RESUMO
OBJECTIVE: Improving patient selection and development of biological therapies such as vedolizumab in IBD requires a thorough understanding of the mechanism of action and target binding, thereby providing individualised treatment strategies. We aimed to visualise the macroscopic and microscopic distribution of intravenous injected fluorescently labelled vedolizumab, vedo-800CW, and identify its target cells using fluorescence molecular imaging (FMI). DESIGN: Forty three FMI procedures were performed, which consisted of macroscopic in vivo assessment during endoscopy, followed by macroscopic and microscopic ex vivo imaging. In phase A, patients received an intravenous dose of 4.5 mg, 15 mg vedo-800CW or no tracer prior to endoscopy. In phase B, patients received 15 mg vedo-800CW preceded by an unlabelled (sub)therapeutic dose of vedolizumab. RESULTS: FMI quantification showed a dose-dependent increase in vedo-800CW fluorescence intensity in inflamed tissues, with 15 mg (153.7 au (132.3-163.7)) as the most suitable tracer dose compared with 4.5 mg (55.3 au (33.6-78.2)) (p=0.0002). Moreover, the fluorescence signal decreased by 61% when vedo-800CW was administered after a therapeutic dose of unlabelled vedolizumab, suggesting target saturation in the inflamed tissue. Fluorescence microscopy and immunostaining showed that vedolizumab penetrated the inflamed mucosa and was associated with several immune cell types, most prominently with plasma cells. CONCLUSION: These results indicate the potential of FMI to determine the local distribution of drugs in the inflamed target tissue and identify drug target cells, providing new insights into targeted agents for their use in IBD. TRIAL REGISTRATION NUMBER: NCT04112212.
Assuntos
Anticorpos Monoclonais Humanizados , Fármacos Gastrointestinais , Doenças Inflamatórias Intestinais , Mucosa Intestinal , Humanos , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/farmacocinética , Fármacos Gastrointestinais/farmacocinética , Fármacos Gastrointestinais/uso terapêutico , Fármacos Gastrointestinais/administração & dosagem , Feminino , Masculino , Adulto , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Pessoa de Meia-Idade , Mucosa Intestinal/metabolismo , Corantes Fluorescentes , Imagem Molecular/métodos , Idoso , Relação Dose-Resposta a Droga , Adulto JovemRESUMO
Dysplasia and intramucosal esophageal squamous cell carcinoma (ESCC) frequently go unnoticed with white-light endoscopy and, therefore, progress to invasive tumors. If suitable targets are available, fluorescence molecular endoscopy might be promising to improve early detection. Microarray expression data of patient-derived normal esophagus (n = 120) and ESCC samples (n = 118) were analyzed by functional genomic mRNA (FGmRNA) profiling to predict target upregulation on protein levels. The predicted top 60 upregulated genes were prioritized based on literature and immunohistochemistry (IHC) validation to select the most promising targets for fluorescent imaging. By IHC, GLUT1 showed significantly higher expression in ESCC tissue (30 patients) compared to the normal esophagus adjacent to the tumor (27 patients) (p < 0.001). Ex vivo imaging of GLUT1 with the 2-DG 800CW tracer showed that the mean fluorescence intensity in ESCC (n = 17) and high-grade dysplasia (HGD, n = 13) is higher (p < 0.05) compared to that in low-grade dysplasia (LGD) (n = 7) and to the normal esophagus adjacent to the tumor (n = 5). The sensitivity and specificity of 2-DG 800CW to detect HGD and ESCC is 80% and 83%, respectively (ROC = 0.85). We identified and validated GLUT1 as a promising molecular imaging target and demonstrated that fluorescent imaging after topical application of 2-DG 800CW can differentiate HGD and ESCC from LGD and normal esophagus.
