REPORT

Diffusion Magnetic Resonance Imaging: an Early Surrogate Marker of Therapeutic Efficacy in Brain Tumors

Thomas L. Chenevert, Lauren D. Stegman, Jeremy M. G. Taylor, Patricia L. Robertson, Harry S. Greenberg, Alnawaz Rehemtulla, Brian D. Ross

Affiliations of authors: T. L. Chenevert (Department of Radiology), L. D. Stegman, B. D. Ross (Departments of Radiology and Biological Chemistry), J. M. G. Taylor (Departments of Biostatistics and Radiation Oncology), P. L. Robertson (Departments of Pediatrics and Neurology), H. S. Greenberg (Department of Neurology), A. Rehemtulla (Department of Radiation Oncology), University of Michigan Medical School, Ann Arbor.

Correspondence to: Brian D. Ross, Ph.D., Center for Molecular Imaging, University of Michigan, 1150 West Medical Center Dr., 9303 MSRB III, Ann Arbor, MI 48109–0648 (e-mail: bdross{at}umich.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Background: A surrogate marker for treatment response that can be observed earlier than comparison of sequential magnetic resonance imaging (MRI) scans, which depends on relatively slow changes in tumor volume, may improve survival of brain tumor patients by providing more time for secondary therapeutic interventions. Previous studies in animals with the use of diffusion MRI revealed rapid changes in tumor water diffusion values after successful therapeutic intervention. Methods: The present study examined the sensitivity of diffusion MRI measurements in orthotopic rat brain tumors derived from implanted rat 9L glioma cells. The effectiveness of therapy for individual brain cancer patients was evaluated by measuring changes in tumor volume on neuroimaging studies conducted 6–8 weeks after the conclusion of a treatment cycle. Results: Diffusion MRI could detect water diffusion changes in orthotopic 9L gliomas after doses of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) that resulted in as little as 0.2 log cell kill, a measure of tumor cell death. Mean apparent diffusion coefficients in tumors were found to be correlated with and highly sensitive to changes in tumor cellularity (r = .78; two-sided P = .041). The feasibility of serial diffusion MRI in the clinical management of primary brain tumor patients was also demonstrated. Increased diffusion values could be detected in human brain tumors shortly after treatment initiation. The magnitude of the diffusion changes corresponded with clinical outcome. Conclusions: These results suggest that diffusion MRI will provide an early surrogate marker for quantification of treatment response in patients with brain tumors.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Primary brain tumors account for more than 26% of childhood cancer deaths and 2% of adult cancer deaths in the United States (1,2). Sadly, improvements in 5-year survival rates for brain tumor patients have been rather modest during the past 20 years (1) despite substantial advances in stereotactic neurosurgery and focal conformal radiation therapy. Progress in adjuvant chemotherapy has also been disappointing, with few demonstrable gains made since the initial trials of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) in 1978 (3). The failure of chemotherapeutic regimens to improve median patient survival may be explained by the fact that fewer than 50% of individual tumors respond to a given agent (4). Adjuvant chemotherapy could be more successful if the responsiveness of a tumor to a given protocol were assessable in a more timely fashion than currently possible, thereby allowing trials of multiple regimens.

At present, comparison of sequential magnetic resonance imaging (MRI) scans is the method of choice for monitoring the response to therapy for central nervous system (CNS) tumors. This method examines changes in the maximal cross-sectional area of the tumor or the product of the maximal perpendicular tumor diameters (59). Gadolinium-enhanced T1 (i.e., spin–lattice relaxation time)-weighted images are often used, but T2 (i.e., spin–spin relaxation time) weighting or other magnetic resonance contrast strategies may be employed. Comparisons of tumor burden are usually made between pretreatment scans and scans obtained weeks to months after the conclusion of a therapeutic protocol (10).

