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1.
Methods Mol Biol ; 2265: 289-304, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704723

RESUMO

Cells release extracellular vesicles (EVs) that can be detected both in vivo and in cell culture medium. Among EVs, exosomes are 50-150 nm vesicles that are systematically packaged into multivesicular bodies for release into the external environment. In cancer, these intentionally packaged exosomes carry a payload of proteins such as RNAs and surface receptors that facilitate the reprogramming of proximal cells to assemble a protumor microenvironment. Exosomes have been implicated as an important intermediary extracellular communication pathway between cells, including in melanoma. Human melanoma-derived exosomes (HMEX) have been demonstrated to modulate the extracellular environment and inhibit immune cell activation. There are many methods to isolate and enrich for exosomes and the method applied can impact yield and purity of the isolates. In this chapter we describe the REIUS (rapid exosome isolation using ultrafiltration and size exclusion chromatography) method to isolate HMEX from melanoma cell cultures and then demonstrate their enrichment using molecular and microscopic approaches.


Assuntos
Exossomos/química , Melanoma/química , Linhagem Celular Tumoral , Cromatografia em Gel , Humanos , Ultrafiltração
2.
PLoS One ; 15(9): e0238591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32886718

RESUMO

Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as "biological adhesion", "cell motility", and "cellular component biogenesis". EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy.


Assuntos
Vesículas Extracelulares/patologia , Neoplasias/patologia , Proteínas/análise , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/química , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Carcinoma Ductal Pancreático/química , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/patologia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Vesículas Extracelulares/química , Humanos , Espectrometria de Massas , Melanoma/química , Melanoma/diagnóstico , Melanoma/patologia , Neoplasias Bucais/química , Neoplasias Bucais/diagnóstico , Neoplasias Bucais/patologia , Neoplasias/química , Neoplasias/diagnóstico , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/patologia , Proteômica
3.
J Nanobiotechnology ; 18(1): 20, 2020 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-31973696

RESUMO

BACKGROUND: Extracellular vesicles (EVs) have shown great potential for targeted therapy, as they have a natural ability to pass through biological barriers and, depending on their origin, can preferentially accumulate at defined sites, including tumors. Analyzing the potential of EVs to target specific cells remains challenging, considering the unspecific binding of lipophilic tracers to other proteins, the limitations of fluorescence for deep tissue imaging and the effect of external labeling strategies on their natural tropism. In this work, we determined the cell-type specific tropism of B16F10-EVs towards cancer cell and metastatic tumors by using fluorescence analysis and quantitative gold labeling measurements. Surface functionalization of plasmonic gold nanoparticles was used to promote indirect labeling of EVs without affecting size distribution, polydispersity, surface charge, protein markers, cell uptake or in vivo biodistribution. Double-labeled EVs with gold and fluorescent dyes were injected into animals developing metastatic lung nodules and analyzed by fluorescence/computer tomography imaging, quantitative neutron activation analysis and gold-enhanced optical microscopy. RESULTS: We determined that B16F10 cells preferentially take up their own EVs, when compared with colon adenocarcinoma, macrophage and kidney cell-derived EVs. In addition, we were able to detect the preferential accumulation of B16F10 EVs in small metastatic tumors located in lungs when compared with the rest of the organs, as well as their precise distribution between tumor vessels, alveolus and tumor nodules by histological analysis. Finally, we observed that tumor EVs can be used as effective vectors to increase gold nanoparticle delivery towards metastatic nodules. CONCLUSIONS: Our findings provide a valuable tool to study the distribution and interaction of EVs in mice and a novel strategy to improve the targeting of gold nanoparticles to cancer cells and metastatic nodules by using the natural properties of malignant EVs.


