Interobserver agreement on definition of the target volume in stereotactic radiotherapy for pancreatic adenocarcinoma using different imaging modalities.

PURPOSE: The aim of this study was to evaluate interobserver agreement (IOA) on target volume definition for pancreatic cancer (PACA) within the Radiosurgery and Stereotactic Radiotherapy Working Group of the German Society of Radiation Oncology (DEGRO) and to identify the influence of imaging modalities on the definition of the target volumes. METHODS: Two cases of locally advanced PACA and one local recurrence were selected from a large SBRT database. Delineation was based on either a planning 4D CT with or without (w/wo) IV contrast, w/wo PET/CT, and w/wo diagnostic MRI. Novel compared to other studies, a combination of four metrics was used to integrate several aspects of target volume segmentation: the Dice coefficient (DSC), the Hausdorff distance (HD), the probabilistic distance (PBD), and the volumetric similarity (VS). RESULTS: For all three GTVs, the median DSC was 0.75 (range 0.17-0.95), the median HD 15 (range 3.22-67.11) mm, the median PBD 0.33 (range 0.06-4.86), and the median VS was 0.88 (range 0.31-1). For ITVs and PTVs the results were similar. When comparing the imaging modalities for delineation, the best agreement for the GTV was achieved using PET/CT, and for the ITV and PTV using 4D PET/CT, in treatment position with abdominal compression. CONCLUSION: Overall, there was good GTV agreement (DSC). Combined metrics appeared to allow a more valid detection of interobserver variation. For SBRT, either 4D PET/CT or 3D PET/CT in treatment position with abdominal compression leads to better agreement and should be considered as a very useful imaging modality for the definition of treatment volumes in pancreatic SBRT. Contouring does not appear to be the weakest link in the treatment planning chain of SBRT for PACA.

Stereotactic radiosurgery and radiotherapy for resected brain metastases: current pattern of care in the Radiosurgery and Stereotactic Radiotherapy Working Group of the German Association for Radiation Oncology (DEGRO).

PURPOSE: Preoperative stereotactic radiosurgery (SRS) of brain metastases may achieve similar local control and better leptomeningeal control rates than postoperative fractionated stereotactic radiotherapy (FSRT) in patients treated with elective metastasectomy. To plan a multicentre trial of preoperative SRS compared with postoperative FSRT, a survey of experts was conducted to determine current practice. METHODS: A survey with 15 questions was distributed to the DEGRO Radiosurgery and Stereotactic Radiotherapy Working Group. Participants were asked under what circumstances they offered SRS, FSRT, partial and/or whole brain radiotherapy before or after resection of a brain metastasis, as well as the feasibility of preoperative stereotactic radiosurgery and neurosurgical resection within 6 days. RESULTS: Of 25 participants from 24 centres, 22 completed 100% of the questions. 24 respondents were radiation oncologists and 1 was a neurosurgeon. All 24 centres have one or more dedicated radiosurgery platform and all offer postoperative FSRT. Preoperative SRS is offered by 4/24 (16.7%) centres, and 9/24 (37.5%) sometimes recommend single-fraction postoperative SRS. Partial brain irradiation is offered by 8/24 (33.3%) centres and 12/24 (50%) occasionally recommend whole-brain irradiation. Two centres are participating in clinical trials of preoperative SRS. SRS techniques and fractionation varied between centres. CONCLUSION: All responding centres currently offer postoperative FSRT after brain metastasectomy. Approximately one third offer single-fraction postoperative SRS and four already perform preoperative SRS. With regard to potential co-investigators, 18 were identified for the PREOP­2 multicentre trial, which will randomise between preoperative SRS and postoperative FSRT.

Improving interinstitutional and intertechnology consistency of pulmonary SBRT by dose prescription to the mean internal target volume dose.

