, around, and through the nerves. It is considered as one of the neoplastic dissemination pathways. Thus, its identification is crucial to establish the prognosis of some malignant skin neoplasms, such as squamous cell carcinoma, and explains the locally aggressive behavior of cutaneous neoplasms, such as microcystic adnexal carcinoma. We have conducted a review of malignant and benign skin tumors in which perineural infiltration has been described, and we also discuss some histopathological findings that may simulate perineural infiltration.
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Carcinoma de Células Escamosas , Neoplasias de Anexos y Apéndices de Piel , Neoplasias Cutáneas , Humanos , Pronóstico , Neoplasias Cutáneas/diagnóstico , Neoplasias Cutáneas/terapia , Neoplasias Cutáneas/patología , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/terapia , Carcinoma de Células Escamosas/patologíaRESUMEN
BACKGROUND: The worldwide outbreak of monkeypox has evidenced the usefulness of the dermatologic manifestations for its diagnosis. OBJECTIVE: To describe the histopathologic and immunohistochemical findings of monkeypox cutaneous lesions. METHODS: This is a retrospective histopathologic and immunohistochemical study of 20 patients with positive Monkeypox virus DNA polymerase chain reaction and immunohistochemical positivity for Vaccinia virus in cutaneous lesions. Four cases were also examined by electron microscopy. RESULTS: The most characteristic histopathologic findings consisted of full-thickness epidermal necrosis with hyperplasia and keratinocytic ballooning at the edges. In some cases, the outer root sheath of the hair follicle and the sebaceous gland epithelium were affected. Intraepithelial cytoplasmic inclusion bodies and scattered multinucleated keratinocytes were occasionally found. Immunohistochemically, strong positivity with anti-Vaccinia virus antibody was seen in the cytoplasm of ballooned keratinocytes. Electron microscopy study demonstrated numerous viral particles of monkeypox in affected keratinocytes. LIMITATIONS: Small sample size. Electron microscopic study was only performed in 4 cases. CONCLUSION: Epidermal necrosis and keratinocytic ballooning are the most constant histopathologic findings. Immunohistochemical positivity for Vaccinia virus was mostly detected in the cytoplasm of the ballooned keratinocytes. These findings support the usefulness of histopathologic and immunohistochemical studies of cutaneous lesions for diagnosis of monkeypox.
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Mpox , Humanos , Mpox/patología , Estudios Retrospectivos , España , Electrones , NecrosisRESUMEN
ABSTRACT: Circumscribed palmar or plantar hypokeratosis is a focal disorder of keratinization that consists of a reduction in the thickness of the corneal layer of the epidermis of palms or soles. Although it is considered a benign entity, the thinning of the stratum corneum facilitates ultraviolet damage in the affected skin, which may result in an increased risk of developing focal epidermal dysplasia. Other factors, such as immunosuppression in transplanted patients, may play a role as well. We present a case of circumscribed palmar or plantar hypokeratosis with features of Bowen disease limited to the hypokeratotic epidermis.
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Neoplasias del Ano , Enfermedad de Bowen , Dermatosis de la Mano , Queratosis , Neoplasias Cutáneas , Humanos , Enfermedad de Bowen/patología , Queratosis/patología , Dermatosis de la Mano/patología , Epidermis/patología , Neoplasias del Ano/patología , Hiperplasia/patología , Neoplasias Cutáneas/patologíaRESUMEN
ABSTRACT: Tattoos are characterized by the introduction of exogenous pigments into the dermis. Tattoos usually serve cosmetic purposes, although they may have other causes, such as traumatic pigment implants in accidents or medical-related tattoos in the context of radiotherapy. Dermatologic adverse reactions are relatively uncommon, and they include infections, immune-mediated reactions, cutaneous lesions secondary to the Koebner phenomenon, exacerbation of preexisting dermatosis, benign and malignant neoplasms, and a miscellaneous group of dermatologic conditions that may appear in a preexisting tattoo. The aim of this study is to review the types of histopathologic reactions that may appear in a preexisting permanent tattoo.
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Enfermedades de la Piel , Tatuaje , Humanos , Enfermedades de la Piel/etiología , Enfermedades de la Piel/patología , Tatuaje/efectos adversosRESUMEN
Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patients.
