RESUMO
Macrophage migration inhibitory factor (MIF) is known to be involved in oncogenic transformation, tumour progression, and immunosuppression and is overexpressed in many solid tumours, including paediatric rhabdomyosarcoma (RMS). We investigated the function of MIF in RMS during treatment with cytotoxic drugs. RMS cell lines were analysed by flow cytometry, immunofluorescence staining, and ELISA. We demonstrated the overexpression of MIF in RMS cells and the enhanced expression and secretion after treatment with cytotoxic agents. Migration assays of RMS cells revealed that inhibitors of MIF (ISO-1, Ant.III 4-IPP, Ant.V, sulforaphane (SF)) and blocking antibodies caused reduced migration, indicating a role for MIF in metastatic invasion. Additionally, we investigated the function of MIF in immune escape. The development of a population containing immunosuppressive myeloid-derived suppressor cells was promoted by incubation in conditioned medium of RMS cells comprising MIF and was reversed by MIF inhibitors but not by antibodies. Although most inhibitors may restore immune activity, Ant.III and 10 µM SF disturbed T cell proliferation in a CFSE assay, whereas T cell proliferation was not reduced by 3 µM SF, ISO-1 or antibodies. However, the inhibition of MIF by blocking antibodies did not increase the killing activity of allogenic PBMCs co-cultured with RMS cells. Our results reveal that MIF may be involved in an immune escape mechanism and demonstrate the involvement of MIF in immunogenic cell death during treatment with cytotoxic drugs. Targeting MIF may contribute to the restoration of immune sensitivity and the control of migration and metastatic invasion.
Assuntos
Antineoplásicos/uso terapêutico , Imunoterapia/métodos , Fatores Inibidores da Migração de Macrófagos/uso terapêutico , Rabdomiossarcoma/terapia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Humanos , Fatores Inibidores da Migração de Macrófagos/administração & dosagem , Fatores Inibidores da Migração de Macrófagos/farmacologiaRESUMO
BACKGROUND: Treatment outcome of children with pediatric hepatocellular carcinoma (pHCC) is poor. Therefore, we evaluated the tyrosine kinase inhibitor sorafenib in a model of pHCC. METHODS: Cell viability after treatment with sorafenib was evaluated in HC-AFW1 cells (pHCC) using MTT assay and compared to an adult HCC (aHCC) and two hepatoblastoma (HB) cell lines. ERK, pERK, E-cadherin, and vimentin expression were investigated using Western Blot. Sorafenib (60 mg/kg) was administered orally to NOD.Cg-Prkdcscid-IL2rgtmWjl/Sz mice bearing subcutaneous HC-AFW1-derived tumors. Tumor progression, viability, and vascularization were monitored by tumor volume, AFP levels, and CD31 immunostaining, respectively. Sensitization to sorafenib was evaluated using the ß-catenin inhibitor ICG001. RESULTS: Sorafenib reduced cell viability in HC-AFW1 (IC50: 8 µM), comparable to HB cells, however less pronounced in aHCC cells (IC50: 23 µM). Sorafenib inhibited ERK signaling in both, HC-AFW1 cells and -xenografts. In vivo, sorafenib treatment only led to a moderate tumor growth inhibition, although significant reduction of vascularization and tumor growth kinetics was observed. Long-term treatment with sorafenib decreased E-cadherin, but showed no induction of vimentin expression. Combining sorafenib with a ß-catenin inhibitor led to an additional reduction of cell viability. CONCLUSION: Sorafenib together with inhibitors of the ß-catenin pathway might be an effective tool in the treatment of pediatric HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Criança , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Niacinamida/uso terapêutico , Sorafenibe , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
