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.

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.

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).

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.

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.

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.

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.

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.

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.

Real-time fluorescence imaging in intraoperative decision making for cancer surgery.

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.

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.

Case report: traumatic displacement of a cochlear implant magnet.

OBJECTIVES: To date, over 200 000 cochlear implants (CIs) have been implanted worldwide and the incidence is still increasing. We present a case of traumatic displacement of CI magnet to raise awareness about this complication and to highlight the need for vigilance during surgery as well as for proper counseling. METHODS: The clinical presentation of a 1.5-year-old boy with a traumatic displacement of a CI magnet was presented and the literature was reviewed for this rare complication. RESULTS: After minor head injury, the sound processor could no longer connect to the CI. X-ray imaging demonstrated displacement of the CI magnet. During revision surgery, the magnet was replaced by a new magnet in the silicon holding cap. Intraoperative impedance measurements were normal and the CI was successfully activated 4 weeks postoperatively. CONCLUSIONS: Clinicians and patients should be aware of the risk of displacement of the CI magnet after (minor) head injury. Young boys tend to have a higher risk for this complication.

Transcutaneous sentinel lymph node detection in skin melanoma with near-infrared fluorescence imaging using indocyanine green.

The aim of the study is to assess whether indocyanine green (ICG) fluorescence can replace technetium in the preoperative detection of sentinel lymph nodes (SLN) from cutaneous melanoma. The current golden standard for SLN detection is the radioisotope technetium. A promising alternative is fluorescence imaging (FLI) using ICG. In this study, we enrolled patients undergoing sentinel lymph node biopsy (SLNB) for skin melanoma at the Erasmus Medical Center between November 2022 and July 2023. The SLNB procedure was performed as a standard of care. After general anesthesia, ICG was injected intradermally around the primary tumor site. Both the patient and the surgeon were not blinded for the location of the SLN. FLI was performed before incision, in vivo after incision, and ex vivo. Fluorescent SLNs were confirmed using the gamma probe in all cases. Thirty-two patients were included in this study, and a total of 39 SLNs were harvested. The transcutaneous detection rate of ICG was 21.9%. The combined ex vivo ICG fluorescence and technetium uptake was 94.9%. One SLN contained only ICG (2.6%) and one SLN contained only technetium-uptake (2.6%). FLI using ICG resulted in a relatively low transcutaneous detection, which means that exclusive use of this technique in its present form is not feasible. However, we did find a high accumulation of ICG in the SLN, indicating the potential of ICG in combination with other imaging techniques.

Micron-resolution fiber mapping in histology independent of sample preparation.

Detailed knowledge of the brain's nerve fiber network is crucial for understanding its function in health and disease. However, mapping fibers with high resolution remains prohibitive in most histological sections because state-of-the-art techniques are incompatible with their preparation. Here, we present a micron-resolution light-scattering-based technique that reveals intricate fiber networks independent of sample preparation for extended fields of view. We uncover fiber structures in both label-free and stained, paraffin-embedded and deparaffinized, newly-prepared and archived, animal and human brain tissues - including whole-brain sections from the BigBrain atlas. We identify altered microstructures in demyelination and hippocampal neurodegeneration, and show key advantages over diffusion magnetic resonance imaging, polarization microscopy, and structure tensor analysis. We also reveal structures in non-brain tissues - including muscle, bone, and blood vessels. Our cost-effective, versatile technique enables studies of intricate fiber networks in any type of histological tissue section, offering a new dimension to neuroscientific and biomedical research.

Tumour Marker Expression in Head and Neck Malignancies to Identify Potential Targets for Intraoperative Molecular Near-Infrared Imaging.