PURPOSE: Dose, fractionation, normalization and the dose profile inside the target volume vary substantially in pulmonary stereotactic body radiotherapy (SBRT) between different institutions and SBRT technologies. Published planning studies have shown large variations of the mean dose in planning target volume (PTV) and gross tumor volume (GTV) or internal target volume (ITV) when dose prescription is performed to the PTV covering isodose. This planning study investigated whether dose prescription to the mean dose of the ITV improves consistency in pulmonary SBRT dose distributions. MATERIALS AND METHODS: This was a multi-institutional planning study by the German Society of Radiation Oncology (DEGRO) working group Radiosurgery and Stereotactic Radiotherapy. CT images and structures of ITV, PTV and all relevant organs at risk (OAR) for two patients with early stage non-small cell lung cancer (NSCLC) were distributed to all participating institutions. Each institute created a treatment plan with the technique commonly used in the institute for lung SBRT. The specified dose fractionation was 3â€¯× 21.5 Gy normalized to the mean ITV dose. Additional dose objectives for target volumes and OAR were provided. RESULTS: In all, 52 plans from 25 institutions were included in this analysis: 8 robotic radiosurgery (RRS), 34 intensity-modulated (MOD), and 10 3D-conformal (3D) radiation therapy plans. The distribution of the mean dose in the PTV did not differ significantly between the two patients (median 56.9 Gy vs 56.6 Gy). There was only a small difference between the techniques, with RRS having the lowest mean PTV dose with a median of 55.9 Gy followed by MOD plans with 56.7 Gy and 3D plans with 57.4 Gy having the highest. For the different organs at risk no significant difference between the techniques could be found. CONCLUSIONS: This planning study pointed out that multiparameter dose prescription including normalization on the mean ITV dose in combination with detailed objectives for the PTV and ITV achieve consistent dose distributions for peripheral lung tumors in combination with an ITV concept between different delivery techniques and across institutions.

The impact of local control on overall survival after stereotactic body radiotherapy for liver and lung metastases from colorectal cancer: a combined analysis of 388 patients with 500 metastases.

BACKGROUND: The aim of this analysis was to model the effect of local control (LC) on overall survival (OS) in patients treated with stereotactic body radiotherapy (SBRT) for liver or lung metastases from colorectal cancer. METHODS: The analysis is based on pooled data from two retrospective SBRT databases for pulmonary and hepatic metastases from 27 centers from Germany and Switzerland. Only patients with metastases from colorectal cancer were considered to avoid histology as a confounding factor. An illness-death model was employed to model the relationship between LC and OS. RESULTS: Three hundred eighty-eight patients with 500 metastatic lesions (lung n = 209, liver n = 291) were included and analyzed. Median follow-up time for local recurrence assessment was 12.1 months. Ninety-nine patients with 112 lesions experienced local failure. Seventy-one of these patients died after local failure. Median survival time was 27.9 months in all patients and 25.4 months versus 30.6 months in patients with and without local failure after SBRT. The baseline risk of death after local failure exceeds the baseline risk of death without local failure at 10 months indicating better survival with LC. CONCLUSION: In CRC patients with lung or liver metastases, our findings suggest improved long-term OS by achieving metastatic disease control using SBRT in patients with a projected OS estimate of > 12 months.

The SBRT database initiative of the German Society for Radiation Oncology (DEGRO): patterns of care and outcome analysis of stereotactic body radiotherapy (SBRT) for liver oligometastases in 474 patients with 623 metastases.

