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Using MRI to quantify skeletal muscle pathology in Duchenne muscular dystrophy: A systematic mapping review

Abstract There is a great demand for accurate non‐invasive measures to better define the natural history of disease progression or treatment outcome in Duchenne muscular dystrophy (DMD) and to facilitate the inclusion of a large range of participants in DMD clinical trials. This review aims to investigate which MRI sequences and analysis methods have been used and to identify future needs. Medline, Embase, Scopus, Web of Science, Inspec, and Compendex databases were searched up to 2 November 2019, using keywords “magnetic resonance imaging” and “Duchenne muscular dystrophy.” The review showed the trend of using T1w and T2w MRI images for semi‐qualitative inspection of structural alterations of DMD muscle using a diversity of grading scales, with increasing use of T2map, Dixon, and MR spectroscopy (MRS). High‐field (>3T) MRI dominated the studies with animal models. The quantitative MRI techniques have allowed a more precise estimation of local or generalized disease severity. Longitudinal studies assessing the effect of an intervention have also become more prominent, in both clinical and animal model subjects. Quality assessment of the included longitudinal studies was performed using the Newcastle‐Ottawa Quality Assessment Scale adapted to comprise bias in selection, comparability, exposure, and outcome. Additional large clinical trials are needed to consolidate research using MRI as a biomarker in DMD and to validate findings against established gold standards. This future work should use a multiparametric and quantitative MRI acquisition protocol, assess the repeatability of measurements, and correlate findings to histologic parameters.


| INTRODUCTION
Although no treatment currently can prevent or reverse the effects of Duchenne muscular dystrophy (DMD), several pharmacologic, cellular, and genetic approaches may reduce disease effects and improve the quality of life for DMD patients. 1 In assessing the outcomes of clinical trials of these treatments, objective biomarkers must be developed and assessed longitudinally in natural history studies. 2 Ideally, these biomarkers should be compared against results from the histological analysis of muscle biopsies, which has historically been used as the gold standard for disease assessment. However, due to the inherent invasive character of biopsy, non-invasive methods to extract information corresponding to biological characteristics of DMD muscle are in great demand.
Several non-invasive imaging modalities have the potential to provide objective insight on DMD disease progression: for example computed tomography (CT), positron emission tomography (PET), ultrasound (US), and magnetic resonance imaging (MRI). 3,4 While CT can be used to detect structural changes in muscle tissue such as fat deposition, 5 its use in DMD is limited by potential side effects of X-ray exposure and insensitivity for differentiation between adipose and connective tissue, especially in younger patients. 6 The use of PET allows identification of reduced metabolism due to replacement of muscle with connective tissue or fat in DMD but this could be obscured by increased uptake of brown fat. 3 Evidence of increased muscle echogenicity is seen early on US in DMD, providing a potential imaging tool to track disease progression. 7 Due to its high soft-tissue contrast, high resolution, and absence of ionizing radiation, MRI has emerged as a promising noninvasive method for imaging skeletal muscles. 8 Various MRI sequences have been widely used to monitor DMD disease qualitatively 9,10 and quantitatively, [11][12][13][14] with a general hypothesis that structural changes in muscle will be reflected in MRI images. Qualitative analysis allows subjective grading of disease features, such as the level of signal intensity on T 1 weighted (T 1 w) and T 2 weighted (T 2 w) MRI protocols. Advances in quantitative MRI (qMRI) sequences (ie, T 1 map, T 2 map, diffusion-weighted imaging [DWI], and Dixon) have provided promising results in objectively monitoring DMD patients longitudinally. 15 Nonetheless, with no consensus on particular imaging sequences or analysis methods and tools to be used, the use of MRI as a DMD biomarker remains underappreciated.
Accordingly, this study aimed to investigate different MRI sequences that have been used for diagnosis and quantification of disease severity in DMD skeletal muscle, to answer two fundamental questions: (a) What are the MRI sequences that have been used to assess changes in skeletal muscle in DMD and pre-clinical models of DMD? (b) What are the methods that have been used to analyze specific MRI sequences in order to differentiate healthy and diseased muscles, assess therapeutic response, or differentiate different stages of DMD? 2 | METHODS

| Data sources and search method
This review was performed in accordance with the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analyses) guidelines, 16 with details summarized in Supporting Information Material S1, which is available online. A systematic search was conducted of the databases of Medline, Embase, Scopus, Web of Science, and Engineering Village with the aid of an experienced librarian on 2 November 2019. The following terms were used for the searches: "magnetic resonance imaging" and "Duchenne muscular dystrophy".
The results from all five searches were combined using EndNote and automatically verified to ensure the exclusion of articles that had the same title or were written by the same authors and/or published in the same journal. The remaining articles were considered for study selection.

