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Introduction: Anastomotic leakage is a severe complication after oesophageal resection with gastric conduit reconstruction. Poor perfusion of the gastric conduit plays an important role in the development of anastomotic leakage. Quantitative near-infrared (NIR) fluorescence angiography with indocyanine green (ICG-FA) is an objective technique that can be used for perfusion assessment. This study aims to assess perfusion patterns of the gastric conduit with quantitative ICG-FA. Methods: In this exploratory study, 20 patients undergoing oesophagectomy with gastric conduit reconstruction were included. A standardized NIR ICG-FA video of the gastric conduit was recorded. Postoperatively, the videos were quantified. Primary outcomes were the time-intensity curves and nine perfusion parameters from contiguous regions of interest on the gastric conduit. A secondary outcome was the inter-observer agreement of subjective interpretation of the ICG-FA videos between six surgeons. The inter-observer agreement was tested with an intraclass correlation coefficient (ICC). Results: In a total of 427 curves, three distinct perfusion patterns were recognized: pattern 1 (steep inflow, steep outflow); pattern 2 (steep inflow, minor outflow); and pattern 3 (slow inflow, no outflow). All perfusion parameters were significantly different between the perfusion patterns. The inter-observer agreement was poor – moderate (ICC:0.345,95%CI:0.164–0.584). Discussion: This was the first study to describe perfusion patterns of the complete gastric conduit after oesophagectomy. Three distinct perfusion patterns were observed. The poor inter-observer agreement of the subjective assessment underlines the need for quantification of ICG-FA of the gastric conduit. Further studies should evaluate the predictive value of perfusion patterns and parameters on anastomotic leakage.

Near-infrared (NIR) fluorescence imaging using exogenous fluorescent agents provides whole-field images in real-time to assist the surgeon in the excision of a tumor. Although the method has high sensitivity, the specificity can sometimes be lower than expected. Raman spectroscopy can detect tumors with high specificity. Therefore, a combination of both techniques can be advantageous. A complication that must be addressed is that the NIR spectral region is favored by both techniques for (in vivo) tissue analysis. When fluorescence and Raman emissions spectrally overlap, it becomes challenging or impossible to detect the Raman signal. In this paper, by avoiding this overlap, we describe a Raman spectroscopy setup capable of recording high-quality Raman spectra from tissue containing NIR exogenous fluorescent agents. We identify an optimal wavelength interval (900-915 nm) for Raman excitation, which avoids both excitation of fluorescent dyes and Raman signal self-absorption by the tissue. In this way, Raman spectroscopy can be combined with the currently most-used NIR fluorescent dyes. This combined novel setup could pave the way for clinical trials benefiting from both fluorescence imaging and Raman spectroscopy to avoid positive margins in cancer surgery.

Purpose The purpose of this study was to assess whether the vascularisation of the meniscus could be visualised intra-operatively using near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) in patients undergoing total knee arthroplasty (TKA). Methods The anterior horn (i.e., Cooper classification: zones C and D) of the meniscus that was least affected (i.e., least degenerative) was removed during TKA surgery in ten patients to obtain a cross section of the inside of the meniscus. Thereafter, 10 mg of ICG was injected intravenously, and vascularisation of the cross section of the meniscus was assessed using the Quest spectrum NIRF camera system. We calculated the percentage of patients in whom vascularisation was observed intra-operatively using NIRF imaging compared to immunohistochemistry. Results Meniscal vascularisation using NIRF imaging was observed in six out of eight (75%) patients in whom vascularisation was demonstrated with immunohistochemistry. The median extent of vascularisation was 13% (interquartile range (IQR) 3–28%) using NIRF imaging and 15% (IQR 11–23%) using immunohistochemistry. Conclusion This study shows the potential of NIRF imaging to visualise vascularisation of the meniscus, as vascularisation was observed in six out of eight patients with histologically proven meniscal vascularisation. Level of evidence IV.

Background: Colorectal cancer is the fourth most diagnosed malignancy worldwide and surgery is one of the cornerstones of the treatment strategy. Near-infrared (NIR) fluorescence imaging is a new and upcoming technique, which uses an NIR fluorescent agent combined with a specialised camera that can detect light in the NIR range. It aims for more precise surgery with improved oncological outcomes and a reduction in complications by improving discrimination between different structures.Methods: A systematic search was conducted in the Embase, Medline and Cochrane databases with search terms corresponding to 'fluorescence-guided surgery', 'colorectal surgery', and 'colorectal cancer' to identify all relevant trials.Results: The following clinical applications of fluorescence guided surgery for colorectal cancer were identified and discussed: (1) tumour imaging, (2) sentinel lymph node imaging, (3) imaging of distant metastases, (4) imaging of vital structures, (5) imaging of perfusion. Both experimental and FDA/EMA approved fluorescent agents are debated. Furthermore, promising future modalities are discussed.Conclusion: Fluorescence-guided surgery for colorectal cancer is a rapidly evolving field. The first studies show additional value of this technique regarding change in surgical management. Future trials should focus on patient related outcomes such as complication rates, disease free survival, and overall survival. (C) 2021 The Authors. Published by Elsevier Ltd.

