Non-invasive imaging of the biodistribution of novel therapeutics including gene therapy vectors in animal models is essential.This study assessed the utility of high-frequency ultrasound (HF-US) combined with biofluoresence imaging (BFI) to determine the longitudinal impact of a Herpesvirus saimiri amplicon on human colorectal cancer xenograft growth.ex vivo volume than mechanical callipers . HF-US showed that the amplicon caused lobe formation. BFI demonstrated retention and expression of the amplicon in the xenografts and quantitation of the fluorescence levels also correlated with tumour volumes.HF-US imaging of xenografts resulted in an accurate and informative xenograft volume in a longitudinal study. The volumes correlated better with final in vivo in real-time. These relatively inexpensive technologies are easy to incorporate into pre-clinical studies.The use of multi-modal imaging provided useful and enhanced insights into the behaviour of gene therapy vectors  In particular, for gene therapy studies, knowledge of maintenance, expression and efficacy of the vector is a fundamental part of the testing process ) was calculated within this using Living Image version 4.2 software.Before sacrifice at day 40, xenografts were imaged in an IVIS Spectrum . Standard settings for GFP were used (excitation 500nm and emission detected at 540nm) in epi-illumination at high intensity. Binning was set at 8, field of view was 13.1cm and f stop was 2. Regions of interest of the same size were drawn around each xenograft and the total radiant efficiency  paraformaldehyde in PBS overnight. After processing and embedding in wax, sections were dewaxed, rehydrated and stained with haematoxylin and eosin. Sections were assessed by an experienced histopathologist.2 value and were considered significant if p < 0.05. Agreement between the methods was then further analysed by Bland-Altman plots where the central line (mean of differences or bias) and 2 standard deviation (SD) limits of agreement were generated. The bias was considered significant if 0 was not included within these standard deviation lines. These calculations were carried out using GraphPad Prism version 5 .Analysis of the tumour volumes and vector expression obtained by these methods used Pearson correlations. Positive correlations produced a positive RHF-US was used to determine the tumour volume during the growth course of the xenografts derived from the parental cell line and amplicon-infected cell line and compared to the volume calculated from mechanical calliper measurements. The tumour volumes generated from the two methods are shown in Figure in vivo callipers on the graphs) to the final ex vivo calliper measurement at the end of the period of xenograft growth which is our most accurate measurement 3/2 for in vivo calliper volumes gave a higher correlation to both HF-US volumes and to mass of tumour than the other two equations.HF-US measurements correlated more closely than mechanical callipers   Figure . The rel) Figure a. Amplic) Figure c.ex vivo calliper volume   . This was similar to our findings with biofluorescence imaging. As with our study, this functional tumour imaging modality is useful for metabolic imaging and should give an indication of the effect of a gene therapy vector on tumour viability. In the current study, HF-US accurately showed the slower tumour growth of the vector-transfected cell line compared to the parental cell line, as predicted from in vitro cell growth curves [ApcMin/+ mouse, where callipers cannot access the tumour. Indeed, gene therapy vectors are also used in non-cancer applications such as diabetes or organ regeneration, where callipers may not be used to measure disease progress or regression. In these cases, HF-US would be invaluable in monitoring progress longitudinally without sacrifice of mice.Multimodal imaging in gene therapy applications is a useful tool to shed light on the behaviour of vectors during y others ,18. The ne paper ). Correl density . They deh curves . However18F-FDG-microPET, for example. Bio-fluorescence is also applicable to patients. It is currently being trialled in surgery on human tumours to define tumour margins for resection [ex vivo tumour sections for vector proteins. Linkage of the therapeutic gene of interest to a fluorescent marker gene via an IRES  sequence or as a fusion protein would yield valuable information on the efficacy of expression during the time course of an in vivo experiment. It may also be used to reduce costs by eliminating animals in which the introduction of a vector by injection has not been successful.In addition to HF-US images, the use of biofluorescence allowed monitoring of tumour growth patterns and correlated well with final tumour volumes . This technique is a simple and very quick method of visualising the tumour and much less expensive than esection . The monin vivo. Addition of imaging to gene therapy protocols would be straightforward especially in the case of relatively inexpensive ultrasound and biofluorescence imaging. The use of multi-modal imaging can give important information on the behaviour of gene therapy vectors in real-time, rather than traditional calliper measurements and final histological examination.In conclusion we believe that multi-modal imaging provides useful and enhanced insights into the behaviour of gene therapy vectors HF-US: High-frequency ultrasound; BFI: Biofluorescent imaging; HVS: Herpesvirus saimiri; GFP: Green fluorescent proteinThe authors declare no competing interests financial or otherwise.NI carried out the experiments, analysed the data and wrote the manuscript. SAM generated the stably-infected cell line. GM imaged xenografts by HF-US and generated tumour volumes. IMC generated the amplicon. NS provided histological information on the resulting xenografts. AFM provided discussion of the results. AW was involved in study design, discussion of results and generation of the amplicon. PLC was involved in study design, discussion of results and manuscript editing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/13/35/prepub


This function was used to calculate the minimum detectable tumour volumes (MDTVs) and the minimum tumour-to-background activity concentration ratio (T:B) for effective delineation of a breast tumour model. The GDP had larger MDTVs and a higher minimum required T:B for tumour delineation with 131I than with 111In or 99mTc. It was shown that for 111In there was a benefit to using a collimator length of 0.5-cm. For the model used, the minimum required T:B required for effective tumour delineation was 5.2 ± 0.4. RIGS has the potential to significantly improve the accuracy of breast-conserving surgery; however, before these benefits can be realized, novel radiopharmaceuticals need to be developed that have a higher specificity for cancerous tissue in vivo than what is currently available.Breast-conserving surgery involves completely excising the tumour while limiting the amount of normal tissue removed, which is technically challenging to achieve, especially given the limited intraoperative guidance available to the surgeon. This study evaluates the feasibility of radioimmunoguided surgery (RIGS) to guide the detection and delineation of tumours intraoperatively. The 3D point-response function of a commercial gamma-ray-detecting probe (GDP) was determined as a function of radionuclide ( In North America, over 60% of breast cancer patients receive breast-conserving surgery . The pri99mTc labelled nanocolloidal tracer peritumorally in the case of ROLL or implant an 125I brachytherapy seed at the center of the nonpalpable tumour in the case of RSL. The surgeon then uses a hand-held gamma-ray-detecting probe (GDP), to guide the excision of the tumour [There is currently only limited intraoperative guidance available for breast procedures. For palpable tumours, the palpable edge is used to guide the complete excision of the tumour. For nonpalpable tumours, wire localization is the standard procedure. During wire placement, radiographic images are obtained to guide the surgeon; however, the wire may shift position within the breast, providing misleading information about the tumour location at the time of breast-conserving surgery. Additionally, the wire only marks the approximate location of the centre of the gross tumour within the breast and provides no information as to its extent. Competing techniques to wire localization are being developed; these techniques include radioguided occult lesion localization (ROLL) and radioactive seed localization (RSL). Radiographic or sonographic guidance is used to inject a e tumour –11. Ultre tumour .Radioimmunoguided surgery (RIGS) differentiates normal and cancerous tissues by exploiting the functional status of cancerous cells. One such functional property is the degree of overexpression of tumour-associated antigens (TAAs). Thus, a targeting vehicle specific to TAAs, such as a monoclonal antibody or an antibody fragment, can be conjugated to a radioactive label to create a radiopharmaceutical that molecularly targets cancerous tissues. For RIGS of breast cancer, TAAs that have been targeted by radiopharmaceuticals include tumour-associated glycoprotein-72 (TAG-72) , human eThe clinical utilization of radioimmunoguidance involves the administration of a radiolabelled monoclonal antibody or antibody fragment two hours to several days prior to the breast-conserving surgery. This allows sufficient time for the radiopharmaceutical to distribute throughout the patient and attach preferentially to the TAAs overexpressed on cancer cells. At the time of the surgery, prior to making an incision and with the patient in the appropriate position, the surgical oncologist takes a measurement of normal background activity using GDP. The GDP is then used to detect the tumour, which is where the measured count rates are significantly greater than normal background count rates. By taking measurements along the skin surface, moving outward from the centre of the tumour, the surgeon can delineate tumour extent as the point where measured count rates are no longer significantly higher than background.In this study, we address two fundamental issues that arise when considering the potential efficacy of RIGS. The first is that there is currently no reference minimum T:B to determine if a radiopharmaceutical would be a promising candidate. The T:B for a particular radiopharmaceutical