Rituximab Treatment before Reduced-Intensity Conditioning Transplantation Associates with a Decreased Incidence of Extensive Chronic GVHD

Article Outline

• Abstract
• Introduction
• Design and Methods
  • Power Analysis and Selection of Patients
  • Determination of B Cell Counts
  • Conditioning Regimen before RICT
  • Immunosuppression after allo-SCT
  • aGVHD and cGVHD
  • Statistical Analysis
• Results
  • Influence of Rtx Treatment Prior to RICT on B Cell Counts post-RICT
  • Impact of Rtx Treatment Prior to RICT on aGVHD and cGVHD
  • Rituximab Prior to RITC Did Not Associate with a Difference in Either PFS or OS
• Discussion
• Acknowledgments
• References
• Copyright

Chronic graft-versus-host-disease (cGVHD) is the major cause of late morbidity and mortality after allogeneic stem cell transplantation. B cells have been reported to be involved in mediating cGVHD. To assess whether preemptive host B cell depletion prevents extensive cGVHD after allogeneic reduced-intensity conditioning transplantation (RICT), 173 patients treated with RICT for various hematologic diseases, who had or had not received Rituximab (Rtx) within 6 month prior to RICT, were analyzed retrospectively. Rtx treatment within 6 months prior to RICT reduced extensive cGVHD significantly from 45.8% to 20.1%. We hypothesize that most likely host B cells initiate cGVHD, and thus, host B cell depletion prior to RICT by Rtx might be a valuable strategy to reduce extensive cGVHD after RICT.

Key Words: Host B cells, RICT, GVHD

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Introduction 

Chronic graft-versus-host-disease (cGVHD) is the major long-term complication of allogeneic stem cell transplantation (allo-SCT), as up to 70% of all survivors of allo-SCT beyond day 100 develop cGVHD 1, 2, 3. Several lines of investigation indicate that B cells are involved in the development of cGVHD 2, 4, and B cell depletion using the monoclonal anti-CD20 antibody Rituximab (Rtx) has demonstrated benefit in the treatment of steroid-refractory cGVHD with a success rate of up to 70% 5, 6, 7, 8. Given poor clinical responses in patients with steroid-refractory cGVHD [2] a potential prophylactic value of Rtx in cGVHD warrants further pursuit. To date, the answer to this question has been approached by others, but remains unanswered. A study of patients after myeloablative allo-SCT suggested no difference in cGVHD in 35 patients treated with Rtx as part of the allo-SCT conditioning regimen, but indicated a possible decrease in overall acute GVHD (aGVHD) incidence [9]. Therefore, we studied whether depletion of host B cells prior to transplantation by Rtx can reduce the incidence of this quality of life and life-threatening complication in patients who underwent allogeneic reduced-intensity conditioning transplantation (RICT). Patients with and without Rtx treatment within 6 months prior to RICT were analyzed retrospectively and were compared for the incidence of aGVHD and cGVHD, graft-versus-leukemia (GVL) effect, and overall survival (OS).

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Design and Methods 

Power Analysis and Selection of Patients 

To calculate the minimum amount of patients needed to see a significant difference in extensive cGVHD between the Rtx pretreated and non-Rtx group an effect size, thus the expected difference between 2 groups was set at 25%. The chi-square calculation indicated that 18 patients per group were needed to achieve a power of 80% (α = 0.05). One hundred seventy-three patients with various hematologic diseases (Table 1), who received an RICT at the University Medical Centre Utrecht, from September 2001 until April 2007, were included in this retrospective study, which allowed us to further increase the number of Rtx-treated patients to 29 Rtx patients and 144 control patients, which increased the power from 80% to >99%. One patient in the Rtx group had been treated with alemtuzumab 6 months prior to RICT. RICT was given as curative or as rescue treatment to patients younger than 70 years with an available HLA-matched related or unrelated donor (1 HLA class I or class II mismatch was allowed). Patients were treated according to clinical protocols approved by the local ethics board and gave their informed consent.

