Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established, potentially curative treatment for a number of hematological malignancies. Originally all HSCTs were preceded by treatment with high doses of systemic chemotherapy, with or without total body irradiation (TBI), in a dual attempt to eradicate malignant cell clones and suppress the host immune response in order to enable grafting (1). Due to its high toxicity, this myeloablative regimen is unsuitable for patients with comorbid medical conditions, such as elderly or severely pretreated patients.
More recently, reduced intensity conditioning (RIC) regimens have been developed and used in these high-risk patients with encouraging results; long-term relapse rates have been shown to be at levels comparable to those after high-dose therapy (2, 3). The mechanism postulated to be behind this observation is the graft-versus-leukemia (GVL) effect, caused by the reaction of donor-derived immune-competent cells against the cells causing disease (4–6). GVL is currently regarded as the single most crucial determining factor for relapse-free survival after HSCT. The non-myeloablative or RIC approach significantly widens the range of patients suitable for HSCT, especially considering the fact that median age at diagnosis for most hematological malignancies is 65–75 years (7).
A histocompatible sibling is the donor of choice, but such is only available in about 30% of all cases where HSCT is indicated. Since the 1980s, human leukocyte antigen (HLA)-matched unrelated donors (MUD) have been used successfully against a number of hematological malignancies including acute leukemia, lymphoma, myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML) and chronic lymphocytic leukemia (8, 9). Registries of volunteer donors have been established worldwide, and they currently include over 10 million stem cell donors. In our own program, the majority of cases involve transplantation preformed with unrelated donors (10, 11).
Unrelated grafts have, however, been associated with a higher rate of transplantation-related mortality (TRM). The main reasons for this are higher frequencies of rejection and acute graft-versus-host disease (aGVHD), as well as a higher incidence of infections due to a prolonged period of immunosuppression as compared to sibling HSCT (12, 13). These differences in outcome are constantly becoming reduced with improvements in techniques for HLA matching. Also, with the increasing number of volunteer donors available, it will be possible to take into account other donor-associated characteristics that are shown to be of importance for the outcome of HSCT. These factors include donor sex, age, immunosuppression, cell dose and cytomegalovirus (CMV) serostatus (14–18).
This is a retrospective analysis of 137 patients with hematological malignancies or solid tumors who underwent RIC prior to HSCT. We compared the outcomes after HSCT in patients grafted with matched unrelated donors or HLA-identical siblings.
PATIENTS AND METHODS
Between April 1999 and February 2005, 137 patients were treated with RIC and HSCT at the Center for Allogeneic Stem Cell Transplantation, Karolinska University Hospital, Huddinge. The outcome in 63 patients receiving a sibling donor graft was compared with the outcome in 74 patients receiving a MUD graft. Patients in the two groups were similar in terms of diagnosis, age, sex and conditioning regimen. Details of patient and donor characteristics are listed in Table 1. GVHD prophylaxis (see below) was also similar in the two groups, with the exception that antithymocyte globulin (ATG) was given to all of the patients receiving an unrelated graft but to only 46% of the patients receiving stem cells from a sibling donor. Ten patients had a liver transplantation prior to HSCT due to advanced primary liver cancer (19).
Donors and Tissue Typing
Peripheral blood stem cells (PBSC) were donated from 61 identical siblings and 59 unrelated donors. The donors of PBSC were treated with 10 μg/kg/day granulocyte-colony-stimulating factor (G-CSF (Neupogen); Amgen, Basel, Switzerland) for four to five days (20). Unstimulated bone marrow (BM) was obtained from two siblings and 15 MUD.
Both HLA class I and II were typed by allele level polymerase chain reaction (PCR) single-stranded polymorphism (21). All donors were at least A, B, and DRβ1 compatible with the recipient.
RIC regimens consisted of fludarabine in combination with TBI and/or other cytostatic agents (Table 1). The most frequently used conditioning was a combination of fludarabine 30 mg/m2/day for six consecutive days followed by busulphan 4 mg/kg/day divided into four daily doses for two days (Flu+Bu) (3). Other conditioning regimens consisting of only chemotherapy were fludarabine 30 mg/m2/day for five consecutive days, in combination with either cyclophosphamide at a dose of 60 mg/kg/day for two days (Flu+Cy) or treosulfan 12–14 g/m2/day for three days (Flu+Treo). Conditioning regimens for patients with solid tumors included TBI 2 Gy combined with fludarabine 30 mg/m2/day for five days in MUD or for three days in sibling HSCT (Flu+TBI) (22). Fludarabine 30 mg/m2/day for six days and cyclophosphamide 30 mg/kg/day for two days combined with fractionated TBI (fTBI) 3Gy for two days (Flu+Cy+fTBI) were given to patients with lymphoma.