Assuntos
Endoscopia/métodos , Carcinoma de Células Escamosas do Esôfago/diagnóstico por imagem , Regulação Neoplásica da Expressão Gênica , Transportador de Glucose Tipo 1/genética , Proteínas de Neoplasias/genética , Diagnóstico Precoce , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Esôfago/diagnóstico por imagem , Esôfago/metabolismo , Fluorescência , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro , Sensibilidade e EspecificidadeAssuntos
Endoscopia Gastrointestinal/métodos , Neoplasias Retais/diagnóstico por imagem , Neoplasias Retais/metabolismo , Reto/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Área Sob a Curva , Quimiorradioterapia Adjuvante , Fibrose , Fluorescência , Humanos , Imageamento por Ressonância Magnética , Terapia Neoadjuvante , Neoplasia Residual , Projetos Piloto , Valor Preditivo dos Testes , Curva ROC , Neoplasias Retais/terapia , Reto/metabolismo , Resultado do TratamentoAssuntos
Adenocarcinoma/diagnóstico , Antineoplásicos Imunológicos/administração & dosagem , Esôfago de Barrett/patologia , Esôfago de Barrett/cirurgia , Bevacizumab/administração & dosagem , Endoscópios Gastrointestinais , Neoplasias Esofágicas/diagnóstico , Esofagoscopia/métodos , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Detecção Precoce de Câncer , Desenho de Equipamento , Feminino , Fluorescência , Humanos , Imuno-Histoquímica , MasculinoRESUMO
Inadequate surgical margins occur frequently in oral squamous cell carcinoma surgery. Fluorescence molecular imaging (FMI) has been explored for intraoperative margin assessment, but data are limited to phase-I studies. In this single-arm phase-II study (NCT03134846), our primary endpoints were to determine the sensitivity, specificity and positive predictive value of cetuximab-800CW for tumor-positive margins detection. Secondary endpoints were safety, close margin detection rate and intrinsic cetuximab-800CW fluorescence. In 65 patients with 66 tumors, cetuximab-800CW was well-tolerated. Fluorescent spots identified in the surgical margin with signal-to-background ratios (SBR) of ≥2 identify tumor-positive margins with 100% sensitivity, 85.9% specificity, 58.3% positive predictive value, and 100% negative predictive value. An SBR of ≥1.5 identifies close margins with 70.3% sensitivity, 76.1% specificity, 60.5% positive predictive value, and 83.1% negative predictive value. Performing frozen section analysis aimed at the fluorescent spots with an SBR of ≥1.5 enables safe, intraoperative adjustment of surgical margins.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Humanos , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/cirurgia , Cetuximab , Corantes , Receptores ErbB , Margens de Excisão , Imagem Molecular , Neoplasias Bucais/diagnóstico por imagem , Neoplasias Bucais/cirurgia , Compostos RadiofarmacêuticosRESUMO
In most oral cancer patients, surgical treatment includes resection of the primary tumor combined with excision of lymph nodes (LNs), either for staging or for treatment. All LNs harvested during surgery require tissue processing and subsequent microscopic histopathologic assessment to determine the nodal stage. In this study, we investigated the use of the fluorescent tracer cetuximab-800CW to discriminate between tumor-positive and tumor-negative LNs before histopathologic examination. Here, we report a retrospective ad hoc analysis of a clinical trial designed to evaluate the resection margin in patients with oral squamous cell carcinoma (NCT02415881). Methods: Two days before surgery, patients were intravenously administered 75 mg of cetuximab followed by 15 mg of cetuximab-800CW, an epidermal growth factor receptor-targeting fluorescent tracer. Fluorescence images of excised, formalin-fixed LNs were obtained and correlated with histopathologic assessment. Results: Fluorescence molecular imaging of 514 LNs (61 pathologically positive nodes) could detect tumor-positive LNs ex vivo with 100% sensitivity and 86.8% specificity (area under the curve, 0.98). In this cohort, the number of LNs that required microscopic assessment was decreased by 77.4%, without missing any metastases. Additionally, in 7.5% of the LNs false-positive on fluorescence imaging, we identified metastases missed by standard histopathologic analysis. Conclusion: Our findings suggest that epidermal growth factor receptor-targeted fluorescence molecular imaging can aid in the detection of LN metastases in the ex vivo setting in oral cancer patients. This image-guided concept can improve the efficacy of postoperative LN examination and identify additional metastases, thus safeguarding appropriate postoperative therapy and potentially improving prognosis.