measurement of perfusion rate in human melanoma xenografts by

Measurement of Perfusion Rate in Human Melanoma Xenografts by Contrast-Enhanced Magnetic Resonance Imaging Heidi Lyng, Gro A. Dahle, Olav Kaalhus, Arne Skretting, Einar K. Rofstad

Reliable methods based on MRI for measurement of the perfusion rate in human tumors are highly warranted. Tumors of two amelanotic human melanoma xenograft lines were subjected to dynamic 'H MRI after i.v. administration of gadopentetate dimeglumine (Gd-DTPA). The aim was to investigate to what extent different perfusion parameters determined from the Gd-DTPA kinetics, i.e., the initial uptake rate, the maximal uptake, the decay rate, and the perfusion rate calculated by use of the Kety equation, can be used as a reliable estimate of tumor perfusion rate. Each parameter was calculated in dual; one calculation was based on relative signal intensity increase (RSII) in T,-weighted MR images and the other on Gd-DTPA concentration determined from the images. The perfusion parameters were compared with the perfusion rates determined from measurement of tumor uptake of =Rb or [14C]iodoantipyrine. The results showed that reliable estimates of tumor perfusion rate can be achieved from analysis of Gd-DTPA kinetics by use of the Kety equation. Gd-DTPA kinetics based on concentration might be used to achieve reliable estimates of absolute tumor perfusion rate, whereas reliable estimates of the relative perfusion rate might also be achieved from Gd-DTPA kinetics based on RSII. The initial uptake rate, the maximal uptake, and the decay rate of Gd-DTPA, however, are not reliable estimates of tumor perfusion rate, mainly because these parameters are highly influenced by the tumor extracellular volume fraction in addition to the perfusion rate. Key words: contrast-enhanced MRI; Kety equation; melanoma xenograft; perfusion rate.

INTRODUCTION

Perfusion rate is important for the growth, progression, and treatment response of tumors (for review, see 1, 2). Development of methods for measurement of the perfusion rate in human tumors is, therefore, highly warranted. MRI is a potentially useful method for this purpose because the method is noninvasive and provides three-dimensional information. The approach most com- monly used clinically involves dynamic imaging to record the kinetics of a freely diffusable contrast agent distributing in the extracellular space of the tumor after i.v. administration. Diffusion of the contrast agent into tumor leads to a decrease in the proton spin-lattice and

MRM 4&89-98 (1998) From the Departments of Biophysics (H.L., G.A.D., O.K., E.K.R.) and Med- ical Physics and Technology (A.S.), The Norwegian Radium Hospital, Oslo, Norway. Address correspondence to: Heidi Lyng, Ph.D., Department of Biophysics, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway. Received July 15, 1997; revised December 22, 1997; accepted December 24, 1997. This work was supported by the Norwegian Cancer Society.

Copyright 0 1998 by Williams & Wilkins All rights of reproduction in any form reserved.

0740-3194/98 $3.00

spin-spin relaxation times, T, and T2, that is linearly related to contrast-agent concentration for relevant contrast-agent doses (3). Most investigators have based their analyses on changes in the signal intensity of T,- or T,-weighted images rather than on changes in TI or T, per se, assuming that the changes in signal intensity are representative of contrast-agent concentration (4-6). However, signal intensity is cxponentially and not linearly related to T, and T, (3). High concentrations of contrast agent may occur at the microregional level in tumors since the contrast agents distribute only extracellularly, suggesting that such analyses can introduce significant errors in the contrast-agent kinetics. It is, therefore, uncertain whether the changes in signal intensity can be used to represent contrast-agent concentration in studies of tumor perfusion rates.

Several methods have been proposed to determine the perfusion rate of tumor tissue from contrast-agent kinetics. Kety (7) presented an equation based on the principle of material conservation that can be used to achieve an estimate of absolute tumor perfusion rate. However, the Kety equation has been of limited use because the method requires knowledge of the extraction fraction of the contrast agent and the contrast-agent kinetics in the arteries supplying the tumor in addition to the kinetics in the tumor. Relative parameters, e.g., the initial uptake rate, the maximal uptake, and the decay rate of the contrast agent in the tumor, have rather been used to represent the perfusion rate (4, 5,8-10). These parameters are determined by considering the contrast-agent kinetics in the tumor only and are, therefore, more practical to use in clinical routine. However, the relevance of these parameters as estimates of tumor perfusion rate is not clear.

In the present work, tumors of two amelanotic human melanoma xenograft lines grown in athymic mice were subjected to dynamic 'H MRI after i.v. administration of gadopentetate dimeglumine (Gd-DTPA). The aim of the study was to investigate to what extent different perfusion parameters determined from the Gd-DTPA kinetics, i.e., the initial uptake rate, the maximal uptake, the decay rate, and the perfusion rate calculated by using the Kety equation, can be used as a reliable estimate of tumor perfusion rate. The validity of using the signal intensity increase in TI-weighted MR images to represent Gd- DTPA concentration in studies of tumor perfusion rate was also investigated by calculating each parameter in dual; one calculation was based on relative signal intensity increase (RSII) and the other was based on concentration. Gd-DTPA concentration was determined from the MR images using the method described by Hittmair et al. (11). The kinetics of Gd-DTPA in the arteries supplying the xenografted tumors was determined from MR images of the left ventricle, and the extraction fraction

89

90 Lyng et al.

was determined graphically from the kinetics of Gd- DTPA in the arteries and the tumors. The perfusion parameters were compared with the perfusion rates determined from measurements of tumor uptake of "Rb and [14C]iodoantipyrine (IAP). The tumor lines were well suited for the present purpose; they differ significantly with respect to tumor perfusion rate and extracellular volume fraction (ECVF), and the tumors contain negligible necrosis at the volumes used here (12).

MATERIALS AND METHODS Mice and Tumor Lines

Female BALBlc-nuhu mice, 8-10 weeks old, were used. They were bred at the animal department of our institute and kept under specific-pathogen-free conditions at constant temperature (2446°C) and humidity (30-50%). Sterilized food and tap water were given ad libitum.

