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BACKGROUND: Advances in precision oncology led to approval of tumour-agnostic molecularly guided treatment options (MGTOs). The minimum requirements for claiming tumour-agnostic potential remain elusive. METHODS: The European Society for Medical Oncology (ESMO) Precision Medicine Working Group (PMWG) coordinated a project to optimise tumour-agnostic drug development. International experts examined and summarised the publicly available data used for regulatory assessment of the tumour-agnostic indications approved by the US Food and Drug Administration and/or the European Medicines Agency as of December 2023. Different scenarios of minimum objective response rate (ORR), number of tumour types investigated, and number of evaluable patients per tumour type were assessed for developing a screening tool for tumour-agnostic potential. This tool was tested using the tumour-agnostic indications approved during the first half of 2024. A taxonomy for MGTOs and a framework for tumour-agnostic drug development were conceptualised. RESULTS: Each tumour-agnostic indication had data establishing objective response in at least one out of five patients (ORR ≥ 20%) in two-thirds (≥4) of the investigated tumour types, with at least five evaluable patients in each tumour type. These minimum requirements were met by tested indications and may serve as a screening tool for tumour-agnostic potential, requiring further validation. We propose a conceptual taxonomy classifying MGTOs based on the therapeutic effect obtained by targeting a driver molecular aberration across tumours and its modulation by tumour-specific biology: tumour-agnostic, tumour-modulated, or tumour-restricted. The presence of biology-informed mechanistic rationale, early regulatory advice, and adequate trial design demonstrating signs of biology-driven tumour-agnostic activity, followed by confirmatory evidence, should be the principles for tumour-agnostic drug development. CONCLUSION: The ESMO Tumour-Agnostic Classifier (ETAC) focuses on the interplay of targeted driver molecular aberration and tumour-specific biology modulating the therapeutic effect of MGTOs. We propose minimum requirements to screen for tumour-agnostic potential (ETAC-S) as part of tumour-agnostic drug development. Definition of ETAC cut-offs is warranted.
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BACKGROUND: Atypical EGFR mutations occur in 10%-30% of non-small-cell lung cancer (NSCLC) patients with EGFR mutations and their sensitivity to classical epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKI) is highly heterogeneous. Patients harboring one group of uncommon, recurrent EGFR mutations (G719X, S768I, L861Q) respond to EGFR-TKI. Exon 20 insertions are mostly insensitive to EGFR-TKI but display sensitivity to exon 20 inhibitors. Clinical outcome data of patients with very rare point and compound mutations upon systemic treatments are still sparse to date. PATIENTS AND METHODS: In this retrospective, multicenter study of the national Network Genomic Medicine (nNGM) in Germany, 856 NSCLC cases with atypical EGFR mutations including co-occurring mutations were reported from 12 centers. Clinical follow-up data after treatment with different EGFR-TKIs, chemotherapy and immune checkpoint inhibitors were available from 260 patients. Response to treatment was analyzed in three major groups: (i) uncommon mutations (G719X, S7681, L861Q and combinations), (ii) exon 20 insertions and (iii) very rare EGFR mutations (very rare single point mutations, compound mutations, exon 18 deletions, exon 19 insertions). RESULTS: Our study comprises the largest thus far reported real-world cohort of very rare EGFR single point and compound mutations treated with different systemic treatments. We validated higher efficacy of EGFR-TKI in comparison to chemotherapy in group 1 (uncommon), while most exon 20 insertions (group 2) were not EGFR-TKI responsive. In addition, we found TKI sensitivity of very rare point mutations (group 3) and of complex EGFR mutations containing exon 19 deletions or L858R mutations independent of the combination partner. Notably, treatment responses in group 3 (very rare) were highly heterogeneous. Co-occurring TP53 mutations exerted a non-significant trend for a detrimental effect on outcome in EGFR-TKI-treated patients in groups 2 and 3 but not in group 1. CONCLUSIONS: Based on our findings, we propose a novel nNGM classification of atypical EGFR mutations.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB , Medicina Genômica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
