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. Author manuscript; available in PMC: 2012 May 18.
Published in final edited form as: Br J Haematol. 2009 Mar 26;145(6):816–824. doi: 10.1111/j.1365-2141.2009.07674.x

Prior Rituximab Correlates with Less Acute Graft-versus-Host Disease and Better Survival in B-Cell Lymphoma Patients Who Received Allogeneic Peripheral Blood Stem Cell Transplantation (PBSCT)

Voravit Ratanatharathorn 1, Brent Logan 2, Dan Wang 3, Mary Horowitz 2, Joseph P Uberti 1, Olle Ringden 4, Robert Peter Gale 5, Hanna Khoury 6, Mukta Arora 7, Stephen Spellman 3, Corey Cutler 8, Joseph Antin 8, Martin Bornhaüser 9, Gregory Hale 10, Leo Verdonck 11, Mitchell Cairo 12, Vikas Gupta 13, Pavletic Steven 14, for the Center for International Blood and Marrow Transplant Research (CIBMTR), Milwaukee, WI, USA
PMCID: PMC3355660  NIHMSID: NIHMS139962  PMID: 19344418

Summary

Prior therapy with rituximab might attenuate disparate histocompatibility antigen presentation by B cells, thus decreased the risk of acute graft-versus-host disease (GVHD) and improved survival. We tested this hypothesis by comparing the outcomes of 435 B-cell lymphoma patients who received allogeneic transplantation from 1999 to 2004 in the Center for International Blood and Marrow Transplant Research database: 179 subjects who received rituximab within 6 months prior to transplantation (RTX cohort) and 256 subjects who did not receive RTX within 6 months prior to transplantation (No-RTX cohort). The RTX cohort had a significantly lower incidence of treatment-related mortality (TRM) (relative risk [RR] = 0.68; 95% confidence interval [CI], 0.47 - 1.0; P = 0.05), lower acute grade II-IV (RR = 0.72; 95% CI, 0.53 - 0.97; P = 0.03) and III-IV GVHD (RR = 0.55; 95% CI, 0.34 - 0.91; P = 0.02). There was no difference in the risk of chronic GVHD, disease progression or relapse. Progression-free survival (PFS) (RR = 0.68; 95% CI 0.50 - 0.92; P = 0.01) and overall survival (OS) (RR = 0.63; 95% CI, 0.46 - 0.86; P = 0.004) were significantly better in the RTX cohort. Prior RTX therapy correlated with less acute GVHD, similar chronic GVHD, less TRM, better PFS and OS.

Keywords: rituximab, lymphoma, GVHD, allogeneic, transplantation

Introduction

Animal models have shown the importance of host-derived antigen presenting cells (APC) in the initiation of graft-versus-host disease (GVHD). In the animal model studied, B6 β2-M-/- chimera (derived from engrafting B6 recipients with marrow from β2-microglobulin knock-out mice) failed to develop GVHD, thus host APC played a pivotal role in the immunopathogenesis of GVHD. (Shlomchik, et al 1999) Although dendritic cells were primarily responsible for the pathogenesis of GVHD in this model, other APC, such as B cells, are also efficient APC for soluble protein antigens and in Friend virus-induced leukemia.(Kurt-Jones, et al 1988, Schultz, et al 1990) Using a B-cell deficient mouse model in which mice received either control rabbit immunoglobulin or rabbit anti-IgM μ chain from birth (B-cell deficient) 3 times per week until the end of the experiment, Schultz et al (1995) reported a lower incidence of GVHD in B-cell deficient animals and the rate of GVHD was even lower if the grafts were depleted of B cells. This study suggested the participating role of host and donor B cells in the immunopathogenesis of acute GVHD.

The anti-CD20 chimeric antibody, rituximab, is an effective therapy for patients with CD20+ non-Hodgkin lymphoma (NHL).(Davis, et al 1999, McLaughlin, et al 1998a, Piro, et al 1999) This antibody is a highly effective B-cell depleting agent.(McLaughlin, et al 1998b) B-cell depletion, resulting from rituximab treatment for lymphoma, may potentially abrogate host B-cell antigen presentation to donor T cells, and thus diminish the risk of acute GVHD for patients undergoing allogeneic stem cell transplantation (alloSCT). To test this hypothesis, we evaluated the impact of rituximab therapy prior to allogeneic transplantation in a large of cohort of NHL patients reported to the Center for International Blood and Marrow Transplant Research (CIBMTR). As the B-cell depleting effect of rituximab therapy lasts up to 6 months after treatment(McLaughlin, et al 1998b), patients receiving rituximab more than 6 months prior to transplantation were categorized as not having prior therapy with rituximab.

