Therefore, we combined these two promising elements, intravenous (IV) Bu/Flu MAC and PTCy, in a multi-institutional study of patients receiving HLA-matched-related or -unrelated bone marrow allografting. We hypothesized that this transplantation platform would provide favorable patient outcomes by preventing grades 3 to 4 aGVHD and cGVHD, lowering regimen-related toxicity, and effectively controlling disease.

Allogeneic blood or marrow transplantation (alloBMT) involves a multifaceted treatment platform, and each component (conditioning regimen, allograft source, and graft-versus-host disease [GVHD] prophylaxis) has significant implications for the overall success of the transplantation. 1 – 5 Busulfan (Bu)/fludarabine (Flu) reduced-toxicity myeloablative conditioning (MAC) has shown encouraging results in several prospective studies with low rates of nonrelapse mortality (NRM) and end-organ toxicity. 6 – 9 Unlike ex vivo or in vivo T-cell depletion, 10 most pharmacologic approaches for preventing acute GVHD (aGVHD) are less effective in preventing chronic GVHD (cGVHD). In contrast, high-dose, post-transplantation cyclophosphamide (PTCy) successfully prevents both aGVHD and cGVHD. 11 This clinical activity was demonstrated first in the reduced-intensity conditioning, human leukocyte antigen (HLA) -haploidentical alloBMT setting 12 , 13 and later as single-agent GVHD prophylaxis after busulfan/cyclophosphamide (Bu/Cy) MAC and HLA-matched-related or -unrelated allografting. 14 In this latter study, the cumulative incidences of grades 3 to 4 aGVHD and cGVHD were each 10%. 14

Unadjusted event time distributions were estimated using the Kaplan-Meier method. 28 Cumulative incidences were estimated using the Gray method. 29 Unadjusted comparisons of DFS and OS between patient groups were performed using the log-rank test. 30 Relationships between covariates and each of NRM, DFS, and OS were assessed using Bayesian survival time regression. 31 Goodness-of-fit was assessed using Bayes Information Criterion, 32 which showed that the log-normal distribution fit the data best for all time-to-event end points. In this model, log(time-to-event | covariates) was assumed to follow a normal distribution with mean β 0 + β 1 (female-into-male alloBMT) + β 2 (patient cytomegalovirus [CMV] seropositive) + β 3 (related donor) + β 4 (CR without MRD at alloBMT) + β 5 (MRD at alloBMT) + β 6 (patient age) + β 7 (total nucleated cell [TNC] dose). For each covariate that had coefficient β in this linear component, Pr(beneficial effect) was the posterior probability Pr(β > 0 | data), which quantifies the strength of association, with large (small) values associated with a longer (shorter) mean event time. Priors and posterior computations are described in the Data Supplement (Methods). All statistical computations were performed using SAS 9.2 for Windows (SAS Institute, Cary, NC) or R 2.15 (R Foundation for Statistical Computing, Vienna, Austria).

NRM was defined as patient death without disease persistence or progression. NRM and relapse were considered competing risks. Events for defining DFS were relapse or death from any cause. Competing risks for GVHD were graft failure, relapse, or death without GVHD. Patient data collection was locked for analysis on February 28, 2013.

Acute GVHD was scored centrally by a third-party reviewer (Paul Martin at FHCRC), using Modified Keystone Criteria. 20 , 21 Chronic GVHD diagnosis and grading were based on National Institutes of Health criteria 22 and were scored by third-party investigators at each participating institution. A pathologic confirmation of clinically suspected GVHD was obtained when feasible. GVHD was treated according to the standard practice at each institution.

Neutrophil engraftment was defined as the first of three consecutive days with an absolute neutrophil count ≥ 500/μL. Platelet engraftment was defined as the first day of achieving a platelet count ≥ 2 × 10 4 /μL, without receiving a platelet transfusion in the preceding 7 days. Primary graft failure was defined as failure to achieve a sustained neutrophil count of ≥ 500/μL in the absence of disease relapse. Secondary graft failure was defined as a decline in the neutrophil count to less than 500/μL after initial engraftment, which was unrelated to infection, medications, or disease relapse and was unresponsive to growth factor stimulation.

