Impact of solitary pulmonary nodule size on qualitative and quantitative assessment using 18F-fluorodeoxyglucose PET/CT: the SPUTNIK trial.

PURPOSE: To compare qualitative and semi-quantitative PET/CT criteria, and the impact of nodule size on the diagnosis of solitary pulmonary nodules in a prospective multicentre trial. METHODS: Patients with an SPN on CT ≥ 8 and ≤ 30 mm were recruited to the SPUTNIK trial at 16 sites accredited by the UK PET Core Lab. Qualitative assessment used a five-point ordinal PET-grade compared to the mediastinal blood pool, and a combined PET/CT grade using the CT features. Semi-quantitative measures included SUVmax of the nodule, and as an uptake ratio to the mediastinal blood pool (SURBLOOD) or liver (SURLIVER). The endpoints were diagnosis of lung cancer via biopsy/histology or completion of 2-year follow-up. Impact of nodule size was analysed by comparison between nodule size tertiles. RESULTS: Three hundred fifty-five participants completed PET/CT and 2-year follow-up, with 59% (209/355) malignant nodules. The AUCs of the three techniques were SUVmax 0.87 (95% CI 0.83;0.91); SURBLOOD 0.87 (95% CI 0.83; 0.91, p = 0.30 versus SUVmax); and SURLIVER 0.87 (95% CI 0.83; 0.91, p = 0.09 vs. SUVmax). The AUCs for all techniques remained stable across size tertiles (p > 0.1 for difference), although the optimal diagnostic threshold varied by size. For nodules < 12 mm, an SUVmax of 1.75 or visual uptake equal to the mediastinum yielded the highest accuracy. For nodules > 16 mm, an SUVmax ≥ 3.6 or visual PET uptake greater than the mediastinum was the most accurate. CONCLUSION: In this multicentre trial, SUVmax was the most accurate technique for the diagnosis of solitary pulmonary nodules. Diagnostic thresholds should be altered according to nodule size. TRIAL REGISTRATION: ISRCTN - ISRCTN30784948. ClinicalTrials.gov - NCT02013063.

Imaging gene delivery in a mouse model of congenital neuronal ceroid lipofuscinosis.

Adeno-associated virus (AAV)-mediated gene replacement for lysosomal disorders have been spurred by the ability of some serotypes to efficiently transduce neurons in the brain and by the ability of lysosomal enzymes to cross-correct among cells. Here, we explored enzyme replacement therapy in a knock-out mouse model of congenital neuronal ceroid lipofuscinosis (NCL), the most severe of the NCLs in humans. The missing protease in this disorder, cathepsin D (CathD) has high levels in the central nervous system. This enzyme has the potential advantage for assessing experimental therapy in that it can be imaged using a near-infrared fluorescence (NIRF) probe activated by CathD. Injections of an AAV2/rh8 vector-encoding mouse CathD (mCathD) into both cerebral ventricles and peritoneum of newborn knock-out mice resulted in a significant increase in lifespan. Successful delivery of active CathD by the AAV2/rh8-mCathD vector was verified by NIRF imaging of mouse embryonic fibroblasts from knock-out mice in culture, as well as by ex vivo NIRF imaging of the brain and liver after gene transfer. These studies support the potential effectiveness and imaging evaluation of enzyme replacement therapy to the brain and other organs in CathD null mice via AAV-mediated gene delivery in neonatal animals.

18F-FDG-PET in guided dose-painting with intensity modulated radiotherapy in oropharyngeal tumours: A phase I study (FiGaRO).

