Identification of differential co-expressed gene networks in early rheumatoid arthritis achieving sustained drug-free remission after treatment with a tocilizumab-based or methotrexate-based strategy

Abstract

Background: Methotrexate is endorsed to be used as first-line treatment in rheumatoid arthritis (RA). However, a large proportion of patients need additional treatment with a biological disease-modifying anti-rheumatic drug (DMARD) to adequately suppress their disease activity. A better understanding of genotypes could help to distinguish between patients with different pathogenic mechanisms. The aim of this study was therefore to identify networks of genes within DMARD-naive early RA patients associated with achieving sustained drug-free remission (sDFR) after initiating tocilizumab plus methotrexate, tocilizumab, or methotrexate therapy.

Methods: Samples were used from 60 patients from the U-Act-Early study who received tocilizumab plus methotrexate, tocilizumab, or methotrexate therapy, and who achieved sDFR (≥3 months in drug-free remission until the end of the study, n = 37) after therapy was tapered and subsequently stopped, or who were not able to discontinue the therapy as controls (n = 23). Whole blood samples were collected and ribonucleic acid (RNA) was isolated from positive cluster of differentiation 4 (CD4+) and CD14+ cells and analysed using high-throughput sequencing. Weighted gene co-expression network analyses were performed to identify clusters (i.e. modules) of differently expressed genes associated with achieving sDFR and which were subsequently used for pathway analyses.

Results: Network analyses within CD4+ cells identified two significant modules in the tocilizumab plus methotrexate arm and four modules in the tocilizumab and methotrexate arms, respectively (p ≤ 0.039). Important pathways in the module best correlating with achieving sDFR were in the tocilizumab plus methotrexate arm related to processes involved with transcription and translation; in the tocilizumab arm, pathways were related to migration of white blood cells and G-protein coupled receptors, and in the methotrexate arm pathways were involved with the response to a bacterial or biotic (i.e. biological material)-related stimulus. No relevant networks could be identified in the sequenced CD14+ cells.

Conclusions: Within networks of co-expressed genes, several pathways were found related to achieving sDFR after initiating therapy with tocilizumab, methotrexate, or the combination. Between the three strategy arms, we identified different networks of predisposing genes which indicates that specific gene expression profiles, depending on the treatment strategy chosen, are associated with a higher chance of achieving sDFR.

Trial registration: Clinicaltrials.gov, NCT01034137 . Registered on 16 December 2009.

Keywords: Drug-free remission; Methotrexate; Rheumatoid arthritis; Tocilizumab; Weighted gene co-expression network analysis.

Conflict of interest statement

Ethics approval and consent to participate

The medical ethics research committee of the University Medical Center Utrecht approved the study for all participating hospitals. All patients signed informed consent before entering the study.

Consent for publication

Not applicable.

Competing interests

JWGJ and JWJB received reimbursements from Roche Nederland BV for including patients to participate in the U-Act-Early trial. JWJB reports grants and fees from Roche, AbbVie, Bristol-Myers Squibb, Merck Sharp & Dohme, Pfizer, and UCB. JMvL received financial contributions from MSD, Pfizer, Eli Lilly, and BMS. FPJL reports grants from Roche. AP-S is an employee of F Hoffmann-La Roche and MEAB is an employee of Roche Nederland BV. XMT and MM declare that they have no competing interests.

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Figures

Fig. 1

A Flowchart of the study. Whole blood samples were collected from 60 patients and thereafter CD4+ and CD14+ cells were extracted using fluorescence-activated cell sorting (FACS). Then RNA was isolated from these cells and, via reverse transcription, the library was prepared and sequenced (RNA-seq). Thereafter, a (standard) pipeline was applied for quality control and processing of the reads; read counts were used for detecting differentially expressed genes (DEGs) and these were then used for detecting networks of co-expressed genes. Pathway analyses in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed in the most important networks

Fig. 2

Hierarchical cluster dendrograms of the differently expressed genes included in the network analysis within the (a) tocilizumab plus methotrexate, (b) tocilizumab, and (c) methotrexate strategy arms. Each line represents an individual gene and the branches correspond to modules of co-expressed genes, which are labelled by colours

Fig. 3

The top five most significantly overrepresented gene ontology (GO) terms within the (a) tocilizumab plus methotrexate, (b) tocilizumab, and (c) methotrexate strategy arms. GO terms with >1 DEGs were included in this overview

Fig. 4

Network visualization of the interaction of gene co-expression within the tocilizumab plus methotrexate (a, salmon module), tocilizumab (b, purple module), and methotrexate (c, black module). Upregulated genes are expressed in green nodes and downregulated genes in red nodes. The rounded rectangular nodes display the highest co-expressed genes within the module (signature genes)