Single-Step 3-D Image Reconstruction in Magnetic Induction Tomography: Theoretical Limits of Spatial Resolution and Contrast to Noise Ratio

Magnetic induction tomography (MIT) is a low-resolution imaging modality for reconstructing the changes of the complex conductivity in an object. MIT is based on determining the perturbation of an alternating magnetic field, which is coupled from several excitation coils to the object. The conductivity distribution is reconstructed from the corresponding voltage changes induced in several receiver coils. Potential medical applications comprise the continuous, non-invasive monitoring of tissue alterations which are reflected in the change of the conductivity, e.g. edema, ventilation disorders, wound healing and ischemic processes. MIT requires the solution of an ill-posed inverse eddy current problem. A linearized version of this problem was solved for 16 excitation coils and 32 receiver coils with a model of two spherical perturbations within a cylindrical phantom. The method was tested with simulated measurement data. Images were reconstructed with a regularized single-step Gauss–Newton approach. Theoretical limits for spatial resolution and contrast/noise ratio were calculated and compared with the empirical results from a Monte-Carlo study. The conductivity perturbations inside a homogeneous cylinder were localized for a SNR between 44 and 64 dB. The results prove the feasibility of difference imaging with MIT and give some quantitative data on the limitations of the method.
Introduction
Magnetic induction tomography (MIT) is a non-invasive and contact-less imaging modality for reconstructing the changes Δκ of the complex conductivity distribution κ = σ + jω ɛ0ɛr in a target object.8,13–16,22 MIT requires an array of excitation (EXC) and receiving coils. Each EXC couples an alternating magnetic field B0 to the object under investigation (see Fig. 1). Changes Δκ of the complex conductivity cause a field perturbation ΔB due to the induction of eddy currents. The perturbation induces voltage changes ΔV in the receiver coils. It is convenient to normalize ΔV to V0, the voltage which is induced by the unperturbed field B0.Figure 1.Schematic of a possible coil system for MIT with 16 excitation coils and 32 receiver coils.
Previous reviews of MIT have been given in8,22,32. The method has been developed for industrial process tomography already more than 10 years ago but is comparatively new in medical imaging. Potential medical applications usually aim at the characterization of biological tissues by means of their complex conductivity. The motivation for measuring the electrical properties is their characteristic dependence on the (patho-) physiological state of tissues, especially hydration and membrane disorders. Medical applications so far suggested are: imaging of limbs,2 imaging of the brain, e.g. for the monitoring of brain edema,14,16,24,26 measurement of human body composition,7 monitoring of wound healing.23
In contrast to electrical impedance tomography (EIT) MIT avoids the ill-defined electrode-skin interface due to its inherently contact-less operation.
Figure 1 shows a schematic MIT coil configuration with rectangular coils as receivers and a cylindrical object space. The solenoid excitation coils are distributed on two different rings in order to obtain a true 3-D-arrangement.
The reconstruction of the absolute conductivity in a target region Ω requires the solution of a complex inverse eddy current problem. Let be
the discretized non-linear forward mapping of the conductivity vector
to the vector of induced voltages y. y contains M = a × b entries, a being the number of EXC and b that of receiving coils. The corresponding inverse problem
is ill-posed and usually underdetermined. Uniqueness of the solution for this inverse boundary value problem was established in21 provided the angular frequency ω of the AC field is not a resonant frequency. The generic approach for the solution of this type of non-linear problem is the application of an iterative scheme such as the regularized Gauss–Newton approach, including an appropriate regularization scheme.
To the knowledge of the authors the full inverse problem of medical MIT in 3-D has not yet been solved satisfactorily, although some approximate solutions, especially for 2-D, have been presented.3,13,33 Some authors9,15 proposed the use of weighted back-projection, similar to EIT. In all published cases the back-projection is done along magnetic flux tubes between excitation and receiving coils, the weights being calculated for the case of conducting perturbations in the empty space. However, own observations17,27,29 suggest that the basic requirements for the applicability of this kind of back-projection are not fulfilled in realistic anatomical structures, so that a more appropriate inverse approach is necessary. This paper is dedicated to demonstrating the feasibility of the 3-D reconstruction of a spherical perturbation within a cylindrical conducting body by means of a regularized one-step Gauss–Newton reconstructor. The conductivities were chosen in the physiological range of human tissues.
Methods
The solution of (2) requires the target region to be discretized into N voxels. Within each voxel i the assigned component κi of the vector of conductivity
is assumed to be constant. A grid of tetrahedral finite elements of second order was employed. In a general setup
is then found with the iterative scheme: Define the forward problem with an initial parameter vector
.
Measure the data vector ym.Solve iteratively for the estimated true parameters
whereby
means the estimated “true” parameter vector. R and λ are a regularization matrix and a regularization parameter, respectively, which are required to stabilize the iteration.
When applying Newton’s method starting from an initial guess the parameter vector
is updated by an increment pk in each iteration step k+1
with the update step
with
. The Jacobian
, also called sensitivity matrix, must be recalculated in each iteration step. This procedure is very time consuming, hence a complete iterative identification run requires significant computing power, the bottleneck being the solution of the forward model. However, in EIT it could be shown that in practice most features of the image can already be recognized very satisfactorily after the first iteration. This fact led to the development of the so-called Newton-one-step reconstructor (NOSER4). NOSER is especially appropriate for so-called dynamical imaging where only the change in the conductivity between two different states of the object under investigation (e.g. lung ventilation) are of interest. In this case the first Newton step corresponds to the solution of the linearized forward problem
whereby
is the change of the conductivity between two states of the observed object, and Δym is the corresponding change of the measured data. In the case of comparatively small changes the inversion of (6) according to (7) yields a fairly correct localization of the perturbed regions. We evaluated the feasibility of this kind of reconstruction in MIT by implementing a NOSER-approach according to (7) under consideration of four different regularization methods.
Calculation of the Forward Solution and the Sensitivity Matrix
The forward mapping
is given by Maxwell’s equations for harmonic excitation:
with H: magnetic field intensity, B magnetic flux density, E electric field strength, J: current density, ɛ: dielectric constant, μ: magnetic permeability, σ: real conductivity, κ: complex conductivity, ω: angular frequency. Ω denotes the interior of the object under investigation.
This forward problem is solved with a previously published finite element program,12,17 which employs an Ar–V, Ar – formulation with edge elements of second order for the reduced magnetic vector potential Ar and nodal elements of second order for the electric scalar potential V. Boundary conditions on the far boundary (normal component of B vanishes) were prescribed on a spherical surface with a radius sufficiently large such that a change of this radius by 50% resulted in a change of the induced voltages by less than 1 %.
Special attention must be paid to the efficient calculation of the Jacobian
. A mathematically rigorous treatment of this topic has been given in.31 In our implementation we exploited the integral formulation published by Mortarelli20, which is based on a physical mutual energy concept. With this approach the absolute sensitivity dy/d for a certain pair of coils is calculated according to (9).
with
Aϕ, AΨ, Vϕ and Vψ denote the total magnetic vector potential and the electric scalar potential in the region Ω due to currents Iϕ and Iψ in the excitation and receiver coils, respectively. The sensitivity matrix
is then obtained by evaluating (9) for all individual elements and all coil pairs. The exact numerical implementation of (9) was described in detail in11. The calculation of the sensitivity map for one pair of coils requires only two forward solutions of the eddy current problem for generating Lϕ and LΨ in (10).
Regularization
In EIT the regularization matrix RTR most frequently used is either the identity matrix I or a discrete spatial derivative operator of first or second order. Such approaches have been discussed extensively in the literature, for a good review see e.g.10 Several regularization matrices can be regarded as simple smoothness criteria for the solution but they have also a more general statistical meaning in the framework of Bayesian estimation theory (see e.g.1). In the case of uncorrelated noise with equal variance for all measurement data the estimator in (7) is a maximum a posteriori (MAP) estimator with RTR being the inverse of the expected covariance matrix of the image E[ppT]. In that sense e.g. the neighbouring matrix accounts for the case that all image values are de-correlated at borders between homogeneous regions with different mean values.
According to our own observations good results can be achieved with variance uniformization5 which imposes a special assumption of the prior distribution. The objective here is to uniformize the expected variance of the reconstructed conductivity changes over the region Ω, thus providing approximately equal image noise in the center as in the periphery. The algorithm has been described in detail in5 and requires singular value decomposition of G according to G = UΣVT. Then the regularization term is expressed as λ RTR = VDVT, with D a diagonal matrix with the entries di whereby σi is the Ith singular value. c is a free scalar tuning parameter.
Alternatively truncated singular value decomposition (TSVD) has been applied in EIT-reconstruction,19 hence this approach was also implemented for MIT. In this case the inverse solution becomes
whereby t denotes the truncation level of the original matrices V, Σ and U, thus removing the contributions of singular values with index > t.
In this paper the results obtained with four different regularization schemes were compared: RTR = I. Using the identity matrix is the most simple Tikhonov-regularization method, penalizing high values of the reconstructed conductivity changes. In the following this method will be abbreviated as ‘IM’.RTR = N with N the neighbouring matrix defined as:
nn is the number of neighbouring elements for element i, whereby only elements with common facets are considered as neighbours. N is an approximation of the spatial derivative operator of second order. Due to the irregular structure of the grid this filter is not spatially invariant, nevertheless it gives good results and is common practice for this type of inverse problem. In the following this method will be abbreviated as ‘NM’. λ RTR = VDVT according to the variance uniformization approach. In the following this method will be abbreviated as ‘VU’.TSVD while choosing the truncation level t such as to remove all singular values below an appropriately chosen threshold.
Methods 1 and 2 require the regularization parameter to be chosen optimally while method 3 in addition implies the choice of the tuning parameter c. In practice it turns out that the value of c is not critical over a very wide range of values because the optimal λ depends on c. That means that c can be fixed at a more or less arbitrary value if a method for the automatic determination of λ is applied. In our case c was set to 0.1.
The regularization parameter λ accounts for the degree of smoothness of the reconstructed image and determines the condition number of the term (GTG + λRTR) in (6). Several methods for the optimal choice of this parameter have been published in the past, the most well-known ones being L-curves, Generalized Cross-Validation and the Morozov-criterion.10 Because of its clear physical interpretation we choose the latter method. In this case the optimal λ is the one where the estimated residuals Gp − Δym have the same variance as the measurement noise. The motivation for this criterion is that it is obviously meaningless to make the residuals lower than expected from the statistics of the data. This method provided always stable images independently of the regularization matrix and was considered as a good basis for a fair comparison between the different regularization methods.
