Tumour size is an important biomarker of the disease development, but to our knowledge, MRI based size measurements have not yet been verified for small tumours (10−2–10−1 g). The aim of this study was to assess the accuracy of MRI based tumour size measurements of small tumours on mice.Animal models are frequently used to assess new treatment methods in cancer research. MRI offers a non-invasive in vivo using a 7 T dedicated animal MR system. For the 3D images the acquired image resolution was varied. The images were exported to a PC workstation where the tumour mass was determined assuming a density of 1 g/cm3, using an in-house developed tool for segmentation and delineation. The resulting data were compared to the weight of the resected tumours after sacrifice of the animal using regression analysis.2D and 3D T2-weighted RARE images of tumour bearing mice were acquired 3 μm3 to 2403 μm3 without affecting the results significantly, thus reducing acquisition time substantially.Strong correlations were demonstrated between MRI- and necropsy determined masses. In general, 3D acquisition was not a prerequisite for high accuracy. However, it was slightly more accurate than 2D when small (<0.2 g) tumours were assessed for inter- and intraobserver variation. In 3D images, the voxel sizes could be increased from 1602D MRI was sufficient for accurate tumour size measurement, except for small tumours (<0.2 g) where 3D acquisition was necessary to reduce interobserver variation. Acquisition times between 15 and 50 minutes, depending on tumour size, were sufficient for accurate tumour volume measurement. Hence, it is possible to include further MR investigations of the tumour, such as tissue perfusion, diffusion or metabolic composition in the same MR session. The efficacy of new tumour treatment methods are usually tested in animal models prior to clinical trialse.g. -3). The . The 3])−2 g) in short acquisition time [MicroCT imaging allows accurate measurement of very small tumour volumes . To our knowledge, there is a lack of information on the accuracy and precision of MRI based size measurements on small tumours.in vivo measurement of tumour size in a small animal tumour model, and 2) to investigate the possibility to reduce acquisition time by reducing image resolution, without affecting the accuracy.The aim of this study was 1) to develop and assess the accuracy of an MRI based method for The study was performed on 17 tumour bearing female, athymic BALB/c and SCID mice s.c. inoculated in the neck/back region with the human midgut carcinoid cell line, GOT1 (n = 15), or the medullary thyroid carcinoma, GOT2 (n = 2) under anaesthesia using tribromoethanol . Animals had free access to water and standard diet.l, width, w and height, h of the tumours were performed. Assuming ellipsoidal tumours, the volume V = (π/6)·l·w·h, was determined and converted to mass, mGB, assuming a tumour density of 1.0 g/cm3. The tumour was then surgically removed and weighed for reference tumour mass, mT, determination. All procedures were approved by the local Animal ethics committee in Gothenburg.Within two days after MR imaging, animals were sacrificed by surgical incision of the heart after i.p. injection of sodium pentobarbital . Gauge block measurements of the length, eff = 30 ms, NSA = 2–5, turbo factor Tf = 4, pixel size: ~150·200 μm2, slice thickness = 700 μm) prior to a 3D volume acquisition . In the 3D acquisition, the matrix size was adjusted to achieve 1603 μm3 voxels with minimal field-of-view (FOV) but full tumour coverage. The 3D sequence was repeated twice using voxel sizes of 2003 μm3 and 2403 μm3 but with maintained FOV. The methods are henceforth designated 3D-160, 3D-200 and 3D-240, respectively. Saturation slices were always used in the phase encoding direction.MRI was performed using a 7 T MR system  equipped with water cooled gradient coils (maximum gradient strength 400 mT/m). A 72 mm volume coil was used for transmission and an actively decoupled 4 channel array rat brain coil was used for signal receiving . Proper animal positioning was assured using a fast gradient-echo localizer scan. All other imaging experiments were based on a respiration triggered and fat suppressed T2-weighted rapid acquisition with relaxation enhancement (RARE) sequence . A 2D seDuring the MRI experiments anaesthesia was maintained using a mixture of air and isoflurane (1.5–2.0 %) . Body temperature was maintained with a heating pad on the animal bed and a pressure sensitive pad was used for respiratory triggering.3. The estimated masses from the 2D and 3D images, denoted m2D and m3D respectively, were compared to mT using regression analysis.The image series were processed on a PC workstation using an in house developed volume estimation tool implemented in MATLAB . Figure m3D-160, m3D-200, and m3D-240) was blinded to images with higher resolution when such existed.The operator determining the masses from the repeated scans with increasing voxel sizes , a medium sized tumour  and a large tumour  were included in the assessment. The operator was blinded to the 3D images when evaluating the 2D images. Learning effects were minimized by presenting the image series in a random order and allowing at least several hours to pass between sessions. The coefficient of variation (CV) was calculated for each image series.Repeated (n = 10) segmentation and mass calculation was performed to investigate the intraobserver variability. Five image series including a small tumour  was considered for two extreme cases of segmentation, where PVE elements were classified either as tumour elements, or as background elements, thus overestimating or underestimating the true tumour volume, respectively.V is the true tumour volume. Voxel sizes of 1003–2503 μm3 and 3–9 mm (diameter) tumours were simulated, and the resulting data were presented as relative volume errors for different number of voxels-per-tumour-diameter ratios.The total influence of PVE on the tumour volume estimation was defined as Regression analyses were performed using the Microsoft Excel (2003) statistics toolkit. Pearson’s r squared correlation coefficient was used.mT, was between 0.01 g and 2.28 g. Tumours with mT <0.2 g are henceforth classified as small.All tumours found at necropsy were well visualized in the MR images. The mass of the resected tumours, m3D-160) and mT  were included.A strong correlation was found between the tumour mass estimated from the 3D-160 MR images . The same subset of tumours analysed with the 3D-160 method yielded R2 = 0.97.In twelve mice, tumours were measured with gauge blocks in addition to MRI and digital balance. The correlation between d Figure a. When am2D to mT  a strong correlation was obtained (R2 = 0.99) , i.e. the 2D method was comparable to the 3D-160 method regarding accuracy.Comparing ) Figure b. Also fm2D was compared directly to m3D-160. The linear relation in the regression and the correlation approached unity m2D = 1.01m3D-160–0.00, R2 = 1.00, n = 15. For small tumours the corresponding relation was m2D =1.04 m3D-160 + 0.00, R2 = 0.99, n = 9.To exclude the inherent uncertainty of the digital balance, m3D-160, m3D-200, and m3D-240 vs. mT did not reveal any obvious voxel size dependence in the range of tumour- and voxel sizes investigated. The acquisition time required for the smallest tumour studied (mT = 0.01 g) was clearly reduced with larger voxels: 55, 34 and 22 minutes for the 1603, 2003 and 2403 μm3 images, respectively.The analysis of The intraobserver variation was in general low for each method and tumour size (CV of 2–3 %), but an indication of increased variability (CV of 7 %) was observed in the 2D image set of the smallest tumour Table .The interobserver results indicated a difference between the 2D and 3D-160 methods  image acquisition at high resolution (in our setup 1603 μm3 voxels) should also be considered. The increased acquisition time using high resolution is compensated for by the smaller FOV needed to cover the tumour. Short acquisition times allow either additional MR investigations on the same animal, such as determining tumour diffusion, perfusion or metabolic parameters within one MR session, or a higher animal throughput.The present study evaluated the possibilities and requirements for accurate measurement of small tumour sizes in mice using MRI. A linear, highly correlated relationship between weighed and MRI measured tumour masses down to 10Initially, the 2D method was included only for anatomical reference since gradient performance limited the minimum slice thickness to 700 μm, resulting in substantial PVE. However, the accuracy of volume estimations based on the 2D images was similar to that based on the 3D images  was adjusted to reduce acquisition time. An increase in Tf results in an increased point spread function (PSF). Computer simulations assuming similar acquisition parameters as those used, and T2 values common at 7 T  showed tThe most time consuming process in the volume determination was probably when adjusting the threshold value in images where the global segmentation had failed. To reduce the time of analysis one could e.g. calculate an average volume based on two extreme segmentations; one including most of the border, and one excluding it. Such a procedure would, however, overestimate the volume, especially for small voxel-to-diameter ratios, where asymmetry between over- and underestimated volume errors is more pronounced Table . In situ3) might be an underestimation that would account for the fact that the relations between the determined masses  and the weight (mT) were less than unity. Another contributing factor could be the inherent uncertainty in the digital balance, since errors in the predictor used in regression analysis are typically manifested as a decrease of the slope coefficient towards zero [m3D-160 and m2D were compared directly to each other, thus excluding the density effect and predictor uncertainties.The tumour density assumption (1.0 g/cmrds zero . These t2 = 0.96 (n = 7) when comparing volumes of pancreatic tumours in mice from T2-weighted 2D MR images (similar to our 2D method), acquired at 4.7 T, for 0.2–2.0 g tumours [2 = 0.96, n = 9) but for smaller tumours (0.01–0.2 g) since we used six times smaller voxel volume, i.e. the influence of PVE might be comparable. Other groups have reported MRI tumour size measurements in mice but without verification with tumour weight, e.g. [3 voxels), but verified the volume determination by one phantom measurement only [Few studies were found in the literature where the accuracy of MRI based tumour size measurement was verified by e.g. comparison with weight after resection. He et al. found a correlation of R0.96 n =  when com3 μm3, voxel volume of 0.0005 mm3) a close correlation was found for 0.02–0.25 g s.c. tumours in mice, verified by weight after resection  [Using high resolution microCT  . Thus, mThe generally faster T1-weighted sequences have also been used for tumour imaging (e.g. ). Often This study shows the feasibility of accurate and precise MRI based size measurement of small tumours in mice. Furthermore, short acquisition times that allow additional MR investigations within the same session can be realized by careful selection of MR sequence parameters, e.g. using 2D instead of 3D methods and optimal voxel size.The authors declare that they have no competing interests.MM designed and carried out the imaging studies, performed the statistical analysis and drafted the manuscript. ML participated in designing and carrying out the imaging study, approved the statistical analysis and helped to draft the manuscript. MH conceived of the study, and participated in its design and coordination. EFA participated in designing the study, performing the statistical analysis and helped to draft the manuscript in addition to supervising the study coordination. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/12/12/prepub


The number of an additional grant from the Ministry of Science and Technology of China was incorrectly omitted from the Funding Statement. The grant number is: 2012ZX10004-904.