OBJECTIVE: Panel-based next-generation sequencing (NGS) is increasingly used for the diagnosis of EGFR-mutated non-small-cell lung cancer (NSCLC) and could improve risk assessment in combination with clinical parameters. MATERIALS AND METHODS: To this end, we retrospectively analyzed the outcome of 400 tyrosine kinase inhibitor (TKI)-treated EGFR+ NSCLC patients with validation of results in an independent cohort (n = 130). RESULTS: EGFR alterations other than exon 19 deletions (non-del19), TP53 co-mutations, and brain metastases at baseline showed independent associations of similar strengths with progression-free (PFS hazard ratios [HR] 2.1-2.3) and overall survival (OS HR 1.7-2.2), in combination defining patient subgroups with distinct outcome (EGFR+NSCLC risk Score, "ENS", p < 0.001). Co-mutations beyond TP53 were rarely detected by our multigene panel (<5%) and not associated with clinical endpoints. Smoking did not affect outcome independently, but was associated with non-del19 EGFR mutations (p < 0.05) and comorbidities (p < 0.001). Laboratory parameters, like the blood lymphocyte-to-neutrophil ratio and serum LDH, correlated with the metastatic pattern (p < 0.01), but had no independent prognostic value. Reduced ECOG performance status (PS) was associated with comorbidities (p < 0.05) and shorter OS (p < 0.05), but preserved TKI efficacy. Non-adenocarcinoma histology was also associated with shorter OS (p < 0.05), but rare (2-3 %). The ECOG PS and non-adenocarcinoma histology could not be validated in our independent cohort, and did not increase the range of prognostication alongside the ENS. CONCLUSIONS: EGFR variant, TP53 status and brain metastases predict TKI efficacy and survival in EGFR+ NSCLC irrespective of other currently available parameters ("ENS"). Together, they constitute a practical and reproducible approach for risk stratification of newly diagnosed metastatic EGFR+ NSCLC.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Estudos Retrospectivos , Medição de RiscoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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INTRODUCTION: We assessed the clinical validity of circulating tumour cell (CTC) quantification for prognostication of patients with advanced non-small cell lung cancer (NSCLC) by undertaking a pooled analysis of individual patient data. METHODS: Nine European NSCLC CTC centres were asked to provide reported/unreported pseudo-anonymised data for patients with advanced NSCLC who participated in CellSearch CTC studies from January 2003 to March 2017. We used Cox regression models, stratified by centres, to establish the association between CTC count and survival. We assessed the added value of CTCs to prognostic clinicopathological models using likelihood ratio (LR) statistics and c-indices. RESULTS: Seven out of nine eligible centres provided data for 550 patients with prognostic information for overall survival. CTC counts of ≥2 and ≥ 5 per 7·5 mL were associated with reduced progression-free survival (≥2 CTCs: hazard ratio [HR] = 1.72, p < 0·001; ≥5 CTCs: HR = 2.21, p < 0·001) and overall survival (≥2 CTCs: HR = 2·18, p < 0·001; ≥5 CTCs: HR = 2·75, p < 0·001), respectively. Survival prediction was significantly improved by addition of baseline CTC count to LR clinicopathological models (log-transformed CTCs p < 0·001; ≥2 CTCs p < 0·001; ≥5 CTCs p ≤ 0·001 for both survival end-points), whereas moderate improvements were observed with the use of c-index models. There was some evidence of between-centre heterogeneity, especially when examining continuous counts of CTCs. CONCLUSIONS: These data confirm CTCs as an independent prognostic indicator of progression-free survival and overall survival in advanced NSCLC and also reveal some evidence of between-centre heterogeneity. CTC count improves prognostication when added to full clinicopathological predictive models.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/secundário , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/mortalidade , Progressão da Doença , Europa (Continente) , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Resistance towards VEGF-centered anti-angiogenic therapy still represents a substantial clinical challenge. We report here that mast cells alter the proliferative and organizational state of endothelial cells which reduces the efficacy of anti-angiogenic therapy. Consequently, absence of mast cells sensitizes tumor vessels for anti-angiogenic therapy in different tumor models. Mechanistically, anti-angiogenic therapy only initially reduces tumor vessel proliferation, however, this treatment effect was abrogated over time as a result of mast cell-mediated restimulation of angiogenesis. We show that mast cells secrete increased amounts of granzyme b upon therapy, which mobilizes pro-angiogenic laminin- and vitronectin-bound FGF-1 and GM-CSF from the tumor matrix. In addition, mast cells also diminish efficacy of anti-angiogenic therapy by secretion of FGF-2. These pro-angiogenic factors act beside the targeted VEGFA-VEGFR2-axis and reinduce endothelial cell proliferation and angiogenesis despite the presence of anti-angiogenic therapy. Importantly, inhibition of mast cell degranulation with cromolyn is able to improve efficacy of anti-angiogenic therapy. Thus, concomitant mast cell-targeting might lead to improved efficacy of anti-angiogenic therapy.Resistance towards VEGF-centered anti-angiogenic therapy is an important clinical challenge. Here, the authors show that mast cells mediate resistance to anti-angiogenetic inhibitors by altering the proliferative and organizational state of endothelial cells through mobilization of FGF-1 and GM-CSF from the tumor matrix and secretion of FGF-2.