Methods of assessing treatment response that are not dependent on relatively slow changes in tumor volume may be capable of providing earlier indications of therapeutic outcome. In pursuit of this goal, attempts have been made to associate changes in brain tumor biochemistry with therapeutic response with the use of magnetic resonance spectroscopy (11,12) and [18F]fluorodeoxyglucose positron emission tomography imaging (13). Diffusion-weighted MRI has proven to be a sensitive technique for identifying regions of ischemic tissue damage in animal models of stroke and in human patients (1416). The sensitivity of diffusion-weighted MRI for detecting ischemic brain injury led us to propose that it would be useful for monitoring the responses of tumors to therapeutic intervention (17). Monte Carlo simulations suggest that changes in tissue water diffusion after tissue damage are predominantly attributable to alterations in the volume and tortuosity of the extracellular space (1820). These properties of the extracellular space are primarily a function of cell density, and recent work (21,22) has shown that tumor water diffusion is associated with tumor cellularity.

We first reported elevation of mean tumor apparent diffusion coefficient (ADC) following BCNU treatment of an orthotopic rat glioma model derived from intracerebrally implanted 9L cells (17). We subsequently demonstrated that regions of coagulative necrosis and recurrent tumor could be distinguished on the basis of their ADC values (12,23). We have also investigated the universal nature of the diffusion response, demonstrating that treatment of orthotopic MCF-7 breast tumors in nude mice with tumor necrosis factor-related apoptosis-inducing ligand (24) and gene therapy for orthotopic 9L gliomas (25) both produce increases in tumor diffusion that precede tumor regression. Other groups have demonstrated therapy-induced diffusion increases in brain tumor models following gene therapy (26,27), in breast tumor models treated with paclitaxel (28), and in murine RIF-1 radiation-induced fibrosarcomas treated with cyclophosphamide (29).

This study investigates the dose-dependent sensitivity of diffusion MRI for detection of early therapeutically induced changes in tumor water diffusion with the use of a rodent brain tumor model. We report preliminary data suggesting that early ADC measures of human brain tumors are indeed predictive of treatment response.


    SUBJECTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Animal Model

All animal experiments, including designations for survival outcomes, were approved by the Committee on Use and Care of Animals of the University of Michigan, Ann Arbor. Rat 9L glioma cells were obtained from the Brain Tumor Research Center at the University of California at San Francisco. The cells were grown as monolayers in minimal essential medium supplemented with 10% fetal calf serum, 100 IU/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine at 37 °C in a 95% air-5% CO2 atmosphere. Intracerebral 9L tumors were induced in male Fischer 344 rats weighing between 125 and 135 g as described previously (30). Briefly, 9L tumor cells (105) were implanted in the right forebrain at a depth of 3 mm through a 1-mm burr hole. The surgical field was cleaned with 70% ethanol, and the burr hole was filled with bone wax to prevent extracerebral extension of the tumor. BCNU was dissolved in absolute ethanol and diluted in saline at the time of treatment and administered by a single intraperitoneal injection at a dose of 0.5 x LD10 (n = 5), 1.0 x LD10 (n = 5), and 2.0 x LD10 (n = 6), where LD10 is the dose at which 10% of the animals died, corresponding to 6.65, 13.3, and 26.6 mg/kg body weight, respectively. Control animals with the 9L tumor (n = 6) received injections of only vehicle (10% ethanol in saline).

Animal Diffusion MRI

We have investigated previously diffusion anisotropy in rat tumors (23). For this study, experiments were designed specifically to measure only the trace of the diffusion tensor because there is little anisotropy in tumors at the resolution typically used in human MRI studies. Imaging was performed every other day, beginning 10 days after implantation of the tumor cells on a Unity Inova system equipped with a 7.0-tesla, 18.3-cm horizontal bore magnet (Varian Inc., Palo Alto, CA) and a quadrature rat head coil (USA Instruments, Inc., Aurora, OH).

