A Pauci-Immune Synovial Pathotype Predicts Inadequate Response to TNFα-Blockade in Rheumatoid Arthritis Patients

Abstract

Objectives: To assess whether the histopathological features of the synovium before starting treatment with the TNFi certolizumab-pegol could predict clinical outcome and examine the modulation of histopathology by treatment. Methods: Thirty-seven RA patients fulfilling UK NICE guidelines for biologic therapy were enrolled at Barts Health NHS trust and underwent synovial sampling of an actively inflamed joint using ultrasound-guided needle biopsy before commencing certolizumab-pegol and after 12-weeks. At 12-weeks, patients were categorized as responders if they had a DAS28 fall >1.2. A minimum of 6 samples was collected for histological analysis. Based on H&E and immunohistochemistry (IHC) staining for CD3 (T cells), CD20 (B cells), CD138 (plasma cells), and CD68 (macrophages) patients were categorized into three distinct synovial pathotypes (lympho-myeloid, diffuse-myeloid, and pauci-immune). Results: At baseline, as per inclusion criteria, DAS28 mean was 6.4 ± 0.9. 94.6% of the synovial tissue was retrieved from the wrist or a metacarpophalangeal joint. Histological pathotypes were distributed as follows: 58% lympho-myeloid, 19.4% diffuse-myeloid, and 22.6% pauci-immune. Patients with a pauci-immune pathotype had lower levels of CRP but higher VAS fatigue compared to lympho- and diffuse-myeloid. Based on DAS28 fall >1.2, 67.6% of patients were deemed as responders and 32.4% as non-responders. However, by categorizing patients according to the baseline synovial pathotype, we demonstrated that a significantly higher number of patients with a lympho-myeloid and diffuse-myeloid pathotype in comparison with pauci-immune pathotype [83.3% (15/18), 83.3 % (5/6) vs. 28.6% (2/7), p = 0.022) achieved clinical response to certolizumab-pegol. Furthermore, we observed a significantly higher level of post-treatment tender joint count and VAS scores for pain, fatigue and global health in pauci-immune in comparison with lympho- and diffuse-myeloid patients but no differences in the number of swollen joints, ESR and CRP. Finally, we confirmed a significant fall in the number of CD68+ sublining macrophages post-treatment in responders and a correlation between the reduction in the CD20+ B-cells score and the improvement in the DAS28 at 12-weeks. Conclusions: The analysis of the synovial histopathology may be a helpful tool to identify among clinically indistinguishable patients those with lower probability of response to TNFα-blockade.

Keywords: anti-TNF; certolizumab-pegol; pathotype; rheumatoid arthritis; synovial tissue.

Figure 1

(A–F) Design of the study and histological characterization of the synovial tissue. (A) Timeline of the study, which included a baseline US-guided needle synovial biopsy at week 0 (37 patients) and a second biopsy of the same joint after 12-weeks of treatment with certolizumab-pegol (28 patients). At the top, representative gray-scale transverse section of an US-guided wrist biopsy showing the needle entering the joint space underneath the IV extensor tendons compartment. (B) Distribution (%) of synovial pathotypes at baseline. (C) Representative images of immunohistochemistry staining of synovial tissue for immune cells markers and classification in three pathotypes: lympho-myeloid, diffuse-myeloid and pauci-immune. Original magnification 4x. (D) Heatmap showing the degree of infiltration of immune cells (CD20, B-cells; CD138, plasma cells; CD3, T-cells; CD68L, macrophages of the lining; CD68SL, macrophages of the sublining) in each pathotype. (E) Distribution (%) of biopsied joints. (F) Histological pathotype according to synovial biopsy site. MCP, metacarpophalangeal. Fisher's test: not significant.

Figure 2

(A–D) Violin plots showing differences in CRP (A), VAS fatigue (0–100) (B), ultrasound (US) Power-Doppler (PD) score (0–3) (C) and US synovial thickening (ST) score measured in gray-scale (0–3) (D) of the biopsied joints between pathotype groups. Median and interquartile ranges are represented by thick and thin dotted lines, respectively. ^**p < 0.01, ^*p < 0.05, Kruskal-Wallis with post-hoc multiple comparison on 31 patients.

Figure 3

(A) Table summarizing clinical response rates by pathotypes to certolizumab-pegol at 12-weeks. ΔDAS28 was calculated by subtracting the baseline-DAS28 value from the 12-weeks-DAS28; DAS28 fall > 1.2 defined “responders.” Distribution of response rates was tested by Fisher's exact test while differences in ΔDAS28 by Kruskal-Wallis with Dunn's test. SD, standard deviation. (B) Comparison of pre- (pre-TH) and post-treatment (post-TH) DAS28 by pathotype. ^***p < 0.001, ^**p < 0.01, Kruskal-Wallis with post-hoc Dunn's multiple comparison test on 31 patients. Red dotted line represents DAS28 5.1 (“high disease activity”); green dotted line represents DAS28 3.2 (“low disease activity”).

Figure 4

(A) Table summarizing the comparison by pathotype of the patients' clinical features at 12-weeks post-treatment (^*Kruskal-Wallis with Dunn's correction). Significantly different variables are in bold. (B) Comparison of pre- (pre-TH) and post-treatment (post-TH) individual parameters of the DAS28 by pathotype. p values were calculated with Wilcoxon matched-pairs rank test (31 patients); ^****p < 0.0001, ^***p < 0.001, ^**p < 0.01, ^*p < 0.05. Green dots and line: “responders”; red dots and lines: “non-responders”. SD, Standard Deviation; ESR, erythrocyte sedimentation rate; CRP, C- Reactive Protein; TJ, Tender Joints; SJ, Swollen Joints; VAS, Visual Analog Scale (0–100); GH, Global Health; pt, patient; phy, physician; tir, tiredness; HAQ, Health Assessment Questionnaire.

Figure 5

(A) Sankey diagram representing the shift of pathotypes from pre- to post-treatment of the 17 patients who had gradable tissue both at baseline and 12-weeks. (B) Comparison of the delta (Δ) CD68 sublining (SL) score between responders and non-responders. ΔCD68SL is the difference in the CD68SL semi-quantitative score (0–4) between post- and pre-treatment. Mean and standard deviation shown. ^**p < 0.01 (Mann-Whitney). (C) Correlation plot showing ΔDAS28^{[12−weeks−baseline]} and ΔCD68SL^{[12−weeks−baseline]}; r Spearman coefficient of correlation, ^**p < 0.01. (D) Comparison of ΔDAS28 between patients with same/higher (ΔCD20≥0) or reduced CD20⁺ B-cells score (ΔCD20 < 0) post treatment. ^**p < 0.01 (Mann-Whitney, mean and standard deviation shown). (E) Correlation plot showing ΔDAS28^{[12−weeks−baseline]} and ΔCD20^{[12−weeks−baseline]}. r Spearman coefficient of correlation, ^**p < 0.01. Only responder lympho-myeloid patients were analyzed in (D,E) (10 patients). (B–E) Individual dots are color-coded by pathotype (blue, lympho-myeloid; red, diffuse-myeloid; orange, pauci-immune).