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Inibidores da Angiogênese/farmacologia , Células Endoteliais/metabolismo , Granzimas/metabolismo , Mastócitos/metabolismo , Animais , Antiasmáticos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromolina Sódica/farmacologia , Células Endoteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fator 1 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Laminina/metabolismo , Mastócitos/efeitos dos fármacos , Camundongos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neovascularização Patológica/metabolismo , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Vitronectina/metabolismoRESUMO
BACKGROUND: Vascular endothelial growth factor (VEGF)-targeting drugs normalise the tumour vasculature and improve access for chemotherapy. However, excessive VEGF inhibition fails to improve clinical outcome, and successive treatment cycles lead to incremental extracellular matrix (ECM) deposition, which limits perfusion and drug delivery. We show here, that low-dose VEGF inhibition augmented with PDGF-R inhibition leads to superior vascular normalisation without incremental ECM deposition thus maintaining access for therapy. METHODS: Collagen IV expression was analysed in response to VEGF inhibition in liver metastasis of colorectal cancer (CRC) patients, in syngeneic (Panc02) and xenograft tumours of human colorectal cancer cells (LS174T). The xenograft tumours were treated with low (0.5 mg kg-1 body weight) or high (5 mg kg-1 body weight) doses of the anti-VEGF antibody bevacizumab with or without the tyrosine kinase inhibitor imatinib. Changes in tumour growth, and vascular parameters, including microvessel density, pericyte coverage, leakiness, hypoxia, perfusion, fraction of vessels with an open lumen, and type IV collagen deposition were compared. RESULTS: ECM deposition was increased after standard VEGF inhibition in patients and tumour models. In contrast, treatment with low-dose bevacizumab and imatinib produced similar growth inhibition without inducing detrimental collagen IV deposition, leading to superior vascular normalisation, reduced leakiness, improved oxygenation, more open vessels that permit perfusion and access for therapy. CONCLUSIONS: Low-dose bevacizumab augmented by imatinib selects a mature, highly normalised and well perfused tumour vasculature without inducing incremental ECM deposition that normally limits the effectiveness of VEGF targeting drugs.
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Bevacizumab/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Mesilato de Imatinib/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Bevacizumab/farmacologia , Linhagem Celular Tumoral , Colágeno Tipo IV/metabolismo , Matriz Extracelular/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologia , Camundongos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Circulating tumor cells (CTCs) were introduced as biomarkers more than 10 years ago, but capture of viable CTCs at high purity from peripheral blood of cancer patients is still a major technical challenge. Here, we report a novel microfluidic platform designed for marker independent capture of CTCs. The Parsortix™ cell separation system provides size and deformability-based enrichment with automated staining for cell identification, and subsequent recovery (harvesting) of cells from the device. Using the Parsortix™ system, average cell capture inside the device ranged between 42% and 70%. Subsequent harvest of cells from the device ranged between 54% and 69% of cells captured. Most importantly, 99% of the isolated tumor cells were viable after processing in spiking experiments as well as after harvesting from patient samples and still functional for downstream molecular analysis as demonstrated by mRNA characterization and array-based comparative genomic hybridization. Analyzing clinical blood samples from metastatic (n = 20) and nonmetastatic (n = 6) cancer patients in parallel with CellSearch(®) system, we found that there was no statistically significant difference between the quantitative behavior of the two systems in this set of twenty six paired separations. In conclusion, the epitope independent Parsortix™ system enables the isolation of viable CTCs at a very high purity. Using this system, viable tumor cells are easily accessible and ready for molecular and functional analysis. The system's ability for enumeration and molecular characterization of EpCAM-negative CTCs will help to broaden research into the mechanisms of cancer as well as facilitating the use of CTCs as "liquid biopsies."
