¹Department of Clinical Hematology, Hospital Duran i Reynals, Institut Català d'Oncologia, Spain

²Cancer Prevention and Control Unit, Hospital Duran i Reynals, Spain

³Department of Endocrinology, Hospital Príncipes de España, Barcelona, Spain

Correspondence to: Dr I Ancín, Department of Clinical Hematology, Hospital Duran i Reynals, Autovía de Castelldefells Km 2.7, 08907 L'Hospitalet de Llobregat, Barcelona, Spain; E-mail: 30.097iaa@comb.es

In a retrospective study, we compared 15 patients who received cyclosporine (CsA), methotrexate (MTX) and prednisone (PDN) and 15 patients who received CsA-MTX for GVHD prophylaxis after allogeneic BMT (HLA-identical sibling (n = 22), related one HLA mismatch (n = 1), unrelated matched donors (n = 6), unrelated one HLA mismatch (n = 1)). The primary objectives of this study were to compare the incidence of GVHD and post-transplantation complications. Secondary objectives were to compare relapse rate, transplant-related mortality and overall survival. The incidence of acute GVHD grade III-IV was similar between the two groups (P = 0.66), as was the incidence of chronic GVHD (P = 0.67). Incidence of arterial hypertension was significantly higher in patients who received prophylactic PDN, (P = 0.03) and more insulin treatment was required in this group (P = 0.003). We observed no differences in the incidence of infections or upper digestive tract bleeding. Musculoskeletal complications appeared earlier in the group which received PDN. With a median follow-up of 4.4 years, patients in the CsA-MTX group had better overall survival, 46.7% vs 13.3% (P = 0.026). Relapse was a more frequent cause of death in the CsA-MTX group, whereas procedure-related mortality was more frequent in the CsA-MTX-PDN group (P = 0.013). These results suggest that prophylactic prednisone when combined with cyclosporine and methotrexate adds no benefit in acute or chronic GVHD prevention and may increase the morbidity of allogeneic transplantation. Corticosteroids may be reserved for GVHD treatment. Bone Marrow Transplantation (2001) 28, 39-45.

allogeneic bone marrow transplantation; acute graft-versus-host disease prophylaxis; corticosteroids

Acute graft-versus-host disease (GVHD) continues to be a major complication after allogeneic bone marrow transplantation and develops in 30-60% of recipients of histocompatible sibling-matched allografts. Mortality due directly or indirectly to GVHD may reach 50%.^1,2

When allogeneic transplants were performed without GVHD prophylaxis, very high rates of GVHD were observed³ and Sullivan et al⁴ reported a case of hyperacute GVHD.

Prophylaxis with methotrexate is associated with a 40-50% incidence of GVHD,^5,6 and several studies demonstrated that cyclosporine A (CsA) and methotrexate (MTX) alone were equally effective in preventing GVHD.^7,8,9

The combination of CsA and MTX decreased the incidence of GVHD in several randomised controlled trials. This combination is associated with a significantly lower rate (20-30%) of II-IV acute GVHD than when using MTX or CsA as single-agent.^{5,10,11,12,13} However, a similar disease-free survival was observed when comparing CsA plus MTX vs CsA alone because there was a more frequent incidence of relapse when MTX was part of GVHD prevention.¹¹

Other investigators added corticosteroids to either MTX, cyclophosphamide or CsA. They found an advantage in CsA plus prednisone compared to the other combinations. This combination allowed for more rapid hematopoietic recovery compared with MTX-containing regimens, albeit at the price of a higher incidence of GVHD than seen with MTX plus CsA.^14,15,16 No advantages were found in the incidence of chronic GVHD. The incidence of chronic GVHD was higher and more frequent when using CsA plus methylprednisolone (MP) than when using cyclosporine alone.¹⁷ When CsA plus MP were compared to CsA plus MTX, the incidence of acute GVHD II-IV was 45% when MP was used, with three GVHD-related deaths. No cases of moderate to severe GVHD were observed when CsA was combined with MTX.¹⁸

In order to reduce the incidence of GVHD three doses of MTX were added to CsA plus PDN.¹⁹ One of the controversies is whether adding corticosteroids provides any benefit to the combination of CsA and MTX. The results of these trials differ.²⁰

