Comprehensive multimodality characterization of hemodynamically significant and non-significant coronary lesions using invasive and noninvasive measures.

BACKGROUND: There is limited knowledge about morphological molecular-imaging-derived parameters to further characterize hemodynamically relevant coronary lesions.
OBJECTIVE: The aim of this study was to describe and differentiate specific parameters between hemodynamically significant and non-significant coronary lesions using various invasive and non-invasive measures.
METHODS: This clinical study analyzed patients with symptoms suggestive of coronary artery disease (CAD) who underwent native T1-weighted CMR and gadofosveset-enhanced CMR as well as invasive coronary angiography. OCT of the culprit vessel to determine the plaque type was performed in a subset of patients. Functional relevance of all lesions was examined using quantitative flow reserve (QFR-angiography). Hemodynamically significant lesions were defined as lesions with a QFR <0.8. Signal intensity (contrast-to-noise ratios; CNRs) on native T1-weighted CMR and gadofosveset-enhanced CMR was defined as a measure for intraplaque hemorrhage and endothelial permeability, respectively.
RESULTS: Overall 29 coronary segments from 14 patients were examined. Segments containing lesions with a QFR <0.8 (n = 9) were associated with significantly higher signal enhancement on Gadofosveset-enhanced CMR as compared to segments containing a lesions without significant stenosis (lesion-QFR>0.8; n = 19) (5.32 (4.47-7.02) vs. 2.42 (1.04-5.11); p = 0.042). No differences in signal enhancement were seen on native T1-weighted CMR (2.2 (0.68-6.75) vs. 2.09 (0.91-6.57), p = 0.412). 66.7% (4 out of 6) of all vulnerable plaque and 33.3% (2 out of 6) of all non-vulnerable plaque (fibroatheroma) as assessed by OCT were hemodynamically significant lesions.
CONCLUSION: The findings of this pilot study suggest that signal enhancement on albumin-binding probe-enhanced CMR but not on T1-weighted CMR is associated with hemodynamically relevant coronary lesions.

Introduction

Atherosclerosis is the major cause of morbidity and mortality in the western world. [1] So far fractional-flow-reserve is considered the gold standard for functional lesion interrogation. It has been shown that clinical outcomes of FFR-guided interventions were superior than those of angiography-guided interventions or conservative medical therapy [2]. Recently, a novel, adenosine-free tool for functional assessment of a coronary lesion—quantitative flow ratio (QFR)—was introduced, which is based on quantitative coronary angiography and computational algorithms. [3]

Several targets for noninvasive imaging have been identified for the detection of vulnerable coronary atherosclerotic plaques. [4,5,6] Non-enhanced and contrast-enhanced cardiovascular magnetic resonance imaging (CMR) provide additional information on plaque morphology and biology. For instance noncontrast—enhanced T1-weighted CMR has shown to be feasible for the identification of intraplaque hemorrhage and thrombus. [7,8] Additionally molecular CMR with the use of target-specific contrast agents highlight certain molecules or cells in order to visualize and characterize a pathological processes on the molecular level, which potentially help to better understand molecular events that contribute to coronary plaque formation.

[4,5,9] The albumin-binding probe (gadofosveset-trisodium) investigated in this study is a clinically approved target-specific molecular MR probe and behaves similarly to Evan’s blue dye, a marker of endothelial permeability. [4,5,6] It reversibly binds to albumin and was originally designed as a blood pool agent for steady-state angiography, before its use for visualization of endothelial permeability and neovascularization was discovered. [4,5,6]) Leaky endothelial junctions may facilitate migration of macromolecules, such as albumin and leucocytes, into the vessel wall, consequently leading to plaque progression. [4]

Hypoxemia within the growing plaque results in an increase in neoangiogenesis and proliferation of new fragile neovessels with increased endothelial permeability. [4] Rupture-prone atherosclerotic plaques are most often characterized by the presence of these intraplaque neo-vessels (i.e. neoangiogenesis). [4,5]

These Information from noninvasive CMR may complement information derived from high-resolution invasive plaque analysis such as optical coherence tomography (OCT) for improved characterization of coronary atherosclerosis

However, there is still limited knowledge about morphological imaging-derived parameters to further characterize hemodynamically–significant and prognostic relevant coronary lesions. [10,11] The purpose of this study was to describe and differentiate hemodynamically significant from non-significant coronary lesions as assessed by QFR-angiography, using different types of noninvasive and invasive tools.

