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Contrast-Enhanced Ultrasound Imaging of Carotid Plaque Neo-vascularization: Accuracy of Visual Analysis
Ultrasound in Med. & Biol., Vol. 40, No. 1, pp. 18–24, 2014 Copyright Ó 2014 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
http://dx.doi.org/10.1016/j.ultrasmedbio.2013.08.012
d
Original Contribution CONTRAST-ENHANCED ULTRASOUND IMAGING OF CAROTID PLAQUE NEO-VASCULARIZATION: ACCURACY OF VISUAL ANALYSIS y ULLER,* AUR ELIEN VIACCOZ,* IGOR KUZMANOVIC,* CHRISTOPHE BONVIN, HUBERTUS FRITZ GEORG M€ z x KARIM BURKHARDT, MARIE-LUCE BOCHATON-PIALLAT, and ROMAN SZTAJZEL*
* Department of Neurology, University Hospital of Geneva and Medical School, Geneva, Switzerland; y Department of Neurology, Hospital of Sion, Sion, Switzerland; z Department of Surgical Pathology, University Hospital of Geneva and Medical School, Geneva, Switzerland; and x Department of Pathology and Immunology, University Hospital of Geneva and Medical School, Geneva, Switzerland (Received 16 January 2013; revised 13 July 2013; in final form 12 August 2013)
Abstract—The aim of our study was to evaluate whether neo-vascularization of the carotid plaque can be accurately assessed by visual analysis of contrast-enhanced ultrasound images and whether these findings correlate with intensity-over-time curve analysis (ITC) and histopathology. Patients with $50% symptomatic or $60% asymptomatic stenosis according to European Carotid Surgery Trial criteria were included. Four investigators evaluated contrast enhancement visually (three grades), with positive agreement when three or more investigators were unanimous. ITC analysis of contrast enhancement was performed in the plaque and in the lumen. Histopathology (microvessel density with CD34 1 staining) was completed when endarterectomy was performed. Visual grading (33 patients, inter-observer agreement 5 94%) correlated significantly with ITC analysis (p 5 0.03). Histopathology (n 5 19) revealed a larger CD34 1 area in patients with grade 1/2 versus grade 0 (p 5 0.03). Visual analysis of neo-vascularization by means of contrast-enhanced ultrasound imaging is accurate and reproducible, with significant correlations with ITC and histopathology. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Neo-vascularization, Contrast-enhanced ultrasound, Vulnerable carotid plaque, Stroke, Visual analysis.
By measuring degree of stenosis and by characterizing its structure, ultrasound is the method of choice for the evaluation of atherosclerotic carotid plaque (Sidhu and Allan 1997) and its clinical risk. Recent work has indicated that neo-angiogenesis may also play an important role in the development of unstable plaque (Fleiner et al. 2004; McCarthy et al. 1999; Moreno et al. 2004). Neo-vascularization arises as a result of chronic hypoxia within plaque, thereby generating new blood vessels (Sluimer et al. 2008). It has been found that ultrasound has the ability to depict neovascularization, particularly when used with contrast enhancement (Feinstein 2006; Shah et al. 2007). Assessment of plaque neo-vascularization is frequently performed by means of a software analysis calculating changes in intensity over time (Faggioli et al. 2011; Hoogi et al. 2011; Xiong et al. 2009). Other studies have added a visual approach to the evaluation of contrast enhancement correlated either with software analysis (Huang et al. 2010) or histopathologic findings (Coli et al. 2008; Giannoni et al. 2009; Shah et al. 2007).
INTRODUCTION Atherosclerotic carotid artery disease accounts for 15% to 20% of all ischemic strokes (Grau et al. 2001; Petty et al. 2000). Its origin is multifactorial and not yet fully understood. The main mechanism of stroke related to pathology of the carotid artery is thought to be embolism from a fissured or ruptured plaque (Kistler et al. 1984). Recent pathologic studies of postmortem and arterectomy specimens have reported that plaque vulnerability is related to the size of the atheromatous core (Redgrave et al. 2006) and the thickness of and inflammation within the fibrous cap (Davies et al. 1993; Munro and Cotran 1988; van der Wal et al. 1994). Unstable plaques usually have a thin fibrous cap with a necrotic core situated near the surface (Hennerici 2004).
Address correspondence to: H. F. G. M€uller, Service de Neurologie, H^ opitaux Universitaires de Geneve, Rue Gabrielle Perret Gentil 4, CH-1211 Geneve 14, Switzerland. E-mail: hubertus.muller@ hcuge.ch 18
CEUI of carotid plaque neo-vascularization d H. F. G. M€ ULLER et al.
However, there is high variability in inter-observer agreement among these studies (Table 1). A uniform and validated scoring system is therefore still needed in clinical practice. The aim of our study was to evaluate whether neo-vascularization of carotid plaque can be accurately assessed by visual analysis of contrast-enhanced ultrasound images and whether these findings correlate with intensity-over-time curve (ITC) analysis as well as with histopathology.