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Based on the French Federation Nationale des Centers de Lutte Contre le Cancer (FNCLCC) grading system, this study assesses the accuracy of conventional and modified core biopsy (CB) systems in predicting the final grade (low vs high) assigned to the resected specimen. Substituting Ki-67 immunoexpression for mitotic count, and radiological for histological assessment of necrosis, we used two modified FNCLCC CB grading systems: (1) Ki-67 immunoexpression alone, and (2) Ki-67 plus radiological assessment of necrosis. We graded 199 soft tissue sarcomas (STS) from nine centers, and compared the results for the conventional (obtained from local histopathology reports) and modified CB systems with the final FNCLCC grading of the corresponding resected specimens. Due to insufficient sample quality or lack of available radiologic data, five cases were not evaluated for Ki67 or radiological assessment of necrosis. The conventional FNCLCC CB grading system accurately identified 109 of the 130 high-grade cases (83.8%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (87.9%) of the 199 resected tumors; overestimating the final grade in three cases and underestimating in 21 cases. Modified system 1 (Ki-67) accurately identified 117 of the 130 high-grade cases (90.0%). The CB grading matched the final FNCLCC grading (low vs high) in 175 (89.7%) of the 195 evaluated cases; overestimating seven and underestimating 13 cases. Modified system 2 (Ki-67 plus radiological necrosis) accurately identified 120 of the 130 high-grade cases (92.3%). This last matched the final FNCLCC grading (low vs high) in 177 (91.2%) of the 194 evaluated cases; overestimating seven and underestimating 10 cases. Modified system 2 obtained highest area under ROC curves, although not statistically significant. Underestimated CB grades did not correlate with histological subtypes, although many of the discrepant cases were myxoid tumors (myxofibrosarcomas or myxoid liposarcomas), leiomyosarcomas or undifferentiated pleomorphic/spindle cell sarcomas. Using modified FNCLCC CB grading systems to replace conventional mitotic count and histologic assessment of necrosis may improve the distinction between low and high-grade STS on CB. Our study confirms that classifying grade 1 as low grade and grades 2 and 3 as high grade improves correlation between CB and final grade by up to 21%, irrespective of CB system used. A higher than expected Ki-67 score in a low-grade sarcoma diagnosed on CB should raise concern that a higher-grade component may not have been sampled. Furthermore, correlation of all clinicopathological and radiological findings at multidisciplinary meetings is essential to assess the histological grade on CB as accurately as possible.
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Antígeno Ki-67/metabolismo , Sarcoma/metabolismo , Neoplasias de los Tejidos Blandos/metabolismo , Adulto , Biomarcadores de Tumor/metabolismo , Biopsia con Aguja Gruesa , Femenino , Humanos , Masculino , Necrosis/metabolismo , Necrosis/patología , Estudios Retrospectivos , Sarcoma/patología , Neoplasias de los Tejidos Blandos/patologíaRESUMEN
ABSTRACT: The prognostic implications of the immunophenotype of the tumor-infiltrating lymphocytes (TILs) in primary cutaneous melanoma are well known. In recent years, the study of this immunophenotype has also resulted in immunotherapeutic consequences. The aims of this study were to characterize the subpopulations of TILs in primary cutaneous melanoma, in cases with and without metastasis, as well as the neovascularization associated with the primary neoplasm, and its influence on the development of metastasis. To this end, the immunophenotype of TILs and the neovascularization of 80 patients with primary cutaneous melanoma (40 each with metastatic and non-metastatic melanoma) were analyzed by immunohistochemistry for CD3, CD4, CD8, FOXP3, PD-1, CD31, and D2-40 antibodies. We found that higher frequencies of TILs with brisk pattern, and CD4+, CD8+, and CD20+ cells in TILs, and a lower frequency of CD31+ vessels were histopathological features associated with better prognosis in primary cutaneous melanoma. Our results support the notion that the immunohistochemical study of TILs and neovascularization in primary cutaneous melanoma may be helpful tools for identifying patients at increased risk of metastasis development.
