Positron Emission Tomography/Computed Tomography Imaging in Therapeutic Clinical Trials in Alzheimer’s Disease: An Overview of the Current State of the Art of Research

INTRODUCTION

Positron emission tomography/computed tomography (PET/CT) is a powerful imaging technique that combines two distinct technologies to provide comprehensive information about the structure and function of tissues within the human body. In PET, a small amount of a radioactive substance, known as radiotracer, is injected to monitor the uptake in targeted body regions. As the radiotracer undergoes decay, it emits positrons, which interact with nearby electrons, leading to the production of gamma rays. These gamma rays are detected by a PET scanner, enabling the creation of detailed three-dimensional images highlighting metabolic and biochemical activity within tissues. On the other hand, CT imaging involves the use of X-rays to generate cross-sectional images of the body’s internal structures, providing anatomical details.

The integration of PET and CT scans in a single imaging modality offers several advantages in medical diagnostics, allowing for a more accurate localization of abnormalities. This is particularly valuable in oncology, where PET/CT is widely used for cancer staging, treatment planning, and monitoring response to therapy. Key applications of PET/CT in medicine also include cardiac imaging, infection and inflammation imaging, and neuroimaging.

In neuroimaging, PET/CT can be used to assess different aspects of brain function, supporting the diagnosis and management of conditions such as epilepsy and neurodegenerative disorders. Although the diagnosis of dementia is still largely clinical, neuroimaging has dramatically increased its diagnostic accuracy and now plays an important role in the diagnosis of dementia. Therefore, it is recommended in most clinical guidelines for investigating the neuropathological basis of cognitive impairment [1].

Alzheimer’s disease (AD) is a complex neurodegenerative disorder characterized by an uncontrollable and progressive cognitive decline. PET/CT imaging emerged as a valuable tool for investigating the underlying molecular and metabolic alterations associated with AD. Indeed, functional AD neuroimaging through PET/CT is able to in vivo reveal alterations causing brain cell toxicity, disruption of neuronal cytoarchitecture, neuronal loss and brain function decline in affected patients. Among these alterations, the impairment of brain glucose metabolism, deposition in the brain of misfolded amyloid-β (Aβ) aggregates and neurofibrillary tangles (NFT) of hyperphosphorylated tau proteins are the three major hallmarks of AD and also the three major areas of application of PET/CT imaging [2]. Practice guidelines and appropriate use criteria reports have already been provided with regards to the use of PET/CT in the imaging of these neuropathological/ pathophysiological targets in AD patients [3–9].

Moreover, the low invasiveness and in vivo applicability of PET are two properties that can be thoroughly exploited in target-specific therapeutic interventions, making this imaging modality an important tool in research studies and clinical trials evaluating new therapies for AD.

The following paragraphs will survey the applications of PET/CT imaging in therapeutic interventional clinical trials in AD recorded up to February 2024 on the National Institutes of Health site “ClinicalTrials.gov”, on the World Health Organization’s International Clinical Trials Registry Platform and the Cochrane Library Trials. To reach our aim, the search focused on interventional studies with treatment as primary purpose and PET/CT as an outcome measure. All trials’ registration codes were then searched on PubMed/Medline to obtain corresponding published results, here discussed.

¹⁸F-FDG PET/CT IN THERAPEUTIC TRIALS IN AD

In clinical practice, one of the most common radiotracers used for PET scanning in AD is 2-deoxy-2-[fluorine-18]fluoro-D-glucose (¹⁸F-FDG). Brain glucose hypometabolism in ¹⁸F-FDG PET scans indicates reduced synaptic activity [10], which commonly results in impaired brain function [11]. Previously conducted studies and literature reviews on the use of ¹⁸F-FDG PET/CT in AD demonstrated consistent findings of temporoparietal glucose hypometabolism (now considered a disease hallmark) in AD patients, already evident in the early stages of the disease. This reduced ¹⁸F-FDG uptake includes frontal brain areas in the late phases of the disease; moreover, a predictive value about the speed of disease progression has been demonstrated when entorhinal cortex glucose hypometabolism also emerges [12, 13]. Such findings prove the value of ¹⁸F-FDG PET across different stages of the disease, emphasizing its usefulness in early diagnosis, refined staging and disease progression monitoring [14–17].