19
Geneva, Switzerland), a suspension of phospholipid stabilized sulfur hexafluoride gas microbubbles, was used as the contrast agent in all cases. We injected 2 mL of Sonovue intravenously for each recording. Sequences of frames in the longitudinal or transversal plane were digitally recorded, using the DICOM format, 800 3 600-pixel resolution and a frame rate of 22 frames/ s, starting a few seconds before the bolus injection and ending when all contrast agents disappeared. Two methods of offline analysis were used: 1. The first method was based on visual evaluation by four different and blinded investigators of plaque contrast enhancement during microbubble circulation. Three of the investigators (A.V., I.K. and R.S.) were highly skilled and certified ultrasonographers with several years of experience, and one (H.M.) was less experienced. The appearance of a hyper-intense signal after lumen enhancement was considered as a positive microbubble when its displacement within the plaque was observed. Contrast enhancement was classified into three grades: 0 5 no enhancement, 1 5 intermediate enhancement and 2 5 extensive enhancement. Plaques with no microbubbles visible were grade 0, plaques with a small number of
METHODS Consecutive patients with $50% symptomatic or $60% asymptomatic internal carotid artery stenosis according to the European Carotid Surgery Trial criteria were included. A local institutional review board for ethics approved the study protocol. Informed consent for the study was obtained from all patients. All patients underwent an ultrasound examination (Antares apparatus, Siemens Medical Solutions, Malvern, PA, USA) including assessment of degree of stenosis according to standard criteria and contrastenhanced imaging using low-mechanical-index cadence contrast pulse sequencing technology. Sonovue (Bracco,
Table 1. Comparison of published studies including visual analysis of contrast-enhanced ultrasound images with this study Visual analysis Correlation Patients/plaques
Histology
TTP
Grading
Analyzers
Inter-observer ratio
With histology
With TTP
With symptoms
Xiong et al. (2009) Shah et al. (2007)
108 15/17
Yes No
2 grades* 4 gradesz
2 3
? ?
— Yesy
? —
Yesy No
Coli et al. (2008)
32/52
No CD31 CD34 CD31 CD34 VEGF MMP3 CD31 CD34 CD34 No No
No
2 gradesx
2
?
Yesy
—
No
Study
Giannoni et al. (2009)
This study Staub et al. (2010) Huang et al. (2010)
77
33 147/111 176
k
No
2 grades
2
?
Yes
—
Yes
Yes No Yes
3 grades 2 grades{ 4 grades#
4 2 2
0.94 0.54 0.66
Yes — —
Yes — ?
No No Yes
* Grade 1: no enhancement within the plaque or enhancement confined to the adventitial side of the plaque and/or the shoulder. Grade 2: enhancement reaching plaque core or extensive contrast enhancement throughout the plaque. y p , 0.05. z Grade 0: no appearance of neo-vascularization within the plaque. Grade 3: images that revealed the presence of a pulsating, arterial vessel within the plaque (implying arteriogenesis). Grade 1: limited appearance of neo-vascularization within the plaque. Grade 2: moderate neo-vascularization with less neo-vascularization than noted in grade 3 and more than that of grade 1. x Grade 1: no bubbles within the plaque or bubbles confined to plaque adventitial side and/or shoulder. Grade 2: bubbles reaching plaque core and/or extensive contrast-agent enhancement throughout the plaque. k Grade I: isolated microvessels, readily identifiable by high-intensity spots of microbubbles running from the adventitial layers toward the vessel lumen. Grade II: a major diffuse area of contrast enhancement appeared at the base of the plaques, due to an agglomerate of many very small microvessels, difficult to differentiate from each other. { Grade 1: no appearance of bubbles within the plaque or bubbles confined to plaque adventitial side. Grade 2: clear visible appearance of bubbles within the plaque moving from the adventitial side or shoulder, reaching plaque core. # Grade I: no enhancement. Grade II: arterial wall vasa vasorum enhancement. Grade III: arterial wall vasa vasorum and plaque shoulder enhancement. Grade IV: extensive and internal plaque enhancement.
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microbubbles were grade 1, and plaques with a large number of microbubbles throughout the plaque with a characteristic scintillating aspect were grade 2 (Fig. 1). Agreement was considered positive when at least three investigators concurred. 2. The second, control method consisted of using a semi-automatic software analysis (program written in-house in MATLAB, Version 2007, Mathworks, Natick, MA, USA). Contrast enhancement was determined on the basis of ITC analysis of the
Volume 40, Number 1, 2014
carotid plaque. Therefore, a fixed-position region of interest (ROI) was drawn manually to outline the plaque on its surface in the longitudinal plane. The ROI was placed so as to avoid contamination by the lumen over time caused by movement of the patient. Intensity was plotted as a function of time, and the change in intensity was calculated as the difference between peak intensity and baseline intensity (DP). A second measurement was performed within the vessel lumen as a reference for intensity changes. Furthermore, histopathologic examination of the carotid plaque by two experienced pathologists (M.L.P., K.B.) was performed when endarterectomy was performed. Pathologists were blinded to the clinical and ultrasonographic data. Plaque samples were examined after formalin fixation and paraffin embedding. Histologic slides (Fig. 2) of transverse sections were stained with hematoxylin and eosin and Miller stain and immunostained with anti-CD34 antibody (DAKO, Glostrup, Denmark). Quantitative immunohistochemistry was performed as previously described (Alizadeh et al. 2007; Hao et al. 2006). Slides for morphometric analysis were scanned at 20 3 magnification and high resolution using a fully automated Mirax Virtual Slide Scanner equipped with a Plan-Apochromat 20 3/0.8 objective (Carl Zeiss,
Fig. 1. Visual analysis of ultrasound images of carotid plaque after ultrasound contrast injection according to our criteria. (a) Grade 0 5 no microbubbles. (b) Grade 1 5 small number of microbubbles. (c) Grade 2 5 large number of microbubbles with a scintillating aspect.
Fig. 2. CD34 1 immunohistochemistry of a carotid plaque. The CD34 1 area with microvessels (arrow) is extensive in this specimen and located near the adventitia.
CEUI of carotid plaque neo-vascularization d H. F. G. M€ ULLER et al.
Jena, Germany). Images were subsequently analyzed using MetaMorph 6.0 software (Universal Imaging, Downingtown, PA, USA). Briefly, ROIs were manually drawn to cover the intimal layer for each slide. Pixels were selected according to hue (i.e., dominant color tone), lightness (i.e., color intensity) and saturation (i.e., color purity) components, and quantitated. Results were calculated as the total area of immunostaining/total intima area (pixels and %). Images for illustrations were taken with a Zeiss Axiophot microscope (Carl Zeiss) equipped with a high-sensitivity, high-resolution digital color camera (Axiocam, Carl Zeiss). A plan Neofluar 20 3/0.50 objective and oil-immersion plan-Neofluar 40 3/1.3 objectives (Carl Zeiss) were used. All statistical analyses were performed with R Software (R Development Core Team 2012). Kappa values were calculated according to the Fleiss algorithm for more than two observers. For correlations, Spearman’s method was used.