Table 1. Patient Characteristics

ALL indicates acute lymphoblastic leukemia; AML, acute myelogenous leukemia; ATG, antithymocyte globulin; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; CR, complete remission; Disease, disease for which RICT was given as treatment; cGVHD, chronic graft-versus-host disease; HLA, human leukocyte antigen; IS, immunosuppression; MDS, myelodysplastic syndrome; MM, multiple myeloma; MRD, matched related donor; NHL, non-Hodgkin lymphoma; No Rtx, no rituximab treatment within 6 months prior to RICT; NR, no remission; PMRD, partial matched related donor; PR, partial remission; RICT, reduced-intensity conditioning transplantation; RT, radiotherapy; Rtx, rituximab treatment within 6 months prior to RICT; SAA, severe aplastic anemia.

∗ P-values: Mann-Whitney U-test for age and follow-up; χ²-test for other factors.

Determination of B Cell Counts 

Absolute B cell counts were determined in whole blood using TRUcount tubes^® (Becton Dickinson, Fullerton, CA), according to the manufacturers’ protocol. In brief, whole blood samples were incubated with antihuman CD19 (fluorescein isothiocyanate [FITC]-labeled; Becton Dickinson) in TRUcount tubes^®. Erythrocytes were lysed with lysing buffer (Becton Dickinson). The samples were acquired on a LSR-II (Becton Dickinson) flow cytometer. Results were analyzed with FACS DIVA software (Becton Dickinson). Absolute B cell numbers were calculated according to manufacturers’ protocol.

Conditioning Regimen before RICT 

For patients with a matched-related donor, the conditioning regimen consisted of fludarabin (Flu; 30 mg/m²/day i.v. for 3 days) and 1 fraction of low-dose total body irradiation (TBI) (2 Gy). The transplantation with granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood hematopoietic stem cells (PBSCs) was performed after TBI. In the case of an HLA-mismatched family donor or any unrelated donor rabbit, antithymocyte globulin (ATG; 2 mg/kg/day for 4 days) was added to the regimen and infused before Flu was given. Patients with multiple myeloma (MM), who were treated with RICT, within 3 months after high-dose melfalan (200 mg/m²) and autologous SCT, only received TBI.

Immunosuppression after allo-SCT 

In the posttransplantation period all patients were treated with the immunosuppressants cyclosporin A (CsA) and mycophenolate mofetil (MMF). Patients received 2 × 4.5 mg/kg/day CSA until day +84 (short) or day +120 (long). Hereafter, CSA was tapered if no GVHD was present. CSA dose was lowered in the case of raised creatinin levels or severe side effects. Patients received 15 mg/kg/day MMF (maximum of 3 g/day) until day +28 (short) or +84 (long), also followed by tapering in the absence of GVHD.

aGVHD and cGVHD 

aGVHD was diagnosed and graded according to the Glucksberg criteria [10]; cGVHD was graded according to the Seattle classification 11, 12. aGVHD greater than grade 1 was treated with prednisone 1-2 mg/kg/day; in case of skin localization, topical prednisone treatment was applied. Additionally, CSA and MMF doses were increased or continued. Steroid-refractory aGVHD was treated with sirolimus, tacrolimus, Rtx, or more experimental drugs, such as alemtuzumab and dacluzimab. cGVHD of the skin was treated with topical prednisone. In severe cases of extensive cGVHD, prednisone 1 mg/kg/day was given. Time to aGVHD and cGVHD was calculated from the date of transplantation until occurrence of aGVHD or cGVHD.