Supplementary figure legends

Supplementary figure 1. A, Immunohistochemical analysis of BMP9 expression in human tumor samples. Supplementary figure 2. A CD31 staining in head and neck model. Images takes from melanomas after treatment with Fc or ALK1-Fc, magnification 200x. Supplementary figure 3. A, Western blotting showing expression of Fc constructs on stable overexpressing cells. Supplementary figure 4. A, Changes in the number of pericytes in breast tumors generated after inoculation of KEP1-11 cells carrying expression constructs for Fc, ALK1-Fc, ALK2-Fc or Flt-Fc. Supplementary figure 5. A, Cytotoxic effect of cisplatin treatment on KEP1-11 cells. Cells were incubated with increasing doses of cisplatin and the number of viable cells was assessed after 72 hours of treatment.

Purpose: Antiangiogenic therapy, mostly targeting VEGF, has been applied in cancer patients for the last decade. However, resistance to anti-VEGF therapy and/or no significant benefit as monotherapeutic agent is often observed. Therefore, new antiangiogenic strategies are needed. In the current study, we investigated the therapeutic effect of interfering with the bone morphogenetic protein (BMP)9/activin receptor–like kinase (ALK)1 signaling pathway by using an ALK1-Fc ligand trap.Experimental Design: We analyzed the potential antiangiogenic and antitumor effects of ALK1-Fc protein as monotherapy and in combination with chemotherapy in vivo in mouse models of melanoma, head and neck cancer, and invasive lobular breast carcinomas. ALK1-Fc sequesters BMP9 and 10 and prevents binding of these ligands to endothelial ALK1, which regulates angiogenesis.Results: Treatment of mice with ALK1-Fc strongly decreased the tumors' microvascular density in the three different mouse cancer models. However, this effect was not accompanied by a reduction in tumor volume. An immunohistochemical analysis of the tumor samples revealed that ALK1-Fc treatment increased the pericyte coverage of the remaining tumor vessels and decreased the hypoxia within the tumor. Next, we observed that combining ALK1-Fc with cisplatin inhibited tumor growth in the breast and head and neck cancer models more efficiently than chemotherapy alone.Conclusions: The addition of ALK1-Fc to the cisplatin treatment was able to enhance the cytotoxic effect of the chemotherapy. Our results provide strong rationale to explore combined targeting of ALK1 with chemotherapy in a clinical setting, especially in the ongoing phase II clinical trials with ALK1-Fc. Clin Cancer Res; 22(1); 96–106. ©2015 AACR.

Background: Radiotherapy (RT) is standard of care for most advanced head and neck squamous cell carcinoma (HNSCC) and results in an unfavorable 5-year overall survival of 40%. Despite strong biological rationale, combining RT with immune checkpoint inhibitors does not result in a survival benefit. Our hypothesis is that the combination of these individually effective treatments fails because of radiation-induced immunosuppression and lymphodepletion. By integrating modern radiobiology and innovative radiotherapy concepts, the patient’s immune system could be maximally retained by: (1) increasing the dose per fraction so that the total-dose and number of fractions can be reduced (HYpofractionation), (2) redistributing the radiation dose towards a higher peak dose within the tumor center and a lowered elective lymphatic field dose (Dose-redistribution), and by (3) using RAdiotherapy with protons instead of photons (HYDRA). Methods: The primary aim of this multicenter study is to determine the safety of HYDRA proton- and photon radiotherapy by conducting two parallel phase-I trials. Both HYDRA arms are randomized with standard of care for longitudinal immune profiling. There will be a specific focus on actionable immune targets and their temporal patterns that can be tested in future hypofractionated immuno-radiotherapy trials. The HYDRA dose prescriptions (in 20 fractions) are 40 Gy elective dose and 55 Gy simultaneous integrated boost on the clinical target volume with a 59 Gy focal boost on the tumor center. A total of 100 patients (25 per treatment group) will be recruited, the final analysis will be performed one year after the last patient has been included. Discussion: In the context of HNSCC, hypofractionation has historically only been reserved for small tumors out of fear for late normal tissue toxicity. To date, hypofractionated radiotherapy may also be safe for larger tumors as both the radiation dose and volume can be reduced by the combination of advanced imaging for better target definition, novel accelerated repopulation models and high-precision radiation treatment planning and dose delivery. HYDRA’s expected immune sparing effect may lead to improved outcome by allowing for future effective combination treatment with immunotherapy. Trial registration: The trial is registered at ClinicalTrials.gov; NCT05364411 (registered on May 6th, 2022).