BACKGROUND: The intent of this pooled analysis as part of the German society for radiation oncology (DEGRO) stereotactic body radiotherapy (SBRT) initiative was to analyze the patterns of care of SBRT for liver oligometastases and to derive factors influencing treated metastases control and overall survival in a large patient cohort. METHODS: From 17 German and Swiss centers, data on all patients treated for liver oligometastases with SBRT since its introduction in 1997 has been collected and entered into a centralized database. In addition to patient and tumor characteristics, data on immobilization, image guidance and motion management as well as dose prescription and fractionation has been gathered. Besides dose response and survival statistics, time trends of the aforementioned variables have been investigated. RESULTS: In total, 474 patients with 623 liver oligometastases (median 1 lesion/patient; range 1­4) have been collected from 1997 until 2015. Predominant histologies were colorectal cancer (n = 213 pts.; 300 lesions) and breast cancer (n = 57; 81 lesions). All centers employed an SBRT specific setup. Initially, stereotactic coordinates and CT simulation were used for treatment set-up (55%), but eventually were replaced by CBCT guidance (28%) or more recently robotic tracking (17%). High variance in fraction (fx) number (median 1 fx; range 1­13) and dose per fraction (median: 18.5 Gy; range 3­37.5 Gy) was observed, although median BED remained consistently high after an initial learning curve. Median follow-up time was 15 months; median overall survival after SBRT was 24 months. One- and 2-year treated metastases control rate of treated lesions was 77% and 64%; if maximum isocenter biological equivalent dose (BED) was greater than 150 Gy EQD2Gy, it increased to 83% and 70%, respectively. Besides radiation dose colorectal and breast histology and motion management methods were associated with improved treated metastases control. CONCLUSION: After an initial learning curve with regards to total cumulative doses, consistently high biologically effective doses have been employed translating into high local tumor control at 1 and 2 years. The true impact of histology and motion management method on treated metastases control deserve deeper analysis. Overall survival is mainly influenced by histology and metastatic tumor burden.

Thyroid tumor formation in the male mouse induced by fluopyram is mediated by activation of hepatic CAR/PXR nuclear receptors.

Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750ppm equating to 105mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh ß transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28days of treatment. In these dose-response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA.

The screening of everyday life chemicals in validated assays targeting the pituitary-gonadal axis.

Ten structurally diverse chemicals (vitamins C, B9, B6, B3, sucrose, caffeine, gingerol, xanthan gum, paracetamol, ibuprofen) deemed intrinsic to modern life but not considered as endocrine active, were tested in vitro using the human estrogen receptor transcriptional activation (hERTa) and the H295R steroidogenesis assays. All were inactive in the hERTa assay but paracetamol, gingerol, caffeine and vitamin C affected steroidogenesis in vitro from 250, 25, 500 and 750 µM respectively. One molecule, caffeine, was further tested in rat pubertal assays at the tumorigenic dose-level and at dose-levels relevant for human consumption. In females pubertal parameters (vaginal opening, estrus cycle), ovarian weight and Fsh and prolactin transcript levels were affected. In males, plasma progesterone levels and prostate and seminal vesicle weights were affected. Although the current regulatory focus is synthetic chemicals that can cause adverse effects on the hypothalamus-pituitary-gonadal axis, our data infer that the range of natural chemicals with the potential to affect this axis may be extensive and is probably overlooked. Thus, to avoid regulation of an overwhelming number of chemicals, a weight of evidence approach, combining hazard identification and characterization with exposure considerations, is needed to identify those chemicals of real regulatory concern.

Tertiary butyl alcohol in drinking water induces phase I and II liver enzymes with consequent effects on thyroid hormone homeostasis in the B6C3F1 female mouse.

Tertiary butyl alcohol (TBA) was administered to groups of 15 female B6C3F1 mice in drinking water at concentrations of 0, 2.0 or 20 mg TBA ml(-1), for 14 days, for assessment of gross and histological changes in the liver and thyroid, thyroid hormones (T3, T4, and TSH), total hepatic cytochrome P450 (Cyp) content, specific Cyp activities and quantitative PCR analysis of specific Cyp enzymes (Cyp1a1, Cyp2b9, Cyp2b10, Cyp3a11), sulfuryltransferases (ST1a1, ST2a2, and STn) and glucuronyltransferases (UGT1a1, UGT2b1, and UGT2b5). Phenobarbital (PB) was administered to a positive control group by oral gavage at a daily dose of 80 mg kg(-1). TBA caused, on day 14, a reduction in circulating T3 (12-15% decrease) and a dose-dependent reduction in T4 (13-22% decrease), with no evidence of thyroid pathology. Two of five livers examined in the 20 mg TBA ml(-1) dose group showed mild, diffuse centrilobular hypertrophy. On day 14, Cyp 7-benzoxyresorufin-O-debenzylase activity was significantly induced 12-fold by TBA at 20 mg ml(-1), and 1.8-fold at the 2.0 mg TBA ml(-1) concentration. Cyp 7-pentoxyresorufin-O-dealkylase activity was slightly induced (2.1-fold) by 20 mg TBA ml(-1) on day 14. Quantitative PCR analysis of gene transcripts showed a significant induction of Cyp2b10 and ST1a1 with both TBA concentrations, and a slight induction of Cyp2b9 at 20 mg TBA ml(-1) only. PB induced all phase I and phase II gene transcripts except for Cyp1a1 and Cyp2b9. These findings suggest that TBA, at and below doses used in chronic studies, is an inducer of phase I and phase II liver enzymes, with resulting decreases in circulating thyroid hormones in B6C3F1 mice.

