Development of an Ex Vivo Functional Assay for Prediction of Irradiation Related Toxicity in Healthy Oral Mucosa Tissue.

Radiotherapy in the head-and-neck area is one of the main curative treatment options. However, this comes at the cost of varying levels of normal tissue toxicity, affecting up to 80% of patients. Mucositis can cause pain, weight loss and treatment delays, leading to worse outcomes and a decreased quality of life. Therefore, there is an urgent need for an approach to predicting normal mucosal responses in patients prior to treatment. We here describe an assay to detect irradiation responses in healthy oral mucosa tissue. Mucosa specimens from the oral cavity were obtained after surgical resection, cut into thin slices, irradiated and cultured for three days. Seven samples were irradiated with X-ray, and three additional samples were irradiated with both X-ray and protons. Healthy oral mucosa tissue slices maintained normal morphology and viability for three days. We measured a dose-dependent response to X-ray irradiation and compared X-ray and proton irradiation in the same mucosa sample using standardized automated image analysis. Furthermore, increased levels of inflammation-inducing factors-major drivers of mucositis development-could be detected after irradiation. This model can be utilized for investigating mechanistic aspects of mucositis development and can be developed into an assay to predict radiation-induced toxicity in normal mucosa.

Uncovering microstructural architecture from histology.

Microstructural tissue organization underlies the complex connectivity of the brain and controls properties of connective, muscle, and epithelial tissue. However, discerning microstructural architecture with high resolution for large fields of view remains prohibitive. We address this challenge with computational scattered light imaging (ComSLI), which exploits the anisotropic light scattering of aligned structures. Using a rotating lightsource and a high-resolution camera, ComSLI determines fiber architecture with micrometer resolution from histological sections across preparation and staining protocols. We show complex fiber architecture in brain and non-brain sections, including histological paraffin-embedded sections with various stains, and demonstrate its applicability on animal and human tissue, including disease cases with altered microstructure. ComSLI opens new avenues for investigating fiber architecture in new and archived sections across organisms, tissues, and diseases.

Intraoperative molecular imaging of colorectal lung metastases with SGM-101: a feasibility study.

PURPOSE: Metastasectomy is a common treatment option for patients with colorectal lung metastases (CLM). Challenges exist with margin assessment and identification of small nodules, especially during minimally invasive surgery. Intraoperative fluorescence imaging has the potential to overcome these challenges. The aim of this study was to assess feasibility of targeting CLM with the carcinoembryonic antigen (CEA) specific fluorescent tracer SGM-101. METHODS: This was a prospective, open-label feasibility study. The primary outcome was the number of CLM that showed a true positive fluorescence signal with SGM-101. Fluorescence positive signal was defined as a signal-to-background ratio (SBR) ≥ 1.5. A secondary endpoint was the CEA expression in the colorectal lung metastases, assessed with the immunohistochemistry, and scored by the total immunostaining score. RESULTS: Thirteen patients were included in this study. Positive fluorescence signal with in vivo, back table, and closed-field bread loaf imaging was observed in 31%, 45%, and 94% of the tumors respectively. Median SBRs for the three imaging modalities were 1.00 (IQR: 1.00-1.53), 1.45 (IQR: 1.00-1.89), and 4.81 (IQR: 2.70-7.41). All tumor lesions had a maximum total immunostaining score for CEA expression of 12/12. CONCLUSION: This study demonstrated the potential of fluorescence imaging of CLM with SGM-101. CEA expression was observed in all tumors, and closed-field imaging showed excellent CEA specific targeting of the tracer to the tumor nodules. The full potential of SGM-101 for in vivo detection of the tracer can be achieved with improved minimal invasive imaging systems and optimal patient selection. TRIAL REGISTRATION: The study was registered in ClinicalTrial.gov under identifier NCT04737213 at February 2021.

Raman spectroscopy to discriminate laryngeal squamous cell carcinoma from non-cancerous surrounding tissue.