is a function of several parameters such as its tumour-targeting ability, tumour perfusion, and normal tissue clearance , 25. TheThis study reports on the results of a systematic phantom-based evaluation of the impact of various critical parameters  on the minimum detectable tumour volume (MDTV) and comments on the feasibility of using RIGS for tumour detection and delineation during breast-conserving surgery.The apparatus used for the experiments consisted of the C-Trak surgical guidance system , with a 3.0 cm long and 1.0 cm diameter cylindrical NaI(Tl) crystal. A simple side shield was manufactured in-house as a 1.5 cm thick, 3.6 cm long hollow cylinder made of lead, so that design specifications could be controlled during the study. It was constructed to fit tightly around the sensitive end of the GDP and completely shielded the NaI(Tl) crystal except for the open face. The thickness of the side shield was chosen such that the penetration was limited to less than 5% at energies up to 364 keV. The side shield was designed to limit interference from photons emitted outside the nominal field of view of the detector. The face of the side shield was constructed so that it would be flushed with the sensitive end of the GDP; consequently, it did not provide any additional collimation. The GDP and the side shield were centred on a water tank phantom and were used to acquire the sensitivity map of a GDP that has robust side shielding, with 0.0 cm of additional collimation .Within t99mTc, 111In, and 131I. These radionuclides are common radiolabels that have been proposed for use in RIGS [99mTc , 111In , and 131I  [The radioactive source was composed of a polymethyl methacrylate capsule with a cavity in the shape of a 2 × 2 mm cylinder filled with radionuclide. The radioactive source was constructed to be of similar diameter to that of milk ducts found in the breast . The wal in RIGS , 27. Eac57Co.The point-response function of the GDP was measured in the water tank phantom for each of the three radionuclides. The sources were moved to a total of 352 positions in a 2D plane, in three overlapping grids: a fine grid with a 2 mm spacing used for the region closest to the GDP where the sensitivity changes rapidly and coarser grid spacings of 5 mm and 10 mm successively further away from the GDP. The total region covered was 70 mm horizontally by 130 mm in depth. For each source position, an energy spectrum was acquired using a multichannel analyzer. A similar technique for characterizing probe response has been used by Kwo et al.  for only99mTc (90–200 keV), 111In (100–350 keV), and 131I (150–460 keV). Photons detected with energies below the Compton backscatter peak are more likely to be a result of multiple scatters in the patient rather than in the detecting crystal and may also arise from the characteristic X-rays emitted from the lead collimator, which contain no useful positional information, thus making the backscatter peak a suitable starting point for the energy range. The collimator response was then calculated and applied to the 2D point-response function, based on the formalism described by Barrett and Swindell [To generate energy-dependent 2D point-response functions, an energy-window threshold was applied to the spectra at each source position. The energy-window thresholds to be evaluated were determined by dividing an energy range (from the Compton backscatter peak to three standard deviations past the photopeak) into ten evenly spaced intervals. The energy ranges that were investigated for each radionuclide were as follows: Swindell . The calThe 3D point-response function was validated experimentally by taking measurements of the radioactivity emitted by the capsule point source at 1 cm intervals along the horizontal axis, at a consistent depth of 1 cm, with a fixed collimator length of 0.5 cm and the energy-window threshold at Compton back scatter peak. This procedure was repeated for each of the radionuclides used in this study.a priori detection limit LD:Current re-excision rates are 15–40%, which can be used as an estimate of the false-negative rates associated with lumpectomy procedures , 3. If aμB is the mean number of counts measured for normal background tissue. In a simulation if a tumour model generated counts equal to or greater than the detection limit + mean background counts, the specificity and sensitivity would be 95% for detecting tumours of that size [hat size .in vivo. A practical range of background activity concentration from 1 − 5kBq/ml was used in this study [mL  of  blood/mL  of  tissue) [The bulk of the normal background activity was assumed to arise from the blood supply within the breast . This was not an ideal model as it did not incorporate nonspecific distribution of the radiopharmaceutical within the interstitium. However, nonspecific distribution is poorly characterized in the literature and, in the absence of relevant clinical data, the foregoing phantom geometry is likely a reasonable approximation of that encountered is study , 20, 22. tissue) .in vivo, accurately. Using a computer simulation, the modelled tumour volume was iteratively increased in size and the detected counts were calculated. The point at which the expected counts exceeded the detection limit determined the MDTV for that combination of input parameters. The modelled tumour volume was increased using a region-growing technique that resulted in horizontal growth of the tumour prior to vertical growth . This rein silico to assess the ability of the GDP to determine the edge of a tumour phantom in a uniform background. To facilitate comparison of all of the parameters being investigated, a simple tumour model was created. Generating a tumour model that captures all of the complexities of actual tumours is difficult; however, the model chosen in this study accounts for some of the main features that are important to the task of RIGS for lumpectomies [in vivo.Tumour edge delineation experiments were conducted ectomies , 5. The Using this tumour model in conjunction with a uniform background activity, a 3D activity distribution was created for different combinations of normal tissue activity (derived from background activity concentrations) and tumour activity . The response of the GDP to each distribution was calculated using the 3D point-response function discussed earlier. The measured edge was determined as the distance from the centre of the tumour model to the point where the calculated counts fell below the detection level. For a range of collimator lengths, background activity concentrations, and energy-window thresholds, the minimum T:B was determined that resulted in the location of the measured edge corresponding to the true edge of the tumour model. This represented the point where the GDP was able to accurately delineate the edge of the tumour model.The calculated 3D point-response function was experimentally validated by measuring the sensitivity profile of the C-Trak GDP at 1 cm depth, with a physical collimator that had a length of 0.5 cm and the energy-window threshold at the Compton back scatter peak for each of the radionuclides used in this study. The maximum deviation between the calculated sensitivity profile and the measured profile was 3.4%, indicating that the method to generate the 3D point-response function was accurate and was suitable for evaluating the physical parameters involved in RIGS.99mTc, 111In, and 131I sources, respectively. From these figures, it was observed that increasing the energy-window threshold reduced the sensitivity of the GDP. The decrease in sensitivity, however, was greater in areas that are further away from the central axis of the probe, where one would prefer the GDP not be sensitive .99mTc, which is representative of all three radionuclides. These images display how increasing the collimator length decreases the field of view, thereby improving the spatial resolution of the system. As shown in 99mTc and 131I occurred when there was 0.0 cm of additional collimation extending beyond the sensitive face of the GDP. Similar trends were observed for 99mTc when the tumour was placed at depths 1 cm, 2 cm, and 3 cm, which verified that the relationship between collimator length and MDTV was relatively unaffected by the tumour depth. For 111In, the smallest MDTV occurred when the collimator length was 0.5 cm. 111In consistently had the smallest MDTV; however, the differences in the smallest MDTV between 111In and 99mTc were not statistically significant.99mTc and 111In have statistically superior performances to 131I at all background activity concentrations (P < 0.05). There also appears to be no significant difference between the performance of 99mTc and 111In. In For each combination of collimator length and energy-window threshold, the minimum T:B required for tumour edge delineation was determined at increasing background activity concentrations for each radionuclide. In this fashion, the effect of the combination of all of these parameters on tumour edge delineation was determined.111In, a 100 keV energy-window threshold and a 0.5 mm collimator length; for 99mTc, a 90 keV energy-window threshold and 0.0 cm collimation; for 131I, a 150 keV energy-window threshold and 0.0 cm collimation. The lowest T:Bs obtained were 6.2 ± 0.5 for 99mTc, 5.2 ± 0.4 for 111In, and 12.0 ± 0.9 for 131I. All were obtained at the highest background activity concentration modelled of 5 kBq/mL. From 111In and 99mTc can be used to accurately delineate the tumour edge at all background activity concentrations at significantly lower T:Bs than is possible with 131I. In addition, there is a trend for 111In to have the lowest required T:B of the three radionuclides evaluated.The parameters used to generate 111In radiolabel for the highest background activity concentration evaluated (5 kBq/mL), a collimator length of 0.5 cm, and an energy window threshold of 100 keV, for detecting tumours in a uniform background at a depth of 3 cm. A tumour must be greater than 8.7 ± 2 cm3 (corresponding to a diameter of 2.5 ± 0.2 cm) to reduce re-excision rates from their current levels of 15–40% [In order to evaluate the feasibility of RIGS of breast cancer, the optimum system parameters were determined. This was found to be an Radiolabel: 99mTc. As expected, the C-Trak GDP was more sensitive to 111In and 99mTc than to 131I. The reason for this was the lower efficiency of the NaI(Tl) crystal used in the