Assuntos
Antineoplásicos/química , Vesículas Extracelulares/química , Ouro/química , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/metabolismo , Melanoma/química , Nanopartículas Metálicas/química , Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/terapia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/terapia , Corantes Fluorescentes/química , Humanos , Pulmão/metabolismo , Melanoma Experimental/diagnóstico por imagem , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Imagem Óptica , Propriedades de Superfície , Distribuição Tecidual
4.
Proc Natl Acad Sci U S A ; 117(6): 2767-2769, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31988118

RESUMO

While single-cell sequencing technologies have revealed tissue heterogeneity, resolving mixed cellular libraries into cellular clones is essential for many pooled screens and clonal lineage tracing. Fluorescent proteins are limited in number, while DNA barcodes can only be read after cell lysis. To overcome these limitations, we used influenza virus hemagglutinins to engineer a genetically encoded cell-surface protein barcoding system. Using antibodies paired to hemagglutinins carrying combinations of escape mutations, we developed an exponential protein barcoding system which can label 128 clones using seven antibodies. This study provides a proof of principle for a strategy to create protein-level cell barcodes that can be used in vivo in mice to track clonal populations.


Assuntos
Anticorpos Monoclonais/análise , Rastreamento de Células/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/análise , Animais , Rastreamento de Células/instrumentação , Feminino , Citometria de Fluxo/métodos , Células HEK293 , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Melanoma/química , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Orthomyxoviridae/química , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo
6.
Am J Surg Pathol ; 44(4): 503-508, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31633488

RESUMO

The distinction of metastatic melanoma from melanocytic nevi in lymph nodes can on occasion be difficult. As diffuse immunohistochemical (IHC) PRAME (PReferentially expressed Antigen in MElanoma) expression is detected in the majority of primary and metastatic melanomas, but rarely in nevi, we reasoned that PRAME could be a useful adjunct marker for the diagnosis of melanocytes in lymph nodes. In this study, we examined 45 nodal melanocytic deposits comprising 30 nodal nevi and 15 melanoma metastases. The latter were diagnostically not straightforward because they either coexisted with nodal nevi or were present in perinodal fibrous tissue. All nodal nevi (30/30) were negative for PRAME, whereas all melanoma metastases (15/15) were diffusely positive for PRAME IHC. We additionally report the novel use of a PRAME/Melan A dual-label immunostain. Our results show that PRAME IHC may be useful in the assessment of diagnostically challenging nodal melanocytic deposits, such as intraparenchymal nodal nevi, metastases confined to the capsular fibrous tissue, or in the setting of small metastases coexisting with a nodal nevus in the same lymph node.


Assuntos
Antígenos de Neoplasias/análise , Biomarcadores Tumorais/análise , Imuno-Histoquímica , Melanoma/química , Nevo/química , Linfonodo Sentinela/química , Neoplasias Cutâneas/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Humanos , Metástase Linfática , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Nevo/patologia , Valor Preditivo dos Testes , Linfonodo Sentinela/patologia , Neoplasias Cutâneas/patologia
7.
Pathol Res Pract ; 215(12): 152668, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31585813

RESUMO

Desmoplastic melanoma (DM) is an uncommon variant of malignant melanoma (MM), histologically characterized by a mainly dermal proliferation of spindled cells within a desmoplastic stroma. Normally, involvement of deeper tissues by DM is the result of direct extension down from the overlying dermis. MM is widely known to harbor a striking potential for morphological and phenotypic variability; among MM morphological variants, pseudoglandular MM is characterized by extensive discohesion within cords and nests of malignant cells and ensuing formation of so-called pseudolumina, thus mimicking adenocarcinoma. We present an exceptional case of DM characterized by intrafascial origin, partly pseudoglandular differentiation, and aberrant experession of cytokeratins in the pseudoglandular component; genetic data from next-generation sequencing supported the final diagnosis of DM, as well as the ontogenetic identity of the pseudoglandular component. Prior to this report, pseudoglandular features had never been described in DM. Additionally, our case is unusual because of the deep origin of the tumor, arising below the subcutaneous fat of the scalp, as well as the aberrant experession of cytokeratins in the pseudoglandular component, thus posing a challenging differential diagnosis with several soft tissue tumors.


