RESUMEN
Sunitinib is an effective treatment for patients with metastatic Renal Cell Carcinoma (mRCC) but ultimately resistance occurs. The aim of this study was to investigate sunitinib resistance in RCCs and to develop therapeutic combination strategies with targeted radioimmunotherapy (RIT). We studied two RCC models, analyzed Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) and AXL/MET expression and performed therapy studies in Balb/cnu/nu mice combining sunitinib and [177Lu]Lu-cG250 RIT (6.5 MBq/10 µg), specifically targeting RCC cells. pAXL and pMET were expressed in sunitinib-resistant SK-RC-52 and absent in sunitinib-sensitive NU12. NGS evaluation showed that expression of VEGFA, VEGFB, VEGFD, PGF and VEGFR1,2,3 was higher and expression of VEGFC and PDGFA was lower in NU12 than in SK-RC-52. Therapy studies combining sunitinib with [177Lu]Lu-cG250 RIT showed that the best response in mice with "resistant" SK-RC-52 tumors was observed with two cycles of Sunitinib and [177Lu]Lu-cG250 RIT, probably due to increased vascular permeability by sunitinib treatment. In the "sensitive" NU12 model, two cycles of [177Lu]Lu-cG250 RIT and two cycles of combination treatment were equally effective. Enhanced therapeutic efficacy was achieved when two agents ([177Lu]Lu-cG250 RIT and sunitinib) that on their own did not induce satisfactory response levels, are combined. Our findings provide a promising new therapeutic strategy for patients with advanced RCC.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Animales , Línea Celular Tumoral , Ratones , Ratones Desnudos , Radioinmunoterapia , Sunitinib , Factor A de Crecimiento Endotelial VascularRESUMEN
BACKGROUND: To identify potential therapeutic target in clear cell renal cell carcinoma (ccRCC), we performed a transcriptome analysis. Our analysis showed that fatty acid binding protein 7 (FABP7) has the highest mean differential overexpression in ccRCC compared to normal kidney. We aimed to investigate the significance of FABP7 in ccRCC. METHODS: Immunohistochemical staining for 40 advanced ccRCC cases was performed to investigate correlation between clinicopathological parameters and FABP7. They were composed of 40-83 years old cases with 33 male, 22 cases with pT ≥ 3, 19 cases with M1, and 16 cases with grade 3. The effect of gene knockdown was analysed by a cell viability assay and invasion assay in FABP7-overexpressing cell lines (SKRC7 and SKRC10). RESULTS: Our immunohistochemical analysis showed that higher FABP7 expression significantly correlated with distant metastasis and poor cancer-specific survival (CSS; both p < 0.05). Functional suppression of FABP7 significantly inhibited SKRC10 cell growth (p < 0.05) and resulted in a significant reduction of the invasive potential (p < 0.01), but did not cause growth inhibition of SKRC7 cells. We found that The Cancer Genome Atlas Research Network (TCGA) database shows FABP6 and 7 as equally overexpressed in the FABP family. Functional suppression of fatty acid binding protein 6 (FABP6) resulted in significant growth inhibition of SKRC7 cells (p < 0.005). CONCLUSIONS: Functional suppression of FABP7 significantly reduced cell viability and invasive potential in a ccRCC cell line. FABP7 may play a role in progression in some metastatic ccRCCs. The suppressed function may be compensated by another FABP family member.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/patología , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hormonas Gastrointestinales/metabolismo , Neoplasias Renales/patología , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/genética , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/mortalidad , Línea Celular Tumoral , Supervivencia Celular , Progresión de la Enfermedad , Proteína de Unión a los Ácidos Grasos 7/antagonistas & inhibidores , Proteína de Unión a los Ácidos Grasos 7/genética , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Hormonas Gastrointestinales/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Humanos , Riñón/patología , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/mortalidad , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Invasividad Neoplásica/patología , Supervivencia sin Progresión , ARN Interferente Pequeño/metabolismo , Tasa de Supervivencia , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genéticaRESUMEN
PURPOSE: Antibodies labeled with both a near-infrared fluorescent dye and a radionuclide can be used for tumor-targeted intraoperative dual-modality imaging. Girentuximab is a chimeric monoclonal antibody against carbonic anhydrase IX (CAIX), an antigen expressed in 95% of clear cell renal cell carcinoma (ccRCC). This study aimed to assess the feasibility of targeted dual-modality imaging with (111)In-girentuximab-IRDye800CW using ex vivo perfusion of human tumorous kidneys. EXPERIMENTAL DESIGN: Seven radical nephrectomy specimens from patients with ccRCC were perfused during 11 to 15 hours with dual-labeled girentuximab and subsequently rinsed during 2.5 to 4 hours with Ringer's Lactate solution. Then, dual-modality imaging was performed on a 5- to 10-mm-thick lamella of the kidney. Fluorescence imaging was performed with a clinical fluorescence camera set-up as applied during image-guided surgery. The