[¹⁸F]FE-PE2I DAT correlates with Parkinson’s disease duration, stage, and rigidity/bradykinesia scores: a PET radioligand validation study

Patients with idiopathic PD, age 45–80, and H&Y stage < 3, were recruited at two local outpatient clinics for movement disorders and through advertisement at the Swedish Parkinson Foundation. Cognitive impairment was excluded through Mini-Mental State Exam (MMSE) ≥ 27; psychiatric comorbidity through structured psychiatric interview; clinically significant physical comorbidity through physical examination, ECG, routine urine and blood tests; significant intracranial deviations through structural MRI. Additional file 2: Table S1 shows summarized exclusion criteria and rules of conduct minimizing possible confounding factors during DAT measurements.

Symptom duration was approximated from the reported onset of motor symptoms. The modified Hoehn and Yahr staging scale was used. In the first cohort, Unified Parkinson’s Disease Rating Scale motor score (UPDRS-III) had been performed at the screening visit while on Parkinson medication for practical reasons. In the remaining patients, the Movement Disorder Society—Unified Parkinson’s Disease Rating Scale motor score (MDS-UPDRS-III) was used and assessed while in practically defined ‘OFF’ state on the PET day, i.e. at least 12 h off dopaminergic medication, or 24 h in case of the longer-lasting Parkinsonian medication. This last improvement in the protocol was implemented to be able to perform correlation analyses between clinical measures and DAT availability in ‘OFF’ state, considering the effect of Parkinsonian medication on the motor scores.

Healthy controls (HC) were recruited and sex and age-matched (± 5 years) to the included patients with Parkinson’s disease, to correct on a group level for age- and sex-related effects on DAT availability when comparing disease-related differences to healthy controls.

T1- and T2-weighted images were acquired with a 3 Tesla MRI system (General Electric, Discovery MR750). MRI acquisition was done as previously described [15, 21], see Additional file 1 for details. The T1-weighted images were used for co-registration with PET images and delineation of the regions of interest (ROI); the T2-weighed images were used to exclude clinically significant pathology.

All PD subjects included were in practically defined ‘OFF’ state during PET acquisition. An individually made plaster helmet was used for head fixation in the PET camera. [¹⁸F]FE-PE2I was produced in-house as previously described [22]. PET acquisition and reconstruction were done as previously described [15]. In short, list mode PET data were acquired for 93 min with a high-resolution research tomograph (HRRT, Siemens Molecular Imaging). A 6-min transmission scan using a ¹³⁷Cs source was performed before radioligand administration for attenuation correction. PET data were reconstructed into 37 frames of increasing duration (8 × 10, 5 × 20, 4 × 30, 4 × 60, 4 × 180, 12 × 360 ms) using 3D OP OSEM with 10 iterations and 16 subsets, including modelling of the point spread function, with a transaxial resolution of ~ 2 mm [23]. The first two minutes of data were used as reference image for frame-to-frame realignment. Frame-to-frame realignment was performed using statistical parametric mapping (SPM, version 12). Realignment plots were evaluated visually to exclude excessive head motion. In the case of translations of more than 3 mm in the z-axis, additional motion correction was performed.

The analysis plan was pre-registered at https://​aspredicted.​org/​f3rq6.​pdf. Three-dimensional (3D) regions of interest were automatically delineated on the T1-weighted MR image and co-registered to PET. The main striatal regions striatum, caudate nucleus, putamen, and accumbens area (in this manuscript referred to as ventral striatum; VS) were delineated using Freesurfer. An FSL template (https://​fsl.​fmrib.​ox.​ac.​uk/​fsl/​fslwiki/​Atlases/​striatumconn) was used for the delineation of the sensorimotor striatum (Fig. 1), and an in-house developed template [21] was used for the delineation of the substantia nigra.

DAT availability (BP_ND) was estimated with wavelet-aided parametric imaging (t* = 27 min), using the Logan graphical analysis and cerebellum as reference region, as described earlier [15]. BP_ND (binding potential) is calculated as distribution volume ratio (DVR) minus 1: DVR-1.

ROI sides were assigned as belonging to the more or less affected hemisphere based on the individual’s BP_ND in the putamen sides, with the lower putamen BP_ND being assigned as the more affected hemisphere (maff), and the higher BP_ND as the less affected hemisphere (laff). These sides were cross-checked with the laterality of the MDS-UPDRS-III scores.

Statistical analysis was performed with R version 3.6.2. The normality of distribution was tested with the Shapiro–Wilk test and density plots. Testing for the difference in variance between the PD and control group was done with Levene’s test. Identification of statistically significant outliers was tested with Grubb’s test, combined with boxplots for visual assessment. The difference between the mean BP_ND of control subjects and PD subjects was tested with an unpaired, one-sided t test. The calculation of the effect size of BP_ND differences between controls and PD patients was done with Cohen's ds, with the formula: (mean(controls)-mean(PD))/SD(controls and PD).

The asymmetry index (AI) in PD was calculated per ROI as:

As reference asymmetry index, the upper limit of the 95% CI of the control group was used. The 95% CI was calculated as follows:which takes the sample distribution into account instead of assuming a normal distribution.