Modeling Setup
The inverse solver was tested with the simple 3-D model comprising a cylindrical conductor with two spherical inhomogeneities placed with their centers at (x =  − 60, y = 0, z = 0) (mm) and (x =  − 30, y = 52 and z = 0) (mm) and the array of 16 excitation coils and 32 receiving coils shown in Fig. 1. The exact geometry is illustrated in Fig. 2. The cylinder had a radius and a height of 100 mm, the perturbing sphere had a radius of 20 mm. The solenoid excitation coils were modeled by cylindrical rings with an inner diameter of 60 mm, a thickness of 1 mm and a width of 21 mm. The exciters were placed in groups of 8 with their centers on two rings with radii of 125 mm in two transversal planes of the cylinder. The lower ring is rotated by 22.5 degrees versus the upper one in order to achieve a lower degree of symmetry. The square receiver coils with an edge length of 40 mm were placed with their centers on two symmetrically arranged parallel rings with a radius of 120 mm, each comprising 16 evenly spaced coils. The orientation of the windings were opposite in both rings so that the 16 vertical pairs can be combined to planar gradiometers as published previously26. The measured data were simulated in terms of induced voltage changes when changing the conductivity of the spherical perturbation from 0.2 S/m (= background conductivity, homogeneous cylinder) to 0.3 S/m. The relative permittivity was kept constant at 80 in all compartments. The excitation frequency was 100 kHz.Figure 2.Schematic of the simulation model. 16 excitation coils and 32 receiver coils are placed on two concentric rings around the tank, respectively. Two spherical perturbations are placed at the shown locations with (x =  − 60, y = 0, z = 0) and (x =  − 30, y = 52, z = 0). All measures are given in (mm).
This arrangement was chosen as the model system for our analysis because of two reasons: It represents a true 3-D-arrangement which delivers theoretically 512 independent measuring combinations, i.e. 512 data points for one image reconstruction.It is similar to our experimental system which employs 16 excitation sites in one plane and 14 planar gradiometers which are formed by connecting in counter-phase the coils in the upper and in the lower receiver plane.
Two different meshes A and B were used for the generation of an artificial dataset and for the reconstruction. Mesh A comprised 11000 elements within the cylinder and approximately 1200 in the spheres while mesh B comprised 17000 elements for the homogeneous cylinder without perturbation. In both cases the diameter of the surrounding spherical surface which approximated the far boundary was chosen as 1m, requiring approximately 30000 elements (mesh A) and 50000 elements (mesh B), respectively. Uncorrelated Gaussian noise was added to the voltage data in order to simulate the noise of the receiver channels. This type of noise, although common practice in simulations of this kind,, is not entirely valid for real situations. In addition the noise of the excitation coils is propagated to all receiver coils, thus resulting in a certain amount of correlated noise in all receiver channels. This phenomenon has been studied in detail for EIT,6 but should be disregarded here for simplicity.
The calculation of the complete sensitivity matrix required 48 forward solutions according to the Mortarelli-approach.
Theoretical Limits of Resolution and Contrast/Noise Ratio
For EIT the theoretical limits of image quality in terms of contrast/noise ratio (CNR) and resolution have been studied carefully in30 while no such study exists for MIT. A similarly rigorous discussion for MIT is certainly beyond the scope of this paper. However, a simplified analysis on resolution and CNR was carried out for our linear reconstruction scheme so as to have a certain theoretical basis for interpreting our empirical results.
There is a fundamental limit for the resolution which depends on the amount of available information in the data. This information depends on the number of data points, i.e. the number of possible sensor–detector-combinations and on the degree of independence between these data points. In the case of EIT and MIT the number of independent data points is usually much lower than the number of voxels, so that the system is under-determined. Moreover the different data are correlated to a certain degree, so that the effective rank is comparatively low. In EIT, e.g. 16 electrodes provide 104 independent data points so that the information is no more than 104 ‘effective pixels’. Including some a priori-information in the form of the regularization terms leads to a defined ‘smearing’ of this information over the imaging plane and provides the typical diffuse images known from EIT.
We characterized the resolution of MIT with the Raleigh criterion. Accordingly two point-shaped perturbations are still separable if their point spread functions (PSF) overlap in such a way that the peak of the first one coincides with the first zero crossing of the second one. In the case of a sinc-shaped PSF the lowest separable distance is equivalent to the 64%-width of the PSF. In contrast to e.g. X-ray CT in MIT the PSF depends on the location of the perturbation and on the geometry of the object under investigation. In this paper the object is the model cylinder which is also used for the numerical reconstruction examples and for the phantoms. The PSF is calculated by mapping the true parameter values p* to the reconstructed ones p via the reconstruction equation
A means the expression
A = (GTG + λ RTR)−1GT for regularized methods 1–3 or A = Vt Σt−1Ut for TSVD.
The ith column of M in Eq. 13 is then the shifted PSF for the ith voxel. The theoretical limit was approximated by applying TSVD with the full set of non-zero singular values, i.e.
We chose TSVD for the estimation of the theoretical limit because it requires the least explicit assumptions about the a-priori distribution of p. In MIT an additional difficulty is that, in general, the 64%-boundary of the three-dimensional PSF is not spherical and thus the resolution is anisotropic. In our simplified analysis we define as Raleigh-width the largest axis of the ellipsoid which best approximates the 64%-boundary.
As CNR at the point x we defined
whereby Δp is the reconstructed difference between perturbation and background and std(np) is the standard deviation of the image noise. We recognize that Δp is not a contrast in the classical sense. Usually contrast is the difference between p in the perturbation and background divided by the background value. However, as our method is a differential one, the background value of the image is always zero and the classical contrast definition is meaningless. Moreover the determinant for the detectability of a perturbation is the difference Δp rather than the contrast. The CNR depends on the size and location of the perturbation as well as on the noise level of the measured data. In contrast to resolution there is no theoretical lower limit in the case of noise-free data and a perfect reconstruction method.
We estimated std(np(x)) by calculating the Cramer-Rao lower bound of the covariance of the parameters. Given the covariance matrix X of the voltage changes Δym the Cramer-Rao lower bound of the covariance matrix of the image noise vector np is
From the diagonal elements of this matrix the expected lower bound of the coefficient of variation (CV) of the reconstructed conductivity in the perturbation can be calculated for any voxel. For the evaluation of Eq. 15 the reconstructed value Δp in voxel i follows from Eq. 13:
whereby P is the set of the indices of all voxels inside the true perturbation. This equation clearly shows that the CNR must decrease when the size of the perturbation shrinks. Assuming a small perturbation and an approximately constant PSF inside this region the CNR is approximately inversely proportional to the volume of the perturbation. The detectability limit can then be defined as the one where the CNR equals 1.
The shown approach is simple and allows the calculation of detectability limits for perturbations with different radii and locations but it is only valid in the linear case. Alternatively Monte-Carlo studies can be carried out for different perturbations varying in size and contrast. Such a study has been published previously for a spherical perturbation in the center of a brain model.18
For simplicity we assumed the noise to be Gaussian with zero mean, although in the general non-linear case this may not be entirely valid. For characterizing the noise level we defined as SNR the ratio max(|ΔV|)/std(V) whereby std(V) is the standard deviation of the noise voltage. ΔV is the vector of voltage changes in all excitation/sensor combinations when a test object is placed into the empty measurement system. In order to be independent on size and location of the perturbation we chose as the test object the homogeneous background cylinder of our model.
Results
The PSF was evaluated at 20 equally spaced points along the x-axis between the center and the border of the cylinder. This set includes also the point (0.6R0,0, 0), i.e. the center of one of the two perturbations in our simulation model. To mitigate discretization errors the PSF was calculated by rotating the coordinate system 8 times about 45° and averaging the data assuming radial symmetry of the true model. Then the normalized resolution was calculated from the PSF as the inverse of the ratio between the Raleigh-width and the cylinder radius. This normalized resolution can be interpreted as the number of points which can be resolved per cylinder radius. The resulting data were plotted in Fig. 3 as a function of the normalized x-coordinate in the xy-plane. Curves are depicted for noise-free data (TSVD with truncation level 512) and TSVD with truncation levels corresponding to a SNR of 44, 50 and 64 dB, respectively. The truncation levels were chosen according to the Morozov-criterion and are listed in table 1. The above three SNR levels were chosen because they correspond to the range in which our current measurement system operates.25Figure 3.Dependence of the theoretical normalized resolution on the noise level. Curves are depicted for noise-free data (TSVD with truncation level 512) and TSVD with truncation levels corresponding to a SNR of 44, 50 and 64 dB, respectively.TABLE 1.Tuning parameters for the regularization, chosen according to the Morozov-criterion.Regularization methodTSVD Truncation levelIM λNM λVU λSNR = 64 dB1822.40E − 202.40E − 202.35E − 11SNR = 50 dB1008.40E − 198.80E − 193.20E − 10SNR = 44 dB681.25E − 171.19E − 177.40E − 10The parameter c for VU was always kept at 0.1.
A clear increase of the resolution with the distance from the center is observable. The theoretical limit for a point-shaped perturbation in the xy-plane is 3.5 (corresponding to 2.9 cm separation) in the center and a maximum resolution of more than 6 (corresponding to 1.7 cm separation) at the periphery. At the locations of the test spheres the resolution is in the range of 4, i.e. 2.5 cm separation. The relative loss of resolution with the noise level is stronger in the center than in the periphery. At a SNR of 44 dB the resolution does not increase any more continuously but levels off above a normalized x position of about 0.6.
In analogy to EIT the PSF depends strongly on the location, showing the broadest distribution in the center of the cylinder. This is reflected by increasing resolution when moving from the center towards the periphery. Increasing the amount of regularization or decreasing the truncation level according to increasing measurement noise the PSF broadens and its center of gravity is shifted towards the borders of the cylinder. Moreover increasingly strong ringing in form of star-like patterns becomes observable close to the border (not shown explicitly in this paper).
In Fig. 4 the four regularization methods are compared at a SNR of 50 dB. Except for few points TSVD, performs poorest which is in accordance with the assumption of least explicit a-priori information.Figure 4.Theoretical normalized resolution for the four regularization methods at a SNR of 50 dB.
