Determinants of 18F-NaF uptake in femoral arteries in patients with type 2 diabetes mellitus

Background The goal of this study was to investigate the potential determinants of 18F-NaF uptake in femoral arteries as a marker of arterial calcification in patients with type 2 diabetes and a history of arterial disease. Methods and Results The study consisted of participants of a randomized controlled trial to investigate the effect of vitamin K2 (NCT02839044). In this prespecified analysis, subjects with type 2 diabetes and known arterial disease underwent full body 18F-NaF PET/CT. Target-to-background ratio (TBR) was calculated by dividing the mean SUVmax from both superficial femoral arteries by the SUVmean in the superior vena cava (SVC) and calcium mass was measured on CT. The association between 18F-NaF TBR and cardiovascular risk factors was investigated using uni- and multivariate linear regression corrected for age and sex. In total, 68 patients (mean age: 69 ± 8 years; male: 52) underwent 18F-NaF PET/CT. Higher CT calcium mass, total cholesterol, and HbA1c were associated with higher 18F-NaF TBR after adjusting. Conclusion This study shows that several modifiable cardiovascular risk factors (total cholesterol, triglycerides, HbA1c) are associated with femoral 18F-NaF tracer uptake in patients with type 2 diabetes. Electronic supplementary material The online version of this article (10.1007/s12350-020-02099-z) contains supplementary material, which is available to authorized users.


INTRODUCTION
Arterial calcification on CT is used as a marker for the presence of intimal calcification (atherosclerosis) and medial arterial calcification. Microcalcifications (\ 50 lm) are observed in the early stages of the calcification process and are possibly associated with plaque rupture, whereas macrocalcifications contribute to stability of atherosclerotic plaques. 1 In addition, microcalcifications in the internal elastic lamina and in the tunica media, 2,3 may be related to arterial stiffening. 4 CT allows to identify macrocalcifications of over 200 lm in diameter. 5 Sodium 18F-fluoride ( 18 F-NaF) PET/CT is a novel imaging technique that can visualize smaller calcifications. [6][7][8][9][10] In the femoral arteries, 18 F-NaF PET can visualize ongoing (medial and intimal) calcification. 11 In the coronaries 18 F-NaF PET is associated with high risk plaque features and plaque rupture. 6 In patients with diabetes, additional to atherosclerosis, medial arterial calcification (MAC or Mönckeberg's sclerosis) is highly prevalent and is associated with increased arterial stiffness, hypertension, distal symmetrical neuropathy, chronic kidney disease, cardiovascular and all-cause mortality. [12][13][14] MAC is observed in arteries of the lower extremity across the entire length. 3 A recent study showed that increased 18 F-NaF PET accumulation in the femoral arteries is associated with diffuse calcium deposition and significantly correlates with cardiovascular risk factors. 11 Several studies investigated determinants of 18 F-NaF uptake in oncologic patients. 11,15 While the use of 18 F-NaF is increasing, the available data are limited regarding its uptake values and potential uptake modifiers in high CVD risk groups such as diabetes patients. The goal of this study is to investigate the potential determinants of 18 F-NaF uptake as a marker of arterial calcification in patients with type 2 diabetes and a history of arterial disease.

METHOD Study Design and Population
The study population consisted of participants of a randomized controlled trial (ClinicalTrials.gov Identifier: NCT02839044). 16,17 Patients were recruited at the University Medical Center Utrecht between June 2016 and September 2018. The goal of the study was to investigate the effect of vitamin K2 supplementation on arterial calcification in patients with type 2 diabetes. Inclusion criteria were age [ 40 years with type 2 diabetes and documented presence of cardiovascular disease. The presence of arterial diseases was defined as coronary artery disease, stroke, peripheral artery disease, abdominal aortic aneurysm or an ankle brachial index (ABI) of \ 0.9. The study protocol was approved by the local medical ethical committee (NL53572.041.15, METC 15/571) and all patients provided written informed consent. In this prespecified analysis, baseline 18 F-NaF PET/CT scans and patient data were used to determine the potential contributors of increased 18 F-NaF uptake. Patient data were collected from questionnaires and available medical records. All participants completed a questionnaire on medical history, medication, smoking history, and a physical examination including height, weight and blood pressure. Non-fasting blood samples were collected to measure cholesterol levels, HbA1c levels and creatinine.