Two amelanotic human melanoma xenograft lines (A-07 and R-18) were included in the study. Xenografted tumors were initiated from exponentially growing monolayer cultures in passages 75-100 (12). Monolayer cells, cultured in RPMI-1640 medium supplemented with 13% fetal calf serum, 250 mg/liter penicillin, and 50 mg/liter streptomycin, were detached by trypsinization. Approx- imately 2.5 X l o 5 A-07 cells or 5.0 X 10" R-18 cells in 10 pl of Caz*- and Mgz'-free Hanks' balanced salt solution were inoculated intradermally in the flanks. Tumor volume, V, was calculated as

[ l l

where 1 and s are the longer and shorter of two perpen- dicular diameters, respectively. The diameters were measured with calipers. Tumors with diameters in the range of 5.0-11.0 mm, corresponding to volumes in the range of 100-600 mm3, were used.

The mice were kept under general anesthesia during MRI and measurement of uptake of sbRb and [14C]IAP. The anesthetic consisted of 80% Sombrevin (Gedeon Richter, Budapest, Hungary), 12% Hypnorm Vet (LEO, Helsingborg, Sweden), and 8% Stetsolid, 5 mg/ml (Dumex, Copenhagen, Denmafk), and was administered i.p. in doses of 0.01 ml/g bbdy weight. The body core temperature of the mice was kept at 36-38°C.

Tumor Uptake of 86Rb and ['4C]IAP

Reference tumor perfusion rates were determined by using established methods based on tumor uptake of 86Rb or [14C]IAP (13). 8"RbC1 or [14C]IAP (Amersham Int., Am- ersham, U.K.), 25 pCi dissolved in 0.2 ml of 0.9% NaC1, was injected into the tail vein of tumor-bearing mice. The tumors were excised 60-75 s after the injection; i.e., within the time period when the concentration of 86Rb or [14C]IAP in the tumor tissue was pseudostable (14), and weighed. The activity of 86Rb was counted in a well-type gamma counter (15). The tumors with [14C]IAP were placed in scintillation vials and prepared as described by Tozer and Shaffi (16). One ml of tissue solubilizer, Solu- ene-350 (Packard Instruments B.V., Groningen, The Netherlands), was added to the scintillation vials. The

vials were left at 50°C overnight before 10 ml of scintil- lant, Hionic-Fluor (Packard Instruments B.V., Groningen, The Netherlands), was added and the activity was counted in a scintillation counter. The perfusion rate per unit tumor weight (ml/g min) was calculated from the tumor uptake of "Rb or [14C]IAP; i.e., the percentage of the injected 86Rb or [14C]IAP taken up per gram of tumor tissue, and the cardiac output

1 [2 ]

A

where CO is the cardiac output (ml/min) and A is the tumor-to-blood partition coefficient of "Rb or [14C]IAP. Cardiac output was determined for each mouse as

Tumor perfusion rate = -*Tumor uptake. CO

CO = 0.762 - (BW)0.776 [31

where BWis the mouse body weight (17). The solubility of "Rb and [14C]IAP in the tumors was probably the same as in blood since the tumors did not contain detectable fat tissue (12). A value of 1 was, therefore, used for A of 86Rb and [14ClIAP.

Magnetic Resonance Imaging

MRI was performed in the central axial plane of the tumors by use of a 1.5 T Signa whole body tomograph (General Electric, CA, USA). A specially designed mouse probe similar to that described earlier (18) was used. The probe consisted of a double foil induction coil of the solenoidal type. The coil was wound around a 5-mm thick polyacrylamide tube with an inner diameter of 40 mm, in which the mice were placed for imaging. The Q factor of the probe was about 250. The probe was insu- lated with Styrofoam to prevent excessive heat loss in the mice during imaging. Contrast agent, 0.5 M Gd-DTPA (Schering, Berlin, Germany), was administered in the tail vein after the mice had been positioned in the magnet. The Gd-DTPA solution was diluted in 0.9% NaCl to a final concentration of 0.03 Mand injected in a bolus dose of 0.01 ml/g body weight by using a neoflon (24G) con- nected to a syringe through a thin polyethylene tubing. Gd-DTPA is an inert substance with a molecular weight of about 550 (19). The diffusion restrictions of Gd-DTPA in tumor interstitium are, therefore, insignificant.

The tumors were positioned in the isocenter of the tomograph. The mice were placed in the prone position with the body length axis transverse to the static field. The excitation field was horizontal, transverse to the static field. The phase encoding axis was set to the ver- tical direction to minimize occurrence of motion artifacts within the tumor region of the images. An image matrix of 256 X 128, a field of view of 8 X 8 cm, a scan thickness of 3 mm, and a number of excitations of 1 were used. A sagittal scan was used for tumor localization and to determine the position of the axial scan in a plane through tumor center. A TI-weighted spoiled gradient-recalled (SPGR) sequence, with a repetition time (TR) of 200 ms, an echo time (TE) of 6 ms, and a flip angle (a) of SOo, was used to record the kinetics of Gd-DTPA in the tumors. The use of a short TE ensured negligible influence of T, on the signal intensity. After a reference image was ob-

MRI and Tumor Pe*sion Rate 91

tained, Gd-DTPA was injected at a constant rate over 5 s, and postcontrast images were acquired continuously every 33 s in 10 min and thereafter every 2 min in 40 min. A proton density-weighted image was acquired before and 50 min after the injection of Gd-DTPA, using an SPGR sequence with a TR/TE/a of 200 ms/6 ms/lO". The proton density-weighted and Tl-weighted images were used to calculate the Gd-DTPA-induced increase in T, relaxation rate, as described below. A Tl-weighted and a proton density-weighted image of two calibration tubes, i.e., perspex tubes containing aqueous Gd-DTPA solutions of different concentrations, were obtained each day experiments were performed and were used to calculate tumor Gd-DTPA concentration from the increase in T, relaxation rate.

T,-weighted and proton density-weighted images were obtained from the left ventricle of three separate mice to determine the kinetics of Gd-DTPA in the arteries. The sequence parameters and imaging procedure were as described above, except that a TR of 50 ms was used. The T,-weighted images were acquired every 13 s in 10 min and thereafter every 2 min in 40 min.

