Autologous haemopoietic stem-cell transplantation followed by allogeneic or autologous haemopoietic stem-cell transplantation in patients with multiple myeloma (BMT CTN 0102): a phase 3 biological assignment trial

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Autologous haemopoietic stem-cell transplantation followed by allogeneic or autologous haemopoietic stem-cell transplantation in patients with multiple myeloma (BMT CTN 0102): a phase 3 biological assignment trial Amrita Krishnan*, Marcelo C Pasquini*, Brent Logan*, Edward A Stadtmauer*, David H Vesole*, Edwin Alyea 3rd, Joseph H Antin, Raymond Comenzo, Stacey Goodman, Parameswaran Hari, Ginna Laport, Muzaffar H Qazilbash, Scott Rowley, Firoozeh Sahebi, George Somlo, Dan T Vogl, Daniel Weisdorf, Marian Ewell, Juan Wu, Nancy L Geller, Mary M Horowitz, Sergio Giralt*, David G Maloney*, on behalf of the Blood and Marrow Transplant Clinical Trials Network (BMT CTN)

Summary Background Autologous haemopoietic stem-cell transplantation (HSCT) improves survival in patients with multiple myeloma, but disease progression remains an issue. Allogeneic HSCT might reduce disease progression, but can be associated with high treatment-related mortality. Thus, we aimed to assess effectiveness of allogeneic HSCT with nonmyeloablative conditioning after autologous HSCT compared with tandem autologous HSCT. Methods In our phase 3 biological assignment trial, we enrolled patients with multiple myeloma attending 37 transplant centres in the USA. Patients (<70 years old) with adequate organ function who had completed at least three cycles of systemic antimyeloma therapy within the past 10 months were eligible for inclusion. We assigned patients to receive an autologous HSCT followed by an allogeneic HSCT (auto-allo group) or tandem autologous HSCTs (auto-auto group) on the basis of the availability of an HLA-matched sibling donor. Patients in the auto-auto group subsequently underwent a random allocation (1:1) to maintenance therapy (thalidomide plus dexamethasone) or observation. To avoid enrolment bias, we classified patients as standard risk or high risk on the basis of cytogenetics and β2-microglobulin concentrations. We used the Kaplan-Meier method to estimate differences in 3-year progression-free survival (PFS; primary endpoint) between patients with standard-risk disease in the auto-allo group and the best results from the auto-auto group (maintenance, observation, or pooled). This study is registered with ClinicalTrials.gov, number NCT00075829. Findings Between Dec 17, 2003, and March 30, 2007, we enrolled 710 patients, of whom 625 had standard-risk disease and received an autologous HSCT. 156 (83%) of 189 patients with standard-risk disease in the auto-allo group and 366 (84%) of 436 in the auto-auto group received a second transplant. 219 patients in the auto-auto group were randomly assigned to observation and 217 to receive maintenance treatment, of whom 168 (77%) completed this treatment. PFS and overall survival did not differ between maintenance and observation groups and pooled data were used. KaplanMeier estimates of 3-year PFS were 43% (95% CI 36–51) in the auto-allo group and 46% (42–51) in the auto-auto group (p=0·671); overall survival also did not differ at 3 years (77% [95% CI 72–84] vs 80% [77–84]; p=0·191). Within 3 years, 87 (46%) of 189 patients in the auto-allo group had grade 3–5 adverse events as did 185 (42%) of 436 patients in the auto-auto group. The adverse events that differed most between groups were hyperbilirubinaemia (21 [11%] patients in the auto-allo group vs 14 [3%] in the auto-auto group) and peripheral neuropathy (11 [6%] in the auto-allo group vs 52 [12%] in the auto-auto group). Interpretation Non-myeloablative allogeneic HSCT after autologous HSCT is not more effective than tandem autologous HSCT for patients with standard-risk multiple myeloma. Further enhancement of the graft versus myeloma effect and reduction in transplant-related mortality are needed to improve the allogeneic HSCT approach. Funding US National Heart, Lung, and Blood Institute and the National Cancer Institute.

Introduction High-dose chemotherapy with autologous haemopoietic stem-cell transplantation (HSCT) improves survival compared with conventional chemotherapy in patients younger than 65 years with multiple myeloma.1,2 However, despite high remission rates and improved survival, a continued risk of disease progression exists after one or two autologous HSCTs, even in patients with so-called standard-risk multiple myeloma (ie, absence of high-risk www.thelancet.com/oncology Vol 12 December 2011

cytogenetic myeloma markers and β2-microglobulin <4·0 mg/L). Investigational strategies that might improve outcomes include tandem autologous HSCT, maintenance strategies after transplantation, and allogeneic HSCT.3,4 Planned sequential autologous HSCT improves responses and survival outcomes compared with one-off autologous HSCT.5–7 Maintenance therapy with thalidomide and corticosteroids after autologous HSCT further prolongs progression-free survival (PFS) and overall survival.5,8

Lancet Oncol 2011; 12: 1195–203 Published Online September 30, 2011 DOI:10.1016/S14702045(11)70243-1 See Comment page 1176 *Authors contributed equally City of Hope Cancer Center, Los Angeles, CA, USA (A Krishnan MD, F Sahebi MD, G Somlo MD); Medical College of Wisconsin, Milwaukee, WI, USA (M C Pasquini MD, B Logan PhD, P Hari MD, M M Horowitz MD); University of Pennsylvania Abramson Cancer Center, Philadelphia, PA, USA (Prof E A Stadtmauer MD, D T Vogl MD); Hackensack University Medical Center, Hackensack, NJ, USA (Prof D H Vesole MD, Prof S Rowley MD); Dana-Farber Cancer Institute, Boston, MA, USA (E Alyea 3rd MD, Prof J H Antin MD); Tufts University and Medical Center, Boston, MA, USA (Prof R Comenzo MD); Vanderbilt University Medical Center and Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN, USA (Prof S Goodman MD); Stanford University, Stanford, CA, USA (G Laport MD); MD Anderson Cancer Research Center, Houston, TX, USA (M H Qazilbash MD); University of Minnesota, Minneapolis, MN, USA (Prof D Weisdorf MD); The EMMES Corporation, Rockville, MD, USA (M Ewell ScD, J Wu MS); National Heart, Lung, and Blood Institute, Bethesda, MD, USA (N L Geller PhD); Memorial Sloan-Kettering Cancer Center, New York, NY, USA (Prof S Giralt MD); and

