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Published online before print November 20, 2006, 10.1148/radiol.2421051355
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(Radiology 2007;242:217-224.)
© RSNA, 2006

Musculoskeletal Imaging

Assessment of Bone Marrow Angiogenesis in Patients with Acute Myeloid Leukemia by Using Contrast-enhanced MR Imaging with Clinically Approved Iron Oxides: Initial Experience1

Lars Matuszewski, MD, Thorsten Persigehl, MD, Alexander Wall, MD, Norbert Meier, PhD, Ralf Bieker, MD, Hendrik Kooijman, PhD, Bernd Tombach, MD, Rolf Mesters, MD, Wolfgang E. Berdel, MD, Walter Heindel, MD and Christoph Bremer, MD

1 From the Departments of Clinical Radiology (L.M., T.P., A.W., N.M., B.T., W.H., C.B.) and Medicine/Hematology and Oncology (R.B., R.M., W.E.B.) and Interdisciplinary Center for Clinical Research (C.B.), University of Muenster, Albert-Schweitzer-Str 33, D-48129 Münster, Germany; and Philips Medical Systems, Hamburg, Germany (H.K.). From the 2003 RSNA Annual Meeting. Received August 13, 2005; revision requested October 20; revision received December 1; accepted January 2, 2006; final version accepted April 10. Address correspondence to C.B. (e-mail: bremerc{at}uni-muenster.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To prospectively assess bone marrow (BM) angiogenesis in patients with acute myeloid leukemia (AML) by using iron oxide–enhanced magnetic resonance (MR) imaging.

Materials and Methods: The study was institutional ethics committee approved. Informed signed consent was obtained from each study participant. The requirement for informed consent for use of data from a reference database was waived. Eleven patients (seven women, four men; mean age, 53 years ± 4.40 [standard deviation]) with an initial diagnosis of AML were enrolled in the study and underwent T2*-weighted two-echo echo-planar MR imaging of the pelvis before and after intravenous injection of a clinically approved iron oxide blood-pool contrast agent. Six healthy control subjects (one woman, five men; mean age, 35 years ± 2.31) were examined with the same MR protocol. The iron oxide–induced change in R2* relaxation rate ({Delta}R2*) was calculated, and the vascular volume fraction (VVF) of the BM was derived by dividing the {Delta}R2* of the BM by the {Delta}R2* of the muscle. Parametric {Delta}R2* maps were calculated to visualize vessel distribution. Patients underwent BM biopsy for correlative determination of microvessel density (MVD) and vascular endothelial growth factor (VEGF). Differences in {Delta}R2*, VVF, VEGF, and MVD were compared by using the Wilcoxon rank sum test.

Results: {Delta}R2* maps showed prominent areas of highly vascularized BM in the patients with AML, whereas the control subjects had moderately vascularized BM with homogeneous vessel distribution. Quantitative analysis revealed VVF values to be significantly higher in patients with AML than in control subjects: The mean VVF in the pelvis was 9.18% ± 1.54 for patients versus 3.91% ± 0.61 for control subjects (P = .010). In accordance with MR results, MVD (P = .009) and VEGF expression (P = .017) were significantly elevated in the AML group compared with values in the control group.

Conclusion: Iron oxide–enhanced MR imaging enables assessment of BM angiogenesis in patients with AML.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
It has been more than 30 years since Folkman et al (1) first proposed that angiogenesis has an important role in the progressive growth, viability, and metastatic spread of solid tumors. Studies have revealed that angiogenesis, apart from having a well-characterized role in the growth and metastasis of solid tumors, is also involved in the pathogenesis of hematologic malignancies (2–6). Higher microvessel density (MVD) levels have been found in the bone marrow (BM) of patients with acute myeloid leukemia (AML) (7).

Moreover, the BM expression of vascular endothelial growth factor (VEGF) and its subfractions is increased in patients with AML compared with that in healthy control subjects (2,6–10). Initial clinical trials have demonstrated that the antiangiogenic treatment of patients with AML with use of thalidomide has resulted in both antiangiogenic and antileukemic effects (9).