For MRI examination, rats were anesthetized with an isoflurane–air mixture and maintained at 37 °C inside the magnet with the use of a heated, thermostated circulating water bath. A single-slice sagittal gradient-echo sequence was used to confirm proper animal positioning and to prescribe subsequent acquisitions.

For time-efficient acquisition, an isotropic, diffusion-weighted sequence (31) was used with two interleaved b-factors ({Delta}b = 1148 seconds/mm2) and the following acquisition parameters: repetition time (TR)/echo time (TE) = 3500/60 milliseconds, 128 x 128 matrix, and a 3 x 3-cm field of view. It has been shown that there are no statistically significant differences in ADC values measured by faster two-point diffusion techniques versus six b-factor methods (32). Thirteen 1-mm-thick slices separated by a 0.2-mm gap were used to cover the whole rat brain. The z-gradient first moment was set to zero to reduce the dominant source of motion artifact. To further reduce motion artifact, a 32-point navigator echo was prepended to each phase-encode echo (33,34). The phase deviation of each navigator echo relative to its mean was subtracted from the respective image echoes before the phase-encode Fourier transform. The low b-factor images were essentially T2 weighted to allow tumor volume measurements, as described previously (30). Images were acquired before treatment and at 2-day intervals thereafter. Isotropic ADC maps were calculated for each image set, and ADC pixel value histograms were generated from tumor regions of interest (ROIs) combined across slices.

For the dose–response studies, localized diffusion measurements were taken from a column of tumor tissue, as described previously (23). Briefly, T2-weighted images were used to prescribe a 2 x 2-mm column through the most homogeneous, noncystic region of the tumor and contralateral brain from which diffusion measurements were made. Motion artifacts were minimized by use of a frequency encode gradient along the column axis for spatial encoding and magnitude processing of Fourier transformed echoes. Diffusion gradients at 42 b-factors ranging from 87 to 1669 seconds/mm2 were independently applied on the x, y, and z axes. ADC values were calculated as the quantity ADC = (ADCx + ADCy + ADCz)/3, which represents a scalar invariant of the 3 x 3 diffusion tensor normally used to fully characterize diffusion in an anisotropic system.

Histologic Analysis

In a separate study, rats (n = 24) were implanted intracerebrally with 9L cells. Twelve days after cell implantation, BCNU was administered intraperitoneally at a dose of 26.6 mg/kg body weight (n = 18), or vehicle only was administered to the controls (n = 6). Rats were killed in groups of three for histologic analysis at days 0 (vehicle only), 2, 4, 8, 16, and 20 (vehicle only and BCNU treated) after treatment. Brain specimens were removed, fixed in 10% buffered formaldehyde, sectioned (6 µm), and stained with hematoxylin–eosin. Randomly selected 400x fields were captured and digitized with a Pixera VCS 110 camera (Pixera Corp., Los Gatos, CA) interfaced with a Macintosh PowerPC computer. Cellularity measurements were made with Scion Image (Scion Corp., Frederick, MD) by segmenting the images based on signal intensity. The intensity and minimum particle size thresholds were set by one individual (L. D. Stegman), who was blinded to the treatment that the animal received and the post-therapy time at which the section was obtained. Comparisons of tumor cellularity with ADC values were made with the use of simple linear regression analysis.

Human Subjects

Preliminary evaluation of the clinical potential of diffusion MRI was performed in two patients with primary CNS tumors. Patients recruited for this study were required to have a malignant brain tumor histologically confirmed as glioblastoma multiforme, anaplastic astrocytoma, anaplastic oligodendroglioma, or primitive neuroectodermal tumor (PNET), either at initial diagnosis or at the time of tumor relapse, and were to undergo radiation therapy, chemotherapy and radiation therapy, or chemotherapy. Of the initial three subjects enrolled in the study, one had a stroke during treatment and was excluded from further study because diffusion MRI can be affected by stroke-induced changes. The remaining two subjects are described here.

Patient 1. A 13-year-old girl underwent a craniotomy for a small partial resection of a supratentorial thalamic PNET. Two baseline MRIs were performed before the start of radiotherapy, which was given in standard daily fractions of 1.8 Gy, 5 days per week, to a total dose of 55.8 Gy. Concurrent carboplatin was given daily as a radiosensitizer, 1 hour before each radiation fraction. Cyclophosphamide was then given as adjuvant chemotherapy in six cycles at a dose of 2 g/cycle, over a 7-month period spanning 3–10 months from the start of therapy. One cycle of salvage chemotherapy with cisplatin, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU or lomustine), and vincristine was given at tumor progression, 11 months after initial treatment. The patient died of her disease 14 months from diagnosis.

Patient 2. A 37-year-old man with an anaplastic oligodendroglioma of the right temporofrontal region that was partially resected 1 month before initiation of treatment with six cycles of procarbazine, CCNU, and vincristine (PCV) chemotherapy. Each cycle consisted of CCNU at a dose of 130 mg/m2 administered on day 1, procarbazine at a dose of 75 mg/m2 administered on days 8–21, and vincristine given at a dose of 1.5 mg/m2 on days 8 and 29. After six cycles of this therapy, the patient's disease progressed based on the MRI scan and was treated with three-dimensional conformal radiation therapy. One year after diagnosis, the patient is doing well clinically without neurologic deficit. He is being followed with serial scans at 4-month intervals.

Standard magnetic resonance sequences including T1-weighted with contrast enhancement, T2-weighted and fluid-attenuated inversion-recovery (FLAIR) contrast were acquired along with diffusion-weighted magnetic resonance images before, during, and 6–8 weeks after treatment to quantitate changes in tumor diffusion values for association with clinical response. Clinical outcomes were classified as complete response (i.e., the complete resolution of tumor contrast enhancement and off all steroids), partial response (i.e., a >50% decrease in tumor volume on stable or decreased dose of steroids), stable disease (i.e., a <50% decrease or a <25% increase in tumor volume on stable or decreased dose of steroids), and progressive disease (i.e., a >25% increase in tumor volume on stable or increased dose of steroids) (35). Written informed consent was obtained from all subjects, and all images and medical records were obtained according to protocols approved by the Institutional Review Board of the University of Michigan Medical School.

Human Diffusion MRI

Water diffusion-sensitive images of the brain were acquired on a 1.5T Human MRI System (General Electric Medical Systems, Milwaukee, WI), capable of single-shot echo-planar imaging (36). The diffusion spin-echo echo-planar imaging sequence (TR/TE = 10 000/100 milliseconds) was set to acquire fourteen 6-mm-thick, contiguous axial-oblique sections through the brain at a given diffusion sensitivity (i.e., "b factors") along all three orthogonal directions. A set of diffusion-weighted images at high diffusion sensitivity (b2 = 1000 seconds/mm2) and low diffusion sensitivity (b1 = 100 seconds/mm2) plus b = 0 (i.e., T2 weighted) were collected in 80 seconds.

Diffusion-weighted images were reduced to ADC diffusion maps according to



where Sb1 and Sb2 are signal intensities at low and high diffusion weighting, respectively, as acquired independently along each orthogonal axis. The quantity, ADCo, a scalar invariant of the diffusion tensor that avoids complexities introduced by anisotropy in brain tissue (3739), was calculated with the use of AVS 5 (Advanced Visual Systems, Inc., Waltham, MA) and MatLab 5 (MathWorks, Inc., Natick, MA) software routines. Tumor water diffusion was summarized by histograms of tumor ADC pixel values across all slices. The neuro-oncologists (H. S. Greenberg and P. L. Robertson) guided ROI definition along tumor boundaries using all available images.

Statistical Analysis

The individual pixel values constituting ADC histograms are derived from image data with inherent spatial relationships and are thus not independent measures. Therefore, to calculate 95% confidence intervals for the mean ADC value of each histogram, we sampled the set of pixels to obtain an approximately independent set. Software was written to randomly sample 1% of pixels in each histogram. The 95% confidence intervals of the means from 25 such random subsamples were used as a conservative estimate of the error of the original histogram. Differences between baseline and follow-up histograms were explored with the use of the Bonferroni corrected t test. For the rodent BCNU dose–response study, the one-way analysis of variance was used to search for statistical differences between localized ADC measurements at progressive time points following treatment and the pretreatment ADC value. Pearson product moment correlation analysis and simple linear regression were used to examine the relationship between mean tumor ADC and cell density measurements. A significance level of P<.05 was used throughout, and all statistical tests were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Rodent Studies

Time-course ADC maps from a representative animal treated with a bolus 2 x LD10 dose of BCNU are shown in Fig. 1Go, A. Although the tumor continued to grow over the first 8 days after BCNU administration, the diffusibility of water in the tumor increased relatively uniformly. The tumor continued to appear hyperintense on ADC maps while it regressed. To quantitate changes in tumor water diffusion, we defined ROIs for the tumor volume and generated histograms of tumor pixel ADC values. The serial ADC histograms shown for this representative tumor (Fig. 1Go, B) demonstrate a right shift in tumor water diffusion beginning by the second day after therapy, despite continued tumor growth reflected by an increase in the area under the histogram. Water diffusibility appeared to peak around day 8, after which the tumor began to regress. Eventually, the small surviving fraction of tumor cells repopulated the tumor volume, resulting in a recurrent tumor with a water environment resembling that of the primary tumor before BCNU treatment. This trend is demonstrated in the plot of mean tumor ADC versus time (Fig. 1Go, D). Such diffusion histograms permit segmentation of tumor pixels that exhibit high diffusion properties, postulated to be sub-elements of the tumor responding to treatment. For example, the region to the right of the vertical lines in the histograms illustrates the volume of tumor pixels above an arbitrarily selected ADC threshold of 1.5 x 10-3 mm2/second, which may indicate the fractional volume of tumor that exhibits a strong therapeutic response. Difference-from-baseline histograms (Fig. 1Go, C), calculated by subtracting the pretreatment histogram from the histograms at each subsequent time point, emphasize the relative shifts in the tumor water diffusion sub-environments. Diffusion histograms and mean ADC results obtained from a representative, vehicle-only-treated 9L tumor are shown in Fig. 1Go, E–G. Note that diffusion values were stable throughout the growth of the intraparenchymal brain tumor.



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Fig. 1. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine) treatment elicits early changes in tumor water diffusion. Panel A: quantitative apparent diffusion coefficient (ADC) maps of a representative rat brain obtained before treatment and 8, 20, and 27 days after administration of a single bolus of BCNU at 26.6 mg/kg body weight. Thresholds set in the ADC calculation routine assign low signal-to-noise pixels as black for exclusion from subsequent analysis. ADC histograms were derived from regions of interest corresponding to the tumor shown in panel A and are displayed in panel B. In panel C, difference histograms were calculated by subtracting the first (pretreatment) histogram from the histograms at each of the other time points to demonstrate the relative shift of pixels between different water environments. Lines at 1.5 represent an arbitrarily selected ADC threshold of 1.5 x 10-3 mm2/second, which may indicate the fractional volume of tumor that exhibits a strong therapeutic response. Panel D: plot of the time course of mean tumor ADC values. ADC histograms (panel E), difference histograms (panel F), and the mean tumor ADC time course (panel G) from a representative control animal, sham-treated with vehicle alone, are also shown. All mean values are shown with 95% confidence intervals. The ADC values of normal contralateral brain in the BCNU-treated and control animals were constant (0.90 ± 0.06 [n = 14 time points] and 0.87 ± 0.01 [n = four time points], respectively) over the course of the experiment.