BACKGROUND & AIMS: Aberrant activation of ß-catenin and Yes-associated protein 1 (Yap1) signaling pathways have been associated with the development of multiple tumor types. Yap functions as a transcriptional coactivator by interacting with TEA domain DNA binding proteins. We investigated the interactions among these pathways during hepatic tumorigenesis. METHODS: We used immunohistochemical analysis to determine expression of ß-catenin and Yap1 in liver cancer specimens collected from patients in Europe and the United States, consisting of 104 hepatocellular carcinoma, 62 intrahepatic cholangiocarcinoma, and 94 hepatoblastoma samples. We assessed ß-catenin and Yap1 signaling and interactions in hepatoblastoma cell lines ((HuH6, HepG2, HepT1, HC-AFW1, HepG2, and HC-AFW1); proteins were knocked down with small interfering RNAs, and effects on proliferation and cell death were measured. Sleeping beauty-mediated hydrodynamic transfection was used to overexpress constitutively active forms of ß-catenin (ΔN90/ß-catenin) and Yap1 (YapS127A) in livers of mice; tissues were collected, and histological and immunohistochemical analyses were performed. RESULTS: We observed nuclear localization of ß-catenin and Yap1 in 79% of hepatoblastoma samples but not in most hepatocellular carcinoma or intrahepatic cholangiocarcinoma samples. Yap1 and ß-catenin coprecipitated in hepatoblastoma but not hepatocellular carcinoma cells. Small interfering RNA-mediated knockdown of Yap1 or ß-catenin in hepatoblastoma cells reduced proliferation in an additive manner. Knockdown of Yap1 reduced its ability to coactivate transcription with ß-catenin; ß-catenin inhibitors inactivated Yap1. Overexpression of constitutively active forms of Yap1 and ß-catenin in mouse liver led to rapid tumorigenesis, with 100% mortality by 11 weeks. Tumor cells expressed both proteins, and human hepatoblastoma cells expressed common targets of their 2 signaling pathways. Yap1 binding of TEA domain factors was required for tumorigenesis in mice. CONCLUSIONS: ß-catenin and the transcriptional regulator Yap1 interact physically and are activated in most human hepatoblastoma tissues; overexpression of activated forms of these proteins in livers of mice leads to rapid tumor development. Further analysis of these mice will allow further studies of these pathways in hepatoblastoma pathogenesis and could lead to the identification of new therapeutic targets.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/metabolismo , Hepatoblastoma/metabolismo , Neoplasias Hepáticas/metabolismo , Fosfoproteínas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Morte Celular , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Colangiocarcinoma/metabolismo , Colangiocarcinoma/patologia , Europa (Continente) , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Fosfoproteínas/genética , Ligação Proteica , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição , Transcrição Gênica , Transfecção , Estados Unidos , Proteínas de Sinalização YAP , beta Catenina/genéticaRESUMO
Drug resistance and metastasis remain major challenges in the treatment of high-risk hepatoblastoma (HB) and require the development of alternative therapeutic strategies. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We investigated the impact of apoptosis sensitisers, BH3-mimetics, on the interaction between the host and HB to reduce tumour growth and dissemination while enhancing immunity. BH3-mimetics, such as obatoclax and ABT-737, enhanced the apoptosis-inducing effect of TRAIL and TNF-α resistant HB cells (HepT1 and HUH6). Tumour cell migration was inhibited by ABT-737 and more markedly by obatoclax. In an orthotopic model of HB, tumour uptake was reduced when the cells were pretreated with low concentrations of obatoclax. Only 1 of 7 mice developed HB in the liver, compared with an incidence of 0.8 in the control group. In summary, our study showed that apoptosis sensitisers had broader effects on HB cells than expected including migration and susceptibility to cytokines in addition to the known effects on drug sensitization. Sensitising HB to apoptosis may also allow resistant HB to be targeted by immune cells and prevent tumour cell dissemination.