RESULTS From 2008 to 2011, 33 patients, 17 symptomatic and 16 asymptomatic, were included in our study. Patients’ demographic characteristics and their vascular risk factors are summarized in Table 2. Visual analysis of plaque contrast enhancement was performed by the four independent observers, with an inter-observer agreement of 94% (Table 3). Only in two cases was no consensus obtained. The k value for our consensus criterion was 0.94, judged as very good agreement. Nevertheless, analyzer H.M., the less experienced ultrasonographer, disagreed considerably more often (eight times between grades 0 and 1, five times between grades 1 and 2). Yet, we decided not to exclude this analyzer so that we could determine to what extent experience may influence inter-observer agreement. See Table 3 for the detailed results. Thirteen plaques were interpreted as having no (grade 0, 39%), 13 as having intermediate (grade 1, 39%) and 5 as having extensive (grade 2, 15%) enhancement. Computerized analysis of the ultrasound image sequences resulted in an ITC for each plaque and lumen. Table 2. Demographic characteristics of the patients
Male gender Hypertension Dyslipidemia Diabetes Smoking
Symptomatic (n 5 17)
Asymptomatic (n 5 16)
p-value
12 (71%) 14 (82) 11 (65) 7 (41) 8 (47)
12 (75%) 16 (100) 9 (56) 9 (56) 5 (31)
ns ns ns ns ns
ns 5 not significant.
21
Table 3. Visual analysis by four independent observers and the consensus as described under Methods Number
A.V.
I.K.
R.S.
H.M.
Consensus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 18 19 20 21 22 23 24 25 26 27 28 29 30 32 33 34 35 36
2 0 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 1 0 1 2 1 1 2 0 2 0 1 1 1 0 2 1
2 0 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 1 0 1 2 1 1 2 0 2 0 1 1 2 0 2 1
2 0 0 1 0 0 1 1 0 0 1 0 0 2 1 1 1 1 0 1 2 1 1 2 0 2 0 1 1 2 0 2 0
1 0 0 1 1 0 1 1 1 0 1 0 1 1 0 0 1 0 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0
2 0 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 1 0 1 2 1 1 2 0 2 0 1 1 No consensus 0 2 No consensus
0 5 no enhancement; 1 5 intermediate enhancement; 2 5 extensive enhancement.
The difference between base level and peak intensity was calculated (DP) (Fig. 3). The overall mean DP was 0.023 (0.00020.139) for plaques and 0.254 (0.01320.800) for lumens. After statistical analysis, we found that the intensity changes assessed by our program revealed a significant distinction between the different groups in visual analysis (Fig. 4). Patients in the no enhancement group had a mean DP value of 0.008, as opposed to 0.029 in group 1 (p 5 0.04) and 0.048 in group 2 (p 5 0.01), or 0.035 if groups 1 and 2 are taken together (p 5 0.02). In the 19 patients for whom a histopathologic examination was carried out, the mean CD34 1 area was larger in patients with grade 2 enhancement than in those with grade 0 enhancement (32,439 pixels vs. 7376 pixels, p 5 0.01) (Fig. 5). The differences between grade 0 and grade 1 or 2 followed a trend, but failed to attain statistical significance. Spearman’s method indicated there was a significant relationship between DP and CD34 1 area, with a r value of 0.508 (p 5 0.026) (Fig. 6).
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Fig. 3. Software analysis of intensity over time curves (ITCs). (a, b) Output (ITC, raw data) of our software analysis for the region of interest in the plaque (blue) and the lumen (red) combined. (a) Example of a plaque with a significant contrast enhancement. (b) Example of no enhancement.
DISCUSSION Our results indicate that visual analysis of neovascularization of the carotid plaque using contrast enhancement is accurate. In fact, we found a good correlation between two different and independent approaches to visual analysis of neo-vascularization, ITC analysis and histologic quantification of vessel density. As seen in Figures 1, 4 and 5, these two methods matched very well.
Furthermore, inter-observer agreement was excellent (k 5 0.94), suggesting our visual method is reproducible. However, as seen in Table 3, observer H.M., the less experienced investigator, disagreed considerably often with the other three ultrasonographers. We explain this by hypothesiaing that a certain skill level and experience in contrast-enhanced ultrasound images are mandatory for analysis. Once the expertise is obtained, visual analysis appears to be reliable.
CEUI of carotid plaque neo-vascularization d H. F. G. M€ ULLER et al.
23
90000
0.10 p=0.01 p=0.04 mean
60000 p=0.02, rho=0.5
0.05 30000
0
0.00 0
1
0.00
2
Fig. 4. Box-and-whisker and dot plot of intensity changes (DP, peak – baseline) of the regions of interest in carotid plaque for visual analysis grades 0, 1 and 2.
Several studies performed so far have used software analyses based on changes in intensity over time to evaluate neo-vascularization. From our experience, this technique has limitations; its use is complicated and may therefore be difficult to apply in clinical practice. Also, the results may be influenced by patients’ movements (i.e., swallowing), consequently inducing contamination of the region of interest by a highly contrast-enhanced lumen or hyper-echogenic parts of the plaque or the adventitia. A motion tracking algorithm should be used to improve accuracy, as already demonstrated (Hoogi et al. 2011), but this is a complicated process and not widely available. Furthermore, diffuse contrast enhancement, as typically assessed by the software method, might be insufficient to characterize neo-vascularization. The presence of regionally condensed (multi-locus) microbubbles in a plaque potentially describes an area at risk, whereas in a large plaque, the absolute change in intensity
120000
90000 p=0.01
60000
mean
30000
0 0
1
0.05
0.10
delta intensity (peak − baseline)
visual grade
CD34+ area (px)
CD34+ area (px)
delta intensity (peak − baseline)
120000
2
visual grade
Fig. 5. Box-whisker and dot plot of CD34 1 area on histology for visual analysis grades 0, 1 and 2.