BACKGROUND AND PURPOSE: The FiGaRO trial assessed the feasibility and safety of using an FDG-PET-based dose-painting technique to deliver a radiotherapy (RT) boostto the FDG-avid primary tumour in patients with locally advanced high and intermediate risk oropharyngeal cancer. MATERIALS AND METHOD: Patients underwent a planning 18FDG-PET-CT scan, immobilised in the treatment position, after one cycle of induction chemotherapy. The volume of persistent FDG-avidity in the primary tumour was escalated to 71.5 Gy in30 fractions delivered using a simultaneous integrated boost Intensity Modulated RT (SIB-IMRT) technique. RT was delivered with concomitant Cisplatin following 2 cycles of induction chemotherapy. The primary outcome was the incidence of grade ≥ 3 late mucosal toxicity 12 months post-treatment, with an excess rate of >10% regarded as unacceptable. RESULTS: Twenty-nine patients were included and twenty-four were treated between 2014 and 2018, in two UK centres. Median follow-up was 36 months (range 4-56 months). Pre-defined planning target volume objectives and organ at risk dose constraints were met in all cases. There were no incidents of acute grade 4 toxicity. There were 4 cases of grade ≥ 3 mucosal toxicity at 12 months post-treatment (19.1%). There were no cases of persistent mucosal ulceration at 12 months. Overall survival at 3-years was 87.5%, 92.9% for intermediate and 70.0% for high risk patients. CONCLUSION: Late toxicity rates, although higher than anticipated, are comparable to contemporary published data for standard dose chemo-IMRT. Results suggest improved 3y survival rates for high risk patients. This approach merits further investigation. ClinicalTrials.gov Identifier: NCT02953197.

Practical issues and limitations of brain attenuation correction on a simultaneous PET-MR scanner.

BACKGROUND: Despite the advent of clinical PET-MR imaging for routine use in 2011 and the development of several methods to address the problem of attenuation correction, some challenges remain. We have identified and investigated several issues that might affect the reliability and accuracy of current attenuation correction methods when these are implemented for clinical and research studies of the brain. These are (1) the accuracy of converting CT Hounsfield units, obtained from an independently acquired CT scan, to 511 keV linear attenuation coefficients; (2) the effect of padding used in the MR head coil; (3) the presence of close-packed hair; (4) the effect of headphones. For each of these, we have examined the effect on reconstructed PET images and evaluated practical mitigating measures. RESULTS: Our major findings were (1) for both Siemens and GE PET-MR systems, CT data from either a Siemens or a GE PET-CT scanner may be used, provided the conversion to 511 keV µ-map is performed by the PET-MR vendor's own method, as implemented on their PET-CT scanner; (2) the effect of the head coil pads is minimal; (3) the effect of dense hair in the field of view is marked (> 10% error in reconstructed PET images); and (4) using headphones and not including them in the attenuation map causes significant errors in reconstructed PET images, but the risk of scanning without them may be acceptable following sound level measurements. CONCLUSIONS: It is important that the limitations of attenuation correction in PET-MR are considered when designing research and clinical PET-MR protocols in order to enable accurate quantification of brain PET scans. Whilst the effect of pads is not significant, dense hair, the use of headphones and the use of an independently acquired CT-scan can all lead to non-negligible effects on PET quantification. Although seemingly trivial, these effects add complications to setting up protocols for clinical and research PET-MR studies that do not occur with PET-CT. In the absence of more sophisticated PET-MR brain attenuation correction, the effect of all of the issues above can be minimised if the pragmatic approaches presented in this work are followed.

Correction to: Practical issues and limitations of brain attenuation correction on a simultaneous PET-MR scanner.

An amendment to this paper has been published and can be accessed via the original article.

Lack of correlation between changes in polyphosphoinositide levels and actin/gelsolin complexes in A431 cells treated with epidermal growth factor.

