Original Research n Gastrointestinal
Imaging
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Esophageal Carcinoma: Ex Vivo Evaluation with Diffusion-Tensor MR Imaging and Tractography at 7 T1 Ichiro Yamada, MD Keigo Hikishima, PhD Naoyuki Miyasaka, MD Tatsuyuki Kawano, MD Yutaka Tokairin, MD Eisaku Ito, MD Daisuke Kobayashi, MD Yoshinobu Eishi, MD Hideyuki Okano, MD
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From the Departments of Diagnostic Radiology and Oncology (I.Y.), Pediatrics, Perinatal and Maternal Medicine (N.M.), Esophagogastric Surgery (T.K., Y.T.), and Pathology (E.I., D.K., Y.E.), Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; Department of Physiology, Keio University School of Medicine, Tokyo, Japan (K.H., H.O.); and Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan (K.H.). Received October 19, 2013; revision requested November 26; revision received January 13, 2014; accepted January 16; final version accepted January 27. I.Y. supported by the Grant-in-Aid for Scientific Research (C) of MEXT, Japan (23591753). Address correspondence to I.Y. (e-mail: [email protected]).
Purpose:
To determine the feasibility of diffusion-tensor magnetic resonance (MR) imaging and tractography as a means of evaluating the depth of mural invasion by esophageal carcinomas.
Materials and Methods:
This study was approved by the institutional review board, and written informed consent was obtained from each patient. Twenty esophageal specimens, each containing a carcinoma, were studied with a 7.0-T MR imaging system equipped with a four-channel phased-array surface coil. Diffusion-tensor MR images were obtained with a field of view of 50–60 mm 3 25–30 mm, matrix of 256 3 128, section thickness of 1 mm, b value of 1000 sec/mm2, and motion-probing gradient in seven noncollinear directions. The MR images were compared with the histopathologic findings as the reference standard. The differences in diffusion-tensor MR imaging parameters between the carcinoma and the layers of the esophageal wall were statistically analyzed by using the Dunnett test.
Results:
In all 20 carcinomas (100%), the diffusion-weighted images, apparent diffusion coefficient (ADC) maps, fractional anisotropy (FA) maps, l1 maps, and directionencoded color FA maps made it possible to determine the depth of tumor invasion of the esophageal wall that was observed during histopathologic examination. The l1 maps showed the best contrast between the carcinomas and the layers of the esophageal wall. The carcinomas had both lower ADC values and lower FA values than the normal esophageal wall; thus, the carcinomas were clearly demarcated from the normal esophageal wall. Diffusiontensor tractography images were also useful for determining the depth of tumor invasion of the esophageal wall.
Conclusion:
Diffusion-tensor MR imaging and tractography are feasible in esophageal specimens and provide excellent morphologic data for the evaluation of mural invasion by esophageal carcinomas. RSNA, 2014
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Online supplemental material is available for this article.
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T
he prognosis of patients with esophageal carcinoma is strictly dependent on the histopathologic stage of the carcinoma (1,2), and accurate preoperative staging has a definitive effect on the selection of appropriate treatment. However, preoperative staging of esophageal carcinoma has been extremely difficult because the imaging modalities that are available do not allow precise evaluation of the depth of tumor invasion of the esophageal wall (1,2). Computed tomography (CT) is used as a method of evaluating extensive tumor spread into adjacent organs, but CT does not allow evaluation of less extensive tumor spread, because the poor soft-tissue contrast makes it impossible to resolve the layers of the esophageal wall, even with new multidetector CT technology (3–5). Endoscopic ultrasonography (US) is widely used to
Advances in Knowledge nn The diffusion-weighted images, apparent diffusion coefficient (ADC) maps, fractional anisotropy (FA) maps, l1 maps, and direction-encoded color FA maps made it possible to determine the depth of invasion of the esophageal wall by all 20 carcinomas (100%). nn The l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall, and the l1 values (0.61 3 1023 mm2/sec) of the carcinomas were significantly lower than the l1 values of all of the layers of the normal esophageal wall (P < .001, except for the epithelium [P = .047]). nn The carcinomas also had lower ADC values (0.54 3 1023 mm2/ sec) and lower FA values (0.20) than the normal esophageal wall; thus, the carcinomas were clearly demarcated from the normal esophageal wall.
evaluate esophageal carcinomas, but it has many inherent problems, including technical failures in stenotic tumors, high operator dependency, artifactual interface echoes in the esophageal wall, and a limited sonographic range (5–7). Magnetic resonance (MR) imaging has been reported to demonstrate mural invasion by esophageal carcinomas, and it is an alternative to CT and endoscopic US (8–10), but conventional MR imaging is still insufficient in depicting the individual layers of the esophageal wall, even by using endocavitary coils (11–16). However, we have recently demonstrated that diffusion-tensor MR imaging and tractography are useful for demonstrating the individual layers of the esophageal wall (17). The purpose of this study was to prospectively determine the feasibility of diffusion-tensor MR imaging and tractography as a means of evaluating the depth of mural invasion by esophageal carcinomas.
Materials and Methods Study Population This study was approved by our institutional review board, and written informed consent with regard to the purpose of this study and the use of clinical and histopathologic data was obtained from each patient. We studied 20 surgical specimens of the esophagus, each of which contained a tumor that was histopathologically diagnosed as squamous cell carcinoma. Normal portions of the esophagus in 12 of these patients were analyzed and findings were reported by using the same techniques as in our previous work (17). No adenocarcinomas or other histologic types of tumors were identified in any of the surgical specimens.
nn Diffusion-tensor tractography images were also useful for determining the depth of tumor invasion of the esophageal wall. Radiology: Volume 272: Number 1—July 2014 n radiology.rsna.org
Implication for Patient Care nn Diffusion-tensor MR imaging and tractography may be useful in the future as a diagnostic tool for the noninvasive assessment of mural invasion by esophageal carcinomas.
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The specimens were obtained from 20 consecutive patients with esophageal carcinoma who were surgically treated at our institution between November 2012 and September 2013. Their ages at the time of surgery ranged from 46 to 83 years (mean age 6 standard deviation, 66 years 6 7; 18 men [mean age, 66 years 6 8; range, 46–83 years] and two women [mean age, 67 years 6 5; range, 62–71 years]). Thirteen patients underwent no treatment prior to surgical resection, five patients underwent only chemotherapy, and two patients underwent both chemotherapy and radiation therapy. All 20 specimens were imaged after fixation in 10% formalin. We did not examine the esophagus in vivo in this series. The time interval between surgery and imaging was approximately 24 hours. We performed MR imaging after the completion of formalin fixation, so partial fixation of the specimen with formalin did not occur during data acquisition. The length of esophageal specimens ranged from 105 to 250 mm (mean length, 164 mm 6 39). The size (ie, the largest diameter) of the resected tumors according to the Response Evaluation Criteria in Solid Tumors ranged from 18 to 80 mm (mean size, 46 mm 6 21).
Imaging Technique Diffusion-tensor MR imaging was performed by using a 7.0-T MR imaging Published online before print 10.1148/radiol.14132170 Content codes: Radiology 2014; 272:164–173 Abbreviations: ADC = apparent diffusion coefficient FA = fractional anisotropy Author contributions: Guarantors of integrity of entire study, I.Y., K.H., N.M., Y.E.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, I.Y., N.M.; clinical studies, I.Y., N.M., Y.T., D.K., Y.E.; experimental studies, I.Y., K.H., T.K.; statistical analysis, I.Y., N.M.; and manuscript editing, I.Y., K.H., N.M., T.K. Conflicts of interest are listed at the end of this article.
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unit (BioSpec 70/16; Bruker BioSpin, Ettlingen, Germany) equipped with actively shielded gradients that had a maximum strength of 700 mT/m. A four-channel phased-array surface coil was used for all measurements. The orientation of diffusion-tensor MR imaging was set longitudinally along the long axis of the resected segment of the esophagus. The specimen was placed directly on the surface coil. Diffusion-tensor MR imaging data sets were acquired by using a diffusionweighted spin-echo pulse sequence. The imaging parameters were repetition time (msec)/echo time (msec), 3000/25; field of view, 50–60 3 25–30 mm; matrix, 256 3 128; section thickness, 1 mm without intersection gaps; voxel size, 0.195–0.234 3 0.195–0.234 3 1 mm (range, 0.038–0.055 mm3); number of signals acquired, two; b value, 0 sec/mm2 (for a reference b0 image without diffusion weighting) or 1000 sec/mm2 (gradient lobe separation = 14 msec); and motion-probing gradient in seven noncollinear directions. The acquisition time was 102 minutes, which was equal to the total imaging time because the diffusion-tensor imaging was the only pulse sequence used in the present study. Since our purpose was to compare the findings of diffusion-tensor MR imaging with the histopathologic findings, T1- and T2weighted sequences were not used in the present study.
