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1.
Recent Results Cancer Res ; 214: 169-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31473853

RESUMO

Treatment of patients with advanced metastatic melanoma has for decades been a story of very limited success. This dramatically changed when therapy with anti-PD-1 checkpoint blocking antibodies was approved in the USA and Europe in 2014 and 2015, respectively. The therapy exploits the capacity of CD8+ T cells to specifically kill tumor cells. Within the tumor microenvironment, CD8+ T cell activity is blocked by suppressive signals received via PD-1, an inhibitory co-receptor and so-called checkpoint of T cell activation. PD-1 binds to its ligand PD-L1 on melanoma cells which dampens the T cell's activity. Antibodies blocking inhibitory PD-1/PD-L1 interaction release T cells from suppression. Treatment of late-stage disease melanoma patients with antibodies targeting the PD-1/PD-L1 axis, termed immune checkpoint blocking therapy (ICBT), yields clinical frequently long-lasting responses in 30-40% of cases. Despite this remarkable breakthrough, still the majority of patients resists ICBT or develops resistance after initial therapy response. Administration of anti-PD-1 antibodies in combination with antibodies targeting CTLA-4, another inhibitory immune checkpoint increased clinical responses rate up to 50% but at costs of higher treatment-related toxicities. Thus, strong efforts are now directed toward the understanding of therapy resistance, the identification of biomarkers predicting therapy response, and the development of alternative PD-1-based combination treatment to improve patient outcomes.


Assuntos
Linfócitos T CD8-Positivos/citologia , Imunoterapia , Melanoma/terapia , Receptor de Morte Celular Programada 1 , Anticorpos Monoclonais , Antígeno B7-H1 , Europa (Continente) , Humanos , Microambiente Tumoral
2.
Theranostics ; 9(21): 6047-6062, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31534536

RESUMO

Extracellular vesicles released by tumor cells contribute to the reprogramming of the tumor microenvironment and interfere with hallmarks of cancer including metastasis. Notably, melanoma cell-derived EVs are able to establish a pre-metastatic niche in distant organs, or on the contrary, exert anti-tumor activity. However, molecular insights into how vesicles are selectively packaged with cargo defining their specific functions remain elusive. Methods: Here, we investigated the role of the chaperone Bcl2-associated anthogene 6 (BAG6, synonym Bat3) for the formation of pro- and anti-tumor EVs. EVs collected from wildtype cells and BAG6-deficient cells were characterized by mass spectrometry and RNAseq. Their tumorigenic potential was analyzed using the B-16V transplantation mouse melanoma model. Results: We demonstrate that EVs from B-16V cells inhibit lung metastasis associated with the mobilization of Ly6Clow patrolling monocytes. The formation of these anti-tumor-EVs was dependent on acetylation of p53 by the BAG6/CBP/p300-acetylase complex, followed by recruitment of components of the endosomal sorting complexes required for transport (ESCRT) via a P(S/T)AP double motif of BAG6. Genetic ablation of BAG6 and disruption of this pathway led to the release of a distinct EV subtype, which failed to suppress metastasis but recruited tumor-promoting neutrophils to the pre-metastatic niche. Conclusion: We conclude that the BAG6/CBP/p300-p53 axis is a key pathway directing EV cargo loading and thus a potential novel microenvironmental therapeutic target.

3.
Eur J Cancer ; 119: 66-76, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31419753

RESUMO

STUDY AIM: Melanomas arising in acral sites are associated with a poorer prognosis than other melanoma subtypes. The aim of this study was to evaluate clinical-pathological and genetic characteristics as well as therapeutic responses of a larger cohort of patients with melanomas arising in acral sites. METHODS: Clinical data of 134 patients with melanomas arising in acral sites from the Dept. of Dermatology Essen were collected and analysed with regard to clinicopathological characteristics and treatment responses. Genetic analysis with targeted next-generation sequencing was done on 50 samples. RESULTS: In our cohort, BRAF (30%), NRAS (28%), TERT promoter (26%), NF1 (14%) and KIT (6%) were frequently identified mutations. Comparing tumours situated on palms and soles with melanomas arising on dorsal acral sites, a higher frequency of NRAS (39.1% versus 25%) and NF1 (17.3% versus 0%) and lower frequencies of BRAF (21.7% versus 75%) and TERT promoter (8.6% versus 50%) mutations were observed. MAPK activating mutations were identified in 64% of tumours. Overall survival was longer in patients treated with immune checkpoint inhibitors as first-line treatment than in patients receiving other systemic therapies (i.e. BRAF/MEK inhibitors and chemotherapy). CONCLUSION: Our data suggest that the genetics of melanomas arising in acral sites varies by tumour location and may influence biological behaviour.

