0449: Parsing Pathophysiology of Rheumatoid Arthritis-associated Lymphoproliferative Disorders via Whole RNA Transcriptome Analysis: Multi-center Study in Japan

ATSUKO TSUJII¹, KAZUKO SAKAI², SHIRO OHSHIMA¹, YUKIHIKO SAEKI¹, MASATO YAGITA³, TOMOYA MIYAMURA⁴, Masao Katayama⁵, YASUSHI HIRAMATSU⁶, SHINJI HIGA⁷, FUMINORI HIRANO⁸, KENJI ICHIKAWA⁹, NORIYUKI CHIBA¹⁰, TAKAO SUGIYAMA¹¹, ATSUSHI IHATA¹², HIROSHI TSUTANI¹³, KOICHIRO TAKAHI¹⁴, KIYOSHI MIGITA¹⁵, SHUNSUKE MORI¹⁶, NORIE YOSHIKAWA¹⁷, ATSUHISA UEDA¹⁸, SHOUHEI NAGAOKA¹⁹, KEIGO SETOGUCHI²⁰, SHOJI SUGII²¹, ASAMI ABE²², TOSHIAKI SUGAYA²³, HIROYUKI SUGAHARA²⁴, SHINICHIRO TSUNODA²⁴, NORISHIGE IIZUKA²⁵, RYOSUKE YOSHIHARA²⁶, HIROKI YABE²⁷, TOMOAKI FUJISAKI²⁸, EIICHI MORII²⁹, KAZUYOSHI SAITO³⁰, Kiyoshi Matsui³¹, YASUHIKO TOMITA³², HIROSHI FURUKAWA³³, Shigeto Tohma³⁴, KAZUTO NISHIO² and YOSHIHIKO HOSHIDA³⁵, ¹National Hospital Organization (NHO), Osaka Minami Medical Center, Kawachinagano, Japan, ²Kindai University School of Medicine Department of Genome Biology, Sayama, Japan, ³Medical Research Institute KITANO HOSPITAL, PIIF Tazuke-kofukai, Osaka, Japan, ⁴NHO Kyushu Medical Center, Fukuoka, Japan, ⁵National Hospital Organization, Nagoya Medical Center, Nagoya, JP, Nagoya, Japan, ⁶Japanese Red Cross Society Himeji Hospital, Himeji, Japan, ⁷Daini Osaka Police Hospital, Osaka, Japan, ⁸NHO Asahikawa Medical Center, Asahikawa, Japan, ⁹Nissei hospital, Sapporo, Japan, ¹⁰NHO Morioka Medical Center, Morioka, Japan, ¹¹NHO Shimoshizu Hospital, Yotsukaido, Japan, ¹²NHO Yokohama Medical Center, Yokohama, Japan, ¹³NHO Awara Hospital, Awara, Japan, ¹⁴NHO Osaka Toneyama Medical Center, Toyonaka, Japan, ¹⁵Department of Rheumatology Fukushima Medical University School of Medicine, Fukushima, Japan, ¹⁶NHO Kumamoto Saishun Medical Center, Koshi, Japan, ¹⁷NHO Miyakonojo Medical Center, Miyakonojo, Japan, ¹⁸Yokohama City University Medical Center, Yokohama, Japan, ¹⁹Yokohama Minami Kyosai Hospital, Yokohama, Japan, ²⁰Tokyo Metropolitan Cancer and Infectious diseases Center Komagome Hospital / Tokyo Metropolitan Komagome Hospital, Tokyo, Japan, ²¹Tokyo Metropolitan Matsuzawa Hospital, Tokyo, Japan, ²²Niigata Rheumatic Center, Shibata, Japan, ²³Fuchu Hospital, Izumi, Japan, ²⁴Sumitomo Hospital, Osaka, Japan, ²⁵Kishiwada City Hospital, Kishiwada, Japan, ²⁶Hyogo Prefectural Kakogawa Hospital, Kakogawa, Japan, ²⁷Ako Central Hospital, Ako, Japan, ²⁸Matsuyama Red Cross Hospital, Matsuyama, Japan, ²⁹Osaka University, Suita, Japan, ³⁰University of Occupational and Environmental Health, Kitakyushu, Japan, ³¹Hyogo Medical University, Nishinomiya, Japan, ³²International University of Health and Welfare, Otawara City, Japan, ³³NHO Tokyo National Hospital, Kiyose, Japan, ³⁴NHO Tokyo National Hospital, Dallas, TX, ³⁵National Hospital Organization Osaka Minami Medical Center, Kawachinagano, Japan

Background/Purpose: Lymphoproliferative disorders (LPD) in rheumatoid arthritis (RA) (RA-LPD) have unique pathophysiological features. More than half of the cases of RA-LPD undergo spontaneous regression after the discontinuation of disease modifying anti rheumatic drugs (DMARDs), whereas approximately 33% of these cases relapse. Epstein-Barr virus (EBV) infection is especially involved in the pathophysiology of RA-LPD. The current study was conducted with whole RNA transcriptome analysis using the peripheral blood of patients developing RA-LPD to investigate the pathophysiology of RA-LPD.

