Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma: Association with Metabolic Tumor Burden Determined with FDG-PET/CT.

BACKGROUND: The detection of circulating tumor DNA (ctDNA) with next-generation sequencing (NGS) in venous blood is a promising tool for the genomic profiling of head and neck squamous cell carcinoma (HNSCC). The association between ctDNA findings and metabolic tumor burden detected with FDG-PET/CT imaging is of particular interest for developing prognostic and predictive algorithms in HNSCC. METHODS: Twenty-six prospectively enrolled HNSCC patients were eligible for further analysis. All patients underwent tumor tissue and venous liquid biopsy sampling and FDG-PET/CT before definitive oncologic treatment. An NGS-based commercial panel was used for a genomic analysis of the samples. RESULTS: Maximum variant allele frequency (VAF) in blood correlated positively with whole-body (WB) metabolic tumor volume (MTV) and total lesion glycolysis (TLG) (r = 0.510, p = 0.008 and r = 0.584, p = 0.002, respectively). A positive liquid biopsy was associated with high WB-TLG using VAF ≥ 1.00% or ≥5.00% as a cut-off value (p = 0.006 or p = 0.003, respectively). Additionally, ctDNA detection was associated with WB-TLG when only concordant variants detected in both ctDNA and tissue samples were considered. CONCLUSIONS: A high metabolic tumor burden based on FDG imaging is associated with a positive liquid biopsy and high maximum VAF. Our findings suggest a complementary role of metabolic and genomic signatures in the pre-treatment evaluation of HNSCC.

Herpesvirus infections in adenoids in patients with chronic adenotonsillar disease.

Adenoids and tonsils have gained interest as a new in vivo model to study local immune functions and virus reservoirs. Especially herpesviruses are interesting because their prevalence and persistence in local lymphoid tissue are incompletely known. Our aim was to study herpesvirus and common respiratory virus infections in nonacutely ill adenotonsillar surgery patients. Adenoid and/or palatine tonsil tissue and nasopharyngeal aspirate (NPA) samples were collected from elective adenoidectomy (n = 45) and adenotonsillectomy (n = 44) patients (median age: 5, range: 1-20). Real-time polymerase chain reaction was used to detect 22 distinct viruses from collected samples. The overall prevalence of herpesviruses was 89% and respiratory viruses 94%. Human herpesviruses 6 (HHV6), 7 (HHV7), and Epstein-Barr virus (EBV) were found, respectively, in adenoids (33%, 26%, 25%), tonsils (45%, 52%, 23%), and NPA (46%, 38%, 25%). Copy numbers of the HHV6 and HHV7 genome were significantly higher in tonsils than in adenoids. Patients with intra-adenoid HHV6 were younger than those without. Detection rates of EBV and HHV7 showed agreement between corresponding sample types. This study shows that adenoid and tonsil tissues commonly harbor human herpes- and respiratory viruses, and it shows the differences in virus findings between sample types.

Persistent human bocavirus 1 infection and tonsillar immune responses.

BACKGROUND: Persistent human bocavirus 1 (HBoV1) infection is a common finding in patients suffering from chronic tonsillar disease. However, the associations between HBoV1 infection and specific immune reactions are not completely known. We aimed to compare in vivo expression of T-cell cytokines, transcription factors, and type I/III interferons in human tonsils between HBoV1-positive and -negative tonsillectomy patients. METHODS: Tonsil tissue samples, nasopharyngeal aspirate (NPA), and serum samples were obtained from 143 immunocompetent adult and child tonsillectomy patients. HBoV1 and 14 other respiratory viruses were detected in NPAs and tonsil tissues by polymerase chain reaction (PCR). Serology and semi-quantitative PCR were used for diagnosing HBoV1 infections. Expression of 14 cytokines and transcription factors (IFN-α, IFN-ß, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-ß, FOXP3, GATA3, RORC2, Tbet) was analyzed by quantitative reverse-transcription (RT)-PCR in tonsil tissues. RESULTS: HBoV1 was detected by PCR in NPA and tonsils from 25 (17%) study patients. Serology results indicated prior nonacute infections in 81% of cases. Tonsillar cytokine responses were affected by HBoV1 infection. The suppression of two transcription factors, RORC2 and FOXP3, was associated with HBoV1 infection (p < 0.05). Furthermore, intratonsillar HBoV1-DNA loads correlated negatively with IFN-λ family cytokines and IL-13. CONCLUSIONS: Our study shows distinctively decreased T-helper17 and T-regulatory type immune responses in local lymphoid tissue in HBoV1-positive tonsillectomy patients. HBoV1 may act as a suppressive immune modulator.

Repeatability of hypoxia dose painting by numbers based on EF5-PET in head and neck cancer.