All patients transplanted from MUD were treated with antithymocyte globulin (ATG; Thymoglobulin; Genzyme, Cambridge, MA) while less than half (46%) of the patients transplanted from siblings received ATG (23). All patients treated with Flu+Bu were administered a total dose of 8 mg/kg. Patients treated with conditioning regimens other than Flu+Bu and transplanted from MUD were administered ATG in a total dose of 4–6 mg/kg (24). One patient treated with Flu+Treo and transplanted from a MUD received ATG-Fresenius (Fresenius AG, Bad Homburg, Germany) at a dose of 10 mg/kg/day for three days.
Supportive Care and Treatment of Infections
Prophylactic Treatment against Infections
The antimycotic agents nystatin and fluconazole were administered orally from the start of the conditioning regimen until the absolute neutrophil count (ANC) reached 0.5×109/L. The partial bowel decontamination also included oral ciprofloxacin, starting one day before transplantation and continuing until the engraftment. Prophylaxis against Pneumocystis carinii with cotrimoxazole was started together with conditioning regimen, stopped two days before transplantation and restarted when ANC was greater than 0.5×109 /L. Acyclovir prophylaxis was used only in patients who had an antibody titer to herpes simplex virus (IgG ≥ 10,000) and administration continued until engraftment (25).
Prevention of Hepatic Complications
All patients were given ursodeoxycholic acid in a total oral dose of 12 mg/kg/day divided into two daily doses (26).
Prophylaxis of Other Side Effects
The prophylaxis against hemorrhagic cystitis included forced diuresis, urine alkalinization, and uromithexane 12 mg/kg/dose at 0, 1, 3, 6, 9, and 12 hr after administration of cyclophosphamide. Granisetron hydrochloride was used as antiemetics. Clonazepam was used as prophylaxis against busulphan-related seizures. The prevention of hyperuricemia and urate nephropathy with allopurinol was employed during the conditioning regimen. The hypersensitivity reaction to ATG was prevented using clemastine and methylprednisolone. Prophylactic G-CSF (5 μg/day) was given to 49 patients from day +10 until engraftment (Table 1). When we found that G-CSF prophylaxis increased the risk of acute GVHD, this regimen was stopped (27, 28).
CMV infection was treated with preemptive therapy using ganciclovir (5 mg/kg i.v. twice daily) or foscarnet (90 mg/kg i.v. twice daily) for two weeks. Alternatively, oral valganciclovir was given as 450 mg/kg twice a day. Doses of these antiviral drugs were reduced according to renal dysfunction. If the PCR test for detection of CMV DNA in peripheral blood became negative, the CMV therapy was discontinued; otherwise, the therapy was continued (once a day) until a negative PCR was found (29). CMV disease was treated with either ganciclovir or foscarnet i.v.
GVHD Prophylaxis and treatment
Cyclosporine (CsA) was given to all patients for at least three months. The CsA doses ranged between 3 and 12 mg/kg to achieve a through level of 100 ng/ml and between 200 and 300 ng/ml in patients with a sibling or unrelated donor, respectively (10). On the day before and on the day of transplantation, and when optimal CsA levels were not achieved by oral medication, intravenous CsA was administered as a continuous infusion in doses ranging from 1 to 5 mg/kg/day. CsA was combined with mycophenolate mofetil (MMF) or methotrexate (MTX) (30, 31). MMF was administered at 15 mg/kg twice daily for 35 days if the donor was a sibling and for 45 days if the donor was unrelated (22, 32). MTX was administered at 15 mg/m2 on day one and 10 mg/m2 on days 3, 6, and 11 after HSCT (33). All 10 patients receiving liver transplantation prior to HSCT were given tacrolimus (Prograf; Fujisawa Pharmaceuticals Ltd., Munich, Germany), 0.1 mg/kg/day administered orally, aiming at 12-hr trough levels between 5 and 15 ng/ml in combination with steroids, 20 mg/day PO, starting from the day of liver transplantation. As for further prevention of GVHD, tacrolimus was combined with either MMF or MTX as described above.
Bacterial septicemia was defined as the first positive blood culture related to a febrile episode (≥38.5°C). Engraftment was defined as a stable level of ANC ≥0.5×109/L after the posttransplantation nadir.