The clinical characterization of tumor angiogenesis is usually determined histologically on the basis of the MVD or by using immunohistochemical assays to visualize the levels of expression of angiogenic promoters such as VEGF (11). However, biopsy specimens—especially those of highly heterogeneous tumors—may not be representative of the overall disease burden (12). Thus, various imaging approaches to noninvasively assess the angiogenic activity of solid tumors have been explored (13–21).

Owing to their minimal leakage into the tumor interstitial space and thus longer plasma half-life, blood pool contrast agents have been shown to be better suited for in vivo assessment of tumor angiogenesis (22). Although some T1 blood pool contrast agents are currently being assessed in clinical approval trials, iron oxide particles have been approved and can be used for blood pool imaging. The grade of angiogenesis of solid tumors was accurately assessed in an experimental setting by using a long-circulating dextran-coated iron oxide and conventional gradient-echo magnetic resonance (MR) imaging sequences (23). In that study, the ultrasmall superparamagnetic iron oxides (SPIOs)-induced change in the R2* relaxation rate ({Delta}R2*) and the extrapolated vascular volume fraction (VVF) showed good correlation with the MVD and VEGF expression of the tumors (23). Thus, the purpose of this study was to prospectively assess BM angiogenesis in patients with AML by using iron oxide–enhanced MR imaging.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
One author (H.K.), an employee of Philips Medical Systems (Hamburg, Germany), was involved in the design of the MR imaging protocol described herein. All other authors, who are not employees of Philips Medical Systems, had control over the inclusion of data and information submitted for publication that might represent a conflict of interest for the author who is an employee.

Study Participants
Eleven patients (seven women, four men) with newly diagnosed AML (Table) who consented to participate in the study were prospectively enrolled between January 2003 and March 2004. To obtain control MR measurements, six healthy (according to medical histories and physical examination results) volunteers (one woman, five men) were recruited. Each study participant gave fully informed signed consent. The study was approved by the institutional ethics committee. The specimens from which control data (histologic and immunohistochemical BM findings) were collected were obtained from a reference database. Our institutional ethics committee granted approval for this retrospective portion of the study; the requirement for informed consent was waived. The mean ages of the patients and the control subjects were 53 years ± 4.40 (standard deviation) (range, 20–75 years) and 35 years ± 2.31 (range, 27–42 years), respectively, at the time of the MR examinations. According to the French-American-British system (24), the disease was classified as AML M1 in three patients, AML M2 in one, AML M4 in two, and AML M5 in five (Table).


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  		Characteristics of Patients with AML

 
SPIO Contrast Agent
For contrast material–enhanced MR imaging, an SPIO contrast agent (SHU 555 A, Resovist; Schering, Berlin, Germany) that is clinically approved in parts of Europe and Asia (Pacific region) was bolus injected. SHU 555 A consists of SPIO particles coated with carboxydextran and has an r1 relaxivity of approximately 25 L · mmol–1 · sec–1 and an r2 relaxivity of approximately 164 L · mmol–1 · sec–1 at 0.47 T and 37°C in water. The size of the iron oxide core at electron microscopy is 3–5 nm. The mean hydrodynamic diameter of the SPIO particle at photon correlation spectroscopy is approximately 65 nm (25). SHU 555 A has an intravascular distribution, and its clearance from the plasma is biphasic—mainly through selective uptake by the reticuloendothelial system of the liver and the spleen. The mean plasma half-life of SHU 555 A is 0.26 hour ± 0.19 (standard deviation) in the initial phase and 4.36 hours ± 0.75 in the terminal phase. All patients and volunteers tolerated the contrast-enhanced MR examination well and showed no signs of side effects after intravenous bolus injection of the SPIO contrast agent.