| Study selection
Two authors (L.A. and J.F.G.) independently reviewed the journal and paper titles and abstracts. The selected papers then underwent fulltext screening and eventually were reviewed to include relevant information. Any discrepancies regarding study inclusion or during the subsequent review process were resolved by full-text screening and discussion. All papers with any of the components missing were passed directly to the full-text screening.

| Inclusion and exclusion criteria
We included only clinical or preclinical studies reporting MRI of skeletal muscles with the following aims: characterization of differences between healthy and DMD muscle; characterization of the natural history of DMD disease progression; correlations between muscle MRI and current standard of care diagnostic methods (blood biochemistry, molecular assessment, clinical assessment, functional assessment, or histology); assessment of the effect of an intervention (medication, supplements, contrast medium, exercise, muscle stimulation, injury). No restrictions were made based on location, size of field of view (FOV), or the number of skeletal muscles imaged.
Only studies written in English were included in this review. Prior to the review, a decision was made to exclude any study with too few participating subjects: <7 for patient studies and <3 for animal studies.
Therefore, all individual case reports and studies with no information on the number of subjects were excluded. In addition, all the following types of studies were excluded: (a) papers describing non-original research (editorials, commentaries, letters, reviews, meta-analyses, opinions, family descriptions, conference summaries, conference abstracts, registries, study protocols, technical notes, pictorial essays), (b) papers not based on in vivo subjects (histology, phantom, ex vivo, synthetic data), (c) papers reporting animal models other than murine (MDX) or canine (golden retriever muscular dystrophy, GRMD), (d) papers reporting imaging of non-skeletal muscles, and (e) papers not differentiating between different dystrophy types.

| Data extraction
A data form was designed to extract the following information: DMD affected or carrier, species (human, murine, canine), population (number of DMD, number of DMD-control, number of healthy-treated, number of healthy-control), MRI field strength (midfield [≤1T], high field [>1T and ≤3T], very high field [>3T and ≤7T], and ultra-high-field [>7T]), MRI sequence, use of MRI contrast agent, the number of muscles or compartments analyzed, gold standard used (none, histology, molecular assessment, functional ability, clinical assessment, biochemistry), the aim of the study (differentiation, natural history, the effect of the intervention, clinical trial), study design (cross-sectional, longitudinal), type of data analysis, and type of performance analysis. All selected papers were independently reviewed by two reviewers, and data extraction was cross-checked by a third reviewer (A.E.). Disagreements between the reviewers were resolved by consensus and arbitrage by another author (J.N.K). The following data were extracted from the full papers: year of publication, human or animal study, type of animal model used, type of study (cross-sectional or longitudinal), number of subjects, number of muscles imaged, MRI sequence(s) used, contrast agent used, image analysis method. All papers were divided into the following categories: (a) cross-sectional studies to differentiate healthy and diseased muscle or to grade disease severity, (b) cross-sectional studies to correlate the imaging results to the current standard of care diagnostics, and (c) longitudinal measurements to characterize the natural history of disease progression or assess changes due to intervention.

