Journal of Neuro-Oncology 14: 177-187, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Clinical Study
In vivo CT measurement of blood-brain transfer constant of iopamidol in human brain tumors
W.T. Ivan Yeung, 1,2,aTing-Yim L e e , 1'2'3 Rolando F. Del Maestro, 4 Roman Kozak 1 and Thomas Brown 1
1Department of Diagnostic Radiology, St. Joseph's Health Centre and University of Western Ontario, 2Department of Medical Biophysics, University of Western Ontario, 3Lawson Family Imaging Research Laboratories, The John P. Robarts Research Institute, 4Brain Research Laboratory, Clinical Research Unit, Department of Clinical Neurological Sciences, University of Western Ontario, London, Ontario, Canada N6A 4G5
Key words: brain tumors, blood-brain barrier, blood-brain transfer constant, x-ray CT, iopamidol, functional images Summary We have developed an in vivo method of measuring the blood-brain transfer constant (K) of iopamidol and the cerebral plasma volume (Vp) in brain tumors using a clinical X-ray CT scanner. In patient studies, Isovue 300 (iopamidol) was injected at a dosage of i ml/kg patient body weight. Serial CT scans of the tumor site and arterial blood samples from a radial artery were taken up to 48 min after injection. The leakage of iopamidol into the brain through the blood-brain barrier was modelled as an exchange process between two compartments, the intravascular plasma space and the tissue interstitial space. Using this model and the concentration measurements in blood plasma and tissue, quantitative estimates of K and Vp in brain tumors were obtained. In addition, distribution of the estimated values of K and Vp in tumors were displayed as false colour functional images overlaid on the conventional CT scan. In a study of twelve patients with anaplastic astrocytoma (n -- 3), glioblastoma multiforme (n = 4) or metastases (n = 5) the mean K and Vp values in tumor were found to be 0.0273 _+ 0.0060ml/min/g and 0.068 _+ 0.11 ml/g respectively. These values were significantly higher than those in grey or white matter in the contralateral 'normal' hemisphere (p < 0.05). The functional images showed variations in K and Vp within the tumor which were difficult to perceive in the original contrast enhanced CT scans.
Introduction The blood-brain barrier (BBB) is a functional interface which regulates the transport of materials from the capillaries to the parenchyma of the brain [1]. The barrier arises from the tight junctions formed by endothelial cells in the capillary wall. In normal brain, it prevents plasma proteins and other solutes from entering the parenchyma. However, in pathological conditions such as brain tumors, the BBB is defective resulting in extravasation of plas-
ma proteins and ultrafiltration of plasma fluid [2]. This is the basis of enhancement of brain tumors with iodinated contrast agent in X-ray computed tomography (CT). In vivo measurement of the BBB permeability or the blood-brain transfer constant of tracers has been attempted by various investigators in order to understand better the pathogenesis of BBB disruption and drug delivery in various brain diseases particularly brain tumors. Most of the in vivo studies employed positron emission tomography (PET)
178 and positron emitting radioisotopes. Hawkins et al. [3] and Iannotti et al. [4] used [6SGa]EDTA and a two-compartment model to measure the bloodbrain transfer constant in brain tumor patients. Lammertsma et al. [5] and Brook et aL [6] measured the same transfer constant with S2Rb+using a steady-state model involving sequential administration of 82Rb+, CO150 and 11CO. Dhawan et al. [7, 8] provided a detailed analysis of the two-compartment model for the measurement of the bloodbrain transfer constant of 82Rb+. The same group also monitored the effect of steroids on the bloodbrain transfer constant of the same tracer in brain tumor [9, 10]. The potential advantages of measuring the blood-brain transfer constant in the treatment of brain diseases and the limited availability of PET scanners prompted us to investigate approaches with other imaging modalities. We report here an in v i v o method of blood-brain transfer constant measurement using X-ray CT and contrast agent (Isovue 300). Recently, Groothuis et al. [11] measured the forward transfer constant of meglumine iothalamate in canine brain tumor with CT using a method similar to our one. We studied twelve patients with primary tumors and metastases with the method. A two-compartment model, same as the one adopted by Hawkins et al. [3] and others [4, 7, 8], was used to calculate the blood-brain transfer constant (K) and cerebral plasma volume (Vp). K and Vp were found to be significantly higher in tumors than in the patients' own control (contralateral 'normal') regions. In addition, functional images showing the distribution of the determined K and Vp values were produced. They were overlaid on regular CT scans such that variations of K and Vp can be correlated with brain anatomy. I n v i v o measurement of cerebral blood volume (CBV) with CT and contrast agent was first attempted by Penn et al. [12] and Zilkha et al. [13]. They determined CBV as the ratio of enhancement in the brain tissue to that in the blood. Their approach was only valid in normal brain [14] where the contrast agent remained in the intravascular space with little egress to the parenchyma. It failed to account for the enhancement of tissue contributed by the interstitial space in diseases in which
the BBB was disrupted. In contrast, our model as discussed below is composed of two compartments which takes into account the enhancement contributed by both the vascular and extravascular extracelluar space in the brain.
Model
In this study, we used the uptake of iopamidol (Isovue 300)in the brain to calculate its bloodbrain transfer constant (K) and the cerebral plasma volume (Vp). Iopamidol is a medium molecular weight (M.W. = 777) hydrophillic molecule that is neutral and biologically inert [15]. It crosses the BBB by passive diffusion. It does not cross cell membranes and therefore is distributed in cerebral plasma space (CPS) as well as in the interstitial or extravascular extracelluar space (EES). A two-compartment model (a special case of Patlak et al. [16, 17]) as shown in Fig. I was used to describe the kinetic behaviour of iopamidol in the brain. The two compartments are the cerebral plasma space (CPS) and the interstitial space (EES) separated by the BBB. Since the interstitial space is assumed to be a compartment, diffusion in it is not included in the model (see Discussion). We have used the following symbols in the subsequent derivation: Cp(t) iopamidol concentration in CPS at time t (mmol/ml), this is equal to the arterial plasma concentration; Ce(t) amount of iopamidol in the EES at time t (mmol/g); Cu(t) amount of iopamidol in the brain (mmol/g); Vp volume of CPS (ml/g); Ve volume of EES (ml/g); K blood-brain transfer constant (ml/min/g); k rate constant (rain -1) of backflux from EES to CPS. The mechanism of exchange of iopamidol between the two compartments is assumed to be passive diffusion only. Solvent-drag [18] is ignored in the transport of iopamidol across the BBB (see Discussion). Therefore, K and k are related by the equation:
179 k = K/V¢
Q(t)
(1)
K is related to the BBB permeability (P) and capillary surface area (S) product, PS [19]: K = F ( 1 - e -Ps/F)
K CPS
(2)
where F is the regional cerebral blood flow (ml/ min/g). PS represents the unidirectional flux of tracer from blood into the parenchyma. If F > > PS, PS can be approximated by K [20]. In brain tumor, the above condition is generally true. By conservation of mass, the change of the amount of iopamidol in the EES can be expressed
EES k BBB
Fig. 1. Schematicdiagram of the 2-compartmentmodel used. Cerebral plasma space (CPS) and extravascular extracellular space (EES) are separatedfunctionallyby the blood-brainbarrier (BBB). Transportof tracer throughthe BBB takesplaceby passive diffusion.
as:
dCe(t) dt - K C p ( t ) -
Method k Ce(t )
(3)
Patient data The solution of Ce(t) with the initial conditions that at t = 0, Ce(t) = 0 is: Ce(t) = K y~ Cp(u) e -k(t-°) du
(4)
CT scanner cannot measure Co(t) per se due to its rather large resolution distance of -700/xm. The amount of tracer as measured by CT in a region of interest (ROI) in the brain has contributions from both CPS and EES, hence: Cb(t) = C.(t)+ Vp Cp(t)
(5)
Substitute (1) into (2), we obtain the expression for Cb(t): Cb(t) = K J'~ Cp(u) e k(t-u)du + Vp Cp(t)
(6)
With multiple measurements of Cb(t ) and Cv(t) at different times following administration of iopamidol, the parameters (K, k and Vp) can be estimated by non-linear regression methods. We have used the constrained quasi-Newton algorithm [21, 22] from the NAG library (Downers Grove, IL). The lower bounds for the parameters were set to be zero as constraints since any negative values are nonphysiological.