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Fred Hutchinson Cancer Research Center, Seattle, WA, USA (Prof D G Maloney MD) Correspondence to: Prof David G Maloney, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D1-100, PO Box 19024, Seattle, WA 98109-1024, USA [email protected]

Allogeneic HSCT, which provides a tumour-free graft, is an alternative treatment approach to autologous transplantation that could offer additional disease control through a graft-versus-myeloma effect. Early studies of allogeneic HSCT with myeloablative conditioning regimens reported an increased frequency of molecular remissions and lower rates of relapse compared with autologous HSCT, but overall benefits were offset by high treatment-related mortality.9–12 Conversely, use of allogeneic HSCT with nonmyeloablative conditioning regimens (which are designed more for immunosuppression than for cytoreduction) after autologous HSCT for cytoreduction might evoke a graftversus-myeloma effect and reduce treatment-related mortality. A multicentre phase 2 clinical trial13 of this approach, which enrolled 54 patients, reported complete response (CR) in 57% of patients and a treatment-related mortality of 15%, which was much lower than that noted with allogeneic HSCT with myeloablative regimens. We aimed to assess a non-myeloablative allogeneic HSCT after autologous HSCT (auto-allo) approach for patients with multiple myeloma in a phase 3 trial with tandem autologous HSCT (auto-auto) as a control, which was chosen on the basis of data suggesting that such an approach had the best PFS of various transplant strategies and potentially offered an overall survival benefit.

Methods Study design and patients For the study protocol see https://web.emmes.com/ study/bmt2/protocol/0102_ protocol/0102_protocol.html See Online for webappendix

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The Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0102 trial was a phase 3 study undertaken at 37 transplant centres in the USA (see webappendix).14 Eligible patients were aged 70 years or younger, were without disease progression after start of initial therapy, and had serum bilirubin concentrations of less than twotimes the upper limit of normal, liver aminotransferase concentrations of less than three-times the upper limit of normal, left ventricular ejection fractions of more than 40%, creatinine clearances of more than 40 mL per min, and Karnofsky performance scores of more than 70%. All patients needed to have received at least three cycles of any initial systemic antimyeloma therapy and were within 10 months of initiation of this therapy; the type of induction therapy was not specified in the protocol. Eligible patients also had to have 4×10⁶ or more autologous CD34+ cells per kg available for infusion. Patients provided written informed consent. We started enrolment after review and approval of the protocol and informed consent by internal review boards at every participating centre. We allocated eligible patients to receive an autologous HSCT followed by an allogeneic HSCT (auto-allo group) or a second autologous HSCT with or without maintenance therapy (auto-auto group). Treatment group allocation was determined by biological criteria: patients were assigned to the auto-allo treatment group if they had an HLA-matched sibling donor. Treatment group allocation occurred when donor availability status was confirmed; assignment could occur

at any time from enrolment to 30 days after the first autologous HSCT. Patients without a suitable sibling donor were assigned to the auto-auto group and subsequently randomly allocated in a one-to-one ratio by use of a computer generated-randomisation list to receive 1 year of maintenance therapy with thalidomide plus dexamethasone or 1 year of observation.

Procedures Enrolled patients received melphalan 200 mg/m² and an autologous peripheral blood stem-cell infusion after 48 h. The day of first autologous HSCT was designated as day 0. Filgrastim was started at day 5 and continued until neutrophil recovery. We defined recovery from autografting as haemopoietic recovery, absence of active infection, resolution of mucositis and gastrointestinal symptoms, and absence of need for hyperalimentation and intravenous hydration. Once patients had recovered, and at least 60 days after the first autologous HSCT, patients received a second transplant according to treatment group assignment. Patients in the auto-allo group received 200 cGy of total body irradiation in one fraction and a subsequent allogeneic peripheral blood stem-cell infusion. The target cell dose for allografts was 2·0×10⁶ CD34+ cells per kg. We used ciclosporin and mycophenolate mofetil as graft-versus-host disease prophylaxis. Mycophenolate mofetil was discontinued 28 days after allogeneic HSCT. In patients without active graft-versus-host disease, use of ciclosporin was reduced (starting at 84 days). We did chimerism and engraftment analyses as previously described.4 Standard infection prophylaxis was given and cytomegalovirus reactivation was monitored and treated with ganciclovir according to institutional guidelines. Patients who did not have an HLA-matched sibling donor received a second autologous HSCT with melphalan 200 mg/m² conditioning. Stem-cell infusion was the same as for the first autologous HSCT. Patients in this group were randomly allocated to observation (for 1 year) or to receive thalidomide (200 mg per day) and dexamethasone (40 mg per day for 4 consecutive days, once a month) orally, at least 60 days after the second HSCT, for 1 year of therapy. We stratified patients by disease risk after enrolment to minimise referral bias as described elsewhere.15 Briefly, disease stage or risk classification can affect enrolment to biological assignment studies; for example, patients at high-risk of relapse are more likely to enrol if they know of a sibling donor than if they do not (due to the perception that a further round of autologous HSCT would be ineffective).15 Thus, the primary aim of our study was to assess outcomes in patients with standard-risk disease. We classified patients as having standard-risk disease after enrolment if their serum β2-microglobulin concentration was less than 4·0 mg/L and no deletion of chromosome 13 was detected by metaphase karyotyping. MCP reviewed karyotype reports centrally. Cytogenetic www.thelancet.com/oncology Vol 12 December 2011

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analysis by fluorescence in-situ hybridisation was not an eligibility prerequisite and was not done routinely at participating centres. Combined analysis of patients with high-risk and standard-risk disease was not prespecified in the planned analysis. Our primary endpoint was PFS at 3 years in patients with standard-risk disease. PFS was defined as time to disease relapse or progression, initiation of non-protocol antimyeloma therapy or death, and was measured from the time of first autologous HSCT, with patients censored at time of last contact. Supporting data for disease status and non-protocol antimyeloma therapy during the trial were centrally reviewed by an outcome adjudication committee who were masked to treatment group assignment. Secondary endpoints were overall survival, incidence of disease relapse or progression, treatment-related mortality, disease response, and incidence of grade 3–5 adverse events (defined by Common Terminology Criteria for Adverse Event version 3). Tertiary endpoints included incidence of acute and chronic graft-versus-host disease, donor–recipient chimerism, and quality of life. We used the International Uniform Response Criteria for multiple myeloma16 to define disease response, with the addition of a category of near CR, which was defined as evidence of disease by immunofixation electrophoresis without morphological evidence of bone marrow involvement by multiple myeloma.16–18 We did disease assessments before the first autologous HSCT, at time of second transplantation, and at 8 weeks, 6 months, and every 6 months after second transplantation to 3 years.