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Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/metabolismo , Melanoma/patología , Neovascularización Patológica/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Adulto , Anciano , Anticuerpos Monoclonales de Origen Murino/metabolismo , Antígenos CD20/metabolismo , Vasos Sanguíneos/metabolismo , Complejo CD3/metabolismo , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/patología , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/patología , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Inmunohistoquímica , Inmunofenotipificación , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Melanoma/secundario , Persona de Mediana Edad , Neovascularización Patológica/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Piel/irrigación sanguíneaRESUMEN
BACKGROUND: Angiolipoma (AL) is considered as a lipoma variant that is characterized by the combination of mature adipocytes and capillary blood vessels diffusely distributed within the tumor. With the exception of recurrent PRKD2 mutations of uncertain pathogenetic significance, the genetic abnormalities of ALs are unknown, in the absence of any of the specific chromosomal aberrations described in other lipoma variants. METHODS: Formalin-fixed and paraffin-embedded blocks of 13 conventional ALs and 5 cellular ALs from 17 individuals were retrieved and analyzed for mutations in exons 9 and 20 of PIK3CA by polymerase chain reaction and Sanger sequencing. RESULTS: Activating PIK3CA mutations were identified in 14 tumors (78%). All PIK3CA-mutated samples carried the same exon 9 mutation, c.1634A>C (p.E545A). No mutation was detected in exon 20 of PIK3CA. No significant difference between PIK3CA-mutated and wild-type samples appeared to exist based on age, gender, and location of the tumor. All 5 cellular ALs carried the p.E545A PIK3CA mutation. CONCLUSION: The high frequency of the p.E545A PIK3CA mutation in both conventional and cellular ALs suggests that activation of the PI3K/AKT pathway plays a key role in AL pathogenesis and reinforces the concept that cellular AL should be regarded as a variant of AL.
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Angiolipoma/genética , Aberraciones Cromosómicas , Fosfatidilinositol 3-Quinasa Clase I/genética , Mutación Missense , Neoplasias Cutáneas/genética , Adulto , Anciano , Sustitución de Aminoácidos , Angiolipoma/enzimología , Angiolipoma/patología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patologíaRESUMEN
We present a case of 15-year old male with solitary fibrofolliculoma on the ear and we demonstrate the use of ultrasound in outlining the features of this rare benign skin tumor with histological correlation. Fibrofolliculoma can be associated with a rare syndrome known as Birt-Hogg-Dubé which affects the skin, lungs and kidneys.
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Síndrome de Birt-Hogg-Dubé , Neoplasias Cutáneas , Adolescente , Síndrome de Birt-Hogg-Dubé/diagnóstico por imagen , Síndrome de Birt-Hogg-Dubé/patología , Oído Externo/diagnóstico por imagen , Oído Externo/patología , Humanos , Masculino , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/patologíaRESUMEN
The application of emerging technologies such as ultrasound, microwaves and high hydrostatic pressure, allows the extraction of compounds in a sustainable manner from a vegetable matrix with a high value such as jackfruit leaf proteins (Artocarpus heterophyllus Lam). Currently, the main method of protein extraction is based on the precipitation with the use of an aggressive solvent, therefore it is necessary to optimize extraction methods with a minimum waste production. In the protein extraction of jackfruit leaves, we obtained a content of 84.1 mg/g using solvents. On the other hand, emergent extractions such as ultrasound, microwaves, and high hydrostatic pressure showed concentrations of 96.3, 95.6 and 147.3 mg/g, respectively. In addition, we found that the best extraction agent was 0.5 M NaCl, offering a range of possibilities that support green technologies as an imperative change in the food industry.
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Deep penetrating nevus (DPN) is an intradermal, sometimes compound benign melanocytic lesion, which involves the reticular dermis, occasionally reaching the subcutis, which can raise concern for melanoma both clinically and histologically. Recently, it has been genetically defined by the combination of MAPK activating and ß-catenin activating mutations. We sought to investigate genetic alterations in 2 cases of combined nevi of congenital melanocytic and DPN. Case 1 was a 16-year-old woman with a pigmented lesion on the trunk since birth, which was completely excised. Histopathological examination revealed a combined congenital nevus with a DPN. Comparative genomic hybridization showed no major genetic alterations, except for gain of 6q11.1 and point mutation of B-RAF V600E. Case 2 was a 62-year-old woman with a congenital pigmented lesion on the back. The lesion was diagnosed as a combined nevus of congenital and DPN. Comparative genomic hybridization showed no genetic alterations, and the NRAS Q61K was detected in both components. DPN is in most cases part of a combined nevus. Our cases showed strong and uniform nuclear expression of ß-catenin and cyclin D1 in the DPN component suggesting the evolution of the congenital nevus to the DPN clone by acquiring ß-catenin activating mutation.
