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0Biology of Blood and Marrow Transplantation 13:560-567 (2007) © 2007 American Society for Blood and Marrow Transplantation l083-879l/07/l305-000l$32.00/0 doi:l0.l0l6/j.bbmt.2006.l2.449
AS BMI
American Society for Blood arid Marrow Transplantation
A Prospective Multicenter Trial of Peripheral Blood Stem Cell Sibling Allografts for Acute Myeloid Leukemia in First Complete Remission Using Fludarabine-Cyclophosphamide Reduced Intensity Conditioning
A. P. Grigg,1 J. Gibson,2 P. G. Bardy,3 J. Reynolds,4 P. Shuttleworth,1 R. L. Koelmeyer,4 J. Szer,1 A. W. Roberts,1 L. B. To,3 G. Kennedy,5 K. F. Bradstock6
department of Clinical Haematology and Bone Marrow Transplant Service, Royal Melbourne Hospital, Melbourne, Australia; 2Institute of Haematology, Blood and Marrow Transplantation, Royal Prince Alfred Hospital, Sydney, Australia; 3Royal Adelaide Hospital, Adelaide, Australia; 4Centre for Biostatistics and Clinical Trials, Peter MacCallum Cancer Centre, Melbourne, Australia; 5Royal Brisbane and Women's Hospital, Brisbane, Australia; 6Blood and Marrow Transplant Service, Westmead Hospital, Sydney, Australia
Correspondence and reprint requests: Andrew Paul Grigg, MD, Department of Clinical Haematology and Bone Marrow Transplant Service, The Royal Melbourne Hospital, Grattan Street, Parkville, Vic. 3050 Australia (e-mail: andrew.grigg@mh.org.au).
Received November 29, 2006; accepted December 22, 2006 ABSTRACT
The role of allogeneic transplantation in patients with de novo acute myeloid leukemia in first complete remission (AML-CR1) is controversial. Aiming to preserve a graft-versus-leukemia effect, but minimize morbidity and mortality from conditioning-related toxicity and graft-versus-host disease (GVHD), we conducted a prospective multicenter study of reduced-intensity conditioning (RIC) as preparation for peripheral blood stem cell sibling allografts in patients with intermediate or poor risk AML-CR1. Conditioning consisted of fludarabine 125 mg/m2 and cyclophosphamide 120 mg/kg. Thirty-four patients were transplanted with a median age of 45 years; 85% had intermediate risk cytogenetics. Early toxicity was minimal. The overall incidence of grade II-IV acute GVHD was low (21%), but the 3 patients (9%) who developed grade IV GVHD died. Donor T cell chimerism was rapid and generally complete, but complete myeloid chimerism was delayed. Thirteen patients (38%) relapsed, 12 within a year of transplant. The estimated disease-free survival (DFS) and overall survival at 2 years was 56% (95% confidence interval [CI] 39%-71%) and 68% (95% CI 50%-81%), respectively. The incidence of extensive chronic GVHD (cGVHD) was low (24% of surviving patients at 12 months) and most survivors had an excellent performance status. These observations justify a prospective comparison of RIC versus myeloablative conditioning allografts for AML-CR1. © 2007 American Society for Blood and Marrow Transplantation
KEY WORDS
AML • Remission • Reduced intensity conditioning
INTRODUCTION
The optimal treatment approach for patients in first complete remission (CR1) of acute myeloid leukemia (AML) remains controversial. For younger patients, options have included an autograft consolidation with high-dose cytosine arabinoside or an allograft from a human leukocyte antigen (HLA)-matched compatible sibling donor using myeloabla-
tive conditioning. Interpretation of the results of randomized studies addressing which of these approaches is superior is complex, as study designs vary and only a minority of patients in remission actually receive the allocated treatment [1]. In general, however, a my-eloablative allograft has produced the lowest risk of relapse but minimal improvement in overall survival (OS) resulting from treatment-related mortality (TRM)
from the conditioning regimen and graft versus host disease (GVHD) [2].