MR Imaging
MR imaging was performed with a 1.5-T clinical MR unit (Gyroscan Intera; Philips Medical Systems, Best, the Netherlands) by using a four-element synergy body coil. All patients and volunteers underwent MR imaging of the pelvis. After a survey examination, high-spatial-resolution T2-weighted fast spin-echo images were acquired by using a repetition time of 4000 msec, an echo time of 100 msec, a 90° flip angle, an echo train length of 15, a 380 x 380-mm field of view, a 384 x 512 matrix, 30 sections, and an acquisition time of 5 minutes 36 seconds. To measure {Delta}R2*, a fast T2*-weighted two-echo echo-planar sequence was performed before and after intravenous injection of the SPIO contrast agent by using a repetition time of 550 msec, echo times of 4.76 and 23.80 msec, a 40° flip angle, an echo-planar imaging factor of three, a 400 x 400-mm field of view, a 128 x 256 matrix, 16 sections, and an acquisition time of 1 minute 30 seconds. Since SPIOs show considerable uptake into the reticuloendothelial system during the initial phase of clearance (discussed earlier), the two-echo echo-planar sequence was chosen on the basis of prior experience, which revealed that this sequence can facilitate fast imaging while maintaining an acceptable signal-to-noise ratio. Before the second acquisition—that of the two-echo echo-planar images—all patients and control subjects received a single manual bolus injection of SHU 555 A (20 µmol per kilogram of body weight) followed by a 10-mL saline flush bolus. The total time for the injection was approximately 10 seconds. The second set of MR data was acquired within 60 seconds after the SPIO injection.

MR Data Analysis
The iron oxide–induced {Delta}R2* of the tumor, {Delta}R2*(t), is proportional to the perfused local blood volume in a given voxel (V) multiplied by a function of the concentration of the applied contrast agent in plasma f(P), as described in detail elsewhere (26): {Delta}R2*(t) = k · f(P) · V, where k is the constant.

Assuming that the SPIO has a steady-state intravascular distribution during MR measurement, the equation can be simplified to a linear relationship between the {Delta}R2* and the perfused blood volume fraction: {Delta}R2*(t) = k · V(t) or V(t) = [{Delta}R2*(t)]/k, where V(t) is the perfused tumor volume and k includes the concentration of the contrast agent in the blood pool and is therefore dose dependent. Assuming a monoexponential signal decay, the R2* relaxation rate can be estimated by using the two-echo echo-planar sequence as follows: R2* = [1/(TE2 – TE1)] · [ln (SITE1/SITE2)], where TE1 and TE2 are echo times 1 and 2 and SITE1 and SITE2 are the signal intensities with echo times 1 and 2. The {Delta}R2* can consequently be calculated as {Delta}R2* = {[1/(TE2 – TE1)] · [ln (SITE1post/SITE2post)]} – {[1/(TE2 – TE1)] · [ln (SITE1pre/SITE2pre)]}, where SITE1post and SITE2post are the signal intensities with echo times 1 and 2, respectively, measured after contrast agent administration and SITE1pre and SITE2pre are the corresponding signal intensities measured before contrast agent administration. The VVF of the tumor, VVFT, can then be calculated by calibrating the {Delta}R2* values of the tumor and muscle tissues—{Delta}R2*T and {Delta}R2*M, respectively—multiplied by 2.0%, which is the known vascular volume of muscle tissue in humans (27): VVFT = ({Delta}R2*T/{Delta}R2*M) · 2.0%.

Measurements of 20–100-pixel regions of interest were performed in the BM of the pelvis (iliac crest, including posterior superior iliac spine), femur (femoral head), and sacrum (ala sacralis), as well as in the gluteal muscle, on corresponding pre- and post-contrast MR images by using identical region-of-interest positions (L.M., C.B., and A.W., with 4, 8, and 6 years experience, respectively). Moreover, parametric {Delta}R2* maps were calculated by using a program developed for visualization of perfusion patterns (28). The maps were created by combining each 3 x 3 median-filtered voxel on four corresponding images with a new image. No post–data collection averaging or smoothing was applied. The resulting real data matrix was then linearly normalized to yield positive integer values that would be saved and viewed in standard Digital Imaging and Communications in Medicine format (N.M., L.M., C.B.).

