CuATSM PET to diagnose age-related diseases: a systematic literature review

Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) positron emission tomography (CuATSM PET) is a non-invasive imaging technique that can be used to detect hypoxia and inform prognosis in cancer. Hypoxia and oxidative stress are also hallmarks of various age-related diseases. Whether CuATSM PET has a role in the evaluation of hypoxia and oxidative stress in age-related diseases has yet to be established. The aim of this systematic review is to evaluate the utility of CuATSM PET in the diagnosis and management of age-related diseases. EMBASE, Medline, Scopus, Web of Science and Psychinfo were systematically searched for articles published between January 1st 1997 and February 13th 2020. We included articles published in English reporting the use of CuATSM PET in the diagnosis and management of age-related diseases in humans or animals. Nine articles were included describing CuATSM PET measures in neurological and cardiovascular disease. There was higher CuATSM uptake in diseased compared to control subjects in Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), myocardial ischemia (MI), cardiac dysautonomia and atherosclerosis. Higher CuATSM uptake was seen in diseased compared to control anatomical areas in PD, cerebrovascular disease (CVD), MI and atherosclerosis. CuATSM uptake was associated with disease severity in PD, ALS, CVD and atherosclerosis. An association between CuATSM uptake and disease duration was shown in atherosclerosis. CuATSM uptake is higher in neurological and cardiovascular diseases and associated with disease severity and duration. Further investigations using CuATSM PET in other age-related diseases are needed.


Introduction
Aging is associated with an increased risk of developing age-related disease [1]. Age-related diseases, such as neurological and cardiovascular diseases, represent the leading cause of death worldwide [2,3]. Many of these diseases share important hallmarks of aging including accumulation of DNA damage [4], mitochondrial dysfunction [5], decreased autophagy [6], and production of reactive oxygen species (ROS) [7]. The latter can lead to oxidative stress and hypoxia [7,8], which in turn can induce ferroptosis [9,10]. This iron-dependent programmed cell death is seen in age-related diseases such as Parkinson's disease (PD) [11], Alzheimer's disease (AD) [12,13] and cardiovascular diseases [14].
Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (CuATSM), first developed as a positron emission tomography (PET) hypoxia marker [15], has been used to evaluate tumor hypoxia, predict disease prognosis and tailor therapy in various types of cancer [16][17][18][19]. More recently, increased CuATSM PET uptake has been seen in PD in the absence of hypoxia [20]. This is thought to be due to CuATSM's ability 1 3 to detect redox changes [21]. Hypoxia and redox changes such as oxidative stress [22] also play an important role in age-related diseases [23][24][25]. Whether CuATSM PET has a role in the diagnosis and management of age-related human diseases has yet to be established.
The aim of this systematic review is to evaluate the utility of CuATSM PET, as a non-invasive imaging technique, in the diagnosis and management of age-related diseases.

Protocol registration
The protocol of the systematic review was registered with the International prospective register of systematic reviews (PROSPERO): CDR/code CRD42020167009. The systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards [26].

Search strategy
A systematic search was performed with an academic senior liaison librarian (P.C.) in five electronic databases: EMBASE, Medline, Scopus, Web of Science and Psychinfo. The search included keywords: "CuATSM", "PET" and other synonyms of CuATSM ("diacetyl-bis(N4-methylthiosemicarbazone) copper (II)", "diacetyl-di (N4-methylthiosemicarbazone) copper") and all radioactive isotopes of CuATSM ( 60, 61, 62,64 CuATSM). The first investigation of CuATSM as a hypoxia marker was conducted in 1997 [15]. Therefore, articles published between January 1st 1997 and February 13th 2020 were included in this search. The full search strategy is available in Appendix 1. The reference section of each included article was also used to identify additional related articles.