 
Dose–Response Studies

To assess the dose dependence and sensitivity of treatment-induced diffusion changes, experiments were performed on intracranial 9L gliomas after administration of three different doses of BCNU. Fig. 2Go, A, is a plot of changes in mean tumor ADC values relative to the mean baseline tumor ADC measured at the beginning of the experiment. There was a clear dose dependence in both the magnitude and duration of tumor diffusion changes. Statistically significant differences between each treatment group and the control group were first apparent at day 4 after treatment. At the 2 x LD10 and 1 x LD10 doses of BCNU, the absolute increases in the diffusion values were similar, but the duration of the increase was slightly longer for the 2 x LD10 dose.



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Fig. 2. Panel A: dose dependence of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU or carmustine)-induced changes in tumor water diffusion. BCNU was given as a single intraperitoneal bolus of 6.6 ({blacksquare}, n = 5), 13.3 ({blacktriangleup}, n = 6), and 26.6 ({bullet}, n = 6) mg/kg body weight, corresponding to 0.5 x LD10, 1 x LD10, and 2 x LD10 doses, respectively, where LD10 is the dose at which 10% of animals died. Control animals ({bigcirc}, n = 5) were sham-treated with vehicle alone. Tumor apparent diffusion coefficient (ADC) values were obtained with the use of the voxel method at 2T (23) and are given as mean change from baseline with the positive half of the 95% confidence interval. Differences between the treatment and control groups became statistically significant at day 4 after treatment, as determined by one-way analysis of variance. Histopathologic changes in hematoxylin–eosin-stained sections of intracerebral 9L tumors treated with a single 2 x LD10 dose of BCNU obtained on the day of treatment and on days 2, 4, 8, 16 and 20 after treatment are shown in panels B, C, D, E, F, and G, respectively (original magnification x400). Panel H: cell density measurements from histologic 9L tumor sections over time before and after treatment with a 2 x LD10 dose of BCNU. Panel I: plot of 9L tumor cell density versus mean ADC with linear regression fit demonstrating a significant correlation (Y = 1.5 – 0.0009X, where Y = ADC and X = cell density; r = .78; two-sided P = .041).

 
Cellularity and Diffusion

Histologic sections of 9L tumors at selected intervals after 2 x LD10 BCNU treatment are shown in Fig. 2Go, B–G. After BCNU treatment, a progressive increase in tumor extracellular space was observed, reaching a maximum at about 8 days after treatment. There was also an increase in pleomorphism, giant cells, and cells with the characteristic morphologic features of apoptosis. A lymphocyte-predominant, mixed inflammatory response was evident 6 days after BCNU administration. At 16 days after BCNU therapy, the extracellular space began to diminish, and regions of dense regrowth of tumor cells became more predominant. The histology of the recurrent tumor was indistinguishable from that of the untreated tumor. Tumor cellularity, defined as the number of nuclei in a 400x field, was measured on digitized sections. Fig. 2Go, H, shows the time course of cell density changes after BCNU treatment. The measured mean tumor cellularity at each time point after BCNU treatment correlated well (r = .78; P = .041) with the mean tumor ADC values measured at that respective time point (Fig. 2Go, I).

Clinical Studies

Shown in Fig. 3Go are sequential FLAIR images (panel A) and ADC maps (panel B) of a large thalamic PNET in a 13-year-old girl. The tumor did not enhance with gadolinium contrast on T1-weighted images. There was marginal shrinkage of the tumor (19%) at 6 months from the start of therapy, which was classified as stable disease (35). However, tumor growth resumed by week 47 after therapy. ADC maps obtained from this patient demonstrated an area of increased water diffusion in the posterior portion of the mass. Diffusion changes were not observed in the left, anterior portion of the mass (right on images). Shown in Fig. 3Go, C, is a stack plot of diffusion histograms of all tumor pixels; the difference histograms obtained by subtracting pretreatment from post-treatment histograms are shown in Fig. 3Go, D. For this patient, there was subtle evidence of an early, weakly positive therapeutic response 4 weeks into treatment identified by an increase in pixel area greater than 1.5 ADC units. The mean ADC plot (Fig. 3Go, E) indicates a 10% increase in ADC 12 weeks from the start of therapy. During tumor regrowth, the mean ADC value declined dramatically. The difference plots accentuate the relative shift between water environments—e.g., the small increase in the quantity of high diffusion pixels (above 1.5 ADC units) with proportional loss in low diffusion pixels suggests a minimal therapeutic response.



