Histopathological And Molecular Pathological Diagnosis Study Of Idiopathic Inflammatory Myopathies

BackgroundIdiopathic inflammatory myopathies(IIM)are a heterogeneous group of autoimmune disorders characterized by subacute onset and proximal muscle weakness.For a long time,IIM were classified into 3 categories:polymyositis(PM);dermatomyositis(DM)and sporadic inclusion body myositis(sIBM)on the basis of distinct clinical and histopathological manifestation.The diagnostic criteria of IIM have long been concerned in order for patients to be treated timely as IIM are regarded as the largest group of potentially treatable myopathies in both children and adults.The diagnostic criteria of IIM were first proposed by Bohan and Peter in 1975.It highlighted that "definite PM" should contain inflammation infiltration in muscle biopsy,those who did not have inflammation infiltration in their muscle biopsy should be classified as "probable PM" or "possible PM" and skin rash was necessary for the diagnose of DM.In 1980s,Arahata et al found CD8+ T cells invading non-necrotic muscle fibers on both light and immunoelectron microscopy in PM.This phenomenon was established as the canonical pathological criterion for the diagnosis of "definite PM" by Dalakas in 1991.However,it is rarely met in muscle biopsies due to the limitation of sampling bias.This strict diagnosis led to the underdiagnosis of PM and even made PM to be a rare subgroup.Myositis-specific antibody(MSA),an important serum marker less limited by muscle sampling and inter-observer bias,was added into the IIM diagnostic criteria revised by Rheumatologist Targoff in 1997.As a molecular marker of antigen presentation,major histocormpatibility complex class I(MHC-I)is an important marker for reflecting autoimmune pathological process."ubiquitous MHC-I expression" was regarded as "probable PM" in the diagnostic criteria revised by Dalakas in 2003.Besides,the understanding of DM also became comprehensive."mild or early DM" which muscle was slightly involved was proposed in the 1991 criteria and the phenomenon that skin rashes were not detected and perifascicular atrophy was found was diagnosed as "probable DM" in the 2003 criteria.In 2004,IIM criteria proposed by European Neuromuscular Center(ENMC)classified "traditional PM" into PM、non-specific myositis(NSM)and immune-mediated necrotizing myopathy(IMNM)according to the expression of MHC-I and inflammatory infiltration.It also classified DM into "definite DM","probable DM","amyopathic DM" and "possible dermatomyositis sine dermatitis"according to clinical and pathological features.The 2004 ENMC classification criteria were updated in 2012 and 2015,with some modifications such as the name change from IMNM to necrotizing autoimmune myopathy(NAM)and efforts to standardize diagnostic work up.The 2004 ENMC criteria have been widely concerned and applied since they were put forward.However,great confusion was raised as the understanding of the nature and extension of PM,NSM and NAM was quitely different among researchers.The practicability and reproducibility of ENMC criteria also caused controversy.On the one hand,the measures of several pathological features which were used for classification(with the description of "surrounding","scattered","sparse","slight,"and "not evident")were not specifically defined.Different researchers have different understanding for the above pathological descriptions,resulting in serious bias in the judgment of subgroup.On the other hand,pathological manifestations that this classification criterion involves,such as the infiltration of inflammatory cells,were mostly localized.Due to the sample limitations resulting from muscle biopsy,it is inevitable that some subgroups of non-DM/sIBM-IIM may be underdiagnosed and the others may be overdiagnosed,which seriously affects the practicability and reproducibility of the judgement of PM,NSM and NAM.In addition,the pathological features of perifascicular atrophy(PFA)and membrane attack complex(MAC)deposition in capillaries can also be influenced by these two above problems summarized as unclearly pathological definition and sample limitation,which also affect the classification of DM subgroup.In recent years,with the rapid development of antibody detection technology,many kinds of MSAs have been found,which greatly improves the efficiency of the diagnosis of IIM.Furthermore,with the in-depth understanding of all kinds of MSAs,it has been found that each kind of MSAs exhibits its characteristic clinical or histopathological features.Therefore,MSA can assist