Assuntos
Biomarcadores Tumorais/análise , Diferenciação Celular , Neoplasias de Cabeça e Pescoço/química , Neoplasias de Cabeça e Pescoço/patologia , Queratinas/análise , Melanoma/química , Melanoma/patologia , Couro Cabeludo/química , Couro Cabeludo/patologia , Neoplasias Cutâneas/química , Neoplasias Cutâneas/patologia , Idoso , Biomarcadores Tumorais/genética , Diagnóstico Diferencial , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Masculino , Melanoma/genética , Melanoma/cirurgia , Valor Preditivo dos Testes , Couro Cabeludo/cirurgia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/cirurgia
8.
Chem Biodivers ; 16(11): e1900322, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31544357

RESUMO

The synthesis of poly[N,N-bis(3-aminopropyl)glycine] (PAPGly) dendrons Gd-based contrast agents (GdCAs) via an orthogonal protection of the different functional groups and an activation/coupling strategy wherein a specific number of synthetic steps add a generation to the existing dendron has been described. The aim of this protocol is to build up two different generations of dendrons (G-0 or dendron's core, and G-1) with peripheral NH2 groups to conjugate a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) derivative and afterwards to chelate with Gd3+ paramagnetic ions. These complexes, which have a well-defined molecular weight, are of relevance to MRI as an attempt to gain higher 1 H relaxivity by slowing down the rotation of molecule compared to monomeric Gd(III) complexes used as contrast agents and to increase the number of paramagnetic centers present in one molecular structure. From the study of their water 1 H longitudinal relaxation rate at different magnetic fields (NMRD, Nuclear Magnetic Relaxation Dispersion) and by evaluating the variable temperature 17 O-NMR data we determined the parameters characterizing the water exchange rate and the rotational correlation time of each complex, both affecting 1 H relaxivity. Furthermore, these two novel PAPGly GdCAs were objects of i) an in vivo study to determine their biodistributions in healthy C57 mice at several time points, and ii) the Dynamic Contrast-Enhanced MRI (DCE-MRI) approach to assess their contrast efficiency measured in the tumor region of C57BL/6 mice transplanted subcutaneously with B16-F10 melanoma cells. The aim of the comparison of these two dendrons GdCAs, having different molecular weights (MW), is to understand how MW and relaxivity may influence the contrast enhancement capabilities in vivo at low magnetic field (1 T). Significant contrast enhancement was observed in several organs (vessel, spleen and liver), already at 5 min post-injection, for the investigated CAs. Moreover, these CAs induced a marked contrast enhancement in the tumor region, thanks to the enhanced permeability retention effect of those macromolecular structures.


Assuntos
Meios de Contraste/química , Gadolínio/química , Melanoma/química , Compostos Organometálicos/química , Animais , Meios de Contraste/síntese química , Meios de Contraste/farmacocinética , Gadolínio/farmacocinética , Humanos , Imagem por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Masculino , Melanoma/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Neoplasias Experimentais/química , Neoplasias Experimentais/diagnóstico por imagem , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacocinética , Distribuição Tecidual
9.
Rev Esp Patol ; 52(4): 234-241, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31530406

RESUMO

Tumors display a high, albeit variable, grade of intratumor heterogeneity, both from a clinical and a morphological viewpoint. Furthermore, recent methods of large-scale molecular analysis demonstrate to what extent tumors can also be heterogeneous from a molecular perspective. This is of paramount importance for patients as it has a great impact on the success of so-called precision therapies and explains the reason for a significant number of therapeutic failures in modern oncology. We present an up-to-date review of the latest findings in a group of tumors with a high social impact, commonly seen in the daily routine of the pathology laboratory.


