Fluorescence-guided Therapy in Oncology : Targeted Imaging and Photodynamic Therapy

This thesis demonstrates the use of fluorescence-guided therapy in oncology. First it shows the preclinical validation of two different targets and probes for fluorescence-guided surgery, results that pave the way towards clinical implementation. Next, the utility of the NIR fluorescence Artemis camera is described. Lastly, the first steps are made towards fluorescence theranostics in oncology: NIR fluorescence-guided surgery and intra-operative targeted photodynamic therapy

Introducing fluorescence guided surgery into orthopedic oncology: A systematic review of candidate protein targets for Ewing sarcoma

Orthopaedics, Trauma Surgery and Rehabilitatio

Higher healing rate after meniscal repair with concomitant ACL reconstruction for tears located in vascular zone 1 compared to zone 2: a systematic review and meta-analysis

Purpose The purpose of this study was to determine and compare the percentage of completely healed meniscal tears after arthroscopic repair combined with anterior cruciate ligament reconstruction (ACLR) for the different vascular zones of the meniscus. Methods PubMed, Embase, Web of Science, Cochrane library and Emcare were searched on 19 May 2020 for articles reporting healing rates after arthroscopic meniscal repair with concomitant ACLR for the different meniscal vascular zones as assessed by second-look arthroscopy. Data on meniscal tears were extracted as located in zones 1, 2 or 3, according to the Cooper classification. Studies were graded in quality using a modified Newcastle-Ottawa Scale. Pooled analyses were performed utilizing a random-effects model. Meta-analyses were performed using R version 3.6.2 and SPSS statistical software version 25.0. The study was registered with PROSPERO (ID:CRD42020176175). Results Ten observational cohort studies met the inclusion criteria, accounting for 758 meniscal tear repairs in total. The pooled overall proportion of healing was 78% (95% CI 72-84%). The mean weighted proportion of healing was 83% (95% CI 76-90%) for studies (n = 10) reporting zone 1 tears and 69% (95% CI 59-79%) for studies (n = 9) reporting zone 2 tears. No study reported healing rates for zone 3 tears. The pooled overall odds ratio was 2.5 (95% CI 1.00-6.02), indicating zone 1 tears as 2.5 times more likely to heal than zone 2 tears. Conclusion This study demonstrates that meniscal tears localized in vascular zone 1 were more likely to heal than those in zone 2.Orthopaedics, Trauma Surgery and Rehabilitatio

Candidate biomarkers for specific intraoperative near-infrared imaging of soft tissue sarcomas: a systematic review

Simple SummaryNear-infrared imaging of tumors during surgery facilitates the oncologic surgeon to distinguish malignant from healthy tissue. The technique is based on fluorescent tracers binding to tumor biomarkers on malignant cells. Currently, there are no clinically available fluorescent tracers that specifically target soft tissue sarcomas. This review searched the literature to find candidate biomarkers for soft tissue sarcomas, based on clinically used therapeutic antibodies. The search revealed 7 biomarkers: TEM1, VEGFR-1, EGFR, VEGFR-2, IGF-1R, PDGFR alpha, and CD40. These biomarkers are abundantly present on soft tissue sarcoma tumor cells and are already being targeted with humanized monoclonal antibodies. The conjugation of these antibodies with a fluorescent dye will yield in specific tracers for image-guided surgery of soft tissue sarcomas to improve the success rates of tumor resections.Surgery is the mainstay of treatment for localized soft tissue sarcomas (STS). The curative treatment highly depends on complete tumor resection, as positive margins are associated with local recurrence (LR) and prognosis. However, determining the tumor margin during surgery is challenging. Real-time tumor-specific imaging can facilitate complete resection by visualizing tumor tissue during surgery. Unfortunately, STS specific tracers are presently not clinically available. In this review, STS-associated cell surface-expressed biomarkers, which are currently already clinically targeted with monoclonal antibodies for therapeutic purposes, are evaluated for their use in near-infrared fluorescence (NIRF) imaging of STS. Clinically targeted biomarkers in STS were extracted from clinical trial registers and a PubMed search was performed. Data on biomarker characteristics, sample size, percentage of biomarker-positive STS samples, pattern of biomarker expression, biomarker internalization features, and previous applications of the biomarker in imaging were extracted. The biomarkers were ranked utilizing a previously described scoring system. Eleven cell surface-expressed biomarkers were identified from which 7 were selected as potential biomarkers for NIRF imaging: TEM1, VEGFR-1, EGFR, VEGFR-2, IGF-1R, PDGFR alpha, and CD40. Promising biomarkers in common and aggressive STS subtypes are TEM1 for myxofibrosarcoma, TEM1, and PDGFR alpha for undifferentiated soft tissue sarcoma and EGFR for synovial sarcoma.Surgical oncolog

Characterization and Evaluation of the Artemis Camera for Fluorescence-Guided Cancer Surgery