Statistical Analysis 

Progression free survival (PFS) was defined as the probability of being alive with no indication of disease progression. OS was defined as the probability of survival without considering the occurrence or nonoccurrence of relapse. PFS and OS were measured in months and calculated from the date of transplantation until the date of the first signs of progression or last date of follow-up. Incidence of aGVHD and cGVHD, Epstein-Barr virus (EBV) and cytomegalovirus (CMV) reactivations, 95% donor chimerism, and probabilities of PFS and OS were calculated using the 1-Kaplan-Meier method. Kaplan-Meier curves were generated to illustrate survival, and the log-rank test was used to compare survival curves between subgroups. Univariate Cox regression was used to determine the prognostic value of various variables for the development of aGVHD and cGVHD, EBV, and CMV reactivation, 95% donor chimerism, PFS, and OS. These variables included sex, age, disease type, HLA-mismatch, sex-mismatch, ATG as part of conditioning regimen, radiotherapy (RT) as part of the treatment regimen of the prior disease, conditioning, remission state prior to RICT, aGVHD grade II-IV or III-IV, limited or extensive cGVHD, status of T cell and non-T cell chimerism, and application of Rtx. For aGVHD and cGVHD, a univariate time-dependent Cox regression analysis was used to assess its predictive value. Variables that had a value of P ≤ .10 in univariate analysis were included in a multivariate Cox regression analysis, because the number of available potential predictors exceeded the maximum allowed number. Rtx treatment was always included in multivariate analysis. For example, the effect of disease stage before RICT was assessed first with univariate Cox regression analysis and when a P-value of <.10 was reached, in multivariate Cox analysis together with Rtx. We thereby corrected for stage of disease. Not more than 7 variables were used at once in multivariate analysis. Thereby, it was guaranteed that the compared groups would not be too small. Variables were entered all at once in the Cox model. We used a backward logistic regression model to calculate the effect hierarchically. Statistical analyses were performed with SPSS 15.0 for Windows (SPSS Inc., Chicago, IL).

The significance of the difference between the B cell counts on time points pre- and posttransplantation of patients who received Rtx and of patients who did not, was assessed by using a Mann-Whitney U-test. Analyses were performed with GraphPad Prism 4.0 for Windows (GraphPad Software Inc., San Diego, CA). A probability level of 5% (P < .05) was considered significant in all analyses.

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Results 

Influence of Rtx Treatment Prior to RICT on B Cell Counts post-RICT 

One hundred seventy-three patients with hematologic malignancies who underwent RICT were analyzed retrospectively (Table 1). Patients were divided into groups that received (Rtx, n = 29) or did not receive (no-Rtx, n = 144) Rtx within 6 months prior to RICT as B cell depletion has been reported to last for up to 6 months 13, 14, 15. The median time of the last administration of Rtx before RICT was 2.0 months (range: 1.0-6.0 months). Patients received 375 mg/m² rituximab per application, and in 27 cases this was administered in combination with chemotherapy (R-CHOP/R-PECC; 6-8 cycles). One patient with EBV reactivation postsolid organ transplantation received 3 single applications of Rtx, 1 patient received twice Rtx for chronic active EBV infection. To investigate whether B cell depletion with Rtx within 6 months prior to RICT effects B cell counts pre- and post-RICT, B cell counts from patients who did (n = 5) and did not receive Rtx (n = 6) within 6 months prior to RICT were investigated from the study cohort when B cell counts were available prior and after RICT at least until 9 months post-RICT. B cell counts from patients were compared to B cell counts from 14 healthy controls (median: 212.5/μL; range: 88-418/μL). Median B cells counts pre-RICT (13.0/μL; n = 4) and 3 months post-RICT (23.5/μL; n = 6) were significantly lower (P < .01) in patients who received Rtx compared to a healthy control group (n = 14) (Figure 1). In contrast, the median of B cell counts in patients who did not receive Rtx prior to RICT did not differ significantly before (182/μL; n = 3) and 3 months after (148.5/μL; n = 6) RICT compared to the healthy control group (n = 14). Six and 9 months after RICT, no significant difference in B cell counts was observed for both the Rtx and the no-Rtx group, when compared to the healthy control group (Figure 1). Thus, Rtx treatment within 6 months prior RICT reduces the total number of B cells post-RICT at least until 3 months after RICT.

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• Figure 1 

  B cell counts pre- and post-RICT in patients who received (white bars) and did not receive (black bars) Rtx within 6 months prior to RICT compared to a healthy control group (gray bar). Statistical analyses compare B cell counts of patients of each time point to B cell counts of the healthy control group. Counts before RICT (within 1 week), or months after RICT are indicated. Bars indicate median and range. Numbers indicate the number of samples measured per time point. Only significant changes (P < .05) are indicated. Statistical analyses were performed with a Mann-Whitney U-test.