in medicine usage and the prognosis of treatment outcome.Due to the low sensitivity of PFA and MAC deposition in capillaries in the diagnosis of DM,DM patients with atypical rash or "possible dermatomyositis sine dermatitis" patients were easily missed diagnosis or misdiagnosis.Recently,the application of high-throughput technology provides technical support for detecting novel diagnostic biomarkers with high sensitivity and specificity for DM.The expression of myxovirus resistance protein A(MxA)gene in muscle tissue of DM patients was significantly higher than that of normal control and other control groups.Whether MxA could be used as a new histological diagnostic biomarker for DM has arisen great attention and exploration.Therefore,our study focuses on the histopathological and molecular pathological diagnosis study of IIM in the following fields:Part I Revisiting the Practicability and Reproducibility of ENMC Criteria for Idiopathic Inflammatory MyopathiesThe European Neuromuscular Centre(ENMC)pathological classification criteria of idiopathic inflammatory myopathies(IIM)are debatable due to the sampling bias and vague pathology definition.The aim of our study was to explore their practicability and reproducibility by in-depth analysis of muscle biopsies and subsequent comparisons of the clinical characteristics among polymyositis(PM),non-specific myositis(NSM)and necrotizing autoimmune myopathy(NAM),We conducted a retrospective analysis of 57 cases of IIM excluding dermatomyositis(DM)and sporadic inclusion body myositis(sIBM).These non-DM/sIBM-ⅡM were diagnosed after a comprehensive work up on muscle biopsies at multiple levels with the specific definition of pathological findings.In 57 non-DM/sIBM-IIM cases,25 were classified as PM,15 as NSM,and 17 as NAM.Among them,51 underwent multilevel sectioning examination of biopsies,with pathological changes at different levels warranting diagnostic rectification in 11 patients(21.57%):4 PM were reclassified as NSM,and 7 NSM as NAM.Applying atypical CD8+T cells surrounding non-necrotic muscle fibers resulted in diagnostic rectification from NSM to PM in 2 patients;using 20 T cells(instead of 10)as the threshold for the perivascular infiltration led to diagnostic rectification from NSM to NAM in 9 patients.The percentage of positive anti-SRP autoantibodies was higher in NAM patients than that in PM patients(p=0.010).The CK levels upon the first visit to our hospital was significantly higher in NAM compared with that in PM(p=0.007)or NSM(p=0.016).There were no differences in disease duration,medicine usage or treatment outcomes among these three subgroups.The strict ENMC pathological classification criteria to distinguish non-DM/sIBM-IIMs are of limited practicability and reproducibility,and may be of limited clinical significance.Thus,new diagnosis and classification criteria are needed to be proposed.Part Ⅱ:Idiopathic Inflammatory Myopathies with Anti-mitochondrial Antibodies:Clinical Features and Treatment Outcomes in a Chinese CohortAnti-mitochondrial antibodies(AMAs),the hallmark of primary biliary cirrhosis(PBC),have been detected in many patients with IIM.Although and these AMA-associated IIM frequently show unique characteristics,the detailed clinical features were different among previous reports.We detected AMA in Chinese IIM and summarized the clinical findings of these AMA-associated patients.Of 136 patients,only seven(5.15%)were found to be AMA-positive.PBC was occurred only in 2 of these 7 patients,chronic disease duration in 2 patients and asymmetrical muscle weakness in 4 patients.The mean disease course was 9.71 months,and the mean creatine kinase level was 2325.00 U/L.Myositis-specific antibodies were found in 3 DM patients.According to clinical features and muscle histopathological findings,3 patients were classified as DM,2 as possible PM and 2 as NAM.Of the 6 AMA-positive patients receiving follow-ups of 22-104 months,they all showed marked clinical improvement.Our study indicates that AMAs are relatively rare in Chinese IIM patients.These patients generally show various clinical features and have favorable treatment outcomes.AMA testing may be helpful to confirm the diagnosis of IIM,especially in patients with atypical manifestations.Part III:The Spectrum of Myositis-specific Autoantibodies and Their Associated Clinical Features in Chinese Patients with Idiopathic Inflammatory MyopathiesMyositis-specific antibodies(MSAs)are a group of importantly serological immune marker of IIM and have a pivotal role in the diagnosis of IIM.Each kind of MSA