High-dose chemotherapy and autologous stem cell transplantation (SCT) are the standard of care for first chemosensitive relapse of aggressive B-cell non-Hodgkin's lymphoma, with estimated 5-year disease-free survival rates of 40–50% (Philip et al, 1987, 1995). Patients with chemorefractory disease do not fare so well, with less than 20% of autologous SCT recipients surviving disease free (Philip et al, 1995). An international consensus conference determined that high-dose chemotherapy and autologous SCT is inappropriate in chemorefractory first or subsequent relapse (Shipp et al, 1999). A small subset of patients who did not achieve a complete remission (primary refractory disease) may benefit from autologous SCT; the majority of them are those who retain some chemosensitivity (Mills et al, 1995; Vose et al, 2001). Allogeneic SCT may offer advantages over autologous SCT by providing a tumour-free graft and most importantly by induction of the graft-versus-lymphoma (GVL) effect (Jones et al, 1991). There are no randomized studies comparing autologous and allogeneic SCT in lymphoma patients, and other comparative studies were often biased by patient selection. However, most of these studies demonstrated that the relapse rate was significantly lower after allogeneic SCT (Chopra et al, 1992; Ratanatharathorn et al, 1994), supporting the existence of the GVL effect. However, long-term survival remained equivalent as better disease control was offset by significantly higher treatment-related mortality (TRM) rates (Chopra et al, 1992; Ratanatharathorn et al, 1994). Non-myeloablative conditioning has been explored in lymphoma patients in an attempt to reduce toxicity while retaining the curative GVL effect (Nagler et al, 2000a; Robinson et al, 2002). Patients with chemorefractory disease continue to do poorly after both myeloablative (Dhedin et al, 1999) and non-myeloablative (Robinson et al, 2002) allogeneic SCT, yet there is still a cure rate of approximately 20% with this approach in this setting. Clearly, novel approaches to reduce relapse risk are needed in these high-risk patients and to improve long-term outcome after both autologous and allogeneic SCT. In this study, we explored the use of rituximab, a humanized anti-CD20 monoclonal antibody, administered after autologous and allogeneic SCT, in high-risk patients with aggressive B-cell lymphoma, as a non-toxic means to reduce the post-transplant relapse rate.
Summary. High-dose chemotherapy and autologous stem cell transplantation (SCT) have limited success in patients with refractory aggressive lymphoma. Allogeneic SCT may offer some advantage in this setting by providing graft-versus-lymphoma effect, but the relapse risk remains substantial. In this study, we evaluated the safety and efficacy of rituximab administration after SCT in patients at high-risk for post-transplant relapse, in order to reduce relapse risk. Twenty-eight patients were included with the intent to treat them with rituximab after autologous (n = 16) or allogeneic (n = 12) SCT. Twenty-four were given rituximab starting a median of 47 d post SCT. Three died of SCT complications prior to therapy. Nine patients not achieving a complete remission (CR) post SCT converted to CR with rituximab and with the onset of graft-versus-host disease (GVHD) in three. With a median follow-up of 12 months (range, 3–33 months) the estimated 2-year overall survival and disease-free survival was 85 ± 7% and 55 ± 13% respectively. When only those patients who were actually treated are analysed, these rates were 95 ± 7% and 64 ± 13% respectively. The relapse risk was 35 ± 14%. Seven patients had recurrent neutropenia episodes associated with severe hypogammaglobulinaemia, which were further prevented with intravenous immunoglobulin. None of the 10 allogeneic SCT recipients treated with rituximab had severe GVHD. Rituximab may be an effective adjuvant therapy after SCT to reduce the relapse rate and improve the outcome in high-risk aggressive lymphoma. Larger scale comparative trials are necessary to better define its role in SCT.