The patient population studied included 12 individuals, 4 (33%) females and 8 (67%) males. All patients had their types of brain tumors confirmed by histopathology. Seven patients had malignant glial tumors: 3 were anaplastic astrocytomas and 4 were glioblastoma multiforme. There were 5 metastatic tumors: 3 malignant melanoma, 1 papillary adenocarcinoma with no primary site identified and 1 non-small cell carcinoma also with no primary site identified. Nine of 12 patients (75%) were taking dexamethasone (dose range 2-4 mg four times daily) at the time of the study. Two of 3 (67%) anaplastic astrocytoma and 3 of 4 (75%) glioblastoma multiforme patients were on steroids as were 4 of 5 (80%) metastatic tumor patients. The study was approved by the University of Western Ontario Review Board for Health Research Involving Human Subjects and informed consent was obtained in each case. No patient suffered any direct complication related to his/her participation in this study.
Study protocol The patient lied on the CT couch with the head strapped in an head holder to minimize movements. An intravenous line was started in one arm
180 for injection of iopamidol. Local anaesthesia (Lidocaine) was applied to the wrist of the contralateral arm where a 20 gauge angiocatheter was placed into the radial artery for arterial blood sampling. Head scans without contrast enhancement were performed using a GE9800 CT scanner at 120 kVp, 170 mA, 2 s scan time, 512 matrix size and 1 cm slice thickness to locate the level through the tumor which had the largest cross sectional area. Another scan without contrast enhancement at the chosen level was obtained with the same technique factors to serve as the baseline scan. In addition, a baseline blood sample of 2ml volume was taken via the arterial line. This was followed by the injection of 1 ml of Isovue 300 (Squibb Diagnostic) per kg of patient body weight via the intravenous catheter over 30 sec. CT scans at 1, 3, 6, 12, 18, 24, 36 and 48 minutes after injection were obtained with the same technique factors as the baseline scan. During the same period of time, enhanced blood samples, each of 2 ml volume, were taken from the arterial line at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 6, 9, 12, 18, 24, 36 and 48 minutes after the injection. All the CT images were transferred to a Sun 4/360 workstation. The contrast enhanced images were registered relative to the baseline image in order to correct for any patient movement during the study. In the registration process, the operator translated and/or rotated the enhanced images using an interactive image manipulation program we have developed until they matched the baseline image at chosen anatomical landmarks. We used the whole skull for matching in our patient studies. The blood samples (baseline and enhanced) were centrifuged to obtain plasma which was then drawn into syringes and placed in a tissue equivalent head phantom (Radiation Measurement Incorporated, Wisconsin). The enhanced plasma samples were scanned with the baseline sample at the same kVp as the head scans in order to determine the arterial plasma concentration of iopamidol.
Data analys& Cb in Hounsfield (CT) number was determined
using a subtraction technique, namely the mean Hounsfield number in a tumor region in the background image was subtracted from that of the enhanced images. Similarly Cp in Hounsfield number was obtained by subtracting the CT number of the ROI corresponding to the baseline plasma sample from ROIs of the other enhanced plasma samples. We have previously shown that the increase in CT number post injection was proportional to the concentration of iodinated contrast agent present [23]. Since the X-ray attenuation of the head phantom and that of the brain tissue was nearly the same, the two proportional relationships between Hounsfield number and concentration of contrast agent, one for plasma and one for brain tissue, were the same. This assumption was based on the observation that the CT number of the material of the head phantom was almost equal to that of brain tissue in patients (unpublished data). We have also shown that the GE9800 CT scanner can be used to measure contrast concentration (mgI/ml) accurately in relative units of (increase in) Hounsfield number in a head phantom [23]. Thus the measurements of Cp(t) and Cb(t) in CT number can be used directly in equation (6) to estimate the parameters of the 2-compartment model we have used. To determine Cb(t), ROI of irregular shape was drawn to include the whole tumor. Any necrotic region (i.e. regions of low CT number in contrast enhanced images) was excluded from the ROI. Rectangular ROIs (between 300 to 400 pixels) defined in the 'normal' grey and white matter on the contralateral side were also examined as controls. The uncertainty (errors) of Cb was contributed by (X-ray) quantum noise and the temporal fluctuations of mean CT number of ROIs. Cb suffered from both of these errors while Cp had errors from quantum noise only because enhanced plasma samples were always scanned with the background plasma sample. The root mean square fluctuations in mean CT number of ROIs was determined to be + 0.4 Hounsfield number by scanning a water phantom repeatedly with the same scanning protocol as the patient studies. Typically the measurement errors of Cb were 4% in tumor and 20% in normal region while Cp had an error of 3%. These errors were used in the calculation of i) K and Vp
181 estimates using the constrained quasi-Newton algorithm, ii) errors associated with the parameters [24], and iii) the chi-square value between the fitted and raw Cb values as a test of goodness of fit. In principle, all three parameters (i.e. K, k and Vp) in equation (6) can be estimated by non-linear regression provided Cb(t) and Cp(t) are known. Nevertheless, k is not sensitive to changes in Cb(t ) [7], only K and Vp can be determined from the model with precision. Student's t-test was used to compare the differences of K or Vp in different tumor types and onetailed paired t-test the difference of these two parameters in tumor and 'normal' (control) regions.
Functional images
The K and Vp estimates can be graphically represented by functional images. Every 4 x 4 pixels in the 512 x 512 CT image were averaged to form an image consisting of 128 x 128 'superpixels'. The choice of 4 x 4 was empirical. We wished to reduce the errors in Cb by averaging over pixels such that stable estimates of K and Vp can be calculated. At the same time, we also wanted to have sufficient spatial resolution in the functional images to correlate them with anatomical features. Averaging over 4 x 4 pixels was a reasonable compromise between these two conflicting requirements. Each of these superpixels was considered as an ROI having unique values of K and Vp. The estimated values of these two parameters in each superpixel were separately encoded with a blue (K) and a red (Vp) colour scale (256 levels) to form functional images. These functional images represented the distribution of the K and Vp values in the brain and could be overlaid on regular CT scans (high resolution, 512 x 512) to show variations of the two parameters within different anatomical structures in the CT scans.
