B lymphocyte-typing for prediction of clinical response to rituximab

Abstract

Introduction: The prediction of therapeutic response to rituximab in rheumatoid arthritis is desirable. We evaluated whether analysis of B lymphocyte subsets by flow cytometry would be useful to identify non-responders to rituximab ahead of time.

Methods: Fifty-two patients with active rheumatoid arthritis despite therapy with TNF-inhibitors were included in the national rituximab registry. DAS28 was determined before and 24 weeks after rituximab application. B cell subsets were analyzed by high-sensitive flow cytometry before and 2 weeks after rituximab administration. Complete depletion of B cells was defined as CD19-values below 0.0001 x10⁹ cells/liter.

Results: At 6 months 19 patients had a good (37%), 23 a moderate (44%) and 10 (19%) had no EULAR-response. The extent of B lymphocyte depletion in peripheral blood did not predict the success of rituximab therapy. Incomplete depletion was found at almost the same frequency in EULAR responders and non-responders. In comparison to healthy controls, non-responders had elevated baseline CD95⁺ pre-switch B cells, whereas responders had a lower frequency of plasmablasts.

Conclusions: The baseline enumeration of B lymphocyte subsets is still of limited clinical value for the prediction of response to anti-CD20 therapy. However, differences at the level of CD95⁺ pre switch B cells or plasmablasts were noticed with regard to treatment response. The criterion of complete depletion of peripheral B cells after rituximab administration did not predict the success of this therapy in rheumatoid arthritis.

Figure 1

Disease activity score using 28 joint counts (DAS28) at baseline and 24 weeks after rituximab application in European League Against Rheumatism (EULAR) responders and non-responders. The lines indicate the border between low disease activity (LDA), moderate disease activity (MDA), and high disease activity (HDA).

Figure 2

Number of B cells 15 days after the first rituximab infusion. Each dot represents one patient. The horizontal lines represent the median.

Figure 3

B cell phenotype distribution before rituximab. Frequencies of (a) CD27^- IgD^- double-negative B cells and (b) CD27⁺⁺ IgD^- plasmablast in patients with rheumatoid arthritis (RA) at baseline and in healthy age-matched controls. The percentages of CD38^hi cells within (c) the post-switch memory B cells and (d) plasmablasts (that is, late plasmablasts [16]) are shown. (e) The frequency of CD95⁺ cells within the pre-switch memory B cells is depicted. Significant differences between the populations using Mann-Whitney-test are indicated. Of note, the difference between non-responders and responders was significant using univariate logistic regression analysis.

Figure 4

Model of B-cell differentiation (adapted from [10,12,16]). *All patients with rheumatoid arthritis (RA) had significantly fewer CD38⁺ post-switch B cells in comparison with healthy age-matched controls. European League Against Rheumatism (EULAR) non-responders (NR) had a significantly higher frequency of CD95⁺ pre-switch memory B cells. Rituximab responders (R) had significantly lower levels of plasmablast before treatment and more B cells differentiated into double-negative B cells that are less likely to generate plasma cells [29].