Ex vivo studies

One of the most important findings of our coronary plaque imaging projects was the identification of a novel radiological sign that we have named as ‘napkin-ring sign’.²⁵⁹ The NRS is a qualitative plaque feature and can be defined in a noncalcified plaque cross-section by the presence of two features: a central area of low CT attenuation that is apparently in contact with the lumen; and a ring-like higher attenuation plaque tissue surrounding this central area.⁷² Interestingly, NRS was present in both native (that is non-contrast-enhanced) and contrast-enhanced ex vivo CT images, suggesting that the feature is the result of differences in CT attenuation between the large necrotic core (a central low CT attenuation) and fibrous plaque tissue (ring-like higher attenuation). However, in vivo, some additional factors (such as the vasa vasorum) might influence the development of NRS.³²⁸ We have demonstrate that the area of necrotic core can be over twice the size in NRS plaques compared with non-NRS plaques (median 1.10 mm² versus 0.46 mm²; P = 0.05).²⁹⁸ These values correlate with other histopathological observations that demonstrate the area of necrotic core in vulnerable plaques is >1.0 mm² in the majority (~80%) of cases.⁵

The current clinically used coronary CTA classification of coronary atherosclerotic plaque composition is based on the presence or absence of calcification and was initially suggested in early coronary CTA studies using 4-slice multidetector CT technology with limited spatial and temporal resolution.^18,78 Although this classification has demonstrated that presence of NCP has some incremental value over the detection of CP in predicting adverse cardiovascular events, its ability to distinguish individual plaques that may be at higher risk for cardiovascular events is limited.^18,86 Therefore, as a next step we have developed a novel plaque classification scheme, which differentiates three types of non-calcified plaques: napkin-ring sign, heterogeneous and homogenous plaque. Our data demonstrates that a qualitative assessment of the attenuation pattern of NCP by coronary CTA under ex vivo conditions

significantly improves diagnostic accuracy for the detection of advanced plaque and TCFA as determined by histopathology compared with the conventional assessment of plaque composition (p<0.05 for both). Remarkably, both heterogeneous appearances of NCP and NRS are highly specific for the presence of both advanced plaque and TFCA in histopathology (specificity: 98.9% and 94.1%, respectively). Previous studies demonstrated the inability of density measurements within the plaque to differentiate reliably between lipid-rich and fibrous plaques because of a significant overlap in attenuation values (30,39–41).79,85,329,330 Significant progress in CT technology with an improvement of spatial resolution reaching 0.3 mm in-plane, allows a more differentiated assessment of the noncalcified portion of plaque. Recent studies suggest that low attenuation (<30 HU) is a hallmark of both culprit lesions in acute coronary syndromes and is more frequently found in plaques that are at high risk for rupture.^84,328,331 In addition, higher spatial resolution may mitigate the “masking” of NCP by CP, which appears at least 4´ larger in CT than its actual size due to blooming artifacts.³³²

Our results demonstrate the increased ability of coronary CTA to differentiate individual plaque characteristics that are specific for advanced atherosclerotic lesions associated with increased vulnerability and subsequent adverse cardiovascular events.5,6,261,262

Whereas the current clinically used classifications of partially calcified and NCP are unable to predict the presence of advanced plaques, NRS demonstrated a 98.9% specificity to identify advanced lesions and a 92.3% specificity for identifying TCFA. Our finding that the frequency of NRS was similar in MP and in NCP may help to resolve some contradictions in published studies, some of them reporting that MP rather than NCP indicates a higher risk for future cardiovascular events.90,102,333-336 Our data suggest that certain qualities of NCP irrespective of the presence of calcium are associated with advanced atherosclerotic lesions.

Prospective clinical studies are warranted to determine the prognostic value of PAP assessment to identify patients with the highest risk of developing cardiovascular events.³³⁷ Whereas quantitative analysis of NCP has been described before, we pursued a qualitative approach to plaque characterization based on initial encouraging findings. The qualitative plaque pattern assessment may be more feasible and easier to implement in clinical practice or large studies. In addition, the quantitative assessment of plaque attenuation might be significantly altered by the coronary lumen enhancement, the reconstruction kernel, and the size and number of regions of interest used for the attenuation assessment.^18,85 The ex vivo coronary CTA imaging was performed in an ideal, motion-free experimental setting. This might limit the direct translation of our findings into in vivo circumstances. However, it is important to note that some recently published studies observed a ring like attenuation pattern similar to

that of NRS in patients presenting with acute coronary syndromes.^338,339 These observations indicate that the qualitative PAP assessment might be feasible in clinical scenarios. Further improvements in acquisition and post-processing techniques (e.g., iterative reconstruction techniques) in combination with reduction in radiation dose may further enhance the ability of coronary CTA to differentiate between individual plaque components and broaden the applicability of coronary CTA for the evaluation of coronary atherosclerosis.