BACKGROUND: Oral and laryngeal squamous cell carcinoma (OSCC and LSCC) and papillary thyroid carcinoma (PTC) are common head and neck cancers (HNCs) typically treated surgically. Challenges in tumour delineation often lead to inadequate resection margins in OSCC and LSCC, and missed multifocality in PTC. Fluorescence imaging (FLI) using near-infrared tumour-targeting tracers may improve intraoperative identification of malignancy, facilitating precise excision. This study evaluates six potential FLI targets in OSCC, LSCC and PTC. MATERIALS AND METHODS: Immunohistochemical staining was performed on OSCC (n = 20), LSCC (n = 10) and PTC (n = 10), assessing CEA, c-Met, EpCAM, EGFR, integrin αvß6 and VEGF-α. Expression was scored (0-12) using the total immunostaining score (TIS) system, and categorized into absent (TIS 0), low (TIS 1-5), moderate (TIS 6-8) or high (TIS 9-12). RESULTS: Integrin αvß6 showed significant overexpression in OSCC (TIS: 12; p < 0.001) and LSCC (TIS: 8; p = 0.002), with 80% of OSCC and 90% of LSCC exhibiting moderate-high expression. Similarly, EGFR expression was moderate-high in most OSCC (87.5%; TIS: 8) and universally high in LSCC (100%; TIS: 12). In PTC, EGFR and VEGF-α expressions were low-moderate, but significantly higher than in healthy tissue (TIS: 6; p < 0.006). CONCLUSION: This study highlights integrin αvß6 and EGFR as viable FLI targets in OSCC and LSCC, especially integrin αvß6 for tumour margin delineation. In PTC, despite lower expressions, the significant overexpression of VEGF-α, c-MET, and EGFR suggests their potential as FLI targets. Our findings support the development of tumour-targeted FLI tracers to improve surgical precision in HNC.

Indocyanine green near-infrared fluorescence bowel perfusion assessment to prevent anastomotic leakage in minimally invasive colorectal surgery (AVOID): a multicentre, randomised, controlled, phase 3 trial.

BACKGROUND: Anastomotic leakage is a severe postoperative complication in colorectal surgery and compromised bowel perfusion is considered a major contributing factor. Conventional methods to assess bowel perfusion have a low predictive value for anastomotic leakage. We therefore aimed to evaluate the efficacy of real-time assessment with near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) in the prevention of anastomotic leakage. METHODS: This multicentre, randomised, controlled, phase 3 trial was done in eight hospitals in the Netherlands. We included adults (aged >18 years) who were scheduled for laparoscopic or robotic colorectal surgery (with planned primary anastomosis) for benign and malignant diseases. Preoperatively, patients were randomly assigned (1:1) to fluorescence-guided bowel anastomosis (FGBA) or conventional bowel anastomosis (CBA) by variable block randomisation (block sizes 4, 6, and 8) and stratified by site. The operating surgeon and investigators analysing the data were not masked to group assignment. Patients were unmasked after the surgical procedure or after study end. In the FGBA group, surgeons marked anastomosis levels per conventional perfusion assessment and then administered 5 mg of ICG by 2 mL intravenous bolus. They assessed bowel perfusion using NIR fluorescence imaging and adjusted (or kept) transection lines accordingly. Only conventional methods for bowel perfusion assessment were used in the CBA group. The primary outcome was the difference in the rate of clinically relevant anastomotic leakage (ie, requiring active therapeutic intervention but manageable without reoperation [grade B] or requiring reoperation [grade C], per the International Study Group of Rectal Cancer) between the FGBA group and the CBA group within 90 days post-surgery. The primary outcome and safety were assessed in the intention-to-treat population. This study was registered with ToetsingOnline.nl (NL7502) and ClinicalTrials.gov (NCT04712032) and is complete. FINDINGS: Between July 2, 2020, and Feb 21, 2023, 982 patients were enrolled, of whom 490 were assigned to FGBA and 492 were assigned to CBA. After excluding 51 patients, the intention-to-treat population comprised 931 (463 assigned FGBA and 468 assigned CBA). Patients had a median age of 68·0 years (IQR 59·0-75·0) and 485 (52%) were male and 446 (48%) were female. Ethnicity data were not available. The overall 90-day rate of clinically relevant anastomotic leakage was not significantly different between the FGBA group (32 [7%] of 463 patients) and the CBA group (42 [9%] of 468 patients; relative risk 0·77 [95% CI 0·50-1·20]; p=0·24). No adverse events related to ICG use were observed. 313 serious adverse events in 229 (25%) patients were at 90-day follow-up (159 serious adverse events in 113 [24%] patients in the FGBA group and 154 serious adverse events in 116 [25%] patients in the CBA group). 18 (2%) people died by 90 days (ten in the FGBA group and eight in the CBA group). INTERPRETATION: ICG NIR fluorescence imaging did not reduce 90-day anastomotic leakage rates in this trial across all types of colorectal surgeries. Further research should be done in subgroups, such as rectosigmoid resections, for which evidence suggests ICG NIR might be beneficial. FUNDING: Olympus Medical, Diagnostic Green, and Intuitive Foundation.

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.