Patients and methods
Patient eligibility. Patients were eligible for this study if they had aggressive B-cell non-Hodgkin's lymphoma, including patients with diffuse large cell lymphoma, mediastinal large cell lymphoma, transformed low-grade lymphoma and mantle cell lymphoma. Patients who failed to achieve a complete response with primary chemotherapy or relapsed after an initial response were eligible for SCT. Patients undergoing SCT as consolidation for lymphoma at first remission were not included. Patients were determined as having high risk for post-transplant relapse based on chemorefractory disease, multiple relapses (more than one prior disease relapse), multiple prior therapies (more than two lines of therapy) and bone marrow or multiple extra-nodal site involvement. Standard SCT candidates with chemosensitive first relapse were not included. Patients aged 18–70 years were eligible. Patients were required to be free of marked organ dysfunction and to have an Eastern Cooperative Oncology Group (ECOG) performance score of 0–1. Patients were allocated to allogeneic or autologous SCT based on age, donor availability, disease status, and patient and attending physician preference. Patients with progressive disease on chemotherapy and most patients with no response to prior chemotherapy were preferentially allocated to allogeneic SCT. Patients given allogeneic SCT had to have a human leucocyte antigen (HLA)-compatible related or unrelated donor willing to donate peripheral blood stem cells or bone marrow. Patients having autologous SCT were required to have an adequate autologous graft by standard institutional criteria. All patients gave written informed consent and the study was approved by the Institution Review Board.
Conditioning regimen. The BEAM regimen was used as conditioning regimen for both autologous and matched sibling allogeneic SCT (Przepiorka et al, 1999), and consisted of carmustine 300 mg/m2 on d −6, etoposide 400 mg/m2 and cytarabine 400 mg/m2 daily on d −5 to −2, and melphalan 140 mg/m2 on d −1. Autologous or allogeneic peripheral blood stem cells were infused on d 0. Allogeneic SCT recipients were eligible for a reduced-intensity regimen if they had undergone a prior autologous SCT (n = 3) or extensive prior therapy (n = 2). The conditioning regimen consisted of fludarabine (a total of 125 mg/m2) and melphalan (140 mg/m2). Patients with an unrelated donor were given high-dose cyclophosphamide and total body irradiation (n = 1) or a reduced-intensity regimen (n = 1). Patients with unrelated donor were also given antithymocyte globulin (ATG; Fresenius, Bad Homburg, Germany) at 5 mg/kg for two to three doses. Prophylaxis against graft-versus-host disease (GVHD) consisted of cyclosporine and a short course of methotrexate (15 mg/m2 on d 1, and 10 mg/m2 on d 3 and 6). Granulocyte colony-stimulating factor (G-CSF) 5 µg/kg was administered routinely until engraftment. The standard institutional regimen of antibiotics was employed for the prevention of infections. Patients could be given radiation therapy to sites of bulky disease after SCT, by standard institutional protocols.
Rituximab therapy. Patients were given rituximab by intravenous infusion for a total of 4 weekly doses at 375 mg/m2 each dose. Premedication included acetaminophen and diphenhydramine, and was infused at the rate recommended by standard package insert instructions. Rituximab was to be started 1–2 months post transplant, and after establishment of a stable graft and resolution of transplant-related toxicities. Patients with late post-transplant relapse were eligible for a further treatment course of rituximab with additional chemotherapy at the attending physician's discretion.
Evaluation of response. Disease status was assessed prior to SCT, at approximately 1 month post SCT (before the start of rituximab), and at 3, 6 and 12 months after SCT or as clinically indicated. Responses were evaluated by physical examination, routine laboratory tests, computerized tomography scans and bone marrow biopsies if indicated, and compared with the prior evaluation. Complete remission (CR) was defined as the resolution of clinical and radiographic evidence of disease. Complete remission with residual imaging abnormalities of uncertain significance (CRu) was included with other CR in this analysis. Partial remission (PR) was defined as a 50% reduction in the product of the bidimensional tumour measurements. No response (NR) was defined as less than a 50% decrease in tumour measurements. Definition of progressive disease required an increase of more than 25% in tumour size or the appearance of new lesions.
Statistical analysis. Overall survival (OS) was calculated from the day of transplantation until death of any cause or last follow-up. Disease-free survival (DFS) was calculated from the day of transplantation until relapse or death of any cause or last follow-up. The probabilities of survival and relapse were estimated and plotted using the Kaplan–Meier method (Kaplan & Meier, 1958). The effect of various patient and disease categorical variables on survival probability were studied with the log-rank test.
