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Clinical and epidemiological research
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Anti-NXP2 autoantibodies in adult patients with idiopathic inflammatory myopathies: possible association with malignancy
  1. Yuki Ichimura1,
  2. Takashi Matsushita2,
  3. Yasuhito Hamaguchi2,
  4. Kenzo Kaji2,
  5. Minoru Hasegawa2,
  6. Yoshinori Tanino3,
  7. Yayoi Inokoshi3,
  8. Kazuhiro Kawai4,
  9. Takuro Kanekura4,
  10. Maria Habuchi5,
  11. Atsuyuki Igarashi5,
  12. Ryosuke Sogame6,
  13. Takashi Hashimoto6,
  14. Tomohiro Koga7,
  15. Ayako Nishino7,
  16. Naoko Ishiguro8,
  17. Naoki Sugimoto9,
  18. Rui Aoki10,
  19. Noriko Ando10,
  20. Tetsuya Abe11,
  21. Takashi Kanda11,
  22. Masataka Kuwana12,
  23. Kazuhiko Takehara2,
  24. Manabu Fujimoto2
  1. 1School of Medicine, Kanazawa University, Kanazawa, Japan
  2. 2Department of Dermatology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
  3. 3Department of Pulmonary Medicine, Fukushima Medical University School of Medicine, Fukushima, Japan
  4. 4Department of Dermatology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
  5. 5Division of Dermatology, Kanto Medical Center NTT EC, Tokyo, Japan
  6. 6Department of Dermatology, Kurume University School of Medicine, Kurume, Japan
  7. 7Unit of Translational Medicine, Department of Immunology and Rheumatology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
  8. 8Department of Dermatology, Tokyo Women's Medical University, Tokyo, Japan
  9. 9Institute of Rheumatology, Tokyo Women's Medical University, Tokyo, Japan
  10. 10Department of Dermatology, Faculty of Medicine, University of Yamanashi, Yamanashi, Japan
  11. 11Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Ube, Japan
  12. 12Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan
  1. Correspondence to Manabu Fujimoto, Department of Dermatology, Kanazawa University Graduate School of Medical Science, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan; fujimoto-m{at}umin.ac.jp

Abstract

Objectives Myositis-specific autoantibodies (MSAs) are useful tools for identifying clinically homogeneous subsets and predicting prognosis of patients with idiopathic inflammatory myopathies (IIM) including polymyositis (PM) and dermatomyositis (DM). Recent studies have shown that anti-NXP2 antibody (Ab) is a major MSA in juvenile dermatomyositis (JDM). In this study the frequencies and clinical associations of anti-NXP2 Ab were evaluated in adult patients with IIM.

Methods Clinical data and serum samples were collected from 507 adult Japanese patients with IIM (445 with DM and 62 with PM). Eleven patients with JDM, 108 with systemic lupus erythematosus, 433 with systemic sclerosis and 124 with idiopathic pulmonary fibrosis were assessed as disease controls. Serum was examined for anti-NXP2 Ab by immunoprecipitation and western blotting using polyclonal anti-NXP2 Ab.

Results Seven patients (1.6%) with adult DM and one (1.6%) with adult PM were positive for anti-NXP2 Ab. Except for two patients with JDM, none of the disease controls were positive for this autoantibody. Among eight adult patients with IIM, three had internal malignancies within 3 years of diagnosis of IIM. Another patient with DM also had a metastatic cancer at the diagnosis. All of the carcinomas were at an advanced stage (stage IIIb–IV).

Conclusions While less common than in juvenile IIM, anti-NXP2 Ab was found in adult IIM. Anti-NXP2 Ab may be associated with adult IIM with malignancy.

https://doi.org/10.1136/annrheumdis-2011-200697

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    Introduction

    Idiopathic inflammatory myopathies (IIM), including polymyositis (PM) and dermatomyositis (DM), are characterised by chronic inflammation of skeletal muscles and/or skin eruptions.1 Although the aetiology remains unclear, they are considered as autoimmune diseases. The presence of disease-specific autoantibodies (autoAbs), known as myositis-specific autoAbs (MSAs), is a prominent feature. Moreover, MSAs are strongly associated with distinct clinical phenotypes and thus classify patients into groups with more homogeneous clinical features.2,–,4 These MSAs include antibodies (Abs) to aminoacyl-tRNA synthetases, the Mi-2 protein, the signal-recognition particle, transcriptional intermediary factor-1 (TIF1; anti-155/140 Ab)5,–,7 and melanoma differentiation-associated gene-5 (MDA5; anti-CADM140 Ab).8

    Oddis and colleagues first described anti-MJ Ab in a US cohort of juvenile DM (JDM),9 and Targoff et al subsequently identified that the antigen of anti-MJ Ab is nuclear matrix protein NXP2 (MORC3).10 Gunawardena et al and Espada et al have demonstrated that anti-NXP2 Ab is among the most common MSAs in JDM.11 12 In this study we evaluated the frequencies and clinical associations of anti-NXP2 Ab in adult patients with IIM.

