Mammary tumors are a heterogeneous group of tumors that commonly develop in dogs. It is important to distinguish the specific types because the tumor type greatly influences the treatment and prognosis for an individual animal. Because it may be difficult to classify canine mammary tumors via cytology, it is of interest to evaluate whether an imaging modality such as ultrasonography can be used as a noninvasive staging method in the evaluation of these tumors. Preliminary studies1,a,b by our group and others have revealed that mammary tumors in dogs are readily imaged ultrasonographically; these images may provide information regarding the size, shape, internal structure, invasion into surrounding tissue, acoustic transmission, and vascularity of the tumor. To evaluate the relevance of the ultrasonographic characteristics, it is important to correlate these with histologic findings. In a previous study,1 the B-mode characteristics of canine mammary tumors were compared with histopathologic findings. To the authors' knowledge, this is the first report of the correlation of Doppler ultrasonographic characteristics with histologic findings in mammary tumors in dogs. Results of some studies2,3 have indicated that the grade of angiogenesis appears to determine the rate of tumor growth and metastasis, and it has been determined that intratumoral MVD is a prognostic sign in canine mammary tumors.4–8 Microvessel density has been used in several studies9–11 to correlate vascularization detected via ultrasonography with histologic findings. Generally, the results of such studies in several human tumor types have been disappointing. The color and power Doppler ultrasonographic approach allows functional assessment of neovascularization by mapping flow signals of the entire tumor volume in real time and thus offers a noninvasive, easily applicable method for direct measurement of the distribution of and flow in intratumoral blood vessels. The hypothesis is that the amount of vascularity in a lesion and the arrangement of blood vessels may help distinguish benign tumors from malignant tumors. The first step is to evaluate whether the blood vessels detected ultrasonographically correlate with findings on histologic examination of tissue sections. The Verhoeff van Gieson method (referred to as the Verhoeff technique) stains elastic fibers, including those forming the internal elastic membrane of blood vessels; thus, the stain can be used to identify the large arterioles, muscular arteries, and some of the medium-sized veins. Typically, arterioles have a luminal diameter of less than approximately 300 μm.12 By use of the Verhoeff technique, vessels of sizes that are detectable via ultrasonography can be evaluated histologically. Because the limiting resolution at ultrasound frequencies that are commonly used for imaging of small body parts in dogs is approximately 2 mm, we hypothesized that it is only necessary to stain blood vessels of this diameter or larger and that the results of Verhoeff staining would more closely match those of ultrasonographic examinations, compared with MVD data. The purpose of the study reported here was to compare and correlate B-mode and color Doppler ultrasonographic characteristics with histologic findings of benign and malignant mammary tumors in dogs.
Materials and Methods
Study population—Forty-nine mammary tumors in 26 dogs were examined in this study. Any dog with ≥ 1 mammary tumor that was admitted to the Small Animal Hospital at the Department of Small Animal Clinical Sciences for staging procedures and surgery was included in the study.
Ultrasonographic procedures—Immediately prior to surgical extirpation, the tumors were examined without a standoff pad by use of 2 ultrasonography machinesc,d and a linear transducer (7 to 14 MHz) in a standard imaging mode for small body parts. The size of the tumor was estimated by measuring the longitudinal and transverse axes. Echogenicity and echopattern, invasiveness of tumor tissue into the surrounding tissue, and presence or absence of distal acoustic enhancement or shadowing were recorded. Tumors were defined as invasive when no distinct margin between the tumor and surrounding normal tissue was observed in the ultrasonographic images. These variables were evaluated by use of B-mode ultrasonography at a depth of 2 to 3 cm.