4.
Cell Rep ; 27(12): 3573-3586.e7, 2019 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216476

RESUMO

The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5' UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential in vivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas.

5.
Cancer Immunol Immunother ; 68(3): 479-488, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30635687

RESUMO

T cell responses are crucial for anti-tumor immunity. In chronic viral infections, anti-tumor T cell responses can be compromised due to various immunological mechanisms, including T cell exhaustion. To study mechanisms of anti-tumor immunity during a chronic viral infection, we made use of the well-established Friend virus (FV) mouse model. Chronically FV-infected mice are impaired in their ability to reject FBL-3 cells-a virus-induced tumor cell line of C57BL/6 origin. Here we aimed to explore therapeutic strategies to overcome the influence of T cell exhaustion during chronic viral infection, and reactivate effector CD8+ and CD4+ T cells to eliminate tumor cells. For T cell stimulation, agonistic antibodies against the tumor necrosis factor receptor (TNFR) superfamily members CD137 and CD134 were used, because they were reported to augment the cytotoxic program of T cells. αCD137 agonistic therapy, but not αCD134 agonistic therapy, resulted in FBL-3 tumor elimination in chronically FV-infected mice. CD137 stimulation significantly enhanced the cytotoxic activity of both CD4+ and CD8+ T cells, which were both required for efficient tumor control. Our study suggests that agonistic antibodies to CD137 can efficiently enhance anti-tumor immunity even in the setting of chronic viral infection, which might have promising therapeutic applications.


Assuntos
Vigilância Imunológica , Neoplasias Experimentais/imunologia , Infecções por Retroviridae/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Doença Crônica , Citotoxicidade Imunológica , Vírus da Leucemia Murina de Friend , Camundongos , Camundongos Endogâmicos C57BL , Receptores OX40/agonistas
6.
Int J Cancer ; 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30230526

RESUMO

Activation of the innate immune receptor retinoic acid-inducible gene I (RIG-I) by its specific ligand 5'-triphosphate RNA (3pRNA) triggers anti-tumor immunity, which is dependent on natural killer (NK) cell activation and cytokine induction. However, to date, RIG-I expression and the functional consequences of RIG-I activation in NK cells have not been examined. Here, we show for the first time the expression of RIG-I in human NK cells and their activation upon RIG-I ligand (3pRNA) transfection. 3pRNA-activated NK cells killed melanoma cells more efficiently than NK cells activated by type I interferon. Stimulation of RIG-I in NK cells specifically increased the surface expression of membrane-bound TNF-related apoptosis-inducing ligand (TRAIL) on NK cells, while activated NK cell receptors were not affected. RIG-I-induced membrane-bound TRAIL initiated death-receptor-pathway-mediated apoptosis not only in allogeneic but also in autologous human leukocyte antigen (HLA) class I-positive and HLA class I-negative melanoma cells. These results identify the direct activation of RIG-I in NK cells as a novel mechanism for how RIG-I can trigger enhanced NK cell killing of tumor cells, underscoring the potential of RIG-I activation for tumor immunotherapy. This article is protected by copyright. All rights reserved.