Figure 5a shows the CNR for TSVD as a function of the normalized x-coordinate and in dependence on the noise level while Fig. 5b compares the four methods at a SNR of 50 dB. As expected the theoretical CNR depends strongly on the location of the perturbation increasing from values around 2 in the center up to about 60 at the cylinder border. At the location of the perturbing spheres the CNR drops from about 26 to 13 when decreasing the SNR from 64 dB to 44 dB. In the simulated images the CNR drops from 24 to 9, i.e. remains fairly in the same range. Figure 5b reveals that IM and NM yield the highest CNR, followed by TSVD and VU, whereby VU is characterized by dramatically lower values. In the center VU yields a CNR around 2 which is already very close to the limit of detectability. When comparing the theoretical values with the reconstructed ones (see table 2) the reconstructions always produce a lower CNR than expected, the discrepancy being stronger at high noise levels.Figure 5.Panel A: CNR for TSVD as a function of the normalized x-coordinate (relative to the cylinder radius) and in dependence on the noise level. Panel B: comparison of the four methods at a SNR of 50 dB.
Figures 6 and 7 show the reconstructed mean images from a Monte-Carlo study with 50 runs for each of the four methods and an SNR of 64 and 50 dB, respectively. Representative cross-sections in the xy-plane and in the xz-plane were selected. The respective regularization parameters are listed in Table 1.Figure 6.Mean images of the Monte-Carlo study. Reconstructed Δ σ (transversal and saggittal section through the origin) for the spherical perturbations with four different regularization matrices and a SNR of 64 dB.Figure 7.Mean images of the Monte-Carlo study. Reconstructed Δ σ (transversal and saggittal section through the origin) for the spherical perturbations with four different regularization matrices and a SNR of 50 dB.
In all cases the two perturbations can be recognized as diffuse bright disks. The dotted circles in the figures delineate the original position of the perturbing spheres.
A number of performance indices were calculated in order to quantitatively assess the results in Figs. 6 and 7. They are summarized in Table 2 and comprise:
Mean and CNR of the pixel values in the center of gravity of each reconstructed perturbation. These parameters quantify the correctness of the reconstructed values as well as their uncertainty. The theoretically expected values are listed for comparison. The center of gravity was chosen as evaluation point because the reconstructed perturbations deviate more or less from the spherical shape and show significant outward shift with increasing noise level.Radial outward shift of the spheres in the reconstructed image (fidelity of the location). This shift was determined by localizing the center of gravity for each spot within a wedge-shaped search region with a height of 2.6 times the sphere’s radius and excluding the outermost 2 mm as well as the innermost 40 mm in radial direction from the center. The restriction of the search region to this volume prevented spurious contributions from outliers and negative image values far away from the real perturbing regions. Also for this parameter we present theoretical values as expected from the PSF.TABLE 2.Summary of the performance indices defined in the text.SNR = 64 dBSNR = 50 dBSNR = 44 dBMethodPerformance indexSphere realSphere theoretBackgroundSphere realSphere theoretBackgroundSphere realSphere theoretBackgroundIMMean central0.0210.0160.000540.0060.00430.00350.001NM0.0220.0160.000560.0060.0040.000510.00320.0010.00035VU0.0240.0240.000550.010.00650.000430.00600.00360.00035TSVD0.0330.0250.000540.010.00740.000500.01100.0020.00055IMCNR42.345.016.123.01220.1NM41.144.016.522.510.719.3VU7.98.25.65.23.43.3TSVD23.925.813.718.49.113IMResolution2.842.602.53NM2.812.582.50VU2.942.462.58TSVD2.582.422.24IMNormalized outward shift*0.100.100.210.250.330.35NM0.100.100.210.250.330.35VU0.100.060.110.150.210.15TSVD0.100.100.180.210.350.33Performance measures for the comparison of the reconstruction methods.*Normalized to the cylinder radius.
In addition Table 2 lists the theoretical resolution limits for all methods and noise levels at the position of the perturbations.
With 64 dB SNR noise the two spheres can be resolved comparatively easily with all four methods. With 50 dB SNR noise the resolution is theoretically still possible for all methods. In the reconstruction the resolution is already somewhat below the limit for IM and VU, the image values in the notch between the two peaks being around 71% of the peak values. TSVD and NM appear to separate the perturbations even worse although theoretically this should not be the case. At higher noise all algorithms tend to shift the objects towards the borders of the cylinder when looking at the parameter ‘normalized outward shift’ in Table 2. Here again the VU performs best by producing the lowest shift. At 64 dB SNR the mean images (not shown here) are in general comparatively poor. IM and NM interestingly still allow a clear separation of two objects, but their localization is very poor, the outward shift being extremely large (see Table 2). VU still provides a much better localization but only at the cost of CNR. TSVD failed to produce a clear image, an observation which was not expected from theory.
Depending on the regularization method the central voxel value of the perturbation at (− 0.6R0,0,0) decreases from 0.02–0.03 S/m to 0.002–0.006 S/m, compared to the true value of 0.1 S/m. This means that even under nearly ideal conditions (64 dB SNR) the conductivity changes are strongly underestimated. NM and TSVD yield nearly the same central voxel values as TSVD while VU produces much lower values.
Figure 8 shows single reconstructions for IM and VU at all three noise levels. Both methods allow a separation of the perturbations in all cases, but the poor CNR of VU implicates a very noisy image at 44 dB. The pronounced difference in outward shift is clearly visible at 44 dB, where VU still allows a fair localization while IM fails completely to reconstruct the perturbations at the right positions.Figure 8.Comparison of single-shot images for VU and IM at the three different noise levels.
IM and NM perform nearly identically, also their optimal regularization parameters are almost identical. VU yields, in general, larger values in the perturbed regions but also a larger STD.
Discussion
The results demonstrate the feasibility of image reconstruction from MIT-data with the same methods as suggested for EIT. This finding is not self-evident, as the sensitivity distribution is significantly different in EIT and MIT.27,29 In EIT the region of maximum sensitivity is located between the equipotential surfaces which meet the surface at the detection electrodes, i.e. within a tube-shaped region which connects injection and detection sites. As shown in27,29 in MIT the sensitivity is not concentrated within a field tube between excitation and receiver coil but increases with the distance from the tube axis, according to the increase of the eddy current density. This may be the main reason why the reconstructed solution tends to be displaced towards the nearest border of the cylindrical tank, especially at higher noise levels. An extreme case for this effect can be observed if the perturbation is placed exactly in the origin and if the senders and receivers are all in the same plane (image not shown due to space restrictions). Instead of the expected spot in the origin two widely separated spots appear on the cylinder axis close to the top and bottom of the cylinder, respectively. In fact such a coil arrangement cannot distinguish between an object in the center and two objects on the cylinder axis placed symmetrically with respect to the origin, because it is always possible to find two corresponding conductivitiy changes so that the field perturbations in the median plane are the same. Obviously, in this ambiguous situation, the algorithm favors the splitted solution according to the sensitivity distribution. A similar ambiguity occurs when using differential sensors, such as the gradiometers employed in our setup. For getting rid of such artifacts it is very important to use a less symmetric transceiver setup which provides enough spatial information in 3-D.
The theoretical resolution limit was calculated from the PSF as derived from the TSVD method. This limit depends on the chosen regularization method, the geometry of the object and on the location within the object. The respective dependences are shown in Fig. 3 and Table 2 for some selected positions inside a cylinder. TSVD was chosen for the calculation of the theoretical limits because it requires no explicit assumptions about any prior distribution of p. In this sense it contains less a-priori information than the other methods and thus describes the worst case, as confirmed by Fig. 3 and Table 2. The calculated PSF shows all basic features of the reconstructed images.
The PSF is a 3-D-distribution similar to a 3-D analog of the sinc function. This means that most of its energy is concentrated in a diffuse cloud around the considered point but that there exist three-dimensional ‘side lobes’ which decay with the distance and show some kind of ‘periodicity’. The ‘bean’-shaped artifacts which are visible in most top views of Figs. 6 and 7 are typical features of the PSF as well as the ‘star-artifact’ in the TSVD-images. Therefore these ringing artifacts do not stem from inaccuracies of the reconstruction method or measurement errors, but, instead, are inherent in the PSF.
The resolution clearly also depends on the contrast in the presence of noise, because the contrast determines the SNR. Increased noise requires more regularization and hence leads to a broadening of the PSF-distributions. Figure 3 shows the dependence of the resolution on the noise in the case of TSVD at one single contrast of 0.5 only. A more complete analysis similar to that given for EIT in30 should also show the dependence of the resolution on contrast, size and location of the perturbation at a given noise level. However, such a comprehensive analysis requires a separate paper and should not be given here.
The CNR depends strongly on the radius and, to a less extent, on the noise level. Obviously IM and NM produce very similar values, followed by TSVD. VU in general yields comparatively small CNR but higher central voxel values. For centrally placed spheres with 4 cm diameter VU yields CNRs close to the detection limit. When comparing the theoretical values with the reconstructed ones (see Table 2) the reconstruction always produces a lower CNR than expected, the discrepancy being stronger at high noise levels. One surprising detail of Fig. 5a is that at higher noise levels the CNR-curves cross the curve for 64 dB. This means that very close to the periphery noisier data yield higher CNR values than less noisy data. The reason for this counter-intuitive effect is not yet entirely clear but may be related to the strong outward shift of the PSF at higher noise. In those cases the evaluation of the CNR at the original position of the perturbation may not be appropriate any more and should be interpreted with caution.
In the case of weak perturbation we can assume that the CNR depends approximately linearly on the conductivity difference Δσ. The dependence on the volume of the perturbation is, in general, more complicated because the PSF depends on the location and is therefore not constant throughout the whole perturbation. Only in the case of small spatial extension of the perturbation an approximately linear dependence on the volume can be assumed.
The low number of significant singular values even at comparatively low noise (64 dB SNR) suggests that, similar as in EIT, a significant amount of sensor combinations does not provide enough independent information. Intuitively one would expect this finding because there exist pairs of excitation/receiving coils which nearly fulfill the reciprocity condition and hence reduce the amount of useful combinations to about half of the number of possible combinations, i.e. to 256 in our case.
Further investigations should determine the maximum ‘useful’ number of sensors in one plane, i.e. that number beyond which additional sensors do not increase the resolution significantly. Adding more sensors off-plane may add more 3-D-information and hence still provide improvement. This possibility should be studied in further research.
When comparing the regularization schemes after application of the Morozov-criterion, the IM and the NM approach yield the smoothest visual appearance and the highest CNR. However, they also tend to displace the perturbations towards the border of the tank. The best localization is obtained with VU, probably because the imposed variance counteracts somewhat the lower sensitivity in the center of the object. However, VU yields also the lowest CNR, i.e. the less homogeneous images and more pronounced ghosts. The failure of TSVD at a SNR of 44 dB was not expected theoretically, although, in general, it produces the poorest theoretical resolution. In terms of separability of the two perturbations VU performs best, especially when also taking into account the correct localization.