Patients and Methods

Subjects

A total of 435 consecutive adult B-cell NHL patients who received allogeneic peripheral blood stem cell transplantation (PBSCT) were reported to the CIBMTR between 1999 and 2004. Recipients of unrelated donor transplantation were facilitated by the National Marrow Donor Program (NMDP). Patients that had received prior therapy with anti-CD52 or anti-T cell antibodies, were recipients of marrow or cord blood grafts, recipients of T-depleted graft from any source or recipients of mismatched sibling graft were excluded from this analysis. Patients receiving rituximab as part of their conditioning regimen were included. Informed consents for patients receiving sibling donor transplantation were obtained prospectively. All surviving unrelated donor transplant recipients were retrospectively contacted to obtain consent for participation in the NMDP research program. Informed consent was waived by the NMDP Review Board for all deceased patients. Surviving patients who did not provide signed informed consent to allow analysis of their clinical data were excluded. To adjust for the potential bias introduced by exclusion of non-consenting surviving patients, a corrective action plan (CAP)- modeling process randomly excluded the same percentage of deceased patients using a biased coin randomization with exclusion probabilities based on characteristics associated with not providing consent for use of the data in survivors. There were 179 patients in the ‘Rituximab’ cohort (RTX), who had received rituximab within 6 months of transplantation, including 17 patients who received rituximab as part of the conditioning regimen. The remaining 256 patients were included as the ‘No-Rituximab’ cohort (No-RTX); they had not received prior rituximab therapy (157 patients) or had received rituximab more than 6 months prior to transplantation (99 patients). Among the 133 patients with follicular lymphoma in the No-RTX cohort, 60 patients received rituximab more than 6 months prior to transplantation and 73 patients did not receive rituximab at any time during the course of their disease. Median follow-up of survivors for the RTX cohort was 30 (3-82) months and 39 (3-88) months for the No-RTX cohort.

The definitions of human leucocyte antigen (HLA)-matching for the unrelated donor transplants shown to have correlation with survival was adopted and included in the prognostic models when applicable.(Weisdorf, et al 2007) The well-matched pairs had no identified HLA mismatch and informative though not necessarily high resolution in all 4 loci (HLA-A, HLA-B, HLA-C and HLA-DRB1) (N=80); partially matched pairs had a defined single locus mismatch at any of the 4 loci and/or missing HLA-C data (N=18) and mismatched pairs had 2 or more identified allele or antigen mismatches (N=7).

Patients were defined as ‘sensitive’ to chemotherapy if the last chemotherapy given with 6 months prior to transplantation had resulted in ≥50% reduction in the bidimensional diameter of all disease sites with no new sites of disease; these included patients who had achieved a complete or partial remission as a result of chemotherapy. ‘Chemotherapy-resistant disease’ was defined as a less than 50% reduction in the diameter of all disease sites or the development of new disease sites. ‘Untreated’ patients were defined as those who did not receive chemotherapy within 6 months prior to the preparative regimen for transplantation.

Study Endpoints

Primary endpoints were grade II-IV acute GVHD and grade III-IV acute GVHD. The GVHD gradings according to Glucksberg criteria (Glucksberg, et al 1974) were captured in the case report forms submitted by the transplant centre. Other endpoints of interests were survival, relapse or disease progression, chronic GVHD and treatment-related mortality (TRM). TRM was defined as death within the first 28 post-transplant days or death while continuously free of relapse or progression. Progression-free survival (PFS) was defined as survival without disease progression or relapse; patients alive without disease progression or relapse were censored at the time of last follow up. Overall survival (OS) was defined as death from any cause and surviving patients were censored at the date of last contact.