Twenty-eight patients with aggressive B-cell lymphomas were included in the study with the intent to treat them with rituximab after haematopoietic stem cell transplantation (SCT). There were 13 men and 15 women, and the median age was 54 years (range, 25–70 years). Fourteen patients had diffuse large cell lymphoma, five patients had mediastinal large cell lymphoma, six patients had transformed low-grade lymphoma and three patients had mantle cell lymphoma. Twelve patients had allogeneic SCT from an HLA-matched sibling (n = 10) or an HLA-matched unrelated donor (n = 2), and 16 patients had autologous SCT. Six patients did not achieve a CR before SCT. In the remaining 22 patients, the median duration of the last response was 6·5 months (range, 1–54 months). The status of disease at SCT was progressive disease (n = 11), no response to last therapy (n = 9) or partial response (n = 8). Patients were considered at high risk for post transplant relapse and, therefore, eligible for this study as a result of chemorefractory disease (n = 20), multiply relapsed disease (n = 9) or multiple extra-nodal site involvement (n = 14) with some patients having more than one of these criteria. Patients were preferentially allocated to allogeneic SCT based on age, disease status and donor availability, such that most of the patients having allogeneic SCT had chemorefractory, multiply relapsed disease and a very high risk for post-transplant relapse. The autologous SCT recipients were also at high risk for relapse based on the above criteria. The different patient characteristics of the two groups are outlined in Table I.
|All patients||Allogeneic SCT||Autologous SCT||P-value|
|Median age, years (range)||54 (25–70)||50 (25–63)||55 (30–70)||NS|
|Number prior therapies (range)||3 (1–7)||4 (2–7)||2 (1–5)||0·03|
|Prior autologous transplants||3 (11%)||3 (25%)||0 (0%)||0·03|
|Prior rituximab therapy||10 (36%)||6 (50%)||4 (25%)||NS|
|Time from diagnosis, months (range)||24 (8–80)||41 (8–80)||19 (8–79)||NS|
|Chemorefractory disease||20 (71%)||11 (92%)||9 (56%)||0·04|
|Multiply relapsed disease||9 (32%)||6 (50%)||3 (19%)||0·08|
|Multiple extra-nodal site involvement||14 (50%)||2 (17%)||12 (75%)||0·03|
Twenty-four patients were given the complete intended course of rituximab, starting at a median of 47 d post transplant (range, 23–182 d). Four patients were not given rituximab. Two allogeneic SCT recipients died early post transplant of therapy-related complications (one of acute GVHD and one of veno-occlusive disease of the liver). One autologous SCT recipient died of pneumonia before starting rituximab therapy, and one patient had insurance difficulties.
At the first post-transplant evaluation, 16 patients were in CR, seven patients had PR, two patients showed NR and three patients were not evaluable because of early death. All of the patients with less than CR achieved CR with post-transplant therapy with rituximab and with the onset of GVHD in three allogeneic SCT recipients. At a median follow-up of 12 months (range, 3–33 months), 24 patients were alive and four had died. Three patients had early treatment-related death (two allogeneic and one autologous SCT recipients), and one autologous SCT recipient died in remission of infection 10 months post transplant. Eighteen patients were disease free and six have relapsed. The projected 2-year OS and DFS were 85 ± 7% and 55 ± 13% respectively (Fig 1). When only the 24 patients that were actually treated with rituximab were analysed, these rates were 95 ± 7% and 64 ± 13% respectively. The estimated relapse risk was 22 ± 9% and 35 ± 14% at 1- and 2-years post transplant (Fig 2). The median time to progression in those who progressed was 5 months (range, 3–33 months). Five of the six relapses occurred within 6 months post transplant. Two relapses occurred in allogeneic SCT recipients at 5 and 19 months post transplant. Both entered a second remission with a second course of rituximab, with additional chemotherapy in one of these patients, withdrawal of immunosuppressive therapy and the onset of chronic GVHD. At the time of writing, both were currently disease free (15 and 8 months later respectively), such that the current DFS for all patients was 67 ± 10%. The four autologous SCT recipients who relapsed have undergone further therapy and were not considered currently disease free in this analysis. There were no significant differences in OS, DFS, TRM and relapse rate among allogeneic and autologous SCT recipients. The paucity of events did not allow the analysis of patient characteristics predicting for adverse outcome (Table II).