Assuntos
Neoplasias/patologia , Neoplasias da Mama/patologia , Carcinoma de Células Renais/química , Carcinoma de Células Renais/patologia , Células Clonais/química , Células Clonais/patologia , Progressão da Doença , Neoplasias do Endométrio/química , Neoplasias do Endométrio/patologia , Feminino , Humanos , Neoplasias Renais/química , Neoplasias Renais/patologia , Melanoma/química , Melanoma/patologia , Mutação , Invasividade Neoplásica , Proteínas de Neoplasias/análise , Neoplasias/química , Neoplasias Primárias Múltiplas/patologia , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/patologia , Sarcoma/química , Sarcoma/patologia , Análise de Célula Única , Microambiente Tumoral , Neoplasias Uterinas/química , Neoplasias Uterinas/patologia
10.
Anticancer Res ; 39(6): 3147-3157, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177161

RESUMO

BACKGROUND/AIM: Regional lymph node recurrence (RLNR) is the most common pattern of recurrence within 2 years from the diagnosis of patients with non-metastatic malignant cutaneous melanoma. However, isolated RLNR without distant metastasis has been rarely studied. PATIENTS AND METHODS: Forty patients with isolated RLNR as a first recurrence were analyzed retrospectively. The clinical outcomes and prognostic impact of clinicopathologic parameters were analyzed. Immunostaining for FOXP3, VEGF, pAKT, and pS6 was also performed. RESULTS: The median disease-free interval from first diagnosis to isolated RLNR and post-recurrence recurrence-free survival (pRFS) were 12 months and 7.2 months, respectively. Distant failure was the most common pattern of failure after isolated RLNR (67.5%). The number of initially harvested lymph nodes (LN) >7 and LN ratio >22.2% at the time of recurrence were prognosticators for pRFS in multivariate analysis. None of the tested biomarkers were significantly related to prognosis. The 5-year post-recurrence overall survival rate was 84.9%. CONCLUSION: Most patients with isolated RLNR will experience a second failure within months, especially distantly. The number of initially harvested LNs and LN ratio at the time of recurrence could predict pRFS.


Assuntos
Melanoma/secundário , Neoplasias Cutâneas/patologia , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Bases de Dados Factuais , Progressão da Doença , Feminino , Fatores de Transcrição Forkhead/análise , Humanos , Excisão de Linfonodo , Metástase Linfática , Masculino , Melanoma/química , Melanoma/mortalidade , Melanoma/cirurgia , Pessoa de Meia-Idade , Fosforilação , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas c-akt/análise , Estudos Retrospectivos , Proteína S6 Ribossômica/análise , Fatores de Risco , Neoplasias Cutâneas/química , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/cirurgia , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/análise , Adulto Jovem
12.
Anal Bioanal Chem ; 411(15): 3309-3319, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31123778

RESUMO

Nowadays, functionalization of the plasmon-supported nanostructured surface is considered as a powerful tool for tumour cell recognition. In this study, the SERS on a surface plasmon polariton-supported gold grating functionalized with folic acid was used to demonstrate an unpretentious recognition of melanoma-associated fibroblasts. Using cultivation media conditioned by different cells, we were able to detect reproducible differences in the secretome of melanoma-associated and normal control fibroblasts. The homogeneous distribution of plasmon energy along the grating surface was proved to provide excellent SERS signal reproducibility, while, to increase the affinity of (bio)molecules to SERS substrate, folic acid molecules were covalently grafted to the gold gratings. As proof of concept, fibroblasts were cultured in vitro, and culture media from the normal and tumour-associated lines were collected and analysed with our proposed SERS substrates. Identifying individual peaks of the Raman spectra as well as comparing their relative intensities, we showed that the proposed functional SERS platform can recognise the melanoma-associated cells without the need for further statistical spectral evaluation directly. We also demonstrated that the SERS chip created provided a stable SERS signal over a period of 90 days without loss of sensitivity. Graphical abstract.