Statistical Analyses

Variables related to patient, disease, and transplant characteristics were compared using the chi-square test for categorical variables and the Kruskal-Wallis test for continuous variables. Probabilities of acute grade II-IV, grade III-IV, and chronic GVHD, TRM, and relapse were calculated using the cumulative-incidence-function.(Gooley, et al 1999) For GVHD endpoints, death without GVHD was the competing event. For TRM, disease progression/relapse was the competing event. For disease progression/relapse, TRM was the competing event. Data on patients without either competing event were censored at last follow-up. Probabilities of OS and PFS were calculated using the Kaplan-Meier estimator, with the variances estimated using Greenwood's formula.(Kaplan & Meier 1958) For analyses of survival, death from any cause was considered an event and data on surviving patients were censored at last follow-up. For analyses of PFS, relapse/progression or death (i.e., treatment failure) was considered an event and data for patients alive without progression/relapse were censored at last follow-up. 95% confidence intervals (95% CI) were calculated with the use of a log-transformation (Klein and Moeschberger 2003), and the chi-square test was used to compare cumulative incidence probabilities or survival probabilities at a fixed time point between the cohorts. All P values were 2 sided, and a value of less than 0.05 was considered statistically significant.

Patient-related, disease-related and treatment-related variables were included in the multivariate analyses using Cox's proportional hazards model.(Cox 1972) This model was built using a stepwise forward selection technique with a P value ≤ 0.05 as the criterion for inclusion in the final models. Patient-related variables were: age at transplantation (21-40 vs. 41-50 vs. 51-70 years), gender (male vs. female), and performance status (<90 vs. ≥ 90). Disease-related variables were: lymphoma histology (small lymphocytic and follicular vs. diffuse large B-cell vs. mantle cell), disease status prior to transplant (complete remission vs. partial remission vs. sensitive relapse vs. other relapse – i.e. resistant/untreated/unknown/progressive disease). Transplant-related variables were: donor type (HLA-identical sibling vs. unrelated donor), interval from diagnosis to transplant, numbers of chemotherapy regimens received prior to transplantation (≤ 2 lines vs. 3-6 lines vs. >6 lines), previous radiation (yes vs. no), time from the last dose of rituximab to transplantation (> 6 months or no prior treatment with rituximab vs. ≤ 6 months), number of prior therapy with rituximab-containing regimens, conditioning regimens (myeloablative vs. non-myeloablative), GVHD prophylaxis (cyclosporine ± others vs. tacrolimus ± others), donor-recipient sex match (M-M vs. M-F vs. F-M vs. F-F), donor parity (male donor vs. nulliparous female donor vs. parous female donor vs. others), donor-recipient cytomegalovirus (CMV) serology (-/- vs. others), year of transplant (1999-2000 vs. 2001-2004) and HLA match (HLA-identical sibling vs. well-matched vs. partially matched/mismatched unrelated). The main effect being tested in this study was the use of rituximab prior to transplantation on various clinical endpoints, therefore this variable was included in all models. All possible risk factors were checked for proportional hazards using a time-dependent covariate approach and there were no violations to the proportionality assumption. There were no first order interactions between prior use of rituximab and these other variables. Completeness of follow-up (the ratio of the sum of the observed follow-up time to the sum of the potential follow-up time for all patients in the study) was 93% (96% for RTX cohort and 89% for No-RTX cohort).(Clark, et al 2002) Analyses were completed with the use of SAS software, version 9.1 (SAS Institute, Cary, NC)

Results

Patients

Patient characteristics of these two cohorts were shown in Table 1. The distribution of baseline characteristics were similar between the two cohorts with the exception of higher proportion of the RTX cohort receiving tacrolimus-containing regimen as GVHD prophylaxis (47% vs 26%, P<0.001)), more unrelated donor recipients (32% vs 19%, P=0.002)), more recent year of transplant (87% vs 66% transplanted in 2001-2004, P<0.001) and shorter time from diagnosis to transplantation (median 22 vs 25 months, P=0.04). There were 225 patients with follicular lymphoma; 133 of these were in the No-RTX cohort and included 60 patients who received rituximab more than 6 months prior to transplantation and 73 patients who did not receive rituximab at anytime during the course of their disease. In order to avoid selection bias, all these patients were included in the analysis.

Table 1. Characteristics of patients receiving HLA-identical sibling or unrelated donor peripheral blood transplant for NHL between 1999 and 2004, reported to CIBMTR.