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Cetuximab , Receptores ErbB , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Excisão de Linfonodo , Linfonodos/diagnóstico por imagem , Linfonodos/patologia , Linfonodos/cirurgia , Metástase Linfática/diagnóstico por imagem , Metástase Linfática/patologia , Imagem Molecular , Neoplasias Bucais/diagnóstico por imagem , Neoplasias Bucais/cirurgia , Estudos RetrospectivosRESUMO
Vascular endothelial growth factor-A (VEGF-A) is assumed to play a crucial role in the development and rupture of vulnerable plaques in the atherosclerotic process. We used a VEGF-A targeted fluorescent antibody (bevacizumab-IRDye800CW [bevacizumab-800CW]) to image and visualize the distribution of VEGF-A in (non-)culprit carotid plaques ex vivo. Freshly endarterectomized human plaques (n = 15) were incubated in bevacizumab-800CW ex vivo. Subsequent NIRF imaging showed a more intense fluorescent signal in the culprit plaques (n = 11) than in the non-culprit plaques (n = 3). A plaque received from an asymptomatic patient showed pathologic features similar to the culprit plaques. Cross-correlation with VEGF-A immunohistochemistry showed co-localization of VEGF-A over-expression in 91% of the fluorescent culprit plaques, while no VEGF-A expression was found in the non-culprit plaques (p < 0.0001). VEGF-A expression was co-localized with CD34, a marker for angiogenesis (p < 0.001). Ex vivo near-infrared fluorescence (NIRF) imaging by incubation with bevacizumab-800CW shows promise for visualizing VEGF-A overexpression in culprit atherosclerotic plaques in vivo.
Assuntos
Bevacizumab/farmacologia , Estenose das Carótidas/diagnóstico , Imagem Óptica/métodos , Placa Aterosclerótica/complicações , Fator A de Crescimento do Endotélio Vascular/análise , Idoso , Doenças Assintomáticas , Benzenossulfonatos/química , Bevacizumab/química , Estenose das Carótidas/etiologia , Estenose das Carótidas/patologia , Estenose das Carótidas/cirurgia , Endarterectomia das Carótidas , Estudos de Viabilidade , Feminino , Corantes Fluorescentes/química , Humanos , Indóis/química , Masculino , Pessoa de Meia-Idade , Imagem Molecular/métodos , Placa Aterosclerótica/patologia , Placa Aterosclerótica/cirurgia , Índice de Gravidade de Doença , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Resection of soft-tissue sarcoma (STS) is accompanied by a high rate of tumor-positive surgical margins (14%-34%), which potentially lead to decreased disease-free survival. Vascular endothelial growth factor A is overexpressed in malignant tumors, including STS, and can be targeted with bevacizumab-800CW during fluorescence-guided surgery for real-time tumor detection. In this phase 1 clinical trial, we determined the feasibility, safety, and optimal dose of bevacizumab-800CW for fluorescence-guided surgery in STS for in vivo and ex vivo tumor detection. Methods: Patients with a histopathologic diagnosis of STS were included. In the dose-escalation phase, patients received bevacizumab-800CW intravenously 3 d before surgery (10, 25, and 50 mg; n = 8). In the subsequent dose-expansion phase, 7 additional patients received bevacizumab-800CW at the optimal dose. Fluorescence images were obtained in vivo and ex vivo during all stages of standard care. The optimal dose was determined by calculating in vivo and ex vivo tumor-to-background ratios (TBR) and correlating these results with histopathology. Results: Fifteen patients with STS completed this study. All tumors could be visualized during in vivo and ex vivo imaging. The optimal bevacizumab-800CW dose proved to be 10 mg, with a median in vivo TBR of 2.0 (±0.58) and a median ex vivo TBR of 2.67 (±1.6). All 7 tumor-positive margins could be observed in real time after surgical resection. Conclusion: GS using 10 mg of bevacizumab-800CW is feasible and safe for intraoperative imaging of STS, potentially allowing tumor detection and margin assessment during surgery. An additional follow-up phase 2 study is needed to confirm the diagnostic accuracy.