In neither case, however, the single-step solution provides the correct values for Δσ. Even at a SNR of 64 dB the reconstructed differences are too low by a factor of at least 5, thus demonstrating that the method yields the correct search direction but not the correct step size.
The highest mean voxel values are provided by VU and TSVD, the drop with the noise levels being lowest. However, on the other hand VU yields the highest standard deviations. Moreover VU tends to produce more pronounced ‘ghost objects’ in the homogeneous region than IM and NM. As expected from the PSF TSVD tends to produce ‘star-artifacts’ at the cylinder border, i.e. a periodic pattern with 16 peaks close to the centers of the receiving coils. This artifact gets worse at increasing noise level.
First experiments with smaller models and at least 10 iterations with an iterative solver show that the solution converges towards the correct voxel values. Nevertheless the single-step method may be completely justified in cases where only qualitative changes are sought for or where proportions are to be reconstructed, e.g. in frequency differential spectroscopic imaging. Therefore the area of applicability of a single-step approach has to be analyzed carefully in future work.
At least for the shown examples MIT appears relatively robust against Gaussian measurement noise. A SNR of 64 dB allows for a stable and distinct solution. Even 44 dB allow the recognition of the two spheres when applying the correct regularization. This result is very important for the practical implementation because, due to technical reasons, MIT is expected to yield low SNR (around 50 dB) at frequencies as low as 100 kHz, which are interesting for the imaging of pathophysiological processes.28,26 However, our results have only been achieved with two single focal perturbations with a relatively large diameter of 20% of the background object. In a more advanced study the stability and the resolution of the images should be investigated for a series of perturbations with different diameter and spacing. For the monitoring of brain edema which usually do not split up in separate sub-regions our approach may be sufficiently stable. This hypothesis has to be tested both theoretically and empirically for centrally placed perturbations (worst case).
As to the detectability of spherical perturbations in a human brain simulation results in18 have shown that a sphere with a diameter of about 40 mm, a background conductivity of 0.1 S/m a contrast of 2 yields a SNR of 24 dB at 100 kHz when applying 1 A to an excitation coil with 45 turns and a receiver coil with 1 turn. The assumed acquisition time was 200 ms, With our present technology single shot images are generated with an acquisition time of 20 ms, an excitation coil with five turns and a current up to 20 A. The receiver coils have 40 turns with otherwise unchanged geometry. This means an overall increase in SNR by 28 dB. Extrapolating the analysis given in,28 an improvement of the SNR by a factor of 5–10 is still technically possible, thus reaching 50–60 dB, which is obviously sufficient for producing fairly acceptable difference images.
Another open question is the influence of the mesh quality on the reconstruction results. We used a comparatively coarse non-uniform grid for the reconstruction. Therefore non-negligible numerical errors are to be expected which may explain the discrepancies between theoretically expected and the reconstructed values for CNR and radial displacement. Also the apparently somewhat worse spatial resolution in the reconstructed images than theoretically expected may be due to such numerical problems. The influence of the mesh and the optimization of mesh quality should be a major issue for further developments.
The results were obtained at a single frequency only. Future work should concentrate on the exploitation of the frequency dependence of the tissue conductivity and measurements at frequencies up to several MHz. A multi-frequency approach is expected to increase significantly the available information and thus the quality of the images. Possible applications may then in fact be the same as for EIT (lung function monitoring, lung edema monitoring) and hydration monitoring in the brain.

magnetic induction tomography
regularization
inverse problem
passive electrical properties
conductivity imaging


Growth charts for Wolf-Hirschhorn syndrome (0–4 years of age)

Wolf-Hirschhorn syndrome is characterized by severe growth and mental retardation, microcephaly, seizures and ‘Greek helmet’ facies, caused by partial deletion of the short arm of chromosome 4. Growth charts are given from 0–4 years of age, based on the study of 101 individuals. Use of these specific growth charts is recommended, because standard growth charts are inapplicable for patients with WHS.
Introduction
Wolf-Hirschhorn syndrome (WHS) is caused by partial deletion of the short arm of chromosome 4 (4p- syndrome) [3–5, 7]. Cardinal features of this rare syndrome are severe growth and mental retardation, microcephaly, seizures, “Greek helmet” facies and closure defects (Fig. 1) [1, 2, 4, 5, 7]. Growth in children with Wolf-Hirschhorn syndrome differs from that of normal children [1]. Up to date, there are no Wolf-Hirschhorn specific growth charts available.
Fig. 1A 7-year-old boy with Wolf-Hirschhorn syndrome
Materials and methods
The study is based on data from 1,057 examinations of 101 children with WHS, 35 males and 66 females, born between 1975 and 2002. Data from 32 children were collected from records of individuals with WHS in the Netherlands. Another set of data was obtained from 69 children with WHS from Great Britain, North America, Germany and Australia who were contacted through WHS support groups. There were no exclusion criteria except growth hormone treatment. Thus, all children regardless of complicating disease such as heart defects, renal disease or feeding problems, were included. The number of observations in the Dutch group of children was higher than in the group of children from other countries. No difference in parameters related to growth was observed between Dutch and non-Dutch patients. The data used for creation of the growth charts were age at examination, height (cm), weight (kg) and head circumference (cm). The growth charts cover the time period from birth until 4 years of age.
The data for each sex were divided into 30 different age groups, with 1-month intervals during the first 2 years of life, 3-month intervals during the 3rd and 4th years of life. Each child contributed only one single set of data for each age group. If data from more than one examination within an interval were available, the data from the first examination were used. The growth data were compared to standard growth charts for healthy children based upon the fourth Dutch growth study of 1997 [6]. As there are no growth charts for children with WHS elsewhere in the world, no comparison could be made with growth charts from children with WHS from other countries.
Data for weight and height were transformed into logarithms before statiscal analysis in order to obtain normal distributions. All growth charts are based on means and standard deviations. The software used was SPSS version 10 and Sigmaplot version 8.02 bij SPSS Inc.
Results
Figures 2 and 3 present growth charts for height for boys and girls. Mean birth length was 43.0 cm in girls and 41.5 cm in boys, corresponding to −3 SDS on growth charts for healthy Dutch children. Mean height at 4 years was 87.2 cm for girls and 85.8 cm for boys, corresponding to −4 and −4.5 SDS, respectively.
Fig. 2Growth chart for height for girlsFig. 3Growth chart for height for boys
Figures 4 and 5 represent growth charts for weight for boys and girls. Mean birth weight was 1.9 kg for girls and 2.1 kg for boys, corresponding to −3 SDS on growth charts for healthy Dutch children. Mean weight at 4 years was 9.8 kg for girls and 9.7 kg for boys, corresponding to −4 SDS and −4.5 SDS respectively. Figure 6 represents the growth chart for head circumference for boys and girls. Mean head circumference at birth was 30.5 cm for girls and boys, corresponding to −3.5 SDS for both sexes on normal growth charts. Mean head circumference at 4 years was 45.8 cm for girls and boys, corresponding to −3.5 SDS, respectively.
Fig. 4Growth chart for weight for girlsFig. 5Growth chart for weight for boysFig. 6Growth chart for head circumference for boys and girls
Discussion
Growth is used in pediatrics as a marker of health and is an important tool in the medical care for children. Severe growth retardation is one of the cardinal features of WHS [1]. It might be caused by genetic factors, poor feeding status, inherited parental factors and associated diseases. Whether patients with WHS suffer from an additional endocrine disorder is not known and is beyond the scope of this study. Up to date there are no WHS-specific growth charts available in contrast to more common chromosomal disorders like Down’s syndrome, Turner syndrome and Prader-Willi syndrome. Although the ideal manner in constructing growth charts would be a prospective, longitudinal study based on a large study group, this method cannot be implied for rare syndromes such as WHS. To increase the number of observations we combined the available longitudinal data with cross-sectional measurements. We have used data from all children with WHS, including those receiving tube-feeding. Several other associated diseases such as heart or renal disease theoretically could influence growth of the patients. Although 33% of children with WHS have congenital heart defects, these defects are in most cases not complex in nature [1]. Closure defects, hypotonia, gastro-eosphageal reflux, epilepsia, repeated infections and surgical procedures all can have a significant influence on growth. Not all patients with WHS have the same deletion size. Until now there has been no evidence of an influence of the deletion size on growth, but all studies have only involved small numbers of children. Deletion size can be expected to be of influence on growth, and therefore we tried not to include patients with a known microdeletion. However, this study inevitably includes patients with different deletion sizes, but the number of included children was too small to show an effect on growth. Based on the available data from the Dutch cohort, we could not identify a major co-morbidity risk factor aggravating growth retardation. Almost all children with WHS showed a marked intra-uterine growth retardation. Despite adequate feeding none of the children with WHS, demonstrated catch-up growth; all remained short with a profound microcephaly. The developed specific growth charts should be used for children with WHS from birth to 4 years of age for the estimation of their growth and health condition as standard growth charts are inapplicable in these patients. At present the authors are constructing growth charts for older children with WHS.

growth
growth charts
wolf-hirschhorn
whs


Chronic kidney disease after liver, cardiac, lung, heart–lung, and hematopoietic stem cell transplant

Patient survival after cardiac, liver, and hematopoietic stem cell transplant (HSCT) is improving; however, this survival is limited by substantial pretransplant and treatment-related toxicities. A major cause of morbidity and mortality after transplant is chronic kidney disease (CKD). Although the majority of CKD after transplant is attributed to the use of calcineurin inhibitors, various other conditions such as thrombotic microangiopathy, nephrotic syndrome, and focal segmental glomerulosclerosis have been described. Though the immunosuppression used for each of the transplant types, cardiac, liver and HSCT is similar, the risk factors for developing CKD and the CKD severity described in patients after transplant vary. As the indications for transplant and the long-term survival improves for these children, so will the burden of CKD. Nephrologists should be involved early in the pretransplant workup of these patients. Transplant physicians and nephrologists will need to work together to identify those patients at risk of developing CKD early to prevent its development and progression to end-stage renal disease.
Introduction
Chronic kidney disease (CKD) is a frequent and increasingly recognized complication of solid-organ and hematopoietic stem cell transplantation (HSCT), increasing the complexity of patient management and impacting survival. The incidence of CKD after cardiac, liver, and HSCT varies from 7–86% in pediatric patients, partly due to the lack of a uniform definition of CKD after transplant (Table 1). Up to 16% of transplant survivors will develop end-stage renal disease (ESRD) [1–19].