distribution of Indium-111 in the slice of tumor tissue was visualized by autoradiography. In two perfusions, an additional dual-labeled control antibody was added to demonstrate specific accumulation of dual-labeled girentuximab in CAIX-expressing tumor tissue. RESULTS: Both radionuclide and fluorescence imaging clearly visualized uptake in tumor tissue and tumor-to-normal tissue borders, as confirmed (immuno)histochemically and by gamma counting. Maximum uptake of girentuximab in tumor tissue was 0.33% of the injected dose per gram (mean, 0.12 %ID/g; range, 0.01-0.33 %ID/g), whereas maximum uptake in the normal kidney tissue was 0.04 %ID/g (mean, 0.02 %ID/g; range, 0.00-0.04 %ID/g). CONCLUSIONS: Dual-labeled girentuximab accumulated specifically in ccRCC tissue, indicating the feasibility of dual-modality imaging to detect ccRCC. A clinical study to evaluate intraoperative dual-modality imaging in patients with ccRCC has been initiated. Clin Cancer Res; 22(18); 4634-42. ©2016 AACR.
Asunto(s)
Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/patología , Diagnóstico por Imagen/métodos , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/patología , Anticuerpos Monoclonales , Bencenosulfonatos , Carcinoma de Células Renales/cirugía , Humanos , Técnicas In Vitro , Indoles , Radioisótopos de Yodo , Neoplasias Renales/cirugía , Microscopía Fluorescente , Estadificación de Neoplasias , Nefrectomía , Imagen de Perfusión/métodos , Carga TumoralRESUMEN
Anti-angiogenic treatment with tyrosine kinase inhibitors (TKI) has lead to an impressive increase in progression-free survival for patients with metastatic RCC (mRCC), but mRCC remains largely incurable. We combined sunitinib, targeting the endothelial cells with Girentuximab (monoclonal antibody cG250, recognizing carbonic anhydrase IX (CAIX) targeting the tumor cells to study the effect of sunitinib on the biodistribution of Girentuximab because combination of modalities targeting tumor vasculature and tumor cells might result in improved effect. Nude mice with human RCC xenografts (NU12, SK-RC-52) were treated orally with 0.8 mg/day sunitinib, or vehicle for 7 to 14 days. Three days before start or cessation of treatment mice were injected i.v. with 0.4 MBq/5 µg (111)In-Girentuximab followed by biodistribution studies. Immunohistochemical analyses were performed to study the tumor vasculature and CAIX expression and to confirm Girentuximab uptake. NU12 appeared to represent a sunitinib sensitive tumor: sunitinib treatment resulted in extensive necrosis and decreased microvessel density (MVD). Accumulation of Girentuximab was significantly decreased when sunitinib treatment preceded the antibody injection but remained unchanged when sunitinib followed Girentuximab injection. Cessation of therapy led to a rapid neovascularization, reminiscent of a tumor flare. SK-RC-52 appeared to represent a sunitinib-resistant tumor: (central) tumor necrosis was minimal and MVD was not affected. Sunitinib treatment resulted in increased Girentuximab uptake, regardless of the sequence of treatment. These data indicate that sunitinib can be combined with Girentuximab. Since these two modalities have different modes of action, this combination might lead to enhanced therapeutic efficacy.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Modelos Animales de Enfermedad , Neoplasias Renales/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Xenoinjertos , Humanos , Indoles/administración & dosificación , Neoplasias Renales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Pirroles/administración & dosificación , Reacción en Cadena en Tiempo Real de la Polimerasa , SunitinibRESUMEN
OBJECTIVES: To explore whether urinary cytokine and chemokine (CK) levels differed between cold mitomycin-C (cold-MMC)-treated patients and chemohyperthermia (C-HT)-treated patients, to shed light on the possible molecular mechanisms that might explain the superior outcome of C-HT. Furthermore, CK-differences were explored between C-HT responders and C-HT non-responders. METHODS: Twelve NMIBC patients were included. Nine received six-weekly C-HT, and three received four-weekly cold-MMC instillations. Urine was collected on 8-12 time points before and after every treatment. MDC, IL-2, IL-6, IL-8, IP-10, MCP-1 and RANTES were determined by Luminex(®)-analysis. RESULTS: Elevated urinary CK levels were observed in both groups after treatment. In general, CK-peaks were lower in the cold-MMC group in comparison with levels in the C-HT group. Significant higher MCP-1 and IL-6 levels were observed in C-HT-treated patients. Additionally, significant cumulative effects were observed for IP-10 and IL-2. However, IP-10 and IL-2 levels did not significantly differ between treatments. MDC levels after the first week of treatment were significantly higher in the C-HT responders compared with the non-responders. CONCLUSION: MMC treatment leads to elevated urinary CK levels with significantly higher MCP-1 and IL-6 levels in C-HT-treated patients. Increased MDC levels after the first C-HT instillation appear to be related to good clinical outcome and might be of additional value to personalize treatment. Studies involving more patients and longer follow-up are needed to substantiate this observation.