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Biomarcadores de Tumor/genética , Mutación con Ganancia de Función , Nevo Pigmentado/congénito , Neoplasias Cutáneas/congénito , beta Catenina/genética , Adolescente , Biomarcadores de Tumor/análisis , Hibridación Genómica Comparativa , Ciclina D1/análisis , Femenino , GTP Fosfohidrolasas/genética , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Proteínas de la Membrana/genética , Persona de Mediana Edad , Invasividad Neoplásica , Nevo Pigmentado/química , Nevo Pigmentado/patología , Nevo Pigmentado/cirugía , Fenotipo , Mutación Puntual , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/química , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/cirugía , beta Catenina/análisisRESUMEN
In mouse models of squamous cell carcinoma, pre-treatment with calcitriol prior to photodynamic therapy with aminolevulinic acid (ALA) enhances tumor cell death. We have evaluated the association between vitamin D status and the response of actinic keratoses to photodynamic therapy with methylaminolevulinate. Twenty-five patients with actinic keratoses on the head received one session of photodynamic therapy with methylaminolevulinate. Biopsies were taken at baseline and six weeks after treatment. Immuno-histochemical staining was performed for VDR, P53, Ki67 and -catenin. Basal serum 25(OH)D levels were determined. Cases with a positive histological response to treatment had significantly higher serum 25(OH)D levels (26.96 (SD 7.49) ngr/mL) than those without response (18.60 (SE 7.49) ngr/mL) (p = 0.05). Patients with a complete clinical response displayed lower basal VDR expression (35.71% (SD 19.88)) than partial responders (62.78% (SD 16.735)), (p = 0.002). Our results support a relationship between vitamin D status and the response of actinic keratoses to photodynamic therapy with methylaminolevulinate.
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OBJECTIVES: Basal cell carcinoma (BCC) is the most common dermatologic malignant skin cancer. Infiltrative histologic variants are more aggressive and require wider surgical margins or Mohs surgery, in contrast with noninfiltrative variants, which are commonly treated with standard surgical excision. Elastography has not been used to date to differentiate between the histologic variants of BCC. The purpose of this study was to differentiate infiltrative from noninfiltrative BCCs on elastography. METHODS: A total of 31 facial BCCs were studied. Preoperatively, color Doppler ultrasound and strain elastographic examinations of the lesions were performed. The size, intralesional vascularization, and presence of hyperechoic dots were considered relevant B-mode and color Doppler variables. Strain ratios of the tumors were obtained with respect to adjacent healthy tissue. Increased marginal stiffness, which was considered hardness in greater than 50% of the tumor margin, was also blindly evaluated. Histologic confirmation and subtyping (infiltrative or noninfiltrative) were performed in all cases. RESULTS: Infiltrative BCCs did not differ in the size, presence or absence of hyperechoic dots, or vascularization from noninfiltrative BCCs. Strain ratios were similar in both infiltrative and noninfiltrative BCCs (mean ± SD, 1.82 ± 0.879 versus 2.2 ± 1.11). However, infiltrative BCCs had statistically increased marginal stiffness in comparison with noninfiltrative BCCs (88.0% versus 18.8%). Increased marginal stiffness had sensitivity and specificity of 0.89 and 0.82 respectively, with a positive predictive value of 0.67 for infiltrative BCCs and a negative predictive value of 0.95 for noninfiltrative BCCs. CONCLUSIONS: Histologic variants of BCC have different elastographic patterns. These differences may be of help in preoperative assessments of the BCC subtype and specific surgical planning, avoiding unnecessary skin biopsies.
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Carcinoma Basocelular/diagnóstico por imagen , Carcinoma Basocelular/patología , Diagnóstico por Imagen de Elasticidad , Neoplasias Faciales/diagnóstico por imagen , Neoplasias Faciales/patología , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/patología , Ultrasonografía Doppler en Color , Anciano , Biopsia , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
The ATPase inhibitory factor 1 (IF1) is an intrinsically disordered protein that regulates the activity of the mitochondrial ATP synthase. Phosphorylation of S39 in IF1 prevents it from binding to the enzyme and thus abolishes its inhibitory activity. Dysregulation of IF1 is linked to different human diseases, providing a relevant biomarker of cancer progression. However, the tissue content of IF1 relative to the abundance of the ATP synthase is unknown. In this study, we characterized the tissue-specific expression of IF1 in human and mouse tissues and quantitated the content of IF1 and of ATP synthase. We found relevant differences in IF1 expression between human and mouse tissues and found that in high-energy-demanding tissues, the molar content of IF1 exceeds that of the ATP synthase. In these tissues, a fraction of IF1 is bound to the enzyme, and the other fraction is phosphorylated and hence is unable to bind the enzyme. Post-transcriptional control accounts for most of the regulated expression of IF1, especially in mouse heart, where IF1 mRNA translation is repressed by the leucine-rich pentatricopeptide repeat containing protein. Overall, these findings enlighten the cellular biology of IF1 and pave the way to development of additional models that address its role in pathophysiology.-Esparza-Moltó, P. B., Nuevo-Tapioles, C., Chamorro, M., Nájera, L., Torresano, L., Santacatterina, F., Cuezva, J. M. Tissue-specific expression and post-transcriptional regulation of the ATPase inhibitory factor 1 (IF1) in human and mouse tissues.