¹⁸F-FDG PET also demonstrated being an effective method for early diagnosing and differentiation of AD from other types of dementia [18]. In a recent meta-analysis, ¹⁸F-FDG PET showed a sensitivity and specificity of, respectively, 0.96 (95% confidence interval [CI], 0.88–0.98) and 0.84 (95% CI, 0.70–0.92) in differentiating AD and frontotemporal dementia, 0.93 (95% CI 0.88–0.98) and 0.92 (95% CI, 0.70–0.92) in differentiating AD and dementia with Lewy bodies, and 0.86 (95% CI, 0.80–0.91) and 0.88 (95% CI, 0.80–0.91) in differentiating AD and non-AD dementias [18]. It is sometimes also used to detect early alterations of brain function even before the onset of cognitive symptoms, in the so called “preclinical AD” stage [16, 20].

Among therapeutical clinical trials with available published results (Tables 1 and 2), ¹⁸F-FDG PET/CT was more often performed only in a subset of the study cohorts, mainly due to the clinical trial’s early termination, treatment discontinuation decided by participant subjects, or logistic issues. Notably, when ¹⁸F-FDG PET/CT was not included among analyzed data, Authors stated that results were speculative and would require detailed analysis of ¹⁸F-FDG PET imaging data coupled with other tracers or functional brain magnetic resonance imaging to further evaluate the consistency of the results [21]. Moreover, it was underlined that ¹⁸F-FDG PET is a feasible imaging modality in multicenter therapeutic trials [22] and the obtained results demonstrated the usefulness of a study protocol incorporating imaging biomarkers for participant inclusion, evaluation, and stratification as a path to increase the probability of success of larger AD trials [23].

In some cases, changes in brain glucose metabolism assessed through ¹⁸F-FDG PET were the primary outcome of the study [22, 24], chosen as such because of known high correlations with clinical disease severity changes [25] and high sensitivity to synaptic changes that are estimated to have begun before the onset of cognitive symptoms [10, 11], even in early stages of AD [26, 27]. A proof of such time sequence finds confirmation in studies reporting changes in brain glucose metabolism but no results in clinical measures of cognitive and global functions [21, 24]. Because abnormal brain glucose metabolism likely precedes deterioration in cognitive function [13, 28], glucose hypometabolism in the affected regions may not be accompanied by related cognitive functional deficits [11, 29]. Therefore, neuropsychological tests in AD clinical studies are believed to require a large sample size and long duration to yield significant results [24, 30]. ¹⁸F-FDG PET represents a timely solution and a feasible early biomarker of functional changes in AD patients, even before clinical manifestations appear.

Among clinical trials using ¹⁸F-FDG PET as a primary outcome, positive treatment results were reported for cilostazol, a phosphodiesterase type III inhibitor, that appeared to prevent the decrease in general cerebral synaptic activity caused by the progression of AD [24]. Indeed, according to ¹⁸F-FDG PET image comparison between pre- and post-medication conditions, glucose uptake was not decreased in the cilostazol group, while a significant decrease in glucose metabolism in the bilateral parietal lobes, posterior cingulate cortex, precuneus, and inferior frontal gyri was reported in the placebo group [24]. In the Authors’ opinion, these results provide strong support for the use of cerebral glucose metabolism as a primary outcome in a proof-of-concept study, as its clinical relevance as a biomarker outcome consisted in a significant correlation with cognitive and functional outcomes both cross-sectionally and longitudinally [24]. Moreover, findings were consistent with longitudinal associations between ¹⁸F-FDG PET and clinical measures in previous observational studies [31] in the context of a therapeutic trial. Additional studies showing an association between ¹⁸F-FDG PET and clinical findings following the therapeutic treatment are needed to provide further support for its value.

Improvement in regional cerebral glucose metabolism secondary to medication was reported for Rasagiline, a monoamine oxidase B (MAO-B) inhibitor, versus placebo in AD [23]. Significantly improved brain glucose metabolism was observed in middle frontal, anterior cingulate, and striatal regions. On the other hand, clinical measures showed no significant benefit except for quality of life. In fact, no effects were observed in AD Assessment Scale-Cognitive Subscale (ADAS-cog) or activities of daily living, digit Span, verbal fluency, and Neuropsychiatric Inventory (NPI) measurements. Therefore, ¹⁸F-FDG PET/CT resulted being the best biomarker to characterize patients and detect treatment response in trials including AD patients [23].