Methods

Study population

Subjects with symptoms suggestive of coronary artery disease such as stable chest pain and acute coronary syndrome (unstable angina or Non-ST-elevation myocardial infarction; NSTEMI) were prospectively recruited between April 2015 and June 2016 and underwent T1-weigthed non-enhanced cardiovascular magnetic resonance imaging (CMR) and 24 and gadofosveset-enhanced CMR within 24 hours. Subsequently invasive coronary angiography and functional lesion interrogation using quantitative flow ration (QFR) was performed in each patient. Hemodynamically unstable patients (patients with cardiogenic shock, rising cardiac enzymes or malignant cardiac arrhythmias, ST-elevation myocardial infarction; STEMI), pregnant women, patients with a history of coronary stenting, patients with renal insufficiency (creatinine clearance <30 ml/min) and patients, who were not able to give their written consent (i.e. <18 years of age, because of mental disorders), have been excluded from the study. Further exclusion criteria were the presence of common contraindication to cardiac magnetic resonance imaging (i.e. allergy to gadolinium-based contrast agents, claustrophobia, specific metallic–items such as cochlear implants, central nervous system aneurysm clips, pacemakers/ defibrillators). Written informed consent was obtained from each subject and the study was approved by the local ethics committee (Ethikkommission, Charité - Universitätsmedizin Berlin) for clinical investigations and performed in accordance with the Declaration of Helsinki.

Cardiac magnetic resonance imaging

A 3-Tesla MRI scanner (Magnetom Skyra, Siemens Healthcare, Erlangen, Germany) using an 18-channel matrix coil was used in this study. Continuous monitoring of vital signs throughout the entire cardiovascular magnetic resonance imaging (CMR) scan was performed with a 4-lead ECG. Patients with elevated cardiac enzymes, were monitored using a CMR-compatible blood pressure monitor and blood oxygenation sensor. In all patients, CMR imaging was performed twice; natively and 24 hours following the administration of gadofosveset trisodium. Gadofosveset-trisodium (0.03 mmol/kg body weight) was administered intravenously through a catheter in an antecubital vein following the first imaging session. In this study, we used a post-contrast imaging time of around 24 hours to reduce the signal from gadofosveset in the coronary lumen and to get optimal wall to lumen contrast as previously demonstrated by Lobbes et al. [6]

After the acquisition of scout scans to identify the major structures of the heart, a cine 4-chamber-view was used to determine trigger delay and acquisition window, followed by a TI scout to determine the patient-specific inversion time to null signal from blood. For whole heart MR coronary angiography a FLASH (fast low angle shot) sequence (T2-preparation) was used including the following imaging parameters: field of view 340 x 340 mm; acquisition matrix 256 x 256; reconstruction matrix 512 x 512; acquisition slice thickness 1.3 mm; acquisition slice number 80–100; (physical) spatial inplane resolution 1.3mm x1.3mm, interpolated inplane spatial resolution 0.65 x 0.65 mm; repetition time/echo time 3.5 ms/1.42 ms; flip angle 20 degrees. An inversion recovery (IR) prepared 3-dimensional (3D) T1W turbo FLASH (fast low angle shot) sequence with fat suppression (FatSat) was used for whole heart coronary vessel wall imaging without an interslice gap. Electrocardiogram–triggering and a navigator-gated free breathing technique in coronal orientation was part of each acquisition. To null blood using a region of interest to determine the most accurate value, we adjusted the patient-specific inversion time (range 270 to 300 ms) using the TI scout sequence. The following acquisition parameters were included: inversion time 250±15 ms; field of view 340 x 340 mm; acquisition matrix 256 x 256; reconstruction matrix 512 x 512; acquisition slice thickness 1.3 mm; acquisition slice number 80–100; reconstruction spatial resolution 0.65 x 0.65 x 0.65 mm; repetition time/echo time 4.1 ms/1.3 ms; flip angle 15 degrees. The navigator gating window width was 1.5–2.5 mm. The respiratory navigator gating window width to compensate for breathing motion was set to an acceptance window between 1.5–2.5 mm Depending on the patient’s heart rate, the data acquisition window duration time varied from 84 to 120 ms depending. The trigger delay and acquisition window were adjusted according to the phase with minimal motion of the right coronary artery (RCA) as determined by cine MR imaging.

Invasive catheterization and assessment of the functional significance of a stenosis using quantitative flow ratio (QFR)

Invasive Catherization (IC) was performed according to standard techniques via a transradial or transfemoral approach. All lesions with a stenosis severity greater than 50 percent as estimated by the interventional cardiologist, were used for quantitative flow ratio (QFR) determination. To assess the functional significance of a stenosis, computation of QFR was performed offline using a special software package (QAngio XA 3D prototype, Medis Medical Imaging System, Leiden, the Netherlands). This novel computational approach to derive fractional flow reserve from diagnostic coronary angiography was described in detail previously [3]: Two angiographic projections—at least 25 degrees apart from each other–were chosen and a 3D reconstruction of the interrogated vessel including 3D QCA data was performed. On basis of the following principles, the QFR software computed three QFR pullbacks: 1) the pressure of the coronary arteries stays constant through healthy coronary arteries. 2) the extent of pressure drop is influenced by the stenosis geometry and the flow through the site of luminal narrowing. 3) the stenosis geometry can be described by the relation of the diseased vessel site to the reference vessel site (i.e. healthy vessel wall). 4) Blood flow velocity is preserved distally to the stenosis in relation to the blood flow velocity proximally to the site of stenosis.5) The mass flow rate drops as the diameter of the coronary artery gets smaller distally due to the presence of side branches. QFR measurements were performed by two experienced cardiologists with more than five years of experience in angiographic software programs such as QCA (LCE and YSA).