Intracellular routing of GLcNAc-bearing molecules in thyrocytes: selective recycling through the Golgi apparatus.

Previous experiments led us to speculate that thyrocytes contain a recycling system for GlcNAc-bearing immature thyroglobulin molecules which prevents these molecules from lysosomal degradation (Miquelis, R., C. Alquier, and M. Monsigny. 1987. J. Biol. Chem. 262:15291-15298). To confirm this hypothesis, the fate of GlcNAc-bearing proteins after internalization by thyrocytes was monitored and compared to that of fluid phase markers. Kinetic internalization studies were performed using 125I-GlcNAc-BSA and 131I-Man-BSA. We observed that the apparent intake rate as well as the amount of hydrolyzed GlcNAc-BSA are smaller than the corresponding values for Man-BSA. These differences were reduced by GlcNAc competitors (thyroglobulin and ovomucoid) or a weak base (chloroquine). Part of the internalized GlcNAc-BSA was released into the extracellular milieu at a higher rate and shorter half life (t1/2 = approximately 30 min) than the Man-BSA (t1/2 = approximately 8 h). Subcellular homing was first studied by cell fractionation after internalization using 125I-ovomucoid and 131I-BSA. During Percoll density gradient fractionation, endogenous thyroperoxidase was used to separate subsets of organelles involved in the biosynthetic exocytotic pathway. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation give rise to a shift in the density of organelles containing 3.5 times more ovomucoid than BSA. Discontinuous sucrose gradient showed that: (a) thyroperoxidase was colocalized with galactosyltransferase-contraining organelles in Golgi-rich subfractions; and (b) that at every time studied from 10 to 100 min, the ovomucoid/BSA ratio was higher in these organelles than in other subfractions. Finally we also observed that: (a) ovomucoid sequestered in the Golgi-rich subfraction incorporated [3H]galactose; and (b) that part of internalized ovomucoid was localized on the Golgi stacks as well as elements of the trans-Golgi, as revealed by immunogold labeling on ultrathin cryosections. These data prove that in thyrocytes GlcNAc accessible sugar moieties on soluble internalized molecules are sufficient to trigger their recycling via the Golgi apparatus.

The design, physical properties and clinical utility of an iris collimator for robotic radiosurgery.