Results
A total of 12 patients were studied. Twelve tumor regions (one from each patient) along with their
corresponding regions of 'normal' white and grey matter were examined. For the tumor regions, the coefficient of variation (CV) of K, defined as the error in the estimated K value divided by the value itself [24], was less than 10% in 8 and 18% in 4 regions. For the parameter Vp, the CV was less than 10% in 10 and 28% in 2 regions. For normal regions, the average CV for K and Vp were 300% and 20% respectively. The extremely high values of CV for K in normal regions were expected because (i) as discussed by Dhawan et aL [7], the model we used was not sensitive in estimating small K values and, (ii) the errors of Cb were high (20%) in normal regions. As for goodness of fit, 11 out of 12 tumor regions had p > 0.05 and one with p = 0.1%. In normal region, the goodness of fit was uniformly good (p > 0.12). The patients could be divided into 3 groups. These were (i) anaplastic astrocytoma (AA, n-3), (ii) glioblastoma multiforme (GM, n = 4), and (iii) metastasis (MA, n = 5). Figure 2 and 3 show in the form of histograms the estimated values of K and Vp in tumor regions together with white and grey matter values in the contralateral 'normal' hemisphere for all 12 patients arranged in their respective groups. The histograms plot the group means with the standard error of the mean represented by error bars. The individual and (group) mean values of K and V v were consistently higher in tumor region than those in 'normal' regions except for Vp in 2 of the 3 AA patients. When all 12 patients were examined together as a group, Student's paired t-test (one-tailed) revealed that mean values of K and Vp in tumor were significantly higher (p < 0.05) than those in control white and grey matter. When tested individually with paired t-test, MA also showed significantly ( p < 0.05) higher values of K and Vp in tumor than in the patients' own 'normal' white and grey matter and so was GM for Vp. The mean values of K or Vp of the three patient groups were found to be not significantly different (p = 0.05) from each other due to the fairly large scatter in the estimated values within each individual group. The possible reasons for this scatter in the estimated values will be discussed in the next section.
182 0.06 0.05
0.12
I
I
White matter
[~
White matter
Tumor
[~
Tumor
Gray m a t t e r
[~
Gray
"-~ 0 . 0 4 c E 0.03 ,~
if-
matter
0.08
IL +
0.02 0.01 0.00
>
o_
I
0.04
i
0.00
AA n=3
GM
MA
n=4
n=5
Fig. 2. Blood-brain transfer constant (K) of iopamidol as determined by the CT method. The mean values for white and grey matter in the contralateral 'normal' hemisphere are plotted beside that of tumor in each of the 3 groups of tumor; namely, anaplastic astrocytoma (AA), glioblastoma multiforme (GM) and metastasis (MA). Error bars are the standard error of the mean and n is the number of patients in each group. Figures 4 and 5 are the functioflal images of K and Vp respectively of a patient with a brain metastasis from malignant melanoma. We used 256 shades of blue and red color to represent a range of 0.001-0.1 ml/g/min and 0.0-0.4 ml/g for K and Vp respectively. The non-zero lower cutoff of the K image was selected such that tumor regions with more leaky blood-brain barrier than normal brain would be highlighted. Figure 4 shows the distribution of K values in the chosen CT slice through the metastasis. Inhomogeneity in the blood-brain transfer constant K in the metastasis is graphically represented as shades of blue colour, with the necrotic centre dark in appearance because of close to zero K values. The functional image of Vp is shown in Fig. 5. The metastasis had a bright red colour because of its elevated Vp. The necrotic centre again was dark in appearance due to nearly zero values of Vp. Normal structures at this level of the brain and the surrounding structures, which were expected to have a high Vp, such as circle of Willis, sagittal sinus and transverse sinus, also had a bright red colour.
AA n=3
GM n=4
MA n=5
Fig. 3. Cerebral plasma volume (Vp) in the 3 groups of tumor studied. Discussion
An in vivo method to measure the blood-brain transfer constant of iopamidol in the brain by CT scanning has been developed. The advantages of this method compared to P E T are (1) the equipment required is clinically more accessible; (2) use of radioactive isotopes and more importantly cyclotron generated isotopes was unnecessary since radiolabelled tracers (S2Rb or [6SGa-EDTA]) were replaced by iopamidol, which is a common nonionic X-ray contrast agent and, (3) CT has a higher in plane resolution than P E T (0.7 mm versus 5 ram) such that it would have less partial volume effect [25]. Hence, CT would provide a better visualization of the inhomogeneity of K or Vp values in tumors as well as in normal brain tissue. The method is not restricted to brain tumor patients but can be applied to other cerebral disorders (such as multiple sclerosis) in which the BBB is disrupted. Its ability to repeatedly measure these two parameters in vivo makes the method a potentially valuable tool in monitoring the progression/ remission of a variety of diseases, or assessing the efficacy of drug treatment programs. The inhomogeneity of these two parameters with respect to brain anatomy can be easily visualized with the functional images. The average values of K and Vp in tumor regions as measured by our method were 0.0273_+ 0.0060 ml/min/g and 0.068 + 0.011 ml/g respective-
183
Fig. 4. Functional image of the blood-brain transfer constant
Fig. 5. Functional image of the cerebral plasma volume (Vp) in
(K) in a CT slice through a brain metastasis of a malignant melanoma patient. The values of K are displayed in a blue colour scale.
the same CT slice as in Fig. 4. The valuesof Vp are displayedin a red colour scale.
ly. The mean K and Vp estimates were in very good agreement with results on canine brain tumors ( K - - 0.028 + 0.003ml/min/g and V p = 0.073 + 0.010 ml/g) by Groothuis et al. [11] with a similar method. Our estimated values for normal grey matter -(K= 0.0019 + 0.0015ml/min/g, Vp = 0.0173 + 0.0013 ml/g) were lower than but in the same order of magnitude as their values ( K = 0.0035 + 0.0007ml/min/g, Vv = 0.022+ 0.003ml/ g) [11]. Blasberg et al. [20] measured a mean K value of 0.0021 + 0.0001 ml/min/g for cortex and 0.00114 + 0.00004ml/min/g for subcortex a-aminoisobutyric acid (AIB) in normal rat brain. Our average K values in 'normal' grey and white matter were 0.0019+ 0.0015ml/g/min and 0.0005+ 0.0002ml/min/g. These values were lower than Blasberg's values as the molecular weight of AIB (104) is less than that of iopamidol (777). The mean Vp values in the 'normal' grey and white regions were found to be 0.0161 + 0.0009 ml/g which translated to a cerebral blood volume of 0.026 ml/g using a hematocrit of 45% and a value of 0.85 for the ratio of small vessel to large vessel hematocrit. Our
estimated CBV was at the lower end of the range measured by Grubb et al. [26] (mean C B V = 0.032) and Penn et al. [12] (mean CBV = 0.030) using iodinated contrast agent with X-ray fluorescence and CT respectively. The discrepancy of our estimated CBV value and the published data could be from the choice of our 'normal' regions. It has been estimated that the distribution of cerebral blood volume is 70% in veins and venules, 10% in arteries and arterioles, and 20% in capillaries [27]. In our studies the 'normal' regions in all cases did not include major arteries and veins in the brain, hence their CBV would be expected to be less than the average CBV of the whole. Significant increases (p < 0.05) were found for K and Vp when tumor regions were compared to the patients' own control regions in 'normal' grey and white matter by one-tailed Student's paired t-test. This finding was consistent with other investigators' reports on [68Ga]EDTA, 8ZRb and meglumine iothalamate blood-brain transfer constant in brain tumors [3, 4, 6, 11]. No significant difference of the mean K or Vp values was found among the three tumor groups.