Assuntos
Materiais Biomiméticos/farmacologia , Compostos de Bifenilo/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Hepatoblastoma/prevenção & controle , Neoplasias Hepáticas/prevenção & controle , Nitrofenóis/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Pirróis/farmacologia , Sulfonamidas/farmacologia , Animais , Materiais Biomiméticos/química , Compostos de Bifenilo/química , Transformação Celular Neoplásica/patologia , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Hepatoblastoma/patologia , Humanos , Indóis , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos Transgênicos , Nitrofenóis/química , Fragmentos de Peptídeos/química , Piperazinas/química , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/química , Pirróis/química , Sulfonamidas/químicaRESUMO
BACKGROUND: Surgery for rhabdomyosarcoma is challenging due to a lack of clear delineation between tumor and surrounding tissue. Mutilating surgery can be necessary in difficult tumor localizations. Therefore, novel diagnostic and therapeutic modalities are required. The aim of this study was to evaluate the in vivo tumor detection of RMS using fluorescence laparoscopy and to analyze the efficacy of hypericin-induced photodynamic therapy in a mouse model. METHODS: Seventeen NOD/LtSz-scid IL2Rγnull-mice were divided into four groups. In group 1, mCherry-expressing tumor cells and in group 2-4 non-transfected tumor cells were xenotransplanted. Three weeks later, one fluorochrome per group (ICG, ICG-cetuximab, hypericin) was injected. Fluorescence laparoscopy was carried out and tumors were resected using fluorescence guidance. In the hypericin group, photodynamic therapy was performed using blue light and apoptosis was evaluated by TUNEL test. RESULTS: A clear discrimination between healthy and tumor tissue was feasible by fluorescending properties with mCherry expressing tumor cells and after injection of hypericin. No fluorescence was detected in mice injected with ICG and ICG-labeled cetuximab. Hypericin photodynamic therapy induced apoptosis of tumor cells after exposure to blue light. CONCLUSIONS: Intraoperative photodynamic diagnosis was feasible using mCherry-transfected tumor cells or hypericin. Additionally, intraoperative photodynamic therapy was possible and effective.
Assuntos
Laparoscopia/métodos , Neoplasias Experimentais , Fotoquimioterapia/métodos , Rabdomiossarcoma Alveolar/patologia , Neoplasias de Tecidos Moles/patologia , Animais , Antracenos , Apoptose , Fluorescência , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos NOD , Perileno/análogos & derivados , Perileno/uso terapêutico , Proteína Quinase C/antagonistas & inibidores , Radiossensibilizantes/uso terapêutico , Rabdomiossarcoma Alveolar/tratamento farmacológico , Rabdomiossarcoma Alveolar/cirurgia , Neoplasias de Tecidos Moles/tratamento farmacológico , Neoplasias de Tecidos Moles/cirurgia , Células Tumorais CultivadasRESUMO
Pediatric hepatoblastoma (HB) is commonly treated by neoadjuvant chemotherapy and surgical tumor resection according to international multicenter trial protocols. Complete tumor resection is essential and survival rates up to 95% have now been achieved in those tumors classified as standard-risk HB. Drug resistance and occurrence of metastases remain the major challenges in the treatment of HB, especially in high-risk tumors. These conditions urgently require the development of alternative therapeutic strategies. One of those alternatives is the modulation of apoptosis in HB cells. HBs regularly overexpress anti-apoptotic proteins of the Bcl-family in comparison to healthy liver tissue. This fact may contribute to the development of chemoresistance of HB cells. Synthetic small inhibitory molecules with BH3-mimetic effects, such as ABT-737 and obatoclax, enhance the susceptibility of tumor cells to different cytotoxic drugs and thereby affect initiator proteins of the apoptosis cascade via the intrinsic pathway. Besides additive effects on HB cell viability when used in combination with cytotoxic drugs, BH3-mimetics also play a role in preventing metastasation by reducing adhesion and inhibiting cell migration abilities. Presumably, including additive BH3-mimetic drugs into existing therapeutic regimens in HB patients might allow dose reduction of established cytotoxic drugs and thereby associated immanent side effects, while maintaining the antitumor activity. Furthermore, reduction of tumor growth and inhibition of tumor cell dissemination may facilitate complete surgical tumor resection, which is mandatory in this tumor type resulting in improved survival rates in high-risk HB. Currently, there are phase I and phase II clinical trials in several cancer entities using this potential target. This paper reviews the available literature regarding the use of BH3-mimetic drugs as single agents or in combination with chemotherapy in various malignancies and focuses on results in HB cells.
