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PURPOSE Squamous cell carcinoma metastasis of the head and neck with unknown primary tumor (CUP) comprises a diagnostic challenge. Human papillomavirus (HPV) testing on cytologic specimens is gaining increasing focus as this may facilitate an early diagnosis of HPV-induced oropharyngeal carcinoma. This study aimed to prospectively assess PCR-based HPV-DNA testing on FNA smears in a clinical setting. METHODS Patients referred to a tertiary Head and Neck Cancer Center with suspected CUP were included from November 2016 to November 2018. Scraped cell material from FNA smears was analyzed for HPV-DNA with PCR using general primers (GP5 + /GP6 +) and correlated with the origin and histology of the primary tumor (oropharynx vs. outside oropharynx or benign tumor). The turn-around time reflecting the workflow for HPV-DNA testing by PCR was also calculated. RESULTS A total of 93 patients were enrolled in the study. The sensitivity and specificity were 86.7% [95% CI 75.4-94.1%] and 92.0% [95% CI 74.0-99.0%], and the positive and negative predictive values were 96.3% [95% CI 87.3-99.0%] and 74.2% [95% CI 59.9-84.7%], respectively. The turn-around time for HPV testing was a mean four calendar days. CONCLUSION HPV-DNA testing on FNA smears can be performed within a reasonable timeframe and can guide for the detection of an HPV-positive oropharyngeal primary tumor in the clinical setting for patients presenting with CUP of the head and neck.

Thyroid nodules are very common and constitute an increasing clinical challenge since improved imaging capabilities and utilisation have led to a higher number of incidental findings. Ultrasound-guided fine-needle aspiration biopsy (FNAB) is the standard diagnostic tool in the work-up of thyroid nodules suspected of malignancy. Non-diagnostic results remain common and require repeated FNAB, leading to increased costs and delayed treatment of thyroid diseases, including treatment of thyroid cancer. If cytological diagnoses cannot be achieved, surgery may be warranted, which may potentially lead to overtreatment. Optimisation of the FNAB procedure is therefore essential. Spinal needles with a stylet have been found to lead to fewer non-diagnostic results, but studies on the subject are few.This is a multicentre, two-arm, randomised clinical trial. Adults with thyroid nodules suspected of malignancy will be included consecutively. A total of 350 patients will be assigned randomly 1:1 to have a FNAB with either a spinal (25G) or a conventional (25G) needle. The primary outcome is the rate of diagnostic cytological samples according to the Bethesda system. Secondary outcomes are patient-experienced pain, complication rate and sensitivity and specificity.This trial will explore whether FNAB from thyroid nodules employing spinal needles compared with conventional fine needles improves diagnostic results, thereby providing evidence-based recommendations for a future choice of the FNAB needle. Secondary outcomes are patient-experienced pain, complication rate and sensitivity and specificity.This trial received funding from Erik and Susanna Olesens Fond. The funding source had no influence on trial design, data collection, analysis or publication.gov Identifier: NCT04879355. Registration date: 07032021; version: 29062022.

Background Calcium electroporation is a novel cancer treatment, which combines temporary cell permeability from electroporation with a high influx of calcium intracellularly resulting in cancer cell necrosis. Methods A phase I trial performing calcium electroporation on 6 patients suffering from recurrent head and neck cancer. In general anesthesia, intratumoral calcium injections were followed by electroporation. Safety was monitored by adverse events registration, serum Ca2+, ECG, and pain scores. Tumor response was measured on PET/MRI scans. Results Procedures were performed without complications. No serious adverse events, signs of hypercalcemia, or cardiac arrhythmias were observed. Two months post-treatment tumor responses on MRI: three partial responses, one stable disease, and two progression. Responses on PET: one partial metabolic disease, four with stable metabolic disease, and one not evaluable. One patient was without clinical evidence of disease after 12 months of observation. Conclusion Calcium electroporation is feasible and safe in head and neck tumors. Clinical responses were observed in three of six patients, warranting further studies. Level of evidence Level 4.

Oropharyngeal squamous cell carcinoma (OPSCC) induced by human papillomavirus (HPV) is highly prevalent in the western part of the world. Patients with HPV-induced OPSCC are often younger and have a much better prognosis than patients with HPV-negative OPSCC. The newly introduced transoral robotic surgery offers the ability to perform en bloc tumour resection with good margin control. It may be an alternative to the traditional radiation therapy for early-stage OPSCC. A Danish randomised clinical trial is now aiming at comparing these two treatments with a focus on long-term functional outcomes.