Fig. 6. Scatterplot illustrating the relationship between CD34 1 area on histology and changes in intensity (DP, peak – baseline) in the region of interest in the carotid plaque. Spearman’s rank correlation r 5 0.508.
might remain low. This may also explain the different results illustrated in Figures 5 and 6, where visual grading of contrast enhancement seemed to correlate better with histopathology than with ITC analysis. Finally, a large variety of software are used that have significant methodological differences, rendering comparisons between different centers difficult. A visually based approach may therefore be useful and of practical interest. Several studies, as summarized in Table 1, have used a semi-quantitative visual method with different scoring methods. A few studies found the visual approach to be correlated with histology, and other studies, with ITC analysis. Our study revealed a relationship between the visual approach, software analysis, and histology. However, it must be mentioned that our cohort was small and that a similar analysis including a larger number of patients is necessary. The k values reported in other studies were lower than those obtained in our work (Table 1). Such differences may be at least partially due to the different scoring systems used. In the present study, we used a very simple grading method: no, moderate or intense enhancement (see above for detail). The location of the microbubbles within the plaque was not taken into account. This aspect was part of the grading approach in other studies and possibly affected the resulting inter-observer agreement rates, which were lower. In contrast to other studies, our results failed to show a clear statistically significant difference between symptomatic and asymptomatic patients. This fact was related not only to our visual approach, but to software and histologic analysis as well. The small study population could be a plausible explanation. As seen in Table 1, studies with larger cohorts of patients were better able to differentiate symptomatic from asymptomatic patients than
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Ultrasound in Medicine and Biology
studies with small numbers of patients. Giannoni et al. (2009) further reported that a pattern of diffuse enhancement located close to the adventitial layer is related to symptomatic plaques. In summary, in our study, visual analysis of contrastenhanced ultrasound images seemed accurate when performed by well-trained sonographers and correlated with ITC analysis. Visual grading did not correlate as well with the histopathologic findings, but at least those plaques with strong enhancement exhibited higher vascularization than those without enhancement. Larger numbers of patients are needed, however, to confirm these results and to confirm that contrast-enhanced ultrasound images combined with a visual analysis may be potentially a valuable method for characterization of vulnerable carotid plaque and its risk stratification. Acknowledgments—Financial support was obtained by a grant of the Swiss Heart Foundation.
REFERENCES Alizadeh N, Pepper MS, Modarressi A, Alfo K, Schlaudraff K, Montandon D, Gabbiani G, Bochaton-Piallat M-L, Pittet B. Persistent ischemia impairs myofibroblast development in wound granulation tissue: A new model of delayed wound healing. Wound Repair Regen 2007;15:809–816. Coli S, Magnoni M, Sangiorgi G, Marrocco-Trischitta MM, Melisurgo G, Mauriello A, Spagnoli L, Chiesa R, Cianflone D, Maseri A. Contrast-enhanced ultrasound imaging of intraplaque neovascularization in carotid arteries: Correlation with histology and plaque echogenicity. J Am Coll Cardiol 2008;52:223–230. Davies MJ, Richardson PD, Woolf N, Katz DR, Mann J. Risk of thrombosis in human atherosclerotic plaques: Role of extracellular lipid, macrophage, and smooth muscle cell content. Heart 1993;69: 377–381. Faggioli GL, Pini R, Mauro R, Pasquinelli G, Fittipaldi S, Freyrie A, Serra C, Stella A. Identification of carotid ‘‘vulnerable plaque’’ by contrast-enhanced ultrasonography: Correlation with plaque histology, symptoms and cerebral computed tomography. Eur J Vasc Endovasc Surg 2011;41:238–248. Feinstein SB. Contrast ultrasound imaging of the carotid artery vasa vasorum and atherosclerotic plaque neovascularization. J Am Coll Cardiol 2006;48:236–243. Fleiner M, Kummer M, Mirlacher M, Sauter G, Cathomas G, Krapf R, Biedermann BC. Arterial neovascularization and inflammation in vulnerable patients: Early and late signs of symptomatic atherosclerosis. Circulation 2004;110:2843–2850. Giannoni MF, Vicenzini E, Citone M, Ricciardi MC, Irace L, Laurito A, Scucchi LF, Di Piero V, Gossetti B, Mauriello A, Spagnoli LG, Lenzi GL, Valentini FB. Contrast carotid ultrasound for the detection of unstable plaques with neoangiogenesis: A pilot study. Eur J Vasc Endovasc Surg 2009;37:722–727. Grau AJ, Weimar C, Buggle F, Heinrich A, Goertler M, Neumaier S, Glahn J, Brandt T, Hacke W, Diener HC. Risk factors, outcome, and treatment in subtypes of ischemic stroke: The German Stroke Data Bank. Stroke 2001;32:2559–2566.