C-Trak GDP for the 364 keV photons emitted by 131I. Barber et al. [111In and 99mTc compared to 131I, therefore, should also be true for other commercially available GDPs. This study has shown that an 111In label results in the best performance; however, a counterintuitive finding from this study is that 111In and 99mTc perform similarly when variations in blood volume within breast tissue are incorporated into the phantom model. For example, similar-sized tumours may be detected by using 99mTc in a patient who has a high activity concentration in their blood as compared to using 111In in a patient with a low activity concentration in their blood. As 111In emits two photons, it would be expected to produce more counts per unit activity, which due to Poisson statistics would reduce the relative error in each measurement compared to 99mTc. However, the two photons emitted by 111In differ in energy (171 keV and 245 keV), and the efficiency of the GDP for the 245 keV photon is low (75% of the efficiency for the 171 keV photon using a NaI(Tl) crystal 3 cm thick). Therefore, the overall benefit of having two photons per decay is diminished. Higher background activity concentrations of 99mTc-labelled radiopharmaceuticals can be achieved for the same patient radiations dose as compared to 111In. Thus, it may be possible to detect smaller tumour volumes using 99mTc. The half-life of 99mTc (6 hours), however, is much shorter than that of 111In (2.83 days), limiting the use of 99mTc  to targeting agents that have rapid clearance from the blood stream.r et al.  have shoBackground Activity Concentration: 5 kBq/mL. An increase in this parameter was assumed to cause proportional increases in normal-tissue background and in tumour activity concentrations. Under these conditions, it was demonstrated that the MDTV is inversely proportional to the square root of the background activity concentration for the assumed tumour model. As stated previously, background activity was assumed to relate directly to administered activity. In principle, the higher the lesion activity, which is associated with a higher background activity, the lower the statistical uncertainty in the measured count rates. This lower statistical uncertainty in turn helps to reduce the minimum detectable tumour volume. This suggests that administering more activity will result in the detection of smaller tumours, as was expected. In principle, the background activity concentration can be translated into administered activity for any radiopharmaceutical, taking into account its specific elimination pharmacokinetics. The radiation dose to the patient per unit of injected activity depends on the radionuclide and the pharmacokinetics of the agent involved. Assuming similar kinetics, the effective dose per unit administered activity dose for 99mTc is considerably lower than those for 111In (which emits photons and Auger electrons) and 131I (which emits photons and beta particles). Since lower radiation doses to the patient would be expected for 99mTc compared to 111In and 131I, it may be possible to administer more activity to patients by using a 99mTc radiolabel; therefore, a higher background activity concentration can be achieved. Of course, 111In's long half-life has the advantage of allowing greater time for clearance of nonspecific uptake than does 99mTc's  short half-life.Collimator Length: 5 mm. The lowest MDTV using 111In resulted when a 5 mm long collimator was used, whereas for 99mTc and 131I 0.0 cm collimation was required. Further investigation of 111In showed that the increase in collimation decreases the number of photons from outside the field of view that penetrate the lead shielding of the GDP and are incorrectly detected. For 111In, 2 mm of lead is required to stop 95% of the 171 keV photons, while the 245 keV photons require 5 mm of lead. A 5 mm extension of the collimator is, therefore, required to provide adequate absorption of both photon energies and may explain why 5 mm collimation resulted in the lowest MDTV for 111In. For 99mTc, 1.2 mm of lead is required to stop 95% of the 140 keV photons. The impact of a collimator extension of 1.2 mm could not be evaluated using the 3D point-response function since it had a spatial resolution of 2 mm. Due to the coarseness of the 3D point-response function, it was found that 0.0 cm collimation was better than a 2 mm collimator length. For 131I, 14.7 mm of lead is required to stop the 364 keV photons. Since the sensitivity of the GDP is already low for these photons, increasing the collimator length only degrades performance by further decreasing the sensitivity of the system. Changing the depth of the tumour from 1 to 3 cm did not alter the fact that increasing the collimator length, beyond the length required to properly shield the GDP, dramatically degraded the GDPs performance. Sensitivity of the GDP seems to be the dominant factor in determining the optimal performance of the system. Energy-Window Threshold: Compton Backscatter Peak. In RIGS the sensitivity of the system is more important than the spatial resolution; as such, the lowest MDTV results when the energy-window threshold was placed at the Compton backscatter peak, indicating that the improved spatial resolution from increasing the energy-window threshold did not compensate for the decrease in sensitivity.Tumour Edge Delineation: This evaluation determined that a minimum T:B of 5.2 ± 0.4 is required in order to delineate a 3 cm diameter tumour from a uniform background using an 111In radiolabel, 5 mm collimator length, and a background activity concentration of 5 kBq/mL. The commonly held paradigm is that a long collimator with no energy-window threshold, thereby a system with high spatial resolution with the highest possible sensitivity, would best suit this application. Our findings show that this is not the case: the impact of collimation, while improving spatial resolution, increases the separation of the probe from the skin surface. This has two effects: (1) sensitivity drops dramatically due to inverse square law and (2) the change in sensitivity with increasing distance becomes less dramatic  to its advantage, by reducing the detected signal from distant organs and tissues that would otherwise confound a conventional GDP. Additionally, the short range of beta particles precludes the need for collimation; thus, sensitivity is retained. The beta particles emitted by ely deep –38.99mTc radiolabel with 0.0 cm collimation and minimal energy-window thresholding or 111In with a collimator length of 0.5 cm. Additionally, it was shown that a T:B of at least 5.2 ± 0.4 is required to accurately delineate the tumour edge of a 3 cm diameter lesion from a simple uniform background. Therefore, the role of RIGS for guidance of breast-conserving procedures is promising; however, novel radiopharmaceuticals need to be developed that have a higher specificity for cancerous tissue in vivo than what is currently available.There are at least two key limitations in the manner in which potential radiopharmaceuticals for RIGS are currently evaluated: (1) the most appropriate set of system parameters has yet to be established; (2) no minimum T:B has been suggested as being required to determine whether a radiopharmaceutical is likely to be a good candidate for use in RIGS. The results reported in this study have shown that the most appropriate combination of parameters included a


Sir,We thank The first question of Another question was which type of surgery was performed. In all cases, the intention was to remove the mesentery desmoid tumour. If this was impossible, then only a bypass was carried out in case of obstruction. Unfortunately, in none of the patients it was possible to remove all desmoid tumour tissue.The final question was about the definition of a progression-free survival (PFS). In our study, PFS was defined as the period between the start of medical treatment or the time of surgery and the beginning of clinical symptoms attributed to growth of the desmoid. We realise that this is not a standardised method for measuring tumour progression. However, we think that the presence of new complaints because of tumour growth is a clinically important parameter. In many cases, tumour growth was confirmed by MRI or by CT scanning. However, we agree with We very much appreciate the critical notes of


This study compared the effect of pulse pressure variation (PPV) and cardiac index (CI)-guided fluid therapy versus mean arterial pressure (MAP)-guided fluid therapy on microcirculatory perfusion in patients undergoing abdominal surgery.n = 11) or a MAP-guided (n = 12) group. PPV, CI and MAP were measured using the non-invasive finger arterial blood pressure measurement device ccNexfin . Tidal volumes were ≥8 ml/kg with PEEP ≥8 mmHg. In both groups, MAP of 70 mmHg was maintained. In the PPV/CI group, an intraoperative algorithm was used keeping the PPV under 12% and CI above 2.5 l/minute/ m2 using fluid therapy and dobutamine and noradrenaline infusion, respectively. Sublingual microvascular perfusion was measured after anesthesia induction, and every subsequent hour using sidestream dark-field imaging . The perfused small vessel density (PVD) values were offline quantified.Patients undergoing elective abdominal surgery were randomized into a PPV/CI-guided group (P = 0.07). At this time point, the PVD was slightly lower in the PPV/CI-guided group (16.7 ± 3.1 mm/mm2) when compared with the MAP-guided group . In both groups the PVD remained stable during the first 2 hours of surgery. However, 2 hours after the start of surgery, the PVD in the PPV/CI group restored and tended to be higher than in the MAP-guided group . After 1 hour of surgery, the administered fluid volume correlated inversely with PVD .The first hour during surgery, the PPV/CI-guided group tended to receive more fluids than the MAP-guided group (1,014 ± 501 ml vs. 629 ± 463 ml; Goal-directed fluid management resulted in a higher administered fluid volume in the beginning of surgery, and this was associated with a slightly reduced microcirculatory perfusion when compared with MAP-guided fluid management. Microcirculatory perfusion tended to improve as surgery progressed in the goal-directed fluid therapy group. Our findings suggest that goal-directed and MAP-guided fluid management are associated with distinct patterns in fluid resuscitation, which may be of consequence for microvascular perfusion.