The therapeutic options for AML-CR1, particularly for older patients, expanded in the late 1990s after the demonstration that reduced-intensity conditioning (RIC) regimens using peripheral blood stem cells (PBSC) produced durable donor engraftment and permitted immune-mediated graft-versus-tumor effects, with a reduction in TRM because of reduced major organ damage during conditioning and lower rates of severe acute GVHD (aGVHD) [3,4]. The lower TRM is particularly relevant when dealing with a malignancy such as AML-CR1 with intermediate risk cytogenetics in which a reasonable cure rate can be expected without transplantation [5]. However, the justification for using RIC regimens, which are truly nonmyeloablative and have only a modest antileuke-mic effect in AML-CR1, depends on demonstration of an acceptably low relapse rate from a graft-versus-leukemia (GVL) effect with acceptable morbidity from chronic GVHD (cGVHD).
In 2001, the Australasian Leukaemia and Lymphoma Group (ALLG) commenced a prospective multicenter study of RIC HLA-compatible sibling PBSC allografts for adult patients with AML-CR1. Three issues were addressed in discussions on study design: (1) eligibility according to risk of relapse, (2) conditioning regimen, and (3) optimal GVHD prophylaxis. The study was largely restricted to patients with intermediate risk cytogenetics [6], based in part on observations from the United Kingdom Medical Research Council (UK MRC) that myeloablative allografting for AML-CR1 was only seen to provide a benefit in disease-free survival (DFS) and OS in this group of patients [7]. Patients with adverse cytogenetics (—5, —7, del[5q], or abnormalities of 3q) were included in this study at the investigator's discretion.
We wished to examine the effect of a highly im-munosuppressive conditioning regimen that was non-myeloablative and caused minimal mucositis on the risk of relapse and GVHD. We selected high-dose cyclophosphamide 120 mg/kg and standard dose flu-darabine 125 mg/m2 (flu-cyclo) as the conditioning regimen, as this regimen caused minimal mucositis and had been demonstrated to produce rapid donor T cell chimerism without a high incidence of severe GVHD in solid tumor patients when used in conjunction with cyclosporine as GVHD prophylaxis [8]. Moreover, high-dose cyclophosphamide was a component of effective myeloablative regimens such as cyclophosphamide- total body irradiation (TBI) and busulphan-cyclophosphamide. It was considered that fludarabine low-dose cyclophosphamide (2 g/m2)3 may not be sufficiently immunosuppressive to reliably facilitate rapid donor engraftment in this group; whereas another widely used RIC regimen at the time, fludarabine-melphalan (flu-mel; 120-140 mg/m2), was
not chosen as it is effectively myeloablative, reliably producing early, complete donor T and myeloid cell engraftment and gastrointestinal toxicity leading to a rate of aGVHD comparable to that of a conventional myeloablative regimen [9].
We elected to use standard GVHD prophylaxis with cyclosporine and short course methotrexate. In vivo or ex vivo T cell depletion was not used to avoid abrogation of any potential T cell-mediated GVL effect.
The primary aims of the study were to evaluate the kinetics of T cell and myeloid chimerism, to relate these to the incidence and severity of GVHD, and to assess the overall tolerability of the RIC regimen with respect to early mucositis, nonhematologic toxicity, and, performance status in long-term survivors. The secondary aims included an assessment of DFS (measured from the date of transplant to the earlier of the date of relapse or the date of death from any cause) and OS.
PATIENTS AND METHODS Eligibility
Patients were enrolled if they met the following eligibility criteria: (a) had a confirmed diagnosis of de novo AML (other than FAB group M3) now in first CR; (b) had at least 1 course of consolidation therapy after achieving CR (patients were not excluded if they required more than 1 induction cycle to achieve CR); (c) were aged between 18-65 years inclusive, and if aged between 55 and 65 years, had an ECOG performance status score <2; (d) had intermediate or adverse risk group cytogenetics and a fully compatible sibling donor; (e) had adequate organ function including creatinine clearance >30 mL/min; and (f) had given written informed consent to participate in the study prior to the commencement of conditioning.
The study was approved by the Human Research Ethics Committees at each of the participating institutions.
Study Protocol
Conditioning therapy consisted of cyclophospha-mide 60 mg/kg intraveneously (i.v.) on days —7 and — 6 and fludarabine 25 mg/m2 i.v. on days —5 to —1 inclusive. In patients with a body mass index (BMI) >27, doses were based on the body weight for the patient's height calculated to give a BMI of 27.