BM Biopsy and Immunohistochemistry
In all patients with AML, a core-needle biopsy specimen from the BM of the iliac crest was obtained for routine clinical work-up 1–3 days after the MR measurements. The biopsies were performed by two hematologists (R.M., 12 years experience; R.B., 6 years experience). The histologic data for healthy subjects were obtained from a reference database. The extracted BM specimens were fixed in paraformaldehyde, decalcified in ethylenediaminetetraacetic acid, and embedded in paraffin. After each biopsy, BM aspiration at a separate puncture site was performed for cytologic analysis.

The histologic specimens were qualitatively assessed for the presence of endothelial cell sprouts and microvessels (with or without visible lumina). The degree of angiogenesis was assessed on the basis of the immunohistochemical identification of microvascular endothelial cells with use of antihuman thrombomodulin antibodies (TM Monoclonal Antibody, clone 1009; Dako, Glostrup, Denmark), as described previously (7). Serial 4-µm-thick sections of each tissue sample were processed with the antihuman thrombomodulin antibodies (working dilution, 1:50).

Semiquantitative analysis of VEGF protein expression in the BM biopsy specimens was performed, as previously described (24), by using a rabbit polyclonal antihuman VEGF (sc-152; Santa Cruz Biotechnology, Santa Cruz, Calif) (working dilution, 1:2000). Control specimens immunostained with nonimmune mouse or rabbit immunoglobulin G (sc-2025 and sc-2027, respectively; Santa Cruz Biotechnology) in substitution for the specific primary antibodies were consistently negative (data not shown).

Immunohistochemical staining was performed by using the alkaline phosphatase–antialkaline phosphatase double-bridge technique (Dako-APAAP Kit; Dako). Briefly, tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series. To promote antigen retrieval, the samples were pretreated in 10 mmol/L sodium citrate (pH 6.0) in a microwave oven at 450 W two times for 7 minutes each. The primary antibodies were added overnight at 4°C. Subsequent steps were performed according to the manufacturer's instructions. Fast red substrate (Dako) supplemented with 0.1% (weight per volume) levamisole was used to reveal phosphatase activity (30-minute incubation at room temperature). The sections were counterstained with 0.1% erythrocin solution.

Semiquantitative analysis of VEGF expression was performed, as described in detail elsewhere (24,29–31). Briefly, the immunostained sections were simultaneously assessed at light microscopy by two independent investigators (R.B., 6 years experience; R.M., 12 years experience). During the growth factor evaluations, the investigators were blinded to the patients' clinical data and the BM MVD counts.

By scoring the proportion (1 indicating fewer than 10%; 2, 10%–50%; and 3, more than 50% positive cells at magnifications of x100 and x250) and intensity (1 indicating very low or no staining; 2, fewer than 50% of cells with moderate staining; 3, more than 49% of cells with moderate staining; 4, fewer than 50% of cells with intense staining; and 5, more than 49% of cells with intense staining at magnification of x500) of stained cells, we semiquantitatively assessed the expression of VEGF. For a reasonable assessment of the protein expression in the entire BM section, we multiplied the mean cellular staining intensity for three representative fields (x500) by the proportion of positive cells in the entire BM section according to the three-grade scale at magnifications of x100 and x250. The results were expressed as arbitrary units (AU). In each biopsy specimen, the expression of VEGF was evaluated on two to three sections independently, and the mean value for each anatomic region, including the data from both readers, was calculated.

Statistical Analyses
All MR data are presented as means ± standard errors of the mean. Differences in age, {Delta}R2*, and VVF between the patients and the control subjects were assessed by using the Wilcoxon rank sum test for independent groups. P ≤ .05 was considered to indicate a significant difference.

MVD and VEGF protein expression are presented as median, minimum (25th percentile), and maximum (75th percentile) percentiles for the AML and control groups. Levels of VEGF and MVD expression in the AML and control groups also were analyzed by using the Wilcoxon rank sum test for independent groups. All calculations were performed (L.M., C.B.) by using commercially available statistics software (GraphPad Prism 4.0; GraphPad Software, San Diego, Calif).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Parametric MR Imaging
Parametric {Delta}R2* maps calculated from the MR data revealed nonhomogeneous vascularization of the BM in the patients with AML, with prominent areas of hypervascularized BM (Fig 1). In contrast, the control subjects had homogeneously vascularized BM with moderate {Delta}R2* values (Fig 1).