Different studies demonstrated that corticosteroids, when added to standard CsA and MTX prophylaxis did not improve or increase acute GVHD incidence and chronic GVHD.^21,22 When patients receive PDN or MP as part of GVHD prophylaxis they have a higher risk of failure of acute GVHD treatment if this develops.²³ On the other hand, relapses are less frequent when corticosteroids are part of GVHD prophylaxis.²¹

Use of corticosteroids may increase risk of infections but it is unclear to what extent. In one study in which corticosteroids were used in the early post-transplant period, the risk of infection was increased by 50%.²⁴ Later complications such as avascular necrosis of the bone appear when corticosteroids are used for treatment of acute GVHD.²⁵

Other possible complications such as hyperglycemia, arterial hypertension or upper digestive tract bleeding have not specifically been compared between prophylactic regimens with or without corticosteroids.

Patients and methods

Patients

We retrospectively studied 30 patients with hematologic malignancies who underwent allogeneic BMT between June 1993 and June 1996. The first 15 patients received CsA-MTX-PDN from June 1993 to September 1994, and the other 15 patients received CsA-MTX with no PDN as part of GVHD prophylaxis from February 1995 to June 1996. Patient characteristics are shown in Table 1. Both groups were comparable for age, underlying disease, pre-transplant status, type of allogeneic transplant, conditioning regimen and T cell depletion. There were 26 males and four females, median age 32 years (range 16-51 years). Twenty-two cases received bone marrow from genotypically HLA-identical siblings, one from a family donor with one HLA antigen mismatch, six from unrelated matched donors, and one from an unrelated one HLA antigen mismatched donor.

BMT

Nineteen patients received CY-TBI as conditioning, consisting of cyclophosphamide 60 mg/kg once daily i.v. on days -6 and -5 (total dose 120 mg/kg) and total body irradiation 13 Gy on days -4 to 0. Eight patients received CY-TBI and antithymocyte globulin (ATG) at a dose of 30 mg/kg i.v..daily for 3 days (total dose 90 mg/kg). One patient in the CsA-MTX group was conditioned with melphalan 140 mg/m² i.v. for 1 day and TBI. One patient in each group received BU-CY consisting of busulphan 4 mg/kg p.o. in four divided doses daily for 4 days (total dose 16 mg/kg) and cyclophosphamide 60 mg/kg i.v. daily for 2 days (total dose 120 mg/kg).

Nine patients in each group received a T cell-depleted inoculum (total depletion of CD4⁺ cells and partial depletion of CD8⁺ cells).

The day of marrow infusion was designated as day 0. All patients received similar supportive care including bacterial and fungal antibiotic prophylaxis and treatment, intravenous immunoglobulin, blood component replacement and care in isolated HEPA filtered positive pressure rooms.

GVHD prophylaxis, grading and treatment

All patients received GVHD prophylaxis after transplantation, with the same dose of CsA and MTX: CsA 5 mg/kg once daily i.v. beginning on day -1 and then tapered on day +3 to 3 mg/kg/once daily and MTX 15 mg/m² i.v. on day +1, and 10 mg/m² i.v. on days +3, +6, +11. In the CsA-MTX-PDN group, PDN was administered at a dose of 2 mg/kg daily i.v. (in the first nine patients) or at a dose of 1 mg/kg daily i.v. (the following six patients) from day +1. The reason for changing dose of prednisone was the increased risk of infections reported by Sayer et al.²⁴ Serum CsA concentrations were measured three times a week with the CsA dose being adjusted to keep whole blood levels between 250 and 450 ng/ml. When patients were able to tolerate oral medication, i.v. CsA and PDN were switched to the oral forms with oral CsA three times the i.v. dose and oral PDN the same dose as i.v. CsA prophylaxis was stopped on day +100. Patients were evaluated for acute GVHD when they had neutrophil engraftment and were alive beyond day +30. Acute GVHD was diagnosed by clinical evaluation and graded according to previously published data.²⁶ Chronic GVHD was evaluated when the patient was alive beyond day +100.