    Methods

    Patients

    Serum samples were obtained from 507 consecutive Japanese adult patients with IIM, 445 with DM and 62 with PM, who had been followed up in the Department of Dermatology, Kanazawa University Hospital and collaborating medical centres from 2003 to 2010. All patients with PM and 365 patients with DM fulfilled Bohan and Peter's criteria.13 14 The remaining 80 did not, but fulfilled Sontheimer's criteria of clinically amyopathic DM (CADM).15 Among the patients with DM, as disease controls, 11 with JDM, 108 with systemic lupus erythematosus, 433 with systemic sclerosis and 124 with idiopathic pulmonary fibrosis were assessed. Clinical information was collected retrospectively by reviewing their clinical medical charts.

    Immunoprecipitation and western blotting

    Serum (10 μl) was incubated with 2 mg protein A-Sepharose beads (Amersham Biosciences, Piscataway, New Jersey, USA) in immunoprecipitation buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.1% Nonidet P-40) for 2 h. Beads were then mixed with 35S-labelled or unlabelled K562 cell extracts derived from 107 cells and rotated at 4°C for 2 h. After five washes, precipitated proteins were fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography or western blotting.

    For western blotting, proteins were transferred onto a nitrocellulose membrane. After blocking, the membrane was incubated with mouse polyclonal anti-human NXP2 Ab (Abcam, Cambridge, UK) and then with horseradish peroxidase-conjugated goat antimouse IgG Ab (Thermo Scientific, Rockford, Illinois, USA). The membrane was developed using an enhanced chemiluminescence kit (Thermo Scientific).

    Additional information regarding methods is available in the online supplement.

    Statistical analysis

    Fisher exact probability tests were used for comparison of frequencies. The Bonferroni test was examined for multiple comparisons of values following normal distribution. Values that were not normally distributed were evaluated by the Mann–Whitney U test. p Values <0.05 were considered significant.

    Results

    Detection of anti-NXP2 Abs

    Serum samples from 507 adult Japanese patients with IIM were screened for anti-NXP2 Ab. From K562 cell extracts, eight serum samples precipitated a 140 kDa protein which was different from other known autoantigens (figure 1A). Additionally, two patients with JDM were positive for this 140 kDa protein. No serum from other diseases precipitated this 140 kDa protein. On SDS-PAGE, the 140 kDa band was slightly higher than the 140 kDa band of anti-TIF1 Ab and was not accompanied by a 155 kDa band (figure 1A). While this 140 kDa protein migrated closely to MDA5, these serum samples were negative for anti-MDA5 Ab by an ELISA using recombinant MDA5 protein as antigen (data not shown). These serum samples were confirmed to react with NXP2 since precipitated proteins were recognised by polyclonal Abs to NXP2 in western blotting (figure 1B). They also reacted with recombinant human NXP2 protein by western blotting (see figure S1 in online supplement). These serum samples were negative for other MSAs and did not have other known autoAbs, except for patient B who was positive for anti-SS-A/Ro and SS-B/La Abs. In indirect immunofluorescence, five showed negative staining while three stained nuclei in speckled pattern at the maximum dilution of ×80.

    Figure 1
    Figure 1

    Detection of anti-NXP2 Ab. (A) Immunoprecipitant from 35S-labelled K562 cell extract using serum samples were subjected to 7% SDS-PAGE. Lanes A–H correspond to anti-NXP2-positive patients shown in table 1. As controls, the prototype serum samples positive for anti-TIF1 Ab and anti-MDA5 Ab and normal control serum were also examined. Molecular weights (MW) are shown on the left. The position of the 140 kDa NXP2 protein is indicated by an arrow. (B) K562 cell extracts were immunoprecipitated with anti-NXP2-positive serum (patients A–H), anti-MDA5-positive serum, anti-TIF1-positive serum or normal control serum and subjected to SDS-PAGE, electro-transferred onto nitrocellulose membranes and probed with polyclonal anti-NXP2 Ab. The MW of NXP2 (140 kDa) is indicated by an arrow.

    View this table:
    Table 1

    Clinical and laboratory profile of adult patients with DM or PM positive for anti-NXP2 Ab

    Clinical and laboratory profiles of patients with IIM with anti-NXP2 Abs

    Among 507 adult patients with IIM, eight were positive for anti-NXP2 Ab: seven (1.6%) in 445 adults with DM and one (1.6%) in 62 adults with PM. All patients with anti-NXP2 Ab showed strong muscle weakness and high elevation of serum creatine kinase levels. Remarkably, internal malignancies found within 3 years of the diagnosis of IIM were present in 37.5% (3/8) (table 2). All these patients had advanced disease (stage IIIb–IV). Additionally, in one patient (C), prostate cancer was found 42 months previously and it was metastatic when the diagnosis of DM was made. However, he was excluded from the statistical analysis since he did not meet the above criteria. Interstitial lung disease (ILD) was not found.

