Brain and tumour blood flow can be measured noninvasively using arterial spin labelling (ASL) magnetic resonance imaging (MRI), but reliable quantification in mouse models remains difficult. Pseudocontinuous ASL (pCASL) is recommended as the clinical standard for ASL and can be improved using multiphase labelling (MP pCASL). The aim of this study was to optimise and validate MP pCASL MRI for cerebral blood flow (CBF) measurement in mice and to assess its sensitivity to tumour perfusion. Following optimization of the MP pCASL sequence, CBF data were compared with gold-standard autoradiography, showing close agreement. Subsequently, MP pCASL data were acquired at weekly intervals in models of primary and secondary brain tumours, and tumour microvessel density was determined histologically. MP pCASL measurements in a secondary brain tumour model revealed a significant reduction in blood flow at day 35 after induction, despite a higher density of blood vessels. Tumour core regions also showed reduced blood flow compared with the tumour rim. Similarly, significant reductions in CBF were found in a model of glioma 28 days after tumour induction, together with an increased density of blood vessels. These findings indicate that MP pCASL MRI provides accurate and robust measurements of cerebral blood flow in naïve mice and is sensitive to changes in tumour perfusion. Reliable and accurate quantification of cerebral blood flow (CBF) by noninvasive methods, such as magnetic resonance imaging (MRI), is of critical importance in many neuropathologies. The measurement of tumour blood flow, for example, is key to both treatment planning and predicting response. Tumour perfusion is widely used as an indicator of tumour microenvironment, stage, and progression, and greatly influences treatment. Clinical studies have shown that tumour perfusion and, consequently, oxygenation are highly correlated with clinical outcome –3. CritiThe MRI method arterial spin labelling (ASL) is often used to image cerebral blood flow (CBF), as it requires no exogenous contrast agent and is easily implemented on clinical scanners. Moreover, this method can provide absolute quantitative measures of blood flow, which are not readily obtained by other modalities. Pseudocontinuous ASL (pCASL), in particular, has been recommended as the methodology of choice for clinical ASL , but no 0 inhomogeneities in high-field preclinical systems. Implementations of ASL in mice have historically produced CBF values that are semiquantitative [ca. 100 mL/100 g/min) in the normal mouse brain [Despite initial reports developing ASL in rodents , 6, mosttitative –9, singltitative , or varytitative –13 and btitative  used. Motitative , 15–17 ase brain –22. Thesse brain , 24. Whise brain –27 or mise brain , pCASL hse brain .The use of multiphase pCASL (MP pCASL) has been shown to reduce these errors in both humans  and ratsThus, the primary aim of this study was to optimise and validate MP pCASL MRI for measuring CBF in naïve mice. Subsequently, we assessed the sensitivity of the optimised MP pCASL sequence to changes in blood flow in early stage tumours, using mouse models of secondary brain cancer (metastasis) and primary glioma.Female BALB/c mice , 7–10 weeks old, were used in all naïve and metastasis model experiments. Female SCID mice , 7–10 weeks old, were used in all primary brain tumour model experiments. All animal experiments were approved by the UK Home Office  Act 1986) and conducted in accordance with the University of Oxford Policy on the Use of Animals in Scientific Research and the ARRIVE guidelines . Animalsn=4) were anaesthetised with 3% isoflurane in O2. Temperature was monitored and maintained at 37.0 ± 0.5°C throughout with a feedback heating system. Respiration was monitored and maintained between 30 and 80 breaths per minute. An electrocardiogram was also acquired. Using a Vevo 3100 ultrasound  and a mouse cardiology probe (MX550D), a respiration-gated 3D ultrasound image was acquired across the whole neck. The left carotid artery was located, and 10 Power Doppler blood flow clips of 30 s duration were acquired per mouse at respiration rates across the range of 30–80 breaths per minute, achieved by varying the concentration of isoflurane between 3 and 1%. Carotid blood flow values from the Power Doppler trace were extracted using an open-source plot digitiser [Mice (Gmax and Gmean (Gmean = 0.05 × Gmax in our MP pCASL sequence) gradient strengths, equivalent to converted labelling plane thicknesses of 1–10 mm in 0.5 mm steps. Blood flow velocities of between 1 and 20 cm/s with steps of 1 cm/s were modelled with T1blood = 2.1 s, T2blood = 0.033 s (based on ex vivo rat blood measurements at 9.4 T) [µT with steps of 0.5 µT for a train of 600 μs Hanning-shaped pulses, beginning every 1.2 ms. Simulations of the theoretical maximum saturation for different label durations were also carried out, and data are available in the Supplementary Information.Bloch simulations were carried out to simulate labelling efficiency over a range of different gradient strengths, radiofrequency (RF) powers, labelling plane thicknesses, and blood flow velocities, as described by Okell . We condt 9.4 T) , and RF n=4) using a 9.4 T MRI spectrometer  with a 26 mm volume transmit-receive birdcage RF coil . Mice were anaesthetised with 3% isoflurane in 30% O2: 70% N2 and positioned in a custom-built cradle in the volume coil. Mouse temperature was monitored and maintained at 37.0 ± 0.5°C with a rectal probe and feedback heating system. Respiration was monitored using a pressure balloon, and breathing rate was maintained at approximately 60 breaths per minute by adjusting isoflurane concentration; stable breathing was typically maintained with an isoflurane concentration of 1.2–1.5%. The respiration trace was recorded and used to inform respiratory triggering.MP pCASL experiments were performed on a separate cohort of mice  = 4.0 s, echo time (TE) = 18.32 ms, spectral width (SW) = 250 kHz, field of view (FOV) = 20 × 20 mm, matrix size = 64 × 64, slice thickness = 1 mm, and number of slices = 8, with the anterior slice positioned just posterior to the olfactory sulcus. Respiratory triggering (with an associated variable post-trigger delay) was also employed to reduce signal fluctuation at the base of the brain caused by respiratory motion.μs, Hanning shape with effective flip angle (FA) = 40°, and slice-refocusing gradient pulse duration 600 μs. The phase of these RF pulses was arrayed from 0° to 315° with incremental steps of 45°: 0°, 45°, 90°, 135°, 180°, 225°, 270°, and 315°. The labelling plane was oriented at −10° relative to the axial plane in order to be perpendicular to the carotid and vertebral arteries  were anaesthetised using the same setup and isoflurane concentrations as for MRI described above. MP pCASL was performed on each animal on the day before autoradiography, owing to restrictions concerning the use of radioisotopes in certain rooms. Care was taken to ensure breathing rate and isoflurane concentration, and duration of anaesthesia prior to measurements was as similar as possible between the ASL and autoradiography sessions. For the MP pCASL MRI, a labelling pulse duration of 0.9 s and a postlabel delay of 0.4 s seconds was used with the parameters described above.For comparison of MP pCASL data with gold-standard autoradiography CBF measurements, naïve mice , and 2 minutes later, they were injected intraperitoneally with an overdose of pentobarbitone then immersed in isopentane on dry ice until frozen. The frozen brain was extracted, and 20 µm sections were collected at 200 µm intervals and dried at 60°C for 10 min. These sections were exposed to film for 24 hours alongside calibrated radioactive standards, and films were scanned . Scanned films were background subtracted and calibrated against the standards. Absolute CBF was then calculated using the equation as described by Sakurada et al. [Autoradiography was performed using the method described by Maeda et al. . Mice wea et al. . Blood shttp://www.fmrib.ox.ac.uk/fsl/BASIL). The raw multiphase data were initially fitted to a modified Fermi function [α=70, β=19) to produce a raw phase map. The phase values were then smoothed and clustered using supervoxel clustering [ASL data analysis and perfusion quantification were performed using a custom version of the BASIL toolbox from the FMRIB Software Library  with 5000 4T1-GFP, metastatic murine mammary carcinoma cells in 0.5 µL sterile saline into the left striatum . Mice underwent MRI at day 7 (n=3), 14 (n=5), 21 (n=6), 28 (n=8), or 35 (n=6) after tumour induction; mice were randomly assigned to each time point group.Sensitivity of the MP pCASL sequence to changes in tumour blood flow over time was first assessed in a brain metastasis model. Female BALB/c mice were anaesthetised with 3% isoflurane in 30% On=10) or 28 (n=6), after tumour induction.To further assess the sensitivity of the MP pCASL method, a primary brain tumour model was also used. Female SCID mice were anaesthetised and injected in the left striatum, as above, with 5000 U87 human glioma cells. Mice underwent MRI at day 14  Gadodiamide  using the T1-weighted scan parameters described above.In addition to MP pCASL acquisitions, Following imaging, mice underwent transcardial perfusion-fixation with 0.9% heparinised saline followed by 20 mL periodate-lysine-paraformaldehyde solution containing 0.025% glutaraldehyde, and the brains were collected for histology.µm autoradiography sections were moderately stained pixel intensity between 180 and 171, and strongly stained pixel intensity lower than 53.Sections were stained for the blood vessel marker CD31  according to the method described by Andreou et al. . Microvet-tests or one-way ANOVA , or two-way repeated measures (paired) ANOVA  followed post hoc by the Bonferroni multiple test correction. An f-test was used to assess heterogeneity between groups.All data are reported as group mean ± group standard deviation. Differences between groups were determined using two-tailed paired µT) and average blood velocity of 124 mm/s, and lies within the linear portion of the feeding arteries in the neck.Blood velocity in the carotid artery was measured to inform Bloch simulations of pCASL. The mean blood velocity in the carotid artery was found to be 124 ± 17 mm/sec, and the maximum blood velocity was found to be 275 ± 58 mm/sec. Mean blood velocity was consistent between animals, but varied with the respiration rate in 3 of 4 animals . Maximum blood velocity varied between animals (range = 214–556 mm/sec), and also varied with the respiration rate in 3 of 4 animals . Using these values to inform the range of blood velocities in the Bloch simulations, a 2 mm label thickness was chosen as it provided sufficient theoretical inversion (labelling) efficiency (78.5%) at the RF pulse amplitude used in our system . The mean absolute reduction in CBF in the tumour-bearing striatum was 15.3 mL/100 g/min. No differences were found between hemispheres at other time points. All tumours were confirmed histologically, and the mean tumour diameter measured by histology at day 35 was 2.14 ± 0.53 mm.Application of the MP pCASL sequence in mice with intracerebral metastases showed a significant, 16%, decrease in CBF in the tumour-bearing striatum (84 ± 16 mL/100 g/min) compared with the contralateral  striatum (99 ± 24 mL/100 g/min) at day 35 (ANOVA n=7), the striatum exhibited gadolinium enhancement throughout the area of metastatic foci and, in these animals, no difference in CBF was evident between the tumour (86 ± 26 mL/100 g/min) and contralateral striatum , a gadolinium-enhancing tumour rim was evident surrounding a nonenhancing tumour core. Analysis of these animals showed a significant reduction in blood flow in the core of the tumour region (69 ± 14 mL/100 g/min) compared with the rim . Analysis of microvessel density within the metastatic foci showed a greatly increased vessel area fraction (13.5 ± 2.5%) in the nonenhancing tumour core compared with the tumour rim  compared with the contralateral striatum at day 28 . The use of respiratory triggering also relies on a reasonably stable respiratory cycle, which was reliably achieved at ∼60 breaths per minute with 1.2–1.5% isoflurane concentrations for up to two hours.Our data show less regional variation in CBF than other autoradiography studies in mice, which may, at least in part, reflect differences in mouse strain and anaesthetic regime used , 21, 39.The MP pCASL technique was found to be sensitive to changes in tumour perfusion in mouse models of both brain metastasis and glioma at later time points. The technique was also sensitive to perfusion heterogeneity within metastatic tumours, showing