At least one reference tumour lesion was included in the fields of view and assessed at both time points. Patients also underwent 18 F-fluorodeoxyglucose (FDG)-PET imaging at baseline and after 2 and 4 weeks of treatment. Radiological response of the reference tumour lesion and overall radiological response were assessed at week 12. Serum pharmacokinetic and biomarker analyses were also performed.Patients with advanced malignancies received sunitinib 50 mg/day orally, once daily for 4 weeks on treatment, followed by 2 weeks off treatment, in repeated 6-week cycles. Quantitative measurement of tumour perfusion was assessed using Data were available for seven patients. Compared with baseline, all patients experienced a decrease in reference tumour blood flow ranging from 20 % to 85 % and also a reduction in the FDG standard uptake value ranging from 29 % to 67 %. Six patients experienced a partial metabolic response based on FDG-PET criteria. Four patients had stable disease defined by radiological response  lasting between 4 and 12 cycles. An association between perfusion change and clinical benefit, and biomarker levels including vascular endothelial growth factor was observed.Administering sunitinib to patients with advanced malignancies is associated with early biological responses, including decreased blood flow in secondary tumour deposits. Once solid tumours have reached an advanced or metastatic stage, treatment is largely palliative rather than curative, with the aim of prolonging survival, relieving pain and other symptoms, and maintaining or improving quality of life -3. PharmTargeted biological therapies with antiangiogenic properties may be particularly beneficial in the palliative care setting by preventing the neovascularisation required for tumour progression beyond a certain size . The earSunitinib  is an orally active, multitargeted tyrosine kinase inhibitor with both antitumour and antiangiogenic effects. Its targets include vascular endothelial growth factor receptors 1, 2, and 3 , platelet-derived growth factor receptors α and β, stem-cell factor receptor, FMS-like tyrosine kinase 3, and glial cell line-derived neurotrophic factor receptor -12. Suni18 F-fluoro-2-deoxy-D-glucose (FDG) as early as the second week of treatment. Both PET response and clinical benefit correlated with trough plasma levels of sunitinib and its principal metabolite (SU12662). The present paper reports on a subset of seven patients from this study who, in addition to standard study procedures, also underwent pharmacodynamic assessment with quantitative 15O-water-PET imaging to assess the effects of sunitinib treatment on tumour perfusion.In a pilot study of 55 adults with advanced malignancies treated with sunitinib, biological activity and antitumour effects were seen in a broad range of tumour types . Seven p15O-water- and FDG-PET imaging. All patients were ≥18 years old, with radiological evidence of metastatic or advanced malignancy for which there was no available therapy with curative potential. Other key eligibility criteria included Karnofsky performance status > 60 %, life expectancy > 12 weeks without rapidly progressive disease, and adequate liver function (aspartate transaminase (AST) or alanine transaminase (ALT) ≤ 2.5 × upper limit of normal (ULN); AST or ALT ≤ 5.0 × ULN or bilirubin ≤ 1.5 × ULN if liver function abnormalities were due to underlying malignancy), renal function , and bone marrow function (absolute neutrophil count (ANC) ≥ 1.5 × 109/L, platelets ≥ 75 × 109/L, and haemoglobin > 10 g/L; ANC ≥ 1.0 × 109/L, platelets ≥ 50 × 109/L, or haemoglobin > 10 g/L for patients with haematological malignancies and bone marrow involvement). At screening, all patients had to have at least one tumour mass detectable by FDG-PET imaging that was suitable for serial quantitative assessment and had not been included in a prior radiotherapy field. Female patients were postmenopausal, surgically sterile, or using effective contraception. Key exclusion criteria included the use of any other anticancer agent or investigational agent within 4 weeks prior to the start of sunitinib treatment, prior specific anti-vascular endothelial growth factor (VEGF) treatment, and any condition requiring treatment that was likely to affect the reliability of the serial PET assessments. Patients were also excluded if they had a medical or psychiatric condition that would limit full compliance with the study. Concomitant treatments necessary for the patients' well-being were given at the discretion of the investigator. The study was carried out in accordance with the International Conference on Harmonisation Good Clinical Practice guidelines and was approved by an institutional review board. All patients provided written informed consent.The use of FDG-PET and other PET-based imaging techniques to assess biological response to sunitinib was investigated in a 12-week, open-label, prospective pilot study involving four groups. The present study describes the group of patients who underwent 9/L and platelet count to 75 × 109/L.Patients received oral sunitinib 50 mg once daily for 4 weeks on treatment, followed by 2 weeks off treatment (schedule 4/2) in repeated 6-week cycles. The primary study period was the first two cycles (12 weeks) of treatment. Patients benefiting from the treatment were permitted to receive additional cycles of sunitinib for up to 1 year. The study drug was discontinued in patients exhibiting grade 4 haematological toxicity, grade 3 thrombocytopenia with haemorrhage of grade 3/4, or grade 3/4 non-haematological toxicity, and only restarted when the ANC recovered to ≥ 1.5 × 1015O-water) and PET imaging. This technique allowed quantification of the arterial concentration of 15O-water and calculation of the perfusion rate. Quantification of blood flow within the tumour (expressed in milliliter per gram per minute) was performed.Tumour perfusion was assessed using a radioactive tracer , employing 16 rings of bismuth germanate oxide block detectors with an axial extent of 108 mm and a spatial resolution of 5.4 mm at the scanner's centre of field of view. At least one viable reference tumour lesion identified by FDG-PET scanning was included in the field of view and was assessed at both time points. Emission scans were acquired dynamically in three-dimensional (3D) mode during bolus intravenous injections of 370 MBq of 15O-water into the antecubital vein. The dynamic acquisition consisted of 11 frames acquired over 3 min with 4 × 5-s, 2 × 10-s, and 4 × 30-s frames. A transmission image (10-min acquisition) was also obtained. Dynamic dataset images were reconstructed with both 3D-filtered back-projection, using a 3D reprojection algorithm (3D-FORE/FBP), and 3D attenuation-weighted ordered-subsets expectation maximisation (AW-OSEM3D) [-OSEM3D) ,18. As aAll patients had an FDG-PET scan at screening, up to 14 days prior to commencing sunitinib treatment. Further FDG-PET scans were performed at baseline and after 2 and 4 weeks of treatment, with SUV measurement of the glucose metabolism of both reference and overall tumour lesions. Whole body FDG-PET scanning was performed using a Philips Allegro dedicated PET scanner  under standardised conditions, following an overnight fast, and with imaging commencing exactly 60 min after FDG administration. All studies were performed with measured attenuation correction and were reconstructed using RAMLA 3D  and reco15O-water studies using the associated baseline and 2-week post-treatment FDG scan-determined VOIs. The entire VOI was used to determine the activity for 15O-water. By using the attenuation image for the FDG and 15O-water studies, the emission images from both studies were co-registered so that the reference tumour VOIs could be applied to the water study  for the reference tumour for each patient was defined using the FDG-PET data by thresholding to approximately 75 % of the maximum SUV in tumour and also by comparing to tumour size on computed tomography scan. The VOIs were defined for the baseline and 2-week post-treatment M(t) denotes the detected activity within a VOI and is made up of a vascular component given by V0Ca(t) and an extra-vascular or tissue component Me(t), where V0 denotes the vascular volume term, and Ca(t), the arterial input term. Such a system is governed by a so-called one-tissue-compartment model dictated by the equation given in Figure K1 denotes the blood perfusion from vascular to tissue compartment, and k2, the washout rate, as illustrated in Figure K1 and k2.A one-tissue compartment model was used to calculate the tumour blood flow and comprised vascular and tissue compartments Figure a. The opαi. By adopting such a spectrum of exponentials, the problem is linearised and, so, is less prone to noise effects in the measured data. The vascular term is incorporated implicitly by ranging the spectrum of exponentials from βi = 0 to a predetermined maximum flow rate. Upon solving this system using standard linear techniques, the blood perfusion + vascular volume of the tumour is determined by summing the spectrum of αi components.For the purposes of this analysis, tumour blood perfusion was derived from this equation by using the tumour VOI to input data into the tissue compartment of this model. Normally, the vascular compartment is neglected in this model as it generally makes a small contribution (approximately 5 %) to the net activity, thus reducing the analysis to a single-compartment model. However, for tumour regions, the high vascular density requires this term to be explicitly modelled . Thus, bRadiological response of the reference tumour lesion and overall radiological response were assessed at week 12 using Response Evaluation Criteria in Solid Tumors (RECIST) . ClinicaBlood samples for analysis of pharmacokinetic parameters were collected on day 1 and every 14 days in cycle 1. Levels of sunitinib and its principal metabolite, SU12662, were determined using liquid chromatography and mass spectrometry techniques. Peripheral blood for biomarker analysis was collected before and 6 h after the first dose on day 1 and at every visit thereafter . Samples were screened by enzyme-linked immunosorbent assays and 2D-gel electrophoresis for proteins whose levels may be altered in association with sunitinib activity or exposure: for example, VEGF and soluble VEGFR-2 (sVEGFR-2).t test, comparing patients with clinical benefit to those without clinical benefit of treatment . Analysis of day 15 trough levels of sunitinib plus SU12662 was performed using a z-test for patients with clinical benefit and patients without clinical benefit and a reference of 50 ng/mL. Linear relationships between change in tumour perfusion or change in FDG-SUV and biomarker levels were determined using Pearson's product moment correlation with 95 % confidence intervals.Data were summarised using descriptive statistics. Analysis of change in tumour perfusion or FDG-SUV was performed using the Student's K1. Partial volume effects were not considered as the tumour VOIs were of similar size for both pre- and post-treatment.Data are reported for seven patients who underwent all study investigations and completed at least two treatment cycles Table . Dosing Table t test, p = 0.05; Figure r = 0.76, p > 0.05 and VEGF: r = −0.75, p > 0.05). In this study, there was no evidence of correlation between the degree of change in tumour perfusion with the degree of FDG-SUV change  compared with those in patients who were not responsive to the therapy (p = 0.38).Further analyses were undertaken in order to investigate potential correlations between percent reduction in tumour perfusion and clinical benefit, duration of therapy, percent reduction in FDG-SUV, plasma levels of sunitinib and SU12662, and changes in plasma levels of VEGF and sVEGFR-2 Table  and 5. TThe results of this study conclusively show that sunitinib treatment of adult patients with a variety of metastatic malignancies is associated with early biological responses, including decreased blood flow in secondary tumour deposits. The reduction in tumour blood flow was associated with a corresponding fall in FDG-SUV, consistent with a decrease in tumour metabolic rate. This is the first study to provide quantitative evidence of the pharmacodynamic effects of sunitinib on tumour blood flow by PET imaging. There was also a possible association between the degree of change in tumour perfusion measured after 2 weeks of therapy and subsequent clinical benefit, duration of therapy, and change in levels of the biomarker VEGF. Blockade of VEGFR signalling by sunitinib may lead to reduced VEGF uptake by VEGFR and, hence, increased circulating VEGF, which was seen in this study.Not every patient who had a fall in tumour perfusion went on to have a conventional tumour response. Possible explanations for this lack of correlation include a mismatched assessment timing for tumour perfusion (at 2 weeks) and radiological response (at 12 