There was an omission in the funding statement. Thomas Fricke (TF) was also supported by National Institutes of Health grant R01 AI087390.There was also an error in Author Contributions. All authors participated in the writing of the manuscript.


We report a patient with significant weakness of the left soft palate, paralysis of the left vocal cord, and left facial nerve palsy. Although the patient showed no herpetic eruption in the pharyngolaryngeal mucosa and auricle skin, reactivation of varicella zoster virus (VZV) was confirmed by serological examination. She was diagnosed with zoster sine herpete. After treatment with antiviral drugs and corticosteroids, her neurological disorder improved completely. When we encounter a patient with associated laryngeal paralysis, we should consider the possibility of reactivation of VZV even when no typical herpetic eruption is observed. Ramsay Hunt syndrome (RHS) is caused by reactivation of varicella zoster virus (VZV) . TypicalAssociated laryngeal paralysis is a clinical condition merged with other cranial nerve disorders associated with vocal cord paralysis. Recently, it has been speculated that a proportion of idiopathic associated laryngeal paralysis are due to VZV . Most caA 50-year-old female presented with hoarseness, dysphagia, and left ear pain on February 10. She was diagnosed with a common cold and received medication from a clinic. Her symptoms did not improve and she visited our hospital on February 14. Physical examination demonstrated significant weakness of the left soft palate with a deviation to the right side during phonation. There was no sign of herpetic eruption within the region surrounding her ear, face, and oral cavity. Her facial sensation was normal and no facial weakness was observed. Laryngoscopy revealed paralysis of the left vocal cord and saliva pooling in the left piriform sinus . There wAt this point, the diagnosis was idiopathic associated laryngeal paralysis. However, we considered the possibility of VZV reactivation from the presence of facial discomfort and ear pain, in spite of the lack of typical skin rash or mucosal eruption. The patient was admitted to our hospital and treatment with antiviral therapy and a short course of oral corticosteroids was started according to our therapeutic strategy for RHS. Seven hundred fifty mg of intravenous acyclovir per day for a week and 60 mg of prednisolone per day for 5 days were administrated and the dose was tapered. On the second hospital day, she complained of left facial weakness, and mild facial nerve palsy with House-Brackmann (HB) grade II was observed. Eight days later, the soft palate and vocal cord were beginning to show movement, the hoarseness was improving, and the facial nerve palsy showed no change from HB grade II. Three weeks later, her facial weakness was improved. Paralysis of the soft palate and vocal cord was completely resolved six weeks later.On the first and 15th day, ELISA for VZV and herpes simplex virus (HSV) was undertaken as HSV can also produce lower cranial nerve palsies. Although ELISA for VZV IgM was negative at the first examination, a slight elevation was observed in the paired sera . Results for VZV IgG showed a significant increase from 10.5 to >128. From these results, we made a diagnosis of reactivation of VZV. Both the first and paired sera were negative for HSV IgM and IgG.A significant elevation in VZV antibody titers is observed in some of the patients diagnosed as idiopathic associated laryngeal paralysis . It is nIn this case, facial nerve palsy followed associated laryngeal paralysis. In patients of RHS, VZV reactivation occurs in the geniculate ganglion and the subsequent neuritis may result in facial nerve palsy . On the In patients with RHS and ZSH, the early administration of antiviral drugs improves the prognosis of facial nerve palsy –12. The


Caenorhabditis elegans (C. elegans) is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS), that are conserved in mammalian cells. To explore C. elegans as a model system to study the regulation of the facilitative glucose transporter (GLUT), we have characterized the GLUT gene homologues in C. elegans: fgt-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1 and Y37A1A.3. The exogenous expression of these gene products in Xenopus oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (Km) of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1-mediated 2-deoxy-D-glucose uptake (P < 0.01), indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in C. elegans, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of fgt-1 resulted in increased fat staining in both wild-type and daf-2  mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by fgt-1 knockdown in wild-type or daf-2 mutants. Our results indicated that in C. elegans, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism. The hGLUT1 cRNA-injected oocytes were used as a positive control, and the water-injected oocytes were used to determine the endogenous levels of glucose uptake in the oocytes. FGT-1 showed significant 2-DG transport activity, although the activity appeared much lower than hGLUT1, whereas all other gene products that were examined showed no activity for 2-DG transport  , D-mannose (79%), D-galactose (33%), or D-fructose (42%)  that was synthesized from exon 6 of the fgt-1 gene, which has limited sequence homologies to other GLUT homologues. The fgt-1 mRNA levels that were measured by real-time RT-PCR did not show a significant reduction of fgt-1 mRNA in the C. elegans that were injected with fgt-1 dsRNA (data not shown). Due to the lack of the antibodies to FGT-1, we confirmed the RNAi effect on fgt-1 protein levels by injecting fgt-1 dsRNA into fgt-1::egfp transgenic animals. F1 progeny were analyzed by Western blot analysis using an anti-GFP antibody. In dsRNA-injected transgenic animals, the FGT-1::eGFP protein level was decreased to 16% of that in the animals injected with buffer, confirming the knockdown effect of our RNAi . We further confirmed this result in an fgt-1(tm3165) mutant animal that showed 9% higher fat storage compared with wild-type animals   animals (daf-2(e1370), also had no effects on dauer formation by fgt-1 RNAi in both wild-type or daf-2(e1370) animals , it is predicted to have only 10 TMs, rather than the 12 TMs predicted in all hGLUTs  with 10 µg/mL penicillin and 10 IU/mL streptomycin (Gibco) for three days and then subjected to the uptake assay using 10 mM 2-DG containing 1 µCi 3H-2-DG. For the phloretin inhibition assay, 0, 10, 25, 50, 75, 100 or 200 µM of phloretin was added to the assays. For the substrate specificity assay, 30 mM of each inhibitor sugar was added to the assays, with L-glucose as a control. For the kinetic assays, increasing concentrations from 0 to 25 mM of 2-DG containing 3 µCi 3H-2-DG were used. For all experiments, ten or more oocytes were used in each assay, and the oocytes were incubated in uptake solutions for 15 min. Each experiment was carried out individually at least three times.2-DG uptake and the kinetic and inhibition analyses of FGT-1 and R09B5.11 were carried out in eviously . cRNA- ofgt-1::eGFP or R09B5.11:eGFP fusion construct that were transcribed in vitro from the plasmid pSP-fgt1g or pSP-R09g plasmid were injected into Xenopus oocytes. The plasma membrane localization of the GFP fusion proteins was observed three days after the injection under a confocal laser microscope (Zeiss LSM 510 META Laser Scanning Microscope).cRNA of the fgt-1 exon 6 was amplified from the pCR-fgt1 plasmid with the primer set fgt1-ds5 and fgt1ds-3  was then applied for 2 hours at room temperature. Immunostained animals were observed under a confocal laser microscope (Zeiss LSM 510 META Laser Scanning Microscope).For the whole-mount immunostaining, mixed-staged worms were settled on Poly-Prep slides (Sigma), fixed and permeabilized using freeze-cracking followed by methanol treatment . An antiC. elegans were collected and fixed for one hour in PBS and an equal volume of 2x MRWB buffer  containing 1% paraformaldehyde. After fixation, the samples were washed with PBS, and Sudan black B staining solution was added and incubated for 16 hours at room temperature. Worms were then washed with PBS containing 0.05% Tween-20 and were observed with an optical microscope. Images taken by the microscope were analyzed with ImageJ software 1.44p (http://rsbweb.nih.gov/ij/) to quantify the levels of Sudan black B staining. The experiment was repeated at least three times.A saturated solution of the fat staining dye Sudan black B in 60% isopropanol was diluted to 50% saturated in 60% isopropanol. Wild-type or RNAi-treated For the brood size assay, ten F1 progenies of dsRNA- or buffer-injected worms were individually placed on NGM agar plates and were transferred to new plates daily. The number of hatched progeny was counted daily. The counting was stopped when the worm produced less than ten progeny per 24 hours.°C for laying eggs. After removing the parent worms, F1 progenies were incubated for 60-72 hours at 20 °C or 25 °C. Dauer worms were scored visually, and scoring was confirmed using SDS. Worms were considered dauers if they survived a several minute incubation in 1% SDS.The dauer formation assay was performed as described previously . Five dsEach experiment was repeated individually at least three times.fgt-1 cRNA-injected oocytes that was lower than three times the mean value of that of water-injected oocytes was considered to be injection failure and was disregarded from the assay. 2-DG uptake in fgt-1 cRNA-injected oocytes was corrected by subtraction of the mean 2-DG uptake of water-injected oocytes at the corresponding concentration and incubation time. Statistical significance was determined by Student’s t-test, Tukey-Kramer honestly significant difference (HSD) test, and Dunnett’s one-way ANOVA, as indicated in the individual figure legends, and the analyses were carried out using JMP 8.0 software (SAS Institute Inc.). Plots and curve fitting analysis for the Michaelis-Menten equation were carried out using GraphPad Prism 5.04 (GraphPad Software Inc.).For the uptake and kinetic analyses, any uptake in Figure S1C. elegans genes encoding FGT-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1, Y37A1A.3 and human GLUT1-4 proteins were performed with the Clustal W program with open gap penalty = 10 and gap extension penalty = 0.05. Residues that are highlighted by a black shaded background represent absolutely conserved amino acids and the gray shading indicates eight or more conserved residues at those positions. Regions of presumed transmembrane domains (TM) are indicated by numbered dashed lines, and the functionally important residues for glucose uptake activity are indicated by the letter “F” at the top of the sequence alignments. In addition, the highly conserved amino acids are shown on the bottom of the sequence alignment.Alignments of the deduced amino acid sequences of (TIF)Click here for additional data file.Figure S2C. elegans GLUT candidates were aligned with Clustal W and a phylogenetic tree was drawn. The scale bar indicates the relative branch lengths obtained from the Clustal W alignment result.Human class I GLUTs and (TIF)Click here for additional data file.Table S1(XLSX)Click here for additional data file.