significantly reduced (but not absent) perfusion in the nonenhancing tumour core compared with normal tissue in the contralateral striatum and increased heterogeneity in perfusion compared with the contralateral striatum. As autoradiography has only been used to validate CBF measurements in the naïve brain, it is possible that differences in tumour arterial transit time or tissue relaxation time may affect the CBF quantification from MP pCASL data in these tumour-bearing animals. However, these results are in line with other preclinical primary brain tumour perfusion studies, which showed reduced perfusion in the tumour core , 40–42. Tumours have a number of distinct vascular phenotypes, each of which may confer changes in blood flow. One such phenotype, which is present in our model, is that of a highly branched network of nonfunctional angiogenic vessels. In this case, despite an increase in the density of vessels, a decrease in CBF may be observed in this region, owing their highly branched and dysfunctional nature. Another vascular phenotype often seen in solid tumours is the rapid growth of the tumour faster than the vasculature can keep up, resulting in a tumour core with low vascularity, and hence a decreased CBF. Both of these phenotypes are characterised by a decrease in CBF, despite the differences in tumour vascularity.Gadolinium contrast enhancement on MRI reports on both vessel permeability and blood flow, and a lack of gadolinium enhancement in tumour core regions is typically taken to reflect tissue necrosis and complete absence of vessels. In contrast, blood vessel density measurements provide no information on whether vessels are perfused, but only whether they are present. However, in both models used in the current study, although core tumour areas showed significant reductions in blood flow in areas devoid of gadolinium enhancement, histological assessment revealed a significant increase in microvessel density and only very small areas of necrosis. Together, these findings suggest that these core vessels are either structurally abnormal or have abnormal flow patterns, as is common in angiogenic tumour vessels –46, and Interestingly, the rim of the metastatic tumour area also showed an increase in microvessel density histologically, but in this region, CBF measured by MP pCASL MRI was normal. Similarly, in those tumours showing enhancement throughout the tumour core, microvessel density was increased, but no change in CBF was evident. These findings suggest that MP pCASL MRI identifies heterogeneity in tumour blood flow that could not be predicted from histological assessment of vascularity alone. Thus, by identifying areas of reduced (but not absent) flow, MP pCASL MRI may enable identification of brain tumours in which vascular normalising therapy or antiangiogenic therapy could be beneficial prior to, or in conjunction with, radiotherapy.Multiphase pCASL MRI has been successfully implemented and validated for imaging cerebral blood flow in naïve mice and has been shown to overcome the limitations inherent in standard nonmultiphase ASL methods. The data show high reproducibility in CBF measurements between animals and concordance with gold-standard autoradiography measurements. Further, this imaging approach is sensitive to changes in perfusion in mouse models of both primary and secondary brain tumours, even in very small tumours, and provides insight into tissue blood flow dynamics that complements vessel density measurements and contrast-enhanced MRI. This method could be of high value for noninvasive measurement of brain tumour blood flow prior to and following therapy, increasing the potential for clinical translation.


Only a few cases of papillary glioneuronal tumour (PGNT) with predominantly focal symptomatology are described in the literature. We report on the clinical, radiological, and histopathological features of PGNT. The intraoperative pathology revealed no tumour in the walls of the cyst, thus surgical resection of the nodule was performed leaving the cyst wall intact. There was no recurrence of tumour at the three-year follow-up, although a long-term follow-up is necessary. Papillary glioneuronal tumour (PGNT) is an uncommon central nervous system tumour type. The first time this name was used by Komori et al. in 1998  and gangA 38-year-old, otherwise healthy, right-handed man presented with a two-year progressive history of motor dysphasia and a three-month history of progressive right-hand weakness. The patient also had one short episode of right leg numbness and weakness. A neurological examination showed reflex asymmetry (right > left), hemihypesthesia, hemiparesis (4/5), and positive Babinski sign on the right. Magnetic resonance imaging (MRI) of the brain revealed a 54 x 54 x 52 mm cystic lesion of the left frontal lobe in front of the precentral gyrus with a septum attached to the posterior wall of the cyst. The cyst was hypointense on T1-weighted images (T1WI)  and 5-μm sections were stained immunohistochemically for glial fibrillary acidic protein (GFAP), synaptophysin, and Ki-67 protein. Histologically, the tumour consisted of morphologically and immunohistochemically distinct components. Small pseudopapillary structures were seen consisting of hyalinized small vessels Figure .The interpapillary space contained cells varying in size from small neurocyte cells to large ganglioid cells  that stained positive for synaptophysin Figure .The Ki-67 labeling index was low Figure .There were scattered hemosiderin deposits observed but no vascular proliferation or necrosis.PGNT predominantly affects the younger population as the patients’ age varied from 11 to 41 years in the series reported by Li et al. . The preNeuroimaging usually shows a cyst lesion with the most frequent pattern being a cystic and solid tumour or cystic tumour with mural nodule , 9, 13. Except for a few reported cases of aggressive behavior , 5, 7, hThe patients diagnosed with PGNT usually undergo gross-total or partial resections, although radiotherapy and chemotherapy for more aggressive tumours were also reported , 9-11. TThe case presented here is rare, as only a few patients with predominantly focal neurological deficits were reported in the literature so far -10. Our Patients with supratentorial, paraventricular cystic lesions, and intracystic septal enhancements should be suspected of having PGNT. The tumour is benign and gross-total resection provides the cure for patients. In the selected cases, a favorable outcome could be achieved by removing only the nodular component, however, a long-term follow-up is important to confirm the true biological behavior of PGNT.


The pericardial fluid may be representative of the interstitium of the heart. The aim of this study was to discriminate in cardiovascular disease patients between adipocytokines that are produced locally by the heart and those supplied by the circulation.Enzyme-linked immunosorbent assays (ELISA) were used to determine levels of N-terminal pro-brain natriuretic peptide (NT-pBNP), fatty acid-binding protein 4 (FABP4), leptin, lipocalin-2, neutrophil elastase, proteinase-3, high sensitivity C-reactive protein (hsCRP) and adiponectin in venous plasma and pericardial fluid harvested during elective cardio-thoracic surgery (n = 132–152).In pericardial fluid compared to plasma, the levels were significantly smaller (p < 0.001) for leptin, lipocalin-2, neutrophil elastase, proteinase-3, hsCRP and adiponectin. For these biomarkers, the ratio of pericardial fluid-to-plasma level ) was 0.65 (0.47–1.01), 0.78 (0.56–1.09), 0.23 (0.11–0.60), 0.17 (0.09–0.36), 0.14 (0.08–0.35), and 0.25 (0.15–0.34), respectively. In contrast, pericardial fluid was significantly enriched (p < 0.001) in NT-pBNP ([PF]/[P]: 1.9 (1.06–2.73)) and even more so for FABP4 ([PF]/[P]: 3.90 (1.47–9.77)). Moreover, in pericardial fluid, the adipocytokines interrelated all significantly positive and correlated negative to hsCRP, whereas for NT-pBNP only a significantly positive correlation with adiponectin was found. These interrelations were distinct from those in the plasma, as were the correlations of the pericardial biomarkers with patient characteristics compared to plasma.In cardiovascular disease patients, the pericardial cavity is a distinct adipocytokine microenvironment in which especially FABP4 is mainly derived from the heart. Adipocytokines are a link between obesity, inflammation and cardiovascular diseases. In obesity, adipose tissue can become dysfunctional, resulting in a change in cellular composition towards a pro-inflammatory phenotype. MoreoveThe aim of this study was to discriminate, in cardiovascular disease patients, between locally produced adipocytokines, related to adipose tissue inflammation and myocardial dysfunction, measured in the pericardial fluid and those supplied by the circulation. We tested the hypothesis that the pericardial cavity of cardiovascular disease patients is a unique adipocytokine microenvironment, the composition of which can only partly be predicted from the circulation. For this, levels of FABP4, leptin, lipocalin-2, neutrophil elastase, proteinase-3, and adiponectin were measured and compared in plasma and pericardial fluid, harvested from a considerably larger group of patients  u2EDTA and aprotinin. Pericardial fluid samples (2–5 mL), also collected in K2EDTA and aprotinin, were obtained right after median sternotomy and section of the pericardium. All samples were centrifuged (3220 G for ten minutes at 4°C) and the supernatants were stored at -80°C until analysis. The patients included in this study were free of pericarditis, pericardial effusion and pericardial tamponade and only pericardial fluid samples without visible blood contaminants were considered for analysis. Routine plasma analyses for HbA1c, lipids and creatinine were performed at the Odense University Hospital Clinical Biochemistry laboratory. Clinical information was obtained from the patients’ medical records and patient characteristics were disclosed to the researchers after finalization of their data collection. These and general patient characteristics are summarized in All experiments were performed in accordance with institutional guidelines and were approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20100044 and S-20140202). Investigations were performed conform the principles outlined in the Declaration of Helsinki and informed written consent was obtained from all patients prior to collection of the biopsies and information from their medical records. The day before elective coronary artery bypass grafting (CABG) and/or cardiac valve replacement (VR) surgery, peripheral venous blood 5 mL) was drawn into pyrogen-free tubes containing K mL was dhttp://www.pharma.hku.hk/sweb/antibody/ELISA.php), as described[Plasma and pericardial fluid levels of NT-pBNP, FABP4, lipocalin-2, neutrophil elastase, proteinase-3, adiponectin and hsCRP were measured using in-house ELISA kits and a hsCRP assay, respectively  are shown as mean ± standard deviation (SD). Concentrations of biomarkers and continuous variables that were not normally distributed are shown as median value and interquartile range. Paired comparisons between observations in plasma and pericardial fluid of the same individuals were performed with Wilcoxon matched-pairs signed rank tests. Associations between the levels of biomarkers in plasma and pericardial fluid and between biomarker concentrations and continuous clinical variables were assessed with Spearman correlation tests. Evaluation between all categorical clinical variables and biomarker levels were performed using Mann-Whitney U tests. Statistical analyses were performed with GraphPad Prism v6.05 . Differences and associations were considered statistically significant at iterature, 17.c < 42 mmol/mol). The creatinine values indicated normal kidney function. Most patients had been prescribed a combination of low-dose aspirin, inhibitors of the renin angiotensin system and diuretics.total protein, median (interquartile range), total protein (5.4 (2.9–9.4) and (10.6 (4.7–26), respectively). Now, leptin (1.8 (1.3–2.8)) and lipocalin-2 (2.2 (1.6–3.1) also seemed significantly enriched in the pericardial cavity. Neutrophil elastase (0.68 (0.39–1.7)), proteinase-3 (0.51 (0.27–1.04)), hsCRP (0.50 (0.21–0.91)), and adiponectin (0.64 (0.45–0.92)), on the other hand, made up a significantly smaller fraction of the total protein concentration in pericardial fluid compared to plasma.In pericardial fluid, the concentrations of all 8 biomarkers and the total protein concentration differed significantly from those in the plasma , NT-pBNPRegardless of the different concentrations in the two compartments, the pericardial