Acute GVHD was treated by increasing the dose of prednisone or by instituting methylprednisolone (MP) at a dose of 2 mg/kg daily i.v. for 2 weeks. Patients who did not respond were treated with MP at a dose of 20 mg/kg daily i.v. for 3 days, tapered to 10 mg/kg daily i.v. for 4 days and then gradually stopped. Patients who did not respond to MP as primary therapy were generally treated with azathioprine.

The total dose of corticosteroids until day 100 post transplantation was clearly higher in the CsA-MTX-PDN group. The median dose of prednisone received in the CsA-MTX-PDN group until day +100 was 9390 mg (2045-14 610) and 0 mg (0-8570) in the CsA-MTX group, P < 0.001. There were no differences in the median dose of methylpredisolone received until day +100 between groups: 0 mg (0-1880) vs 0 mg (0-8160) P = 0.7894.

Engraftment and chimerism

Engraftment was defined as an absolute neutrophil count >500 ´ 10⁹/l for more than 3 consecutive days.

Chimerism was assessed by karyotype differences of sex between donor and host and in other cases by the variable nucleotide tandem repeats method (VNTR_s). Full chimerism was defined as 100% analyzed cells of donor origin.

Complications

We analyzed complications until day +100. Arterial hypertension was defined as an arterial tension value more than 150/90 mm Hg in three or more determinations requiring medical treatment with anti-hypertensive drugs. Insulin treatment was initiated when glycemia values exceeded 200 mg/dl.²⁷ Infection data were collected retrospectively by reviewing patient microbiologic data. Bacteremia was defined as one or more blood cultures with any bacterial organism regardless of associated symptoms. Septicemia was defined as bacteremia in conjunction with either hypotension or disseminated intravascular coagulation occurring within 24 h of positive blood cultures, or documented local infection caused by the same organisms recovered from the blood. Upper digestive bleeding was a clinical diagnosis.

Later complications such as avascular necrosis of bone, myopathy, pathological fractures or dementia were diagnosed by standard clinical and radiological criteria.

Procedure-related mortality was defined as a cause of death other than relapse.

Statistical analysis

Both groups were statistically compared by the Pearson chi-squared test (Fisher's exact test in some cases) or the Mann-Whitney test depending on whether the variable was categorical or continuous, respectively. Standard survival analysis methods (Kaplan-Meier curves, log-rank statistics) were used to analyze survival.²⁸ A level of <0.05 was accepted as statistically significant. Multivariate analysis was performed by means of the Cox proportional hazards method.²⁹

Results

Engraftment and chimerism

All but two patients in the CsA-MTX group of patients were evaluable for engraftment. The first patient died on day +15 due to interstitial pneumonitis, when hematologic recovery commenced. The second patient died on day +8 of sepsis caused by Streptococcus mitis. All patients (100%) in the CsA-MTX-PDN and 12 of 13 (92%) evaluable patients in the CsA+MTX group engrafted (P = 0.189). The only patient who did not engraft was a 28-year-old man with CML in first chronic phase who received related, T cell-depleted bone marrow with one HLA-locus mismatch. A second peripheral stem cell transplant without T cell depletion from the same donor was performed. Engraftment was observed but the chimerism analysis showed autologous reconstitution.

Chimerism was evaluated in 11 patients in the CsA-MTX-PDN group and 100% had full chimerism. Of the 10 patients evaluated in the CsA-MTX group, four (40%) had autologous reconstitution, five (50%) had full chimerism and one patient (10%) had mixed chimerism (P = 0.061).

Incidence of acute and chronic GVHD

Thirteen patients in the CsA-MTX group and all 15 patients in the CsA-MTX-PDN group were evaluable for development of acute GVHD. There was no difference in the incidence of acute or chronic GVHD between the two groups

No GVHD was observed in four of 15 patients (26%) vs six of 13 patients (46%). Grade I-II developed in seven (46%) vs four (30%), and grade III-IV in four (26%) vs three (23%) in the CsA-MTX-PDN and CsA-MTX groups, respectively (P = 0.339).

Three patients died before day 100 in each group and were therefore not evaluable for chronic GVHD. Three out of 12 patients in the CsA-MTX-PDN group (25%) and five out of 12 patients in the CsA-MTX group (41%) developed chronic GVHD (P = 0.687).

