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Cervical Lymph Node Metastases: MR Imaging of Gadofluorine M and Monocrystalline Iron Oxide Nanoparticle–47 in a Rabbit Model of Head and Neck Cancer¹

¹ From the Department of Radiology and Institute of Radiation Medicine, Seoul National University College of Medicine, Clinical Research Institute, Seoul National University Hospital, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea (S.H.C., M.H.H., W.K.M., K.R.S., J.H.K., B.J.K., D.G.N., K.H.C.); Department of Pathology, Seoul National University Hospital, Seoul, Korea (J.K.W.); and Department of Contrast Media Research, Schering, Berlin, Germany (H.J.W.). Received December 7, 2005; revision requested January 27, 2006; revision received February 8; final version accepted March 22. Supported by a grant from the Seoul National University Hospital Research Fund (04-2005-075-0). Address correspondence to M.H.H. (e-mail: hanmh{at}radcom.snu.ac.kr).

	ABSTRACT

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

 
Purpose: To prospectively compare the accuracy of gadofluorine M with that of monocrystalline iron oxide nanoparticle (MION)-47 for the depiction of cervical lymph node metastases at magnetic resonance (MR) imaging in a rabbit model of head and neck cancer by using histologic analysis as the reference standard.

Materials and Methods: Experiments were approved by the animal care committee. VX2 carcinomas were implanted in both ears of 11 rabbits 4 weeks before MR imaging. T2-weighted, T2*-weighted, and T1-weighted MR images were acquired, and sequential T1-weighted MR imaging was performed immediately and 30 minutes after administration of gadofluorine M (0.05 mmol gadolinium per kilogram body weight). T2-weighted and T2*-weighted MR imaging were performed 24 hours after administration of MION-47 (2.6 mg iron per kilogram body weight). Gadofluorine M– and MION-47–enhanced MR imaging were performed separately and independently by two radiologists who had no knowledge of histopathologic results, and the presence of metastases in lymph nodes was evaluated. A receiver operating characteristic analysis was conducted to compare the diagnostic value of gadofluorine M– and MION-47–enhanced MR imaging.

Results: Metastases were confirmed in 20 of 77 lymph nodes at histopathologic analysis. The area under the curve was significantly greater for gadofluorine M–enhanced MR imaging (0.997 and 0.981 for readers 1 and 2, respectively) than for MION-47–enhanced MR imaging (0.889 and 0.846 for readers 1 and 2, respectively). For gadofluorine M–enhanced MR imaging, sensitivity was 100% for both readers and specificity was 89.5% for reader 1 and 87.7% for reader 2. For MION-47–enhanced MR imaging, sensitivity was 80.0% for both readers and specificity was 75.4% for reader 1 and 71.9% for reader 2.

Conclusion: Gadofluorine M–enhanced MR imaging has higher accuracy for depicting lymph node metastases than does MION-47–enhanced MR imaging.

© RSNA, 2006

	INTRODUCTION

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

 
The identification of metastases in lymph nodes of the neck has a major effect on the prognosis and treatment of head and neck cancer (1). Several imaging modalities, including computed tomography (CT), magnetic resonance (MR) imaging, and nuclear medicine imaging, are used to help diagnose metastatic involvement in the lymph nodes (2). Anatomic imaging (CT and routine MR imaging), however, will not depict small metastatic deposits in normal-sized nodes. Also, size is a poor criterion when there is no extracapsular extension or focal nodal necrosis to rely on (3). Likewise, positron emission tomography with fluorine 18 fluorodeoxyglucose may not depict small deposits below the resolution of the scanner. Thus, there is a need for functional methods that can be reliably used to identify small metastases.

Intravenous contrast material–enhanced MR lymphography offers a noninvasive means of potentially analyzing the lymphatic system (4). The first intravenous contrast medium used for MR lymphography was dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles (5,6). In several studies, investigators have demonstrated enhanced sensitivity and specificity for lymph node evaluation after the administration of USPIO particles for pelvic, head and neck, and chest malignancies (5–11).