weeks), allowing for tumour progression after an initial early response. Dosing was delayed in one patient, and the dose was reduced in another of the three patients with progressive disease at 12 weeks, which may have influenced the clinical tumour response despite an earlier decrease in tumour perfusion. Mixed responses, with the reference tumour showing a response but other tumour deposits showing progression, and stable or enlarging tumour masses despite central necrosis, could also account for the observed lack of correlation between tumour perfusion and conventional response. In this context, PET imaging may be informative regarding initial pharmacodynamic changes in response to sunitinib treatment, but may not be predictive of the final response.15O-water-PET without requiring an arterial line. The use of 15O-water-PET for the quantitation of blood flow is well established; it is a reliable method to assess the pharmacodynamics of therapeutic agents with a mechanism of action involving angiogenesis inhibition [15O-water does restrict its use to PET centres with on-site cyclotrons. Additional limitations of this study that preclude definitive conclusions include the small sample size and heterogeneous nature of the cases. In addition, there may be a sampling error associated with the selection of target lesions when multiple lesions are present.The methodology used in this study for assessing tumour perfusion changes was based on established techniques and adapted to enable quantitative analysis of tumour perfusion with hibition . The sho89Zr-ranibizumab in mouse xenograft models of human cancer. VEGF-PET demonstrated dynamic changes during sunitinib treatment, with a strong decline in signal in the tumour centre and only minimal reduction in the tumour rim, with a pronounced rebound after sunitinib discontinuation. In addition, VEGF-PET results corresponded with tumour growth and immunohistochemical vascular and tumour markers.Blood flow in solid tumours is often increased relative to the surrounding tissue -25. MoreThe results presented here indicate that sunitinib therapy is associated with early biological responses and measurable pharmacodynamic changes in tumour blood flow and metabolism. These data provide important new insights into the antitumour effects of sunitinib in this patient population.EML4-ALK mutations in lung cancer patients. RJH received funding support from Pfizer for the Translational Lab and PET Centre via his institution . CB, NB, and TJM are Pfizer employees and hold Pfizer stock. GCT acted as a consultant for Pfizer, received honoraria for attending international and Australian advisory board meetings, and received travel sponsorship to attend international meetings, including meetings at which this study was reported; his institution  received funding support from Pfizer for the Translational Lab and PET Centre. The other authors have no competing interests to declare.PLM received funding from Pfizer for a laboratory-based research on the prevalence of 15O-water studies would be acquired over and aid in subsequent image analysis. CB was responsible for the clinical development of sunitinib and was therefore involved in the conception, design, and execution of the study, and data evaluation. NB was involved in conceiving of the study and participated in the study design and coordination. TJM reviewed the data and the manuscript. GCT was involved in the conception of the project, protocol writing, patient accrual, and data analysis. All authors were involved in the drafting of the manuscript and approved the final version for submission.AMS participated in the design of the molecular imaging components of the study and coordination of molecular imaging data acquisition and analysis. PLM participated in the design and coordination of the study, and analysis. GO'K was involved in the design of the imaging studies and data analysis. TS performed the PET data collection and image analysis. RJH was involved in the conception and design of the molecular imaging components of the clinical trial of which this study represents a subgroup. AP assessed the site of the metabolically active malignancy on the patients' baseline FDG-PET studies and then decided on the tumour site where the


Early therapy-induced increase of the tumour tracer uptake correlates with favourable outcome, whereas stable or decreased uptake correlates with stable disease or tumour progression. Therefore sequential 99mTc-Hynic-rh-Annexin V scintigraphy could be used to predict therapy outcome on a patient-to-patient basis within 48 hours after the start of treatment. However, moderate tumour-to-background ratio and therapy-induced changes in normal tissues could confound image analysis. To assure accurate interpretation of Annexin V scans, the awareness of the biophysiological and biochemical properties contributing to the tracer distribution is essential. In with manuscript we discuss the patterns of Annexin V tumour uptake and illustrate the most frequent pitfalls associated with Annexin V imaging in correlation with CT and MRI imaging.Radionuclide detection of apoptosis with of  Defects in apoptotic pathways are now known to contribute to a wide range of human diseases, varying from neurodegenerative disorders to malignanciesIf disruption of the apoptosis process contributes to pathogenesis of cancer, then it is not surprising that existing anticancer agents work by triggering apoptosisDespite extremely complicated genetic regulation of apoptotic pathways, morphological cell changes are quite predictable. The morphological hallmarks of apoptosis are cell membrane blebbing, cytoplasm shrinkage, chromatin condensation and fragmentation into membrane-closed vesicles2+ and phospholypid-binding siteAnnexin V is a member of the annexin family of calcium and phospholipids binding family of proteinsMost of the known biological activities of Annexin V are attributed to its high affinity for 99mTechnetium by hydrazine-nicotinamide (HYNIC) method is so far the most widely investigated and extensively used tracer for in vivo visualisation of programmed cell death. It is successfully used in animal models of myocardial ischemiaRecombinant Annexin V labelled with 99mTc-Hynic-rh-Annexin V scintigraphy is performed 4 hours after the injection of 555–925 MBq (15–25mCi) of 99mTc-labeled Annexin V to allow clearance of the radiotracer from the soft tissues, resulting in a higher tumour-to-background ratio. A gamma camera equipped with low-energy, high-resolution collimator provides the best quality images. Standard acquisition includes anterior and posterior static whole body images, obtained with the matrix 1024 × 512 and SPECT of the region of interest acquired by the step-and-shoot mode, one step per 3 degrees, 30 seconds per frame, matrix size 128 × 128 × 16. For SPECT reconstructions an iterative algorithm and postfiltering using a Butterworth filter  are recommended.The time interval between the initiation of therapy and early posttreatment scan can be a critical point for correct interpretation of the tumour uptake changes. Currently posttreatment scans are performed between 4 and 48 hours after the start of treatment and depends on both the tumour morphology and treatment modality.99mTc-Hynic-rh-Annexin V binds selectively to phosphatidylserine (PS) residuals in the presence of Ca2+ ions independently of the apoptosis-inducing agent or cell type. After intravenous injection it is distributed with the bloodstream and binds to the apoptotic cells or cell debris in normal tissues and tumours. Imaging is typically started at least four hours after injection to allow sufficient blood pool clearance. Normal physiologic uptake is seen in the liver, spleen, and bone marrow. Because of predominantly renal clearance, intense accumulation is always seen in renal parenchyma, collecting system and bladder99mTc-Hynic-rh-Annexin V is illustrated on It is widely accepted that therapy-induced changes in Annexin V tumour uptake, detected shortly after the start of anticancer treatment correlate well with therapy outcome in various malignant tumours. In our previous publications we reported the pattern of the tracer uptake in low grade lymphomas, non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma. In general, therapy-induced increase of the tumour uptake within 48-hours after the start of treatment correlated with favourable response, stable or decreased tracer accumulation indicated stable disease or progression. Interestingly, the pattern of Annexin V tumour uptake depended to a large extent on tumour morphology and treatment regimen. Low grade lymphomas, highly radio-sensitive tumours, responding to radiotherapy predominantly through therapy-induced apoptosisThus the general pattern of therapy-induced tumour uptake changes in malignant tumours seems to be constant. Therapy-induced increase of tracer uptake correlates with favourable outcome, stable or decreased uptake—with stable disease or progression, however the intensity and grade of tracer uptake could vary depending on tumour morphology.Annexin V accumulation before the start of treatment represents a combination of spontaneous apoptosis and necrosis as part of tumour cell turnover. Consequently, tracer accumulations outside the known tumour sites or areas of physiological uptake on the total body and SPECT could indicate unknown tumour localisations as illustrated on Tumour uptake evaluation could be compromised by intense tracer accumulation in the surrounding normal tissues. This phenomenon is of high importance for the evaluation of the posttreatment scans, when normal tracer distribution is disturbed by therapy-induced increase of uptake in the salivary glands and bone marrow.Introduction of such hybrid SPECT/CT systems has addressed this problem; however in absence of hybrid systems, accurate software based matching could deliver additional information about the tumour localisation and its relation with normal structures.Increased Annexin V uptake can be observed in many benign processes, including inflammation and infectious diseases , instable atherosclerotic plaques. Another possible pitfall we have observed was the tracer accumulation in capillary haemangioma.Infectious foci contain both necrotic and apoptotic cells, thus providing the substrate for Annexin V binding as illustrated in Annexin V was first introduced as a platelet-directed thrombus-targeting agent. In vivo visualisation of acute platelet-rich thrombi by means of Annexin V scintigraphy is widely discussed in the literatureAnnexin V accumulation in atherosclerotic plaques is suggested to represent plaque instability and may be used to identify patients at risk for acute vascular eventsHaemangioma is a common benign vascular neoplasm that closely resembles normal vessels and that can be found in all organs of the human body. The mechanism of the Annexin V accumulation in haemangioma is not completely clear yet, however it is suggested that spontaneous apoptosis in the endothelial cells, reflecting involuntary changes in the lesion, could be responsibly for this phenomenon as illustrated in Anticancer treatment causes prominent changes in tumour tracer accumulation and affects tracer distribution in normal tissues. Both early and late therapy induced changes could influence tracer distribution in normal tissues. The most prominent changes occur in salivary glands and bone marrow. As reported in our previous publication, salivary glands show intense increase of tracer accumulation confined to the radiation field. It could represent radiation-induced apoptosis in the serous gland cells as demonstrated by Stephens et al Chemotherapy and radiotherapy-induced bone marrow tracer accumulation could reflect intramedullary apoptosis of haematopoietic tissue. As shown by Blankenberg et al, increase of Annexin V uptake in the bone marrow shortly after the start of chemotherapy, correlated with decrease in marrow cellularity affecting white and red blood cell nuclei with increasing atypia and nuclear fragmentation (apoptosis)Absence of tracer uptake in previously irradiated bone marrow could, thus be related to different radiosensitivity and  regrowthSequential evaluation of early therapy-induced apoptosis is a promising method to predict tumour response within 48 hours after the start of treatment in various malignant tumours. Therapy-induced increase of the tracer uptake correlates well with short-term outcome, decrease or stable tracer uptake is characteristic for unfavourable prognosis. Intensity and time-window for apoptosis imaging varies from tumour type and therapy regimen. Moreover, tumour uptake evaluation could be compromised by tracer accumulation in normal structures and non-malignant lesions. Knowledge of pattern of tracer uptake and recognition of the pitfalls of Annexin V-based apoptosis imaging could improve accurate identification and interpretation of tracer uptake to allow patient-tailored therapy.