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Dispositivos Lab-On-A-Chip , Células Neoplásicas Circulantes , Linhagem Celular Tumoral , Separação Celular/instrumentação , Forma Celular , Tamanho Celular , Sobrevivência Celular , HumanosRESUMO
P38α/ß has been described as a tumor-suppressor controlling cell cycle checkpoints and senescence in epithelial malignancies. However, p38α/ß also regulates other cellular processes. Here, we describe a role of p38α/ß as a regulator of acute lymphoblastic leukemia (ALL) proliferation and survival in experimental ALL models. We also report first evidence that p38α/ß phosphorylation is associated with the occurrence of relapses in TEL-AML1-positive leukemia. First, in vitro experiments show that p38α/ß signaling is induced in a cyclical manner upon initiation of proliferation and remains activated during log-phase of cell growth. Next, we provide evidence that growth-permissive signals in the bone marrow activate p38α/ß in a novel avian ALL model, in which therapeutic targeting can be tested. We further demonstrate that p38α/ß inhibition by small molecules can suppress leukemic expansion and prolong survival of mice bearing ALL cell lines and primary cells. Knockdown of p38α strongly delays leukemogenesis in mice xenografted with cell lines. Finally, we show that in xenografted TEL-AML1 patients, ex vivo p38α/ß phosphorylation is associated with an inferior long-term relapse-free survival. We propose p38α/ß as a mediator of proliferation and survival in ALL and show first preclinical evidence for p38α/ß inhibition as an adjunct approach to conventional therapies.
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Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Adolescente , Animais , Proliferação de Células , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Camundongos , Fosforilação , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidade , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapiaRESUMO
Multiple myeloma is a mostly incurable malignancy characterized by the expansion of a malignant plasma cell (PC) clone in the human bone marrow (BM). Myeloma cells closely interact with the BM stroma, which secretes soluble factors that foster myeloma progression and therapy resistance. Growth arrest-specific gene 6 (Gas6) is produced by BM-derived stroma cells and can promote malignancy. However, the role of Gas6 and its receptors Axl, Tyro3 and Mer (TAM receptors) in myeloma is unknown. We therefore investigated their expression in myeloma cell lines and in the BM of myeloma patients and healthy donors. Gas6 showed increased expression in sorted BMPCs of myeloma patients compared with healthy controls. The fraction of Mer(+) BMPCs was increased in myeloma patients in comparison with healthy controls whereas Axl and Tyro3 were not expressed by BMPCs in the majority of patients. Downregulation of Gas6 and Mer inhibited the proliferation of different myeloma cell lines, whereas knocking down Axl or Tyro3 had no effect. Inhibition of the Gas6 receptor Mer or therapeutic targeting of Gas6 by warfarin reduced myeloma burden and improved survival in a systemic model of myeloma. Thus, the Gas6-Mer axis represents a novel candidate for therapeutic intervention in this incurable malignancy.
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Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mieloma Múltiplo/genética , Plasmócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Animais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Transplante de Neoplasias , Plasmócitos/patologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Células Estromais/patologia , Análise de Sobrevida , Varfarina/farmacologia , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlAssuntos
Leucemia Mieloide Aguda/patologia , Proteínas Serina-Treonina Quinases/análise , Receptores de Fatores de Crescimento Transformadores beta/análise , Receptores de Activinas Tipo II/análise , Adulto , Feminino , Humanos , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptor do Fator de Crescimento Transformador beta Tipo I , Resultado do Tratamento , Adulto JovemRESUMO
Strategies to alter angiogenesis have been successfully translated from the bench to bedside. With an estimated number of more than 500 million patients worldwide potentially benefiting from it, it is a prime example of targeted therapy that is increasingly changing the face of clinical medicine. Most efforts to stimulate or inhibit angiogenesis in the past were focused on the key angiogenic factor vascular endothelial growth factor (VEGF), resulting in the approval by the Food and Drug Administration of several drugs for the treatment of cancer and ocular disease. However, mounting clinical evidence reveals that inhibition of VEGF causes resistance and class-specific side effects, while therapeutic angiogenesis by delivering VEGF protein is more challenging than anticipated in human patients. Hence, alternatives are needed, and modulation of oxygen-sensitive enzymes (prolyl hydroxylase domain proteins) and of hypoxia induced transcription factors has recently emerged as a potential novel strategy to treat cancer and ischemic diseases. Furthermore, placental growth factor is a disease-specific angiogenic target, whose inhibition reduces cancer growth without causing major side effects, while its delivery induces revascularization of ischemic tissues. In this review, we summarize recent developments and discuss questions that arise in the exciting, rapidly developing field of angiogenic medicine, including a brief description of its possible implications in neurodegenerative diseases.