Image Processing The diffusion-tensor MR imaging data were processed with Diffusion Toolkit software (Massachusetts General Hospital, Boston, Mass) (18) by using monoexponential fitting. The three eigenvalues (l1 l2 l3) and corresponding eigenvectors were calculated for each voxel. The formulas (17) were used to calculate the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) for each voxel from the eigenvalues. As shown in our previous report (17), isotropic diffusion-weighted images, ADC maps, FA maps, l1 maps, l2 maps, and l3 maps were generated on a voxel-by-voxel basis. We also generated 166
direction-encoded color FA maps that showed anisotropy in different colors according to the direction of the major axis. Color FA maps make it possible to differentiate fiber tracts on the basis of their orientation. As shown in our previous report (17), diffusion-tensor tractograpy images were computed with TrackVis software (Massachusetts General Hospital) (18). The software performed fiber tracking by reading in all of the tracking data of the esophageal specimen, and it displayed all fiber tracts contained in the specimen. Then we used the two regions of interest method to selectively display specific fiber tracts (19,20). One of the authors (I.Y., with 23 years of experience in reading MR images) set two regions of interest on opposite sides of the specific fiber tracts in the specimen. For the fiber tracts oriented along the long axis and short axis of the esophagus, the size of the regions of interest was approximately equal to the thickness of each layer, multiplied by the width and length of the displayed tractography image, respectively.
Image Analysis An independent, blinded evaluation of diffusion-tensor MR images in each surgical specimen was performed by two observers (I.Y. and N.M., with 23 and 19 years of experience in reading MR images, respectively) who had no knowledge of the results of the histopathologic examination. When the observers could not fully agree on the findings, consensus was achieved by means of discussion. This occurred in two (10%) of 20 specimens in the present study. Isotropic diffusion-weighted images, ADC maps, FA maps, l1 maps, and direction-encoded color FA maps were reviewed for the presence, signal intensity, uniformity, and thickness of each layer of the esophageal wall. As described in our previous report (8), the contour and signal intensity of the carcinoma were also analyzed. The degree of carcinoma penetration into the esophageal wall was categorized according to the layer invaded: mucosa,
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submucosa, muscularis propria, or adventitia (8). For the ADC maps, FA maps, and l1 maps, regions of interest were placed on the carcinoma and on the layers of the esophageal wall. The size of the regions of interest was approximately equal to the cross-section area of the carcinoma and to the thickness of each layer, and one of the observers (I.Y.) shaped and placed the regions of interest. The number of the regions of interest was three or four for the carcinoma and for each layer per specimen, and the mean value of the three or four regions of interest in the carcinoma and in each layer of the esophageal wall was calculated. All quantitative diffusiontensor measurements reported were obtained by analyzing the regions of interest with ImageJ 1.47 software (available at http://imagej.nih.gov/ij). Fiber tracts on the diffusion-tensor tractography images were also evaluated for the presence, orientation, color, continuity, uniformity, and thickness of each layer of the esophageal wall. The diffusion-tensor MR imaging findings in the 20 esophageal specimens were compared with the histopathologic findings, which served as the reference standard. The MR images were compared with specific histopathologic sections on a slice-by-slice level, and correlations were made by means of visual inspection. For matching the MR images and the specimens, spatial correlation was achieved by identifying anatomic landmarks (eg, esophageal contour and blood vessels) that were depicted.
Histologic Preparations and Examination After MR imaging, each surgical specimen was sectioned longitudinally so that the orientation of the sections corresponded to the orientation of the MR images. The sectioned specimens were embedded in paraffin and cut into 6-mm-thick slices with a microtome. These slices were then stained with hematoxylin-eosin stain and elastica–van Gieson stain. An experienced pathologist (E.I., with 15 years of experience in histopathology) who did not have any
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knowledge of the MR imaging findings identified carcinoma invasion into each layer of the esophageal wall.
Statistical Analysis Means 6 standard deviations were calculated for the ADC values, FA values, and l1 values in the carcinoma and in each layer of the esophageal wall. All statistical analyses were performed with a commercial software package (SPSS Statistics, version 20; IBM SPSS Japan, Tokyo, Japan). The differences in diffusion-tensor MR imaging parameters between the carcinoma and the layers of the esophageal wall were statistically analyzed by using the Dunnett test. A P value less than .05 was considered to indicate a significant difference. Furthermore, box plots were used for comparing the parameters of the carcinoma and the esophageal wall layers, and scatterplots were used for comparing the pixels of the carcinoma and the esophageal wall layers. The Wilcoxon matched-pairs signed rank test was used for analysis of the box plots. Diffusion-tensor imaging was the only pulse sequence used, and a definitive diagnosis of the depth of tumor invasion was assessed on the basis of the combined findings on the diffusion-weighted images, ADC maps, FA maps, l1 maps, and color FA maps, so it was difficult to apply a receiver operating characteristic curve analysis to each map. Results Diffusion-weighted Images, ADC Maps, FA Maps, l1 Maps, and Direction-encoded Color FA Maps of Esophageal Carcinomas and Layers of the Esophageal Wall The diffusion-weighted images, ADC maps, FA maps, l1 maps, and direction-encoded color FA maps of all 20 specimens (100%) clearly depicted the esophageal wall as consisting of the eight layers (Fig E1 [online], Fig 1). These eight layers clearly corresponded to the layers of the esophageal wall observed in histologic sections. As shown in Figure E1 (online) and Figure 1, the esophageal carcinomas were seen as areas of high signal
intensity on diffusion-weighted images; as areas of low values on ADC maps, FA maps, and l1 maps; and as variable colors on direction-encoded color FA maps. The carcinomas on the diffusionweighted images appeared as areas of higher signal intensity than the lamina propria mucosae, submucosa, intermuscular connective tissue, and adventitia. On the ADC maps, the carcinomas had lower ADC values than the lamina propria mucosae, submucosa, intermuscular connective tissue, and adventitia. On the FA maps, the carcinomas also had lower FA values than the epithelium, muscularis mucosae, inner circular muscle, and outer longitudinal muscle. On the l1 maps, however, the carcinomas had lower l1 values than all of the layers of the esophageal wall; thus, the l1 maps provided the best contrast between the carcinoma and the layers of the esophageal wall. The reason they provided the best contrast was that the lamina propria mucosae, submucosa, intermuscular connective tissue, and adventitia had high l1 values, and the epithelium, muscularis mucosae, inner circular muscle, and outer longitudinal muscle had intermediate l1 values.
ADC Values, FA Values, and l1 Values of Esophageal Carcinomas and Layers of the Esophageal Wall As shown in the Table, the ADC values of the esophageal carcinomas were significantly lower than the ADC values of the lamina propria mucosae, muscularis mucosae, submucosa, intermuscular connective tissue, and adventitia (P < .001). The FA values of the carcinomas, on the other hand, were significantly lower than the FA values of the epithelium, muscularis mucosae, inner circular muscle, and outer longitudinal muscle (P < .001) and slightly higher than the FA of the submucosa (P = .012). The l1 values of the carcinomas, however, were significantly lower than the l1 values of all of the layers of the esophageal wall (P < .001, except for the epithelium [P = .047]). These findings explain why the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall.
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We also compared the ADC values, FA values, and l1 values of the carcinomas, solid layers, and loose layers of the esophageal wall (Fig E2 [online]). The ADC values of the carcinomas were significantly lower than the ADC values of the loose layers of the esophageal wall (P < .001). The FA values of the carcinomas were significantly lower than the FA values of the solid layers of the esophageal wall (P < .001). The l1 values of the carcinomas, however, were significantly lower than the l1 values of both the loose layers and the solid layers of the esophageal wall (P < .001 for both).