Purpose: Near-infrared (NIR) fluorescence imaging can provide the surgeon with real-time visualization of, e.g., tumor margins and lymph nodes. We describe and evaluate the Artemis, a novel, handheld NIR fluorescence camera.Procedures: We evaluated minimal detectable cell numbers (FaDu-luc2, 7D12-IRDye 800CW), preclinical intraoperative detection of sentinel lymph nodes (SLN) using indocyanine green (ICG), and of orthotopic tongue tumors using 7D12-800CW. Results were compared with the Pearl imager. Clinically, three patients with liver metastases were imaged using ICG.Results: Minimum detectable cell counts for Artemis and Pearl were 2 × 105 and 4 × 104 cells, respectively. In vivo, seven SLNs were detected in four mice with both cameras. Orthotopic OSC-19-luc2-cGFP tongue tumors were clearly identifiable, and a minimum FaDu-luc2 tumor size of 1 mm3 could be identified. Six human malignant lesions were identified during three liver surgery procedures.Conclusions: Based on this study, the Artemis system has demonstrated its utility in fluorescence-guided cancer surgery

Evaluation of potential targets for fluorescence-guided surgery in pediatric Ewing sarcoma: a preclinical proof-of-concept study

Simple Summary The only cure for children with Ewing sarcoma (ES) is surgery. Unfortunately, surgeons are often not able to differentiate healthy from malignant tissue. Fluorescent imaging during the operation will facilitate recognition of malignant cells, but unfortunately there are no ES specific tracers available yet. We searched for proteins on ES cells that could be used as a target against which specific tracers could be developed. The most promising proteins, CD99, CD117, and GD2, were found in paraffin-embedded tissue samples collected from ES patients. Tracers against CD99 and CD117, consisting of monoclonal antibodies attached with a fluorescent dye, showed positive signals on cultured ES cells. In a proof-of-concept study, these tracers were topically applied on fresh ES tissue, showing a signal in the tumor. Our results indicate the applicability for fluorescence-guided surgery of ES-based tracers, but these data have to be confirmed in a larger cohort of pediatric ES patients. Fluorescence-guided surgery (FGS), based on fluorescent tracers binding to tumor-specific biomarkers, could assist surgeons to achieve complete tumor resections. This study evaluated potential biomarkers for FGS in pediatric Ewing sarcoma (ES). Immunohistochemistry (IHC) was performed to assess CD99, CXCR4, CD117, NPY-R-Y1, and IGF-1R expression in ES biopsies and resection specimens. LINGO-1 and GD2 evaluation did not work on the acquired tissue. Based on the immunoreactive scores, anti-CD99 and anti-CD117 were evaluated for binding specificity using flow cytometry and immunofluorescence microscopy. Anti-GD2, a tracer in the developmental phase, was also tested. These three tracers were topically applied to a freshly resected ES tumor and adjacent healthy tissue. IHC demonstrated moderate/strong CD99 and CD117 expression in ES tumor samples, while adjacent healthy tissue had limited expression. Flow cytometry and immunofluorescence microscopy confirmed high CD99 expression, along with low/moderate CD117 and low GD2 expression, in ES cell lines. Topical anti-CD99 and anti-GD2 application on ES tumor showed fluorescence, while anti-CD117 did not show fluorescence for this patient. In conclusion, CD99-targeting tracers hold promise for FGS of ES. CD117 and GD2 tracers could be potential alternatives. The next step towards development of ES-specific FGS tracers could be ex vivo topical application experiments on a large cohort of ES patients.Orthopaedics, Trauma Surgery and Rehabilitatio

EpCAM as multi-tumour target for near-infrared fluorescence guided surgery

Background: Evaluation of resection margins during cancer surgery can be challenging, often resulting in incomplete tumour removal. Fluorescence-guided surgery (FGS) aims to aid the surgeon to visualize tumours and resection margins during surgery. FGS relies on a clinically applicable imaging system in combination with a specific tumour-targeting contrast agent. In this study EpCAM (epithelial cell adhesion molecule) is evaluated as target for FGS in combination with the novel Artemis imaging system. Methods: The NIR fluorophore IRDye800CW was conjugated to the well-established EpCAM specific monoclonal antibody 323/A3 and an isotype IgG1 as control. The anti-EpCAM/800CW conjugate was stable in serum and showed preserved binding capacity as evaluated on EpCAM positive and negative cell lines, using flow cytometry and cell-based plate assays. Four clinically relevant orthotopic tumour models, i.e. colorectal cancer, breast cancer, head and neck cancer, and peritonitis carcinomatosa, were used to evaluate the performance of the anti-EpCAM agent with the clinically validated Artemis imaging system. The Pearl Impulse small animal imaging system was used as reference. The specificity of the NIRF signal was confirmed using bioluminescence imaging and green-fluorescent protein. Results: All tumour types could clearly be delineated and resected 72 h after injection of the imaging agent. Using NIRF imaging millimetre sized tumour nodules were detected that were invisible for the naked eye. Fluorescence microscopy demonstrated the distribution and tumour specificity of the anti-EpCAM agent. Conclusions: This study shows the potential of an EpCAM specific NIR-fluorescent agent in combination with a clinically validated intraoperative imaging system to visualize various tumours during surgery

Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging

Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage. Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT. Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h com

Preclinical Studies on Tumor-Specific Fluorescent Targeting Agents: the Need for a Gold Standard of Tumor Localization

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Shifting Focus in Optical Image-Guided Cancer Therapy

Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas