Biochemical Markers of Musculoskeletal Health and Aging to be Assessed in Clinical Trials of Drugs Aiming at the Treatment of Sarcopenia: Consensus Paper from an Expert Group Meeting Organized by the European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases (ESCEO) and the Centre Académique de Recherche et d'Expérimentation en Santé (CARES SPRL), Under the Auspices of the World Health Organization Collaborating Center for the Epidemiology of Musculoskeletal Conditions and Aging

Literature Reviews and Meeting Consensus

The European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Disorders (ESCEO ASBL) jointly with the Centre Académique de Recherche et d’Expérimentation en Santé (CARES SRL) organized in September 2022, under the auspices of the World Health Organization Collaborating Center for Epidemiology of Musculoskeletal Health and Aging, a working group including scientists, specialists in laboratory medicine and clinician experts in the field of biochemical markers and sarcopenia as well as representatives of the regulatory bodies. The methodology employed in several other publications emerging from different ESCEO working groups was replicated [7, 11,12,13,14]. Three members of the present working group (EC, AL and AM) first prepared, before the meeting, a literature review to identify potential biochemical markers of sarcopenia to be assessed in clinical trials of drugs and possibly in clinical practice in the future. Literature reviews and original studies (either observational or interventional) published until September 2022 were searched on Medline and Scopus using a combination of the following MeSH terms and keywords: (Biomarkers (MeSH) OR [biologic* OR biochemical or laboratory or clinical or serum or surrogate) AND marker*] OR biomarker*) AND (Sarcopenia (MeSH) OR sarcopeni* OR EWGSOP). Additional studies were identified by a manual search of the bibliographies of relevant papers. Experts in the field were also requested to provide additional references. Finally, a free web search on Google Scholar was also performed.

The three members involved in this literature search were asked to prepare a presentation summarizing their findings and to make some preliminary recommendations to be discussed during the meeting.

Then, all experts met during a face-to-face meeting to discuss recommendations for the selection of biochemical markers of musculoskeletal status and the pathophysiological mechanisms of sarcopenia to be assessed in clinical trials of drugs aimed at the management of sarcopenia. The discussion ended when all experts reached a consensus and agreed on the conclusion of the manuscript. The general plan of the manuscript was also discussed and agreed upon by all. The core writing group (AL, CB, RR and EC) provided the first version of the manuscript, and all experts were invited to offer comments and corrections and ultimately approve its contents.

Clinical and Analytical Performances of Biochemical Markers

To be recommended in this paper, the biochemical markers examined needed to encounter clinical evidence as well as analytical performance. Among clinical evidence, the considered biochemical markers were required to be either increased or decreased in patients suffering from sarcopenia or either modified by non-pharmacological strategies aimed at sarcopenia management in older adults. Biochemical markers showing correlations with outcome that are available for phase II studies aimed at sarcopenia management as defined in our previous ESCEO recommendations [7] were also considered. Additionally, the selected biochemical markers were classified into one of the following four categories: useful for stratification of the disease, for monitoring of the disease, to assess response to treatment or to assess drug mode of action. As the current consensus paper did not aim to update the definition of sarcopenia, biochemical markers that only offer a diagnostic perspective of sarcopenia without any known physio-pathological explanation were not listed in the recommendations. Regarding analytical performances, to be selected for recommendations, the biomarkers should be measurable in an accurate and reproducible manner with a widely available method. The blood matrix was preferred over other matrices. Additional but not mandatory criteria were the existence of a standardized method, suitability for high-throughput analysis, feasibility of preanalytical conditions, existence of literature defining biological variation (available through https://biologicalvariation.eu) and other technical limitations.

Selection of Biochemical Markers

The group has identified two sets of biochemical markers to be assessed in phase II or phase III pharmacological trials on sarcopenia. The first category of biochemical markers evaluating musculoskeletal status comprises biochemical markers of muscle mass, neuro-muscular junction, muscle turnover and myokines. The second set comprises biochemical markers evaluating nonmuscle-specific pathophysiological mechanisms, also referred to hereafter as causal factors. This second set includes three subclasses: adipokines, hormones, and inflammatory biochemical markers. At least one biochemical marker per subclass for each set, except for muscle mass biochemical markers (see related section), should be selected if the pharmacological trial is a phase II or a phase III trial. Recommendations for the selection of these chemical biochemical markers, as well as their time point assessment, are summarized in Table 1.