To mobilize PBSC, donors received filgrastim (Neupogen, AMGEN, Thousand Oaks, CA) 8 ^g/kg twice daily by subcutaneous injection with the first leukapheresis performed on the fourth day of filgras-tim. The intent was to collect a minimum of 3 X 106 CD34 cells/kg recipient weight and preferably at least 6 X 106 CD34 cells/kg.
Graft-versus-host-disease prophylaxis consisted of methotrexate 15 mg/m2 on day 1, 10 mg/m2 on days 3, 6, and 11, and cyclosporine 3 mg/kg IV from day — 1, switched to oral administration from days 10-20. Cyclosporine was tapered according to day 30 T cell chimerism results. If >50% donor, cyclosporine was tapered from day 100 to day 180 in the absence of significant GVHD. If <50% donor, cyclosporine was tapered over the next 30. aGVHD requiring therapy was treated with 2 mg/kg methylprednisolone i.v. in addition to full-dose cyclosporine. Antimicrobial prophylaxis was at the discretion of each investigator according to local institution policy.
Patients were eligible to receive donor leukocyte infusions (DLIs) if they had ceased cyclosporine at least 2 weeks prior, had experienced less than grade 2 GVHD and had (1) successive blood samples 4 weeks apart within the first 6 months posttransplant demonstrating progressive loss of donor T cell chimerism (or persistently <50% donor T cells), or (2) recurrent disease.
Laboratory Investigations
Chimerism studies on T cell and myeloid subsets of peripheral blood samples were scheduled in remission patients at days 30, 90, 180, 270, and 365 and performed as previously published [9]. In patients with <50% donor T cell chimerism at day 30 undergoing a rapid cyclosporine taper, chimerism was repeated at day 60. Serial fertility assessments posttransplant were performed in a subset of nonvasectomized males and premenopausal females.
Statistics
Estimates of cumulative incidences and their standard errors of achievement of "full" (>95%) donor chimerism (T cell or myeloid), were calculated. The association between donor chimerism and the incidence and severity of aGVHD was studied by comparing the maximum percentage of donor cells observed in the first 100 days following allograft with the worst grade of aGVHD recorded at the day 100 assessment. Maximum donor chimerism percentages for patients in the 3 aGVHD severity groups (0, I-II, and III-IV) were compared using the exact form of the Kruskal-Wallis Test and the exact form of the Wil-coxon 2-sample test for patients in the 2 aGVHD incidence groups (0 and I-IV). Correlations were calculated to assess the association between maximum donor chimerism percentages and transfused CD34 levels.
For incidences such as the rate of aGVHD, Blyth-Still-Casella 95% confidence intervals were calculated. The Kaplan-Meier product limit method was used to estimate OS, DFS, and also remission duration (the latter being censored by deaths without prior
relapse). Accrual closed in December 2004, and the status of surviving patients was assessed between May and August 2006. Event times were censored at the study close-out date (May 15, 2006) for patients who were still being followed up without having experienced the relevant events by the close-out date. Statistical analyses were carried out using SAS, S-Plus, and StatXact statistical software. Transplant toxicities were accessed using the Bearman criteria [10].
RESULTS
Thirty-eight patients were enrolled in the study. The protocol specified that the primary group of patients were those eligible patients who underwent a transplant. Three patients relapsed soon after registration and did not proceed to transplant; a fourth was subsequently rediagnosed as acute lymphoblastic leukemia (ALL) and removed from the study. The basic demographic details of the 34 evaluable patients who proceeded to transplant are summarized in Table 1.
Eighteen donors (53%) underwent 1 leukapheresis and 16 donors (47%) underwent a second. The median number of CD34 cells (1) collected per apheresis was 3.8 X 106/kg recipient body weight (range 1.214.6), and (2) infused was 5.3 X 106/kg (range 2.414.6).