Characteristics No Rituximab N (%) Rituximab N (%) P-valued
Number of patients 256 179
Age, median (range), years 50 (22-70) 50 (22-67) 0.42
Age at transplant (years) 0.65
 21-40 43 (17) 34 (19)
 41-50 80 (31) 60 (34)
 51-70 133 (52) 85 (47)
Male sex 169 (66) 121 (68) 0.71
Performance score 0.32
 <90 89 (35) 54 (30)
 90-100 163 (64) 119 (66)
 Unknown 4 (2) 6 (3)
Histology of lymphoma 0.07
 Small lymphocytic 18 (7) 4 (2)
 Follicular 133 (52) 92 (51)
 Diffuse large cell 45 (18) 46 (26)
 Mantle cell 53 (21) 32 (18)
 Othera 7 (3) 5 (3)
Disease status prior to transplant 0.07
 Partial remission 90 (35) 63 (35)
 Complete remission 55 (21) 47 (26)
 Relapse sensitive 58 (23) 50 (28)
 Relapse resistant/ untreated/unknown/ 51 (20) 18 (10)
 progressive disease
 Missing 2 (1) 1 (1)
Donor 0.002
 HLA-identical siblings 208 (81) 122 (68)
 Unrelated donor 48 (19) 57 (32)
Number of chemotherapy regimen 0.69
 < 3 lines 95 (37) 72 (40)
 3-6 lines 153 (60) 100 (56)
 > 6 lines 8 (3) 7 (4)
Radiation therapy, yes 54 (21) 40 (22) 0.75
Conditioning regimen 0.84
 Myeloblative 117 (46) 80 (45)
 Non-myeloblative 139 (54) 99 (55)
GVHD prophylaxis <0.001
 CSA+MTX+/-other 103 (40) 46 (26)
 CSA+/-other 84 (33) 45 (25)
 FK506+MTX+/-other 40 (16) 63 (35)
 FK506+/-other 25 (10) 22 (12)
 Otherb 4 (2) 3 (2)
Donor/recipient sex match 0.76
 Male -> Male 92 (36) 75 (42)
 Male -> Female 46 (18) 32 (18)
 Female -> Male 77 (30) 46 (26)
 Female -> Female 39 (15) 25 (14)
 Unknown 2 (1) 1 (1)
Donor pregnancy 0.37
 Male donor 138 (54) 107 (60)
 Female, no pregnancy 17 (7) 14 (8)
 1 or more pregnancies 60 (23) 30 (17)
 Unknown 41 (16) 28 (16)
Donor/recipient CMV match 0.05
 D(-)/R(-) 66 (26) 57 (32)
 D(-)/R(+) 51 (20) 43 (24)
 D(+)/R(-) 32 (13) 30 (17)
 D(+)/R(+) 92 (36) 42 (23)
 Unknown 15 (6) 7 (4)
Number of rituximab-containing regimen received N/A
 1 regimen -- 83 (46)
 2 regimen -- 64 (36)
 3 regimen -- 20 (11)
 4 regimen -- 9 (5)
 5 regimen -- 3 (2)
Year of transplant < 0.001
 1999-2000 87 (34) 24 (13)
 2001-2004 169 (66) 155 (87)
Time from last dose of rituximab to transplant N/A
 < 3 months -- 120 (67)
 3-6 months -- 59 (33)
HLA-matchc 0.01
 HLA-identical sibling 208 (81) 122 (68)
 Well-matched 38 (15) 42 (23)
 Partially matched 8 (3) 10 (6)
 Mismatched 2 (1) 5 (3)
Median (range) time from diagnosis to transplant, months 25 (2-194) 22 (4-196) 0.04
Median follow-up of survivors, months 39 (3-88) 30 (3-82)
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Abbreviations: CSA = Cyclosporine, MTX = Methotrexate, FK506 = Tacrolimus.

a

Other includes (N=12)

  1. Diffuse, mixed, small and large cell (n=2)
  2. Nodal marginal zone B-cell lymphoma (n=6)
  3. Splenic marginal zone B-cell lymphoma (n=1)
  4. B-cell lymphoma, not otherwise specified (n=2)
  5. Missing (n=1)
b

Other includes (N=7)

  1. No GVHD prophylaxis (n=4)
  2. Mycophenolate mofetil (MMF) (n=1)
  3. Missing (n=2)
c

HLA-identical sibling transplants are identified by 8/8 matched at HLA-A, B, C and DRB1 allele-level typing and/or antigen-level typing.

d

The chi-square test was used for discrete covariates; the Kruskal-Wallis test was used for continuous covariates.

