Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer.

OBJECTIVES: Establishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis. MATERIAL AND METHODS: To determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated. RESULTS: All human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150µg) and 0.34nmol (50µg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes. CONCLUSION: This study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.

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.

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 × 10(5) and 4 × 10(4) 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 mm(3) 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.

Real-time intraoperative detection of breast cancer using near-infrared fluorescence imaging and Methylene Blue.

BACKGROUND: Despite recent developments in preoperative breast cancer imaging, intraoperative localization of tumor tissue can be challenging, resulting in tumor-positive resection margins during breast conserving surgery. Based on certain physicochemical similarities between Technetium((99m)Tc)-sestamibi (MIBI), an SPECT radiodiagnostic with a sensitivity of 83-90% to detect breast cancer preoperatively, and the near-infrared (NIR) fluorophore Methylene Blue (MB), we hypothesized that MB might detect breast cancer intraoperatively using NIR fluorescence imaging. METHODS: Twenty-four patients with breast cancer, planned for surgical resection, were included. Patients were divided in 2 administration groups, which differed with respect to the timing of MB administration. N = 12 patients per group were administered 1.0 mg/kg MB intravenously either immediately or 3 h before surgery. The mini-FLARE imaging system was used to identify the NIR fluorescent signal during surgery and on post-resected specimens transferred to the pathology department. Results were confirmed by NIR fluorescence microscopy. RESULTS: 20/24 (83%) of breast tumors (carcinoma in N = 21 and ductal carcinoma in situ in N = 3) were identified in the resected specimen using NIR fluorescence imaging. Patients with non-detectable tumors were significantly older. No significant relation to receptor status or tumor grade was seen. Overall tumor-to-background ratio (TBR) was 2.4 ± 0.8. There was no significant difference between TBR and background signal between administration groups. In 2/4 patients with positive resection margins, breast cancer tissue identified in the wound bed during surgery would have changed surgical management. Histology confirmed the concordance of fluorescence signal and tumor tissue. CONCLUSIONS: This feasibility study demonstrated an overall breast cancer identification rate using MB of 83%, with real-time intraoperative guidance having the potential to alter patient management.

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent anti-epidermal growth factor receptor nanobody.

Intraoperative near-infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real-time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)-targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti-EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12-800CW and R2-800CW). Orthotopic tongue tumors were induced in nude mice using the OSC-19-luc2-cGFP cell line. Tumor-bearing mice were injected with 25 µg 7D12-800CW, R2-800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12-800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12-800CW. A significantly higher tumor-to-background ratio (TBR) was observed in mice injected with 7D12-800CW compared to mice injected with R2-800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR-specific nanobody. After injection of 75 µg 7D12-800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR-targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.

Continuous femoral nerve block after total knee arthroplasty?

BACKGROUND: A continuous femoral nerve block is frequently used as an adjunct therapy after total knee arthroplasty (TKA). However, there is still debate on its benefits. METHODS: In this prospective, randomized study, patients received a basic analgesic regimen of paracetamol and dicloflenac for the first 48 h postoperatively. In addition, the study group received a continuous femoral nerve block. A morphine patient-controlled analgesia pump was also available as a rescue analgesic to all the patients. Patients' numeric rating scores for pain, the amount of morphine consumed and its side effects during the first 48 h were recorded. Knee flexion angles achieved during the first week were registered. Three months postoperatively, patients completed Western Ontario and McMaster Universities Osteoarthritis Index and Knee Society Score. RESULTS: The study group (n=27) had less pain (P=0.0016) during the first 48 h, was more satisfied with the analgesia (P<0.001) and used less morphine (P=0.007) compared with the control group (n=26). Fewer patients were nauseated, vomited or were drowsy in the study group (P=0.001). Also, the study group achieved better knee flexion in the first 6 days after surgery (P=0.001), with more patients reaching 90 degrees flexion than the control group. However, after 3 months, there were no significant functional differences between the groups. CONCLUSION: A continuous femoral nerve block leads to better analgesia, less morphine consumption and less morphine-related side effects after TKA. Early functional recovery is improved, resulting in more patients reaching 90 degrees knee flexion after 6 days. However, after 3 months, no significant functional benefits were found.

How to quantify knee function after total knee arthroplasty?

Total knee arthroplasty (TKA) is being undertaken in a younger population than before and as a result the functional demands on the knee are likely to be increasing. As a consequence, it is important to define quantitative functional knee tests that can monitor any increase. A valuable functional knee test has to be able to distinguish small differences (selectivity) and has to be independent of pain (content validity). In this study, patient-based questionnaires (WOMAC and Knee Society score) and performance-based tests (sit-to-stand movement, maximal isometric contraction and timed-up-and-go) were used to assess which of these tests are selective and valid to measure knee function. Tests were considered to be selective if they could discriminate between knee patients and healthy control subjects, and to have functional content validity if they were relatively independent of pain. Twenty-eight patients were measured 16 months after surgery and compared to a healthy control group of 31 subjects. The sit-to-stand movement and timed-up-and-go test were both selective and functionally content valid. The timed-up-and-go test can be used for a quick initial assessment of global function and the sit-to-stand movement as a more biomechanical instrument identifying how the knee function of the patient is affected.

Functional evaluation of the TKA patient using the coordination and variability of rising.

A kinematic analysis of the knee function is important for the evaluation of total knee arthroplasties (TKA). We used the coordination and variability of rising from a chair as functional knee parameters. Twelve knee patients were measured prior to surgery (=pre-TKA group) and one year after surgery (=post-TKA group). A group of 15 healthy, age-matched subjects was selected as control group. The WOMAC questionnaire, frequently used by orthopaedic surgeons, was administered prior to the test. The test consisted of 10 times rising from a low chair and 10 times from a high chair. Knee and hip angles and angular velocities were measured with electrogoniometers. The relative phase (=MRP) between hip and knee was a measure for the coordination of rising and the standard deviation of the relative phase of the 10 trials (=SRP) was a measure for the variability. The coordination and variability of rising of the TKA patients were compared to the control group, and the relationship with the WOMAC questionnaire was calculated. The coordination of rising from a high chair and the variability of rising from both chair heights were significantly different for the pre-TKA group compared to the control group (p<0.05). The post-TKA group showed no significant differences with the control group, which indicates a functional recovery after TKA implantation. The functional parameters correlated adequately with the subjective WOMAC questionnaire. This study showed that our method is an objective measure of functionality and it will be worthwhile to use it as an additional evaluation tool.