Image Analysis

Region of interest, corresponding to the tumor or the left ventricle, was defined with a cursor in the images, as illustrated in Fig. 1. The tumor diameters derived from the MR images did not differ from those measured with calipers. Artifacts were occasionally seen in the images, probably because the SPGR sequence is highly sensitive to inhomogeneities in the radiofrequency pulse and in the magnetic field (20). The artifacts always occurred in the outer rim of the tumors, and the outer rim was, therefore, not included in the region of interest (Figs. l a and Ic). Region of interest was held constant in size and position in all images of a dynamic series. Median signal intensity was calculated for each region of interest and used in the calculation of RSII and Gd-DTPA concentration. The RSII was calculated for each postcontrast image as

[41 AS = * 100

where A S is the RSII and SRef and S,, are the signal intensities in the reference and the postcontrast T,- weighted image, respectively.

The Gd-DTPA concentration in the tumors and left ventricles was determined from the increase in T, relaxation rate, as described previously (11). Shortly, the increase in T, relaxation rate was calculated for each postcontrast image as

sPC - SRsf

SRef

1 SMax - SRef ARl = const . C- -* K - TR ln( SMax - Spc) 15]

where AR, is the increase in T, relaxation rate, const is a proportionality constant, C is the Gd-DTPA concentration, K is a flip angle correction factor, and SMax is the maximal signal intensity for the Tl-weighted SPGR sequence, i.e., the signal intensity achieved if full relaxation had occurred during the time between excitation and signal detection. A value of 1.13 was used for K (11).

S,, depends on the proton density and the flip angle, and was determined from the proton density-weighted images:

sar=lo"

sin 10' sin 80" I61 SMax = N(H) sin 80" = -.

where N(H) is the proton density and S,=,,, is the average signal intensity based on the two proton density- weighted images. The value of const in Eq. [51 was determined from the Tl-weighted and the proton density- weighted images of the two calibration tubes

[71

where (ARJCalib refers to the difference in Tl relaxation rate between the two calibration tubes, and C, and C, are the Gd-DTPA concentrations. (ARl)Calib was calculated from Eqs. [5] and [6] by using the signal intensity of the calibration tube with the lowest and the highest contrast- agent concentration as SRef and Spcl respectively. The use of aqueous Gd-DTPA rather than Gd-DTPA dissolved in blood plasma or tumor extracellular fluid to determine const is justified by the observation that the relaxivity of Gd-DTPA is similar in water and tissue fluids (3). A minor systematic error in const, if present, would not have influenced the numerical values of the perfusion parameters determined in the present work significantly.

RSII and Gd-DTPA concentration, calculated for region of interest in each image of a dynamic series, were plotted as a function of time after injection. These RSII and concentration curves were subjected to further analysis for determination of perfusion parameters. Analysis of Contrast-Agent Kinetics

The initial uptake rate, PERFInit, the maximal uptake, PERF,,,, and the decay rate, PERFDecay, of Gd-DTPA were determined for each tumor by use of an exponential function, g(t), fitted to the tumor RSII and concentration

g( t ) = A * (1 - e-"t) e-b* [81

where t is time after injection of Gd-DTPA and A, a, and b are constants (8). The derivative of g(t) at time zero was used to represent PERFhit, the value of g(t) at the time when the derivative of g(t) was zero was used to represent PERF,,, and b was used to represent PERFDecay.

The perfusion rate, PERFKety, was determined for each tumor by use of the Kety equation fitted to the tumor RSII and concentration curves

curves

J O

where CJT) is the RSII or Gd-DTPA concentration in the tumor tissue at time T, E is the extraction fraction of Gd-DTPA, PERF,,,, is the perfusion rate per unit tumor weight (ml/g min), C,(t) is the time course of the RSII or Gd-DTPA concentration in the arteries, i.e., the arterial input function, and A is the tumor-to-blood partition coefficient of Gd-DTPA (7). CJt) was determined by use of a double exponential function fitted to the left ventri-

92 Lyng et al.

FIG. 1. Reference (a, b) and postcontrast (c, d) T,-weighted MR images from the axial plane through the center of an A-07 human melanoma xenografted tumor (a, c) and the left ventricle (b, d) of a mouse. The postcontrast images were obtained 7 min (c) and immediately (d) after the injection of Gd-DTPA. Region of interest is outlined in each image.

cle RSII and concentration curves addition to the Gd-DTPA concentration (22). Equation [lo] rather than the actual experimental data was used to represent the arterial input function to minimize possible errors due to blood movement. Numeric values of A and E - PERF,,,,, were determined by fitting Eq. [91 to the tumor RSII and concentration curves.

The h represents the solubility of Gd-DTPA in tumor tissue relative to blood. Since Gd-DTPA distributes only

C,(t) = X. e-"' + Y - e-fl [ l o ]

where X , x, Y, and y a r e constants (21). A mean C,(t) was calculated from the values of X , x, Y, and y for three mice and used to represent C,(t) in Eq. [9]. The signal intensity of the ventricle is influenced by blood movement in

MRI and Tumor Perfusion Rate 93

extracellularly, A depends on the tumor ECVF, the blood plasma volume fraction (BPVF), and the solubility of Gd-DTPA in the tumor extracellular fluid and in the blood plasma. 'The solubility of Gd-DTPA in the extracellular fluid of the tumors studied here was about the same as in blood plasma because Gd-DTPA is a highly hydrophilic substance (19) and none of the tumors con- tained detectable fat tissue (12). The A can, therefore, be expressed as

ECVF

BPVF A=------ [111

The BPVF differs little among individual BALBIc-nuhu mice. Consequently, differences in A among individual tumors should mainly reflect differences in tumor ECVF.