Moreover, treatment of nude mice with repeated intraperitoneal or subcutaneous injections of Erufosine is well tolerated and yields drug concentrations in the brain tissue that are higher than the concentrations required for cytotoxic drug effects on glioblastoma cell lines in vitro.Erufosine is a promising anticancer drug that increases the efficacy of radiotherapy in glioblastoma cell lines xenograft-tumours in NMRI nu/nu mice.In the present study we aimed to evaluate the effects of a combined treatment with radiotherapy and Erufosine on growth and local control of T98G subcutaneous glioblastoma We show that repeated intraperitoneal injections of Erufosine resulted in a significant drug accumulation in T98G xenograft tumours on NMRI nu/nu mice. Moreover, short-term treatment with 5 intraperitoneal Erufosine injections caused a transient decrease in the growth of T98G tumours without radiotherapy. Furthermore, an increased radiation-induced growth delay of T98G xenograft tumours was observed when fractionated irradiation was combined with short-term Erufosine-treatment. However, no beneficial drug effects on fractionated radiotherapy in terms of local tumour control were observed.We conclude that short-term treatment with Erufosine is not sufficient to significantly improve local control in combination with radiotherapy in T98G glioblastoma xenograft tumours. Further studies are needed to evaluate efficacy of extended drug treatment schedules. Despite recent advances in therapy of high-grade glioma the prognosis of these highly aggressive tumours is still poor. The high morbidity upon resection of the primary tumour, the early and widespread infiltration of malignant cells into the surrounding tissue, the rapid progress and the high intrinsic resistance against chemotherapy and radiotherapy are major biological factors responsible for treatment failure. Therefore, researchers aim at the identification of drugs that cross the blood–brain barrier and overcome therapy resistance for improving clinical outcome for patients suffering from high-grade glioma.in vitro[Preclinical data suggest alkylphosphocholines (APC) as promising compounds for the treatment of brain tumours, including malignant glioma: Intravenously applicable APCs like Erufosine cross the blood–brain barrier of rats and mice upon repeated parenteral administration of tolerable drug-doses ,2 and exin vitro. Agents in vitro. Clinicain vitro-8. Moreoin vitro,10 suggein vitro and in animal experiments [Interestingly, APC increase the efficacy of chemotherapy and radiotherapy eriments . Althougeriments , recent eriments -14 as weeriments ,16. Of neriments .in vitro activity on glioblastoma cell lines alone and in combination with ionizing radiation, and its ability to cross the blood–brain barrier and to accumulate in the brain tissue we aimed to evaluate the effects of a combination of Erufosine and fractionated irradiation on growth and local control of T98G glioblastoma xenograft-tumours in immunodeficient mice. The T98G glioblastoma cell line was selected as experimental model because these highly apoptosis-resistant cells were particularly sensitive to Erufosine-treatment alone and in combination with radiotherapy in vitro[While Perifosine represents an orally applicable APC, Erufosine is the first clinically relevant intravenously applicable APC. Due to the cis-double-bond in the alkyl chain, Erufosine and the closely related Erucylphosphocholine form lamellar instead of micellar structures in aqueous solutions and therefore lack haemolytic activity. Because of modifications in the polar part of the molecule Erufosine exhibits a better solubility in aqueous solutions compared to Erucylphosphocholine thereby facilitating its parenteral injection. On the basis of its potent  in vitro.ad libitum.Animal experiments were conducted according to German animal welfare regulations and approved by the local authorities (registration number RO 1/05). Immunodeficient NMRI-(nu/nu)-nude mice were purchased from the University Hospital Essen (age 6–12 weeks). Animals were housed in an individually ventilated cage rack system . They were fed with sterile high calorie laboratory food . Drinking water was supplemented with chlorotetracycline and potassium sorbate acidified to a pH of 3.0 with hydrochloric acid and was provided 6 cells in 300 μl medium (2+1 mixture of RPMI1640/10% FCS with matrigel (BD Biosciences)) into the axilla and serial passage of the resulting tumours. To obtain optimal suppression of residual immune activity in the donor mice a whole-body-irradiation with 4 Gy was performed 2 days before injection of the tumour cell suspension and before the first serial passage. For the experiments a source tumour was excised and tumour pieces of about 2 mm diameter were transplanted subcutaneously into the right hind limb of ether-anaesthetized mice (approximately 70% engraftment rate).Xenograft tumours of the human glioblastoma cell line T98G  were generated by injection of 3×102)/2, where a and b are the long tumour axis and the short tumour axis, respectively. Body weight was monitored twice a week except for the treatment time; here, the body weight was recorded daily. Mice were randomly allocated to the treatment groups when the tumour volume reached 80–100 mm3. In the present experiments the median volume doubling time from the inclusion volume was 6.5 days .For the determination of tumour growth, tumour size was quantified with calipers in two perpendicular diameters twice a week. The tumour volume (V) was calculated as V =  is the -propylammoniumester of erucyl-phosphoric acid. It was kindly provided by H. Eibl, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany. For aqueous solutions Erufosine was dissolved at 60°C in a mixture of destilled water and 1.2-Propandiol  to a final concentration of 24 mg/ml Erufosine and stored at 5°C after sterile filtration. For intraperitoneal or subcutaneous injection this stock solution was diluted with 0.9% sodium-chlorid solution in the appropriate ratio to obtain the required Erufosine-concentration in 100 μl for a body weight of 30 g. Difference in body weight of the mice were adjusted with injection volume.xenograft tumours in preliminary experiments. Eight intraperitoneal injections of Erufosine every 48h at doses of 20 mg/kg BW or 40 mg/kg BW yielded tumour concentrations of 197 ± 52 nmol/g or 626 ± 76 nmol/g, respectively  at the end of Erufosine-treatment. Such concentrations are above the drug concentrations  known to potently induce tumour cell death when given as single drug and for increasing the cytotoxic efficacy of ionizing radiation in vitro[To establish the schedule of Erufosine-treatment for the present study we analyzed drug-accumulation in  in vitro.intent to treat” (1% of injections). The descision was taken by the responsible scientist (G.H) on the basis of weight measurement and control of the animal behavior before each injection. Control animals received an intraperitoneal injection of the solvent.Accordingly, in the experimental study mice were treated by eight intraperitoneal injections of 40 mg/kg body weight (BW) Erufosine to a cumulative dose of 320 mg/kg, respectively. Intraperitoneal injection was used as standard administration route because we showed in our earlier investigation that intraperitoneal injection was generally well tolerated whereas repeated subcutaneous injections of high-dose Erufosine regularly caused local reactions at the injection site at similar bioavailability although less gastrointestinal side effects were observed . If a weFor the quantification of Erufosine in plasma and tissue samples liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed with a deuterium labelled analogue  as internal standard as described in detail elsewhere .For the analysis of Erufosine-concentrations in subcutaneous xenograft tumours, the tumours were removed after diethylether anaesthesia and immediate cervical distortion 1 day after the end of the Erufosine treatment. The tumours were weighed and stored at −20°C until analysis. The Erufosine-concentration was then analyzed using the frozen tissue samples.-1 dose rate) to the tumour on the right hind limb of the animals as described earlier [Fractionated irradiation was given under ambient conditions with a linear accelerator . Follow up was discontinued after 120 days from treatment, in case of intercurrent death or if the volume of the progressive or recurrent tumours reached 8-fold the starting volume. An exponential regression model was used to interpolate a modified growth delay endpoint as time after onset of irradiation treatment needed to achieve 4- and 8-fold size of the initial tumour volume at the start of treatment for each single tumour. Animals that appeared to suffer were killed before reaching these endpoints and were considered as censored at this time-point.To estimate the irradiation dose for the combination experiments animals were randomized to 4 treatment groups receiving no irradiation (n=7), fractionated irradiation of 5x 2 Gy (n=8), 5x 3.5 Gy (n=7) and 5x 4.5 Gy (n=7) alone  compared to the first experiment in order to achieve comparable tumour volumes at the onset of irradiation treatment on day 5. Follow-up was discontinued after 200 days from treatment of the last set of animals, in case of intercurrent death or if the volume of the progressive or recurrent tumours reached 8-fold the starting volume. Growth delay after the onset of Erufosine treatment was determined as described above.In experiment 2 Figure B, fractiin vitro. ErufosiVolume doubling times (VDT) were defined as time interval between the start of treatment to the day when the tumour reached twice the initial tumour volume in case of continuously growing tumours. If a transient regression occurred upon treatment the volume doubling time was calculated as interval from the first day of increase of tumour volume to the day when the tumour reached twice the minimal tumour volume.3 . Follow up was similar to the procedure described above. Tumour control rates at day 200 after the end of treatment were determined with respect to censored animals as described by Walker and Suit [Experiment 3 was designed to determine the tumour control probability. For this, a graded top irradiation - estimated to achieve local tumour control with the higest top-up dose from experiment 1 - was added after fractionated irradiations (5 × 3.3 Gy) with and without 40 mg/kg BW Erufosine . P -values of p ≤ 0.05 were considered statistically significant.Growth delay is presented according to the method of Kaplan and Meier considering achievement of the 4- and 8-fold size of the initial tumour volume as event and taking animals without the respective tumour volume at the end of the observation period or undergoing intercurrent death as censored at that time-point. Differences between groups were tested for significance by the Wilcoxon-test. Median growth delay with 95%-confidence intervals was calculated with lognormal approximation implemented in JMP 9.0.0® software (SAS institute Inc.).For estimation of the dose dependent tumour control probabilities including 95%-confidence intervals (CI), the results of the top up dose experiments including the data for control tumours (no cures at 0 Gy) were modelled by using the logit module of a commercial software package (XLSTAT2011® by Addinsoft SARL).Arithmetic means with standard deviation are presented and student′s t-test was applied when appropriate.We first established the appropriate doses of fractionated radiotherapy for the combination experiments Figure A. FractiIn the experimental study mice were treated by eight intraperitoneal injections to a cumulative dose of 320 mg/kg. In order to achieve an appropriate Erufosine concentration within the tumour tissue during the course of radiotherapy mice were pretreated with 5 daily injections of 40 mg/kg body weight (BW) Erufosine followed by 3 injections of Erufosine every 48h