In this single-center study, a consecutive cohort of 59 adult patients transplanted with HLA-identical bone marrow and receiving graft-versus-host disease (GVHD) prophylaxis with either standard cyclosporine/methotrexate (n = 33) or partial T cell depletion (E-rosetting) (TCD, n = 26 were analyzed). Only patients with chronic myeloid leukemia in first chronic phase or acute leukemia/myelodysplasia in first or second remission were included. Except for age (median 28 vs 42 years), both groups were comparable in terms of diagnosis, conditioning regimen and growth factor support. TCD significantly reduced >grade II acute GVHD (0 vs 24%, P = 0.02), chronic GVHD (8.5 vs 45%, P = 0.007) and other major bone marrow transplant (BMT)-related complications (4 vs 36%, P = 0.005). TCD decreased overall transplant-related mortality (11.5 vs 36%, P = 0.04). In the TCD group faster neutrophil (13 vs 22 days, P = 0.02) and platelet recoveries (18 vs 26 days, P < 0.001) were noted. The relapse risk was higher after TCD (57.5 vs 21.5%, P = 0.04). Overall survival probability at 10 years was identical in both groups (54 vs 53.5%, P = 0.33). We found a relationship between the number of T cells in the graft and the occurrence of major complications (P < 0.001) and relapse (P = 0.03). This comparative analysis shows that graft-derived T cells have a major role in overall BMT-related toxicity and that partial TCD is an acceptable approach in terms of survival for patients between 40 and 50 years of age. Bone Marrow Transplantation (2001) 28, 917–922.
Despite continuous improvements in supportive care, the practice of allogeneic bone marrow transplantation (BMT) remains limited by its toxicity. Considerable transplant-related mortality (TRM) is caused by graft-versus-host disease (GVHD) and other severe transplant-related complications including infections, veno-occlusive liver disease (VOD), endothelial leakage syndrome and pneumonitis. The age of the recipient is an independent risk factor for TRM after BMT for leukemia.1,2 This is at least partly due to the relationship between patient age and the incidence and severity of GVHD.3,4 Ex vivo removal of T cells from the graft or T cell depletion (TCD) reduces the risk of acute as well as chronic GVHD.5 The impact of TCD on other BMT-related complications is less well documented. In some series, TCD has led to a decreased incidence of neutropenic fever, mucositis and infections.6,7 TCD has been associated with a 10–15% risk of graft failure, occurring more frequently with the use of pan-T monoclonal antibodies for TCD.8 In the absence of graft failure, neutrophil recovery appears to occur more rapidly after TCD, especially after G-CSF administration.9,10 The effect of TCD on platelet recovery is less clear.
The major disadvantage of TCD is its interference with graft-versus-leukemia (GVL) mechanisms, leading to an increased relapse rate in leukemia patients.5 The impact of TCD on disease recurrence is more evident in chronic as compared to acute leukemia where low relapse rates in T cell-depleted marrow recipients have been reported.11,12,13 Also, the loss of the GVL effect appears to be related to the method of TCD and is less pronounced when physical techniques such as elutriation or E-rosetting are used.5 Our local policy was to use TCD for patients older than 40 years because of the higher risk of GVHD. We applied a method of partial TCD (E-rosetting) in an attempt to minimize relapse and graft failure. We compared engraftment kinetics, BMT-related morbidity and mortality as well as relapse and survival with a similar group of younger patients receiving marrow which was left unmanipulated (UNM). The analysis was limited to adult leukemia patients of standard risk at time of BMT who had an HLA-identical sibling donor. In addition, we studied the relationship between the number of T cells in the graft and transplant-related complications as well as relapse.
Patients and methods
Patients and transplant procedure
A total of 59 consecutive adult (>15 years) patients with acute leukemia (n = 27) or myelodysplastic syndrome (n = 2) in first or second complete remission and chronic myeloid leukemia in first chronic phase (n = 30) were analyzed. All patients were grafted in our BMT-Unit between April 1989 and April 1999 with bone marrow from an HLA-identical sibling donor. Only those receiving a first BMT were included. At the time of analysis, the minimum follow-up was 24 months from BMT. TCD was performed when patients were older than 40 years but a few exceptions were made according to the patient's or physician's decision. Patient characteristics are summarized in Table 1. Median age was substantially higher in the TCD group (42 vs 28 years, P < 0.001) and most (73%) of the patients in the TCD group were between 40 and 50 years. Both groups were similar with regard to sex, proportion of patients with acute and chronic leukemia and those receiving G-CSF which was administered from the first day after BMT until stable neutrophil recovery. All conditioning regimens included the same schedule of TBI (2 × 5 Gy), which was preceded with high-dose cytosine-arabinoside (4 g/m2) and cyclophosphamide (120 mg/kg) in 88% of cases. In the UNM group, graft-versus-host prevention consisted of cyclosporine and methotrexate and in the case of TCD, only cyclosporine was given.