OCT image analysis

Optical coherence tomography (OCT) was performed in the culprit vessel of patients where possible (glomerular filtration rate >30 ml/min). OCT images were assessed by two experienced readers (M.J. and L.C.E.), who were at the time of OCT analysis blinded to the cardiovascular magnetic resonace images (CMR), using proprietary software (St. Jude medical). Consensus reading was done by a third investigator (B.B.) in case of disagreement between the two OCT readers. Plaques were evaluated using validated OCT criteria. [12]

CMR image analysis

We used a dedicated image analysis software for cardiovascular magnetic resonance imaging (CMR) image analysis (Osirix 3.6.1, Geneva, Switzerland). Signal intensity of coronary segments was determined in Gadofosveset-enhanced CMR and the T1-weigthed non-enhanced CMR scan according to a 9-segment model as previously. [5,6] Contrast-to-noise ratio (CNR) was defined as the difference in signal enhancement between the coronary segment and blood divided by the background noise (SI lesion–SI blood /noise). The standard deviation of the signal enhancement in a region of interest ventrally to the patient´s chest was used to obtain the background noise.

Statistics

We used the SPSS software (IBM SPSS Statistics Version 24) for statistical analysis. An unpaired Student t-test and Mann-Whitney U-Test was applied for comparison of continuous and non-normally distributed variables respectively. Nominal variables were compared among the three groups using chi-square and Fisher exact tests, when appropriate. Continuous variables were reported as mean standard deviation or median with interquartile range (25th and 75th percentiles). Nominal variables were reported as percentage or frequencies, as appropriate. A 2-tailed p-value less than 0.05 was reported as statistically significant.

Results

Out of 26 patients who underwent T1-weighted non-contrast enhanced cardiovascular magnetic resonance imaging (CMR), Gadofosveset-enhanced CMR and invasive coronary angiography between April 2015 and June 2016, we identified 17 patients who had at least one lesion of 50% stenosis on invasive coronary angiography. These lesions (n = 49 segments) were taken for further functional assessment using quantitative flow ratio (QFR). Coronary segments, which were not correctly visible on invasive coronary angiography due to overlap of other vessels of foreshortening (i.e. wrong projections) and coronary segment which did not fulfill the requirement for QFR determination (i.e. two angiographic projections at least 25 degrees apart from each other), were excluded. As a result, overall n = 14 patients and 28 coronary segments were included in the final analysis. Mean age of all patients was 74.1±10.7. The majority of patient was male (64.3%). Other baseline characteristics such as cardiovascular risk factors, lab values, and medication are shown in Table 1.

Table 1

Baseline patients´ characteristics and medical treatment upon admission.

	All patients(n = 14)
Age, y	74.1±10.7
Male, n (%)	9 (64.3)
Risk factors
Hypercholesterolemia, n (%)	6 (42.9)
Hypertension, n (%)	12 (85.7)
Diabetes mellitus, n (%)	5 (35.7)
Smoking, n (%)	5 (35.7)
Family history of CAD, n (%)	3 (21.4)
Laboratory findings
Troponin T, ng/ml	153.1±376.6
CK, UI/l	167.7±122.0
CK-MB, UI/l	38.3±40.6
Creatinine, mg/dl	1.1±0.35
C-reactive protein, mg/dl	25.5±30.4
Platelets, x 109	253.7±59.2
Total cholesterol, mg/dl	166.5±43.4
Triglyceride, mg/dl	180.0±133.1
HDL cholesterol, mg/dl	44.1±15.5
LDL cholesterol, mg/dl	94.6±35.0
Hemoglobin A1c, %	6.5±1.2
Medication
Aspirin	10 (71.4)
Statin	5 (35.7)
Beta-blocker	7 (50.0)
ACEI and/or ARB	10 (71.4)

Angiographic and quantitative flow ratio (QFR) data

The total amount of patients in this study having 1-vessel, 2-vessel and 3-vessel coronary artery disease were n = 4, n = 8 and n = 1, respectively. Overall n = 4 patients had one lesion with a vessel-QFR ≤0.8, n = 2 patients had both a lesion with a vessel-QFR≤0.8 and a lesion with a vessel-QFR>0.8 and n = 8 patients had only one lesion with a vessel-QFR>0.8. There were 9 out of 28 stenotic segments which were associated with a vessel-QFR ≤0.8 while 19 out of 28 stenotic segments were associated with a vessel-QFR >0.8. From all hemodynamically significant lesions, n = 4 were located the RCA, n = 5 were located in the LAD and n = 0 were located in the LCX. Hemodynamically non-significant lesions were present in all three coronary vessels (LAD, n = 8; LCX, n = 5; RCA, n = 7). Quantitative flow ratio (QFR) data can be seen in Table 2. Mean length of lesions with a QFR ≤0.8 was 21.7±9.2 mm while mean length of lesions with a QFR >0.8 was 14.1±6.4 mm. Percent area stenosis 71.2±11.5% for hemodynamic significant lesions (QFR<0.8) and 52.3±17.9% for hemodynamically non-significant lesions (p = 0.0017).