without or with concomitant fractionated irradiation Figure B. To evaTo detect a beneficial effect of a combined treatment with signal transduction inhibitors and radiotherapy it is important to use a radiation dose which induces a significant but moderate tumour growth delay without causing prominent local control as single treatment. On the basis of the results from Experiment 1 we selected a dose of 3.3 Gy per fraction. To gain insight into treatment-related toxicity we recorded the median relative weight of the nude mice in the four treatment groups. Radiation alone failed to induce a relevant weight loss whereas treatment with 40 mg/kg BW Erufosine alone or in combination with fractionated irradiation led to a transient median weight loss of about 10% (data not shown). Also the number of Erufosine-treated mice that died during treatment was equal in the nonirradiated and irradiated treatment groups . These observations reveal that the addition of fractionated irradiation to the Erufosine treatment does not cause unexpected additional toxicity.The course of the median tumour volume in the four treatment groups is depicted in Figure In line with these results, the median growth delay until completion of the 4- and 8-fold initial tumour volume significantly increased after fractionated irradiation. Again, laying emphasis on the early observation period, a trend (Wilcoxon-Test p=0.08) to an increased tumour growth delay was observed when Erufosine treatment was used in combination with fractionated irradiation , 6.6 , 9.2  and 8.6  days for untreated controls, Erufosine alone, fractionated irradiation alone and combination therapy, respectively . Since the growth delay described by the authors was more pronounced compared to the results obtained in our study we speculate that the higher cumulative dose (475 mg/kg) of Perifosine upon oral administration may be responsible for the improved drug action [in vivo efficacy of these compounds may depend on an extended treatment schedule.We show that repeated parenteral injections of 20 or 40 mg/kg BW Erufosine result in a significant accumulation of this membrane-targeted agent in T98G xenograft tumours. This corroborates findings of Vink and co-workers showing accumulation of the closely related compound Perifosine in KB squamous cell carcinoma xenograft tumours upon repeated oral drug administration . Interesg action . Similarg action  detectedin vivo[Otherwise, the heterogeneous outcome of preclinical and clinical studies with Perifosine implicates that the efficacy of treatment with the membrane-targeted APC also largely depends on the cell type. Whereas extended treatment with Perifosine was without single drug effect in prostate cancer xenografts in vivo and no ein vivo-28, the in vivo and to sin vivo,31.in vivo radiation sensitivity of this glioblastoma cell line but is in contrast to the clinical experience of high radiation resistance in glioblastoma tumours [We also found that T98G xenograft tumours respond to fractionated irradiation with a dose-dependent tumour growth delay. Already with doses of 5 × 3.5 Gy  we observed a significant tumour growth delay compared to the non-irradiated controls. This corroborates earlier findings showing exceptional  tumours -34.The combination of fractionated irradiation with 8 parenteral injections of 40 mg/kg BW Erufosine 4 days before and during the fractionated irradiation was obviously able to intensify the radiation-induced decrease in tumour volume. The failure to detect more than borderline significant differences in the growth-delay of tumours treated with fractionated irradiation alone and fractionated irradiation plus Erufosine may at least partially be due to the unexpected high rate of local tumour control rates that were observed in response to fractionated irradiation alone in the combination experiment 1): Only a low rate of local controls (14%) had been observed in the dose-finding experiment in the highest dose-group (5 × 4.5 Gy) and no local controls had been observed in the 5 × 3.5 Gy dose-group. In contrast, a largely increased rate of local controls (28.6%) was observed in the combined treatment experiment (experiment 2) upon fractionated irradiation with a dose of 5 × 3.3 Gy. The reason for the discrepancy between the two experiments is unknown. Only 66% of transplanted animals developed subcutaneous T98G tumours which may be indicative of a residual antitumour immunoreactivity in the NMRI nu/nu mice . But KraFinally, when analyzing whether the addition of short-term Erufosine treatment to fractionated irradiation would improve tumour control probability by measuring the tumour control rates after additional top up irradiations we found that short-term Erufosine failed to improve the outcome of fractionated radiotherapy in terms of local tumour control. This suggests that although tumour regrowth was slightly retarded by co-treatment with erufosine no additional effect on the eradication of clonogenic tumour stem cells could be achieved.Notably, the TCD50 value for T98G xenografts detected in the present study  was in the range of the TCD50 values reported in earlier studies for T98G xenografts after single dose or fractionated irradiation ,34,36 alin vivo. However, those studies were restricted to the use of the orally available APC Perifosine and the evaluation of tumour growth delay. Consistent with our observations with the novel intravenously applicable APC Erufosine, De la Pena et al. [in vivo radiosensitivity of the two glioblastoma cell lines T89G and U251 [Up to now, only few studies are available that tested a potential benefit of APC in combination with radiotherapy a et al.  observedand U251  the irraIn contrast, the same protocol of Perifosine-treatment combined with 2 × 5 Gy on days 2 and 4 caused a significant growth delay in a prostate cancer xenograft model compared to untreated controls whereas the single modality treatment was not effective . NotablyIn vitro radiosensitivity does not necessarily correlate to the tumour control probabilities determined in vivo[in vivo[in vivo, e.g. orthotopic or spontaneous tumours, is still sparse because of the limited availablity of image-guided radiation systems for small animals.Also, the following limitations of the preclinical models have to be carefully considered: (i) d in vivo. (ii) Tro[in vivo. In thiso[in vivo-42. Howein vivo but fails to improve efficacy of fractionated irradiation in terms of local tumour control. Further in vivo studies are needed to evaluate whether extended Erufosine treatment may be more effective in terms of radiosensitization and whether the drug may interfere with the known adverse biological factors that limit the efficacy of radiotherapy in vivo.In conclusion, the intervenously applicable APC Erufosine with proven ability to cross the blood brain barrier causes a transient decrease in the growth of T98G glioblastoma tumours The authors declare that they have no competing intereststhat could inappropriately influence this work.GH contributed significantly to the design of the study, data acquisition, data analysis and drafting the manuscript. VM contributed significantly to data acquisition, data analysis and drafting the manuscript. LHL contributed significantly to data acquisition and data analysis. HE synthesized and provided ErPC and ErPC3 for the analysis. MB performed critical revision of the manuscript. CB participated in the conception of the study and interpretation of data. WB substantially contributed to evaluation and interpretation of data and performed critical revision of the manuscript. VJ performed conception and design of the study and substantially contributed to interpretation of data, drafting of the manuscript, critical revision of the manuscript and final approval. All authors read and approved the final manuscript.


Sertoliform cystadenoma of the rete testis represents an uncommon benign tumour. They appear in patients from 26 to 62 years of age. We describe a case of a 66-year-old man with a tumour in the area of the epididymal head. The tumour markers were not increased. Under the assumption of a malignant testicular tumour an inguinal orchiectomy was performed. The cut surface of this tumour was of grey/white color and showed small cysts. The tumour consisted of two compartments. The epithelial like tumour cells showed a sertoliform growth pattern and cystic dilatations. In between the tumour cells repeatedly actin expressing sclerotic areas could be recognized as the second tumour component. Proliferative activity was not increased. Immunohistochemically the tumour cells were positiv for inhibin, S-100, and CD 99. Alpha feto protein (AFP), human chorionic gonadotropin (ß-HCG) and placental alkaline phosphatase (PLAP) as well as synaptophysin, epithelial membrane antigene (EMA), and BCL-2 were not expressed. As far as we know this is the sixth reported case of this tumour. Because of the benign nature of this tumour the correct diagnosis is important for the intra- and postoperative management. Here we present a case of this rare tumour and discuss potential differential diagnosis.http://www.diagnosticpathology.diagnomx.eu/vs/1956026143857335The virtual slide(s) for this article can be found here:  A 66 year old man was presented in the urological clinic because of slightly increased blood level of prostatic specific antigen. The investigation of the prostate revealed a benign hyperplasia. Further urological examinations showed a mass of approximately 2 cm in diameter in the area of the epididymal head Figure A+B. On uOn macroscopical examination the testis measured 8.5 × 4.5 × 1.5 cm. The epidymidis measured 5 × 2 × 1.5 cm and the spermatic cord was 10 cm in length. The cutting surface of the testis showed a homogenous brown colour. Within the area of the tunica albuginea/rete testis a 2.1 × 1.8 × 1,4 cm tumour was detectable. The cut surface of this tumour was of grey/white color and showed small cysts of about 5 mm in diameter. The tumour exhibited an expansive growth pattern into the testis parenchym.The tumourfree testicular tissue showed regular tubules, regular spermatogenesis, and normal interstitial tissue. The tumour consisted of two compartments. The epithelial like tumour cells showed a sertoliform growth pattern and cystic dilatations. The uniform tumour cells were ordered in tubules and acini. The cytoplasm of the tumour cells was eosinophilic, the nuclei showed prominent nucleoli Figure A-F. ProlSo far only five cases of a sertoliform cystadenoma of the rete testis have been reported. They appear in patients from 26 to 62 years of age. The tumors range from 1 to 3 cm in size showing cystic and solid masses arising from the rete testis [Because of the benign nature of sertoliform cystadenomas the correct diagnosis is important for the intra- and postoperative management. Clinically and on ultrasound examination a clear diagnosis can not be ultimatively made.Because of the striking sclerotic tumour component three possible differential diagnoses exist morphologically: 1) Rete testis cystadenoma: This rare tumour also shows cystic dilatations and sclerotic areas within the rete testis, in addition the cysts are also lined by cuboidal epithelium. In contrast to sertoliform cystadenoma, however a sertoliform growth pattern is missing  Rete tes. (3) DesA sertoliform cystadenoma of the rete tesis is an extremly rare tumour of the testis. As far as we know this is the sixth reported case of this tumour. Because of the benign nature of this tumour the correct diagnosis is important for the intra- and postoperative management.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.FB and CLB drafted the manuscript and arranged histological pictures. FB, SS and HJR were responsible for microscopic and histopathologic elements. MB cared for the patient and provided clinical information. HJR was responsible for the critical revision of the manuscript. All authors read and approved the final manuscript.