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Inibidores da Angiogênese/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Oftalmopatias/tratamento farmacológico , Humanos , Hipóxia , Isquemia/tratamento farmacológico , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Increased vessel density in the bone marrow of patients with acute myeloid leukemia as well as elevated expression of proangiogenic factors by leukemic cells implies a central role of angiogenesis in hematological malignancies. Endostatin (ES), a fragment of collagen XVIII, is an endogenous inhibitor of angiogenesis that has shown therapeutic activity in solid tumors in various preclinical models. Using microencapsulation technology, we studied the therapeutic effect of ES in AML. While ES had no effect on proliferation of M1 murine leukemic cells in vitro, ES producing microbeads significantly inhibited growth of subcutaneous chloromas in SCID mice as compared to controls. In a leukemia model using M1 cells the concomitant treatment of mice with ES microbeads prolonged median survival significantly. Histological analysis revealed a decreased microvessel density and a reduced number of CD31-positive single cells, putatively endothelial progenitor cells, in the bone marrow of treated animals. Taken together, ES has inhibitory effects on neo-angiogenesis in the bone marrow and on progression of leukemia in vivo. These experiments suggest a possible therapeutic role of antiangiogenic gene therapy with ES in AML.
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Inibidores da Angiogênese/uso terapêutico , Endostatinas/uso terapêutico , Doença Aguda , Inibidores da Angiogênese/farmacologia , Animais , Exame de Medula Óssea , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Composição de Medicamentos , Endostatinas/farmacologia , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Camundongos , Camundongos SCID , Neoplasias Experimentais , Neovascularização Patológica/tratamento farmacológico , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Taxa de SobrevidaRESUMO
Liver-kidney microsomal antibodies type 1 (LKM) are a diagnostic marker for autoimmune hepatitis type 2 (AIH-2), however, LKM autoantibodies are also detected in a small percentage of patients with chronic hepatitis C. The major target of LKM antibodies as evidenced by indirect immunofluorescence is cytochrome P4502D6 (CYP2D6). Anti-CYP2D6 titers of 62 LKM positive sera, 196 sera of patients with hepatic and rheumatic diseases and 33 sera of healthy blood donors (BD) were determined by an in vitro transcription/in vitro translation assay (ITT). Twenty five out of 26 AIH-2 sera and 33/36 LKM positive hepatitis C virus (HCV) sera were anti-CYP2D6 positive by ITT and antibody titers were similar in both patient groups. Epitope mapping experiments were performed by a series of truncated CYP2D6 proteins and by single epitopes of 257-269, 321-351, 373-389 and 410-419 amino acid (aa) expressed as DHFR-fusion proteins in Escherichia coli. The major linear epitope consists of 257-269 aa. This epitope is recognized with a significantly higher prevalence (64%) in AIH-2 than in LKM sera from patients with chronic hepatitis C (24%) (p < 0.001). None of the other autoepitopes showed significant differences in the prevalence of recognition by sera from both patient groups. Minor binding sites consisted of 321-351 aa, which was recognized by less than 20% of LKM sera and in the C-terminal region of 350-494 aa, which was recognized by less than 5% of LKM sera Our study revealed an epitope of 321-379 an on CYP2D6, which was shown to be conformation dependent. It was recognized by the vast majority of LKM sera, specifically by 76% of sera from HCV positive LKM patients and also by 76% of sera from patients with AIH-2. This epitope is homologous to three-dimensional epitopes detected by autoantibodies directed against hepatic cytochromes P450s in drug induced hepatitis and to an autoepitope on CYP21B associated with adrenal failure.