Assuntos
Imagem Óptica , Sarcoma/diagnóstico por imagem , Sarcoma/metabolismo , Cirurgia Assistida por Computador , Fator A de Crescimento do Endotélio Vascular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Bevacizumab , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doses de Radiação , Sarcoma/cirurgiaRESUMO
Negative circumferential resection margins (CRM) are the cornerstone for the curative treatment of locally advanced rectal cancer (LARC). However, in up to 18.6% of patients, tumor-positive resection margins are detected on histopathology. In this proof-of-concept study, we investigated the feasibility of optical molecular imaging as a tool for evaluating the CRM directly after surgical resection to improve tumor-negative CRM rates. Methods: LARC patients treated with neoadjuvant chemoradiotherapy received an intravenous bolus injection of 4.5 mg of bevacizumab-800CW, a fluorescent tracer targeting vascular endothelial growth factor A, 2-3 d before surgery (ClinicalTrials.gov identifier: NCT01972373). First, for evaluation of the CRM status, back-table fluorescence-guided imaging (FGI) of the fresh surgical resection specimens (n = 8) was performed. These results were correlated with histopathology results. Second, for determination of the sensitivity and specificity of bevacizumab-800CW for tumor detection, a mean fluorescence intensity cutoff value was determined from the formalin-fixed tissue slices (n = 42; 17 patients). Local bevacizumab-800CW accumulation was evaluated by fluorescence microscopy. Results: Back-table FGI correctly identified a tumor-positive CRM by high fluorescence intensities in 1 of 2 patients (50%) with a tumor-positive CRM. For the other patient, low fluorescence intensities were shown, although (sub)millimeter tumor deposits were present less than 1 mm from the CRM. FGI correctly identified 5 of 6 tumor-negative CRM (83%). The 1 patient with false-positive findings had a marginal negative CRM of only 1.4 mm. Receiver operating characteristic curve analysis of the fluorescence intensities of formalin-fixed tissue slices yielded an optimal mean fluorescence intensity cutoff value for tumor detection of 5,775 (sensitivity of 96.19% and specificity of 80.39%). Bevacizumab-800CW enabled a clear differentiation between tumor and normal tissue up to a microscopic level, with a tumor-to-background ratio of 4.7 ± 2.5 (mean ± SD). Conclusion: In this proof-of-concept study, we showed the potential of back-table FGI for evaluating the CRM status in LARC patients. Optimization of this technique with adaptation of standard operating procedures could change perioperative decision making with regard to extending resections or applying intraoperative radiation therapy in the case of positive CRM.