We previously reported that the primary tumour/vessel tumour/nodal tumour (PVN) classification is significantly superior to the UICC pTNM classification and the Nottingham Prognostic Index for accurately predicting the outcome of patients with invasive ductal carcinoma of the breast in a manner that is independent of the nodal status and the hormone receptor status.n=1042) using multivariate analyses by the Cox proportional hazard regression model.The purpose of the present study was to compare the outcome predictive power of a modified PVN classification to that of the newly devised pathological UICC pTNM classification and the reclassified Nottingham Prognostic Index in a different group of patients with invasive ductal carcinoma (The modified PVN classification clearly exhibited a superior significant power, compared with the other classifications, for the accurate prediction of tumour recurrence and tumour-related death among patients with invasive ductal carcinoma in a manner that was independent of the nodal status, the hormone receptor status, and adjuvant therapy status.The modified PVN classification is a useful classification system for predicting the outcome of invasive ductal carcinoma of the breast. We previously reported that the primary tumour/vessel tumour/nodal tumour (PVN) classification is significantly superior to the UICC pTNM classification , the NotThe purpose of this study was to compare the outcome predictive power of the modified PVN classification with that of the newly devised pathological UICC pTNM classification , the Allred scores for oestrogen receptor and progesterone receptor and the category of HER2 expression in the tumour cells, and the type of adjuvant therapy . Factors that were significantly associated with outcome in univariate analyses were entered together into a multivariate analysis performed using the Cox proportional hazard regression model. The case-wise, step-down, and two-sided method was applied until all the remaining factors were significant at a P-value of <0.05. All analyses were performed using Statistica/Windows software .We previously reported that a grading system for lymph vessel tumour emboli is a very useful histological classification in the accurate prediction of the outcome of patients with invasive ductal carcinoma independent of nodal status using multivariate analyses with well-known clinicopathological factors . FurtherThe following 12 histological factors of the primary invasive ductal carcinomas were evaluated: (1) invasive tumour size , (2) tubule formation  , (3) numThe nodal metastases were evaluated using single sections of each node or half of each node stained with hematoxylin and eosin. The following 10 histological parameters of metastatic mammary carcinoma to the lymph nodes were evaluated: (1) number of nodal metastases , (2) maximum dimension , (3) tubule formation , (4) nuclear features , (5) number of mitotic figures , 2011, , 2011, , 2011, aThe following existing histological classifications were compared with our modified classification with regard to the prediction of disease-free survival and overall survival: (1) the UICC pTNM classification , the Allred scores for oestrogen receptor and progesterone receptor and the category of HER2 expression in the tumour cells, and the type of adjuvant therapy  were evaluated using univariate analyses with the Cox proportional hazard regression model according to nodal status, hormone receptor status, and adjuvant therapy status. The classifications and factors that were significantly associated with outcome in the univariate analyses were then entered together into the multivariate analyses that were performed using the Cox proportional hazard regression model. The case-wise, step-down and two-sided method was applied until all the remaining factors were significant at a P-value of <0.05. The survival curves were drawn using the Kaplan–Meier method. All the analyses were performed with Statistica/Windows software (StatSoft).The predictive powers for the disease-free and overall survivals of each classification, age  until March 2011. Of the 1042 invasive ductal carcinoma patients, 858 patients were alive and well, 184 had developed tumour recurrences , and 89 had died of their disease. The tumour recurrence-free survival and overall survival periods were calculated using the time of surgery as the starting point. Tumour relapse was considered to have occurred whenever evidence of distant-organ metastasis or local recurrence was found.Among all the patients with invasive ductal carcinoma, a fibrotic focus diameter >8 mm, lymph vessel tumour embolus grades 2 and 3, lymph node metastases with a dimension of >20 mm, >2 apoptotic figures in blood vessel tumour emboli, and >5 mitotic figures in metastatic carcinoma to the lymph nodes had significantly higher hazard ratios for tumour recurrence and tumour-related death in multivariate analyses . Marked Among patients with invasive ductal carcinoma without nodal metastasis, lymph vessel tumour grade 3 and >19 mitotic figures in primary invasive ductal carcinomas had a significantly higher hazard ratio for tumour recurrence and tumour-related death in multivariate analyses . Lymph vAmong patients with invasive ductal carcinoma with nodal metastases, a fibrotic focus diameter >8 mm, lymph vessel tumour embolus grades 2 and 3, lymph node metastases with a dimension of >20 mm, >2 apoptotic figures in blood vessel tumour emboli, >2 extranodal blood vessel tumour emboli, and >5 mitotic figures in metastatic carcinoma to the lymph nodes had significantly higher hazard ratios for tumour recurrence and tumour-related death in multivariate analyses . A severWe refined our previously proposed PVN classification . Four faFurthermore, we also attempted to modify the PVN classification using the weight given to each factor based on its median hazard ratio obtained using the multivariate analyses in this study. The weights that were given for each factor were as follows: (1) 2.0 for a fibrotic focus diameter >8 mm, (2) 1.5 for the marked variation of nuclear features of primary invasive ductal carcinoma, (3) 7.2 for >19 mitotic figures in primary invasive ductal carcinoma, (4) 2.9 for lymph vessel tumour embolus grade 2 and 4.2 for lymph vessel tumour embolus grade 3, (5) 3.2 for >2 apoptotic figures in blood vessel tumour emboli, (6) 1.9 for a severe grade of stromal fibrosis in metastatic carcinoma to the lymph nodes, (7) 1.9 for lymph node metastases with a dimension of >20, (8) 1.9 for >2 extranodal blood vessel tumour emboli, and (9) 3.1 for >5 mitotic figures in metastatic carcinoma to the lymph nodes. The total factor weights for individual patients were calculated  and we classified all the patients into the following five classes based on the total factor weight for each patient: (1) 484 patients with a total factor weight of 0, class 0; (2) 192 with a total factor weight of >0 to ⩽5, class 1; (3) 321 with a total factor weight of >5 to ⩽10, class 2; (4) 31 with a total factor weight of >10 to ⩽18, class 3; and (5) 14 with a total factor weight of >18, class 4.P<0.001; tumour-related death: trend hazard ratio, 2.2, P<0.001) had higher trend hazard ratios and lower trend P-values for tumour recurrence and tumour-related death than the weight-modified PVN classification . Therefore, we adopted the former classification for a comparison with the other two classifications in this study.Next, we performed multivariate analyses for tumour recurrence and tumour-related death between the score-modified PVN and the weight-modified PVN classification as a whole. Although both classifications significantly increased the hazard ratios for tumour recurrence and tumour-related death, the score-modified PVN classification  and tumour-related death (P<0.001). Although the UICC pTNM classification showed a significant association with tumour recurrence (P=0.018), it failed to show a significant association with tumour-related death (P=0.165). HER2 category 3 had a significant association with tumour recurrence (P=0.033). In model 2 multivariate analyses, the modified PVN classification significantly increased the trend hazard ratios for tumour recurrence (P<0.001) and tumour-related death (P<0.001). The Nottingham Prognostic Index also showed significant associations with tumour recurrence (P=0.003) and tumour-related death (P=0.006). HER2 category 3 failed to significantly increase the hazard ratio for tumour recurrence in model 2 multivariate analyses.In model 1 multivariate analyses of all the patients, the modified PVN classification significantly increased the trend hazard ratios for tumour recurrence (P<0.001) and tumour-related death (P<0.001). In model 2 multivariate analyses, the modified PVN classification was significantly associated with tumour recurrence and tumour-related death, but the Nottingham Prognostic Index was not significantly associated with tumour recurrence or tumour-related death . In model 1 multivariate analyses, the modified PVN classification was significantly associated with tumour recurrence  but failed to significantly increase the trend hazard ratios for tumour recurrence .In model 1 and 2 multivariate analyses of patients with invasive ductal carcinoma who were positive for one or two hormone receptors, the modified PVN classification exhibited significantly increasing trend hazard ratios for tumour recurrence and tumour-related death . The NotP=0.048) but failed to significantly increase the trend hazard ratio for tumour recurrence in a model 2 multivariate analysis (P=0.093). As only five patients died as a result of their disease in this series, a multivariate analysis for tumour-related death could not be performed.In model 1 and 2 multivariate analyses of patients with invasive ductal carcinoma not treated with adjuvant therapy, the modified PVN classification significantly increased the hazard ratios for tumour recurrence . The