Statistical analysis Our study had two primary hypotheses to be tested in patients with standard-risk multiple myeloma: first, does maintenance therapy improve outcomes after auto-auto transplantation; and, second, does an auto-allo approach prolong PFS compared to an auto-auto approach. We aimed to enrol 150 patients with standard-risk multiple myeloma to the auto-allo group. We estimated the probability of available HLA-matched sibling donors to be 20–30%, and therefore expected 350–600 standard-risk patients would be enrolled to the auto-auto group. This sample size would provide at least 80% power to detect a difference of 15% between outcomes for the auto-allo group and the autoauto group in terms of 3-year probability of PFS, assuming a baseline probability of 45% PFS in the auto-auto group.3 We did the primary analysis for all patients according to their original assigned treatment even if they did not receive all prescribed interventions (intention-to-treat analysis). All outcomes were measured from the time of first autologous HSCT. We assessed the primary endpoint in two stages. First, 3-year PFS was compared between patients in the maintenance groups (thalidomide plus dexamethasone vs observation) in terms of percentage difference in KaplanMeier estimates at 3 years. If no significant differences www.thelancet.com/oncology Vol 12 December 2011

were noted, we planned to pool data for the auto-auto group and compare 3-year PFS between the autoauto group and the auto-allo group. Otherwise, the maintenance group with the best results would be compared with the auto-allo group. We estimated survival distributions with the Kaplan-Meier method and treatmentrelated mortality, relapse and progression, and acute and chronic graft-versus-host disease in terms of cumulative incidence. We compared percentage differences in survival at 3 years with Gray’s test of cumulative incidence. To address concerns of covariate imbalance in our biological assignment trial, we did a protocol-specified secondary analysis of PFS with a Cox proportional hazards model.19 This model included some or all of the prospectively defined variables if their distribution differed (p<0·1) between the auto-auto and auto-allo groups at baseline; these variables were β2-microglobulin concentration, Karnofsky performance status scores, and age. Monitoring for accrual and toxicity according to sequential probability ratio stopping guidelines was undertaken by the US National Heart, Lung, and Blood Institute (NHLBI) appointed data and safety monitoring board. Patients assigned to the auto-allo group at every centre were monitored as described elsewhere.15 Treatment-related mortality was monitored with the null hypotheses that the frequency at 6 months would be less than 5% after autologous HSCT and less than 20% after an allogeneic HSCT, and that the frequency at 9 months would be less than 20% in patients aged 65 years or older. Specific toxic effects were monitored separately in both treatment groups, including deep-vein thrombosis, sensory neuropathy, and ciclosporin-associated renal and hepatic toxic effects. We did an unplanned post-hoc analysis to assess the effect of graft-versus-host disease on relapse or progression 710 patients met eligibility criteria and received first autologous HSCT 625 were at standard risk 85 were at high risk

226 had an HLA-matched sibling donor (auto-allo group) 189 were at standard risk 37 were at high risk

484 did not have an HLA-matched sibling donor (auto-auto group) 436 were at standard risk 48 were at high risk

185 received allogeneic HSCT 156 were at standard risk 29 were at high risk

397 received second autologous HSCT 366 were at standard risk 31 were at high risk

234 randomly allocated to observation 219 were at standard risk 15 were at high risk

233 randomly allocated to thalidomide plus dexamethasone 217 were at standard risk 16 were at high risk

Figure 1: Trial profile HSCT=haemopoietic stem-cell transplantation.

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Standard risk

Sex (male)

High risk

Auto-auto group (n=436)

Auto-allo group (n=189)

p value

260 (60%)

111 (59%)

0·833

Median age (years)

p value

Auto-auto group (n=48)

Auto-allo group (n=37)

27 (56%)

21 (57%)

57 (32–70)

51 (32–66) 0·012

0·963

55 (22–70)

53 (29–68) 0·006

Black

77 (18%)

18 (10%)

··

8 (17%)

3 (8%)

··

White

335 (77%)

161 (85%)

··

38 (79%)

33 (89%)

··

Other

24 (6%)

10 (5%)

··

2 (4%)

1 (3%)

··

Karnofsky performance score ≥90%

334 (77%)

148 (78%)

0·642

27 (56%)

26 (70%)

0·186

Median β2-microglobulin concentrations (mg/L)

2·0 (IQR 1·6–2·4)

2·0 (IQR 1·6–2·5)

0·949

4·4 (IQR 2·8–5·1)

3·7 (IQR 2·0–4·8)

0·035

I–II

142 (33%)

59 (31%)

··

10 (21%)

9 (24%)

III

294 (67%)

130 (69%)

··

38 (79%)

28 (76%)

7 (3–55)

7 (4–35)

7 (5–22)

7 (3–38)

1 (2%)

3 (8%)

Ethnic origin

0·032

Durie-Salmon stage

0·458

0·739

Median time from diagnosis to transplantation (months)

0·925

0·702 ·· ·· 0·683

Disease status* Complete response

41 (9%)

24 (13%)

Near complete response

65 (15%)

22 (12%)

··

1 (2%)

2 (5%)

··

Very good partial response

79 (18%)

32 (17%)

··

1 (2%)

7 (19%)

··

158 (36%)

76 (40%)

··

21 (44%)

14 (38%)

··

31 (7%)

17 (9%)

··

10 (21%)

3 (8%)

·· ··

Partial response Minimal response

··

Stable disease

21 (5%)

6 (3%)

··

6 (13%)

4 (11%)

Not assessable†

41 (9%)

12 (6%)

··

8 (17%)

4 (11%)

Median time from first to 98 second transplantation (days) (58–193)

105 (61–262)

0·004

101 (63–155)

111 (61–155)

··

·· 0·397

Data are n (%) or median (range), unless otherwise stated. Patients in the auto-allo group received autologous followed by allogeneic haemopoietic stem-cell transplants. Patients in the auto-auto group received two autologous haemopoietic stem-cell transplants. *Disease status at time of first autologous transplantation was assigned at the initial multiple myeloma treatment received before trial enrolment; details of induction regimens were not collected; for standard-risk patients, p=0·678 of very good partial response or better differing between auto-auto and auto-allo patients and for high-risk patients p=0·028. †Patients who did not have available comprehensive disease assessment at time immediately before initial multiple myeloma treatment.