Table 1Percentage of patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) based on transplant typeTransplant typeCKDESRDReferenceLiver28–86%0–8%[1–5]Cardiac7–54%2%[6–10]Heart–lung34%7–16%[11–13]Hematopoietic stem cell transplantation18–42%5–8%[14–19]
This review focuses on the epidemiology, risk factors, and outcomes of children who develop CKD after liver, cardiac, lung, heart–lung, and stem cell transplant.
Liver transplant
Epidemiology
The majority of studies of renal disease after liver transplantation has been done in adults, and the cumulative incidence of CKD varies from 5% to 30% over 1–5 years after transplant [20]. In a retrospective study of 117 pediatric liver transplant patients who survived 3 years after transplant (median 7.6, range 3–14.6 years), the prevalence of CKD, defined as a glomerular filtration rate (GFR) <70 ml/min per 1.73 m2 at last follow-up was 32% [1]. In a 10-year follow-up study of 12 children undergoing a liver transplant in France, mild to moderate CKD (mild = GFR 60–80 ml/min per 1.73 m2; moderate = GFR 20–60 ml/min per 1.73 m2) developed in six of seven patients. Early deterioration of renal function is usually seen within the first year following transplant, followed by a period of stabilization, then with progression on long-term follow-up [2, 3]. In contrast, in a study of 50 pediatric liver transplant patients in Poland, CKD stages 2 and 3 as defined by the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF/KDOQI) (Table 2) developed at 1 year after transplant and remained stable after 3 years of follow-up [4]. In this study, GFR was calculated using both diethylenetriamine pentaacetic acid (DTPA) plasma clearance and estimated using the Schwartz formula. Little correlation was found between the two methods with the Schwartz formula, consistently overestimating GFR [4]. Therefore, the prevalence of CKD is likely underestimated in this patient population if estimated measures of GFR or serum creatinine are used to define CKD.
Table 2National Kidney Foundation Kidney Disease Outcomes Quality Initiative definition of chronic kidney disease by stageStageDescriptioneGFR (ml/min per 1.73 m2)1Kidney damage with normal or ↑ GFR≥ 902Kidney damage with mild ↓ GFR60–893Moderate ↓ GFR30–594Severe ↓ GFR15–295Kidney failure<15 or dialysiseGFR estimated glomerular filtration rate
Risk factors for developing CKD
Many patients will have renal disease at the time of liver transplant secondary to hepatorenal syndrome and/or acute tubular necrosis. In one study, renal histology pretransplant revealed glomerulosclerosis and other mild glomerular changes with mesangial matrix expansion, capillary-wall changes, and mesangial immunoglobulin (Ig) G, IgA, and IgM in the majority of cases despite normal serum creatinines [21]. Ultrasonographic findings included nephromegaly and increased echogenicity, which improved in the majority of cases after transplant [22]. In pediatric patients, there can be renal involvement from the primary liver disease, as in patients with primary hyperoxaluria, autosomal recessive polycystic kidney disease, Alagille’s syndrome, and tyrosinemia (Table 3). Renal dysfunction in patients with these disorders often improves after liver transplant. However, the long-term affects of preexisting renal disease on developing CKD after orthotopic liver transplant has not been studied systematically in pediatric patients. In contrast, in adult liver transplant patients who develop acute renal failure during the peri- and postoperative periods, there is an increased risk for developing CKD after transplant [23] and progression to CKD stage 5 requiring dialysis [24].
Table 3Liver diseases associated with preexisting renal diseaseTyrosinemiaAutosomal recessive polycystic kidney diseaseAlagille’s syndromePrimary hyperoxaluriaHepatitis B- and C-related glomerulonephritis
Other identified risk factors (Table 4) for CKD include GFR <70 ml/min per 1.73 m2 at 1 year after transplant and cyclosporine use [1]. In this study, there was an inverse relationship between hypertension at 1 year after transplant and development of renal dysfunction. In addition, in adult patients, preexisting diabetes, pre- and postoperative renal failure, hypertension, age, female gender, and hepatitis C infection increased the risk of CKD and ESRD [20]. Though the majority of renal dysfunction after liver transplant is attributed to the use of cyclosporine and/or tacrolimus, there have been reports of other types of pathology present leading to CKD. Studies have found preexisting renal disease such as focal segmental glomerulosclerosis (FSGS) and hepatitis-C-related injury including membranoproliferative glomerulonephritis, unresolved hepatorenal syndrome, and diabetic nephropathy on renal biopsy after liver transplant [25, 26].
Table 4Risk factors for developing chronic kidney disease by type of transplantRisk factor common to all transplant typesCalcineurin inhibitor useLiverGFR of <70 ml/min per 1.73 m2 at 1 year after transplantPretransplant renal dysfunctionAcute renal failurePreexisting diabetesAgeFemale genderHepatitis CCardiacPretransplant dialysisHypertrophic cardiomyopathyAfrican American racePrevious transplantPretransplant diabetesExtracorporeal membrane oxygenation useHeart–lungHypertension posttransplantElevated serum creatinine at 1 month posttransplantHematopoietic stem cell transplantAcute graft-versus-host disease grades II–IVOlder ageTransplant from an unrelated donorAcute renal failureChronic graft vs. host diseaseTotal body irradiation
Calcineurin inhibitors
The use of cyclosporine (CSA) and tacrolimus in managing liver transplant patients has greatly improved outcomes. However, the improvement in survival is associated with an increased development of CKD. The manifestations of CSA toxicity range from asymptomatic azotemia and proteinuria to fulminant multiorgan failure [27]. In addition to the commonly described striped fibrosis and arterial and vascular lesions seen in patients on these medications, thrombotic microangiopathy has also been described in as many as 50% of patients after liver transplant [25]. These lesions are typically characterized by mesangiolysis, thrombus formation within glomerular capillaries, and widening of the subendothelial spaces. The nephrotoxic effects of CSA correlate with drug serum levels and therapy duration. CSA is also known to cause arteriolar injury, glomerulosclerosis, and interstitial fibrosis, as well as diffuse expansion of the mesangial matrix [28, 29]. The hypothesized mechanisms behind this injury appears to be vasoconstriction secondary to an imbalance between the vasodilatory hormones such as prostaglandin E1 and vasoconstrictive ones such as thromboxane A2 [30]. Increased synthesis of transforming growth factor (TGF)-β1 by calcineurin inhibitors also contributes to the development of CKD in patients after transplant, and genetic polymorphisms in the TGF-β1 gene have been associated with the development of ESRD after cardiac transplant [31].
Reducing calcineurin inhibitor levels while adding mycophenolate mofetil (MMF) in patients with CKD 5 years after liver transplant resulted in renal function improvements as measured by serum creatinine and creatinine clearance after 24 months of combined therapy, with only minor changes noted in immune function in these patients. Decreases in microalbuminuria were also noted[32]. Some authors have suggested that thrice-daily dosing of cyclosporine leads to decreased nephrotoxicity and only mild histopathologic changes in the kidney after 3 years of follow-up [33]. The authors attribute their findings to more constant trough blood levels and lower, less toxic, peak levels as well as the use of a calcium-channel blockers to manage their patients’ hypertension [33]. In a case series, three children who underwent combined liver and kidney transplant were switched to sirolimus because of prolonged renal failure requiring dialysis and acute calcineurin inhibitor toxicity on biopsy. All three demonstrated improved renal function with cessation of dialysis [34]. These studies suggest that decreasing calcineurin inhibitor exposure by adding MMF or eliminating long-term exposure by switching to sirolimus can have beneficial effects on renal function in children after liver transplantation.
Cardiac transplantation
Epidemiology
Approximately 350 pediatric cardiac transplants are performed annually in the USA [7]. In a 2006 report from the International Society for Heart and Lung Transplantation, the incidence of renal dysfunction (defined as an abnormal serum creatinine) was 10% at 5 years after cardiac transplant [6]. Sixty-three percent of patients had hypertension. After 8 years of follow-up, the percentage of patient with renal dysfunction did not change, but 2% of patients were on dialysis or had received a renal transplant. In reports from single centers, the incidence of CKD at approximately 10 years after cardiac transplant in pediatric patients ranged from 7% to 54%; however, only a small percentage of these patients progress to ESRD requiring dialysis and/or renal transplantation [7–10]. Hypertension is a more common finding, occurring in approximately 70% of patients after transplant [7–10]. Some centers actually report an improvement in kidney function in the first year following cardiac transplant and stabilization thereafter [9]. In adult studies, there is an increased risk of mortality associated with development of CKD [35, 36].
Risk factors for development of CKD after cardiac transplant
A recent study of 2,032 pediatric cardiac transplant patients transplanted between 1990 and 1999 identified pretransplant dialysis, hypertrophic cardiomyopathy, African American race, and previous transplant as risk factors for developing CKD (defined in this study as a creatinine >2.5 mg/dl) [37]. Additional risk factors for developing ESRD included pretransplant diabetes and intensive care unit stay or extracorporeal membrane oxygenation [37]. In the adult population, several risk factors for CKD development have been identified and include older age at transplant, pretransplant serum creatinine, preexisting diabetes, abnormal GFR at 1 year after transplant, hypertension after transplant, and cyclosporine immunosuppression within the first 6 months after transplant [12, 35, 36, 38, 39]. Risk factors for progression to ESRD include postoperative development of hypertension and proteinuria of >1 g/24 h [13, 40].
Calcineurin inhibitors
Though the nephrotoxic effects of calcineurin inhibitors are well known, data in the pediatric cardiac transplant population are conflicting. After 18 months of triple immunosuppression including cyclosporine, patients’ GFR remained stable, and renal biopsy specimens in four patients did not show signs of cyclosporine toxicity [41]. Minor abnormalities in tubular function resulting in hyperuricemia and hyperkalemia were reported. Similarly, in a study of adult cardiac transplant patients treated with cyclosporine for 5 years, GFR (measured by inulin clearance) remained stable at 66 ml/min per 1.73 m2, as did tubular function. The only abnormalities noted were hypertension and the presence of microalbuminuria. However, other studies of pediatric cardiac transplant patients found an association between cyclosporine use within the first 2 months after transplant and decreases in GFR years after transplant [42]. High cyclosporine levels (>500 μg/L) in the first 6 months after cardiac transplant were associated with developing ESRD at anytime after cardiac transplant [43]. In a pediatric study of 14 patients with progressive decline in renal function over 2–5 years, inulin clearance declined from 84 ml/min per 1.73 m2 at 1 year to 49.8 ml/min per 1.73 m2 at 5 years. Biopsies performed on 13 patients revealed chronic tubulointerstitial lesions of grade II associated with acute changes of vacuolization of the proximal tubules. Arteriolar lesions were also present, along with focal glomerular scarring and fibrosis. Lesion extent correlated with calcineurin therapy duration. After reduction in calcineurin inhibitor dose by 50% and change from azathioprine to MMF, a 67% improvement in GFR (77 ml/min per 1.73 m2) was noted 1 year after the change. The authors suggest that this improvement may be related to a decrease in preglomerular vasoconstriction [44]. In a study of pediatric patients treated with tacrolimus after transplant, 41% of patients had elevations in serum creatinine of 1–2 mg/dl approximately 2–3 years after transplant [45].