184 There were three possible reasons: firstly, the sample size of each group was small due to the limited number of patients (12) in this series. Secondly, it might reflect the large inherent variability of K values in individual tumor types as shown by Groothuis et al. [28] for brain tumors in rat. Thirdly, Jarden et al. [9, 10] showed that dexamethasone reduced K and Vp in some brain tumors. However, not all of the patients in each group were treated with dexamethasone at the time of the CT study. This nonuniformity of steroid treatment would contribute to additional variability in the measured values of K and Vp within each tumor group. In order to assess the effect of dexamethasone on the values of K and Vp, and elucidate the correlation between these two parameters and tumor types, it will be necessary to study the patient before and after dexamethasone treatment in a prospective study protocol [9, 10]. As discussed in the Model section, we have ignored the bulk flow term in the transport of iopamidol across BBB. The blood-brain transfer constant K is related to the actual PS and bulk flow by [29]: K = F ( 1 - exp(-
PS + f(1 + o)/2_) F
after injection. The loss of iopamidol in tumor region to the surrounding due to diffusion would be misinterpreted by the 2-compartment model as the lack of tracer because of lower BBB permeability, hence underestimating K. Conversely, K would be overestimated in the surrounding regions with intact BBB due to the influx of tracer from the tumor. In order to study the significance of diffusion on the estimated K values, we recalculated the K estimates by using data only up to 12 minutes when less tracer would have diffused out of the ROI. The estimated K values from 12-minute data were higher than those from 48-minute data by 10 to 40%. A better study protocol to minimize the inaccuracy in estimated K due to diffusion is one in which the data collection will be completed in 6 instead of 48 rain. As a conservative estimate of the diffusion coefficient (D) for iopamidol, we use the value of D-sucrose (7 x 10-6cm 2 s-1) [31]. Iopamidol is expected to have a lower D because of its higher molecular weight. Assuming a tortuosity factor (T) of 0.6 [30] for iopamidol in human brain, the mean diffusion distance (d) in two dimensions [32] in 6 rain is:
(7)
where f is a size-independent volume flux representing the bulk fluid flow rate and o is the reflection coefficient of the tracer. Nakagawa et al. [30] determined a bulk flow rate of 0.0008 ml/min/g in RG-2 gliomas in rats. Assuming the worst case of o(= 0) and an average K value we measured in tumor region, ie. 0.0273 ml/min/g, as PS. The apparent K (ie. with bulk flow term) would be 0.0272 ml/min/g whereas the 'true' K (ie. without bulk flow term) would have been 0.0268. Therefore, for the average tumor values we have found in this study, we may have overestimated K by 1.5% by ignoring the bulk flow term. Diffusion in the EES was not considered in this model. However, its effect can be clearly visualized in our patient studies. Figure 6 was a series of scans from a typical patient. It clearly showed that spreading of iopamidol due to diffusion from regions of enhancement into neighbouring nonenhanced regions occurred between 6 and 12 minutes
d = (4× D × T × 6rain) 1/2 = 0.78 mm Since the GE9800 CT scanner has a spatial resolution of 0.7mm, diffusion of iopamidol in brain in 6 min would have little effect on the K estimates. Patient movement is a technical limitation which may affect the accuracy of the K and Vp estimates. Movement in the (x-y) plane of the CT image was corrected by image registration as described above. However, movement and tilting in the longitudinal (z) direction could not be corrected and would lead to error in the estimation of K and Vp. In fact, movement in the z-direction of more than 2mm would lead to changes in anatomical landmarks present in 10 mm thick head slices taken before and after the movement. No such change was observed in the patient studies we presented. Therefore, error due to patient movement in z-direction was minimal and would not have a significant contribution to the overall errors of K and Vp estimates. Another factor which may cause error in the K
185
Fig. 6. A series of CT scans from a typical patient study. The spreading of iopamidol due to diffusion from regions of enhancement to surrounding nonenhanced regions occurred between 6 and 12 minutes after intravenous injection.
and Vp estimates is the undesirable side effects caused by the X-ray contrast agent itself. The X-ray contrast agent we used, Isovue 300, is a hyperosmolar solution (0.79 M) of iopamidol. Hyperosmolarity of plasma solutes is known to induce osmotic opening of BBB [1], increase cerebral blood flow (CBF) [33] as well as CBV [34]. Rapoport et al. [35] reported that carotid infusion of 1.4M arabinose solution for 30s into 300g rats for a dosage of 17 mmol/kg produced no significant elevation of K measured with [14C]sucrose compared to the control group. We used intravenous injection of Isovue 300 (0.79M) at a dosage of 0.79mmol/kg and therefore would expect negligible osmotic opening of BBB. Ravussin et al. [34] infused intravenously a 1.1 M
mannitol solution for 4min at a dosage of 5.5 mmol/kg into human subjects; they obtained a maximum increase in CBV of 14%. Direct scaling of their findings suggests that an increase in CBV of less than 2% would be expected in our situation. Assuming the extreme cause in which the increase of CBV occurs entirely by dilation of the capillaries, the increase of capillary surface area (S), hence K by Eq. 2, would be less than 2%. Hyperosmolar solutions had been shown to increase CBF. Jafar et al. [33] employed infusion of 1.1M mannitol with a dosage of 8.2mmol/kg in stroke patients; they observed an average CBF increase of about 25% in ischemic brain tissue. Even an increase of 25% of CBF in our case would raise K only by less than 1% according to Eq. 2 for
186 an average K value we have determined in tumors. This result was consistent with Dhawan et al. 's [8] report that K was independent of CBF.
Fund, Radiology Department, St. Joseph's Health Centre, London are gratefully acknowledged. The authors also acknowledged the technical assistance of Ms Rosemary Millar and Cathy Greenaway in the patient studies.
Conclusions An in vivo method of measuring the BBB permeability using iopamidol and X-ray CT scanning is described. Twelve brain tumor patients divided into 3 groups (ie. anaplastic astrocytoma, glioblastoma multiforme and metastases) were studied to determine the values of the blood-brain transfer constant of iopamidol (K) and cerebral plasma volume (Vv). There was a significant increase in K and Vp in tumor regions against the patients' own control. No significant difference of the mean tumor K or Vp was found among the three groups of patients possibly due to the effect of nonuniform steroid administration and the inherent variability in tumors. By displaying the estimated values with a false colour scale, red for K and blue for Vp, functional images of blood-brain transfer constant of iopamidol and cerebral plasma volume were obtained. These images, when overlaid on an ordinary CT image, can be used to correlate the distribution of K and Vp with anatomy in the CT image. Although we have only studied brain tumor patients, this method is useful in monitoring the involvement of the blood-brain barrier in various cerebral diseases and its responses to drug therapy.