Robotic radiosurgery using more than one circular collimator can improve treatment plan quality and reduce total monitor units (MU). The rationale for an iris collimator that allows the field size to be varied during treatment delivery is to enable the benefits of multiple-field-size treatments to be realized with no increase in treatment time due to collimator exchange or multiple traversals of the robotic manipulator by allowing each beam to be delivered with any desired field size during a single traversal. This paper describes the Iris variable aperture collimator (Accuray Incorporated, Sunnyvale, CA, USA), which incorporates 12 tungsten-copper alloy segments in two banks of six. The banks are rotated by 30 degrees with respect to each other, which limits the radiation leakage between the collimator segments and produces a 12-sided polygonal treatment beam. The beam is approximately circular, with a root-mean-square (rms) deviation in the 50% dose radius of <0.8% (corresponding to <0.25 mm at the 60 mm field size) and an rms variation in the 20-80% penumbra width of about 0.1 mm at the 5 mm field size increasing to about 0.5 mm at 60 mm. The maximum measured collimator leakage dose rate was 0.07%. A commissioning method is described by which the average dose profile can be obtained from four profile measurements at each depth based on the periodicity of the isodose line variations with azimuthal angle. The penumbra of averaged profiles increased with field size and was typically 0.2-0.6 mm larger than that of an equivalent fixed circular collimator. The aperture reproducibility is < or =0.1 mm at the lower bank, diverging to < or =0.2 mm at a nominal treatment distance of 800 mm from the beam focus. Output factors (OFs) and tissue-phantom-ratio data are identical to those used for fixed collimators, except the OFs for the two smallest field sizes (5 and 7.5 mm) are considerably lower for the Iris Collimator. If average collimator profiles are used, the assumption of circular symmetry results in dose calculation errors that are <1 mm or <1% for single beams across the full range of field sizes; errors for multiple non-coplanar beam treatment plans are expected to be smaller. Treatment plans were generated for 19 cases using the Iris Collimator (12 field sizes) and also using one and three fixed collimators. The results of the treatment planning study demonstrate that the use of multiple field sizes achieves multiple plan quality improvements, including reduction of total MU, increase of target volume coverage and improvements in conformality and homogeneity compared with using a single field size for a large proportion of the cases studied. The Iris Collimator offers the potential to greatly increase the clinical application of multiple field sizes for robotic radiosurgery.

Stereotactic body radiotherapy (SBRT) for multiple pulmonary oligometastases: Analysis of number and timing of repeat SBRT as impact factors on treatment safety and efficacy.

BACKGROUND: Stereotactic body radiotherapy (SBRT) for oligometastatic disease is characterized by an excellent safety profile; however, experiences are mostly based on treatment of one single metastasis. It was the aim of this study to evaluate safety and efficacy of SBRT for multiple pulmonary metastases. PATIENTS AND METHODS: This study is based on a retrospective database of the DEGRO stereotactic working group, consisting of 637 patients with 858 treatments. Cox regression and logistic regression were used to analyze the association between the number of SBRT treatments or the number and the timing of repeat SBRT courses with overall survival (OS) and the risk of early death. RESULTS: Out of 637 patients, 145 patients were treated for multiple pulmonary metastases; 88 patients received all SBRT treatments within one month whereas 57 patients were treated with repeat SBRT separated by at least one month. Median OS for the total patient population was 23.5 months and OS was not significantly influenced by the overall number of SBRT treatments or the number and timing of repeat SBRT courses. The risk of early death within 3 and 6 months was not increased in patients treated with multiple SBRT treatments, and no grade 4 or grade 5 toxicity was observed in these patients. CONCLUSIONS: In appropriately selected patients, synchronous SBRT for multiple pulmonary oligometastases and repeat SBRT may have a comparable safety and efficacy profile compared to SBRT for one single oligometastasis.

Stereotactic body radiotherapy for oligo-metastatic liver disease - Influence of pre-treatment chemotherapy and histology on local tumor control.

INTRODUCTION: Stereotactic body radiation therapy (SBRT) is applied in the oligometastatic setting to treat liver metastases. However, factors influencing tumor control probability (TCP) other than radiation dose have not been thoroughly investigated. Here we set out to investigate such factors with a focus on the influence of histology and chemotherapy prior to SBRT using a large multi-center database from the German Society of Radiation Oncology. METHODS: 452 SBRT treatments in 363 patients were analyzed after collection of patient, tumor and treatment data in a multi-center database. Histology was considered through random effects in semi-parametric and parametric frailty models. Dose prescriptions were parametrized by conversion to the maximum biologically effective dose using alpha/beta of 10Gy (BEDmax). RESULTS: After adjusting for histology, BEDmax was the strongest predictor of TCP. Larger PTV volumes, chemotherapy prior to SBRT and simple motion management techniques predicted significantly lower TCP. The model predicted a BED of 209±67Gy10 necessary for 90% TCP at 2years with no prior chemotherapy, but 286±78Gy10 when chemotherapy had been given. Breast cancer metastases were significantly more responsive to SBRT compared to other histologies with 90% TCP at 2years achievable with BEDmax of 157±80Gy10 or 80±62Gy10 with and without prior chemotherapy, respectively. CONCLUSIONS: Besides dose, histology and pretreatment chemotherapy were important factors influencing local TCP in this large cohort of liver metastases. After adjusting for prior chemotherapy, our data add to the emerging evidence that breast cancer metastases do respond better to hypofractionated SBRT compared to other histologies.