Color flow mapping was used in different planes to evaluate the vascular supply within each tumor. Doppler imaging was performed by use of machine settings designed to optimize detection of slow flow in small vessels. The color Doppler gain was increased until background noise appeared and then was decreased slightly to eliminate noise and retain maximal sensitivity to flow. The presence and distribution of vascular flow was recorded and classified into 1 of 3 groups: central, peripheral, or mixed central and peripheral flow. The central region was defined as the central one third of the total tumor area, whereas the peripheral region was defined as the remaining two thirds of the total tumor area. Tumor blood flow was classified as peripheral flow when at least 80% of the detected flow was localized to the peripheral region, as central when at least 80% of the detected flow was localized to the central region, and as mixed central and peripheral flow when flow ratios < 80% were detected in both the peripheral and central parts. The number of vessels in the image (recorded as individual Doppler signals) was also determined. Images were recorded and digitally stored as individual images and 3- to 5-second cinematic loops on a magnetic optic disc in a standard imaging format.e Computer softwaref was used to view the images initially and to export images in formats suitable for analyses.g For each tumor, the image with the highest number of vessels was chosen for quantification of vascularity by use of software.g The number of color pixels was expressed as a percentage of the total pixel count for the tumor.
Histologic evaluation—All of the extirpated tumors were examined histologically. A 0.5-cm-thick slice of tissue from the midline region of each tumor was placed in ≥ 1 capsules (depending on the size of the node); tissue samples were fixed in neutral-buffered 4% formaldehyde for 24 hours, stored for another 24 to 72 hours in 70% ethanol, and then embedded in paraffin wax. The tissues were cut into 4-μm sections and underwent deparaffinization in xylene and rehydration in alcohol and water. The tissue sections were stained with H&E or via the Verhoeff technique.13 The tumors were classified according to the WHO criteria14 by one of the authors (EH); tumors were grouped as benign, atypical benign, or malignant. The H&E-stained sections were evaluated for presence or absence of areas of necrosis, cysts, cartilage, bone, mineralization, infiltrative growth, and overall tissue heterogeneity; for these aforementioned features, detection of an area of at least 1 mm2 was considered a positive finding. The overall tissue heterogeneity was evaluated subjectively and defined as heterogenic if at least 3 tissue types were interspaced within the section. Slides prepared by use of the Verhoeff technique were evaluated for the number of blood vessels with a stained internal elastic lamina; as with the ultrasonographic images, the central part of the tumor was defined as the inner third of the area, whereas the periphery was defined as the outer two thirds of the area.
Statistical analysis—Categorical variables are described as relative proportions expressed in percentages, whereas continuous data are expressed as mean values. The ultrasonographic findings were correlated with the histopathologic findings. Logistic regression analysis was used to correlate echogenicity with tissue heterogeneity. The κ statistic was used to assess the agreement between ultrasonographic and histologic findings with respect to tumor invasiveness. The κ statistic was also used to assess the agreement between acoustic transmission detected via ultrasonography and the histologic presence of areas of cysts, necrosis, cartilage, bone, and mineralization, respectively. Linear regression analysis and Bland-Altman plots were used to evaluate the correlation between the numbers of vessels detected ultrasonographically and the number of Verhoeff-stained vessels in the tissue sections and to correlate both the width and length of the tumor with the amount of vascularity present. The Bland-Altman plot was also used to correlate the distribution of flow determined ultrasonographically with that determined histologically. The Bland-Altman plot compared the difference of the results obtained by each measure with the mean of the 2 measurements. This is used to determine agreement between 2 tests that involve modes of measurement for which the true value for the measurement is not known and determine whether any difference in results is a function of the magnitude of the result (ie, to detect bias). A value of P < 0.05 was considered significant.
Results
Twenty-six dogs with mammary tumors were included in the study; 49 tumors were examined via Bmode and color Doppler ultrasonography prior to excision. After surgical removal, tissue sections from all tumors were examined histologically (Table 1).
Histologic classification of 49 mammary tumors in 26 dogs.
| Tumor group | Histologic type* | No. of tumors |
|---|---|---|
| Benign (n = 11) | Complex adenoma | 6 |
| Hyperplasia† | 3 | |
| Simple adenoma | 1 | |
| Benign mixed tumor | 1 | |
| Atypical‡ (8) | Atypical complex adenoma | 5 |
| Atypical cystic adenoma | 2 | |
| Atypical simple adenoma | 1 | |
| Malignant (30) | Tubulopapillary carcinoma | 9 |
| Complex carcinoma | 6 | |
| Carcinosarcoma | 4 | |
| Carcinoma in benign tumor | 3 | |
| Solid carcinoma | 3 | |
| Lipid-rich carcinoma | 3 | |
| Basaloid carcinoma | 1 | |
| Lymphoma | 1 |
Modified from the WHO mammary tumor classification system.