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Carcinoma de Células Transicionales/terapia , Citocinas/orina , Hipertermia Inducida/métodos , Mitomicina/administración & dosificación , Neoplasias de la Vejiga Urinaria/terapia , Administración Intravesical , Anciano , Anciano de 80 o más Años , Antibióticos Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/orina , Carcinoma de Células Transicionales/patología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Instilación de Medicamentos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is characterized by high constitutive vascular endothelial growth factor A (VEGF-A) production that induces a specific vascular phenotype. We previously reported that this phenotype may allow shedding of multicellular tumor fragments into the circulation, possibly contributing to the development of metastasis. Disruption of this phenotype through inhibition of VEGF signaling may therefore result in reduced shedding of tumor fragments and improved prognosis. To test this hypothesis, we investigated the effect of neoadjuvant sorafenib treatment on tumor cluster shedding. PATIENTS AND METHODS: Patients with renal cancer (n = 10, of which 8 have ccRCC) received sorafenib for 4 weeks before tumor nephrectomy. The resection specimens were perfused, and the perfundate was examined for the presence of tumor clusters. Effects of the treatment on the tumor morphology and overall survival were investigated (follow-up of 2 years) and compared with a carefully matched control group. RESULTS: Neoadjuvant sorafenib treatment induced extensive ischemic tumor necrosis and, as expected, destroyed the characteristic ccRCC vascular phenotype. In contrast to the expectation, vital groups of tumor cells with high proliferation indices were detected in postsurgical renal venous outflow in 75% of the cases. Overall survival of patients receiving neoadjuvant treatment was reduced compared to a control group, matched with regard to prognostic parameters. CONCLUSIONS: These results suggest that neoadjuvant sorafenib therapy for ccRCC does not prevent shedding of tumor fragments. Although this is a nonrandomized study with a small patient group, our results suggest that neoadjuvant treatment may worsen survival through as yet undefined mechanisms.
Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Neoplasias Renales/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Anciano , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/cirugía , Femenino , Humanos , Estimación de Kaplan-Meier , Riñón/irrigación sanguínea , Riñón/efectos de los fármacos , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Neoplasias Renales/cirugía , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Nefrectomía , Niacinamida/uso terapéutico , Sorafenib , Resultado del Tratamiento , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
UNLABELLED: Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of metastatic clear cell renal cell carcinoma (RCC). Although TKIs have demonstrated good clinical efficacy, the lack of complete responses, the chronic nature of the treatment, and the side effects are clear disadvantages. An interesting new approach in the treatment of clear cell RCC is antibody-mediated therapy with the chimeric anti-carbonic anhydrase IX (CAIX) antibody girentuximab (cG250). As the results of several girentuximab trials become available, the question arises of whether TKI treatment can be combined with girentuximab-based therapy. In this study, we assessed the effect of the widely used TKI sorafenib on the tumor-targeting potential of (111)In-labeled girentuximab. METHODS: (111)In-girentuximab imaging was performed on 15 patients suspected of having a renal malignancy, with surgery being part of their treatment plan. Of these, 10 patients were treated in a neoadjuvant setting with sorafenib (400 mg orally twice daily). Five patients received treatment during 1 wk, and 5 patients received treatment during 4 wk. In both sorafenib-treated groups, baseline and posttreatment tumor targeting of (111)In-girentuximab were compared. Surgery was performed 3 d after the last image acquisition. Five additional patients were included as a control group and had only a single (111)In-girentuximab injection and scintigraphy without any treatment. Distribution of (111)In-girentuximab was determined scintigraphically ex vivo in a 1-cm lamella of the resected tumorous kidney. Expression of CAIX and of the vascular marker CD31 was determined immunohistochemically on specimens of both tumor and normal kidney tissue. RESULTS: Treatment with sorafenib resulted in a marked decrease of (111)In-girentuximab uptake in the tumor in clear cell RCC patients, especially in the group treated for 4 wk (mean change in both sorafenib-treated groups, -38.4%; range, +9.1% to -79.4%). Immunohistochemical analysis showed markedly reduced CD31 expression and vessel density in the sorafenib-treated groups but no differences in CAIX expression between the sorafenib-treated groups and the nontreated patients. CONCLUSION: Treatment with sorafenib resulted in a treatment duration-dependent significantly decreased uptake of (111)In-girentumab in clear cell RCC lesions. These results indicate that the efficacy of antibody-mediated treatment or diagnosis modalities is hampered by TKI treatment.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/tratamiento farmacológico , Radioisótopos de Indio/farmacocinética , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Anciano , Sinergismo Farmacológico , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante/métodos , Niacinamida/uso terapéutico , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Cintigrafía , SorafenibRESUMEN
Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.
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Carcinoma de Células Renales/secundario , Neoplasias Renales/patología , Neoplasias Pulmonares/secundario , Células Neoplásicas Circulantes/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Femenino , Humanos , Neoplasias Renales/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIXG250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250MN promoter constructs revealed strong promoter activity in G250MN -positive RCC cell lines, but no activity in G250MN -negative cell lines. DNase-I footprint and band-shift analysis demonstrated that Sp1 and HIF-1alpha proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1alpha expression and G250MN expression was observed. In contrast, no relationship between HIF-2alpha expression and G250MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1alpha and Sp1, in combination with CBP/p300, are crucial elements for G250MN expression in ccRCC, and CAIXG250 can be regarded as a unique HIF-1alpha target gene in ccRCC.
Asunto(s)
Adenocarcinoma de Células Claras/genética , Antígenos de Neoplasias/genética , Anhidrasas Carbónicas/genética , Carcinoma de Células Renales/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/genética , Factores de Transcripción/metabolismo , Secuencia de Bases , Anhidrasa Carbónica IX , ADN de Neoplasias/metabolismo , Histona Acetiltransferasas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Datos de Secuencia Molecular , Coactivador 3 de Receptor Nuclear , Unión Proteica , Mapeo Restrictivo , Factor de Transcripción Sp1/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Transfección , Células Tumorales CultivadasRESUMEN
OBJECTIVES: In renal cell carcinoma (RCC) cell lines, expression of the RCC-associated antigen G250 correlates with hypomethylation of the investigated CpG dinucleotides in the G250 promoter region, despite the absence of a CpG island. To gain insight into the molecular mechanism leading to G250 expression in vivo, we ascertained whether this correlation between G250 gene expression and the methylation status of the G250 gene also existed in primary RCC and normal kidney tissue. METHODS: G250 mRNA and protein expression was determined by reverse transcriptase-polymerase chain reaction, fluorescence activated cell sorting analysis, and immunohistochemistry in 15 RCC cell lines and 13 paired primary RCC/normal kidney tissue specimens. The methylation status of the G250 gene was determined by bisulfite genomic sequencing. RESULTS: RCC cell lines revealed a clear correlation between G250 expression and hypomethylation. In contrast, no hypomethylation was observed in primary RCC compared with normal kidney tissue. The CpG dinucleotides investigated were generally completely methylated in RCC, as well as in normal kidney tissue. Furthermore, a primary culture of RCC tissue revealed increasing hypomethylation of the G250 gene in successive passages, suggesting that the G250 hypomethylation observed in vitro is tissue culture induced. CONCLUSIONS: The methylation status of the G250 gene correlated with G250 expression in vitro but not in vivo.