Sirolimus in combination with cyclosporine or tacrolimus plus methotrexate for prevention of graft-versus-host disease following hematopoietic cell transplantation from unrelated donors

Abstract

In two consecutive prospective clinical trials, we evaluated the efficacy of sirolimus together with a calcineurin inhibitor (cyclosporine or tacrolimus) and low-dose methotrexate for prevention of graft-versus-host disease (GVHD) after unrelated hematopoietic cell transplantation (HCT). Nine patients received sirolimus with cyclosporine, and 17 received sirolimus with tacrolimus. The incidence of Grade II–IV GVHD was 77%, with the median onset at day 7 after HCT. Because of toxicity, administration of sirolimus was discontinued earlier than planned in 11 patients, but after the onset of GVHD. Three patients developed renal failure requiring hemodialysis. Accrual in both studies was terminated due to lack of efficacy. In these studies, the addition of sirolimus to regimens containing a calcineurin inhibitor and methotrexate appeared to cause toxicity and provided no detectable improvement in preventing GVHD.

Introduction

Acute graft-versus-host disease (GVHD) is a major cause of morbidity and non-relapse mortality after allogeneic hematopoietic cell transplantation (HCT) [1]. Current pharmacologic approaches to prevent acute GVHD generally involve the use of a combination of immunosuppressive medications, such as cyclosporine (CSP) or tacrolimus (TAC) together with an antimetabolite, such as methotrexate (MTX) [2–6]. Despite the use of these regimens, 35–80% of recipients develop acute GVHD, often requiring systemic immunosuppressive treatment [7–10].

Sirolimus is a macrocyclic lactone fermentation product with antifungal and immunosuppressive activity. The immunosuppressive effect of sirolimus is mediated through a mechanism different from TAC or CSP. Sirolimus blocks IL-2-induced proliferation of T cells but does not affect signals that result in activation-induced apoptosis. The proliferation of T cells driven by other lymphokines, such as IL-4, IL-12, IL-7 and IL-15, is also inhibited by sirolimus [11]. Sirolimus inhibits lipopolysaccharide-induced proliferation of B cells resistant to TAC and CSP [11,12]. Although both TAC and sirolimus have the same intracellular receptor, it is hypothesized that the drug-immunophilin complexes interact differently with other molecules to form functionally distinct complexes and specific immunosuppressive effects peculiar to each drug. Calcineurin inhibitors used in combination with sirolimus cause an additive or synergistic suppression of immune cell function [13].

Sirolimus is effective for preventing rejection of renal and hepatic allografts [14–17] and has been evaluated for treatment of acute [18] and chronic GVHD [19,20]. Results of several recent studies have suggested that sirolimus is effective for preventing GVHD after allogeneic HCT [21–23]. In two phase II clinical trials, we attempted to confirm the efficacy and safety of sirolimus for preventing acute GVHD after unrelated HCT, first in combination with CSP and MTX and subsequently in combination with TAC and MTX.

Patients and Methods

The first study evaluated the immunosuppressive regimen of CSP, sirolimus and low-dose MTX (CSP Group). Enrollment began in December 2001 and was terminated in May, 2002. The second study evaluated the combination of TAC, sirolimus and low-dose MTX (TAC Group). Patients were enrolled between February 2003 and March 2005. Results for both studies were analyzed as of March 3, 2007. The trials were approved by the Institutional Review Board at Fred Hutchinson Cancer Research Center (FHCRC), and all patients signed consent documents.

Patients

The primary inclusion criteria for patient selection were 1) the use of a preparative conditioning regimen containing total body irradiation (TBI) followed by cyclophosphamide or busulfan followed by cyclophosphamide, 2) an estimated creatinine clearance ≥ 70 mL/min, 3) total serum bilirubin concentration within the normal range, and 4) hepatic transaminase levels less than twice the upper limit of normal. Patients with diagnoses of chronic myelogenous leukemia in chronic phase, de novo acute leukemia in first remission, and refractory anemia were excluded. HLA-mismatching at DRB1 or DQB1 was not allowed, although a single Class 1 allele disparity was allowed. Histocompatibility with the respective donors was determined by high-resolution HLA-A, B, C and DRB1 typing and by intermediate-resolution HLA-DQB1 typing.