Incidence of transplant-related complications until day +100 post transplantation

Data on complications are shown in Table 2. All patients (100%) in the CsA-MTX-PDN group developed arterial hypertension compared with 11 patients in the CsA-MTX group (73%) and this difference was statistically significant (P = 0.031).

There was more requirement for insulin in the first 100 days post transplantation in the CsA-MTX-PDN group (73%) than in the CsA-MTX group (20%) (P = 0.003).

No differences were observed in the incidence of upper digestive tract bleeding between the two groups of patients.

Although there were no differences in the number of patients requiring antibiotic treatment for documented infection until day +100 (10 vs 12, P = 0.409), there was a trend toward more bacterial infectious episodes in the group receiving prednisone as part of their GVHD prophylaxis. Thirty-three of 35 infectious episodes (94%) in the CsA-MTX-PDN group and 17 of 21 (80%) in the CsA-MTX group were bacterial (P = 0.118). There was a trend toward more bacteremia episodes in the CsA-MTX-PDN group: 23 vs 13 episodes in each group. Gram-positive bacterial pathogens were the most common (data not shown).

Late complications

Late complications due to treatment with corticosteroids appeared in five patients. In the CsA-MTX-PDN group one patient developed corticosteroid myopathy on day +69 when he had received a total of 8715 mg of prednisolone; the other patient in this group developed vertebral fractures on day +98 after receiving 7350 mg of prednisone. Three patients in the CsA-MTX group developed late complications. One patient developed avascular necrosis of the bone on day +274 with a total dose of 11 325 mg of prednisone, another developed polytopic avascular necrosis of bone on day +389 with a total dose of 11 785 mg of prednisone and the third patient developed corticosteroid dementia on day +81 when he had received 6760 mg of prednisone for GVHD treatment.

Causes of death

At the time of the study six (40%) patients in the CsA-MTX group and two (13%) in the CsA-MTX-PDN group were alive.

Procedure-related mortality was a more frequent cause of death in the group receiving prednisone (92% vs 55%, P = 0.013), whereas relapse was more frequent in the group not receiving prednisone as part of GVHD prophylaxis (7% vs 44%, P = 0.013).

Five patients (38%) in the CsA-MTX-PDN group and only one patient (11%) in the other group died of acute GVHD, P = 0.158.

Septicemia as a cause of death was similar between the two groups but patients who died of septicemia in the context of severe GVHD were slightly more frequent in the CsA-MTX-PDN group (53% vs 22%, P = 0.138).

Survival

The median follow-up for the censored patients was 4.4 years (range 3.7-6.4 years). At the time of analysis, two patients in the CsA-MTX-PDN group and six in the CsA-MTX group were alive. The actuarial survival of the CsA-MTX-PDN group was 13.3% at 6.5 years, median survival 4.2 months, (95% CI, 2.6-5.7). Survival of the CsA-MTX group was 46.7% at 5 years, median survival 18.7 months (95% CI, 15.9-20.4), (P = 0.026). Figure 1 shows the actuarial survival of both groups.

Multivariate analysis

As the incidence of acute GVHD and survival could be influenced by other factors such as T cell depletion and the use of ATG, we performed a multivariate analysis (Table 3). The use of prednisone as part of GVHD prophylaxis was the only factor associated with a poorer survival and an increased incidence of acute GVHD. No influence was observed with the use of T cell depletion or ATG. There was a three-fold increase in risk of death (HR: 3.40, IC95% 1.20, 9.52) and a two-fold increase in risk of acute GVHD (HR: 2.27, IC95% 0.42, 12.48) in the CsA-MTX-PDN (Table 3).

Discussion

We performed this study to compare the incidence of acute and chronic GVHD using two different prophylactic schemes and to compare the incidence of post-transplant complications when corticosteroids are part of prophylactic regimens.

Our results suggest that prednisone when added to a standard CsA and MTX regimen did not aid in the prevention of either acute or chronic GVHD or improve long-term survival, and increased the morbidity of allogeneic transplantation. Although patient numbers in each of the treatment groups are limited and clearly inadequate to eliminate beta-error, our overall results did not warrant more extensive comparisons.

We observed the same incidence of acute GVHD reported by other groups,^{5,10,11,12,13} with no improvement in the incidence of acute GVHD when prednisone is added to the CsA and MTX standard prophylactic regimen.