Table 2

Quantitative flow ratio (QFR) data.

	Lesion-QFR> 0.8(n = 19)	Lesion-QFR ≤0.8(n = 9)	P-Value
Lesion length	14,1±6,4	21,7±9,2	0.010
Area stenosis*	52,3±17.9	71,2±11,5	0.004
Bending angle	22.8±15.1	34.1±22.8	0.090

OCT analysis

Overall 12 segments were analyzed using optical coherence tomography (OCT) of which 50% (n = 6 segments) were functionally relevant (QFR≤0,8) lesions. 66,7% (4 out of 6) of all vulnerable plaque (i.e. thin-cap fibroatheroma) and 33.3% (2 out of 6) of all non-vulnerable plaque (fibroatheroma) as assessed by OCT were hemodynamically significant lesions. In 66,7% (4/6) of all hemodynamically significant lesions, coronary artery calcification (CAC) was present, whereas in 33.3% (2/6) of all hemodynamically non-significant lesions CAC was found.

CMR analysis

Segments containing lesions with a QFR ≤0.8 (n = 9) were associated with significantly higher signal enhancement on Gadofosveset-enhanced CMR as compared to segments containing a lesion without significant stenosis (lesion-QFR>0.8; n = 19) (5.32 (4.47–7.02) vs. 2.42 (1.04–5.11); p = 0.042). No differences in signal enhancement were seen on native T1-weighted CMR (2.2 (0,68–6.75) vs. 2.09 (0.91–6.57), p = 0.412) (Fig 1). Signal enhancement of segments containing atherosclerotic plaques was 4.0 (2.3–6.4) on gadofosveset-enhanced CMR and 0.7 (-0.9–4.2) on T1-weighted CMR.

Fig 1

Comparison of contrast-to-noise ratios (CNR) between functionally relevant and non-relevant lesions in A) gadofosveset-enhanced CMR (i.e. albumin-binding probe enhanced) CMR and B) non-contrast-enhanced T1-weighted CMR.

Discussion

Quantitative flow ratio (QFR) was recently introduced as an angiography-based method for deriving fractional flow reserve (FFR) without the use of a pressure wire and demonstrated high diagnostic accuracy in identifying hemodynamically significant coronary stenosis. [3] The main finding of this study is that hemodynamically-significant lesions as assessed by QFR-angiography, were associated with significantly higher signal enhancement on Gadofosveset-enhanced cardiovascular magnetic resonance imaging (CMR) as compared to non-hemodynamically significant lesions (Figs 2 and 3), whereas on native T1-weighted CMR, no differences between the groups were observed. On a pathophysiological level, these findings suggest that functionally relevant lesions have a higher grade of endothelial permeability and / or a higher density of intraplaque neovessels taken into account the findings of prior gadofosveset-enhanced CMR studies, while the presence of intraplaque hemorrhage may not be an appropriate predictor for functionally relevant lesions.

Fig 2

Sample case of a 64-year-old woman presenting with typical chest pain.

A) Cardiovascular magnetic resonance imaging demonstrated signal enhancement in the mid to distal right coronary artery (RCA). B) Invasive catheterization revealed significant stenosis at the site of CMR signal enhancement. C) Further analysis using quantitative flow ratio (QFR) graded the stenosis as functionally relevant with a vessel QFR and an index QFR of 0.71 and 0.75 respectively. D) Optical coherence tomography, which was performed during invasive catheterization, revealed a fibroatheroma at the site of maximal luminal narrowing.

Fig 3

Representative images of comprehensive multimodality assessment of coronary lesions with a quantitative flow ratio <0.8 (first row), >0.8 (second row), and non-or minimal diseased vessel wall (third row) using non-enhanced T1-weigthed cardiovascular magnetic resonance imaging (A, A´, A`), albumin-binding probe–enhanced CMR (B, B´, B´´), invasive catheterization (C, C´, C”) and quantitative flow ratio assessment (D, D´, D”). While T1-weigthed CMR showed no clear difference in signal enhancement between the different lesion categories, gadofosveset-enhanced CMR demonstrated strongest signal enhancment at the site of the hemodynamic-relevant stenosis as compared to the lesion with a QFR below 0,8 and the minimal diseased vessel area; (Ao, aorta; LAD, left anterior descending; RCA, right coronary artery; QFR, quantitative flow ratio).