The polyphosphoinositides, PIP and PIP2, have been proposed to regulate actin polymerization in vivo because they dissociate actin/gelsolin complexes in vitro. We tested this hypothesis by comparing the ability of EGF and bradykinin to affect PI metabolism and the actin cytoskeleton in A431 cells. EGF, but not bradykinin, was found to induce ruffling and dissociation of actin/gelsolin complexes in these cells. However, both EGF and bradykinin stimulated the accumulation of inositol phosphates in [3H]inositol-labeled cells indicating that stimulation of PI turnover is not sufficient for the induction of changes in actin/gelsolin complex levels. EGF stimulated a twofold increase in the level of PIP in A431 cells. Other phosphoinositide levels were not markedly altered. Treatment of the cells with cholera toxin abrogated the EGF-induced rise in PIP levels without altering the dissociation of actin from gelsolin. These data indicate that increases in PIP and/or PIP2 are not necessary for dissociation of actin/gelsolin complexes. Overall, these experiments suggest that in A431 cells, the effects of EGF on the actin cytoskeleton are unlikely to be mediated through changes in PIP or PIP2 levels.

Phosphatidylinositol 4-kinases and the role of polyphosphoinositides in cellular regulation.

Phosphoinositides play a central role in the transduction of signals for a variety of hormone and growth factor receptors. Multiple derivatives of phosphatidylinositol are present within the cell including phosphatidylinositol 4,5-bisphosphate, the phosphorylated derivative that is hydrolyzed by phospholipase C to produce the two intracellular second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. The synthesis, degradation, and subsequent resynthesis of the phosphoinositides form a metabolic cycle known as the phosphoinositide cycle. The phosphoinositide cycle begins with the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a reaction catalyzed by phosphatidylinositol 4-kinase. Phosphatidylinositol kinase activity has been reported to be present in a variety of cellular membranes, and multiple isozymes of phosphatidylinositol 4-kinase are present within the cell, suggesting that the product of this reaction may have more than one biological function. The activity of phosphatidylinositol 4-kinase is regulated by growth factors, further underscoring the importance of this enzyme in cellular regulation. Recent data suggest that in addition to serving as substrates for phospholipase C, the polyphosphoinositides may themselves function as intracellular mediators of hormone action. For example, polyphosphoinositides have marked effects on the activity of certain actin binding proteins that may allow these lipids to participate in the regulation of actin polymerization. This review focuses on the properties of the phosphatidylinositol 4-kinases and the potential role of polyphosphoinositides in the regulation of cellular processes.

The acoustic properties, centered on 20 MHZ, of an IEC agar-based tissue-mimicking material and its temperature, frequency and age dependence.

The purpose of this study was to characterize the ultrasonic properties of agar-based tissue-mimicking materials (TMMs) at ultrasound frequencies centered around 20 MHz. The TMM acoustic properties measured are the amplitude attenuation coefficient alpha (dB cm(-1)MHz(-1)), the speed of sound (ms(-1)) and the backscattered power spectral density (distribution of power per unit frequency normalized to the total received power) characteristics of spectral slope (dB MHz(-1)), y-axis intercept (dB) and reflected power (dB). The acoustic properties are measured over a temperature range of 22 to 37 degrees C. An intercomparison of results between two independent ultrasound measurement laboratories is also presented. A longitudinal study of the acoustic properties over a period of two years is also detailed, and the effect of water immersion on the acoustic properties of TMM is measured. In addition, the physical parameters of mass density rho (kg m(-3)) and specific heat capacity C (J kg(-1) K(-1)) are included. The measurement techniques used were based on the substitution technique using both broadband and narrowband pulses centered on 20 MHz. Both the attenuation coefficient and speed of sound (both group and phase) showed good agreement with the expected values of 0.5 dB cm(-1) MHz(-1) and 1540 ms(-1), respectively, with average values over the three-year period of 0.49 dBcm(-1)MHz1 (SD +/- 0.05) and 1540.9 ms(-1) (SD +/- 8.7). These results also showed agreement between the two independent measurement laboratories. Speed of sound and attenuation coefficient were shown to change with temperature with rates of + 2.1 m s(-1) degrees C(-1) and -0.005 dB cm(-1) MHz(-1) degrees C(-1), respectively. Attenuation changed linearly with frequency at the high frequency range of 17 to 23 MHz, and speed of sound was found to be independent of frequency in this range. The spectral slope of relative backscattered power for the material increased with frequency at typically 1.5 dB MHz(-1). This compared favorably with theoretical spectral slope values, calculated for a variety of scatterer sizes, albeit at a lower frequency range. It is also noticed that, on extrapolation back to lower frequencies, the backscatter is comparable with that measured at 7 MHz. Overall, this non-commercial agar-based TMM is shown to perform as expected at the higher frequency range of 17 to 23 MHz and is seen to retain its acoustic properties of attenuation and speed of sound over a three-year period.

Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains.

Recent evidence has implicated caveolae/DIGs in various aspects of signal transduction, a process in which polyphosphoinositides play a central role. We therefore undertook a study to determine the distribution of phosphoinositides and the enzymes that utilize them in these detergent-insoluble domains. We report here that the polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low density, Triton-insoluble membrane fraction that contains caveolin. This fraction is also enriched in polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P2. Treatment of cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter the distribution of polyphosphoinositides or the polyphosphoinositide phosphatase. However, PMA treatment did lead to a decrease in the mitogen-activated protein kinase and actin present in these domains. PMA also induced the recruitment of protein kinase C alpha to the caveolae/DIGs fraction. These findings suggest that polyphosphoinositides, the polyphosphoinositide phosphatase and protein kinase C play an important role in the structure or function of detergent-insoluble membrane domains.

Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.

Accuracy and cost-effectiveness of dynamic contrast-enhanced CT in the characterisation of solitary pulmonary nodules-the SPUtNIk study.

INTRODUCTION: Solitary pulmonary nodules (SPNs) are common on CT. The most cost-effective investigation algorithm is still to be determined. Dynamic contrast-enhanced CT (DCE-CT) is an established diagnostic test not widely available in the UK currently. METHODS AND ANALYSIS: The SPUtNIk study will assess the diagnostic accuracy, clinical utility and cost-effectiveness of DCE-CT, alongside the current CT and 18-flurodeoxyglucose-positron emission tomography) (18FDG-PET)-CT nodule characterisation strategies in the National Health Service (NHS). Image acquisition and data analysis for 18FDG-PET-CT and DCE-CT will follow a standardised protocol with central review of 10% to ensure quality assurance. Decision analytic modelling will assess the likely costs and health outcomes resulting from incorporation of DCE-CT into management strategies for patients with SPNs. ETHICS AND DISSEMINATION: Approval has been granted by the South West Research Ethics Committee. Ethics reference number 12/SW/0206. The results of the trial will be presented at national and international meetings and published in an Health Technology Assessment (HTA) Monograph and in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ISRCTN30784948; Pre-results.

Stimulation of purified phosphatidylinositol 4-kinase by cobra venom cardiotoxin.

Cobra venom cardiotoxin was found to stimulate the phosphatidylinositol kinase activity present in A431 cell membranes and in detergent extracts of these membranes. Incubation of highly purified phosphatidylinositol 4-kinase from this source with cardiotoxin resulted in a 2- to 3-fold stimulation of phosphatidylinositol kinase activity. The activation of the purified phosphatidylinositol 4-kinase by cardiotoxin was time- and dose-dependent and appeared to be associated with a decrease in the Km apparent of the enzyme for phosphatidylinositol with no change in the Vmax apparent of the enzyme. The data suggest that the phosphatidylinositol 4-kinase is activated by direct interaction of the enzyme with cobra venom cardiotoxin.

Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes.