Assuntos
Fibroblastos Associados a Câncer/patologia , Ácido Fólico/química , Ouro/química , Melanoma/patologia , Nanopartículas Metálicas/química , Análise Espectral Raman/métodos , Fibroblastos Associados a Câncer/química , Células Cultivadas , Humanos , Melanoma/química , Propriedades de Superfície , Células Tumorais Cultivadas
13.
Am J Surg Pathol ; 43(6): 755-765, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31091203

RESUMO

Although immunohistochemistry (IHC) has improved our ability to detect melanoma metastases in sentinel lymph nodes (SLN), the American Joint Committee on Cancer (AJCC) does not provide a lower threshold for determining if a SLN is positive for metastasis. Existing literature suggests that even a small aggregate or an enlarged, abnormal cell detectable by IHC can be associated with an adverse outcome. In our experience, however, some SLNs contain small solitary cells the size of neighboring lymphocytes demonstrable only by IHC. We sought to determine their clinical significance. A total of 821 patients underwent a SLN biopsy at our institution over a 12-year period. In all, 639 (77.8%) were SLN-negative, 125 (15.2%) were SLN-positive, and 57 (6.9%) had rare IHC-positive cells of undetermined clinical significance with no disease progression over a mean 59-month follow-up. Kaplan-Meier method with pair-wise comparisons revealed no significant difference in disease-specific survival and recurrence-free survival between SLN-negative and rare IHC-positive groups. There were significant differences in survival and recurrence between patients in the rare IHC-positive group and those with melanoma metastases, including those with solitary melanoma cells and those with tumor burdens ≤0.2 mm. While the lower diagnostic threshold for metastatic melanoma on IHC-stained sections needs to be studied further, our data suggest that rare IHC-positive cells lacking cytomorphologic features of overt malignancy are equivocal for melanoma and could impart a similar prognosis as patients with no evidence of SLN involvement.


Assuntos
Biomarcadores Tumorais/análise , Imuno-Histoquímica , Melanoma/química , Recidiva Local de Neoplasia , Linfonodo Sentinela/química , Neoplasias Cutâneas/química , Adulto , Feminino , Humanos , Metástase Linfática , Masculino , Melanoma/mortalidade , Melanoma/secundário , Melanoma/terapia , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Intervalo Livre de Progressão , Estudos Retrospectivos , Fatores de Risco , Linfonodo Sentinela/patologia , Biópsia de Linfonodo Sentinela , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Fatores de Tempo
14.
Hum Pathol ; 88: 78-86, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30965022

RESUMO

We observed that non-tumor-infiltrating inflammatory cells are often present in the stroma of melanoma. The role of these stromal inflammatory cells (SIC) in cancer has not been studied. We evaluated the prognostic significance of SIC in 299 patients with vertical growth phase primary melanomas with at least 10 years of clinical follow-up. Lymphatic density and lymphatic invasion in the areas with SIC was quantified. The prognostic significance of these factors was evaluated using univariable and multivariable Cox models for melanoma-specific death and the time to first recurrence. Of the 299 melanomas, 161 exhibited areas with SIC. Percentages of vertical growth phase tumor-infiltrating lymphocytes and radial growth phase regression were significantly higher in cases with SIC compared to those without SIC (P = .005); lymphatic invasion was also detected more frequently in cases with SIC (P = .001). Lymphatic density in SIC areas was higher than that in other areas of the melanomas. Patients with SIC had poorer clinical outcome. Vascular endothelial growth factor-C (VEGFC) staining in a subset of these melanoma patients showed that VEGFC expression in the stromal macrophages was associated with lymphatic invasion in SIC areas. In conclusion, SIC in melanoma is associated with poorer prognosis, and the prognostic effect is partially mediated through induction of lymphangiogenesis with increased lymphatic invasion.


