We initiated a randomized study of amifostine (the organic thiophosphate formerly known as WR-2721) given to patients during myeloablative conditioning therapy for allogeneic bone marrow transplantation. Amifostine was given at a dose of 1000 mg/day of conditioning and was well tolerated if attention was given to serum calcium levels, blood pressure and antiemetics. Since August 1998, 60 patients (30 on each arm) have completed the study. There was no significant difference in the days to neutrophil or platelet engraftment in either arm of the study. Significantly, the duration of grade I–IV mucositis was decreased in the group that received amifostine (P=0.02). Also grade III or IV infections (P=0.008), duration of antibiotic therapy (P=0.03) and duration of fever (P=0.04) were significantly reduced with amifostine. However, there were no differences in the incidence of grade III or IV mucositis, liver toxicity or renal toxicity. There were also no differences in early mortality, relapse and long-term survival. We conclude that amifostine, while reducing the duration of mucositis and infections (possibly through some preservation of gut mucosal integrity), has a modest effect in allogeneic bone marrow transplants given the multiplicity of factors influencing organ toxicity and survival in this setting.
Amifostine (the organic thiophosphate formerly known as WR-2721), originally designed as a radioprotective agent in event of a nuclear fallout,1 was later also found to protect normal tissues and cells from the toxicity of radiation and cytotoxic agents of alkylating and platinum classes.2, 3 Implanted tumors and leukemia cells are not similarly protected4 and studies suggest that the cytoprotection afforded to normal cells is due to the fact that normal cells have higher membrane alkaline phosphatase activity (required for the metabolism of the amifostine prodrug to the active thiol WR-1065) than tumor cells (Figure 1).5, 6 Since amifostine reduces the toxicities associated with chemotherapy as well as radiotherapy, it has been used in autologous hematopoietic stem cell transplantation (HSCT), where it appears to reduce the toxic effects of myeloablative conditioning regimens.7, 8, 9 In allogeneic bone marrow transplants, there has been no randomized trial exploring the role of amifostine. In this study, we undertook a randomized controlled trial to determine the efficacy of amifostine in reducing the regimen-related toxicities associated with myeloablative preparative regimens in patients receiving allogeneic HSCT and to determine their impact on overall survival.
Patients and methods
Patients undergoing allogenic marrow or peripheral blood stem cell transplantation from human leukocyte antigen-identical siblings were eligible for the study. Patients were excluded if there was evidence of significant hepatic or renal dysfunction (defined as a level of bilirubin, alanine aminotransferase, aspartate aminotransferase, or alkaline phosphatase that was more than three times the upper limit of normal and a serum creatinine level of more than two times the upper limit of normal). Written informed consent was obtained from all patients randomized to receive amifostine. This study protocol was approved by the local ethics committee.
Study design and drug administration
The study was a single-institution, randomized, open-label trial. Patients were recruited from 1998 to 2003. Amifostine was given at a daily dose of 740 mg/m2 daily intravenous (i.v.) on each day of conditioning. The drug was given as a single daily dose when total body irradiation (TBI) or chemotherapy was delivered to the patient only once a day. Alternatively, the daily dose of 740 mg/m2 was given in two divided doses when the patient received chemotherapy or radiation more than once a day. Amifostine doses were rounded off to the nearest 500 mg. Each infusion was given over 15 min i.v., starting within 30 min prior to chemotherapy or TBI. Antiemetics were administered prior to amifostine infusion and comprised dexamethasone 8 mg i.v. and ondansetron 8 mg i.v. 30 min prior to the initiation of amifostine.
Patients were watched closely for hypotension during infusion of the drug. A measure of 500 ml of saline were given i.v. prior to drug delivery and blood pressure monitoring was instituted every 3–5 min during the infusion until 5 min after the infusion. In circumstances when there was fall in systolic blood pressure more than 20% below the baseline, the infusion was temporarily interrupted and normal saline infused until normalization of blood pressure. The patient was placed in a Trendelenberg position if symptomatic.
Calcium levels were monitored daily and calcium supplementation was given whenever the corrected calcium levels fell below normal.
Toxicity evaluation and monitoring
The extent of organ toxicity was graded daily by the WHO grading system until patients were discharged from hospital. The 1979 WHO Handbook for Reporting Results for Cancer Treatment was initially used when the trial started in 1998,10 but as the 1998 and 1999 Common Toxicity Criteria in the Cancer Therapy Evaluation Program (CTEP) became available, toxicity grading was, whenever possible, retrospectively defined according to the new criteria.11 The 2003 Common Terminology Criteria for Adverse Events v3.0 (CTCAE) was not used as it became available only after completion of the trial.