Another clinical trial evaluated the safety of transcranial electromagnetic treatment (TEMT) in patients with mild to moderate AD. This intervention caused cognitive enhancement, changes to cerebrospinal fluid (CSF)/blood markers, and evidence of stable/enhanced brain functionality in AD patients [32]. Specifically, more positive percent changes in brain ¹⁸F-FDG PET results following treatment were correlated with greater patients’ cognitive performance [32].

In a study using intravenous immunoglobulins (IVIG) for treating AD, a significant dose-dependent attenuation of decreased glucose metabolism in the temporal/hippocampal brain regions within 24 weeks of treatment was documented by brain ¹⁸F-FDG PET [33]. However, significantly increased glucose metabolism in parietal and frontal brain regions was displayed by IVIG-treated patients at follow-up, whereas placebo-treated patients did not show any increase. Such findings remained unexplained and require further investigations [33].

¹⁸F-FDG PET was used as a surrogate measure of neuronal and synaptic activity in a trial testing the effects of CT1812, a novel brain penetrant sigma-2 receptor ligand that interferes with the binding of Aβ oligomers to neurons, in AD patients [34]. However, in this trial, only volumetric MRI findings suggested a trend towards brain tissue preservation following treatment.

No significant changes in ¹⁸F-FDG brain uptake were reported in the liraglutide [35], rosiglitazone [22], crenezumab [36], saracatinib [37, 38], or semagacestat trials [39, 40], all involving AD patients at different disease stages, also in the mild cognitive impairment (MCI) –which is considered an early stage of the disease.

Overall, these results suggest mixed outcomes for these therapeutic interventions in modifying AD-related PET measurements, with some showing promising effects on specific brain regions or glucose metabolism parameters and others failing to demonstrate significant impact on brain function.

AMYLOID PET/CT IN THERAPEUTIC TRIALS IN AD

The accumulation of Aβ peptides outside neuronal cells, forming plaques, is a distinctive pathological feature of AD. Since the first observation of such phenomenon, researchers put forward the hypothesis that the in vivo verification of Aβ deposition in individuals and the continuous monitoring of changes in Aβ accumulation in the brain could be crucial in setting therapeutic approaches specifically aimed at eliminating Aβ deposits. The recent advancement in molecular imaging tracers designed to target Aβ plaques in the brain facilitated their in vivo identification through PET scans. Molecular imaging findings have in fact proven to match histopathological reports of Aβ accumulation in brain areas of patients with AD.

The Amyloid Imaging Task Force of the Alzheimer’s Association and the Society of Nuclear Medicine and Molecular Imaging provided appropriate use criteria for amyloid PET for particular clinical scenarios: persistent or progressive unexplained MCI; unclear clinical presentation despite core clinical criteria for possible AD; progressive dementia with atypically early age of onset [4]. Moreover, amyloid PET has enabled clinicians to stratify patients in clinical trials aimed at delaying or preventing the symptomatic phase ofAD [1].

So far, three compounds have received approval for imaging Aβ plaques from both the U.S. Food and Drug Administration and the European Medicines Agency. These are: ¹⁸F-florbetapir (Amyvid; Eli Lilly), ¹⁸F-flutemetamol (Vizamyl; GE Healthcare), and ¹⁸F-florbetaben (NeuraCeq; Piramal Pharma). Of note, rare side effects have been reported for these three pharmaceuticals, namely headache (2%) and musculoskeletal pain, fatigue, blood pressure increase, and nausea (<1%) for florbetapir [41], flushing (2%), increased blood pressure (1%), headache (1%), nausea (1%), and dizziness (1%) for flutemetamol [42], and injection site pain (4%), injection site erythema (2%), and injection site irritations (1%) for florbetaben [43]. The prototype for these ¹⁸F-compounds was the initially developed ¹¹C-Pittsburgh compound B (PIB) [44], with measurements made in cortical regions found to have the highest burden of fibrillar amyloid at autopsy in participants diagnosed as having AD pathology. However, the short half-life of this compound became the reason for the new ¹⁸F-amyloid tracers to be promoted and used.

Among interventional clinical trials in AD with available published results (Tables 3 and 4), the most frequent use of amyloid PET was intended for treatment response assessment in settings testing molecules targeting Aβ plaques.