Simple Summary The treatment outcomes in patients with head and neck cancer vary greatly, and serious side effects are often observed. Being able to predict therapy effects is therefore crucial for choosing the best treatment option for each patient. In this study, we developed an assay to evaluate how head and neck tumor cells respond to radiation and chemotherapy. Treatment of thin patient-derived cancer tissue slices in the laboratory (in vitro) resulted in large differences in individual tumor's reactions to treatment. In the sensitive tumors, cancer cells repaired the DNA damage inflicted by therapy only partially, stopped multiplying, and showed increased levels of cell death. On the other hand, resistant tumors were able to recover from the damage caused by the treatment. The next crucial step is to investigate whether the differences we observed in vitro can indeed predict the treatment outcomes; this is currently being tested in an ongoing clinical trial. Background: Head and neck squamous cell carcinoma (HNSCC) displays a large heterogeneity in treatment response, and consequently in patient prognosis. Despite extensive efforts, no clinically validated model is available to predict tumor response. Here we describe a functional test for predicting tumor response to radiation and chemotherapy on the level of the individual patient. Methods: Resection material of 17 primary HNSCC patients was cultured ex vivo, irradiated or cisplatin-treated, after which the effect on tumor cell vitality was analyzed several days after treatment. Results: Ionizing radiation (IR) affected tumor cell growth and viability with a clear dose-response relationship, and marked heterogeneity between tumors was observed. After a single dose of 5Gy, proliferation in IR-sensitive tumors dropped below 30% of the untreated level, while IR-resistant tumors maintained at least 60% of proliferation. IR-sensitive tumors showed on average a twofold increase in apoptosis, as well as an increased number and size of DNA damage foci after treatment. No differences in the homologous recombination (HR) proficiency between IR-sensitive and -resistant tumors were detected. Cisplatin caused a decrease in proliferation, as well as induction of apoptosis, again with marked variation between the samples. Conclusions: Our functional ex vivo assay discriminated between IR-sensitive and IR-resistant HNSCC tumors, and may also be suitable for predicting response to cisplatin. Its predictive value is currently under investigation in a prospective clinical study.

Fluorescence-guided surgery is an intraoperative optical imaging method that provides surgeons with real-time guidance for the delineation of tumours. Currently, in phase 1 and 2 clinical trials, evaluation of fluorescence-guided surgery is primarily focused on its diagnostic performance, although the corresponding outcome variables do not inform about the added clinical benefit of fluorescence-guided surgery and are challenging to assess objectively. Nonetheless, the effect of fluorescence-guided surgery on intraoperative decision making is the most objective outcome measurement to assess the clinical value of this imaging method. In this Review, we explore the study designs of existing trials of fluorescence-guided surgery that allow us to extract information on potential changes in intraoperative decision making, such as additional or more conservative resections. On the basis of this analysis, we offer recommendations on how to report changes in intraoperative decision making that result from fluorescence imaging, which is of utmost importance for the widespread clinical implementation of fluorescence-guided surgery.

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.

Photodynamic therapy (PDT) has a great therapeutic potential because it induces local cellular cytotoxicity upon application of a laser light that excites a photosensitizer, leading to toxic reactive oxygen species. Nevertheless, PDT still is underutilized in the clinic, mostly because of damage induced to normal surrounding tissues. Efforts have been made to improve the specificity. Nanobody-targeted PDT is one of such approaches, in which the variable domain of heavy-chain antibodies, i.e., nanobodies, are used to target photosensitizers selectively to cancer cells. In vitro studies are certainly very valuable to evaluate the therapeutic potential of PDT approaches, but many aspects such as bio-distribution of the photosensitizers, penetration through tissues, and clearance are not taken into account. In vivo studies are therefore essential to assess the influence of such factors, in order to gain more insights into the therapeutic potential of a treatment under development. This chapter describes the development of an orthotopic model of head and neck cancer, to which nanobody-targeted PDT is applied, and the therapeutic potential is assessed by immunohistochemistry one day after PDT.

Introduction: A challenge in the treatment of patients with head and neck cancer is the management of occult cervical lymph node (LN) metastases. Single-fiber reflectance (SFR) spectroscopy has the potential to detect physiological tissue changes that occur in a positive LN. This pilot study aimed to investigate whether SFR spectroscopy could serve as an alternative or additional technique to detect cervical lymph node metastases. Materials and Methods: We performed intraoperative SFR spectroscopy measurements of LNs with and without malignancies. We analyzed if physiological and scattering parameters were significantly altered in positive LNs. Results: Nine patients with a total of nineteen LNs were included. Three parameters, blood volume fraction (BVF), microvascular saturation (StO2), and Rayleigh amplitude, were significantly lower in positive LNs. They were combined into one optical parameter ‘delta’, using discriminant analysis. Delta was significantly decreased in positive LNs, p = 0,0006. It had a high diagnostic accuracy where the sensitivity, specificity, PPV, and NPV were 90,0%, 88.9%, 90,0%, and 88.9%, respectively. The area under the ROC curve was 96.7% (95% confidence interval 89.7–100.0%). Conclusion: This proof of principle study is a first step in the development of an SFR spectroscopy technique to detect LN metastases in real time. A next step towards this goal is replicating these results in LNs with smaller metastases and in a larger cohort of patients. This future study will combine SFR spectroscopy with fine-needle aspiration, using the same needle, to perform preoperative in vivo measurements.

