1270 - Clinical Response and B Cell Analysis by High-Sensitive Flowcytometry After the Second Course of Rituximab

Monday, November 7, 2011: 9:00 AM-6:00 PM
Hall F2 - Poster Hall (McCormick Place West)
Hans-Peter Brezinschek1, Franz Rainer2, Kerstin Brickmann1, Raimund Lunzer2 and Winfried B. Graninger1, 1Medical University Graz, Graz, Austria, 2Hospital Barmherzige Brueder, Graz- Eggenberg, Austria
Presentation Number: 1270

Background/Purpose: Recently, we have shown that in an Austrian rituximab-registry RA-patients did not have a better clinical response when they completely depleted B cells 15 days after the first infusion. In addition, our results suggested that RA patients with a low number of transitional 1 B cells and/or low percentage of CD95+ post switch cells will benefit more profoundly from a B cell depleting therapy. The purpose of this study was to correlate the effect of a second course of rituximab (RTX) with the number of B cells after 15 days as well as B cell subsets.

Methods: Patients in the Austrian B Cell surveillance (ABS)-register were included in the study when they had a second course of RTX. DAS28 was determined before, 2 and 24 weeks after rituximab application. Peripheral blood mononuclear cells were isolated at the same time points and cells were stained with monoclonal antibodies directed towards CD19, CD24, CD27, CD38, CD45 and IgD. To exclude T cells and monocytes CD3 and CD14 were utilized. Five hundred thousand cells were acquired and analyzed using a seven-channel flow cytometry (BD Canto II cytometer, Software FACSDiva). According to their surface staining B cells were divided in naive (CD19+, IgD+, CD27-); IgD memory (CD19+, IgD+, CD27+), post switch (CD19+, IgD-, CD27+) and double negative (CD19+, IgD-, CD27-) cells. In addition, B cells were further characterized using CD95 and CD80. Complete depletion was defined as 0,002%, i.e. 10 events or less in 500.000 CD45+ cells. 

Results: Until now, 105 RA patients have been included in the ABS registry and 13 patients have undergone the week 24-analysis of a second course of RTX. There was a significant reduction in the DAS28 (mean ± SE) before the first and the second therapy course (5,92 ± 0.25 versus 4.23 ± 0.25, p < 0.0001). Interestingly, patients who did not respond to RTX after the 1st course still had a reduction in DAS28 after the second treatment, but it was smaller compared to 1st cycle responders (-0.8 ± 0.8 versus -1.9 ± 0.4; respectively). Similar to the results from the 1st cycle, no correlation between the EULAR response and complete depletion at day 15 was found. Thus, there was no significant difference in the mean number of B cells (mean ± SE) in responders and non-responders (33.0 ± 22.6 versus 19.5 ± 4.6, respectively). Interestingly, 2 out of four non-responder had an improvement of their DAS28 after the second cycle. Non-responder had an elevated frequency of post switch B cells compared to responders (29.8% 5.9 versus 15.0% ± 7.6; respectively). No significant differences were found for CD80 or CD95 expression on B cell subsets for responders and non-responders. Furthermore, in responders the numbers of transitional (T1) B cells were lower compared to non-responders  or healthy controls (45 ± 23, 67 ± 40 and 384 83, respectively), but this was not significant.

Conclusion: Our preliminary results suggest again that the enumeration of B cells on day 15 will not help to discriminate Rituximab-responders from non-responders. Furthermore, a second cycle of RTX is warranted in RA-patient with an inappropriate response to the first therapy-course.


Keywords: rituximab

Disclosure: H. P. Brezinschek, None; F. Rainer, None; K. Brickmann, None; R. Lunzer, None; W. B. Graninger, None.