Nomogram based overall survival prediction in stereotactic body radiotherapy for oligo-metastatic lung disease.

BACKGROUND: Radical local treatment of pulmonary metastases is practiced with increasing frequency due to acknowledgment and better understanding of oligo-metastatic disease. This study aimed to develop a nomogram predicting overall survival (OS) after stereotactic body radiotherapy (SBRT) for pulmonary metastases. PATIENTS AND METHODS: A multi-institutional database of 670 patients treated with SBRT for pulmonary metastases was used as training cohort. Cox regression analysis with bidirectional variable elimination was performed to identify factors to be included into the nomogram model to predict 2-year OS. The calibration rate of the nomogram was assessed by plotting the actual Kaplan-Meier 2-year OS against the nomogram predicted survival. The nomogram was externally validated using two separate monocentric databases of 145 and 92 patients treated with SBRT for pulmonary metastases. RESULTS: The median follow up of the trainings cohort was 14.3months, the 2-year and 5-year OS was 52.6% and 23.7%, respectively. Karnofsky performance index, type of the primary tumor, control of the primary tumor, maximum diameter of the largest treated metastasis and number of metastases (1 versus >1) were significant prognostic factors in the Cox model (all p<0.05). The calculated concordance-index for the nomogram was 0.73 (concordance indexes of all prognostic factors between 0.54 and 0.6). Based on the nomogram the training cohort was divided into 4 groups and 2-year OS ranged between 24.2% and 76.1% (predicted OS between 30.2% and 78.4%). The nomogram discriminated between risk groups in the two validation cohorts (concordance index 0.68 and 0.67). CONCLUSIONS: A nomogram for prediction of OS after SBRT for pulmonary metastases was generated and externally validated. This tool might be helpful for interdisciplinary discussion and evaluation of local and systemic treatment options in the oligo-metastatic setting. KEY MESSAGE: A nomogram for prediction of overall survival after stereotactic body radiotherapy (SBRT) for pulmonary metastases was developed and externally validated. This tool might be helpful for interdisciplinary discussion and evaluation of local and systemic treatment options in the oligo-metastatic setting.

Real time tracking in liver SBRT: comparison of CyberKnife and Vero by planning structure-based γ-evaluation and dose-area-histograms.

The purpose of this study was to evaluate and compare two clinical tracking systems for radiosurgery with regard to their dosimetric and geometrical accuracy in liver SBRT: the robot-based CyberKnife and the gimbal-based Vero. Both systems perform real-time tumour tracking by correlating internal tumour and external surrogate motion. CyberKnife treatment plans were delivered to a high resolution 2D detector array mounted on a 4D motion platform, with the platform simulating (a) tumour motion trajectories extracted from the corresponding CyberKnife predictor log files and (b) the tumour motion trajectories with superimposed baseline-drift. Static reference and tracked dose measurements were compared and dosimetric as well as geometrical uncertainties analyzed by a planning structure-based evaluation. For (a), γ-passing rates inside the CTV (γ-criteria of 1% / 1 mm) ranged from 95% to 100% (CyberKnife) and 98% to 100% (Vero). However, dosimetric accuracy decreases in the presence of the baseline-drift. γ-passing rates for (b) ranged from 26% to 92% and 94% to 99%, respectively; i.e. the effect was more pronounced for CyberKnife. In contrast, the Vero system led to maximum dose deviations in the OAR between +1.5 Gy to +6.0 Gy (CyberKnife: +0.5 Gy to +3.5 Gy). Potential dose shifts were interpreted as motion-induced geometrical tracking errors. Maximum observed shift ranges were -1.0 mm to +0.7 mm (lateral) /-0.6 mm to +0.1 mm (superior-inferior) for CyberKnife and -0.8 mm to +0.2 mm /-0.8 mm to +0.4 mm for Vero. These values illustrate that CyberKnife and Vero provide high precision tracking of regular breathing patterns. Even for the modified motion trajectory, the obtained dose distributions appear to be clinical acceptable with regard to literature QA γ-criteria of 3% / 3 mm.