Background Calcium electroporation is a novel cancer treatment, which combines temporary cell permeability from electroporation with a high influx of calcium intracellularly resulting in cancer cell necrosis. Methods A phase I trial performing calcium electroporation on 6 patients suffering from recurrent head and neck cancer. In general anesthesia, intratumoral calcium injections were followed by electroporation. Safety was monitored by adverse events registration, serum Ca2+, ECG, and pain scores. Tumor response was measured on PET/MRI scans. Results Procedures were performed without complications. No serious adverse events, signs of hypercalcemia, or cardiac arrhythmias were observed. Two months post‐treatment tumor responses on MRI: three partial responses, one stable disease, and two progression. Responses on PET: one partial metabolic disease, four with stable metabolic disease, and one not evaluable. One patient was without clinical evidence of disease after 12 months of observation. Conclusion Calcium electroporation is feasible and safe in head and neck tumors. Clinical responses were observed in three of six patients, warranting further studies. Level of Evidence Level 4

Vascular Endothelial Growth Factor (VEGF) plays a prominent role in tumor angiogenesis and plasma VEGF concentration may carry prognostic information in colorectal cancer. The VEGF receptor 1 (VEGFR-1) is a regulatory receptor which is shredded into plasma of patients with colorectal cancer. For both molecules, large biological variation and lack of standardization of assay procedures are major challenges.We investigated pre-analytical, analytical, as well as short term and long term biological variation of plasma VEGF and VEGFR-1 in volunteers. In addition, we evaluated plasma VEGF and VEGFR-1 as markers of colorectal disease in a case-control study on four groups of 77 individuals undergoing bowel endoscopy. Groups were categorized as 'no findings', 'non-malignant findings', 'adenoma', or 'colorectal cancer'.In the studies on variation, temperature and delay before centrifugation significantly influenced plasma VEGF and, to a minor extent, plasma VEGFR-1 concentrations. In addition, we found large biological variations with CV up to 69.2% for VEGF and CV up to 35.9% for VEGFR-1. For both molecules the intra-subject variation exceeded the inter-subject variation. In the case control study neither plasma VEGF nor VEGFR-1 was able to differentiate between the four groups of individuals although plasma VEGFR-1 was significantly lower in patients with 'no findings'.There was no difference in plasma VEGF or VEGFR-1 between patients with no findings, benign disease, pre-malignant findings, and malignant findings after endoscopy. The poor discrimination between patients may be explained by the large inter- and intra-subject variations found for both molecules in volunteers.

Background: Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality in developed countries. It is known that early detection results in improved survival, and consequently there is a need for improved diagnostic tools in CRC. The plasma level of soluble urokinase plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer. Methods: In a case–control study comprising 308 patients undergoing endoscopical examination following CRC-related symptoms, 77 CRC patients with adenocarcinoma were age and gender matched to: 77 patients with adenomas; 77 with other non-malignant findings, and 77 with no findings. The different uPAR forms were measured in citrate plasma collected before endoscopical examination, using three different Time Resolved – Fluorescence Immuno Assays (TR-FIA's). Results: All soluble uPAR forms were found to be significantly higher in cancer patients than in patients presenting with other non-malignant findings; uPAR(I) P=0.0006, suPAR(I–III) P

BACKGROUNDThe liberated domain I of the urokinase plasminogen activator receptor [uPAR(I)] is a significant prognostic marker in lung and ovarian cancer, although the uPAR(I) concentration is below the limit of quantification (LOQ) in a substantial proportion of patient samples (Lung Cancer 2005;48:349–55; Clin Cancer Res 2008;14:5785–93; APMIS 2009;117:755–61). This study was undertaken to design an immunoassay with improved functional sensitivity for measuring uPAR(I) and to evaluate the prognostic value of uPAR(I) for colorectal cancer (CRC) patients.METHODSSurface plasmon resonance analysis identified 2 monoclonal antibodies, R3 and R20, that simultaneously bind to the liberated uPAR(I) but not to intact uPAR. We used R3 for capture and Eu-labeled R20 for detection in designing a 2-site sandwich time-resolved fluorescence immunoassay (TR-FIA 4) for measuring liberated uPAR(I). TR-FIA 4 was validated for use with citrated plasma. The prognostic value of the uPAR(I) concentration was evaluated in 298 CRC patients. The Cox proportional hazards model was used for the uni- and multivariate survival analyses.RESULTSThe LOQ was 0.65 pmol/L. Liberated uPAR(I) was measurable in all patient samples with TR-FIA 4. In the multivariate analysis that included sex, age, tumor stage, tumor localization, and adjuvant treatment, the uPAR(I) concentration measured with TR-FIA 4 (hazard ratio, 1.72; 95% CI, 1.15–2.57; P = 0.009), as well as the concentration of intact soluble uPAR plus the cleaved uPAR fragment containing domains II and III, tumor stage, and age were independent predictors of prognosis.CONCLUSIONSTR-FIA 4 has a functional sensitivity improved 4-fold over that of the previous uPAR(I) assay. The uPAR(I) concentration measured with TR-FIA 4 is an independent predictor of prognosis in CRC patients.