The E is defined as the degree of diffusional equilibrium between the blood plasma and the tumor extracellular fluid during passage of blood through the tumor capillary network. The ratio of tumor extracellular fluid to blood plasma Gd-DTPA concentration thus represents a reasonable estimate of E (23). Previous studies have indicated that the extraction fraction is not constant during the time period when tissue uptake of tracers occurs; it increases with increasing time and approaches a constant value asymptotically (24,251. An average value of E based on the whole uptake period was, therefore, determined for each tumor. Concentration curves for the tumor extracellular fluid and blood plasma were established by dividing the tumor concentration curves by A BPVF and the arterial input function by BPVF, assuming a value of 0.4 for BPVF. The Ewas calculated as

where I , and Ib are the integrals of the concentration curves for tumor extracellular fluid and blood plasma, respectively, from time zero to the time when the blood plasma and tumor extracellular fluid concentrations were the same (Fig. 2). A value of E based on RSII was also determined for each tumor by performing a similar analysis of RSII curves. It should be noted that the choice of numeric value of BPVF had no influence on the calculated value of E, since the ratio of the arterial input function divided by BPVF to the tumor concentration curves divided by A BPVF was considered. The E of Gd-DTPA depends on tumor ECVE the time period before diffusional equilibrium between tumor extracellular fluid and blood plasma occurs is longer in tumors with a high ECVF than in tumors with a low ECVF because more Gd-DTPA is taken up in the former tumors. This leads to a lower E in tumors with a high ECVF. Consequently, differences in E among individual tumors should partly reflect differences in tumor ECVF. The PERFKcty was determined from E PERFKety and E.

Phantoms

To investigate the linearity between RSII and Gd-DTPA concentration, MR images were acquired from phantoms, i.e., perspex tubes containing Gd-DTPA solutions with known concentrations, using the same sequence param-

0 E E u 1.5

. . . . . . . Tumor extracellular fluid

t: 0 .- -w !! -w 6 1.0 - 0 c 0 0

0.5 - 4 m 0

0.0 - I

I I I t I I

0 2 4 6 8

Time [min]

FIG. 2. Schematic diagram of Gd-DTPA concentration in blood plasma and tumor extracellular fluid versus time after a bolus injection of Gd-DTPA. I , and It are the integrals of the concentration curves for blood plasma and tumor extracellular fluid, respectively, from time zero to the time when blood plasma and tumor extracellular fluid concentrations are the same.

eters as for the tumors and left ventricles. Moreover, the Gd-DTPA concentrations of the phantoms were calculated from the MR images by using the method described above, to verify that the calculated values represented the true Gd-UTPA concentrations. Concentrations in the range from 0.0 to 3.5 mmol/liter were used. Two series of images were considered, one with a TR of 50 ms and one with a TR of 200 ms.

Statistics

Statistical comparisons of data were performed by using a two-tailed t test. Significant correlations between two parameters were searched for by linear regression analysis. A significance criterion of P < 0.05 was used.

RESULTS Tumor Uptake of 86Rb and ['4C]IAP

Tumor perfusion rate determined from the uptake of "Rb was highly consistent with that determined from the uptake of [14C]IAP. The mean (2SE) perfusion rate (ml/g min) was 0.17 (?0.01) for the A-07 and 0.14 (20.01) for the R-18 line, i.e., 1.2 times higher for A-07 than for R-18, regardless of whether the uptake of 86Rb or [l*C]IAP was considered ( P < 0.05) (Table 1).

RSll and Gd-DTPA Concentration

RSII calculated from phantom MR images was analyzed against true phantom Gd-DTPA concentration for a TR of 50 and 200 ms. A continuously curved relationship was found, regardless of whether a TR of 50 or 200 ms was used (data not shown), consistent with the well-established curved relationship between signal intensity and

94 Lyng et al.

TI (3). Thus, there was no linear relationship between RSII and Gd-DTPA concentration for the SPGR sequence used here. A linear relationship was, however, found between Gd-DTPA concentration calculated from phantom MR images and true phantom Gd-DTPA concentration, when both a TR of 50 and 200 ms were used (data not shown). The relationship was linear for concentrations up to 3.5 mmol/liter at a TR of 50 ms and for concentrations up to 2.0 mmol/liter at a TR of 200 ms (P < 0.001).

The Gd-DTPA dose administered to the mice was suf- ficiently high to ensure a satisfactory signal-to-noise ratio for the tumors with the lowest Gd-DTPA uptake. Gd- DTPA concentrations up to 0.4 mmol/liter were achieved in tumors, and the highest value of the arterial input function was 1.7 mmol/liter. The tissue concentrations achieved in the present work were, therefore, well within the range where calculated and true concentrations were linearly related. However, significant deviations from linearity were introduced by using RSII as a measure of Gd-DTPA concentration. This is illustrated in Fig. 3, showing RSII versus calculated Gd-DTPA concentration for the arterial input function (Fig. 3a) and a tumor (Fig. 3b). Continuously curved relationships were found, consistent with the results hom the phantom studies.

Perfusion Parameters

RSII and concentration curves for an A-07 and an R-18 tumor are shown in Fig. 4. The Gd-DTPA kinetics based on RSII differed little from that based on concentration. All tumors showed a sharp initial increase in the RSII and concentration due to uptake of Gd-DTPA from the supplying arteries. The initial uptake rate differed little among tumors of the same line. After a time period ranging from 0.5 to 5.5 min for the R-18 tumors and from 4.0 to 9.0 min for the A-07 tumors, the RSII and concentration reached a maximal level and thereafter decreased, approaching the value of the reference image. No pseudostable period for Gd-DTPA occurred in the tumors.

Data similar to those presented in Fig. 4 were used in Eq. [81 to determine PERFInit, PERF,,,, and PERFD,,,,, for each tumor (Table 1). Satisfactory fitting of Eq. [8] to the

Table 1 Perfusion Parameters for Human Melanoma Xenograft Lines

300

250

200

150

I00

50

0

6 a 0.0 0.4 0.8 1.2 1.6 2.0 =

Tumor 120

100

80

60

40

20

0

0.0 0.1 0.2 0.3 0.4

b Gd-DTPA concentration [mmol/l]

FIG. 3. Relative signal intensity increase versus Gd-DTPA concentration for the arterial input function (a) and a typical A-07 human melanoma xenografted tumor (b). Each point is based on a single MR image in a dynamic series. ------, linear curve through the first two data points.