|All patients||28||85 ± 7%||55 ± 13%|
|Allogeneic||12||83 ± 11%||NS||36 ± 27%||NS|
|Autologous||16||86 ± 9%||63 ± 12%|
|DLCL||14||91 ± 9%||62 ± 13%|
|Transf LG||6||50 ± 20%||NS||50 ± 20%||NS|
|Mediastinal||5||100%||40 ± 30%|
|Sensitive||8||70 ± 18%||NS||60 ± 18%||NS|
|Refractory||20||90 ± 18%||51 ± 17%|
|Time from diagnosis|
|> 24 months||14||68 ± 13%||NS||67 ± 16%||NS|
|< 24 months||14||100%||49 ± 17%|
|Number prior therapies|
|≤ 2||11||90 ± 9%||NS||82 ± 12%||0·07|
|> 2||17||82 ± 9%||35 ± 17%|
|Prior rituximab therapy|
|Yes||10||80 ± 13%||NS||67 ± 16%||NS|
|No||18||87 ± 8%||49 ± 17%|
|Yes||14||84 ± 10%||NS||64 ± 13%||NS|
|No||14||86 ± 9%||34 ± 25%|
Transfusion reactions occurred frequently during rituximab therapy but were transient and easily controlled, and all patients completed the intended therapy. Seven patients had severe neutropenic episodes (absolute neutrophil count < 0·5 × 109/l), with a median absolute neutrophil count of 0·3 × 109/l (range, 0·07–0·5 × 109/l). There was a median of two such episodes (range, 1–6), and all patients responded to growth factor therapy. There were no additional changes in haemoglobin or platelet counts. This complication occurred in six of the 10 allogeneic SCT recipients treated with rituximab post transplant, and in one of 14 autologous SCT recipients treated. The first episode occurred a median of 45 d (range, 33–108 d) after the first dose of rituximab. This phenomenon was associated with severe hypogammaglobulinaemia in all patients. The mean immunoglobulin (Ig)G level was 5·1 ± 3·0 g/l (range, 0·7–11·0) and 6·93 ± 2·64 (range, 4·2–11·7) in allogeneic and autologous SCT recipients respectively (P = NS). However, severe hypogammaglobulinaemia (IgG level < 5·0 g/l) occurred in six allogeneic SCT recipients (60%) and only three autologous SCT recipients (21%). Moreover, the mean IgG level at approximately 6 weeks after the start of rituximab (or at the time of first episode of neutropenia) was 3·3 ± 1·44 g/l (range, 0·7–4·5) in the patients with rituximab-associated neutropenia and 7·41 ± 2·43 g/l (range, 4·5–11·7) in those without neutropenia (normal range 6·5–16·0 g/l) (P < 0·001). Neutropenic episodes did not recur in four patients with two or more such episodes after starting monthly intravenous immunoglobulin therapy. The course of a typical patient is presented in Fig 3.
Acute GVHD occurred in five of 12 patients having allogeneic SCT. It was grade I–II in four patients and fatal grade IV in one patient. Chronic GVHD occurred in four of 10 evaluable patients, and was limited and easily controlled in all of them.
