Radiological Characteristics of Patients With Anti-MDA5–Antibody-Positive Dermatomyositis in 18F-FDG PET/CT: A Pilot Study

Objective: To elucidate the 18F-fluorodeoxyglucose (FDG) PET/CT characteristics and its prognostic value in the patients with anti-melanoma differentiation associated protein 5 antibody positive (anti-MDA5+) dermatomyositis (DM). Methods: This retrospective cross-sectional study included 26 patients with anti-MDA5+ DM and 43 patients with anti-MDA5 negative (anti-MDA5–) idiopathic inflammatory myopathy (IIM) who were examined by 18F-FDG PET/CT from January 1, 2017 to December 31, 2020. The maximum standardized uptake value (SUVmax) of multiple organs and other clinical characteristics of the patients were measured and analyzed. Results: Compared with the anti-MDA5– group, the patients in the anti-MDA5+ group showed higher bilateral lung SUVmax (p = 0.029), higher SUVmax of spleen (p = 0.011), and bone marrow (p = 0.048). Significant correlations between the spleen SUVmax and serum ferritin levels (r = 0.398, p < 0.001), erythrocyte sedimentation rate (ESR) (r = 0.274, p = 0.023), platelet count (r = −0.265, p= 0.028), myositis disease activity assessment score (r = 0.332, p = 0.005), bone marrow SUVmax (r = 0.564, p < 0.001), and bilateral lung SUVmax (r = 0.393, p < 0.001) were observed. Conclusion: 18F-FDG PET/CT was found valuable in quantifying the pulmonary focal inflammation and potentially unveil the distinctive characteristics and pathophysiological mechanisms in the patients with anti-MDA5+ DM.


INTRODUCTION
Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune diseases, which display several clinical manifestations, such as muscle, skin, or articular involvement, interstitial lung disease (ILD), and sometimes associated with malignancy (1). The myositis-specific autoantibodies contribute to delineate the homogenous subgroups of IIM (2,3). Dermatomyositis (DM) associated with anti-melanoma differentiation associated protein 5 antibody (anti-MDA5+) is typically defined by the presence of characteristic skin lesions (Gottron's papules, heliotrope rash) and ILD, whereas the clinical signs of myositis are usually mild or absent (4). Rapidly progressive ILD (RP-ILD), which is a serious and life-threatening condition, leads to high mortality in anti-MDA5+ DM (5)(6)(7).
The studies on the predictive risk factors for RP-ILD in the patients with anti-MDA5+ DM revealed that biomarker, such as ferritin, Krebs von den Lungen-6 (KL-6), MDA5 antibody titers, and baseline forced vital capacity (FVC) were considered to be important in evaluating the disease activity and prognosis (8,9). The image techniques were widely used in the evaluation of anti-MDA5+ DM. High resolution computed tomography (HRCT) was often performed to verify the pathological classification of ILD in the patients with anti-MDA5+ DM patients (5,10). 18 F-fluorodeoxyglucose (FDG) PET/CT had proven to be a useful, hybrid technique (combining nuclear and CT imaging) for detecting the morphologic and functional changes in a variety of diseases. PET/CT was usually performed to screen malignancy in the patients with IIM (11). Moreover, abnormal increases in 18 F-FDG uptake on the PET/CT images were observed in proximal muscles, which are correlated to inflammatory lesions in the patients with IIM (12,13). A good correlation was also found between the maximal 18 F-FDG standardized uptake value (SUVmax) and the results of muscle biopsy in the patients with myositis patients (14). Li et al. (15) reported that 18 F-FDG uptake on the PET/CT images is observed not only in the proximal muscles but also in interstitial lung disease. In this study, high sensitivity and specificity for detecting rapidly progressive interstitial lung disease were reported when the 18 F-FDG SUVmax was higher than 2.5 on PET/CT (15). Our previous study reported that higher 18 F-FDG uptake of the interstitial lesions of the patients with IIM on PET/CT images is significantly associated with RP-ILD and poor outcome (16).
However, the PET/CT characteristics remain unclear in the patients with anti-MDA5 positive DM. Thus, this retrospective study was conducted to elucidate the PET/CT characteristics and their effect on the clinical course and its prognostic value in anti-MDA5+ DM. In addition, we compared the patients with anti-MDA5+ with a group of patients with IIM without anti-MDA5 antibody (anti-MDA5-) to confirm the specificity of the PET/CT characteristics.