Tumor line A-07, R-18'

Isotopes Uptake of "Rb [mug min] 0.14 ? 0.01 Uptake of ['4C]IAP [ml/g min] 0.14 5 0.01

Gd-DTPA kinetics based on RSlld PfRF,,,,, [%/min] 72.2 t 7.5 325.5 t 16.0 PERF,, [%I 76.0 t 11 .O 30.0 2 8.0

PERF,,, [ml/g min] 0.52 * 0.03 0.38 5 0.02

PfRF,,,, [mmol/liter min] 0.17 2 0.02 0.86 t 0.16 Pf RF,,, [m mol/l iter] 0.07 2 0.02 PERFDecay [min-'1 0.024 * 0.010 0.024 t 0.01 0 PERF,,,, [ml/g min] 0.18 0.01 0.14 2 0.01

0.17 2 0.01' 0.17 * 0.01

PERF,--,, [min-'1 0.020 t 0.010 0.022 2 0.010

Gd-DTPA kinetics based on concentration

0.22 t 0.05

a Based on 13-15 tumors Based on 15-1 6 tumors Mean ? SE Relative signal intensity increase

data was generally achieved, as illustrated by the stippled curves in Fig. 4. PERFInit based on RSII was 4.5 times lower for A-07 than for R-18, whereas PERFIni, based on concentration was 5.1 times lower for A-07 than for R-18 (P < 0.0001) [Table 1). PER- F,, based on RSII was 2.5 times higher for A-07 than for R-18, whereas PERF,, based on concentration was 3.2 times higher for A-07 than for R-18 (P < 0.0001) (Table 1). PERFDecay was not different for the two lines (Table 1). Thus, the relative

MRI and Tumor Pedusion Rate

70 -

60 -

5 0 -

95

.r .. - a

'.a

e. ' a.

'

a 0 0.24

= 5

g 0.20 E .- 0.16 E 5 0.12

a a I- n 0.04

0.00 B

Y

+ - 0 c

0.08

0 10 20 30 40 50 60

b Time [minl

FIG. 4. Relative signal intensity increase (a) and Gd-DTPA concentration (b) versus time after i.v. administration of Gd-DTPA for a typical A-07 and a typical R-18 human melanoma xenografted tumor. Each point is based on a single MR image in a dynamic series. ------, exponential curves fitted to the data.

difference in PEIWInit, PERF,,, or PERFDecay between the tumor lines was not consistent with the relative difference in perfusion rate determined from measurement of tumor uptake of 86Rb or [I4C]IAP, regardless of whether the RSII or concentration curves were considered in the analysis.

The Gd-DTPA kinetics of the left ventricle differed little among mice. The mean of the left ventricle curves was determined and used to represent the mean arterial input function. The mean RSII and concentration arterial input functions are shown in Fig. 5, together with RSII and concentration curves of an A-07 and an R-18 tumor. Curves showing the SE in the arterial input functions are also plotted. The arterial input functions showed a rapid decrease immediately after the injection of Gd-DTPA followed by a slower decrease approaching zero after 50 min. The initial uptake of Gd-DTPA in the tumors was not delayed compared with the arterial input function, suggesting that the time difference in the Gd-DTPA kinetics between

the left ventricle and the arteries supplying the tumors was less than the temporal resolution of the dynamic MR series. This is consistent with a recirculatory time for blood in mice of only about 20 s (17).

Data similar to those presented in Fig. 5 were used in Eq. [9] to determine E PERF,,,, and A for each tumor. Satisfactory fitting of Eq. [9] to the tumor RSII and concentration curves was generally achieved with only one combination of h and E * PERFKety for each curve. The fitted curves were usually indistinguishable from the experimental data the first 10 min of the observation period. E * PERFKety did not differ between the tumor lines (Table 2). h based on RSII was 3.2 times higher for

Mean arterial input Mean arterial input +/- SE 8 m

--c A-07 150 --C R-18 C .-

a 2.0

5 5

i? 1.5

c 0 .- -I-

t! c 1.0 8 8

4 s

44

C

2 0.5 I-

0.0

Mean arterial input Mean arterial input +/- SE

--t A-07

0 10 20 30 40 50

Time [min] b

FIG. 5. Relative signal intensity increase (a) and Gd-DTPA concentration (b) versus time for arteries L, mean; ------, mean ? SE), a typical A-07 and a typical R-18 human melanoma xenografted tumor. Each point is based on a single MR image in a dynamic series. The artery curves represent the arterial input functions and are described by Xe-* + where X, x, Y, and y(meanrSE)are291 ~ 1 1 1 , 2 . 4 ~ 0 . 9 , 9 8 . 6 ~ 3 . 4 , a n d 0 . 0 4 ~ 0.02, respectively (a), and 5.8 ? 2.2, 4.4 t 1.1, 0.7 ? 0.2, and 0.05 ? 0.02, respectively (b). The use of the stippled curves to represent the arterial input function in the Kety equation resulted in values for PERF,,, that differed <45% (RSII) and 15% (concentration) from the values achieved when using the mean arterial input function.

96

0 - a

Lyng et al.

1 I, ’ I I I I I I I I

A-07 than for R-18, whereas A based on concentration was 4.0 times higher for A-07 than for R-18 ( P < 0.05) (Table 2). The difference in A between the tumor lines reflected mainly a difference in the tumor ECVF [Eq. [Ill). The ECVF of the tumor lines has been determined previously from the volume and density of the tumor cells (unpublished results). These measurements showcd that A-07 had about 4.0 times higher ECVF than R-18. The A based on concentration was, therefore, consistent with earlier studies of tumor ECVF performed in our laboratory.

The tumor RSII and concentration curves were divided by A BPVF, and the arterial input functions were divided by BPVF to compare the kinetics of Gd-DTPA in the tumor extracellular fluid and blood plasma. This is illustrated in Fig. 6 for the same tumors as those depicted in Fig. 5 . During the first minutes after the injection of Gd-DTPA, the blood plasma concentration was higher than the concentration in the tumor extracellular fluid, and Gd-DTPA diffused into the tumors (Fig. 6b). The extracellular fluid concentration reached a maximal level approximately at the time when the plasma and extracellular fluid concentrations were the same. After this time, the plasma concentration was always lower than the extracellular fluid concentration, and Gd-DTPA diffused out of the tumors. The kinetics based on RSII was similar to that based on Gd-DTPA concentration [Fig. 6a).