Scatterplots of the ADC Values, FA Values, and l1 Values of Esophageal Carcinomas and Layers of the Esophageal Wall As shown in Figure E3a (online), the pixels of the carcinoma on the scatterplot of the ADC values and FA values were clearly demarcated from the layers of the normal esophageal wall, because the pixels of the carcinoma had lower ADC values and lower FA values than the other pixels of the esophageal wall. As shown in Figure E3b (online), the pixels of the carcinoma were clearly demarcated from the layers of the normal esophageal wall on the three-dimensional scatterplot, because the pixels of the carcinoma had lower l1 values than the other pixels of the esophageal wall. The two scatterplots clearly demonstrated that diffusion-tensor MR imaging makes it possible to determine the extent of esophageal carcinoma by identifying the areas that have low ADC values and low FA values and/or the areas that have low l1 values in the esophageal wall. Evaluation of Esophageal Carcinoma Invasion on Diffusion-Tensor MR Images Histopathologic examination of the 20 esophageal carcinomas showed that two of the carcinomas were confined to the mucosa (stage T1a), six carcinomas had invaded the submucosa (stage T1b), three carcinomas had involved the muscularis propria (stage T2), and nine carcinomas had extended into the adventitia (stage T3 or T4). The depth 167
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Figure 1
Figure 1: Images in a patient with esophageal carcinoma invading the submucosa. (a) Diffusion-weighted MR image (3000/25) depicts the carcinoma (T) as an area of high signal intensity. (b) ADC map (3000/25) depicts the carcinoma as a low-ADC area. (c) FA map (3000/25) depicts the carcinoma as a low-FA area. (d) l1 map (3000/25) clearly depicts the carcinoma as a low-l1 area. The l1 map shows better contrast between the carcinoma and the layers of the esophageal wall than the diffusion-weighted image, ADC map, FA map, and direction-encoded color FA map. (e) Direction-encoded color FA map (3000/25) depicts the carcinoma as a variable-color area. Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (f) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 310) shows carcinoma that has invaded the submucosa, which has disrupted the muscularis mucosae layer (arrows). Adv = adventitia, Epi = epithelium, ICM = inner circular muscle, IMCT = intermuscular connective tissue, LPM = lamina propria mucosae, MM = muscularis mucosae, OLM = outer longitudinal muscle, SM = submucosa, T = tumor.
ADC Values, FA Values, and l1 Values in Esophageal Carcinoma and Layers of the Esophageal Wall at Diffusion-Tensor MR Imaging Tissue Carcinoma Epithelium Lamina propria mucosae Muscularis mucosae Submucosa Inner circular muscle Intermuscular connective tissue Outer longitudinal muscle Adventitia
ADC Values (31023 mm2/sec) FA Values
l1 Values (31023 mm2/sec)
0.54 6 0.06 0.63 6 0.06 1.51 6 0.19* 0.80 6 0.09* 1.90 6 0.20* 0.70 6 0.09 1.46 6 0.20* 0.69 6 0.08 1.91 6 0.18*
0.61 6 0.07 0.82 6 0.08* 1.84 6 0.26* 0.95 6 0.16* 2.27 6 0.22* 1.10 6 0.14* 1.88 6 0.28* 1.09 6 0.15* 2.31 6 0.18*
0.20 6 0.03 0.58 6 0.06* 0.16 6 0.04 0.58 6 0.06* 0.13 6 0.03* 0.64 6 0.07* 0.20 6 0.04 0.69 6 0.06* 0.15 6 0.04
Note.—Data are mean values 6 standard deviations. * Values were significantly different from the corresponding value of the esophageal carcinoma.
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of invasion of the esophageal wall by all 20 carcinomas (100%) was clearly demonstrated by the diffusion-weighted images, ADC maps, FA maps, l1 maps, and direction-encoded color FA maps. Since the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall, the l1 maps were the most useful means of assessing tumor invasion of the esophageal wall. In the present study, there were no cases of false-positive or falsenegative findings with respect to assessment of invasion of the different esophageal layers. However, since there was disagreement in two cases (10%) that was resolved by means of consensus, it may be unlikely that the technique radiology.rsna.org n Radiology: Volume 272: Number 1—July 2014
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Figure 2
Figure 2: Images depict an esophageal carcinoma confined to the mucosa. (a) l1 map (3000/25) clearly shows discrete thickenings of the mucosal layer (arrows) that have lower l1 values than other parts of the esophageal wall. (b) FA map (3000/25) shows that the muscularis mucosae layer (arrows) is intact. (c) Diffusion-tensor tractography image (3000/25) shows that the following four layers appear to be intact: epithelium (blue fiber tracts), muscularis mucosae (green fiber tracts), inner circular muscle (blue fiber tracts), and outer longitudinal muscle (green fiber tracts). It is difficult to differentiate the normal epithelium from the epithelium involved by carcinoma, owing to the superficial nature of the tumor, so this point seems to be a limitation of the technique. Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (d) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 320) shows the carcinoma confined to the mucosa (arrows).
would actually have perfect diagnostic performance, and this issue may constitute a limitation in the present study. Carcinomas confined to the mucosa appeared as discrete thickening of the mucosal layer that had lower l1 values than other parts of the esophageal wall (Fig 2). Carcinomas that had invaded the submucosa appeared as irregular masses with low l1 values that had disrupted the muscularis mucosae layer (Fig 3). Carcinomas that had involved the muscularis propria appeared
as low-l1 masses that had partially replaced the muscularis propria layer (Fig E4 [online]). Carcinomas that had extended into the adventitia appeared as low-l1 masses that had completely disrupted the muscularis propria layer and invaded the adventitial layer (Fig 4).
Evaluation of Esophageal Carcinoma Invasion on Diffusion-Tensor Tractography Images Three-dimensional diffusion-tensor tractography images of all 20 specimens
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(100%) clearly depicted the four layers (Figs 2–4, Fig E4 [online]). The diffusion-tensor tractography images were also found to be useful for determining the depth of tumor invasion of the esophageal wall. When the carcinoma was confined to the mucosa, all four layers appeared to be intact (Fig 2). When the carcinoma had invaded the submucosa, the muscularis mucosae layer (green fiber tracts) was observed to have been disrupted by the tumor (Fig 3). When the 169
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Figure 3
Figure 3: Images depict an esophageal carcinoma that has invaded the submucosa. (a) l1 map (3000/25) clearly shows a mass lesion (arrows) that has lower l1 values than other parts of the esophageal wall. (b) FA map (3000/25) shows that the mass lesion has disrupted the muscularis mucosae layer (arrows). (c) Diffusion-tensor tractography image (3000/25) shows that the muscularis mucosae layer (green fiber tracts) has been disrupted by the tumor (arrows). Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (d) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 310) shows carcinoma that has invaded the submucosa, which has disrupted the muscularis mucosae layer (arrows).
carcinoma had involved the muscularis propria, the muscularis propria layer (blue fiber tracts and/or green fiber tracts) was observed to have been partially disrupted by the tumor (Fig E4 [online]). When the carcinoma had extended into the adventitia, the muscularis propria layer (both blue fiber tracts and green fiber tracts) was observed to have been completely disrupted by the tumor (Fig 4).