¹¹C-PIB and ¹⁸F-Florbetapir PET showed no statistically significant changes in global cortical amyloid standardized uptake value ratio versus placebo in trials testing crenezumab [36], bapinezumab [45], semagacestat [40], solanezumab [46], ACC-001 [47], stereotactic intracerebroventricular injection of human umbilical cord blood mesenchymal stem cells [48, 49], intranasal administration of allogenic human adipose mesenchymal stromal cells-derived exosomes [50], a GLP-1 analog [35], and candesartan (that though had a positive effect on the hippocampal region alone) [51].

However, PET results helped by excluding subjects with non-amyloid forms of dementia [45], or providing information on specific AD populations such as Presenilin-1 E280A mutation carriers with autosomal dominant AD, who might experience cerebellar Aβ deposition about a decade prior to the estimated onset of MCI [52]. Moreover, there were hints of possible therapeutical differentiated response among Apolipoprotein ɛ4 (APOEɛ4) carriers and noncarriers [53].

Positive outcomes were demonstrated in studies conducted over extended periods, assessing the long-term effects of treatments like lecanemab [54–56], aducanumab [57–59], donanemab [60–64], gantenerumab [65–67], verubecestat [68], and lanabacestat [69] in reducing the brain Aβ plaques load measured by amyloid PET.

In these cases, brain Aβ plaque burden, measured by ¹⁸F-Florbetapir PET imaging, reduced in a dose- and time-dependent fashion at second and often third timepoints from baseline evaluations. Moreover, as it happened in previously described ¹⁸F-FDG PET-CT, amyloid PET results showed significant therapeutic effects earlier than clinical effects, that were not apparent until one year [57], at least [59, 64–68]. Considering that it may have taken up to 20 years for Aβ to have accumulated to the levels in clinically diagnosed AD patients at study entry [70], the observed kinetics of Aβ removal within a 12-month time period appears encouraging for a disease-modifying treatment for patients with AD [57].

PET imaging data were also often analyzed to explore associations between amyloid plaque clearance and downstream effects on tau pathology progression and clinical outcomes. Reductions in amyloid plaque levels were associated with slower tau deposition and clinical decline in some cases, providing insights into the relationship between PET findings and underlying AD pathology. Early significant changes on brain amyloid PET scans, CSF or plasma tau species and neurogranin levels, also suggested opportunities for clinical monitoring of therapy [51, 71]. Such data have been very recently confirmed by a systematic review on prognostic and predictive factors in AD [72].

Regarding safety concerns of studies on anti-Aβ monoclonal antibodies, amyloid-related imaging abnormalities-edema (ARIA-E) often was dose-dependent and more common in APOEɛ4 carriers [57, 72].

Some studies explored different reference regions and methodological approaches for PET analysis to enhance sensitivity and accuracy in detecting changes in amyloid burden [45, 73]. The use of smaller reference regions such as the pons against the entire cerebellar gray matter might reduce the noise and thereby improve sensitivity to detect changes [45].

Of interest, one study used ¹⁸F-Florbetapir maps to personalize transcranial alternating current stimulation on the basis of individual Aβ accumulation maps, with promising results [74].

PET data were also used to describe amyloid removal and the occurrence of amyloid-related imaging abnormalities (ARIA), and consequently develop pharmacometrics (pharmacokinetic/pharmacodynamic) models to guide dose selection and optimize dosing regimens for anti-amyloid therapies, avoiding toxicity [55, 76]. For example, favorable Open Label Extension trials data, that matched well with model predictions, supported the decision to continue the gantenerumab clinical development program, and further apply model-based analytical techniques to optimize the design of new phase III studies [75].

Overall, the findings highlight the usefulness of PET imaging in tracking amyloid pathology, assessing treatment response, and predicting clinical outcomes in AD research. They also underscore the importance of integrating PET data with other biomarkers for a comprehensive understanding of disease progression and treatment effects.

TAU PET/CT IN THERAPEUTIC TRIALS IN AD

Tau PET enables the visualization of NFT of tau proteins in the brain, another characteristic neuropathological aspect of AD. Tau PET studies demonstrated promise in visualizing tau pathology, providing valuable insights into the spatial distribution and severity of tau aggregates, therefore aiding in AD staging and prognosis [1]. Emerging evidence suggested the potential of tau PET in predicting cognitive decline and offering a more precise characterization of AD progression.