Data similar to those presented in Fig. 6 were used in Eq. [12] to determine E for each tumor. E based on RSII was 1.3 times higher for R-18 than for A-07, whereas E based on concentration was 1.4 times higher for R-18 than for A-07 ( P < 0.05) (Table 2). The difference in E between the tumor lines reflected partly a difference in the tumor ECVF, as explained above. The lower E of A-07 than of R-18 was, therefore, consistent with the higher

The mean ( t S E ) PERFKcty (ml/g/min) based on RSII was 0.52 (20.03) for A-07 and 0.38 (20.02) for R-18 [Table 11, i.e., 1.4 times higher for A-07 than for R-18 (P < 0.05). The numeric values of this parameter were higher than the perfusion rates determined from measurement of tumor uptake of “Rb or [14C]IAP. However, the relative difference in the parameter between the lines was consistent with the relative difference in the perfusion rate determined from measurement of tumor uptake of “Rb or [14C]IAP. The mean (2SE) PERFKety (ml/g/min) based on concentration was 0.18 (20.01) for A-07 and 0.14 (20.01) for R-18 (Table I), i.e., 1.3 times higher for A-07 than for R-18 ( P < 0.05). The numeric values of this parameter were almost identical to the perfusion rates

Table 2

ECVF of A-07,

- 500

s a $ 400 C .-

- A-07 extracellular fluid

4.0 = L

E - 3.0 C 0 .- * !!! cu 2.0

4- C

0 C 0 0

2 1.0 6 s

0.0

- A-07 extracellular fluid - R-18 extracellular fluid

I I I I I I I I

0 I 0 20 30 40 50

b Time [min]

FIG. 6. Relative signal intensity increase (a) and Gd-DTPA concentration (b) versus time for blood plasma and the extracellular fluid of a typical A-07 and a typical R-18 human melanoma xenograft4 tumor. The tumors are the same as those depicted in Fig. 5. Each point is based on a single MR image in a dynamic series.

determined from measurement of tumor uptake of 86Rb or [14C]IAP, regardless of whether A-07 or R-18 was considered.

DISCUSSION

The maximal tumor uptake of 86Rb or [14C]IAP was used to determine a reference for perfusion rate in the present work. Highly consistent results were achieved with the

two substances, in accor- Parameters of the Kety Equation for Human Melanoma Xenograft Lines

Gd-DTPA kinetics based on relative signal intensity

increase

Gd-DTPA kinetics based on concentration

Tumor line A-07“ R-1 8b A-07= R-18’ h 0.95 i 0.04“ 0.30 z 0.03 0.44 2 0.01 0.11 i 0.03 E * PERFKety [mVg min] 0.38 * 0.02 0.35 i 0.03 0.13 -c 0.01 0.12 i 0.01 E 0.72 % 0.01 0.92 + 0.03 0.71 2 0.01 0.85 5 0.03

a Based on 13 tumors Based on 15 tumors Mean ? SE

dance with previous reports (26). The kinetics of “Rb in tumors is similar to that of

stances diffuse easily across capillary walls and cell membranes and distribute throughout the whole tumor. “Rb is a water-soluble substance that is trapped intra- cellularly, whereas [14C11AP

[14C]IAP (13). Both sub-

MRI and Tumor Pqfuusion Rate 97

is a lipid-soluble substance with no diffusion restrictions at membranes. The large distribution volume of the substances ensures that a pseudostable period of several min, in which the concentration of 86Rb and [14C]IAP remains fairly constant, occws before the substances diffuse out of the tumor (13, 14). Diffusion equilibrium within the tumor is established during the pseudostable period. The maximal uptake of '"Rb and [14C]IAP, therefore, depends only on tumor perfusion rate and cardiac output.

The kinetics of Gd-DTPA in tumors differs from that of "Rb and ['*C]IAP. Gd-DTPA does not penetrate cell membranes and distributes only within the extracel lular space (19). The small distribution volume of the substance entails that no pseudostable period, in which the Gd-DTPA concentration remains fairly constant, is established. Due to the steep arterial input function, Gd-DTPA diffuses out of the tumor immediately after the extracellular fluid concentration has reached the concentration in blood plasma. The Gd-DTPA kinetics, therefore, depends on tumor ECVF and the arterial input function in addition to tumor perfusion rate.

PERF,,, was not consistent with tumor perfusion rate determined from the maximal uptake of 86Rb or [14C]IAP in the present work. The A-07 had a higher ECVF than R-18. More Gd-DTPA could, therefore, be taken up in A-07 than in R-18 tumors. The PERFM, was 2.5 (RSII) and 3.2 [concentration) times higher and the ECVF was 4.0 times higher for A-07 than for R-18, suggesting that the difference in PERF,, between the lines was mainly due to a difference in the ECVF rather than in the perfusion rate. Maximal uptake of Gd-DTPA is, therefore, not a reliable estimate of the relative perfusion rate in tumors. In fact, maximal uptake of Gd-DTPA has been used previously to determine cellular volume fraction in tumors and normal tissues (27).

Furthermore, neither PERFIni, nor PERFDecay was consistent with tumor perfusion rate determined from the maximal uptake of 86Rb or [14C]IAP. The uptake rate of Gd-DTPA in tumors depends on the extraction fraction, and hence on the ECVF, in addition to the perfusion rate. Thus, PERFrnil was lower for A-07 than for R-18, probably because this line had a higher ECVF. The initial uptake rate of Gd-DTPA is, therefore, not a reliable estimate of relative tumor perfusion rate. It should also be noted that the numeric value of the initial uptake rate is highly sensitive to the method of calculation. Initial uptake rates determined from only the precontrast and a few postcontrast MR images of the tumors differed from the numerical values presented in this report, depending on the number of postcontrast images included in the calculations [data not shown).

The decay rate of Gd-DTPA in tumors depends on the steepness of the arterial input function during the decay in addition to the perfusion rate. Tumors with a low ECVF reach maximal Gd-DTPA uptake earlier than tumors with a high ECVF. Consequently, since the steepness of the arterial input function decreases with time, the arterial input function has a stronger influence on the Gd-DTPA decay rate in tumors with a low ECVF than in tumors with a high ECVF. No difference in PEWDecay between the tumor lines was found in the present work.

R-18 had a lower ECVF than A-07, and hence a steeper arterial input function during the decay. Howcver, the perfusion rate was also lower for R-18, leading to the same decay rate for the two lines. The decay rate of Gd-DTPA is, therefore, not a reliable estimate of the relative pcrfusion rate in tumors.