Clinical positron emission tomography imaging has demonstrated the vast majority of human cancers exhibit significantly increased glucose metabolism when compared with adjacent normal tissue, resulting in an acidic tumour microenvironment. Recent studies demonstrated reducing this acidity through systemic buffers significantly inhibits development and growth of metastases in mouse xenografts.in vivo in mice. We parameterise the model to humans to determine the translational safety and efficacy, and predict patient subgroups who could have enhanced treatment response, and the most promising combination or alternative buffer therapies.We apply and extend a previously developed mathematical model of blood and tumour buffering to examine the impact of oral administration of bicarbonate buffer in mice, and the potential impact in humans. We recapitulate the experimentally observed tumour pHe effect of buffer therapy, testing a model prediction in vivo experiments. Simulations predict limited efficacy of bicarbonate, especially in humans with more aggressive cancers. We predict buffer therapy would be most effectual: in elderly patients or individuals with renal impairments; in combination with proton production inhibitors (such as dichloroacetate), renal glomular filtration rate inhibitors , or with an alternative buffer reagent possessing an optimal pK of 7.1–7.2.The model predicts a previously unseen potentially dangerous elevation in blood pHe resulting from bicarbonate therapy in mice, which is confirmed by our Our mathematical model confirms bicarbonate acts as an effective agent to raise tumour pHe, but potentially induces metabolic alkalosis at the high doses necessary for tumour pHe normalisation. We predict use in elderly patients or in combination with proton production inhibitors or buffers with a pK of 7.1–7.2 is most promising. In fact, extensive clinical experience in imaging human cancers through 18F-deoxy-D-glucose PET has demonstrated the vast majority of human malignancies exhibit increased glycolysis  to severe combined immunodeficient mice bearing MDA-MB-231 and PC3M tumours  Can the experimental bicarbonate therapy results be safely and effectively translated into clinical application as a therapeutic intervention against malignant cancer? (2) Can tumour pHe normalisation be enhanced or more safely delivered through strategies that increase buffer concentration, improve buffering capacity, decrease H2/HCO3− buffer system present in blood and tissues. In this analysis, we examine the impact of administration of bicarbonate on blood and tumour pHe in mice and humans. A schematic of the model is shown in To examine the effect of buffer administration on blood and tumour pHe, we apply and draw clinical insights from a previously developed simple, but realistic mathematical model of the COWe use a two-compartment model, representing, respectively, the arterial blood and tumour tissue with a diffusively dominated transport coupling given the small molecules under consideration . The ai2=40 mm Hg, HCO3−=24 mmol l−1, and pHe=7.4). Mouse and human parameters used for the simulations are listed in The system of equations is solved in MATLAB using the stiff solver ode 15s, a variable order multistep solver. Initial conditions are normal arterial biological values of the variables . Model predictions were compared with the experimentally observed pHe, which was monitored using fluorescence ratio imaging of SNARF-1 in the dorsal skin-fold window chamber tumour xenografts =0.007184 × height (cm)0.725 × weight (kg)0.425 (To examine whether the buffering impact seen in the n=20) received orthotopic injections of 5 × 106 MDA-MB-231 tumour cells in a mammary fatpad. Animals were monitored and maintained by the Experimental Mouse Shared Services core facility of the Arizona Cancer Center, Tucson Arizona. Six days after injection, mice were randomised into two groups (n=10 in each group): the control was provided drinking water and the treated was provided sodium bicarbonate (200 mmol l−1) ad libitum, which continued for the duration of the experiment (3 weeks). At intervals, mice from both groups were removed for collection of urine specimens to obtain pH measurements. This was achieved by applying gentle pressure to the abdomen over a plastic membrane.The effect of bicarbonate on blood pHe was indirectly measured by sampling urine pHe in tumour-bearing mice. It is well established that a rise in blood pHe because of bicarbonate loading causes a proportionally larger rise in urine pHe (http://www.graphpad.com). Unpaired, two-tailed t-tests were used to determine if means of urine pHe  at each sample time-point  were significantly different between untreated and treated groups. A P-value of <0.05 was considered to be statistically significant. Data are represented as mean±s.e.m.Statistical calculations were determined using the analysis feature in GraphPad Prism version 4.03. for Windows , we develop an extended mathematical model, incorporating the administration and reaction kinetics of a hypothetical exogenously administered buffer. The equations and parameters for this system are detailed in the To determine promising combination therapies and potential patient groups who would benefit from treatment, we analyse results of a one-way sensitivity analysis . The elevated urine pHe in the bicarbonate treatment group was observable at the first data point after treatment initiation . As shown in P<0.001 at hour 6, P=0.02 at day 1, P=0.01 at day 2, P<0.001 at day 5, P<0.001 at day 7, P<0.001 at day 12, P=0.04 at day 21). Similarly, the urine pHe of the treated group remained significantly (P<0.01) elevated at each sample time-point as compared with before treatment initiation (at t=0).Before initiation of bicarbonate therapy, the urine pHe was not statistically different between each group  is approximately 12.5–15.0 g per day in a 70 kg human. Simulations indicate the equivalent human bicarbonate dose will have a substantially lower quantitative effect than in mice (rising the tumour pHe to 7.04 in humans vs 7.07 in a mouse), shown in An equivalent dose to mice maintained daily on 200 m2 or bicarbonate levels, we focus primarily on the dangers of altered blood pHe. An optimal treatment would maintain the blood pHe within normal levels (7.35–7.45) but raise tumour pHe to normal levels (7.2–7.4). Predicted dose response plots are shown in To examine the safety for various doses of chronically administered bicarbonate in humans, we predict dose response curves with respect to blood pHe. As the human body is resilient to altered COMetabolic alkalosis is characterised by a blood pHe of above 7.45 . In order to maintain the blood pHe below 7.46, the model predicts a maximum dose of 35 g per day of bicarbonate for a 70 kg human, leading to a tumour pHe of 7.10. Maximum daily dose recommendations of oral administration of bicarbonate are around 5 level teaspoons per day, or around 20–25 g per day, and little is known about the safety of long-term chronic ingestion of bicarbonate above this level. The modest predicted tumour pHe change in humans at the maximum safe dose emphasises the importance of using bicarbonate in conjunction with other therapies.If the alternative buffer is administered as a solution of acid and conjugate base, titrated to the blood pHe of 7.4, it will increase the buffering capacity of the blood. In contrast to bicarbonate therapy, the model predicts it will not raise the blood pHe, and could hypothetically be given at much higher doses. Simulations indicate the ideal buffer pKa is halfway between the pHe of the tumour and the blood; for a blood pHe of 7.4 and tumour pHe of 6.8–7.0, the optimal buffer pK is 7.1–7.2 . At equiA sensitivity analysis of the mathematical model indicates kidney filtration terms have the largest effect on tumour pHe . ConsequBased on the sensitivity analysis indicating the importance of renal function on tumour pHe, we examine the potential treatment effect in elderly patients with impaired renal function, and also in patients with chronic renal failure.−1, corresponding to a 35% loss in nephron number and equivalent reduction in acid secretion rate  is an analogue of acetic acid that increases the flux of pyruvate into the mitochondria, promoting glucose oxidation over glycolysis. Experimental studies have shown DCA selectively targets tumour cells, decreasing acid production and inducing apoptosis In combination, simulations with bicarbonate and proton inhibition (such as through the administration of DCA) show additive beneficial effects . Althoug3 tumour , was effective at reducing metastases in a PC3M mouse model, but resulted in only a marginal increase in tumour pH , and angiotensin-converting enzyme inhibitors (commonly prescribed for high blood pressure). As it is highly probable that late middle-aged and elderly patients will already be on these drugs, it is likely that the bicarbonate therapy would have a better effect on them. The use of drugs modifying renal filtration is likely to have significant side effects as well such as electrolyte imbalance, so treatments should be monitored closely. It is also important to note that the blood pHe is exquisitely sensitive to renal function , and theAging and other diseases that affect renal function contribute to higher concentrations of protons in the blood supply. These physiological states would hypothetically be advantageous for users of oral bicarbonate to treat acid-producing cancers because our model indicates they could tolerate higher and more effective doses with lower risk of developing systemic alkalosis. One study reports good tolerance of a chronic high dose (60 g per day) oral administration of sodium bicarbonate in a 79-year-old man with metastatic renal cancer, though blood pHe measurements were not reported . The bicarbonate-buffering effect is likely to vary between different tumour types. In our experiments, we use a spontaneously metastatic, highly aggressive tumour model. Therefore, experiments using a less aggressive cell line such as MCF7 could overestimate the potential bicarbonate benefit, but the true effect is unknown as bicarbonate buffering has not been examined in many cell lines. Previous bicarbonate-buffering experiments (Chronic oral bicarbonate clinical studies have previously been conducted, but not in cancer patients. In these studies, the doses and durations ranged from 12.6 g per day for 46 days to 1.8 g per day for 2 years (There is a compelling relationship between the adverse tumour microenvironment and cancer progression. Malignant tumour cells have a critical role in modulating the extracellular environment and there is evidence that this is advantageous for tumour invasion and metastases. The findings presented here show how the administration of exogeneous buffers could modify the tumour microenvironment in humans, specifically in terms of pHe. We elaborate how this process has limitations and may introduce other dangers, but suggest that modest changes in tumour pHe may be sufficient in some cases or helpful in other circumstances to reduce chemoresistance to specific therapies that are stifled by tumour acidity.