Table 1 Recommended biochemical markers for the conduct of any new phase II or phase III pharmacological trial in sarcopeniaSet 1. Musculoskeletal biochemical markers

This first set of biochemical markers is intended to evaluate the musculoskeletal status of sarcopenic patients. Ideally, biochemical markers included in this set should be highly specific to muscle, associated with muscle mass or strength, and sensitive to interventional trials. Additionally, the previous “ESCEO update on recommendations for the conduct of clinical trials for drugs aiming at the treatment of sarcopenia” identified several biochemical markers of “muscle-bone interaction” as potentially applicable outcomes for phase II studies [7]. Major clinical evidence and analytical constraints for using this set of musculoskeletal biochemical markers in pharmacological trials are reported in Tables 2 and 3.

Table 2 Clinical evidence for the use of musculoskeletal biochemical markers in the conduct of pharmacological trials in sarcopeniaTable 3 Analytical aspects for the use of biochemical markers for the conduct of pharmacological trial in sarcopeniaBiochemical Markers Specific to Muscle Mass

In addition to imaging techniques, a few biochemical tools, namely, the deuterium-labelled creatine (D3-Cr) dilution test and sarcopenia index (SI), were developed to evaluate muscle mass. In pharmacological trials, biochemical markers specific to muscle mass are expected to provide additional information that may help in patient risk stratification. Of note, given the poor specificity of SI to muscle mass, this ratio has been reclassified in the muscle turnover subclass.

The D3 creatine dilution test has been developed to ensure an analytical quantification of muscle mass, where imaging techniques are more considered to reflect fat-free mass or bone- and fat-free lean mass [15]. Thus, although well correlated, dual-energy X-ray absorptiometry (DXA) and D3-Cr dilution tests should not be considered equivalent [16]. The D3-Cr dilution test is based on the ingestion of an oral solution of deuterium-labelled creatine (D3-Cr) by a fasting patient followed by the measurement of both the labelled and total creatine and creatinine by liquid chromatography coupled with tandem mass spectrometry (LC‒MS/MS) in urine before and four days after ingestion [17]. As creatine directly enters the muscles where a certain amount is nonenzymatically converted to creatinine to be excreted in urine, an algorithm based on the ratio of D3-Cr to unlabelled creatine can provide an accurate measurement of muscle mass.

The D3-Cr dilution test has shown interesting clinical evidence, such as associations with “hard” clinical outcomes. Indeed, D3-Cr muscle mass appears to be an independent predictor of self-reported incident mobility disability together with walking speed [18] but also a predictor of self-reported disabilities in activities of daily living [19] and risk of hip fracture [20]. However, most of these observations have been made in the same cohort of older men (the osteoporotic fracture in men cohort) [18,19,20], and the D3-Cr dilution test was only evaluated in one cohort of postmenopausal women [21]. Furthermore, most often D3-Cr muscle mass was divided by body weight in the abovementioned studies, whereas body weight itself is associated with these difficult clinical outcomes.

From an analytical perspective, this method has some limitations that make the D3-Cr dilution test impractical for use in pharmacological trials. Indeed, it is only available in a few highly specialized laboratories without any external quality assessment or ring-test that allows lab-to-lab data comparison. Of note, this method requires a standardized procedure for D3-Cr ingestion and urine collection.

Given the analytical limitations of the D3-Cr dilution test, the group recommends not using the method in pharmacological trials at present. In addition to the need for a more widely available method, the group also identified that the method should be validated in other cohorts before any recommendation in clinical trials. Therefore, also taking into account that it is presently the only analytical test highly specific to muscle mass, the group concluded that muscle mass should be assessed according to the EWGSOP2 revised definition without additional biochemical markers Myokines

Myokines are muscle-secreted small proteins (5–20 kDa) with autocrine, paracrine or endocrine effects. Myokines contribute to muscle maintenance, acting, for example, on metabolism, angiogenesis, and inflammation [22]. In aging, myokine secretion and the sensitization of the muscle to these myokines are altered, leading to a disturbance in the balance between anabolic and catabolic effects with consequent age-related muscle atrophy [23]. Therefore, the group agreed that myokines are interesting tools in pharmacological trials to decipher the drug mode of action and physio-pathological mechanisms of muscle protein turnover. Myokines might also be helpful in evaluating secondary outcomes for phase II studies. However, it is premature to determine whether myokines are useful for patient stratification, monitoring of therapies or monitoring side effects.