Patients Population
This retrospective cross-sectional study was conducted in the First Affiliated Hospital, Zhejiang University School of Medicine (FAHZJU) in China. In-hospital databases were searched for 18 F-FDG PET/CT scans of the patients with IIM that were performed between January 1, 2017 and December 31, 2020. The inclusion criteria for this study were: (1) age over 18 years old. (2) The diagnosis of DM, polymyositis (PM), or amyopathic dermatomyositis (ADM) was based on the 2017 the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) classification criteria (17). (3) A 18 F-FDG PET/CT scan was performed during hospitalization. The exclusion criteria were: (1) newly identified or unsolved malignancies; (2) clarified overlap syndrome with other connective tissue diseases; (3) myopathy related to thyroid dysfunction, strenuous exercise, inherited metabolic disorders, and drug-induced myositis; (4) loss of follow-up for any cause within 3 months after hospitalization. A written informed consent was acquired from all the included patients and the study was approved by the Institutional Review Board of FAHZJU (No. IIT202100194).

ILD and HRCT Imaging Assessments
In each patient with IIM, the HRCT scan was performed within 1 month before or after the 18 F-FDG PET/CT scans. Thin-section CT images were obtained in the supine position during breathholding and deep inspiration. The chest HRCT images were reassessed by an experienced radiologist and a respiratory specialist who were blinded to the clinical information of the patients. The radiological ILD patterns were defined as per the American Thoracic Society/European Respiratory Society criteria, which was a multidisciplinary decision made by the rheumatologists, respirologists, and radiologists (19). RP-ILD was defined as follows in this study: previous or concurrent diagnosis of ILD, presenting with progressive dyspnea or hypoxemia, and a worsening of interstitial change on chest HRCT radiograph within 1 month after the initial visit or onset of respiratory symptoms (20,21).
FDG-PET/CT Imaging 18 F-FDG PET/CT had to be performed within 2 weeks of diagnosis. The PET/CT acquisitions were carried out according to the European Association of Nuclear Medicine procedure guidelines (22). The patients were instructed to avoid exercise for at least 24 h and fast for at least 6 h prior to PET/CT detection. The mean serum glucose level was 5.5 mmol/L (range 5.2-6.1 mmol/L) and confirmed before the injection of 4.0 MBq/kg 18 F-FDG. Then, 1 h after the intravenous injection, whole-body FDG-PET images were obtained using a dedicated PET scanner (Biograph, Sensation 16, Siemens systems, Germany). Low-dose CT was performed with 120 kV and 30 mAs prior to emission scanning. 18 F-FDG uptake was calculated by the following formula: SUV (g/ml) = regional radioactivity concentration (Bq/ml)/[injected dose (Bq)/body weight (g)].
Visual examinations for the detection of increased 18 F-FDG uptake were retrospectively performed by a single trained radiologist who was blinded to the diagnosis, complications, and outcome of all the patients involved. FDG uptake was calculated by using SUVmax of a circular region of interest (ROI) with a fixed diameter of 20 mm, containing lung, liver, spleen, bone marrow, heart, digestive system, cerebellum, and muscle. Bilaterally proximal muscle observed by PET/CT contained deltoideus, biceps brachii, trapezius, iliopsoas, gluteus maximus, gluteus medius, and quadriceps.

Statistical Analysis
All the statistical analyses were performed using SPSS 22.0 (Chicago, IL, USA), R 3.6.1 and Graphpad Prism 8.0 (San Diego, CA, USA). The variables were expressed as mean ± SD or median (quartiles). The categorical variables were expressed as frequencies and percentages. The comparisons were performed   by using the t-test or Mann-Whitney U-test for the quantitative variables. Unordered categorical variables were compared using the chi-square test or Fisher's exact. The survival curves were drawn by using the Kaplan-Meier method and compared by using the log-rank tests. The correlation between the two continuous variables was assessed utilizing Pearson's linear analysis. All the statistical tests were performed two-sided, and a P-value < 0.05 was considered statistically significant.