Simple linear regression was used for the correlation analysis between DAT availability and clinical measures, as described in the pre-registration. In the case of categorical or ordinal variables (such as H&Y stage), a generalized linear model (GLM) was used. LEDD (levodopa equivalent daily dose), age, and sex were tested as covariates.

A priori hypotheses had a p value threshold of < 0.05 for statistical significance; other comparisons were adjusted for multiple comparisons with Meff-correction [24, 25].

Data are available upon reasonable request.

In PD patients, the mean total striatal DAT availability (BP_ND) was 60% lower than in controls (see Fig. 2; Table 2). The largest difference was observed in the sensorimotor striatum and putamen (79% and 71% lower than controls), followed by the caudate nucleus (45%), and the substantia nigra and ventral striatum (31% and 25% lower than controls). Effect sizes for the distinction of PD from controls ranged between 1.18 (ventral striatum) and 1.86 (putamen) (Table 2). The putamen-to-caudate ratio was 0.79 ± 0.20 in PD patients and 1.44 ± 0.16 in controls. The ventral striatum-to-caudate ratio was 1.32 ± 0.40 in PD patients and 0.91 ± 0.17 in controls.

In PD patients, we observed that the lower putamen BP_ND side indeed corresponded to the contralateral most affected MDS-UPDRS-III side in all except one case. This case had similar BP_ND values in right and left putaminal ROIs, as well as identical left/right total scores of the MDS-UPDRS-III. For this reason, the asymmetry indexes (AI) were calculated excluding this patient (Table 2, Additional file 2: Table S3). The regions with the largest mean AI were the sensorimotor striatum and the putamen (around 35–40%). The mean AI in the caudate nucleus and substantia nigra was less than 20% and in the ventral striatum less than 10%. However, we observed a wide range of AI values for some of the regions, particularly in the ventral striatum, sensorimotor striatum, and substantia nigra, including negative AI values. Additional analysis on the asymmetry index was, therefore, performed excluding the cases with a negative asymmetry index or in which the asymmetry index was below the upper limit of the reference asymmetry index (see Table 2). With these criteria, the striatum and putamen were the regions in which all PD subjects (n = 40) had an asymmetry index above the threshold, whereas in the sensorimotor striatum, substantia nigra, and ventral striatum, this was the case for only 88%, 58, and 28% of the PD sample, respectively (Additional file 2: Tables S2, S3).

The relationships between symptom duration, H&Y stage, and MDS-UPDRS-III scores and [¹⁸F]FE-PE2I BP_ND in the a priori-defined regions and additional exploratory regions are presented in Fig. 3. Correlational analysis with MDS-UPDRS-III was only done with the PD subgroup that had this measure assessed in practically defined ‘OFF’, i.e. n = 23. Regarding relationships in a priori-defined regions, statistically significant negative correlations were found for symptom duration and DAT availability in the sensorimotor striatum, but not in the substantia nigra. H&Y stage and DAT correlated in all hypothesized regions (caudate nucleus, putamen, substantia nigra), and even in substantia nigra; in the subgroup of patients assessed in ‘OFF’, only a trend was observed between BP_ND in substantia nigra and H&Y stage. MDS-UPDRS-III score correlated negatively only with BP_ND in the sensorimotor striatum, but when subtracting the tremor from the total score, the correlation was significant also in the putamen.

The relationship between symptom duration and BP_ND in caudate nucleus and putamen was better described using an exponential function (Fig. 4). Exponential fitting was not superior to linear fitting for the relationship between MDS-UPDRS-III and the DAT availability in the four main ROIs (results not shown).

In post hoc analyses, significant negative correlations were also found for symptom duration and DAT availability in putamen (Fig. 3, n = 41), less affected putamen and less affected sensorimotor striatum (Additional file 2: Table S3a/b). Correlations between the H&Y stage and DAT availability in less affected caudate nucleus, more and less affected putamen, and more and less affected sensorimotor striatum were also statistically significant in the post hoc analysis (Additional file 2: Table S3a). No additional statistically significant negative correlations of MDS-UPDRS-III scores with any of the other areas were found.

[¹⁸F]FE-PE2I BP_ND data in the whole group of 41 PD patients were compared to those obtained in a group of 37 control subjects with similar age and sex distribution. To our knowledge, this is the largest reported cohort of PD patients examined with [¹⁸F]FE-PE2I PET. We confirmed our earlier findings of a PD-related pattern of DAT deficit and extended the quantification also to the sensorimotor striatum. Overall, the findings of this study corroborate the knowledge that dopaminergic neurodegeneration in PD follows a retrograde pattern, affecting neuronal terminals first (striatum) before the cell bodies (substantia nigra) [21]. Within the striatum, the posterior putamen (sensorimotor striatum) is affected first, followed by the anterior putamen, caudate nucleus, and ventral striatum.