Summary Patients with head and neck squamous cell carcinoma (HNSCC) have an increased risk of developing esophageal second primary tumors (ESPTs). We aimed to determine the incidence, stage, and outcome of synchronous ESPTs in patients with HNSCC in a Western population. We performed a prospective, observational, and cohort study. Patients diagnosed with HNSCC in the oropharynx, hypopharynx, any other sub-location in combination with alcohol abuse, or patients with two synchronous HNSCCs, between February 2019 and February 2020 underwent screening esophagogastroduodenoscopy (EGD). ESPT was defined as presence of esophageal squamous cell carcinoma (ESCC) or high grade dysplasia (HGD). Eighty-five patients were included. A lesion suspected for ESPT was detected in 14 of 85 patients, which was pathologically confirmed in five patients (1 ESCC and 4 HGD). The radiotherapy field was extended to the esophagus in two of five patients, HGD was treated with endoscopic resection in three of five patients. None of the ESPTs were detected on MRI and/or CT-scan prior to EGD. Of the remaining nine patients, three had low grade dysplasia on histology whereas the other six patients had benign lesions. Incidence of synchronous ESPT was 5.9% in our cohort of HNSCC patients. All ESPTs were diagnosed at an early stage and treated with curative intent. We recommend that screening for synchronous ESPTs should be considered in a selected group of patients with HNSCC.

The goal of head and neck oncological surgery is complete tumor resection with adequate resection margins while preserving acceptable function and appearance. For oral cavity squamous cell carcinoma (OCSCC), different studies showed that only 15%-26% of all resections are adequate. A major reason for the low number of adequate resections is the lack of information during surgery; the margin status is only available after the final histopathologic assessment, days after surgery. The surgeons and pathologists at the Erasmus MC University Medical Center in Rotterdam started the implementation of specimen-driven intraoperative assessment of resection margins (IOARM) in 2013, which became the standard of care in 2015. This method enables the surgeon to turn an inadequate resection into an adequate resection by performing an additional resection during the initial surgery. Intraoperative assessment is supported by a relocation method procedure that allows accurate identification of inadequate margins (found on the specimen) in the wound bed. The implementation of this protocol resulted in an improvement of adequate resections from 15%-40%. However, the specimen-driven IOARM is not widely adopted because grossing fresh tissue is counter-intuitive for pathologists. The fear exists that grossing fresh tissue will deteriorate the anatomical orientation, shape, and size of the specimen and therefore will affect the final histopathologic assessment. These possible negative effects are countered by the described protocol. Here, the protocol for specimen-driven IOARM is presented in detail, as performed at the institute.

Objectives: To compare the impact on survival and late radiation toxicity of patients with squamous cell carcinoma of unknown primary who were treated with ipsilateral neck dissection and ipsilateral postoperative radiotherapy (PORT) and patients treated with ipsilateral neck dissection and PORT to both sides of the neck plus the pharyngeal axis. Methods: Retrospective cohort study performed at the Erasmus University Medical Center in which 78 patients with squamous cell carcinoma of unknown primary were identified. Thirty-nine patients received PORT to both sides of the neck plus the pharyngeal axis (BILAX) and another 39 patients were irradiated on the ipsilateral neck (IPSI) only. The endpoints of the present study were 3-year overall survival (OS), 3-year disease-free survival (DFS), and overall late radiation toxicity. Results: The 3-year OS rate of the entire group of 78 patients was 74.2% and the 3-year DFS rate was 72.7%. The 3-year OS rates for the IPSI and the BILAX groups were 74.4% and 74.1%, respectively (P =.654). The most common late radiation toxicity experienced was xerostomia (64.8%), which was significantly more often seen in the BILAX group than in the IPSI group (83.8% and 44.1%, respectively, P =.001). Overall late radiation toxicity was significantly higher in the BILAX group (P =.003). Conclusion: There was no significant difference in OS and DFS rates between the IPSI and the BILAX group. Late radiation toxicity was significantly higher in the BILAX group. Level of Evidence: Level 2b: Individual retrospective cohort study.

