DOI: 10.5152/eurjrheum.2019.18109 Original Article Rituximab levels are associated with the B cell homeostasis but not with the clinical response in patients with rheumatoid arthritis Cèsar Díaz‐Torné1 , Maria Àngels Ortiz2 , Mónica Sarmiento1,3 , César Díaz‐López1 , Hèctor Corominas1 , Jordi Casademont4 , Silvia Vidal2 Abstract Objective: To study the levels of rituximab (RTX) and anti-RTX antibodies (ARAs) in patients with rheu- matoid arthritis (RA) at 30, 90, and 180 days after the first infusion, in relation to clinical and serological parameters and B cell homeostasis. Methods: Thirty-four patients with RA who failed to respond to anti-tumor necrosis factor therapy received RTX. At baseline, 4, 12, and 24 weeks after the first infusion of RTX, we performed a clinical assessment and determined the levels of RTX, ARAs, B cells, rheumatoid factors, anti-cyclic citrullinat- ed peptide antibodies, immunoglobulins, and complements. Results: RTX levels varied widely among patients. No ARAs were detected during the follow-up. Pa- tients with lower levels of RTX presented with higher decreases in erythrocyte sedimentation rate, immunoglobulins, and complement 6 months after the first infusion. Patients with higher levels of RTX showed a higher B cell depletion at 90 days but an earlier B cell recovery than those with lower ORCID IDs of the authors: levels of RTX. No differences in clinical response were observed between the two groups at 6 months C.D.T. 0000-0001-6275-7699; M.A.O. 0000-0002-1574-9861; after starting the treatment. M.S. 0000-0001-5212-9904; Conclusion: Our findings suggest that RTX levels in the serum of patients with RA are related to B C.D.L. 0000-0002-8416-1068; cell homeostasis and the severity of immunological parameters but not to the clinical response at 6 H.C. 0000-0002-7738-6787; J.C. 0000-0002-8100-1827; months. S.V. 0000-0002-3909-6682. Keywords: Rheumatoid arthritis, rituximab, B cells Cite this article as: Díaz‐Torné C, Ortiz MA, Sarmiento M, Díaz‐López C, Corominas H, Casademont J, Vidal S. Rituximab levels are associated with the B cell homeostasis Introduction but not with the clinical response in patients with rheumatoid arthritis. Eur J Advances in knowledge of rheumatoid arthritis (RA) have shown that this disease is not a syndrome Rheumatol 2019; 6(2): 81-4. as different subsets of patients have different prognoses and responses to treatment. Our greater 1 Department of Rheumatology, Hospital understanding of the pathogenesis of the disease has also led to the development of biological de la Santa Creu i Sant Pau, Barcelona, therapies. One such therapy is rituximab (RTX), a glycosylated chimeric mouse/human monoclonal Spain 2 Department of Immunology, IIB‐Institut antibody directed toward CD20, a pan‐B surface marker that depletes B lymphocytes. B cell deple- de Recerca de l’Hospital de Sant Pau, tion after RTX treatment differs in distinct diseases, tissues, and from patient to patient (1‐2). As in Barcelona, Spain 3 Department of Rheumatology, other diseases, such as lymphoma (3), and with other biological therapies (4), higher serum levels of Colmédica, Bogota, Spain RTX should predict a better B cell depletion and clinical response. However, to date, studies of RTX 4 Departament of Internal Medicine, Hospital de la Santa Creu i Sant Pau. levels in patients with RA have not found an association between RTX levels and clinical outcomes. Universitat Autònoma de Barcelona, None of these studies completed the B cell count or determined RTX levels beyond 3 months. We Barcelona, Spain prospectively analyzed clinical outcomes and B cell and RTX levels in an RA cohort during a com- Address for Correspondence: plete course of treatment. Cèsar Díaz-Torné; Department of Rheumatology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain Methods E-mail: cesardiaztorne@gmail.com Peripheral blood samples were obtained from 34 patients who met the American College of Rheuma- Submitted: 5 July 2018 tology