Gadofluorine M (Schering, Berlin, Germany) is the first T1 contrast agent that shows rapid lymph node–specific enhancement after intravenous administration (4). Promising results with gadofluorine M were reported for the evaluation of malignant nodes in a rabbit cancer model (4). Thus, the purpose of our study was to prospectively compare the accuracy of gadofluorine M with that of monocrystalline iron oxide nanoparticle (MION)–47 (Schering) for the depiction of cervical lymph node metastases at MR imaging in a rabbit model of head and neck cancer by using histologic analysis as the reference standard.

	MATERIALS AND METHODS

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

 
Gadofluorine M was provided by Schering, and one author (H.J.W.) is an employee of Schering. The authors who are not employees of Schering had full control of the inclusion of any data or information that might have presented a conflict of interest for the author who was an employee of Schering.

Animal Preparation
Experiments were performed by using 13 New Zealand white rabbits weighing 2.5–3.0 kg. The experimental protocol was approved by the animal care committee at Seoul National University Hospital. Animals were allowed food and water ad libitum.

The 13 rabbits were divided into control (n = 2) and tumor-bearing (n = 11) groups. In the control group, changes in T1-weighted, T2-weighted, and T2*-weighted signal intensities (SIs) in the cervical lymph nodes were determined before and after gadofluorine M administration; changes in T1-weighted SI in the salivary glands were also evaluated. The performance of gadofluorine M– and MION-47–enhanced MR imaging for the depiction metastatic lymph nodes was evaluated in the tumor-bearing group. The number of tumor-bearing rabbits was selected on the basis of preliminary sample size calculations that yielded an estimated power of 80% for the detection of a significant difference between gadofluorine M– and MION-47–enhanced MR imaging with respect to metastatic lymph node detection, with the assumption that such a difference existed (12).

In the tumor-bearing group, tumor implantation was performed with an aseptic technique by two radiologists (S.H.C., K.R.S.), who worked together. VX2 carcinoma was inoculated concentrically between the lateral auricle edge and the central auricular artery into the cranial section of the lower third of both auricles by injecting 0.5 mL of tumor suspension with an 18-gauge needle. The details of preparing this experimental VX2 rabbit carcinoma have been previously reported (13).

Contrast Material
Gadofluorine M is an amphiphilic gadolinium complex with a molecular weight of about 1530 g/mol and a concentration of 200 mmol gadolinium per liter (4). The contrast medium is synthesized by adding a perfluoroctyl chain to a gadolinium-containing macrocycle. In 40°C water, the R1 and R2 relaxation rates at 0.47 T were about 15.8 mmol^–1 · sec^–1 and 19.5 mmol^–1 · sec^–1, respectively (4). In rabbits, the plasma elimination half-life of gadofluorine M is 8 hours, and most of the injected dose of gadofluorine M is eliminated from the body 7 days after intravenous injection (4). A median lethal dose of gadofluorine M is 5 mmol per kilogram body weight when administered intravenously in mice (4).

MION-47 consists of USPIO particles coated with dextran, with R1 and R2 relaxation rates of about 21.9 mmol^–1 · sec^–1 and 44.6 mmol^–1 · sec^–1, respectively, at 0.47 T in 37°C water (14). The iron has a concentration of 11.22 mg/mL and is contained in 20 mmol sodium citrate, with a pH 8. The mean particle size is 27.3 nm.

MR Imaging
At MR imaging, the animals were sedated with an intramuscular injection of 50 mg ketamine hydrochloride (Ketalar; Yuhan Yanghang, Seoul, Korea) and 20 mg xylazine hydrochloride (Rompun; Bayer, Seoul, Korea) at 0.5 mL per kilogram of body weight. All examinations were performed by using a 1.5-T MR imager (Signa; GE Medical Systems, Milwaukee, Wis) with a knee coil to improve resolution. All images were acquired in the coronal plane, and animals were imaged in the supine position. Gadofluorine M and MION-47 were administered to each animal via an ear vein. Administration of gadofluorine M was performed manually by means of a fast bolus injection, and administration of MION-47 was performed by means of an approximately 10-minute infusion of 10 mL normal saline.