Acute Graft-versus-Host Disease

Cumulative incidence of grade II-IV acute GVHD for the No-RTX and RTX cohorts were 48% (95% CI, 41%-54%) and 36% (95% CI, (29% - 43%) (P = 0.01), respectively. Cumulative incidence of grade III-IV acute GVHD for the No-RTX and RTX cohorts were 23% (95% CI, 18% - 29%) and 12% (95% CI, 8% - 18%) (P = 0.002), respectively. (See Figure 1A, B)

Figure 1. Panel A Cumulative incidence of grade 2-4 acute GVHD; Panel B Cumulative incidence of grade 3-4 acute GVHD; Panel C Progression-free survival; Panel D Overall survival.

Figure 1

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In multivariate analysis, prior rituximab therapy remained a significant variable associated with lower risk of grade II-IV acute GVHD (relative risk [RR] = 0.72; 95% CI, 0.53 - 0.97; P = 0.033). Other significant covariates associated with lower risk of grade II-IV acute GVHD were GVHD prophylaxis with tacrolimus (RR = 0.68; 95% CI, 0.49 – 0.93; P = 0.018) and female recipient who received either male (RR = 0.44 compared to male donor/male recipient; 95% CI, 0.27 - 0.72; P = 0.001) or female (RR = 0.61 compared to male donor/male recipient; 95% CI, 0.39 - 0.98; P = 0.039) stem cell grafts. Similarly, prior therapy with rituximab was associated with lower risk of grade III-IV acute GVHD (RR = 0.55; 95% CI, 0.34 - 0.91; P = 0.019). Other covariates associated with lower risk of grade III-IV acute GVHD were GVHD prophylaxis with tacrolimus (RR = 0.56; 95% CI, 0.33 - 0.94; P = 0.029) and higher risk of grade III-IV acute GVHD in patients with diffuse large cell lymphoma (DLBCL) (RR = 1.88 compared to small lymphocytic/follicular lymphoma; 95% CI, 1.12 - 3.14; P = 0.016).

Chronic Graft-versus-Host Disease

There was no difference in the cumulative incidence of chronic GVHD in univariate analysis. The cumulative incidence at 3 years was 53% (95% CI, 47% - 60%) for the No-RTX cohort and 56% (95% CI, 48% - 64%) for the RTX cohort (P = 0.58).

In the multivariate models, lower risk of chronic GVHD was associated with GVHD prophylaxis with tacrolimus (RR = 0.69; 95% CI, 0.51 - 0.93; P = 0.015); higher risks of chronic GVHD were associated with well-matched unrelated donor (RR = 1.97 compared to matched sibling donor; 95% CI, 1.38 – 2.82; P < 0.001), partially matched or mismatched unrelated donor (RR = 3.92 compared to matched sibling donor; 95% CI, 2.20 -6.97; P < 0.001), and increased donor parity (for ≥ 1 pregnancy, RR = 1.78 compared to male donor; 95% CI, 1.25 - 2.51; P = 0.001).

Treatment-Related Mortality

In the univariate analysis, TRM was not different between the No-RTX (37%; 95% CI, % 30% - 44% at 5 years) and RTX cohorts (30%; 95% CI, 23% - 38% at 5 years) (P = 0.24). However, multivariate analyses identified prior treatment with rituximab to be associated with lower TRM (RR = 0.68; 95% CI, 0.47 - 1.00; P = 0.05). Other significant covariates associated with higher TRM were recipients of unrelated donor graft (P < 0.001) and non-complete remission status of disease at the time of transplantation (P < 0.001).