The protocols restricted enrollment to patients with high-risk diagnoses, allowed a single Class 1 allele disparity and did not restrict the type of donor graft used, since it was felt that these criteria would not significantly affect the protocol endpoints. For similar reasons, the eligibility criteria allowed the use of either of our standard myeloablative preparative conditioning regimens.

Treatment Plan and Supportive Care

Table 1 describes the treatment plan for each group. Four pediatric patients in the TAC Group received 13.2 Gy TBI, and all other patients received 12.0 Gy TBI. The first study used CSP as the calcineurin inhibitor since CSP/MTX was our standard GVHD prophylaxis regimen. Since we initiate CSP therapy one day before the infusion of the donor graft, sirolimus was also started on the same day. Sirolimus blood levels were targeted at 4–14 ng/mL, which was slightly higher than the levels targeted by the investigators at the Dana Farber Cancer Institute (DFCI). Due to the high incidence of early-onset GVHD observed in the CSP Group, the second study substituted TAC for CSP, with the treatment plan and dosing schedule made identical to the regimen used at the DFCI. When early enrollment in this study showed a similar pattern to that seen in the CSP study, there was a concern that immunosuppression with sirolimus may have been inadequate. Therefore, the protocol was revised to increase the target level of sirolimus to 5–12 ng/mL. Sirolimus trough levels were measured 2–3 times weekly by chromatography-tandem mass spectrometry, and dose adjustments made to maintain levels within the targeted range. The tablet formulation of sirolimus was used throughout both studies. In the CSP Group, in the absence of toxicity, administration of sirolimus was discontinued on day 30. In the TAC Group, the dose of sirolimus was gradually tapered beginning on day 57, and administration was discontinued on day 180, unless the patient required continued treatment for GVHD or experienced toxicity related to sirolimus.

Table 1.

Treatment Plan

	Cyclosporine Group	Tacrolimus Group
Conditioning Regimen
Total Body Irradiation	12 Gy (6 fractions)	12–13.2 Gy (6–8 fractions)
Cyclophosphamide	120 mg/kg (2 divided doses)	120 mg/kg (2 divided doses)
or
Busulfan	16 doses (targeted 800–900 ng/mL)	16 doses (targeted 800–900 ng/mL)
Cyclophosphamide	120 mg/kg (2 divided doses)	120 mg/kg (2 divided doses)
GVHD Prophylaxis
Sirolimus*	12 mg PO loading dose day -1	12 mg PO loading dose day -3
	followed by 4 mg PO daily	followed by 4 mg PO daily
Calcineurin inhibitor	Cyclosporine - 1.5 mg/kg/Q12h IV start day -1	Tacrolimus - 0.02 mg/kg/day IV start day-3
Methotrexate	5mg/m2 IV days 1,3,6,11	5mg/m2 IV days 1,3,6,11
Targeted levels
Sirolimus	4–14 ng/mL	3–12 ng/mL or 5–12 ng/mL
Calcineurin inhibitor	Cyclosporine	Tacrolimus - 5–10 ng/mL

^*sirolimus dosing for patients < 1.5 m² was 6 mg/m² loading dose followed by 2 mg/m² daily

All patients received antifungal prophylaxis with fluconazole. Tacrolimus and CSP levels were drawn 1–3 times per week, and dose adjustments were made to maintain therapeutic levels. In the absence of GVHD, TAC and CSP doses were tapered starting days 50–57 after transplantation, and administration was discontinued on day 180. This tapering schedule is similar to our standard practice. If patients developed GVHD, tapering of the calcineurin inhibitor was delayed and left to the discretion of the attending physician. Typically, tapering of CSP or TAC doses was not begun until patients had discontinued primary treatment for acute or chronic GVHD.