Simple Summary Laryngeal cancer is a prevalent head and neck malignancy, with poor prognosis and low survival rates for patients with advanced disease. The recurrence rate for advanced laryngeal cancer is between 25 and 50%. In order to improve surgical resection of laryngeal cancer and reduce local recurrence rates, various intraoperative optical imaging techniques have been investigated. In this systematic review we identify these technologies, evaluating the current state and future directions of optical imaging for this indication. Evidently, the investigated imaging modalities are generally unsuitable for deep margin assessment, and, therefore, inadequate to guide resection in advanced laryngeal disease. We discuss two optical imaging techniques that can overcome these limitations and suggest how they can be used to achieve adequate margins in laryngeal cancer at all stages. Abstract Laryngeal cancer is a prevalent head and neck malignancy, with poor prognosis and low survival rates for patients with advanced disease. Treatment consists of unimodal therapy through surgery or radiotherapy in early staged tumors, while advanced stage tumors are generally treated with multimodal chemoradiotherapy or (total) laryngectomy followed by radiotherapy. Still, the recurrence rate for advanced laryngeal cancer is between 25 and 50%. In order to improve surgical resection of laryngeal cancer and reduce local recurrence rates, various intraoperative optical imaging techniques have been investigated. In this systematic review, we identify these technologies, evaluating the current state and future directions of optical imaging for this indication. Narrow-band imaging (NBI) and autofluorescence (AF) are established tools for early detection of laryngeal cancer. Nonetheless, their intraoperative utility is limited by an intrinsic inability to image beyond the (sub-)mucosa. Likewise, contact endoscopy (CE) and optical coherence tomography (OCT) are technically cumbersome and only useful for mucosal margin assessment. Research on fluorescence imaging (FLI) for this application is sparse, dealing solely with nonspecific fluorescent agents. Evidently, the imaging modalities that have been investigated thus far are generally unsuitable for deep margin assessment. We discuss two optical imaging techniques that can overcome these limitations and suggest how they can be used to achieve adequate margins in laryngeal cancer at all stages.

The purpose of this study was to assess whether the vascularisation of the meniscus could be visualised intra-operatively using near-infrared fluorescence (NIRF) imaging with indocyanine green (ICG) in patients undergoing total knee arthroplasty (TKA).The anterior horn (i.e., Cooper classification: zones C and D) of the meniscus that was least affected (i.e., least degenerative) was removed during TKA surgery in ten patients to obtain a cross section of the inside of the meniscus. Thereafter, 10 mg of ICG was injected intravenously, and vascularisation of the cross section of the meniscus was assessed using the Quest spectrum NIRF camera system. We calculated the percentage of patients in whom vascularisation was observed intra-operatively using NIRF imaging compared to immunohistochemistry.Meniscal vascularisation using NIRF imaging was observed in six out of eight (75%) patients in whom vascularisation was demonstrated with immunohistochemistry. The median extent of vascularisation was 13% (interquartile range (IQR) 3-28%) using NIRF imaging and 15% (IQR 11-23%) using immunohistochemistry.This study shows the potential of NIRF imaging to visualise vascularisation of the meniscus, as vascularisation was observed in six out of eight patients with histologically proven meniscal vascularisation.IV.

Background In head and neck oncological surgery the goal is to achieve a complete tumor resection with acceptable remaining function and appearance. For oral cavity squamous cell carcinoma (OCSCC) only 15% of the resections are reported as adequate. Since 2013, we have performed intraoperative assessment of resection margins (IOARM) in our institute, based on palpation and visual inspection of the resected specimens by pathologist and surgeon. This has resulted in an improvement of adequate resection margins from 15% to 50%, underlining the importance of IOARM. However, this method is subjective, labor intensive, and logistically challenging. Objective Our aim is to develop an objective method for fast and reliable IOARM based on Raman spectroscopy (RS). Methods RS is a non-destructive objective optical technique that provides information about the molecular composition of tissues. It can discriminate between healthy tissue and tumors. We developed a prototype Raman instrument employing a fiber-optic needle probe. The fiber-optic needle is driven into the OCSCC specimen, from the resection surface towards the tumor. Based on the Raman spectra collected along the insertion path, the location of the tumor border can be determined. From this the resection margin can be determined. Results First tests of the method show that the instrument accurately predicts the achieved resection margins. Per location the measurement and assessment takes 5 seconds. Conclusions This development signifies an important step towards a fast and objective IOARM. The fast measurement time enables an objective inspection of the margins achieved at a large number of locations of the resection surface.