Assuntos
Compostos de Bifenilo/uso terapêutico , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Nitrofenóis/uso terapêutico , Pirróis/uso terapêutico , Sulfonamidas/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Criança , Resistencia a Medicamentos Antineoplásicos , Hepatoblastoma/patologia , Humanos , Indóis , Neoplasias Hepáticas/patologia , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirróis/farmacologia , Sulfonamidas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Advanced stages of tumour and development of metastases are the two major problems in treating liver tumours such as hepatoblastoma (HB) and hepatocellular carcinoma (HCC), in paediatric patients. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We analysed the effect of apoptosis modulators, BH3 mimetics, on mechanisms of dissemination such as adhesion or migration of HB and HCC cells. BH3 mimetics such as ABT-737 and obatoclax can reduce cell migration in a scratch assay as well as adhesion of HB and HCC cells to matrigel. Immunofluorescence staining of F-actin demonstrated that development of lamellipodia, which are important for migration, decreased. BH3 mimetics increase the level of activated caspases 3 and 7 in HUH6 cells. This results in the degradation of GTPase Cdc42, which can be determined by western blot analysis. A pan-caspase inhibitor can block the migration and degradation of Rho-GTPase. In summary, our study showed that BH3 mimetics not only enhance drug sensitivity but also may prevent metastasis by inhibiting HB and HCC cell motility.
Assuntos
Compostos de Bifenilo/farmacologia , Carcinoma Hepatocelular/metabolismo , Movimento Celular/efeitos dos fármacos , Hepatoblastoma/metabolismo , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Actinas/química , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Inibidores de Caspase/farmacologia , Adesão Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática , Imunofluorescência , Hepatoblastoma/patologia , Humanos , Indóis , Metástase Neoplásica , Oligopeptídeos/farmacologia , Piperazinas/farmacologia , Proteólise , Pseudópodes/química , Pirróis/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Ovarian cancer is one of the most common cancers among women and the most lethal malignancy of all gynecological cancers. Surgery is promising in the early stages; however, most patients are first diagnosed in the advanced stages, where treatment options are limited. Here, we present a 49-year-old patient who was first diagnosed with stage III ovarian cancer. After the tumor progressed several times under guideline therapies with no more treatment options available at that time, the patient received a fully individualized neoantigen-derived peptide vaccine in the setting of an individual healing attempt. The tumor was analyzed for somatic mutations via whole exome sequencing and potential neoepitopes were vaccinated over a period of 50 months. During vaccination, the patient additionally received anti-PD-1 therapy to prevent further disease progression. Vaccine-induced T-cell responses were detected using intracellular cytokine staining. After eleven days of in vitro expansion, four T-cell activation markers (namely IFN-É£, TNF-α, IL-2, and CD154) were measured. The proliferation capacity of neoantigen-specific T-cells was determined using a CFSE proliferation assay. Immune monitoring revealed a very strong CD4+ T-cell response against one of the vaccinated peptides. The vaccine-induced T-cells simultaneously expressed CD154, TNF, IL-2, and IFN-É£ and showed a strong proliferation capacity upon neoantigen stimulation. Next-generation sequencing, as well as immunohistochemical analysis, revealed a loss of Beta-2 microglobulin (B2M), which is essential for MHC class I presentation. The results presented here implicate that the application of neoantigen-derived peptide vaccines might be considered for those cancer stages, where promising therapeutic options are lacking. Furthermore, we provide more data that endorse the intensive investigation of B2M loss as a tumor escape mechanism in clinical trials using anti-cancer vaccines together with immune-checkpoint inhibitors.