PET/CT Imaging and Quantification
All patients underwent a full body 18 F-NaF PET/CT scan, performed on a Siemens Biograph 40 scanner (Siemens Healthineers, Erlangen, Germany). Imaging was performed 90 min after intravenous injection of 2.0 MBq/kg 18 F-NaF, with a maximum dosage of 200 MBq. Circular regions of interest (ROI) were drawn bilaterally around the superficial femoral arteries (see Figure 1A and B) from the bifurcation of the femoral artery to the femur condyles on every sequential axial slice to determine the 18 F-NaF uptake. To derive the average maximum standardized uptake value (SUV max ), maximum values measured per ROI were summed and averaged. Target-to-background ratio (TBR) was calculated by dividing the mean SUV max from both femoral arteries by the SUV mean from three sequential slices in the SVC (see Figure 1C), the circular ROI size was not fixed as the vena cava size differs between individuals. The femoral artery TBR was used as the measure of femoral 18 F-NaF PET activity. To evaluate interrater reproducibility on radiotracer uptake ten participants were randomly selected and analyzed by thee blinded raters.

Calcification Quantification
Non-contrast-enhanced CT scans were visually evaluated using in-house built software to identify calcium scores in the femoral arteries. A threshold of C 130 Hounsfield units was used and calcification was quantified using the calcium mass equivalent score which considers calcification volume and density.

Statistical Analyses
Normality of the data was tested using Quantile-Quantile plots. Descriptive data were expressed as mean ± standard deviation (SD) for normally distributed continuous variables, and median (Q1-Q3) for non-normally distributed continuous variables. Categorical variables are displayed as count (percentage). Reliability for TBR measurements was assessed using intraclass correlation coefficient. Univariate linear regression (unstandardized betas) was used to identify associations between all variables and 18 F-NaF TBR. Multivariate linear analysis was performed to correct for age and sex. No adjustments were made for multiplicity of testing, and no imputation was used for missing values (only 3 patients had missing data on calcium mass). A p value \ 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software (IBM Corp, Version 24.0. Armonk, NY, USA).

Patient Characteristics
The study population consisted of 68 participants (76% male) with a mean age 69 ± 8 years. Glucose lowering drugs were used by 60 (88%) patients, while insulin was used in 32 patients (47%). In total 25 patients used both insulin and glucose lowering drugs and only one patient was neither on insulin nor glucose lowering drugs. Fifty-two participants (77%) were treated with statins. As defined by the inclusion criteria all patients had a history of cardiovascular disease. Femoral arterial calcification on CT was present in 97% of the study population. Arterial 18 F-NaF was measured on average on 76 slices per patient (38 per side). Baseline characteristics of the patients are reported in Table 1.

F-NaF PET Activity
Higher femoral calcium mass on CT, total cholesterol, and HbA1c were associated with higher 18 F-NaF TBR (Table 2). Age, blood pressure, creatine and body mass index (BMI) were not associated to 18 F-NaF TBR. When applying multivariate linear regression adjusting for age and sex, CT femoral calcium mass, total cholesterol and HbA1c remained significantly associated ( Table 2). A trend towards significance was seen for higher triglycerides and higher 18 F-NaF PET TBR (p = 0.052). 18 F-NaF PET TBR was lower in patients on statins with a trend towards significance (p = 0.058). No significant differences in TBR were observed for smokers, male sex or other medication ( Table 2). The intraclass correlation coefficient for inter-rater reliability of radiotracer uptake (measured as TBR) was excellent with a value of 0.98 (95% CI 0.94-0.99).