Objectives: To define metastatic categories based on their prognostic significance. We hypothesized that oligometastasis in patients with head and neck squamous cell carcinoma (HNSCC) is associated with better post-distant metastasis disease specific survival (post-DM DSS) compared to patients with polymetastasis. Furthermore, the impact on survival of synchronous versus metachronous distant metastasis (DM) occurrence was assessed. Materials and methods: Retrospective cohort study in which patients with DM were stratified into three groups: oligometastasis (maximum of 3 metastatic foci in ≤2 anatomic sites), explosive metastasis (≥4 metastatic foci at one anatomic site) and explosive-disseminating metastasis (spread to ≥3 anatomic sites or >3 metastatic foci in 2 anatomic sites). In addition, patients were divided into synchronous versus metachronous DM. Results: Between January 1, 2006 and December 31, 2013, a total of 2687 patients with HNSCC were identified, of which 324 patients developed DM. In this group, 115 (35.5%) patients had oligometastasis, 64 (19.8%) patients had explosive metastasis and 145 (44.8%) patients had explosive-disseminating metastasis. Their median post-DM DSS were 4.7 months, 4.1 months and 1.7 months respectively (p

In oral cavity cancer only 15% of operations succeed in removing the whole tumor with the required margin of more than 5 mm of surrounding healthy tissue. This negatively affects prognosis. Clearly the hands and eyes of the surgeon do not suffice. We use a fiber optic needle probe for high-wavenumber Raman spectroscopic analysis of the freshly resected tissue to determine if the distance between the resection surface and the tumor is sufficient. A system is under development to inspect the resected tissue in under 15 minutes, while the patient is still in the OR.

Depth of invasion (DOI) is the most important predictor for lymph node metastasis (LNM) in early stage (T1-T2) oral cancer. The aim of this study is to validate the cut-off value of 4 mm on which the decision to perform an Elective Neck Dissection (END) is made.We performed a retrospective study in patients with pathologically proven early stage oral cavity squamous cell carcinoma (OCSCC) without clinical or radiological signs of LNM, who were treated between 2013 and 2018. An END was performed when DOI was ≥ 4 mm and a watchful waiting protocol was applied in patients with DOI 4 mm.Three hundred patients were included. END was performed in 77% of patients with DOI ≥ 4 mm, of which 36% had occult LNM (pN+). Patients in the watchful waiting group (48%) developed a regional recurrence in 5.2% for DOI 4 mm and 24.1% for DOI ≥ 4 mm. For DOI ≥ 4 mm, regional recurrence free survival was higher for patients who were treated with END compared to watchful waiting (p = 0.002). A Receiver-Operator-Curve -analysis showed that a DOI cut-off value of 4.0 mm was the optimal threshold for the prediction of occult LNM (95.1% sensitivity, 52.9% specificity).A DOI of ≥ 4 mm is an accurate cut-off value for performing an END in early stage OCSCC. END results in higher survival rates and lower regional recurrence rates in patients with DOI ≥ 4 mm.

Background Oral cavity squamous cell carcinoma (OCSCC) is the most frequent head and neck cancer. Surgery is the mainstay treatment for patients with OCSCC. In case of bone involvement, the affected bone needs to be resected. Cancer-free bone resection margins (BRMs) are of crucial importance: patients with cancer-free BRMs have a 2 times higher chance of survival. Currently, there is no standard method for intraoperative assessment of BRMs. Bone margin status is only known after tissue decalcification, which takes 1 to 2 weeks. After that time reoperations are highly undesirable, because the surgical defect has healed. Therefore, it is crucial to achieve tumor-free resection surfaces, which requires the possibility of intraoperative assessment of BRMs. Objective The aim of this study was to investigate the potential of Raman spectroscopy (RS) for detection of OCSCC in bone resection surfaces during mandibulectomy. RS is a nondestructive objective optical technique that provides information about the molecular composition of tissues. Methods Raman mapping experiments were performed on fresh mandible resection specimens from patients treated with mandibulectomy for OCSCC. A tumor detection algorithm was created based on water concentration and the high-wavenumber range (2800 cm−1-3050 cm−1) of the Raman spectra. Results Results show that RS can detect OCSCC in bone resection surfaces with a high sensitivity (96%) and specificity (83%; 26 mapping experiments, 22 patients). Conclusions These results form the basis for further development of an RS tool as an objective method for intraoperative assessment of BRMs.

Objectives Tumour-positive resection margins are a major problem during oral cancer surgery. gGlu-HMRG is a tracer that becomes fluorescent upon activation by gamma-glutamyltranspeptidase (GGT). This study aims to investigate the combination of gGlu-HMRG and a clinical fluorescence imaging system for the detection of tumour-positive resection margins. Materials and methods The preclinical Maestro and clinical Artemis imaging systems were compared in vitro and ex vivo with cultured human head and neck cancer cells (OSC19, GGT-positive; and FaDu, GGT negative) and tumour-bearing nude mice. Subsequently, frozen sections of normal and oral cancer tissues were ex vivo sprayed with gGlu-HMRG to determine the sensitivity and specificity. Finally, resection margins of patients with suspected oral cancer were ex vivo sprayed with gGlu-HMRG to detect tumour-positive resection margins. Results Both systems could be used to detect gGlu-HMRG activation in vitro and ex vivo in GGT positive cancer cells. Sensitivity and specificity of gGlu-HMRG and the Artemis on frozen tissue samples was 80% and 87%, respectively. Seven patients undergoing surgery for suspected oral cancer were included. In three patients fluorescence was observed at the resection margin. Those margins were either tumour-positive or within 1 mm of tumour. The margins of the other patients were clear (≥8 mm). Conclusion This study demonstrates the feasibility to detect tumour-positive resection margins with gGlu-HMRG and a clinical fluorescence imaging system. Applying this technique would enable intraoperative screening of the entire resection margin and allow direct re-resection in case of tumour-positivity.