Rituximab is a chimaeric monoclonal antibody directed against CD20. It has an established role in the treatment of follicular lymphoma (Maloney et al, 1997; McLaughlin et al, 1998). Rituximab is also effective in aggressive lymphoma, especially when included in combination chemotherapy (Coiffier et al, 1998, 2002). The role of rituximab in SCT is not yet well determined. Rituximab has been used for the in vivo purging of autografts (Buckstein et al, 1999). It has been shown to induce complete and partial responses in small series of patients with follicular (Kaya et al, 2002) or aggressive lymphoma (Tsai et al, 1999; Pan et al, 2002) relapsing after autologous SCT. In the largest of these series, nine of 17 patients (53%) with aggressive lymphoma relapsing after autologous SCT responded, four CRs and five PRs, lasting a median of 13 months (Pan et al, 2002). These results compare favourably with other modes of therapy of post-SCT relapse. The effectiveness of rituximab in this setting provides a rationale for the use of post-transplant rituximab as adjuvant therapy to improve responses and prevent disease recurrence. In our series, nine of 25 evaluable patients were not in CR at the time of the first post-transplant evaluation (seven had PR and two had NR). All of these patients achieved CR with post-transplant therapy with rituximab and with the onset of GVHD in three allogeneic SCT recipients. Thus, adjuvant rituximab may convert patients with only PR to CR after SCT. The expected DFS after high-dose chemotherapy and autologous SCT of standard-risk patients, in chemosensitive first relapse, is approximately 40–50% but only 0–20% in high-risk patients such as patients with refractory disease. In our series of patients, who were all at high risk for post-transplant relapse, 10 of 14 autologous SCT recipients treated with adjuvant rituximab remained disease free at 2-years post SCT (estimated 2-year DFS 71 ± 12%). Similarly, in one series of patients with aggressive lymphoma of unreported risk, treated post-SCT with rituximab, the 2-year progression-free survival was 86% (Horwitz et al, 2001). These are encouraging results, suggesting that rituximab may reduce recurrence rate and improve survival after autologous SCT by controlling minimal residual disease in both standard- and high-risk patients.
Allogeneic SCT offers some advantages over autologous SCT by providing a GVL effect, but the expected long-term DFS in patients with refractory disease is approximately 20% as the effect of better disease control is offset by treatment-related toxicity. There are very little data on the use of rituximab during conditioning for allogeneic SCT (Khouri et al, 2001) or as salvage therapy for post-SCT relapse (Milojkovic et al, 2000; Ratei et al, 2000). The strategy of rituximab as an adjuvant therapy after allogeneic SCT has not been reported before. The group of allogeneic SCT recipients in this study included 12 very high-risk patients, 11 were chemorefractory, and most were also heavily pretreated and multiply relapsed. All 10 patients that were actually given rituximab remain alive, eight have been continuously disease free, and all are currently disease free. These results are encouraging and better than expected in such high-risk patients. The results of allogeneic and autologous SCT in this study were equivalent, however, as the patients allocated to allogeneic SCT were at higher risk, these results support allogeneic SCT for patients with refractory lymphoma. As also seen in other series of such high-risk patients (Philip et al, 1987), relapse and TRM events occurred within the first 6 months post SCT such that, although the median follow-up was only 1 year, it is expected that long-term outcome will also be favourable.
There are several potential mechanisms involved in the rituximab antilymphoma effect (Maloney et al, 2002). The most significant mechanism is probably antibody-dependant cytotoxicity (ADCC), involving the activation of natural killer (NK) cells and macrophages. Other mechanisms include complement activation and direct effects resulting from the binding of the CD20 molecules on lymphoma cells, such as induction of apoptosis and growth inhibition. Rituximab synergies with chemotherapy to induce apoptosis. Rituximab may have a more prominent effect in a minimal disease state such as that achieved after high-dose chemotherapy. Patients with refractory lymphoma also responded, suggesting that there is no cross resistance with standard chemotherapy. An additional mechanism may have occurred in the allogeneic SCT group. These patients had potent GVL effects that were disproportionate to the relatively mild degree of GVHD. One of the mechanisms of tumour escape from rituximab therapy relates to the lack of effective host immune effector cells. Donor NK cells are among the earliest immune effector cells to recover after allogeneic SCT (Nagler et al, 2000b) and rituximab may have amplified the GVL effect by inducing donor cell ADCC or by synergism with other allogeneic responses, thereby inducing apoptosis of lymphoma cells.