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Proteínas/fisiología , Procesamiento Postranscripcional del ARN , Animales , Línea Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/genética , Proteína Inhibidora ATPasaRESUMEN
AIM: To compare the immunological and clinical changes induced by allergen-specific immunotherapy (AIT) using different adjuvants. MATERIALS & METHODS: Olea europaea pollen-sensitized mice were treated with olea plus aluminum hydroxide, calcium phosphate, monophosphoryl lipid A (MPL) or immunostimulatory sequences (ISS). RESULTS: Aluminum hydroxide seems to drive initially to a Th2-type response. Bacteria-derived adjuvants (MPL and ISS) skew the immune response toward Th1 and Treg pathways. Specific-IgE production was lower after AIT with MPL and ISS. Moreover, IgG2a production significantly increased in ISS-treated mice. Bacteria-derived adjuvants also improved the Th1 cytokine response due to IFN-γ higher secretion. In addition, they improved bronchial hyper-reactivity and lung inflammation. CONCLUSION: Bacteria-derived adjuvants may enhance the efficacy of AIT.
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Adyuvantes Inmunológicos/uso terapéutico , Alérgenos/uso terapéutico , Desensibilización Inmunológica/métodos , Hipersensibilidad/terapia , Extractos Vegetales/uso terapéutico , Alérgenos/inmunología , Hidróxido de Aluminio/uso terapéutico , Animales , Antígenos de Plantas/inmunología , Fosfatos de Calcio/uso terapéutico , Modelos Animales de Enfermedad , Femenino , Humanos , Hipersensibilidad/inmunología , Lípido A/análogos & derivados , Lípido A/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Olea/inmunología , Extractos Vegetales/inmunología , Polen/inmunología , Pruebas de Función RespiratoriaRESUMEN
Synovial sarcoma (SS) accounts for 5%-10% of all soft tissue sarcomas. It is a well-defined soft tissue neoplasm with biphasic and monophasic histologic subtypes and unknown histogenesis. It usually occurs in the extremities, especially the thigh-knee region of young adults. Recurrences are frequent and distant metastasis developed in approximately half of the patients. SSs are characterized by a recurrent nonrandom chromosomal translocation, t(X; 18) (p11; q11), which is considered the primary genetic event in more than 90% of cases. Only 4 cases of cutaneous and subcutaneous SSs have been published in the literature so far. We report a case of primary subcutaneous SS in the forearm of a young woman and discuss the histopathologic differential diagnosis with other similar neoplasms. This is the first reported case of primary cutaneous SS showing immunoreactivity for TLE1 in the nuclei of neoplastic cells, supporting the use of this marker for diagnosis of this rare cutaneous neoplasm.
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Biomarcadores de Tumor/inmunología , Inmunohistoquímica , Proteínas Represoras/inmunología , Sarcoma Sinovial/inmunología , Neoplasias de los Tejidos Blandos/inmunología , Tejido Subcutáneo/inmunología , Adulto , Biomarcadores de Tumor/genética , Biopsia , Proteínas Co-Represoras , Diagnóstico Diferencial , Femenino , Antebrazo , Reordenamiento Génico , Humanos , Hibridación Fluorescente in Situ , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Sarcoma Sinovial/genética , Sarcoma Sinovial/patología , Sarcoma Sinovial/cirugía , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/patología , Neoplasias de los Tejidos Blandos/cirugía , Tejido Subcutáneo/patología , Tejido Subcutáneo/cirugíaRESUMEN
Acral persistent papular mucinosis is a subtype of localized lichen myxedematosus. It presents as acrally located papules with a benign, but persistent course. It is a scarcely reported disease. We present a female with both the clinical and histopathological described criteria.