In the substantia nigra, there was no clear asymmetry of BP_ND between the less and more affected hemispheres. DAT density in the substantia nigra is much lower than the DAT in the striatal regions, and we have also previously shown that in the substantia nigra BP_ND estimates have lower reliability than in the striatal regions, making accurate measurement of DAT in this area challenging. Neuronal loss in the substantia nigra is mainly observed in the pars compacta, with the ventrolateral tier being relatively spared [26]. The substantia nigra ROI template that we used in this study covers the entire region and is based on the distribution of uptake of [¹⁸F]FE-PE2I [22]. Therefore, it is possible that we were not able to detect an asymmetry of BP_ND because DAT was measured in the whole region rather than in the part of the substantia nigra that is most severely affected. Partial volume effect on the small area might have influenced the results as well. In a previous study with [¹¹C]PE2I PET in mild-to-moderate PD patients [20], BP_ND was measured in subdivisions of the substantia nigra. Although it was found that the DAT deficit was larger in the ventral tier than the dorsal tier (p < 0.001) [20], no significant difference in BP_ND was observed between the more vs. less affected substantia nigra, in agreement with the findings of this study.

In the publication of the first cohort [21], no statistically significant correlation was found between BP_ND and disease duration, H&Y, or UPDRS motor score. Our improved sample size and clinical data collection protocol enabled a more powered assessment of the correlations.

In agreement with other previous DAT imaging studies [12, 27], we observed a significant negative correlation between symptom duration and DAT availability measured with [¹⁸F]FE-PE2I in the putamen, and more specifically the sensorimotor striatum. This correlation was not significantly influenced by the inclusion of the patient with a statistical outlier symptom duration (14 years). The relation between symptom duration and DAT availability in the putamen, caudate nucleus, and sensorimotor striatum was better described by exponential fitting than linear fitting. This non-linear relationship between symptom duration and dopaminergic deficit has been previously described in a large cohort of PD patients examined with the vesicular monoamine transporter type 2 PET radioligand [¹⁸F]FP-DTBZ [28]. The findings are not surprising, since it is known that clinical signs of Parkinson’s disease do not appear until about 50–70% of the nerve terminals are already lost [26, 29, 30, 31, 32]. This might result in larger inter-individual variability of the dopaminergic deficit at the onset of the disease, with a more uniform decline as the disease progresses. The exponential correlation suggests that early on in the disease course, there is some degree of variability of DAT availability that probably reflects the fact that patients develop symptoms at different levels of dopaminergic deficit, probably due to different motor or dopamine reserve.

A significant negative correlation was found between the H&Y stage and [¹⁸F]FE-PE2I BP_ND in all nigrostriatal regions (caudate nucleus, putamen, and substantia nigra). The H&Y stage is a measure of the patient’s global motor disability. These findings suggest that the H&Y stage is the clinical measure that is most directly related to the overall nigrostriatal dysfunction, compared to the other clinical variables used.

A significant negative correlation between the MDS-UPDRS-III score and DAT availability in the putamen was only found when the tremor score was subtracted from the total score. It has to be highlighted, though, that one subject presented BP_ND values that were relatively high compared to their MDS-UPDRS-III score (Fig. 3), was identified as an outlier by the Grubb’s test, and indeed influenced the statistical results significantly. Methodological reasons for this subject’s relatively high BP_ND values were not found, and, therefore, the subject was not excluded from the analysis, despite being an outlier from a statistical point of view. Exploratory re-analysis with the removal of this subject gave correlations of BP_ND with total MDS-UPDRS-III scores in the putamen and sensorimotor striatum both with linear fitting (r_s between -0.43 and -0.56, p values between 0.006 and 0.05, see Additional file 2: Fig. S1a), as well as with exponential fitting (p-values < 0.05, see Additional file 2: Fig. S1b).

Previous [¹⁸F]FE-PE2I studies in PD patients have reported no significant correlations between striatal DAT and UPDRS-III [21, 33]. Possible explanations for these differences might be related to whether UPDRS-III was measured ‘ON’ or ‘OFF’ medication, or whether the motor score included also the tremor score or only the bradykinesia and rigidity scores. Two PET studies performed with the close analogue [¹¹C]PE2I found that striatal DAT-binding significantly correlated with total UPDRS-III (OFF) and bradykinesia-rigidity scores [3] or with bradykinesia, rigidity, and axial symptoms, but not with tremor score [20]. Overall, the findings of this study support the latter and suggest that the tremor score should be subtracted from the total MDS-UPDRS-III score in studies that intend to examine the correlation between DAT and motor symptoms.

There was no correlation of DAT availability in the substantia nigra with any of the clinical measures in patients examined ‘OFF’ medication, thereby not confirming our initial hypothesis. A possible explanation is that in the early stages of PD, there is still a relative sparing of neurodegeneration in substantia nigra, and the relationship might be more clearly expected later on in the disease process. Furthermore, a floor effect due to the low density of DAT could also be an explanation for the lack of correlation findings. However, in the combined group (n = 41), the correlation between BP_ND in the substantia nigra and H&Y stage became statistically significant likely due to increased sample size.

When examining the correlation between BP_ND in unilateral regions and symptom duration and H&Y stage, statistically significant correlations were found in those regions that also displayed significant correlation in the bilateral BP_ND (main analysis). In the case of MDS-UPDRS-III scores, no statistically significant correlations were found with more or less affected ROI sides specifically.