Table 1: Demographic characteristics of enrolled patients

Auto-auto group (n=436) Auto-allo group (n=189) Number not receiving second transplant

70*

33

Reasons for no second transplant Adverse event (grade 3–5)

4 (6%)

Death

4 (6%)

1 (3%)

10 (14%)

6 (18%)

Progression or relapse

1 (3%)

Insurance coverage denied

2 (3%)

Inadequate physical recovery from first transplant

2 (3%)

2 (6%)

43 (61%)

19 (58%)

Patient refused or withdrew consent

0

Other

4 (6%)†

4 (12%)‡

Unknown

1 (1%)

0

*One patient assigned to the auto-auto group did not receive the first transplant because of disease progression. †One nocardia infection and three due to doctor’s decision. ‡One donor did not mobilise sufficient cell dose according to protocol, one cardiac amyloidosis diagnosed after the first transplantation, one pyschosocial issues, and one donor deemed not medically eligible to donate.

Table 2: Reasons for not proceeding to second transplantation or maintenance therapy in patients with standard-risk disease

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in patients receiving an allogeneic HSCT. We analysed development of chronic graft-versus-host disease as a time dependent covariate in a Cox regression model of disease relapse or progression. Statistical analyses were done with with SAS version 9.2 and R version 2.10.0. This study is registered with ClinicalTrials.gov, number NCT00075829.

Role of the funding source Statisticians from the US NHLBI were members of the protocol team and assisted in the development of the study design. The NHLBI and National Cancer Institute had access to data provided to the data and safety monitoring board. The NHLBI revised and offered input to this report. DGM, MCP, BL, and JW had full access to all the data in the study and DGM had final responsibility for the decision to submit for publication.

Results Between Dec 17, 2003, and March 30, 2007, we enrolled 710 patients with multiple myeloma who had completed initial antimyeloma therapy. The median follow-up was 40 months (IQR 38–43). No patient progressed or died before assignment to treatment groups. Figure 1 shows the trial profile, including treatment-group assignment and disease-risk classifications. Table 1 shows demographic data for all patients. Patients in the auto-allo group were more likely to be white or younger than were those in the auto-auto group (table 1). Patients at standard risk in the auto-allo group had longer waits from first to second transplantation than did those in the auto-auto group (table 1). However, we noted no difference in proportions of patients with disease statuses of very good partial response or better before the first autologous HSCT (table 1). 156 (83%) of the 189 standardrisk patients in the auto-allo group received the second transplant, as did 366 (84%) of 436 standard-risk patients in the auto-auto group (figure 1). Table 2 lists reasons for not undergoing a second transplantation. 59 (27%) of 217 standard-risk patients randomly allocated to receive thalidomide and dexamethasone refused to start maintenance and 168 (77%) did not complete this course of treatment. Cumulative incidence of premature discontinuation of maintenance therapy was 84% (95% CI 79–90). The Kaplan-Meier probability of 3-year PFS after first autologous HSCT for standardrisk patients randomly allocated to thalidomide plus dexamethasone group was 49% (95% CI 43–57) compared with 43% (37–50) in the observation group (p=0·080); 3-year overall survival was 80% (75–86) in the thalidomide plus dexamethasone group and 81% (75–86) in the observation group (p=0·817). Therefore, the maintenance groups were combined into one autoauto group for comparison with the auto-allo group. For the 116 standard-risk patients who tolerated thalidomide plus dexamethasone, the median duration of maintenance therapy was 256 days (range 3–466). www.thelancet.com/oncology Vol 12 December 2011

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A

B

100

Auto-auto group Auto-allo group

90 Progression-free survival (%)

80

3-year OS 80% (77–84)

3-year PFS 46% (95% CI 42–51)

3-year OS 77% (72–84) Overall survival (%)

70 60 50 40

3-year PFS 43% (36–51)

30 20 10 p=0·191

p=0·671 0 Number at risk Auto-auto group Auto-allo group

436 189

395 165

348 138

292 117

242 105

213 89

178 71

54 23

42 16

436 189

424 183

406 167

395 160

370 156

348 143

305 124

107 43

79 27

D

C 100

p=0·402

p<0·0001

90 Treatment-related mortality (%)

Progression or relapse (%)

80 70 60

3-year progression or relapse 50% (46–55)

50 40

3-year progression or relapse 46% (39–54)

30 20

3-year TRM 11% (7–16)

10

3-year TRM 4% (2–5)

0 Number at risk Auto-auto group Auto-allo group

0

6

12

18

24 Months

30

36

42

48

0

436 189

395 165

348 138

292 117

242 105

213 89

178 71

54 23

42 16

436 189

6

12

18

395 165

348 138

292 117

24 30 Months 242 105

213 89

36

42

48

178 71

54 23

42 16

Figure 2: Progression-free survival (A), overall survival (B), cumulative incidence of disease progression (C), cumulative incidence of transplant-related mortality (D) in patients with standard-risk disease in auto-auto and auto-allo groups PFS=progression-free survival. OS=overall survival. TRM=treatment-related mortality.

