The Utility Of Myositis-specific Autoantibodies In Clinical Phenotyping Of Idiopathic Inflammatory Myopathies And Their Pathogenic Role In Cancer-associated Myositis

Backgroud:Idiopathic inflammatory myopathies(IIM)are a systemic autoimmune disease with the etiology and pathogenesis are still not well understood.Their clinical manifestations are diverse and heterogeneous and they often involve a variety of organs and are associated with an increased risk of cancer.The previous studies reported that myositis-specific antibodies(MSAs)could distinguish myositis patients into more homogeneous groups and were of great significance in judging the clinical phenotype,efficacy response and prognosis of IIMs.This study was divided into three parts.The first part was to explore the clinical phenotype related to MSAs(anti-NXP-2 antibodies),and to clarify their relevance to the disease activity index..The second part was to systematically study the association of MSAs with cancer-associated myositis.The third part was to analyze the influence of genetic alterations and differential expression of transcription intermediary factor 1(TIF1)-γ genein the pathophysiology of cancer-associated myositis(CAM).Part Ⅰ.Differential clinical associations of anti-nuclear matrix protein-2 autoantibodies in patients with idiopathic inflammatory myopathiesObjective:To investigate the associations between anti-NXP-2 autoantibody levels and disease activity as well as calcinosis severity.Methods:The serum levels of anti-NXP-2 autoantibodies were determined in 709 idiopathic inflammatory myopathies(IIMs)and also serially measured by an in-house enzyme-linked immunosorbent assay using recombinant MORC3.Patients with anti-NXP-2 autoantibodies were divided into two subgroups:with or without calcinosis.The associations of anti-NXP-2 autoantibody levels with organ-specific disease activity,serum creatine kinase(CK)levels,and calcinosis severity were investigated in cross-sectional and longitudinal analyses.Non-parametric Mann-Whitney U test,Fisher’s exact test,Spearman correlation coefficient analysis and mixed linear regression model were used for statistical analysis.Results:A cross-sectional analysis of 56 patients(38 without calcinosis and 18 with calcinosis)with anti-NXP-2 autoantibodies showed that the levels of anti-NXP-2 autoantibody were positively correlated with the physician global assessment visual analog scale(PGA VAS),muscle VAS,and CK levels in patients without calcinosis,while no such association was found in patients with calcinosis.The longitudinal study revealed strong correlations between the anti-NXP-2 antibody levels and PGA,constitutional,cutaneous,gastrointestinal,muscle VAS,and serum CK levels in patients without calcinosis,but only modest correlation with PGA and constitutional VAS in patients with calcinosis.Of note,in patients without calcinosis,the anti-NXP-2 autoantibodies could turn negative in clinical remission;while reappeared with disease relapse.No association was observed between anti-NXP-2 levels and calcinosis severity.Conclusion:This study indicated that anti-NXP-2 autoantibodies served as a useful marker for disease activity,especially in patients without calcinosis.The differential associations between anti-NXP-2 autoantibody levels and disease activity suggested a phenotypic difference between patients with and without calcinosis.Part Ⅱ.Study on the relationship between myositis-specific autoantibodies profiles and cancer-associated myositisObjective:Cancer is a significant complication contributing to increased mortality of idiopathic inflammatory myopathies(IIMs),and the association between IIMs and cancer has been extensively reported.Myositis specific autoantibodies(MSAs)can help to stratify patients into more homogeneous groups and may be used as a biomarker for cancer-associated myositis.In this study,we aimed to systematically define the cancer associated MSAs in IIMs.Methods:Serum anti-Mi-2a,anti-Mi-2β anti-TIF1-γ,anti-NXP2,anti-SAE1,anti-MDA5,anti-SRP,anti-Jo-1,anti-PL-7,anti-PL-12,anti-OJ,anti-EJ and anti-HMGCR were detected by commercial line dot assays and enzyme-linked immunosorbent assay(ELISA).Through long-term follow-up to understand whether IIMs patients will develop cancer and their outcomes.The cancer risk with different MSAs was estimated by standardized incidence ratio(SIR).Paraneoplastic manifestation,such as the close temporal relationship between myositis onset and cancer diagnoses in patients