Assuntos
Bevacizumab , Margens de Excisão , Imagem Óptica , Neoplasias Retais/diagnóstico por imagem , Neoplasias Retais/patologia , Cirurgia Assistida por Computador , Adulto , Idoso , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Retais/cirurgia , Resultado do TratamentoRESUMO
Optical molecular imaging using fluorescently labeled monoclonal antibodies is of significant added value in guiding surgical or endoscopic procedures. However, development of tracers for clinical trials is complex, and implementation in the clinic is therefore slow. We present a roadmap for development and translation of monoclonal antibody tracers into a drug product compliant with current good manufacturing processes (cGMPs). Methods: The production process for cetuximab-800CW and trastuzumab-800CW was optimized with regard to dye-to-protein ratio and formulation buffer. Promising formulations were produced under cGMP conditions and advanced to a full-scale stability study. Tracers were analyzed for stability by size-exclusion high-pressure liquid chromatography, pH measurement, osmolality, visual inspection, and sterility, as required by the European Pharmacopeia and cGMP guidelines. Results: Seven formulations were investigated for cetuximab-800CW and 10 for trastuzumab-800CW. On the basis of the formulation study results, we chose 2 formulations per antibody for investigation during the full-scale stability study. These formulations all performed well, showing good compliance with the acceptance criteria set for each product. Conclusion: We designed a roadmap to standardize the development, formulation, and cGMP translation of molecular fluorescent tracers. Using our standardized approach, we developed 2 stable antibody-based tracers for clinical use. The proposed roadmap can be used to efficiently develop a cGMP-compliant formulation and improve the translation of newly developed optical tracers to first-in-human use.
Assuntos
Cetuximab/química , Imagem Óptica/métodos , Pesquisa Translacional Biomédica , Trastuzumab/química , Cetuximab/isolamento & purificação , Descoberta de Drogas , Controle de Qualidade , Trastuzumab/isolamento & purificaçãoRESUMO
During the last decade, the emerging field of molecular fluorescence imaging has led to the development of tumor-specific fluorescent tracers and an increase in early-phase clinical trials without having consensus on a standard methodology for evaluating an optical tracer. By combining multiple complementary state-of-the-art clinical optical imaging techniques, we propose a novel analytical framework for the clinical translation and evaluation of tumor-targeted fluorescent tracers for molecular fluorescence imaging which can be used for a range of tumor types and with different optical tracers. Here we report the implementation of this analytical framework and demonstrate the tumor-specific targeting of escalating doses of the near-infrared fluorescent tracer bevacizumab-800CW on a macroscopic and microscopic level. We subsequently demonstrate an 88% increase in the intraoperative detection rate of tumor-involved margins in primary breast cancer patients, indicating the clinical feasibility and support of future studies to evaluate the definitive clinical impact of fluorescence-guided surgery.
Assuntos
Benchmarking , Neoplasias da Mama/diagnóstico por imagem , Corantes Fluorescentes/administração & dosagem , Imagem Molecular/métodos , Cirurgia Assistida por Computador/métodos , Idoso , Ácidos Alcanossulfônicos/administração & dosagem , Ácidos Alcanossulfônicos/química , Animais , Bevacizumab/administração & dosagem , Bevacizumab/química , Neoplasias da Mama/cirurgia , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Corantes Fluorescentes/química , Humanos , Indóis/administração & dosagem , Indóis/química , Margens de Excisão , Mastectomia/métodos , Pessoa de Meia-Idade , Imagem Óptica/métodosRESUMO
Staphylococcus aureus infections are a major threat in healthcare, requiring adequate early-stage diagnosis and treatment. This calls for novel diagnostic tools that allow noninvasive in vivo detection of staphylococci. Here we performed a preclinical study to investigate a novel fully-human monoclonal antibody 1D9 that specifically targets the immunodominant staphylococcal antigen A (IsaA). We show that 1D9 binds invariantly to S. aureus cells and may further target other staphylococcal species. Importantly, using a human post-mortem implant model and an in vivo murine skin infection model, preclinical feasibility was demonstrated for 1D9 labeled with the near-infrared fluorophore IRDye800CW to be applied for direct optical imaging of in vivo S. aureus infections. Additionally, 89Zirconium-labeled 1D9 could be used for positron emission tomography imaging of an in vivo S. aureus thigh infection model. Our findings pave the way towards clinical implementation of targeted imaging of staphylococcal infections using the human monoclonal antibody 1D9.