fluid concentrations correlated positively with the plasma concentrations for each biomarker investigated except NT-pBNP .In pericardial fluid, the levels of FABP4, leptin, lipocalin-2, neutrophil elastase, proteinase-3 and adiponectin were positively interrelated and each displayed an inverse correlation to the local concentrations of hsCRP . In the In the pericardial cavity, NT-pBNP levels were significantly positively correlated to age and cardiac valve disease. FABP4 levels correlated positively with plasma creatinine concentrations. Leptin related positively to BMI and circulating triglycerides. Lipocalin-2 correlated positively to age and plasma creatinine. Neutrophil elastase was positively linked to cardiac valve disease. Adiponectin correlated negatively with ischemic heart disease, type 2 diabetes, plasma triglycerides, and use of a biguanide, but positively with age, and circulating HDL cholesterol .In plasma, NT-pBNP concentrations were inversely correlated with being diabetic, use of statins and a biguanide. FABP4 was positively linked to cardiac valve disease, age and HDL cholesterol. Leptin correlated positively with BMI and triglycerides. The concentration of lipocalin-2 was positively related to age, creatinine and the use of inhibitors of the renin angiotensin system. Neutrophil elastase was negatively linked to HDL cholesterol and biguanide use. Proteinase-3 was also negatively linked to plasma HDL cholesterol levels, but positively linked to circulating creatinine. hsCRP was negatively correlated to HDL cholesterol levels whereas adiponectin was negatively correlated to BMI, type 2 diabetes, HbA1c concentrations, triglycerides and use of statins, but positively to cardiac valve disease, age and HDL cholesterol .The main findings of this study are that i) in the pericardial cavity, the concentrations of all 8 biomarkers significantly differed from those in the plasma with NT-pBNP and FABP4 being more abundant and the remaining biomarkers less abundant, ii) the pericardial fluid concentrations of all biomarkers, except NT-pBNP, correlated positively with their plasma levels, iii) the pericardial fluid levels of FABP4, leptin, lipocalin-2, neutrophil elastase, proteinase-3 and adiponectin were strongly interrelated and negatively related to the pericardial fluid concentration of hsCRP, whereas in the venous plasma less and only positive interrelationships were observed, and that iv) pericardial fluid biomarker concentrations correlated with many patient characteristics for which no correlations were found in the plasma. These findings demonstrate that the pericardial cavity is a distinct adipocytokine microenvironment and indicate that especially FABP4 is produced by the heart in CVD patients.Dysfunctional adipose tissue, such as hypertrophic adipose tissue, is associated with increased release of pro-inflammatory adipocytokines from adipocytes and immune cells, 3. ThesThe composition of the pericardial fluid may both reflect and contribute to the pathophysiology of the heart and its coronary vasculature.We previously obtained suggestive evidence that the pericardial cavity in cardiovascular disease patients is a pro-inflammatory atherogenic micro-environment, distinct from the plasma. In the Pericardial fluid was sampled from patients with coronary artery or cardiac valve disease. During cardiothoracic surgery, opening of the pericardial cavity enables easy and safe sampling of pericardial fluid, 8. PatiOnly a few studies compared pericardial fluid and plasma from patients suffering from heart failure or coronary artery disease, 8–11. IThe pericardial fluid is regarded by some as an ultrafiltrate of the plasma, whereashsCRP, an important risk factor and independent predictor of cardiovascular events is mainlIn the systemic circulation of coronary artery disease or heartOur study is a cross-sectional observational study in very diverse patients with the aim to obtain broad ranges of local and circulating concentrations of adipocytokines and markers of heart failure and inflammation. For obvious ethical reasons, pericardial fluid could not be obtained from healthy volunteers. Our results may therefore be affected by for instance presence of comorbidities or the severity of chronic cardiovascular condition. The associations that we identified could be accidental and coincident rather than indicative of causal relationships. We determined pericardial fluid concentrations but could not consider the volume of the pericardial fluid. Echocardiography information on the pericardial space was not Despite these limitations, our suggestive evidence of the pericardial space as a distinct adipocytokine micro-environment adds to the proposal that the pericardial space holds potential for improved diagnosis and local treatment. It inviIn conclusion, the results demonstrate significant differences of the concentrations of all biomarkers investigated between the pericardial fluid and plasma, and significant different interplays between adipocytokines and inflammatory signals within each fluid compartment, indicating that the pericardial cavity is a distinct micro-environment of the heart in which leptin, lipocalin-2 and especially FABP4 seem to be derived from the heart.


Jackson Pollock’s most celebrated abstract paintings were produced with the so-called dripping technique. By pouring liquid paint with the help of a stick or from a can, Pollock deposited viscous fluid filaments on a horizontal canvas, rhythmically moving around it. The intricate webs of lines, ubiquitous in his compositions, have fascinated art historians and scientists. Based on image analysis of historical video recordings, we experimentally reproduced the painting process. We conclude that Pollock avoided the appearance of the hydrodynamic instabilities, contrary to what was argued by previous studies. Pollock selected the physical properties of the paint to prevent filament fragmentation before deposition, and applied it while moving his hand sufficiently fast and at certain heights to avoid fluid filaments from coiling into themselves. An understanding of the physical conditions at which these patterns were created is important to further art research and it can be used as a tool in the authentication of paintings. His pet al. [Jackson Pollock described the technique himself . In summet al. .It is important to emphasize that the technique used by Pollock has been incorrectly named ‘dripping.’ The term, in the fluid mechanics’ literature, refers to the break up of a fluid jet onto drops resulting from a surface tension instability . As discet al. [A clear illustration of the technique can be observed in ‘Number 14: Gray’ . Fig 1 iet al.  argued, et al. .et al. [The phenomenon of liquid rope coiling is central to understand the dripping technique. It can be summarized as follows: the gravitational motion of a slender fluid filament falling into itself becomes unstable, under certain conditions, developing a coiling motion. Due to its relevance to many related subjects, it has been studied extensively –16. In pet al.  summarizν is the fluid kinematic viscosity, g is the gravitational acceleration, H is the height from which the fluid is dispensed, Q is the fluid volumetric flow rate, and d is the filament diameter at the height H. Hence, the following functional relation can be expected:For the simplest case of a Newtonian filament with negligible surface tension effects, four dimensionless groups are relevant :Ω*=Ω. For thρ is the fluid density and σ is the surface tension. If the value of Oh is large, surface tension effects can be considered negligible. The value of the Ohnesorrge number also determines whether or not a long viscous filament will fragment or not, as discussed by [Furthermore, if the surface tension effect is relevant and is also included in the analysis, an additional dimensionless group emerges. One reasonable option is to consider the Ohnesorge number:ussed by . Note, hOh > 1 and Πσ > 1, then surface tension effects can be considered negligible.If both Another property which can be relevant for the case of paints is the viscoelasticity of the liquid . If the De ≪ 1, the viscoelastic effect can be discarded. Furthermore, the viscoelastic relaxation time can also be compared to the characteristic gravitational time leading to:If λ and De would have to be small.For viscoelastic effects to be considered unimportant both Πet al. [In the context of the present study, it is important to consider the case when the substrate and the nozzle issuing the fluid have a relative velocity. Chiu-Webster and Lister , for exaet al.  evaluateHhand = 20 cm.We conducted a series of measurements of the speed and height at which Pollock painted. These measurements were obtained from the documentary video from Hans Namuth , in whicUhand, the height from which the paint drips, H, depicted in Uload (the speed at which the stick is retrieved from the can of paint). Uload is used to determine the amount of fluid and the flow rate dripping from the stick.We analyzed side-view sequences for which the distance to the canvas could be measured in time. In parts of the movie, Pollock painted on top of a transparent glass sheet, as shown schematically in H, above the horizontal substrate onto which the paint is dripped. The syringe is mounted on a syringe pump such that it produces a constant flow rate, Q. The syringe exit nozzle has a diameter d. As the fluid leaves the syringe, it forms a continuous steady filament that thins as it falls under gravity, reaching a diameter do as it reaches the substrate. The mean fluid velocity leaving the nozzle, U, can be calculated as U = 4 Q/(πd2). To recreate the motion of the hand . The paint was nearly Newtonian; it had nearly constant viscosity, μo, for very low shear rates. Its shear-thinning nature, quantified by the power index, n, is small (n ≈ 1). To assess the viscoelastic nature of the fluid, oscillatory rheological tests were conducted. Considering the technique used by [τ, which was found to be of O(10−3) s, indicating weak viscoelastic effects for low shear rates. used by , we fittH, and the speed of the substrate, Uhand. Hence, for each campaign, the values of the nozzle diameter, flow rate, and fluid properties were kept fixed. The nozzle diameter was d = 2.0 mm for all cases, while the flow rate varied slightly for each case . The histogram of the hand speed is presented in Due to the low resolution of the videos, we were not able to obtain simultaneous height and hand speed measurements. Therefore, it was not possible to correlate these two quantities.g cos 30° would drive the downward flow, the additional effect due to the inclination of the stick is not substantial and was neglected in the analysis.It is also relevant to note that, from the historical videos, the stick used to manipulate the fluid was not held vertically. During the application of the paint to the canvas, the stick would be swung from roughly -30° to +30° . Since, at a maximum inclination, the flow rate is expected to decrease by 13%, as only d1 ≈ 3 mm. Comparing this thickness to the mean height from which the filament is being deposited, we calculate their aspect ratio, Γ = H/d1. This metric indicates the slenderness of the fluid filaments, which can be used to assess if they will fragment or not. From the measurement of the mean height, we estimate ΓPollock ≈ 50. According to Driessen et al. [Oh2 would remain stable, where Oh is the Ohnesorge number Pollock ≈ 120, indicating that gravitational forces are much larger than surface tension ones, which confirms the fact that fragmentation of fluid filaments rarely occurred in Pollock’s traces.For the case of Pollock’s paintings, considering the physical properties of paint B1 see , and a ton value . TherefoOh2 from , valid fDe)Pollock ≈ 1.2 × 10−4 and (Πλ)Pollock ≈ 7.9 × 10−3, which indicates that viscoelastic effects were not relevant for Pollock’s painting action.Also, from the video measurements, considering the mean values we calculate 1/3 is a normalized speed and Q* = Q(g/ν5)1/3 is the normalized flow rate. Therefore, the transition from curled to straight lines would be expected when:Therefore, the critical condition for transition can be expressed in terms of other physical variables of the problem:H, above a horizontal substrate that moved at a constant speed Uhand. The setup emulates Pollock’s painting action, with the difference that in this case the ‘hand’ is fixed and the substrate steadily translates. Experiments were conducted for several fluids, and for a range of heights and speeds to produce traces which were either straight or coiled.Now, to obtain the proportionality constant in H* > 8, a line is fitted through the transition considering