The treatment schema is shown in Figure 1 . The busulfan dose of 130 mg/m 2 IV daily for 4 days was adjusted based on measured pharmacokinetics. We used a test dose of Bu to estimate a targeted daily systemic exposure of 4,600 μMol-min at Johns Hopkins Hospital (JHH; with an acceptable range of 3,600 to 5,600 μMol-min) and a targeted daily systemic exposure of 5,000 μMol-min at MD Anderson Cancer Center (with an acceptable range of 4,700 to 5,800 μMol-min). Bu levels were adjusted in real time to a targeted steady-state concentration goal of 900 ± 100 ng/mL at Fred Hutchinson Cancer Research Center (FHCRC). Patients received 40 mg/m 2 /day IV Flu immediately before Bu on all four days to achieve synergistic cytotoxicity. 17 – 19 One or two days of rest were allowed after conditioning and before infusion of a T-cell–replete bone marrow allograft. Patients received 50 mg/kg/day IV cyclophosphamide on days +3 and +4, with the first dose starting 62 to 72 hours after the start of allograft infusion. Mesna was administered in four doses each on days +3 and +4 at a total daily dose of 80% of the cyclophosphamide dose. Supportive care measures were administered according to standard practices at each institution. Growth factors were not allowed, except in cases of delayed engraftment or secondary graft failure.

Eligibility criteria included the following: patient age 65 years or younger; bone marrow donor who was either a genotypically HLA-identical sibling, HLA-matched first-degree relative, or unrelated donor (10/10 HLA-matched at HLA-A, -B, -C, -DRB1, and -DQB1); and a high-risk hematologic malignancy as defined (Data Supplement; Table A2). Patients with Eastern Cooperative Oncology Group performance status 16 of 3 or higher, active serious infections, significant end-organ dysfunction (Data Supplement; Table A2), positive serology for HIV or human T-cell lymphotropic virus (HTLV), or pregnancy were excluded. The institutional review boards of all participating institutions approved this study. All patients provided written informed consent before enrollment.

This phase II study (clinical trial No. NCT00809276) was designed to determine whether cyclophosphamide on days +3 and +4 after transplantation would provide sufficient postgrafting immunosuppression in patients with high-risk hematologic malignancies conditioned with fludarabine and once-daily IV busulfan. To assess this in a nonrandomized fashion, three possible GVHD prophylactic regimens were considered (Data Supplement; Table A1 [online-only]). The primary end point was the cumulative incidence of grades 3 to 4 aGVHD at 100 days after alloBMT. A sequentially adaptive Bayesian design, 15 defined in terms of the 100-day grades 3 to 4 aGVHD rate compared with the historic rate of 11%, 7 , 14 was followed by a data safety monitoring board (Data Supplement; Methods). Enrollment was begun using PTCy on days +3 and +4 as the sole GVHD prophylaxis. Because there were no excess grades 3 to 4 aGVHD, all 92 patients enrolled were treated using PTCy as the only GVHD prophylaxis. Predefined secondary end points included primary and secondary graft failure, NRM, cGVHD, disease-free survival (DFS), and overall survival (OS).