Towards real-time MRI-guided 3D localization of deforming targets for non-invasive cardiac radiosurgery.

Radiosurgery to the pulmonary vein antrum in the left atrium (LA) has recently been proposed for non-invasive treatment of atrial fibrillation (AF). Precise real-time target localization during treatment is necessary due to complex respiratory and cardiac motion and high radiation doses. To determine the 3D position of the LA for motion compensation during radiosurgery, a tracking method based on orthogonal real-time MRI planes was developed for AF treatments with an MRI-guided radiotherapy system. Four healthy volunteers underwent cardiac MRI of the LA. Contractile motion was quantified on 3D LA models derived from 4D scans with 10 phases acquired in end-exhalation. Three localization strategies were developed and tested retrospectively on 2D real-time scans (sagittal, temporal resolution 100 ms, free breathing). The best-performing method was then used to measure 3D target positions in 2D-2D orthogonal planes (sagittal-coronal, temporal resolution 200-252 ms, free breathing) in 20 configurations of a digital phantom and in the volunteer data. The 3D target localization accuracy was quantified in the phantom and qualitatively assessed in the real data. Mean cardiac contraction was ⩽ 3.9 mm between maximum dilation and contraction but anisotropic. A template matching approach with two distinct template phases and ECG-based selection yielded the highest 2D accuracy of 1.2 mm. 3D target localization showed a mean error of 3.2 mm in the customized digital phantoms. Our algorithms were successfully applied to the 2D-2D volunteer data in which we measured a mean 3D LA motion extent of 16.5 mm (SI), 5.8 mm (AP) and 3.1 mm (LR). Real-time target localization on orthogonal MRI planes was successfully implemented for highly deformable targets treated in cardiac radiosurgery. The developed method measures target shifts caused by respiration and cardiac contraction. If the detected motion can be compensated accordingly, an MRI-guided radiotherapy system could potentially enable completely non-invasive treatment of AF.

The N-acetylglucosamine-specific receptor of the thyroid: purification, further characterization, and expression patterns on normal and pathological glands.

The N-acetylglucosamine receptor of the thyroid has been putatively described as both a prohormonal receptor that could play a role in the intrafollicular retention of immature thyroglobulin and a vectorial conveyor of these immature molecules to the iodination site. To further characterize this receptor, we have developed a purification procedure yielding nanomolar amounts of N-acetylglucosamine receptor. This thyroid lectin appeared to have an isoelectric point near 5.2 and to be composed of 51-kilodalton monomers with no Asn-linked glycoconjugates. Recognition of the receptor by antipeptide antibodies (Ab/ROV1) raised against a preselected sequence of cation-dependent lectins indicated immunological kinship with the Gal/GalNAc-specific hepatic lectin. Affinity-purified Ab/ROV1 and polyclonal antibodies against the purified receptor (TGRD-Ab) were used to study the location and expression pattern of the receptor on animal and human thyroid tissue. On porcine slices, positive labeling was observed in various intracellular vesicular compartments with both antibodies and was particularly intense in the apical membrane and subapical compartments. The same pattern was observed in normal human thyroid. In contrast, the receptor 1) could not be found on epithelial cells from thyroid papillary carcinoma; 2) was abundant, but concentrated in the subnuclear region of the thyrocytes in adenomatous goiter; and 3) was almost exclusively located at the basolateral membrane in follicular carcinoma as well as in thyrocytes from glands treated with antithyroid drug before surgery. These observations indicate that expression of the N-acetylglucosamine receptor is characteristic of the fully differentiated phenotype, and its potential function as a thyroglobulin conveyor back to the lumen would be either impaired or abolished in some disease processes.

On the relationship between completion of N-acetyllactosamine oligosaccharide units and iodine content of thyroglobulin: a reinvestigation.

