RESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence. METHODS: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival. RESULTS: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003). CONCLUSIONS: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.
Assuntos
Carcinoma Ductal de Mama/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Receptores ErbB/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , PrognósticoRESUMO
Exploratory clinical trials using therapeutic anti-HER3 antibodies strongly suggest that neuregulin (NRG1; HER3 ligand) expression at tumor sites is a predictive biomarker of anti-HER3 antibody efficacy in cancer. We hypothesized that in NRG1-expressing tumors, where the ligand is present before antibody treatment, anti-HER3 antibodies that do not compete with NRG1 for receptor binding have a higher receptor-neutralizing action than antibodies competing with the ligand for binding to HER3. Using time-resolved-fluorescence energy transfer (TR-FRET), we demonstrated that in the presence of recombinant NRG1, binding of 9F7-F11 (a nonligand-competing anti-HER3 antibody) to HER3 is increased, whereas that of ligand-competing anti-HER3 antibodies (H4B-121, U3-1287, Ab#6, Mab205.10.2, and MOR09825) is decreased. Moreover, 9F7-F11 showed higher efficacy than antibodies that compete with the ligand for binding to HER3. Specifically, 9F7-F11 inhibition of cell proliferation and of HER3/AKT/ERK1/2 phosphorylation as well as 9F7-F11-dependent cell-mediated cytotoxicity were higher in cancer cells preincubated with recombinant NRG1 compared with cells directly exposed to the anti-HER3 antibody. This translated in vivo into enhanced growth inhibition of NRG1-expressing BxPC3 pancreatic, A549 lung, and HCC-1806 breast cell tumor xenografts in mice treated with 9F7-F11 compared with H4B-121. Conversely, both antibodies had similar antitumor effect in NRG1-negative HPAC pancreatic carcinoma cells. In conclusion, the allosteric modulator 9F7-F11 shows increased anticancer effectiveness in the presence of NRG1 and thus represents a novel treatment strategy for NRG1-addicted tumors. Mol Cancer Ther; 16(7); 1312-23. ©2017 AACR.
Assuntos
Anticorpos Monoclonais Murinos/administração & dosagem , Biomarcadores Tumorais/imunologia , Neoplasias/tratamento farmacológico , Neuregulina-1/genética , Receptor ErbB-3/imunologia , Células A549 , Animais , Anticorpos Anti-Idiotípicos/administração & dosagem , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais Murinos/imunologia , Biomarcadores Tumorais/genética , Proliferação de Células/efeitos dos fármacos , Feminino , Transferência Ressonante de Energia de Fluorescência , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neuregulina-1/imunologia , Fosforilação , Ligação Proteica , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The human EGF receptor (HER/EGFR) family of receptor tyrosine kinases serves as a key target for cancer therapy. Specifically, EGFR and HER2 have been repeatedly targeted because of their genetic aberrations in tumors. The therapeutic potential of targeting HER3 has long been underestimated, due to relatively low expression in tumors and impaired kinase activity. Nevertheless, in addition to serving as a dimerization partner of EGFR and HER2, HER3 acts as a key player in tumor cells' ability to acquire resistance to cancer drugs. In this study, we generated several monoclonal antibodies to HER3. Comparisons of their ability to degrade HER3, decrease downstream signaling, and inhibit growth of cultured cells, as well as recruit immune effector cells, selected an antibody that later emerged as the most potent inhibitor of pancreatic cancer cells grown as tumors in animals. Our data predict that anti-HER3 antibodies able to intercept autocrine and stroma-tumor interactions might strongly inhibit tumor growth, in analogy to the mechanism of action of anti-EGFR antibodies routinely used now to treat colorectal cancer patients.
Assuntos
Anticorpos Monoclonais/imunologia , Receptor ErbB-3/imunologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , HumanosRESUMO
Activation of receptor tyrosine kinases (RTK), such as those belonging to the human epidermal growth factor receptor (HER) family, occurs only after receptor dimerization, which is a crucial step for cellular signal transduction and diversification. The HER family includes four members (EGFR/HER1, HER2, HER3, and HER4) that can homodimerize or heterodimerize. Here, we describe immunoassays based on time-resolved Förster resonance energy transfer (TR-FRET) to profile EGFR-EGFR, HER2-HER2, and EGFR-HER2 dimers directly in tumor samples.