Kaplan-Meier estimates of 3-year PFS for standard-risk patients did not differ between auto-allo and auto-auto groups (p=0·671; figure 2). Of the three prespecified baseline variables, only age differed significantly between treatment groups and was included in the multivariate Cox regression analysis. The hazard ratio (HR) of treatment failure (death or relapse or progression), adjusted for age, for patients assigned to the auto-allo group versus the auto-auto group was 1·17 (95% CI 0·94–1·46, p=0·17). Overall survival did not differ between groups at 1 year (91% [95% CI 87–95] in the auto-allo group vs 95% [93–97] in the auto-auto group, p=0·085), 2 years (85% [80–91] vs 89% [86–92], p=0·176), or 3 years (77% [72–84] vs 80% [77–84], p=0·191; figure 2). Cumulative incidences of disease relapse or progression did not differ between groups at 3 years; however, corresponding incidence of 3-year treatment-related mortality was higher in the auto-allo group than it was in the auto-auto group (p<0·0001; figure 2). www.thelancet.com/oncology Vol 12 December 2011

Nearly 10% more standard-risk patients in the auto-allo group than the auto-auto group had a CR, but rates of very good partial responses or better did not differ between groups (table 3). For 415 patients not in CR before the second transplantation, almost half of the patients in the auto-allo group and just over a third of those in the autoauto group upgraded their disease status to CR (table 3). Within 3 years, 87 (46%) of 189 standard-risk patients in the auto-allo group had grade 3–5 adverse events (78 [41%] grade 3, 29 [15%] grade 4, and 12 [6%] grade 5), as did 185 (42%) of 436 standard-risk patients in the auto-auto group (156 [36%] grade 3, 39 [9%] grade 4, and 21 [5%] grade 5). Table 4 lists specific grade 3–5 toxic effects seen in the standard-risk population at 1 year and 3 years after the second transplantation. Grade 3–5 toxic effects for patients in the auto-auto group were much the same apart from grade 3–5 neuropathy in 39 (18%) of 217 patients allocated to the thalidomide plus dexamethasone group and 24 (11%) of 219 patients assigned to observation. 1199

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Complete responses

Very good partial response* or better

Auto-auto group

Auto-allo group

p value

Auto-auto group

Auto-allo group

p value

All patients (n=625)

175/436 (40%)

94/189 (50%)

Patients not in complete response (n=560)†

142/395 (36%)

74/165 (45%)

0·026

285/436 (65%)

118/189 (62%)

0·482

0·049

252/395 (64%)

98/165 (59%)

0·327

All patients (n=522)

164/366 (45%)

Patients not in complete response (n=415)‡

102/295 (35%)

90/156 (58%)

0·007

272/366 (74%)

113/156 (72%)

0·655

58/120 (48%)

0·009

210/295 (71%)

81/120 (68%)

0·457

First haemopoietic stem-cell transplantation

Second haemopoietic stem-cell transplantation

*Includes very good partial response, near complete response, and complete response. †Patients with less than complete response at study entry. ‡Patients who were not in complete remission immediately before the second transplantation.

Table 3: Disease responses in patients with standard-risk disease, by treatment group

1 year

Abnormal liver enzymes

3 years

Auto-auto group (n=436)

Auto-allo group (n=189)

Auto-auto group (n=436)

Auto-allo group (n=189)

Grade 3–4

Grade 3–4

Grade 5

Grade 3–4

Grade 3–4

25 (6%)

11 (3%)

Grade 5 0

9 (5%)

0

Grade 5 0

17 (9%)

Grade 5 0

Ataxia

1 (<1%)

0

1 (1%)

2 (1%)

1 (<1%)

0

1 (1%)

Capillary leak syndrome*

0

0

3 (2%)

1 (1%)

0

0

3 (2%)

1 (1%)

Cardiac arrhythmia

6 (1%)

1 (<1%)

1 (1%)

1 (1%)

6 (1%)

1 (<1%)

1 (1%)

1 (1%)

Deep-vein thrombosis

14 (3%)

0

Dyspnoea

24 (6%)

1 (<1%)

6 (3%)

1 (1%)

14 (3%)

10 (5%)

7 (4%)

41 (9%)

0 15 (3%)

2 (1%)

6 (3%)

1 (1%)

25 (13%)

10 (5%)

Haemorrhage

1 (<1%)

0

0

1 (1%)

1 (<1%)

0

0

Haemorrhagic cystitis

2 (<1%)

0

3 (2%)

0

4 (1%)

3 (1%)

3 (2%)

Hyperbilirubinemia*

9 (2%)

0

20 (11%)

0

14 (3%)

0

21 (11%)

0

Hyperglycaemia*

20 (5%)

0

20 (11%)

0

20 (5%)

0

20 (11%)

0

Hypertension*

10 (2%)

0

17 (9%)

0

10 (2%)

0

17 (9%)

0

Hypotension

10 (2%)

2 (<1%)

5 (3%)

3 (2%)

10 (2%)

2 (<1%)

Hypoxia

19 (4%)

0

7 (4%)

5 (3%)

27 (6%)

Left ventricular dysfunction

1 (<1%)

1 (1%)

2 (1%)

Mucositis*

32 (7%)

1 (<1%)

0

4 (2%)

0

1 (<1%)

Peripheral motor neuropathy

10 (2%)

0

5 (3%)

Peripheral sensory neuropathy*

33 (8%)

0

3 (2%)

0

0

0

1 (<1%)

14 (3%)

1 (1%) 1 (1%)

5 (3%)

3 (2%)

14 (7%)

9 (5%)

1 (<1%)

1 (1%)

2 (1%)

32 (7%)

0

4 (2%)

0

1 (1%)

20 (5%)

3 (1%)

15 (8%)

1 (1%)

49 (11%)

3 (1%)

9 (5%)

2 (1%)

0

0

0

2 (1%)

Seizures

1 (<1%)

Somnolence

5 (1%)

1 (<1%)

4 (2%)

3 (2%)

5 (1%)

1 (<1%)

4 (2%)

3 (2%)

Thrombotic microangiopathy

1 (<1%)

0

1 (1%)

1 (1%)

1 (<1%)

0

1 (1%)

1 (1%)

Data are for toxic effects reported from day 0 of second transplantation to 1 year and 3 years. *Incidence differs significantly (p<0·05).