UICP<0.001) but failed to significantly increase the trend hazard ratios for tumour recurrence .In model 1 and 2 multivariate analyses of patients with invasive ductal carcinoma treated with endocrine therapy, the modified PVN classification significantly increased the trend hazard ratios for tumour recurrence and tumour-related death . The UICIn model 1 and 2 multivariate analyses of patients with invasive ductal carcinoma treated with chemoendocrine therapy, the modified PVN classification significantly increased the hazard ratios for tumour recurrence and tumour-related death . The UICIn model 1 and 2 multivariate analyses of patients with invasive ductal carcinoma treated with chemotherapy, although the modified PVN classification significantly increased the trend hazard ratios for tumour recurrence and tumour-related death, the UICC pTNM classification and the Nottingham Prognostic Index failed to show significant associations with tumour recurrence or tumour-related death .We previously reported that the PVN classification can accurately predict the outcome of patients with invasive ductal carcinoma in a manner that is independent of the nodal status or hormone receptor status (The factors included in the modified PVN classification were selected based on the precise analyses of this study using well-known clinicopathological factors, such as histologic grade, invasive tumour size, and nodal status. Among the nine factors in the modified PVN classification, seven of them were the histological factors that we proposed for primary invasive ductal carcinoma, carcinomas in vessels, and metastatic carcinoma to the lymph nodes , 2011. TThis study also strongly suggests that the tumour characteristics of invasive ductal carcinomas matter more than the quantity of tumour with regard to the accurate prediction of the outcome of patients with invasive ductal carcinoma. Both the UICC pTNM stage classification and the Nottingham Prognostic Index evaluate the malignant potential of invasive ductal carcinomas based on the invasive tumour size and the number of nodal metastases. These factors reflect the quantity of invasive ductal carcinoma cells. In contrast, almost all the factors in the modified PVN classification, exception of the maximum diameter of lymph node metastases, represent the tumour characteristics of invasive ductal carcinomas. In addition, we previously showed that mitotic figures and apoptotic figures in tumour cells of lymph vessel tumour emboli have significantly stronger outcome predictive powers than the number of lymph vessels that have been invaded . As the We used the modified PVN classification for patients with invasive ductal carcinoma because our previous studies clearly demonstrated that the factors included in this classification were significant outcome predictors only for patients with invasive ductal carcinoma , 2011. TIn conclusion, the current study clearly confirmed that the modified PVN classification is a useful histological classification for predicting the outcome of patients with invasive ductal carcinoma of the breast. Thus, pathologists should attempt to assess the true malignant potential of invasive ductal carcinomas using the criteria of the modified PVN classification.


Source localization identified the Ncl in the lateral occipital object recognition area. Patients showed a significant decrease in the Ncl, but not N1, compared to controls, and this was associated with impaired behavioral ability to identify contours. In addition, an earlier negative peak was found at ~120 ms (termed N120) that differentiated jitter conditions, had a dorsal stream source, and differed between patients and controls. Patients also showed a deficit in the dorsal stream sensory P1 component. These results are in accord with impairments in distributed circuitry contributing to perceptual organization deficits and provide an ERP analog to the behavioral contour integration task.Perceptual organization, which refers to the ability to integrate fragments of stimuli to form a representation of a whole edge, part, or object, is impaired in schizophrenia. A contour integration paradigm, involving detection of a set of Gabor patches forming an oval contour pointing to the right or left embedded in a field of randomly oriented Gabors, has been developed for use in clinical trials of schizophrenia. The purpose of the present study was to assess contributions of early and later stages of processing to deficits in contour integration, as well as to develop an event-related potential (ERP) analog of this task. Twenty-one patients with schizophrenia and 28 controls participated. The Gabor elements forming the contours were given a low or high degree of orientational jitter, making it either easy or difficult to identify the direction in which the contour was pointing. ERP results showed greater negative peaks at ~165 (N1 component) and ~270 ms for the low-jitter versus the high-jitter contours, with a much greater difference between jitter conditions at 270 ms. This later ERP component was previously termed N Visual integration, also referred to as “perceptual organization,” is impaired in schizophrenia , occurs at ~300 ms, is impaired in schizophrenia, and has been found to have a source in LOC  are ideally suited to assess different stages of processing due to their high temporal resolution. Previous ERP studies have looked at visual integration in other paradigms such as perceptual closure, which involves identification of fragmented objects  but that the later components, particularly the Nn = 15) or schizoaffective disorder (n = 6) and 28 healthy volunteers. Patients were recruited from inpatient and outpatient facilities associated with the Nathan Kline Institute for Psychiatric Research. Diagnoses were obtained using the Structured Clinical Interview for DSM-IV (SCID)  criteria for schizophrenia (p = 0.76) or gender . As expected, patients had significantly lower IQ  . Individual IQ data were missing for one patient. Patients were ill for a mean of 13.5 ± 10.2 years and were receiving a mean antipsychotic dose equivalent to 988.59 ± 460.4 mg CPZ per day. Duration of illness data were missing for two patients. Mean Positive and Negative Syndrome Scale (PANSS)  of 2000 ms. Participants were instructed to look at the center of the screen, and to press the left or right mouse button if the contour pointed toward the left or right, respectively. Practice trials were given with the low-jitter stimuli in blocks of twenty trials until participants scored above chance on at least one block. During the ERP experiment, all 80 unique stimuli were viewed once in each block, and the blocks were approximately 3 min long. Eight blocks were conducted overall, allowing participants to view 320 trials each of low- and high-jitter stimuli. Participants were periodically prompted to stay focused. The behavioral outcome measure was the percent of correct responses.t(47) = 2.9, p = 0.006). Epochs were averaged for each participant for each jitter condition. A spherical spline algorithm was implemented to compute the current source density (CSD) of the EEG. The CSD represents an improvement over more commonly used voltage measures as it decreases the effects of volume conduction and acts as a high-pass spatial filter, which reduces the overlap between ERPs at different sites , along with digital stimulus timing-tags. Data were digitized online at 512 Hz. All data were recorded relative to a common average reference  online. Epochs (−100 to 400 ms) were created off-line. Data were baseline corrected from −100 ms to stimulus onset and an artifact rejection of ±120 μv was applied to all electrodes. Trials containing eye-movements, identified as deflections of >10 μ V lasting >25 ms appearing on both eye channels, were rejected offline. The number of trials that survived artifact rejection for patients was 206 ± 68.9 and for controls was 255 ± 51.8 and was significantly different between groups , N120 over central electrodes (P0z and Oz), and N1 and Ncl over lateral, ventral-occipital sites . The mean CSD was obtained for each component over specific latency windows . Grand average waveforms were constructed separately for low and high jitter for patients and controls. Figures show filtered waveform data .CSD topography maps were used to select the electrode sites with most prominent P1, N120, N1, and Ncl) were performed separately using mixed-model analyses of variance (ANOVAs) with group  as the between-subjects factor, and with jitter  and hemisphere  as within-subject factors. Relationships between measures were assessed using Pearson correlation coefficients. Behavioral data were not collected from 1 patient and 2 controls in the low-jitter condition and 2 patients and 2 controls in the high-jitter condition. Effect sizes for ANOVAs are reported as partial eta squared (η2p) to be consistent with past studies of contour integration  and each ERP component  = 12.7, p = 0.001, η2p = 0.228]. Both groups performed better on the low-jitter condition than on the high-jitter condition   = 4.1, p = 0.049, η2p = 0.087], with a greater between-group difference in the low- than high-jitter condition.Controls performed significantly better than patients   = 1.5, p = 0.23, η2p = 0.031] and group × jitter × hemisphere  interactions were not significant, reflecting similar P1 CSD to low- and high-jitter stimuli. There was no significant group × hemisphere interaction  or main effect of hemisphere .P1 CSD was significantly reduced in patients compared to controls  Figure . Group ×F = 4.4, p = 0.04, η2p = 0.085], indicating differential activity across groups to low- versus high-jitter stimuli  = 4.1, p = 0.05, η2p = 0.081], indicating lack of differential CSD to low versus high jitter in patients  but differential CSD to the jitter conditions in controls   = 0.29, p = 0.59, η2p = 0.006], group × jitter , and group × jitter × hemisphere  interactions