Heart–lung and lung transplantation
Epidemiology
The cumulative incidence of CKD, defined as a doubling of serum creatinine, after lung or heart–lung transplantation varies from 34% at 1 year after transplant to 53% by 5 years after transplant [11]. ESRD occurs in 7.3–16% of patients 5 years after transplant [11–13]. The majority of patients, 51%, developed CKD stage 3 by 1 year posttransplant [12].
Risk factors for CKD after heart–lung and lung transplant
Risk factors identified for developing CKD in a study of 219 patients undergoing a lung or a heart–lung transplant include cumulative periods of a diastolic blood pressure >90 mmHg and serum creatinine value at 1 month posttransplant [11]. In this same study, the authors found that tacrolimus use in the first 6 months posttransplant decreased the risk of CKD compared with those who received cyclosporine alone. Other studies found GFR at 1 month to be a predictor of later CKD [12]. Development of postoperative hypertension is associated with an increased risk of ESRD after lung and heart–lung transplants [13].
Hematopoietic stem cell transplant
Epidemiology
The cumulative incidence of CKD varies from 13% to 60% in adult studies [15–17] to as high as 62% in children [14]. CKD usually becomes apparent 6–12 months after HSCT, although it has been described as early as 2 months and as late as 10 years posttransplant. Though this section discusses injury that occurs after HSCT, baseline or pretransplant renal function can impact the results [46]. Therefore, baseline assessments not only of serum creatinine but also urinalyses and more formal estimations of GFR are warranted. Accurate assessment of baseline renal function can help guide later medication dosing.
There are three distinct clinical manifestations of renal disease that can occur in the HSCT patient: thrombotic microangiopathy (TMA), typically hemolytic uremic syndrome (HUS), graft-versus-host-disease (GVHD)-related CKD, and nephrotic syndrome (NS). TMA syndromes represent a spectrum of clinical diseases characterized by systemic or intrarenal platelet aggregation, thrombocytopenia, and microvascular fragmentation of erythrocytes. Platelet aggregation can result in ischemia and organ injury. When renal injury is predominant, a diagnosis of HUS is usually rendered, whereas the presence of extensive extrarenal manifestations leads to a diagnosis of thrombotic thrombocytopenic purpura (TTP) (reviewed in [47]).
Histopathology of TMA after HSCT
Microscopic examination of kidney biopsy specimens from patients with TMA-associated CKD demonstrates mesangiolysis and loss of endothelial cells with expansion of the subendothelium and occlusion of capillary loops. On electron micrographs, there is extensive widening of the space between the glomerular basement membrane and the subendothelium, with amorphous deposits that are not immune complexes.
Risk factors for HUS after HSCT
Though no clear relationships have been found to date for the development of TMA after HSCT, a number of risk factors have been examined. In earlier studies, where HUS was the primary diagnosis, risk factors identified were total body irradiation (TBI) [14, 15, 48, 49] and calcineurin inhibitor use [15, 27, 50–54]. However, in more recent studies of TMA, acute graft-versus-host disease (GVHD) grades 2–4, older age, and transplant from an unrelated donor are the primary risk factors identified [55, 56]. Other investigators identified sinusoidal obstruction syndrome, matched unrelated donors or haploidentical donors, and lymphoid malignancy as significant predictors of TMA after HSCT in addition to the above risk factors [17, 57–59]. However, in children who develop HUS after HSCT, the presumptive risk factor in these studies was TBI used as part of the conditioning regimen. For example, Tarbell et al. studied 44 children (aged 3–15 years) with acute lymphocytic leukemia (ALL) or neuroblastoma (NB) who underwent HSCT [14]. Twenty-nine of these patients were alive and in remission 3 months after HSCT and were evaluated in the study. Eleven patients developed increases in blood urea nitrogen (BUN) and creatinine, and ten were anemic and thrombocytopenic with evidence of hemolysis on peripheral blood smear; they also had elevated lactate dehydrogenase (LDH) levels. In every patient except one, the hemolytic process resolved, yet renal insufficiency persisted. The pathologic findings of mesangiolysis with intraglomerular capillary aneurysm formation in conjunction with laboratory abnormalities support the diagnosis of HUS in these children. In another small study, Antignac et al. described seven children referred to their nephrology clinic with CKD approximately 5–10 months after TBI followed by HSCT [48]. All seven children had leukemia, and all received cyclophosphamide alone or with cytosine arabinoside and vepeside, in addition to single-dose TBI as part of their conditioning regimen. Three patients developed CKD without hypertension, and four developed HUS with severe hypertension and microangiopathic hemolytic anemia. Of these, two of the four had normalization of their renal function. Follow-up biopsies, however, showed extensive scarring of the renal parenchyma but almost complete resolution of the mesangiolysis. The glomeruli were globally sclerotic, ischemic, or demonstrated mesangial hypercellularity. Thus, there was evidence of persistent and progressive renal damage in these patients despite normalization of serum creatinine and urinalysis. The occurrence of two different clinical presentations, CKD without hypertension and HUS with hypertension, but similar pathology in these children supports the notion that this is a spectrum of disease rather than distinct pathophysiologic processes.
GVHD-related CKD
GVHD-related CKD in this patient population is usually defined as an elevated serum creatinine or an abnormal GFR 6–12 months after transplant. The incidence of GVHD-related CKD in children after HSCT varies from 11% to 41% [60–63]. In one recent study, the incidence of CKD (GFR <70 ml/min per 1.73 m2) changed over time, with 41% of children having CKD at 1 year, 31% at 3 years, and only 11% 7 years after transplant [63]. In approximately 19% of patients, hematuria and proteinuria persisted up to 10 years after HSCT. Berg and Bolme followed 44 children with acute lymphocytic leukemia (ALL), acute myeloblastic leukemia (AML), and severe aplastic anemia (SAA) and found a significant decrease in GFR 1–2 years after HSCT when compared with their baseline GFR (ALL and AML groups) or with a healthy control group despite serum creatinines that remained within normal limits. An initial decrease in GFR was followed by stabilization up to 5 years posttransplant [64]. This study supports the contention that serum creatinine is not an accurate measure of kidney function. Serum creatinine level and related estimating equations, routinely used clinical measures to estimate kidney function, are dependent on muscle mass and are influenced by age, race, gender, and weight [65, 66]. Patients undergoing HSCT may have large fluctuations in their nutritional status, muscle mass, and weight that will influence GFR based on estimation equations or serum creatinine levels. Proximal tubular dysfunction has also been described in 14–45% of pediatric patients 1–2 years after HSCT, with initial injury to the proximal tubules being nonspecific as reflected by elevated urinary excretion of alpha-1 microglobulin and beta-N-acetylglucosaminidase (β-NAG) followed by more specific damage manifested by decreases in phosphate reabsorption [67].
Risk factors for GVHD-related CKD
The risk factors for GVHD-related CKD in children are similar to those identified in adult studies. Kist-van Holthe et al. also retrospectively identified risk factors for developing both acute and chronic renal insufficiency in a cohort of 142 children undergoing transplant over a period of 5 years in the Netherlands [68]. All children received allogeneic transplants. Ninety-one children received radiation, and 82 of these 91 received TBI. Twenty-five children (18%) had CKD (defined as a GFR <85 ml/min per 1.73 m2). These authors found no correlation between radiation dose used and renal insufficiency at 1 year. In a later study from the same group, only acute renal insufficiency predicted the later development of CKD in patients after HSCT [62]. These studies contradict others in the literature that found TBI to be associated with renal injury [60, 61, 69]. However, the doses used here (5–8 Gy in a single fraction) were much lower than described elsewhere. In a study of 92 pediatric HSCT patients by van Why et al., late renal insufficiency developed in 18 of 64 (28%) patients; in half of these patients, the renal disease persisted for 3 months to 3 years [60]. Amphotericin B use, cyclosporine, and TBI were associated with the later development of CKD. In a large retrospective review of 1,635 children and adults, risk factors for developing CKD after HSCT included acute renal failure and acute and chronic GVHD [17]. In this study, TBI was not associated with development of CKD.
Nephrotic syndrome (NS) after HSCT
Chronic GVHD may manifest itself in the kidney as NS with or without renal insufficiency (reviewed in [70]). Patients usually present with proteinuria, edema, and hypoalbuminemia. The majority of these case reports demonstrate membranous nephropathy (MN) with subepithelial deposits on biopsy; it is postulated that these deposits are antigen/antibody complexes representing GVHD in the kidney. However, cases of minimal-change disease (MCD), which is thought to be a T-cell-mediated process, have also been described [70]. Comparisons between case reports of MN and MCD after HSCT found that MN occurs in 61% of cases compared with 22% of cases having MCD [71]. The majority of reported patients with MN were slightly older males with a history of acute and chronic GVHD. Both MCD and MN occur later after transplant, at 8 and 14 months, respectively, and tend to occur within 1–5 months of GVHD development and/or the tapering of immunosuppression for their chronic GVHD. MN is more difficult to treat, with only 27% of patients reportedly achieving remission compared with 90% of patients with MCD [71]. Others have reported cases of diffuse proliferative glomerulonephritis, anti-nuclear-cytoplasmic-antibody (ANCA)-related glomerulonephritis, focal segmental glomerulosclerosis, and IgA nephropathy [72–76] occurring after HSCT. The development of each of these diseases seems to be associated with chronic GVHD and/or immunosuppression tapering. Treatment with high-dose prednisone and/or reinstitution of calcineurin inhibitors usually results in resolution of NS. Some physicians have used rituximab successfully in patients with NS after HSCT, typically in cases of MN [77].