Figure 1
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  		Figure 1: A, B, Transverse T2-weighted MR images (4000/100, 90° flip angle; echo train length of 15), and, C, D, corresponding parametric {Delta}R2* maps obtained in 32-year-old healthy man (A and C) and 37-year-old woman with AML (B and D). Note the homogeneous distribution of moderate {Delta}R2* values in C as opposed to the prominent areas of hypervascularized BM in the pelvis in D.

 
Quantitative analysis of the MR data revealed significantly higher {Delta}R2* values for the pelvic, femoral, and sacral BM in the patients with AML compared with corresponding values in the control subjects (for pelvic BM, 0.0062 msec–1 ± 0.0012 in control group vs 0.0190 msec–1 ± 0.0038 in AML group; P = .003) (Fig 2).


Figure 2
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  		Figure 2a: Bar graphs show (a) {Delta}R2* and (b) VVF data in different anatomic regions in healthy volunteers (white bars) and patients with AML (black bars). (a) Significantly higher {Delta}R2* values were seen in all anatomic regions in the AML group compared with values in the control group (P < .05 [*]). (b) Similarly, BM VVF values in the pelvis and the sacrum were significantly increased in the AML group compared with values in the control group (P < .05 [*]). Data are means ± standard errors of the mean calculated at Wilcoxon rank sum testing.

 

Figure 2
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  		Figure 2b: Bar graphs show (a) {Delta}R2* and (b) VVF data in different anatomic regions in healthy volunteers (white bars) and patients with AML (black bars). (a) Significantly higher {Delta}R2* values were seen in all anatomic regions in the AML group compared with values in the control group (P < .05 [*]). (b) Similarly, BM VVF values in the pelvis and the sacrum were significantly increased in the AML group compared with values in the control group (P < .05 [*]). Data are means ± standard errors of the mean calculated at Wilcoxon rank sum testing.

 
Accordingly, the overall VVF was significantly higher in patients than in control subjects. VVF values were significantly higher in the pelvis (3.91% ± 0.61 in control group vs 9.18% ± 1.54 in AML group, P = .010) (Fig 2) and the sacrum (2.81% ± 0.64 in control group vs 6.36% ± 1.49 in AML group, P = .047) (Fig 2). However, differences in femoral VVF between the AML (6.01% ± 1.40) and control (3.14% ± 0.66, P = .119) groups were not significant (Fig 2).

Immunohistochemistry
Immunohistochemical staining of the BM sections of patients with AML revealed areas of intense neovascularization widely distributed among cellular regions of the specimens (Fig 3). The control specimens retrieved from the reference database (7), however, showed distinctively lower levels of MVD (Fig 3). Compared with the normal MVD (12.99 counts per field ± 0.53) and well-shaped vessel lumina seen in the control group specimens, endothelial cell sprouts, microvessels without visible lumina, and increased MVD (17.88 counts per field ± 1.59, P = .009) were observed in the AML group specimens (Fig 3) (7). Furthermore, compared with the control subjects, the patients with AML had elevated VEGF levels (4.00 AU ± 1.04 vs 1.95 AU ± 0.16 in control group, P = .017) (7).


Figure 3
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  		Figure 3a: BM sections from (a) control subject and (b) patient with AML immunohistochemically stained with antihuman thrombomodulin antibodies (magnification, x400). In b, note the increased MVD, as represented by the BM hypercellularity in association with multiple collapsed endothelial cell sprouts (arrowheads). There are fewer vessels in a, however, and the vessels are well differentiated.

 

Figure 3
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  		Figure 3b: BM sections from (a) control subject and (b) patient with AML immunohistochemically stained with antihuman thrombomodulin antibodies (magnification, x400). In b, note the increased MVD, as represented by the BM hypercellularity in association with multiple collapsed endothelial cell sprouts (arrowheads). There are fewer vessels in a, however, and the vessels are well differentiated.