with different MSAs were also evaluated.Meanwhile,we investigated the prognoses of patients with cancer-associated myositis(CAM)with different MS As.Kruskal-Wallis test,Chi-square test and Log-rank test were used for statistical analysis.Results:A total of 72 patients in 617 IIMs patients had cancer,with a positive rate of 11.7%.Among these 72 individuals,38 tested positive for anti-TIF1-γ,three for anti-NXP2,four for anti-SAE1,10 for anti-aminoacyl-tRNA-synthetase(anti-ARS)antibodies(included five anti-Jo-1,three anti-PL-12,one anti-PL-7 and one anti-EJ antibodies);one each for anti-MDA5,anti-HMGCR,and anti-SRP,14 for MSAs-(patients who carried none of these MS As,hereinafter referred as the "MSAs-" group).Compared with the general Chinese population,IIMs patients with anti-TIF1-γ antibodies(SIR=17.28,95%CI:11.94 to 24.14);anti-NXP2 antibodies(SIR=8.14,95%Cl:1.63 to 23.86);or anti-SAE1 antibodies(SIR=12.92,95%CI:3.23 to 32.94),or who were MSAs-negative(SIR=3.99,95%CI:1.96 to 7.14)faced an increased risk for cancer.There was no association between specific MSAs subtypes and certain types of cancer(p>0.05).The median duration of IIMs at cancer-diagnosis did not differ significantly between the groups:+0.19 years in the anti-TIF1-γ group(a plus sign signifies cancer developing after myositis onset;a minus sign signifies cancer developing before myositis);+0.5 years in the anti-NXP2 group;+0.46 years in the anti-SAE1 group;-3.00 years in the anti-Jo-1 group;+0.25 years in the anti-PL-12 group;and +0.67 years in the"MSAs-" group(p>0.05).Myositis complicated with cancer can be classified as CAM and cancer-unrelated to myositis.There were no prognostic differences among the cancer-associated myositis(CAM)patients from different MSAs subgroups.However,in comparison to those with cancer-unrelated to myositis,CAM had a worse prognosis,with an age-and sex-adjusted Cox hazard ratio(HR)of 10.8[95%CI:1.38-84.5,P = 0.02]for all-cause mortality.Conclusion:Our study demonstrates,in what is to our knowledge the largest population examined to date,that anti-NXP2 antibodies,anti-SAE antibodies,MSAs",and previously reported anti-TIF1-γ antibodies,are all associated with an increased risk of cancer in IIMs patients.Moreover,our data suggest that in some cases,anti-HMGCR,anti-Jo-1 and anti-PL-12 antibodies production might also be driven by malignancy.This can aid in the etiologic research of paraneoplastic myositis and clinical management.Part Ⅲ.A preliminary study on the mechanism of cross immunity induced by tumor-associated antigen TIFl-y in cancer-associated dermatomyositisObjective:To investigate the possible mechanisms of genetic alteration and differential expression of transcription intermediary factor 1(TIF1)-γ in the development of CAM.Methods:Paired tumor tissues and blood samples from 6 anti-TIF1-γ-positive CAM patients and 5 anti-TIF1-γ-negative CAM were obtained.The target region capture and sequencing technique was used to analyze the mutation type of TRIM33 in cancer cells.The protein level of TIF1-γ in tumor,muscle and skin tissues of CAM patients was detected by immunohistochemistry.Results:In the tumors of 6 anti-TIF1-γ-positive CAM patients,four cases had somatic mutations and 4 cases exhibited loss of heterozygosity(these four cases included two cases that did not contain a detectable somatic mutation).Only 1 of the 5 anti-TIF1-γ-negative CAM patients had somatic mutations and none of patients exhibited loss of heterozygosity.Compared with the anti-TIF1-γ-negative group,the mutation probability of TRIM3 3 was significantly higher in the anti-TIF1-γ-positive group,and the difference was statistically significant(100%vs 20%,P=0.03).Four cases of anti-TIF1-γ-positive CAM patients had muscle specimens and TIF-1-γ protein was expressed in their muscle tissues.Meanwhile,the protein was expressed in one of three anti-TIF1-γ-negative CAM patients.Notably,TIF1-γprotein was expressed in the skin tissue regardless of whether the anti-TIF1-γ antibody is positive or negative.Conclusion:The tumor tissue of anti-TIF1-γ-positive CAM patients is more susceptible to gene mutation of TRIM33,thus inducing the body to develop an immune response against TIF1-γ to produce the mutated TIF-y protein,and the expression of TIF1-γ protein in muscle and skin tissue may become the target of the above immune response.The results provide some evidence that myositis may be caused by the tumor associated antigen TIF-γ-induced cross-immune responses.