Assuntos
Receptores ErbB/química , Regulação Neoplásica da Expressão Gênica , Imunoensaio , Neoplasias Ovarianas/química , Receptor ErbB-2/química , Animais , Anticorpos Monoclonais Humanizados/química , Linhagem Celular Tumoral , Cetuximab , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Humanos , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Inibidores de Proteínas Quinases/química , Multimerização Proteica , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais , Trastuzumab , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The anti-HER2 antibody pertuzumab inhibits HER2 dimerization and affects HER2/HER3 dimer formation and signaling. As HER3 and its ligand neuregulin are implicated in pancreatic tumorigenesis, we investigated whether HER3 expression could be a predictive biomarker of pertuzumab efficacy in HER2low-expressing pancreatic cancer. We correlated in vitro and in vivo HER3 expression and neuregulin dependency with the inhibitory effect of pertuzumab on cell viability and tumor progression. HER3 knockdown in BxPC-3 cells led to resistance to pertuzumab therapy. Pertuzumab treatment of HER3-expressing pancreatic cancer cells increased HER3 at the cell membrane, whereas the anti-HER3 monoclonal antibody 9F7-F11 down-regulated it. Both antibodies blocked HER3 and AKT phosphorylation and inhibited HER2/HER3 heterodimerization but affected differently HER2 and HER3 homodimers. The pertuzumab/9F7-F11 combination enhanced tumor inhibition and the median survival time in mice xenografted with HER3-expressing pancreatic cancer cells. Finally, HER2 and HER3 were co-expressed in 11% and HER3 alone in 27% of the 45 pancreatic ductal adenocarcinomas analyzed by immunohistochemistry. HER3 is essential for pertuzumab efficacy in HER2low-expressing pancreatic cancer and HER3 expression might be a predictive biomarker of pertuzumab efficacy in such cancers. Further studies in clinical samples are required to confirm these findings and the interest of combining anti-HER2 and anti-HER3 therapeutic antibodies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Receptor ErbB-3/metabolismo , Animais , Biomarcadores Tumorais , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologia , Distribuição Aleatória , Receptor ErbB-3/antagonistas & inibidores , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Following the development of targeted therapies against EGFR and HER2, two members of the human epidermal receptor (HER) family of receptor tyrosine kinases, much interest has been focused on their expression in tumors. However, knowing the expression levels of individual receptors may not be sufficient to predict drug response. Here, we describe the development of antibody-based time-resolved Förster resonance energy transfer (TR-FRET) assays for the comprehensive analysis not only of EGFR and HER2 expression in tumor cryosections, but also of their activation through quantification of HER homo- or heterodimers. First, EGFR and HER2 expression levels were quantified in 18 breast tumors and the results were compared with those obtained by using reference methods. The EGFR number per cell determined by TR-FRET was significantly correlated with EGFR mRNA copy number (P<0.0001). Moreover, our method detected HER2 overexpression with 100% specificity and sensibility, as confirmed by the standard IHC, FISH and qPCR analyses. EGFR and HER2 dimerization was then assessed, using as controls xenograft tumors from cell lines with known dimer expression profiles. Our results show that quantification of HER dimerization provides information about receptor activation that cannot be obtained by quantification of single receptors. Quantifying HER expression and dimerization by TR-FRET assays might help identifying novel clinical markers for optimizing patients' treatment in oncology.
Assuntos
Neoplasias da Mama/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Receptor ErbB-2/química , Receptor ErbB-2/metabolismo , Animais , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Humanos , Técnicas In Vitro , Camundongos , Multimerização ProteicaRESUMO
In oncology, simultaneous inhibition of epidermal growth factor receptor (EGFR) and HER2 by monoclonal antibodies (mAbs) is an efficient therapeutic strategy but the underlying mechanisms are not fully understood. Here, we describe a time-resolved fluorescence resonance energy transfer (TR-FRET) method to quantify EGFR/HER2 heterodimers on cell surface to shed some light on the mechanism of such therapies. First, we tested this antibody-based TR-FRET assay in NIH/3T3 cell lines that express EGFR and/or HER2 and in various tumor cell lines. Then, we used the antibody-based TR-FRET assay to evaluate in vitro the effect of different targeted therapies on EGFR/HER2 heterodimers in the ovarian carcinoma cell line SKOV-3. A simultaneous incubation with Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) disturbed EGFR/HER2 heterodimers resulting in a 72% reduction. Cetuximab, Trastuzumab or Pertuzumab (anti-HER2) alone induced a 48, 44, or 24% reduction, respectively. In contrast, the tyrosine kinase inhibitors Erlotinib and Lapatinib had very little effect on EGFR/HER2 dimers concentration. In vivo, the combination of Cetuximab and Trastuzumab showed a better therapeutic effect (median survival and percentage of tumor-free mice) than the single mAbs. These results suggest a correlation between the extent of the mAb-induced EGFR/HER2 heterodimer reduction and the efficacy of such mAbs in targeted therapies. In conclusion, quantifying EGFR/HER2 heterodimers using our antibody-based TR-FRET assay may represent a useful method to predict the efficacy and explain the mechanisms of action of therapeutic mAbs, in addition to other commonly used techniques that focus on antibody-dependent cellular cytotoxicity, phosphorylation, and cell proliferation.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Transferência Ressonante de Energia de Fluorescência , Neoplasias/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Receptor ErbB-2/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Cloridrato de Erlotinib , Humanos , Lapatinib , Camundongos , Células NIH 3T3 , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/genéticaRESUMO
Probing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin. Then we directly labeled MutS with the long-lived fluorescent donor molecule europium tris-bipyridine cryptate ([TBP(Eu(3+))]) and demonstrated by electrophoretic mobility shift assay that this chemically labeled protein retained DNA mismatch binding property. Consequently, we used [TBP(Eu(3+))]-MutS to develop a faster and simpler semidirect HTRF assay.
