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Epstein–Barr virus infection and gene promoter hypermethylation in rheumatoid arthritis patients with methotrexate-associated B cell lymphoproliferative disorders

Abstract

We analyzed CpG-island hypermethylation status in 12 genes of paraffin-embedded tissues from 38 rheumatoid arthritis (RA) patients with methotrexate (MTX)-associated large B cell lymphoproliferative disorder (BLPD), 11 RA patients with non-MTX-associated BLPD (non-MTX-BLPD), 22 controls with diffuse large B cell lymphoma (DLBCL), and 10 controls with Epstein–Barr virus (EBV)+ DLBCL. Among them, tumor cells from EBV+ MTX-BLPD patients and control EBV+ DLBCL patients had significantly lower median incidence of CpG island methylator phenotype (CIMP) than those from non-MTX-BLPD and control DLBCL groups (2.3 and 1.7 vs. 4.3 and 4.4; P < 0.01 for each). In the MTX-BLPD group, EBV+ patients showed lower median CIMP than EBV patients (2.3 vs. 3.2); they also had significantly lower hypermethylation incidence in four apoptosis-related genes, especially death-associated protein kinase (14 vs. 55 %), higher incidence of massive tumor necrosis (86 vs. 27 %), and lower BCL2 protein expression (19 vs. 86 %) than did the control DLBCL group (P < 0.01 for all). In all clinical stages, EBV+ MTX-BLPD patients had better prognoses than the EBV MTX-BLPD (P = 0.011), non-MTX-BLPD (P = 0.002), and control DLBCL groups (P = 0.015). MTX-BLPD patients without hypermethylated RAS-associated domain family-1A (RASSF1A) or O 6 -methyl guanine-DNA methyltransferase (MGMT) had significantly better prognosis than those with hypermethylation of those genes (P = 0.033). We conclude that in RA patients with MTX-BLPD, EBV infection is associated with a lower incidence of CIMP, apoptosis-related gene hypermethylation, and BCL2 expression, which can induce tumor regression by MTX withdrawal and lead to better prognoses.

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Abbreviations

APC :

Adenomatous polyposis coli

BLPD:

Large B cell lymphoproliferative disorder

CDH1 :

Cadherin-1

CIMP:

CpG island methylator phenotype

DAPK :

Death-associated protein kinase

DLBCL:

Diffuse large B cell lymphoma

EBER:

EBV-encoded RNA

EBV:

Epstein–Barr virus

hMLH1 :

Human MutL homolog-1

LMP:

Latent membrane protein

MGMT :

O 6-Methyl guanine-DNA methyltransferase

ML:

Malignant lymphoma

MTX:

Methotrexate

MTX-LPD:

MTX-associated lymphoproliferative disorders

non-MTX:

Non-MTX associated

RA:

Rheumatoid arthritis

RASSF1A :

RAS-associated domain family-1A

SHP1 :

Src homology region-2 domain-containing phosphatase-1

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Acknowledgments

We are grateful to Dr. Tetsuo Hamada for his skillful advice and to Tomomi Okabe, Kaori Saga, and Masako Ishiguro for their technical assistance with immunohistochemistry and PCR analysis.

Authors’ contributions

K. Y.-E. and M.T. designed the experiments. S.O., T.F., E.S., and K.T. retrieved and reviewed clinical data. K. Y.-E., Y.O., Y.M., and M.T. contributed histological diagnoses and collected patient samples. K. Y.-E. and M.T. performed immunohistochemistry scoring. K. Y.-E., T.F., and M.T. contributed DNA analysis. Statistical analysis was performed by K. Y.-E. and M.T. The manuscript was written by K.Y. and M. T. and approved by all contributing authors.

Author information

Correspondence to Morishige Takeshita.

Ethics declarations

The study was approved by the Institutional Review Board of Fukuoka University Hospital. Institutional ethical approval was obtained in compliance with the Declaration of Helsinki.

Conflict of interest

The authors declare that they have no competing interests.

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Ejima-Yamada, K., Oshiro, Y., Okamura, S. et al. Epstein–Barr virus infection and gene promoter hypermethylation in rheumatoid arthritis patients with methotrexate-associated B cell lymphoproliferative disorders. Virchows Arch 470, 205–215 (2017). https://doi.org/10.1007/s00428-016-2030-x

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Keywords

  • Rheumatoid arthritis
  • Methotrexate
  • B cell lymphoproliferative disorder
  • Epstein–Barr virus
  • CpG islands
  • DNA methylation