Volume 40, Number 1, 2014 Hao H, Gabbiani G, Camenzind E, Bacchetta M, Virmani R, Bochaton-Piallat M-L. Phenotypic modulation of intima and media smooth muscle cells in fatal cases of coronary artery lesion. Arterioscler Thromb Vasc Biol 2006;26:326–332. Hennerici MG. The unstable plaque. Cerebrovasc Dis 2004;17(Suppl 3): 17–22. Hoogi A, Adam D, Hoffman A, Kerner H, Reisner S, Gaitini D. Carotid plaque vulnerability: Quantification of neovascularization on contrast-enhanced ultrasound with histopathologic correlation. AJR Am J Roentgenol 2011;196:431–436. Huang PT, Chen CC, Aronow WS, Wang XT, Nair CK, Xue NY, Shen X, Li SY, Huang FG, Cosgrove D. Assessment of neovascularization within carotid plaques in patients with ischemic stroke. World J Cardiol 2010;2:89–97. Kistler J, Ropper A, Heros R. Therapy of ischemic cerebral vascular disease due to atherothrombosis. N Engl J Med 1984; 311:27–34. McCarthy MJ, Loftus IM, Thompson MM, Jones L, London NJ, Bell PR, Naylor AR, Brindle NP. Angiogenesis and the atherosclerotic carotid plaque: An association between symptomatology and plaque morphology. J Vasc Surg 1999;30:261–268. Moreno PR, Purushothaman KR, Fuster V, Echeverri D, Truszczynska H, Sharma SK, Badimon JJ, O’Connor WN. Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: Implications for plaque vulnerability. Circulation 2004;110:2032–2038. Munro JM, Cotran RS. The pathogenesis of atherosclerosis: Atherogenesis and inflammation. Lab Invest 1988;58:249–261. Petty GW, Brown RD, Whisnant JP, Sicks JD, O’Fallon WM, Wiebers DO. Ischemic stroke subtypes: A population-based study of functional outcome, survival, and recurrence. Stroke 2000;31: 1062–1068. R Development Core Team. R: A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing. URL: http://www.R-project.org/; 2012. Redgrave JNE, Lovett JK, Gallagher PJ, Rothwell PM. Histological assessment of 526 symptomatic carotid plaques in relation to the nature and timing of ischemic symptoms: The Oxford plaque study. Circulation 2006;113:2320–2328. Shah F, Balan P, Weinberg M, Reddy V, Neems R, Feinstein M, Dainauskas J, Meyer P, Goldin M, Feinstein SB. Contrast-enhanced ultrasound imaging of atherosclerotic carotid plaque neovascularization: A new surrogate marker of atherosclerosis? Vasc Med 2007;12:291–297. Sidhu PS, Allan PL. Ultrasound assessment of internal carotid artery stenosis. J Clin Ultrasound 1997;52:654–658. Sluimer JC, Gasc JM, Van Wanroij JL, Kisters N, Groeneweg M, Sollewijn Gelpke MD, Cleutjens JP, Van den Akker LH, Corvol P, Wouters BG, Daemen MJ, Bijnens APJ. Hypoxia, hypoxiainducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis. J Am Coll Cardiol 2008;51:1258–1265. Staub D, Patel MB, Tibrewala A, Ludden D, Johnson M, Espinosa P, Coll B, Jaeger KA, Feinstein SB. Vasa vasorum and plaque neovascularization on contrast-enhanced carotid ultrasound imaging correlates with cardiovascular disease and past cardiovascular events. Stroke 2010;41:41–47. Van der Wal AC, Becker AE, Van der Loos CM, Tigges AJ, Das PK. Fibrous and lipid-rich atherosclerotic plaques are part of interchangeable morphologies related to inflammation: a concept. Coronary Artery Dis 1994;5:463–469. Xiong L, Deng Y, Zhu Y, Liu Y, Bi X. Correlation of carotid plaque neovascularization detected by using contrast-enhanced US with clinical symptoms. Radiology 2009;251:583–589.
http://dx.doi.org/10.1016/j.ultrasmedbio.2013.08.012
d
Original Contribution CONTRAST-ENHANCED ULTRASOUND IMAGING OF CAROTID PLAQUE NEO-VASCULARIZATION: ACCURACY OF VISUAL ANALYSIS y ULLER,* AUR ELIEN VIACCOZ,* IGOR KUZMANOVIC,* CHRISTOPHE BONVIN, HUBERTUS FRITZ GEORG M€ z x KARIM BURKHARDT, MARIE-LUCE BOCHATON-PIALLAT, and ROMAN SZTAJZEL*
* Department of Neurology, University Hospital of Geneva and Medical School, Geneva, Switzerland; y Department of Neurology, Hospital of Sion, Sion, Switzerland; z Department of Surgical Pathology, University Hospital of Geneva and Medical School, Geneva, Switzerland; and x Department of Pathology and Immunology, University Hospital of Geneva and Medical School, Geneva, Switzerland (Received 16 January 2013; revised 13 July 2013; in final form 12 August 2013)
Abstract—The aim of our study was to evaluate whether neo-vascularization of the carotid plaque can be accurately assessed by visual analysis of contrast-enhanced ultrasound images and whether these findings correlate with intensity-over-time curve analysis (ITC) and histopathology. Patients with $50% symptomatic or $60% asymptomatic stenosis according to European Carotid Surgery Trial criteria were included. Four investigators evaluated contrast enhancement visually (three grades), with positive agreement when three or more investigators were unanimous. ITC analysis of contrast enhancement was performed in the plaque and in the lumen. Histopathology (microvessel density with CD34 1 staining) was completed when endarterectomy was performed. Visual grading (33 patients, inter-observer agreement 5 94%) correlated significantly with ITC analysis (p 5 0.03). Histopathology (n 5 19) revealed a larger CD34 1 area in patients with grade 1/2 versus grade 0 (p 5 0.03). Visual analysis of neo-vascularization by means of contrast-enhanced ultrasound imaging is accurate and reproducible, with significant correlations with ITC and histopathology. (E-mail: [email protected]) Ó 2014 World Federation for Ultrasound in Medicine & Biology. Key Words: Neo-vascularization, Contrast-enhanced ultrasound, Vulnerable carotid plaque, Stroke, Visual analysis.