Control group.—Sequential MR imaging was performed by using T1-weighted spin-echo (400/12 [repetition time msec/echo time msec], 2-mm section thickness, 12-cm field of view, 256 x 256 matrix, and two signals acquired), T2-weighted spin-echo (5000/85, echo train length of 16, 2-mm section thickness, 12-cm field of view, 256 x 256 matrix, and three signals acquired), and T2*-weighted two-dimensional gradient-echo (400/24, 20° flip angle, 2-mm section thickness, 12-cm field of view, 256 x 256 matrix, and two signals acquired) MR imaging sequences before, immediately after, and 15, 30, 60, and 1440 minutes after gadofluorine M administration (0.05 mmol gadolinium per kilogram body weight).

Tumor-bearing group.—MR images were obtained in all 11 rabbits 4 weeks after inoculation with tumor cells. T1-weighted spin-echo, T2-weighted spin-echo, and T2*-weighted two-dimensional gradient-echo MR images were acquired. T1-weighted spin-echo MR imaging was performed immediately after and 30 minutes after gadofluorine M administration (0.05 mmol gadolinium per kilogram body weight), and MION-47 (2.6 mg iron per kilogram body weight) was administered after gadofluorine M–enhanced MR imaging. MR imaging was performed 24 hours after MION-47 administration by using T2-weighted spin-echo and T2*-weighted two-dimensional gradient-echo MR imaging.

Isolation of Lymph Nodes and Histopathologic Results
After MR imaging, the 11 rabbits in the tumor-bearing group were sacrificed with a lethal dose (90 mg per kilogram body weight) of intravenously administered sodium pentobarbital (Pentothal; Choong Wae Pharmacy, Seoul, Korea). The regional draining lymph nodes (ie, parotid, caudal mandibular, and rostral mandibular lymph nodes) were isolated by one author (S.H.C.) in a manner previously reported (13). MR images were reviewed by the same author who evaluated lymph node location before the dissection of each rabbit. The lymph nodes that were harvested from each rabbit were placed in individual trays for processing, and tray numbers were annotated against the nodes on the MR images. The orientation of the lymph nodes was also labeled. The dissected lymph nodes were fixed with 10% formalin. The lymph nodes were then embedded in paraffin, and prepared sections (approximately 5-µm thick) that were obtained at 0.5-mm intervals were stained with hematoxylin-eosin for microscopic examination by a pathologist (J.K.W., 4 years of experience). The pathologist examined the lymph nodes with respect to the presence of metastasis, size, and location. Lymph node and metastasis sizes were recorded with regard to the long- and short-axis diameters of the largest section.

Image Analysis
Images were analyzed by two radiologists (M.H.H. and B.J.K., with 20 and 5 years of experience, respectively, in the interpretation of MR images of the head and neck). The investigators were told which group (control or tumor-bearing group) each rabbit belonged to. All MR images were reviewed on a picture archiving and communication system (PACS; Marotech, Seoul, Korea) workstation.

Control group.—In each rabbit, SI values were determined by choosing an appropriate region of interest (about 1.0–5.0 mm² [placed in consensus by M.H.H. and B.J.K.]) in the nodal functional tissues (ie, parotid, rostral mandibular, and caudal mandibular lymph nodes), mandibular and parotid salivary glands, and right sternomastoid muscle tissues. SI values were related to the SI of a water phantom that contained 0.25 mmol/L gadopentetate dimeglumine (Magnevist; Schering). T1-weighted, T2-weighted, and T2*-weighted SI ratios that compared the SI of the lymph nodes with that of the muscle tissue and T1-weighted SI ratios that compared the SI of the salivary glands with that of the muscle tissue before and after gadofluorine M administration were evaluated.

Tumor-bearing group.—Image analysis for the evaluation of the presence of metastasis in the parotid, rostral mandibular, and caudal mandibular lymph nodes of each rabbit was performed separately and independently by two radiologists (M.H.H., B.J.K.) before the pathologic results were known. Two different sets of readings were performed in each rabbit in the following order: gadofluorine M set (unenhanced T1-weighted and T2-weighted MR images with gadofluorine M–enhanced T1-weighted MR images) and MION-47 set (unenhanced T1-weighted, T2-weighted, and T2*-weighted MR images with MION-47–enhanced T2-weighted and T2*-weighted MR images). To minimize recall bias, images were presented in random order to each of the readers at each reading session. The interval between reading sessions was 1 week.