Assuntos
Inflamação/patologia , Linfócitos do Interstício Tumoral/patologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Feminino , Humanos , Macrófagos/química , Masculino , Melanoma/química , Melanoma/mortalidade , Pessoa de Meia-Idade , Prognóstico , Neoplasias Cutâneas/química , Neoplasias Cutâneas/mortalidade , Células Estromais/patologia , Fator C de Crescimento do Endotélio Vascular/análise
15.
Artigo em Inglês | MEDLINE | ID: mdl-30897406

RESUMO

Determination of eumelanin and pheomelanin in melanomas that exhibit different pigmentation was carried using a solid-phase extraction (SPE) preparation method based on weak anion exchange chemistry. This extraction significantly enhanced the chromatographic profile obtained by reverse phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD). The SPE method was developed using aqueous standards of melanin markers: thiazole-2,4,5-tricarboxylic acid (TTCA), thiazole-4,5-dicarboxylic acid (TDCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and non-pigmented cell lines spiked with those markers. An excellent average recovery, above 90%, was obtained for the four markers with a relative standard deviation below 7%. We have also optimized the stationary phase and the mobile phase (phosphate concentration and pH) to improve sensitivity and to reduce the analysis time. Elution of the four markers is achieved in 5 min and total analysis of biological samples is completed in 15 min. The quantification limits for TDCA, TTCA, PDCA and PTCA are 60, 50, 47 and 48 ng/mL respectively. Furthermore, DAD detection improves the marker identification in complex matrices through the analysis of UV spectra. We have successfully applied this method to melanoma tumors and cells. Murine B16BL6 tumor are highly pigmented with mostly eumelanin (98.1% of eumelanin) while human SK-MEL-3 tumor contain about 30% pheomelanin. B16BL6 and B16F10 are eumelanic cells lines and NHEM melanocytes contain about 24% of pheomelanin.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Melaninas/análise , Melanoma/química , Extração em Fase Sólida/métodos , Animais , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Modelos Lineares , Masculino , Melaninas/química , Melaninas/isolamento & purificação , Camundongos , Reprodutibilidade dos Testes
16.
Arch Pathol Lab Med ; 143(9): 1126-1130, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30855172

RESUMO

CONTEXT.­: Currently, no universal protocol exists for the assessment of sentinel lymph nodes (SLNs) in cutaneous melanoma. Many institutions use a multistep approach with multiple hematoxylin-eosin (H&E) and immunohistochemical stains. However, this can be a costly and time- and resource-consuming task. OBJECTIVE.­: To assess the utility for multistep protocols in the analysis of melanoma SLNs by specifically evaluating the Calgary Laboratory Services (CLS) protocol (which consists of 3 H&E slides and 1 S100 protein, 1 HMB-45, and 1 Melan-A slide per melanoma SLN block) and to develop a more streamlined protocol. DESIGN.­: Histologic slides from SLN resections from 194 patients with diagnosed cutaneous melanoma were submitted to the CLS dermatopathology group. Tissue blocks were processed according to the CLS SLN protocol. The slides were re-reviewed to determine whether or not metastatic melanoma was identified microscopically at each step of the protocol. Using SPSS software, a decision tree was then created to determine which step most accurately reflected the true diagnosis. RESULTS.­: We found with Melan-A immunostain that 337 of 337 negative SLNs (100%) were correctly diagnosed as negative and 55 of 56 positive nodes (98.2%) were correctly diagnosed as positive. With the addition of an H&E level, 393 of 393 SLNs (100%) were accurately diagnosed. CONCLUSIONS.­: We recommend routine melanoma SLN evaluation protocols be limited to 2 slides: 1 H&E stain and 1 Melan-A stain. This protocol is both time- and cost-efficient and yields high diagnostic accuracy.