Management of CKD after liver, cardiac, lung, and HSCT
Patients who develop CKD after liver, cardiac, lung, and hematopoietic stem cell transplants are at increased risk of mortality [18, 26, 78]. Transplant physicians and nephrologists should work together starting from the time prior to transplant to monitor these patients closely. More accurate measures of kidney function are needed, and baseline and follow-up iohexol or iothalamate studies to measure GFR may be indicated to identify patients with underlying CKD prior to transplant and to allow early identification and intervention in patients with mild changes in GFR posttransplant. New markers to estimate GFR, such as cystatin C, may be more informative in certain patient populations than is serum creatinine. Cystatin C is a cysteine protease inhibitor expressed by all nucleated cells and is freely filtered by the glomerulus. Serum cystatin C correlates well with measured GFR and more accurately measures kidney function than does serum creatinine in the elderly, cancer patients, diabetics, and renal transplant recipients [79–83]. Normal ranges for cystatin C have been validated in children. The normal reference range for children older than 1 year is 0.7–1.38 mg/dl [84]. An equation to estimate GFR based on cystatin C levels in children has also been created: log (GFR) = 1.962 + [1.123*log(1/cystatin C)] [85]. Urinalyses including a microalbumin to creatinine ratio should also be part of the pretransplant workup, and urinalyses should be monitored closely following transplant.
Animal models of HSCT, and specifically of radiation-induced HUS, offer potential interventions for patients with HUS after HSCT. Angiotensin-converting enzyme inhibitors (ACEI) have been used in rodent models of HSCT-related renal injury. The use of captopril or enalapril at the time of TBI in these animals resulted in less azotemia, lower blood pressures, decreased proteinuria, and long-term preservation of renal function [86]. ACEI and angiotensin receptor blockers (ARBs) also help reduce inflammation and inflammatory markers and reduce circulating levels of TGF-β1 in patients after transplant [87–90]. These agents have also been shown to slow CKD progression and decrease proteinuria in patients with renal disease from various causes [91, 92].
Hypertension management early after transplant is important to prevent CKD development and progression to ESRD in certain transplant populations. ARBs and ACEIs have been shown to be effective and safe in managing hypertension in cardiac transplant patients [93], and ACEIs have been shown to stabilize renal function over 2 years of follow-up [94]. These drugs should be considered as first-line agents to manage hypertension in patients after transplant. In addition, hyperlipidemia management may be important to prevent CKD after transplant. In a study of adult patients undergoing a cardiac transplant, statin use was associated with a decreased risk of development of CKD after transplant [78]. The difficult decision is when to intervene (Fig. 1). Should patients be started on these medications prior to transplant, at the first signs of hypertension and microalbuminuria, or at some set time point after transplant to help protect their kidneys? These decisions need to be made on an individual basis and will vary based on the type of transplant. Frequent discussions between the transplant physician and nephrologist are required to optimize the management of patients with CKD after transplant.
Fig. 1Timing of intervention. Bold numbers are estimated glomerular filtration rate based on the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines. Adapted with permission from [102]
In addition to the above medications, immunosuppression adjustment is important in managing the CKD that develops after transplant, further supporting the need for close collaboration between transplant physicians and nephrologists. In two reported series of lung transplant recipients, sirolimus was used to decrease and eventually stop calcineurin inhibitors in patients with CKD after transplant. Patients had a sustained improvement in kidney function as reflected by a decrease in serum creatinine for 1–12 months [95, 96]. However, prior to changes in immunosuppression, a kidney biopsy may be indicated to define the etiology of the kidney injury and better tailor therapy to prevent or slow disease progression.
Management of patients with ESRD after transplant include the use of peritoneal and/or hemodialysis. Caring for these patients also involves management of complications associated with ESRD, which include anemia, bone disease, hypertension, and metabolic abnormalities. There have been case series of patients undergoing renal transplantation successfully after cardiac, liver or HSCT; renal transplantation is a viable option for patients with ESRD after transplant and can improve outcomes [19, 97–101].
Questions
(Answers appear following the reference list)
Which of the following are risk factors for developing CKD after cardiac, liver, and HSCT?
Calcineurin inhibitorsTotal body irradiationAcute renal failureHypertensionAll of the following are manifestations of CKD seen in patients after HSCT except:
Nephrotic syndromeAcute glomerulonephritisThrombotic microangiopathyGVHD-related CKDWhat pretransplant factors increase the risk of CKD in pediatric cardiac and liver transplant patients?
Preexisting renal diseaseRacePretransplant diabetesAll of the aboveManagement of pediatric patients who develop CKD after transplant should include all of the following except:
Reducing exposure to calcineurin inhibitorsACEI and/or ARBCalcium-channel blockersStatin useWhat is the most common cause for developing TMA after transplant in patients receiving a heart, liver, or stem cell transplant?
Graft-versus-host diseaseTotal body irradiationCalcineurin inhibitor useDiabetes
Answers:
a and c
b
d
c
c

chronic kidney disease
hematopoietic stem cell transplant
calcineurin inhibitors
risk factors
epidemiology
liver transplant
cardiac transplant
lung transplant


The influence of thermo-chemotherapy on bladder tumours: an immunohistochemical analysis

To study the influence of microwave induced thermo-chemotherapy on high-grade urothelial cell carcinomas. Five groups of each three patients were formed of whom initial biopsies and cystectomy samples were collected. Patients were treated 2 days prior to cystectomy with mitomycin-C (group 1), hyperthermia (group 2) or thermo-chemotherapy (group 3). Group 4 patients had been treated with a cycle of six thermo-chemotherapy treatments prior to cystectomy and group 5 patients served as control (no treatment). Tumour samples were stained with Haematoxylin and Eosin, monoclonal antibody Ki-67 and the monoclonal antibody p53. In six out of the nine patients treated with hyperthermia a decrease in proliferation activity in the tumour was found. Seven out of nine patients treated with hyperthermia showed a decrease in p53 activity. A decrease in proliferation activity and p53 activity illustrate the potential role of thermo-chemotherapy as a promising intravesical treatment.
Introduction
Bladder cancer is the seventh most common cancer worldwide, accounting for 3.2% of all cancers. In 2000 an estimated 260,000 new cases in men and 76,000 in women were found worldwide [5]. The highest incidence rates of bladder cancer in both sexes are observed in Europe, North America and Australia [12]. Bladder cancer incidence is still rising moderately in most developed countries. Of all these malignant bladder tumours, more than 90% are transitional cell carcinomas of which two third is superficial and one third is muscle invasive. The management of these two types of transitional cell carcinoma differs enormously. The management of superficial bladder cancer consists of transurethral resection in the first place, whereas the primary treatment for muscle invasive bladder cancer consists of cystectomy, a complete different approach.
After transurethral resection of a superficial bladder tumour 30–85% of patients develop recurrences, despite the most accurate resection technique. This high risk of recurrences makes bladder cancer one of the most prevalent human tumours. To decrease the number of recurrences after transurethral resection, patients are treated subsequently with chemotherapeutic or immunotherapeutic agents. Immunotherapy, usually BCG treatment, is more effective than any intravesical chemotherapeutic agent, but has more serious and more frequent side effects. So, there is a need for more effective treatment options or treatment options with less frequent and less severe side effects. A new treatment option in patients with intermediate to high risk tumours is the combination of intravesical hyperthermia and intravesical chemotherapy [2, 6, 15].
The endocavitary location and ease of accessibility by the urethra makes thermo-chemotherapy a good therapeutic option for superficial bladder cancer. Local hyperthermia at temperatures of 40–44°C in combination with selected cytostatic agents in several tumours, including transitional cell carcinoma, results in a synergistic anti-tumour effect [10].
In this descriptive study the influence of different treatment modalities on bladder tumours (thermo-chemotherapy, chemotherapy and thermotherapy) was investigated by several immunohistochemical stainings.
Methods
Subjects
The study included 15 patients with high-grade transitional cell carcinoma of the bladder. All patients signed a patient informed consent and all these patients were facing cystectomy. The 15 patients were divided in five groups with each three patients. Group 1 patients were treated 2 days before cystectomy with an intravesical mitomycin-C (MMC) instillation. After the bladder was emptied, 20 mg MMC (Kyowa Hakko Kogyo Co., Tokyo, Japan) in 50 ml saline was instilled. In order to stabilize the MMC concentration in the bladder throughout the entire session, the bladder was emptied after 30 min and with urine diluted solution was replaced by a new solution containing 20 mg MMC. Group 2 patients were treated 2 days before cystectomy with sterile water and local hyperthermia. To standardize treatments the instillation was replaced after 30 min. The local microwave induced hyperthermia was delivered by the SB-TS 101 system as described by Colombo et al. [3]. This system consists of a 915 MHz intravesical microwave applicator that delivers hyperthermia of the bladder walls via direct irradiation. The applicator is part of the specially designed 20F transurethral catheter. The catheter also contains five thermocouples. Two thermocouples measure the temperature in the prostatic urethral tract; the other three are spread out and pushed tangentially against the posterior and lateral walls of the bladder. To avoid urethral overheating, the solution is continuously pumped out of the bladder and re-instilled after being cooled. Hyperthermia was delivered within a temperature range of 41–42°C.
Group 3 patients were treated 2 days before cystectomy with two times 30 min intravesical MMC (20 mg in 50 ml sterile water) combined with local microwave induced hyperthermia delivered by the SB-TS 101 system. Group 4 patients were treated with a cycle of six thermo-chemotherapy treatments during the last 3 months. Finally, group 5 patients served as control group. These patients did not receive any intravesical instillations within 3 months prior to cystectomy.
Immunohistochemistry
All tumour tissue samples were fixed in a 10% buffered formaldehyde solution. The specimens were embedded in paraffin blocks and sections of 4 μm were cut. All specimens were deparaffinized and stained with Haematoxylin and Eosin. A microscopic examination of the samples was performed and the extend of inflammation (1+ to 3+) haemorrhage (1+ to 3+) were semi quantitatively scored.
Ki-67, a nuclear protein present during phases G1, S, G2 and M of cycling cells, is accepted as a good indicator of cell proliferation [11]. Sections were deparaffinized and immersed in a 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for two times 5 min to enhance antigen retrieval. After washing, the slides were incubated with 0.3% H2O2 in methanol to quench endogenous peroxidase activity. After incubation at room temperature for 2 h with the anti-Ki67 monoclonal antibody clone MIB-1 (BioGenex) diluted 1:30, a biotinylated antibody that recognises murine IgG (BioGenex) was applied for 20 min, followed by incubation in streptavidin-peroxidase complex (BioGenex) for 20 min. The peroxidase reaction was developed using 0.5 mg/ml diaminobenzidine tetrahydrochloride (Sigma) in 0.01% H2O2. Haematoxylin was used as a light counter stain. The slides were dehydrated and mounted with a xylene-soluble mounting medium.