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Fig. 3. Example of a patient with a primary neuroectodermal tumor who displayed a weak therapeutic response to radiation therapy with carboplatin radiosensitization. Panel A: representative fluid-attenuated inversion recovery (FLAIR)-weighted magnetic resonance images obtained before treatment and 7, 13, and 57 weeks after a 55.8-Gy dose to the tumor field and a 3600-cGy dose to the craniospinal axis. Panel B: corresponding quantitative apparent diffusion coefficient (ADC) maps at the same time intervals. At 24 weeks after treatment, the tumor volume had shrunk by 19%, and the patient was classified as having stable disease. Rapid regrowth of the tumor was evident by week 47. Panel C shows tumor ADC histograms; panel D shows difference histograms, and panel E shows the mean ADC changes over time with 95% confidence intervals. Lines at 1.5 represent an arbitrarily selected ADC threshold of 1.5 x 10-3 mm2/second, which may indicate the fractional volume of tumor that exhibits a strong therapeutic response. Pre-Tx = before treatment.

 
Gadolinium-enhanced T1 images and ADC maps of a 37-year-old man with a deep midline, bilateral oligodendroglioma treated with PCV are shown in Fig. 4Go, A and B, respectively. Three weeks after the start of the PCV therapy, the area of gadolinium contrast enhancement decreased. The mass effect observed in the FLAIR and T2-weighted magnetic resonance images (not shown) decreased during the next 10 weeks. Serial diffusion histograms (Fig. 4Go, C), difference histograms (Fig. 4Go, D), and tumor mean ADC values (Fig. 4Go, E) demonstrated the magnitude of the water diffusion change. A peak diffusion increase of 86% was observed at 6 weeks. The histograms shown in Fig. 4Go were derived from ROIs thought to represent residual tumor on the basis of their contrast-enhancing character. Similar results were obtained with the use of larger ROIs defined by hyperintense areas on FLAIR.



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Fig. 4. Example of a patient with an oligodendroglioma who responded to procarbazine/1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU or lomustine)/vincristine (PCV) chemo therapy. Contrast-enhanced T1-weighted magnetic resonance images (A) and apparent diffusion coefficient (ADC) maps (B) obtained before treatment and at 3, 6, and 13 weeks after initiation of the PCV protocol. The treatment resulted in reduction of the tumor volume by 25%–50%, and the patient was classified as having a minimal response. ADC histograms (C), difference histograms (D), and the mean ADC changes over time with 95% confidence intervals (E) are also shown. Lines at 1.5 represent an arbitrarily selected ADC threshold of 1.5 x 10-3 mm2/second, which may indicate the fractional volume of tumor that exhibits a strong therapeutic response. Note the greater rate of tumor water diffusion observed 3 weeks after the start of therapy.

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We reported previously that serial measurements of tumor volume obtained from standard T2-weighted images of intracerebral 9L gliomas can be used to accurately quantitate tumor cell kill in individual animals (30,40). Treatment of tumors with 0.5 x LD10, 1 x LD10, and 2 x LD10 doses of BCNU resulted in log cell kill values of 0.2, 0.8, and 1.7, respectively (30). In this study, we observed a dose-dependent change in tumor diffusion that appeared to saturate at the 1 x LD10 dose, corresponding to approximately 1 log kill. The observation of statistically significant changes in tumor ADC values at 4 days after administration of a 0.5 x LD10 dose, producing a cell kill of 0.2 log, reveals that diffusion is very sensitive to small cell kill values. The apparent saturation at 1 log kill suggests that the dynamic range is greatest at the lower cell kills. This feature could provide for detection of small, therapy-induced tissue changes required for clinical monitoring of fractionated therapies for brain tumors.