Lack of completion of N-acetyllactosamine-type glycosylation on thyroglobulin (Tg) has been implicitly considered as an etiological factor of some thyroid disorders, i.e. goiter and hypothyroidism. However, there is some evidence that Tg with incompletely processed N-acetyllactosamine glycans occurs in the normal gland. Recent findings demonstrated that exposed N-acetylglucosamine (GlcNAc) residues present on internalized glycoprotein in the thyrocyte may act as a retention signal that prevents lysosomal homing and triggers recycling of GlcNAc-bearing molecules through galactosyltransferase- and thyroperoxidase-containing compartments of the Golgi apparatus. This finding raises the possibilities 1) that exposed GlcNAc residues are not randomly distributed, but are mainly present on immature Tg; and 2) that this process promotes elongation of complex glycans, thereby eliminating the retention signal. To further validate this hypothesis, we reinvestigated the relationship between the iodine content and the glycan completion of porcine Tg of luminal origin. Tg subpopulations were separated according to their iodine content on rubidium chloride centrifugation gradients, and their interactions with various plant and animal lectins were analyzed in solid phase assays. Iodine content used as an index of age ranged from 0.6-1.2%. There was no significant correlation between iodine content and either neutral sugar or oligosaccharide content, as judged by chemical methods or interaction with [125I]Solanum tuberosum and [125I]Pisum sativum agglutinins. In contrast, the number of GlcNAc-accessible residues (as judged by interaction with [125I]Bandeiraea simplificolia II) decreased as iodine content increased. These changes were concomitant with an increase in galactose (measured by interaction with [125I]R-icinus communis and [125I]galactosidase (Gal)/GalNAc rat hepatic lectin) and sialic acid content. Related experiments using a Tg subpopulation depleted in GlcNAc-exposed residues by passage through a B. simplificolia II affinity column showed that the capacity of this subpopulation to bind to membranes was lowered compared to that of the total Tg. These results support the following conclusions: 1) in normal glands, all or part of the Tg molecules are secreted in an incompletely glycosylated form; and 2) iodine organification is correlated with glycan completion. Therefore, asialoagalactothyroglobulin appears to be a physiological precursor for an efficient recycling mediated by the GlcNAc receptor to the iodination site. New insights in thyroid disorders are discussed.

Carbohydrate and protein determinants are involved in thyroglobulin recognition by FRTL 5 cells.

To avoid premature lysosomal degradation, thyrocytes have a system able to recycle internalized immature thyroglobulin molecules (Tg) to the follicular lumen via the Golgi apparatus. It has been shown that this quality control system depends on recognition of exposed N-acetylglucosamine (GlcNAc) determinants (Miquelis et al., J Cell Biol, 1993, 123, 1695) present on immature Tg (Bastiani et al., 1995, Endocrinology, 1995, 136, 4204). However, the same in vitro kinetics studies also showed that GlcNAc residues alone induce only weak recycling. The latter finding led us to investigate the possibility that protein determinants might also be involved in binding. For this purpose, we studied binding of Tg to FRTL 5 cells, a widely available TSH-dependent cell line and found that binding to plasma membranes occurred at acidic pH in the presence of calcium, i.e. under conditions previously reported for binding of GlcNAc-BSA to porcine thyroid cell membranes. As expected, binding was GlcNAc- and oligosaccharide-dependent because Bandeiraea Simplificiola II affinity column analysis indicated that GlcNAc-bearing Tg were preferentially bound and N-glycanase treatment of Tg inhibited interaction. Ovomucoid, GlcNAc-BSA, and porcine Tg oligosaccharides did not inhibit binding, indicating that carbohydrates were not the sole determinants for binding. The fact that pronase digestion of Tg totally abolished binding implied that peptide determinants were involved in the interaction. This involvement is supported by the observation that porcine, rat, bovine, and human Tg bound FRTL 5 cell membranes and that monoclonal antibodies raised against human Tg interfered with the binding of both human and porcine Tg. Based on these findings we conclude that, besides the involvement of GlcNAc-bearing oligosaccharides, Tg receptors form a stable bond with peptide determinants.