The median follow-up period of surviving patients was 794 days (2.2 years; range, 0.61 to 3.52 years). At 2 years, the cumulative incidence of relapse by competing-risk analysis was 22% (95% CI, 13% to 31%), DFS probability was 62% (95% CI, 52% to 73%; Fig 3 A), and OS probability was 67% (95% CI, 58% to 77%; Fig 3 B). Relapse, DFS, and OS outcomes did not differ by donor type ( Figs 3 C and 3 D); however, disease status did have a significant impact. The 2-year cumulative incidence of relapse for patients in CR without MRD was 10% (95% CI, 2% to 19%) compared with 33% for patients in CR with MRD (95% CI, 11% to 56%) and 38% for patients with active disease (95% CI, 18% to 58%; P = .03). Correspondingly, patients in CR without MRD had a 2-year DFS of 80% (95% CI, 69% to 92%) compared with 50% for patients in CR with MRD (95% CI, 32% to 79%) and 33% for patients with active disease (95% CI, 18% to 59%; P = .0005; Fig 3 E). Likewise, patients in CR without MRD had an 80% 2-year OS (95% CI, 68% to 92%) compared with 50% for patients in CR with MRD (95% CI, 32% to 79%) and 54% for patients with active disease (95% CI, 38% to 78%; P = .019; Fig 3 F). Multivariable Bayesian analysis showed that predictors for beneficial effects on DFS were female-into-male alloBMT and being in CR without MRD at the time of alloBMT with posterior probabilities 0.93 and > 0.99, respectively ( Table 3 ). Multivariable Bayesian analysis for OS showed that being in CR without MRD was the only predictor of improved OS with posterior probability > 0.99 ( Table 3 ).

The cumulative incidence of cGVHD at 2 years was 14% (95% CI, 7% to 21%) for all patients and was lower in patients receiving HLA-matched-related (7%; 95% CI, 0% to 14%) versus HLA-matched-unrelated allografts (22%; 95% CI, 10% to 34%; P = .041; Fig 2 D). All instances of cGVHD occurred within the first year. By National Institutes of Health criteria, 22 cGVHD was moderate in 58% of patients and severe in 42% of patients. Two patients had bronchiolitis obliterans syndrome as the only manifestation of cGVHD.

Acute GVHD was treated with corticosteroids alone in 10 patients (21%), corticosteroids plus a calcineurin inhibitor (CNI) in 27 patients (57%), or a CNI alone in one patient (2%). Corticosteroids and a non-CNI agent were used in eight patients (17%), including seven treated with mycophenolate mofetil versus placebo on the Blood or Marrow Transplantation Clinical Trials Network study 0802 (randomization has not been unblinded). One patient (2%) with grade 2 aGVHD did not receive any systemic immunosuppression. Sixty-five percent of patients with grade 2 aGVHD who were alive at 1 year had stopped receiving all immunosuppressive therapy by that time and never required further immunosuppression. Overall, 35% of all patients and 39% of patients alive at last follow-up never required immunosuppression after PTCy on day +4 for any reason.

The cumulative incidence of grades 2 to 4 aGVHD at 100 days was 51% (95% CI, 41% to 61%) and was significantly higher in patients receiving HLA-matched-unrelated (60%; 95% CI, 45% to 74%) versus HLA-matched-related allografts (42%; 95% CI, 28% to 57%; P = .027; Fig 2 B). The onset of grades 2 to 4 aGVHD occurred at a median of 34 days (range, 15 to 88) after alloBMT. The cumulative incidence of grades 3 to 4 aGVHD at 100 days was 15% (95% CI, 8% to 23%) for all patients and was similar between patients who received HLA-matched-related (11%; 95% CI, 2% to 20%) or HLA-matched-unrelated allografts (19%; 95% CI, 8% to 31%; P = .44; Fig 2 C). Acute GVHD was the cause of death in two patients (2%).

The cumulative incidences of NRM were 9% and 16% at 100 days and 1 year, respectively, and did not differ by donor type ( Figure 2 A) nor by time to neutrophil engraftment (Data Supplement; Tables A4 and A5). 38 Of the 15 patients experiencing NRM ( Table 2 ), seven patients died as a result of infections (five bacterial and two respiratory viral). Of the five patients who died as a result of bacterial infections, none died before neutrophil engraftment, three died during aGVHD treatment, and one died after receiving a contaminated platelet product. Multivariable Bayesian analysis showed that patients in CR with or without MRD at alloBMT had posterior probabilities 0.92 and > 0.99, respectively, of having lower NRM compared with patients with active disease, suggesting that active disease at transplantation was associated with higher NRM ( Table 3 ). No patients died as a result of invasive fungal infection or CMV disease. Detectable peripheral blood CMV reactivation occurred in 31 patients (34%) at a median of 42 days (range, 12 to 324) after transplantation. Consistent with a recent report, 39 there were no incidents of post-transplantation lymphoproliferative disease.