Comparison of tumor histology with magnetic resonance-measured mean tumor ADC values demonstrated a statistically significant correlation between therapy-induced changes in cellularity and water diffusion. Previous studies have reported similar correlations in human tumors (21) and in xenografts (22,41). The regression line indicates that cell-free tumor, e.g., tumor tissue with no nuclei per field, would exhibit an ADC of 1.7 x 10-3 mm2/second, which is lower than the ADC of cerebrospinal fluid at body temperature (3.0 x 10-3 mm2/second). This diffusion restriction is likely due to cell remnants and mucoid substance in the necrotic spaces. Decreases in cellularity reflect an increased volume of water in the extracellular compartment, which could be due to vasogenic edema or shifts of intracellular water to the extracellular space associated with tumor cell necrosis. Our study does not provide specific data regarding the relative roles of these processes; however, a previous study (30) has demonstrated that BCNU kills approximately 40% of 9L tumor cells at the 0.5 x LD10 dose. Moreover, vasogenic edema was not observed in contralateral normal brain tissue. These observations are consistent with the hypothesis that water liberated by cell necrosis is a major mechanism for therapy-induced diffusion increase.

Preliminary evaluation of the clinical potential of diffusion MRI was performed in two patients with different types of primary CNS tumors. A modest, early increase in diffusion, followed by a drop in tumor ADC with increased tumor volume, was observed during the therapy for the patient with a PNET. We argue that this pattern of diffusion changes was consistent with the clinical response of stable disease followed by rapid progressive disease. The data obtained from this patient indicate that diffusion MRI is a very sensitive technique that is able to detect relatively low levels of tumor cell kill. A stronger diffusion shift would be anticipated in patients more responsive to treatment, parallel to the trends observed in the rodent brain tumor studies. A more pronounced diffusion increase was observed in the patient with the oligodendroglioma. This change reflects the 25%–50% reduction in apparent tumor volume. These data support the hypothesis that the magnitude of change in tumor water diffusion assessed with the use of MRI is related to the numbers of cells killed and, hence, to the therapeutic efficacy. Moreover, the maximal diffusion change preceded changes in tumor volume by weeks to months, suggesting that the diffusion parameters could be useful as an early predictor of therapeutic response in human brain tumors. Regional changes in tumor water diffusion were evident on ADC maps in both cases, suggesting that this technique will be able to characterize regional heterogeneity in the response of a tumor. The quantitative histogram analysis approach reported in this study does not address regional variation in response, since the histograms were derived from the whole tumor mass. However, this novel approach yields a quantitative display that provides a measure of overall tumor response. Spatial differences in tumor response are likely to occur because of tumor heterogeneity; thus, both tumor histograms and ADC diffusion maps provide complementary and important information.

These results show that diffusion imaging has potential for early assessment of treatment response in individual patients. Early, reliable prognostic information may help physicians to tailor treatment plans to individual patients and allow alternative therapies to be attempted in a more timely fashion if a tumor appears resistant. The sensitivity of diffusion MRI to changes in tissue structure may also advance experimental therapeutics by providing new tools for analyzing therapeutic outcomes both in laboratory models and in clinical trials. Further work is required to determine whether the observed changes in tumor diffusion are a universal response to the successful killing of tumor cells and to delineate more fully the kinetics of diffusion changes and the prognostic value of diffusion MRI.


    NOTES
 
Supported in part by a research grant from the Charles A. Dana Foundation; by Public Health Service grants R24CA83099 and P20CA86442 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; and by the University of Michigan Clinical Research Partnership Fund.

We thank Annette Betley for assistance with the patient data collection and Phil Kish for assistance with digitizing the histological sections.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Manuscript received March 30, 2000; revised September 22, 2000; accepted October 6, 2000.


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