Patient Characteristics
A total of 69 patients who satisfied the inclusion/exclusion criteria were included in the study (Figure 1). As characterized in Table 1, 26 patients with anti-MDA5+ DM were incorporated into this study, encompassing 17 with DM and 9 with ADM. Eighteen of them (69.2%) were women and the mean age was 55.92 ± 7.68 years old. The medium followup time was 11.9 (4.00, 23.80) months. The anti-MDA5+ and the anti-MDA5-group were similar with respect to age, gender, course of disease, and duration of diagnosis delay. Among the patients with anti-MDA5+ DM, 53.8% had fever, 38.5% had splenomegaly, and 15.4% had gastrointestinal hemorrhage. We found no difference in the prevalence of co-existing infectious complications between the anti-MDA5+ and the anti-MDA5-group. However, the prevalence of preceding carcinoma was higher in the anti-MDA5-group (p = 0.005). The majority of the patients with anti-MDA5+ DM had ILD (96.2%) and a considerable proportion had RP-ILD (42.3%). About 83.7% patients had ILD in the anti-MDA5-group, with 23.3% patients had RP-ILD, owing to a considerable proportion of anti-synthetase syndrome (19/43). Furthermore, compared with the anti-MDA5-group, the patients in the anti-MDA5+

FDG PET/CT Scan Findings
By comparing the patients with anti-MDA5+ DM and anti-MDA5-, we found a higher bilateral lung SUVmax (p = 0.029) in anti-MDA5+ group (Figures 3A,D). Besides, a significant positive correlation between serum ferritin and bilateral lung SUVmax (r = 0.449, p < 0.001, Figure 4A) in the patients with anti-MDA5+ DM. However, no significant correlation was observed between the ESR, serum CRP levels, MYOACT score, and bilateral lung SUVmax (Figures 4B-D, respectively). These results suggest that FDG uptake (SUVmax) in the lungs by FDG-PET/CT imaging may be useful to evaluate the location and activity of ILD in the patients with anti-MDA5+ DM.
Interestingly, the patients with anti-MDA5+ DM were found to have significantly higher SUVmax of spleen (p = 0.011) and bone marrow (p = 0.048) than those in the patients with anti-MDA5-IIM (Figures 3B,C,E,F, respectively). Significant correlations between the spleen SUVmax and serum ferritin levels (r = 0.398, p < 0.001), ESR (r = 0.274, p = 0.023), platelet count (r = -0.265, p = 0.028), and MYOACT score (r = 0.332, p = 0.005) were as well observed (Figures 5A,B,D,E, respectively). However, no significant correlation was identified between the spleen SUVmax and serum CRP/WBC levels (Figures 5C,F, respectively). In addition, we recognized a significantly positive correlation between the spleen SUVmax and bone marrow SUVmax (r = 0.564, p < 0.001, Supplementary Figure 1A), as well as bilateral lung SUVmax (r = 0.393, p < 0.001, Supplementary Figure 1B). However, there existed no significant correlation between the bone marrow SUVmax and bilateral lung SUVmax (Supplementary Figure 1C) levels. These results suggested that the spleen FDG uptakes (SUVmax) might be valuable in the assessment of disease activity in the patients with anti-MDA5+ DM.
The patients with anti-MDA5-IIM had higher SUVmax of bilateral biceps (p = 0.006). In addition, we could not detect significant differences in SUVmax of the other organs: liver, heart, digestive system, cerebellum, and other proximal limb muscles between the patients with anti-MDA5+ and anti-MDA5-IIM ( Table 1).
To explore the predictive value of PET/CT scans for the unfavorable short-term outcome (death within 3 months), we further analyzed the clinical data of 69 patients with IIM. The spleen SUVmax was found to be significantly correlated with the unfavorable short-term outcome (p = 0.001) (Supplementary Table 2). A ROC curve analysis revealed an area under the curve of 0.732 and a cutoff value of 2.319 ( Supplementary Figure 2A). In addition, the spleen SUVmax was found to be significantly correlated with RP-ILD (p = 0.031) (Supplementary Table 3