In contrast, malignant mesothelioma (MM) is a non-immunogenic tumour and because only a few mesothelioma-specific TAA are known to date, we investigated whether mesothelioma-derived exosomes could be used as antigen source in DC-based immunotherapy.In 2001, it was postulated that tumour-derived exosomes could be a potent source of tumour-associated antigens (TAA). Since then, much knowledge is gained on their role in tumorigenesis but only very recently tumour-derived exosomes were used in dendritic cell (DC)-based immunotherapy. For this, DCs were cultured Mouse MM AB1 cells were used to generate tumour lysate and tumour-derived exosomes. Tumour lysate was generated by 5 cycles of freeze–thawing followed by sonication of AB1 cells. Tumour exosomes were collected from the AB1 cell culture supernatant and followed a stepwise ultracentrifugation. Protein quantification and electron microscopy were performed to determine the protein amount and to characterise their morphology. To test whether MM derived exosomes are immunogenic and able to stimulate an anti-tumoral response, BALB/c mice were injected with a lethal dose of AB1 tumour cells at day 0, followed by intraperitoneal injection of a single dose of DCs loaded with tumour exosomes, DCs loaded with tumour lysate, or phosphate buffered saline (PBS), at day 7.Mice which received tumour exosome-loaded DC immunotherapy had an increased median and overall survival compared to mice which received tumour lysate-loaded DC or PBS.In this study, we showed that DC immunotherapy loaded with tumour exosomes derived from non-immunogenic tumours improved survival of tumour bearing mice. Exosomes are 30–100 nm extravesicular membrane particles that are released by virtually all cells in the body as well as tumour cells . PreviouDue to these potential immunogenic properties, studies have been performed to take advantage of tumour exosomes as an antigen source for dendritic cell (DC) immunotherapy in melanoma, renal cell carcinoma, and glioma –11. The Malignant mesothelioma (MM) is traditionally considered as a non-immunogenic tumour, meaning that they have lost the ability to trigger a strong immune response by the host. Because not all known MM associated antigens expressed in all MM cases, our previous studies both in mice and patients , 14 usedTherefore, in this study, we investigated the feasibility of using mesothelioma cell line-derived exosomes to load DCs in a mouse mesothelioma immunotherapy model.The AB1 mouse mesothelioma cell line was a gift from Prof. Bruce W. Robinson, Queen Elizabeth II Medical Centre, Australia. This highly immunosuppressive and non-immunogenic cell line was derived from BALB/c mice that developed mesothelioma after exposure to asbestos fibres. The cells were cultured in exosome-free medium (EFM) culture media consisting of RPMI 1640 supplemented with 25 mM HEPES, GlutaMax, 50 µg/ml gentamycin  and 5% exosome-free foetal bovine serum (FBS) . Exosome-free FBS was prepared by first heating the serum to 56°C for 30 minutes in a water bath to destroy heat-labile complement proteins. FBS was then centrifuged at 400×g for 10 minutes and at 2,000×g for 20 minutes (Hereaus Instruments). Finally, the FBS was centrifuged at 100,000×g  using ultra clear centrifuge tubes  at 4°C for at least 16 hours to remove all vesicles and exosomes. The supernatant containing exosome-free FBS was stored at −20°C until use.6 cells according to previously described ultracentrifugation methods  for 10 seconds on ice followed by 50 seconds rest, repeated for 3 times, to produce a homogeneous lysate.AB1 cells from the cell culture were collected and resuspended at a concentration of 100×102B4O7 10 H2O, pH 9.3). Potassium cyanide (KCN) and CBQCA were added to the solution to start the reaction with the primary amines to form highly fluorescent derivatives. After an incubation period of 2 hours, the samples were analysed in a fluorescence microplate reader  using excitation/emission wavelength of 465/550 nm, respectively. Bovine serum albumin (BSA) was used as a protein standard according to manufacturer's suggestion. The optical density (OD) of the dilution series was used to calculate a standard curve. The protein concentration was determined by using the standard curve and the OD value of the samples.The CBQCA-protein quantification kit (Invitrogen) was used to determine the amount of protein contained in tumour exosomes and necrotic tumour lysate. The CBQCA protein quantification kit uses the ATTO-TAG CBQCA reagent (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde) for the quantitation of proteins in solution. The protein samples were diluted into a sodium borate buffer  for 2 minutes each, samples were negatively stained with uranyl acetate and examined with a Philips CM 100 electron microscope (EM) at 80 kV .2. After 8 days in culture, DCs were loaded with exosomes or tumour lysate and after 8 hours, 100 µg/ml Lipopolysaccharide (LPS) [E. coli Serotype R515 (Re): Enzo Life Science] was added to the DC culture to allow complete maturation while incubated overnight at 37°C. Exosome-loaded DCs or tumour lysate-loaded DCs were washed in PBS at 400×g for 7 minutes with a Varifuge 3. OR centrifuge (Heraeus Instruments) at 4°C and resuspended in 3 ml PBS. Next, these DCs were separated and purified from free tumour material using 3 ml lympholyte-Mammal  density gradient centrifugation. After centrifugation at 1,200×g (Heraeus Instruments) for 20 minutes at room temperature, DCs were collected from the interphase using a pipette. DCs were washed in PBS at 400×g for 7 minutes at 4°C and resuspended in a concentration of 1.6×106 cells/500 µl PBS for in vivo DC immunotherapy.DCs were generated using modification of a previously described procedure . InsteadDCs were analysed by the following markers: CD11c (PE Texas Red) and MHCII (AF700) CD40 (APC), CD80 (PerCP-Cy5.5), and CD86 (PE Cy7). DAPI was added to exclude dead cells and relevant isotype antibodies were used as control. Antibodies were diluted in a ratio of: 1:200 CD40, 1:800 CD80, 1:1,600 CD86, 1:400 MHCII, 1:100 CD11c and 1:300 2.4.G2 (to prevent non-specific binding). Live/dead marker DAPI (Invitrogen) was added in a 1:5,000 dilution. After 30 minutes of incubation at 4°C, cells were washed twice in binding buffer and the cells were subsequently measured on the FACS LSR II.6 AB1 tumour cells. On day 7, 6 mice received an injection with 500 µl PBS as control, 6 mice received DCs loaded with tumour lysate, and 6 mice received DCs loaded with tumour exosomes. Each vaccine contained 1.6×106 DCs diluted in 500 µl PBS. Tumour growth, physical well-being, body temperature and survival were monitored up to 52 days after tumour injection. Mice were killed if profoundly ill according to UKCCCR regulations, and were scored as a death in survival analysis. At day 52, surviving mice were sacrificed.Eighteen female BALB/c mice 6–8 weeks old  were housed at the animal care facility of the Erasmus MC, Rotterdam. The local Ethical Committee for Animal Welfare approved the experiment. At day 0, all BALB/c mice were injected intraperitoneally with a lethal dose of 0.5×10−1, Sigma-Aldrich) and new fuchsine (160 mg·mL−1 in Sodium Nitrite) were used as substrate. Finally, the tissue sections were stained with Mayer's hematoxylin for nuclear staining.Tumour material was collected from mesothelioma bearing mice that were euthanised at day 15 (untreated mice) and day 52 (DC-treated mice). Tissue sections (7 µm) were cut on a HM-560 cryostat  and washed in acetone for 10 minutes before adding Normal Goat Serum (1:10 diluted) in blocking buffer 1%. Immunostaining was performed using CD3 , CD4 , CD 8  and CD11c  antibodies, with a 1-hour incubation period. Binding of antibodies was detected using immune-alkaline phosphatase (AP) on the secondary antibodies Goat-anti-Rat (1:50) and Goat-anti-Hamster (1:50). Naphtol-AS-MX-phosphate , we harvested DCs and injected 1.6×10In the survival curve, within 15 days after tumour injection, all mice from the PBS control group died In contrast, DC treated group had an increase in survival; 16.7% of mice from necrotic tumour lysate-loaded DC group and 33.3% of mice from tumour exosome-loaded DC group survived  until th+ T cells, CD8+ T cells and DCs infiltration within the tumour of the DC vaccine treated group (The surviving tumour exosome-loaded DC treated mice did develop tumours, but we noticed a remarkable difference between the tumours developed in these mice and tumours developed in control group. Immunohistochemistry results on the tumour isolated from the 2 groups demonstrated distinct cellular composition inside the tumour nest. We observed the presence of abundant CD4ed group . In contAltogether, these findings suggest that mesothelioma exosome-loaded DC vaccine was capable of stimulating immune responses in mesothelioma bearing mice resulting in an increased survival compared to untreated group and the tumour lysate-loaded DC group.Although in this experiment we did not include a group of mesothelioma bearing mice which received unloaded DC to show that this effect was indeed mesothelioma exosome dependent, our previous study  showed tin vivo. Although mesothelioma-derived exosomes contained less proteins than necrotic tumour lysate (Exosomes derived from non-immunogenic and highly suppressive mesothelioma tumours are enriched with tumour antigens and are capable in inducing anti-tumour responses when presented by DCs to the T cells r lysate , when loTo confirm the immunogenicity of mesothelioma-derived exosomes, future studies are required to further define the composition of these exosomes to optimise and standardise the technique. Here, we show that tumour exosomes from the non-immunogenic mesothelioma tumour improve the efficacy of DC-based immunotherapy for MM.


