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Introduction

Type 2 diabetes is hallmarked by systemic inflammation [1], which is a pivotal process driving atherogenesis [2]. Specifically, diabetes is associated with activation of the cardio–haematopoietic axis [3], wherein inflammatory monocytes produced by the haematopoietic organs migrate to atherosclerotic plaques, accelerating atherosclerotic disease. With new anti-inflammatory therapeutics, such as ziltivekimab [4], entering the clinical stage of testing, accurate surrogate markers of vascular inflammation that reflect activation of the cardio–haematopoietic axis are needed to prevent large scale exposure to ineffective immunosuppressive drugs. A highly promising tool to meet this end is Gallium-68-labelled [1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid]-d-Phe1, Tyr3-octreotate (68Ga-DOTATATE), a positron emission tomography (PET) tracer with high affinity for the somatostatin type 2 receptor (SSTR2) that is highly expressed in activated (M1) macrophages within atherosclerotic plaques [5]. A crucial issue for surrogate imaging markers is the amenability of the signal towards therapeutic interventions. In the present study, we sought to investigate whether statin treatment, an established intervention to reduce cardiovascular events and with anti-inflammatory activity [6] is able to reduce 68Ga-DOTATATE uptake in the coronary arteries and aorta, and in the bone marrow and spleen as the key haematopoietic organs.

Methods

Detailed methods are included in the electronic supplementary material (ESM).

In short, individuals with type 2 diabetes from the Amsterdam UMC were eligible, if they were >50 years old, statin-naive for at least 6 weeks, had HbA1c levels <65 mmol/mol (8.1%) and no changes in glucose-lowering medication within 3 months of inclusion. All patients provided written informed consent. Atorvastatin 40 mg once daily was initiated after the first 68Ga-DOTATATE PET/computed tomography (CT) scan, for a period of 3 months. After statin therapy was completed, the patients were subjected to a follow-up 68Ga-DOTATATE PET/CT scan. Blood was collected at baseline and follow-up visits, to determine lipid, metabolic and inflammatory variables. To quantify uptake of 68Ga-DOTATATE in coronary arteries, we used the maximum target-to-background ratio (TBRmax). We reported both the per vessel TBRmax, as well as the overall coronary tree TBRmax. The maximum standardised uptake value (SUVmax) in bone marrow and spleen was assessed by drawing volumes of interest (VOIs) around each respective structure. Methods regarding the measurement of uptake in lung and muscle tissue can be found in the ESM Methods. The study protocol was approved by the local medical ethics committee and performed in accordance with the Declaration of Helsinki

Results

Patient characteristics

Of the 24 patients included, one patient withdrew from the study prior to first scan and another patient discontinued study participation owing to myalgia and did not complete follow-up PET/CT. Accordingly, 22 patients (mean age 63±6 years, 82% male, HbA1c 55 mmol/mol [7.2%]) were included in subsequent analyses. A flowchart of the inclusions can be found in ESM Fig. 1. The baseline and follow-up characteristics after 12 weeks of statin therapy are listed in Table 1. Of note, LDL-cholesterol levels decreased by 58% and C-reactive protein (CRP) by 20%, while the HbA1c did not decrease at follow-up.

Table 1 Baseline characteristics and changes in laboratory variables and imaging parameters after 12 weeks of atorvastatin treatment
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Changes in 68Ga-DOTATATE uptake in the cardio–haematopoietic axis after atorvastatin treatment

The imaging parameters at baseline and follow-up are shown in Table 1. Overall, we observed a consistent and significant decrease of 68Ga-DOTATATE TBRmax in the coronary arteries (−31%) and ascending aorta (−25%), and SUVmax in bone marrow (−15%) and spleen (−17%) (Fig. 1 and Table 1). The difference in TBRmax within the ascending aorta correlated with the difference in 68Ga-DOTATATE within the coronary arteries (r=0.49, p=0.025). In contrast, no correlations were found between changes in CRP (r=0.24, p=0.289) or LDL-cholesterol levels (r=0.214, p=0.349) with changes in coronary 68Ga-DOTATATE.