7.
Oncoimmunology ; 7(8): e1450127, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30221038

RESUMO

The profound but frequently transient clinical responses to BRAFV600 inhibitor (BRAFi) treatment in melanoma emphasize the need for combinatorial therapies. Multiple clinical trials combining BRAFi and immunotherapy are under way to further enhance therapeutic responses. However, to which extent BRAFV600 inhibition may affect melanoma immunogenicity over time remains largely unknown. To support the development of an optimal treatment protocol, we studied the impact of prolonged BRAFi exposure on the recognition of melanoma cells by T cells in different patient models. We demonstrate that autologous CD8+ tumor-infiltrating lymphocytes (TILs) efficiently recognized short-term (3, 7 days) BRAFi-treated melanoma cells but were less responsive towards long-term (14, 21 days) exposed tumor cells. Those long-term BRAFi-treated melanoma cells showed a non-proliferative dedifferentiated phenotype and were less sensitive to four out of five CD8+ T cell clones, present in the preexisting TIL repertoire, of which three recognized shared antigens (Tyrosinase, Melan-A and CSPG4) and one being neoantigen-specific. Only a second neoantigen was steadily recognized independent of treatment duration. Notably, in all cases the impaired T cell activation was due to a time-dependent downregulation of their respective target antigens. Moreover, combinatorial treatment of melanoma cells with BRAFi and an inhibitor of its downstream kinase MEK had similar effects on T cell recognition. In summary, MAP kinase inhibitors (MAPKi) strongly alter the tumor antigen expression profile over time, favoring evolution of melanoma variants cross-resistant to both T cells and MAPKi. Our data suggest that simultaneous treatment with MAPKi and immunotherapy could be most effective for tumor elimination.

8.
J Natl Cancer Inst ; 110(6): 677-681, 2018 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-29917141

RESUMO

Poor clinical responses to checkpoint blockade with anti-CTLA-4 and anti-PD-1 antibodies in melanoma have recently been associated with acquired IFNγ resistance that protects tumor cells from the antiproliferative and pro-apoptotic cytokine activity. IFNγ-resistant melanoma cells very often lack functional expression of the IFNγ signaling pathway gene JAK2 due to gene deletions or inactivating gene mutations. Analyzing melanoma cell lines (n = 46, applying next-generation targeted sequencing and single nucleotide polymorphism arrays) as well as available genomic data sets from The Cancer Genome Atlas (TCGA) tumor tissue samples (cutaneous melanoma n = 367, lung squamous cell carcinoma n = 501, bladder urothelial carcinoma n = 408, breast invasive carcinoma n = 768, colorectal adenocarcinoma n = 257), we demonstrate that the frequent chromosomal losses of the tumor suppressor CDKN2A in melanoma and other tumor entities enhance the susceptibility to IFNγ resistance by concomitant deletion of the JAK2 gene (odds ratio = 223.17, 95% confidence interval = 66.91 to 1487.38, two-sided P = 7.6×10-46). Tumors with JAK2 mutations or homozygous JAK2 deletions demonstrate allelic losses covering both CDKN2A and JAK2. This suggests that patients with tumor chromosomal CDKN2A losses are susceptible to developing immunotherapy resistance and should be screened for JAK2 deficiency prior to and under immune checkpoint blocking therapy.

9.
J Nanobiotechnology ; 16(1): 47, 2018 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-29720199

RESUMO

BACKGROUND: Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. METHODS: Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. RESULTS: Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. CONCLUSIONS: These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.


Assuntos
Antígenos de Neoplasias/sangue , Exossomos/química , Antígenos de Histocompatibilidade Classe I/sangue , Imunoensaio/métodos , Melanoma/sangue , Linhagem Celular Tumoral , Humanos , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Nanopartículas/química , Tetraspaninas/sangue
10.
Oncoimmunology ; 7(2): e1392426, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29308322