Assuntos
DNA/metabolismo , Transferência de Energia , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Pareamento Incorreto de Bases , Sequência de Bases , DNA/genética , Escherichia coli , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
A series of fluorescent ligands designed for vasopressin and oxytocin G protein-coupled receptors was synthesized and characterized to develop fluorescence polarization or homogeneous time-resolved fluorescence (HTRF) binding assays. These ligands, labeled with europium pyridine-bis-bipyridine cryptate or with Alexa 488,546,647 selectively bound to the vasopressin V1a and oxytocin receptors with high affinities and exhibited antagonistic properties. The affinities of several unlabeled ligands determined by our homogeneous assays on membrane preparations or on intact cells into 96- and 384-well plate formats were similar to those determined by usual radioligand binding methods. Compared to other binding assays, the polarization and HTRF binding assays are nonradiaoactive, therefore safer to perform, yet very sensitive and homogeneous, therefore easier and faster to automate. These methods are thus suitable for efficient drug high-throughput screening procedures and can easily be applied to other G protein-coupled receptor models.
Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos , Corantes Fluorescentes/química , Oligopeptídeos/síntese química , Receptores de Ocitocina/antagonistas & inibidores , Animais , Ligação Competitiva , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , AMP Cíclico/análogos & derivados , AMP Cíclico/química , Fluoresceínas/química , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência , Ligantes , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Compostos Organometálicos/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Compostos de Quinolínio/química , Ensaio Radioligante , Receptores de Ocitocina/agonistas , Receptores de Vasopressinas/agonistasRESUMO
BACKGROUND: Blockade of costimulation and adhesion signaling is an attractive approach to interfere with graft rejection METHODS: Between January 1997 and May 1999, forty adults having benign liver diseases were included in a prospective, randomized study comparing tacrolimus plus low-dose short-term steroids without (n=20, TAC group) or with a 10-day course of antihuman CD2 monoclonal antibody (n=20, BTI group). RESULTS: At day 7, histological rejection expressed by mean Banff scores (2.3+/-1.6 vs. 5.4+/-1.6 in the TAC group; P<0.0001) and incidence of moderate to severe rejection (score>or=6) (0 vs. 10 [50%] in the TAC group; P<0.001) were significantly lower in the BTI group. Rejection was treated in 10% (two patients) of BTI patients during the first 3 months and in 15% during the whole follow-up and in 25% (five patients) of TAC patients (P=NS). None of the BTI-patients presented with an adverse event. Three-month, 1-year, and 5-year actual patient survival rates were 100%, 95%, and 95% in the BTI group and 100%, 100%, and 85% in the TAC group. Graft survival rates were 100%, 90%, and 90% in the BTI group and 95%, 95%, and 80% in the TAC group (P=NS). The mAb had no negative impact on infectious or tumor events. CONCLUSIONS: Antihuman CD2 monoclonal antibody is a safe immunosuppressive drug which has a favorable impact on early immunological follow-up of liver transplanted patients. The antibody had no impact on late patient and graft survival.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antígenos CD2/imunologia , Imunossupressores/uso terapêutico , Transplante de Fígado/imunologia , Tacrolimo/uso terapêutico , Adulto , Idoso , Relação Dose-Resposta a Droga , Esquema de Medicação , Rejeição de Enxerto/epidemiologia , Sobrevivência de Enxerto , Humanos , Incidência , Infecções/complicações , Falência Hepática/complicações , Falência Hepática/cirurgia , Contagem de Linfócitos , Pessoa de Meia-Idade , Neoplasias/complicações , Período Pós-Operatório , Esteroides/administração & dosagem , Esteroides/uso terapêutico , Análise de SobrevidaRESUMO
Single nucleotide primer extension reaction has been widely used in DNA testing, and several detection methods based on this core allelic discrimination have been developed. Most of the reported formats are based on a two step protocol involving first, a liquid phase extension reaction, then a physical separation process (chromatography, electrophoresis, capture on solid support, mass spectrometry). Here we describe a new strategy based on homogeneous time-resolved fluorescence (HTRF), which does not involve any separation process and which allows a simple "mix and measure" protocol. In this approach, a 5'-(europium) cryptate-labeled primer is elongated by a biotinylated dideoxynucleoside-triphosphate, followed by the addition of a streptavidin-acceptor conjugate, which gives rise to a long-life fluorescence resonance energy transfer (FRET) signal between the cryptate donor and the acceptor. We present the development of HTRF technology as applied to the diagnosis of tumor suppressor gene p53 (TP53) mutations, and its application to the analysis of genomic DNA from human tumoral samples. The sensitivity of the reported method is compared to the corresponding fluorescent polarization assay.