RESUMO
BACKGROUND: Chemoresistance and advanced tumour stage at time of diagnosis are the major reasons for poor treatment results in hepatoblastoma (HB) and paediatric hepatocellular carcinoma (HCC). Positive results with transplantation of liver and bone marrow revealed the impact of the immune system on the treatment of liver malignancies. AIM: Cytotoxic-immune-cells-like natural killer (NK) and T cells are major player in the defence against developing tumours. This study aimed to specifically analyse the ability of ex-vivo expanded γδ T cells to recognise and lyse HB and HCC cell lines in coculture assays. METHODS: Cell viability after treatment with γδ T cells was evaluated with two HB (HUH6 and HepT1) and one HCC cell line (HC-AFW1) using a MTT-based cytotoxicity assay. The binding of T cells to target cells was monitored using immunofluorescence microscopy. RESULTS: Incubation of hepatic tumour cell lines with γδ T cells led to a significant decrease in tumour cell viability. This was enhanced by zoledronic acid and histone deacetylase inhibitors. MT110, an EpCAM/CD3-bispecific BiTE antibody could bluntly enhance tumour cell lysis close to completion. γδ T cells efficiently interacted with HB and HCC cells in a spheroid culture model. CONCLUSION: Bispecific antibodies such as MT110 might be used to intensify the antitumoural effect of γδ T cells in context of adoptive immune cell transfer. Optimised immunotherapeutic strategies might therefore improve the outcome of high risk hepatoblastoma and hepatocellular carcinoma.
Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Citotoxicidade Imunológica , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Difosfonatos/farmacologia , Molécula de Adesão da Célula Epitelial , Hepatoblastoma/imunologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Imidazóis/farmacologia , Neoplasias Hepáticas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Microscopia de Fluorescência , Anticorpos de Cadeia Única/farmacologia , Esferoides Celulares , Linfócitos T/efeitos dos fármacos , Ácido ZoledrônicoRESUMO
Alveolar rhabdomyosarcoma (RMA) and malignant rhabdoid tumor (MRT) have a frequent metastatic spread and a poor prognosis. Aberrant miRNA expression is often found in metastatic tumors. The aim of this study was to identify specific miRNA expression patterns in these tumors. We analyzed the expression of miRNAs in RMA and MRT in tissue samples and in the rhabdomyosarcoma (RMS) cell lines (Rh30 and RD). Selected target miRNAs were modulated with mimic or inhibitor oligonucleotides. Functional analysis was monitored by flow cytometry and migration assays. A set of 107 differentially expressed miRNAs showed tissue-specific clustering of RMA and MRT. Comparison with the Sarcoma microRNA Expression Database revealed RMA- and MRT-specific miRNAs. Metastatic invasion associated miRNA miR-9 was overexpressed in RMA. miR-200c-inhibiting migration-was lower expressed in RMA than in MRT. Transient transfection of RMS cells with a miR-200c mimic and miR-9( inhibitor did neither increase the expression of the known target E-cadherin nor decrease migration. Expression of E-cadherin could be induced in RD cells using decitabine, but demethylation did not influence cell migration. Despite a comparable high rate of metastatic invasion pediatric RMA and MRT show a different pattern of miRNA expression possibly allowing risk stratification.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , MicroRNAs/genética , Tumor Rabdoide/genética , Rabdomiossarcoma Alveolar/genética , Sarcoma/genética , Neoplasias de Tecidos Moles/genética , Adulto , Linhagem Celular Tumoral , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , MicroRNAs/biossíntese , Tumor Rabdoide/secundário , Rabdomiossarcoma Alveolar/secundário , Sarcoma/patologia , Neoplasias de Tecidos Moles/patologia , Adulto JovemRESUMO
PURPOSE: Duplex drugs are promising anticancer agents. After in vivo cleavage into active nucleoside analogues, they exert their anti-tumour activity with reduced toxicity and side effects. Here we evaluated the impact of two duplex drugs on the viability of hepatoblastoma (HB) cells lines and their toxicity against human fibroblasts. METHODS: The duplex drugs 2'-deoxy-5-fluorouridylyl-(3'-5')- 3'-C-ethynylcytidine (5-FdU(3'-5')ECyd) and 3'-C-ethynylcytidinylyl-(5'â1-O)-2-O-octadecyl-sn-glycerylyl-(3'-Οâ5')-2'-deoxy-5-fluorouridine (ECyd-lipid-5-FdU) were analysed in two HB cell lines (HUH6, HepT1) and fibroblasts by MTT assay. The treatment potential was compared to the single substances 2'-deoxy-5-fluorourindine (5-FdU), 3'-C-ethynylycytidine (ECyd) and an equimolar mixture of both. Cell cycle