Assuntos
Técnicas Histológicas/métodos , Melanoma/patologia , Biópsia de Linfonodo Sentinela/métodos , Linfonodo Sentinela/patologia , Neoplasias Cutâneas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Corantes , Amarelo de Eosina-(YS) , Feminino , Hematoxilina , Humanos , Imuno-Histoquímica/métodos , Antígeno MART-1/análise , Masculino , Melanoma/química , Antígenos Específicos de Melanoma/análise , Pessoa de Meia-Idade , Melhoria de Qualidade , Proteínas S100/análise , Sensibilidade e Especificidade , Neoplasias Cutâneas/química
17.
Ann Dermatol Venereol ; 146(5): 372-376, 2019 May.
Artigo em Francês | MEDLINE | ID: mdl-30910333

RESUMO

BACKGROUND: Myxoid melanoma is a rare variant of melanoma that must be recognised. Herein we describe a new metastatic case. PATIENTS AND METHODS: A 78-year-old woman consulted for a firm, pinkish nodule measuring 25mm and present for six months on her left leg. Analysis of the biopsy revealed achromic fusiform tumour cells separated by large myxoid plaques. Labeling of SOX10, HMB45 and PS100 was diffuse and of moderate to strong intensity. A diagnosis of myxoid melanoma was considered, with Breslow thickness of 9mm. Surgery was carried out with a 2-cm margin and confirmed the diagnosis. Dermatological follow-up at one year revealed metastatic spread to the ganglia, pleura, liver and bone. DISCUSSION: Few cases of primary myxoid melanoma have been described, and the condition is probably underdiagnosed. The classic clinical presentation of this condition consists of a solitary achromic nodule found chiefly on the limbs. The microscopic appearance is relatively non-specific. Immunohistochemical analysis may indicate melanocytic involvement: cells exhibit expression of SOX10, diffuse expression of protein S100, and less consistent and more variable expression of HMB45. The increasingly common use of anti-SOX10 is of value since it is expressed in the nucleus of melanocytes. Mastocytes and TGF-ß secretion appear to be involved in myxoid stroma production. In the absence of specific codification, management of myxoid melanoma is comparable to that of other types of melanoma. There is uncertainty about the prognosis, with the involvement of TGF-ß possibly indicating the aggressive potential of this type of tumour.


Assuntos
Perna (Membro) , Melanoma/patologia , Neoplasias Cutâneas/patologia , Idoso , Biomarcadores Tumorais/análise , Biópsia , Feminino , Humanos , Melanócitos/química , Melanoma/química , Antígenos Específicos de Melanoma/análise , Proteínas S100/análise , Fatores de Transcrição SOXE/análise , Neoplasias Cutâneas/química
18.
In Vivo ; 33(2): 567-572, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30804143

RESUMO

AIM: To investigate the molecular structural disorders of cancerous skin. MATERIALS AND METHODS: Human malignant melanoma and basal cell carcinoma biopsies were used for the investigation. Fourier transform infrared (FT-IR), Raman spectroscopy, and scanning electron microscopy were utilized. Spectral differences between healthy, basal cell carcinoma and melanoma tissues were recorded. RESULTS: The FT-IR bands of vasCH2, vsCH2 and Raman vsCH3 of cell membrane lipids were increased in intensity in melanoma due to an increased lipophilic environment. The FT-IR band at 1,744 cm-1 assigned to malondialdehyde can be used as a band diagnostic of cancer progression. The amide I bands at 1,654 cm-1 and 1,650 cm-1 for Raman and FT-IR, respectively were broader in spectra from melanoma, reflecting changes of protein secondary structure from α-helix to ß-sheet and random coil. The intensity of the FT-IR band at 1,046 cm-1 was increased in melanoma, suggesting glycosylation of the skin upon cancer development. Another band that might be considered as diagnostic was found at about 815 cm-1 in melanoma and was attributed to Z-DNA configuration. As far as we know, this is the first time that scanning electron microscopy revealed that metal components of titanium alloys from tooth implants were transferred to melanoma tissue taken from the back of one patient. CONCLUSION: Vibrational spectroscopy highlighted increased glycosylation in melanoma.