Transplant Toxicity
Transplants were extremely well tolerated. The median times to neutrophil and platelet recovery (>0.5 and >20 X 109/L, respectively) posttransplant were 17 and 8.5 days, respectively. The platelet nadir was >20 X 109/L in 6 of the 34 study patients (18%; 95% confidence interval [CI] 7-33%) and 12 patients (35%; 95% CI 20-54%) did not require a platelet transfusion. Only 1 patient required parenteral narcotic analgesia for mucositis. Four patients required
Table 1. Demographic Details of the 34 Study Patients
Male:female 23:11
Median age in years (range) 45 (19-60)
FAB group
MI-2 20 (59%)
M4 7 (21%)
Other 7 (21%)
Cytogenetics
Intermediate risk 29 (85%)
Adverse risk 5 (15%)
CR after
First induction 29 (85%)
Second induction 5 (15%)
No. of consolidation cycles
One 11 (32%)
Two 23 (68%)
Median time (days) from start of induction
to transplant (range) 164 (77-497)
CR indicates complete remission.
parenteral nutrition (1 for 3 days only and 3, in the context of acute gastrointestinal GVHD, for 13 to 16 days). In the first 4 weeks grade II renal toxicity occurred in 3 patients, grade II stomatitis in 2 patients, and grade II and III hepatotoxicity in 2 patients. There was no cardiac, bladder, pulmonary, neurologic or gastrointestinal toxicity in 97% of the study patients.
Acute GVHD occurred as follows: grade 0: 24 patients (71%), grade I: 3 patients (9%); grade II: 4 patients (12%); grade III: 0 patients; and grade IV: 3 patients (9%). GVHD was fatal in all 3 patients with grade IV disease. The incidence of cGVHD was assessed at 12 months posttransplant in 21 patients; 9 were not evaluable because of death, 3 had early relapses (2 of whom underwent a second allograft), and 1 was nonassessable (failed to attend appointments). Seven patients (33%) experienced no cGVHD; 9 (43%) had limited and 5 (24%) had extensive cGVHD. No patient in ongoing remission has subsequently developed extensive cGVHD.
Chimerism
Data on the percentage of donor chimerism (my-eloid and T cell) were available at some stage for 28 of the 34 study patients and were predominantly based on peripheral blood samples. The median donor T cell chimerism in the 24 patients in remission evaluated in the vicinity of day 30 (days 20-35 inclusive) was >90%. These levels were subsequently sustained in the absence of relapse: median (range) values in the vicinity of day 90 (days 60-120 inclusive, n = 21) were 94 (36%-90)% and in the vicinity of day 180 (days 150-220, n = 13) were 100 (67%-100)%. No patient required an early cyclosporine taper on the basis of low levels of donor T cell chimerism.
Myeloid engraftment was generally less rapid with median (range) values in the vicinity of day 30 (n = 24) of 81 (10-100)%, in the vicinity of day 90 (n = 21) of 90 (90-100)%, and in the vicinity of day 180 (n = 12) of 100 (13-100)%. Patients relapsing prior to these time points were not included in this analysis.
Correlation coefficients indicated no close association between CD34 levels transfused and the achieved maximum percentage donor T cell (r = 0.33) or myeloid (r = 0.31) chimerism at day 100. There was no statistically significant relationship between the severity of aGVHD and maximal levels of donor chimerism in the first 100 days following transplant.
Relapse, Outcome, and Survival
At the study close-out date, 18 patients were alive in ongoing CR from the time of transplant. The
median follow-up by the reverse Kaplan-Meier method was 2.9 years (observed range, 1.6-5.3 years). Thirteen patients had relapsed, 6 before 4 months, 6 between 4 and 12 months, and 1 at 46 months. The relapse rates at 6 months, 1, and 2 years following transplant (with death without relapse regarded as censoring) were 21% (95% CI 10%-38%), 34% (95% CI 20%-52%), and 37% (95% CI 25%-55%), respectively. In exploratory analyses, no significant differences in the 1-year relapse-free rates were observed according to (1) the number of induction cycles required to achieve CR (1: 71%, n = 29 versus 2: 40%, n = 5; P = .19); (2) cytogenetic risk group (adverse 53%, n = 5 versus intermediate 68%, n = 24; P = .32); and (3) the number of cycles of consolidation (1: 73%, n = 11 versus 2: 63%, n = 23; P = .31).