Assessment of GVHD

Acute GVHD was graded according to previously described criteria [24]. Biopsy samples were obtained, when appropriate, to corroborate the clinical diagnosis of GVHD. Follow-up was censored for evaluation of acute GVHD at the time of hematologic relapse or death. Chronic GVHD was diagnosed according to criteria closely similar to those recommended by the NIH Consensus Conference [25].

Toxicities

Assessment of safety focused on identifying toxicities attributable to sirolimus. These included cytopenia, hyperlipidemia, hypercholesterolemia, hemolytic uremic syndrome (HUS) and transaminase elevation. The onset of engraftment was defined as the first of three consecutive days after HCT when the absolute neutrophil count surpassed 0.5 × 10⁹/L.

Results

Patient and Transplant Characteristics

All patients had high-risk hematologic malignancies. Twenty-four patients had HLA-matched donors, and 22 received G-CSF-mobilized peripheral blood stem cells. Nine patients were given sirolimus with CSP, and 17 were given sirolimus with TAC (Table 2).

Table 2.

Patient and transplant characteristics according to treatment group^*

	CSP Group	TAC Group
	(n = 9)	(n = 17)
Patient age, median years (range)	39 (20 – 65)	33 (5 – 52)
Disease type and stage, n
AML > CR1 or persistent disease	0	3
ALL > CR1 or persistent disease	1	7
CML – beyond chronic phase	2	3
AML from MDS	1	1
RAEB†	3	3
NHL	2	0
HLA typing, n
HLA-matched	8	16
HLA-mismatched	1	1
Stem cell source, n
G-CSF-mobilized blood cells	7	15
Marrow	2	2
Conditioning regimen, n
TBI/cyclophosphamide	5	12
Busulfan/cyclophosphamide	4	5
Donor age, median years (range)	37 (18 – 48)	33 (27 – 58)
Gender match (donor/recipient)
Male/Male	3	8
Male/Female	1	4
Female/Male	1	5
Female/Female	4	0

^*Abbreviations: AML, acute myeloid leukemia; CR1, first complete remission; ALL, acute lymphoblastic leukemia; CML, chronic myeloid leukemia; MDS, myelodysplastic syndrome; RAEB, refractory anemia with excess blasts; NHL, non-Hodgkin lymphoma.

^†One patient had RA after treatment of RAEB2 with 5-azacytidine.

Graft-versus-host disease

Grade II–IV acute GVHD was observed in 20 (77%) of the 26 patients, 7 of 9 in the CSP Group and 13 of 17 in the TAC Group (Table 3). Grade II GVHD was observed in 14 patients (54%), Grade III in 6 (23%) and Grade IV in none. GVHD was diagnosed in all 9 patients who received busulfan and cyclophosphamide. The median onset of GVHD occurred at day 6 in the CSP Group and day 7 in the TAC Group. Sixteen of the 20 patients with GVHD had histological confirmation of the diagnosis, including 13 with gastrointestinal involvement. Ten patients had positive skin or intestinal biopsies at the time of onset of GVHD or within one week after the onset. All patients diagnosed with GVHD required glucocorticoid therapy, and 4 required secondary therapy. Due to the need for treatment of acute GVHD, only four patients began tapering CSP or TAC doses as planned at week 8 after HCT. Chronic GVHD requiring systemic immunosuppressive treatment developed in 6 of 7 patients in the CSP group and in 13 of 14 patients in the TAC group who could be evaluated. Two patients in the CSP group and 3 patients in the TAC group could not be evaluated because of early death (n = 4) or recurrent malignancy (n = 1).

Table 3.