Acknowledgements This project is supported by grants from the National Cancer Institute of Canada, the London UpJohn Neurosciences Program, the Up John Company, the Medical Research Council of Canada and Medical Systems Division, General Electric of Canada. W.T.I. Yeung is supported by a studentship from the Medical Research Council of Canada. Dr. Del Maestro is a recipient of an Ontario Ministry of Health Career Scientist Award. The financial assistance from the Brain Research Fund Foundation and the Academic Development
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Address for offprints: T.Y. Lee, Lawson Family Imaging Research Laboratories, The John P. Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8
Clinical Study
In vivo CT measurement of blood-brain transfer constant of iopamidol in human brain tumors
W.T. Ivan Yeung, 1,2,aTing-Yim L e e , 1'2'3 Rolando F. Del Maestro, 4 Roman Kozak 1 and Thomas Brown 1
1Department of Diagnostic Radiology, St. Joseph's Health Centre and University of Western Ontario, 2Department of Medical Biophysics, University of Western Ontario, 3Lawson Family Imaging Research Laboratories, The John P. Robarts Research Institute, 4Brain Research Laboratory, Clinical Research Unit, Department of Clinical Neurological Sciences, University of Western Ontario, London, Ontario, Canada N6A 4G5
Key words: brain tumors, blood-brain barrier, blood-brain transfer constant, x-ray CT, iopamidol, functional images Summary We have developed an in vivo method of measuring the blood-brain transfer constant (K) of iopamidol and the cerebral plasma volume (Vp) in brain tumors using a clinical X-ray CT scanner. In patient studies, Isovue 300 (iopamidol) was injected at a dosage of i ml/kg patient body weight. Serial CT scans of the tumor site and arterial blood samples from a radial artery were taken up to 48 min after injection. The leakage of iopamidol into the brain through the blood-brain barrier was modelled as an exchange process between two compartments, the intravascular plasma space and the tissue interstitial space. Using this model and the concentration measurements in blood plasma and tissue, quantitative estimates of K and Vp in brain tumors were obtained. In addition, distribution of the estimated values of K and Vp in tumors were displayed as false colour functional images overlaid on the conventional CT scan. In a study of twelve patients with anaplastic astrocytoma (n -- 3), glioblastoma multiforme (n = 4) or metastases (n = 5) the mean K and Vp values in tumor were found to be 0.0273 _+ 0.0060ml/min/g and 0.068 _+ 0.11 ml/g respectively. These values were significantly higher than those in grey or white matter in the contralateral 'normal' hemisphere (p < 0.05). The functional images showed variations in K and Vp within the tumor which were difficult to perceive in the original contrast enhanced CT scans.
Introduction The blood-brain barrier (BBB) is a functional interface which regulates the transport of materials from the capillaries to the parenchyma of the brain [1]. The barrier arises from the tight junctions formed by endothelial cells in the capillary wall. In normal brain, it prevents plasma proteins and other solutes from entering the parenchyma. However, in pathological conditions such as brain tumors, the BBB is defective resulting in extravasation of plas-
ma proteins and ultrafiltration of plasma fluid [2]. This is the basis of enhancement of brain tumors with iodinated contrast agent in X-ray computed tomography (CT). In vivo measurement of the BBB permeability or the blood-brain transfer constant of tracers has been attempted by various investigators in order to understand better the pathogenesis of BBB disruption and drug delivery in various brain diseases particularly brain tumors. Most of the in vivo studies employed positron emission tomography (PET)
178 and positron emitting radioisotopes. Hawkins et al. [3] and Iannotti et al. [4] used [6SGa]EDTA and a two-compartment model to measure the bloodbrain transfer constant in brain tumor patients. Lammertsma et al. [5] and Brook et aL [6] measured the same transfer constant with S2Rb+using a steady-state model involving sequential administration of 82Rb+, CO150 and 11CO. Dhawan et al. [7, 8] provided a detailed analysis of the two-compartment model for the measurement of the bloodbrain transfer constant of 82Rb+. The same group also monitored the effect of steroids on the bloodbrain transfer constant of the same tracer in brain tumor [9, 10]. The potential advantages of measuring the blood-brain transfer constant in the treatment of brain diseases and the limited availability of PET scanners prompted us to investigate approaches with other imaging modalities. We report here an in v i v o method of blood-brain transfer constant measurement using X-ray CT and contrast agent (Isovue 300). Recently, Groothuis et al. [11] measured the forward transfer constant of meglumine iothalamate in canine brain tumor with CT using a method similar to our one. We studied twelve patients with primary tumors and metastases with the method. A two-compartment model, same as the one adopted by Hawkins et al. [3] and others [4, 7, 8], was used to calculate the blood-brain transfer constant (K) and cerebral plasma volume (Vp). K and Vp were found to be significantly higher in tumors than in the patients' own control (contralateral 'normal') regions. In addition, functional images showing the distribution of the determined K and Vp values were produced. They were overlaid on regular CT scans such that variations of K and Vp can be correlated with brain anatomy. I n v i v o measurement of cerebral blood volume (CBV) with CT and contrast agent was first attempted by Penn et al. [12] and Zilkha et al. [13]. They determined CBV as the ratio of enhancement in the brain tissue to that in the blood. Their approach was only valid in normal brain [14] where the contrast agent remained in the intravascular space with little egress to the parenchyma. It failed to account for the enhancement of tissue contributed by the interstitial space in diseases in which
the BBB was disrupted. In contrast, our model as discussed below is composed of two compartments which takes into account the enhancement contributed by both the vascular and extravascular extracelluar space in the brain.
Model
In this study, we used the uptake of iopamidol (Isovue 300)in the brain to calculate its bloodbrain transfer constant (K) and the cerebral plasma volume (Vp). Iopamidol is a medium molecular weight (M.W. = 777) hydrophillic molecule that is neutral and biologically inert [15]. It crosses the BBB by passive diffusion. It does not cross cell membranes and therefore is distributed in cerebral plasma space (CPS) as well as in the interstitial or extravascular extracelluar space (EES). A two-compartment model (a special case of Patlak et al. [16, 17]) as shown in Fig. I was used to describe the kinetic behaviour of iopamidol in the brain. The two compartments are the cerebral plasma space (CPS) and the interstitial space (EES) separated by the BBB. Since the interstitial space is assumed to be a compartment, diffusion in it is not included in the model (see Discussion). We have used the following symbols in the subsequent derivation: Cp(t) iopamidol concentration in CPS at time t (mmol/ml), this is equal to the arterial plasma concentration; Ce(t) amount of iopamidol in the EES at time t (mmol/g); Cu(t) amount of iopamidol in the brain (mmol/g); Vp volume of CPS (ml/g); Ve volume of EES (ml/g); K blood-brain transfer constant (ml/min/g); k rate constant (rain -1) of backflux from EES to CPS. The mechanism of exchange of iopamidol between the two compartments is assumed to be passive diffusion only. Solvent-drag [18] is ignored in the transport of iopamidol across the BBB (see Discussion). Therefore, K and k are related by the equation:
179 k = K/V¢
Q(t)
(1)
K is related to the BBB permeability (P) and capillary surface area (S) product, PS [19]: K = F ( 1 - e -Ps/F)
K CPS
(2)
where F is the regional cerebral blood flow (ml/ min/g). PS represents the unidirectional flux of tracer from blood into the parenchyma. If F > > PS, PS can be approximated by K [20]. In brain tumor, the above condition is generally true. By conservation of mass, the change of the amount of iopamidol in the EES can be expressed
EES k BBB
Fig. 1. Schematicdiagram of the 2-compartmentmodel used. Cerebral plasma space (CPS) and extravascular extracellular space (EES) are separatedfunctionallyby the blood-brainbarrier (BBB). Transportof tracer throughthe BBB takesplaceby passive diffusion.
as:
dCe(t) dt - K C p ( t ) -
Method k Ce(t )
(3)
Patient data The solution of Ce(t) with the initial conditions that at t = 0, Ce(t) = 0 is: Ce(t) = K y~ Cp(u) e -k(t-°) du
(4)
CT scanner cannot measure Co(t) per se due to its rather large resolution distance of -700/xm. The amount of tracer as measured by CT in a region of interest (ROI) in the brain has contributions from both CPS and EES, hence: Cb(t) = C.(t)+ Vp Cp(t)
(5)
Substitute (1) into (2), we obtain the expression for Cb(t): Cb(t) = K J'~ Cp(u) e k(t-u)du + Vp Cp(t)
(6)
With multiple measurements of Cb(t ) and Cv(t) at different times following administration of iopamidol, the parameters (K, k and Vp) can be estimated by non-linear regression methods. We have used the constrained quasi-Newton algorithm [21, 22] from the NAG library (Downers Grove, IL). The lower bounds for the parameters were set to be zero as constraints since any negative values are nonphysiological.
