Patient registry
This was a single-center, retrospective, observational cohort study including 48 Chinese patients who were diagnosed with ANM-SRP according to clinical, serological, and pathological criteria. Demographic data, clinical features, and initial drug treatment information were collected (Supplementary Materials). Written informed consent was obtained from all patients (or an appropriate family member where the patient was unable to provide consent). The procedures were performed in accordance with the ethical standards of the responsible committee on human experimentation and approved by the Institutional Review Board.
Serum myositis antibody test
An immunoblot kit was used to determine the serum antibodies to SRP by detecting the 54kD subunit. The membrane strips were pretreated, incubated with patient serum, and subjected to enzyme binding. The results were scanned using EUROlineScan software, and were recorded as negative, weak positive (+), positive (++), and strong positive (+++). All the patients recruited were strong positive (+++).
Muscle MRI
Thirty-six patients underwent bilateral thigh MRI (tMRI) (3.0 T, GE 1.5 Sigma Twin Speed; GE Healthcare, Waukesha, WI, USA). Axial T1-weighted MRIs were used to evaluate the degree of fatty infiltration according to the modified Mercuri scale (0–5 scale; from normal appearance to complete fatty infiltration). Axial short T1 inversion recovery sequences were used to assess the degree of edema (0–5 scale, from normal to moderate intrafascicular global edema)[11]. Edema and fatty infiltration scores were calculated in the gluteus maximus at the pelvic level and thigh muscles (vastus intermedius, vastus medialis, vastus lateralis, rectus femoris, biceps femoris, semitendinosus, semimembranosus, adductor magnus, sartorius, long adductor, and gracilis) at the mid-thighs. Additionally, we compared the thigh MRI of 36 patients with 36 controls (autoimmune necrotizing myopathy patients with negative anti-SRP antibody) to observe the damage in the tMRI. Total edema and the degree of fatty infiltration were compared before and after treatment as an indicator of therapeutic effect.
Muscle pathology
Muscle biopsies were taken from the biceps brachii or quadriceps femoris of all patients. Serial frozen sections were stained with hematoxylin and eosin (HE), modified Gomori trichrome, periodic acid-Schiff, oil red O, adenosinetriphosphate (ATP) enzyme (pH 4.5 and 10.8), NADH-tetrazolium reductase, succinate dehydrogenase (SDH), and cytochrome C oxidase (COX) stains. The sections were immunohistochemically stained with primary antibodies against human CD3, CD4, CD8, CD20, CD68, major histocompatibility complex class-I (MHC-I), membrane attack complex (MAC), dystrophin, sarcoglycans, and dysferlin.
BAFF and BAFF-R measurement
To further clarify the role of B lymphocytes in the therapeutic effect on ANM-SRP, immunohistochemical staining of B cell activating factor (BAFF), B cell activating factor receptor (BAFF-R) and CD19 was performed in 29 patients. The positive cellular expression of BAFF, BAFF-R and CD19-positive cells (numbers of positive cells / numbers of muscle fibers) was calculated per high-power microscopic field: 10 high-power microscopic fields (400×) were randomly selected from each section and the numbers of staining-positive cells and muscle fibers were counted under each high-power microscopic field (those with more than ½ area were included and those with less than ½ area were omitted). The average value of 10 high-power fields was calculated. Correlation of the positive cellular expression of BAFF, BAFF-R, CD19-positive cells was analyzed.
Additionally, muscle tissues of 14 patients with ANM-SRP and four healthy controls were sampled for semi-quantitative analysis of BAFF and BAFF-R. Radioimmunoprecipitation assay (RIPA) lysate (including protease inhibitor and phosphatase inhibitor) was added to the tissue samples for homogenization and the protein was extracted after lysis on ice for 30 min. An equal amount of protein (80 μg) was suspended in loading buffer, denatured at 100°C for 5 minutes, and loaded on an SDS-PAGE gel. After being electrophoresed in 10% polyacrylamide gel, transferred electrophoretically to nitrocellulose membrane, the membrane was blocked with non-fat milk buffer for 1 hour and then incubated with the primary antibodies overnight at 4°C. The primary antibodies were IgG-specific for BAFF (Abcam, ab16081, 1:500), BAFF-R (Abcam, ab5965, 1:500), and actin (Abcam, ab8226, 1:3000). After three 5-minute washes (20-mmol/L Tris, pH 7.6, 8g sodium chloride, 0.05% Tween-20), blots were incubated for 50 minutes with horseradish peroxidase-conjugated goat anti-rabbit IgG or rabbit anti-rat IgG (1:5000). After washing, bound IgG was detected auto-radiographically by enhanced chemiluminescence (Quantity One v.4.6.2).
Clinical follow-up and efficacy evaluation
The modified Rankin Scale score (mRS) was evaluated at 3, 6, and 12 months after treatment to evaluate the therapeutic effect on the basis of glucocorticoid therapy combined with other immunosuppressive agents or intravenous immunoglobulins (IVIg) in the study period. Patients were divided into non-refractory and refractory groups. If patients’ muscle strengths returned to normal or was close to normal, the mRS scores were 0–2 points and those patients were placed in the non-refractory group. If there was still obvious limb weakness after 12 months of immunotherapy, the mRS scores were 3–5 points, or patients had a relapse, then the patients were placed into the refractory group [8, 13]. The relapse criteria were that the serum creatine kinase(CK) returned to above the patient’s baseline level, rose to more than three times the reference upper limit, and limb weakness worsened again (excluding other causes such as exercise, electrolyte disturbance, thyroid dysfunction, or drug induction) [9].
Twenty-five patients with ANM-SRP were followed up by tMRI. We monitored the muscle fatty infiltration and edema changes by tMRI for 3, 6, 12, 18, and 24 months after treatment. Imaging indicators were the average change rates of thigh muscle fatty infiltration and edema (the scale gap of tMRI / interval time between before and after treatment) for 3, 6, 12, 18, and 24 months after treatment.
Statistical analysis
All analyses were performed using SPSS 17.0 software. Single factor and binary logistic regression analyses were used to compare the clinical, tMRI changes and pathological features between the two groups. The Mann–Whitney U test or Kruskal–Wallis test was used for the continuous variables and Chi-square test for categorical variables. Multivariate logistic regression analysis was performed on related predictors for refractory disease. Explanatory variables were selected using a liberal criterion (p<0.10) for inclusion in the multivariate regression model. For all statistical analyses, significance was accepted as p<0.05.