The levels of the soluble urokinase plasminogen activator receptor (suPAR) in blood have been shown to correlate with prognosis in various cancers. Plasma levels of the combined suPAR forms have previously shown to be a strong prognostic marker in the present cohort of CRC patients and could potentially identify high-risk patients among those with early stage disease. In order to investigate whether the individual suPAR forms are stronger prognostic markers than the combined amount we measured the different uPAR forms in serum from the same cohort and evaluated their prognostic significance.The different suPAR forms were measured in serum preoperatively collected from 518 patients. Patients were followed up to nine years (median 7.9 years) and the primary endpoint was overall survival. The different suPAR forms were measured using Time Resolved Fluorescence Immunoassays(TR-FIAs): Intact, suPAR(I-III) by TR-FIA 1; intact and cleaved, suPAR(I-III)+(II-III) by TR-FIA 2; and liberated uPAR(I) by TR-FIA 3.All three uPAR variants demonstrated prognostic significance when evaluated individually. In a multivariable analysis suPAR(I-III)+(II-III) and the liberated uPAR(I) were shown to be independent markers of prognosis (HR=1.74, CI:1.33-2.26; p0.0001 and HR=1.32; CI:1.02-1.71; p=0.036 respectively), and independent of the clinical baseline variables: age, gender, tumor stage and localization.This study demonstrated that suPAR(I-III)+(II-III) and the liberated uPAR(I) in serum are independent prognostic markers in CRC.

Colorectal cancer (CRC) accounts for about 1 million new cases per year worldwide and is the third most prevalent cancer in Europe, and the second most important in relation to cancer-specific death. Outcome in patients with CRC relates to the stage of disease at diagnosis. The staging is based on histopathological characteristics and has limitations especially concerning the prediction of prognosis for patients presenting with the intermediate tumour stages (II and III). A number of publications have suggested tissue inhibitor of metalloproteinases 1 (TIMP-1) as a new and valid prognostic marker in CRC. This chapter reviews the literature on TIMP-1 as a prognostic marker in CRC.

Every year colorectal cancer (CRC) is diagnosed in more than one million persons world-wide. Despite improved treatment modalities, only half of the patients will survive the next five years. Since...

Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) may be a valuable biological marker in Colorectal Cancer (CRC). However, prospective validation of TIMP-1 as a biological marker should include a series of pre-analytical considerations. TIMP-1 is stored in platelets, which may degranulate during collection and storage. The aim of this study was to evaluate the influence of platelet TIMP-1 contamination on plasma TIMP-1 levels in healthy volunteers.All four parts of this study were done on EDTA-plasma. 1: The effect of stasis was evaluated in plasma collected with and without tourniquet. The collected whole blood was centrifuged at three different g-values. The effect of cellular contamination was evaluated 2: by adding plasma from just above the buffy-coat to one of four tubes containing plasma from the same sample and 3: by separating the plasma into three layers: upper, middle and lower. 4: The effect of temperature was studied by collection and handling of corresponding samples on ice and at room temperature. Prior to analysis samples were stored at -80 degrees C. TIMP-1 was determined using a validated in-house ELISA.1: TIMP-1 levels in plasma collected with or without stasis were not significantly different. Similarly TIMP-1 levels were not affected by the studied differences in centrifugation force. 2: TIMP-1 levels were significantly increased in plasma potentially contaminated with platelets (p0.0001). 3: Separation of plasma into an upper, middle and lower layer did not affect the levels of plasma TIMP-1. 4: Samples kept at room temperature following collection showed significantly higher plasma TIMP-1 levels than samples kept on ice (p0.0001).Contamination with platelets during handling and storage of plasma may have significant effect on TIMP-1 levels. The results can define a standard operating procedure for sample collection and handling, which is important in obtaining uniform, comparable and reproducible plasma TIMP-1 levels.