Fig. 1
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68Ga-DOTATATE uptake throughout the cardio–haematopoietic axis at baseline and follow-up. The uptake of the 68Ga-DOTATATE within the coronary arteries, ascending aorta, bone marrow and spleen, at baseline and after 12 weeks of atorvastatin treatment in patients with type 2 diabetes. Paired t tests were performed to test for statistical significance: *p<0.05, **p<0.01

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Discussion

We provide evidence of therapeutic modulation of vascular 68Ga-DOTATATE uptake in individuals with type 2 diabetes. We discovered significant reductions in 68Ga-DOTATATE uptake in the coronary arteries and ascending aorta after a 12 week regimen of atorvastatin 40 mg daily (Fig. 1). These changes did not correlate with CRP, which currently is the most used surrogate marker of vascular inflammation. Interestingly, 68Ga-DOTATATE uptake in haematopoietic organs was also reduced substantially, suggesting that 68Ga-DOTATATE PET/CT could be used to assess inflammation in other key organs that contribute to atherosclerotic cardiovascular disease. Collectively, these data show that 68Ga-DOTATATE PET/CT holds promise as a surrogate marker to non-invasively evaluate the treatment response of inflammatory activity throughout the cardio–haematopoietic axis in individuals with type 2 diabetes.

We observed a significant change in the coronary TBRmax after statin treatment, indicative of a reduction in inflammatory activity in the coronary arteries [5]. We substantiated this finding by demonstrating a similar effect in the ascending aorta, while the background uptake of 68Ga-DOTATATE measured in the left atrium did not change after statin treatment. Comparing this imaging modality with the currently used 18F-fluorodeoxyglucose (18F-FDG) PET/CT, not only can 68Ga-DOTATATE readily be used to evaluate vascular inflammation in the coronary arteries without being affected by myocardial spillover, but also greater effect sizes are observable. This allows future studies to have smaller study populations when using 68Ga-DOTATATE instead of 18F-FDG. A previous study examining changes in arterial 18F-FDG uptake after 12 weeks of atorvastatin treatment reported a mean reduction of 14.4% in TBRmax [7], whereas our study demonstrated a mean reduction of 31% in 68Ga-DOTATATE TBRmax.

We observed a notably higher uptake of 68Ga-DOTATATE in the bone marrow and spleen in individuals with type 2 diabetes, at a similar background signal as was previously reported as physiological uptake in apparently healthy non-diabetic individuals [8]. Notably, we also identified a lower 68Ga-DOTATATE uptake within the bone marrow and spleen after statin treatment. Future studies, including bone marrow biopsies in tandem with 68Ga-DOTATATE PET/CTs, are required to determine whether a decrease in 68Ga-DOTATATE uptake is caused by a decreased production of M1 macrophages, polarisation towards an M2 phenotype, or a combination of the two. In mice both chronic and transient intermittent hyperglycaemia promote myelopoiesis [3]. The fact that 68Ga-DOTATATE uptake reduction in the haematopoietic organs bears striking resemblance to a reduction in the coronary arteries is in accordance with the hypothesis that haematopoietic activation may be a contributing factor for atherosclerosis in individuals with type 2 diabetes. In support of this, 18F-FDG studies have demonstrated that haematopoietic uptake in apparently healthy individuals is also associated with early atherosclerosis [9].

Limitations of our study include the lack of a placebo group. Since international guidelines recommend statin therapy for all patients with diabetes mellitus of 40 years and older [10], it was not considered ethical to include a placebo group. Nonetheless, the interpretation of our results is unlikely to be affected by lack of a control group, as we do not consider spontaneous resolution of atherosclerotic inflammation to be a likely phenomenon. Second, we may have underestimated the total coronary plaque burden because vascular PET imaging has relatively low spatial resolution. Therefore, to limit the challenges of identifying the coronary arteries, we performed ECG-gated, breathing-corrected PET/CT scans. Accordingly, we drew VOIs along the grooves of the coronary tree, to approximate the TBRmax of the coronary arteries as closely as possible. Despite the spatial limitations inherent to current PET/CT techniques, we were clearly able to detect a strong decrease in coronary uptake of 68Ga-DOTATATE.

In conclusion, we show that 68Ga-DOTATATE PET can be used to identify changes in arterial wall inflammation, as well as activity in haematopoietic organs, providing a rapid readout after just 3 months of drug therapy. Therefore, 68Ga-DOTATATE holds promise as a surrogate marker in upcoming intervention trials that dampen inflammation. However, owing to limitations of the study design, these results will require further confirmation.