However, it remains to be clarified in a larger study how the albumin leakage sign on molecular CMR can add information to QFR or FFR in cases of borderline obstructive lesions, when decision whether to perform percutaneous coronary intervention (PCI) in invasive coronary angiography or not is unclear.

So far—out of all noninvasive imaging modalities—mainly coronary computed tomography angiography (CCTA) with its relatively high spatial resolution has been able to reliably give quantitative information on plaque characteristics. This allows us to get a clearer understanding of the complex relationship between luminal narrowing and plaque characteristics which defines whether a lesion is functionally–relevant or not. [13] To the best of our knowledge, CMR has not been used to investigate the relationship between the hemodynamic significance of a lesion and its morphologic characteristics but certain CMR sequences have demonstrated to successfully identify high-risk coronary atherosclerotic plaques. [5,14] High-intensity plaques as seen on non-enhanced t1-weighted sequences, for instance, have been considered to be vulnerable lesions. [14] As carotid MRI studies suggested, high-intensity lesions of T1-weighted sequences indicate the presence of methemoglobin in intraplaque hemorrhage which leads to an extensive shortening in T1-relaxation time. [15] Similarily lipid-rich necrotic plaques are associated with strong signal enhancemenet on T1-weighted images, since intraplaque hemorrhage often happens within lipid-rich necrotic cores. [14,15] In a different approach, MRI in combination with an albumin-binding probe (Gadofosveset) has the potential to identify advanced atherosclerotic plaque based to the presence of endothelial permeability. [4]

Up to now it was believed that the detection of high-risk plaque however—based on either the presence of intraplaque hemorrhage or the presence of endothelial permeability–may not necessarily answer the question whether this lesion was hemodynamically significant, since these plaque were considered as lesions which are not associated with stenosis. In a recent multivariate analysis, however. Driessen et al. were able to demonstrate that certain vulnerable plaque features on CT such as the presence of non-calcified plaque, low-attenuation plaque, positive remodelling, and spotty calcifications, were the only independent predictors of FFR. [16]

In line with this, we actually found in a subset of patients that the majority of vulnerable plaques (i.e. thin-cap fibroatheroma) as assessed using the means of optical coherence tomography (OCT), were hemodynamically-significant lesions with a degree of stenosis of at least 50%.

Our observation that functionally-relevant lesions are associated with high signal enhancement after application of an albumin-binding probe suggests the presence of increased endothelial permeability and endothelial dysfunction at these coronary sites. In fact, this has been shown before in study using IVUS with radiofrequency spectral analysis, where low density plaques with necrotic cores were associated with local endothelial dysfunction. [17,18] Taken together, we hypothesize that local endothelial dysfunction do not allow for sufficient vasodilation during physiological or pharmacological stress, consequently resulting in ischemia, as opposed to coronary lesions without necrotic cores that maintain a vasodilatory capacity.

Our subanalysis using optical coherence tomography (OCT) revealed that coronary artery calcification (CAC) was more common in segments containing lesions with a QFR <0.80. The reason for this observation may be that plaques without CAC were simply smaller and therefore less stenotic than plaques associated with CAC which can be regarded as a marker for plaque size. However the assumption that CAC is associated with a lower FFR/QFR is still controversial. [10,11] For instance in a recent CCTA study, Baraskan et al. concluded that CAC was not an independent predictor of hemodynamically significant stenosis, even though it closely correlated with total and non-calcified plaque volume. [10]

This study has some limitations. The current study consisted only of a relatively small number of patients. Functional lesion interrogation was only performed using QFR-angiography and not using the gold standard adenosin-induced FFR.

Finally, GE-CMR is still associated with a relatively long scan time for the assessment of the coronary enhancement in vivo, which currently limits the applicability of this technique in a wide clinical setting. Using more advanced motion correction techniques in combination with undersampled image reconstruction (e.g. compressed sensing), this limitation may be solved in the future. [19]

Conclusion

The findings of this pilot study suggest that signal enhancement on albumin-binding probe-enhanced CMR but not on T1-weighted CMR is associated with hemodynamically relevant coronary lesions. Larger studies are needed to validate our findings.

11 Sep 2019

PONE-D-19-21636

Comprehensive Multimodality Characterization of Hemodynamically Significant and Non-Significant Coronary Lesion using Invasive and Noninvasive Measures

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Reviewer #1: Authors presents results on the important topic of the analysis of atherosclerotic malformation in coronaries. The methodology based on validation of the result of contrast enhanced MRI using the albumin-bind extra-vascular contrast media by the measurements of QFR is technically sound. Yet the approach used by the authors in terms of MRI techniques does not look fully adequate to the problem.

