The Discovery Of Novel Serological Biomarkers For Malignancy In Patients With Idiopathic Inflammatory Myopathy

Objectives1.To investigate the expression levels of soluble programmed death-ligand 1(sPD-L1)and evaluate its association with malignancy in adult-onset dermatomyositis(DM)patients.2.To investigate the prevalence and clinical significance of anti-calreticulin autoantibodies(anti-CRT Ab),and to evaluate the association of anti-CRT Ab with malignancy in a large cohort of adult-onset idiopathic inflammatory myopathy(ⅡM)patients.Methods1.sPD-L1 levels were measured in serum from 88 DM patients without malignancies(sDM),40 with cancer-related DM(CRDM),and 30 healthy controls(HC)by using enzyme-linked immunosorbent assay(ELISA).The CRDM subjects were divided into new-onset cancers(nCRDM)and stable cancers(sCRDM).ROC curve analysis was performed to determine the cut-off sPD-L1 value that distinguished nCRDM from sDM patients.Serum anti-transcription intermediary factor 1γ antibodies(anti-TIF-ly Ab)were detected by using immunoblot and the diagnostic values for malignancy were compared with sPD-L1 levels in DM patients.The skeletal muscle tissues were collected from 10 anti-TIF-1γ Ab-positive DM patients and 9 normal controls,and the muscle transcriptomics was analyzed using RNA-sequencing.The commercial myoblasts and DM patient-derived myoblasts were cultured in vitro,after stimulation with recombinant IFN-β protein(concentration at 50ng/mL)for 24 hours,the expression levels of PD-L1 mRNA were detected using real-time quantitative PCR.2.Sera from 469 patients with IIM,196 with other connective tissue diseases(including 72 systemic lupus erythematosus,70 rheumatoid arthritis,and 54 primary Sjogren’s syndrome),28 with solid tumors,and 81 healthy controls were screened for anti-CRT Ab by ELISA using recombinant human CRT protein.Sera from 35 ⅡM patients were tested using an immunoprecipitation assay to confirm the presence of anti-CRT Ab.IIM-cancer patients were identified and divided into new-onset,remission,and recurrent groups based on their cancer status.The relationships between anti-CRT Ab levels and IIM disease activity were also investigated.Results1.Serum sPD-L1 levels were significantly higher in sDM[median 12.3ng/ml(IQR 8.4-16.2)]than in HC[median 1.3ng/ml(IQR 0.4-2.2),P<0.001].Extremely high sPD-L1 levels were found in nCRDM[median 18.5ng/ml(IQR 13.8-22.4)],much higher than that in sCRDM(median 8.5ng/ml(IQR 6.8-11.8),P<0.001).The sPD-L1 levels in four nCRDM patients decreased after curative cancer treatment(P=0.013).ROC curve analysis revealed that the sPD-L1 value distinguishing nCRDM from sDM was 16.1 ng/ml,with an area under curve value of 0.72 ± 0.04(P<0.001).The combination of sPD-L1 and anti-TIF-1γ Ab yielded greater specificity and positive predictive value in diagnosing cancer,reaching to values of 95%and 70%respectively.RNA-seq results showed that the PD-L1 mRNA levels were significantly up-regulated in muscle tissues of anti-TIF-ly Ab-positive DM patients compared to that of normal controls.The PD-L1 mRNA levels positively correlated with the inflammatory infiltration scores(r=0.68,P=0.031)in muscle biopsies.Moreover,the IFN scores positively correlated with PD-L1 mRNA levels(r=0.79,P=0.006)and serum sPD-L1 concentration(r=0.84,P=0.002)in DM patients.There was an interaction relationship between interferon-stimulated genes and PD-1/PD-L1 signaling pathway.In vitro,IFN-β up-regulated the expression levels of PD-L1 in both commercial myoblasts and DM patient-derived myoblasts.2.Serum anti-CRT Ab were detected positive in 81 of 469(17.3%)IIM patients.Immunoprecipitated bands at a molecular weight of approximately 60kDa,corresponding to the CRT protein,were detected in ELISA-positive sera.IIM patients with anti-CRT Ab more frequently had cancer and less frequently had arthritis/arthralgia compared to patients without anti-CRT Ab.Moreover,the prevalence of anti-CRT Ab differentiated concerning cancer status.IIM patients with recurrent cancer had a much higher prevalence of anti-CRT Ab than that of patients with cancer in remission.Also,serum anti-CRT Ab levels positively correlated with disease activity at baseline and at longitudinal visits.Conclusion1.Serum sPD-L1 levels increased significantly and markedly high sPD-L1 levels may serve as a diagnostic indicator for malignancies in DM patients,especially in those with anti-TIF-1γ Ab.The expression levels of PD-L1 were abnormally up-regulated in muscle tissues of anti-TIF-1γ Ab-positive DM patients,and positively correlated with the degree of IFN pathway activation and the degree of inflammatory infiltration in muscle biopsies.2.We reported the prevalence and clinical significance of serum anti-CRT Ab in a large cohort of adult-onset IIM patients.Serum anti-CRT Ab could be a novel candidate marker of cancer in IIM.Furthermore,the detection of anti-CRT Ab could be valuable in monitoring cancer recurrence in IIM patients with a history of solid tumors.Also,serum anti-CRT Ab levels may serve as a potential marker of disease activity in IIM patients.Further studies are required to characterize the role of anti-CRT Ab in the tumorigenesis and pathogenesis of ⅡM.