RESUMO

Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAFV600E. In general, patients experience remarkable clinical responses under BRAF inhibitor (BRAFi) treatment but eventually progress within 6-8 months due to resistance development. Responding metastases show an increased immune cell infiltrate, including also NK cells, that, however, is no longer detectable in BRAFi-resistant lesions, suggesting NK cell activity should be exploited to prevent disease progression. Here, we examined the effects of BRAFi on the expression of ligands targeting activating NK cells receptors immediately after treatment onset, prior to resistance development. We demonstrate that BRAFV600E mutant melanoma cells cultured in the presence of vemurafenib, strongly decreased surface expression of ligands for NK activating receptors including the NKG2D-ligand, MICA, and the DNAM-1 ligand, CD155, and became significantly less susceptible to NK cell attack. NKG2D-ligand protein downregulation was due to a significant decrease in mRNA levels, already detectable 24 h after drug treatment. Interestingly, vemurafenib-induced MICA downregulation could be counteracted by treatment of melanoma cells with the histone deacetylase (HDAC) inhibitor (HDACi) sodium butyrate, that also upregulated the DNAM1-ligand, Nectin-2. HDACi treatment enhanced surface expression of NKG2D-ligands in the presence of BRAFi, accompanied by recovery of NK cell recognition, but only upon simultaneous drug application. These results suggest that co-administration of BRAFi and HDAC inhibitors as well as having direct effects on melanoma cell survival, could also synergise to improve NK cell recognition and avoid tumour immune evasion.

11.
Sci Rep ; 7(1): 7785, 2017 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798348

RESUMO

CD4+ helper T cells and cytotoxic CD8+ T cells are key players for adaptive immune responses against acute infections with retroviruses. Similar to textbook knowledge the most important function of CD4+ T cells during an acute retrovirus infection seems to be their helper function for other immune cells. Whereas there was no direct anti-viral activity of CD4+ T cells during acute Friend Virus (FV) infection, they were absolutely required for the control of chronic infection. During chronic FV infection a population of activated FV-specific CD4+ T cells did not express cytotoxic molecules, but Fas Ligand that can induce Fas-induced apoptosis in target cells. Using an MHC II-restricted in vivo CTL assay we demonstrated that FV-specific CD4+ T cells indeed mediated cytotoxic effects against FV epitope peptide loaded targets. CD4 + CTL killing was also detected in FV-infected granzyme B knockout mice confirming that the exocytosis pathway was not involved. However, killing could be blocked by antibodies against FasL, which identified the Fas/FasL pathway as critical cytotoxic mechanism during chronic FV infection. Interestingly, targeting the co-stimulatory receptor CD137 with an agonistic antibody enhanced CD4+ T cell cytotoxicity. This immunotherapy may be an interesting new approach for the treatment of chronic viral infections.

12.
Stem Cell Reports ; 9(3): 985-998, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28781075

RESUMO

The interaction of mesenchymal stromal cells (MSCs) with natural killer (NK) cells is traditionally thought of as a static inhibitory model, whereby resting MSCs inhibit NK cell effector function. Here, we use a dynamic in vitro system of poly(I:C) stimulation to model the interaction of NK cells and tissue-resident MSCs in the context of infection or tissue injury. The experiments suggest a time-dependent system of regulation and feedback, where, at early time points, activated MSCs secrete type I interferon to enhance NK cell effector function, while at later time points TGF-ß and IL-6 limit NK cell effector function and terminate inflammatory responses by induction of a regulatory senescent-like NK cell phenotype. Importantly, feedback of these regulatory NK cells to MSCs promotes survival, proliferation, and pro-angiogenic properties. Our data provide additional insight into the interaction of stromal cells and innate immune cells and suggest a model of time-dependent MSC polarization and licensing.


Assuntos
Células Matadoras Naturais/citologia , Células-Tronco Mesenquimais/citologia , Regeneração , Apoptose/efeitos dos fármacos , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Comunicação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Inflamação/patologia , Interleucina-6/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Mucosa Nasal/citologia , Fenótipo , Poli I-C/farmacologia , Receptores CXCR4/metabolismo , Regeneração/efeitos dos fármacos , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/efeitos dos fármacos
13.
Invest Ophthalmol Vis Sci ; 58(9): 3464-3470, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28700778

RESUMO

Purpose: The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods: In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results: Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions: In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.