Acute GVHD was diagnosed from clinical symptoms and/or biopsies from skin, oral mucosa, liver and gastrointestinal tract, and graded from 0 to IV as previously reported (34). Chronic GVHD (cGVHD) was evaluated in patients surviving more than 90 days after HSCT, and was classified as limited or extensive (35).
Asymptomatic CMV infection was defined as isolation of CMV or detection of viral proteins or nucleic acid in any body fluid or tissue specimen. Primary CMV infection was defined for individuals previously found to be CMV seronegative. CMV disease was defined as a symptomatic organ involvement such as CMV pneumonia, hepatitis, colitis, or retinitis (36).
Invasive fungal infection (IFI) was defined as positive blood culture and/or positive cultures from at least two organs for Candida or Aspergillus species. Aspergillus pneumonia was defined as pulmonary infiltrates and positive cultures or direct microscopy of bronchoalveolar lavage fluid (BAL), sputum, or autopsy specimens.
The probability of survival and relapse-free survival (RFS) was calculated using the Kaplan-Meier method and compared with the log-rank test. The incidence rates of GVHD and TRM were estimated using a parametric estimator of cumulative incidence curves to accommodate competing risks. Death within 100 days without GVHD and relapse (TRM) was considered as a competing event. Censored data were taken into account.
The Cox proportional hazard regression model was used for univariate and multivariate analyses. This analytic approach is designed to generate predictive factors for the events (GVHD, TRM, CMV infection) after HSCT. Factors affecting day of engraftment were analyzed using the multiple linear regression method. Analyses were performed using the cmprsk package (developed by Gray, June 2001), Splus 6.2 software, and Statistica software.
Engraftment and Transfusions
Graft rejection occurred in 15 patients (6 sibling, 9 MUD) and secondary graft failure occurred in one patient (MUD). Nine of the rejections occurred in patients receiving the Flu+TBI (2 Gy) conditioning regimens. This conditioning regimen was associated with a significantly higher rejection rate as compared to the other regimens, 9/25 vs. 7/112 (P<0.001). ANC engraftment was faster after sibling HSCT (median time 14 days; range 0–34) than after MUD HSCT (median 18 days; range 0–28) (P<0.001). The time to reach a platelet count of greater than 50×109/L was a median of 14 days after both sibling and unrelated HSCT.
In multivariate analysis, the use of MMF instead of MTX, the absence of ATG in the conditioning regimen, and treatment of G-CSF posttransplant were factors associated with faster ANC engraftment (Table 2).
The sibling transplants required a median of two (range 0–23) units per patient of erythrocyte transfusions, as compared to four (range 0–48) units per patient in MUD transplants (NS). The median requirement for platelet transfusions was zero (range 0–24) units per patient and one (range 0–28) unit per patient in the two groups, respectively (P=0.03). In multivariate analysis, the use of ATG was associated with a higher number of platelet transfusions (P<0.01).
Bacterial septicemia was diagnosed in 10 (16%) of the sibling transplants, as compared to 17 (23%) of the MUD transplants. CMV infection was observed in 29 (46%) of the sibling transplants and in 48 (65%) of the MUD transplants (P=0.04). In multivariate analysis, factors associated with a higher incidence of CMV infection were MUD HSCT, the use of G-CSF, acute GVHD II-IV and transplantation from a seronegative donor to a seropositive patient. CMV disease was diagnosed in five patients (1 sibling, 4 MUD).
The cumulative incidences of acute GVHD grades II–IV and grades III–IV was similar after both sibling and MUD HSCT (Fig. 1,A and B). However, a higher incidence of cGVHD was seen after sibling HSCT (Fig. 1C).
In multivariate analysis, the use of MMF instead of MTX, higher doses of CD34+ cells in the graft, and female to male transplants were significantly associated with a higher incidence of aGVHD grades II–IV (Table 2). For cGVHD, only higher donor age was found to be a significant risk factor (P=0.02).
The cumulative incidence of TRM was 14% after sibling HSCT and 34% after MUD HSCT (P<0.01) (Fig. 1D). Risk factors for TRM included aGVHD grades II–IV, MUD HSCT, and bacterial septicemia (Table 2).
Survival and Death
RFS for hematological malignancies and overall survival for all patients were no different between sibling and MUD transplants (Fig. 2, A and B). The incidence of relapse did not differ between sibling and MUD transplants in patients with hematological malignancies (data not shown). Causes of death in both groups are shown in Table 3. The major cause of death after sibling HSCT was relapse/disease progression while transplant-related complications were the cause of death in the majority of MUD transplants.