Study selection
The articles obtained using the search strategy were assessed for eligibility against predefined inclusion and exclusion criteria independently by two authors (J.J.Y. and N.M.) by screening titles and abstracts. The same reviewers then screened the full text of resulting articles. A third reviewer (A.B.M.) resolved any disagreements between the authors regarding eligibility. Inclusion criteria were: articles using CuATSM PET in the diagnosis and management of agerelated diseases in humans or animals, published in English . Opinion articles, conference abstracts, editorials, letters to the editor, and case reports were excluded.

Data extraction and quality assessment
The following variables were extracted independently by two reviewers (J.J.Y. and N.M.) from the included studies: first author, year of publication, title, study design, study population, sample size, age, proportion of females, country, agerelated disease studied, intervention, primary purpose of the scan, type of PET tracer, duration of the scan, scan results, clinical outcomes measured, other reported outcomes, adverse events related to intervention and statistical analysis used.
Articles were assessed for their risk of bias independently by two reviewers (J.J.Y. and N.M.) using the Newcastle Ottawa Scale (NOS) for cohort studies [27,28]. A system of points was given to the following categories: (i) selection of the study population, (ii) comparability, and (iii) description of the outcome (Appendix 2). A study was given a maximum of one point in each item within the "selection" and "outcome" categories and a maximum of two points in the "comparability" category. The scores ranged from 0 to 9. A score of seven or more was classified as high quality.

Analysis
We summarized the difference between CuATSM uptake in diseased and control subjects, and between diseased and control anatomical areas. Additionally, the association between CuATSM uptake and severity and duration of agerelated disease was summarized.

Search strategy
The search retrieved 9633 articles. After exclusion of duplicates (n = 5774), articles were screened for title and abstracts of which 244 were screened for full text and nine articles were included in this review (Fig. 1).

Study characteristics
The included articles were classified according to the type of age-related disease: neurological (four articles) and cardiovascular disease (five articles) ( Table 1). A total of 46 humans with neurological disease (mean age 62.7 ± 6.58 years, n = 19 female) and 22 controls (mean age 43.6 ± 8.33 years, n = 3 female) as well as seven humans with cardiovascular disease (aged 63-80 years, n = 3 female (no controls)) were included. A range of animal models including dog, macaque, rabbit and mouse served to study cardiovascular disease (total n = 40 diseased subjects and n = 7 controls). The aim of all included articles was to use CuATSM PET to diagnose or prognosticate 1 3 age-related diseases. The Cu isotope, type, duration and anatomical location of the scan differed according to the disease and type of subjects studied. All articles describing neurological diseases used a 20 min dynamic 62 CuATSMPET scan and measured standard uptake value (SUV) in the brain: striatum for PD [20,29], whole-brain for ALS [30] (Fig. 2a) and cerebrovascular disease [31] (Fig. 2b). All cardiovascular disease articles used dynamic 30-60 min PET scan except one which used static 10 min PET scan data [32] (Fig. 2c). The 64 Cu isotope was used to image atherosclerosis in rabbit and mouse [33,34] and myocardial ischemia (MI) in dogs [35]. The 61 Cu isotope was used to image cardiac dysautonomia in macaques [36] and 62 Cu for coronary artery disease (CAD) in humans [32].

Quality assessment
The total NOS scores are shown in Appendix 3. Three articles [30,33,35] were scored as high quality and the remaining as medium quality. Table 2 shows the difference in CuATSM uptake in diseased compared to control subjects, and diseased compared to control anatomical areas, as well as the association of CuATSM uptake with disease severity and duration. Compared to healthy controls, CuATSM uptake was higher in the  [20,29,30]; (b) human cerebrovascular diseases studies [31]; (c) human, dog, macaque, mouse and rabbit cardiovascular disease studies [32][33][34][35][36] striatum of PD patients and the bilateral cortices around the central sulcus, including the motor cortex and right superior parietal lobule, of ALS patients [20,30]. Higher CuATSM uptake was seen in affected compared to non-affected hemispheres in PD patients [29]. CuATSM uptake in the brain was significantly associated with disease severity expressed as the total and motor unified Parkinson's disease rating scale (UPDRS) in PD patients [20,29] and revised amyotrophic lateral sclerosis functional rating scale (ALSSFR-S) in ALS patients [30]. No association was observed between CuATSM uptake and ALS disease duration [30], while a positive trend was observed between CuATSM uptake and PD duration [20].

Qualitative description of CuATSM PET uptake and age-related diseases
Compared to controls, higher CuATSM uptake was detected in the myocardium of dogs after MI, the anterior ventricle of macaques with cardiac dysautonomia and in the aortic arch of mice with atherosclerosis [34][35][36]. CuATSM uptake was greater in affected compared to non-affected hemispheres in patients with unilateral major cerebral artery occlusion [31], in ischemic compared to normal tissue in dog myocardium and in injured (modelling atherosclerosis) compared to the non-injured femoral artery of rabbits [33,35]. CuATSM uptake was significantly associated with disease severity expressed as the oxygen extraction fraction (OEF) in patients with unilateral major cerebral artery occlusion [31] and with the degree of thickening of the vessel wall in rabbits with atherosclerosis [33]. CuATSM uptake increased significantly from 14 to 23 weeks after atherosclerosis induction in mice [34] suggesting an association between CuATSM uptake and duration of atherosclerosis. Finally, CuATSM uptake was enhanced in one patient with unstable angina compared to patients with stable coronary artery disease [32]. The heterogeneity of study designs and outcome measures precluded a meta-analysis.

Discussion
CuATSM uptake was higher in human and animal subjects with neurological and cardiovascular disease compared to controls and in diseased compared to non-diseased anatomical areas. CuATSM uptake was positively associated with disease severity and duration.

CuATSM PET and neurological diseases
Currently, neurodegenerative diseases like PD and ALS can only be diagnosed when symptoms manifest, while biomarkers allowing earlier detection remain elusive [37][38][39]. Hypoxia and oxidative stress are associated with cell death mechanisms involved in ALS and PD [40,41]. Thus, using CuATSM as a redox change biomarker could lead to earlier detection of neurodegenerative diseases [42]. Due to its sensitivity to disease severity in ALS, PD and cerebrovascular disease [20,[29][30][31], CuATSM PET could also be used as a tool for monitoring therapy. Additionally, vascular cognitive impairment, in part caused by chronic cerebral hypoperfusion [43], is generally not detected until patients present with cognitive decline [44]. Therefore, using CuATSM PET to detect chronic hypoperfusion may result in earlier diagnosis and intervention in cognitive impairment and dementia [45].

CuATSM PET and cardiovascular diseases
CuATSM PET provides a quantitative assessment of myocardial ischemia in research [35] and clinical settings [32] and shows promise in diagnosing systemic atherosclerosis in research settings [33,34,46], but its utility has not yet been translated into clinical practice. Animal and human studies show the presence of hypoxia in atherosclerotic plaques and its link to plaque rupture [47,48]. Thus, using CuATSM PET to image hypoxia in atherosclerosis may provide important pathophysiological insight and help identify vulnerable atherosclerotic plaques, which could leave to improved patient management [49].

CuATSM PET and other age-related diseases
No evidence of the use of CuTASM PET in the detection of hypoxia and oxidative stress in age-related diseases was found other than in neurological and cardiovascular diseases. Other age-related diseases associated with redox changes include chronic kidney disease [50], urinary tract infections [51], type 2 diabetes mellitus [52] and sarcopenia [53]. Hypoxia in tubulointerstitium and glomerular oxidative stress play a role in chronic kidney disease progression [54,55], although blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) has failed to demonstrate this association in the past [55][56][57]. Given the ability of CuATSM PET to detect much lower concentrations of molecules than MRI [58], it may be useful in predicting the progression of chronic kidney disease before any clinical manifestations and hence identify patients who require more intense primary prevention [59,60]. Urinary tract infections are associated with oxidative stress [61] and the accumulation of hypoxia-inducible factor-1α subunit (HIF-1α) [62]. HIF-1α is also induced by hyperglycemia in diabetes [63,64], where inhibition of HIF-1α leads to decreased insulin resistance [65]. Thus, CuATSM PET may also be useful in research into the pathogenesis of recurrent urinary tract infections [66,67] and insulin resistance [68]. Recent evidence suggests that oxidative stress and hypoxia play a role in the development of sarcopenia [53,69]. Given the current need for new imaging tools for sarcopenia [70], the potential Table 2 Standard uptake values of CuATSM and its association with severity and duration of age-related disease stratified by species of CuATSM PET as a non-invasive diagnostic tool for identifying sarcopenia in older adults should be explored.