Acute and chronic GVHD

	CSP Group	TAC Group
Characteristic	(n = 9)	(n = 17)
Onset of acute GVHD, median day after HCT (range)	6 (5 – 23)	7 (3 – 100)
Acute GVHD peak grade, n
0 – I	2	4
II	4	10
III – IV	3	3
Chronic GVHD requiring systemic treatment, n	6	13
None	1	1
Not evaluated	2	3

Sirolimus, CSP and Tacrolimus Levels

Twenty-three patients had sirolimus blood levels drawn on the day the donor graft infusion was finished or within 24 hours. Eighteen patients had sirolimus levels within or above the range targeted for each group, including 7 of 8 patients in the CSP Group, in whom sirolimus therapy was started on the day before transplantation. Mean sirolimus blood levels through day 30 after transplantation (or through the time of discontinued administration, if earlier) were 11.7 ng/mL in the CSP Group and 8.8 ng/mL in the TAC Group, with only 2% and 11% of all levels below the targeted range in each group, respectively. At the onset of GVHD, 5 patients (26%) had sirolimus levels below the target range. The mean CSP and TAC levels for the same 30 day period were 324 ng/mL and 9.6 ng/mL, respectively. Thirteen percent of all CSP levels were below the targeted range, and no patients had levels below the targeted range at the onset of GVHD. Twelve percent of all TAC levels were below the targeted range, and two patients had levels below the targeted range at the onset of GVHD (Table 4).

Table 4.

Sirolimus and calcineurin inhibitor levels and toxicity

	CSP Group	TAC Group
	(n = 9)	(n = 17)
Sirolimus level (ng/mL), mean*	11.7	8.8
Sirolimus levels,*n(%)
> Target range	26 (24)	35 (18)
Within target range	80 (74)	133 (71)
< Target range	2 (2)	20 (11)
Sirolimus level at onset of GVHD, n (%)+
Patients with level > target range	3 (43)	0 (0)
Patients with level within target range	3 (43)	8 (67)
Patients with level < target range	1 (14)	4 (33)
Cyclosporine level (ng/mL), mean*	324	-
Cyclosporine levels,*n (%)
> Target range	22 (22)	-
Within target range	65 (65)	-
< Target range	13 (13)	-
Cyclosporine level at onset of GVHD, n (%)
Patients with level > target range	1 (14)	-
Patients with level within target range	6 (86)	-
Patients with level < target range	0 (0)	-
Tacrolimus level (ng/mL), mean*	-	9.6
Tacrolimus levels,*n (%)
> Target range	-	81 (32)
Within target range	-	140 (56)
< Target range	-	30 (12)
Tacrolimus level at onset of GVHD, n (%)
Patients with level > target range	-	2 (15)
Patients with level within target range	-	9 (70)
Patients with level < target range	-	2 (15)
Reasons for discontinuation of sirolimus, n
Cytopenia	2	3
Renal failure	1	3
Abnormal liver function tests	0	1
Elevated sirolimus level	1	0
Hyperlipidemia	0	1

^*Sirolimus, TAC and CSP levels include all measurements through day 30 after HCT or until administration was discontinued, if earlier. The mean levels are for the same time period.

⁺One patient in the TAC Group stopped sirolimus administration before the onset of acute GVHD

Toxicity and Early Discontinuation of Sirolimus

Eleven of 26 patients (42%) discontinued administration of sirolimus early because of adverse events, including cytopenias (5), hemolytic-uremic syndrome (HUS) or renal failure (4), abnormal liver function tests (1), hyperlipidemia (1). The administration of sirolimus was discontinued in one patient due to an elevated blood level (Table 4). Nine patients had sirolimus levels drawn at the time of the adverse event, and 3 had levels above the targeted range. In two patients, who discontinued sirolimus therapy on days 277 and 351, the levels of sirolimus were unknown. Four of five patients with cytopenias had resolution of neutropenia or thrombocytopenia after the administration of sirolimus was discontinued. One patient with neutropenia had recurrent malignancy 21 days after the administration of sirolimus was discontinued. Another patient with thrombocytopenia also had evidence of mild HUS, and this likely contributed to the thrombocytopenia. One patient had elevated alanine and asparate transaminase values in the blood in the absence of GVHD and with normal blood levels of TAC and sirolimus. After discontinuing the administration of sirolimus, the transaminase values returned to the normal range. One patient in the CSP Group developed rhabdomyolysis with uncertain attribution to sirolimus.