Assuntos
Anticorpos Monoclonais/metabolismo , Imagem Óptica/métodos , Infecções Estafilocócicas/diagnóstico por imagem , Infecções Cutâneas Estafilocócicas/diagnóstico por imagem , Staphylococcus aureus/isolamento & purificação , Animais , Anticorpos Monoclonais/química , Antígenos de Bactérias/metabolismo , Cadáver , Modelos Animais de Doenças , Corantes Fluorescentes/química , Humanos , Camundongos , Infecções Estafilocócicas/microbiologia , Infecções Cutâneas Estafilocócicas/microbiologiaRESUMO
Adenoma miss rates in colonoscopy are unacceptably high, especially for sessile serrated adenomas / polyps (SSA/Ps) and in high-risk populations, such as patients with Lynch syndrome. Detection rates may be improved by fluorescence molecular endoscopy (FME), which allows morphological visualization of lesions with high-definition white-light imaging as well as fluorescence-guided identification of lesions with a specific molecular marker. In a clinical proof-of-principal study, we investigated FME for colorectal adenoma detection, using a fluorescently labelled antibody (bevacizumab-800CW) against vascular endothelial growth factor A (VEGFA), which is highly upregulated in colorectal adenomas. Methods: Patients with familial adenomatous polyposis (n = 17), received an intravenous injection with 4.5, 10 or 25 mg of bevacizumab-800CW. Three days later, they received NIR-FME. Results: VEGFA-targeted NIR-FME detected colorectal adenomas at all doses. Best results were achieved in the highest (25 mg) cohort, which even detected small adenomas (<3 mm). Spectroscopy analyses of freshly excised specimen demonstrated the highest adenoma-to-normal ratio of 1.84 for the 25 mg cohort, with a calculated median tracer concentration in adenomas of 6.43 nmol/mL. Ex vivo signal analyses demonstrated NIR fluorescence within the dysplastic areas of the adenomas. Conclusion: These results suggest that NIR-FME is clinically feasible as a real-time, red-flag technique for detection of colorectal adenomas.
Assuntos
Adenoma/diagnóstico , Neoplasias Colorretais/diagnóstico , Endoscopia/métodos , Fluorescência , Técnicas de Diagnóstico Molecular/métodos , Adulto , Idoso , Bevacizumab/administração & dosagem , Feminino , Corantes Fluorescentes/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Fator A de Crescimento do Endotélio Vascular/análise , Adulto JovemRESUMO
Esophageal carcinoma (EC) is a global health problem, with disappointing 5-year survival rates of only 15-25%. Near-infrared targeted photodynamic therapy (NIR-tPDT) is a novel strategy in which cancer-targeted phototoxicity is able to selectively treat malignant cells. In this in vitro report we demonstrate the applicability of antibody-based NIR-tPDT in esophageal adenocarcinoma (EAC), using the phototoxic compounds cetuximab-IRDye700DX and trastuzumab-IRDye700DX, targeting respectively epidermal growth factor receptor 1 (EGFR) and 2 (HER2). Furthermore, we demonstrate that NIR-tPDT can be made more effective by tyrosine kinase inhibitor (TKI) induced growth receptor upregulation. Together, these results unveil a novel strategy for non-invasive EAC treatment, and by pretreatment-induced receptor upregulation its future clinical application may be optimized.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias Esofágicas/metabolismo , Fotoquimioterapia , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Linhagem Celular Tumoral , Células Cultivadas , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/farmacologia , Luz , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Regulação para CimaRESUMO
In vivo tumor labeling with fluorescent agents may assist endoscopic and surgical guidance for cancer therapy as well as create opportunities to directly observe cancer biology in patients. However, malignant and nonmalignant tissues are usually distinguished on fluorescence images by applying empirically determined fluorescence intensity thresholds. Here, we report the development of fSTREAM, a set of analytic methods designed to streamline the analysis of surgically excised breast tissues by collecting and statistically processing hybrid multiscale fluorescence, color, and histology readouts toward precision fluorescence imaging. fSTREAM addresses core questions of how to relate fluorescence intensity to tumor tissue and how to quantitatively assign a normalized threshold that sufficiently differentiates tumor tissue from healthy tissue. Using fSTREAM we assessed human breast tumors stained in vivo with fluorescent bevacizumab at microdose levels. Showing that detection of such levels is achievable, we validated fSTREAM for high-resolution mapping of the spatial pattern of labeled antibody and its relation to the underlying cancer pathophysiology and tumor border on a per patient basis. We demonstrated a 98% sensitivity and 79% specificity when using labeled bevacizumab to outline the tumor mass. Overall, our results illustrate a quantitative approach to relate fluorescence signals to malignant tissues and improve the theranostic application of fluorescence molecular imaging. Cancer Res; 77(3); 623-31. ©2016 AACR.