analyses were performed using flow cytometry after 7-AAD staining. RESULTS: Both duplex drugs achieve a potent cytotoxic effect at low µM concentrations, which was more pronounced than the mixture of ECyd + 5-FdU. Further, both substances exert toxicity on fibroblasts of tumour samples, with less toxicity in foreskin fibroblasts cultures. Cell cycle analyses revealed a shift towards apoptotic cells for both drugs in HB cells. CONCLUSION: 5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU exert a highly potent anti-tumoural effect on HB cells and might therefore be a treatment option in HB. Pharmacological formulations of both duplex drugs have to be evaluated in vivo to reduce possible side effects.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Monofosfato de Citidina/análogos & derivados , Fluordesoxiuridilato/análogos & derivados , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Oligonucleotídeos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Monofosfato de Citidina/administração & dosagem , Citometria de Fluxo/métodos , Fluordesoxiuridilato/administração & dosagem , Humanos , Células Tumorais CultivadasRESUMO
BACKGROUND: High tumor mutational burden (TMB) is associated with a favorable outcome in metastatic melanoma patients treated with immune checkpoint inhibitors. However, data are limited in the adjuvant setting. As BRAF mutated patients have an alternative with targeted adjuvant therapy, it is important to identify predictive factors for relapse and recurrence-free survival (RFS) in patients receiving adjuvant anti-PD-1 antibodies. METHODS: We evaluated 165 melanoma patients who started adjuvant anti-PD-1 antibody therapy at our center between March 2018 and September 2019. The initial tumor stage was assessed at the beginning of therapy according to the 8th edition of the AJCC Cancer Staging Manual. Tumor and normal tissue of the high-risk stages IIIC/D/IV were sequenced using a 700 gene NGS panel. RESULTS: The tumor stages at the beginning of adjuvant anti-PD-1 therapy were as follows: N = 80 stage IIIA/B (48%), N = 85 stage IIIC/D/IV (52%). 72/165 patients (44%) suffered a relapse, 44/72 (61%) with only loco regional and 28/72 (39%) with distant metastases. Sequencing results were available from 83 to 85 patients with stage IIIC/D/IV. BRAF mutation status (HR 2.12, 95% CI 1.12-4.08; p = 0.022) and TMB (HR 7.11, 95% CI 2.19-23.11; p = 0.001) were significant and independent predictive factors for relapse-free survival (RFS). CONCLUSION: BRAF mutation status and TMB were independent predictive factors for RFS. Patients with BRAF V600E/K mutation and TMB high had the best outcome. A classification based on BRAF mutation status and TMB is proposed to predict RFS in melanoma patients with adjuvant anti-PD-1 therapy.
Assuntos
Melanoma , Neoplasias Cutâneas , Humanos , Neoplasias Cutâneas/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Adjuvantes Imunológicos , MutaçãoRESUMO
BACKGROUND: Sequencing of tumour tissue with comprehensive gene panels is increasingly used to guide treatment in precision oncology. Analysis of tumour-normal pairs allows in contrast to tumour-only assessment direct discrimination between somatic and germline alterations, which might have important implications not only for the patients but also their families. METHODS: We performed tumour normal sequencing with a large gene panel in 1048 patients with advanced cancer to support treatment decision. Sequencing results were correlated with clinical and family data. RESULTS: We identified 156 likely pathogenic or pathogenic (LP/P) germline variants in cancer predisposition genes (CPGs) in 144 cases (13.7%). Of all patients, 8.8% had a LP/P variant in autosomal-dominant cancer predisposition genes (AD-CPGs), most of them being genes with high or moderate penetrance (ATM, BRCA2, CHEK2 and BRCA1). In 48 cases, the P/LP variant matched the expected tumour spectrum. A second variant in tumour tissue was found in 31 patients with AD-CPG variants. Low frequency mutations in either TP53, ATM or DNMT3A in the normal sample indicated clonal haematopoiesis in five cases. CONCLUSIONS: Tumour-normal testing for personalised treatment identifies germline LP/P variants in a relevant proportion of patients with cancer. The majority of them would not have been referred to genetic counselling based on family history. Indirect functional readouts of tumour-normal sequencing can provide novel links between CPGs and unexpected cancers. The interpretation of increasingly complex datasets in precision oncology is challenging and concepts of interdisciplinary personalised cancer prevention are needed to support patients and their families.