Commonly employed tracers include the FDA approved ¹⁸F-flortaucipir (also known as ¹⁸F-AV-1451, mostly used for later disease stages) and ¹⁸F-RO948 [1, 9]. ¹⁸F-PI-2620 is a next-generation tau PET tracer with a high binding affinity for pathological tau depositions and low off-target binding for an improved detection of tau deposition at earlier stages [1, 77].

Clinical therapeutical trials with tau PET as an outcome measure, and corresponding published available results found up to February 2024 are shown in Tables 5 and 6.

Tau PET in clinical trials was mostly used for treatment response assessment [23, 78] and/or for patient stratification at baseline or according to inclusion criteria [50, 79]. One study specifically noticed high association between a positive tau PET with a positive amyloid PET in patients with early symptomatic AD, while the opposite relation was not true, thus suggesting the performance of tau PET alone to confirm candidates for AD trials, with consequent benefits on clinical trials and health care expenses, radiation exposure and participant time [79].

No brain global or regional effects were observed on ¹⁸F-flortaucipir PET after candesartan treatment [51], zagotenemab treatment [78], or intranasal administration of allogenic human adipose mesenchymal stromal cells-derived exosomes [50].

Rasagiline treatment, instead, showed uniform longitudinal brain tau load decreases in subcortical regions, particularly accumbens and putamen [23].

Patients from a trial sub-study were scanned with ¹⁸F-PI-2620 PET to assess tau deposits in an early AD population [77]. Results suggested that quantifiable tau load and its corresponding increase in early AD cases could be a relevant target engagement marker in clinical trials of anti-amyloid and anti-tau agents. Other serum biomarkers of AD progression were described in association with changes in tau PET in the donanemab trial [62].

Tau PET evaluations were mostly performed in sub-studies or for secondary study endpoints, often in anti-Aβ treatment trials. The lack of effect on the global tau load prompts questions about whether targeting Aβ reduction affects biologic disease progression.

In summary, tau PET currently plays an important role in assessing disease severity, treatment effects, and target engagement in AD research in clinical trials.

PET/CT VERSUS CSF FOR QUANTIFYING AMYLOID AND TAU CEREBRAL PATHOLOGY IN THERAPEUTIC TRIALS IN AD

Low CSF concentrations of Aβ₄₂ with normal levels of Aβ₄₀, and high CSF levels of total tau (t-tau) and phosphorylated tau (p-tau181) are currently recognized markers of AD pathology and can also help in predict cognitive decline [80]. Indeed, CSF level reduction of Aβ₄₂ and increase of t-tau and p-tau181, or increased uptake of amyloid and tau tracers visualized by PET/CT are used as in vivo measurements of brain amyloid and tau accumulation [81]. Several studies have shown that low CSF levels of Aβ₄₂ and high CSF levels of t-tau and p-tau181 correlated with high PET/CT amyloid and tau load, respectively [82]. However, CSF and PET/CT may provide partially different and independent information. Indeed, PET/CT evidence of amyloid and tau pathology is directly related to the deposition of amyloid plaques and NFT of tau proteins, respectively [83], whereas CSF Aβ₄₂ and tau levels are markers of soluble Aβ and neuronal injury, respectively, and only indirectly related to amyloid plaques and tau-NFT pathology. Moreover, CSF Aβ₄₂ levels can decrease due to other mechanisms not related to amyloid plaque deposition, such as variations in amyloid-β protein precursor processing and production (which reasons the usefulness of Aβ₄₀ addition measurement, since it is not reduced by AD pathology), and neuroinflammation [84]. On the other hand, tau levels can increase following brain tissue damage, such as traumatic brain injury, seizures, or stroke [85].

Moreover, it has been suggested that CSF Aβ₄₂ levels decrease and CSF tau proteins increase before the PET/CT amyloid and tau cerebral load, although not all studies supported such relation [86, 87]. Therefore, from a clinical point of view, giving substance to the establishment of indications for therapeutic clinical trials, CSF and PET/CT amyloid and tau measurements can provide partially independent information since CSF levels of Aβ₄₂, t-tau and p-tau can pathologically change before cerebral amyloid and tau deposition, measured by PET/CT. Moreover, isolated CSF Aβ₄₂ positivity has been reported as a common feature in cognitively normal subjects and individuals with subjective memory complaints or cognitive decline, who did not yet show cerebral amyloid deposition at PET/CT [82]. Therefore, concordance between CSF and PET/CT amyloid and tau positivity was common in patients at late disease stage (late MCI and AD) [82]. Based on this evidence, measurement of both CSF and PET/CT amyloid levels are required in therapeutic clinical trials in order to better candidate subjects to experimental treatments.