The patient population studied included 12 individuals, 4 (33%) females and 8 (67%) males. All patients had their types of brain tumors confirmed by histopathology. Seven patients had malignant glial tumors: 3 were anaplastic astrocytomas and 4 were glioblastoma multiforme. There were 5 metastatic tumors: 3 malignant melanoma, 1 papillary adenocarcinoma with no primary site identified and 1 non-small cell carcinoma also with no primary site identified. Nine of 12 patients (75%) were taking dexamethasone (dose range 2-4 mg four times daily) at the time of the study. Two of 3 (67%) anaplastic astrocytoma and 3 of 4 (75%) glioblastoma multiforme patients were on steroids as were 4 of 5 (80%) metastatic tumor patients. The study was approved by the University of Western Ontario Review Board for Health Research Involving Human Subjects and informed consent was obtained in each case. No patient suffered any direct complication related to his/her participation in this study.
Study protocol The patient lied on the CT couch with the head strapped in an head holder to minimize movements. An intravenous line was started in one arm
180 for injection of iopamidol. Local anaesthesia (Lidocaine) was applied to the wrist of the contralateral arm where a 20 gauge angiocatheter was placed into the radial artery for arterial blood sampling. Head scans without contrast enhancement were performed using a GE9800 CT scanner at 120 kVp, 170 mA, 2 s scan time, 512 matrix size and 1 cm slice thickness to locate the level through the tumor which had the largest cross sectional area. Another scan without contrast enhancement at the chosen level was obtained with the same technique factors to serve as the baseline scan. In addition, a baseline blood sample of 2ml volume was taken via the arterial line. This was followed by the injection of 1 ml of Isovue 300 (Squibb Diagnostic) per kg of patient body weight via the intravenous catheter over 30 sec. CT scans at 1, 3, 6, 12, 18, 24, 36 and 48 minutes after injection were obtained with the same technique factors as the baseline scan. During the same period of time, enhanced blood samples, each of 2 ml volume, were taken from the arterial line at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 6, 9, 12, 18, 24, 36 and 48 minutes after the injection. All the CT images were transferred to a Sun 4/360 workstation. The contrast enhanced images were registered relative to the baseline image in order to correct for any patient movement during the study. In the registration process, the operator translated and/or rotated the enhanced images using an interactive image manipulation program we have developed until they matched the baseline image at chosen anatomical landmarks. We used the whole skull for matching in our patient studies. The blood samples (baseline and enhanced) were centrifuged to obtain plasma which was then drawn into syringes and placed in a tissue equivalent head phantom (Radiation Measurement Incorporated, Wisconsin). The enhanced plasma samples were scanned with the baseline sample at the same kVp as the head scans in order to determine the arterial plasma concentration of iopamidol.
Data analys& Cb in Hounsfield (CT) number was determined
using a subtraction technique, namely the mean Hounsfield number in a tumor region in the background image was subtracted from that of the enhanced images. Similarly Cp in Hounsfield number was obtained by subtracting the CT number of the ROI corresponding to the baseline plasma sample from ROIs of the other enhanced plasma samples. We have previously shown that the increase in CT number post injection was proportional to the concentration of iodinated contrast agent present [23]. Since the X-ray attenuation of the head phantom and that of the brain tissue was nearly the same, the two proportional relationships between Hounsfield number and concentration of contrast agent, one for plasma and one for brain tissue, were the same. This assumption was based on the observation that the CT number of the material of the head phantom was almost equal to that of brain tissue in patients (unpublished data). We have also shown that the GE9800 CT scanner can be used to measure contrast concentration (mgI/ml) accurately in relative units of (increase in) Hounsfield number in a head phantom [23]. Thus the measurements of Cp(t) and Cb(t) in CT number can be used directly in equation (6) to estimate the parameters of the 2-compartment model we have used. To determine Cb(t), ROI of irregular shape was drawn to include the whole tumor. Any necrotic region (i.e. regions of low CT number in contrast enhanced images) was excluded from the ROI. Rectangular ROIs (between 300 to 400 pixels) defined in the 'normal' grey and white matter on the contralateral side were also examined as controls. The uncertainty (errors) of Cb was contributed by (X-ray) quantum noise and the temporal fluctuations of mean CT number of ROIs. Cb suffered from both of these errors while Cp had errors from quantum noise only because enhanced plasma samples were always scanned with the background plasma sample. The root mean square fluctuations in mean CT number of ROIs was determined to be + 0.4 Hounsfield number by scanning a water phantom repeatedly with the same scanning protocol as the patient studies. Typically the measurement errors of Cb were 4% in tumor and 20% in normal region while Cp had an error of 3%. These errors were used in the calculation of i) K and Vp
181 estimates using the constrained quasi-Newton algorithm, ii) errors associated with the parameters [24], and iii) the chi-square value between the fitted and raw Cb values as a test of goodness of fit. In principle, all three parameters (i.e. K, k and Vp) in equation (6) can be estimated by non-linear regression provided Cb(t) and Cp(t) are known. Nevertheless, k is not sensitive to changes in Cb(t ) [7], only K and Vp can be determined from the model with precision. Student's t-test was used to compare the differences of K or Vp in different tumor types and onetailed paired t-test the difference of these two parameters in tumor and 'normal' (control) regions.
Functional images
The K and Vp estimates can be graphically represented by functional images. Every 4 x 4 pixels in the 512 x 512 CT image were averaged to form an image consisting of 128 x 128 'superpixels'. The choice of 4 x 4 was empirical. We wished to reduce the errors in Cb by averaging over pixels such that stable estimates of K and Vp can be calculated. At the same time, we also wanted to have sufficient spatial resolution in the functional images to correlate them with anatomical features. Averaging over 4 x 4 pixels was a reasonable compromise between these two conflicting requirements. Each of these superpixels was considered as an ROI having unique values of K and Vp. The estimated values of these two parameters in each superpixel were separately encoded with a blue (K) and a red (Vp) colour scale (256 levels) to form functional images. These functional images represented the distribution of the K and Vp values in the brain and could be overlaid on regular CT scans (high resolution, 512 x 512) to show variations of the two parameters within different anatomical structures in the CT scans.