In contrast to the calculation of PERF,,,, PERFrnit, and PERFDecay, the calculation of PERFKety takes into consid- eration the arterial input function and the tumor ECVF. Thus, both A and E in the Kety equation depend on ECVF. PERFKety based on RSII was found to give reliable estimates of relative tumor perfusion rate, whereas PERFKety based on concentration was even found to give reliable estimates of the absolute perfusion rate. The use of RSII to represent concentration in the calculations underesti- mated the concentration significantly. The error was most pronounced for tissues with a high Gd-DTPA uptake and was, therefore, larger for the arterial input function than for the tumors. Consequently, PERFKcty based on RSII was increased compared with the true tumor perfusion rate. RSII should, therefore, not be used to represent concentration in studies where estimates of absolute perfusion rates are needed. On the other hand, RSII might be used in studies where the perfusion rates of different tumors are compared. It should be emphasized, however, that the use of RSII rather than concentration in the analysis leads to considerable uncertainties in the numerical values of A and E.

The use of the Kety equation to determine tissue perfusion rate is based on the assumption that the capillary structure of the tissue is homogeneous (7). The capillary structure in most tumors is heterogeneous, and regions with large intercapillary distances and high resistance to flow frequently occur (1). The heterogeneity in the capillary structure was minimized in the present work by only including tumors without necrosis. Herscovitch et al. (28) found that heterogeneity in the capillary structure of brain tissue caused an error in the calculated perfusion rates of <3.7%. Although the heterogeneity in the capillary structure of most tumors is somewhat larger than that of brain tissue, the error in the perfusion rate caused by capillary heterogeneity is probably not unacceptably large, at lcast for tumors without necrosis.

The initial uptake rate, the maximal uptake, and the decay rate of Gd-DTPA have kequently been used as estimates of relative tissue perfusion rate in clinical studies (4, 5, 29-31). The present work suggests that these parameters are influenced significantly by the ECVF in addition to the perfusion rate. The use of these parameters as estimates of relative tumor perfusion rate can lead to erroneous ranking of tumors, as tumors differ consid- erably in ECVF due to differences in cell density and amount of necrosis and stroma. However, it cannot be excluded that the initial uptake rate, the maximal uptake, or the decay rate of Gd-DTPA may be useful perfusion parameters in studies of specific normal tissues, as specific normal tissues differ little in ECVF from patient to patient. Thus, Wilke et al. (32) found a significant correlation between perfusion rate and initial increase in signal intensity after administration of Gd-DTPA in a study of the dog myocardium.

98 Lyng et al.

In contrast, the present work suggests that analysis of tumor Gd-DTPA kinetics by use of the Kety equation results in reliable estimates of tumor perfusion rates, at least for tumors without significant necrosis. Gd-DTPA kinetics based on concentration might be used to achieve reliable estimates of absolute tumor perfusion rate, whereas reliable estimates of the relative perfusion rate might also be achieved from Gd-DTPA kinetics based on RSII. The procedure used here can easily be imple- mented clinically; the arterial input function can be determined from MR images of the left ventricle or a large artery, and the extraction fraction can be determined graphically from the arterial input function and the Gd- DTPA kinetics in the tumor. The present method can be improved in clinical studies by determining the arterial input function individually for each patient using the dual flash technique (33) and by enhancing the quality of the MR images of the left ventricles or arteries using flow compensation (34). Moreover, possible errors in the perfusion rate due to the presence of necrosis may be minimized by using a two-component model to separate the Gd-DTPA kinetics in viable and necrotic tissue (9).

ACKNOWLEDGMENT

The authors thank Kristin Nilsen for skillful technical assis- tance.

REFERENCES 1. R. K. Jain, Determinants of tumor blood flow: a review. Cancer Res.

2. P. Vaupel, F. Kallinowski, P. Okunieff, Blood flow, oxygen and nu- trient supply, and metabolic microenvironment of human tumors: a review. Cancer Res. 49, 6449-6465 (1989).

3. K. M. Donahue, D. Burstein, W. J. Manning, M. L. Gray, Studies of Gd-DTPA relaxivity and proton exchange rates in tissue. Magn. Re- son. Med. 32, 66-76 (1994).

4. K. Fujii, N. Fujita, N. Hirabuki, T. Hashimoto, T. Miura, T. Kozuka, Neuromas and meningiomas: evaluation of early enhancemenl with dynamic MR imaging. Am. I. Neuro. Radio]. 13, 1215-1220 (1992).

5. H. J. Feldmann, K. Sievers, J. Fuller, M. Molls, E. Lohr, Evaluation of tumor blood perfusion by dynamic MRI and CT in patients undergo- ing thermoradiotherapy. Eur. I. Radio]. 16, 224-229 (1993).

6. T. Nagele, D. Petersen, U. Klose, W. Grodd, H. Opitz, E. Gut, J. Martos, K. Voigt, Dynamic contrast enhancement of intracranial tumors with snapshot FLASH MR imaging. Am. J. Neuro. Radiol. 14, 89-98 (1993).

7. S. S. Kety, Theory of blood-tissue exchange and its application to measurement of blood flow. Mefh. Med. Res. 8, 223-227 (1960).

8. C. A. Belfi, L.-L. Ting, S . J. Hassenbusch, M. Tefft, F. Q. H. Ngo, Determination of changes in tumor blood perfusion after hydralazine treatment by dynamic paramagnetic-enhanced magnetic resonance imaging. Int. J. Radiat. Oncol. Biol. Pbys. 22, 477-482 (1992).

9. F. Goda, G. Bacic, J. A. O’Hara, B. Gallez, H. M. Swartz, J, F. Dunn, The relationship between partial pressure of oxygen and perfusion in two murine tumors after x-ray irradiation: a combined gadopentetate dimeglumine dynamic magnetic resonance imaging and in vivo elec- tron paramagnetic resonance oximetry study. Cuncer Res. 56, 3344- 3349 (1996).

10. M.-Y. Su, 2. Wang, G. M. Roth, X. Lao, M. K. Samoszuk, 0. Nalcioglu, Pharmacokinetic changes induced by vasomodulators in kidneys, livers, muscles, and implanted tumors in rats as measured by dynamic Gd-DTPA enhanced MRI. Mugn. Reson. Med. 36, 868-877 (1996).

