Patient Recruitment
This multicenter hybrid imaging study was conducted between December 2015 and June 2018 at two sites: The British Heart Foundation centre for Cardiovascular Science at the University of Edinburgh, UK and the Icahn School of Medicine in New York, USA. All participants were older than 50 years. Patients with cardiac amyloid were recruited from outpatient clinics and inpatient wards over both sites. The diagnosis of AL or TTR amyloid was established on histological analysis of biopsy samples in all patients bar one who had Multiple Myeloma. This patient had CMR features of amyloid (included in the AL group) but did not have a tissue biopsy. Age/sex-matched subjects were recruited as a negative control group as were patients with aortic stenosis (peak aortic jet velocity of > 2.5 m/s, with no clinical suspicion of amyloid).
Exclusion criteria for all cohorts included inability to receive iodinated contrast, renal impairment (estimated glomerular filtration rate ≤ 30 mL/min/1.73 m2) or women of child-bearing potential.
For those recruited in Edinburgh, the study was approved by the Scottish Research Ethics Committee and the United Kingdom (UK) Administration of Radiation Substances Advisory Committee. It was performed in accordance with the Declaration of Helsinki and all patients provided written informed consent prior to any study procedures. Subjects recruited in New York had ethical and Institutional Review Board approval for the study (GCO#01-1032). All participants provided written informed consent. The study was registered on Clinicaltrials.gov (NCT03626584). We did not directly include patient and public involvement (PPI) in this study, but the patient information sheet used in the study was developed with PPI and was reviewed by a committee that includes patient representatives.
Imaging Protocols
18F-fluoride positron emission tomography and coronary magnetic resonance angiography
This was a multicenter PET/MR study, but where possible protocols were standardized between centers with all patients undergoing simultaneous PET and MR imaging using the same hybrid PET/MR system (Biograph mMR, Siemens Healthcare GmbH, Erlangen, Germany). The MR protocol at each site was the same including long axis cine imaging (3-chamber, 2-chamber, 4-chamber), a short axis cine stack (8 mm thickness, 1.6 mm gap) coronary magnetic resonance angiography (CMRA for accurate co-registration) performed with 0.2 mmol/kg of intravenous gadobutrol contrast (Gadovist, Bayer Pharma AG, Germany) and late gadolinium enhancement imaging 10 to 15 minute post-contrast administration. Importantly, the same radial gradient recalled echo (GRE, Siemens work-in-progress #793F) sequences was acquired for MR attenuation correction at both sites as previously described,6. PET imaging was performed 60 to 120 minutes post administration of 125-350MBq 18F-fluoride. List mode PET data were then reconstructed using e7tools (Siemens Healthcare) applying the radial GRE sequence (2 tissue classes: background [air and lung] and soft tissue [soft tissue and fat]).18,19 An Ordered Subsets Expectation Maximization (OSEM) algorithm with the following parameters was employed: 256 × 256 matrix, 4 iterations, 21 subsets, 5 mm Gaussian filter in Edinburgh and 344 × 344 matrix, 6 iterations, 21 subsets, 2 mm Gaussian filter in New York.
PET/MR image analysis
All PET/MR images were analyzed at the University of Edinburgh Core Lab by two expert readers (JA, MRD). Accurate co-registration was achieved by aligning 18F-fluoride activity in the blood pool and ascending aorta with the corresponding anatomical structures on the CMRA.20 Qualitative and semi-quantitative analysis of the PET images was performed using FusionQuant software (Cedars-Sinai Medical Center, Los Angeles). Radiotracer uptake was analyzed using a standardized protocol (supplemental data). For myocardial analysis two approaches were employed. First, based on previous research showing amyloid infiltration and septal hypertrophy in 79% of ATTR cases we sampled septal uptake.21 This was calculated using cylinders of 3 mm radius and 15 mm length set within the septum at mid cavity level on the co-registered co-axial image to generate volumes of interest (VOI, Figure 1, supplementary protocol). Standardized uptake values (SUVMEAN and SUVMAX) were calculated for these septal VOIs and corrected for blood pool activity (measured in the right atrium)22 to provide tissue-to-background ratio (TBRMEAN and TBRMAX). Second, in patients with cardiac amyloid I8F-fluoride uptake was assessed in areas of myocardial LGE. Equal sized volumes of interest (VOI) were placed within areas of LGE demonstrating the greatest visual uptake on short axis slices with SUVMEAN and TBRMEAN values recorded (Figure 1D).
Throughout the study TBR values were used in preference for comparison. In principle the correction of tissue uptake for blood pool measurements made on the same scan adjusts for potential differences in PET values acquired at different scanners. In order to test this principle, we compared TBRMEAN values for both healthy volunteers and amyloid patients imaged in Edinburgh to the equivalent subjects imaged in New York.
Statistical Analysis
All statistical analyses were performed using GraphPad Prism Version 8.0. A two-sided P < 0.05 was considered statistically significant. The distribution of all continuous variables was assessed using the Shapiro-Wilk test, which were presented using mean ± standard deviation or median [interquartile range]. Comparisons between groups were performed using the two-sample t test, Mann-Whitney test or ordinary one-way ANOVA as appropriate. Receiver operating characteristic (ROC) curves were generated to determine area under curve (AUC) and perform sensitivity and specificity analyses. We presented all categorical variables as percentages.