were not significant. However, the low-jitter condition produced a more negative peak than the high-jitter condition .N1 amplitude was not significantly different between groups  Figure . Group ×F = 47.8, p < 0.001, η2p = 0.504], indicating differential activity across groups to low- versus high-jitter stimuli  = 8.2, p = 0.006, η2p = 0.148], indicating reduced differential activity to low versus high jitter in patients  compared to controls . Group × hemisphere , jitter × hemisphere , and group × jitter × hemisphere  interactions were not significant.There was a significant main effect of jitter . For N120, the significant group × jitter interaction  remained, but the main effect of jitter was no longer significant . For N1, the main effect of jitter remained significant . For Ncl, a significant main effect of jitter was still found  as was a group × jitter interaction . This indicates that even when only participants who were clearly performing the low-jitter task above chance were included, there was still a large difference in Ncl response to low- versus high-jitter stimuli overall and patients showed a reduced difference between jitter conditions compared to controls. However, while the behavioral deficit was less pronounced than when all participants were included, patients still showed an impaired behavioral response to low-  and high-jitter  conditions compared to controls.To assess whether the ERP results were due to difficulty of patients in identifying the contour and/or to a general deficit, analyses were also carried out with only those participants reaching at least 70% correct in the behavioral low-jitter condition. This resulted in the inclusion of 12 out of 21 patients and 22 out of 28 controls. The general pattern of results was similar to that seen with the entire cohort. For P1, CSD for patients was lower than controls, with a trend for a significant difference between groups [x, ±18; y, −89; z, 13). The N120 component was fitted to a window of 115–135 ms to obtain a clear temporal separation from the P1 component, and accounted for 99% of the variance in controls and 98% of the variance in patients. The generator sources were localized to extrastriate Brodmann Area 18 . The N1 component was fitted to a window of 150–180 ms accounting for 96% of the variance in controls and 98% in patients. The Ncl was fitted to a window of 250–290 ms accounting for 98% of the variance in controls and 92% in patients. These two components were co-localized to Brodmann Area 37 (fusiform gyrus) .Inverse dipole modeling was carried out on the grand average waveforms using the brain electrical source algorithm Scherg, . Models cl, high-jitter CSD was subtracted from low-jitter CSD because these components showed a differential response to jitter. P1 responses were significantly correlated with the Ncl CSD difference for controls  but not patients . In addition, controls showed a trend for  and patients showed a significant  correlation between N1 and Ncl CSD differences.ERP components were collapsed across hemisphere because there were no significant main effects or interactions with hemisphere. For P1, correlations were performed for low and high jitter separately. For N120, N1, and Nr = −0.47, n = 19, p = 0.04) and Ncl CSD , replicating past findings that poorer contour integration is related to greater disorganization in other domains , but not other components.No significant relationships were found between any ERP component or results on the behavioral task and CPZ equivalents or duration of illness for patients. However, using the 5-factor PANSS, patients showed a significant relationship between the Cognitive (Disorganized) factor and both low-jitter behavior , suggesting that this component is important in the actual recognition of contours. The Ncl, like the N1, was source localized to ventral stream fusiform gyrus, and this region was shown to be involved in contour integration in a prior fMRI study  and later  processes appear to differentiate between recognizability of the Gabor-defined contours.The present ERP results for Gabor-defined contours represent something of a hybrid of results found for perceptual closure, which involves actual objects , patients still showed a significant impairment in NA further limitation of the study was that all patients were receiving antipsychotic medication. However, CPZ equivalents were not correlated with any ERP component or behavioral performance. In addition, lack of relationship with medication has been found in previous studies of contour integration and a deficit has also been found in un-medicated patients (Silverstein and Keane, The present results extend previous behavioral and fMRI contour integration findings to the ERP domain. Further, the current contour integration paradigm demonstrated similar ERP signatures as were found in perceptual closure studies (Doniger et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tumour size in breast cancer influences therapeutic decisions. The purpose of this study was to evaluate sizing of primary breast cancer using mammography, sonography and magnetic resonance imaging (MRI) and thereby establish which imaging method most accurately corresponds with the size of the histological result.Data from 121 patients with primary breast cancer were analysed in a retrospective study. The results were divided into the groups “ductal carcinoma in situ (DCIS)”, invasive ductal carcinoma (IDC) + ductal carcinoma in situ (DCIS)”, “invasive ductal carcinoma (IDC)”, “invasive lobular carcinoma (ILC)” and “other tumours” . The largest tumour diameter was chosen as the sizing reference in each case. Bland-Altman analysis was used to determine to what extent the imaging tumour size correlated with the histopathological tumour sizes.Tumour size was found to be significantly underestimated with sonography, especially for the tumour groups IDC + DCIS, IDC and ILC. The greatest difference between sonographic sizing and actual histological tumour size was found with invasive lobular breast cancer. There was no significant difference between mammographic and histological sizing. MRI overestimated non-significantly the tumour size and is superior to the other imaging techniques in sizing of IDC + DCIS and ILC.The histological subtype should be included in imaging interpretation for planning surgery in order to estimate the histological tumour size as accurately as possible. The key importance of imaging methods in breast diagnostics lies in the detection and sizing of areas suspicious for malignancy. Breast results are classified using the BI-RADS (Breast Imaging Reporting and Data System) classification ,2.Exact pre-therapeutic tumour sizing using imaging methods plays a central role. For example, the possibility of breast conserving treatment significantly depends on the relationship between the tumour-to-breast size. Furthermore, the indication for primary systemic treatment is made, amongst other things, from the tumour size.The aim of this study was to analyse which of the given imaging methods is the most accurate in the pre-therapeutic sizing of primary breast cancer.121 patients with primary breast cancer who presented preoperatively in the University Breast Centre of Tuebingen between June 2005 and July 2007 were retrospectively analysed. All patients fulfilled the following criteria: primary breast cancer, no neoadjuvant chemotherapy, complete documentation of the tumour size from mammography, sonography and magnetic resonance imaging (MRI) as well as the postoperative histological tumour size. The largest tumour diameter was chosen as the sizing reference in each case.Mammography was performed using two digital full field instruments .Sonography was performed using a linear transducer with a 50 mm width and a frequency of 12 MHz. Models iU22 and HD 11 from Philips  were used. The examination was carried out using “Spatial Compound Imaging” and “XRES Adaptive Image Processing”. Measurement of tumor size took the echopoor centre of the lesion and the echogenic halo into account. The clinical examiner held a minimum of the Degum Level 2 qualification.Magnetic resonance imaging (MRI) was performed using a 1.5 Telsa system . A T1-weighted dynamic gradient echo sequence (T1w-FFE = Fast Field Echo) with a native and 7 post-contrast medium series was used. Automated bolus injection of 0.16 mmmol Gadobutrol per kg body weight followed by 10 ml saline were infused intravenously. Image post-processing included the generation of subtraction series and reconstruction of a MIP (maximum intensity projection). Imaging analysis was carried out using the digital data with the help of a suitable workstation .Only physicians who were specialized on breast diagnostics performed and reviewed each imaging.The histological results were divided into the groups “ductal carcinoma in situ (DCIS)”, “invasive ductal carcinoma (IDC) + ductal carcinoma in situ (DCIS)”, “invasive ductal carcinoma (IDC)”, “invasive lobular carcinoma (ILC)“ and “other tumours” . The largest tumour diameter was chosen as the sizing reference in each case. Bland-Altman analysis was used to determine to what extent the imaging tumour size correlated with the histopathological tumour sizes.The mean difference between the imaging and the histological results was calculated and related to the interval in which 95% of the calculated differences were found (LOA = limits of agreement). Size variation on imaging versus pathology were reported as median and interquartile range, using Box plots. The statistical analysis was performed using SPSS® for Windows . The level of significance was defined as a p-value of <0.05.There was no objection against the study from the local ethic committee.121 patients with primary breast cancer were evaluated in a retrospective analysis. The median age was 57 years (range 35–92). An IDC was present in 33.9% of the cases. 