During reading of the gadofluorine M set, a lymph node was considered malignant if it showed either a focal area with lower SI than that of the well-enhanced surrounding functional lymph node tissue or no enhancement on T1-weighed MR images obtained 30 minutes after gadofluorine M administration (4).

Criteria for the review of the MION-47 set were as follows: A lymph node with an area of high SI on T2-weighted or T2*-weighted MR images after MION-47 administration was considered malignant, and a lymph node with a fatty hilum, complete signal void, and speckles of granularity without a definite focus of high SI on T2-weighted or T2*-weighted MR images after MION-47 administration was considered nonmalignant (11,15). A lymph node with central low SI on T2*-weighted MR images after MION-47 administration was also considered nonmalignant (16).

The investigators separately and independently assigned a confidence level to the diagnosis of metastasis for each lymph node. Diagnostic confidence was subjectively scored on a five-point scale (score of 0, no metastasis; 1, metastasis probably absent; 2, metastasis possibly present; 3, metastasis probably present; and 4, metastasis definitely present). Before interpreting the images, the two investigators were informed that a confidence level of 2 or higher represented a positive diagnosis of lymph node metastasis.

Statistical Analysis
For all statistical analyses, a two-tailed P value of less than .05 was considered to indicate a statistically significant difference. Statistical analyses were performed by using commercially available software (Instat, version 3.05, GraphPad, San Diego, Calif, and MedCalc, version 8.0.0.1, MedCalc, Mariakerke, Belgium).

Control group.—Tests for significant differences between T1-weighted, T2-weighted, and T2*-weighted SI ratios of nodal functional tissues before, immediately after, and 15, 30, 60 and 1440 minutes after gadofluorine M administration were performed by using a repeated measurements analysis of variance. The Tukey-Kramer test was used for multiple comparisons and for post hoc comparisons. Differences in the T1-weighted SI ratio between the functional nodal tissues and the salivary glands at each time point were compared by using an unpaired t test.

Tumor-bearing group.—Interobserver variability that was based on a five-point scale was evaluated by calculating the statistic for two readers with the weighted binary statistic. A value of 0.01–0.20 was judged as minor agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, high; and 0.81–1.00, excellent agreement.

A receiver operating characteristic analysis was performed to compare the diagnostic value of the gadofluorine M and MION-47 sets for each reader. The diagnostic accuracy of imaging for each reading session for each reader and his or her composite data were estimated by calculating the area under the receiver operating characteristic curve. Paired receiver operating characteristic curves were compared by means of a z test, as described by Hanley and McNeil (17).

Sensitivities and specificities with 95% confidence intervals (CIs) were calculated for the gadofluorine M and MION-47 sets. Data clustering (ie, more than one lesion per rabbit) was accounted for by using the method of Rao and Scott (18). Statistical differences were evaluated by comparing the sensitivities and specificities of the gadofluorine M and MION-47 sets by using a z test. At z testing, estimates of variance and covariance were weighted for data clustering in rabbits (19).

	RESULTS

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Histopathologic Results
Seventy-seven lymph nodes (31 parotid, 24 rostral mandibular, and 22 caudal mandibular lymph nodes) were isolated and evaluated. Isolated metastasis was found in only 20 of 31 parotid lymph nodes. In the parotid lymph nodes, the mean long-axis nodal diameters of malignant and benign lymph nodes were 7.2 mm (range, 5.3–10.0 mm) and 7.0 mm (range, 5.0–8.0 mm), respectively, and the mean short-axis nodal diameters of malignant and benign lymph nodes were 5.3 mm (range, 4.0–7.3 mm) and 5.1 mm (range, 4.2–6.0 mm), respectively. All 11 benign parotid lymph nodes showed reactive hyperplasia at histopathologic analysis. The mean long- and short-axis diameters of the metastases were 4.6 mm (range, 2.0–8.0 mm), and 3.5 mm (range, 1.5–6.0 mm), respectively.