Discussion As we have shown in our previous work, the diffusion-weighted images, ADC maps, FA maps, l1 maps, and 170
direction-encoded color FA maps clearly depicted the normal esophageal wall as consisting of eight layers, thereby clearly demonstrating that diffusion-tensor MR images are able to depict the individual layers of the esophageal wall (17). We have proposed a new schematic illustration of the normal esophageal wall to explain the diffusion-tensor MR imaging and tractography findings in the normal esophageal wall (17). On the basis of our schematic illustration, alternating levels of cellularity (cell density), anisotropy, and directionality between the adjacent layers account for the clear depiction of the layers of the normal esophageal wall on the ADC maps, FA maps, and
direction-encoded color FA maps, respectively. Furthermore, the high signalto-noise ratios obtained by using 7.0-T MR imaging may have contributed to the clear depiction of the esophageal wall layers, because the signal-to-noise ratio increases approximately linearly with the field strength (21). In our current study, carcinomas on the l1 maps appeared darker than all of the layers of the esophageal wall; thus, the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall. The l1 values of the carcinomas were found to be significantly lower than the l1 values of all of the layers of the esophageal wall. Thus,
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Figure 4
Figure 4: Images depict an esophageal carcinoma that has extended into the adventitia. (a) l1 map (3000/25) clearly shows an irregularly shaped mass lesion (arrows) with low l1 values. (b) FA map (3000/25) shows that the mass lesion has completely disrupted the muscularis propria layer and invaded the adventitial layer (arrows). (c) Diffusion-tensor tractography image (3000/25) shows that the muscularis propria layer (both blue fiber tracts and green fiber tracts) has been completely disrupted by the tumor (arrows). Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (d) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 31) shows carcinoma that has extended into the adventitia (arrows).
the l1 values of the carcinomas being the lowest explains why the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall. The l1 values of the solid layers appeared to be considerably higher than the ADC values of the solid layers because of local fiber orientation,
while the l1 values of the carcinomas and the loose layers were only slightly higher than the respective ADC values because of no local fiber orientation. These differences were responsible for the carcinomas having the lowest l1 values, thereby accounting for the l1 maps having the best contrast between
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the carcinomas and the esophageal wall layers. Our findings demonstrated that diffusion-tensor MR imaging makes it possible to determine the extent of esophageal carcinomas by identifying areas that have both low ADC values and low FA values and/or areas that have low l1 171
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values in the esophageal wall. Since the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall, the l1 maps were found to be most useful for assessing tumor invasion of the esophageal wall. The diffusion-tensor tractography images were also found to be useful for determining the depth of tumor invasion of the esophageal wall. Gilbert et al (22) used diffusion spectrum imaging and tractography to resolve the myoarchitecture of the bovine esophageal wall. Gaige et al (23) reported using diffusion-tensor imaging and tractography in vivo to visualize the structural anatomy of the human tongue, and their findings suggested the feasibility of invivo diffusion-tensor MR imaging and tractography in the human esophagus. Conventional MR imaging has recently been used for preoperative staging of esophageal carcinoma. Riddell et al (13–16) performed high-spatialresolution MR imaging with an external surface coil in patients with esophageal carcinoma. Dave et al (12) performed endoluminal MR imaging with a surface coil incorporated into the tip of an endoscope in patients with esophageal carcinoma. Therefore, we believe that by using an external surface coil technique or endoluminal surface coil technique, it may be possible to perform diffusion-tensor MR imaging and tractography in patients with esophageal carcinoma. There were some limitations to our study. First, this study was performed ex vivo, and the specimens were imaged after fixation in formalin. However, since previous studies of other organs showed that the diffusion anisotropy indexes measured in fixed and in vivo tissues provided essentially the same information regarding the underlying microstructure (24–28), using diffusiontensor MR imaging and tractography in fixed tissues would appear to be a valid means of evaluating the diffusion anisotropy of tissues in vivo. The same studies also showed that although the ADC values of fixed tissues were lower than those in vivo, the relative in vivo ADC difference between different tissue types was preserved in fixed tissues, and the 172
relative ADC difference showed no significant difference between fixed and in vivo tissues (26,27,29). Furthermore, the temperature effect of ex vivo imaging may influence ADC, FA, and other metrics, so it should be noted that the indicated values cannot be used as absolute thresholds for in vivo imaging. Second, the imaging time in this study was considerably long (102 minutes), so shortening the imaging time would be necessary to translate our data into in vivo diffusion-tensor MR imaging and tractography. At present, clinical application of whole-body 7-T MR imaging to esophageal carcinoma may be technically difficult because of motion effects (peristalsis, respiratory movement, and patient motion) and the deep location in the thorax. For this purpose, modifications of pulse sequences, the development of faster MR imaging techniques, or the application of higher field strengths to clinical settings may be required in the future. In conclusion, the results of the present study have demonstrated that diffusion-tensor MR imaging and tractography are able to clearly depict the individual tissue layers of the esophageal wall ex vivo. Diffusion-tensor MR imaging and tractography in these ex vivo specimens provided excellent diagnostic accuracy for evaluating mural invasion by esophageal carcinomas. Disclosures of Conflicts of Interest: I.Y. No relevant conflicts of interest to disclose. K.H. No relevant conflicts of interest to disclose. N.M. No relevant conflicts of interest to disclose. T.K. No relevant conflicts of interest to disclose. Y.T. No relevant conflicts of interest to disclose. E.I. No relevant conflicts of interest to disclose. D.K. No relevant conflicts of interest to disclose. Y.E. No relevant conflicts of interest to disclose. H.O. Financial activities related to the present article: none to disclose. Financial activities not related to the present article: author received payment for consultancies with San Bio, Easai, and Daiichi Sankyo; author and institution have received payment for patents relating to the introduction of foreign genes into early embryos of primates and the production of transgenic animals; and author has stock and/or stock options in San Bio. Other relationships: none to disclose.
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esophagogastric junction: data-driven staging for the seventh edition of the American Joint Committee on Cancer/International Union Against Cancer Cancer Staging Manuals. Cancer 2010;116(16):3763–3773. 2. Edge SB, Byrd DR, Compton CC, Fritz AG, Greene FL, Trotti A, eds. AJCC Cancer Staging Manual. 7th ed. New York, NY: Springer, 2010; 103–115. 3. Panebianco V, Grazhdani H, Iafrate F, et al. 3D CT protocol in the assessment of the esophageal neoplastic lesions: can it improve TNM staging? Eur Radiol 2006;16(2): 414–421. 4. Onbaş O, Eroglu A, Kantarci M, et al. Preoperative staging of esophageal carcinoma with multidetector CT and virtual endoscopy. Eur J Radiol 2006;57(1):90–95. 5. Wakelin SJ, Deans C, Crofts TJ, Allan PL, Plevris JN, Paterson-Brown S. A comparison of computerised tomography, laparoscopic ultrasound and endoscopic ultrasound in the preoperative staging of oesophago-gastric carcinoma. Eur J Radiol 2002;41(2): 161–167. 6. Preston SR, Clark GW, Martin IG, Ling HM, Harris KM. Effect of endoscopic ultrasonography on the management of 100 consecutive patients with oesophageal and junctional carcinoma. Br J Surg 2003;90(10):1220–1224. 7. Schlick T, Heintz A, Junginger T. The examiner’s learning effect and its influence on the quality of endoscopic ultrasonography in carcinoma of the esophagus and gastric cardia. Surg Endosc 1999;13(9):894–898. 8. Yamada I, Murata Y, Izumi Y, et al. Staging of esophageal carcinoma in vitro with 4.7-T MR imaging. Radiology 1997;204(2): 521–526. 9. Yamada I, Izumi Y, Kawano T, et al. Superficial esophageal carcinoma: an in vitro study of high-resolution MR imaging at 1.5T. J Magn Reson Imaging 2001;13(2):225–231. 10. Yamada I, Izumi Y, Kawano T, et al. Esophageal carcinoma: evaluation with high-resolution three-dimensional constructive interference in steady state MR imaging in vitro. J Magn Reson Imaging 2006;24(6):1326–1332. 11. Feldman DR, Kulling DP, Hawes RH, et al. MR endoscopy: preliminary experience in human trials. Radiology 1997;202(3): 868–870. 12. Dave UR, Williams AD, Wilson JA, et al. Esophageal cancer staging with endoscopic MR imaging: pilot study. Radiology 2004;230(1):281–286. 13. Riddell AM, Richardson C, Scurr E, Brown G. The development and optimization of
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high spatial resolution MRI for imaging the oesophagus using an external surface coil. Br J Radiol 2006;79(947):873–879. 14. Riddell AM, Hillier J, Brown G, et al. Potential of surface-coil MRI for staging of esophageal cancer. AJR Am J Roentgenol 2006;187(5):1280–1287. 15. Riddell AM, Allum WH, Thompson JN, Wotherspoon AC, Richardson C, Brown G. The appearances of oesophageal carcinoma demonstrated on high-resolution, T2weighted MRI, with histopathological correlation. Eur Radiol 2007;17(2):391–399. 16. Riddell AM, Davies DC, Allum WH, Wotherspoon AC, Richardson C, Brown G. High-resolution MRI in evaluation of the surgical anatomy of the esophagus and posterior mediastinum. AJR Am J Roentgenol 2007;188(1):W37–W43. 17. Yamada I, Hikishima K, Miyasaka N, et al. Diffusion-tensor MRI and tractography of the esophageal wall ex vivo. J Magn Reson Imaging 2013. 18. Wang R, Benner T, Sorensen AG, Wedeen VJ. Diffusion Toolkit: a software package for diffusion imaging data processing and tractography [abstr]. In: Proceedings of the Fifteenth Meeting of the International Society for Magnetic Resonance in Medicine. Berke-
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in live and formalin-fixed mouse brains. Magn Reson Med 2003;50(4):743–748.