criteria for RA. Treatment with RTX was started following standard clinical practice. All patients Accepted: 18 January 2019 Copyright@Author(s) - Available online at were refractory to treatment with synthetic disease‐modifying antirheumatic drugs, including metho- www.eurjrheumatol.org. trexate, and tumor necrosis factor antagonists. The study was approved by the ethics committee of the Content of this journal is licensed under a Creative Hospital de Sant Pau according to the Declaration of Helsinki. Written consent was obtained from all Commons Attribution-NonCommercial 4.0 International License. patients. Heparinized blood samples were collected on days 0 (baseline, before treatment), 30, 90, and 180 days after the first RTX infusion. Every course cycle consisted of two doses of RTX (1000 mg) 2 weeks apart. Nine patients had minor infusion reactions that required a slower infusion rate, but completed 81 Díaz-Torné et al. Rituximab, B cells, and clinical response in RA Eur J Rheumatol 2019; 6(2): 81-4 treatment. One patient presented a major phycoerythrin (PE)/CD8‐PE‐Texas red (ECD)/ Statistical Analysis infusion. Treatment was terminated, and the CD3‐PE‐Cy5) and Natural Killer (NK) cocktail Calculations were performed using Statis- patient was excluded. At all visits, we deter- (anti‐CD45‐FITC/CD19‐ECD/CD3‐PE‐Cy5/ tical Packages of Social Sciences version 18 mined erythrocyte sedimentation rate (ESR), CD56‐PE) (Beckman Coulter). Analysis was (SPSS Inc.; Chicago, IL, USA). Results were C‐reactive protein (CRP), immunoglobulins performed on a Cytomics FC 500 using CXP expressed as mean±SEM. Mann–Whitney U (IgG, IgA, and IgM), complements (C3 and C4), software (Beckman Coulter). Sphero Rainbow test and Wilcoxon’s test were used for inde- rheumatoid factor (RF), anti‐cyclic citrullinat- Calibration particles (Spherotech, Lake Forest, pendent and related variables respectively, ed peptide antibodies (ACPAs), and RTX and IL, USA) were used for periodic calibration of at different time‐points during the follow-up. A p‐value of ≤0.05 was considered statistical- anti‐RTX levels. channels. ly significant. Flow cytometry RTX and anti-RTX levels in serum Results Venous blood was processed for flow cytom- Sera were tested for RTX (PG‐M‐127‐01‐ A total of 34 patients were included in the etry as described previously, within 2 h of col- PROMONITOR®‐RTX) and anti‐RTX levels study. The mean age of the patients was 62.4 lection (5). Monoclonal antibodies used were PG‐M‐127‐02‐PROMONITOR®‐RTX) using spe- (37–81) years, and 93.1% were women. Dis- as follows: Cyto‐STAT tetrachrome system cific ELISAs according to the manufacturer’s ease duration was 15.7 (10.7) years. Only 9.09% lymphocyte cocktail (anti‐CD45‐FITC/CD4‐ instructions. were RF and ACPA negative. Of the 34 patients, 55.6% were on monotherapy, and 33.3% were Table 1. Decrements of clinical and analytical values at 180 days after the first rituximab infusion on methotrexate. Group A Group B (>56,000 pg/mL) (<56,000 pg/mL) p RTX and anti-RTX levels As shown in Figure 1, the peak of RTX lev- ▼180 DAS28 1.7±1.3 1.7±1 0.96 els was observed 30 days after initiating treatment. As in previous studies (6‐9), ▼180 ESR (mm/h) RTX levels were highly variable and ranged ▼180 ESR (mm/h) 9.1±9.1 37.1±8 0.038 from 13,000 pg/mL to 115,000 pg/mL. After treatment for 90 days, RTX levels decreased ▼180 IgG (mg/dL) 3±3.6 23.3±6.9 0.018 significantly but were detectable in 80% of ▼180 IgA (mg/dL) 0.8±16.3 16.3±22.6 0.092 patients. No RTX was detected on day 180. As in other studies, no anti‐RTX antibodies ▼180 IgM (mg/dL) 24.2±4.5 39.2±4.2 0.03 (ARAs) were detected during the follow-up ▼180 RF (UI/mL) 56.7±6.4 53.3±10.9 0.7 (9). ▼180 C3 (mg/dL) −13±4.7 6±3.7 0.005 Effects of RTX levels on clinical and laboratory pa- ▼180 C4 (mg/dL) −20±4.6 −3.7±7.8 0.07 rameters and B cell recovery To analyze the association between RTX B cells on day 180 (cells/mL) 9.7±3.2 1.3±0.4 0.03 levels and clinical and immunological