MR Imaging Analysis
Control group.—The repeated measurements analysis of variance test revealed no significant changes in SI ratio for functional lymph node tissue on T2-weighted (P = .104) or T2*-weighted (P = .893) MR images obtained before, immediately after, and 15, 30, 60 and 1440 minutes after gadofluorine M administration; however, a statistically significant difference in SI ratio was observed for functional lymph node tissue on T1-weighted MR images (P < .001) (Table 1, Fig 1). T1-weighted SI ratios for functional lymph node tissue increased to a maximum value of 2.934 ± 0.020 at 30 minutes after gadofluorine M injection (P < .001, Tukey-Kramer test). A maximum T1-weighted SI difference between functional lymph node tissues (1.601 ± 0.013) and the salivary glands (2.504 ± 0.009) was observed on MR images obtained immediately after gadofluorine M injection (P < .001, unpaired t test). A combination of T1-weighted MR images obtained immediately after and 30 minutes after gadofluorine M administration allowed excellent depiction of cervical lymph nodes located adjacent to the salivary glands (Fig 1).

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 1: Unenhanced (Baseline) and contrast-enhanced coronal T1-weighted spin-echo MR images (400/12) obtained immediately after and 15, 30, 60, and 1440 minutes after gadofluorine M administration (0.05 mmol gadolinium per kilogram body weight). Images show caudal mandibular lymph nodes (arrows) and mandibular salivary glands (arrowheads) in one rabbit from the control group. Functional lymph node tissues show strong enhancement 15 minutes after gadofluorine M administration, and salivary glands show strong enhancement immediately after injection.

For both readers (Table 2), the gadofluorine M set showed significantly greater areas under the receiver operating characteristic curve than did the MION-47 set (P = .033 and .022, respectively).