31.4% of the patients were allocated to the IDC + DCIS tumour group, and a DCIS alone or ILC alone were found in 12.4% and 14.9% respectively. “Other tumours” occurred in 7.4% of the cases.The density level of the breast tissue was graded using mammography according to the American College of Radiology (ACR)  classifiAs demonstrated in Table The mean difference between mammography and histology was -1 mm , IDC (p = 0.008) and ILC (p = 0.001). The greatest mean difference between the sonographically measured tumour size and the actual histological tumour size was found for invasive lobular breast cancer Table . Pritt eNo significant differences between tumour types and histologically established tumour sizes could be found in our study for mammography and for MRI.In contrast to sonography and MRI, the sensitivity of mammography is significantly negatively affected by increasing breast tissue density -15. MammIf imaging malignancy assessment with reference to the individual tumour groups is considered, isolated DCIS is clearly more commonly classified as BIRADS 5 Table  with mamWhen considering ILC, the detection of clinical findings must be regarded as separate from sizing. In a study by Butler et al. , 39% of Sizing of ILC using sonography reveals a significant underestimation of tumour size compared to mammography Table . In thisModern ultrasound equipment also usually operates with complex image processing software. A danger of the image processing is that clinical findings are modified or so embellished that the interpretation of the classical malignancy criteria done up to now is no longer possible . This coIn contrast to mammography and sonography, all tumours were correctly preoperatively classified as requiring further clarification (> BI-RADS IV) with MRI, Table , and 38%Investigator influence during the malignancy assessment of the results due to previous knowledge of the results of other imaging techniques cannot be excluded. 38% of the MRI results were BI-RADS 6-lesions and were therefore histologically confirmed first of all. However, this study considered sizing and not malignancy assessment; therefore this does not appear to have any influence on the results. Moreover study population was retrospectively analysed and limited to only those patients with cancer visible on all three imaging modalities.According the data from this study (see Table 1. IDC can be measured well with all three imaging methods; MRI and mammography are the more exact methods, whilst sonography showed a significant underestimation of the results.2. IDC with extensive DCIS involvement can be most accurately measured with MRI. Ultrasound leads to a significant size underestimation on average.3. According to our data, DCIS alone can be most accurately measured using mammography. Mammography and MRI show no significant variations from the mean tumour size compared to histology.4. ILC is measured most accurately using MRI and mammography, provided that the results are visible with mammography. Sonography leads to a significant underestimation of the mean tumour size.From these results, we conclude that for surgical planning, the histological subtype should be included in the imaging interpretation in order to estimate the tumour size as accurately as possible.ACR: American College of Radiology; BI-RADS: Breast Imaging Reporting and Data System; DCIS: Ductal carcinoma in situ; IDC: Invasive ductal carcinoma; ILC: Invasive lobular carcinoma; LOA: Limits of agreement; MIP: Maximum intensity projection.The authors declare that they have no competing interests.IG carried out imaging and measurements. MR carried out measurements. KK participated in the design of the study and performed the statistical analysis. AS participated in the histological workup. KS carried out imaging and measurements. AH participated in the design of the study and performed the statistical analysis. DW participated in the study design and its coordination. MH participated in the study design and its coordination, imaging, measurements and surgery. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/328/prepub


We are often confronted with severe cases–patients with very aggressive tumours that suppose a complex and in the same time radical approach–in our medical practice. The correct approach and management of such cases ensure both the surgical success and the patient survival. In this paper, we present the case of a young woman, who has been admitted in our clinic with a giant, irradiated tumour involving left axilla, shoulder and scapula. Due to the vast size of the tumour and to the fact that surgical biopsy revealed a poorly differentiated sarcoma; other clinics considered that the case above belongs to surgical therapy.  After the clinical examination, blood tests and diagnostic imaging, which allowed the correct evaluation of the case–tumour sizes and neighbouring tissue reports–we decided to perform tumour radical excision, respectively forequarter amputation, when the patient presented a satisfactory metabolic status. The presented case supports the idea that radical excision which might involve even mutilating amputations for extensive cancers can give patients a chance, even in desperate cases.  Synovial sarcomas represent 8–10% of all sarcomas and they most frequently affect adults in their decades from three to five. The median age of appearance is 31, 3 years old.  They are frequently misdiagnosed as benign tumours due to their small sizes and fine definition of their aspect. They arWe present the case of a patient, ML, aged 33, who was admitted in our clinic for an arm and axilla radionecrotic tumour. The tumour was first seen in the patient 9 months ago. It presented as a round mass, with 0.75 cm diameter. The tumour had an explosive growing in the past 3 months. She was investigated in an Orthopaedic Clinic, where the diagnostic imaging included MRI, and which revealed a heterogeneous tumour in close contact with the axilla vascular and nervous bundle, invading the rhomboids and deltoid.The differential diagnosis included chondrosarcoma, liposarcoma, malignant fibrous histiocytoma, musculoskeletal tumors (percutaneous needle biopsy), osteochondroma and osteochondromatosis, osteosarcoma and synovial osteochondromatosis, etc.Further, a tumour biopsy was performed–tissue fragments representing malignant tumour proliferation with small, round cells, having a reduced cytoplasm, hyperchromatic nuclei with a compact disposition and large areas of tumoral necrosis. These hystopathologic aspects are synonymous with a Ewing PNET tumour, but do not exclude a synovial sarcoma or a rabdomiosarcomaImmunohistochemical studies revealed: EMA: positive in rare tumour cells; CK cocktail positive in tumour cells; S 100 negative in tumour cells; CD57 negative; CD 34 negative in tumour cells and positive in vessels; CLA negative in tumour cells and positive in small lymphocytes.  Thus, these tests are compatible with a low'grade synovial sarcoma diagnostic. The tumour had a substantial growth from the day of biopsy. Further, due to the tumour's large volume, surgical intervention was preceded by radiotherapy and chemotherapy (ifosfamide and doxorubicine). The tumour regressed a lot. When the patient was admitted in our clinic, she presented a 10/10 cm mass, involving the posterior left arm and axilla, fixed on the deep planes. The tumour was heterogeneous, ulcerated, with radiation necrosis, redness, oedema and tissue debris. Diagnostic methods included; blood tests, ultrasonography and pulmonary radiology that revealed no metastases. Local infection (staphylococcus aureus and Raoultella (K) ornithinolytica) control was made possible by pre and postoperative administration of medaxone and gentamicyn.Radical surgical procedure was imposed by: 1) the tumour's large dimension, 2) shoulder girdle muscle invasion, 3) close contact and invasion of axilla vascular and nervous bundle and 4) histopathological form–low–grade sarcoma–more aggressive and a higher rate of metastases[etastases,6,7,8.Forequarter amputation was imposed as a life saving measure. Surgical intervention was carefully planned so as to allow long time cover of the amputation stump.We decided to perform an anterior approach, the patient being placed in a right lateral position.The surgical technique consisted of an incision on the preoperative markings. These were deepened to the musculofascial layers. Platysma and supraclavicular nerves were cut. The clavicle was exposed and cut laterally to the sternocleidomastoidian with a Gigli saw. The tendons of pectoralis major, coracobrachialis and short head of the biceps were cut. Then, the subclavicular vessels were ligated.Surgical technique: After the subclavicular vessel ligature, brachial plexus branches were isolated, severed and let to retract. Scapula, trapezius, rhomboids, latissimus dorsi and seratus anterior were removed as the dissection progressed in the posterior plane [or plane .The histopathology revealed a sarcoma–like tumour proliferation, with small, round cells presenting radiation necrosis changes. Axillary lymph nodes presented degenerative changes with massive reactive sinus histiocytosis.The ideal attitude would be tumour rapid diagnostic, establishing its gravity and, last but not least, immediate and correct therapeutic approach. By evaluating the tumour's extension both at surface and in depth, we shall be able to perform a radical excision. Synovial sarcomas need an aggressive therapeutic attitude. Whenever possible the approach would be a limb sparing surgery. The election treatment is a margin free disease large excision. This supposes, quite often, the neighbouring muscle resection or total amputation.In our case amputation was synonym with radical excision, being the only measure for a prognostic improvement.‘Forequarter amputation’ represents a mutilating surgical intervention. Therapeutic option was imposed both as a need for radical excision and as a life saving measure.