The catecholamine-sensitive adenylate cyclase system appears to be comprised of at least three components; the beta-adrenergic receptor (R component), the catalytic unit of adenylate cyclase (C component) and a nucleotide regulatory protein (N component), responsible for mediating the effects of guanine nucleotides on the system. Cell fusion techniques were used to investigate the role of these three components in the process of homologous desensitization in the frog erythrocyte. Dicyclohexylcarbodiimide (DCCD) was used to inhibit beta-receptor function in one population of frog erythrocytes, whilst phenyl glyoxal was employed to inactivate the N and C components in a second population of frog erythrocytes. Using Sendai virus to fuse the two types of modified cell, heterologous beta-adrenergic receptor-adenylate cyclase systems were constructed which contained components from each cell type. When beta receptors from cells previously desensitized to catecholamines were coupled to N-C components derived from fresh erythrocytes, the resulting hybrid exhibited a densitized response to isoproterenol. By contrast, when beta-adrenergic receptors from fresh cells were coupled to N-C components derived from desensitized erythrocytes, no decreased responsiveness to isoproterenol was apparent in the hybrid. That this resensitization was the result of the addition of fresh beta-adrenergic receptors was demonstrated in a control experiment. Frog erythrocytes were desensitized simultaneously to catecholamines and prostaglandin E1 and modified with DCCD which inactivates the beta-adrenergic receptor but not the prostaglandin receptor. When fresh beta-adrenergic receptors were supplied by cell fusion to these doubly desensitized erythrocytes, only the beta-adrenergic response was restored to control levels. The response to prostaglandin remained desensitized in the hybrids, indicating that the observed resensitization of catecholamine-stimulated adenylate cyclase activity was specific and was due to the addition of fresh beta-adrenergic receptors. These data suggest that in the frog erythrocyte, homologous desensitization is primarily the result of receptor-related alterations.

Differential effects of GTP on the coupling of beta-adrenergic receptors to adenylate cyclase from frog and turkey erythrocytes. Application of new methods for the analysis of receptor-effector coupling.

A detailed comparison of the interaction of beta-adrenergic receptors with adenylate cyclase stimulation and modification of this interaction by guanine nucleotides has been made in two model systems, the frog and turkey erythrocyte. Objective analysis of the data was facilitated by the development of new graphical methods which involve the use of logit-logit transformations of percent receptor occupancy versus percent enzyme stimulation plots (coupling curves). Receptor-cyclase coupling in turkey erythrocyte membranes demonstrates a proportional relationship between receptor occupancy and adenylate cyclase activation and is unaffected by exogenous guanine nucleotides. By comparison, the proportional relationship of receptor occupancy and adenylate cyclase activation observed in frog erythrocyte membranes in the absence of guanine nucleotides is modified by the addition of exogenous guanine nucleotides such that a greater fractional enzyme stimulation is elicited by low receptor occupancy. Methodological criteria crucial for valid comparison of receptor occupancy and adenylate cyclase activity are delineated. In addition, the possible molecular mechanisms of receptor-cyclase coupling which might give rise to the coupling curves observed are discussed.

Desensitization of the EGF receptor alters its ability to undergo EGF-induced dimerization.

Treatment of A431 cells with EGF has been shown to induce the formation of EGF receptor dimers. Sucrose density gradient centrifugation as well as surface radio-iodination followed by crosslinking were used to study further the properties of EGF receptor monomers and dimers as well as the regulation of dimer formation. We have shown previously that treatment of A431 cells with high doses of EGF at 37 degrees C leads to the desensitization of the EGF receptor without a significant loss of cell surface 125I-EGF binding [Kuppuswamy and Pike (1989) J. biol. Chem. 264, 3357-3363; Cunningham et al. (1989) J. biol. Chem. 264, 15351-15356]. Desensitization of the EGF receptor led to a decrease in the ability of receptor monomers to be induced to form dimers by EGF both in vivo and in vitro. These data suggest that the sensitivity of a cell to EGF may be modulated by altering the capacity of the EGF receptor to form oligomers.

Histoplasmosis in Belize, Central America.