Disease Progression or Relapse

There was a significant difference in rate of disease progress or relapse between the No-RTX and RTX cohorts at 5 years in the univariate analysis (P = 0.04), but this was not significant in the multivariate analyses (P = 0.19). The cumulative incidence of disease progression or relapse at 5 years for the No-RTX and RTX cohorts were 22% (95% CI, 17% - 28%) and 14% (95% CI, 9% - 20%), respectively (P = 0.04). Other independent variables associated with disease progression or relapse were patients with diffuse large cell (DLC) histology, patients who were transplanted in partial remission or those who were transplanted in relapse with chemotherapy-resistant disease.

Survival

Kaplan-Meier estimates for PFS and OS for the No-RTX and RTX cohorts are shown in Figure 1 (C, D). PFS at 5 years for the No-RTX and RTX cohorts were 41% (95% CI, 34% - 49%) and 56% (95% CI, 47% - 64%), respectively (P = 0.01). OS at 5 years for the No-RTX and RTX cohorts were 44% (95% CI, 37% - 51%) and 57% (95% CI, 49% - 65%), respectively (P = 0.02). Prior therapy with rituximab was a strong independent predictor of favorable PFS (RR = 0.68; 95% CI = 0.5 – 0.92; P = 0.013) and OS (RR = 0.63; 95% CI = 0.46 – 0.86; P = 0.004). Other independent prognostic variables associated with poor PFS and OS were Karnofsky performance status < 90, DLC histology, recipients of unrelated donor transplant and disease status not in complete remission at the time of transplantation. There were 129 deaths out of 256 patients in the No-RTX cohort, and 65 deaths out of 179 patients in the RTX cohort (Table 2). There was a significantly higher number of deaths resulting from infections in the RTX cohort (21 out of 65 deaths) than in the No-RTX cohort (20 out of 129 deaths) (P = 0.007).

Table 2. Causes of death.

There were 129 deaths out of 256 patients in the No-RTX cohort (50.4%), and 65 deaths out of 179 patients in the RTX cohort (36.3%). (Chi square P = 0.03)

Primary Cause of Death No-RTX (N = 129) RTX (N = 65)
Lymphoma 48 (37.2%) 14 (21.5%)
GVHD 17 (13.2%) 6 (9.2%)
Pulmonary syndromes 10 (7.8%) 3 (4.6%)
Infection 20 (15.5%) 21 (32.3%)
Organ failures 19 (14.7%) 15 (23.1%)
Others 15 (11.6%) 8 (12.3%)
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GVHD, graft-versus-host disease.

Subgroup Analyses

We conducted three subgroup analyses to examine the robustness of our findings in a retrospective analysis. First, because tacrolimus has been demonstrated to reduce the risk of GVHD in this study and others,(Nash, et al 2000, Ratanatharathorn, et al 1998) we examined the effect of rituximab on the risk of grade II-IV acute GVHD separately for patients receiving cyclosporine vs. those receiving tacrolimus. The RR of grade II-IV acute GVHD for RTX cohort vs. No-RTX cohort was observed to be lower in the cyclosporine group (RR=0.61, 95% CI: 0.41-0.91) than in the tacrolimus group (RR=0.96, 95% CI: 0.56-1.62), although the numbers of patients receiving tacrolimus were smaller. Second, because patients exposed to rituximab were more likely to be transplanted in more recent years, we examined the effect of rituximab in the cohort of patients transplanted between 2001-2004 (148 in the RTX cohort and 161 in the No-RTX cohort). The RR of this recent cohort for grade II-IV acute GVHD was 0.72 (95% CI: 0.50-1.03), similar to the overall cohort. In the third subgroup analysis, we examined the dose effect of rituximab. Patients with rituximab exposure within three months of transplantation had a RR of 0.63 (95% CI: 0.44-0.90) compared to the No-RTX cohort, while patients with rituximab exposure between 3 and 6 months prior to transplant had a RR of 0.93 (95% CI: 0.6-1.43).

Discussion

In a lethal minor histocompatibility complex -incompatible acute GVHD model, Schultz et al (1995) employed C57BL/6 recipients continuously depleted with rabbit anti-μ from birth that received marrow from LP/J donor. B-cell depleted recipients had a lower frequency of GVHD compared to control mice receiving normal rabbit Ig. Recently, Iori et al (2008) reported the concentration of B cells in the granulocyte colony-stimulating factor-mobilized stem cell grafts correlated with higher incidence of acute GVHD in recipients of allogeneic HLA-identical sibling transplantation who received myeloablative therapy for hematological malignancies.

