Imaging of cardiac fibroblast activation in a patient after acute myocardial infarction using 68Ga-FAPI-04

Our previous study has demonstrated the feasibility of noninvasive imaging of fibroblast activation protein (FAP)-expression after myocardial infarction (MI) in MI-territory in a rat model with 68Ga-FAPI-04-PET. In the current extended clinical case, we sought to delineate cardiac uptake of 68Ga-FAPI-04 in a patient after MI with clinical indication for the evidence of fibroblast activation. Carcinoma patients without cardiac disease underwent 68Ga-FAPI-04-PET/CT as control. The patient with one-vessel disease underwent dynamic 68Ga-FAPI-04-cardiac-PET/CMR for 60 minutes. Correlation of cardiac 68Ga-FAPI-04 uptake with clinical findings, ECG, echocardiography, coronary-arteriography and enhanced cardiac-MRI with T1 MOLLI and ECV mapping were performed. No uptake was found in normal myocardium and in mature scar. A focal intense 68Ga-FAPI-04 uptake with continuous wash-out in the infarct territory of coronary occlusion correlating with T1 and ECV mapping was observed. The uptake of 68Ga-FAPI-04 extends beyond the actual infarcted area and overestimates the infarct size as confirmed by follow-up CMR.


INTRODUCTION
Following myocardial infarction (MI), an orchestrated inflammatory response and reparative pathways are initiated, aiming to produce a robust and collagenrich scar. 1,2 The phenotype conversion of cardiac fibroblasts to their overly active counterparts, myofibroblasts, is the critical event in cardiac remodeling. 3,4 Post-MI, a high abundance of extracellular matrix proteins is synthesized by myofibroblasts to replace myocyte loss and form a reparative scar. A balanced turnover of extracellular matrix via extracellular matrix synthesis by activated fibroblasts and degradation by matrix metalloproteinases is crucial for proper scar formation. 4 The excessively increased activity of the fibroblasts resulting in excessive fibrosis within the myocardium is associated with poor patient prognosis. A diagnostic strategy targeted at detecting active ongoing fibrosis may provide critical insights into pathogenesis of heart failure. In addition, early detection of cardiac remodeling and fibrosis may be essential to prevent development of apparent heart failure.
The serine protease fibroblast activation protein (FAP) is a membrane-anchored enzyme which is specifically expressed in fibroblasts activated to differentiate to (proto-)myofibroblasts, but not in dormant fibroblasts or mature fibrocytes. 5 68 Gallium-labeled FAP-inhibitor (FAPI) compound 04 ( 68 Ga-FAPI-04) was initially introduced for PET imaging of cancerassociated fibroblasts. 6 In our previous work, we demonstrated that image derived FAP expression after MI in a rat model allowed noninvasive PET imaging of activated fibroblasts. 7 Further retrospective evaluations of cardiac FAPI distribution in a heterogeneous patient population with metastasized cancer associated with preexisting coronary artery disease, cardiovascular risk factors or metabolic disease were reported. 8,9 However, 68 Ga-FAPI-04-PET investigations in patients after MI have not been reported yet. For this case presentation, we retrospectively evaluated fibroblast activation in a patient after MI und carcinoma patients without cardiac disease.

CASE PRESENTATION
All examinations had clinical indications and complied to the conditions of the updated Declaration of Helsinki (Section 37, unproven interventions in clinical practice) and the German Pharmaceutical Law (Section 13, 2b). In the controls, the indications for 68 Ga-FAPI-04-PET/CT was the possible compassionate use of 177 Lu-FAPI-radiotherapy 10 and staging. In a patient after MI, the indications for 68 Ga-FAPI-04-PET/MR was the compassionate use for chimeric antigen receptor T-celltherapy 11 of myocardial fibrosis and clarification of inflammation and viability after MI. Informed written consent for the investigation and scientific analyses were achieved in all patients.