    View this table:
    Table 2

    Comparison of clinical and laboratory profile of adult patients with dermatomyositis with anti-NXP2 Ab, anti-TIF1 Ab and anti-Mi-2 Ab*

    During the follow-up period which varyied from 2 to 61 months, seven of eight patients (all except patient H) were treated with systemic corticosteroid therapy. Patient A also received methotrexate and patient D also underwent intravenous immunoglobulin therapy. While the response to the treatment was favourable in all patients, two (patients C and G) died of malignancy.

    Comparison with other MSAs

    In addition to seven patients positive for anti-NXP2 Ab, adult patients with DM in this study included 74 patients with anti-TIF1 Ab and 15 with anti-Mi-2 Ab. They also included 51 with anti-MDA5 Ab, 26 with anti-PL-7 Ab, 18 with anti-Jo-1 Ab and 8 with anti-PL-12 Ab. Since anti-TIF1 Ab and anti-Mi-2 Ab are specifically associated with DM, the clinical features of patients with anti-NXP2 Ab were compared with those with anti-TIF1 Ab and anti-Mi-2 Ab (table 2).

    Anti-NXP2 Ab was predominantly found in men. Although Gottron's sign was slightly less frequent, there was no significant difference in the frequency of each cutaneous manifestation. The frequency of fever was significantly higher than in patients with anti-TIF1-positive DM. The highest creatine kinase level was similar to that in patients with anti-Mi-2 Ab-positive DM and significantly higher than in patients with anti-TIF1 Ab. The frequency of internal malignancy in those with anti-NXP2 Ab was lower than in those with anti-TIF1 Ab and higher than in patients with anti-Mi-2 Ab, although the differences were not statistically significant.

    Discussion

    Two studies have recently detected anti-NXP2 Ab in 23% and 25% of patients with JDM in the UK and Argentina, respectively.11 12 While the number of patients was small, anti-NXP2 Ab was detected in 18% in the control JDM population in this study. Anti-NXP2 Ab is therefore likely to be a major MSA in JDM across racial groups as well as anti-TIF1 Ab, which is detected in 23–29% of patients with JDM.16 In addition to JDM, Espada et al reported that two (28%) of seven patients with juvenile PM were also positive for anti-NXP2 Ab.12 In this study we identified eight anti-NXP2 Ab-positive adult patients in a Japanese cohort of IIM. The frequencies were 1.6% in both adult DM and adult PM. Therefore, while the population sizes of DM and PM are substantially different, anti-NXP2 Ab has been found at similar frequencies both in juvenile and adult IIM.12 In contrast, in a preliminary report in a UK cohort, Betteridge et al detected anti-NXP2 Ab in 13 (5%) patients with DM but not in patients with PM.17

    Intriguingly, 37.5% of adult IIM patients positive for anti-NXP2 Ab had malignancy which was found within 3 years, in addition to a patient with metastatic prostate cancer found 42 months previously. Moreover, all of these carcinomas were at an advanced stage. Anti-TIF1 Ab also has a strong association with internal malignancy in adult DM, as it is found in 50–70% of patients with cancer-associated DM.5 6 18 Therefore, while anti-NXP2 Ab may not be restricted to DM, anti-TIF1 Ab and anti-NXP2 Ab may have a shared property in that they represent two clinical subsets of cancer-associated adult DM and JDM. However, in contrast to our study, Betteridge et al reported that anti-NXP2 Ab was not associated with malignancy but with ILD.17 This may be due to ethnical differences. It may also have resulted from a different distribution of patients between dermatology and rheumatology clinics. Since both studies had relatively small numbers of patients, more studies are needed to evaluate more precisely the clinical relevance of anti-NXP2 Ab in patients with IIM.

    Among adult patients with DM, anti-NXP2 Ab appeared to have strong muscle involvement while we could not find any particular cutaneous manifestations related to anti-NXP2 Ab. Unlike anti-NXP2-positive JDM,11 no adults with DM with anti-NXP2 Ab had cutaneous calcinosis. This may be due to the relatively low incidence of calcinosis in adult DM compared with JDM.

    In summary, this study shows that anti-NXP2 Ab occurs in adult patients with IIM and suggests that anti-NXP2 Ab may be correlated with cancer-associated myositis. NXP2 is involved in the activation and localisation of a tumour suppressor gene, p53.19 TIF1 proteins also have a functional relationship with p53.20 Since these two autoAbs may share similar clinical characteristics, especially the association with cancer, they may develop during antitumour immune responses.

    Acknowledgments

    The authors thank Masako Matsubara, Tomoko Hayashi and Natsuho Yoshifuji for technical assistance.

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    View Abstract

    Supplementary materials

    • Supplementary Data

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    Footnotes

    • Ethic approval This study was conducted with the approval of Kanazawa University Graduate School of Medical Science and Kanazawa University Hospital.

    • Funding This work was supported by a grant of Research on Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan.

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.