Grades 3 to 4 toxicities occurring during the first 100 days after alloBMT are listed in Table 2 . The most common toxicities were grade 3 transaminase elevation and mucositis. Veno-occlusive disease occurred in two patients (2.2%) and was mild in one patient but fatal in the other. 36 , 37

The median times to neutrophil and platelet engraftment were 21 days (range, 15 to 42) and 24 days (range, 12 to 65), respectively, for patients with primary engraftment. Chimerism studies, performed in a subset of patients, showed that the vast majority of these patients were fully donor when assessed between days +60 to 100 (Data Supplement; Fig A1). Primary graft failure occurred in five patients (5.4%). All three patients who received MUD allografts and experienced primary graft failure had allograft transit times of longer than 24 hours, and two of these three allografts had low TNC doses of ≤ 1.3 × 10 8 /kg, variables that have been suggested individually to be risk factors for graft failure in unrelated allografting. 33 Four patients with primary graft failure achieved full neutrophil and platelet engraftment after second alloBMTs using standard institutional protocols. 34 , 35 The fifth patient was found to have disease relapse on day +83. Secondary graft failure occurred in two additional patients (2.2%), both of whom received MUD allografts. One fully engrafted after a reduced-intensity conditioning, HLA-haploidentical alloBMT as previously described, 13 and the other had documented disease relapse 1 month after secondary graft failure occurred.

Patient, donor, and allograft characteristics are summarized in Table 1 . A total of 92 adult patients with high-risk hematologic malignancies were treated between July 2009 and September 2011 at three institutions: JHH (42 patients), FHCRC (38 patients), and MD Anderson Cancer Center (12 patients). Median patient age was 49 years (range, 21 to 65 years). Sixty-seven patients (73%) were in morphologic CR at alloBMT, 25 patients (27%) had active disease, and 18 patients (20%) had evidence of minimal residual disease. The allograft source was a genotypically identical HLA-matched-sibling donor for 44 patients (48%), a 10/10 HLA-matched familial donor for one patient (1%), and a 10/10 HLA-matched-unrelated donor (MUD) for 47 patients (51%; Table 1 ).

DISCUSSION Section: Choose Top of page Abstract INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION << REFERENCES

In this multi-institutional study, the combination of IV Bu/Flu MAC, T-cell–replete bone marrow allografting, and PTCy as sole GVHD prophylaxis was a well-tolerated and effective platform for facilitating HLA-matched-related or -unrelated alloBMT. Although approximately half of the patients developed grades 2 to 4 aGVHD, only 15% and 14% developed grades 3 to 4 aGVHD and cGVHD, respectively. The low incidences of severe aGVHD and cGVHD observed in our study are consistent with previous trials using PTCy GVHD prophylaxis after bone marrow allografting, regardless of the specific transplantation platform used.13,14,40 Importantly, despite the low incidence of cGVHD, disease control remained excellent.

In a randomized study that compared cyclophosphamide with cyclosporine in the 1980s, cyclophosphamide was more effective at preventing cGVHD.41 But cyclophosphamide-treated patients displayed slower hematopoietic reconstitution, higher aGVHD rates, and worse overall survival primarily because of toxicity; thus, that cyclophosphamide GVHD prophylactic approach was abandoned. However, cyclophosphamide was employed quite differently in that study, being administered serially (days +1, +3, +5, +7, +9, and weekly thereafter until day +100) at a low dose (7.5 mg/kg) rather than the high-dose (50 mg/kg) on days +3 and +4 as we used in our study. The effectiveness of high-dose PTCy in several studies including this current one likely reflects the timing and intensity of dosing and the absence of concurrent prophylactic methylprednisolone, which could ablate regulatory T-cell persistence after PTCy.42