Three patients in the TAC Group required hemodialysis. One of the patients who developed HUS had previously experienced renal injury attributed to the cyclophosphamide conditioning regimen. Among 5 patients who developed HUS or renal failure, 3 had blood levels of sirolimus or the calcineurin inhibitor above the specified target range at the onset of renal insufficiency.

Five patients in the CSP Group discontinued administration of sirolimus as planned at day 30 after HCT, and the remaining 4 patients discontinued administration of sirolimus on days 13, 24, 26 and 27 because of presumed toxicity. In the TAC Group, administration of sirolimus was discontinued at a median of 86 days (range 9 – 474 days) after HCT. With one exception, all patients developed GVHD before sirolimus administration was stopped. Among the 25 patients who could be evaluated, engraftment of neutrophils occurred at a median of 16 days (range 10 – 28) after HCT.

Nine patients received targeted oral busulfan therapy as part of their preparative conditioning regimen, with levels targeted, in most cases, at 800–900 ng/mL. Seven patients had mean busulfan levels within the targeted range. Two patients had mean levels of 923 ng/mL and 952 ng/mL.

Outcome

Nineteen of 21 patients (90%) who could be evaluated developed chronic GVHD. Twenty-one (81%) and 16 patients (62%) were alive at 6 months and one year after HCT, respectively (Figure 1). Twelve (46%) remain alive at a median of 1106 days after HCT. Three patients had recurrent malignancy and died. Eleven patients died from other causes: respiratory failure (not infection) (4), cardiac arrest (2), hepatic failure (2), GVHD (1), infection (1) and multi-organ failure (1).

Figure 1.

Relapse and overall and event-free survival for all patients.

Discussion

In these two studies, the addition of sirolimus to a standard calcineurin inhibitor plus MTX regimen provided no detectable improvement in preventing GVHD after unrelated HCT. Accrual in the CSP Group was terminated when 7 of 9 patients developed acute GVHD at a median of 6 days after HCT. When 13 of 17 patients in the TAC Group developed acute GVHD at a median of 7 days after HCT, the protocol was closed to further enrollment.

The immunosuppressive regimen of sirolimus, TAC and MTX for patients in the TAC Group of our study was identical to that used at the Dana Farber Cancer Institute (DFCI), where the incidence of acute GVHD after unrelated HCT was 26% [22]. It is difficult to explain why our results differ so greatly from the DFCI experience. Review of patient selection criteria and treatment methods has identified several differences between the two studies, but none of these differences can be easily invoked as likely explanations for the disparate results. The DFCI study enrolled only adults, whereas we enrolled adults and children. The DFCI patients received a conditioning regimen of cyclophosphamide followed by fractionated TBI, whereas patients who received a TBI-based regimen at our center were given TBI followed by cyclophosphamide. It is unlikely that this scheduling of therapy would explain the high incidence of acute GVHD seen in this study, since all patients who received busulfan and cyclophosphamide also developed acute GVHD. The dose of cyclophosphamide was slightly lower for DFCI patients than for FHCRC patients, but the TBI exposure in the DFCI study (14 Gy) was slightly higher than in our study. The DFCI patients were given clotrimazole and nonabsorbable antibiotics until engraftment, whereas FHCRC patients were given fluconazole and levofloxacin (adults) or ceftazidime (children) to prevent infection during neutropenia. There were no obvious differences in sirolimus blood levels between the two studies and the ranges used in our studies for targeting sirolimus levels were identical to or higher than those used in the DFCI study. Nearly all patients in our studies had levels of CSP or TAC within or above the targeted range at the onset of GVHD. Of note, the dose of MTX used in these studies was lower than the standard dose used at our center for GVHD prophylaxis after HCT with myeloablative conditioning regimens. Although early blood levels of CSP or TAC and sirolimus were within the targeted range in most patients, it is possible that the reduced dosing of MTX contributed to inadequate immunosuppression early after transplantation.

