B Cell Synovitis and Clinical Phenotypes in Rheumatoid Arthritis: Relationship to Disease Stages and Drug Exposure

Abstract

and the PEAC-R4RA Investigators Objective. To define the relationship of synovial B cells to clinical phenotypes at different stages of disease evolution and drug exposure in rheumatoid arthritis (RA).Methods. Synovial biopsy specimens and demographic and clinical data were collected from 2 RA cohorts (n = 329), one of patients with untreated early RA (n = 165) and one of patients with established RA with an inadequate response to tumor necrosis factor inhibitors (TNFi-IR; n = 164). Synovial tissue was subjected to hematoxylin and eosin and immunohistochemical staining and semiquantitative assessment for the degree of synovitis (on a scale of 0-9) and of CD20+ B cell infiltrate (on a scale of 0-4). B cell scores were validated by digital image analysis and B cell lineage-specific transcript analysis (RNA-Seq) in the early RA (n = 91) and TNFi-IR (n = 127) cohorts. Semiquantitative CD20 scores were used to classify patients as B cell rich (≥2) or B cell poor (<2).Results. Semiquantitative B cell scores correlated with digital image analysis quantitative measurements and B cell lineage-specific transcripts. B cell-rich synovitis was present in 35% of patients in the early RA cohort and 47.7% of patients in the TNFi-IR cohort (P = 0.025). B cell-rich patients showed higher levels of disease activity and seropositivity for rheumatoid factor and anti-citrullinated protein antibody in early RA but not in established RA, while significantly higher histologic synovitis scores in B cell-rich patients were demonstrated in both cohorts.Conclusion. We describe a robust semiquantitative histologic B cell score that closely replicates the quantification of B cells by digital or molecular analyses. Our findings indicate an ongoing B cell-rich synovitis, which does not seem to be captured by standard clinimetric assessment, in a larger proportion of patients with established RA than early RA.

Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy-driven, multicentre, open-label, phase 4 randomised controlled trial

“…CD20 staining was evaluated at three cutting levels on a minimum of six biopsies, which is recommended for use in clinical trials and reported to be representative of the whole joint tissue. 27 Although the semi-quantitative score used for balanced stratification (before randomisation) had been validated both against digital image analysis and the transcript concentrations, determined using the FANTOM5-derived B-cell related gene set, 20 , 25 because no published gold standard is available, the cutoff of 0–1 for B-cell poor classification and 2–4 for B-cell rich classification was set arbitrarily on the basis of a previous pilot study and might not have been at an optimal level for the whole trial. Furthermore, considering that these cutoffs were determined by physically counting the number of CD20 B cells, miscounting had the potential for misclassification.…”

“…The sections were then stained for haematoxylin and eosin and immune-histochemical markers ( appendix p 13 ), as previously described. 3 , 20 Sections underwent semi-quantitative scoring to determine expression of CD20 B cells, CD3 T cells, CD138 plasma cells, and CD68 lining and sublining macrophages ( appendix p 13 ). The scoring process was adapted from a previously described and validated score.…”

“…The scoring process was adapted from a previously described and validated score. 20 , 21 , 22 Following histological assessment of CD20 semi-quantitative scores, patients were classified as B-cell rich or B-cell poor at Barts Health NHS Trust by a consultant pathologist (HR), which was confirmed in an independent evaluation by a second expert in synovial pathology, according to a validated algorithm ( appendix p 14 ). Synovial tissues with a CD20 score less than two were classified as B-cell poor; tissues with CD20 score of two to four and CD20 B-cell aggregates were classified as B-cell rich.…”

“…A minimum of six synovial samples per patient were immediately immersed in RNA-Later for RNA sequencing. The RNA from these samples was extracted as described in the appendix (p 15) 20 and sequenced at Genewiz (Bishop's Stortford, UK) according to the standard operating procedure ( appendix p 15 ). 184 paired-end RNA sequenced samples of 150 base pairs were trimmed to remove the Illumina adaptors with bbduk from the BBMap package (version 37.93) according to default parameters, using R (version 3.6.0).…”

Persistence of Mast Cell-Positive Synovitis in Early Rheumatoid Arthritis Following Treatment With Conventional Synthetic Disease Modifying Anti-Rheumatic Drugs

“…Synovial sections underwent standard H&E staining and semi-quantitative (SQ) assessment of synovitis according to a previously validated score (Krenn) ( Krenn et al., 2006 ). Sequentially cut sections underwent Immunohistochemical (IHC) staining and upon SQ scoring (0‑4), sections were stratified into synovial pathotypes according to the degree of immune cell infiltration: i) Lymphoid- grade 2/3 B cell aggregates, CD20≥ 2 and/or CD138>2, ii) Myeloid- CD68 SL≥ 2, CD20 ≤ 1 and/or CD3≥1, CD138 ≤ 2, and iii) Fibroid- CD68 SL<2 and CD3, CD20, CD138<1) ( Humby et al., 2009 ; Rivellese et al., 2019a ). Following IHC staining for CD117, patients were classified as MC+/MC-, based on the presence/absence of synovial mast cells and MC density (n of cells/mm 2 ) was calculated by automated cell counting (cellSens, Olympus).…”