T cell depletion
On the day of transplant, donor bone marrow was aspirated from the iliac crests into heparinized syringes. After preparation of the buffy coat, mononuclear cells were isolated by Ficoll–Isopaque density centrifugation and incubated with sheep red blood cells, pretreated with AET (2-aminoethylisothio-uronium bromide). Rosetted T cells were removed by a second Ficoll–Isopaque density centrifugation. The level of T cells before and after the depletion procedure was determined by immunogold–silver staining on cytospin preparations using the anti-CD3 monoclonal antibody Leu-4 (Becton Dickinson, Erembodegerm, Belgium).14 Slides were evaluated by light microscopy and 103 nucleated cells per slide were counted.
Clinical outcome variables
Time to PMN recovery was defined as the number of days to reach an absolute number of 0.5 × 109/l in the blood. Primary graft failure was defined as failure to reach stable PMN recovery within 25 days after BMT and in the presence of an empty marrow. Late marrow failure was defined as the occurrence of marrow aplasia after an episode of stable PMN and platelet recovery or evidence of autologous reconstitution without signs of relapse. Serum levels of C-reactive protein (CRP) were measured at least every 48 h so that a maximal value (CRPmax) within the first 6 weeks post BMT could be determined. Early bacteremia was defined as the finding, within 30 days after BMT, of at least two positive blood cultures, taken at two different time points during the same episode of fever, with identification of the bacterial species having the same antimicrobial susceptibility pattern. Acute and chronic GVHD were diagnosed according to published criteria.15,16 All patients were considered evaluable for acute GVHD. Patients alive and with evidence of graft function at 90 days after BMT were evaluable for chronic GVHD. Only patients with chronic GVHD requiring systemic corticosteroids were scored as positive. Major transplant-related complications (MTC) were considered: >grade II acute GVHD, severe endothelial leakage syndrome (ELS), VOD or pneumonitis. The definitions of ELS and pneumonitis have been previously reported in detail.17 In short, endothelial leakage syndrome was diagnosed as the occurrence of fever, fluid retention and weight gain of >3%, in the absence of cardiac failure with insufficient response to diuretics, requiring fluid restriction and dopamine therapy. Pneumonitis was defined as the presence of fever and respiratory symptoms that could not be related to cardiac failure or generalized fluid retention and with clear demonstration of pulmonary infiltrates on chest X-ray. A diagnosis of veno-occlusive liver disease was made with the presence of at least two of the following features: jaundice, hepatomegaly and right upper quadrant pain, ascites and/or unexplained weight gain.18 Treatment-related mortality was defined as death due to any reason in a patient without evidence of relapse. Patients surviving at least 30 days after BMT were considered evaluable for relapse. Relapse in CML patients was defined hematologically.
All statistical tests were carried out two-tailed, at the 5% level of significance, using the Prism 3.0 software for statistics. For comparison of continuous variables the Mann–Whitney test was used. Association between categorical variables was investigated by the chi-square test or the Fisher's exact test. The duration of survival and the time to relapse (risk) were estimated by the Kaplan–Meier method and for comparison between groups the log-rank test was used.
The median number of T cells infused after TCD was 0.8 × 105/kg (range 0.1–14.0) as compared to 1.68 × 107/kg (range 0.23–4.47) in the case of UNM-BMT, corresponding to a 2–3 log depletion. TCD led to a ±78% reduction of mononuclear cells: medians 0.40 × 108/kg (range 0.12–0.98) vs 1.82 ± 108/kg (range 0.34–3.20) and to a ±38% loss of graft progenitor cells as assessed by the CFU-GM assay: medians 6.60 × 104/kg (range 1.10–15.00) vs 10.55 × 104/kg (range 1.70–29.60) (P < 0.001).