Table 4: Grade 3–5 toxic effects in patients with standard-risk disease, by treatment group after second transplantation

10 (6%) of the 156 standard-risk patients in the auto-allo group who received allogeneic HSCT had graft failure after such transplantation. Two patients failed to engraft and eight had secondary graft failure. Six secondary failures occurred before 100 days and two before 365 days. Overall donor chimerism was 91% at 28 days, 97% at 56 days, 98% at 84 days, 100% at 180 days, and 100% at 365 days. For standard-risk patients in the auto-allo group, the cumulative incidence of grade II–IV acute graft-versus-host disease at 100 days was 26% (95% CI 19–33) and for grade III–IV disease was 9% (4–14); cumulative incidence of chronic graft-versus-host disease was 47% (39–56) at 1 year and 54% (46–63) at 2 years. The relative risk of disease relapse or progression was 0·44 (95% CI 0·24–0·81, 1200

p=0·008) for patients with chronic graft-versus-host disease compared with those without such disease. For recipients of autologous and allogeneic transplants who were alive 6 months after the second transplantation, 29 patients who developed chronic graft-versus-host disease in the first 6 months had a cumulative incidence of disease relapse or progression of 17% (6–37) after 3 years compared with 39% (30–48) for 99 patients who did not develop chronic graftversus-host disease by 6 months (p=0·012). 60 (32%) of the 189 standard-risk patients in the autoallo group and 108 (25%) of the 436 standard-risk patients in the auto-auto group died. The main cause of death was multiple myeloma, accounting for 22 (37%) of 60 deaths in the auto-allo group and 78 (72%) of 108 deaths in the www.thelancet.com/oncology Vol 12 December 2011

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A

B

Auto-auto group Auto-allo group

100 90

3-year OS 67% (54–82)

70

Overall survival (%)

Progression-free survival (%)

80

60 50

3-year PFS 40% (95% CI 27–60)

40

3-year OS 59% (45–78)

30 20 3-year PFS 33% (22–50)

10

p=0·743

p=0·460

0 Number at risk Auto-auto group Auto-allo group

0

6

12

18

24 Months

30

36

42

48

0

6

12

18

48 37

39 30

33 20

23 18

20 15

17 14

14 13

3 4

2 4

48 37

42 35

40 25

37 24

24 30 Months 36 22

31 22

36

42

48

27 20

8 5

5 5

Figure 3: Progression-free survival (A) and overall survival (B) in patients with high-risk disease in auto-auto and auto-allo groups PFS=progression-free survival. OS=overall survival.

auto-auto group. Other causes of death in the auto-allo group were organ failure (12 patients [20%]), infection (10 patients [17%]), graft-versus-host disease (eight patients [13%]), idiopathic pneumonia syndrome (three patients [5%]), acute respiratory distress syndrome (two patients [3%]), graft failure (one patient [2%]), secondary malignancy (one patient [2%]), and suicide (one patient [2%]). In the auto-auto group, other causes were organ failure (15 patients [14%]), infection (two patients [2%]), graft-versus-host disease (two patients [2%]), secondary malignancy (one patient [1%]), pneumonia (one patient [1%]), pulmonary haemorrhage (one patient [1%]), toxic megocolon (one patient [1%]), post-transplant lymphoproliferative (one patient [1%]), suicide (one patient [1%]), accidental death (one patient [1%]), and unknown causes (four patients [4%]). Table 1 shows demographics of patients with high-risk disease. Patients with high-risk disease in the auto-allo group were younger than were those in the auto-auto group and progressive response was the most frequent disease status at study entry in both treatment groups. Kaplan-Meier estimates of 3-year PFS did not differ between treatment groups in patients with high-risk disease (figure 3). The cumulative incidence of disease relapse or progression at 3 years was 38% (95% CI 22–54) in patients with high-risk disease in the auto-allo group and 57% (42–71) in the auto-auto group (p=0·079); corresponding incidences of 3-year treatment-related mortality were 22% (95% CI 8–35) and 11% (2–19, p=0·311).

Discussion In our trial of autologous HSCT followed by allogeneic HSCT with a non-myeloablative conditioning regimen in patients with standard-risk multiple myeloma, we did not show a benefit in 3 year PFS or overall survival compared with tandem autologous transplantation. We show indirect evidence of a graft-versus-myeloma effect www.thelancet.com/oncology Vol 12 December 2011

with a 60% reduction in risk of relapse in patients diagnosed with chronic graft-versus-host disease. Treatment-related mortality was higher in the auto-allo group (11%) than the auto-auto group (4%), despite use of a non-myeloablative conditioning regimen; chiefly due to graft-versus-host disease and infections. Risks of progression or relapse did not differ between groups. Although much the same proportion of patients in both groups had very good partial responses or better, more patients in the auto-allo group upgraded their response to a CR. Differences in toxicity and causes of death are inherent to the transplantation approach. Recipients of the allogeneic transplant had more complications related to organ dysfunction and immune deregulation as a result of the use of chronic immunosuppression and the development or treatment of graft-versus-host disease. Recipients of autologous HSCT had more neuropathy related to thalidomide use. Our present analysis did not assess quality of life between treatment groups, which is a planned future analysis. We noted no benefit of maintenance therapy in this trial, but because compliance with thalidomide plus dexamethasone was poor, definitive conclusions about the role of drug maintenance cannot be made. Previous trials showed benefit of thalidomide maintenance on survival outcomes,5,20,21 but compliance with maintenance was also a challenge in those trials. For example, in the Intergroupe Français du Myélome (IFM) 99-02 trial5 (in which most patients were not exposed to thalidomide at induction), two-fifths of patients discontinued thalidomide due to toxic effects. Thalidomide maintenance in the IFM 99-02 trial was planned to continue until disease progression, however the median time on maintenance was 15 months, which is much shorter than the median event-free survival of 37 months.5 Thus, presumed previous thalidomiderelated neuropathy could be a reason for an increased rate of non-compliance in our trial. 1201