DISCUSSION
This study provides several observations that contribute to our understanding of the determinants of 18 F-NaF PET in the femoral arteries of patients with type 2 diabetes and a history of arterial disease. Firstly, we showed that femoral calcium mass on CT is positively associated with 18 F-NaF uptake. Second, several cardiovascular risk factors including total cholesterol, triglycerides and HbA1c were associated with 18 F-NaF uptake in patients with type 2 diabetes. Together, these data suggest that 18 F-NaF uptake is an important imaging biomarker of arterial disease burden both related to dyslipidemia and diabetes control even in extensively treated patients.
Previous research showed that multiple cardiovascular risk factors are associated with 18 F-NaF uptake. Derlin et al 15 examined 18 F-NaF in the carotid arteries in 269 oncologic patients. 18 F-NaF uptake was significantly associated with age, male sex, hypertension and hypercholesterolemia. In a different study among 409 oncologic patients, the association between 18 F-NaF accumulation in femoral arteries with cardiovascular risk factors and calcified plaque burden was demonstrated. 11 In both studies, hypercholesterolemia was associated with increased 18 F-NaF uptake, which is in line with our study. An association between HbA1c and 18 F-NaF uptake, had been observed in healthy controls. 18 In patients with diabetes, high HbA1c is known to be associated with osteocalcin, which supports the concept of vascular calcification in patients with diabetes. 19 Blomberg et al 18 investigated 18 F-NaF uptake in the coronary arteries of 89 healthy adults. According to their results age, female sex and BMI were independent determinants of increased coronary 18 F-NaF uptake. In our study we did not find an association between age, sex or BMI and 18 F-NaF uptake. The differences in associations between cardiovascular determinants and 18 F-NaF uptake compared to our study could be explained by the differences in study population (our cohort consisted predominantly out of males with a high BMI), investigated vessels beds 20 and inclusion criteria. 18 F-NaF uptake is significantly higher in microcalcifications compared to macrocalcifications. 21 Irkle et al 21 demonstrated using preclinical lPET/lCT that microcalcifications have no barriers for penetration, while 18 F-NaF cannot penetrate into the deeper layers of macrocalcifications. Also, microcalcifications have a relatively big surface area compared to their volume. In a prior longitudinal study, we observed that areas without calcification on CT, but with increased 18 F-NaF uptake, had more arterial calcification at followup. 17 These could represent microcalcifications not yet detectable on CT. 17 In the current study CT detected arterial calcification was related to 18 F-NaF uptake, suggesting a significant effect of large calcium depositions on tracer uptake with clinical PET/CT systems.
An important strength of the current study is that images were acquired 90 min after tracer injection. The 90 min time-point is considered to be advantageous, because of the balance between signal-to-background ratio and patient comfort. For comparability with other studies we used TBR as our measure of 18 F-NaF uptake for research. 15 There is debate on how to quantify arterial calcification with NaF PET. [22][23][24] We calculated Values are represented as mean ± SD, median [Q1-Q3] or n (%) TBR, target-to-background ratio; LDL, low-density lipoprotein; HDL, high-density lipoprotein; HbA1c, glycated hemoglobin; CAD, coronary artery disease; CVA, TIA, cerebrovascular disease, transient ischemic attack; PAD, peripheral artery disease; ABI \ 0.9, ankle brachial index \ 0.9; AAA, abdominal aortic aneurysm the TBR using the average of the SUVmax of each femoral artery slice. Though SUVmax is sensitive to noise, it can also be a good measure of the maximum disease burden in a slice. 22,23 By averaging (76 slices per patient) the noise effect is reduced to some extent. Others advocate the SUVmean for an individual slice, 24 which is less limited by noise, but also averages nondiseased parts of the artery and the blood pool. Differences in PET SUV quantification will influence the observed result therefore it would be important to achieve uniform quantification methods. Other limitations are the relative small sample size and the lack of prospective outcome data.
In conclusion, we showed that cardiovascular risk factors including total cholesterol, triglycerides and HbA1c are associated with 18 F-NaF uptake and calcification in the femoral arteries in patients with type 2 diabetes mellitus and a history of arterial disease. Our findings show that 18 F-NaF uptake can be an imaging biomarker of arterial disease burden both related to dyslipidemia and diabetes control. Larger studies are needed to investigate whether these findings are generalizable to non-diabetic patients with cardiovascular diseases and if (further) modification of these risk factors is beneficial for 18 F-NaF uptake and outcome.

NEW KNOWLEDGE GAINED
18 F-NaF uptake may be an important arterial imaging biomarker related to dyslipidemia and diabetes control (HbA1c) even in extensively treated patients with type 2 diabetes.

Disclosure
RAPT, RvA, JWB, JMW, CC and PAdJ report no financial support or conflict of interest. SRZ and JWB are supported by the Senior Dr. Dekker grant (2013T120) from the Dutch Heart Foundation.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent was obtained from all individual participants included in the study.

Open Access
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