BACKGROUND: Hypoxia dose painting is a radiotherapy technique to increase the dose to hypoxic regions of the tumour. Still, the clinical effect relies on the reproducibility of the hypoxic region shown in the medical image. 18F-EF5 is a hypoxia tracer for positron emission tomography (PET), and this study investigated the repeatability of 18F-EF5-based dose painting by numbers (DPBN) in head and neck cancer (HNC). MATERIALS AND METHODS: Eight HNC patients undergoing two 18F-EF5-PET/CT sessions (A and B) before radiotherapy were included. A linear conversion of PET signal intensity to radiotherapy dose prescription was employed and DPBN treatment plans were created using the image basis acquired at each PET/CT session. Also, plan A was recalculated on the image basis for session B. Voxel-by-voxel Pearson's correlation and quality factor were calculated to assess the DPBN plan quality and repeatability. RESULTS: The mean (SD) correlation coefficient between DPBN prescription and plan was 0.92 (0.02) and 0.93 (0.02) for sessions A and B, respectively, with corresponding quality factors of 0.02 (0.002) and 0.02 (0.003), respectively. The mean correlation between dose prescriptions at day A and B was 0.72 (0.13), and 0.77 (0.12) for the corresponding plans. A mean correlation of 0.80 (0.08) was found between plan A, recalculated on image basis B, and plan B. CONCLUSION: Hypoxia DPBN planning based on 18F-EF5-PET/CT showed high repeatability. This illustrates that 18F-EF5-PET provides a robust target for dose painting.

The FLUKA Monte Carlo code coupled with an OER model for biologically weighted dose calculations in proton therapy of hypoxic tumors.

INTRODUCTION: The increased radioresistance of hypoxic cells compared to well-oxygenated cells is quantified by the oxygen enhancement ratio (OER). In this study we created a FLUKA Monte Carlo based tool for inclusion of both OER and relative biological effectiveness (RBE) in biologically weighted dose (ROWD) calculations in proton therapy and applied this to explore the impact of hypoxia. METHODS: The RBE-weighted dose was adapted for hypoxia by making RBE model parameters dependent on the OER, in addition to the linear energy transfer (LET). The OER depends on the partial oxygen pressure (pO2) and LET. To demonstrate model performance, calculations were done with spread-out Bragg peaks (SOBP) in water phantoms with pO2 ranging from strongly hypoxic to normoxic (0.01-30 mmHg) and with a head and neck cancer proton plan optimized with an RBE of 1.1 and pO2 estimated voxel-by-voxel using [18F]-EF5 PET. An RBE of 1.1 and the Rørvik RBE model were used for the ROWD calculations. RESULTS: The SOBP in water had decreasing ROWD with decreasing pO2. In the plans accounting for oxygenation, the median target doses were approximately a factor 1.1 lower than the corresponding plans which did not consider the OER. Hypoxia adapted target ROWDs were considerably more heterogeneous than the RBE1.1-weighted doses. CONCLUSION: We realized a Monte Carlo based tool for calculating the ROWD. Read-in of patient pO2 and estimation of ROWD with flexibility in choice of RBE model was achieved, giving a tool that may be useful in future clinical applications of hypoxia-guided particle therapy.

Intratonsillar detection of 27 distinct viruses: A cross-sectional study.

Palatine tonsils have been observed to harbor several distinct respiratory and herpesviruses in separate studies. In this study, the presence of these viruses in palatine tonsils was comprehensively studied in both children and adults. A cross-sectional analysis of 181 patients (median age 22 years; range, 2.6-66) operated for a benign tonsillar disease was conducted. Real-time polymerase chain reaction was performed to detect 27 distinct viruses in all: eight human herpesviruses, 16 respiratory viruses, parvo B19, and polyoma BK/JC viruses. Clinical characteristics of the patients and underlying conditions were evaluated. In total, 92% of patients had virus detected in tonsils (Epstein-Barr virus 72%, human herpesvirus 7, and 6B 54% and 16%, respectively, enterovirus 18%, parvovirus B19 7% and the rest <4%). No herpes simplex virus 2, varicella zoster virus, polyoma JC virus, parainfluenza-, metapneumo-, or coronaviruses were found. Enterovirus was more common in children and was frequently observed in the presence of HHV6B. None of the viruses showed a positive association to the tonsillar disease. Respiratory symptoms were not associated with the prevalence of viruses. This study comprehensively reports a cross-sectional view of intratonsillar virus infections in elective tonsillectomy patients in a wide age range cohort. Tonsils are a major virus reservoir for distinct herpes and respiratory viruses without a positive association with tonsillar disease or respiratory symptoms.

Rhinovirus species and tonsillar immune responses.