19. Mori S, van Zijl PC. Fiber tracking: prin ciples and strategies—a technical review. NMR Biomed 2002;15(7-8):468–480.
25. Sun SW, Neil JJ, Liang HF, et al. Formalin fixation alters water diffusion coefficient magnitude but not anisotropy in infarcted brain. Magn Reson Med 2005;53(6):1447–1451.
20. Konomi T, Fujiyoshi K, Hikishima K, et al. Conditions for quantitative evaluation of injured spinal cord by in vivo diffusion tensor imaging and tractography: preclinical longitudinal study in common marmosets. Neuroimage 2012;63(4):1841–1853. 21. van der Kolk AG, Hendrikse J, Zwanenburg JJ, Visser F, Luijten PR. Clinical applications of 7 T MRI in the brain. Eur J Radiol 2013;82(5):708–718. 22. Gilbert RJ, Gaige TA, Wang R, et al. Re solving the three-dimensional myoarchitecture of bovine esophageal wall with diffusion spectrum imaging and tractography. Cell Tissue Res 2008;332(3):461–468. 23. Gaige TA, Benner T, Wang R, Wedeen VJ, Gilbert RJ. Three dimensional myoarchitecture of the human tongue determined in vivo by diffusion tensor imaging with tractography. J Magn Reson Imaging 2007;26(3):654–661.
26. Xu J, Humphrey PA, Kibel AS, et al. Magnetic resonance diffusion characteristics of histologically defined prostate cancer in humans. Magn Reson Med 2009;61(4):842–850. 27. Bourne R, Kurniawan N, Cowin G, Sved P, Watson G. 16 T diffusion microimaging of fixed prostate tissue: preliminary findings. Magn Reson Med 2011;66(1):244–247. 28. Bourne RM, Kurniawan N, Cowin G, Sved P, Watson G. Microscopic diffusion anisotropy in formalin fixed prostate tissue: preliminary findings. Magn Reson Med 2012;68(6):1943–1948. 29. Williams MC, Does MD, Price RR. ADC mapping of whole excised human prostate [abstr]. In: Proceedings of the Twelfth Meeting of the International Society for Magnetic Resonance in Medicine. Berkeley, Calif: International Society for Magnetic Resonance in Medicine, 2004; 2064.
24. Sun SW, Neil JJ, Song SK. Relative indices of water diffusion anisotropy are equivalent
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Imaging
Note: This copy is for your personal non-commercial use only. To order presentation-ready copies for distribution to your colleagues or clients, contact us at www.rsna.org/rsnarights.
Esophageal Carcinoma: Ex Vivo Evaluation with Diffusion-Tensor MR Imaging and Tractography at 7 T1 Ichiro Yamada, MD Keigo Hikishima, PhD Naoyuki Miyasaka, MD Tatsuyuki Kawano, MD Yutaka Tokairin, MD Eisaku Ito, MD Daisuke Kobayashi, MD Yoshinobu Eishi, MD Hideyuki Okano, MD
1
From the Departments of Diagnostic Radiology and Oncology (I.Y.), Pediatrics, Perinatal and Maternal Medicine (N.M.), Esophagogastric Surgery (T.K., Y.T.), and Pathology (E.I., D.K., Y.E.), Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; Department of Physiology, Keio University School of Medicine, Tokyo, Japan (K.H., H.O.); and Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan (K.H.). Received October 19, 2013; revision requested November 26; revision received January 13, 2014; accepted January 16; final version accepted January 27. I.Y. supported by the Grant-in-Aid for Scientific Research (C) of MEXT, Japan (23591753). Address correspondence to I.Y. (e-mail: [email protected]).
Purpose:
To determine the feasibility of diffusion-tensor magnetic resonance (MR) imaging and tractography as a means of evaluating the depth of mural invasion by esophageal carcinomas.
Materials and Methods:
This study was approved by the institutional review board, and written informed consent was obtained from each patient. Twenty esophageal specimens, each containing a carcinoma, were studied with a 7.0-T MR imaging system equipped with a four-channel phased-array surface coil. Diffusion-tensor MR images were obtained with a field of view of 50–60 mm 3 25–30 mm, matrix of 256 3 128, section thickness of 1 mm, b value of 1000 sec/mm2, and motion-probing gradient in seven noncollinear directions. The MR images were compared with the histopathologic findings as the reference standard. The differences in diffusion-tensor MR imaging parameters between the carcinoma and the layers of the esophageal wall were statistically analyzed by using the Dunnett test.
Results:
In all 20 carcinomas (100%), the diffusion-weighted images, apparent diffusion coefficient (ADC) maps, fractional anisotropy (FA) maps, l1 maps, and directionencoded color FA maps made it possible to determine the depth of tumor invasion of the esophageal wall that was observed during histopathologic examination. The l1 maps showed the best contrast between the carcinomas and the layers of the esophageal wall. The carcinomas had both lower ADC values and lower FA values than the normal esophageal wall; thus, the carcinomas were clearly demarcated from the normal esophageal wall. Diffusiontensor tractography images were also useful for determining the depth of tumor invasion of the esophageal wall.
Conclusion:
Diffusion-tensor MR imaging and tractography are feasible in esophageal specimens and provide excellent morphologic data for the evaluation of mural invasion by esophageal carcinomas. RSNA, 2014
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Online supplemental material is available for this article.
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T
he prognosis of patients with esophageal carcinoma is strictly dependent on the histopathologic stage of the carcinoma (1,2), and accurate preoperative staging has a definitive effect on the selection of appropriate treatment. However, preoperative staging of esophageal carcinoma has been extremely difficult because the imaging modalities that are available do not allow precise evaluation of the depth of tumor invasion of the esophageal wall (1,2). Computed tomography (CT) is used as a method of evaluating extensive tumor spread into adjacent organs, but CT does not allow evaluation of less extensive tumor spread, because the poor soft-tissue contrast makes it impossible to resolve the layers of the esophageal wall, even with new multidetector CT technology (3–5). Endoscopic ultrasonography (US) is widely used to
Advances in Knowledge nn The diffusion-weighted images, apparent diffusion coefficient (ADC) maps, fractional anisotropy (FA) maps, l1 maps, and direction-encoded color FA maps made it possible to determine the depth of invasion of the esophageal wall by all 20 carcinomas (100%). nn The l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall, and the l1 values (0.61 3 1023 mm2/sec) of the carcinomas were significantly lower than the l1 values of all of the layers of the normal esophageal wall (P < .001, except for the epithelium [P = .047]). nn The carcinomas also had lower ADC values (0.54 3 1023 mm2/ sec) and lower FA values (0.20) than the normal esophageal wall; thus, the carcinomas were clearly demarcated from the normal esophageal wall.
evaluate esophageal carcinomas, but it has many inherent problems, including technical failures in stenotic tumors, high operator dependency, artifactual interface echoes in the esophageal wall, and a limited sonographic range (5–7). Magnetic resonance (MR) imaging has been reported to demonstrate mural invasion by esophageal carcinomas, and it is an alternative to CT and endoscopic US (8–10), but conventional MR imaging is still insufficient in depicting the individual layers of the esophageal wall, even by using endocavitary coils (11–16). However, we have recently demonstrated that diffusion-tensor MR imaging and tractography are useful for demonstrating the individual layers of the esophageal wall (17). The purpose of this study was to prospectively determine the feasibility of diffusion-tensor MR imaging and tractography as a means of evaluating the depth of mural invasion by esophageal carcinomas.