Assuntos
Bevacizumab/farmacocinética , Neoplasias da Mama/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Imagem Molecular/métodos , Imagem Óptica/métodos , Idoso , Antineoplásicos/farmacocinética , Benzenossulfonatos/farmacocinética , Feminino , Corantes Fluorescentes/farmacocinética , Humanos , Indóis/farmacocinética , Pessoa de Meia-IdadeRESUMO
Purpose: To provide proof of principle of safety, breast tumor-specific uptake, and positive tumor margin assessment of the systemically administered near-infrared fluorescent tracer bevacizumab-IRDye800CW targeting VEGF-A in patients with breast cancer.Experimental Design: Twenty patients with primary invasive breast cancer eligible for primary surgery received 4.5 mg bevacizumab-IRDye800CW as intravenous bolus injection. Safety aspects were assessed as well as tracer uptake and tumor delineation during surgery and ex vivo in surgical specimens using an optical imaging system. Ex vivo multiplexed histopathology analyses were performed for evaluation of biodistribution of tracer uptake and coregistration of tumor tissue and healthy tissue.Results: None of the patients experienced adverse events. Tracer levels in primary tumor tissue were higher compared with those in the tumor margin (P < 0.05) and healthy tissue (P < 0.0001). VEGF-A tumor levels also correlated with tracer levels (r = 0.63, P < 0.0002). All but one tumor showed specific tracer uptake. Two of 20 surgically excised lumps contained microscopic positive margins detected ex vivo by fluorescent macro- and microscopy and confirmed at the cellular level.Conclusions: Our study shows that systemic administration of the bevacizumab-IRDye800CW tracer is safe for breast cancer guidance and confirms tumor and tumor margin uptake as evaluated by a systematic validation methodology. The findings are a step toward a phase II dose-finding study aimed at in vivo margin assessment and point to a novel drug assessment tool that provides a detailed picture of drug distribution in the tumor tissue. Clin Cancer Res; 23(11); 2730-41. ©2016 AACR.
Assuntos
Benzenossulfonatos/administração & dosagem , Bevacizumab/administração & dosagem , Neoplasias da Mama Masculina/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Indóis/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Benzenossulfonatos/efeitos adversos , Bevacizumab/efeitos adversos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Neoplasias da Mama Masculina/diagnóstico , Neoplasias da Mama Masculina/diagnóstico por imagem , Neoplasias da Mama Masculina/patologia , Linhagem Celular Tumoral , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Estudos de Viabilidade , Feminino , Humanos , Indóis/efeitos adversos , Masculino , Imagem Óptica , Tomografia por Emissão de Pósitrons , Distribuição Tecidual/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
There is a dire need for better visualization of cancer and analysis of specific targets in vivo. Molecular imaging with fluorescence is gaining more and more attention, as it allows detection of these targets and has advantages over radioactivity, such as no radiation dose, and lower costs. A key challenge in optical imaging however, is translation of the newly developed tracers from pre-clinical phase to clinical application. We describe the development and safety testing of clinical grade bevacizumab-800CW, an antibody-based targeted agent for non-invasive imaging of vascular endothelial growth factor A (VEGF-A). Development included implementing the manufacturing process and analytical methods according to current Good Manufacturing Practice (cGMP), formulation studies, extended characterization and stability testing. For safety pharmacology an extended single dose toxicity study in mice was performed. Bevacizumab-800CW was formulated in isotonic phosphate buffered sodium chloride solution at pH 7. The production was robust and showed a reproducible labeling efficiency, and no impurities. The binding affinity to VEGF-A remained intact. The optimized product meets all release specifications, is stable up to at least 3months and its characteristics did not significantly differ from the unlabeled bevacizumab. Toxicity testing in mice showed no remarkable findings. In conclusion, sterile bevacizumab-800CW (6mg=6ml) can be produced in stock according to current Good Manufacturing Practice. It is ready for first-in-human use.