Our results for chronic GVHD differ from previous studies,^21,22 we did not observe an increased incidence of chronic GVHD when corticosteroids were part of GVHD prevention.

We observed a higher incidence of complications until day 100 post transplantation. Arterial hypertension (AHT) was the major complication and affected all patients receiving PDN. This fact was not observed by Gondo et al,¹⁸ who saw the same incidence of AHT when comparing CsA-MPD and CsA-MTX. New approaches such as the use of nifedipine along with continuous infusion CsA, short-course MTX and steroids reduced toxicity in MUD transplants.³⁰

Hyperglycemia was also an important early complication in the corticosteroid prophylaxis arm. Patients needed insulin by continuous i.v. infusion with strict control of sugar levels.

Bacterial infections were slightly more frequent in the CsA-MTX-PDN group and contributed to a higher procedure-related mortality rate in patients who developed acute GVHD.

Musculoskeletal and neurologic complications due to prophylactic use and treatment with corticosteroids appeared in both groups with total accumulated doses of prednisone superior to 6500 mg. These complications appeared earlier (before day +100) in the CsA-MTX-PDN group. This could be explained by the high accumulated doses of corticosteroids, due to their early introduction, and the higher intensity of GVHD treatment in those patients. We found that patients receiving prophylaxis with steroids were resistant to corticosteroid-based GVHD treatment. This fact is consistent with the previously reported experience in the difficulty of controlling GVHD when corticosteroids are part of GVHD prophylaxis.²³ The hypothesis for this observation could be the selection of resistant T lymphocyte clones.

The clearly inferior survival in the CsA-MTX-PDN group can be explained by the higher procedure-related mortality in this group. Acute GVHD and septicemia were the maior causes of death. The explanation could be corticosteroid resistance to acute GVHD treatment in patients receiving prednisone from day +1 of BMT and the higher susceptibility to septicemia of these patients due to both GVHD and immunosuppression due to corticosteroids.

In conclusion, our results suggest that steroids increase morbidity when used as part of GVHD prophylaxis and should not be used until GVHD appears. Other strategies to prevent GVHD can be used without impairment of the graft-versus-leukemia effect.

T cell depletion is an effective method of preventing severe GVHD after allogeneic BMT, but is associated with an increased risk of graft failure,³¹ mixed chimerism,^32,33 and leukemic relapse.^34,35,36,37 Total depletion of CD4⁺ and partial depletion of CD8⁺ cells with infusion of a fixed number of CD8⁺ cells ensures adequate engraftment, and decreases GVHD rate.³⁸ Recent experiences with negative purging with CD2 and CD7 MoABs and re-infusion of a fixed quantity of CD2⁺ peripheral blood T cells, associated with an intensified conditioning regimen, decreased severe acute GVHD and graft failure but was associated with a high risk of relapse and procedure-related mortality.³⁹

Approaches to reduce relapses due to T cell depletion have included the use of MoAB against IL-2 receptor combined with standard CsA plus MTX prophylaxis but no advantages were found, confirming the importance of T cells in transplantation for leukemia.⁴⁰

Tacrolimus (FK506) seems to be more effective than CsA in preventing grade II to IV acute GVHD,^41,42 with the same relapse rates, but with worse disease-free survival and overall survival. The principal adverse effects of FK506 are nephrotoxicity, arterial hypertension, and hyperglycemia.^43,44

Recent studies have revealed that the use of low doses of CsA (1 mg/kg daily) is associated with a statistically significant reduction of relapse but at the price of increasing the incidence of acute GVHD.^45,46,47

Further studies must be directed toward strategies for tolerance or anergy of donor T cells against host cells but without impairment of the graft-versus-leukemia effect and without important side-effects which could affect long-term survival.

Acknowledgements

We thank all the nurses and residents in the Department of Clinical Hematology, Hospital Duran i Reynals. This study has been supported by a grant: FIJC/00.

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Figure 1 Probability of overall survival according to GVHD prophylactic group. Tick marks represent patients who are currently alive.

Table 1 Patient characteristics (n = 30)

Table 2 Complications until day +100

Table 3 Multivariate analysis using Cox proportional hazard models