| Data synthesis and analysis
The following MRI sequences were identified: T 1 w, T 2 w, T 1 -mapping, T 1 w MRI sequences use spin-lattice relaxation by using a short repetition time (T R ) and a short echo time (T E ), while T 2 w MRI sequences assess spin-spin relaxation by using a long T R and a long T E . PD-weighted MRI created by a long T R (reduce T 1 ) and a short T E (minimize T 2 ) to reflect the actual density of protons. 17 PD-weighted MRI sequences share some features of both T 1 w and T 2 w MRI. T 1 -mapping, also referred to as native T 1 -mapping, measures pixel-wise T 1 relaxation time or spin-lattice or longitudinal relaxation time, the decay constant for the recovery of the z-component of the nuclear spin magnetization towards its thermal equilibrium value. T 1 -mapping traditionally uses a series of independent single-point T 1 w measurements at different inversion times.
As this acquisition method is rather time-consuming, several MRI sequences were developed to speed up the acquisition, eg, inversion recovery spin echo, echo planar imaging, inversion recovery spoiled gradient echo, and variable flip angle. 18 T 2 -mapping measures pixel-wise T 2 relaxation time or spin-spin or transverse relaxation time, which represents the time constant that the transverse components of magnetization decay or diphase toward their thermal equilibrium value. T 2 map is typically reconstructed from a series of T 2 w images at various T E. 19 DTI is an MRI technique that assesses restricted (anisotropic) water diffusion using bi-polar preparation gradients. It is often visualized using pseudo-colors and can be used to produce fiber tract images. Additionally, DTI provides structural information about muscle. 20 The Dixon technique is an MRI method for fat suppression and/or fat quantification that uses a simple spectroscopic imaging technique for water and fat separation. 21 Image analysis methods to assess muscle quality were divided into five categories: semi-qualitative scoring (QS), first-order statistics (FOS), volume/cross-sectional area (V/CSA), fractions (FR), and texture methods (TM).

| QS
The semi-qualitative assessment of DMD imaged by MRI is usually graded by two or more independent observers using several ordinal scales with gradually increasing muscle involvement. Often, for purposes of anatomical clarity and synergistic function, muscles have been graded together as a muscle group. These methods provide an overall impression of the degree of increased signal intensity in T 1 w and/or T 2 w MRI: 1. Grading of fat infiltration using T1w MRI without fat suppression: infiltration, less than 30% of the muscle was infiltrated; 3, moderate infiltration with 30%-60% infiltrated muscle; 4, severe infiltration with more than 60% infiltrated muscle. 6 2. Grading of edema patterns using T2w MRI (without fat suppression) originally proposed by Borsato 26

| FOS
These methods characterize muscle by non-spatial descriptors, that is, the gray-level frequency distributions as mean, median, SD, skewness, maximum, minimum, and range.

| V/CSA
Under the assumption that structural and compositional changes in skeletal muscle affected by DMD alter cross-sectional area (and consequently also the muscle volume), some studies characterize muscle by V/CSA. The methods for V/CSA assessment vary from completely manual annotations to automatic segmentation protocols.

| FR
The muscle composition is assessed with intramuscular fat fraction using 1 H-MRS 28 or chemical shift-based imaging techniques 29 by the integration of the phase-corrected spectra from the lipid and 1 H 2 O parts of the spectrum. Additionally, two-point Dixon 21 or three-point Dixon 22 methods acquire images at identical positions with water and fat protons in-phase and opposed-phase, respectively. The intramuscular fat fraction, also referred to as (relative) fat content map, is generated from the pixel-wise fat and water fraction.

| Risk of bias assessment
Conclusions reached through systematic reviews are influenced by the quality of the papers included, largely related to various sources of potential bias. 34 In an effort to minimize selection bias, we performed an initial wide search without limitations and conducted the review according to PRISMA guidelines. 16 Given the heterogeneous nature of this review, for instance, including both human and animal studies and a range of study designs, we did not systematically assess the potential for bias across all 127 papers. Since the recommendations generated by this review are most important for patient care, we assessed longitudinal DMD studies reporting clinical data. The included papers were subjected to rigorous appraisal by two authors (L.A. and A.E.) using the extended Newcastle-Ottawa Quality Assessment Scale (NOS) 35,36 adapted to include assessment of outcome, the validity of follow-up, and dropout rate. This assessment awards points across four domains to a total of 10 points: selection (4), comparability (1), exposure (3), and outcome (2). Based on the score, the datasets were categorized as high (score ≥9), moderate (6 ≤ scores ≤8), or low (scores ≤5). Disagreements were arbitrated by a third author (J.F.G.). Figure 1 presents details on literature inclusion after reviewing the paper title, the journal-title, abstract, and full paper. In summary, of the 5238 potentially relevant articles, 482 (9.2%) were considered for inclusion. After a full-text screening for all relevant papers, an additional 352 were excluded. At this stage, three additional papers were excluded as they reported the results on the same dataset as that used in another paper included. For these publications, the most recent one was included in the analysis. Therefore, the data from 127 original papers 6,[8][9][10][11]15,23,[27][28][29] were extracted for further analysis.  15,65,100,104,105,107,108,125,140,145 The studies included MRI field strengths ranging from midfield to ultra-high field. The majority of studies were cross-sectional. A variety of methods were used to analyze the MRI data in decreasing order: FOS, followed by FR, V/CSA, SQ, raw MRI data, and TX.