the trend of κ = 3.04 fits the transition relatively well.Uf is the speed of the flow when it reaches the ground. Interestingly, Ribe et al. [ΩR and Uf scale in the same manner. Hence, the two criteria would lead to similar transition conditions.Another, more direct, manner to assess the cessation of coiling resulting from the relative displacement from the hand and ground can be formulated considering the conditionhandUf≥1,where UfQstick. The experimental results were fitted to a scaling , leading to Γ = 56.3 which is very close to the fragmentation onset. One would expect some of of the fluid filaments to lose their stability and fragment. In fact, a closer inspection of Pollock’s works in the dripping period also reveals the occasional occurrence of drops (fragmented filaments). Notwithstanding the precise values of the properties of the paint, for the measured values of speed and height, the large majority of the traces would be straight lines. If we could argue that Pollock always painted with the same action (as that in videos), we can conclude that very few traces would coil. In other words, the painting action was conducted such that the appearance of the coiling instability would rarely appear.Since the actual properties of Pollock’s paints are not known, we can recalculate the region of painting action considering variations in the viscosity of the paints. Note that the change in viscosity changes the normalization of U* would be larger. Therefore, with less viscous fluids, coiling traces could be produced without having to move the hand too quickly. A slowly moving hand would then produce more uniform lines. Also, if the viscosity is reduced, the value of H* would increase. The filaments would thus have to be deposited from lower heights to prevent the formation of curled lines. These arguments are in good agreement with the way in which Pollock painted, as per the recordings and the measurements in Painters, including Pollock, commonly manipulate the fluid properties by adding solvents. Although changes in surface tension and density do occur, the viscosity is significantly affected by the addition of such additives. In general, the addition of fluid-thinners, leads to a reduction of viscosity. Considering the map shown in By locating the painting action in the general map of behavior, in the appropriate dimensionless form, we demonstrate that Pollock aimed to prevent the coiling instability. This result could be of importance for authentication: a painting with too many coiled traces would indicate that the painting was not created by Pollock. Furthermore, understanding the conditions for which the coiling instability can be prevented could have implications in practical applications where such an effect needs to be prevented, as in the case of ink-jet printing or the fabrication of optic fibers.S1 File(TXT)Click here for additional data file.


In a genomic screen for determinants of the tumour vasculature, we identified insulin receptor (INSR) to mark the tumour endothelium. As a functional role for insulin/INSR in cancer has been suggested and markers of the tumour endothelium may be attractive therapeutic targets, we investigated the role of INSR in angiogenesis.In a genomic screen for determinants of the tumour vasculature we identified insulin receptor to mark the tumour endothelium.The current report demonstrates the following: (i) the heavy overexpression of INSR on angiogenic vasculature in human tumours and the correlation to short survival, (ii) that INSR expression in the tumour vasculature is mainly representing the short oncofoetal and non-metabolic isoform INSR-A, (iii) the angiogenic activity of insulin on endothelial cells (EC) in vitro and in vivo, (iv) suppression of proliferation and sprouting of EC in vitro after antibody targeting or siRNA knockdown, and (v) inhibition of in vivo angiogenesis in the chicken chorioallantoic membrane (CAM) by anti-INSR antibodies. We additionally show, using preclinical mouse as well as patient data, that treatment with the inhibitor sunitinib significantly reduces the expression of INSR-A.The current study underscores the oncogenic impact of INSR and suggests that targeting the INSR-A isoform should be considered in therapeutic settings. Previously identified tumour EC targets7 were most often neither fully specific for the tumour endothelium, nor were they absent in physiologically activated endothelium, rendering them inferior for anticancer strategies in patients. Therefore, the identification of unique targeting molecules on tumour EC is urgently needed.Angiogenesis is an intricately regulated process required during normal development and a large array of different pathologies. Strategies to inhibit blood vessel formation are therefore considered a promising therapeutic approach.9 Using cDNA libraries from freshly isolated colorectal tumour EC, patient-matched normal colon tissue EC and placenta derived EC, a broad profile of differentially expressed genes in tumour endothelium, but not physiologically activated placenta EC, was generated.9 Furthermore, we have shown that targeting these markers can have therapeutic effects in animal models.13We have previously identified several markers of angiogenic tumour EC.14 A large body of research has been performed on IGF1R and its function in cancer is well documented.15 Anti-IGF1R strategies have been clinically used to treat cancer. The role of INSR in cancer is less well studied but a role for insulin and INSR has been suggested.17 Moreover, associations between insulin treatment and the occurrence of cancer have been observed.19 A role for INSR in cancer therapy is becoming eminent since it has been suggested that the non-metabolic, oncofoetal isoform INSR-A is the variant of INSR that is overexpressed in cancer.21 Targeting only INSR-A would then become possible without intervention in the metabolic functions of the insulin/INSR pathway. Since INSR-A is a shorter isoform, lacking exon 11, which is present in the full-length protein INSR-B, it might be difficult but not impossible to specifically target INSR-A.In this study, we demonstrate that the insulin receptor (INSR) is one of these tumour EC markers having a clear role in tumour angiogenesis and a potential impact for the clinical management of cancer therapy. The insulin signalling axis consists of, next to insulin, two structurally related ligands, i.e. insulin-like growth factor (IGF)-1 and -2, a set of receptors, i.e. INSR, IGF1R and IGF2R and a family of seven insulin-like growth factor-binding proteins (IGFBP).The current study describes the overexpression of INSR, which is mainly INSR-A, in the vasculature in a variety of human cancer types, where it is observed to predict decreased patient survival. Since mice are metabolically insensitive to insulin overstimulation and deprivation, extensive mechanistic investigation of specific targeting of INSR-A in a mouse model is not feasible. We present here for the first time the finding on the embryonic origin of the INSR-A isoform and its overexpression in the tumour vasculature. Our results on intervention in the insulin/INSR signalling axis by siRNA and antibody-mediated targeting in vitro and in vivo demonstrate the capacity of this pathway to regulate tumour angiogenesis. The current insights suggest that an INSR-A targeting strategy may be an attractive angiostatic cancer treatment.22 Similar procedures were applied to isolate EC from murine tumours  to enable detection of alternative splicing. Samples were sequenced on an Illumina HiSeq 2000  with HCS 2.2.68 software suite (Illumina). RNA sequencing experiments and analysis of the data was performed by the genomics core facility at the Netherlands Cancer Institute. The obtained reads (50 million 50 bp paired end reads per sample) were mapped to the mouse reference genome (GRCm38). Genome mapping and differential expression analysis was performed with Cufflinks software.23 For detection of splice variants, the reads were mapped to the whole mouse genome, including exon and intron DNA.Next generation sequencing was performed to analyse the transcriptomes of sorted mouse tumour endothelial cells (TEC), whole embryos and adult mice to profile re-expression of embryonic genes in TEC. PolyA 25 Quantification was performed by blinded evaluation of coded samples by two independent observers, following a score ranging from lack of expression (score 0) to very high expression (score 4). The expression was quantified in 11 different tumour types with up to 20 samples per tumour type of both in-house stained tissues and tissue staining from the human protein atlas (www.proteinatlas.org).Immunohistochemical staining of INSR in paraffin sections of human tumours and corresponding normal tissues was performed using mouse monoclonal anti-INSR antibody  according to previously described protocols.http://r2.amc.nl) was employed on three available colorectal cancer Affymetrix gene expression data sets27 using average INSR gene expression as selection parameter. Relapse-free survival at 120 months was taken as evaluation point.Kaplan–Meier scanner functionality in R2  were seeded in gelatine-coated 96-well cell culture plates as described previously.10 Briefly, 24 h after seeding, culture medium with or without compounds was added and cells were grown for an additional 72 h. Cell viability was assessed using the CellTiter-Glo® luminescent Cell Viability Assay  according to the manufacturer’s instructions. Tritium-thymidine incorporation to measure cell proliferation by DNA synthesis was performed as previously described.28Endothelial cells (5 × 10T = 0 h and T = 6 h.29 Wound closure (μm2) was expressed as a percentage of control wells. EC spheroids were created using the hanging drop technology,30 as detailed in the 31Endothelial cell migration was performed using a guided 96-well pin tool  and Leica DMI3000 microscope  with Universal Grab 6.3 software , at time points N = 21) were used for the evaluation as described previously.24 The tissues of 12 primary tumours came from EudraCT 2006-006491-38 (https://eudract.ema.europa.eu/) phase II trial where the main objective was the investigation of the response rate of the primary RCC tumour to sunitinib at 50 mg/day for 2 cycles of each 4 weeks on treatment followed by 2 weeks off treatment. At completion of the 2nd cycle patients underwent cytoreductive nephrectomy as per protocol 1 day after discontinuation of sunitinib. Remaining 9 primary tumours were provided from a second phase II study (NCT00715442) of prior to surgery treatment with sutent in patients with primary clear cell mRCC. These patients were restaged after one cycle of systemic therapy, began a second cycle of systemic therapy with sunitinib, and discontinued therapy 1 day before nephrectomy. Clear cell RCC tissues from non-treated patients were used as controls.Primary tumours from patients with RCC treated prior to surgery with sutent  at the indicated concentrations. Vasculature was visualised and analysed on embryo development day 9 as previously described.29Detailed methods on growth, handling and treatments of the eggs are described in the ®-photodynamic therapy (PDT) was performed32 on embryo development day 11. Within PDT-treated areas, 20 μl anti-INSR monoclonal or polyclonal antibodies (10 μg/ml) were administered topically twice, immediately after PDT and 24 h later. Quantification based on the fluorescence angiographies was performed on embryo development day 13.33Visudyne34 Four- to six-week-old male severe combined immunodeficient (SCID) mice were housed under pathogen-free conditions. Mice were injected subcutaneously with 5 × 106 HT29 cells. Mice received treatment with sunitinib malate (40 mg/kg) or a corresponding amount of vehicle, once daily, 7 days a week, by oral gavage. After 6–8 weeks of treatment, animals were sacrificed; tumours were harvested and subsequently snap-frozen in liquid nitrogen for qPCR.Animal experiments were approved by the local Ethical Review Committee and performed as described previously.N = 5) and their paired healthy kidney tissues (N = 5) were also collected and snap-frozen in liquid nitrogen. Visudyne®-PDT-treated zones (see below) of the CAM were fixed. Samples were processed for RNA isolation using TRIzol Reagent. Cultured cells and mouse HT29 tumours were subject to RNA isolation using RNeasy mini columns  according to the manufacturers’ instructions. Primers were designed according to previously published guidelines,35 to allow detection of species-specific transcripts. This was, however, not possible for the variant-specific detection due to (i) absence of the long (INSR-B homologous) isoform in chicken and (ii) too extensive homology between mouse and