Myostatin, also called growth and differentiation factor 8 (GDF-8), is a member of the transforming growth factor-β (TGF-β) superfamily and is mostly seen as a muscle growth suppressor [22]. Indeed, when binding activin type IIA and IIB receptors or TGF-β receptors, myostatin suppresses mammalian target of rapamycin (mTOR)-mediated protein synthesis [24]. However, some argue that myostatin can promote muscle growth through different downstream pathways [22]. Myostatin is considered specific to skeletal muscle even if it is also expressed in adipose and cardiac tissues [22].

Myostatin is probably the most studied myokine. However, many studies have yielded conflicting data on the relationship between myostatin and its role in age-related muscle atrophy. Indeed, while most studies found an association between higher myostatin blood concentration and higher muscle mass, this association is not systematically observed [25]. Nevertheless, myostatin is a good predictor of one-year mortality as a “hard” clinical outcome in patients on hemodialysis [26]. Regarding the association between myostatin and muscle function, studies have shown either increased myostatin concentrations in participants with better muscle function or no (or only men-specific) association [25]. Part of these conflicting data may stem from a sex-dependent expression pattern [27, 28]. Regarding its variability with age, myostatin assessment through a highly specific LC‒MS/MS method showed a decline in myostatin concentration with aging in men but not in women [29]. However, many other studies have shown a relatively steady state or increased serum myostatin level with age [25, 30].

Another part of these discrepancies may come from the analytical heterogeneity of the assays. Indeed, myostatin is highly similar to its homologous growth and differentiation factor 11 (GDF-11) [29]. Thus, older kits are known to present significant cross-reactivity with several TGF-β superfamily members [27]. Therefore, whenever possible, LC‒MS/MS methods to measure myostatin should be favoured.

Follistatin is an antagonist of TGF-β ligands, including myostatin and activin A, by acting on myogenic transcription factors. Follistatin has been correlated with muscle mass and muscle function in women in a small number of studies [28, 31], but this association was not confirmed in men [28]. However, in mid- to long-term resistance training intervention trials, follistatin and/or follistatin/myostatin ratio were increased. The same observation was not consistently found for myostatin alone [32,33,34,35]. Analytically, several ELISA and RIA assays are easily available to measure follistatin, but preanalytical considerations and biological variability are not properly described [36].

The myostatin-follistatin system has long been considered a possible target for sarcopenia therapies, and several clinical trials are ongoing, as reviewed by Skrzypczak and colleagues [37]. Although it is unclear whether these proteins are good biochemical markers to monitor the disease or the drug efficacy, myostatin and follistatin should be considered as a couple that helps decipher the physio-pathological drug mode of action.

The group recommends that clinical trials with drugs directly targeting the myostatin/follistatin system should follow the two proteins in both phase II and phase III trials with a precise statistical analysis for men and women. To this end, the follistatin/myostatin ratio can be optionally calculated. However, when the target is an entirely different system, these biochemical markers should be measured in phase II and considered optional in phase III if no changes have been observed in the phase II study. Additionally, when physical training is associated with pharmacological therapies, the standardized procedure for blood collection should include a delay of at least 24 h between the last exercise and venepuncture. Indeed, myostatin and follistatin are acutely increased several hours after exercise but return to baseline after approximately 24 h [38]

Activin A is another member of the TGF-β superfamily that preferentially binds the activin type IIA receptor. Activin A is also considered a negative regulator of muscle growth, acting through the same pathways as myostatin. However, Activin A’s contribution to sarcopenia physiopathology is still a theoretical concept with an evident lack of cohort-based evidence.