Materials and Methods Study Population This study was approved by our institutional review board, and written informed consent with regard to the purpose of this study and the use of clinical and histopathologic data was obtained from each patient. We studied 20 surgical specimens of the esophagus, each of which contained a tumor that was histopathologically diagnosed as squamous cell carcinoma. Normal portions of the esophagus in 12 of these patients were analyzed and findings were reported by using the same techniques as in our previous work (17). No adenocarcinomas or other histologic types of tumors were identified in any of the surgical specimens.
nn Diffusion-tensor tractography images were also useful for determining the depth of tumor invasion of the esophageal wall. Radiology: Volume 272: Number 1—July 2014 n radiology.rsna.org
Implication for Patient Care nn Diffusion-tensor MR imaging and tractography may be useful in the future as a diagnostic tool for the noninvasive assessment of mural invasion by esophageal carcinomas.
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The specimens were obtained from 20 consecutive patients with esophageal carcinoma who were surgically treated at our institution between November 2012 and September 2013. Their ages at the time of surgery ranged from 46 to 83 years (mean age 6 standard deviation, 66 years 6 7; 18 men [mean age, 66 years 6 8; range, 46–83 years] and two women [mean age, 67 years 6 5; range, 62–71 years]). Thirteen patients underwent no treatment prior to surgical resection, five patients underwent only chemotherapy, and two patients underwent both chemotherapy and radiation therapy. All 20 specimens were imaged after fixation in 10% formalin. We did not examine the esophagus in vivo in this series. The time interval between surgery and imaging was approximately 24 hours. We performed MR imaging after the completion of formalin fixation, so partial fixation of the specimen with formalin did not occur during data acquisition. The length of esophageal specimens ranged from 105 to 250 mm (mean length, 164 mm 6 39). The size (ie, the largest diameter) of the resected tumors according to the Response Evaluation Criteria in Solid Tumors ranged from 18 to 80 mm (mean size, 46 mm 6 21).
Imaging Technique Diffusion-tensor MR imaging was performed by using a 7.0-T MR imaging Published online before print 10.1148/radiol.14132170 Content codes: Radiology 2014; 272:164–173 Abbreviations: ADC = apparent diffusion coefficient FA = fractional anisotropy Author contributions: Guarantors of integrity of entire study, I.Y., K.H., N.M., Y.E.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, I.Y., N.M.; clinical studies, I.Y., N.M., Y.T., D.K., Y.E.; experimental studies, I.Y., K.H., T.K.; statistical analysis, I.Y., N.M.; and manuscript editing, I.Y., K.H., N.M., T.K. Conflicts of interest are listed at the end of this article.
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unit (BioSpec 70/16; Bruker BioSpin, Ettlingen, Germany) equipped with actively shielded gradients that had a maximum strength of 700 mT/m. A four-channel phased-array surface coil was used for all measurements. The orientation of diffusion-tensor MR imaging was set longitudinally along the long axis of the resected segment of the esophagus. The specimen was placed directly on the surface coil. Diffusion-tensor MR imaging data sets were acquired by using a diffusionweighted spin-echo pulse sequence. The imaging parameters were repetition time (msec)/echo time (msec), 3000/25; field of view, 50–60 3 25–30 mm; matrix, 256 3 128; section thickness, 1 mm without intersection gaps; voxel size, 0.195–0.234 3 0.195–0.234 3 1 mm (range, 0.038–0.055 mm3); number of signals acquired, two; b value, 0 sec/mm2 (for a reference b0 image without diffusion weighting) or 1000 sec/mm2 (gradient lobe separation = 14 msec); and motion-probing gradient in seven noncollinear directions. The acquisition time was 102 minutes, which was equal to the total imaging time because the diffusion-tensor imaging was the only pulse sequence used in the present study. Since our purpose was to compare the findings of diffusion-tensor MR imaging with the histopathologic findings, T1- and T2weighted sequences were not used in the present study.
Image Processing The diffusion-tensor MR imaging data were processed with Diffusion Toolkit software (Massachusetts General Hospital, Boston, Mass) (18) by using monoexponential fitting. The three eigenvalues (l1 l2 l3) and corresponding eigenvectors were calculated for each voxel. The formulas (17) were used to calculate the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) for each voxel from the eigenvalues. As shown in our previous report (17), isotropic diffusion-weighted images, ADC maps, FA maps, l1 maps, l2 maps, and l3 maps were generated on a voxel-by-voxel basis. We also generated 166
direction-encoded color FA maps that showed anisotropy in different colors according to the direction of the major axis. Color FA maps make it possible to differentiate fiber tracts on the basis of their orientation. As shown in our previous report (17), diffusion-tensor tractograpy images were computed with TrackVis software (Massachusetts General Hospital) (18). The software performed fiber tracking by reading in all of the tracking data of the esophageal specimen, and it displayed all fiber tracts contained in the specimen. Then we used the two regions of interest method to selectively display specific fiber tracts (19,20). One of the authors (I.Y., with 23 years of experience in reading MR images) set two regions of interest on opposite sides of the specific fiber tracts in the specimen. For the fiber tracts oriented along the long axis and short axis of the esophagus, the size of the regions of interest was approximately equal to the thickness of each layer, multiplied by the width and length of the displayed tractography image, respectively.
Image Analysis An independent, blinded evaluation of diffusion-tensor MR images in each surgical specimen was performed by two observers (I.Y. and N.M., with 23 and 19 years of experience in reading MR images, respectively) who had no knowledge of the results of the histopathologic examination. When the observers could not fully agree on the findings, consensus was achieved by means of discussion. This occurred in two (10%) of 20 specimens in the present study. Isotropic diffusion-weighted images, ADC maps, FA maps, l1 maps, and direction-encoded color FA maps were reviewed for the presence, signal intensity, uniformity, and thickness of each layer of the esophageal wall. As described in our previous report (8), the contour and signal intensity of the carcinoma were also analyzed. The degree of carcinoma penetration into the esophageal wall was categorized according to the layer invaded: mucosa,
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submucosa, muscularis propria, or adventitia (8). For the ADC maps, FA maps, and l1 maps, regions of interest were placed on the carcinoma and on the layers of the esophageal wall. The size of the regions of interest was approximately equal to the cross-section area of the carcinoma and to the thickness of each layer, and one of the observers (I.Y.) shaped and placed the regions of interest. The number of the regions of interest was three or four for the carcinoma and for each layer per specimen, and the mean value of the three or four regions of interest in the carcinoma and in each layer of the esophageal wall was calculated. All quantitative diffusiontensor measurements reported were obtained by analyzing the regions of interest with ImageJ 1.47 software (available at http://imagej.nih.gov/ij). Fiber tracts on the diffusion-tensor tractography images were also evaluated for the presence, orientation, color, continuity, uniformity, and thickness of each layer of the esophageal wall. The diffusion-tensor MR imaging findings in the 20 esophageal specimens were compared with the histopathologic findings, which served as the reference standard. The MR images were compared with specific histopathologic sections on a slice-by-slice level, and correlations were made by means of visual inspection. For matching the MR images and the specimens, spatial correlation was achieved by identifying anatomic landmarks (eg, esophageal contour and blood vessels) that were depicted.
Histologic Preparations and Examination After MR imaging, each surgical specimen was sectioned longitudinally so that the orientation of the sections corresponded to the orientation of the MR images. The sectioned specimens were embedded in paraffin and cut into 6-mm-thick slices with a microtome. These slices were then stained with hematoxylin-eosin stain and elastica–van Gieson stain. An experienced pathologist (E.I., with 15 years of experience in histopathology) who did not have any
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knowledge of the MR imaging findings identified carcinoma invasion into each layer of the esophageal wall.