Three of the 13 patients who relapsed remain alive in second CR. Seven patients were treated with further chemotherapy (myeloablative [n = 3] or nonmy-eloablative [n = 4]) and stem cell infusion, 2 of whom remain alive in CR2 (8 and 11 months after relapse) and 2 who died of GVHD in CR2. Another patient remains alive in remission after 14 months of singleagent dasatinib for Ph+ AML. Eight patients who relapsed subsequently died of progressive leukemia.
Thirteen patients have died, 8 of relapse, and 5 of GVHD in CR1 (n = 3) or in CR2 (n = 2). The estimated DFS rates at 1 and 2 years following transplant were 59% (95% CI 41%-74%) and 56% (95% CI 39%-71%), respectively, as presented in Figure 1. Of the patients transplanted on study, an estimated 74% (95% CI 56%-86%) survived for 1 year following transplant and 68% (95% CI 50%-81%) survived 2 years (Figure 2).
Fertility
Posttransplant semen analysis was performed in 4 nonvasectomized males aged 19-51 years at transplant. Sperm counts were 9 and 27 X 106/mL at 24 and 39 months, 6.4 X 106/mL at 19 months, 28.6 X 106/mL at 36 months, and 36 X 106/mL at 24 months, respectively (normal count >20 X 106/mL).
Serial posttransplant hormonal measurements were available for 2 premenopausal females. In 1 patient, aged 40 years at transplant, spontaneous menstruation occurred at 13 months posttransplant, 1 month after ceasing the oral contraceptive pill (OCP), with a hormonal profile consistent with a premenopausal state. Estradiol levels were 445, 1378, and 275 pmol/L at 24, 38, and 50 months. Her periods became irregular after 4 years, and stopped completely in the fifth year (age 45) with a postmenopausal hormonal profile. The second patient, aged 32 years at transplant, ceased the OCP at 8 months posttrans-plant, with a return of menses within a month. Testing confirmed a premenopausal hormonal profile and
Figure 1. DFS, with 95% CI, for all patients who underwent a transplant.
menses have been maintained for 3 years posttrans-plant.
Performance Status
The Karnofksy score was 100% in 16 of the 18 surviving patients who never relapsed and 80% and 90% in the remaining 2 patients.
DISCUSSION
This prospective, multicenter study using a uniform RIC protocol demonstrated that RIC was well tolerated, with minimal early morbidity, a low incidence of severe aGVHD, and extensive cGVHD and excellent performance status in the relapse-free survi-
vors. The OS and DFS rates are comparable with those reported with myeloablative conditioning [11] and broadly consistent with the published literature of RIC allografts for AML-CR1, reviewed by Lazarus and Rowe [12], together with a number of recent publications [13,14], in which in general the 2-3 year DFS was <50%.
A key issue in this field is whether the intensity of the conditioning preallograft has a substantial impact on the relapse rate of AML in remission and OS. Retrospective studies comparing levels of conditioning intensity, although difficult to interpret because of small numbers and imbalances in prognostic factors, have not demonstrated a consistent survival advantage with more intensive conditioning [12,15,16]. A diffi-
Figure 2. OS, with 95% CI, for all patients who underwent a transplant.
culty is that the contribution of the low intensity of the preparative regimen to relapse cannot be definitively assessed because of the confounding effect of GVHD-GVL interactions. In comparison with my-eloablative conditioning, RIC is generally associated with a lower incidence of both aGVHD and extensive cGVHD [17]. This is relevant as a recent report highlighted the importance of GVHD in reducing relapse after RIC for AML and myelodysplasia [18]. A European Group for Blood and Marrow Transplantation study comparing RIC (n = 315) with myeloablative allografts (n = 407) in AML patients over 50 years demonstrated a statistically significant, higher relapse rate in the RIC group, perhaps related in part to a lower incidence of grade II-IV aGVHD [19]. This GVHD-GVL interaction makes the intrinsic antileu-kemic potency of the conditioning regimen potentially difficult to delineate; it could be argued that the lower relapse rates after "more intensive" RIC regimens such as flu-mel or flu-busulfan, which generally result in early full donor T cell and myeloid chimer-ism and a substantial rate of aGVHD [9,20], predominantly relate to an early GVL effect rather than cy-toreduction of residual leukemia by the conditioning protocol.