Assuntos
DNA de Neoplasias/genética , Fator de Iniciação 1 em Eucariotos/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Neoplasias da Íris/genética , Melanoma/genética , Mutação , Idoso , Análise Mutacional de DNA , Fator de Iniciação 1 em Eucariotos/metabolismo , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Neoplasias da Íris/metabolismo , Neoplasias da Íris/patologia , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade
14.
Nat Commun ; 8: 15440, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28561041

RESUMO

Melanoma treatment has been revolutionized by antibody-based immunotherapies. IFNγ secretion by CD8+ T cells is critical for therapy efficacy having anti-proliferative and pro-apoptotic effects on tumour cells. Our study demonstrates a genetic evolution of IFNγ resistance in different melanoma patient models. Chromosomal alterations and subsequent inactivating mutations in genes of the IFNγ signalling cascade, most often JAK1 or JAK2, protect melanoma cells from anti-tumour IFNγ activity. JAK1/2 mutants further evolve into T-cell-resistant HLA class I-negative lesions with genes involved in antigen presentation silenced and no longer inducible by IFNγ. Allelic JAK1/2 losses predisposing to IFNγ resistance development are frequent in melanoma. Subclones harbouring inactivating mutations emerge under various immunotherapies but are also detectable in pre-treatment biopsies. Our data demonstrate that JAK1/2 deficiency protects melanoma from anti-tumour IFNγ activity and results in T-cell-resistant HLA class I-negative lesions. Screening for mechanisms of IFNγ resistance should be considered in therapeutic decision-making.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Interferon gama/imunologia , Melanoma/genética , Neoplasias Cutâneas/genética , Linfócitos T/imunologia , Evasão Tumoral/genética , Apresentação do Antígeno/genética , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Biópsia , Linhagem Celular Tumoral , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Resistencia a Medicamentos Antineoplásicos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia/métodos , Interferon gama/metabolismo , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Melanoma/tratamento farmacológico , Melanoma/imunologia , Melanoma/patologia , Mutação , Taxa de Mutação , Modelagem Computacional Específica para o Paciente , Medicina de Precisão/métodos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Pele/patologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Linfócitos T/metabolismo , Resultado do Tratamento , Sequenciamento Completo do Genoma
15.
Sci Rep ; 7(1): 2290, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28536458

RESUMO

Merkel cell carcinoma (MCC) is a rare and aggressive, yet highly immunogenic skin cancer. The latter is due to its viral or UV-associated carcinogenesis. For tumor progression MCC has to escape the host's immuno-surveillance, e.g. by loss of HLA class-I expression. Indeed, a reduced HLA class-I expression was observed in MCC tumor tissues and MCC cell lines. This reduced HLA class-I surface expression is caused by an impaired expression of key components of the antigen processing machinery (APM), including LMP2 and LMP7 as well as TAP1 and TAP2. Notably, experimental provisions of HLA class-I binding peptides restored HLA class-I surface expression on MCC cells. Silencing of the HLA class-I APM is due to histone deacetylation as inhibition of histone deacetylases (HDACs) not only induced acetylation of histones in the respective promoter regions but also re-expression of APM components. Thus, HDAC inhibition restored HLA class-I surface expression in vitro and in a mouse xenotransplantation model. In contrast to re-induction of HLA class-I by interferons, HDAC inhibitors did not interfere with the expression of immuno-dominant viral proteins. In summary, restoration of HLA class-I expression on MCC cells by epigenetic priming is an attractive approach to enhance therapies boosting adaptive immune responses.

16.
Mod Pathol ; 30(7): 928-939, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28409567

RESUMO

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called 'blue nevus-like melanoma', which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.


Assuntos
Melanoma/diagnóstico , Nevo Azul/diagnóstico , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Neoplasias Cutâneas/diagnóstico , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Diagnóstico Diferencial , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Humanos , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Mutação , Nevo Azul/genética , Nevo Azul/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Adulto Jovem
17.
Oncotarget ; 8(17): 28312-28327, 2017 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-28423700

RESUMO

T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient's HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy.


Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Genes MHC Classe I , Melanoma/genética , Melanoma/imunologia , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Alelos , Animais , Apresentação do Antígeno , Linhagem Celular Tumoral , Biologia Computacional/métodos , Modelos Animais de Doenças , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Melanoma/patologia , Camundongos , Metástase Neoplásica , Peptídeos/química , Peptídeos/genética , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Transcriptoma
18.
Mod Pathol ; 30(3): 350-356, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27934878

RESUMO

Blue nevi are common melanocytic tumors arising in the dermal layer of the skin. Similar to uveal melanomas, blue nevi frequently harbor GNAQ and GNA11 mutations. Recently, recurrent CYSLTR2 and PLCB4 mutations were identified in uveal melanomas not harboring GNAQ or GNA11 mutations. All four genes (GNAQ, GNA11, CYSLTR2, and PLCB4) code for proteins involved in the same signaling pathway, which is activated by mutations in these genes. Given the related functional consequences of these mutations and the known genetic similarities between uveal melanoma and blue nevi, we analyzed a cohort of blue nevi to investigate whether CYSLTR2 and PLCB4 mutations occur in tumors lacking GNAQ or GNA11 mutations (as in uveal melanoma). A targeted next-generation sequencing assay covering known activating mutations in GNAQ, GNA11, CYSLTR2, PLCB4, KIT, NRAS, and BRAF was applied to 103 blue nevi. As previously reported, most blue nevi were found to harbor activating mutations in GNAQ (59%, n=61), followed by less frequent mutations in GNA11 (16%, n=17). Additionally, one BRAF (1%) and three NRAS (3%) mutations were detected. In three tumors (3%) harboring none of the aforementioned gene alterations, CYSLTR2 mutations were identified. All three CYSLTR2 mutations were the same c.386T>A, L129Q mutation previously identified in uveal melanoma that has been shown to lead to increased receptor activation and signaling. In summary, our study identifies CYSLTR2 L129Q alterations as a previously unrecognized activating mutation in blue nevi, occuring in a mutually exclusive fashion with known GNAQ and GNA11 mutations. Similar to GNAQ and GNA11 mutations, CYSLTR2 mutations, when present, are likely defining pathogenetic events in blue nevi.


Assuntos
Mutação , Nevo Azul/genética , Receptores de Leucotrienos/genética , Neoplasias Cutâneas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Análise Mutacional de DNA , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Nevo Azul/patologia , Fosfolipase C beta/genética , Neoplasias Cutâneas/patologia , Adulto Jovem
19.
J Extracell Vesicles ; 5: 31803, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27527605

RESUMO

Exosomes are cell-secreted nanovesicles (40-200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×10(5) exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.

20.
Cancer Res ; 76(15): 4347-58, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27261508

RESUMO

Melanoma often recurs after a latency period of several years, presenting a T cell-edited phenotype that reflects a role for CD8(+) T cells in maintaining metastatic latency. Here, we report an investigation of a patient with multiple recurrent lesions, where poorly immunogenic melanoma phenotypes were found to evolve in the presence of autologous tumor antigen-specific CD8(+) T cells. Melanoma cells from two of three late recurrent metastases, developing within a 6-year latency period, lacked HLA class I expression. CD8(+) T cell-resistant, HLA class I-negative tumor cells became clinically apparent 1.5 and 6 years into stage IV disease. Genome profiling by SNP arrays revealed that HLA class I loss in both metastases originated from a shared chromosome 15q alteration and independently acquired focal B2M gene deletions. A third HLA class I haplotype-deficient lesion developed in year 3 of stage IV disease that acquired resistance toward dominant CD8(+) T-cell clonotypes targeting stage III tumor cells. At an early stage, melanoma cells showed a dedifferentiated c-Jun(high)/MITF(low) phenotype, possibly associated with immunosuppression, which contrasted with a c-Jun(low)/MITF(high) phenotype of T cell-edited tumor cells derived from late metastases. In summary, our work shows how tumor recurrences after long-term latency evolve toward T-cell resistance by independent genetic events, as a means for immune escape and immunotherapeutic resistance. Cancer Res; 76(15); 4347-58. ©2016 AACR.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Melanoma/genética , Proteínas de Neoplasias/imunologia , Humanos , Melanoma/patologia , Metástase Neoplásica
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