Assuntos
Neoplasias Hematológicas , Neoplasias , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisão , Mutação em Linhagem Germinativa , Mutação , Genes BRCA2 , Predisposição Genética para Doença , Testes Genéticos/métodosRESUMO
BACKGROUND: Genomic characterisation has led to an improved understanding of adult melanoma. However, the aetiology of melanoma in children is still unclear and identifying the correct diagnosis and therapeutic strategies remains challenging. METHODS: Exome sequencing of matched tumour-normal pairs from 26 paediatric patients was performed to study the mutational spectrum of melanomas. The cohort was grouped into different categories: spitzoid melanoma (SM), conventional melanoma (CM), and other melanomas (OT). FINDINGS: In all patients with CM (n = 10) germline variants associated with melanoma were found in low to moderate melanoma risk genes: in 8 patients MC1R variants, in 2 patients variants in MITF, PTEN and BRCA2. Somatic BRAF mutations were detected in 60% of CMs, homozygous deletions of CDKN2A in 20%, TERTp mutations in 30%. In the SM group (n = 12), 5 patients carried at least one MC1R variant; somatic BRAF mutations were detected in 8.3%, fusions in 25% of the cases. No SM showed a homozygous CDKN2A deletion nor a TERTp mutation. In 81.8% of the CM/SM cases the UV damage signatures SBS7 and/or DBS1 were detected. The patient with melanoma arising in giant congenital nevus (CNM) demonstrated the characteristic NRAS Q61K mutation. INTERPRETATION: UV-radiation and MC1R germline variants are risk factors in the development of conventional and spitzoid paediatric melanomas. Paediatric CMs share genomic similarities with adult CMs while the SMs differ genetically from the CM group. Consistent genetic characterization of all paediatric melanomas will potentially lead to better subtype differentiation, treatment, and prevention in the future. FUNDING: Found in Acknowledgement.
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A growing number of druggable targets and national initiatives for precision oncology necessitate broad genomic profiling for many cancer patients. Whole exome sequencing (WES) offers unbiased analysis of the entire coding sequence, segmentation-based detection of copy number alterations (CNAs), and accurate determination of complex biomarkers including tumor mutational burden (TMB), homologous recombination repair deficiency (HRD), and microsatellite instability (MSI). To assess the inter-institution variability of clinical WES, we performed a comparative pilot study between German Centers of Personalized Medicine (ZPMs) from five participating institutions. Tumor and matched normal DNA from 30 patients were analyzed using custom sequencing protocols and bioinformatic pipelines. Calling of somatic variants was highly concordant with a positive percentage agreement (PPA) between 91 and 95% and a positive predictive value (PPV) between 82 and 95% compared with a three-institution consensus and full agreement for 16 of 17 druggable targets. Explanations for deviations included low VAF or coverage, differing annotations, and different filter protocols. CNAs showed overall agreement in 76% for the genomic sequence with high wet-lab variability. Complex biomarkers correlated strongly between institutions (HRD: 0.79-1, TMB: 0.97-0.99) and all institutions agreed on microsatellite instability. This study will contribute to the development of quality control frameworks for comprehensive genomic profiling and sheds light onto parameters that require stringent standardization.
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Background: The clinical utility of molecular profiling and targeted therapies for neuro-oncology patients outside of clinical trials is not established. We aimed at investigating feasibility and clinical utility of molecular profiling and targeted therapy in adult patients with advanced tumors in the nervous system within a prospective observational study. Methods: molecular tumor board (MTB)@ZPM (NCT03503149) is a prospective observational precision medicine study for patients with advanced tumors. After inclusion of patients, we performed comprehensive molecular profiling, formulated ranked biomarker-guided therapy recommendations based on consensus by the MTB, and collected prospective clinical outcome data. Results: Here, we present initial data of 661 adult patients with tumors of the nervous system enrolled by December 31, 2021. Of these, 408 patients were presented at the MTB. Molecular-instructed therapy recommendations could be made in 380/408 (93.1%) cases and were prioritized by evidence levels. Therapies were initiated in 86/380 (22.6%) cases until data cutoff. We observed a progression-free survival ratio >1.3 in 31.3% of patients. Conclusions: Our study supports the clinical utility of biomarker-guided therapies for neuro-oncology patients and indicates clinical benefit in a subset of patients. Our data might inform future clinical trials, translational studies, and even clinical care.