human in the region of exon 11.Tumour specimens from patients with kidney malignancies (35qPCR was performed using SYBR green reagent (Bio-Rad), in a 2-step protocol at a Tm of 60°C on a CFX96 thermal cycler (Bio-Rad) and runs were analysed using CFX manager software (Bio-Rad). PCR primers are listed in Supplementary Table t-test, Mann–Whitney U-test or one-way ANOVA in GraphPad Prism. Where relevant, the Bonferroni post-test was used to correct for multiple comparisons. P values < 0.05 were considered statistically significant.All values are given as mean values ± SEM. Statistical analyses were done using In a genomic search for specific tumour endothelial markers, we compared the transcriptomes of sorted colon carcinoma tumour endothelial cells (TEC) and normal colon endothelial cells (NEC). Endothelial cells (EC) of human placenta (PLEC) were used for comparison to a source of physiologically activated EC. We identified insulin receptor (INSR) as a specifically and highly overexpressed gene in the tumour vasculature of colorectal carcinoma (CRC) and validated this by qPCR. A similar result was found for EC from renal cell cancer tissue (RCC) Fig. . Immunohp < 0.01, Supplementary Fig. p < 0.01, Supplementary Fig. The overexpression of INSR in tumour vessels was further assessed and quantified in a series of 11 different tumour types. A strong to very strong expression was observed in the vasculature of these tumours, while vessels in the corresponding normal tissues showed only negligible expression  patients. In a recent phase II clinical study to investigate the need for removal of the primary tumour in patients with metastasised disease, surgical debulking was performed after two cycles of sunitinib treatment. We previously published that these two cycles of sunitinib treatment effectively inhibited angiogenesis and enhanced the number of mature blood vessels.ney Fig. , two cycney Fig. . In thes 24 Fig. . This su 24 Fig. . As we h 24 Fig. . Suppres 24 Fig. , right,  24 Fig.  left, re 24 Fig. .Fig. 3Al36 While the induced angiogenesis response by PDT resulted in an approximately 10-fold stimulation of INSR expression  model was used.28 HUVEC were used for these assays. A significantly enhanced sprouting was observed in the presence of insulin. The enhanced in vitro sprouting was based on an increase in the number of sprouts . Sprouting  and the average sprout length . This significantly reduced the cell number (**p < 0.01), as compared to a scrambled siRNA .Independent verification of the role of INSR in the biology of EC was generated by knockdown of INSR by siRNA technology . HUVEC w® and subsequent exposure to light.32 At the molecular level, qPCR showed that expression of INSR was significantly induced during this angiogenic stimulation, a feature that was also visible for VEGFR2. A similar trend for VEGFR1 was observed , as quantified in four concentric areas . In this model angiogenesis was induced by photodynamic vaso-occlusion after injection of photosensitiser VisudyneWe identified insulin receptor (INSR) as a marker of the tumour vasculature in a genomic search analysing RNA preparations from isolated colorectal carcinoma (CRC) endothelial cells (EC) and their counterparts from normal colorectal tissue samples. In this study, markers of physiologically activated endothelium were excluded by co-analysis of isolated placental endothelial cells. In an independent deep sequencing study for identification of embryo-specific genes that become re-expressed in tumour endothelial cells, INSR was also found to be a specific marker of the tumour vasculature. This overexpression of INSR suggests a function in the process of tumour angiogenesis, and points to a relationship with tumour aggressiveness and prognosis. Indeed, an inverse correlation between the expression of INSR and relapse-free survival was observed in CRC expression profiles. For these reasons we investigated the role of INSR in the biology of EC and in the formation of (tumour) vessels. This knowledge would be instrumental for the assessment of the relevance of INSR and the application of INSR targeting for angiostatic cancer therapy. Interestingly, immunohistochemical analysis of human CRC samples revealed that expression of INSR was almost exclusively found in the vasculature of colorectal tumours. Similar results were observed in ten other tumour types. We show in the current report that INSR was induced in EC by angiogenic stimulation. We present several lines of evidence for the pro-angiogenic function of INSR in EC. Firstly, INSR functions as a growth factor receptor, as exposure to insulin stimulated growth and tube formation of EC in vitro and in vivo. Secondly, targeting of INSR on activated EC displayed effective anti-angiogenic activity. Thirdly, anti-angiogenic treatment of tumours was shown to suppress the expression of vascular INSR.14 Enhanced complexity comes from alternative splicing of INSR in INSR-A and -B on the one hand and heterodimerisation of INSR and IGF1R on the other.Insulin receptor is part of a complex signalling axis, in which insulin-like growth factor-1 receptor (IGF1R) and IGF2R take part, together with the INSR ligands insulin and the insulin-like growth factors (IGF)-1 and -2, as well as a series of seven insulin-like growth factor-binding proteins (IGFBP1-7).14 This signalling axis has pleiotropic and versatile functions in many cells and its role in EC is gradually becoming unveiled. It is important to realise that the ligands, insulin and the IGFs, although binding to their cognate receptors with the highest affinity, can all bind to the other two receptors as well, albeit with lower affinity. The stimulatory effect of insulin on sprouting of EC has been reported previously,41 and these observations may explain why patients with type 2 diabetes and those that are treated with daily insulin supplementation have an increased risk of developing malignant neoplasms.18 Our observation of the correlation between INSR expression and cancer patient survival, as shown in Fig. 42 Together, these considerations suggest that an angiostatic INSR targeting approach may constitute a valid anti-cancer strategy in the clinic.Differences in the function of INSR-A and INSR-B now suggest that INSR targeting may have clinical impact in the field of oncology. We reviewed the background of this pathway recently.21 who recently described INSR-A as the main isoform in tumour endothelium and endothelial cells in vitro. We furthermore provide functional studies on the role of INSR-A in vitro and in vivo and on the regulation during angiostatic treatment of patients. INSR-A is the main isoform in foetal tissues  and in cancer, and binds insulin-like growth factor 2 (IGF-2) with high affinity, which elicits mitogenic effects,20 thereby contributing not only to organism development, but also to cancerous growth.The most interesting finding of the current study was that the major isoform of INSR in the tumour vasculature is the proliferation associated isoform INSR-A and not the metabolic isoform INSR-B. Furthermore, RNA sequencing demonstrated INSR-A to be the dominant embryonic form, confirming its oncofoetal characteristics. Our study independently verifies the recent results by Roudnicky et al.,45 Several neutralising antibodies and small molecule receptor kinase inhibitors have been developed, such as dalotuzumab and OSI-906.47 However, therapies targeting IGF1R alone or in combination with other drugs were tested but eventually revealed contradictory results.51 Resistance to IGF1R targeting agents is believed to be mediated by upregulation of INSR in tumours, especially the oncofoetal INSR-A variant, which has a high affinity for IGF2 and as such sustains proangiogenic signalling despite inhibition of IGF1R.52 Moreover, INSR-A is less involved in metabolic activities of insulin than INSR-B.53 It is interesting to note here that Gallus gallus (chicken) expresses only one isoform of INSR, being the shorter INSR-A variant. We showed that targeting of INSR in this model is extremely effective at inhibition of angiogenesis , EGFR , HER2  and PDGFR . Hormone receptors are growth factor receptors as well, as many hormones signal to stimulate cell growth. Although insulin is known as a hormone produced in the beta-cells of the pancreas with liver-, muscle- and fat cells as its targets, it also has growth factor functions. The strong overexpression of INSR (mainly INSR-A) in the tumour vasculature and its functional role in activation signals suggests that this signalling axis may serve as a target for therapy. Relatedly, a similar selective overexpression was found for the follicle stimulating hormone (FSH) receptor. Application of targeting the FSH receptor for therapeutic purposes was also previously suggested.24 We now show that, in analogy to VEGFRs, INSR is also suppressed by angiostatic treatment with sunitinib. The rapid rebound of angiogenesis upon halting the treatment with sunitinib before surgery, but supposedly also in the treatment rests that occur after each treatment period of 4 weeks, is clearly associated to relative induction of INSR-A vs. INSR-B expression clinical evidence on the importance of INSR in tumour angiogenesis was provided by the demonstration that anti-angiogenic treatment reduces vascular INSR expression. Sunitinib is a tyrosine kinase inhibitor that blocks the signalling pathways of a series of growth factor receptors, among which are VEGFR1 and VEGFR2, PDGFR-b, c-KIT, and FLT-3. In our CAM and mouse models, treatment with sunitinib significantly decreased the expression levels of endothelial INSR  IGF-IR cannot be distinguished from phosphorylated INSRs by IHC. Overall, though theoretically, targeting the INSR-A is an exciting possibility, currently tools and technologies are lacking to accomplish this. Further insight on the relative roles of these isoforms could be generated by the development of genetically engineered mouse models (GEMMs) that allow for  expression of either isoform in the endothelium. This may open avenues to develop tools to target INSR-A variant through either genetic means, or the development of targeting moieties with specific effects on (hybrid) receptors of this isoform.Summarising, we have demonstrated that overexpression of INSR is associated with increased angiogenesis. We therefore suggest that direct targeting of endothelial INSR, supposedly most beneficial through targeting of only INSR-A, may present an interesting treatment strategy.Supplementary Material


Juvenile nasopharyngeal angiofibroma is a benign disease affecting young males with a propensity to invade intracranially and into the orbit along preformed pathways. Complete surgical excision is the mainstay of management. Patients with multiple recurrences along with tumour extension into skull base and orbit can be considered for external beam radiation as either adjuvant or definitive treatment. Possibility of radiation-induced malignancy has been speculated by many authors, proof of which exists in only two studies so far. This report focuses on radiation-induced leiomyosarcoma in a patient with recurrent juvenile nasopharyngeal angiofibroma. Juvenile nasopharyngeal angiofibroma is a benign tumour originating in the nasopharynx, presenting in adolescent males with nasal mass, epistaxis, nasal obstruction, cheek swelling, and proptosis. Though benign, it has a capacity for local invasion along preformed pathways. Thus, JNA can encroach into critical structures like optic nerve, internal carotid artery and present intracranially. Surgery is the main modality of treatment, preceded by embolisation of arterial feeders in case of extensive tumours involving infratemporal fossa or intracranial involvement. For tumours infiltrating into infratemporal fossa, sphenoid sinus, base of pterygoid, cavernous sinus, foramen lacerum, anterior and middle cranial fossa , subtotaOur patient is a 37-year-old male who was previously diagnosed as a case of juvenile nasopharyngeal angiofibroma. He was diagnosed with the condition at 30 years of age during which he underwent his first surgery by the lateral rhinotomy approach. Tumour was seen to involve the nasopharynx and sphenoid sinus, eroding the basisphenoid. Optic nerve and carotid artery were not involved. He underwent endoscopic excision 2 years later for tumour involving nasopharynx and sphenoid sinus. The patient remained symptom-free for 2 years, following which lateral rhinotomy excision was performed for a recurrent tumour involving nasopharynx, sphenoid sinus, eroding basisphenoid, and vidian canal. Postoperative histopathology confirmed the presence of angiofibroma and ruled out the presence of any sarcomatous element. In view of the frequently