Finding in Patients with No History of Cardiac Diseases (Control)
Patient population of control group is summarized in Table 1. Normal myocardium showed activity uptake of 68 Ga-FAPI-04 of similar intensity as blood pool activity indicating no specific uptake ( Figure 1). The averages of maximum and mean standard uptake values (SUV max , SUV mean ) are summarized in Table 2A.

Finding in Patient After Myocardial Infarction
A 33-year-old male was referred to intensive cardiac care unit due to acute STEMI followed by ventricular fibrillation. Return of spontaneous circulation after defibrillation. The present clinical findings at admission are summarized in Table 3. His coronary angiography showed severe sub-occlusive stenosis of medial LAD. Immediately, percutaneous coronary intervention and insertion of one drug eluting stent was performed. The pre-and post-interventional electrocardiograms are summarized in Table 4. Transthoracic echocardiography 5 days after MI showed normal left ventricular diameters, moderate myocardial thickening (interventricular septum 1.3 cm, posterior wall 0.9 cm), normal ventricular wall motion, but moderate left ventricular ejection fraction (LVEF) of 50%. CRP was persistently elevated at 80 mgÁL -1 . The patient complained about persistent dyspnea and fatigue. To investigate the cardiac fibroblast activation, a dynamic PET/MR imaging was performed 6 days after STEMI immediately after intravenous injection of 165 MBq 68 Ga-FAPI-04. 68 Ga-FAPI-04-PET of this patient showed focal intense uptake in the anterior and anterior-septal wall, which correlated well with the sub-occluded LAD territory (Figures 2A-D). Almost no uptake was registered in the remote remaining left ventricular wall with activity similar to blood pool. The assessed SUVs are summarized in Table 2B. A small mature scar in inferior apex (arrowhead in Figure 2D in CMR) showed almost no corresponding uptake. Tracer kinetics revealed after rapid peak accumulation a continuous wash-out of the activity in the anterior wall and in its subendocardial border zone ( Figure 2F).
The corresponding cardiac magnetic resonance (CMR) revealed in cine sequences a myocardial thickening of the anterior septum (14 mm end-diastolic) and thinning of the inferior apex with hypokinesia. The left ventricular function was moderately reduced (46%). In T2 weighted imaging, the anterior wall showed increased signal, suggesting myocardial edema. In dynamic myocardial perfusion imaging moderate hyperemia of the anterior wall could be observed. Early gadolinium enhancement sequences demonstrated a transmural enhancement of the anterior wall and adjacent septal segments, while late gadolinium enhancement revealed a sub-endocardial enhancement anterior-septal, but also a sub-endocardial enhancement in inferior apex. In T1 Modified Look-Locker Inversion Recovery (MOLLI) ( Figure 3A) the anterior wall and adjacent septal segments showed in pre-and postcontrast enhancement pathological T1-relaxation-time and extracellular volume (ECV)-fraction in contrast to remote posterior-basal segments ( Table 5). The corresponding 68 Ga-FAPI-04-PET/CMR image fusion of the infarcted myocardium presented in Figures 2B-D showed a good correlation of the extent of CMR findings and 68 Ga-FAPI-04 intense uptake with the LAD territory. But the extent of intense 68 Ga-FAPI-04 uptake was slightly larger than the extent of the pathological CMR findings.
Control CMR 6 months after MI revealed discrete sub-endocardial late enhancement in the inferior apex and anterior-septal wall, indicating scar tissue which is significantly smaller than the extent in the sub-acute of 68 Ga-FAPI-04 scan. The left ventricular function recovered to almost normal with LVEF 60%. No hypokinetic ventricular wall motion was detected, except a discrete hypokinesia in the apex. Apart from the anterior-septal sub-endocardial scar (arrowhead in Figure 3B), the assessed pre-and post-contrast T1 MOLLI and ECV  ( Figure 3B) showed normalization of the findings (Table 5).