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Bevacizumab/uso terapêutico , Inibidores da Angiogênese/efeitos adversos , Inibidores da Angiogênese/química , Animais , Bevacizumab/efeitos adversos , Bevacizumab/química , Corantes Fluorescentes/química , Humanos , Camundongos , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Optimum cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is essential for the curative treatment of peritoneal carcinomatosis of colorectal origin. At present, surgeons depend on visual inspection and palpation for tumour detection. Improved detection of tumour tissue using molecular fluorescence-guided surgery could not only help attain a complete cytoreduction of metastatic lesions, but might also prevent overtreatment by avoiding resection of benign lesions. METHODS: For this non-randomised, single-centre feasibility study, we enrolled patients with colorectal peritoneal metastases scheduled for cytoreductive surgery and HIPEC. 2 days before surgery, 4·5 mg of the near-infrared fluorescent tracer bevacizumab-IRDye800CW was administered intravenously. The primary objectives were to determine the safety and feasibility of molecular fluorescence-guided surgery using bevacizumab-IRDye800CW. Molecular fluorescence-guided surgery was deemed safe if no allergic or anaphylactic reactions were recorded and no serious adverse events were attributed to bevacizumab-IRDye800CW. The technique was deemed feasible if bevacizumab-IRDye800CW enabled detection of fluorescence signals intraoperatively. Secondary objectives were correlation of fluorescence with histopathology by back-table imaging of the fresh surgical specimen and semi-quantitative ex-vivo analyses of formalin-fixed paraffin embedded (FFPE) tissue on all peritoneal lesions. Additionally, VEGF-α staining and fluorescence microscopy was done. This study is registered with the Netherlands Trial Registry, number NTR4632. FINDINGS: Between July 3, 2014, and March 2, 2015, seven patients were enrolled in the study. One patient developed an abdominal sepsis 5 days postoperatively and another died from an asystole 4 days postoperatively, most probably due to a cardiovascular thromboembolic event. However, both serious adverse events were attributed to the surgical cytoreductive surgery and HIPEC procedure. No serious adverse events related to bevacizumab-IRDye800CW occurred in any of the patients. Intraoperatively, fluorescence was seen in all patients. In two patients, additional tumour tissue was detected by molecular fluorescence-guided surgery that was initially missed by the surgeons. During back-table imaging of fresh surgical specimens, a total of 80 areas were imaged, marked, and analysed. All of the 29 non-fluorescent areas were found to contain only benign tissue, whereas tumour tissue was detected in 27 of 51 fluorescent areas (53%). Ex-vivo semi-quantification of 79 FFPE peritoneal lesions showed a tumour-to-normal ratio of 6·92 (SD 2·47). INTERPRETATION: Molecular fluorescence-guided surgery using the near-infrared fluorescent tracer bevacizumab-IRDye800CW is safe and feasible. This technique might be of added value for the treatment of patients with colorectal peritoneal metastases through improved patient selection and optimisation of cytoreductive surgery. A subsequent multicentre phase 2 trial is needed to make a definitive assessment of the diagnostic accuracy and the effect on clinical decision making of molecular fluorescence-guided surgery. FUNDING: FP-7 Framework Programme BetaCure and SurgVision BV.