In the WHO classification, hyperplasia is a separate group; in this study, it was included in the benign tumor group.
Atypical tumors are not part of the WHO classification but were included as a separate group because they were difficult to classify otherwise.
Overall, the mean tumor length was 19 mm (range, 3.9 to 60 mm) and mean tumor width was 11 mm (range, 2.6 to 48 mm; Table 2). There was a significant (P < 0.05) difference in the size, with respect to both length and width, of the tumors between benign and malignant tumors; malignant tumors were larger than benign tumors.
B-mode and Doppler ultrasonographic characteristics of 49 mammary tumors in 26 dogs.
| Characteristic | Tumor group | ||
|---|---|---|---|
| Benign (n = 11) | Atypical (8) | Malignant (30) | |
| B-mode ultrasonography | |||
| Length (mm) | 15 | 14 | 22 |
| Width (mm) | 8 | 7 | 13 |
| Echogenicity (No. of nodes) | |||
| Hypoechoic (uniform) | 2 | 3 | 3 |
| Isoechoic (uniform) | 4 | 5 | 3 |
| Varied | 5 | 0 | 24 |
| Appearance of tissue echopattern (No. of nodes) | |||
| Homogenous | 6 | 8 | 6 |
| Heterogenous | 5 | 0 | 24 |
| Invasion of surrounding tissue (No. of nodes) | 4 | 2 | 16 |
| Acoustic transmission (No. of nodes) | |||
| Shadowing | 1 | 0 | 4 |
| Enhancement | 5 | 5 | 22 |
| Doppler ultrasonography | |||
| Mean No. of vessels in tumor | 5.5 | 4.8 | 7.1 |
| Total flow area in tumor (%) | 16.2 | 11 | 10.9 |
| Distribution of flow (No. of nodes) | |||
| Central | 0 | 1 | 2 |
| Peripheral | 0 | 1 | 6 |
| Mixed central and peripheral | 11 | 6 | 22 |
Examination of the H&E-stained sections revealed that approximately 70% of benign and malignant tumors had tissue heterogeneity, as determined subjectively. Linear regression analysis revealed that tissue heterogeneity correlated with a varied echogenicity on ultrasonographic images; the odds ratio was 18.8 (P < 0.001), meaning that there was an increased odds of tumor tissue appearing heterogeneous rather than homogeneous on examination of tissue sections when the echogenicity of the tissue in situ was heterogeneous. Benign mammary tumors were more often uniformly isoechoic or hypoechoic, whereas malignant tumors had a more varied echogenicity.
Invasiveness of the tumor into the surrounding tissue as determined ultrasonographically did not correlate with the histologic findings (κ = 0.05; 95% CI, −0.21 to 0.31; Figure 1). Invasion of surrounding tissue was only detected histologically in association with malignant tumors (Table 3); however, via ultrasonography, invasion was detected in association with 16 of 30 malignant tumors, 2 of 8 atypical tumors, and 4 of 11 benign tumors.
Histologic characteristics of 49 mammary tumors in 26 dogs.