| General characteristics
The number of imaging studies assessing DMD is increasing steadily, that is, from 13 studies published up to 1994 to 54 papers in the period 2014-2019 (see Figure 2A). In the latest 5 y were murine and 11% were canine. Figure 2B illustrates the composition of subjects used, with a clear trend of increasing numbers, especially in clinical studies. Recent pre-clinical studies have tended to include vehicle-treated control animals. Figure 2C shows the distribution of studies according to the MRI sequence used. Generally, the contribution of T 1 w images has been historically high. However, T 2 map, DWI, and Dixon sequences have gained interest in the latest 10 y. Dixon maps have increasingly been used since their introduction in 1984. 21 In the most recent 5 y (2014-2019), the most-used MRI sequences included: weighted images (40%) and MRI maps F I G U R E 1 Results of the literature search. PRISMA flow diagram for study collection, 16 showing the number of studies identified, screened, eligible, and included in the systematic review T A B L E 1 Characteristics of the included papers (n = 127) detailing MRI sequences and magnetic field, subjects used, and study type

| Papers reporting correlation experiments
Of 127 papers included in this review, 29 (23%) correlated the current standard of care for assessment of DMD (eg, clinical functional F I G U R E 3 Distribution of studies (subject type, population, and number of muscles assessed) by different field strengths (A-C) and different MRI sequences (D-F). HF, high field strength; MF, medium field strength; T 1 w, T 1 -weighted; T 2 w, T 2 -weighted; T 1 m, T 1 -mapping; T 2 m, T 2mapping grading or functional ability) with the semi-quantitative MRI assessment, 10,37,47 or quantitative MRI assessment. 6,11,38,39,44,54,56,59,63,73,76,79,88,89,113,122,123,126,137,138,[143][144][145]153 These papers reported a statistically significant correlation between fatty infiltration of muscle on MRI and functional status. 10,37,47 Only five papers correlated histologic findings with either semiqualitative or quantitative assessment 8 Studies have not established the optimal number of MRI slices to assess. One paper compared findings from local (one slice) and multislice approaches in semi-qualitatively assessing T 1 w images. 9 They concluded that caution needs to be taken when using single-slice acquisitions, as they may not appropriately represent overall disease status.
Consistent with this result, another paper found that manual segmentation of every fifth slice, with subsequent interpolation to the muscle length, more accurately predicted effects than mid-muscle belly analysis. 8 Seven papers reported correlations between different quantitative parameters. In general, all papers used Spearmanʼs rank or Pearsonʼs correlation coefficient, but the use of these methods was not rationalized. The time of creatine rephosphorylation, reflecting mitochondrial oxidative capacity) and assessed using 31 P-NMR, 82 showed a tight correlation with perfusion parameters, ranging from 0.66 (maximum perfusion) to 0.99 (total perfusion in the first 30 s).  58 Even with a such small number of publications, there were controversies. For example, the correlation between T2map and fatty infiltration assessed using 3D gradient-echo Dixon sequences was assessed as significant (0.7) in one study 142 but not in another. 90

| Papers reporting agreement data
The agreement between two graders in a semi-qualitative study was for intra-observer to 13% for interobserver assessment. 97,98 Considering intra-observer variability, redrawing ROIs was shown to change the average signal intensity up to 21%, but no significant interobserver differences between the three observers were revealed. 57 In addition, Bland-Altman methods were used to assess the agreement between the two repeated measurements, 66 so as to determine the difference between the fat fraction calculated at the central slices and fat fraction calculated over the whole muscle, 67 the degree of inter-rater agreement, 90 or between two different analysis methods 29,91,141 or two different sequences. 87,110 Considering intra-observer variability, 31 (Table 2). Three studies included two types of controls: non-treated patients and age-matched healthy subjects. 91,135,138 As illustrated in Table 2, several studies lack critical information to assess and interpret the risk of bias. For instance, in intervention have also become more prominent, in both clinical 44,111 and animal model subjects. 88,127,147 Further studies that quantitatively document the validity of MRI as a longitudinal biomarker to track DMD disease progression in clinical trials, so as to complement functional testing, are critically needed. The publications included are too heterogeneous in their methodology and reporting to perform either meta-analysis or even assess the risk of bias because quantitative inputs needed to assess the risk of bias are not available.