By measuring degree of stenosis and by characterizing its structure, ultrasound is the method of choice for the evaluation of atherosclerotic carotid plaque (Sidhu and Allan 1997) and its clinical risk. Recent work has indicated that neo-angiogenesis may also play an important role in the development of unstable plaque (Fleiner et al. 2004; McCarthy et al. 1999; Moreno et al. 2004). Neo-vascularization arises as a result of chronic hypoxia within plaque, thereby generating new blood vessels (Sluimer et al. 2008). It has been found that ultrasound has the ability to depict neovascularization, particularly when used with contrast enhancement (Feinstein 2006; Shah et al. 2007). Assessment of plaque neo-vascularization is frequently performed by means of a software analysis calculating changes in intensity over time (Faggioli et al. 2011; Hoogi et al. 2011; Xiong et al. 2009). Other studies have added a visual approach to the evaluation of contrast enhancement correlated either with software analysis (Huang et al. 2010) or histopathologic findings (Coli et al. 2008; Giannoni et al. 2009; Shah et al. 2007).
INTRODUCTION Atherosclerotic carotid artery disease accounts for 15% to 20% of all ischemic strokes (Grau et al. 2001; Petty et al. 2000). Its origin is multifactorial and not yet fully understood. The main mechanism of stroke related to pathology of the carotid artery is thought to be embolism from a fissured or ruptured plaque (Kistler et al. 1984). Recent pathologic studies of postmortem and arterectomy specimens have reported that plaque vulnerability is related to the size of the atheromatous core (Redgrave et al. 2006) and the thickness of and inflammation within the fibrous cap (Davies et al. 1993; Munro and Cotran 1988; van der Wal et al. 1994). Unstable plaques usually have a thin fibrous cap with a necrotic core situated near the surface (Hennerici 2004).
Address correspondence to: H. F. G. M€uller, Service de Neurologie, H^ opitaux Universitaires de Geneve, Rue Gabrielle Perret Gentil 4, CH-1211 Geneve 14, Switzerland. E-mail: hubertus.muller@ hcuge.ch 18
CEUI of carotid plaque neo-vascularization d H. F. G. M€ ULLER et al.
However, there is high variability in inter-observer agreement among these studies (Table 1). A uniform and validated scoring system is therefore still needed in clinical practice. The aim of our study was to evaluate whether neo-vascularization of carotid plaque can be accurately assessed by visual analysis of contrast-enhanced ultrasound images and whether these findings correlate with intensity-over-time curve (ITC) analysis as well as with histopathology.
19
Geneva, Switzerland), a suspension of phospholipid stabilized sulfur hexafluoride gas microbubbles, was used as the contrast agent in all cases. We injected 2 mL of Sonovue intravenously for each recording. Sequences of frames in the longitudinal or transversal plane were digitally recorded, using the DICOM format, 800 3 600-pixel resolution and a frame rate of 22 frames/ s, starting a few seconds before the bolus injection and ending when all contrast agents disappeared. Two methods of offline analysis were used: 1. The first method was based on visual evaluation by four different and blinded investigators of plaque contrast enhancement during microbubble circulation. Three of the investigators (A.V., I.K. and R.S.) were highly skilled and certified ultrasonographers with several years of experience, and one (H.M.) was less experienced. The appearance of a hyper-intense signal after lumen enhancement was considered as a positive microbubble when its displacement within the plaque was observed. Contrast enhancement was classified into three grades: 0 5 no enhancement, 1 5 intermediate enhancement and 2 5 extensive enhancement. Plaques with no microbubbles visible were grade 0, plaques with a small number of
METHODS Consecutive patients with $50% symptomatic or $60% asymptomatic internal carotid artery stenosis according to the European Carotid Surgery Trial criteria were included. A local institutional review board for ethics approved the study protocol. Informed consent for the study was obtained from all patients. All patients underwent an ultrasound examination (Antares apparatus, Siemens Medical Solutions, Malvern, PA, USA) including assessment of degree of stenosis according to standard criteria and contrastenhanced imaging using low-mechanical-index cadence contrast pulse sequencing technology. Sonovue (Bracco,
Table 1. Comparison of published studies including visual analysis of contrast-enhanced ultrasound images with this study Visual analysis Correlation Patients/plaques
Histology
TTP
Grading
Analyzers
Inter-observer ratio
With histology
With TTP
With symptoms
Xiong et al. (2009) Shah et al. (2007)
108 15/17
Yes No
2 grades* 4 gradesz
2 3
? ?
— Yesy
? —
Yesy No
Coli et al. (2008)
32/52
No CD31 CD34 CD31 CD34 VEGF MMP3 CD31 CD34 CD34 No No
No
2 gradesx
2
?
Yesy
—
No
Study
Giannoni et al. (2009)
This study Staub et al. (2010) Huang et al. (2010)
77
33 147/111 176
k
No
2 grades
2
?
Yes
—
Yes
Yes No Yes
3 grades 2 grades{ 4 grades#
4 2 2
0.94 0.54 0.66
Yes — —
Yes — ?
No No Yes
* Grade 1: no enhancement within the plaque or enhancement confined to the adventitial side of the plaque and/or the shoulder. Grade 2: enhancement reaching plaque core or extensive contrast enhancement throughout the plaque. y p , 0.05. z Grade 0: no appearance of neo-vascularization within the plaque. Grade 3: images that revealed the presence of a pulsating, arterial vessel within the plaque (implying arteriogenesis). Grade 1: limited appearance of neo-vascularization within the plaque. Grade 2: moderate neo-vascularization with less neo-vascularization than noted in grade 3 and more than that of grade 1. x Grade 1: no bubbles within the plaque or bubbles confined to plaque adventitial side and/or shoulder. Grade 2: bubbles reaching plaque core and/or extensive contrast-agent enhancement throughout the plaque. k Grade I: isolated microvessels, readily identifiable by high-intensity spots of microbubbles running from the adventitial layers toward the vessel lumen. Grade II: a major diffuse area of contrast enhancement appeared at the base of the plaques, due to an agglomerate of many very small microvessels, difficult to differentiate from each other. { Grade 1: no appearance of bubbles within the plaque or bubbles confined to plaque adventitial side. Grade 2: clear visible appearance of bubbles within the plaque moving from the adventitial side or shoulder, reaching plaque core. # Grade I: no enhancement. Grade II: arterial wall vasa vasorum enhancement. Grade III: arterial wall vasa vasorum and plaque shoulder enhancement. Grade IV: extensive and internal plaque enhancement.