Methods: Peripheral blood samples of the patients with RA-LPD were obtained between May 2013 and October 2018 from 30 hospitals in Japan. Whole transcriptome sequencing was performed using the Ion AmpliSeq Human Gene Expression Kit (Thermo Fisher Scientific) and an IonS5 XL sequencer (Thermo Fisher Scientific). The kit covers the expression levels of 20,802 human RefSeq genes (based on UCSC hg19). Differential gene expression analysis was performed by Transcriptome Analysis Console (TAC) software (ver. 4.0.3, Thermo Fisher Scientific) with |FC| > 2-fold difference and at p < 0.1. A Gene Set Enrichment Analysis (GSEA) was performed to identify pathways enriched in the Molecular Signatures Database (MSigDB) Hallmark gene set. A nominal p value of < 0.05 and an FDR (false discovery rate) q value of < 0.25 were considered statistically significant.

Results: In total, 211 cases (191 RA-LPD cases and 20 non-LPD RA cases) were analyzed. The flow diagram of the study is shown in Figure 1. We grouped diffuse large B-cell lymphoma (DLBCL) cases into two groups; regression cases (n = 43), which regressed spontaneously after stopping DMARDs and non-regression cases (n = 9), which did not regress. Gene expression levels were compared between the two groups. GSEA analysis showed that interferon alpha and gamma pathways were enhanced in regression cases (Figure 2a). To investigate mechanisms of regression in EBV-negative cases, we performed further analysis according to EBV infection status. (EBV positive, n= 22; EBV negative, n= 15). The interleukin-2-STAT5 (IL2-STAT5) pathway was enhanced in regression cases without EBV infection, which was suggested to be a unique mechanism for regression in EBV-negative RA-LPD (Figure 2b, c). The heatmap (Figure 3) shows that genes related to IL2-STAT5 were expressed highly in regression cases without EBV infection. Additionally, expression levels of some genes related to the IL2-STAT5 pathway were significantly different between regression and non-regression EBV-negative cases. This result suggests that these genes may serve as new liquid markers for regression in EBV-negative RA-LPD.

Conclusion: The current study showed that interferon-alpha and gamma pathways were upregulated in the regression cases with DLBCL of RA-LPD. Moreover, the study revealed a unique mechanism related to IL2-STAT5 for regression of EBV-negative cases. Our research further suggested new potential markers for regression in EBV-negative cases.








A. TSUJII: None; K. SAKAI: Chugai Pharmaceutical, 6, Life Technologies Japan, 6, Qiagen, 6, Takeda Pharmaceutical, 6; S. OHSHIMA: None; Y. SAEKI: None; M. YAGITA: Medical & Biological Laboratories, CO., Ltd, 9, ONK therapeutics, 9; T. MIYAMURA: None; M. Katayama: None; Y. HIRAMATSU: None; S. HIGA: None; F. HIRANO: None; K. ICHIKAWA: None; N. CHIBA: None; T. SUGIYAMA: None; A. IHATA: None; H. TSUTANI: None; K. TAKAHI: None; K. MIGITA: None; S. MORI: AbbVie GK, 6, 6, Asahikasei Pharma Corp, 6, Boehringer Ingelheim Japan, 6, Chugai Pharmaceutical Co. Ltd., 6, Chugai Pharmaceutical Co., Ltd., 6, Eli Lilly Japan K.K., 6, Janssen Pharmaceutical K.K., 6, Pfizer Japan Inc., 6, Taisho Pharma Co., Ltd., 6; N. YOSHIKAWA: None; A. UEDA: None; S. NAGAOKA: None; K. SETOGUCHI: None; S. SUGII: Eisai CO., Ltd., 6, MOCHIDA PHARMACEUTICAL CO>, 6; A. ABE: AbbVie/Abbott, 6, Bristol-Myers Squibb(BMS), 6; T. SUGAYA: None; H. SUGAHARA: None; S. TSUNODA: None; N. IIZUKA: None; R. YOSHIHARA: None; H. YABE: None; T. FUJISAKI: None; E. MORII: None; K. SAITO: None; K. Matsui: None; Y. TOMITA: None; H. FURUKAWA: None; S. Tohma: AbbVie/Abbott, 5, AsahiKASEI Co., Ltd., 6, Chudai Pharmaceutical Co., Ltd., 5, Mitsubishi Tanabe Pharma Corporation, 5, Pfizer Japan Inc., 6; K. NISHIO: Amgen, 6, AstraZeneca, 6, Bristol-Myers Squibb, 6, Chugai, 6, DAIICHI SANKYO, 6, Eli Lilly Japan, 5, 6, FUJIREBIO, 6, Guardant Health, 6, Hitachi, 5, Invitae Japan, 6, Janssen Pharmaceutical, 6, Merck Biopharma, 6, MSD, 6, Nichirei Biosciences, 5, Nippon Boehringer Ingelheim, 5, 6, Novartis Pharma, 6, Ono, 6, Otsuka Pharmaceutical, 5, 6, Pfizer, 6, SymBio Pharmaceuticalsっっｖ, 6, SYSMEX, 5, Takeda, 6, Yakult Honsha, 6; Y. HOSHIDA: None.