Mucositis was defined as grade 0 when absent; grade I when erythema and sore throat were found on daily examination; grades II and III when ulceration was present with or without the ability to eat, respectively; and grade IV when symptoms were associated with severe ulceration requiring prophylactic intubation or resulting in documented aspiration pneumonia.
Emesis was defined as grade 0 when absent; grade I if the frequency was one episode a day; grade II if frequency was two to five episodes; grade III when patients were unable to retain food or liquids with six or more episodes a day; and grade IV when dehydration and electrolyte disorders occurred with the potential life-threatening consequences.
Diarrhea was defined as grade 0 when absent; grade I when there was an increase with less than four stools a day (or less than 1 l); grade II when there was an increase with less than four to six stools a day (or more than 1 l); grade III when there were more than seven stools a day and treatment with intravenous fluids was needed; and grade IV when hemorrhage, electrolyte disorders and dehydration occurred.
Infections were defined as grade III when specific treatment was required and as grade IV when septicemia or life-threatening complications occurred.
Full blood count analysis was performed daily until white cell (defined as absolute neutrophil count, ANC, >500/μl) and platelet count (defined as platelet count, Plt >20 000/μl) recovery. Liver and renal functions were assessed at least three times a week. Mucositis was graded by the physicians looking after the patients and the grading double-checked by our trial coordinator. Patients were also assessed for the use of opiate analgesics and duration of opiate usage was recorded. As severe mucositis could predispose to graft-versus-host disease (GVHD) and infections, the occurrence and grading of GVHD and infections were noted, as was the duration of antibiotic usage and hospital stay. Patients were also assessed for other parameters, including fever, nausea, vomiting and diarrhea. Special note was made of amifostine-associated toxicities (hypocalcemia, hypotension and nausea or vomiting) during the period of myeloablative conditioning. Patients were subsequently followed up until death or last contact.
All patients were given oral ciprofloxacin 500 mg twice daily for Gram-negative bacterial prophylaxis, which was begun from the beginning of conditioning until neutrophil engraftment. All patients were treated in conventional private, nonlaminar airflow rooms. All patients were also randomly assigned to either itraconazole oral solution 200 mg/day or low-dose i.v. amphotericin B 0.2 mg/kg/day up to a maximum of 10 mg/day, for prophylaxis against fungal infections, as part of our other ongoing trial. Broad-spectrum antibiotics were used to treat initial episodes of fever. High-dose amphotericin B (0.5–1.0 mg/kg/day) was initiated for patients with suspected or proven fungal infections. All patients were given subcutaneous granulocyte colony-stimulating factor (G-CSF) 5 μg/kg/day until the ANC exceed 500/cm3. All blood products were irradiated (2500 cGy) and filtered before infusion. Immuno-globulin was administered to all patients at a dose of 500 mg/kg weekly from days −7 to +54.
The proportions of patients with a given characteristic were compared by the χ2 test or by Fisher's exact test. Differences in the mean of continuous measurements were tested by either Student's t-test or Mann–Whitney U-test. All tests were two tailed. Probabilities of overall survival were calculated by the method of Kaplan and Meier12 and the levels of significance were calculated by the log-rank statistic.13 All statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 10.0.
Patient characteristics (Table 1)
Between August 1998 and October 2003, a total of 60 patients were enrolled into the trial, with 30 patients assigned to receive amifostine (treatment group) and another 30 patients not given amifostine (control group). There was no significant difference among the two treatment arms for any of the clinical features with regard to age, gender, underlying disease, disease status, preparative regimen or prophylactic antifungal usage.
The median age was 28 years (range, 15–47 years) and 30 years (range, 14–46 years) for the treatment group and control group, respectively. In all, 22 (73%) patients in each arm received a busulfan/cyclophosphamide (BuCy) regimen, which consisted of Bu 4 mg/kg p.o. in divided doses daily for 4 days (total 16 mg/kg) in combination with Cy 60 mg/kg once daily i.v. on days 1 and 2 (total dose 120 mg/kg). Six (20%) patients and four (13%) patients received the cyclophosphamide/total body irradiation (CyTBI) regimen, which consisted of Cy 60 mg/kg once daily i.v. on days 1 and 2 (total dose 120 mg/kg) in combination with fractionated TBI 2 Gy twice daily for 3 days. An etoposide-containing regimen was given to three patients (treatment arm: n=1; control arm: n=2) who had mildly impaired cardiac function and were deemed unfit to receive full dose Cy. All patients in both arms were given cyclosporine in combination with methotrexate as GVHD prophylaxis.14
Amifostine-related toxicities (Table 2)
Toxicities experienced by the amifostine-treated patients were analyzed and compared with the control group. The most common side effect reported during the infusion of amifostine was nausea or vomiting. This occurred in 29 (97%) patients, with 14 (47%) patients with grade ⩾III severity. However, the incidence of vomiting in these amifostine-treated patients was not significantly different from that in the control arm (P=0.18).