As for many solid cancers, laryngeal cancer is treated surgically, and adequate resection margins are critical for survival. Raman spectroscopy has the capacity to accurately differentiate between cancer and non-cancerous tissue based on their molecular composition, which has been proven in previous work. The aim of this study is to investigate whether Raman spectroscopy can be used to discriminate laryngeal cancer from surrounding non-cancerous tissue. Patients surgically treated for laryngeal cancer were included. Raman mapping experiments were performed ex vivo on resection specimens and correlated to histopathology. Water concentration analysis and CH-stretching region analysis were performed in the high wavenumber range of 2500-4000 cm-1. Thirty-four mapping experiments on 22 resection specimens were used for analysis. Both laryngeal cancer and all non-cancerous tissue structures showed high water concentrations of around 75%. Discriminative information was only found to be present in the CH-stretching region of the Raman spectra of the larynx (discriminative power of 0.87). High wavenumber region Raman spectroscopy can discriminate laryngeal cancer from non-cancerous tissue structures. Contrary to the findings for oral cavity cancer, water concentration is not a discriminating factor for laryngeal cancer.

Intraoperative assessment of resection margins by Raman spectroscopy to guide oral cancer surgery.

Patients with oral cavity cancer are almost always treated with surgery. The goal is to remove the tumor with a margin of more than 5 mm of surrounding healthy tissue. Unfortunately, this is only achieved in about 15% to 26% of cases. Intraoperative assessment of tumor resection margins (IOARM) can dramatically improve surgical results. However, current methods are laborious, subjective, and logistically demanding. This hinders broad adoption of IOARM, to the detriment of patients. Here we present the development and validation of a high-wavenumber Raman spectroscopic technology, for quick and objective intraoperative measurement of resection margins on fresh specimens. It employs a thin fiber-optic needle probe, which is inserted into the tissue, to measure the distance between a resection surface and the tumor. A tissue classification model was developed to discriminate oral cavity squamous cell carcinoma (OCSCC) from healthy oral tissue, with a sensitivity of 0.85 and a specificity of 0.92. The tissue classification model was then used to develop a margin length prediction model, showing a mean difference between margin length predicted by Raman spectroscopy and histopathology of -0.17 mm.

Endoscopic screening of the upper gastrointestinal tract for second primary tumors in patients with head and neck cancer in a Western country.

BACKGROUND: Patients with head and neck squamous cell carcinoma (HNSCC) can develop second primary tumors (SPTs) in the esophagus. Endoscopic screening could lead to detection of SPTs at early stages and improve survival. METHODS: We performed a prospective endoscopic screening study in patients with curably treated HNSCC diagnosed between January 2017-July 2021 in a Western country. Screening was performed synchronously (< 6 months) or metachronously (≥ 6 months) after HNSCC diagnosis. Routine imaging for HNSCC consisted of flexible transnasal endoscopy with positron emission tomography/computed tomography or magnetic resonance imaging, depending on primary HNSCC location. The primary outcome was prevalence of SPTs, defined as presence of esophageal high grade dysplasia or squamous cell carcinoma. RESULTS: 202 patients (mean age 65 years, 80.7 % male) underwent 250 screening endoscopies. HNSCC was located in the oropharynx (31.9 %), hypopharynx (26.9 %), larynx (22.2 %), and oral cavity (18.5 %). Endoscopic screening was performed within 6 months (34.0 %), 6 months to 1 year (8.0 %), 1-2 years (33.6 %), and 2-5 years (24.4 %) after HNSCC diagnosis. We detected 11 SPTs in 10 patients (5.0 %, 95 %CI 2.4 %-8.9 %) during synchronous (6/85) and metachronous (5/165) screening. Most patients had early stage SPTs (90 %) and were treated with curative intent with endoscopic resection (80 %). No SPTs in screened patients were detected with routine imaging for HNSCC before endoscopic screening. CONCLUSION: In 5 % of patients with HNSCC, an SPT was detected with endoscopic screening. Endoscopic screening should be considered in selected HNSCC patients to detect early stage SPTs, based on highest SPT risk and life expectancy according to HNSCC and comorbidities.

Quantification of indocyanine green near-infrared fluorescence bowel perfusion assessment in colorectal surgery.

