A novel autoantibody targeting calreticulin is associated with cancer in patients with idiopathic inflammatory myopathies

Abstract

Objectives: To investigate the prevalence and clinical significance of anti-calreticulin autoantibodies (anti-CRT Ab) in a large cohort of idiopathic inflammatory myopathy (IIM) patients.

Methods: Sera from 469 patients with IIM, 196 patients with other connective tissue diseases, 28 patients with solid tumors and 81 healthy controls were screened for anti-CRT Ab by enzyme-linked immunosorbent assay using human recombinant CRT protein. Sera from 35 IIM patients were tested using an immunoprecipitation assay to confirm the presence of anti-CRT Ab. Subsequently, 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.

Results: Serum anti-CRT Ab was detected positive in 81 of the 469 (17.3%) IIM patients. Immunoprecipitated bands were observed at a molecular weight of 60 kDa corresponding to the CRT protein. The IIM patients with anti-CRT Ab more frequently had cancers compared to the patients without anti-CRT Ab. Moreover, the prevalence of anti-CRT Ab differed according to the cancer status. The IIM patients with recurrent cancers had a much higher prevalence of anti-CRT Ab than those with cancers in remission. Also, serum anti-CRT Ab levels positively correlated with disease activity at baseline and at follow-up visits.

Conclusion: We report the existence of serum anti-CRT Ab in IIM patients and demonstrate the possible association of anti-CRT Ab with malignancy in IIM patients. Serum anti-CRT Ab could serve as a novel candidate marker of cancer in IIM patients.

Keywords: anti‐calreticulin autoantibodies; biomarkers; idiopathic inflammatory myopathy; malignancy.

Figure 1

Detection of serum anti‐CRT Ab in IIM patients. (a) Serum anti‐CRT Ab was screened by ELISA in patients with various connective tissue diseases and solid tumors. The broken line indicates the cut‐off value calculated as the mean value of 81 HC samples plus a threefold standard deviation. The prevalence of anti‐CRT Ab in IIM (81/469, 17.3%), SLE (13/72, 18.1%), RA (12/70, 17.1%), pSS (9/54, 16.7%), solid tumors (7/28, 25%) and HC (1/81, 1.2%). (b) Patient sera were used to immunoprecipitate the CRT protein in K562 cell extracts. The immunoprecipitated bands represent the CRT protein bound by the antibody. Inputs used for immunoprecipitation include Lane 1, healthy control sera; Lanes 2‐4, anti‐CRT Ab‐positive sera from IIM patients, which immunoprecipated bands at a molecular weight of approximately 60 kDa; Lane 5, commercial rabbit polyclonal anti‐CRT antibody (Abcam, Cambridge, UK) as a positive control, which immunoprecipitated bands at a molecular weight of approximately 64 kDa. anti‐CRT Ab, anti‐calreticulin autoantibodies; ELISA, enzyme‐linked immunosorbent assay; IIM, idiopathic inflammatory myopathies; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; pSS, primary Sjogren’s syndrome; HC, healthy controls.

Figure 2

Association between serum anti‐CRT Ab with malignancy in IIM patients. (a) The prevalence of anti‐CRT Ab in IIM patients (81/469, 17.3%), IIM patients without cancer (63/405, 15.6%) and IIM–cancer patients (18/64, 28.1%). IIM patients with cancer had a higher prevalence of anti‐CRT Ab when compared to IIM patients (28.1% vs. 17.3%, P = 0.036) and IIM patients without cancer (28.1% vs. 15.6%, P = 0.013). (b) The prevalence of anti‐CRT Ab in IIM patients with new‐onset cancers (9/34, 26.5%), patients with cancers in remission (4/22, 18.2%) and patients with recurrent cancers (5/8, 62.5%). Patients with recurrent cancers presented a much higher prevalence of anti‐CRT Ab than patients with cancers in remission (62.5% vs. 18.2%, P = 0.032). anti‐CRT Ab, anti‐calreticulin autoantibodies; *P < 0.05; NS: not significant.

Figure 3

Positive correlations between serum anti‐CRT Ab levels and disease activity in IIM patients. (a, b) Cross‐sectional analyses of 81 anti‐CRT Ab‐positive IIM patients demonstrated a positive correlation between serum anti‐CRT Ab levels and MYOACT scores (Spearman r = 0.28, P = 0.009) and PGA scores (Spearman r = 0.29, P = 0.008). (c) Longitudinal analyses of 16 anti‐CRT Ab‐positive IIM patients demonstrated that the variations in anti‐CRT Ab titres positively correlated with the changes in MYOACT scores (β = 0.03, P < 0.001). Patient 14 to Patient 16 were IIM–cancer patients. anti‐CRT Ab, anti‐calreticulin autoantibodies; MYOACT, myositis disease activity assessment visual analogue scales; PGA, physician global assessment of disease activity.