The group considers that future research is required before any recommendation for using Activin A in clinical trials for sarcopenia can be made

Growth factor differentiation-15 (GDF-15) is also a member of the TGF-β superfamily, and its expression is induced by stress or myocardial infarction [39]. Several cohorts have shown increased GDF-15 expression in sarcopenic participants [40, 41]. GDF-15 was also sometimes, but not systematically, associated with handgrip strength, skeletal muscle index (SMI) [42,43,44] and physical performance tests [45, 46]. Myostatin and follistatin expression is induced upon acute physical exercise [47]. However, the few long-term interventional trials that tested GDF-15 expression upon resistance training in a sarcopenic cohort failed to observe any longitudinal change in GDF-15 expression [32, 35]. The GDF-15 could also not predict sarcopenia occurrence or evolution in a 2-year follow-up period [40, 48].

Several ELISAs are available on the market for GDF-15 measurement. Nevertheless, GDF-15 is increased with age and chronic kidney disease [49], and doubts have been raised regarding the existence of a circadian rhythm [39]. Given these analytical and clinical considerations, GDF-15 does not appear to be a good biomarker to assess response to treatment. However, adding GDF-15 concentrations to physical performance tests may have some added value to understanding the therapies' effects on physio-pathological mechanisms.

Given that GDF-15 has shown some interesting associations with muscle mass, muscle strength and performance tests but has failed thus far to show modifications in longitudinal follow-up, the group recommends the optional use of GDF-15 in phase II or phase III considering that baseline levels may help in stratifying patients. The standardized measurement procedure should include a 24 h free exercise period before venepuncture and a defined time for blood collection

Irisin is a myokine secreted by skeletal muscles under physical exercise, although it is not a member of the TGF-β superfamily. Irisin is responsible for the browning of white adipose tissue [50], and irisin injection in mice induces muscle hypertrophy with increased protein synthesis [51]. This mechanism might be at least partially regulated by the myostatin/follistatin couple, as irisin is increased in myostatin knock-out mice or after recombinant follistatin injection [50].

Irisin is decreased in sarcopenic persons in several cohorts with very few discordant data [52,53,54]. Irisin is also regularly associated with muscle strength or mass [55, 56]. Nevertheless, irisin has not been studied thus far either in non-pharmacological interventional trials or as a prognostic biomarker of outcomes. Analytically, the analytical precision of ELISA is not accurate enough, and physiological variation is poorly characterized [57].

The group considers it premature to recommend irisin use in clinical trials at present Neuromuscular Junction

By including performance tests and muscle strength in the definition of sarcopenia, the EWGSOP group states that sarcopenia is “a multidimensional concept that not only involves muscles but also central and peripheral nervous function, including balance” [1]. Therefore, not only muscle-specific biochemical markers but also biochemical markers of the neuromuscular junction are required to evaluate muscle integrity, as any impairment to the neuromuscular junction could lead to decreased capacities in using muscles and producing volitional tasks. In pharmacological trials, any modification of these biochemical markers should be integrated with performance tests to understand potential drug modes of action. It is premature to determine whether these biochemical markers may help monitor the disease. However, it is conceivable that these biochemical markers could reflect positive effects on cognition or neurological side effects of therapies.

C-terminal agrin fragment (CAF), also called CAF-22 when referring to the smaller fragment of 22 kDa, is a byproduct of agrin released during the remodeling of the neuromuscular junction. CAF is increased in sarcopenic patients in various cohorts and is associated with the skeletal muscle index [58,59,60,61,62]. It has also been reported to be lower in older dancers than in their sedentary counterparts [63]. Clinically, this biomarker looks very promising. However, there is currently no commercially available assay for CAF.

Given that the appropriate technology is not available at the moment, the group does not currently recommend this biomarker in clinical trials. Nevertheless, as a clinically promising biomarker, developing a widely available and accurate method for CAF determination could modify this recommendation

Brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factors (GDNF) are other biochemical markers of neuromuscular junctions and neuroinflammation. BDNF and GDNF are neurotrophic factors expressed by motor neurons [64]. BDNF and GDNF participate in motor axonal regeneration and neuronal plasticity through a paracrine effect [

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