| Risk of bias
To better promote the wider application of MRI in assessing DMD in clinical trials across centers and among radiologists, agreement studies are needed to establish the reliability of imaging results. Our review indicated that only 16% of studies assessed agreement. Even though this is slightly higher than the average numbers reported in radiology research, 154  On the other hand, Spearmanʼs rank correlation coefficient is a nonparametric measure of rank and assesses how well the relationship between two variables can be described using a monotonic function (no assumption on linearity). With no reasoning behind the use of either of these statistical instruments, the choice appeared rather arbitrary.
The characteristic histopathological lesions of DMD, including myofiber necrosis and regeneration, inflammation, fat deposition, and fibrosis, lend themselves to assessment by texture analysis methods.
In this systematic review, a comparison between the performance of different methods for a certain classification task was not possible due to the large variety in the datasets used and the classification tasks posed. Identification of new, optimal heterogeneity features, including combinations, will require validation against large welldefined datasets from other clinical outcomes. The design of future studies should also take into account requirements from pattern recognition, that is, a balanced number of subjects and features, crossvalidation, independent test datasets, and prospective study design.
Satisfying these requirements would allow a more reliable evaluation of the value of heterogeneity features.
We were particularly concerned about the bias in the studies included 36   Note: Medication: *all DMD patients on medication, no baseline without medication; ** part of the DMD population on steroids, no baseline without medication; NT, no treatment, natural history study.
Gold standard: CA, clinical assessment; FA, functional ability.
high or moderate values, consistent with a good quality paper. During the period our study was under review, an additional systematic review was published 155 that assessed papers correlating muscle MRI and function in DMD patients. Ropars et al. 155 assessed the potential for bias and associated quality of these papers using the Joanna Briggs Institute checklist with three of 17 included papers appraised as low quality.
Clearly, in our study and this other recent DMD systematic review, conclusions and associated recommendations should be weighted toward high-quality papers. However, these studies often suffered from other design problems, including small sample sizes that could result in chance findings. There was also the potential for prevalence-incidence (Neyman) bias that arises due to patients with either mild or severe disease being excluded. 156 For instance, individuals with severe DMD are often not studied, causing a potential error in the estimated association between treatment and an outcome. Similarly, the impact of corticosteroids was not always considered, introducing possible performance bias.
Furthermore, the choice of functional ability as the main outcome in many studies introduced the potential for apprehension bias. 157 Finally, with regard to bias, given the natural tendency to not publish negative findings, the results of this systematic review are probably skewed towards positive results. Studies in which MRI did not track with disease severity likely were underreported.
Despite the limitations of this study, we established that the broader use of qMRI in DMD has led to a desirable increase in quantitative versus semi-qualitative biomarkers, and a tendency to validate findings against objective histopathological and functional outcome parameters. However, without the incorporation of MRI into larger clinical trials, this effort will have minimal impact.

| Recommendations for the clinician
Since the available data are insufficient to propose a specific acquisition protocol or method of image analysis, we recommend that future work emphasize MRI techniques aimed at measuring fat deposition, edema, myofiber necrosis and regeneration, inflammation, and fibrosis. This necessitates a multiparametric and quantitative approach.
This will likely include T1-mapping, T2-mapping, Dixon methods (for fat and water quantification), DTI, and/or MRS. Specifically, the clinician should aim to quantify fat deposition and edema using sequences with and without chemical fat saturation or short tau inversion recovery. Clinicians are encouraged to include functional MRI in order to better understand the features of the disease and detect the therapeutic effect. For example, information obtained from DTI and MRS provides exciting insights into skeletal muscle substructure and metabolism. Emerging MRI methods should also be considered. The development and refinement of semi-automated image analysis techniques are expected to further improve precision. Importantly, efforts should be made to assess and improve the repeatability of measurements and to correlate MRI findings with histology at the local level.