View larger version (95K): [in this window] [in a new window] [Download PPT slide]   Figure 2a: Left parotid lymph node in rabbit in the prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph node (arrowheads) with low SI and VX2 tumor (arrow) with slightly high SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph node, with slight enhancement in malignant tissue (straight arrow). High contrast between functional lymph node tissue and malignant tissue in b enables more obvious detection of metastasis than in d. VX2 tumor (curved arrow) with peripheral rim enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows left parotid lymph node (arrowheads) with high SI and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows functional tissue (arrowheads) in left parotid lymph node with uniform low SI and peripheral malignant tissue (straight arrow) with high SI that was not detected during prospective readings because of poor contrast between malignant tissue and surrounding parenchymal tissue. High SI in VX2 tumor (curved arrow) with peripheral low SI is noted. (e) Histopathologic specimen shows malignant tissue (arrows) with maximum diameter of 2 mm in the subcapsular portion of left parotid lymph node. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (102K): [in this window] [in a new window] [Download PPT slide]   Figure 2b: Left parotid lymph node in rabbit in the prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph node (arrowheads) with low SI and VX2 tumor (arrow) with slightly high SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph node, with slight enhancement in malignant tissue (straight arrow). High contrast between functional lymph node tissue and malignant tissue in b enables more obvious detection of metastasis than in d. VX2 tumor (curved arrow) with peripheral rim enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows left parotid lymph node (arrowheads) with high SI and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows functional tissue (arrowheads) in left parotid lymph node with uniform low SI and peripheral malignant tissue (straight arrow) with high SI that was not detected during prospective readings because of poor contrast between malignant tissue and surrounding parenchymal tissue. High SI in VX2 tumor (curved arrow) with peripheral low SI is noted. (e) Histopathologic specimen shows malignant tissue (arrows) with maximum diameter of 2 mm in the subcapsular portion of left parotid lymph node. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (120K): [in this window] [in a new window] [Download PPT slide]   Figure 2c: Left parotid lymph node in rabbit in the prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph node (arrowheads) with low SI and VX2 tumor (arrow) with slightly high SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph node, with slight enhancement in malignant tissue (straight arrow). High contrast between functional lymph node tissue and malignant tissue in b enables more obvious detection of metastasis than in d. VX2 tumor (curved arrow) with peripheral rim enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows left parotid lymph node (arrowheads) with high SI and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows functional tissue (arrowheads) in left parotid lymph node with uniform low SI and peripheral malignant tissue (straight arrow) with high SI that was not detected during prospective readings because of poor contrast between malignant tissue and surrounding parenchymal tissue. High SI in VX2 tumor (curved arrow) with peripheral low SI is noted. (e) Histopathologic specimen shows malignant tissue (arrows) with maximum diameter of 2 mm in the subcapsular portion of left parotid lymph node. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (128K): [in this window] [in a new window] [Download PPT slide]   Figure 2d: Left parotid lymph node in rabbit in the prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph node (arrowheads) with low SI and VX2 tumor (arrow) with slightly high SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph node, with slight enhancement in malignant tissue (straight arrow). High contrast between functional lymph node tissue and malignant tissue in b enables more obvious detection of metastasis than in d. VX2 tumor (curved arrow) with peripheral rim enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows left parotid lymph node (arrowheads) with high SI and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows functional tissue (arrowheads) in left parotid lymph node with uniform low SI and peripheral malignant tissue (straight arrow) with high SI that was not detected during prospective readings because of poor contrast between malignant tissue and surrounding parenchymal tissue. High SI in VX2 tumor (curved arrow) with peripheral low SI is noted. (e) Histopathologic specimen shows malignant tissue (arrows) with maximum diameter of 2 mm in the subcapsular portion of left parotid lymph node. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (126K): [in this window] [in a new window] [Download PPT slide]   Figure 2e: Left parotid lymph node in rabbit in the prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph node (arrowheads) with low SI and VX2 tumor (arrow) with slightly high SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph node, with slight enhancement in malignant tissue (straight arrow). High contrast between functional lymph node tissue and malignant tissue in b enables more obvious detection of metastasis than in d. VX2 tumor (curved arrow) with peripheral rim enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows left parotid lymph node (arrowheads) with high SI and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows functional tissue (arrowheads) in left parotid lymph node with uniform low SI and peripheral malignant tissue (straight arrow) with high SI that was not detected during prospective readings because of poor contrast between malignant tissue and surrounding parenchymal tissue. High SI in VX2 tumor (curved arrow) with peripheral low SI is noted. (e) Histopathologic specimen shows malignant tissue (arrows) with maximum diameter of 2 mm in the subcapsular portion of left parotid lymph node. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (103K): [in this window] [in a new window] [Download PPT slide]   Figure 3a: Two left parotid lymph nodes in rabbit in prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with low SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph nodes, with slight enhancement in malignant tissue (straight arrows). High contrast between functional lymph node tissue and malignant tissue in b enables detection of metastasis. VX2 tumor (curved arrow) with heterogeneous enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows two left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows two left parotid lymph nodes (arrowheads) with high SI and a VX2 tumor (arrow) with heterogeneous high and low SI. (e) Histopathologic specimen shows two left parotid lymph nodes almost completely replaced by malignant tissue (arrows). MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (110K): [in this window] [in a new window] [Download PPT slide]   Figure 3b: Two left parotid lymph nodes in rabbit in prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with low SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph nodes, with slight enhancement in malignant tissue (straight arrows). High contrast between functional lymph node tissue and malignant tissue in b enables detection of metastasis. VX2 tumor (curved arrow) with heterogeneous enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows two left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows two left parotid lymph nodes (arrowheads) with high SI and a VX2 tumor (arrow) with heterogeneous high and low SI. (e) Histopathologic specimen shows two left parotid lymph nodes almost completely replaced by malignant tissue (arrows). MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (126K): [in this window] [in a new window] [Download PPT slide]   Figure 3c: Two left parotid lymph nodes in rabbit in prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with low SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph nodes, with slight enhancement in malignant tissue (straight arrows). High contrast between functional lymph node tissue and malignant tissue in b enables detection of metastasis. VX2 tumor (curved arrow) with heterogeneous enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows two left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows two left parotid lymph nodes (arrowheads) with high SI and a VX2 tumor (arrow) with heterogeneous high and low SI. (e) Histopathologic specimen shows two left parotid lymph nodes almost completely replaced by malignant tissue (arrows). MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (120K): [in this window] [in a new window] [Download PPT slide]   Figure 3d: Two left parotid lymph nodes in rabbit in prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with low SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph nodes, with slight enhancement in malignant tissue (straight arrows). High contrast between functional lymph node tissue and malignant tissue in b enables detection of metastasis. VX2 tumor (curved arrow) with heterogeneous enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows two left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows two left parotid lymph nodes (arrowheads) with high SI and a VX2 tumor (arrow) with heterogeneous high and low SI. (e) Histopathologic specimen shows two left parotid lymph nodes almost completely replaced by malignant tissue (arrows). MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (154K): [in this window] [in a new window] [Download PPT slide]   Figure 3e: Two left parotid lymph nodes in rabbit in prone position 4 weeks after VX2 tumor inoculation. Coronal T1-weighted spin-echo MR images (400/12) (a) before and (b) 30 minutes after gadofluorine M injection. In a, left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with low SI are seen. In b, strong enhancement is seen in functional tissues (arrowheads) of left parotid lymph nodes, with slight enhancement in malignant tissue (straight arrows). High contrast between functional lymph node tissue and malignant tissue in b enables detection of metastasis. VX2 tumor (curved arrow) with heterogeneous enhancement is also seen. (c) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) before MION-47 injection shows two left parotid lymph nodes (arrowheads) and VX2 tumor (arrow) with high SI. (d) T2*-weighted gradient-echo MR image (400/24, 20° flip angle) 1440 minutes after MION-47 injection shows two left parotid lymph nodes (arrowheads) with high SI and a VX2 tumor (arrow) with heterogeneous high and low SI. (e) Histopathologic specimen shows two left parotid lymph nodes almost completely replaced by malignant tissue (arrows). MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (100K): [in this window] [in a new window] [Download PPT slide]   Figure 4a: Right caudal mandibular lymph node in rabbit in prone position 4 weeks after VX2 tumor inoculation. (a) Coronal T1-weighted spin-echo MR image (400/12) 30 minutes after gadofluorine M injection shows less enhancement in central functional tissue (straight arrow) of right caudal mandibular lymph node relative to peripheral functional tissue (arrowheads), which was considered malignant during prospective readings. VX2 tumor (curved arrow) with peripheral enhancement is also noted. (b) Histopathologic specimen shows a right caudal mandibular lymph node without malignant tissue. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 4b: Right caudal mandibular lymph node in rabbit in prone position 4 weeks after VX2 tumor inoculation. (a) Coronal T1-weighted spin-echo MR image (400/12) 30 minutes after gadofluorine M injection shows less enhancement in central functional tissue (straight arrow) of right caudal mandibular lymph node relative to peripheral functional tissue (arrowheads), which was considered malignant during prospective readings. VX2 tumor (curved arrow) with peripheral enhancement is also noted. (b) Histopathologic specimen shows a right caudal mandibular lymph node without malignant tissue. MR images and histopathologic specimen do not exactly align because section orientation is not completely identical. (Hematoxylin-eosin stain; original magnification, x5.)