Results
A total of 12 patients were studied. Twelve tumor regions (one from each patient) along with their
corresponding regions of 'normal' white and grey matter were examined. For the tumor regions, the coefficient of variation (CV) of K, defined as the error in the estimated K value divided by the value itself [24], was less than 10% in 8 and 18% in 4 regions. For the parameter Vp, the CV was less than 10% in 10 and 28% in 2 regions. For normal regions, the average CV for K and Vp were 300% and 20% respectively. The extremely high values of CV for K in normal regions were expected because (i) as discussed by Dhawan et aL [7], the model we used was not sensitive in estimating small K values and, (ii) the errors of Cb were high (20%) in normal regions. As for goodness of fit, 11 out of 12 tumor regions had p > 0.05 and one with p = 0.1%. In normal region, the goodness of fit was uniformly good (p > 0.12). The patients could be divided into 3 groups. These were (i) anaplastic astrocytoma (AA, n-3), (ii) glioblastoma multiforme (GM, n = 4), and (iii) metastasis (MA, n = 5). Figure 2 and 3 show in the form of histograms the estimated values of K and Vp in tumor regions together with white and grey matter values in the contralateral 'normal' hemisphere for all 12 patients arranged in their respective groups. The histograms plot the group means with the standard error of the mean represented by error bars. The individual and (group) mean values of K and V v were consistently higher in tumor region than those in 'normal' regions except for Vp in 2 of the 3 AA patients. When all 12 patients were examined together as a group, Student's paired t-test (one-tailed) revealed that mean values of K and Vp in tumor were significantly higher (p < 0.05) than those in control white and grey matter. When tested individually with paired t-test, MA also showed significantly ( p < 0.05) higher values of K and Vp in tumor than in the patients' own 'normal' white and grey matter and so was GM for Vp. The mean values of K or Vp of the three patient groups were found to be not significantly different (p = 0.05) from each other due to the fairly large scatter in the estimated values within each individual group. The possible reasons for this scatter in the estimated values will be discussed in the next section.
182 0.06 0.05
0.12
I
I
White matter
[~
White matter
Tumor
[~
Tumor
Gray m a t t e r
[~
Gray
"-~ 0 . 0 4 c E 0.03 ,~
if-
matter
0.08
IL +
0.02 0.01 0.00
>
o_
I
0.04
i
0.00
AA n=3
GM
MA
n=4
n=5
Fig. 2. Blood-brain transfer constant (K) of iopamidol as determined by the CT method. The mean values for white and grey matter in the contralateral 'normal' hemisphere are plotted beside that of tumor in each of the 3 groups of tumor; namely, anaplastic astrocytoma (AA), glioblastoma multiforme (GM) and metastasis (MA). Error bars are the standard error of the mean and n is the number of patients in each group. Figures 4 and 5 are the functioflal images of K and Vp respectively of a patient with a brain metastasis from malignant melanoma. We used 256 shades of blue and red color to represent a range of 0.001-0.1 ml/g/min and 0.0-0.4 ml/g for K and Vp respectively. The non-zero lower cutoff of the K image was selected such that tumor regions with more leaky blood-brain barrier than normal brain would be highlighted. Figure 4 shows the distribution of K values in the chosen CT slice through the metastasis. Inhomogeneity in the blood-brain transfer constant K in the metastasis is graphically represented as shades of blue colour, with the necrotic centre dark in appearance because of close to zero K values. The functional image of Vp is shown in Fig. 5. The metastasis had a bright red colour because of its elevated Vp. The necrotic centre again was dark in appearance due to nearly zero values of Vp. Normal structures at this level of the brain and the surrounding structures, which were expected to have a high Vp, such as circle of Willis, sagittal sinus and transverse sinus, also had a bright red colour.
AA n=3
GM n=4
MA n=5
Fig. 3. Cerebral plasma volume (Vp) in the 3 groups of tumor studied. Discussion
An in vivo method to measure the blood-brain transfer constant of iopamidol in the brain by CT scanning has been developed. The advantages of this method compared to P E T are (1) the equipment required is clinically more accessible; (2) use of radioactive isotopes and more importantly cyclotron generated isotopes was unnecessary since radiolabelled tracers (S2Rb or [6SGa-EDTA]) were replaced by iopamidol, which is a common nonionic X-ray contrast agent and, (3) CT has a higher in plane resolution than P E T (0.7 mm versus 5 ram) such that it would have less partial volume effect [25]. Hence, CT would provide a better visualization of the inhomogeneity of K or Vp values in tumors as well as in normal brain tissue. The method is not restricted to brain tumor patients but can be applied to other cerebral disorders (such as multiple sclerosis) in which the BBB is disrupted. Its ability to repeatedly measure these two parameters in vivo makes the method a potentially valuable tool in monitoring the progression/ remission of a variety of diseases, or assessing the efficacy of drug treatment programs. The inhomogeneity of these two parameters with respect to brain anatomy can be easily visualized with the functional images. The average values of K and Vp in tumor regions as measured by our method were 0.0273_+ 0.0060 ml/min/g and 0.068 + 0.011 ml/g respective-
183
Fig. 4. Functional image of the blood-brain transfer constant
Fig. 5. Functional image of the cerebral plasma volume (Vp) in
(K) in a CT slice through a brain metastasis of a malignant melanoma patient. The values of K are displayed in a blue colour scale.
the same CT slice as in Fig. 4. The valuesof Vp are displayedin a red colour scale.
ly. The mean K and Vp estimates were in very good agreement with results on canine brain tumors ( K - - 0.028 + 0.003ml/min/g and V p = 0.073 + 0.010 ml/g) by Groothuis et al. [11] with a similar method. Our estimated values for normal grey matter -(K= 0.0019 + 0.0015ml/min/g, Vp = 0.0173 + 0.0013 ml/g) were lower than but in the same order of magnitude as their values ( K = 0.0035 + 0.0007ml/min/g, Vv = 0.022+ 0.003ml/ g) [11]. Blasberg et al. [20] measured a mean K value of 0.0021 + 0.0001 ml/min/g for cortex and 0.00114 + 0.00004ml/min/g for subcortex a-aminoisobutyric acid (AIB) in normal rat brain. Our average K values in 'normal' grey and white matter were 0.0019+ 0.0015ml/g/min and 0.0005+ 0.0002ml/min/g. These values were lower than Blasberg's values as the molecular weight of AIB (104) is less than that of iopamidol (777). The mean Vp values in the 'normal' grey and white regions were found to be 0.0161 + 0.0009 ml/g which translated to a cerebral blood volume of 0.026 ml/g using a hematocrit of 45% and a value of 0.85 for the ratio of small vessel to large vessel hematocrit. Our
estimated CBV was at the lower end of the range measured by Grubb et al. [26] (mean C B V = 0.032) and Penn et al. [12] (mean CBV = 0.030) using iodinated contrast agent with X-ray fluorescence and CT respectively. The discrepancy of our estimated CBV value and the published data could be from the choice of our 'normal' regions. It has been estimated that the distribution of cerebral blood volume is 70% in veins and venules, 10% in arteries and arterioles, and 20% in capillaries [27]. In our studies the 'normal' regions in all cases did not include major arteries and veins in the brain, hence their CBV would be expected to be less than the average CBV of the whole. Significant increases (p < 0.05) were found for K and Vp when tumor regions were compared to the patients' own control regions in 'normal' grey and white matter by one-tailed Student's paired t-test. This finding was consistent with other investigators' reports on [68Ga]EDTA, 8ZRb and meglumine iothalamate blood-brain transfer constant in brain tumors [3, 4, 6, 11]. No significant difference of the mean K or Vp values was found among the three tumor groups.