Statistical Analysis Means 6 standard deviations were calculated for the ADC values, FA values, and l1 values in the carcinoma and in each layer of the esophageal wall. All statistical analyses were performed with a commercial software package (SPSS Statistics, version 20; IBM SPSS Japan, Tokyo, Japan). The differences in diffusion-tensor MR imaging parameters between the carcinoma and the layers of the esophageal wall were statistically analyzed by using the Dunnett test. A P value less than .05 was considered to indicate a significant difference. Furthermore, box plots were used for comparing the parameters of the carcinoma and the esophageal wall layers, and scatterplots were used for comparing the pixels of the carcinoma and the esophageal wall layers. The Wilcoxon matched-pairs signed rank test was used for analysis of the box plots. Diffusion-tensor imaging was the only pulse sequence used, and a definitive diagnosis of the depth of tumor invasion was assessed on the basis of the combined findings on the diffusion-weighted images, ADC maps, FA maps, l1 maps, and color FA maps, so it was difficult to apply a receiver operating characteristic curve analysis to each map. Results Diffusion-weighted Images, ADC Maps, FA Maps, l1 Maps, and Direction-encoded Color FA Maps of Esophageal Carcinomas and Layers of the Esophageal Wall The diffusion-weighted images, ADC maps, FA maps, l1 maps, and direction-encoded color FA maps of all 20 specimens (100%) clearly depicted the esophageal wall as consisting of the eight layers (Fig E1 [online], Fig 1). These eight layers clearly corresponded to the layers of the esophageal wall observed in histologic sections. As shown in Figure E1 (online) and Figure 1, the esophageal carcinomas were seen as areas of high signal
intensity on diffusion-weighted images; as areas of low values on ADC maps, FA maps, and l1 maps; and as variable colors on direction-encoded color FA maps. The carcinomas on the diffusionweighted images appeared as areas of higher signal intensity than the lamina propria mucosae, submucosa, intermuscular connective tissue, and adventitia. On the ADC maps, the carcinomas had lower ADC values than the lamina propria mucosae, submucosa, intermuscular connective tissue, and adventitia. On the FA maps, the carcinomas also had lower FA values than the epithelium, muscularis mucosae, inner circular muscle, and outer longitudinal muscle. On the l1 maps, however, the carcinomas had lower l1 values than all of the layers of the esophageal wall; thus, the l1 maps provided the best contrast between the carcinoma and the layers of the esophageal wall. The reason they provided the best contrast was that the lamina propria mucosae, submucosa, intermuscular connective tissue, and adventitia had high l1 values, and the epithelium, muscularis mucosae, inner circular muscle, and outer longitudinal muscle had intermediate l1 values.
ADC Values, FA Values, and l1 Values of Esophageal Carcinomas and Layers of the Esophageal Wall As shown in the Table, the ADC values of the esophageal carcinomas were significantly lower than the ADC values of the lamina propria mucosae, muscularis mucosae, submucosa, intermuscular connective tissue, and adventitia (P < .001). The FA values of the carcinomas, on the other hand, were significantly lower than the FA values of the epithelium, muscularis mucosae, inner circular muscle, and outer longitudinal muscle (P < .001) and slightly higher than the FA of the submucosa (P = .012). The l1 values of the carcinomas, however, were significantly lower than the l1 values of all of the layers of the esophageal wall (P < .001, except for the epithelium [P = .047]). These findings explain why the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall.
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We also compared the ADC values, FA values, and l1 values of the carcinomas, solid layers, and loose layers of the esophageal wall (Fig E2 [online]). The ADC values of the carcinomas were significantly lower than the ADC values of the loose layers of the esophageal wall (P < .001). The FA values of the carcinomas were significantly lower than the FA values of the solid layers of the esophageal wall (P < .001). The l1 values of the carcinomas, however, were significantly lower than the l1 values of both the loose layers and the solid layers of the esophageal wall (P < .001 for both).
Scatterplots of the ADC Values, FA Values, and l1 Values of Esophageal Carcinomas and Layers of the Esophageal Wall As shown in Figure E3a (online), the pixels of the carcinoma on the scatterplot of the ADC values and FA values were clearly demarcated from the layers of the normal esophageal wall, because the pixels of the carcinoma had lower ADC values and lower FA values than the other pixels of the esophageal wall. As shown in Figure E3b (online), the pixels of the carcinoma were clearly demarcated from the layers of the normal esophageal wall on the three-dimensional scatterplot, because the pixels of the carcinoma had lower l1 values than the other pixels of the esophageal wall. The two scatterplots clearly demonstrated that diffusion-tensor MR imaging makes it possible to determine the extent of esophageal carcinoma by identifying the areas that have low ADC values and low FA values and/or the areas that have low l1 values in the esophageal wall. Evaluation of Esophageal Carcinoma Invasion on Diffusion-Tensor MR Images Histopathologic examination of the 20 esophageal carcinomas showed that two of the carcinomas were confined to the mucosa (stage T1a), six carcinomas had invaded the submucosa (stage T1b), three carcinomas had involved the muscularis propria (stage T2), and nine carcinomas had extended into the adventitia (stage T3 or T4). The depth 167
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Figure 1
Figure 1: Images in a patient with esophageal carcinoma invading the submucosa. (a) Diffusion-weighted MR image (3000/25) depicts the carcinoma (T) as an area of high signal intensity. (b) ADC map (3000/25) depicts the carcinoma as a low-ADC area. (c) FA map (3000/25) depicts the carcinoma as a low-FA area. (d) l1 map (3000/25) clearly depicts the carcinoma as a low-l1 area. The l1 map shows better contrast between the carcinoma and the layers of the esophageal wall than the diffusion-weighted image, ADC map, FA map, and direction-encoded color FA map. (e) Direction-encoded color FA map (3000/25) depicts the carcinoma as a variable-color area. Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (f) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 310) shows carcinoma that has invaded the submucosa, which has disrupted the muscularis mucosae layer (arrows). Adv = adventitia, Epi = epithelium, ICM = inner circular muscle, IMCT = intermuscular connective tissue, LPM = lamina propria mucosae, MM = muscularis mucosae, OLM = outer longitudinal muscle, SM = submucosa, T = tumor.
ADC Values, FA Values, and l1 Values in Esophageal Carcinoma and Layers of the Esophageal Wall at Diffusion-Tensor MR Imaging Tissue Carcinoma Epithelium Lamina propria mucosae Muscularis mucosae Submucosa Inner circular muscle Intermuscular connective tissue Outer longitudinal muscle Adventitia
ADC Values (31023 mm2/sec) FA Values
l1 Values (31023 mm2/sec)
0.54 6 0.06 0.63 6 0.06 1.51 6 0.19* 0.80 6 0.09* 1.90 6 0.20* 0.70 6 0.09 1.46 6 0.20* 0.69 6 0.08 1.91 6 0.18*
0.61 6 0.07 0.82 6 0.08* 1.84 6 0.26* 0.95 6 0.16* 2.27 6 0.22* 1.10 6 0.14* 1.88 6 0.28* 1.09 6 0.15* 2.31 6 0.18*
0.20 6 0.03 0.58 6 0.06* 0.16 6 0.04 0.58 6 0.06* 0.13 6 0.03* 0.64 6 0.07* 0.20 6 0.04 0.69 6 0.06* 0.15 6 0.04
Note.—Data are mean values 6 standard deviations. * Values were significantly different from the corresponding value of the esophageal carcinoma.
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of invasion of the esophageal wall by all 20 carcinomas (100%) was clearly demonstrated by the diffusion-weighted images, ADC maps, FA maps, l1 maps, and direction-encoded color FA maps. Since the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall, the l1 maps were the most useful means of assessing tumor invasion of the esophageal wall. In the present study, there were no cases of false-positive or falsenegative findings with respect to assessment of invasion of the different esophageal layers. However, since there was disagreement in two cases (10%) that was resolved by means of consensus, it may be unlikely that the technique radiology.rsna.org n Radiology: Volume 272: Number 1—July 2014
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Figure 2
Figure 2: Images depict an esophageal carcinoma confined to the mucosa. (a) l1 map (3000/25) clearly shows discrete thickenings of the mucosal layer (arrows) that have lower l1 values than other parts of the esophageal wall. (b) FA map (3000/25) shows that the muscularis mucosae layer (arrows) is intact. (c) Diffusion-tensor tractography image (3000/25) shows that the following four layers appear to be intact: epithelium (blue fiber tracts), muscularis mucosae (green fiber tracts), inner circular muscle (blue fiber tracts), and outer longitudinal muscle (green fiber tracts). It is difficult to differentiate the normal epithelium from the epithelium involved by carcinoma, owing to the superficial nature of the tumor, so this point seems to be a limitation of the technique. Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (d) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 320) shows the carcinoma confined to the mucosa (arrows).
would actually have perfect diagnostic performance, and this issue may constitute a limitation in the present study. Carcinomas confined to the mucosa appeared as discrete thickening of the mucosal layer that had lower l1 values than other parts of the esophageal wall (Fig 2). Carcinomas that had invaded the submucosa appeared as irregular masses with low l1 values that had disrupted the muscularis mucosae layer (Fig 3). Carcinomas that had involved the muscularis propria appeared
as low-l1 masses that had partially replaced the muscularis propria layer (Fig E4 [online]). Carcinomas that had extended into the adventitia appeared as low-l1 masses that had completely disrupted the muscularis propria layer and invaded the adventitial layer (Fig 4).