Assuntos
Carcinoma Basocelular/química , Melanoma/química , Proteínas de Neoplasias/química , Neoplasias Cutâneas/química , Idoso , Biópsia , Carcinoma Basocelular/patologia , Humanos , Malondialdeído/química , Melanoma/patologia , Pessoa de Meia-Idade , Estrutura Secundária de Proteína , Pele/química , Pele/patologia , Neoplasias Cutâneas/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman
19.
Biochem Biophys Res Commun ; 509(4): 1034-1040, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30660359

RESUMO

Malignant melanoma typically metastasizes to lymph nodes (LNs) as a primary or in-transit lesion before secondary metastasis occurs, and LN biopsy is a common procedure to diagnose melanoma progression. Since cancer metastasis is a complex process where various interactions between tumor cells and the stroma play key roles in establishing metastatic lesions, the exact mechanisms underlying melanoma metastasis to LNs remains unknown. It has been known that focal adhesion kinase (FAK) activity promotes the expression of proinflammatory vascular cell adhesion molecule-1 (VCAM-1). As VCAM-1 is a major receptor for α4 integrin and plays a key role in leukocyte recruitment, we reasoned that inhibition of FAK activity may reduce VCAM-1 expression within LNs and thus reduce metastasis of α4 integrin-expressing melanoma to LNs. First, we found that a pharmacological FAK inhibitor, PF-271, blocked tumor necrosis factor-α (TNF-α)-mediated VCAM-1 expression on human dermal lymphatic endothelial cells (HDLECs). In vitro, PF-271 significantly decreased B16F10 melanoma adhesion to and transmigration through HDLECs compared to TNF-α treated cells. Furthermore, in vivo FAK inhibition by oral PF-271 administration reduced VCAM-1 expression in inguinal, cervical, and popliteal LNs compared to vehicle treated mice. Finally, in a footpad metastasis model, B16F10 melanoma cells were injected into the right footpad of C57BL/6 mice, and PF-271 (50 mg/kg, twice daily for 6 days) was orally administrated after 1 week of tumor transplantation. While untreated mice exhibited significant metastatic melanoma lesions in popliteal LNs, PF-271 treated mice showed only marginal melanoma metastasis. These results support the possibility that FAK inhibitors may be a novel preventative option in melanoma metastasis by blocking VCAM-1 expression in LNs.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Integrina alfa4/metabolismo , Linfonodos/patologia , Melanoma/patologia , Metástase Neoplásica/prevenção & controle , Molécula 1 de Adesão de Célula Vascular/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Melanoma/química , Melanoma Experimental , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Molécula 1 de Adesão de Célula Vascular/metabolismo
20.
Turk Patoloji Derg ; 35(3): 237-241, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-28832076

RESUMO

The term collision tumor is used to describe two neoplasms occurring in the same anatomic location with juxtaposition of different tumor elements. Such a coexistence of tumors anywhere in the body is relatively rare. We report a case of 32-year-old female with collision tumor of rectum. The tumor showed two distinct histological patterns with predominant component consisting of malignant melanoma and a minor component of squamous cell carcinoma. The morphological picture of collision was further confirmed by specific immunohistochemical profile of the two tumors. Collision tumors of rectum are uncommon with most of the reported cases comprising adenocarcinoma and neuroendocrine tumors. To the best of our knowledge this is the first case of collision tumor of malignant melanoma and squamous cell carcinoma in the rectum.


Assuntos
Carcinoma de Células Escamosas/patologia , Melanoma/patologia , Neoplasias Primárias Múltiplas/patologia , Neoplasias Retais/patologia , Biomarcadores Tumorais/análise , Biópsia , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Melanoma/química , Melanoma/cirurgia , Neoplasias Primárias Múltiplas/química , Neoplasias Primárias Múltiplas/cirurgia , Neoplasias Retais/química , Neoplasias Retais/cirurgia
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