The physical spatial resolution of the GRE-based sequence used by authors is above is 1.3 mm /pixel. Additionally , authors use a non-selective single inversion recovery (for 3D sequence)with fat suppression and breath-free navigation for the full-heart vessel -wall imaging. This approach is know for introducing significant quantitative bias in results based on signal intensity analysis due to incomplete suppression of fat signal and related problems of identification vessel wall and especially adequate segmenting of the wall malformation. For this reason, number of researches introduced double and even quadruple IR methods for separation of signal from blood, tissue , fat and contrast media in order to make quantitative analysis more reliable. In this work when relatively simple approach is used it would be important to demonstrate clearly the adequate quality of CMR images for two considered cases ( QFR>0.8 and QFR<0.8) especially the quality of blood and fat suppression and to show the example of identified lesion segments images along with non-lesion vessel wall. This would support the capability of methodology to provide conclusive and diagnostically relevant results. Currently paper contains only single CMR image with relatively low spatial resolution and quality of blood suppression. The methodology of data processing on this point should be uncovered in more details. The same is for the quality of the respiratory gating which is known to be drastic problem for the CMR and demands at least demonstration of reliability and comparison with breath-hold technique to be able to estimate the severity of introduced artifacts.

Paper has relative broad Discussion in which however is difficult to identify own result of author from the results from literature. On the other hand Introduction to very concise and no adequate overview of the existing works on coronary vessel wall MRI and existing problems of analysis and quantification is given whereas this topic is covered by numerous publication in the specialized MRI journals ( Magnetic Resonance in Medicine in particular). Most part of the discussion would be reasonable to transfer to the Introduction and update the later one with more references to the key works in vessel wall MR-imaging and especially its technical challenges.

The Conclusion is short, but fussy and does not really match to the formulated objectives of the paper. With limited amount of scanned patients and being a pilot study the focus should be given to the workflow and technical details of the approach to prove the feasibility of methods and evaluate reasoning for the further patient recruitment or necessity to work on technical side.

Some specific remarks

Line 63 – 65 abbriviation CMR, OCT, CAD are not defined.

Line 81 - What does it mean “more advanced” . “Conclusion” does not really match to the goal formulated in “Objective”. Which experimentally measured parameters _parameters_ and values allows or does not allow do make conclusions regarding significance of lesions ?

Line 104. Why the role of CMR measurements is not mentioned in Introduction ? Why it is mentioned in Abstract but no details on the capabilities of MRI for atherosclerosis detection are given here and very little overview of the existing methods of atherosclerotic plaques detection and analysis ( especially contrast enhanced vessel wall imaging). The same is about role MR-angiography and MR-based flow quantification – no or very poor literature overview for the Introduction for paper where CMR supposed to provide the key results.

Line 166 „T2-prepared“ ?

Line 170 : 340mm FOV at matrix 256x256 provides resolution (physical )1.3mm. 0.65mm is interpolated resolution.

169: What was the interslice gap ? Was it 2D ( slice selective RF-pulses) or 3D-encoded sequence ?

170: 3.5ms TR time is quite short and hardware demanding. Was it a standard vendor -provided protocol ( e.g TWIST or VIBE ) or any inhouse-developed sequence ?

182: What means “navigator gating window” in “mm”. Usually “window” for the navigator is time available for measurements the same as cardiac trigger window. Proper adjustment of navigator position is essential part of such measurements – some data demonstrating the navigation quality ( as shown by scanner) and dependence of image quality on the position of navigator “pencil” would be very helpful for the illustration of method robustness and role of motion artifacts in the results.

Line 211: The scheme illustrating the segmenting of coronary is key point of data processing. It would be helpful to demonstrate 1 or 2 examples with significant and non-significant lesion for understanding of the methodology and it reliability.

278: The statistics of quantitative results does not look very reliable for CMR. The standard deviation of enhancement is close or even exceeds ( for native T1) the mean value. What is explanation of such a major variation. How reliable is this data and are they reproducible within single patient ?

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25 Dec 2019

Dear Prof. Bauer, M.D., Ph.D.,

Here we are submitting the revision of the above mentioned manuscript to “PLOS ONE”.

We have addressed all remaining concerns and suggestions that you and the reviewers have provided and hope that these changes would increase the priority for publication in PLOS ONE.

As mentioned in the initial submission, the current study consists of 11 patients, who were also part of our prior study "Novel Approach for In Vivo Detection of Vulnerable Coronary Plaques Using Molecular 3-T CMR Imaging With an Albumin-Binding Probe" where - unlike the current study - CMR results were solely correlated to optical coherence tomography and which was published in JACC cardiovasc imaging in Feb 2019. On the contrast, this current study correlates findings of both Gadofosveset-enhanced CMR and non-enhanced T1-weighted CMR to a novel angiographic-based method to assess the hemodynamic relevance of a coronary lesion (i.e. quantitative flow ratio; QFR). Therefore we believe that this study provide a does not constitute dual publication.

Again, we want to express our appreciation for the time and effort you and each of the reviewers have dedicated to provide insightful feedback on ways to strengthen our manuscript.

To facilitate your review of our revisions, the following is a point-by-point response to the reviewers’ questions and comments delivered in your letter dated on September 29 th, 2019.