| Characteristic | Tumor group | ||
|---|---|---|---|
| Benign (n = 11) | Atypical (8) | Malignant (30) | |
| H&E-stained sections (No. of tumors) | |||
| Necrosis | 4 | 1 | 22 |
| Cysts | 6 | 4 | 13 |
| Cartilage | 4 | 1 | 6 |
| Bone or mineralization | 3 | 0 | 4 |
| Invasion of surrounding tissue | 0 | 1 | 16 |
| Tissue heterogeneity | 8 | 1 | 21 |
| Verhoeff-stained sections | |||
| Mean No. of stained vessels in the entire tumor | 7.2 | 14.4 | 7.9 |
| Mean No. of stained vessels in the center of the tumor | 1.9 | 4 | 1.6 |
| Mean No. of stained vessels in the periphery of the tumor | 5.3 | 10.4 | 6.3 |
Ultrasonographic and histologic characteristics of a metastatic mammary tubulocarcinoma in a 10-year-old sexually intact mixed-breed dog. A—Color Doppler ultrasonographic image. Notice that the tumor has varied echogenicity with acoustic enhancement and shadowing and a mixed vascular distribution pattern. The undefined borders in the horizontal plane of the tumor are classified as invasion of the tumor into surrounding tissue. B—Photomicrograph of a tissue section of the same tumor. Notice the infiltrative growth of tumor cells into the mammary tissue (arrow). H&E stain; bar = 200 μm.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.985
Acoustic enhancement was correlated with presence of necrotic and cystic areas, either separately or in combination, in the tumor sections (κ = 0.4; 95% CI, 0.1 to 0.7; Figures 1 and 2). Enhancement was seen in association with a larger proportion of the malignant tumors (22/30 tumors), compared with the benign tumors (5/11 tumors); histologically, necrosis was detected in malignant tumors more frequently than in benign tumors. Distal acoustic shadowing was seen in equal proportions of benign and malignant tumors, but the numbers were too small to perform correlative statistical analysis. However, 4 of 5 tumors with shadowing in ultrasonographic images contained bone or mineralization (Figure 3). Compared with malignant tumor sections, cartilage and bone were more often detected histologically in benign tumor sections, whereas cysts were present in similar proportions among the 3 tumor types.
Ultrasonographic and histologic characteristics of a mammary carcinosarcoma in a 12-year-old sexually intact female German Shepherd Dog. A—Gray scale ultrasonographic image. Notice that the tumor has varied echogenicity; slight acoustic enhancement is present deep in the hypoechoic focal area to the left. B—Photomicrograph of a tissue section of the same tumor. Notice that there are areas of necrosis that correspond to the hypoechoic area in the ultrasonographic image. H&E stain; bar = 200 μm.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.985
Ultrasonographic and histologic characteristics of a mixed malignant mammary tumor in an 11-year-old spayed female Staffordshire Bull Terrier. A—Color Doppler ultrasonographic image. Notice that the tumor has varied echogenicity and acoustic shadowing is present. B—Photomicrograph of a tissue section of the same tumor. Notice the area of cartilage that corresponds to the hyperechoic area causing shadowing in the ultrasonographic image (arrow). H&E stain; bar = 200 μm.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.985
Blood flow sufficient to permit ultrasonographic measurement was present in all the tumors. A positive correlation (P < 0.001) was determined between tumor vascularity assessed via ultrasonographic image analyses and the size of the tumors; large tumors had smaller vascular supplies than small tumors. An overall correlation (P = 0.015) between the number of vessels detected ultrasonographically and the number detected histologically was identified. There was also a significant (P < 0.02) correlation between the distribution of flow determined ultrasonographically and that determined histologically (Figure 4). There was no significant difference in the distribution of flow within the tumors of the different groups, although mixed flow was detected in all benign tumors and only 28 of the 38 atypical benign and malignant tumors.
Ultrasonographic and histologic characteristics of a complex mammary carcinoma in a 9-year-old sexually intact female Poodle. A—Color Doppler ultrasonographic image. Notice that multiple vessels with a mixed flow distribution are present. The vessels have different diameters and bifurcations and trifurcations, all of which are considered signs of malignancy. It is difficult to delineate the tumor borders in the horizontal plane of the tumor because of invasive growth of the tumor into the surrounding tissue (verified via histologic examination). B—Photomicrograph of a tissue section of the same tumor. By use of the Verhoeff staining technique, the elastic fibers in the internal elastic lamina of the arteries have stained black. Verhoeff stain; bar = 50 μm.