In this retrospective study, we compared the outcomes after RIC of sibling and MUD HSCT. Although the patient material was heterogeneous regarding diagnosis and conditioning regimens, the sibling and MUD groups were comparable.
Engraftment was successful in most patients, with no significant difference between sibling and MUD HSCT. The incidence of rejections was 12% in this patient group. The major cause of rejection was the use of Flu/TBI (2 Gy) as the conditioning regimen. In a previous report from our center regarding RIC transplant, the Flu/TBI (2 Gy) regimen was found to be associated with a higher probability of graft failure, as compared to other RIC regimens (37). In a European study of RIC in patients with chronic myeloid leukemia, the Flu/2 Gy TBI regimen was associated with a worse outcome (38). On the other hand, an excellent outcome has also been reported with this regimen (7).
Time to ANC engraftment was found to be faster after sibling HSCT as compared to MUD HSCT in univariate analysis. This difference was probably due to the use of ATG in MUD patients, since the use of ATG was associated with slower engraftment in multivariate analysis. Furthermore, MTX delayed engraftment of ANC compared to MMF, which might be expected. Fast engraftment after G-CSF treatment has been shown in several randomized studies (39). That ATG delays engraftment of ANC and platelets is due to the fact that these are broadly reacting against not only T-cells, but also against other cell surface structures on lymphocytes and platelets. Furthermore, ATG can be detected in serum several weeks after transplantation (23).
Infections are usually more common after MUD transplants than after sibling transplants (40). This is most probably due to differences in histocompatibility antigens in MUD transplants, whereas all major HLA antigens are the same in HLA-identical sibling transplants. We could not find a significant difference in the incidence of bacterial septicemia in the two groups. There was, however, a higher incidence of CMV infection after MUD transplantation. Besides MUD transplants, the use of G-CSF, acute GVHD grades II-IV and a transplant from a seronegative donor to a seropositive patient were risk factors for CMV infection. Early posttransplant use of G-CSF has previously been found to increase the risk of acute GVHD grades II–IV (27, 28). There is a close correlation between GVHD and CMV infection. Herpesvirus immunity and latency in recipient and donor are associated with an increased risk of acute GVHD (41). Furthermore, GVHD and its treatment are immunosuppressive events and pave the way for CMV infection and CMV associated disease (42). A higher incidence of CMV infection after MUD transplantation has also been reported by others (43).
With the introduction of RIC transplantation, it was first thought that the incidence of severe GVHD should decrease as compared to myeloablative conditioning. This has not been found to be the case in our institution, although it has been found by others (44). The incidence of acute GVHD grades II–IV was 33% in this study, which is similar to that after myeloablative conditioning in our center (10). Interestingly, in the present study there was no difference between sibling and MUD transplants. The comparable incidence of severe acute GVHD in patients after MUD transplantation may be the result of high-resolution HLA-typing using PCR for both HLA class I and II alleles. The use of ATG as well as early treatment of acute GVHD grade I with high-dose steroids is also believed to reduce the incidence of severe acute GVHD after MUD HSCT.
Unexpectedly, a higher incidence of chronic GVHD was found after sibling HSCT (56%) than after MUD HSCT (29%). This difference was not significant in multivariate analysis where only donor age proved to be a significant factor. A higher donor age in sibling transplants (median 52 years) than in MUD transplants (median 36 years) may explain the difference in the incidence of chronic GVHD. High donor and recipient age are well known factors associated with chronic GVHD (1).
As expected, TRM was significantly higher after MUD HSCT than after sibling HSCT. This is probably due to a higher degree of histoincompatibility, which may lead to GVHD and infections. To reduce the risk of GVHD in MUD transplants, we use ATG (10, 23). Indeed, the risk of acute GVHD was the same in the two groups. However, by delaying T-cell recovery, ATG increases the risk of infections, which is a dose-dependent phenomenon (24). Despite this, the overall survival was no different between the two groups. Relapse/disease progression was the major cause of death after sibling HSCT, while MUD transplant patients more often died from infections.
In conclusion, despite the fact that there was a higher incidence of TRM after MUD transplants, the overall survival was similar whether using unrelated or related donors.
We thank Inger Hammarberg for help in the preparation of this manuscript. We are indebted to the staff at the Center for Allogeneic Stem Cell Transplantation, Departments of Hematology and Pediatrics for compassionate and competent patient care.
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