Our schedule for discontinuing administration of sirolimus, particularly in the CSP Group, was different from that used in the DFCI study. All patients in the CSP Group had sirolimus therapy discontinued on or before day 30 after transplantation. In the TAC Group administration of sirolimus was discontinued at a median of 86 days after transplantation. The impact of this schedule on the incidence of acute GVHD is not significant, as all patients, except one, in both studies developed GVHD before the discontinuation of sirolimus therapy.

It is possible that the grading of GVHD differs between centers, and the threshold severity of GVHD required to prompt treatment may also differ. The incidence of Grades II – IV acute GVHD after unrelated HCT with myeloablative conditioning at our center has been approximately 80%, substantially higher than the rates reported from other centers [10]. We have published evidence suggesting that sensitivity for making the diagnosis of gastrointestinal GVHD might be higher at our center than at other centers [10]. The consistent early presentation of GVHD in the present studies was highly atypical in our experience. The early onset of symptoms might suggest the development of an engraftment syndrome. If so, treatment with a short course of high-dose glucocorticoids should have been adequate. This approach was not tested, and the patients were treated with glucocorticoids per our standard practice for management of acute GVHD. Of note, a high incidence of engraftment syndrome was not mentioned in the report from the DFCI.

Risk factors for the development of hyperacute GVHD following HCT have been identified by Saliba et al [26]. These factors include a donor/recipient (female/male) gender mismatch, the use of HLA-matched unrelated donor grafts, the use of a myeloablative preparative conditioning regimen and treatment with more that five prior chemotherapy regimens prior to transplantation. All of the patients in these studies received unrelated donor grafts following a myeloablative preparative conditioning regimen. All had high-risk hematologic malignancies likely requiring multiple courses of chemotherapy prior to transplantation. Six male patients received grafts from female donors. Although the demographic characteristics of our study cohort included many of these risk factors, this group is similar to the population of patients who receive unrelated HCT at our institution, yet the onset of GVHD among the patients receiving sirolimus was distinctly earlier than what we normally expect. In a multicenter trial comparing CSP/MTX versus TAC/MTX for GVHD prophylaxis following unrelated donor transplantation [3], the median time of onset of GVHD was much later than that observed in the current studies.

In the setting of myeloablative HCT for patients with high-risk malignancies, it is difficult to attribute the occurrence and severity of any specific adverse event to sirolimus alone. On the other hand, some of the adverse events that we observed in the current study have been previously reported in GVHD treatment and renal transplant studies with sirolimus [15, 18–20]. For example, neutropenia has been reported to occur in 5–19% of patients and thrombocytopenia in 9–57% of patients. In the current studies, 4 of the 5 patients who experienced neutropenia or thrombocytopenia had resolution of the cytopenia following discontinuation of sirolimus therapy. In a study evaluating the use of sirolimus for treatment of chronic GVHD, 7 of 19 patients discontinued sirolimus therapy because of adverse events [20]. Six of these 7 patients developed HUS or acute renal insufficiency, and the study was closed to accrual earlier than originally planned due to a high incidence of toxicity. Even with close monitoring of blood levels, combination regimens of sirolimus and calcineurin inhibitors may have a very narrow therapeutic index.

Eleven of the 14 deaths in our study were related to causes other than relapse, with 4 deaths within the first 100 days after transplantation. With the exception of 4 patients who died with non-infectious pulmonary complications, the other causes of death varied, with no single dominant cause to explain the high rate of non-relapse deaths. The high rate of non-relapse mortality observed in this study may be related to effects of intensive chemotherapy before referral for transplantation, limited ability to tolerate the conditioning and immunosuppressive regimens, as well as the early onset of acute GVHD.

In conclusion, we found no evidence for improved prevention of GVHD after adding sirolimus to the combination of methotrexate and a calcineurin inhibitor after unrelated HCT. Moreover, we found an unusually high incidence of complications that we believe were caused by administration of sirolimus. Our results sound a note of caution in future clinical trials testing the safety and efficacy of sirolimus for GVHD prophylaxis after unrelated HCT.