Impact of Rtx Treatment Prior to RICT on aGVHD and cGVHD 

The incidence of aGVHD was analyzed by both univariate and multivariate analysis. There was no significant difference in the incidence of grade II-IV and grade III-IV between the Rtx (48.2% grade II-IV, n = 14; 17.9% grade III-IV, n = 5) and the no-Rtx group (45.7% grade II-IV, n = 64; and 15.6% grade III-IV, n = 22) (Figure 2A and B). However, the median onset of grade III-IV aGVHD was significantly earlier in the Rtx compared to the no-Rtx group (Rtx group 0.60 months, no-Rtx group 1.60 months; P = .013) (Figure 2C). Only ATG prevented grade III-IV aGVHD significantly (P = .041; hazard ratio [HR] 2.57; 95% confidence interval [CI]: 1.04-6.38) in multivariate analysis with Cox regression (Table 2).

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• Figure 2 

  1-Kaplan-Meier curves of aGVHD grade II-IV (A) and grade III-IV (B) in patients who received Rtx prior to RICT and in patients who did not. (C) Onset of aGVHD grade III-IV in patients who received Rtx prior to RICT and in patients who did not.

Table 2. Multivariate Cox Regression Analysis of Outcome in Terms of aGVHD Grade II-IV, aGVHD Grade III-IV, Total cGVHD, Extensive cGVHD, Limited cGVHD, PFS, and Nonrelapse Mortality

ATG indicates antithymocyte globulin; CI, confidence interval; GVHD, graft-versus-host disease; aGVHD, acute graft-versus-host disease; cGVHD, chronic graft-versus-host disease; HLA, human leukocyte antigen; HR, hazard ratio; PFS, progression-free survival; RT, radiotherapy; Rtx, rituximab within 6 months prior to RICT.

P-values <.05 are considered significant.

To assess whether Rtx treatment prior to RICT prevents the development of limited and extensive cGVHD, first the overall frequency of limited and extensive cGVHD was determined and was not significantly different between the Rtx and the no-Rtx group (42.5% [n = 10] versus 54.9% [n = 69], P = .468), in neither uni- nor multivariate analysis (Figure 3A and Table 2). However, in the Rtx group 20.1% of patients developed extensive cGVHD (n = 5), whereas in the no-Rtx group, 45.8% of patients developed extensive cGVHD (n = 58) (P = .053) (Figure 3B). The main sites affected were skin, liver, mucosae, and lung, with no profound difference between the Rtx and the no-Rtx group (Table 1). After multivariate analysis significantly less extensive cGVHD was detected in patients treated with Rtx (P = .035, HR 2.67 [95% CI: 1.07-6.68]) (Table 2). Only the application of ATG as part of the conditioning regimen (P < .001, HR 4.35 [95% CI: 2.26-8.38]), but no other analyzed factors, also decreased the incidence of extensive cGVHD in a multivariate analysis. Vice versa, limited cGVHD was increased in the Rtx group (25.6% [n = 5]) compared to the no-Rtx group (12.9% [n = 11], P = .088) (Figure 3C). Again, this difference reached significance after multivariate analysis (P = .040, HR 0.33 [95% CI: 0.11-0.95]) (Table 2). In summary, Rtx treatment prior to RICT did not affect the frequency of grade II-IV or III-IV aGVHD, but substantially attenuated extensive cGVHD and thereby presumably increased the frequency of limited cGVHD.

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• Figure 3 

  1-Kaplan-Meier curves of total cGVHD (A) extensive cGVHD (B), and limited cGVHD (C) in patients who received Rtx prior to RICT and in patients who did not.

Rituximab Prior to RITC Did Not Associate with a Difference in Either PFS or OS 

Frequently, manipulations that result in a reduction of GVHD are also associated with a reduction of GVL and vice versa 1, 2. Therefore, it was assessed whether host B cell depletion by Rtx treatment might not only impair GVHD, but also influence GVL. As surrogate marker for GVL, PFS was assessed separately in the Rtx and no-Rtx group. PFS was decreased by HLA mismatch (P = .038, HR 0.51 [95% CI: 0.27-0.96]) and radiotherapy as part of the treatment regimen prior to RICT (P = .009, HR 0.43 [95% CI: 0.23-0.81]). Although Rtx treatment was associated with a significantly reduced incidence of cGVHD, PFS was not influenced (P = .306) (Figure 4A and Table 2) by any other analyzed factors including type of disease. Also, OS at 40 months did not differ significantly (P = .159) between the Rtx group (n = 18; 61.4%) and no-Rtx group (n = 103; 67.4%) (Figure 4B). This observation is surprising, as PFS and OS [16], in contrast to aGVHD and cGVHD 7, 17, might have been heavily influenced by the imbalance in the type of disease in this study cohort. However, the low number for certain entities in this study cohort might have hampered the analysis. In summary, our data demonstrate a reduced cGVHD in Rtx treated patients, whereas GVL is not affected.