recurring nature of the tumour, the patient was given 45 Gy, 25 fractions of conformal radiotherapy. During the routine follow-up nasal endoscopy after 3 years, a fleshy vascular mass was seen in the nasopharynx. The patient underwent subtotal excision at another centre by lateral rhinotomy.The specimen block was reviewed. Sections examined showed spindle-shaped tumour cells arranged in long intersecting fascicles, with moderate nuclear pleomorphism and increased mitosis . Tumour Contrast-enhanced computed tomogram showed heterogeneously enhancing soft tissue mass lesion in the right posterolateral wall of nasopharynx measuring 6 × 3.7 × 4 cm with erosion of the adjacent bone with involvement of the pterygoid muscles .In view of the extensive involvement of skull base, surgery was not considered to be suitable for providing a negative margin. Patient has thus been planned for neoadjuvant chemotherapy (gemcitabine based) followed by chemoradiation.Surgery is undoubtedly the definitive treatment option for juvenile nasopharyngeal angiofibroma. Dilemma arises when there are multiple recurrences, extensive intracranial, intraorbital, or infratemporal fossa involvement. There are multiple case series and case reports citing the use of radiation therapy for this benign condition. Cure rates of 80% have been reported for both these modalities of treatment . The recThe tumour arose in a previously irradiated field.The new tumour is histologically different from the original condition.There was no evidence of the new tumour at the time of radiation therapy.A latency period existed between irradiation and the development of the new tumour.Radiation-induced malignancy (RIM) is thus the rarest and the most feared complication following irradiation for a benign condition like JNA. Our patient fulfilled the criteria for RIM as stated by Cahan et al.  and MurrRIM in head and neck has an incidence of 0.9% and develops after a mean latency of 22 years with a mean radiation dose of 6035 cGy, most commonly located in paranasal sinuses . The samThe mechanism behind the occurrence of RIM is not well established. One hypothesis as proposed by Brenner and Shuryak et al. , 23 inclSurgery is generally the preferred modality for RIM; however, as in our case due to the involvement of critical structures like skull base, nonsurgical modality including radiation have also been employed for management by other authors . But in Though current radiation techniques have been significantly refined in terms of limiting exposure to uninvolved tissues and putative organs at risk, it is essential to bear in mind this small but potentially life-threatening risk while irradiating benign diseases in the case of children.Sarcomatous change in JNA following irradiation is the rarest and the most dangerous complication ever reported. This case report aims to lay emphasis on this aspect of managing JNA which recur frequently. Multiple recurrences and skull base involvement precludes surgery as an option in our patient. Though both the modalities of treatment have equal recurrence rates, morbidity and mortality, use of conformal radiotherapy should be carefully weighed against the possible occurrence of such complications. The long-term effects of IMRT and stereotactic radiosurgery also need to be evaluated in detail before these modalities can be used in this setting without being fraught with such complications.


Introduction The underlying assumptions of the CyberKnife®  fiducial tracking system are: i) fiducial positions are accurately detected; ii) inter-fiducial geometry remains consistent (rigid); iii) inter-fiducial geometric array changes are detected and either accommodated with corrections or treatment is interrupted. However: i) soft-tissue targets are deformable & fiducial migration is possible; ii) the accuracy of the tracking system has not previously been examined with fiducial displacement; iii) treatment interruptions may occur due to inter-fiducial geometric changes, but there is little information available to assist subsequent troubleshooting. The purpose of this study was to emulate a clinical target defined with a two, three, or four-fiducial array where one fiducial is displaced to mimic a target deformation or fiducial migration scenario. The objectives: evaluate the fiducial positioning accuracy, array interpretation, & corresponding corrections of the CyberKnife system, with the aim of assisting troubleshooting following fiducial displacement.MethodsA novel solid-water phantom was constructed with three fixed fiducials  & one moveable fiducial (F4), arranged as if placed to track an imaginary clinical target. Using either two fiducials , different combinations of three fiducials  or four fiducials , repeat experiments were conducted where F4 was displaced inferiorly at 2-mm intervals from 0-16 mm. Data were acquired at each position of F4, including rigid body errors (RBE), fiducial x, y, & z coordinate displacements, six degrees of freedom (DOF) corrections, & robot center-of-mass (COM) translation corrections.ResultsMaximum positioning difference (mean±SD) between the reference and live x, y, & z coordinates for the three fixed fiducials was 0.08±0.30 mm, confirming good accuracy for fixed fiducial registration. For two fiducials , F4 registration was accurate to 14-mm displacement and the F4 x-axis coordinate change was 2.0±0.12 mm with each 2 mm inferior displacement validating the phantom for tracking evaluation. RBE was >5 mm (system threshold) at 6-14 mm F4 displacement: however, F1 was misidentified as the RBE main contributor. Further, F1/F4 false-lock occurred at 16 mm F4 displacement with corresponding RBE <3 mm & COM corrections >13 mm. For combinations of three fiducials, F4 registration was accurate to 10-mm displacement. RBE was >5 mm at 6-16 mm F4 displacement: however, F4 false-lock occurred at 12-16 mm with RBE 5-6 mm. For four fiducials, F4 registration was accurate to 4 mm displacement: however, F4 false-lock occurred at 6-16 mm displacement with concerning RBE <2 & <5 at 6 & 8-mm F4 displacement, respectively. False-locks were easily identified in the phantom but frequently uncorrectable.ConclusionsResults indicate fiducial positioning accuracy and system output following fiducial displacement depends on the number of fiducials correlated, displacement distance, and clinical thresholds applied. Displacements ≤4 mm were accurately located, but some displacements 6-16 mm were misrepresented, either by erroneous main contributor  or by false-locks and misleading RBE, which underestimated displacement. Operator vigilance and implementation of our practical guidelines based on the study findings may help reduce targeting error and assist troubleshooting in clinical situations. On-treatment tracking using implanted fiducials for surrogate tumor positioning is commonly applied to soft-tissue targets to overcome X-ray visualization limitations during stereotactic ablative body radiotherapy (SABR) using the CyberKnife® system  -2. AccurThe relative positions of the fiducials to the target and adjacent organs-at-risk (OAR) are established at the planning computed tomography (CT) scan when orthogonal reference digitally reconstructed radiographs (DRR) are generated. Before CyberKnife treatment begins and periodically during treatment  orthogonal live X-ray image pairs are simultaneously acquired by the integrated system and compared with the reference DRR images. The CyberKnife fiducial tracking process is often considered the "gold standard," with a reported success rate of >99% -5. Its oDuring treatment, fiducial displacements may occur, as soft-tissue targets are deformable and undergo inter or intrafraction motion and/or deformation. Interfraction fiducial displacement may also be due to migration, where a fiducial independently moves within tissues to a new anatomical position that is less representative of the target position. Interpretation of fiducial displacement may be difficult, as motion/deformation may not be distinguishable from migration on X-ray imaging. Following a post-implantation "settling" period, fiducial migration is usually considered to be small (≤2.00 mm) and relatively uncommon in most tissues in which CyberKnife fiducial tracking is commonly used (such as the prostate); as such, it is generally considered unlikely to affect tracking accuracy on treatment, with little relevance for intrafraction motion -15. On tBy tracking the fiducial array COM, CyberKnife is considered to have an excellent ability to correct for fiducial array COM offsets and rotations . However, the limits of the CyberKnife fiducial tracking system regarding target array deformations are not well-characterized – even though soft tissue targets are known to be non-rigid and treatment interruptions do occur due to breached RBE thresholds. Other factors known to inhibit fiducial displacement recognition and interpretation in clinical situations include X-ray visualization limitations and the fact that the clinical fiducial array placement may not conform to all recommended ideals. Nonetheless, fiducial array positioning interpretation is critical. In practice, an operator first needs to be notified of any significant fiducial displacement and then needs to determine whether the displaced fiducial represents an unreliable surrogate for a target position that requires the fiducial to be disabled or whether the fiducial is a reliable surrogate for target shape deformation that requires COM corrections to accommodate the new fiducial position — where incorrect interpretation carries the risks of inadequate treatment of the tumor target or increased treatment toxicity due to adjacent OAR irradiation, respectively.The CyberKnife fiducial tracking system has previously been tested in various ways, including in phantoms with static fiducials -5,19-21.An experimental system was designed to model a clinical target. A solid water 30.00x30.00x3.00 cm phantom block was used that contained three fixed fiducials and one moveable fiducial. The fiducial-bearing phantom was placed horizontally between two further solid water phantom blocks, each 30.00x30.00x5.00 cm, to simulate positioning within the body Figure .Within the central fiducial-bearing phantom block, three gold fiducials , termed F1, F2, and F3, were drilled and glued into fixed positions. A fourth moving fiducial (F4) was similarly fixed into the end of a moveable tube and inserted into a 2.00 cm diameter channel within the central phantom. The vertical and horizontal marks on the tube and the phantom were aligned to assure a reproducible orientation of the tube. When fully inserted into the channel, the tube protruded inferiorly from the phantom by 10.37 mm when measured by digital calipers . This was the baseline/CT planning reference position of F4. In this position, the fixed fiducials were arranged in a triangular shape around F4 in the superior and right/left lateral positions . An isocentric plan was subsequently created by outlining F4 as a dummy target, purely so the system would generate reference DRRs for a comparative analysis of fiducial positioning.To ensure a reproducible setup at the start of each experiment, the phantom was arranged on the CyberKnife treatment RoboCouch as per the planning computed tomography (CT) with F4 in the baseline position. A pair of live X-ray images was acquired with the in-room imaging system  and the "start" couch corrections were automatically generated and applied to assure accurate and reproducible phantom positioning consistent with the baseline. The default setting of 60% fiducial extraction confidence level (FECL) was used for fiducial registration throughout.Independent inferior (X-axis) displacement of F4 was simulated by sliding the tube out of the phantom, using digital caliper measurement to increase its protrusion by 2.00 mm. A further pair of live images was acquired and the console information was recorded, including automatically generated x, y, and z coordinate positions of each fiducial, distances between fiducials, 6DOF corrections and RBE values, and the x, y and z COM translation corrections. This procedure was repeated following 2.00 mm incremental shifts of the tube position, up to a total inferior F4 displacement of 16.00 mm. To explore the relationship between fiducial migration and overall translational correction of the system in situations where they may be withheld , an overall COM translational shift analogous to an automated robot correction was calculated at each position of F4 using the root of the sum of the squares formula.Systematic repeat experiments were conducted to investigate the following fiducial combinations:· Two fiducials: one fixed and one moving fiducial (F1 and F4)· Three fiducials: three different combinations each of two fixed fiducials and one moving fiducial .