BACKGROUND: Indocyanine green near-infrared fluorescence bowel perfusion assessment has shown its potential benefit in preventing anastomotic leakage. However, the surgeon's subjective visual interpretation of the fluorescence signal limits the validity and reproducibility of the technique. Therefore, this study aimed to identify objective quantified bowel perfusion patterns in patients undergoing colorectal surgery using a standardized imaging protocol. METHOD: A standardized fluorescence video was recorded. Postoperatively, the fluorescence videos were quantified by drawing contiguous region of interests (ROIs) on the bowel. For each ROI, a time-intensity curve was plotted from which perfusion parameters (n = 10) were derived and analyzed. Furthermore, the inter-observer agreement of the surgeon's subjective interpretation of the fluorescence signal was assessed. RESULTS: Twenty patients who underwent colorectal surgery were included in the study. Based on the quantified time-intensity curves, three different perfusion patterns were identified. Similar for both the ileum and colon, perfusion pattern 1 had a steep inflow that reached its peak fluorescence intensity rapidly, followed by a steep outflow. Perfusion pattern 2 had a relatively flat outflow slope immediately followed by its plateau phase. Perfusion pattern 3 only reached its peak fluorescence intensity after 3 min with a slow inflow gradient preceding it. The inter-observer agreement was poor-moderate (Intraclass Correlation Coefficient (ICC): 0.378, 95% CI 0.210-0.579). CONCLUSION: This study showed that quantification of bowel perfusion is a feasible method to differentiate between different perfusion patterns. In addition, the poor-moderate inter-observer agreement of the subjective interpretation of the fluorescence signal between surgeons emphasizes the need for objective quantification.

HYpofractionated, dose-redistributed RAdiotherapy with protons and photons to combat radiation-induced immunosuppression in head and neck squamous cell carcinoma: study protocol of the phase I HYDRA trial.

BACKGROUND: Radiotherapy (RT) is the 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 (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 the 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 immunoradiotherapy 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, and 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 outcomes by allowing for future effective combination treatment with immunotherapy. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov; NCT05364411 (registered on May 6th, 2022).

Development of a near-infrared Raman spectroscopy setup compatible with fluorescence-guided surgery.

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.

Ex Vivo Functional Assay for Evaluating Treatment Response in Tumor Tissue of Head and Neck Squamous Cell Carcinoma.

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.

The role of patient characteristics and the effects of angiogenic therapies on the microvasculature of the meniscus: A systematic review.

BACKGROUND: Considerable interindividual variation in meniscal microvascularization has been reported. The purpose of this review was to identify which patient characteristics affect meniscal microvascularization and provide a structured overview of angiogenic therapies that influence meniscal neovascularization. METHODS: A systematic literature search was undertaken using PubMed, Embase, Web of Science, Cochrane library and Emcare from inception to November 2021. Studies reporting on (1) Patient characteristics that affect meniscal microvascularization, or (2) Therapies that induce neovascularization in meniscal tissue were included. Studies were graded in quality using the Anatomical Quality Assessment (AQUA) tool. The study was registered with PROSPERO(ID:CRD42021242479). RESULTS: Thirteen studies reported on patient characteristics and eleven on angiogenic therapies. The influence of Age, Degenerative knee, Gender, and Race was reported. Age is the most studied factor. The entire meniscus is vascularized around birth. With increasing age, vascularization decreases from the inner to the peripheral margin. Around 11 years, blood vessels are primarily located in the peripheral third of the menisci. There seems to be a further decrease in vascularization with increasing age in adults, yet conflicting literature exists. Degenerative changes of the knee also seem to influence meniscal vascularization, but evidence is limited. Angiogenic therapies to improve meniscal vascularization have only been studied in preclinical setting. The use of synovial flap transplantation, stem cell therapy, vascular endothelial growth factor, and angiogenin has shown promising results. CONCLUSION: To decrease failure rates of meniscal repair, a better understanding of patient-specific vascular anatomy is essential. Translational clinical research is needed to investigate the clinical value of angiogenic therapies.

Investigation of the Therapeutic Potential of Nanobody-Targeted Photodynamic Therapy in an Orthotopic Head and Neck Cancer Model.

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.

Intra-operative assessment of the vascularisation of a cross section of the meniscus using near-infrared fluorescence imaging.

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.

Fluorescence-guided surgery in colorectal cancer; A review on clinical results and future perspectives.

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.

Detecting head and neck lymph node metastases with white light reflectance spectroscopy; a pilot study.

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.

Screening for synchronous esophageal second primary tumors in patients with head and neck cancer.

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.

Dissemination patterns and chronology of distant metastasis affect survival of patients with head and neck squamous cell carcinoma.

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 < .001). Synchronous DM was associated with more favorable survival rates in univariable and multivariable analyses than metachronous DM with recurrence of the index tumor (6-month post-DM DSS probability of 0.51 vs 0.17, p < .001). CONCLUSION: Oligometastasis in HNSCC signifies a better prognosis than a polymetastatic pattern. Metachronous DM occurrence with recurrence of the primary index tumor is associated with an unfavorable prognosis.