 
Comparison of MR and Histologic Data
MR data showed VVF values for the pelvis, femur, and sacrum in the patients with AML to be 2.4-, 1.9-, and 2.3-fold higher than those in the healthy control subjects. These values corresponded to a 1.4-fold higher MVD and a 2.1-fold overexpression of VEGF in the patients with AML compared with values in the healthy control subjects.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
For several years, parametric imaging approaches involving the use of clinically available low-molecular-weight contrast agents, fast MR imaging, and, occasionally, complex pharmacologic modeling have been proposed for measurement of the physiologic and molecular surrogate markers of angiogenesis (21–23,25,32,33). The specificity of this imaging approach is inherently limited because the first-pass extravasation of small molecules is high in both tumorous and nontumorous tissue (34). For example, in breast cancer detection, there is a substantial overlap between malignant and benign tissue. In this setting, the use of so-called blood pool contrast agents has been shown to substantially improve the capability to differentiate benign from malignant tissue. SPIOs represent the first class of clinically available blood pool contrast agents that have the potential to be used as intravascular tracers and thus to image tissue angiogenesis noninvasively. Our study results show that {Delta}R2* and VVF measurements are significantly higher in the BM of patients with AML than in the BM of healthy subjects. The MR results closely resembled the histologic data.

Although histologic tissue assessment remains the reference standard for assessment of tumor angiogenesis, the MVD markedly differs among various tumor and BM regions, and, thus, some specimens may not be representative of the overall disease burden (12). This tissue heterogeneity was seen on the parametric maps in our study and was represented in the relatively large standard errors of the mean in the patient group. This biologic variability has also been observed by others (2). Although MR data yield an average VVF for the entire BM volume, histologic examinations cover only a specific subset of the diseased tissue volume. Thus, it is not surprising that MR data do not exactly match MVD count or VEGF protein expression analysis results. Similar observations have been described elsewhere (35).

In a study conducted by Rahmouni et al (36), BM infiltration was assessed in patients with lymphoproliferative diseases such as myeloma and lymphoma. In that study, the patients were imaged with fast T1-weighted MR imaging and a bolus-injected extracellular gadolinium-based contrast agent. Rahmouni et al observed markedly higher maximum BM enhancement (529%) in the patients compared with the enhancement in the control subjects (82%). Moreover, treatment resulted in reduced BM enhancement (36). Although the use of T1-weighted imaging is an interesting approach, it can be problematic because changes in T1 are generally less sensitive to minor variations in MVD. BM enhancement is frequently imperceptible on T1-weigted spin-echo images (37).

Moreover, the results of dynamic imaging protocols can vary greatly, depending on the timing and sequences used. In particular, the rapid first-pass extravasation of a low-molecular-weight contrast agent can hamper the exact quantification of tissue perfusion parameters. Because blood pool contrast agents have less leakage into the tumor interstitial space and thus a higher plasma half-life, they have also been shown by other investigators to be better suited for in vivo assessment of tumor angiogenesis (22). Moreover, with use of steady-state T2*-weighted imaging, quantitative data, which can also be helpful in longitudinal (eg, treatment-monitoring) studies, can be extrapolated. In contrast to our study, the Rahmouni et al study did not include a direct comparison of the MR data and the MVD or other surrogate parameters of tissue angiogenesis such as VEGF (36).

A different aspect of BM enhancement was assessed by Montazel et al (37), who compared the BM enhancement patterns among healthy adults of different ages. They reported that BM enhancement decreased substantially with increasing age—by more than 75% in individuals older than 60 years compared with that in individuals younger than 30 years—in association with conversion of the BM to fat tissue. Gadolinium enhancement 60 seconds after injection was markedly lower in patients aged 40 years or older than in those younger than 40 years.