pa- rameters, patients were segregated into two groups according to RTX levels at 30 days. Patients with RTX levels >56,000 pg/ mL (group H: 51.5% of patients) and those with RTX levels <56,000 pg/mL (group L: 48.5% of patients). Patients from both groups were comparable before treatment. As shown in Table 1, decreases in DAS28, CRP, and RF were similar in both groups at 180 days. However, group L patients pre- sented a larger decrease of ESR, IgG, IgA, IgM, C3, and C4. As shown in Figure 2, depletion kinetics and recovery of B cells were different in the two groups of patients. Patients from group H showed a greater initial B cell depletion than those from group L. This greater depletion was maintained up to day 90. B cell recovery was significantly faster in group H patients. By day 180, B cells were still depleted in group L Figure 1. Rituximab levels evolution from baseline to 180 days after RTX first infusion. RTX levels (20% of group H patients and 44% of group were highly variable from 13,000 to 115,000 pg/mL. After the first infusion for 90 days, RTX levels L patients presented <1 CD19+ cell/mL) were still detectable in 80% of patients (p=0.028). 82 Eur J Rheumatol 2019; 6(2): 81-4 Díaz-Torné et al. Rituximab, B cells, and clinical response in RA This hypothesis could also explain why the treatment response in patients with RA with higher levels of RTX in sera was comparable with those with lower levels of RTX. It also could suggest that patients with lower RTX levels could be candidates of RTX treatment on demand instead of every 6 months. More studies should be conducted to clarify this issue. Ethics Committee Approval: Ethics committee approv- al was received for this study from the Ethics Com- mittee of Hospital de la Santa Creu i Sant Pau. Informed Consent: Written informed consent was ob- tained from patients who participated in this study. Peer-review: Externally peer-reviewed. Author Contributions: Concept - C.D.T., C.D.L., J.C., S.V.; Design - C.D.T., M.A.O., M.S., C.D.L., S.V.; Supervision Figure 2. B cell evolution in both groups. Group with higher levels of rituximab showed earlier - C.D.L., J.C., S.V.; Data Collection and/or Processing - peripheral B cell depletion and also an earlier B cell recovery C.D.T., M.A.O., M.S., H.C., S.V.; Analysis and/or Interpre- tation - C.D.T., M.A.O., C.D.L., S.V.; Writing Manuscript - Discussion depletion levels on days 30 and 90 after the C.D.T., M.A.O., S.V.; Critical Review - C.D.T., M.A.O., M.S., We found that higher decreases of immuno- first infusion (7). Unfortunately, the authors did H.C., C.D.L., J.C., S.V. logical parameters were associated with low- not show results further than 90 days, the time er levels of RTX. In addition, the recovery of B when we observed that peripheral B cell recov- Conflict of Interest: The authors have no conflict of cells was delayed in patients with lower levels ery began in patients with higher levels of RTX. interest to declare. of RTX. This finding appears to contradict pre- There are several explanations for highly vari- Financial Disclosure: The authors declared that this vious studies in patients with lymphoma or lu- study has received no financial support. pus treated with RTX that higher levels of RTX able levels of RTX in patients with RA: (1) differ- in sera were associated with better outcomes ences in RTX metabolism, (2) polymorphisms in Fc neonatal receptor, a molecule involved in References and a stronger B cell depletion (3). Differenc- 1. Leandro MJ, Cooper N, Cambridge G, Ehren- es in RTX levels and in B cell recovery pace the recycling of IgG1 (11), and (3) differences stein MR, Edwards JC. Bone marrow B‐lineage between patients with RA and lymphoma are in RTX distribution in the body compartments. cells in patients with rheumatoid arthritis fol- not surprising because doses of the drug and The complex distribution of RTX in different lowing rituximab therapy. 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