11. K. Hittmair, G. Gomiscek, K. Langenberger, M. Recht, H. Imhof, J. Kramer, Method for quantitative assessment of contrast agent uptake in dynamic contrast-enhanced MRI. Magn. Reson. Med. 31, 567-571 (1994).

12 . E. K. Rofstad, Orthotopic human melanoma xenograft model systems

48, 2641-2658 (1988).

for studies of tumor angiogeuesis, pathophysiology, treatment sensi- tivity, and metastatic pattern. Br. I. Cancer 70, 804-812 (1994).

13. L. K. Appelgren, Methods of recording tumor blood flow, in “Tumor Blood Circulation: Angiogenesis, Vascular Morphology and Blood Flow of Experimental and Human Tumors” (H.4 Peterson, Ed.), pp. 87-101, CRC Press, Inc., Boca Raton, FL, 1979.

14. 6. D. Zanelli, J. F. Fowler, The measurement of blood perfusion in expen- mental tumors by uptake of 06Rb. Cancer Res. 34, 1451-1456 (1974).

15. H. Lyng, A. Skretting, E. K. Rofstad, Blood flow in six human melanoma xenograft lines with different growth characteristics. Cancer Res. 52, 584-592 (1992).

16. G. M. Tozer, K. M. Shaffi, Modification of tumor blood flow using the hypertensive agent, angiotensin 11. Br. 1. Cancer 67, 981-988 (1993).

17. H. M. Kaplan, N. R. Brewer, W. H. Blair, Physiology, in “The Mouse in Biomedical Research (H. L. Foster, J. D. Small, J. G. Fox, Eds.). pp. 247-292, Academic Press, New York, 1983.

18. E. K. Rofstad, E. Steinsland, 0. Kaalhus, Y. B. Chang, B. Hevik, H. Lyng, Magnetic resonance imaging of human melanoma xenografts in vivo: proton spin-lattice and spin-spin relaxation times versus frac- tional tumor water content and fraction of necrotic tumor tissue. Int. J. Radiat. B i d . 65, 387-402 (1994).

19. H.-J. Weinmann, R. C. Brasch, W.-R. Press, G. E. Wesbey, Character- istics of gadolinium-DTPA complex: a potential NMR contrast agent. Am. J. Roentgenol. 142, 619-624 (1984).

20. E. M. Haacke, J, A. Tkach, Fast MR imaging: techniques and clinical applications. Am. J, Roentgenol. 155, 951-964 (1990).

21. L. D. Diesbourg, F. S. Prato, G. Wisenberg, D. J. Drost, T. P. Marshall, S. E. Carroll, B. O’Neill, Quantification of myocardial blood flow and extracellular volumes using a bolus injection of Gd-DTPA: kinetic modeling in canine ischemic disease. M a p . Reson. Med. 23, 239- 253 (1992).

22. L. Axel, Blood flow effects in magnetic resonance imaging. Am. J . Roentgenol. 143, 1157-1166 (1984).

23. C. Crone, D. G. Levitt, Capillary permeability to small solutes, in “Handbook of Physiology,” Vol. 4 (E. M. Renkin, C. C. Michel, Eds.), pp. 411-466, American Physiology Society, Bethesda, MD, 1984.

24. J. A. Johnson, T. A. Wilson, A model for capillary exchange. Am. J. Physiol. 210, 1299-1303 (1966).

25. W. W. Eckman, R D. Phair, J. D. Fenstermacher, C. S. Patlak, C. Kennedy, L. Sokoloff, Permeability limitation in estimation of local brain blood flow with [“’Clantipyrine. Am. J. Physiol. 229, 215-221 (1975).

26. I. Kjartansson, Tumor circulation. An experimental study in the rat with a comparison of different methods for estimation of tumor blood flow. Actu Cbir. Scand. [Suppl.) 471, 1-74 (1976).

27. K. M. Donahue, R. M. Weisskoff, D. J. Parmelee, R. J. Callahan, R. A. Wilkinson, J. B. Mandeville, B. R. Rosen, Dynamic Gd-DTPA enhanced MRI measurement of tissue cell volume fraction. Magn. Re- son. Med. 34,423-432 (1995).

28. P. Herscovitch, J. Markham, M. E. Raichle, Brain blood flow measured with intravenous H,150. I. Theory and error analysis. J. Nucl. Med. 24, 782-789 (1983).

29. D. Burstein, E. Taratuta, W. J. Manning, Factors in myocardial “perfusion” imaging with ultrafast MRI and Gd-DTF’A administration. Mogn. Reson. Med. 20, 299-305 (1991).

30. N. A. A. Matheijssen, A. de Roos, E. E. van der Wall, J. Doornbos, P. R. M. van Dijkman, A. V. G. Bruschke, A. E. van Voorthuisen, Acute myocardial infarction: comparison of T,-weighted and Tl- weighted gadolinium-DTPA enhanced MR imaging. Magn. Reson. Med. 17, 460-469 (1991).

31. F. P. van Rugge, J. J. Boreel, E. E. van der Waal, P. R. M. van Dijkman, A. van der Laarse, J. Doornbos, A. de Roos, J. A. den Boer, A. V. G. Bruschke, A. E. van Voorthuisen, Cardiac first-pass and myocardial perfusion in normal subjects assessed by subsecond Gd-DTPA enhanced MR imaging. J. Comput. Assist. Tomogr. 15, 959-965 (1991).

32. N. Wilkc, C . Simm, J. Zhang, J. Ellerman, X Ya, H. Merkle, G. Path, H. Liidemann, R. J. Bache, K. Ugurbil, Contrast-enhanced first-pass myocardial perfusion imaging: correlation between myocardial blood flow in dogs at rest and during hypercmia. Magn. Reson. Med. 29, 485-497 (1993).

33. W. H. Pernian, M. H. Gado, K. B. Larson, J. S. Perlmutter, Simulta- nenus MR acquisitinn of arterial and brain signal time curves. Mugn. Reson. Med. 28, 74-83 (1992).

34. H. Kostler, H. Becker, The value of gating and flow compensation for measurements of the arterial input funL$on in dynamic susceptibility con- baqt MRI. M a s . Reson. Mafer. Phys. Biol. Med. [Suppl.) 4, 68 (1996).

measurement of perfusion rate in human melanoma xenografts by

Documents