We look forward to hearing from you regarding our revision. We would be glad to respond to any further questions and comments that you may have.

Signed respectfully on behalf of all authors,

Leif-Christopher Engel, MD &

Marcus Makowski, MD &

Boris Bigalke, MD, MBA

Reviewer: 1

Comment1 / reviewer#1

The authors have done a great effort and the manuscript has improved notably but unfortunately there are still issues that need to be corrected. To help finishing this task in the hope that the authors provide a high quality report that the journal may consider I provide a detailed recipe of corrections:

Response to comment 1 / reviewer #1

We are grateful for the effort and time the reviewer spent on improving our manuscript. In the following, we have addressed all of the remaining concerns.

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

comment 2 / reviewer #1

Reviewer #1: Authors presents results on the important topic of the analysis of atherosclerotic malformation in coronaries. The methodology based on validation of the result of contrast enhanced MRI using the albumin-bind extra-vascular contrast media by the measurements of QFR is technically sound.

• Response to comment 1 / reviewer #1

We are grateful for the effort and time the reviewer spent on improving our manuscript and appreciate this evaluation. In the following, we have addressed all of the remaining concerns.

comment 3 / reviewer #1

Yet the approach used by the authors in terms of MRI techniques does not look fully adequate to the problem. The physical spatial resolution of the GRE-based sequence used by authors is above is 1.3 mm /pixel. Additionally, authors use a non-selective single inversion recovery (for 3D sequence) with fat suppression and breath-free navigation for the full-heart vessel - wall imaging. This approach is know for introducing significant quantitative bias in results based on signal intensity analysis due to incomplete suppression of fat signal and related problems of identification vessel wall and especially adequate segmenting of the wall malformation. For this reason, number of researches introduced double and even quadruple IR methods for separation of signal from blood, tissue, fat and contrast media in order to make quantitative analysis more reliable.

• Response to comment 3 / reviewer #1

We agree with the reviewer, that double and even quadruple IR methods are more advance methods for this type of acquisition. In the patient collective we investigated the non-selective single inversion recovery (for 3D sequence) with fat suppression and breath-free navigation for the full-heart vessel - wall imaging however was a reliable approach and we did not observe significant issues with fat suppression in the mediastinum in this study. We however fully agree that for future studies a double and quadruple IR could improve the image quality further.

comment 4 / reviewer #1

In this work when relatively simple approach is used it would be important to demonstrate clearly the adequate quality of CMR images for two considered cases ( QFR>0.8 and QFR<0.8) especially the quality of blood and fat suppression and to show the example of identified lesion segments images along with non-lesion vessel wall. This would support the capability of methodology to provide conclusive and diagnostically relevant results.

• Response to comment 4 / reviewer #1

We have included sample pictures for multimodality assessement for overall three different lesions types (QFR>0.8, QFR<0.8 and minimal diseased vessel wall), emphasizing the potential of gadofosveset-enhanced CMR to identify hemodynamic-relevant coronary lesions.

comment 5 / reviewer #1

Currently paper contains only single CMR image with relatively low spatial resolution and quality of blood suppression. The methodology of data processing on this point should be uncovered in more details. The same is for the quality of the respiratory gating which is known to be drastic problem for the CMR and demands at least demonstration of reliability and comparison with breath-hold technique to be able to estimate the severity of introduced artifacts.

• Response to comment 5 / reviewer #1

We thank the reviewer for this suggestion. As written before, we have included more samples of multimodality assessment of lesions of different hemodynamic relevance. We acknowledge the limitations of the current CMR protocol. However, a comparable CMR protocol was successfully used in several prior CMR studies

comment 6 / reviewer #1

• Paper has relative broad Discussion in which however is difficult to identify own result of author from the results from literature. On the other hand Introduction to very concise and no adequate overview of the existing works on coronary vessel wall MRI and existing problems of analysis and quantification is given whereas this topic is covered by numerous publication in the specialized MRI journals (Magnetic Resonance in Medicine in particular). Most part of the discussion would be reasonable to transfer to the Introduction and update the later one with more references to the key works in vessel wall MR-imaging and especially its technical challenges.

• Response to comment 6 / reviewer #1

We appreciate the comment of the reviewer and have accordingly shortened the discussion. The introduction was extended with information on CMR plaque evaluation.

comment 7 / reviewer #1

The Conclusion is short, but fussy and does not really match to the formulated objectives of the paper. With limited amount of scanned patients and being a pilot study the focus should be given to the workflow and technical details of the approach to prove the feasibility of methods and evaluate reasoning for the further patient recruitment or necessity to work on technical side.

• Response to comment 7 / reviewer #1

We have changed the conclusion section accordingly, better matching now to the objectives of the current study.

comment 8 / reviewer #1

Line 63 – 65 abbriviation CMR, OCT, CAD are not defined.