Other than this comparison, there are other concerns about the use of CSF quantification of AD biomarkers, related to some analytical and pre-analytical factors that can hamper the use of CSF in both the diagnosis and monitoring of AD pathology, particularly in clinical trials. Moreover, the lack of standardization of CSF biomarkers’ measurement coupled with a lack of consensus on ranges of normality and the use of different kits and methods, further limit the use of CSF in some memory clinics [88, 89]. Finally, CSF is obtained by lumbar puncture, requiring hospitalization, and it is discouraged in some clinical settings and conditions, since there is a risk for local infection, hemorrhage and spinal hematoma formation, up to very rare cases of central nervous system herniation [90]. On the other hand, a PET/CT examination is expensive, requires preparation and managing of radiotracers, and a nuclear medicine department equipped to perform this exam.

Hence, there are advantages and criticisms for both methodologies and their combination is now considered as the gold standard for patients’ recruitment in therapeutic clinical trials.

FUTURE PERSPECTIVES

Neuroinflammation activation, cholinergic deficit, and synaptic dysfunction represent other pathological aspects of interest in neurodegenerative disorders. They can become targets of molecular neuroimaging and will most probably become outcome measures in future clinical trials in AD.

Regarding neuroinflammation, for example, a recent metanalysis searched the literature for case-control studies examining levels of translocator protein levels, representing neuroinflammation, using PET/CT in regional analyses between healthy controls and MCI or AD subjects. Findings support the association of increased neuroinflammation during the progression of MCI and AD, relative to healthy controls [91]. On the side of cholinergic deficit, it has recently been found that nicotinic cholinergic receptor binding in specific limbic and subcortical regions is lower in MCI and further reduced in AD, compared to cognitively unimpaired older adults, and is related to cognitive deficits. Therefore, contemporary modification of the cholinergic deficit of aging and AD may reveal opportunities to prevent or improve clinical symptoms [92]. Finally, very recently, PET/CT imaging of synapses is being developed, accelerating the focus on the role of synaptic loss in AD and other conditions [93].

The design of new radiotracers for the above-mentioned aspects of neuroimaging in AD, dedicated brain PET/CT scanners with, e.g., high spatial resolution, high depth-of-interaction and time-of-flight resolutions, and a built-in system for marker-less continuous motion tracking [94] and the employment of Artificial-Intelligence-powered software will also improve quality, sensitivity and diagnostic power of molecular neuroimaging [95–97].

CONCLUSIONS

PET/CT imaging plays a crucial role in the evaluation of AD by providing valuable insights into the underlying pathophysiological processes.

Specifically, various PET tracers, including ¹⁸F-FDG for brain glucose metabolism assessment, amyloid PET and tau PET tracers, offer distinct advantages in elucidating different aspects of AD pathology. This overview highlights the multifaceted utility of PET/CT across various therapeutic trials in AD. While some interventions yielded promising effects on specific brain regions or metabolic parameters, others failed to demonstrate significant impacts. Notably, on the one hand, treatments targeting amyloid plaque load showed significant reductions over time, offering hope for disease-modifying therapies. Tau PET imaging, on the other hand, provided valuable insights into tau-NFT deposition, aiding in disease staging and treatment response assessment.

As such, PET/CT remains at the forefront of AD research, offering unparalleled opportunities for unravelling the complexities of the disease and advancing therapeutic interventions. However, the need for a comprehensive evaluation of AD pathology may include PET/CT exams but possibly also other biomarkers of AD pathology, such as those in the CSF. Indeed, the combination of CSF analysis with PET/CT quantification of amyloid and tau cerebral load may help clinicians in recognizing candidates, showing early signs of AD pathology, to be recruited in therapeutic clinical trials evaluating the clinical potential of disease-modifying strategies against AD.

AUTHOR CONTRIBUTIONS

Elizabeth Katherine Anna Triumbari (Investigation; Methodology; Writing – original draft); Agostino Chiaravalloti (Conceptualization; Methodology; Supervision); Orazio Schillaci (Supervision; Validation); Nicola Biagio Mercuri (Supervision; Validation); Claudio Liguori (Conceptualization; Validation; Visualization; Writing – review & editing).