DISCUSSION
In the present retrospective analysis of cardiac 68 Ga-FAPI-04 uptake in patients with no prior history of cardiac disease and in a patient after acute MI, we found substantial different results (Figures 1A, 2A). In patients without history of cardiac disease, the myocardium showed a homogenous tracer uptake similar to the blood pool activity (Figure 1). Whereas, in the patient with acute MI, a focal intense uptake of 68 Ga-FAPI-04 was registered in the infarct territory of the occluded coronary artery, indicating local enhanced fibroblasts activation. The tracer uptake in the remote myocardium  (the myocardium appearing unremarkable in CMR) was similar to normal myocardium of the control group, indicating the absence of active fibroblasts in healthy myocardium 5 (Figure 2; Table 2). A mature myocardial scar in inferior apex detected in CMR showed no uptake of 68 Ga-FAPI-04 (arrowhead in Figure 2D), indicating no further FAP expression in established disease with fixed fibrosis because of significant reduction of activated fibroblast density during infarct maturation. 12 A major determinant of post-MI remodeling severity is the infarct size. In the present study, we observed that the extent of 68 Ga-FAPI-04 uptake in the infarcted area overestimates the actual infarct size. This finding is in line with recent data obtained from different animal  studies showing increased fibroblast activity in adjacent MI (MI border zone). 7,13 This finding indicates that the extent and the intensity of 68 Ga-FAPI-04 uptake do not represent solely myocardial scaring process. But it also indicates more likely overlapping enhanced myocardial fibroblast migration in an inflammatory, proliferative process, such as in viable but ischemic jeopardized border zone or hibernating myocardium. The presence of active fibroblasts in the cardiac interstitium of the hibernating myocardium is reported to be an important  indicator in determining recovery of function after revascularization. 14 As confirmed by the control CMR investigation performed 6 months after MI, it is remarkable that the extent of the resulting scar is definitely smaller than in the first examination performed 6 days after MI. Contrast-enhanced CMR offers high spatial resolution and can identify acute myocardial infarction and myocardial scar. 15 CMR assessment and differentiation of pathological cellular, vascular and interstitial myocardial alterations with T1 and ECV mapping may be useful in the estimation of the degree of fibrosis or volume of myocardial collagen. The concordance of the focal intense myocardial 68 Ga-FAPI-04 uptake with the alteration in T1 and ECV mapping registered in our patient is remarkable (Figures 2, 3). However, the wide range of reported correlations between T1 and ECV estimates and histo-morphological parameters, which vary from poor to excellent, limits the potential of CMR. 16 More importantly, the measured ECV values reflect an indirect evidence of fibrosis. But other pathologies resulting in an expansion of extracellular space, such as inflammatory edema or protein deposition, can lead to increased ECV values. Therefore, CMR techniques seem not to be specific for fibrosis. 16 Furthermore, these measurements tend to reflect a relatively late product of fibroblast activation. Noninvasive imaging of activated fibroblasts with 68 Ga-FAPI-04-PET, however, may have a potential to specifically visualize fibrotic process already at its onset. It could therefore provide unique opportunities to study cardiac remodeling at molecular level over time and to monitor therapeutic interventions that aim to prevent a progressive decline of ventricular function. 17 The simultaneous 68 Ga-FAPI-04-PET/CMR may boost the diagnostic potential of 68 Ga-FAPI-04-PET by additional information achieved from CMR in identification of early manifestation of remodeling amenable to preventive intervention. To confirm our preliminary results and to further investigate the comprehensive pathophysiology, further studies with a larger population are encouraged.

CONCLUSION
As shown in this case of a patient after STEMI, the enhanced FAP activation in acutely injured myocardium was identified und visualized with 68 Ga-FAPI-04-PET. Noninvasive assessment of activated fibroblasts may provide unique opportunities to identify early manifestation of cardiac remodeling amenable to preventive intervention.

Funding
Open Access funding enabled and organized by Projekt DEAL. Alberto Villagran Asiares received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie Grant Agreement No 764458.

Disclosure
No conflict of interest.

Open Access
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