· Four fiducials: three fixed and one moving fiducial .Accuracy and validationMean (±SD) distances measured between fixed fiducials were F1–F2=103.20±0.05 mm, F1–F3=73.10±0.06 mm and F2–F3=95.70±0.00 mm. Good registration accuracy was observed for fixed fiducials: overall mean x, y, and z positioning differences for fixed fiducials were -0.08±0.23, -0.07±0.30, and -0.01±0.08 mm, respectively; and mean (±SD) positioning differences between reference and live positioning for x, y, and z coordinates for F1, F2, and F3 were -0.17±0.00, 0.17±0.27, and 0.05±0.05 mm; -0.27±0.00, -0.23±0.22, and -0.03±0.00 mm; and 0.20±0.16, -0.14±0.28 and -0.04±0.10 mm, respectively, representing the inherent registration error in defining the center of each fiducial . For theResults summary The results obtained for two fiducials, three fiducials, and four fiducials are presented in Table Two fiducialsFor a single suboptimal two-fiducial array comprising F1 and F4, the mean COM translational shifts and mean RBE values obtained with a two-fiducial array, for each 2.00 mm x-axis displacement of F4, are illustrated in Figure Three fiducialsThe mean COM translation shifts and mean RBE values obtained with three different three-fiducial arrays, for each 2.00 mm x-axis displacement of F4, are illustrated in Figure Four fiducialsThe calculated mean COM translation shifts and mean RBE values obtained for a single four-fiducial array , for each 2.00 mm X-axis displacement of F4, are illustrated in Figure Additional observationsAn overall pattern for COM translations was observed following the displacement of one fiducial, whereby the COM translational correction approximated the migration distance of F4 (mm) divided by the total number of fiducials correlated, i.e. shift correction ≈ migration distance/number of fiducials. However, this pattern begins to break down when false-locks occurred and is unreliable from 10.00 mm F4 displacement. A further analysis of RBE values showed an overall mean±SD RBE between fixed fiducials F1 and F2, F2 and F3, and F1 and F3 were 0.24±0.02, 0.23±0.13, and 0.58±0.24 mm, respectively, with overall range 0.19–0.70 mm, showing low inherent registration variation for fixed fiducials. Maximum RBE values were smaller than the fiducial displacement distance in all cases, illustrating that because of relative fiducial positioning, RBE magnitude is a distinct measure to  fiducial displacement distance. In addition, when pooled RBE values obtained between fiducial pairs F1 and F4, F2 and F4, and F3 and F4 were examined with 0.00–16.00 mm F4 displacement distance , RBE values between F3 and F4 were consistently smaller compared with F1 and F4 and F2 and F4, indicating relative fiducial positioning affects the RBE values generated.SummaryIf the phantom study results are extrapolated to clinical situations, the following points are notable.• For arrays of two, three, and four fiducials, all displacements of one fiducial ≤ 4.00 mm were accurately detected.• Displacement of one fiducial ≤2.00 mm resulted in RBE and COM values that would not breach commonly used 2.00–5.00 mm RBE and COM clinical thresholds, indicating that they would not result in treatment pause but would likely be accommodated as a small target deformation with an appropriate COM adjustment.• Displacement of one fiducial by 4.00 mm resulted in RBE <5.00 mm, indicating that whether treatment interruption occurred would depend on clinical thresholds employed. To ensure automatic treatment pause with the displacement of one fiducial ≥4.00 mm for arrays of two, three, and four fiducials, the minimum RBE clinical thresholds indicated to be necessary are 2.76, 3.63, and 1.88 mm, respectively.• For larger displacements of one fiducial (>4.00 mm), the two-fiducial array showed better tracking results compared with the four-fiducial array. The two-fiducial array showed a greater range of detection accuracy following F4 displacement (from 0.00–14.00 mm vs 0.00–4.00 mm), and thus increased displacement distance before false-lock occurred (16.00 mm vs 6.00 mm). In addition, RBE values that would induce treatment interruption due to RBE system threshold breach occurred at a lower displacement distance (6.00 mm vs 10.00 mm).• For many instances where displacements would definitely result in treatment pause due to system threshold breach (i.e. RBE >5.00), the system output information is either erroneous  and/or would involve relatively large COM robot translation corrections that are notably greater than typical margins applied for motion uncertainties, thereby increasing risk if there is array interpretation uncertainty, which may make operator interpretation and troubleshooting difficult.Overall, the results showed good accuracy for fixed fiducial localization, with low inherent variation. With the displacement of one fiducial in one direction , the total number of fiducials in an array, the displacement distance, and the clinical COM/RBE thresholds applied were found to determine system response.Smaller displacements of one fiducial of 0.00–4.00 mm showed good fiducial registration accuracy for arrays of two, three, or four fiducials, with reassuring, approximately representative RBE and appropriate compensatory COM corrections. The results additionally indicate that in a clinical situation, small displacements of ≤2.00 mm are likely to be accommodated within typical planning margins as a target deformation, whereas displacements of 4.00 mm may either be accommodated as a target deformation or may lead to treatment interruption for operator investigation, depending on the clinical thresholds employed.However, system limitations were shown for larger fiducial displacements of 6.00–16.00 mm. The accuracy of fiducial positioning and thus reported console information was found to depend on the number of fiducials correlated in the array and displacement distance, with accuracy affected by the occurrence of false RBE main contributor  and the incidence of false-locks that underestimated fiducial displacement and gave misleading RBE and COM . When a false-lock occurred, the reported RBE did not accurately reflect the displacement and, in some instances, despite fiducial displacement >5.0 mm, resulted in RBE that was less than the 5.00 mm RBE system threshold. This suggests that treatment may not be interrupted following larger displacements unless sufficiently small clinical thresholds are used. Furthermore, tracking range correction limitations were experienced when the investigators attempted to help the system recognize the true position of the displaced fiducial. All of these factors may inhibit the recognition, interpretation, and appropriate correction of fiducial displacement in vivo. This is because a false-lock is not reported on the operator console and, similar to the identification of a true RBE main contributor, may not be identifiable without a close inspection of treatment and planning images and a comparison with anatomical landmarks, where image clarity may additionally be limited in a clinical situation. Thus, without sufficiently small clinical thresholds and operator vigilance, it is possible that a false-lock may go unnoticed or may be recognized despite misleading information and be considered to represent a deformation but may not be correctable. Furthermore, as false-locks were shown for larger displacements that resulted in RBE <5.00 mm , it raises the possibility that larger fiducial displacements (e.g. due to intrafraction target deformation) may occur more frequently in clinical situations than may be accurately detected by the tracking system.The main limitations of the study were of the phantom model system. The first was that the study was wholly conducted in a phantom and, as is the case with all model/phantom studies, the results may only be extrapolated/inferred to clinical situations. The second is that only the specific geometry relating to the phantom model was studied. This was intended to model a "typical" clinical scenario, with placement and sizing similar to a moderate liver lesion. Although a total of five baseline geometries were investigated, each with nine different positions of the moveable fiducials, and it is notable that three different combinations of three fiducials gave very similar results, it is possible that other relative positions of fiducials may give different results. The third limitation is that the fiducials were in a homogenous solid water phantom, and the y/z axis positions of all fiducials were as expected throughout. This does not accurately reflect a clinical situation, as fiducial registration and positioning are unlikely to be as accurate in vivo due to concurrent y and z-axis alterations or the displacement of more than one fiducial, leading to overall 3D array orientation changes. Other important factors, including less soft-tissue contrast, multiple tissue densities in the region of interest, deformation of adjacent tissue, and X-ray attenuation, are also likely to inhibit fiducial positioning in a clinical situation ,19-23. IMany of the study findings were reassuring. First, the sub-millimeter level of tracking accuracy obtained for fixed fiducials and for smaller fiducial displacements ≤4.0 mm confirms the fiducial registration accuracy shown in previous studies ,21,24. SFor larger displacements (>4.00 mm), some findings showed system limitations may be considered counterintuitive or unexpected or were contrary to other data, indicating that a four-fiducial array improves tracking accuracy and interpretation -21. FirsThe study findings have several important implications. They indicate that although tracking range accuracy is excellent for single fiducial displacements ≤2.00 mm, which appear to be typically accommodated as a target deformation, there is reduced tracking range accuracy for larger displacements >4.0 mm. These are likely conferred by the rigid body assumptions and positional verification with DRR positioning that underlie the systems fiducial registration processes. The accuracy of fiducial registration following displacement is affected by a number of factors, including the number of fiducials correlated and fiducial displacement distance: X-ray discrimination, FECL, and sampling rate are additional factors ,20,24-25Based on our results and their potential implications for clinical situations, we have developed recommendations and troubleshooting guidelines, as given in Table CyberKnife fiducial tracking is often considered the "gold standard" in the performance evaluation of other registration algorithms. In this novel experimental phantom model with one, two, or three fixed fiducials and one migrating fiducial, we found that the imaging of fiducial position by the in-room imaging system was reassuringly highly accurate (to within 0.24 mm) and reproducible (SD<0.09mm), as long as fiducials were correctly identified.Although acknowledged to be limited, the study indicated that the accuracy of fiducial positioning, corresponding console information, and likelihood of treatment interruption depended on the number of fiducials correlated in an array, fiducial displacement distance, and clinical thresholds used. Rigid fiducials and smaller (<4.00 mm) single fiducial displacements were consistently detected and appropriately reported when using an array of two, three, or four fiducials. However, larger (≥ 4.00 mm) displacements of one fiducial were not always accurately detected or reported. This was due to either false RBE main contributor information  or because of false-locks that underestimated displacement, gave corresponding misleading RBE, and were resistant to correction . When extrapolated to clinical situations, the results indicate that without operator vigilance and sufficiently tight pre-set clinical parameters, displacements of 6.00–16.00 mm that may occur in vivo due to target deformation or fiducial migration may not lead to treatment interruption and may not be recognized or adequately interpreted and corrected following treatment interruption. Strategies to mitigate system limitations, such as optimizing fiducial arrays, frequent visual inspection of treatment images, careful selection of treatment parameters, and operator vigilance, are, therefore, recommended for optimal targeting, particularly for target structures subject to larger on-treatment deformations. The implementation of our troubleshooting guidance based on the study findings, including temporary fiducial deselection to inspect tracking accuracy, may additionally assist array interpretation and decision-making when issues arise with fiducial tracking in clinical situations.