Introduction. We systematically evaluated the use of transthoracic echocardiography in the assessment of dynamic markers of preload to predict fluid responsiveness in the critically ill adult patient. Methods. Studies in the critically ill using transthoracic echocardiography (TTE) to predict a response in stroke volume or cardiac output to a fluid load were selected. Selection was limited to English language and adult patients. Studies on patients with an open thorax or abdomen were excluded. Results. The predictive power of diagnostic accuracy of inferior vena cava diameter and transaortic Doppler signal changes with the respiratory cycle or passive leg raising in mechanically ventilated patients was strong throughout the articles reviewed. Limitations of the technique relate to patient tolerance of the procedure, adequacy of acoustic windows, and operator skill. Conclusions. Transthoracic echocardiographic techniques accurately predict fluid responsiveness in critically ill patients. Discriminative power is not affected by the technique selected. Our primary concern in the management of the critically ill patient is the optimisation of tissue oxygen delivery. Insufficient intravascular loading in the early resuscitation of acute sepsis results in tissue underperfusion, organ dysfunction, and acidosis. Excessive fluid administration has also been shown to be detrimental in the perioperative setting and in acute lung injury, prolonging both time on mechanical ventilation and time in intensive care .It has been reported that as few as 40 percent of critically ill patients thought to be intravascularly deplete gain an improvement in cardiac output after a standard fluid bolus, exposing more than half of patients to the risks of excessive fluid administration .Knowledge of static measures of preload such as central venous pressure, pulmonary artery wedge pressure, end-diastolic volumes, and intrathoracic blood volume has not translated into patient benefit –6. This Contemporary investigation has therefore focussed on the search for clinical markers which predict a useful response to a fluid bolus. These “dynamic” markers make use of provoked cardiac reaction assessed without the need for a fluid bolus, instead utilizing either the consequences of heart-lung interaction during ventilation or the response to postural change to mimic the effect of a fluid bolus on stroke volume.Firstly, in mechanically ventilated patients who have no spontaneous respiratory effort, the change in intrathoracic pressure has a cyclical effect on both the left and right heart . The degree of collapse during tidal volume breaths is known to reflect the right atrial pressure with reasonable accuracy in health . To a deThirdly, raising the legs from the horizontal position to 45 degrees causes the gravitational movement of lower limb venous blood towards the heart. This provides a transient volume load of between 150 and 300 millilitres to the central circulation, lasting for a few minutes , 8. The Modern intensive care is increasingly concerned with the avoidance of unnecessary invasive procedures which contribute to patient morbidity either directly or more often through the associated risk of catheter-related bloodstream infection . Transoesophageal echocardiography may provide superior image quality in some cases and is increasingly utilised for cardiovascular monitoring on intensive care units. Nonetheless, it requires equipment, time, and skills that are less abundant on many intensive care units. It is contraindicated in some patients with upper airway or oesophageal surgery and also usually necessitates sedation which is not always achieved without adverse consequence. Accordingly, the objective of this review is to systematically evaluate the literature examining the use of transthoracic echocardiography in the assessment of dynamic markers of preload used to predict fluid responsiveness in the critically ill patient.An electronic literature search was carried out using Medline, EMBASE, CINAHL, and the Cochrane database of systematic reviews. The search terms used were ((fluid) OR (volume) OR (preload) OR (filling)) AND ((respons*) OR (status) OR (assess*)) AND ((echocardiograph*) OR (echog*)). The search was limited to “human” and “English language.” Transoesophageal echocardiography studies were excluded, as were those in which the sample group, the equipment used and the reference test cut-off criteria implied the conclusions were not applicable to the critically ill patient in a high dependency of critical care environment.“Fluid responsiveness” refers to a predefined rise in stroke volume or cardiac output after rapid fluid loading with a predetermined volume of fluid. Between investigators, there are inevitable differences in the choice of stroke volume or cardiac output, the volume of fluid given, the duration over which the fluid load was given, and the type of fluid given.The diagnostic test or equivalent of the index test in these studies is defined as the echocardiographic test done to give a prediction of fluid responsiveness. In this review, this test will be termed the “predictive test.” The test done to assess the response to a fluid bolus once given is similar to a reference test but for the purposes of this review will be called the “response test.”Responders are those patients in whom the cardiac output or stroke volume rises by the threshold amount after a given bolus of fluid.The Standards for Reporting of Diagnostic Accuracy (STARD) initiative developed a guide for assessing the quality of reporting of studies of diagnostic accuracy . In thisQ star statistics, and the simple average of sensitivity and specificity data is not an informative approach. Furthermore, the usage of a fixed-effects model such as SROC would be expected to produce exaggeratedly high levels of reported accuracy for a test that is to be put to use in the complex environment of the critically ill patient [The results of the selected studies were not meta-analysed due to the heterogeneity of methodologies, as well as the differences in patient selection, modes of ventilation and definition of fluid response. There was insufficient data for the construction of summary receiver-operator characteristic (SROC) curves or for the calculation of  patient . Of the 3138 articles identified through the search terms, eight studies were included for review. The quality scores ranged between 13 and 15, indicating an adequate standard throughout. The studies were all small but of appropriate intensive care unit setting Tables  and 3. Five studies used transaortic stroke volume variation to predict fluid responsiveness using passive leg raising to mimic a fluid bolus –16. OverImportant differences between studies were evident in the study protocols. Maizel et al.  and Prèa All studies showed good sensitivity (77 to 100 percent) and specificity (88 to 99 percent) using a threshold of 10 to 15 percent increment of stroke volume or cardiac output.Strikingly, stroke volume change with PLR predicted the correct response to volume expansion in 16 of the 18 patients with arrhythmia [2 bolus of 4% albumin. Stroke volume variation was calculated using the formula:A single study by Biais et al. looked at the use of stroke volume variation for prediction of fluid responsiveness . In thisThe area under the receiver operator characteristic (ROC) curve was used to ascertain a threshold of nine percent stroke volume variation as being the most useful for discerning responders from nonresponders. Using this cut-off, there was excellent sensitivity and specificity .Dmax and Dmin, respectively  provides an accurate estimation of diastolic function of the left ventricle irrespective of preload changes , 24. PulAlthough detailed examination of the heart requires an experienced echocardiography practitioner, there is an increasing acceptance of the value of focussed echocardiographic assessments to answer common clinical questions arising in critical illness. This has arisen in tandem with the emergence of a number of courses and training programmes centred on evaluation of the critically ill patient by those less experienced in echocardiography. Jensen showed that with only limited training, a diagnostic transthoracic window was achieved 97 percent of the time when used in the evaluation of shock . In the This review was restricted to the specific question of fluid response. In reality, echocardiographic assessment of the critically ill aims to gain as complete a picture as possible of the cardiovascular state. Ideally, this should also involve a full structural study in addition to inspection of left ventricular filling state and perhaps even ultrasonic examination of the lungs.Furthermore, studies using transoesophageal echocardiography (TOE) were not selected for this review and, although it would seem intuitive that flow or diameter measurements techniques taken with one kind of echocardiography could be safely extrapolated to another, this ignores the differing technical restrictions of each technique. Transoesophageal echocardiography has its own growing evidence base for its application in intensive care and clearly where it is available provides invaluable haemodynamic information to inform clinical decisions.A significant limitation of this review is the small size of the study groups since only a single study included more than 40 patients ; this isIt is conspicuous that only one article reported on the time between the initial predictive test and the subsequent assessment of a response to a fluid bolus . PatientThe amount of fluid used, the type used, and the rate at which it was given all impact upon the response test in these studies. Unfortunately, there is no agreed formulation for a standard fluid load although almost all studies use approximately the same formulation.Although no specific details were given about the qualifications of the echocardiography operator or reader most studies inferred they were experienced. Furthermore, blinding of the operator or reader, to the measurements taken after volume loading was rare and this is, therefore, a source of observer bias within the data.Intraobserver variability was considered by the majority of studies and attempts were made to measure it with variable success. An intuitively more useful measurement of reproducibility was achieved by examining the variability of repeated measurements of distensibility by Feissel et al. . This shOf note, whilst the effects of varying tidal volumes on echocardiographic parameter assessment are minimal, the impact of raised intra-abdominal pressure and of different positive end expiratory pressure is largely unstudied .The clinical question that was not addressed in any of the articles was that of the “real-world” value of echocardiographic approaches to assessing fluid responsiveness. The studies reviewed do not provide us with information about translation into effects on morbidity or mortality, nor is there yet such a current evidence base in the literature. This evidence may well originate in the context of future investigation into the dilemma of conservative versus liberal fluid management.Transpulmonary microsphere contrast has already been shown to dramatically improve volumetric assessment and its use in the critically ill would intuitively improve the clinical utility of the modality still further . Three-dTransthoracic echocardiography is becoming a powerful noninvasive tool in the daily care of the critically ill. This review brings together the evidence for employing TTE to predict fluid responsiveness. Assuming there is equipment and local expertise TTE is a repeatable and reliable method of predicting volume responsiveness in the critically ill.Transaortic stroke volume variation with the respiratory cycle, stroke volume difference following passive leg raising, and IVC diameter changes with respiration all provide good prediction of the likelihood of a response to a fluid bolus. The techniques can be used individually to address the needs of different patients and in combination to triangulate clinical information where uncertainties may occur.The studies reviewed form a robust platform of physiological data on which to base further studies involving larger numbers of patients which engage with clinically relevant outcomes, such as inotrope use, blood pressure, length of stay, and time to weaning from mechanical ventilation.Improved access to clinician-echocardiographers through a defined training process will facilitate such clinical studies and give patients access to accurate noninvasive information in answer to the daily clinical conundrum of fluid responsiveness.


Fluid responsiveness is not equal to a clinical need for fluid therapy. The aim of our study was to assess the incidence of microcirculatory flow alterations, according to a predefined arbitrary cut-off value, in patients with clinical signs of impaired organ perfusion. The secondary endpoint was to establish the correlation between the microcirculatory and macrocirculatory response to a fluid challenge.®) solution in 30 minutes. Before and after fluid challenge, systemic hemodynamics and direct in vivo observation of the microcirculation were obtained with sidestream dark-field imaging. Assessment of microcirculatory parameters of convective oxygen transport (microvascular flow index (MFI) and proportion of perfused vessels), and diffusion distance  was done using a semiquantitative method.We performed a prospective, single-centre, observational study. Included were ICU patients ≥18 years with invasive hemo-dynamic monitoring and clinical signs of impaired organ perfusion, as the principal reason for fluid administration. Fluid challenge was performed by the infusion of 500 ml crystalloid or a balanced colloid  at baseline to 2.7 (2.4 to 2.8) after fluid challenge, P = 0.003, but its change was only significant in fluid-responsive patients.We enrolled 50 patients. MFI <2.6 was present in 66% of the patients. After fluid challenge, signs of impaired organ perfusion reduced from 100% to 68% of the patients, These data demonstrate a relationship between clinical signs of impaired organ perfusion and MFI <2.6. Fluid responsiveness did not discriminate between patients with and without clinical signs of impaired organ perfusion or MFI <2.6. However, significant improvement of microvascular alterations and attenuation of clinical signs of impaired organ perfusion was restricted to patients who were fluid responsive. Noninvasive assessment of microvascular perfusion may help to define patients with potential need for fluid therapy, and to evaluate its effect.