exhibits its characteristic clinical and pathological manifestations,and its associated IIM can be regarded as a distinct clinical subgroup.Our study aimed to explore the positive rate of each kind of MSA and its unique clinical features and treatment outcomes.We detected anti-3-hydroxy-3-methylglutaryl-CoA reductase(HMGCR)by enzyme-linked immunosorbent assay(ELISA)in 153 patients with IIM and other MSAs by line immunoblot in 219 patients with IIM.Of these 219 patients,more than 60%(133/2 1 9)were detected to be MSA-positive.Specially,the positive rate of anti-signal recognition particle(SRP)antibody was the highest,about 23%(50/2 1 9),followed by anti-nuclear matrix protein 2(NXP2)antibody,anti-HMGCR antibody,anti-Mi-2 antibody,anti-transcriptional intermediary factor(TIF)1 gamma antibody and anti-Jo-1 antibody,about 5-9%and the positive rate of other types of MSA was relatively low.The frequency of dysphagia was higher in patients with anti-SRP antibody than in patients with anti-ARS antibody and in patients with anti-MDA5 antibody.The frequency of skin rash was lower in patients with anti-SRP antibody than in patients with other MSA.The frequency of interstitial lung disease(ILD)was higher in patients with anti-MDA5 antibody than in patients with other MSA.The frequency of arthritis was higher in patients with anti-MDA5 antibody than in patients with anti-SRP antibody,in patients with anti-Mi-2 antibody,in patients with anti-TIF1γ antibody and in patients with anti-NXP2 antibody.Compared with patients with anti-SRP antibody,patients with anti-TIF1γ antibody had a higher frequency of myalgia.Compared with patients with anti-SRP antibody,patients with anti-ARS antibody had a higher frequency of arthritis.Patients with anti-TIF1γantibody and patients with MDA5 antibody often had a lower CK level than patients with anti-SRP antibody,patients with anti-HMGCR antibody and patients with anti-Mi-2 antibody.A major of patients with anti-SRP antibody were classified NAM.Patients with anti-HMGCR antibody showed various ENMC pathological subgroups.Patients with anti-Mi-2 antibody,patients with anti-TIF1γ antibody,patients with anti-NXP2 antibody and patients with anti-MDA5 antibody were mostly classified into DM subgroup.Compared with anti-Mi-2 antibody positive patients,anti-HMGCR antibody positive patients often needed corticosteroid and another immunotherapy.Our study suggests that MSAs,especially anti-SRP antibody are commonly seen in Chinese IIM patients.These MSA-positive patients generally show unique clinical and histopathological features.Part IV:Diagnostic Value of Sarcoplasmic Myxovirus Resistance A Expression for DermatomyositisCurrently,pathological features that PFA and MAC deposition in capillaries have a low sensitivity in the diagnosis of dermatomyositis,which can easily lead to missed diagnosis or misdiagnosis in DM patients without skin rash or with atypical skin rash.We aimed to evaluate sarcoplasmic myxovirus resistance A(MxA)expression in DM and the diagnostic value of sarcoplasmic myxovirus resistance A(MxA)expression of DM.We assessed the sensitivity and specificity of the sarcoplasmic expression of MxA in muscles with 25 DM patients,36 non-DM-IIM patients(9 with ASS,7 with PM,5 with NSM and 11 with NAM),and 163 non-IIM patients(42 with mild myogenic damage,16 with Duchenne muscular dystrophy,14 with Baker muscular dystrophy,10 with limb-girdle muscular dystrophy,6 with facioscapulohumeral muscular dystrophy,30 with Metabolic myopathy diseases,7 with myotonic dystrophy,26 with neurogenic disease and,12 with selective type Ⅱfiber atrophy)by immunohistochemistry and compared them with conventional pathologic hallmarks such as PFA and MAC deposition in endomysial capillaries of DM.The sensitivity of sarcoplasmic MxA expression was quite different from that of PFA(p=0.016)and MAC deposition in endomysial capillaries specificity(p=0.022).The specificity of sarcoplasmic MxA expression was quite different from that of MAC deposition in endomysial capillaries(p=0.001).Sarcoplasmic MxA expression showed a high sensitivity(72.00%)than PFA(44.00%)and MAC deposition in endomysial capillaries(36.00%)for the diagnose of DM.The specificity for the diagnose of DM of both sarcoplasmic MxA expression and PFA was 99.50%,higher than that of MAC deposition in endomysial capillaries(91.45%).All 3 DM patients with anti-MDA5 antibody did not show sarcoplasmic MxA expression,while other DM patients with MSA all had sarcoplasmic MxA expression.Sarcoplasmic MxA expression have a higher sensitivity for the diagnostic of DM.