• Response to comment 8 / reviewer #1

The above mentioned abbreviation are now explained (i.e. CMR, cardiovascular magnetic resonance imaging; OCT, optical coherence tomography; CAD, coronary artery disease)

comment 9 / reviewer #1

Line 81 - What does it mean “more advanced”

• Response to comment 9 / reviewer #1

More advanced in this context means that plaque associated the a QFR<0,8 may be associated with a higher cardiovascular risk since OCT revealed a higher proportion of vulnerable plaques in these lesion

comment 10 / reviewer #1

“Conclusion” does not really match to the goal formulated in “Objective”.

• Response to comment 10 / reviewer #1

We have changed the conclusion part so that it matches the objectives section

comment 11 / reviewer #1

Which experimentally measured parameters _parameters_ and values allows or does not allow do make conclusions regarding significance of lesions ?

• Response to comment 11 / reviewer #1

Obviously lesion length, area stenosis and bending angle were parameter that significantly different between hemodynamically significant and non-significant lesions. Other – mainly qualitative parameters such as the presence of coronary artery calcification within the lesion – did not differ between lesions with a QFR greater or smaller than 0,8.

comment 12 / reviewer #1

Line 104. Why the role of CMR measurements is not mentioned in Introduction? Why it is mentioned in Abstract but no details on the capabilities of MRI for atherosclerosis detection are given here and very little overview of the existing methods of atherosclerotic plaques detection and analysis ( especially contrast enhanced vessel wall imaging). The same is about role MR-angiography and MR-based flow quantification – no or very poor literature overview for the Introduction for paper where CMR supposed to provide the key results.

• Response to comment 12 / reviewer #1

Thank you for this comment. We have now edited and extended the introduction to include more background information on CMR and its role on plaque evaluation and vessel wall imaging.

comment 13 / reviewer #1

Line 166 „T2-prepared“ ?

• Response to comment 13 / reviewer #1

We apologize for this typo. „T2-prepared“ was changed to „T2- preparation “

comment 14 / reviewer #1

Line 170 : 340mm FOV at matrix 256x256 provides resolution (physical) 1.3mm. 0.65mm is interpolated resolution.

• Response to comment 14 / reviewer #1

We apologize for this inaccuracy and are now clearly stating the resolutions achieved:

“…following imaging parameters: field of view 340 x 340 mm; acquisition matrix 256 x 256; reconstruction matrix 512 x 512; acquisition slice thickness 1.3 mm; acquisition slice number 80 - 100; (physical) spatial inplane resolution 1.3mm x1.3mm, interpolated inplane spatial resolution 0.65 x 0.65 mm.

comment 15 / reviewer #1

169: What was the interslice gap ? Was it 2D ( slice selective RF-pulses) or 3D-encoded sequence ?

• Response to comment X / reviewer #1

It was a 3D-encoded sequence sequence without an interslice gap. We are now clarifying this in the manuscript.

comment 16 / reviewer #1

170: 3.5ms TR time is quite short and hardware demanding. Was it a standard vendor -provided protocol ( e.g TWIST or VIBE ) or any inhouse-developed sequence ?

• Response to comment 16 / reviewer #1

The sequence was based on a standard vendor-provided protocol.

comment 17 / reviewer #1

182: What means “navigator gating window” in “mm”. Usually “window” for the navigator is time available for measurements the same as cardiac trigger window. Proper adjustment of navigator position is essential part of such measurements – some data demonstrating the navigation quality ( as shown by scanner) and dependence of image quality on the position of navigator “pencil” would be very helpful for the illustration of method robustness and role of motion artifacts in the results.

• Response to comment 17 / reviewer #1

We apologize for being not clearer regarding the naviator. We are now claryfing this in the manuscript: The respiratory navigator gating window width to compensate for breathing motion was set to an acceptance window between 1.5-2.5 mm.

comment 18 / reviewer #1

Line 211: The scheme illustrating the segmenting of coronary is key point of data processing. It would be helpful to demonstrate 1 or 2 examples with significant and non-significant lesion for understanding of the methodology and it reliability.

• Response to comment 18 / reviewer #1

We thank the reviewer fort his suggestion and have included a corresponding figure.

comment 19 / reviewer #1

278: The statistics of quantitative results does not look very reliable for CMR. The standard deviation of enhancement is close or even exceeds ( for native T1) the mean value. What is explanation of such a major variation. How reliable is this data and are they reproducible within single patient ?

• Response to comment 19 / reviewer #1

We thank the reviewer fort his suggestion. The data is not normally distributed. Therefore we have used a „mann whitney-U-Test“ comparing median values.

13 Jan 2020

Comprehensive Multimodality Characterization of Hemodynamically Significant and Non-Significant Coronary Lesions using Invasive and Noninvasive Measures

PONE-D-19-21636R1

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Academic Editor

PLOS ONE

24 Jan 2020

PONE-D-19-21636R1

Comprehensive Multimodality Characterization of Hemodynamically Significant and Non-Significant Coronary Lesions using Invasive and Noninvasive Measures

Dear Dr. Engel:

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