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Ultrasound in Medicine and Biology
microbubbles were grade 1, and plaques with a large number of microbubbles throughout the plaque with a characteristic scintillating aspect were grade 2 (Fig. 1). Agreement was considered positive when at least three investigators concurred. 2. The second, control method consisted of using a semi-automatic software analysis (program written in-house in MATLAB, Version 2007, Mathworks, Natick, MA, USA). Contrast enhancement was determined on the basis of ITC analysis of the
Volume 40, Number 1, 2014
carotid plaque. Therefore, a fixed-position region of interest (ROI) was drawn manually to outline the plaque on its surface in the longitudinal plane. The ROI was placed so as to avoid contamination by the lumen over time caused by movement of the patient. Intensity was plotted as a function of time, and the change in intensity was calculated as the difference between peak intensity and baseline intensity (DP). A second measurement was performed within the vessel lumen as a reference for intensity changes. Furthermore, histopathologic examination of the carotid plaque by two experienced pathologists (M.L.P., K.B.) was performed when endarterectomy was performed. Pathologists were blinded to the clinical and ultrasonographic data. Plaque samples were examined after formalin fixation and paraffin embedding. Histologic slides (Fig. 2) of transverse sections were stained with hematoxylin and eosin and Miller stain and immunostained with anti-CD34 antibody (DAKO, Glostrup, Denmark). Quantitative immunohistochemistry was performed as previously described (Alizadeh et al. 2007; Hao et al. 2006). Slides for morphometric analysis were scanned at 20 3 magnification and high resolution using a fully automated Mirax Virtual Slide Scanner equipped with a Plan-Apochromat 20 3/0.8 objective (Carl Zeiss,
Fig. 1. Visual analysis of ultrasound images of carotid plaque after ultrasound contrast injection according to our criteria. (a) Grade 0 5 no microbubbles. (b) Grade 1 5 small number of microbubbles. (c) Grade 2 5 large number of microbubbles with a scintillating aspect.
Fig. 2. CD34 1 immunohistochemistry of a carotid plaque. The CD34 1 area with microvessels (arrow) is extensive in this specimen and located near the adventitia.
CEUI of carotid plaque neo-vascularization d H. F. G. M€ ULLER et al.
Jena, Germany). Images were subsequently analyzed using MetaMorph 6.0 software (Universal Imaging, Downingtown, PA, USA). Briefly, ROIs were manually drawn to cover the intimal layer for each slide. Pixels were selected according to hue (i.e., dominant color tone), lightness (i.e., color intensity) and saturation (i.e., color purity) components, and quantitated. Results were calculated as the total area of immunostaining/total intima area (pixels and %). Images for illustrations were taken with a Zeiss Axiophot microscope (Carl Zeiss) equipped with a high-sensitivity, high-resolution digital color camera (Axiocam, Carl Zeiss). A plan Neofluar 20 3/0.50 objective and oil-immersion plan-Neofluar 40 3/1.3 objectives (Carl Zeiss) were used. All statistical analyses were performed with R Software (R Development Core Team 2012). Kappa values were calculated according to the Fleiss algorithm for more than two observers. For correlations, Spearman’s method was used.
RESULTS From 2008 to 2011, 33 patients, 17 symptomatic and 16 asymptomatic, were included in our study. Patients’ demographic characteristics and their vascular risk factors are summarized in Table 2. Visual analysis of plaque contrast enhancement was performed by the four independent observers, with an inter-observer agreement of 94% (Table 3). Only in two cases was no consensus obtained. The k value for our consensus criterion was 0.94, judged as very good agreement. Nevertheless, analyzer H.M., the less experienced ultrasonographer, disagreed considerably more often (eight times between grades 0 and 1, five times between grades 1 and 2). Yet, we decided not to exclude this analyzer so that we could determine to what extent experience may influence inter-observer agreement. See Table 3 for the detailed results. Thirteen plaques were interpreted as having no (grade 0, 39%), 13 as having intermediate (grade 1, 39%) and 5 as having extensive (grade 2, 15%) enhancement. Computerized analysis of the ultrasound image sequences resulted in an ITC for each plaque and lumen. Table 2. Demographic characteristics of the patients
Male gender Hypertension Dyslipidemia Diabetes Smoking
Symptomatic (n 5 17)
Asymptomatic (n 5 16)
p-value
12 (71%) 14 (82) 11 (65) 7 (41) 8 (47)
12 (75%) 16 (100) 9 (56) 9 (56) 5 (31)
ns ns ns ns ns
ns 5 not significant.
21
Table 3. Visual analysis by four independent observers and the consensus as described under Methods Number
A.V.
I.K.
R.S.
H.M.
Consensus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 18 19 20 21 22 23 24 25 26 27 28 29 30 32 33 34 35 36
2 0 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 1 0 1 2 1 1 2 0 2 0 1 1 1 0 2 1
2 0 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 1 0 1 2 1 1 2 0 2 0 1 1 2 0 2 1
2 0 0 1 0 0 1 1 0 0 1 0 0 2 1 1 1 1 0 1 2 1 1 2 0 2 0 1 1 2 0 2 0
1 0 0 1 1 0 1 1 1 0 1 0 1 1 0 0 1 0 0 1 1 1 1 1 0 1 0 0 0 0 0 1 0
2 0 0 1 0 0 1 1 0 0 1 0 0 1 0 1 1 1 0 1 2 1 1 2 0 2 0 1 1 No consensus 0 2 No consensus
0 5 no enhancement; 1 5 intermediate enhancement; 2 5 extensive enhancement.