Citation: American Journal of Veterinary Research 67, 6; 10.2460/ajvr.67.6.985
Discussion
The present study was designed to correlate Bmode and color Doppler ultrasonographic characteristics of canine mammary tumors with findings of histologic examinations of H&E- and Verhoeff-stained tumor sections. Results of our study have confirmed the heterogenic nature of mammary tumors in dogs. The ultrasonographic finding of varied echogenicity correlated with tissue heterogeneity detected histologically. The varied echogenicity observed particularly in larger tumors may be a consequence of the development of necrosis, cysts, edema, hemorrhage, or increased interstitial pressure or cartilage and bone formation within the tumor. Cysts are easily detected ultrasonographically and correlated with the histopathologic findings, but the proportion of tumors with cystic lesions appeared to be similar among all tumor types. Therefore, detection of cystic areas on ultrasonographic images cannot be used to classify canine mammary tumors as benign or malignant. Typically, malignant tumors contain more areas of necrosis than benign tumors, and this may be the reason for the greater frequency of acoustic enhancement associated with malignant tumors in the present study. Cartilage was detected in some tumors of all tumor types, whereas bone or mineralization was identified in some benign and malignant tumors but not in any of the atypical tumors. Among the benign tumors, the proportion of tumors with a homogenous ultrasonographic appearance of the tissue echopattern was similar to the proportion with a heterogenous appearance; almost half of these tumors had varied echogenicity. All the atypical mammary tumors had a homogenous appearance ultrasonographically and were primarily hypoechoic, whereas 24 of the 30 malignant tumors had a varied echogenicity. In another study,1 all the malignant canine mammary tumors evaluated had varied echogenicity. However, that study involved only 11 malignant tumors. In humans, it has been suggested that echogenicity may change as a result of hormonal influences.15 It is not known whether this also occurs in dogs. Certain tissue compositions may result in enhancement or shadowing as the ultrasonic beams propagate through tissues. Shadowing distal to a structure may result from attenuation, beam spreading due to refraction, or reflection related to that structure. The degree of shadowing by an object is in part dependent on its size relative to the ultrasonic beam,16 and it is therefore possible that tumors of the same histologic type may have different acoustic transmission patterns. Acoustic shadowing was present in 1 of 11 benign tumors that contained bone or mineralization and 4 of 30 malignant tumors that contained bone or mineralization. These findings contradict those of studies16–18 in humans with breast tumors, which suggest that acoustic shadowing is a typical sign of malignancy and acoustic enhancement is accepted as a sign that a tumor is benign. The explanation for this difference in findings may be that canine mammary tumors are much more heterogenous than human tumors and the fact that benign tumors often contain more bone or larger areas of cartilage and fibrous tissue than malignant tumors in dogs. The presence and arrangement of fibrous connective tissue has also been suggested to result in acoustic shadowing.16 However, the amount of fibrous tissue was not quantified in the present study.
In ultrasonographic images, the tumors were rarely seen invading tissue layers beneath the mammary tissue; invasiveness was more commonly observed with malignant tumors than benign tumors, but the difference was not significant. This lack of correlation is surprising, as a distinct difference between the tumor groups was expected. It can be argued that the reason for this may be that we should have used another scanning definition of invasion or that reactive, hyperplastic, or inflammatory tissue surrounding some tumors may affect delineation of the tumor borders. In the present study, it was found that larger tumors were more malignant, contained larger areas of necrosis, and had a lower total flow area. These findings confirm the WHO classification of mammary tumor stages by size (as T1 [< 3 cm], T2 [> 3 but < 5 cm], and T3 [> 5 cm]) in that the greater the tumor size the worse the prognosis.