After two small outbreaks of histoplasmosis in Belize, an epidemiologic survey was carried out. Forty percent of 141 persons in two groups tested demonstrated a positive histoplasmin reaction. In one study group there was a significant association between visiting caves and histoplasmin positivity. Histoplasma capsulatum was not isolated in 20 soil specimens collected from outbreak-associated caves, but was isolated from 1 of 26 bats collected from the same caves. The presence of histoplasmosis in Belize, C.A. is documented, and this disease should be considered in differential diagnosis in patients, both residents and visitors in Belize, with compatible clinical presentations.

The effects of verapamil and propranolol on quality of life in hypertension.

Quality of life was evaluated in a four-month randomised double-blind trial of verapamil compared with propranolol in the treatment of hypertension in 94 patients in the UK. Scores on a health status index, measuring activity and perceived health, increased in verapamil patients compared to a decrease in propranolol patients (P = 0.01). Measures of psychiatric morbidity also tended to improve with verapamil and deteriorate with propranolol. Propranolol patients reported more symptoms overall compared with verapamil (P less than 0.05). The prevalence of certain symptoms--headaches, weak limbs and slower walking pace, increased significantly with propranolol compared with verapamil, but constipation was more common in verapamil patients (P less than 0.05). After four months, diastolic blood pressure averaged 86.2 mmHg with verapamil and 90.3 mmHg with propranolol (P = 0.02). However, this difference in final blood pressure did not explain the more favourable quality of life scores with verapamil, and the data suggest that health-related well-being is higher with this drug.

Macro-alkaline phosphatase due to IgG kappa complex: demonstration with polyethylene glycol precipitation and immunofixation.

An otherwise unexplained, persistently elevated plasma alkaline phosphatase concentration in a 71-year-old woman was found to be attributable to the presence of macro-alkaline phosphatase using polyethylene glycol precipitation. Gel filtration showed two high MW peaks with masses of about 330 kDa and 560 kDa. The alkaline phosphatase (ALP) complex was characterized by immunoelectrophoresis as a complex with IgG with kappa light chains.

Value of written health information in the general practice setting.

The value of a library for patients as a way of providing written health information in the general practice setting has been investigated using a questionnaire. In 15 months, 243 books were borrowed from one library. Each book contained a questionnaire and 163 questionnaires were returned (67%). It was found that 106 respondents (65%) would not have sought the information elsewhere and 159 respondents (98%) found the books they had read to be very useful or of some use. The perceived level of anxiety after reading was raised in nine respondents (6%) but reduced in 71 (44%). Patients read books on 53 separate topics overall, although 73 respondents (45%) read on 10 recurring topics. A patients' library thus enables most patients to gain useful information from their general practice without increasing their anxiety.

Effect of multislice scanners on patient dose from routine CT examinations in East Anglia.

As part of the dose optimization process, the Ionising Radiation (Medical Exposure) Regulations 2000 include requirements relating to the assessment of patient dose, and the setting and subsequent review of diagnostic reference levels. In East Anglia, audits of effective dose in CT have been carried out in 1996, 1999 and 2002. In the 2002 audit, nine of the 14 scanners assessed had been replaced since the previous audit. Eight of the new scanners were multislice scanners, acquiring up to 16 slices in a single rotation. The objective of the 2002 audit was to investigate the effect of the introduction of these multislice scanners on patient doses from routine CT examinations. Exposure parameters were collected for 10 different types of routine CT examination. In excess of 550 sets of patient data were obtained. For each of these, effective doses were calculated using the results of Monte Carlo simulations published by the National Radiological Protection Board. Averaged across all 10 examinations, regional mean effective doses are 34% higher than in 1999. The multislice scanners in the region give, on average, 35% more effective dose than the single-slice scanners. The effect of collimation in multislice scanners makes these effective dose differences most notable for examinations that use narrow slice widths. Further optimization of exposures on multislice scanners has the potential to reduce the differences observed between single-slice and multislice doses. However, when taken in combination with the increased use of CT in many hospitals, the effective dose increases observed are likely to result in a significant increase in the already substantial collective radiation dose from CT.