Hypotension, which was defined as a decrease in systolic blood pressure of ⩾20 mmHg from baseline value, was observed in nine (30%) patients who were given amifostine and required interruption at some point during chemotherapy. In six of these patients with hypotension, the blood pressure recovered with infusion of normal saline and they were able to complete the infusion as planned, without dose reduction. Transient reduction in blood pressure did not recur in subsequent cycles. Three of the nine patients, however, were unable to proceed with the infusion as the hypotension was not reversed with corrective measures other than discontinuation of the amifostine. No patients became symptomatic during the hypotension or had any clinical sequelae from it.
Hypocalcemia occurred significantly more in patients receiving amifostine infusion than in the control arm (P<0.001). In all, 25 (83%) patients receiving amifostine developed hypocalcemia of varying severity, with grade ⩾III seen in five (17%) patients.
Hematological toxicity and engraftment kinetics (Table 2)
One of the 30 patients receiving amifostine died on day 2 after bone marrow infusion from acute respiratory distress syndrome, leaving only 29 patients in the treatment arm evaluable for transplant-related toxicity. Hematological toxicity was not statistically different the two arms. All patients in both arms experience grade III–IV neutropenia and thrombocytopenia requiring transfusion support. There was no significant difference in the days to achieve neutrophil engraftment (defined as a neutrophil count of >500/mm3) between the amifostine-treated group (median 20 days, range 14–21 days) and the control group (median 17 days, 11–27 days) (P=0.32). Similarly, amifostine treatment did not affect platelet recovery. The median time to achieve platelet >20 000/mm3 was 23 days (range, 10–33 days) for the treatment group and 20 days (range, 10–41 days) for the control group (P=0.55). This suggests that amifostine, while it may protect normal cells, does not reduce the effects of conditioning therapy such as to cause rejection or delayed engraftment.
Transfusion requirement was also not significantly different in both arms. The median number of red blood cells given was 6 U (range, 0–56 U) and 8 U (range, 0–46 U) in the treatment arm and control arm, respectively (P=0.21). There was also no statistically significant difference in the number of platelets transfused between the two arms (median 33 U, range 5–180 U vs median 34 U, range 3–275 U; P=0.44). The median duration of hospitalization was 32 days for both arms (P=0.99).
Extrahematological toxicity (Table 3)
Incidence and severity of renal toxicity, hepatic toxicity and GVHD were not significantly different between the two arms. Patients receiving amifostine showed a trend toward a lower incidence of grade ⩾III mucositis (41%) as compared to the control group (63%), although this difference did not reach statistical difference (P=0.44). Notably, the duration of mucositis of all grades was significantly shorter among patients receiving amifostine (median 16 days, range 0–42 days) as compared to the control group (median 21 days, range 9–49 days) (P<0.02). However, while there was a trend towards a shorter duration of grade ⩾III mucositis in the amifostine arm: 0 days in the amifostine group (range, 0–23 days) vs 5 days in the control group (range, 0–16 days); the difference was not statistically significant (P=0.3). Similarly, the median duration of opiate analgesic administration was not significantly different between the two groups: 14 days in the amifostine arm (range, 3–30 days) and 15 days in the control arm (range, 6–43 days) (P=0.17).
Patients who received amifostine had a significantly lower incidence of grade ⩾III infections (65%) as compared to the control group (97%) (P=0.008). The median duration of antibiotic therapy was significantly shorter among patients receiving amifostine (17 days; range 7–47 days) than in the control group (24 days; range 7–66 days) (P=0.03). Fever ⩾37.5°C occurred in all patients in both arms with a median duration of 11 days (range, 1–26 days) among the patients receiving amifostine and 14 days (range, 3–34 days) in the control group (P=0.04).
Survival and outcome
In all, 18 (60%) patients in each arm died after transplantation. The median follow-up after transplantation was 240 days (range, 1–1917 days) for amifostine recipients and 116 days (range, 20–1751 days) for the control group. Relapse of underlying hematological malignancies, infection, hemorrhage and graft-versus-disease were the predominant causes of death in these patients, and the incidence was not statistically significantly different between the two arms. Death was attributed to relapse in seven (23%) amifostine recipients and five (7%) patients from the control group (P=0.52). Kaplan–Meier analysis of survival at 3 years after transplantation was similar between the two arms (amifostine 37% vs control 37%, P=0.80) (Figure 2).