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Autoanticorpos/imunologia , Citocromo P-450 CYP2D6/imunologia , Hepatite C/imunologia , Hepatite Autoimune/imunologia , Sequência de Aminoácidos , Doença Crônica , Mapeamento de Epitopos , Epitopos/imunologia , Humanos , Dados de Sequência Molecular , Testes de PrecipitinaRESUMO
BACKGROUND & AIMS: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations of both copies of the autoimmune regulator (AIRE) gene. It is characterized by susceptibility to mucocutaneous candidiasis and multiple autoimmune lesions. A serious disease component is hepatitis. To identify diagnostic autoantibodies for APECED hepatitis, sera from 64 patients with APECED were screened for autoantibodies established in the diagnosis of idiopathic autoimmune hepatitis, and for autoantibodies against 10 cytochrome P450s. METHODS: Screening methods were indirect immunofluorescence, Western blot, Ouchterlony gel diffusion, enzyme-linked immunosorbent assay, and immunoprecipitation. RESULTS: Anti-liver microsomal antibodies were detected in 50% of the patients with APECED hepatitis and 11% of those without hepatitis. Prevalences of antinuclear, smooth muscle, anti-liver cytosol, anti-soluble liver protein/liver pancreas, and anti-CYP2D6 autoantibodies were 9%, 6%, 3%, 0%, and 0%, respectively. CYP1A1, CYP2B6, CYP1A2, and CYP2A6 were identified as autoantigens. Thirty percent of patients with anti-CYP2A6 and 100% of patients with anti-CYP1A2 were affected by hepatitis. Despite the high specificity of anti-CYP1A2 for APECED hepatitis, its sensitivity was low (50%). Anti-CYP2A6 and anti-CYP1A2 were not detected in patients with autoimmune hepatitis (N = 68) or nonhepatitic controls (N = 81). CONCLUSIONS: Anti-CYP1A2 is a highly specific but insensitive marker for APECED hepatitis. No clinical correlation was observed for anti-CYP2A6. Autoimmune hepatitis and APECED hepatitis are characterized by different molecular targets of autoantibodies with no overlap.
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Autoanticorpos/sangue , Autoantígenos/imunologia , Fígado/imunologia , Poliendocrinopatias Autoimunes/imunologia , Adolescente , Adulto , Idoso , Biomarcadores , Criança , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Hepatite/diagnóstico , Hepatite/imunologia , Humanos , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Poliendocrinopatias Autoimunes/diagnóstico , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND/AIMS: Primary biliary cirrhosis is an autoimmune liver disease which is characterized by the presence of autoantibodies directed against mitochondrial components which belong to the pyruvate dehydrogenase enzyme complex. Apart from antibodies against mitochondrial components, primary biliary cirrhosis patients often show antibodies against nuclear components, of which anti-Sp100 and anti-gp210 are considered to be disease specific. We investigated the incidence and course of antibodies against nuclear components in primary biliary cirrhosis patients before and after liver transplantation. METHODS: Sera from 42 primary biliary cirrhosis patients were studied using indirect immunofluorescence to detect antibodies against mitochondrial components and antibodies against nuclear components, ELISA to detect anti-Sp100, and immunoblot analysis to detect anti-gp210 and antibodies against nuclear components subtypes. RESULTS: Ninety-three percent of primary biliary cirrhosis patients in our study were antimitochondrial antibody positive. Forty-three percent of the patients were antinuclear antibody positive. Of these, 35% had antibodies against Sp100 and 36% were positive for anti-gp210. After transplantation, antimitochondrial antibody titers as well as antinuclear antibody titers decreased in all patients. Autoantibodies in low titer persisted for up to 13 years. The pattern of nuclear autoantigens recognized by patient sera was unchanged after liver transplantation. However, the antinuclear antibody pattern was very different between the individual patients. Anti-Sp100 and anti-gp210 were not detected in sera of patients with autoimmune hepatitis, hepatitis C infection, inflammatory bowel disease, connective tissue diseases, or primary sclerosing cholangitis. The serum alkaline phosphatase level was not different in antinuclear antibody negative or positive patients before or after transplantation. CONCLUSIONS: We conclude that the persistence of antibodies against mitochondrial components, and anti-Sp100 and anti-gp210 in primary biliary cirrhosis patients after liver transplantation is disease specific, but that this does not reflect recurrent disease activity in the graft.