Purpose Hyperbaric oxygen therapy (HBOT) is used in the treatment of radiation-induced tissue injury but its effect on (residual) tumor tissue is indistinct and therefore investigated in this study. Procedures Orthotopic FaDu tumors were established in mice, and the response of the (irradiated) tumors to HBOT was monitored by bioluminescence imaging. Near infrared fluorescence imaging using AngioSense750 and Hypoxisense680 was applied to detect tumor vascular permeability and hypoxia. Results HBOT treatment resulted in accelerated growth of non-irradiated tumors, but mouse survival was improved. Tumor vascular leakiness and hypoxia were enhanced after HBOT, whereas histological characteristics, epithelial-to-mesenchymal transition markers, and metastatic incidence were not influenced. Conclusions Squamous cell carcinoma responds to HBOT with respect to tumor growth, vascular permeability, and hypoxia, which may have implications for its use in cancer patients. The ability to longitudinally analyze tumor characteristics highlights the versatility and potential of optical imaging methods in oncological research. Electronic supplementary material The online version of this article (doi:10.1007/s11307-015-0834-8) contains supplementary material, which is available to authorized users.

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 1 nmol (150 μg) and 0.34 nmol (50 μg) dose groups (n = 4, p

During the European Molecular Imaging Meeting (EMIM) 2013, the fluorescence-guided surgery study group held its inaugural session to discuss the clinical implementation of fluorescence-guided surgery. The general aim of this study group is to discuss and identify the steps required to successfully and safely bring intraoperative fluorescence imaging to the clinics. The focus group intends to use synergies between interested groups as a tool to address regulatory and implementation hurdles in Europe and operates within the intraoperative focus group of the World Molecular Imaging Society (WMIS) that promotes the same interests at the WMIS level. The major topics on the critical path of implementation identified within the study group were quality controls and standards for ensuring accurate imaging and the ability to compare results from different studies, regulatory affairs, and strategies to increase awareness among physicians, regulators, insurance companies, and a broader audience. These hurdles, and the possible actions discussed to overcome them, are summarized in this report. Furthermore, a number of recommendations for the future shape of the fluorescence-guided study group are discussed. A main driving conclusion remains that intraoperative imaging has great clinical potential and that many of the solutions required are best addressed with the community working together to optimally promote and accelerate the clinical implementation of fluorescence imaging towards improving surgical procedures.

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.

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.

Navigation with fluorescence guidance has emerged in the last decade as a promising strategy to improve the efficacy of oncologic surgery. To achieve routine clinical use, the onus is on the surgical community to objectively assess the value of this technique. This assessment may facilitate both Food and Drug Administration approval of new optical imaging agents and reimbursement for the imaging procedures. It is critical to characterize fluorescence-guided procedural benefits over existing practices and to elucidate both the costs and the safety risks. This report is the result of a meeting of the International Society of Image Guided Surgery (www.isigs.org) on February 6, 2015, in Miami, Florida, and reflects a consensus of the participants' opinions. Our objective was to critically evaluate the imaging platform technology and optical imaging agents and to make recommendations for successful clinical trial development of this highly promising approach in oncologic surgery.

Purpose: Antiangiogenic therapy, mostly targeting VEGF, has been applied in cancer patients for the last decade. However, resistance to anti-VEGF therapy and/or no significant benefit as monotherapeutic agent is often observed. Therefore, new antiangiogenic strategies are needed. In the current study, we investigated the therapeutic effect of interfering with the bone morphogenetic protein (BMP)9/activin receptor–like kinase (ALK)1 signaling pathway by using an ALK1-Fc ligand trap. Experimental Design: We analyzed the potential antiangiogenic and antitumor effects of ALK1-Fc protein as monotherapy and in combination with chemotherapy in vivo in mouse models of melanoma, head and neck cancer, and invasive lobular breast carcinomas. ALK1-Fc sequesters BMP9 and 10 and prevents binding of these ligands to endothelial ALK1, which regulates angiogenesis. Results: Treatment of mice with ALK1-Fc strongly decreased the tumors' microvascular density in the three different mouse cancer models. However, this effect was not accompanied by a reduction in tumor volume. An immunohistochemical analysis of the tumor samples revealed that ALK1-Fc treatment increased the pericyte coverage of the remaining tumor vessels and decreased the hypoxia within the tumor. Next, we observed that combining ALK1-Fc with cisplatin inhibited tumor growth in the breast and head and neck cancer models more efficiently than chemotherapy alone. Conclusions: The addition of ALK1-Fc to the cisplatin treatment was able to enhance the cytotoxic effect of the chemotherapy. Our results provide strong rationale to explore combined targeting of ALK1 with chemotherapy in a clinical setting, especially in the ongoing phase II clinical trials with ALK1-Fc. Clin Cancer Res; 22(1); 96–106. ©2015 AACR.