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• Figure 4 

  Kaplan-Meier curves of PFS (A) and OS (B) in patients who received Rtx prior to RICT and in patients who did not.

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Discussion 

B cells play an important, but yet unclear role in the pathogenesis of cGVHD 2, 4, and B cell depletion of donor B cells after allo-SCT with the monoclonal anti-CD20 antibody, Rtx, has been shown to improve steroid refractory cGVHD 5, 6, 7, 8. We asked whether B cell depletion by Rtx prior to RICT could decrease the incidence of cGVHD, and preemptive B cell depletion could therefore be a strategy to prevent this major complication after allo-SCT. Our data indeed indicate that Rtx treatment prior to RICT decreases the incidence of extensive cGVHD, whereas relapse rate was not influenced. This result is in contrast to a previous study that retrospectively compared aGVHD and cGVHD in 35 leukemia patients who received Rtx, and 31 control patients [9]. The authors reported no influence of rituximab on the incidence of cGVHD, and a possible, but not significant, decrease of the incidence of aGVHD. The main explanation for this difference might be the myeloablative conditioning regimen, which depletes the majority of immune cells, thus also B cells. Consequently, the reported overall incidence of cGVHD in both arms was very low (∼11%).

A significantly shorter follow-up period in Rtx patients than in patients who did not receive Rtx might have influenced the incidence of cGVHD. However, the latest time point of onset of extensive cGVHD was 18.2 months (median 5.0 months [range: 1.5-18.2 months]), and the median follow-up was in both groups >20 months. So, even though the follow-up of the Rtx group was shorter than the follow-up of the no-Rtx group, it was still long enough for extensive cGVHD to develop.

B cells have been suggested to be the final effector cells in cGVHD by secreting interleukin (IL) 6, a known fibroblast growth factor [18]. However, in patients of whom B cell counts were available at longer follow-up (6 or 9 months post-RICT), no significant differences could be observed in total B cell counts at the onset of cGVHD, and in most patients a full donor chimerism was reached (data not shown). This suggests that the early depletion of most likely host B cells rather than donor B cells are important for the development of cGVHD. Thus, alternative mechanisms must be responsible for the reduced incidence of extensive cGVHD if patients are treated with Rtx prior to RICT. Host B cells have been proposed to serve as professional antigen-presenting-cells (APCs), present minor histocompatibility antigens, and induce GVL and GVHD [19]. In this light, our data suggest that host antigen-presenting B cells are under certain conditions [20] required for initiating priming of donor T cells, which mediate maximum GVHD but not substantially GVL. Thus, host B cells might rather influence shaping of a self-reactive donor T cell repertoire than directly mediating cGVHD.

We can only speculate as to why Rtx-treated patients who developed grade III-IV aGVHD developed it earlier, and conclusions have to be drawn very cautiously because of the very low number of patients in the Rtx arm (n = 5). One explanation for this more rapid course of aGVHD could be that B cells serve as regulatory B cells, as reported in mice, and provide IL-10 to attenuate the course of the disease [21]. However, we cannot exclude that also other modulators of the immune system such as regulatory T cells 22, 23 or Th17 [24] cells were indirectly affected by B cell depletion.

We are aware of major limitations of this study, such as an imbalance in disease, which primarily might have influenced the analysis of PFS and OS, but not the incidence of aGVHD and cGVHD. Thus, our data suggest that most likely host B cells are important in the pathogenesis of cGVHD. Preemptive B cell depletion might therefore be a valuable strategy to substantially reduce extensive cGVHD after RICT.

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Acknowledgments 

Financial disclosure: This work was supported by a grant from the Dutch Cancer Society, KWF 2006-3685. The authors have no conflicting financial interests.

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