The mutated p53 tumour suppressor gene product can be detected immunohistochemically and is associated with a lower survival in patients with bladder cancer [7]. Sections were deparaffinized and immersed in a 10 mM sodium citrate buffer (pH 6.0) in a microwave oven for two times 5 min to enhance antigen retrieval. After cooling down, 0.6% H2O2 in a 40% methanol solution was used for 30 min to block endogenous peroxidase activity. Slides were pre-treated with 10% normal swine serum for 10 min to block non-specific staining followed by adding primary antibody DO-7 for 1 h at 20°C. After extensive rinsing, sections were incubated for 30 min with biotinylated swine anti mouse antibody (1:200 dilution) and then for 30 min with avidin-biotin complexes (1:50 dilution) at 20°C. Diaminobenzidine (DAB) staining were finally followed by haematoxylin nuclear counter staining. The slides were dehydrated and mounted with a xylene-soluble mounting medium.
Immunoreactivity scoring
The screening of tumour tissue samples was performed by two independent investigators (CH and AH). The sections were screened for positive cells, defined as cells with nuclear staining. The amount of Ki67 or p53 staining is scored in percentages. The threshold for p53 “positivity” is ≥20% positive staining. The areas with maximal immunohistochemical staining were used for scoring. In total, 300–500 tumour cells were scored. In the visual estimation only definitely brown nuclei were recorded as positive. The results were expressed as percentage of immunoreactive tumour cell nuclei.
Results
Fifteen patients (11 males, 4 females) signed a patient informed consent and participated in this study. Ages ranged from 42 to 75 years (mean 63.2 years). All patients underwent cystectomy for transitional cell carcinoma of the bladder. Eight patients had a de novo invasive bladder tumour, whereas seven patients had a history of high-risk superficial disease. Patient details are summarized in Table 1.
Table 1Patient details containing number of previous occurrences, interval period between initial diagnosis and cystectomy in months, pathology data of biopsy and cystectomy (grading according to WHO 2002)GroupsPatient no/age/sexN previous occurrencesInterval biopsy–cystectomy (in months)Histopathology initial biopsyHistopathology cystectomy1 (MMC)1/75/M02,0≥pT2aGIIIpT3aGIIIN22/46/F04,4pT2GIIpT3bGIIIN13/67/F01,2≥pT2GIII≥pT2GIII 2 (HT)4/64/F01,9pT1GIIIpT2aGIII5/71/M338,7pT1GIII + CISpT1GIII + CIS6/42/M10121,9pTaGIIpTaGII3 (MMC + HT)7/55/M02,6pT1GIII≥pT2GIII8/67/M01,9pT2GIIIpT3aGIIIN19/48/F13,3pT1GIIpT2bGIIIN14 (History of MMC + HT)10/54/M18107,5pTaGIIpTaGII11/73/M222,3CISpT4aGIIIN212/75/M1057,7pTaGIIpTaGII5 (Control)13/71/M9115,5CISpT2GIII14/71/M03,1pT2GIIIpT2GIII15/71/M03,3pT2GIIIpT2GIIIN1HT microwave induced hyperthermia
The initial biopsies and tumour tissues obtained with cystectomy were used for histopathological diagnosis and immunohistochemical analysis of Ki-67 and p53. The results from the different treatment groups are summarized in Table 2. In one patient of the MMC group no residual tumour could be retrieved in the cystectomy specimen. The proliferation activity and p53 activity could not be scored in that patient.
Table 2The p53 and Ki67 immunoreactivity scorings in percentages of all patients divided in five different treatment groupsGroupsMaterialInflammationProliferation (%)P53 (%)Haemorrhage1 (MMC) aBiopsy+++3020+Cystectomy+++7520++ bBiopsy++2075−Cystectomy+++1575+ cBiopsy+++4075+++CystectomyNANANANA2 (HT) aBiopsy+6080−Cystectomy+++2040++ bBiopsy+3090−Cystectomy++2040− cBiopsy+10-−Cystectomy++10-+3 (MMC + HT) aBiopsy+++40>75−Cystectomy+++2575− bBiopsy+++>75>75+Cystectomy+++6015− cBiopsy+++30>75+Cystectomy+++1575++4 (History of MMC + HT) aBiopsy+1075−Cystectomy+20−−  bBiopsy+++3025−Cystectomy+++3025− cBiopsy++5020−Cystectomy++2010+5 (Control) aBiopsy+15>75−Cystectomy+++40>75− bBiopsy++4020−Cystectomy++4020− cBiopsy++3575+Cystectomy+++3575−The extend of inflammation and haemorrhage is semi quantitatively scored (1+ to 3+)NA not applicable due to absence of residual tumour
The intensity of inflammation increased in three out of nine patients treated with hyperthermia. In three out of five patients from the MMC group and control group an increase was seen. The intensity of haemorrhage increased in four out of nine patients treated with hyperthermia, one patient showed a decrease and four patients did not show any difference. In the MMC group and in the control group two patients showed an increase, one patient a decrease and two patients did not show any difference.
In six out of the nine patients treated with hyperthermia a decrease in proliferation activity in the tumour tissue sample was found (Fig. 1a). In the MMC group one patient showed a decrease and one showed an increase in proliferation activity. In the control group two out of three patients showed no changes, whereas in one patient an increase in proliferation activity was seen.
Fig. 1The p53 and Ki67 positive staining in patient no. 4 (hyperthermia group). a1 shows 60% Ki67 positivity before treatment; a2 shows 20% Ki67 positivity after treatment; b1 shows 80% p53 positivity before treatment and b2 shows 40% p53 positivity after treatment
As regards p53, seven out of nine patients treated with hyperthermia showed a decrease in p53 activity (Fig. 1b). In one patient no change was found. The control group and the group treated with MMC did not show any differences concerning p53.
Discussion
Thermo-chemotherapy has shown to be a promising method for treating several kinds of malignant tumours including superficial bladder cancer [15]. While hyperthermia is important in cancer therapy, it can also damage normal tissues adjacent to the tumour. Fajardo [4] reviewed the effects of hyperthermia from several studies on various tissues of mammals and humans. Hyperthermia with a maximum applied temperature of 44.5°C showed no gross or microscopic alterations of the bladder of dogs and rabbits. Subsequently, Rath-Wolfson et al. [13] studied the effect of hyperthermia, with a maximum applied temperature of 46°C, and simultaneous MMC treatment in sheep. In this study thermo-chemotherapy showed no significant macroscopic or microscopic differences in the bladder wall as compared to a control group with untreated sheep.
In the current study the effect of the combination thermo-chemotherapy is compared with solely hyperthermia, solely chemotherapy and no treatment (control group) in human patients with high-grade urothelial cell carcinoma of the bladder. The system SB-TS 101 used to deliver local microwave induced hyperthermia makes it possible to dose the hyperthermia very precisely [1]. In this study hyperthermia was delivered within a temperature range of 41–42°C, the clinical situation. This makes comparison between the different groups more accurate.
The sample size of the group studied is small due to the fact that recruitment of patients was difficult. Patients had to agree on an extra treatment session only two days before cystectomy, which did not give them any benefit at all.
The degree of inflammation increased in the group treated with solely hyperthermia (N = 3), solely MMC (N = 1) and the control group (N = 2). In the group treated with thermo-chemotherapy 2 days before surgery the degree of inflammation was initially already maximal. In the group treated with a cycle of six thermo-chemotherapy treatments during the last 3 months the degree of inflammation apparently had returned to baseline again.
At least one patient per group (except the control group) showed an increase in the degree of haemorrhage. Patients from group 1 (solely MMC) showed the highest degree of haemorrhage.
Group 3 patients (treated with thermo-chemotherapy prior to cystectomy) show in one patient an increase, in one patient a decrease and in one patient a stable extend of haemorrhage. This is in line with the results from Rath-Wolfson et al. [13]. Nevertheless, since solely MMC and solely hyperthermia gives an increase in haemorrhage, it would have been logical to find a higher extend of haemorrhage after the combination of both treatments. Possibly, this is not found due to the small sample size.
The inhibition of proliferation activity is one of the most important goals in cancer treatment. All groups treated with hyperthermia showed to a different extend a decrease in proliferation activity. Most interesting are the three patients treated with thermo-chemotherapy preceding transurethral resection. All three patients showed a decrease in proliferation activity. The control group on the other hand shows in two patients no difference and in one patient an increase in proliferation activity. In earlier in vitro studies using hyperthermia and bladder cancer cell lines, this decrease in cell proliferation due to hyperthermia combined with chemotherapeutic agents was already shown [14, 16]. However, in these in vitro studies solely hyperthermia did not cause a significant decrease in cell proliferation. Nevertheless, there is a difference between in vitro and in vivo studies. This in vivo study is the first one showing a decreased proliferation activity in humans treated with microwave-induced hyperthermia with or without MMC. The effect of hyperthermia was seen in six out of nine patients treated with hyperthermia preceding cystectomy.
Furthermore, the group treated with solely MMC did not show a significant decrease in proliferation activity. This is probably due to the limited penetration properties of this intravesical used drug, especially after one single treatment.
p53 is known to be responsible for repair or apoptosis in response to DNA damage. The p53 activity, in other words the expression of mutant p53, decreased exclusively in patients treated with hyperthermia with or without MMC.
Previous experiments showed that the p53 pathway is heat sensitive and that the p53 protein is inactivated at temperatures above 41°C [8, 9]. When cells were heated to 42.5°C and returned to normal temperatures, a strong p53 response with an increase in protein levels was observed. In our study we found the opposite, but the time interval between treatment and cystectomy was with 2 days (group 3) and several months (group 4) significantly longer than the time interval used by Guan et al. [8]. Furthermore the results of this study are based on a small number of patients, which makes statistic analysis unreliable. On the other hand, the current results suggest that thermo-chemotherapy could be an effective treatment in patients with a p53 tumour suppressor gene mutated tumour. In all, the results show several trends and encourage carrying out larger experimental studies.
Conclusion
The degree of inflammation and haemorrhage in bladder tumours did not increase in patients treated with hyperthermia. This, in combination with a decrease in proliferation activity and a decrease in p53 activity, implies that thermo-chemotherapy is a safe and promising treatment.