Morbidity and mortality
TCD was associated with less (4% vs 28%, P = 0.02) and less severe (stage >II, 0% vs 24%, P = 0.007) acute GVHD (Figure 1). Also, TCD led to a lower incidence of early bacteremia (11.5% vs 36%, P = 0.04) and MTC, other than acute GVHD >II: 4% vs 36% (P = 0.005). This was due to a decrease in cases with VOD (4% vs 24%, P = 0.06), ELS (0% vs 15%, P = 0.06) and pneumonitis (0% vs 18%, P = 0.03). Finally, TCD significantly reduced the incidence of chronic GVHD (8.5% vs 45%, P = 0.006). The extent of systemic inflammation during the early post-BMT episode, as evidenced by the CRPmax, was much lower in the case of TCD (median 61 vs 221 mg/l, P < 0.001). Overall, treatment-related mortality was lower (11.5% vs 36%, P = 0.04) after TCD. Causes of death (n = 10) in the TCD group were relapse (n = 7), late graft failure (n = 2) and late infection (n = 1). In the UNM group, 15 patients died as a result of CGVHD (n = 5), relapse (n = 3), AGVHD (n = 2), late infection (n = 2), VOD (n = 1), pneumonitis (n = 1) and CGVHD with hepatitis B (n = 1). TRM occurred significantly more frequently (44% vs 9%, P = 0.003) in patients developing at least one MTC and/or chronic GVHD (n = 27). In all 59 patients, MTC and/or chronic GVHD accounted for 40% of overall and 67% of BMT-related mortality. TRM was observed beyond 4 months and up to more than 3 years after BMT in 11 patients (eight UNM and three TCD).
There were no cases of early graft failure. Among 24 (TCD) and 31 (UNM) patients evaluable in each group, PMN recovery occurred on average 9 days earlier after TCD (13 vs 22 days, P = 0.02) (Figure 2a). G-CSF led to a significant reduction in time to recovery in the TCD-group (12 vs 21 days, P < 0.001) and this effect was more pronounced compared with the UNM group (19.5 vs 24 days, P = 0.01). Without G-CSF administration, the difference in PMN recovery between TCD and UNM groups was no longer significant (Figure 2b). Platelet recovery was not evaluable in six patients, due to early death or prolonged thrombopenia in the context of GVHD. For the other patients, time to platelets >20 × 109/l was significantly shorter after TCD (18 vs 26 days, P < 0.001) (Figure 2c). Late failure occurred in three patients at 3, 5 and 11 months post BMT, respectively. All had received a T cell-depleted graft, corresponding to an incidence of 12% after TCD. The primary diagnosis in these patients was CML and only one had a relatively poor graft with 1.4 × 104 CFU-GM/kg. Two patients died of toxicity after a second BMT and one had gradual autologous recovery after G-CSF administration, as determined by karyotype analysis.
Relapse and survival
The median survival of patients alive at time of analysis was 4.3 years (range 2–10.2). The relapse risk was significantly higher in the TCD group: 57.5% vs 21.5% (P = 0.04) (Figure 3a). After TCD, relapse occurred in 12 patients, four with acute and eight with chronic leukemia. The four patients with acute leukemia died of relapse. Of the eight patients with recurrent chronic leukemia, five were rescued with donor lymphocyte infusions, one died of GVHD after donor lymphocyte infusion and two died of relapse. Only four relapses occurred in the UNM group. All had acute leukemia and three of the four patients died of relapse. The fourth patient was rescued with a second BMT from the same donor and is alive in remission more than 6 years after BMT. Overall survival probability at 10 years did not differ between the TCD and UNM groups (54% vs 53.5%, P = 0.33) (Figure 3b).
T cells and transplant outcome
We analyzed the relationship between the number of residual T cells in the graft and major transplant outcome variables including the occurrence of MTC and/or chronic GVHD as well as relapse. Patients were assigned to one of three groups according to the number of residual T cells in their grafts: <105/kg, 105–107/kg and >107/kg (Table 2). The incidence of complications increased from 0% to 37% to 78% (P < 0.001) whereas relapse rate decreased from 54% to 31.5% to 13% (P = 0.03).
Over a 10-year period we grafted two groups of leukemia patients, either with or without TCD. Both groups were comparable with respect to type and status of disease, type of transplant, conditioning regimen, general supportive care and growth factor support. The only major difference was age of the patients who were, on average, more than 10 years older in the TCD group. Without TCD, the TRM in this older group would have been significantly higher.1,2 We found that TCD led to a more than 50% reduction of TRM and thus effectively compensated for the more advanced age as a known risk factor. This effect was due not only to a reduction in GVHD but also to a decreased incidence of major complications, other than GVHD, including VOD, ELS and early pneumonitis. We hypothesize that T cells in the graft have a major contribution not only in allo-reactive processes but also in systemic inflammation that can be triggered by infections and regimen-related toxicity. This hypothesis is supported by our observation that CRP release, as reflected by maximal values reached during the early post-BMT episode, was markedly lower after TCD.