DISCUSSION
To the best of our knowledge, this is the first retrospective cohort study reported the PET/CT characteristics of the patients with anti-MDA5+ DM. A previous case report mentioned that 18 F-FDG PET/CT was capable of detecting bilateral pulmonary inflammatory 1 month prior to the development of RP-ILD in a patient with an anti-MDA5+ DM (23). However, the PET/CT characteristics remain unclear in the patients with anti-MDA5+ DM.
F-Fluorodeoxyglucose PET/CT had shown multiple diagnostic values for the patients IIM, the main interest of which focused on cancer screening and measures of myositis activity (24). In the patients with IIM, a good correlation was found between the proximal muscle SUVmax and the serum creatine kinase, muscle strength, muscle biopsy findings in several studies (13,14,25). 18 F-FDG PET/CT can help to verify the muscle inflammation activity in IIM. However, lower sensitivity for the myositis diagnosis was reported for 18 F-FDG PET/CT imaging compared with the conventional examinations, In this retrospective study, the patients with anti-MDA5+ DM were found to suffer more prominent pulmonary inflammation in 18F-FDG PET/CT than those with anti-MDA5-. The evaluating role of PET/CT in pulmonary focal inflammation had been demonstrated in the preceding studies. Uehara et al. reported that the 18 F-FDG FDG SUVmax changed after treatment and was consistent with the activity of ILD (26). The patients with progressive disease in Granulomatous-Lymphocytic ILD had significantly higher SUVmean on 18 F-FDG-PET/CT which suggested that this modality may be valuable for identifying the patients with active pulmonary inflammation and progressive disease (27). 18 F-FDG-PET/CT was found to be useful to evaluate the pulmonary inflammation in other connective tissue disease associated ILD, such as systemic sclerosis (28,29).
Notably, the patients with anti-MDA5+ DM had a higher FDG uptake of the spleen which was significantly associated with myositis disease activity and pulmonary inflammation. Likewise, these findings might suggest that the patients with anti-MDA5+ DM had distinctive characteristics, and potentially represents unique pathophysiological mechanisms. Given that serum ferritin has been used as a marker of macrophage activation (30), we presumed that spleen SUVmax of the patients with anti-MDA5+ may be related to macrophage activation.
In this study, we observed that the patients with anti-MDA5+ showed some similarities with macrophage activation syndrome (MAS), such as leukocytopenia, thrombocytopenia, elevated serum ferritin, and splenomegaly, but were not fully compliant with the diagnostic criteria of MAS. Zuo et al. (10) reported that the infiltration of CD163-positive macrophages into alveolar spaces was significantly higher in RP-ILD group of the patients with DM, which as well-indicated that macrophage activation might be involved in the pathogenesis of RP-ILD in DM.
Although the pathophysiology of anti-MDA5+ DM is still not fully understood, a widely accepted guess is an abnormal inflammatory response which led to multi-system involvement (lungs in particular). On the one hand, the alveolar cell damage is a consequence of systemic hyper-inflammation, on the other hand, if the integrity of the epithelial-endothelial lining is damaged, the alveolar macrophages will produce proinflammatory cytokines and chemokines, thus resulting in a cytokine storm. More recently, the association between the choline phospholipid metabolism and macrophage immune responsiveness has been identified. 18 F-Fluorocholine was reported to image and quantify the macrophage activity in pulmonary interstitial infiltrates of coronavirus disease 2019 (COVID-19) pneumonia (31,32), which might inspire similar investigation in the patients with anti-MDA5+ DM. In addition, Fibroblast Activation Protein specific PET/CT imaging was performed as a promising new imaging modality for fibrotic ILD and lung cancer (33). Its potential clinical value for monitoring and therapy evaluation of CTD-ILD should be investigated in the future studies.
The present study has several limitations, such as retrospective nature and the small sample size. The HRCT scans were evaluated qualitatively, instead of quantitative analyses. 18 F-FDG PET/CT was identified as an important diagnostic tool when evaluating active inflammation and disease progression in the patients with anti-MDA5+ DM. A prospective cohort study with larger sample will be essential to confirm our findings and fill in the gaps.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Institutional Review Board of the First Affiliated Hospital, Zhejiang University School of Medicine. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
JLin: conceptualization and methodology. HC: data curation and writing-original draft preparation. DX and JLia: verification of IIM diagnosis. YL: evaluation of FDG uptake in multiple organs. HC and YY: identification of ILD and RP-ILD. YS: writing and reviewing. YH: investigation and validation. All authors contributed to the article and approved the submitted version.