	DISCUSSION

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

In our study, maximum SI in the functional lymphatic tissue was observed 30 minutes after injection in the control group; thus, pre- and postcontrast MR imaging of the lymph nodes could be performed in one imaging session. In addition, our results from the control group show that T1-weighted MR images obtained immediately after gadofluorine M administration allow straightforward detection of the cervical lymph nodes because of the high contrast between the slightly enhanced functional lymphatic tissues and the strongly enhanced adjacent salivary glands. As a result, we included T1-weighted MR images obtained immediately after and 30 minutes after gadofluorine M administration in the gadofluorine M set as the optimal gadofluorine M–enhanced MR imaging protocol for the evaluation of cervical lymph node metastasis.

The results of our receiver operating characteristic analysis showed that the gadofluorine M set (area under the curve, 0.997 and 0.981) had a significantly higher accuracy for metastatic lymph node depiction than did the MION-47 set (area under the curve, 0.889 and 0.846) (P < .05). In addition, gadofluorine M enabled the additional detection of 20 metastatic lymph nodes that were not detected with MION-47. Histopathologic results revealed that these metastases were small (long-axis diameter of 3 mm or less) and were located at the subcapsular portion of lymph nodes. We believe that high contrast between the enhanced functional lymph node tissue and the only slightly enhanced malignant tissue on gadofluorine M–enhanced T1-weighted MR images enabled metastasis detection.