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BACKGROUND: Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients AIMS: This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours. METHODS: Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence. RESULTS: Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment. CONCLUSION: Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.
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Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Hepatoblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Benzenossulfonatos/uso terapêutico , Western Blotting , Caspase 3 , Linhagem Celular Tumoral , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Fatores de Transcrição Forkhead/genética , Marcação In Situ das Extremidades Cortadas , Camundongos , Neovascularização Patológica/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Sorafenibe , Sais de Tetrazólio , Tiazóis , Transplante Heterólogo , Transplantes , alfa-Fetoproteínas/metabolismoRESUMO
PURPOSE: Drug resistance remains a major challenge for the treatment of high-risk hepatoblastoma (HB). To enhance effectiveness of chemotherapy we modulate apoptosis in HB cells in vitro. METHODS: Viability was monitored in HB cells (HuH6, HepT1) and fibroblasts in monolayer and spheroid cultures treated with ABT-737, obatoclax, HA14-1, and TW-37 and each in combination with CDDP, etoposide, irinotecan, paclitaxel, and DOXO in a MTT assay. Western blot analyses were performed to determine expressions of pro- and anti-apoptotic proteins. RESULTS: Obatoclax and ABT-737 led to a dose-dependent decrease of viability in HB cells at concentrations above 0.3 µM. TW-37 and HA14-1 were less effective. ABT-737 and obatoclax had additive effects when combined with CDDP, etoposide, irinotecan, paclitaxel, or DOXO. This was also observed for fibroblast, however, for higher drug concentrations. In spheroid cultures, relative expression of Bcl-XL was increased, Bax was decreased, Mcl-1 was low, and Bcl-2 was not detected compared to 2D cultures, denoting an anti-apoptotic state in spheroids. Obatoclax and ABT-737 have overcome the resistance to CDDP. HuH6 cells have shown higher susceptability for apoptosis sensitizers than HepT1. CONCLUSION: The data provide evidence that ABT-737 and obatoclax might improve treatment results in children with HB.
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Apoptose , Benzamidas/farmacologia , Benzopiranos/farmacologia , Compostos de Bifenilo/farmacologia , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Nitrilas/farmacologia , Nitrofenóis/farmacologia , Pirróis/farmacologia , Sulfonamidas/farmacologia , Sulfonas/farmacologia , Western Blotting , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Hepatoblastoma/tratamento farmacológico , Humanos , Indóis , Neoplasias Hepáticas/tratamento farmacológico , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
PURPOSE: Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors. METHODS: Viability was monitored in HB cells (HUH6, HepT1) in monolayer and spheroid cultures treated with kinase inhibitors VX-680, Wee1-InhibitorII, and SU11274 alone or in combination with cisplatin (CDDP) using MTT assays. Apoptosis was revealed by Caspase-3 assay. Western blot and immunohistochemical analyses were performed to determine histone H3 phosphorylation. RESULTS: Among the kinase inhibitors strongest anti-proliferative effect on HB cells was documented for VX-680. HUH6 cells responded more sensitively to the Aurora kinase inhibitor as HepT1 cells (IC(50) 8 and 16.6 µM, respectively). While VX-680 and CDDP showed no additive effects, the combination of VX-680 and histone deacetylase inhibitor SAHA had a synergistic effect on the proliferation of HUH6 cells. The inhibition with VX-680 led to reduced histone H3 phosphorylation, to an increase of apoptotic cells, and to morphological changes such as vacuolization and swelling of the cells and nuclei. CONCLUSION: The data provide evidence that VX-680 might improve treatment results in HB with increased Aurora kinase activity by inhibiting cell proliferation and induction of apoptosis.