The flu high-dose cyclophosphamide regimen used in this study is more lymphoablative than my-eloablative, and robust early donor T cell engraftment was observed in the majority of patients, with myeloid engraftment being slower and more gradual. There was no relationship between the risk of aGVHD and maximum levels of donor lymphoid chimerism, with a low overall incidence of moderate to severe aGVHD perhaps from minimal tissue damage and cytokine release [21]. Accordingly, it is difficult to clearly attribute the relatively high early relapse rate to suboptimal chemotherapy intensity or to inadequate GVL-GVHD effects. Inadequate induction and consolidation chemotherapy is unlikely to be relevant, as most study patients had received at least 1 course of high-dose cytarabine and idarubicin in induction or consolidation, and the relapse rate was not obviously higher in the 3rd of patients who received only 1 course of consolidation therapy.
The incidence of grade III-IV aGVHD in the study was low (9%), but fatal in the 3 affected patients. This suggests that although a nonmucositic regimen may have an impact on the overall incidence of moderate to severe GVHD [18], some recipients are still destined to develop refractory GVHD. T cell depletion has permitted the safe application of the more aggressive RIC allografts to older AML patients with unrelated donors [22,23]. Whether the relapse rate after T cell depletion in this context is increased to the extent that it offsets any benefit from a reduction in GVHD-related mortality is still open to question.
The "time-lag" bias in which patients have to survive long enough in remission to reach transplant, and hence are a selected group, is well recognized as a problem in nonrandomized transplant studies [2]. There was a degree of selection in our study as the median time from the start of induction therapy to transplant was reasonably long at 5.5 months. Of note, 3 patients initially registered for the trial relapsed prior to the anticipated time of transplant; their inclusion in an intention-to-treat analysis would have increased the estimated relapse rate at 1 year from 34% to 39%.
Options for relapse after an RIC allograft include 1 or a combination of: withdrawal of immunosuppression, infusions of donor leukocytes or PBSC, conventional dose chemotherapy, or a myeloablative allo-graft. One theoretical attraction of an RIC allograft is that the modest organ toxicity may allow for a myeloablative regimen to be used for subsequent relapse. This approach was infrequently used in our study, as only 3 of 13 patients who relapsed subsequently had a myeloablative allograft, 1 of whom is alive in CR2. The available literature thus far suggests that prolonged DFS after therapy for relapse in this context is unusual [24]. DLI alone does not seem effective [22], but late relapses may occasionally be effectively treated with nonmyeloablative cytoreductive therapy and further PBSC infusions [25]. CR2 was achieved in all 4 study patients treated in this way with a durable remission in 1 and death from GVHD in 2 patients.
The flu-cyclo conditioning regimen was extremely well tolerated. Mucositis and other significant nonhe-matologic toxicities were rare and transfusion requirements were minimal. Delayed-onset aGVHD reported to occur with other RIC regimens [26] was not observed, perhaps because of the continuation of cy-closporine to 6 months after transplant in most patients. The incidence of extensive cGVHD in evaluable patients in the first year was low and the majority of survivors who maintained remission have a normal performance status and excellent quality of life. The limited data we collected on posttransplant fertility suggest that spermatogenesis is not abolished, with normal sperm counts in all 3 men tested beyond 2 years. Estradiol levels were within the premenopausal range in the 2 eligible women tested posttransplant with restoration of menses, although in 1 of these menopause occurred at a relatively young age.
The results of our study are encouraging, but only a modest number of patients were enrolled. Moreover, as the median age was somewhat lower than most RIC studies in this context, only sibling donors were used, few patients had adverse cytogenetics, and there was a substantial time from induction therapy to transplant. Arguably, this represented a better risk group than those reported in other studies in this context.
Ultimately the benefits or otherwise of RIC versus myeloablative conditioning for AML in remission will only be proved in prospective randomized trials in which the comparison groups are balanced for relevant prognostic factors, including cytogenetic risk groups, mutations within and overexpression of various genes [27], and duration of CR1. The results of the current Cooperative German Transplant Study Group trial in this context comparing flu-TBI 8Gy with conventional cyclophosphamide-TBI 12Gy will be of great interest.
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