Color Doppler ultrasonography can be used to detect angiogenesis within solid tumors, and several authors have suggested the possibility for differentiating malignant and benign tumors.19–23 Measurements of MVD have been used to assess tumor angiogenesis as an independent prognostic factor for predicting tumor growth and metastasis. Reports9–11 concerning the correlation between MVD and ultrasonography are contradictory. This may be explained by the fact that MVD estimates the amount of microvessels, which are not detected via ultrasonography, and that the number of capillaries is counted in areas containing the highest numbers of capillaries and small venules (ie, in socalled hot spots). It is arguable whether this is the most suitable method for direct correlation of vascularity on tissue sections with ultrasonographic findings; malignant tumors will most likely have a high MVD as a result of an angiogenic response, particularly in certain areas of the tumor. However, as the tumor is growing, large necrotic areas often develop resulting in decreased total flow in the tumor, as detected ultrasonographically. This decrease may be undetected when estimating vascularity through assessment of hot spots. Benign tumors are not expected to have as high an MVD as malignant tumors because they are usually growing more slowly. Nevertheless, they may contain larger, more-developed vessels; ultrasonographically, this would appear as a large total flow area and would not correlate with the histologic findings in tissue sections. Therefore, it was hypothesized that a closer correlation would be achieved with a staining method that stains the types of vessels that are detected via ultrasonography. The Verhoeff technique stains the elastic fibers in the internal elastic membrane of arterioles and arteries that are > 250 to 300 μm in diameter. These blood vessels are expected to be detected via ultrasonography because the image resolution in the frequency range used is approximately 2 to 3 mm. In this type of correlation, one needs to realize that many potentially variable factors determine whether a particular vessel will be displayed and identified on the color Doppler image. Thus, counting the number of vessels displayed in an individual image provides only a crude index of vascular activity, and the vascular activity will always be underestimated by an amount that varies from ultrasonographic scan to scan. The same is true when evaluating tissue sections histologically because the staining results are dependent on factors such as tissue treatment before fixation and staining procedures. Because Doppler ultrasonography can detect smaller vessels than those that become stained with the Verhoeff technique, there will be a risk of underestimating vessels counted on tissue sections, compared with the estimate made ultrasonographically. This was not a problem in the present study. It was also found that the ultrasonographic and histologic assessments of the distribution of flow within the tumors were correlated. Therefore, it can be concluded that the Verhoeff staining technique can be used to correlate ultrasonographic and histologic assessments of vascularity and that this staining method appears to be more suitable for this purpose than assessment of MVD. However, the technique has not been evaluated for the purpose of differentiating benign and malignant tumors. In the present study, a mixture of vessels of different sizes was detected among the groups of tumors, although a systematic characterization of the individual vessels within each tumor was not performed. Results of other studies24,25 have suggested that bifurcations and trifurcations of vessels may be an indication of malignancy. In our study, multiple divisions from 1 original vessel were identified in benign and malignant tumors. This needs further evaluation to elucidate whether this is a sign of malignancy in canine mammary tumors.
Overall, the results of our study indicate that intratumoral blood flow can be detected and evaluated via color Doppler ultrasonography; the use of Doppler ultrasonography offers a noninvasive method of assessing characteristics of vascularization in naturally occurring mammary tumors in dogs. Our data have suggested that there is a correlation between the echogenicity and acoustic transmission associated with a mammary tumor and histopathologic findings. In sections of tumors, the number and distribution of Verhoeffstained vessels (although underestimated) correlated with the ultrasonographic findings; this staining technique can therefore be recommended for use in such correlation studies instead of assessment of MVD.
ABBREVIATIONS
| MVD | Microvessel density |
| WHO | World Health Organization |
| CI | Confidence interval |
Müller F, Kiefer I, Himmelsbach P, et al. Ultrasound of the canine mammary gland (abstr), in Proceedings. 11th Annu Sci Conf Eur Assoc Vet Diagn Imaging 2004;28.
Nyman HT, McEvoy FJ, Kristensen AT. Characterization of superficial tumors using ultrasonography (abstr), in Proceedings. 11th Annu Sci Conf Eur Assoc Vet Diagn Imaging 2004;40.
Siemens Sequoia, Siemens, Denmark.
Acuson Aspen, Acuson, Sweden.
DICOM (digital imaging and communications in medicine format), Nema, Rosslyn, Va. Available at: medical.nema.org/dicom/geninfo/Strategy.htm. Accessed June 13, 2005.
Power ShowCase software, Trillium Technology, Ann Arbor, Mich.
Image J 1.33, Rasband, WS, Image J, National Institutes of Health, Bethesda, Md. Available at: rsb.info.nih.gov/ij/index.html. Accessed June 13, 2005.
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