Background In oral cancer surgery, intraoperative optical imaging could help the surgeon to determine adequate tumor-free margins. Methods Tumor-specific near-infrared fluorescence agents targeting epidermal growth factor receptor (CW800 EGF) or glucose transporter system (CW800 2-DG) were administered to mice with tongue carcinoma and cervical lymph node metastases. Tumor growth was followed by bioluminescence imaging. Fluorescence signals were compared with a control group of healthy animals. Results Significantly higher fluorescence was found in tongue tumors and cervical lymph node metastases compared with that in control animals. Fluorescence correlated with histopathology. Tumor-to-background ratio of CW800 EGF in the tongue was 13.8 (SD = 6.1) and in the lymph nodes 15.7 (SD = 8.8). For CW800 2-DG, the tumor-to-background ratio in the tongue was 4.6 (SD = 2.1) and in the lymph nodes 33.9 (SD = 18.4). Conclusions Optical imaging can be used to detect oral cancer and cervical lymph node metastases and could potentially improve complete surgical resection by real-time image-guided surgery. © 2011 Wiley Periodicals, Inc. Head Neck, 2012

Background and Objectives Near-infrared (NIR) fluorescence optical imaging is a promising technique to assess the tumor margins during cancer surgery. This technique requires targeting by specific fluorescence agents to differentiate tumor from normal surrounding tissue. We assessed the feasibility of cancer detection using NIR fluorescence agents that target either αvβ3 integrins or the enhanced permeability and retention (EPR) effect in an orthotopic mouse model of oral cancer. Methods Binding of the integrin-targeted agent to tumor cells was assessed in vitro. Oral cancer was induced in 6 BALB/c nu/nu mice by submucosal inoculation of human OSC19-luc cells into the tongue. Tumor growth was followed with bioluminescence imaging. A combination of agents targeting integrins or EPR effect was injected followed by fluorescence imaging in vivo and ex vivo after resection of the tongues. Results Oral cancer was clearly demarcated in vitro; in vivo; and on histological analysis with sufficient tumor-to-background ratios of the contrast agents. Conclusion This study demonstrates the feasibility of optical imaging of oral squamous cell carcinoma based on targeting of αvβ3 integrins and the EPR effect. Once these NIR fluorescence agents become available for clinical testing, optical image-guided surgery could reduce residual disease after oral cancer surgery. J. Surg. Oncol. 2012; 105:714–718. © 2011 Wiley Periodicals, Inc.

Chemoradiation (CRT) is a valuable treatment option for advanced hypopharyngeal squamous cell cancer (HSCC). However, long-term toxicity and quality of life (QOL) is scarcely reported. Therefore, efficacy, acute and long-term toxic effects, and long-term QOL of CRT for advanced HSCC were evaluated,using retrospective study and post-treatment quality of life questionnaires. in a tertiary hospital setting. Analysis was performed of 73 patients that had been treated with CRT. Toxicity was rated using the CTCAE score list. QOL questionnaires EORTC QLQ-C30, QLQ-H&N35, and VHI were analyzed. The most common acute toxic effects were dysphagia and mucositis. Dysphagia and xerostomia remained problematic during long-term follow-up. After 3 years, the disease-specific survival was 41%, local disease control was 71%, and regional disease control was 97%. The results indicated that CRT for advanced HSCC is associated with high locoregional control and disease-specific survival. However, significant acute and long-term toxic effects occur, and organ preservation appears not necessarily equivalent to preservation of function and better QOL.

In cancer surgery, intra-operative assessment of the tumor-free margin, which is critical for the prognosis of the patient, relies on the visual appearance and palpation of the tumor. Optical imaging techniques provide real-time visualization of the tumor, warranting intra-operative image-guided surgery. Within this field, imaging in the near-infrared light spectrum offers two essential advantages: increased tissue penetration of light and an increased signal-to-background-ratio of contrast agents. In this article, we review the various techniques, contrast agents, and camera systems that are currently used for image-guided surgery. Furthermore, we provide an overview of the wide range of molecular contrast agents targeting specific hallmarks of cancer and we describe perspectives on its future use in cancer surgery.