Rituximab is efficacious as single agent for B-cell lymphoma(Grillo-Lopez, et al 1999) and acts synergistically with combination chemotherapy to enhance the efficacy of chemotherapy, thus rituximab is included in the first line treatment of adult CD-20 positive lymphoma.(Coiffier, et al 2002, Marcus, et al 2005) Most patients who received rituximab would have B-cell lymphopenia for about 6 months.(McLaughlin, et al 1998b) Therefore, B-cell antigen presentation might be impaired for at least several months after rituximab therapy.

Based on these concepts, we reported a pilot study comparing two contemporaneous groups of patients who had or had not been pretreated with rituximab, suggesting prior rituximab treatment might result in less acute GVHD.(Ratanatharathorn, et al 2000a) In this current study, we included large sample size from the CIBMTR database spanning from 1999 to 2004. Due to nature of this retrospective study, most patients in the latter years received rituximab as part of first-line or salvage therapy while patients who did not receive rituximab had transplantation in the earlier years. We categorized patients who received rituximab less than 6 months prior to transplantation as patients who had prior rituximab therapy. The reason for using 6 months as the cut-off was the duration of B cell depletion achievable with 4 doses of rituximab therapy.(McLaughlin, et al 1998b) Obviously, one could not be absolutely certain about the level of B cell depletion, which also depended on the size of the ‘antigen sink’, as patients with large tumor burden might be less likely to achieve a high level of B cell depletion, therefore normal host B cells might have been spared to function as APC.(Reff, et al 1994) However, the half-life of rituximab, despite the influence of the tumor burden, could range from several days to more than a week. (Berinstein, et al 1998, Maloney, et al 1997) Therefore, in patients treated with rituximab close to the time of transplantation or given as a part of preparative regimen (Khouri, et al 2008) might have derived greatest GVHD protection with additional depletion of B cells in the stem cell graft, as suggested by the animal model (Schultz, et al 1995) and recently in the report by Iori et al (2008) Multivariate analysis of the subgroup of patients with rituximab exposure within 3 months of transplantation in this study also indicated lower risk of acute GVHD than in the No-RTX group, but not different from those with rituximab exposure between 3-6 months. Despite these limitations, we were able to demonstrate the lower risk of grade II-IV and III-IV acute GVHD in patients who received rituximab compared to patients who did not receive rituximab prior to transplantation. The other independent variable that predicted for lower acute GVHD was the use of tacrolimus for GVHD prophylaxis, which is consistent with prior prospective randomized studies in sibling donor and unrelated donor transplantation.(Nash, et al 2000, Ratanatharathorn, et al 1998) The association of DLBCL with higher risk of acute GVHD was not apparent. Prior rituximab therapy had no significant impact on the risk of chronic GVHD, but female donor and unrelated donor were associated with higher chronic GVHD risk. It is not surprising that prior rituximab therapy did not prevent chronic GVHD; probably because this immunological complication occurred later, after transplantation, when the anti-CD20 antibody level was no longer detectable. On the other hand, there is mounting evidence for the participation of B cells in the immunopathogenesis of chronic GVHD and treatment with rituximab has resulted in significant clinical responses.(Canninga-van Dijk, et al 2004, Cutler, et al 2006, Okamoto, et al 2006, Ratanatharathorn, et al 2001, Ratanatharathorn, et al 2003, Ratanatharathorn, et al 2000b, Zaja, et al 2007) Taken together, the lower risk of acute GVHD in patients receiving prior rituximab had resulted in significantly lower TRM despite an insignificant impact on chronic GVHD. The risk of relapse or disease progression was not higher in patients previously treated with rituximab. Thus, the lower risk of GVHD did not offset the graft-versus-lymphoma effect, resulting in improved OS and PFS.

The relative risks of the RTX and No-RTX cohorts were summarized in Table 3. We speculate that rituximab therapy prior to transplantation or as part of conditioning regimen resulting in host B-cell, and perhaps donor B-cell depletion, thus attenuated donor T-cell activation in the early phase of transplantation via antigen presentation. Later emergence of APC from engrafted lymphohematopoietic system might be crucial for donor T-cell activation to perpetuate clinical GVHD independent of B cells. On the other hand, T-cell dysregulation in patients with acute GVHD may be modulated by target B-cell depletion, such as those seen in patients with idiopathic thrombocytopenic purpura treated with rituximab. (Stasi, et al 2008, Stasi, et al 2007) Future prospective studies with biological marker correlates will be needed to ascertain the role of B-cell in acute GVHD immunopathogenesis.