Assuntos
Análise Mutacional de DNA/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Linhagem Celular Tumoral , Polarização de Fluorescência/métodos , Genes p53/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: In the pig-to-baboon model, acute vascular rejection remains the main hurdle for successful long-term xenograft survival. The production of galactosyl knockout pigs could solve concomitantly the problem of hyperacute and acute vascular rejection. This work studies in vitro the cell-mediated cytotoxicity of natural killer (NK) and T cells after priming of baboon peripheral blood lymphocytes (PBLs) with pig antigens to evaluate whether cytotoxicity is galactosyl-dependent. MATERIAL AND METHODS: PBLs from naive and primed baboons were used as effectors on primary porcine aortic endothelial cells (PAECs) to assess cytotoxicity. Untreated or galactosidase-digested PAECs were used to evidence the role of galactosyl residues on cell-mediated cytotoxicity. Two rat-anti baboon monoclonal antibodies were tested to inhibit either T+NK cells (LO-CD2b) or NK cells alone (LO-CD94). RESULTS: When using PBLs from naive animals, spontaneous lysis occurred and was inhibited by both LOCD-2b and LO-CD94. In comparison, lysis of PAECs was significantly higher when baboon PBLs were first primed in vivo with pig xenoantigens. In this case, cytotoxicity was completely inhibited by LO-CD2b but only partially by LO-CD94. Reduction of galactosyl residues by galactosidase digestion showed that PAEC lysis almost completely disappeared with naive baboon PBLs but not with primed baboon PBLs, thereby indicating that anti-pig T-cell response is not dependent on galactosyl residues. CONCLUSION: Galactosyl knockout pigs could solve hyperacute rejection and also prevent the activation of NK cells even after xenogeneic priming. T cells will then be the next hurdle for the success of xenografting.
Assuntos
Antígenos Heterófilos/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/transplante , Imunoglobulina G/imunologia , Linfócitos/imunologia , Linfócitos T Citotóxicos/imunologia , Transplante Heterólogo/imunologia , Animais , Aorta , Sobrevivência Celular , Células Cultivadas , Endotélio Vascular/citologia , Modelos Animais , Papio , Ratos , Suínos , Condicionamento Pré-Transplante/métodos , alfa-GalactosidaseRESUMO
BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is closely linked to primary Epstein-Barr virus (EBV) infection and a defect of EBV specific cellular immunity is supposed to be the basis of PTLD. However, EBV load is so far the only marker proposed to evaluate PTLD risk, and no study has investigated the role of specific anti-EBV T lymphocytes (EBV-TL). METHODS: We therefore prospectively measured the EBV-TL by enzyme-linked immunospot (elispot) assay, in correlation to EBV load by real-time quantitative PCR and lymphoproliferation occurrence in 45 liver transplanted children. RESULTS: EBV load at the time of primary infection was high in all patients irrespective to subsequent emergence of PTLD. Seven patients developed PTLD, all of them following primary EBV infection. All seven had low EBV-TL (<2/mm3) associated with high viral load (>25,000 copies/microg DNA). Both parameters can be combined in a 100% positive predictive index. Healing from lymphoma was characterized by rapid EBV-TL increase concomitant to decreasing viral load. EBV-TL follow-up helped to adapt immunomodulation. No patient had PTLD whenever EBV-TL were above 2/mm3. CONCLUSIONS: We conclude that high viral load is systematic in patients who underwent primary EBV infection and is indicative of the PTLD risk only if there is low concomitant cellular immune response. Healing from PTLD requires modulation of immunosuppression, and appearance of EBV-TL.