The potential clinical use of CuATSM PET in age-related diseases
This review has demonstrated the ability of CuATSM PET to delineate hypoxic tissue and showed the association between CuATSM uptake with disease severity in neurologic and cardiac diseases in clinical and pre-clinical models. Given these results, potential future clinical applications of CuATSM PET include disease detection prior to symptom manifestation allowing improved risk factor management, earlier disease diagnosis and sensitive monitoring of disease activity. To achieve clinical translation, future research should focus on studies in humans with larger sample sizes and robust study designs in the diseases already studied, as well as examining a broader range of age-related diseases in which hypoxia and oxidative stress are implicated.

Ionic Cu(II) salts as imaging tracers for age-related diseases
PET CuATSM uptake in cancer and age-related diseases has been demonstrated in preclinical and clinical studies. Interestingly, PET uptake of ionic copper Cu(II) salts, such as [ 64 Cu]CuCl 2 , has also been seen in various cancer xenografts [71] and was found to be comparable to CuATSM in imaging and therapeutic cancer studies [72]. The mechanism of tissue uptake of CuATSM and ionic Cu(II) salts, however, remain controversial [73]. Given the redox changes common to both cancer and age-related diseases, future studies focusing on these mechanisms may clarify to whether Cu(II) salts could also be used in research into age-related diseases [73].

Strengths and limitations
To our knowledge, this is the first systematic review examining the utility of CuATSM PET in the diagnosis and management of age-related diseases. So far, there are only a few studies in this area with heterogeneous study design and small number of study subjects which precluded a meta-analysis. All included articles reported on the diagnostic capacity of CuATSM PET but not on its utility in the management of age-related disease. Given the specificity of CuATSM as an imaging biomarker of hypoxia has been challenged previously [74], we would have liked to report on the sensitivity and specificity of the CuATSM PET scans used in the articles included in this review. However, only one of these articles reported these parameters [31]. This limits the translational potential of the results to clinical and other research settings. Finally, healthy controls in many of these studies were not matched for age and sex.

Conclusions
Hypoxia, oxidative stress and ferroptosis are important hallmarks of various age-related diseases [11-14, 23, 75, 76]. CuATSM PET is a non-invasive imaging technique that detects hypoxia and oxidative stress in neurological and cardiovascular diseases [23-25, 77, 78]. The clinical potential of CuATSM PET in early diagnosis, disease prognostication and monitoring disease activity [79,80] and its utility in research settings in other age-related diseases, such as chronic kidney disease, recurrent urinary tract infections, diabetes and sarcopenia, should be investigated further. Author contributions All authors made substantial contributions to the design of the review, the acquisition, analysis, and interpretation of data; drafted the work or revised it critically for important intellectual content; approved the version to be published; and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Funding This study was supported by an unrestricted grant of the University of Melbourne, Australia received by Professor Andrea B. Maier.
Data availability Not applicable.

Compliance with ethical standards
Conflict of interest None of the authors reported any conflict of interest. Selection 1) Representativeness of the exposed cohort a) truly representative of the average age-related disease in human or animal subjects* b) somewhat representative of the average age-related disease in human or animal subject * c) selected group of users eg nurses, volunteers d) no description of the derivation of the cohort 2) Selection of the non exposed cohort a) drawn from the same community as the exposed cohort* b) drawn from a different source c) no description of the derivation of the non exposed cohort