B cells may function as antigen-presenting cells and thus have an important role in the pathogenesis of GVHD (Schultz et al, 1995). Initial studies provide evidence that host B-cell depletion with rituximab administered prior to transplantation may reduce the incidence of acute GVHD (Ratanatharathorn et al, 2000). There are no data on the role rituximab may have in chronic GVHD. In our series of 10 patients who were treated early post allogeneic SCT with rituximab, no patient had extensive chronic GVHD, yet the relapse rate was lower than expected, suggesting that the GVL effect was retained. Rituximab may have limited the severity of GVHD by the depletion of host or donor B cells, however, this observation needs confirmation in a larger scale study.
We noted a unique syndrome that occurred in patients treated with rituximab after SCT. Patients had several episodes of severe isolated neutropenia occurring over several months after treatment. Some, but not all, of these episodes were associated with fever. All of these episodes in all of the patients responded promptly to growth factor therapy and were relatively benign with no associated mortality or severe infections. This syndrome was relatively common after allogeneic SCT, occurring with variable severity in six of 10 patients, but was relatively rare after autologous SCT. IgG levels tended to be subnormal among SCT recipients but this was not statistically different between the allogeneic and autologous groups. However, severe hypogammaglobulinaemia was more common after allogeneic SCT. Moreover, IgG levels were more severely depressed in patients having rituximab-associated neutropenia, and neutropenia occurred almost exclusively in patients with severe hypogammaglobulinaemia after SCT. Neutropenia is a rare complication of rituximab therapy in lymphoma patients (Maloney et al, 1997; McLaughlin et al, 1998). There are a few reports of prolonged and late-occurring neutropenia when rituximab is administered after autologous SCT (Tsai et al, 1999; Flinn et al, 2000; Horwitz et al, 2001; Pan et al, 2002) but there are no data on the safety of rituximab after allogeneic SCT. Rituximab therapy may lead to severe depression of B-cell function but is usually not associated with hypogammaglobulinaemia (McLaughlin et al, 1998). There is only one case report, where prolonged neutropenia, which followed rituximab therapy (not in a SCT setting), occurred in association with severe hypogammaglobulinaemia and was corrected with intravenous immunoglobulin (IVIG) (Saikia et al, 2001). Recovery of B-cell function and immuonoglobulin production after allogeneic SCT is often delayed (Parkman & Weinberg, 1999). The addition of rituximab post SCT may further delay B-cell recovery and explain why severe hypogammaglobulinaemia was more common after allogeneic SCT than after autologous SCT. The pathogenesis of neutropenia and its relationship to hypogammaglobulinaemia are unclear. Bone marrow aspiration was performed in four of seven patients with rituximab-associated neutropenia and showed normocellularity to hypercellularity with maturation arrest in the myeloid series rather than marrow hypoplasia in all of them. Nine patients had marrow involvement with disease at some point prior to SCT but none of the patients had lymphoma in the marrow at the time of neutropenia, and all were in clinical CR at that time. Neutropenia may relate to underlying infections such as viral infections in the hypogammaglobulinaemic patient or to an immune phenomenon. There is one report of T-large granular lymphocyte (LGL) expansion associated with neutropenia after rituximab therapy (Papadaki et al, 2002). Although LGL expansion may occur after allogeneic SCT (Mohty et al, 2002), it could not be documented as the cause of neutropenia in our series (data not shown). Also, LGL expansion is usually associated with marrow hypoplasia rather than maturation arrest. IVIG therapy corrected neutropenia in our series and it may be useful to administer IVIG routinely in allogeneic SCT recipients treated with rituximab in an attempt to prevent this syndrome.
Rituximab may be effective adjuvant therapy after autologous and allogeneic SCT to reduce the recurrence rate and improve outcome in high-risk aggressive lymphoma. Rituximab was relatively safe in this setting with the exception of induction of prolonged and recurrent neutropenia, however, this was not life threatening and could be prevented with IVIG administration. Rituximab merits further study also in standard-risk patients. Larger scale comparative trials are necessary to better define the role of rituximab in SCT.