This study explores the immune cell repertoire present in ganglia during PHN and provides evidence for an ongoing immune cell inflammation years after HZ.Varicella-zoster virus (VZV) is a human herpesvirus which causes varicella (chicken pox) during primary infection, establishes latency in sensory ganglia, and can reactivate from this site to cause herpes zoster (HZ) (shingles). A major complication of HZ is a severe and often debilitating pain called post-herpetic neuralgia (PHN) which persists long after the resolution of the HZ-associated rash. The underlying cause of PHN is not known, although it has been postulated that it may be a consequence of immune cell mediated damage. However, the nature of virus-immune cell interactions within ganglia during PHN is unknown. We obtained rare formalin fixed paraffin embedded sections cut from surgically excised ganglia from a PHN-affected patient years following HZ rash resolution. VZV DNA was readily detected by qPCR and regions of immune infiltration were detected by hematoxylin and eosin staining. Immunostaining using a range of antibodies against immune cell subsets revealed an immune cell response comprising of CD4 Varicella zoster virus (VZV) is a highly species specific human herpesviruses responsible for two clinically distinct human diseases. Primary infection results in varicella (chickenpox), during which the virus establishes latency within neurons in the sensory ganglia . LatencyThe underlying pathology and pathogenesis of PHN is unknown, and investigations into PHN are complicated by the rare nature of relevant clinical samples and the non-availability of a suitable animal model. PHN may result from damage to the sensorineural pain pathway during HZ, resulting in malfunctioning of sensory neurons causing the transmission of pain signals . An alte+ and cytolytic CD8+ T cells may play an important role in the ganglia during active HZ  which innervate the painful area , 1991a. ctive HZ . HoweverAs a result of our very limited understanding of the ongoing pathology of PHN, treatment options are very limited. There is a range of pharmacological, interventional and surgical options available. However, in most cases the pain can often be severe and intractable  and someThe aim of this study was to investigate VZV antigen expression and nucleic acid levels within ganglion samples from a PHN-affected patient (years following rash resolution) and a HZ-affected patient (presence of HZ rash). These rare clinical samples were also used to define for the first time the nature of the immune cell subsets present within human ganglia years after HZ rash resolution in an individual suffering from PHN.One of the greatest obstacles to the study of PHN and HZ is the difficulty in obtaining appropriate human ganglia samples. For this study we obtained very rare surgically excised PHN-affected ganglia, as well as post-mortem ganglia from a patient suffering HZ at the time of death, and two control cases consisting of ganglia from individuals without any evidence of VZV disease.The PHN-affected patient was a 59 year old female who was diagnosed with acute myeloid leukemia, and underwent a bone marrow transplant. During recovery the patient experienced HZ in the right thoracic region (T9–T12), and following the resolution of the rash experienced prolonged pain (>5 years) around the same anatomical site. After exhausting all conservative treatment options a ganglionectomy was performed in an attempt to control the pain, with right thoracic T11 ganglion removed. Nine months later, a second ganglionectomy was performed, with right thoracic T10 removed. Surgery was performed when the patient was 60 years of age. Both of the right T10 and right T11 ganglia  were utilized in this study.The HZ patient was a 93 year old male with frontotemporal dementia who died due to aspiration pneumonia. This patient developed a HZ rash approximately 17 days prior to death. At autopsy there was a hemorrhagic vesicular and ulcerated rash in a left lumbar dermatome. The ganglion innervating the site of the rash (left lumbar L2) was collected, and noted to be inflamed.The first control patient thoracic ganglion (CON1) was from a 73 year old female who died due to metastatic adenocarcinoma. The second control patient ganglion (CON2) was from a 73 year old female who died due to a head injury. Medical records indicate that these patients experienced no clinical VZV reactivation prior to their death.Histological examination of ganglia samples utilized a hematoxylin and eosin (HE) stain on 5 μm formal fixed paraffin embedded (FFPE) sections with representative images shown in Viable DNA was successfully isolated from all ganglion samples. The presence of VZV DNA was confirmed in all PHN and HZ-affected material via PCR, yet the closely related virus HSV-1 was not detected (data not shown). Thus, the immune cell infiltration observed in these ganglionic samples was not a consequence of HSV-1 infection.5 copies of VZV DNA per 105 cells in PHN2 and 4.27 × 104 copies of VZV DNA per 105 cells in HZ. No VZV DNA was detected in CON2. This result demonstrates that VZV DNA was readily detectable in ganglia of a PHN-affected patient years after the resolution of their HZ rash and may provide evidence of an ongoing virological process during PHN.To further investigate VZV DNA load a real-time PCR approach for VZV open reading frame 28 (ORF28) was employed. Nucleic acids were extracted from sections and the VZV DNA ganglia load calculated. Sufficient DNA was obtained from the PHN2, HZ and CON2 samples only. Human albumin is present in tissue as one copy per cell, and provides a standard against which the copy number of VZV DNA was calculated. There were 2.95 × 10Detection of VZV antigen expression in FFPE material has proven to be problematic . Thus weVaricella zoster virus IE63 staining was not detected in PHN1 and PHN2 affected ganglia . In cont+ T cells present when compared to the control samples but the number of CD3/CD4 lymphocytes was greatest in the HZ sample. Representative images are shown in + T cells was greatest in the HZ sample and these were observed throughout the neuronal regions of the ganglion. The PHN1 sample contained CD3+ T cells and CD20+ B cells in all regions of the ganglion, but with infiltrating cells spread throughout both neuronal and nerve bundle regions of the ganglion. PHN2 ganglion had an infiltration of CD3+ T and CD20+ B cells mainly restricted to the neuronal region. The PHN ganglia showed comparable frequencies of CD4+ and CD8+ T cells. This contrasted to that observed in HZ affected ganglia where there was a predominance of CD4+ T cells over CD8+ T cells  cells, as we have previously reported to be present in ganglia months after VZV reactivation . In the absence of detectable VZV antigen expression this may represent a latent pool of virus. In this respect, previous studies have reported in VZV latently infected ganglia a copy number of 258 of VZV DNA per 105 cells  and from as per manufacturer’s instructions.The human albumin-specific primer pair were as previously published . The VZVStandard curves were created using serial dilutions of a known amount of linearized plasmid constructs. Plasmid constructs consisted of pGEM-T Easy backbone  containing small coding regions of human albumin or VZV ORF28 containing the region detected by the corresponding primer pairs.All samples were processed using a Rotorgene 6000 qRT-PCR machine . Data was analyzed using Rotorgene 6000 software . qRT-PCR for human albumin was performed using the SYBR Green system  as per manufacturer’s instructions. Cycling conditions were as follows: 50°C for 2 min, 95°C for 2 min, 45 cycles of 95°C for 10 s, 62°C for 15 s, 72°C for 20 s (acquiring fluorescence levels during this step). qRT-PCR for VZV ORF28 was performed using the Rotor-Gene Probe PCR kit . Cycling conditions were as follows: 95°C for 3 min, 50 cycles of 95°C for 3 s and 60°C for 10 s (acquiring fluorescence).Single and dual immunofluorescence staining was performed as previously described .1, mouse IgG2a , normal rabbit and normal goat IgG , were diluted to match primary antibody concentrations. Secondary antibodies were AlexaFluor labeled antibodies  at a dilution of 1:200.The following primary antibodies and dilutions were used: mouse anti-human CD3  (20 μg/mL), goat anti-human CD4  (10 μg/mL), Rabbit anti-human CD8  (2 μg/mL), mouse anti-human CD20  (18 μg/mL), mouse anti-human T cell intracellular antigen 1 (TIA-1)  (20 μg/mL), predilute rabbit anti-cow S100 , rabbit anti-VZV IE63 polyclonal antibody  and mouse anti-VZV gE:gI . Isotype controls were mouse IgGImaging was performed using a Zeiss AxioPlan 2 upright microscope with an AxioCam camera . To perform cell counts 5–20 (depending on tissue size) random fields of view were captured and manual cell counting performed using the AxioVision image acquisition software. Counts from at least two independent stains were averaged. Tissue area for each field of view was calculated using the “Measure Outline” function of the AxioVisionLE microscope software. The data represents the average number of positive cells per square millimeter of regions of neuronal cell bodies and regions of nerve bundles.All datasets generated for this study are included in the manuscript and/or supplementary files.The studies involving human participants were reviewed and approved by The University of Sydney Ethics approval. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.JS planned, performed the experiments, and analyzed the data. AA, BS, and MS planned the experiments and analyzed the data. MB and MR provided the samples. BS and AA received funding for the study. All authors contributed to writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.