Even without prophylactic immunosuppression beyond day +4, the incidence of grades 2 to 4 aGVHD was similar in our patients to that observed using standard GVHD prophylaxis.43,44 Moreover, most of our patients had grade 2 disease, which had little impact on their ultimate clinical course. Only 15% of patients with grade 2 aGVHD went on to develop cGVHD, and approximately two thirds of patients had been taken off all immunosuppression permanently by 1 year after transplantation. Furthermore, patients with grade 2 aGVHD had encouraging 2-year DFS and OS rates of 64% (95% CI, 51% to 79%) and 70% (95% CI, 58% to 85%), respectively.

Outcomes for patients receiving MUD allografts were similar to those receiving HLA-matched-related allografts, including low rates of cGVHD despite no patients receiving prophylactic antithymocyte globulin. Furthermore, among all patients, only two patients died as a result of infections after day +100 and there were no incidents of post-transplantation lymphoproliferative disease, suggesting that PTCy may promote favorable immune reconstitution when compared with strategies utilizing either T-cell depletion or prolonged pharmacologic immunosuppression.44–46

NRM was higher in our study than was observed in initial reports of Bu/Flu conditioning with standard GVHD prophylaxis.6,7 These results suggest that the effectively higher intensity conditioning of adding PTCy to Bu/Flu may be associated with additional toxicity, but does provide cGVHD protection without requiring antithymocyte globulin. The low cGVHD rates observed in our study may be partly a result of the exclusive use of bone marrow allografts, but these cGVHD rates are still markedly lower than those reported when using bone marrow allografting without PTCy.2 Given the higher but still favorable rates of cGVHD when using peripheral blood stem cells for HLA-haploidentical alloBMT with PTCy,47 further study is needed to determine whether additional immunosuppression beyond PTCy is required when using peripheral blood stem-cell allografting for MAC, HLA-matched alloBMT.

Despite the promising outcomes seen for our patients, the optimal myeloablative conditioning approach for HLA-matched alloBMT in the context of PTCy GVHD prophylaxis is unknown. Using standard GVHD prophylaxis, retrospective data suggested a possible advantage for Bu/Flu over Bu/Cy MAC.8,48,49 However, recent prospective studies have failed to confirm this benefit with inferior results for Bu/Flu in one study50 and similar outcomes in a second study.51 When comparing our results with those of our previous trial of Bu/Cy MAC alloBMT followed by PTCy,14 mucositis rates were higher, hepatotoxicity rates including veno-occlusive disease were lower, and GVHD rates were similar.14 Primary graft failure seemed to be slightly higher using Bu/Flu than with Bu/Cy MAC and PTCy,14 but overall are in the range reported using bone marrow MUD allografting2 and may have been affected by long transit times and low TNC counts for the individual cases of primary graft failure observed in this study. DFS and OS for patients in our current study were better than those observed for patients treated with Bu/Cy MAC followed by PTCy, but the included patient populations differed, with 54% of patients in the Bu/Cy MAC study not being in CR at the time of alloBMT.14 Indeed, outcomes for patients with active disease were similar between the two studies. Prospective, randomized studies are necessary to compare these two conditioning strategies using PTCy.

Our current study, as a collaborative effort by three academic centers, has confirmed the clinical efficacy of both PTCy and IV Bu/Flu reduced-toxicity MAC as previously seen in single-institutional studies. Moreover, the lack of a requirement for immunosuppression in a significant subset of patients can facilitate the early integration of adjunct therapies to prevent relapse.52,53 This advantage of PTCy single-agent GVHD prophylaxis is critical, given our finding, similar to a recent retrospective study,54 that any amount of detectable disease at the time of alloBMT for patients in morphologic CR portends a worse prognosis. As PTCy is a promising strategy for GVHD prophylaxis in both HLA-matched and HLA-mismatched alloBMT,14,40,47 its efficacy relative to other approaches requires further study through randomized clinical trials.