Table 3. Summary of relative risks of key clinical endpoints in patients who received rituximab prior to transplantation. Baseline was patients who did not receive rituximab prior to transplantation.

Variables Relative Risks (95% CI) P-Value
Grade II-IV acute GVHD 0.72 (0.53-0.97) 0.03
Grade III-IV acute GVHD 0.55 (0.34-0.91) 0.02
Chronic GVHD 0.89 (0.67-1.18) 0.41
TRM 0.68 (0.47-1.0) 0.05
Disease progression or relapse 0.7 (0.42-1.19) 0.19
Progression-free survival 0.68 (0.50-0.92) 0.01
Overall survival 0.63 (0.46-0.86) 0.004
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GVHD, graft-versus-host disease.

Acknowledgments

This project has been supported by funding from the National Marrow Donor Program and the Department of the Navy, Office of Naval Research Grant #N00014-05-1-0859 to the NMDP. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the Office of Naval Research or the National Marrow Donor Program. The CIBMTR is supported by Public Health Service Grant U24-CA76518 from the National Cancer Institute, the National Institute of Allergy and Infectious Diseases, and the National Heart, Lung and Blood Institute; Office of Naval Research; Health Services Research Administration (DHHS); and grants from Abbott Laboratories; Aetna; American International Group, Inc.; American Red Cross; Amgen, Inc.; Anonymous donation to the Medical College of Wisconsin; AnorMED, Inc.; Astellas Pharma US, Inc.; Baxter International, Inc.; Berlex Laboratories, Inc.; Biogen IDEC, Inc.; BloodCenter of Wisconsin; Blue Cross and Blue Shield Association; Bristol-Myers Squibb Company; BRT Laboratories, Inc.; Cangene Corporation; Celgene Corporation; CellGenix, Inc.; Cell Therapeutics, Inc.; CelMed Biosciences; Cylex Inc.; Cytonome, Inc.; CytoTherm; DOR BioPharma, Inc.; Dynal Biotech, an Invitrogen Company; Enzon Pharmaceuticals, Inc.; Gambro BCT, Inc.; Gamida Cell, Ltd.; Genzyme Corporation; Gift of Life Bone Marrow Foundation; GlaxoSmithKline, Inc.; Histogenetics, Inc.; HKS Medical Information Systems; Kirin Brewery Co., Ltd.; Merck & Company; The Medical College of Wisconsin; Millennium Pharmaceuticals, Inc.; Miller Pharmacal Group; Milliman USA, Inc.; Miltenyi Biotec, Inc.; MultiPlan, Inc.; National Marrow Donor Program; Nature Publishing Group; Novartis Pharmaceuticals, Inc.; Osiris Therapeutics, Inc.; Pall Medical; Pfizer, Inc.; Pharmion Corporation; PDL BioPharma, Inc; Roche Laboratories; Sanofi-aventis; Schering Plough Corporation; StemCyte, Inc.; StemSoft Software, Inc.; SuperGen, Inc.; Sysmex; The Marrow Foundation; THERAKOS, Inc.; University of Colorado Cord Blood Bank; ViaCell, Inc.; ViraCor Laboratories; Wellpoint, Inc.; and Zelos Therapeutics, Inc. The views expressed in this article do not reflect the official policy or position of the National Institute of Health, the Department of the Navy, the Department of Defense, or any other agency of the U.S. Government.

Footnotes

Authorship: Contribution: VR designed the study, interpreted the data, and wrote the paper. BL and DW performed data analyses and wrote part of the paper. MH assisted in the design of the study and interpretation of data. JU assisted in the design of the study and critical review of the paper. OR, RPG, MA, SS, CC, MB GH, HK, LV, MC and VG provided critical review of the paper. SP assisted in study design, data interpretation and critical review of the paper.

Conflict-of-interest disclosure: The authors declared no financial interests.

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