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Panel: Research in context Systematic review We searched PubMed for articles published in English with the search terms “multiple myeloma”, “allogeneic transplantation”, “autologous transplantation”, “maintenance therapy”, and “thalidomide therapy”. Previous trials3,9–14 investigating haemopoietic stem-cell transplantation (HSCT) for treatment of patients with multiple myeloma have suggested potential benefits of autologous5–7 and allogeneic strategies.9–12 Other trials addressing the role of autologous followed by allogeneic HSCTs have been reported, but our investigation is the largest phase 3 trial to date to assess the effectiveness of such treatment compared with two autologous HSCTs with or without pharmacological maintenance. Interpretation On the basis of previous trials, we aimed to assess allogeneic HSCT as follow-up treatment to autologous HSCT for patients with standard-risk multiple myeloma, which was shown to be feasible and had promising early results.15 Choice of the most appropriate comparative treatment group was based on data for a tandem autologous HSCT strategy used by the Intergroupe Francais du Myelome (IFM).3 During the trial-design phase of our study, data for use of thalidomide became available suggesting a potential maintenance role.5 Early reports from investigators from the IFM group also suggested that thalidomide administration after HSCT was possible and well tolerated,5 and thus we included a pharmacological randomisation in the control arm. An Italian group randomly allocated all patients with available sibling donors but had a substantial dropout of patients from enrolment to start of intervention. Nevertheless, this trial22 showed an event-free survival and overall survival benefit of the allogeneic approach, albeit in a small population. Conversely, although again in a small population, a Spanish group did not report a survival benefit with an autologous followed by allogeneic approach.23 In 2011, the European Blood and Marrow Transplant group trial24 updated their long-term results and showed autologous followed by allogeneic approach was better than one-off or double autologous HSCT. This study24 analysed patients irrespective of disease risk and differences in survival outcomes did not emerge until beyond 24 months. However, the overall survival benefit in the intervention group was minimised in the as-treated analysis, and there was a significant benefit in risk reduction with time in the intervention group. Overall these trials suggest that the outcome of an auto-allo approach in multiple myeloma is at a minimum the same as a tandem autologous HSCT approach. Some studies with longer follow up showed additional survival benefit with auto-allo. Although outcomes still need improvement to further decrease transplant-related mortality and to reduce disease progression by maximising the graft-versus-myeloma effect seen with allogeneic HSCT. Clinicians need to understand the differences in all these trials to correctly interpret the results before deciding to offer an allogeneic HSCT to patients with available sibling donors.

We did a biological assignment rather than a randomisation procedure and used several methods to reduce selection bias,15 including separation of the cohort by disease risk, use of planned multivariate analysis adjustments for prespecified demographic characteristics, and active monitoring of auto-allo enrolment at every centre. Despite limitations of biological assignment trials, both groups had comparable demographic characteristics, and adjusted and unadjusted comparisons of the primary endpoint had equivalent results. Notably, separation by disease risk after enrolment resulted in a more balanced ratio of donor versus no-donor cohorts in the standard-risk group compared with high-risk group. Three biological assignment trials22–24 have compared auto-allo with auto-auto HSCT approaches in multiple myeloma (panel). Bruno and colleagues22,25 showed 1202

improved event-free survival (EFS) and overall survival in 58 patients in the auto-allo group and 46 patients in the auto-auto group. Biological assignment in their trial was done in patients with siblings. However, there was a fairly high dropout rate, with only 46 of 82 patients in the autoauto group receiving two transplants. Superiority of the auto-allo group in this trial could be attributed to a small number of patients at risk beyond 2 years. Furthermore, the second transplant-conditioning regimen consisted of varying doses of melphalan (as low as 100 mg/m²). These factors might account for the low CR rate (26%) in the auto-auto group. In the Spanish group trial (PETHEMA),23 biological assignment occurred after the first transplantation in 110 patients not in CR or near CR and 25 patients received an allogeneic HSCT. Median EFS and overall survival were equivalent in the allo-auto and autoauto group. Bjorkstrand and colleagues24 showed extended PFS and overall survival with an auto-allo approach with a combination of fludarabine and total-body irradiation of 200 cGy in patients followed up beyond 3 years. Outcomes in the autologous HSCT group seemed inferior to those in our trial at 48 months, possibly because of a combination of one and tandem autologous HSCT as the control group, and inclusion of patients with high-risk disease as defined in our trial. Additionally, in their as-treated analysis, overall survival in 91 recipients of auto-allo transplants was much the same as in the autologous HSCT group. Differences in EFS reported in the Italian group trial22 occurred after 30 months. CR rates were higher in the auto-allo group in both the Italian group22 and BMT CTN14 trials, and thus longer follow up of patients will be important to assess whether the reported differences in CR rates will translate into improved long-term survival, as shown in other multiple myeloma trials.1,3,24 The IFM ran two parallel phase 2 trials in patients with high-risk multiple myeloma, defined by deletion of chromosome 13 or β2-microglobulin of more than 3 mg/L. IFM 99-03 analysed patients with available HLAmatched sibling donors who received an allogeneic HSCT with reduced intensity conditioning after an autologous HSCT. Outcomes for these 65 patients were compared with 219 patients enrolled in IFM 99-04, which was a trial of tandem autologous HSCT. Akin to our trial, EFS and overall survival did not differ between patients receiving auto-allo transplants and auto-auto transplants in the IFM trials.26 The transplant-related mortality of 10·9% was also very similar to our trial; however, the relapse or progression rate was strikingly high at 56·5% in the autoallo group. Despite the definition of high-risk disease in our trial not including the most recent cytogenetic markers of poor prognosis, patients with high-risk disease had worse outcomes than did patients with standard risk. Although restricted by a small number of patients, the comparison in high-risk disease group showed much the same survival outcomes. In conclusion, we reported no improvement in PFS or overall survival in patients with standard-risk multiple www.thelancet.com/oncology Vol 12 December 2011