Evaluation of Esophageal Carcinoma Invasion on Diffusion-Tensor Tractography Images Three-dimensional diffusion-tensor tractography images of all 20 specimens
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(100%) clearly depicted the four layers (Figs 2–4, Fig E4 [online]). The diffusion-tensor tractography images were also found to be useful for determining the depth of tumor invasion of the esophageal wall. When the carcinoma was confined to the mucosa, all four layers appeared to be intact (Fig 2). When the carcinoma had invaded the submucosa, the muscularis mucosae layer (green fiber tracts) was observed to have been disrupted by the tumor (Fig 3). When the 169
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Figure 3
Figure 3: Images depict an esophageal carcinoma that has invaded the submucosa. (a) l1 map (3000/25) clearly shows a mass lesion (arrows) that has lower l1 values than other parts of the esophageal wall. (b) FA map (3000/25) shows that the mass lesion has disrupted the muscularis mucosae layer (arrows). (c) Diffusion-tensor tractography image (3000/25) shows that the muscularis mucosae layer (green fiber tracts) has been disrupted by the tumor (arrows). Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (d) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 310) shows carcinoma that has invaded the submucosa, which has disrupted the muscularis mucosae layer (arrows).
carcinoma had involved the muscularis propria, the muscularis propria layer (blue fiber tracts and/or green fiber tracts) was observed to have been partially disrupted by the tumor (Fig E4 [online]). When the carcinoma had extended into the adventitia, the muscularis propria layer (both blue fiber tracts and green fiber tracts) was observed to have been completely disrupted by the tumor (Fig 4).
Discussion As we have shown in our previous work, the diffusion-weighted images, ADC maps, FA maps, l1 maps, and 170
direction-encoded color FA maps clearly depicted the normal esophageal wall as consisting of eight layers, thereby clearly demonstrating that diffusion-tensor MR images are able to depict the individual layers of the esophageal wall (17). We have proposed a new schematic illustration of the normal esophageal wall to explain the diffusion-tensor MR imaging and tractography findings in the normal esophageal wall (17). On the basis of our schematic illustration, alternating levels of cellularity (cell density), anisotropy, and directionality between the adjacent layers account for the clear depiction of the layers of the normal esophageal wall on the ADC maps, FA maps, and
direction-encoded color FA maps, respectively. Furthermore, the high signalto-noise ratios obtained by using 7.0-T MR imaging may have contributed to the clear depiction of the esophageal wall layers, because the signal-to-noise ratio increases approximately linearly with the field strength (21). In our current study, carcinomas on the l1 maps appeared darker than all of the layers of the esophageal wall; thus, the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall. The l1 values of the carcinomas were found to be significantly lower than the l1 values of all of the layers of the esophageal wall. Thus,
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Figure 4
Figure 4: Images depict an esophageal carcinoma that has extended into the adventitia. (a) l1 map (3000/25) clearly shows an irregularly shaped mass lesion (arrows) with low l1 values. (b) FA map (3000/25) shows that the mass lesion has completely disrupted the muscularis propria layer and invaded the adventitial layer (arrows). (c) Diffusion-tensor tractography image (3000/25) shows that the muscularis propria layer (both blue fiber tracts and green fiber tracts) has been completely disrupted by the tumor (arrows). Green indicates the long-axis direction of the esophagus; blue, the short-axis direction parallel to the esophageal wall; and red, the short-axis direction perpendicular to the esophageal wall. (d) Corresponding histopathologic section (hematoxylin-eosin stain; original magnification, 31) shows carcinoma that has extended into the adventitia (arrows).
the l1 values of the carcinomas being the lowest explains why the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall. The l1 values of the solid layers appeared to be considerably higher than the ADC values of the solid layers because of local fiber orientation,
while the l1 values of the carcinomas and the loose layers were only slightly higher than the respective ADC values because of no local fiber orientation. These differences were responsible for the carcinomas having the lowest l1 values, thereby accounting for the l1 maps having the best contrast between
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the carcinomas and the esophageal wall layers. Our findings demonstrated that diffusion-tensor MR imaging makes it possible to determine the extent of esophageal carcinomas by identifying areas that have both low ADC values and low FA values and/or areas that have low l1 171
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values in the esophageal wall. Since the l1 maps provided the best contrast between the carcinomas and the layers of the esophageal wall, the l1 maps were found to be most useful for assessing tumor invasion of the esophageal wall. The diffusion-tensor tractography images were also found to be useful for determining the depth of tumor invasion of the esophageal wall. Gilbert et al (22) used diffusion spectrum imaging and tractography to resolve the myoarchitecture of the bovine esophageal wall. Gaige et al (23) reported using diffusion-tensor imaging and tractography in vivo to visualize the structural anatomy of the human tongue, and their findings suggested the feasibility of invivo diffusion-tensor MR imaging and tractography in the human esophagus. Conventional MR imaging has recently been used for preoperative staging of esophageal carcinoma. Riddell et al (13–16) performed high-spatialresolution MR imaging with an external surface coil in patients with esophageal carcinoma. Dave et al (12) performed endoluminal MR imaging with a surface coil incorporated into the tip of an endoscope in patients with esophageal carcinoma. Therefore, we believe that by using an external surface coil technique or endoluminal surface coil technique, it may be possible to perform diffusion-tensor MR imaging and tractography in patients with esophageal carcinoma. There were some limitations to our study. First, this study was performed ex vivo, and the specimens were imaged after fixation in formalin. However, since previous studies of other organs showed that the diffusion anisotropy indexes measured in fixed and in vivo tissues provided essentially the same information regarding the underlying microstructure (24–28), using diffusiontensor MR imaging and tractography in fixed tissues would appear to be a valid means of evaluating the diffusion anisotropy of tissues in vivo. The same studies also showed that although the ADC values of fixed tissues were lower than those in vivo, the relative in vivo ADC difference between different tissue types was preserved in fixed tissues, and the 172
relative ADC difference showed no significant difference between fixed and in vivo tissues (26,27,29). Furthermore, the temperature effect of ex vivo imaging may influence ADC, FA, and other metrics, so it should be noted that the indicated values cannot be used as absolute thresholds for in vivo imaging. Second, the imaging time in this study was considerably long (102 minutes), so shortening the imaging time would be necessary to translate our data into in vivo diffusion-tensor MR imaging and tractography. At present, clinical application of whole-body 7-T MR imaging to esophageal carcinoma may be technically difficult because of motion effects (peristalsis, respiratory movement, and patient motion) and the deep location in the thorax. For this purpose, modifications of pulse sequences, the development of faster MR imaging techniques, or the application of higher field strengths to clinical settings may be required in the future. In conclusion, the results of the present study have demonstrated that diffusion-tensor MR imaging and tractography are able to clearly depict the individual tissue layers of the esophageal wall ex vivo. Diffusion-tensor MR imaging and tractography in these ex vivo specimens provided excellent diagnostic accuracy for evaluating mural invasion by esophageal carcinomas. Disclosures of Conflicts of Interest: I.Y. No relevant conflicts of interest to disclose. K.H. No relevant conflicts of interest to disclose. N.M. No relevant conflicts of interest to disclose. T.K. No relevant conflicts of interest to disclose. Y.T. No relevant conflicts of interest to disclose. E.I. No relevant conflicts of interest to disclose. D.K. No relevant conflicts of interest to disclose. Y.E. No relevant conflicts of interest to disclose. H.O. Financial activities related to the present article: none to disclose. Financial activities not related to the present article: author received payment for consultancies with San Bio, Easai, and Daiichi Sankyo; author and institution have received payment for patents relating to the introduction of foreign genes into early embryos of primates and the production of transgenic animals; and author has stock and/or stock options in San Bio. Other relationships: none to disclose.
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