184 There were three possible reasons: firstly, the sample size of each group was small due to the limited number of patients (12) in this series. Secondly, it might reflect the large inherent variability of K values in individual tumor types as shown by Groothuis et al. [28] for brain tumors in rat. Thirdly, Jarden et al. [9, 10] showed that dexamethasone reduced K and Vp in some brain tumors. However, not all of the patients in each group were treated with dexamethasone at the time of the CT study. This nonuniformity of steroid treatment would contribute to additional variability in the measured values of K and Vp within each tumor group. In order to assess the effect of dexamethasone on the values of K and Vp, and elucidate the correlation between these two parameters and tumor types, it will be necessary to study the patient before and after dexamethasone treatment in a prospective study protocol [9, 10]. As discussed in the Model section, we have ignored the bulk flow term in the transport of iopamidol across BBB. The blood-brain transfer constant K is related to the actual PS and bulk flow by [29]: K = F ( 1 - exp(-
PS + f(1 + o)/2_) F
after injection. The loss of iopamidol in tumor region to the surrounding due to diffusion would be misinterpreted by the 2-compartment model as the lack of tracer because of lower BBB permeability, hence underestimating K. Conversely, K would be overestimated in the surrounding regions with intact BBB due to the influx of tracer from the tumor. In order to study the significance of diffusion on the estimated K values, we recalculated the K estimates by using data only up to 12 minutes when less tracer would have diffused out of the ROI. The estimated K values from 12-minute data were higher than those from 48-minute data by 10 to 40%. A better study protocol to minimize the inaccuracy in estimated K due to diffusion is one in which the data collection will be completed in 6 instead of 48 rain. As a conservative estimate of the diffusion coefficient (D) for iopamidol, we use the value of D-sucrose (7 x 10-6cm 2 s-1) [31]. Iopamidol is expected to have a lower D because of its higher molecular weight. Assuming a tortuosity factor (T) of 0.6 [30] for iopamidol in human brain, the mean diffusion distance (d) in two dimensions [32] in 6 rain is:
(7)
where f is a size-independent volume flux representing the bulk fluid flow rate and o is the reflection coefficient of the tracer. Nakagawa et al. [30] determined a bulk flow rate of 0.0008 ml/min/g in RG-2 gliomas in rats. Assuming the worst case of o(= 0) and an average K value we measured in tumor region, ie. 0.0273 ml/min/g, as PS. The apparent K (ie. with bulk flow term) would be 0.0272 ml/min/g whereas the 'true' K (ie. without bulk flow term) would have been 0.0268. Therefore, for the average tumor values we have found in this study, we may have overestimated K by 1.5% by ignoring the bulk flow term. Diffusion in the EES was not considered in this model. However, its effect can be clearly visualized in our patient studies. Figure 6 was a series of scans from a typical patient. It clearly showed that spreading of iopamidol due to diffusion from regions of enhancement into neighbouring nonenhanced regions occurred between 6 and 12 minutes
d = (4× D × T × 6rain) 1/2 = 0.78 mm Since the GE9800 CT scanner has a spatial resolution of 0.7mm, diffusion of iopamidol in brain in 6 min would have little effect on the K estimates. Patient movement is a technical limitation which may affect the accuracy of the K and Vp estimates. Movement in the (x-y) plane of the CT image was corrected by image registration as described above. However, movement and tilting in the longitudinal (z) direction could not be corrected and would lead to error in the estimation of K and Vp. In fact, movement in the z-direction of more than 2mm would lead to changes in anatomical landmarks present in 10 mm thick head slices taken before and after the movement. No such change was observed in the patient studies we presented. Therefore, error due to patient movement in z-direction was minimal and would not have a significant contribution to the overall errors of K and Vp estimates. Another factor which may cause error in the K
185
Fig. 6. A series of CT scans from a typical patient study. The spreading of iopamidol due to diffusion from regions of enhancement to surrounding nonenhanced regions occurred between 6 and 12 minutes after intravenous injection.
and Vp estimates is the undesirable side effects caused by the X-ray contrast agent itself. The X-ray contrast agent we used, Isovue 300, is a hyperosmolar solution (0.79 M) of iopamidol. Hyperosmolarity of plasma solutes is known to induce osmotic opening of BBB [1], increase cerebral blood flow (CBF) [33] as well as CBV [34]. Rapoport et al. [35] reported that carotid infusion of 1.4M arabinose solution for 30s into 300g rats for a dosage of 17 mmol/kg produced no significant elevation of K measured with [14C]sucrose compared to the control group. We used intravenous injection of Isovue 300 (0.79M) at a dosage of 0.79mmol/kg and therefore would expect negligible osmotic opening of BBB. Ravussin et al. [34] infused intravenously a 1.1 M
mannitol solution for 4min at a dosage of 5.5 mmol/kg into human subjects; they obtained a maximum increase in CBV of 14%. Direct scaling of their findings suggests that an increase in CBV of less than 2% would be expected in our situation. Assuming the extreme cause in which the increase of CBV occurs entirely by dilation of the capillaries, the increase of capillary surface area (S), hence K by Eq. 2, would be less than 2%. Hyperosmolar solutions had been shown to increase CBF. Jafar et al. [33] employed infusion of 1.1M mannitol with a dosage of 8.2mmol/kg in stroke patients; they observed an average CBF increase of about 25% in ischemic brain tissue. Even an increase of 25% of CBF in our case would raise K only by less than 1% according to Eq. 2 for
186 an average K value we have determined in tumors. This result was consistent with Dhawan et al. 's [8] report that K was independent of CBF.
Fund, Radiology Department, St. Joseph's Health Centre, London are gratefully acknowledged. The authors also acknowledged the technical assistance of Ms Rosemary Millar and Cathy Greenaway in the patient studies.
Conclusions An in vivo method of measuring the BBB permeability using iopamidol and X-ray CT scanning is described. Twelve brain tumor patients divided into 3 groups (ie. anaplastic astrocytoma, glioblastoma multiforme and metastases) were studied to determine the values of the blood-brain transfer constant of iopamidol (K) and cerebral plasma volume (Vv). There was a significant increase in K and Vp in tumor regions against the patients' own control. No significant difference of the mean tumor K or Vp was found among the three groups of patients possibly due to the effect of nonuniform steroid administration and the inherent variability in tumors. By displaying the estimated values with a false colour scale, red for K and blue for Vp, functional images of blood-brain transfer constant of iopamidol and cerebral plasma volume were obtained. These images, when overlaid on an ordinary CT image, can be used to correlate the distribution of K and Vp with anatomy in the CT image. Although we have only studied brain tumor patients, this method is useful in monitoring the involvement of the blood-brain barrier in various cerebral diseases and its responses to drug therapy.
Acknowledgements This project is supported by grants from the National Cancer Institute of Canada, the London UpJohn Neurosciences Program, the Up John Company, the Medical Research Council of Canada and Medical Systems Division, General Electric of Canada. W.T.I. Yeung is supported by a studentship from the Medical Research Council of Canada. Dr. Del Maestro is a recipient of an Ontario Ministry of Health Career Scientist Award. The financial assistance from the Brain Research Fund Foundation and the Academic Development
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Address for offprints: T.Y. Lee, Lawson Family Imaging Research Laboratories, The John P. Robarts Research Institute, 100 Perth Drive, London, Ontario, Canada N6A 5K8