Articles

myeloma assigned to auto-allo HSCT versus auto-auto HSCT at 3 years. Disease relapse is a key difficulty in both groups suggesting that additional treatments before and after transplantation to reduce the risk of relapse are warranted.27–29 Our trial is the largest to date to address the role of allogeneic HSCT in standard-risk multiple myeloma and absence of superiority compared with an auto-auto approach should form part of the treatment decision discussions with patients. Nonetheless, this trial offers glimpses of the anti-tumour activity of the allogeneic HSCT approach as seen in the reduced risk of relapse in patients with chronic graft-versus-host disease. Further improvements in allogeneic HSCT to reduce treatment-related mortality and augment graft-versusmyeloma effects will prompt future investigation. Contributors DHV, FS, EAS, ME, NLG, MMH, SGi, and DGM developed the protocol. AK, MCP, EAS, DHV, ME, MMH, SGi, and DGM undertook the study. AK, EA, JHA, RC, SGo, PH, GL, MHQ, SR, GS, DTV, DW, SGi, and DGM enrolled patients. AK, MCP, DHV, EAS, NLG, JW, and DGM were the endpoint review committee. MCP, BL, and JW did the data analysis. AK, MCP, DHV, EAS, NLG, MMH, DGM, and DTV interpreted the results. AK, MCP, JHA, EAS, DHV, DTV, MMH, NLG, and DGM prepared the manuscript; AK, MCP, DHV, and DGM did the literature review; and MCP, JW, and BL drew the figures and tables. Conflicts of interest AK is on the speakers’ bureau for Celgene Pharmaceuticals and Millennium Pharmaceutics and has consulted for Celgene Pharmaceuticals. DGM is a consultant for Celgene Pharmaceuticals. GS is on the Advisory of Board of Celgene Pharmaceuticals. SGi is on the Advisory of board of Celgene, Millennium, AMGEN and ONYX Pharmaceuticals; and receives research support from Celgene Pharmaceuticals. The other authors declare that they have no conflicts of interest. References 1 Attal M, Harousseau JL, Stoppa AM, et al, for the Intergroupe Français du Myélome. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. N Engl J Med 1996; 335: 91–97. 2 Child JA, Morgan GJ, Davies FE, et al. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 2003; 348: 1875–83. 3 Attal M, Harousseau JL, Facon T, et al. Single versus double autologous stem-cell transplantation for multiple myeloma. N Engl J Med 2003; 349: 2495–502. 4 Lokhorst HM, van der Holt B, Zweegman S, et al. A randomized phase 3 study on the effect of thalidomide combined with adriamycin, dexamethasone, and high-dose melphalan, followed by thalidomide maintenance in patients with multiple myeloma. Blood 2010; 115: 1113–20. 5 Attal M, Harousseau JL, Leyvraz S, et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood 2006; 108: 3289–94. 6 Barlogie B, Tricot GJ, van Rhee F, et al. Long-term outcome results of the first tandem autotransplant trial for multiple myeloma. Br J Haematol 2006; 135: 158–64. 7 Cavo M, Tosi P, Zamagni E, et al. Prospective, randomized study of single compared with double autologous stem-cell transplantation for multiple myeloma: Bologna 96 clinical study. J Clin Oncol 2007; 25: 2434–41. 8 Barlogie B, Tricot G, Anaissie E, et al. Thalidomide and hematopoietic-cell transplantation for multiple myeloma. N Engl J Med 2006; 354: 1021–30. 9 Bensinger WI, Buckner CD, Anasetti C, et al. Allogeneic marrow transplantation for multiple myeloma: an analysis of risk factors on outcome. Blood 1996; 88: 2787–93.

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Gahrton G, Tura S, Ljungman P, et al. Prognostic factors in allogeneic bone marrow transplantation for multiple myeloma. J Clin Oncol 1995; 13: 1312–22. Gahrton G, Tura S, Ljungman P, et al, and for the European Group for Bone Marrow Transplantation. Allogeneic bone marrow transplantation in multiple myeloma. N Engl J Med 1991; 325: 1267–73. Reynolds C, Ratanatharathorn V, Adams P, et al. Allogeneic stem cell transplantation reduces disease progression compared to autologous transplantation in patients with multiple myeloma. Bone Marrow Transplant 2001; 27: 801–07. Maloney DG, Molina AJ, Sahebi F, et al. Allografting with nonmyeloablative conditioning following cytoreductive autografts for the treatment of patients with multiple myeloma. Blood 2003; 102: 3447–54. Weisdorf D, Carter S, Confer D, Ferrara J, Horowitz M. Blood and marrow transplant clinical trials network (BMT CTN): addressing unanswered questions. Biol Blood Marrow Transplant 2007; 13: 257–62. Logan B, Leifer E, Bredeson C, et al. Use of biological assignment in hematopoietic stem cell transplantation clinical trials. Clin Trials 2008; 5: 607–16. Durie BG, Harousseau JL, Miguel JS, et al. International uniform response criteria for multiple myeloma. Leukemia 2006; 20: 1467–73. Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 2005; 352: 2487–98. Lahuerta JJ, Martinez-Lopez J, Serna JD, et al. Remission status defined by immunofixation vs. electrophoresis after autologous transplantation has a major impact on the outcome of multiple myeloma patients. Br J Haematol 2000; 109: 438–46. Cox D. Regression models and life tables. J R Stat Soc Series B Stat Methodol 1972; 4: 187–200. Barlogie B, Shaughnessy JD, Crowley J. Duration of survival in patients with myeloma treated with thalidomide. N Engl J Med 2008; 359: 210–12. Barlogie B, Tricot G, Anaissie E, et al. Thalidomide and hematopoietic-cell transplantation for multiple myeloma. N Engl J Med 2006; 354: 1021–30. Bruno B, Rotta M, Patriarca F, et al. A comparison of allografting with autografting for newly diagnosed myeloma. N Engl J Med 2007; 356: 1110–20. Rosiñol L, Perez-Simon JA, Sureda A, et al. A prospective PETHEMA study of tandem autologous transplantation versus autograft followed by reduced-intensity conditioning allogeneic transplantation in newly diagnosed multiple myeloma. Blood 2008; 112: 3591–93. Bjorkstrand B, Iacobelli S, Hegenbart U, et al. Tandem autologous/ reduced-intensity conditioning allogeneic stem-cell transplantation versus autologous transplantation in myeloma: long-term follow-up. J Clin Oncol 2011; 29: 3016–22. Bruno B, Storer B, Patriarca F, et al. Long-term follow up of a comparison of non-myeloablative allografting with autografting for newly diagnosed myeloma. Blood 2011; 117: 6721–27. Garban F, Attal M, Michallet M, et al. Prospective comparison of autologous stem cell transplantation followed by dose-reduced allograft (IFM99-03 trial) with tandem autologous stem cell transplantation (IFM99-04 trial) in high-risk de novo multiple myeloma. Blood 2006; 107: 3474–80. Kroger N, Shimoni A, Zagrivnaja M, et al. Low-dose thalidomide and donor lymphocyte infusion as adoptive immunotherapy after allogeneic stem cell transplantation in patients with multiple myeloma. Blood 2004; 104: 3361–63. Lioznov M, El-Cheikh J Jr, Hoffmann F, et al. Lenalidomide as salvage therapy after allo-SCT for multiple myeloma is effective and leads to an increase of activated NK (NKp44(+)) and T (HLA-DR(+)) cells. Bone Marrow Transplant 2010; 45: 349–53. Minnema MC, van der Veer MS, Aarts T, Emmelot M, Mutis T, Lokhorst HM. Lenalidomide alone or in combination with dexamethasone is highly effective in patients with relapsed multiple myeloma following allogeneic stem cell transplantation and increases the frequency of CD4+Foxp3+ T cells. Leukemia 2009; 23: 605–07.

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