The use of SPIO for imaging BM infiltration has been described by Daldrup-Link et al (38). In their study, the BM infiltration in patients with Hodgkin lymphoma was assessed. Daldrup-Link et al found that SPIOs are taken up by normal and hypercellular reconverted BM but not by neoplastic BM lesions; they therefore concluded that MR is sensitive for imaging BM infiltration in this patient population. In their study, MR imaging was performed 45–60 minutes after contrast agent injection to allow SPIO uptake by the reticuloendothelial system. However, we investigated the intravascular SPIO distribution early (ie, 60 seconds) after contrast agent injection and thus used the SPIOs as an intravascular tracer. Nonetheless, one could easily design an imaging protocol that involved a supplementary late-enhancement data set. This combined approach might enable one to obtain information on both BM MVD and the functionality (and thus recovery) of normal BM.

The use of SPIOs, as opposed to ultrasmall SPIOs, to image angiogenesis, as was done in our study, involves several imaging protocol requirements. Since SHU 555 A has a biexponential half-life with rapid initial clearing (mean, 0.26 hours ± 0.19), a fast T2*-weighted two-echo sequence should be applied to provide a sufficient signal-to-noise ratio. The acquisition time for the sequence applied in our study was 90 seconds, so well over 90% of the SPIO should have still been intravascular during the data acquisition. Therefore, the steady-state distribution of the SPIO, being the basis of the VVF calculation, was a valid assumption for the time of data acquisition.

The use of bolus-injectable ultrasmall SPIOs, as have been applied for T1-weighted MR angiography (39), would greatly enhance the time frame for data acquisition and therefore help to image a larger body volume and/or increase the signal-to-noise ratio. Moreover, the use of higher field strengths (eg, 3.0 T) should further enhance the sensitivity of {Delta}R2* values.

More recently, the use of MR imaging techniques that consist of multiecho gradient-echo sequences with echo train lengths greater than 100 has been proposed. These sequences allow more precise measurement of tissue relaxivity properties. Moreover, several algorithms that help to reduce susceptibility artifacts and to broaden the dynamic range of the sequence could be implemented (40,41). These technical developments should greatly enhance the applicability of T2* relaxometry in clinical scenarios.

There were limitations to this study. There was a significant difference in mean age between the patients with AML (53 years ± 4.40) and the healthy volunteers (35 years ± 2.31, P < .03). According to results of the Montazel et al study, one should expect substantially lower {Delta}R2* and VVF values in an age-matched control population and thus even greater differences between the patient and control groups (37). Moreover, a potential bias arose from the comparison of histologic specimens from a reference database control group with specimens from prospectively enrolled patients. Thus, the control group MR and histologic data were not directly comparable because they were obtained from different groups of individuals. However, even the histologic and MR data obtained from the same individuals (in this study, from patients with AML) may differ substantially because the tissue volumes examined are not identical. Finally, the study was not statistically designed in terms of power: It was intended to keep the number of healthy control subjects as small as possible. Thus, the overall study population was relatively small.

In summary, our study results show that fast T2*-weighted MR imaging with bolus-injectable SPIO can yield high-spatial-resolution maps of the BM VVF in patients with AML. Because the VVF is a surrogate marker for angiogenesis, this imaging technique can be applied to visualize and quantify BM infiltration. Further studies to explore whether the antiangiogenic treatment effects in AML can be monitored by using this technique are warranted. Thus, determination of VVF with contrast-enhanced MR imaging might be useful in selecting the antiangiogenic drugs that warrant further testing in clinical studies and in monitoring these clinical trials.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: AU = arbitrary unit • AML = acute myeloid leukemia • BM = bone marrow • MVD = microvessel density • SPIO = superparamagnetic iron oxide • VEGF = vascular endothelial growth factor • VVF = vascular volume fraction • {Delta}R2* = change in R2* relaxation rate

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantors of integrity of entire study, L.M., C.B.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, L.M., T.P., A.W., N.M., R.B., H.K., B.T., R.M., W.E.B., C.B.; clinical studies, L.M., T.P., A.W., R.B., H.K., B.T., R.M., W.E.B., W.H., C.B.; statistical analysis, L.M., N.M., B.T., C.B.; and manuscript editing, all authors


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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