In our third ex vivo investigation, we have described a robust approach for data acquisition, co-registration, and systematic comparison of non-invasive (coronary CTA) and invasive (IVUS and OFDI) imaging modalities with the standard of reference (histologic examination). In this study we have demonstrated that (a) Various imaging features of plaques are associated with early plaque (normal cross section at coronary CT angiography and fibrous at OFDI) and advanced plaque (mixed at coronary CT angiography, any calcified plaque at intravascular US, and lipid-rich plaque at OFDI); (b) The overall performance of OFDI for differentiating early from advanced plaque is significantly better than that of IVUS and coronary CTA; (c) We found excellent interobserver agreement for OFDI and coronary CTA and good agreement for IVUS.

Overall, the performance of each modality for differentiating early from advanced plaques reflects the differences in the physics behind these imaging methods. Not surprisingly, OFDI, with its excellent spatial resolution and tissue characterization, rendered the strongest associations with histologic examinations as compared with IVUS and coronary CTA. This is reflected by several significant associations between plaque type at OFDI and histopathologic examination. At OFDI, fibrous plaques were associated with early lesions, while lipid-rich plaques were associated with advanced lesions. In addition, OFDI yielded a sensitivity of 100%

and a specificity of 81% for identifying TCFA, confirming the previously described potential of OFDI to help identify or confirm high-risk lesions.^340-342 In contrast, TCFA composition was heterogeneous and non-discriminatory at intravascular US and coronary CTA. Normal IVUS findings precluded the presence of TCFA at histologic examination. However, the overall modest association of IVUS-based plaque composition with early or advanced plaques emphasizes the known limitations of gray-scale IVUS for tissue differentiation using the conventional classification scheme.^343-345 Hence, IVUS has a higher potential in plaque size and volume assessment than for the presence of high-risk plaque.^346,347 However, the newest IVUS methods, using, for example, virtual histologic examination techniques, and more advanced plaque classification may overcome these limitations.^344,348 Our data suggest that conventional IVUS and coronary CT angiography are not significantly different in their ability to help predict

lesion stage at histologic examination. Importantly, both ex vivo studies demonstrated that the absence of plaque in coronary CTA excludes the presence of advanced atherosclerotic lesion or TFCA. This finding is consistent with clinical studies demonstrating the rarity of cardiovascular events in patients without coronary artery disease as described by coronary CTA.90,101,349,350

Although non-invasive imaging is evolving rapidly, IVUS and OFDI still offer the highest accuracy regarding the differentiation of plaque components, and IVUS remains the clinical standard of plaque burden assessment. However, coronary CTA is able to reliably help exclude relevant coronary artery disease and offers a high sensitivity for the detection of significant coronary artery stenosis.³⁵¹ Furthermore, as the presence of mixed plaques and other high-risk plaque features are associated with the presence of advanced coronary atherosclerotic lesions at histologic examination.90,102,259,350

It is important to note that these studies were performed in an ideal ex vivo setting providing motion-free image-based plaque characterization in a limited number of hearts. Thus, our results cannot be directly translated to in vivo circumstances. At histopathologic examination, a modified nomenclature for coronary atheroslcerosis was applied.⁶ We believe that the modified stratification is useful as it differentiates plaques that have a higher risk of causing cardiac events from those with lower risk while accommodating the limited spatial and contrast resolution of intravascular US and of coronary CT angiography. Moreover, our experimental setup resulted in no exclusion of vessel segments because of artifacts and allowed the analysis of 379 histologic cuts from three donor hearts. In contrast, previously published ex vivo studies included a much higher number of cadaver hearts (11-30 hearts), on the other hand because of methodologic limitations and air bubble formation, much fewer cross sections were suitable for analysis (17-312 sections).^352-354 Furthermore, the generally used plaque classification schemes for coronary CTA, IVUS, and OFDI were developed independently;

thus, a direct comparison between modalities is limited.

A sequential imaging strategy using coronary CTA to identify high-risk plaque features such as the NRS, followed by invasive imaging tools to confirm the presence of vulnerable plaques might provide a framework suitable to identify individuals with the highest risk to develop acute coronary syndromes. Further ex vivo and in vivo research is warranted to assess the generalizability of our findings.