BACKGROUND: Rhinovirus A and C infections are important contributors to asthma induction and exacerbations. No data exist on the interaction of local immune responses in rhinovirus infection. Therefore, we aimed to determine the tonsillar immune responses according to rhinovirus A, B and C infections. METHODS: We collected tonsillar samples, nasopharyngeal aspirates and peripheral blood from 42 rhinovirus positive tonsillectomy patients. Fifteen respiratory viruses or their types were investigated from nasopharynx and tonsil tissue, and rhinovirus species were typed. The expression of 10 cytokines and 4 transcription factors (IFN-α, IFN-ß, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-ß, FOXP3, GATA3, RORC2 and Tbet) were studied from tonsil tissue by quantitative PCR. A standard questionnaire of respiratory symptoms and health was filled by the patient or his/her guardian. The patients were divided into three groups by the determination of rhinovirus species. RESULTS: Overall, 16 patients had rhinovirus A, 12 rhinovirus B and 14 rhinovirus C infection. In rhinovirus B positive group there were significantly less men (P = 0.0072), less operated in spring (P = 0.0096) and more operated in fall (P = 0.030) than in rhinovirus A or C groups. Rhinovirus A positive patients had more respiratory symptoms (P = 0.0074) and particularly rhinitis (P = 0.036) on the operation day. There were no significant differences between the groups in virus codetection. In adjusted analysis, rhinovirus C infections were associated with increased IFN-α (P = 0.045) and decreased RORC2 expression (P = 0.025). CONCLUSIONS: Rhinovirus species associated differently with clinical characteristics and tonsillar cytokine responses.

Repeatability of tumour hypoxia imaging using [18F]EF5 PET/CT in head and neck cancer.

PURPOSE: Hypoxia contributes to radiotherapy resistance and more aggressive behaviour of several types of cancer. This study was designed to evaluate the repeatability of intratumour uptake of the hypoxia tracer [18F]EF5 in paired PET/CT scans. METHODS: Ten patients with newly diagnosed head and neck cancer (HNC) received three static PET/CT scans before chemoradiotherapy: two with [18F]EF5 a median of 7 days apart and one with [18F]FDG. Metabolically active primary tumour volumes were defined in [18F]FDG images and transferred to co-registered [18F]EF5 images for repeatability analysis. A tumour-to-muscle uptake ratio (TMR) of 1.5 at 3 h from injection of [18F]EF5 was used as a threshold representing hypoxic tissue. RESULTS: In 10 paired [18F]EF5 PET/CT image sets, SUVmean, SUVmax, and TMR showed a good correlation with the intraclass correlation coefficients of 0.81, 0.85, and 0.87, respectively. The relative coefficients of repeatability for these parameters were 15%, 17%, and 10%, respectively. Fractional hypoxic volumes of the tumours in the repeated scans had a high correlation using the Spearman rank correlation test (r = 0.94). In a voxel-by-voxel TMR analysis between the repeated scans, the mean of Pearson correlation coefficients of individual patients was 0.65. The mean (± SD) difference of TMR in the pooled data set was 0.03 ± 0.20. CONCLUSION: Pretreatment [18F]EF5 PET/CT within one week shows high repeatability and is feasible for the guiding of hypoxia-targeted treatment interventions in HNC.

Radiotherapy volume delineation using dynamic [18F]-FDG PET/CT imaging in patients with oropharyngeal cancer: a pilot study.

PURPOSE: Delineation of gross tumour volume in 3D is a critical step in the radiotherapy (RT) treatment planning for oropharyngeal cancer (OPC). Static [18F]-FDG PET/CT imaging has been suggested as a method to improve the reproducibility of tumour delineation, but it suffers from low specificity. We undertook this pilot study in which dynamic features in time-activity curves (TACs) of [18F]-FDG PET/CT images were applied to help the discrimination of tumour from inflammation and adjacent normal tissue. METHODS: Five patients with OPC underwent dynamic [18F]-FDG PET/CT imaging in treatment position. Voxel-by-voxel analysis was performed to evaluate seven dynamic features developed with the knowledge of differences in glucose metabolism in different tissue types and visual inspection of TACs. The Gaussian mixture model and K-means algorithms were used to evaluate the performance of the dynamic features in discriminating tumour voxels compared to the performance of standardized uptake values obtained from static imaging. RESULTS: Some dynamic features showed a trend towards discrimination of different metabolic areas but lack of consistency means that clinical application is not recommended based on these results alone. CONCLUSIONS: Impact of inflammatory tissue remains a problem for volume delineation in RT of OPC, but a simple dynamic imaging protocol proved practicable and enabled simple data analysis techniques that show promise for complementing the information in static uptake values.

Uptake of [18F]EF5 as a Tracer for Hypoxic and Aggressive Phenotype in Experimental Head and Neck Squamous Cell Carcinoma.