The difference between base level and peak intensity was calculated (DP) (Fig. 3). The overall mean DP was 0.023 (0.00020.139) for plaques and 0.254 (0.01320.800) for lumens. After statistical analysis, we found that the intensity changes assessed by our program revealed a significant distinction between the different groups in visual analysis (Fig. 4). Patients in the no enhancement group had a mean DP value of 0.008, as opposed to 0.029 in group 1 (p 5 0.04) and 0.048 in group 2 (p 5 0.01), or 0.035 if groups 1 and 2 are taken together (p 5 0.02). In the 19 patients for whom a histopathologic examination was carried out, the mean CD34 1 area was larger in patients with grade 2 enhancement than in those with grade 0 enhancement (32,439 pixels vs. 7376 pixels, p 5 0.01) (Fig. 5). The differences between grade 0 and grade 1 or 2 followed a trend, but failed to attain statistical significance. Spearman’s method indicated there was a significant relationship between DP and CD34 1 area, with a r value of 0.508 (p 5 0.026) (Fig. 6).
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Ultrasound in Medicine and Biology
Volume 40, Number 1, 2014
Fig. 3. Software analysis of intensity over time curves (ITCs). (a, b) Output (ITC, raw data) of our software analysis for the region of interest in the plaque (blue) and the lumen (red) combined. (a) Example of a plaque with a significant contrast enhancement. (b) Example of no enhancement.
DISCUSSION Our results indicate that visual analysis of neovascularization of the carotid plaque using contrast enhancement is accurate. In fact, we found a good correlation between two different and independent approaches to visual analysis of neo-vascularization, ITC analysis and histologic quantification of vessel density. As seen in Figures 1, 4 and 5, these two methods matched very well.
Furthermore, inter-observer agreement was excellent (k 5 0.94), suggesting our visual method is reproducible. However, as seen in Table 3, observer H.M., the less experienced investigator, disagreed considerably often with the other three ultrasonographers. We explain this by hypothesiaing that a certain skill level and experience in contrast-enhanced ultrasound images are mandatory for analysis. Once the expertise is obtained, visual analysis appears to be reliable.
CEUI of carotid plaque neo-vascularization d H. F. G. M€ ULLER et al.
23
90000
0.10 p=0.01 p=0.04 mean
60000 p=0.02, rho=0.5
0.05 30000
0
0.00 0
1
0.00
2
Fig. 4. Box-and-whisker and dot plot of intensity changes (DP, peak – baseline) of the regions of interest in carotid plaque for visual analysis grades 0, 1 and 2.
Several studies performed so far have used software analyses based on changes in intensity over time to evaluate neo-vascularization. From our experience, this technique has limitations; its use is complicated and may therefore be difficult to apply in clinical practice. Also, the results may be influenced by patients’ movements (i.e., swallowing), consequently inducing contamination of the region of interest by a highly contrast-enhanced lumen or hyper-echogenic parts of the plaque or the adventitia. A motion tracking algorithm should be used to improve accuracy, as already demonstrated (Hoogi et al. 2011), but this is a complicated process and not widely available. Furthermore, diffuse contrast enhancement, as typically assessed by the software method, might be insufficient to characterize neo-vascularization. The presence of regionally condensed (multi-locus) microbubbles in a plaque potentially describes an area at risk, whereas in a large plaque, the absolute change in intensity
120000
90000 p=0.01
60000
mean
30000
0 0
1
0.05
0.10
delta intensity (peak − baseline)
visual grade
CD34+ area (px)
CD34+ area (px)
delta intensity (peak − baseline)
120000
2
visual grade
Fig. 5. Box-whisker and dot plot of CD34 1 area on histology for visual analysis grades 0, 1 and 2.
Fig. 6. Scatterplot illustrating the relationship between CD34 1 area on histology and changes in intensity (DP, peak – baseline) in the region of interest in the carotid plaque. Spearman’s rank correlation r 5 0.508.
might remain low. This may also explain the different results illustrated in Figures 5 and 6, where visual grading of contrast enhancement seemed to correlate better with histopathology than with ITC analysis. Finally, a large variety of software are used that have significant methodological differences, rendering comparisons between different centers difficult. A visually based approach may therefore be useful and of practical interest. Several studies, as summarized in Table 1, have used a semi-quantitative visual method with different scoring methods. A few studies found the visual approach to be correlated with histology, and other studies, with ITC analysis. Our study revealed a relationship between the visual approach, software analysis, and histology. However, it must be mentioned that our cohort was small and that a similar analysis including a larger number of patients is necessary. The k values reported in other studies were lower than those obtained in our work (Table 1). Such differences may be at least partially due to the different scoring systems used. In the present study, we used a very simple grading method: no, moderate or intense enhancement (see above for detail). The location of the microbubbles within the plaque was not taken into account. This aspect was part of the grading approach in other studies and possibly affected the resulting inter-observer agreement rates, which were lower. In contrast to other studies, our results failed to show a clear statistically significant difference between symptomatic and asymptomatic patients. This fact was related not only to our visual approach, but to software and histologic analysis as well. The small study population could be a plausible explanation. As seen in Table 1, studies with larger cohorts of patients were better able to differentiate symptomatic from asymptomatic patients than
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Ultrasound in Medicine and Biology
studies with small numbers of patients. Giannoni et al. (2009) further reported that a pattern of diffuse enhancement located close to the adventitial layer is related to symptomatic plaques. In summary, in our study, visual analysis of contrastenhanced ultrasound images seemed accurate when performed by well-trained sonographers and correlated with ITC analysis. Visual grading did not correlate as well with the histopathologic findings, but at least those plaques with strong enhancement exhibited higher vascularization than those without enhancement. Larger numbers of patients are needed, however, to confirm these results and to confirm that contrast-enhanced ultrasound images combined with a visual analysis may be potentially a valuable method for characterization of vulnerable carotid plaque and its risk stratification. Acknowledgments—Financial support was obtained by a grant of the Swiss Heart Foundation.
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