The limitations of MION-47–enhanced MR imaging for the detection of small peripherally located metastases are, in our opinion, due to (a) the "blooming effect," which is why very small lesions go undetected (T2*-weighted imaging is extremely sensitive to magnetic susceptibility effects) (22,23) and (b) the relatively poor contrast between the metastasis and the surrounding extralymphatic tissue on T2-weighted and T2*-weighted MR images, which makes the detection of subcapsular metastases difficult. It has also been suggested that micrometastases in the germinal center may not be seen due to the scarcity of macrophages in this area (24,25).

We also encountered false-positive cases for the gadofluorine M and MION-47 sets. It is well known that false-positive cases with USPIO-enhanced MR imaging are related to an insufficient dose of contrast agent or delivery problems (6,26). On gadofluorine M–enhanced MR images, the lower enhancement of the medullary sinus compared with that of the cortex is responsible for false-positive cases.

Our study has some limitations. Sequential imaging was performed in the same tumor-bearing group of rabbits rather than in two animals groups that could be separated to minimize the number of animals in accordance with our institution guidelines; MION-47–enhanced MR imaging was performed approximately 25 hours after gadofluorine M administration (24 hours after MION-47 administration), which might be a disadvantage for the detection of lymph node metastases by using MION-47–enhanced MR imaging. As was described in a previous report, malignant lymph node tissue demonstrated enhancement on T1-weighted MR images 24 hours after gadofluorine M administration (4), and our results in the control group also demonstrated the residual enhancement in functional nodal tissues on T1-weighted MR images 24 hours after injection.

In addition, the MR imaging protocol used in our study raises the issues of a potential interaction between MION-47 and gadofluorine M for the lymph node uptake of these materials. However, the results obtained in the control group show that no significant T2-weighted and T2*-weighted SI changes occurred in the lymph nodes after gadofluorine M administration. Also, MION-47 and gadofluorine M are known to have different pathways for lymph node uptake, as described above (4,6,20). Thus, we believe that there was no significant decrease in the detection of nodal metastases for the MION-47 set.

Although it might be reasonable to perform MION-47–enhanced MR imaging after most of the injected dose of gadofluorine M is eliminated from the body (7 days after intravenous injection), thereby avoiding the potential interaction between gadofluorine M and MION-47, the VX2 tumor grows too rapidly to expect an equivalent lymph node condition after 1 week.

The other drawback is that the gadofluorine M set included two sessions of postcontrast MR imaging (immediately after and 30 minutes after injection), but the MION-47 set included only one session of postcontrast imaging (24 hours after injection). This might be an advantage for the detection of lymph node metastases in the gadofluorine M set; however, we performed optimized MR imaging protocols for the gadofluorine M and MION-47 sets, which provided a useful and fair comparison (5–11). Finally, we did not consider the effectiveness of gadofluorine M in the evaluation of T and M staging; thus, future study is warranted.

Practical application: In terms of MR lymphography, gadofluorine M has two benefits compared with USPIO particles. One is that a complete pre- and postcontrast MR imaging examination is performed in one session, and the other is that the high contrast between malignant and functional lymph node tissues facilitates the detection of lymph node metastases. In oncologic imaging, differentiation between malignant and benign lymph nodes is of major interest when determining the therapeutic plan. Thus, we believe that gadofluorine M might be useful for such differentiation and that future experimental and clinical investigations seem warranted.

	ADVANCE IN KNOWLEDGE

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

 

• The results of our receiver operating characteristic analysis showed that the gadofluorine M set (area under the curve, 0.997 and 0.981) had a significantly higher accuracy for metastatic lymph node depiction than did the monocrystalline iron oxide nanoparticle–47 set (area under the curve, 0.889 and 0.846) (P < .05).
  

	FOOTNOTES

 

Abbreviations: CI = confidence interval • MION = monocrystalline iron oxide nanoparticle • SI = signal intensity • USPIO = ultrasmall superparamagnetic iron oxide

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantors of integrity of entire study, S.H.C., M.H.H., H.J.W.; 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, S.H.C., M.H.H., W.K.M.; experimental studies, S.H.C., M.H.H., W.K.M., K.R.S., J.K.W., H.J.W.; statistical analysis, S.H.C., M.H.H., B.J.K.; and manuscript editing, S.H.C., M.H.H., W.K.M., J.H.K., B.J.K., D.G.N., K.H.C.

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