PURPOSE: This study aims to investigate whether the uptake of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide ([18F]EF5) and 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) is associated with a hypoxia-driven adverse phenotype in head and neck squamous cell carcinoma cell lines and tumor xenografts. METHODS: Xenografts were imaged in vivo, and tumor sections were stained for hypoxia-inducible factor 1α (Hif-1α), carbonic anhydrase IX (CA IX), and glucose transporter 1 (Glut-1). Tracer uptakes and the expression of Hif-1α were determined in cell lines under 1% hypoxia. RESULTS: High [18F]EF5 uptake was seen in xenografts expressing high levels of CA IX, Glut-1, and Hif-1α, whereas low [18F]EF5 uptake was detected in xenografts expressing low amounts of CA IX and Hif-1α. The uptake of [18F]EF5 between cell lines varied extensively under normoxic conditions. A clear correlation was found between the expression of Hif-1α and the uptake of [18F]FDG during hypoxia. CONCLUSIONS: The UT-SCC cell lines studied differed with respect to their hypoxic phenotypes, and these variations were detectable with [18F]EF5. Acute hypoxia increases [18F]FDG uptake in vitro, whereas a high [18F]EF5 uptake reflects a more complex phenotype associated with hypoxia and an aggressive growth pattern.

Evaluation of repeated [(18)F]EF5 PET/CT scans and tumor growth rate in experimental head and neck carcinomas.

BACKGROUND: Tumor hypoxia is linked to invasion and metastasis but whether this associates with tumor growth rate is not well understood. We aimed to study the relationship between hypoxia evaluated with the positron emission tomography (PET) tracer [(18)F]EF5 and tumor growth. Our second goal was to assess the variability in the uptake of [(18)F]EF5 in tumor between two scans. METHODS: Four human head and neck squamous cell carcinoma (UT-SCC) cell lines were xenografted in flank or neck of nude mice, and tumor size was closely monitored over the study period. The tumors were clearly visible when the first [(18)F]EF5 scan was acquired. After an exponential growth phase, the tumors were imaged again with [(18)F]EF5 and also with (18)F-fluorodeoxyglucose ([(18)F]FDG). RESULTS: There was a clear correlation between the percentage of tumor growth rate per day and the [(18)F]EF5 uptake in the latter scan (r = 0.766, p = 0.01). The uptake of [(18)F]EF5 in the first scan and the uptake of [(18)F]FDG did not significantly correlate with the tumor growth rate. We also observed considerable variations in the uptake of [(18)F]EF5 between the two scans. CONCLUSIONS: The uptake of [(18)F]EF5 in the late phase of exponential tumor growth is associated with the tumor growth rate in mice bearing HNC xenografts.

Radiation dosimetry and biodistribution of the hypoxia tracer (18)F-EF5 in oncologic patients.

UNLABELLED: The primary goals of this study were to determine the biodistribution and excretion of (18)F-EF5 in oncologic patients, to estimate the radiation-absorbed dose and to determine the safety of this drug. METHODS: Sixteen patients with histologically confirmed malignancy received a mean intravenous infusion of 217 MBq (range 107-364 MBq) of (18)F-EF5. Over a 4-6-hour period, four to five serial positron emission tomography (PET) or PET/computed tomography (CT) scans were obtained. To calculate the radiation dosimetry estimates, volumes of interest were drawn over the source organs for each PET scan or on the CT for each PET/CT scan. Serial blood samples were obtained to measure (18)F-EF5 blood clearance. Bladder-wall dose was calculated based on urine activity measurements. RESULTS: The urinary bladder received the largest radiation-absorbed dose, 0.12 ± 0.034 mSv/MBq (mean ± SD). The average effective dose equivalent and the effective dose of (18)F-EF5 were 0.021 ± 0.003 mSv/MBq and 0.018 ± 0.002 mSv/MBq, respectively. (18)F-EF5 was well tolerated in all subjects. CONCLUSIONS: (18)F-EF5 was demonstrated to be safe for patients, and the radiation exposure is clinically acceptable. As with any radiotracer with primary excretion in the urine, the bladder-wall dose can be minimized by active hydration and frequent voiding.

Parotidectomy in the treatment of pleomorphic adenoma -- analysis of long-term results.

CONCLUSION: Parotidectomy is an efficient surgical treatment modality for pleomorphic adenoma of the parotid gland, although some morbidity may occur. In this study, the median time interval between primary surgery and the presentation of the recurrent tumor was observed to be 14.4 years. OBJECTIVE: Analysis of the long-term results of patients undergoing lateral or total parotidectomy as first-line treatment of parotid pleomorphic adenoma at our institution between the years 1979 and 1996. METHODS: The individual patient charts of 230 patients were feasible for retrospective analysis in 2007. RESULTS: In all, 42 patients had dysfunction of the facial nerve after the primary surgery, but only 14 of them had permanent dysfunction. A recurrent tumor occurred in nine cases (3.9%). The time interval between primary surgery and the first recurrence ranged from 7.1 to 24.5 years. Recurrent tumors were treated with surgery, two patients received additional radiotherapy.