The effects of amifostine as a cytoprotectant of normal tissue during the administration of chemotherapy,15 radiotherapy16 or both17 have been well studied, albeit they are a source of some controversy.18 It is has been shown that normal tissue is selectively protected over cancerous tissue.2, 3, 4 While normal cells are somewhat protected from the effects of cytotoxic therapy, tumor cells that have deficient cell surface intestinal-type alkaline phosphatase are not.5, 19
A number of trials have explored the effects of amifostine as a cytoprotectant in autologous HSCT,20, 21 and amifostine may allow chemotherapy dose increases beyond those permitted by autologous HSCT.22 The drug has also been studied in the in vivo or ex vivo purging of leukapheresis collections23 and there seems to be a role for amifostine in protecting stem cells during salvage chemotherapy, thus facilitating stem cell collection.24
However, there are no published randomized trials of its use in allogeneic transplantation. Indeed, such a trial could be difficult to undertake and interpret, as there may be many factors that could contribute to mucosal and organ toxicity after allogeneic transplantation.
However, we showed that amifostine given together with conditioning therapy for HSCT adds little to the toxicity of the procedure (albeit mild hypotension, nausea and correctable hypocalcemia). While some of the initial patients on amifostine developed symptomatic hypocalcemia with tetany, which was corrected with calcium infusion, with prophylactic calcium supplementation, subsequent patients were spared this complication.
In addition, while amifostine may protect normal cells from the effects of myeloablative chemo/radiotherapy, there may be concerns as to whether it also protects recipient lymphocytes to such an extent that they would be poised to mediate rejection of the donor allograft. In order to ascertain whether this phenomenon could result in significant differences in hematopoietic recovery, we analyzed neutrophil and platelet recovery on the patients in our study. In our study, there were no significant differences in engraftment between the group given amifostine and the group that was not. This suggests that amifostine does not block the immunosuppressive effects of the conditioning regimen to the extent of having any significant effect on engraftment. In another trial, it was also shown that amifostine in autologous transplants did not significantly influence reconstitution of lymphocyte subpopulations.25 Thus hematopoietic and immunological recovery do not appear to be adversely affected by the administration of this drug.
Despite the potential confusion of data by multiple variables, we were able to find a significant reduction in duration of mucositis in our patients (16 days with amifostine vs 21 days for controls, P=0.02). However, there were patients receiving amifostine who still developed severe mucositis and the duration of grade III or IV mucositis was not different between groups. We believe that the lack of protection in some patients is due to the long half-life of many of the chemotherapeutic drugs given (such as Bu and Cy) and the much shorter half-life of amifostine. In all, 90% of the drug is cleared from the plasma and taken up by normal tissues within 6 min of administration.26 The narrow window of time for which amifostine is able to exert its cytoprotective effects would limit its efficacy. For regimens that rely entirely on TBI or drugs with very short half-lives, the cytoprotective effects could possibly be more apparent.
There were no significant differences in liver/renal toxicity in this study and we believe this is because, in addition to the above confounding factors, there are many other factors that contribute to these organ dysfunctions in allogeneic transplants. These include GVHD as well as the effects of immunosuppressive drugs such as cyclosporine.
Of great interest was the very significant reduction in maximal grade of infections (P=0.008) as well as a statistically significant reduction in the duration of fever (P=0.04) and antibiotic therapy (P=0.03). As loss of gastrointestinal mucosal integrity leads to the translocation of gut pathogens into the circulatory system,27, 28 the administration of amifostine could result in preservation of the normal mucosal barrier and a consequent reduction in all these indicators of infection.
In conclusion, amifostine can be safely given in allogeneic HSCT without excess toxicity or nonengraftment. It appears to have a significant impact on duration of all grades of mucositis, although there is also the absence of mucosal protection seen in some patients as discussed above. Of greatest significance was the reduction in all parameters of infection in the group receiving amifostine, and this may be due to a preservation of gut mucosal integrity against intraluminal pathogens. However, as there was no significant difference in survival, transplant-related mortality or relapse, our study suggests that the overall role of amifostine is modest in allogeneic bone marrow transplantation where multiple factors may influence the outcome and survival.
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We thank the nursing staff in the Department of Haematology, Singapore General Hospital for excellent nursing care and Schering-Plough Singapore for sponsoring this clinical trial.
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Hwang, W., Koh, L., Ng, H. et al. A randomized trial of amifostine as a cytoprotectant for patients receiving myeloablative therapy for allogeneic hematopoietic stem cell transplantation. Bone Marrow Transplant 34, 51–56 (2004) doi:10.1038/sj.bmt.1704521
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