Despite a 38% loss of CFU-GM, TCD did not impair early graft function. Primary graft failure has been a problem after TCD with broad-range monoclonal antibodies such as Campath.8 It has been suggested that donor lymphoid cells play a role in facilitating allogeneic marrow engraftment.19 Our study shows that the average of 1 × 105/kg residual T cells left in the graft after E-rosetting is sufficient to elaborate this effect. Moreover, we found that TCD led to a significant enhancement of early PMN and platelet recovery. This can, in part, be explained by the administration of methotrexate as GVH prophylaxis in the UNM group. In addition, indirect inhibitory effects on hematopoiesis related to enhanced systemic inflammation and cytokines may also have contributed to the prolonged neutrophil and platelet recovery after UNM-BMT.
Stable long-term engraftment after TCD with E-rosetting remains a problem since late failure occurred in 12% of our series. We found no clear relationship with the number of CFU-GM infused into the patient. We assume that the number of stem cells with long-term engraftment potential may be reduced by TCD procedures in a small but significant subset of patients. This problem may be overcome by augmenting T cell-depleted marrow preparations with CD34-positive progenitor cells.20
The role of TCD as GVH prophylaxis for BMT in leukemia patients is controversial because of concerns about an increased relapse rate. This information is mainly derived from large statistical analyses based on registry data as well as from single-center studies where different methods of TCD in various patient populations were used.5,21,22 We also observed an increased relapse rate after TCD so that it appears that a mean of 1 × 105/kg residual T cells in the graft of our patients was not enough to ensure sufficient GVL effect. This finding is somewhat in contrast with the results obtained after BMT with a low fixed number of 1 × 105/kg T cells where relapse rates of only 11% have been reported.11 The differences may be explained by the fact that this result was found in a series that included few CML patients. In our series, the graft-versus-leukemia effect also appeared most prominent in this type of leukemia since all CML patients who relapsed had received a TCD-BMT. It is clear that, at least in this particular subset, higher numbers of T cells should be left-in or added-to the transplant. We found a clear relationship between the number of T cells in the graft and relapse on the one hand, as well as severe complications on the other hand. Our data suggest that, especially for CML, a 2–3 log TCD followed by a strategy of donor lymphocyte infusions, thereby reducing the risk of transplant-related complications, seems to be a reasonable option. As a technique for more precise TCD, E-rosetting could be replaced by the combination of CD34-positive cell selection and add-back of fixed numbers of, eg CD3-positive T cells in the graft. The precise number of T cells to be added remains unclear. Based on our data we estimate it to be somewhere between 105 and 106 per kg, but further study is needed before definite conclusions can be made. For AML/ALL, the situation is more complicated because the benefit of acute GVHD to prevent, or donor lymphocyte infusions to treat a relapse post transplant is less evident in acute leukemia.
Despite the higher risk of relapse, overall survival in our patients was not inferior after TCD. This is due to the decreased TRM on the one hand, and to the possibility of rescuing CML patients who relapse after BMT by donor lymphocyte infusions on the other hand. The same observation has been reported by other groups.23,24,25
In summary, this comparative single-center analysis with long-term follow-up has led to the following findings: (1) graft T cells contribute to BMT-related complications, other than GVHD and are a major factor in all causes of post-transplant morbidity and mortality; (2) partial TCD has a positive indirect effect on early neutrophil and platelet recovery. Furthermore, our data suggest that risks of toxicity and relapse in this transplant setting could be better balanced by adjusting the dose of T cells in the graft. Finally, this study shows that partial TCD is an acceptable approach in terms of survival for patients between 40 and 50 years with standard risk leukemia in the HLA-identical sibling donor situation.
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We thank Mrs A Willekens and her nursing staff from the BMT-Unit for the excellent care of our patients. This work was supported by a grant from the scientific Fund W Gepts AZ-VUB.
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Schots, R., Van Riet, I., Ben Othman, T. et al. The impact of partial T cell depletion on overall transplant-related toxicity, graft function and survival after HLA-identical allogeneic bone marrow transplantation in standard risk adult patients with leukemia. Bone Marrow Transplant 28, 917–922 (2001). https://doi.org/10.1038/sj.bmt.1703268
- allogeneic BMT
- T cell depletion
- adult leukemia
- BMT-related complications
Differential sensitivity of T lymphocytes and hematopoietic precursor cells to photochemotherapy with 8-methoxypsoralen and ultraviolet A light
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