Assuntos
Anticorpos Antinucleares/sangue , Antígenos Nucleares , Autoantígenos/imunologia , Cirrose Hepática Biliar/imunologia , Cirrose Hepática Biliar/cirurgia , Transplante de Fígado/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas Nucleares/imunologia , Adulto , Idoso , Fosfatase Alcalina/sangue , Doenças do Tecido Conjuntivo/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Células HeLa , Hepatite Autoimune/imunologia , Hepatite Autoimune/cirurgia , Humanos , Doenças Inflamatórias Intestinais/imunologia , Masculino , Pessoa de Meia-Idade , Complexo de Proteínas Formadoras de Poros Nucleares , Valores de ReferênciaRESUMO
Microsomal antigen autoantibodies are typical of type 2 autoimmune hepatitis, and a strong association with chronic hepatitis C virus (HCV) infection has been reported in certain geographical areas. These autoantibodies have been denominated LKM-1 to differentiate them from those associated with thienylic acid-induced hepatitis (LKM-2) and from those seen in patients with chronic delta hepatitis (LKM-3). To investigate the antigenic specificity of autoantibodies associated with chronic hepatitis C and delta, we analyzed 52 LKM-1 positive serum samples from patients with chronic hepatitis C and 17 LKM-3 positive serum samples from patients with chronic delta hepatitis by indirect immunofluorescence and Western blotting (immunoblotting). Reactivity of subjects with chronic hepatitis C was heterogeneous: only 5 out of 52 LKM-1 positive patients, tested by Western blot, recognized a single protein of 50 kD, previously identified by Manns et al. with an immunogenic epitope of cytochrome P450IID6. Thirteen of the 52 patients also reacted with a 70 kD microsomal protein, and 12 out of 52 reacted only with a 59 kD protein. Twenty-two sera, notwithstanding the high titer in immunofluorescence, did not evidence any reactivity when tested by Western blot. The same sera tested positive in LKM-1 ELISA when solubilized human microsomal proteins were used. Fourteen out of 17 LKM-3 positive sera from patients with chronic hepatitis delta recognized a 55 kD microsomal protein in Western blot; three sera, HCV and HIV positive, did not react with any protein by Western blot. None of these sera was positive in ELISA LKM-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Autoanticorpos/análise , Hepatite C/imunologia , Hepatite D/imunologia , Microssomos/imunologia , Especificidade de Anticorpos , Western Blotting , Doença Crônica , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Imunofluorescência , Humanos , Rim/imunologia , Masculino , Microssomos Hepáticos/imunologiaRESUMO
BACKGROUND/AIMS: Anti-liver-kidney microsomal (LKM) autoantibodies occur in a proportion of patients with chronic hepatitis C and D infections. Because of different immunofluorescence patterns, antibodies in hepatitis C and D were termed LKM-1 and LKM-3, respectively. The aim of the present study was to evaluate the different specificities of LKM-1 and LKM-3 antibodies. METHODS: Forty-nine samples of LKM-1 sera and 16 samples of LKM-3 sera were studied for reactivity against rat and human liver microsomal proteins by immunofluorescence, enzyme-linked immunosorbent assay, and Western blot. RESULTS: Thirty-four percent of the LKM-1 sera reacted with 50-kilodalton cytochrome P4502D6 in Western blot. In addition, a proportion of the sera recognized either a 59- or 70-kilodalton antigen, and 45% of the sera did not react in Western blot. Recently, the major LKM-3 antigen was identified as an autoepitope expressed on uridine diphosphate-glucuronosyltransferases (UGT). Seven LKM-3-positive sera reacted with recombinant rabbit family one UGT. None of the anti-LKM-1-positive hepatitis C sera reacted with UGT. Antibody reactivity against liver microsomal proteins in enzyme-linked immunosorbent assay ended when antigens were pretreated with sodium dodecyl sulfate, confirming that antibodies recognize conformational epitopes. CONCLUSIONS: LKM-1 antibodies in hepatitis C are more heterogeneous and react with different antigens compared with LKM-3 antibodies in hepatitis D.